key: cord- -c jxj b authors: memish, ziad a.; al-tawfiq, jaffar a. title: middle east respiratory syndrome coronavirus infection control: the missing piece? date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: c jxj b nan since the initial occurrence of middle east respiratory syndrome coronavirus (mers-cov) in , , the disease had caused cases, with a case fatality rate of . %. as with any emerging infectious diseases of pandemic potential there is a concern of the global spread of the disease. it is therefore the first priority of the global public health community to develop and implement the required infection control practices to prevent the dissemination of these emerging organisms within health care facilities (hcfs) and worldwide based on the best available evidence and previous experience with similar or related groups of pathogens. the world health organization (who) through its expert technical committees was prompt in developing its first infection control guidelines based on available knowledge on the new emerging virus, but it mostly drew on experience from a similar virus, severe acute respiratory syndrome coronavirus (sars). these guidelines were updated based on accumulating experience from reports of mers-cov cases and clusters in the community and hcfs in affected countries and regions. this became particularly important with the third and largest wave of hcf-associated mers-cov cases reported from the kingdom of saudi arabia and united arab emirates in april-may . the initial cases were reported in hospital a in jeddah with subsequent appearance of cases in other hospitals. during the initial jeddah outbreak, there were involved hospitals: a-e. hospital a had cases: a community case and health care workers (hcws) assumed to have been infected from the first case. hospital b had community-acquired infections. hospital c reported community-acquired case. hospital d had fatal case in an hcw. the source of infection was believed to be the community; however, hospital-acquired infection could not be ruled out. hospital e was where the main nosocomial cluster occurred. hospital e is a large and busy referral teaching hospital where staff share accommodation. the emergency room is busy with an occupancy rate of % for the emergency beds. there were initially community cases reported. extensive screening of their contacts among hcws and family members identified laboratory-confirmed cases among hcws ( were asymptomatic), family member, and patient contacts. careful review of the recent increase in the number of cases revealed that about % were among hcws. of the initial recent mers-cov infected patients in jeddah, kingdom of saudi arabia, most ( %) were infected in the health care setting. of those, were hcws. , these cases occurred between february , , and april , , and were treated in hospitals in jeddah. most hospitals had or patients, and hospital e had cases. of these cases, % were primary cases. of the cases, % (including hcws) were from health careeacquired infection. an extensive screening of contacts showed that of household contacts ( . %) were polymerase chain reaction positive for mers-cov compared with . % of family contacts screened during - . because the largest percentage of secondary human-to-human transmission occurs in hcfs, the critical question remains of whether the recent large multi-hcf clustering in was caused by failure of the evidence-based recommended infection control measures outlined by the who or failure of its strict application in the affected facilities. the who continues to stress different approaches to infection control: contact precautions, droplet precautions, and airborne isolation. the use of contact precautions is thought to be needed because of the presence of diarrhea and vomiting in approximately % of cases. , , although, there was no documented transmission of mers-cov through this route, the virus was isolated from stool in a few patients. in addition, studies investigating environmental stability of mers-cov have revealed that mers-cov was stable at different temperature and humidity conditions and could still be recovered after hours, which supports the potential of mers-cov to be transmitted via contact or fomite transmission because of prolonged environmental presence. drawing on data from similar viruses, the potential transmission of viral respiratory infections by contacts was highlighted previously. because mers-cov is similar to sars in many aspects, patients with sars had high virus concentrations and prolonged virus excretion in stools. [ ] [ ] [ ] assessment of infection control practices applied in the affected hcfs during the peak of the outbreak conducted by local and international agencies concluded that the jeddah outbreak was related to poor compliance with the recommended basic infection control measures. the finding is supported by similar observations from sars. during the sars outbreak, the infection in health care settings was further exaggerated by overcrowding, short distance between patient beds, inadequate ventilation, use of aerosol-generating techniques, and during cardiopulmonary resuscitation. , the who report on infection control measures for severe acute respiratory infections also elucidated the major risks associated with high-risk aerosol-generating procedures. the recommendations to use droplet precautions for all patients admitted with confirmed or suspected mers-cov, except in aerosol-generating procedures, come from the understanding of the al-hasa outbreak. during that outbreak, a total of patients were recorded across health care settings. the outbreak was controlled with implementation of the basic infection control measures without airborne isolation. , the recommendation also relies on the fact that viral respiratory tract infections (eg, sars) spread by large ( mm in diameter) respiratory droplets. there is a clear seasonal disease activity because the zarqa, jordan, outbreak was in in april , the al-hasa outbreak was in april-may , and the jeddah and united arab emirates outbreak was in april-may (fig ) . therefore, exposure of hcws is more likely during these months because of increased community cases. , airborne infection isolation (aii) precautions should be applied during any aerosol-generating procedures as recommended by the who. , during the sars outbreak, aerosol-generating procedures associated with increased risk of transmission of sars were intubation, tracheotomy, and manual ventilation. indeed, in the only study addressing the risk of mers-cov transmission among hcws, the reported staff were involved in at least of the following highrisk procedures: intubation, airway suctioning, and sputum induction. the centers for disease control and prevention in the united states and european centre for disease prevention and control continue to recommend the application of aii precautions when caring for patients with mers-cov. these recommendations and the recommendations from other experts rely on the fear of the disease and high case fatality rate. in a recently published debate, the presented evidence supported the use of droplet precautions, not aerosol-generating procedures. a recent study showed that mers-cov rna was isolated from the barn of camels linked to a human mers-cov case suggesting possible aerosolization of mers-cov. the study had several limitations, including the following: it was only positive sample; there was a lack of internationally acceptable sampling strategies of the air, and the sequences are all % identical to all other sequences from the patient, camel, and laboratory, which suggests contamination; there was a lack of proof of the causation of aerosol dissemination of the virus; and finally, the viral load in the air sample was higher than the viral load in the camel's nose. in addition, there are no established methods for sampling airborne exposures. for the transmission of sars, and this is likely true with mers-cov, multiple factors play a role in the propagation of infection in a health care setting. these factors include the following: lax basic infection control procedures, aerosol-generating procedures, improper use of personal protective equipment, and mouth exposure to patients' body fluids and excretions. [ ] [ ] [ ] [ ] [ ] [ ] [ ] teasing out the most important factors contributing to hcw infection of sars, mers-cov, or emerging respiratory viruses is of paramount importance. in addition, the utilization of maximum respiratory protection is easily applied when there are a few cases, but this strategy puts a burden on any health care system when the number of cases increases substantially. when resources are available, using aii precautions in conjunction with contact precautions would provide the best protection for hcws. isolation of a novel coronavirus from a man with pneumonia insaudi arabia hospital outbreak of middle east respiratory syndrome coronavirus global alert and response (gar): middle east respiratory syndrome coronavirus (mers-cov) e update world health organization. global alert and response (gar): coronavirus infections european centre for disease prevention and control. epidemiological update middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov) summary and literature updateeas of screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study assessment of potential risk factors of infection of middle east respiratory syndrome coronavirus (mers-cov) among health care personnel in a health care setting global alert and response (gar): infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions infection prevention and control measures for acute respiratory infections in healthcare settings: an update identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome the severe acute respiratory syndrome world health organization. who concludes mers-cov mission in saudi arabia sars: experience at prince of wales hospital, hong kong detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units hospital-associated middle east respiratory syndrome coronavirus infections infection control and mers-cov in health-care workers travel implications of emerging coronaviruses: sars and mers-cov coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers global alert and response (gar): middle east respiratory syndrome coronavirus (mers-cov) e update aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review middle east respiratory syndrome coronavirus infections in health care workers european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov) debate on mers-cov respiratory precautions: surgical mask or n respirators detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient commentary: protecting health workers from airborne mers-covdlearning from sars which preventive measures might protect health care workers from sars? clinical management and infection control of sars: lessons learned cluster of cases of severe acute respiratory syndrome among toronto healthcare workers after implementation of infection control precautions: a case series transmission of severe acute respiratory syndrome during intubation and mechanical ventilation illness in intensive care staff after brief exposure to severe acute respiratory syndrome sars transmission among hospital workers in hong kong risk factors for sars transmission from patients requiring intubation: a multicenter investigation in toronto key: cord- - aqi jg authors: gray, j. w. title: his middle east infection prevention summit date: - - journal: journal of hospital infection doi: . /j.jhin. . . sha: doc_id: cord_uid: aqi jg nan in june the healthcare infection society held a twoday middle east infection prevention summit with the aim of uniting colleagues across the globe in driving down infection rates and improving infection prevention and control practice. in many ways the middle east is a microcosm for the challenges in infection prevention and control across the world. the economies range from underdeveloped countries such as egypt, with a low per-capita gross domestic product, to the wealthy hydrocarbon-exporting, and increasingly diversified, economies of the gulf cooperation council countries; of course these vast economic differences impact on the resources available for healthcare. moreover, there are large ex-patriot populations in many middle eastern countries and international travel to and from the middle east is frequent, meaning that infections emerging there can quickly have a global impact. two of the themes of the conference that have particular international relevance are the huge challenges presented by multidrug-resistant gram-negative bacteria in middle eastern hospitals, and local experience with middle east respiratory syndrome coronavirus (mers-cov). coverage of the latter was particularly opportune, given the outbreak of mers-cov in south korea in may originating from a traveller returning from visiting several middle eastern countries. this special section of the journal includes three papers relating to the summit. drs omrani and shalhoub present a review of mers-cov, and dr jon otter and professor nesrene omar describe their british and egyptian perspectives of the summit. e middle east respiratory syndrome coronavirus (mers-cov); what lessons can we learn? the inaugural healthcare infection society middle east summit: a local perspective the inaugural healthcare infection society middle east summit: 'no action today. no cure tomorrow key: cord- - ub j x authors: bleibtreu, a.; bertine, m.; bertin, c.; houhou-fidouh, n.; visseaux, b. title: focus on middle east respiratory syndrome coronavirus (mers-cov) date: - - journal: med mal infect doi: . /j.medmal. . . sha: doc_id: cord_uid: ub j x since the first case of human infection by the middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia in june , more than cases of confirmed mers-cov infection and related deaths have been reported since the th of october . the vast majority of these cases ( %) were reported in saudi arabia but the epidemic has now spread to countries and has not ceased years later, unlike sars-cov that disappeared a little less than years after emerging. due to the high fatality rate observed in mers-cov infected patients ( %), much effort has been put into understanding the origin and pathophysiology of this novel coronavirus to prevent it from becoming endemic in humans. this review focuses in particular on the origin, epidemiology and clinical manifestations of mers-cov, as well as the diagnosis and treatment of infected patients. the experience gained over recent years on how to manage the different risks related to this kind of epidemic will be key to being prepared for future outbreaks of communicable disease. the first case of infection attributed to middle east respiratory syndrome coronavirus (mers-cov) was detected in saudi arabia in june [ ] . mers-cov then spread to several neighboring countries, mainly jordan and qatar (see fig. ), and imported cases of the disease were reported throughout the world in asia, africa, europe and the americas [ ] . by the th of october , con- the first two coronaviruses demonstrated to cause respiratory infections in humans, the coronaviruses e and oc , were identified in the s. they were held responsible for respiratory infections of moderate severity in humans. despite these viruses being identified in several reports as causing lower respiratory tract infections, it was generally accepted that coronaviruses were of low pathogenicity until the emergence of sars-cov (severe acute respiratory syndrome coronavirus) in , a virus with a fatality rate estimated at %. the sars outbreak that resulted in more than cases was finally contained two years later, in , and the virus has not been detected again since [ ] . there was renewed interest in coronavirus research following the sars epidemic, and two novel endemic human coronaviruses were identified, nl and hku respectively in and , but could not be replicated in cell culture. both of these new viruses were responsible for respira-tory infections of moderate seriousness like the coronaviruses e and oc . great effort has been made to identify coronaviruses in animal populations, both before and after the sars outbreak, in order to better understand and control the risk of animal-to-human transmission. this resulted in the discovery of coronaviruses in numerous animal species, with a few exceptions such as sheep and goats, fish and non-human primates [ ] . the first case of mers-cov infection was reported in jeddah, saudi arabia, in june [ ] . the patient, a -year-old man, died from lung and kidney failure days after being admitted to hospital. very shortly afterwards, in september , a second patient was admitted to hospital in the united kingdom for severe respiratory infection related to a novel coronavirus following travel to the middle east. the new virus was found to replicate in a tissue culture model and was rapidly isolated and identified for both cases [ , ] . retrospectively, other cases of the disease were found to have occurred before the aforementioned cases: in april , an outbreak at zarqa hospital in jordan affected the staff of the intensive care unit, with two fatal cases. the respiratory samples collected were later confirmed to be positive for mers-cov [ ] . these initial cases were rapidly followed by a series of outbreaks in all saudi arabian provinces that were characterized by the infection of health professionals in direct contact with the patients. other similar outbreaks were observed in several neighboring countries: qatar, bahrain, kuwait, jordan and tunisia. health authorities reacted quickly to the reports of these epidemics and the strong resemblance with observed clinical features of sars-cov infections. indeed, although a few patients developed mild infections, the fatality rate for patients infected with mers-cov was over % [ ] . following the identification of mers-cov, great effort was put into finding which animal species it originated from in order to stop the further spread of the disease to humans. mers-cov was very rapidly determined to be genotypically closely related to the betacoronavirus lineage c viruses identified in bats [ ] . based on these findings, and the major role of bats in the genetic diversity and spread of coronaviruses, much of the initial work aiming at finding the natural reservoir of mers-cov focused on bats. however, no conclusive evidence demonstrating that bats were the natural reservoir of mers-cov in the arabian peninsula were found, despite the identification of closely related viruses in bats in sub-saharan africa [ ] , far from the existing outbreaks. very strong epidemiological links were identified between the human cases and camels and resulted in the isolation in camels of viruses that were directly related to mers-cov and that could replicate in cultured human cells [ ] . the investigation of dromedary camel serum collections, some of which collected as early as , demonstrated that the virus was already widespread (seropositivity rate > %) in the east african countries (somalia, sudan and egypt). these countries export dromedary camels to arabian countries, but also in kenya, nigeria, tunisia, ethiopia, burkina faso and morocco [ ] [ ] [ ] . phylogenetic analysis revealed distinct coronavirus lineages in dromedary camels, including one recombinant lineage that led to the mers-cov epidemic in humans [ ] . mers-cov is a betacoronavirus belonging to lineage c. it is an enveloped virus with a positive-sense single-stranded rna genome of about kb. under electron microscopy, virions are generally spherical with surface projections (spikes) formed by the surface protein s creating an image reminiscent of a crown or solar corona. the positive-sense single-stranded rna genome acts as messenger rna (mrna) with a cap and a polyadenylated tail. it plays three roles during the host cell cycle: (i) it acts as the initial rna molecule for the infection cycle; (ii) it is the template for replication and transcription; (iii) it is the substrate that is packaged into the newly assembled viral particles [ ] . the mers-cov genome is organized in the same way as other coronavirus species. the first two thirds of the mers-cov genome contain two overlapping reading frames (orf a and orf b) that translate into the replication-transcription complex including non-structural proteins. the remaining third of the genome encodes the four structural proteins, the spike (s), envelop (e), membrane (m) and nucleocapsid (n) proteins, as well as five accessory proteins (orf , orf a, orf b, orf and orf b) that are not required for genome replication but are probably involved in virulence. the flanking sequences, on both ends of the genome, contain untranslated and regions (utr) (fig. ) [ ] . the viral particle can enter the cell in two ways, which probably contribute to the broad tissue tropism of this virus that replicates mainly in respiratory epithelial cells but can also infect many other cell types. via the endosomal pathway, the s domain of the mers-cov spike protein (s) binds its receptor, dipeptidyl peptidase (dpp ) [ ] , induces endocytosis of the viral particle and a change in the conformation of the s subunit of the s protein that then mediates virus-host membrane fusion and uncoating of virus rna. mers-cov can also enter host cells via a non-endosomal mechanism by direct fusion of the virus with the plasma membrane following s protein cleavage by human proteases [ ] . following entry into the cytoplasm and uncoating of the virus nucleocapsid, the viral genomic rna is translated to produce two polypeptides, pp a and pp b, that form the replicase-transcriptase complex. this initial replicase-transcriptase complex uses the genomic rna to produce non-structural proteins that assemble into the replication complex. the replication complex then replicates the genomic rna and produces other subgenomic rnas that ensure the translation of the structural proteins. virions are assembled at the endoplasmic reticulum membrane as viral proteins and genomic rna are grouped together and then bud into the lumen of the endoplasmic reticulum. the virions are then exported via the secretory pathway of the endoplasmic reticulum into the golgi intermediate compartment and then into the extracellular environment. the m protein drives the packaging process by selecting and organizing the viral envelop components at the assembly sites and interacting with the nucleocapsid to allow budding [ ] . several large serology studies suggest that cases of asymptomatic or mild mers-cov infection occur regularly, although infrequently. the importance of such cases is difficult to assess [ ] . it is therefore difficult to determine whether these cases are due to or take part in human-to-human transmission. several studies suggest that less than % of infected patients transmit the virus to individuals they come into contact with, even at the beginning of an outbreak [ ] . the disease therefore seems to spread due to frequent animal-to-human transmission, from camels to humans, with limited subsequent human-to-human transmissions [ ] . there are unfortunately exceptions to this observation and local outbreaks caused by human-to-human transmission have been observed on a regular basis, mostly in hospitals. to date, the most poignant example is the outbreak that occurred in south korea in which the index case caused secondary cases, among whom were care providers, leading to fatalities [ ] . this outbreak was characterized by the key role of a few "super spreaders", delayed diagnosis, high doctor shopping behavior and the importance of confined spaces (waiting room, hospital room, ambulance). in this example, the resemblance with sars-cov's spreading mechanisms is striking, despite lower degrees of transmission to care providers for mers-cov [ ] . these regular cases of imported-mers, the most recent was reported in england in august [ ] , represent a real threat of local epidemics outside saudi arabia and special screening and isolation procedures need to be implemented in units likely to receive patients suspected of mers-cov infections. when possible, the first measure to be taken is to delay departure, in particular for individuals over or with chronic disease, and for pregnant women or children. such measures are nevertheless challenging to maintain today as that the virus is still present years after its apparition. all other preventive measures aim at preventing both animalto-human transmission and human-to-human transmission. it is therefore recommended to avoid any contact with domestic animals (firstly dromedary camels), their secretions, raw milk and insufficiently cooked meat. it is also advised to avoid eating fruit and vegetables that might have been in contact with animal secretions if not washed and peeled by oneself. to avoid human-to-human transmission, the usual recommendations for preventing the spread of any respiratory virus should be applied: hand washing with soapy water or an alcohol-based solution, covering one's nose and mouth when sneezing, refraining from shaking hands and touching one's mouth and nose with one's hands, avoiding contact with people with respiratory symptoms. finally, a last series of recommendations focus on how to behave in case of suspicious symptoms: (i) consult a doctor as soon as symptoms occur during travel and delay the return until symptoms disappear; (ii) if symptoms occur with days of returning home, consult a doctor and tell him/her about the recent travel [ ] . pcr-based detection methods are currently the preferred option for detecting the virus in respiratory samples and making a diagnosis of mers-cov infection. serology tests can also be performed and are often used for second-line diagnostic investigation in patients with a high suspicion of mers-cov but negative results by direct pcr testing. various respiratory matrices can be used: nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal), and even in some cases, induced sputum. the deepest samples, tracheal aspirates and bals, show the greatest sensitivity and significantly higher viral loads [ ] . the genome amplification and detection methods used (pcr) were initially mostly developed in situ and performed in biosafety level (bsl- ) reference facilities. the time to results is generally relatively long, - h, due to the usual time required for conventional pcr testing to which must be added the additional preparation and sample neutralization time needed to protect the laboratory staff against this virus. the pcr methods used are generally semi-quantitative and some studies suggest a correlation between the amount of virus detected and the severity of the symptoms [ ] . nevertheless, no consensus has been reached yet regarding a threshold level that could actually predicts clinical severity. targeting the envelop gene upe is recommended with confirmatory testing for orf a or b or the n gene. if results diverge, sequencing is sometimes required to obtain conclusive results [ ] . today, an increasing number of commercial tests are becoming available (altona diagnostics, fast track diagnostics, primerdesing ltd.) some even with a time to results of less than hour (biofire-biomérieux). some of these tests are point-of-care, or can be performed in bsl facilities or a standard laboratory following sample neutralization in a bsl facility. these commercial tests must always be validated before use to check their sensitivity and compare their performance with reference methods. as with any other acute viral infection, antibodies can only generally be detected about days after the onset of symptoms. in some patients, especially those with severe infections, the time interval to antibody detection may be even longer [ ] . serological testing is therefore of little help for the initial diagnosis of symptomatic patients, but can be useful for epidemiological investigations. the highly immunogenic s and n viral proteins are widely used targets for serological tests and are found on all coronaviruses. various approaches have been developed: serum neutralization assays [ ] , microarrays [ ] , or more recently elisa confirmed by a microneutralization test [ ] . all methods are technically complex and require a high level of expertise that restrict their use to a few highly specialized facilities. the first cases of infection with mers-cov were reported in [ ] . hospital-acquired mers-cov infections have been described worldwide and represented a third of all cases reported in saudi arabia in the early stages of the epidemic [ , , ] . clustered hospital-acquired infections were frequently observed during the first outbreaks and probably contributed to spreading the disease from the primary site of virus infection to the whole arabian peninsula, the most striking example of hospital-acquired outbreak being the korean outbreak in [ ] . care providers are often affected and represent - % of cases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most of the cases are described in middle east countries, in particular saudi arabia ( %), with a predominance of male patients ( - % in various studies) and a mean patient age ranging from to years [ , , ] . comorbidities are found in - . % of patients, in particular diabetes and high blood pressure, followed by other heart conditions and finally obesity [ , , , ] . the mean incubation time is to . days. the generation interval (time between the onset of symptoms of the first case and those of the second case) is . days, which is identical to that of the respiratory syncytial virus (rsv) but threefold more than the influenza virus [ , [ ] [ ] [ ] . the main challenge of mers-cov infection is the absence of specific clinical features for differential diagnosis with other viral respiratory diseases [ , ] . this difficulty, combined with precautionary action taken to avoid potential secondary contamination with mers-cov [ ] , can result in medical confusion and inappropriate patient management due to prolonged, difficult isolation that makes it impossible to perform the necessary complementary tests while waiting for pcr results [ ] . the clinical features of mers-cov infection are extremely variable, ranging from an absence of symptoms ( - % of cases) to a flu-like syndrome, pneumonia and acute respiratory distress syndrome (ards) [ , ] . the three most frequent symptoms are: fever ( % [iqr: - ]), cough ( % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ), and dyspnea ( % ). many other secondary symptoms have been reported, such as sputum production ( %), odynophagia ( %), digestive system signs ( %), hemoptysis ( . %), myalgia ( %) and headache ( %) [ , , , ] . diarrhea is significantly more frequent in patients infected with mers-cov than in patients with another acute, febrile respiratory conditions [ ] . severe mers is characterized predominantly by ards, acute kidney failure, and in the most severe cases, by multiple organ failure that can be fatal [ , ] . one third of patients develop pneumonia and % develop ards [ ] . the median time to respiratory failure is days after the onset of symptoms. depending on studies, to % of hospitalized patients are admitted to an intensive care unit (icu) [ , ] . since the first mers outbreak, who had documented, in october , cases of mers-cov infection confirmed by laboratory testing and related deaths in different countries. the retrospective fatality rate varies between outbreaks, ranging from . to % [ , , , , ] . the mortality rate of . % observed for the korean outbreak is probably the most reliable epidemiologically due to the comprehensive investigations carried out [ ] . the death rate is highest among patients admitted to an icu, ranging from % to % [ , ] . in the only cohort study performed in saudi arabia, the fatality rate for mers-cov patients was of only % ( / ). however, the patients of this cohort were younger, had less symptoms, showed less radiological features and only % were admitted to an icu [ ] . the findings of the latter study diverge therefore with the situations observed in other hospitals, but are perhaps a better reflection of the infection profile in the general population in which younger subjects are less symptomatic and therefore less frequently admitted to hospital. the time interval between the onset of symptoms and death ranges from . to days [ , , ] . finally, co-infection with other respiratory viruses, in particular influenza, has been described although the impact of such combined infections have not been evaluated [ , ] . co-infections with bacteria have also been reported in the patients developing the most severe disease [ , ] . there are no specific laboratory findings related to mers-cov infection. nevertheless in patients with acute respiratory infection in mers-endemic areas, mers-cov infections have been associated with normal leukocyte and/or polymorphonuclear neutrophil counts but elevated transaminases [ , ] . moreover, hyperleukocytosis, lymphocytopenia, thrombocytopenia, hypoalbuminemia, elevated serum creatinine, ldh and crp levels, and hypoxemia (pao /fio < ) have been repeated reported in mers-cov infected patients and are associated with severity and death [ , ] . imaging (chest x-ray and sometimes chest ct) has revealed infection-related features in - % of cases. the lesions observed are uni-or multi-focal ground glass opacifications, of subpleural and lower lobe predominance, with sometimes bilateral bi-basal involvement or features of organizing pneumonia [ , , , ] . mortality is highest in elderly, male patients with comorbidities, especially diabetes [ , , ] . patients from saudi arabia and the middle east have an increased mortality rate compared with patients from korea or other countries [ , ] . in contrast, being a medical professional significantly reduces the risk of mortality [ , ] . other factors associated with a higher mortality risk have been described in various studies: digestive symptoms, prolonged delay between the onset of symptoms and admission to hospital, smoking, low blood pressure, impaired gas exchange, leukopenia, anemia, disturbance of liver or kidney function, use of mechanical ventilation and prolonged stay in the icu [ , ] . for the korean outbreak in , the independent risk factors for mortality were: age > years, dyspnea, diabetes, chronic lung disease, systolic blood pressure at admission < mmhg, hyperleukocytosis at admission (> , /mm ) and the use of mechanical ventilation [ ] . positive pcr results for mers-cov in blood at diagnosis are associated with an increased risk of requiring mechanical ventilation, extracorporeal membrane oxygenation (ecmo) or to lead to death [ , ] . the lack or delayed detection of mers antibodies (elisa igg and iga, or prnt) in the blood or airways is a poor prognostic factor [ , ] . it should however be noted that no seroconversion is observed in asymptomatic mers-infected patients [ ] . finally, the mers-cov viral loads in distal lung samples were higher among deceased patients [ ] . in a study including patients in a tertiary referral hospital in south korea: • the predictive factors for pneumonia in mers-cov patients were: age > years, body temperature > . • c on day , platelet counts < , /mm , lymphocytopenia (< /mm ), crp ≥ mg/l and high viral loads (ct value < . ); • the predictive factors for respiratory failure were male sex, high blood pressure, thrombocytopenia, lymphocytopenia, hypoalbuminemia < g/l and crp ≥ mg/l. the patients with at least two, one and none of the predictive pneumonia factors developed pneumonia in %, % and % of cases, respectively [ ] . several therapeutic options targeting various viral elements are currently available or under development (fig. ) [ ] . the different classes of available treatment are (i) immunotherapy with specific anti-mers-cov antibodies, (ii) molecules with antiviral activity, (iii) symptomatic treatment. few molecules have shown real curative action and the reports in the literature generally describe isolated cases or small series of cases. more studies have focused on associated treatment and supportive care. at this time, preventive therapies are still in preclinical stages. the efficacy and safety of plasma from convalescent patients have not been assessed. three separate reports concluded that such therapeutic approaches were inappropriate [ ] . one trial is listed on www.clinicaltrials.gov. two cases of therapy with intravenous polyclonal iggs have been reported. in one of them, the iggs originated from donors in regions negative for mers specific antibodies. several monoclonal antibodies were tested and seemed to show anti-mers-cov activity in vitro [ ] . no clinical trials are currently underway. recently, a phase i placebo-controlled, dose escalation study evaluated the efficacy of polyclonal iggs produced by transchromosomal cattle with human immunoglobulin genes immunized with the mers-cov spike (s) protein [ ] . the primary outcome of tolerance to a single dose was reached. the secondary pharmacodynamic endpoint (serum neutralization activity) showed efficacy with a dose of mg/kg. no phase ii trials are currently underway. a phase i study has been registered to assess the immunogenicity and tolerance of a combination of two monoclonal anti-mers-cov antibodies. the study has not yet started recruiting patients. infection with mers-cov reduces the host's interferon response. mers-cov is times more sensitive to ifn-␣. treatment with ifn-␣ has been reported for many clinical cases and several retrospective cohort studies have been performed, in combination with ribavirin, lopinavir or mycophenolate mofetil (mmf). none of these studies have demonstrated increased overall survival. one study reported increased survival at d but not at d for critically ill intubated and ventilated patients [ ] . a ifn/mmf combination trial is currently underway (see below). high doses of ribavirin have shown anti-mers-cov activity in vitro. ribavirin has been used to treat patients in saudi arabia as well as in france for the most severe cases managed in icus [ ] . no significant effects were demonstrated either on the mortality rate or the time spent in the icu. ritonavir-boosted lopinavir has shown efficacy against mers-cov in vitro. as a result, the fda has extended the indications of lopinavir to patients infected with mers-cov. two case reports (in greece and korea) have described improvement in patients treated with lopinavir, type interferon and ribavirin [ ] . a phase ii-iii clinical trial is registered on clinicaltrials.gov. the aim of this study is to evaluate the feasibility, efficacy and safety of the combination lopinavir/ritonavir/recombinant ifn␤- b vs. a placebo in patients with confirmed mers receiving optimal symptomatic care. chloroquine is among the molecules approved by the fda following in vitro studies. no clinical data or studies support its use in vivo at the present time. in vitro, anti-mers-cov activity has been demonstrated for doses of nitazoxamide that could be reached with two daily oral doses. no clinical data or studies support its use in vivo at the present time [ ] . in vitro, anti-mers-cov activity has been demonstrated for doses of mmf that are acceptable for use in humans. mmf seems to show a synergistic effect with ifn-␤ b in vitro [ ] . but in a non human primate common marmosets model, animals treated with mmf developed more severe lesions and showed a higher case fatality rate compared with untreated animals [ ] . in contrast with animal model, the combination ifn-␤ b/mmf was administered to patients in saudi arabia. all the patients survived but had lower apache ii scores that other patient groups [ ] . alisporivir has been shown to provide additive in vitro anti-mers-cov activity when used in combination with ribavirin. no clinical data or studies support its use in vivo at the present time [ ] . silvestrol is a molecule of the flavagline family found in plants. it binds to eif a and enhances the affinity of eif a for mrna. this blocks helicase activity and inhibits protein translation. a recent in vitro study demonstrated that silvestrol has anti-mers-cov activity [ ] . no clinical data or studies support its use in vivo at the present time. corticosteroid therapy is currently the most widely studied therapeutic option. in a retrospective study, arabi et al. [ ] compared the outcome of patients with confirmed mers-cov infection managed in an icu setting and treated with ( ) or without ( ) corticosteroid therapy. the overall fatality rate was %. univariate analysis showed that mortality in the icu, during the hospital stay or at days was higher in the corticosteroid group. then, following adjustment using a marginal structural model for causal inference, corticosteroid therapy was shown not to be associated with mortality, but delayed virus clearance. these findings, together with the absence of any description of the adverse effects caused by corticosteroid treatment, argue against the use of corticosteroids. a retrospective study was recently carried out in saudi arabia in mers-cov patients with refractory respiratory failure [ ] . the patients were included in the study from to in five icus. the study consisted of two patient groups: ecmo versus conventional treatment. the primary endpoint was inhospital mortality. secondary endpoints included the length of stay in the icu and in hospital. thirty-five patients were included: were treated with ecmo and received conventional care. both groups had similar baseline characteristics. inhospital mortality was lower in the ecmo group ( vs. %; p = . ) although they stayed longer in the icu (median stay of days vs. days; p < . ). the overall time in hospital was similar in both groups (median stay of vs. days; p = . ). in addition, patients in the ecmo group showed improved pao /fio values at and days after admission into the icu ( vs. , and vs. , respectively; p < . ), and lower levels of vasoactive amines at d and d ( vs. %, and vs. %, respectively; p < . ). the results of this study support the use of ecmo as salvage treatment for mers patients with respiratory failure, as is the case for other respiratory infections. two trials with candidate vaccines are currently registered at https://clinicaltrials.gov/ct /home. a phase-i clinical trial on healthy volunteers was set up to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls- ) that expresses the s protein of mers-cov. this trial was planned to last one year and started in . no results are available yet. a second phase-i trial was started by oxford university in january . it uses a chimpanzee adenovirus vector containing the mers-cov s protein gene [ ] . patient inclusion is currently underway. many other candidate vaccines using various different technologies are at a less advanced stage of development. the mers epidemic started in . in contrast with sars-cov that disappeared years after it first appeared, mers-cov continues to persist in the middle east years later. although the disease has not become pandemic, outbreaks have occurred worldwide. today, it is impossible to predict with certainty whether mers-cov will disappear or continue to remain a threat for human populations. efficient vaccine development for host ani-mals and humans could play a key role in tilting the balance from potentially-pandemic to mers-cov elimination. furthermore, the epidemiological and viral determinants of the emergence of mers-cov in the middle east are difficult to comprehend, due to the high seropositivity rate of african dromedary camels but no similar disease in local human populations. the constant increase of transcontinental travel, in particular towards the main focal points of mers outbreaks with religious pilgrimages and mass tourism, raises the problem of the management of patients suspected of mers-cov infection and the absence of efficient treatment options to this date. the main problem in non-epidemic countries is to detect a mers-cov case among a great number of non-mers patients. in france, with the exception of the first cases, no further cases have been detected. the current strategy is to isolate any suspicious cases as rapidly as possible to contain the infection and prevent local outbreaks as seen in south korea. the ability to rapidly test patients suspected to have mers-cov infection is the cornerstone of this strategy. the experience gained over the last few years by the health community will also help deal with any respiratory infections that will emerge in the future. the authors declare that they have no competing interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov). who mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) molecular evolution of human coronavirus genomes genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans severe respiratory illness caused by a novel coronavirus who | background and summary of novel coronavirus infection-as of comparative analysis of twelve genomes of three novel group c and group d coronaviruses reveals unique group and subgroup features presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological stud human infection with mers coronavirus after exposure to infected camels, saudi arabia mers coronavirus neutralizing antibodies in camels antibodies against mers coronavirus in dromedary camels risk factors for mers coronavirus infection in dromedary camels in co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov accessory orfs play key role for infection and pathogenesis host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein a structural analysis of m protein in coronavirus assembly and morphology east respiratory syndrome coronavirus (mers-cov)-update. who transmission characteristics of mers and sars in the healthcare setting: a comparative study new case of mers-cov identified in the united kingdom middle east respiratory syndrome coronavirus (mers-cov): prevention in travelers respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome association of higher mers-cov virus load with severe disease and death, saudi arabia a roadmap for mers-cov research and product development: report from a world health organization consultation presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study specific serology for emerging human coronaviruses by protein microarray inclusion of mers-spike protein elisa in algorithm to determine serologic evidence of mers-cov infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from to clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea mers-cov outbreak in jeddah-a link to health care facilities mers outbreak in korea: hospital-to-hospital transmission predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia the predictors of -and -day mortality in mers-cov patients middle east respiratory syndrome coronavirus infections in health care workers estimating survival rates in mers-cov patients and days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome a comparative study of clinical presentation and risk factors for adverse outcome in patients hospitalised with acute respiratory disease due to mers coronavirus or other causes rapid risk assessment: severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov), nd update clinical management of respiratory syndrome in patients hospitalized for suspected middle east respiratory syndrome coronavirus infection in the paris area from outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus infection: a short note on cases with renal failure problem state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia serologic responses of mers-coronavirus-infected patients according to the disease severity the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea ifn-( a or ifn-( a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients prevention and treatment of respiratory viral infections: presentations on antivirals, traditional therapies and host-directed interventions at the th isirv antiviral group conference feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase randomised, double-blind, single-dose-escalation study a review of treatment modalities for middle east respiratory syndrome corticosteroid therapy for critically ill patients with the middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-( b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model broad-spectrum antiviral activity of the eif a inhibitor silvestrol against corona-and picornaviruses extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice the chapters on the origin, emergence, structure, transmission mechanisms, prevention and diagnostic methods were mainly written mb, nh, and bv.bv produced the figures. the chapters on clinical presentation, prognosis and available treatment options were mainly written by ab and cb.all authors read, amended and agreed with the entire final manuscript. key: cord- -wyamc k authors: leung, chi hung czarina; gomersall, charles david title: middle east respiratory syndrome date: - - journal: intensive care med doi: . /s - - -y sha: doc_id: cord_uid: wyamc k nan middle east respiratory syndrome (mers) is due to rna betacoronavirus (mers-cov) infection. by february , the world health organization (who) had received reports of laboratory-confirmed cases in the middle east, europe and northern africa, with all having connections to the middle east. the median age is years, with male predominance ( . %) [ ] . severe cases deteriorate rapidly: median time from symptom onset to icu admission is days and to intubation is . - days, with an icu admission rate of . % [ , ]. mortality amongst critically ill ventilated patients is high with a -day mortality of % and a -day mortality of %. the median icu stay is days [ ] . the apparent epidemiology may be biased by selective reporting of more severe cases and the small total number of patients makes it susceptible to distortion by individual outbreaks. for example, of the cases were in a single outbreak involving three healthcare facilities, including a haemodialysis unit [ ] , and more than half of all secondary cases have been nosocomial [ ], increasing the prevalence of co-morbidity ( . %). bats [ ] and camels [ ] have been implicated as primary animal hosts; however, the data are not conclusive. human to human transmission occurs with a relatively low basic reproductive number (r ) of . - . [ , ] and an incubation period of approximately days [ , ] but up to days [ ] . the mode of transmission is unknown, but relatively simple infection control measures effectively controlled a nosocomial outbreak [ ]. in an ex vivo model mers-cov rapidly achieved a high viral load, infecting type i and ii alveolar cells. cell entry appears to be via proline exopeptidase (dpp ) receptors, which are expressed in lung and kidney. histological changes include detachment of type ii cells from basement membrane, disruption of alveolar tight junctions and changes consistent with apoptosis [ ] . viral nucleic acid in patients' faeces and urine suggests direct involvement of gastrointestinal and urinary tracts [ ] . the presentation is non-specific with fever, chills, sore throat, myalgia, arthralgia and dyspnoea. vomiting and diarrhoea are common. chest x-ray changes are consistent with viral pneumonitis and acute respiratory distress syndrome. clinical suspicion, therefore, depends on vigilance and, for the present time, on a history of travel to the middle east or contact with a patient with respiratory disease and an appropriate travel history [ ] . the prevalence of organ failure is difficult to determine owing to reporting of limited icu data and possible duplicate reporting of cases [ , ]. however, almost all icu admissions required mechanical ventilation [ ] [ ] [ ] ] . a recent case series reported critically ill patients with confirmed mers and one with probable mers [ ]. all required invasive mechanical ventilation. the median (range) p/f ratio on day was ( - ). six patients required high frequency oscillation, nitric oxide or prone ventilation with four requiring a combination. median duration of ventilation was days. five received noninvasive ventilation but all progressed to invasive ventilation, in keeping with other reports [ , , ] . acute kidney injury was common, with / critically ill patients requiring renal replacement therapy [ ], consistent with other reports [ , ] . shock developed in / patients and appeared to be moderately severe [ ]. routine investigations are neither specific nor sensitive. lymphopenia and thrombocytopaenia occur in about onethird of patients, but approximately % have lymphocytosis. lactate dehydrogenase is raised in about half and hepatic transaminases in - % [ ]. lower respiratory tract specimens (preferably, due to higher viral load) and oropharyngeal and nasopharyngeal samples should be obtained [ ] . viral shedding may vary with time and repeated sampling is recommended for patients with high suspicion of mers-cov infection, even if initial results are negative [ ] or other organisms are identified [ ]. blood, urine and stool specimens tested by real-time reverse-transcription polymerase chain reaction for mers-cov rna targets, along with conjunctival swabs and cerebral spinal fluid if conjunctivitis or encephalitis are suspected. there is no effective disease-specific treatment or vaccine. early ribavirin and interferon reduced severity of respiratory symptoms, radiology, inflammatory markers and viral load in rhesus macaque monkeys but was ineffective in humans with mers and ards. however this may be due to late administration and severity of disease [ ] . there is no evidence of benefit from high dose steroid [ ] and sars data indicate a significant risk of harm [ ] . a management algorithm is given in fig. . currently, the epidemic or pandemic risk appears to low. the estimated r (the number of secondary infections generated by a primary infection in a susceptible population) ranges from . to . . if this number is less than , then transmission is guaranteed to fade away. if greater than , there is a risk of an epidemic; but if it is only a little over (i.e. - . ), then transmission may fade away anyway [ ] . however, there are a number of caveats. firstly, r is difficult to estimate [ ] . secondly, coronaviruses rapidly adapt to new hosts and in the process may become more infectious. thirdly, the existence of mild, perhaps unidentified, cases makes infection control measures less likely to be effective. approximately % of mers cases may have been undiagnosed [ ] . fourthly, a significant epidemic will pose a major challenge to intensive care, owing to the prolonged duration of mechanical ventilation and high requirement for renal replacement therapy. non-invasive ventilation does not appear to be useful, except possibly to delay intubation, and may increase disease transmission and risk undue delay in intubation: four patients who received cpap suffered six cardiac arrests [ ]. as the mode of transmission is unknown implement airborne and contact precautions until proved unnecessary [ ] . personal protective equipment against airborne transmission includes a fit-tested ffp (or equivalent) face mask. fit testing is time-consuming and should be carried out in advance of hospital admission of a patient with mers as the delay between onset of symptoms and icu admission [ ] is very short. visiting without full personal protective equipment has been associated with nosocomial transmission [ ] and visitors should also be fit-tested. middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs middle east respiratory syndrome: new disease, old lessons clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids assessing the pandemic potential of mers-cov key: cord- -q exf s authors: toosy, arshad haroon; o'sullivan, sean title: an overview of middle east respiratory syndrome in the middle east date: - - journal: fowler's zoo and wild animal medicine current therapy, volume doi: . /b - - - - . - sha: doc_id: cord_uid: q exf s nan middle east respiratory syndrome (mers) is an emerging infectious zoonotic disease caused by a novel coronavirus (cov). mers was first reported in in jeddah, kingdom of saudi arabia (ksa), and in jordan, respectively. the disease was considered a potential pandemic threat to public health in the persian gulf region. (see also chapter .) most known covs infect and circulate in animals, mainly bats, but a number of covs are known to cause human disease (see also chapter ). [ ] [ ] [ ] the rapid emergence of mers-cov coupled with its limited geographic distribution has led to the suspicion that this is a zoonotic disease with an animal reservoir, and the evidence supports the hypothesis that dromedary camels (dcs) are the reservoir host. in dcs mers-cov causes a mild, transient upper respiratory tract (urt) infection. [ ] [ ] [ ] a mild or asymptomatic disease has also been reported in humans, but this is not always the case. mers-cov infection in humans often results in a severe, life-threatening disease of the lower respiratory tract (lrt), with high mortality. , , immunocompromised, elderly people and those with comorbidities, usually with a history of close contact with infected dcs, are particularly susceptible. is a positive-sense enveloped single-stranded rna virus and is the first lineage of c betacoronavirus known to infect humans. , it is more closely related to bat covs hku and hku (lineage c) than to the severe acute respiratory syndrome cov (sars-cov, (lineage b). , recent genome sequencing analysis reported the genomic evolution rate ( . × substitutions per site), suggesting that mers-cov diverged from its viral ancestor in march . analysis of human mers-cov sequences has identified several circulating genotypes. these distinct genotypes are phylogenetically classified into clades a, b, and, most recently, c, which correlate with outbreaks of mers among humans. , , , the emergence of divergent mers-cov clades in humans since is consistent with several independent sporadic introductions into the human population from an animal reservoir, of which the camel was unquestionably the source. , , , pathogenesis host cell entry of mers-cov is mediated by the binding of mers spike (s) proteins to a specific cellular receptor known as dipeptidyl peptidase (dpp ). dpp is expressed on the epithelial and endothelial cells of most human organ tissues in ex vivo studies using human tissue culture lines; this may account for the multisystem clinical spectrum of the mers-cov infection. , a strain cultured from a fatal human case was experimentally inoculated into three dcs using intratracheal, intranasal, and conjunctival routes. a mild transient disease resulted in submucosal inflammation and necrosis in the urt and lrt, but the alveoli remained unaffected. experimental inoculation of rhesus macaques (macaca mulatta) and common marmosets (callithrix jacchus) resulted in mild to severe lrt disease causing multifocal interstitial pneumonia in the macaques and extensive fatal pneumonia in the marmosets. , , since at least . several mers-cov serologic surveys confirm that the disease is not present in domesticated livestock (namely, horses, sheep, goats, and cattle) and is enzootic in the dc population across the arabian peninsula as well as in north and east africa. , , , , [ ] [ ] [ ] [ ] [ ] historically, the camel was the mainstay of land-based trade transportation and was used extensively as a food source across the entire region prior to industrialization during the latter part of the th century. , the free movement of humans and animals across the region supports the widespread prevalence and genetic diversity of mers-cov in the dc populations of arabia and east africa today. , the temporal dynamics of mers infection in dcs in al-ahsa, ksa, was examined by collecting nasal swabs and lung tissue during postmortem examination from two independent groups of animals over the course of a year and testing these for mers-cov rna by real-time reverse-transcriptase polymerase chain reaction (rt-rtpcr). positive samples were typically associated with young immunologically naive animals (< years of age) rather than adults (> years of age). seasonal peaks were detected during the winter months and coincided with the calving season, less extreme environmental conditions, cooler ambient temperatures, and higher relative humidity, for the transmission of infection amongst susceptible individuals. this seasonal peak has also been described in epidemic nosocomial outbreaks in humans that occur more frequently during the winter months. , extensive virologic evidence has been accumulated since supporting the epidemiologic link between dcs and humans in the transmission of mers-cov, although epidemiology mers-cov belongs to a lineage commonly associated with bats, the closest relatives of which lineage were recently identified in vesper bats (i.e., various species of the family vespertilionidae) from europe, asia, and south africa. initial research efforts have focused on establishing an epidemiologic link between bats and humans. , , , there is no conclusive evidence to support the theory that bats are the source of human infection, although there is consensus that bats are the ancestral hosts of the disease. , , a related mers-like cov virus, isolated from an african pipistrelle bat (pipistrellus hesperidus) in uganda, has shown high divergence of the s protein nucleotide sequence compared with an index mers-cov s protein sequence ( % amino acid identity divergence). this suggests that the two viruses differ significantly in receptor binding properties, implying that the mers-like cov virus is not a zoonotic threat and supporting the theory of a common ancestry. to date the only direct link between bats and human disease was a single instance when an rna sequence from a fatal human mers index case showed a % nucleotide match of a polymerase chain reaction (pcr)-amplified sequence of a fecal pellet from an egyptian tomb bat (taphozous perforates) collected in the same area of bisha, ksa. the human fatality was an owner of four dcs, which also tested positive for the same strain of mers-cov. at present, bat-to-human infection by mers-cov is considered to be purely speculative. surveillance of dcs in ksa has shown that mers-cov clade b has been enzootic in the camel population in arabia genetic deep sequencing methods (i.e., high-throughput sequencing) have been readily available to researchers since the disease was first reported. sequenced data have been used in these cases to successfully construct the phylogenetic tree between related viruses and hosts. , , , direct mers-cov antigen detection is possible but has been rarely performed. immunochromatography assays and monoclonal antibody-based capture elisas targeting the mers-cov nucleocapsid protein have been described. since the virus was first reported in , a range of comprehensive laboratory tests has been developed. , to better understand the disease, it has been important to collate sampling methodology data, laboratory results, and analyses in combination with clinical and epidemiologic data. until laboratory assays are fully validated, a combination of molecular and serologic laboratory tests is required to improve confidence in laboratory diagnosis during outbreaks. in cases of mild or asymptomatic infection, full validation of serologic assays is required to rule out false-negative results. validation is also required to successfully apply newly developed diagnostic serology algorithms to inform public health decisions. , , treatment therapeutic options for mers-cov are limited. supportive treatment is indicated for hospitalized patients, but vigilance for complications is essential. empirical use of antimicrobial agents or steroids has not succeeded in reversing the progression of severe disease. , , no specific drug or vaccine is currently available to treat mers. indeed, it has been stated that the complexity and time required for the development and registration process of drugs for human use impedes the ability to counter the rapid threat against an emerging infectious agent. for example, there is no vaccine available against sars-cov because of the brevity of the threat to the public health. , it is likely that a mers-cov vaccine for human use may not be developed due to a lack of commercial interest, or if the threat posed by mers-cov declines in the meantime. nevertheless, given the prevalence of mers-cov infection in the middle east's dc population and due to the potential for spillover to the human population in direct contact with dcs, the development of a vaccine for use in dcs may be feasible. , , a recent successful trial of a mers orthopoxvirus vaccine has conferred mucosal immunity in the urts of dcs. eradication of mers-cov from herds may be possible, if vaccines are administered to young, immunologically naive camels prior to exposure. , , identification of the zoonotic source of mers guides control strategies at the human-animal interface. , by preventing spillover of mers-cov from animals to humans, the risk of nosocomial and familial outbreaks in the middle east could be eliminated. strategic serosurveys of humans using samples collected after have been infrequent. , , there is a paucity of baseline data to describe the proportion of the potentially infected human population for much of the arabian peninsula and all of east africa, including the horn of africa. the exact mechanism of transmission from camels to human remains uncertain. sustained close contact is most probably necessary for transmission by aerosolized droplets, as mers-cov viral rna has been detected in air samples from a barn housing infected dcs in qatar, and the virus may remain viable in aerosol for up to minutes. , [ ] [ ] [ ] the potential public health risk resulting from aerosol-generating activities ranges from contamination of a room occupied by a symptomatic patient to slaughter practices. , , aerosolized transmission of mers-cov has been attributed to hospital outbreaks in ksa and south korea. , , mers-cov spreads inefficiently from human to human, but transmission is effective in a hospital environment, where susceptible individuals are concentrated and the risks are amplified by poor infection prevention and control (ipc) protocols. in some reported cases of mers, direct contact with camels was not apparent. , camel-to-human transmission through other routes is, however, possible owing to the consumption of unpasteurized camel milk or raw camel meat and in traditional medicine, when camel urine is consumed as a natural remedy for a variety of ailments. , a recent survey has found that infected camels may shed mers-cov virus in milk and urine, and the virus has been shown to remain infectious for days in milk stored at °c. , the transmission risks associated with the handling of camel products, raw milk, urine, and meat during animal slaughter are yet to be fully elucidated. further studies are needed to demonstrate the potential of camel-to-human transmission. serologic methods with high sensitivity and specificity to detect mers-cov antibodies have been developed for use in seroepidemiologic studies. methods include indirect immunofluorescence assays, enzyme-linked immunosorbent assays (elisas), protein microarray technology, and microneutralization (mn) assays. , [ ] [ ] [ ] pseudoparticle virus neutralization tests (ppnts) and conventional mn assays have also been used to detect neutralizing antibodies to mers-cov. validated molecular assays have been developed. , , rt-rtpcr is the preferred diagnostic method for the detection of mers-cov and has been endorsed by the who. confirmation of mers in suspected cases requires the screening of samples targeting a number of genes specific to mers-cov, namely up e, orf a, orf b, and n genes. , , , its infancy. aside from bats, the role that other wildlife may play in the ecology of mers-cov in east africa and arabia is yet to be elucidated. at present the implementation of intensive ipc measures in human health care is vital, including improving education and awareness among healthcare workers. , most human cases have been linked to lapses in ipc, as one-fifth of viral infections have been reported among healthcare workers. , stringent precautions while handling suspected mers-cov patients include the use of personal protective equipment (ppe) (i.e., disposable gloves, gowns, respiratory protection, and eye protection). , , immunocompromised individuals and those with preexisting medical conditions should avoid close contact with dcs. , public health authorities should adopt a standardized public health response protocol to include standardized case reporting methodology as defined by the who. , standardization of case definitions aids accurate calculation of a case fatality ratio by including mild or asymptomatic cases. the health authority of abu dhabi in the united arab emirates recently implemented a standardized reporting option for mers, successfully incorporating it into existing epidemiologic surveillance systems with the aim of enhancing surveillance, educating healthcare workers, and ensuring laboratory capacity. in countries where mers-cov is enzootic in dcs, mers control at the animal-human interface is unlikely to succeed unless appropriate preventive strategies are implemented. these should include the following: • strict regulation of camel movement with imposition of a requirement for mers clearance prior to the importation and transport of camels, including animals presented for slaughter. • camels with detectable mers-cov rna should be quarantined and tested at regular intervals. • use of appropriate ppes while handling dcs. • increased awareness among camel owners and the general public of the risks of consuming unpasteurized camel milk and urine. this may prove challenging given the depth of customs and beliefs in some areas. • accelerated development of safe and effective mers vaccines for animal and human use. , conclusions mers-cov has been observed for only years, and vigilance is vital for the containment of the disease due to the high case fatality rate in humans and possible genetic instability of the virus. continued laboratory testing, genetic sequencing, analysis, timely data sharing, and clear communication are essential if such vigilance is to be effective. nonetheless, despite the potential for a pandemic outbreak at multiple mass gatherings during the islamic calendar (hajj, eid, and umrah) there were no reported outbreaks of mers during or immediately after these events. as such mers-cov is not a virus of pandemic concern. since our understanding of mers has increased greatly although gaps in knowledge still exist. the understanding of the disease's ecology-especially the interplay between camels, humans, and the environment-is still in who mers-cov global summary and risk assessment middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd who emergencies: mers-cov. available at mers coronavirus: diagnostics, epidemiology and transmission dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia pathogenesis of middle east respiratory syndrome coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels response to emergence of middle east respiratory syndrome coronavirus transmission of middle east respiratory syndrome coronavirus infections in healthcare settings hospital outbreak of middle east respiratory syndrome coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient mers-cov outbreak in jeddah-a link to health care facilities mers coronavirus: data gaps for laboratory preparedness asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels centers for disease control and prevention: interim prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). available at an animal model of mers produced by infection of rhesus macaques with mers coronavirus further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia antibodies against mers coronavirus in dromedaries middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of mers coronavirus from a dromedary camel mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia the authors wish to thank the following: h. e. ghanim mubarak al hajeri and the senior management of al ain zoo for their encouragement and support, dr. ahsan ul haq of dubai camel hospital for his technical insight, and dr. andrew higgins, fellow of the zoological society of london and honorary editor in chief of the veterinary journal, who reviewed the manuscript. key: cord- -nvgz su authors: li, kun; mccray, paul b. title: development of a mouse-adapted mers coronavirus date: - - journal: mers coronavirus doi: . / - - - - _ sha: doc_id: cord_uid: nvgz su first identified in , middle east respiratory syndrome coronavirus (mers-cov) is a novel virus that can cause acute respiratory distress syndrome (ards), multiorgan failure, and death, with a case fatality rate of ~ %. an animal model that supports mers-cov infection and causes severe lung disease is useful to study pathogenesis and evaluate therapies and vaccines. the murine dipeptidyl peptidase (dpp ) protein is not a functional receptor for mers-cov; thus, mice are resistant to mers-cov infection. we generated human dpp knock-in (hdpp ki) mice by replacing exons – at the mouse dpp locus with exons – from the human dpp gene. the resultant human dpp ki mice are permissive to mers-cov (hcov-emc/ strain) infection but develop no disease. to generate a mouse model with associated morbidity and mortality from respiratory disease, we serially passaged hcov-emc/ strain in the lungs of young hdpp ki mice. after in vivo passages, an adapted virus clone was isolated and designated mers(ma) . . . this virus clone produced significantly higher titers than the parental clone in the lungs of hdpp ki mice and caused diffuse lung injury and a fatal respiratory infection. in this chapter, we will describe in detail the procedures used to mouse adapt mers-cov by serial passage of the virus in lungs. we also describe the methods used to isolate virus clones and characterize virus infection. middle east respiratory syndrome (mers) is a fatal respiratory illness that first appeared on the saudi arabian peninsula in mid- . it is caused by a novel betacoronavirus, mers coronavirus (mers-cov) [ ] . shortly after the identification of the virus, its receptor dipeptidyl peptidase (dpp ) was discovered [ ] . as of september , the world health organization (who) has reported laboratory-confirmed cases of mers in countries, including associated deaths (fatality rate: %). mers-cov does not currently have pandemic potential [ ] [ ] [ ] . however, mers-cov is still epidemic in the middle east and remains a cause for significant concern due to the potential spread by global travel as demonstrated by the outbreak in south korea in [ ] [ ] [ ] . to date, there are only two published reports on autopsy findings from subjects who died from mers [ , ] and our understanding of mers-cov pathogenesis in humans is still limited. animal models are useful for the study of viral diseases and play an important role in the investigation of pathogenesis and evaluation of antiviral therapies and vaccines. an ideal animal model should be permissive to the viral infection and develop disease and pathology with similarities to that observed in humans. mers-cov infection has been evaluated in two nonhuman primate (nhp) models, the rhesus macaque and common marmoset [ ] [ ] [ ] [ ] [ ] . both species are susceptible to mers-cov infection. however, mers-cov caused only a transient lower respiratory tract infection without mortality in rhesus macaques [ ] [ ] [ ] . in common marmosets, the consequences of mers-cov infection are controversial. falzarano et al. reported the common marmoset reproduced several features of mers-cov infection in humans including progressive severe pneumonia [ ] , while another group observed only mild to moderate nonlethal respiratory disease following mers-cov infection [ ] . thus, the common marmoset is potentially a model to study pathogenesis and evaluate antiviral therapies and vaccines. however, nhps are expensive, their availability limited, and their use may raise ethical concerns. in contrast, small animal models provide advantages over nhps, including reduced cost, availability in large numbers, ease of handling, and species-specific reagents, especially for the studies of highly pathogenic viruses like mers-cov in the biosafety level (bsl- ) laboratory. unfortunately, common small laboratory animals like mice [ , ] , ferrets [ ] , guinea pigs [ ] , and hamsters [ ] are not susceptible to mers-cov infection because their homologous dpp cannot be bound and utilized by mers-cov as a host receptor for entry [ , ] . haagmans et al. detected mers-cov rna in the respiratory tract of new zealand white rabbits following inoculation but found no clinical signs of disease [ ] . several strategies have been used to overcome this receptor incompatibility and develop mouse models of mers-cov infection. in , we developed the first mouse model of mers-cov infection [ ] . we delivered a recombinant adenovirus encoding human dpp (hdpp ) to the lungs of mice. transient expression of hdpp by adenovirus transduction made the mice temporarily permissive for mers-cov infection, but animals developed only mild lung disease. generation of transgenic mice expressing a virus receptor is a common strategy to make mice permissive to infection. several groups in addition to ours developed mice with transgenic expression of hdpp using different promoters [ ] [ ] [ ] [ ] . the disease severity following mers-cov infection in transgenic mice correlated with the cellular distribution and expression level of hdpp . in transgenic mice expressing hdpp driven by the cytokeratin promoter [ ] or a ubiquitous promoter [ , , ] , mers-cov replicates and causes respiratory disease and mortality. however, the lethality was found to be secondary to overwhelming central nervous system (cns) disease or multiorgan damage. this was also observed in mice transgenic for human ace driven by the cytokeratin promoter and infected with sars-cov [ ] . in transgenic mice expressing hdpp under the human surfactant protein c (spc) promoter, which restricts expression to bronchiolar and alveolar epithelia, mers-cov infection caused only mild disease [ ] . thus, these transgenic mice do not reproduce a severe lung disease phenotype that resembles mers. alternative strategies for the creation of mouse models of mers-cov infection are generation of dpp humanized mice and adaptation of the virus to the animals. pascal et al. reported a model in which all of the mouse dpp exons had been humanized and also generated humanized monoclonal antibodies against the mers-cov s protein using a novel strategy [ ] . mers-cov infection in this model caused pulmonary edema, vascular cuffing, and alveolar septal thickening with an associated~ % weight loss, necessitating euthanasia [ ] . another mers mouse model was engineered by changing two amino acids in the mouse dpp locus using crispr-cas technology [ ] . this model supported mers-cov replication without severe disease. similarly, our human dpp knock-in mouse model supported mers-cov replication but did not lead to a severe lung disease phenotype [ ] . two mouse-adapted (ma) strains of mers-cov were subsequently developed independently by serial passage of the hcov-emc/ strain [ ] in the lungs of the two humanized mouse models [ , ] . the resultant mers- and mers ma . . mouse-adapted mers-cov strains replicated to high titers in the lungs of the crispr-cas genetically engineered mouse model and the hdpp knock-in mouse model, respectively. the respiratory disease that developed in both mouse models and the associated mortality shared similarities with severe cases of mers [ , ] . mouse adaptation was also successfully used to generate several sars-cov strains capable of modeling severe sars-cov lung disease in mice [ ] [ ] [ ] . thus, the adaption of the virus to enhance virulence in the mouse is a very useful approach to generate mouse models for coronavirus-associated lung disease. these materials can be altered to fit the requirements for other viruses of interest. . hcov-emc/ strain. . hdpp knock-in mouse (c bl/ strain with mouse dpp exons - replaced with the human codons). all procedures are performed under bsl- laboratory conditions and must follow the standard operating protocol of a bsl- facility and regulatory agencies. all manipulations of infectious specimens, samples, and mice must be performed within a biosafety cabinet or within a contained device such as a centrifuge. all tissue culture media and waste must be bleached and autoclaved prior to disposal. all instruments must be disinfected with virex plus or % bleach. . insert surgical scissors under the sternum and cut the diaphragm following the costal arch. remove the rib cage using scissors and forceps, exposing the lungs and heart. . fill a ml syringe with cold sterile dpbs using a g  / inch needle. insert the needle into the apex of the left ventricle and make a small incision in the right atrium. slowly perfuse ! ml cold sterile dpbs into the left ventricle. next, insert the needle into the apex of the right ventricle and perfuse ! ml cold sterile dpbs (see note ). . remove the lungs and heart from the thoracic cavity. remove the liver, kidney, spleen, and small intestine. place organs in a small polystyrene weighing dish. remove remaining connective tissue. . to harvest the brain, turn the mouse over and wet the fur of the head with % ethanol. grasp the ears with forceps and cut off the skin and fur to expose the skull. remove remaining skin at the base of the neck to further expose the skull. immobilize the mouse and make an incision along the sagittal suture of the skull using a single edge razor blade (see note ) . wedge one prong of the curved serrated forceps into the now opened sagittal suture. slowly pry up the skull, grasp the piece of skull with forceps and peel outward to remove. repeat on the other side. use curved forceps to lift the brain from the skull. place brain tissue in the small polystyrene weighing dish. . carefully place each organ into a ml disposable tissue grinder filled with ml sterile cold dpbs for homogenization or into ml of trizol for rna extraction (see note ). . grind the lung tissue and transfer the homogenates or rna samples into . ml sterile screw-top tubes with o-ring cap (see note ) . store samples at À c. thaw and spin down the cell debris in lung homogenates before use. the virulence of the virus should be evaluated in groups of mice by weight loss and survival after every - in vivo passages. after the virulence of the virus has been significantly enhanced, single plaques of the adapted virus should be purified and evaluated. . plate vero cells in d media in -well plates one day before infection. . rapidly thaw lung homogenates from the selected passage. mix the lung homogenates from two mice in a . ml sterile screwtop tube with o-ring cap on ice. . serially dilute the mixed lung homogenates tenfold in . ml sterile screw-top tubes with o-ring caps, using ice-cold serumfree dmem (see note ) . keep the dilutions on ice. . remove the medium from each well and add diluted samples (in a volume of μl) to each well. . place the plates in the c incubator for h and rotate gently every min. . melt % low melting point agarose and maintain in a c water bath. . mix overlay media and % low melting point agarose at a volume ratio of : . rotate the tube several times to fully mix. overlay cells with . ml of mixed media using ml stripettes. . let the plates sit in the hood for~ min at rt or until the agarose overlay turns solid. add . ml d medium on the top of solidified agarose. . place the plates in the c incubator. . after days, plaques should be visible. remove the liquid on the top of the agarose. circle the visible plaques on the underside of the plates using a permanent marker (see note ). . vertically penetrate the agarose and pipette the circled plaque several times with a ml graduated transfer pipette. the agarose above the plaque will be pulled into the pipette. . transfer the agarose above the plaque into a ml conical tube filled with μl dmem by pipetting up and down several times. . transfer the μl dmem containing the agarose into a . ml sterile screw-top tube with o-ring cap. . repeat the procedure and pick six single plaques. store the tubes at À c. . animal euthanasia should follow the institution's animal care and use guidelines. there may be differences in institutional requirements regarding when euthanasia is required based on weight loss. . use the lid of an insulated foam shipping box. cut absorbent bench underpad (  cm) into small pieces that fit the lid. . carefully keep the tip of the needle in the lumen of the ventricle. be sure to puncture the right atrium as this will help to drain blood during the perfusion. the lung will turn white after perfusion. . make sure that the incision does not exceed the thickness of the skull; avoid cutting into the brain tissue. . the individual organs can be divided into pieces and transferred to grinders with pbs or trizol separately. . lung homogenates are immediately transferred to tubes on ice. rna samples are transferred to a tube and incubated for at least min at room temperature per our bsl- specific standard operating procedures. . at days post infection with pfu/mouse, hcov-emc/ strain replicates in the lung of the hdpp ki mice to a titer of around  pfu/ml, which equals  pfu in μl. we chose the pfu/mouse inoculum to begin because we wanted to use a similar dose range during in vivo serial passage while skipping the titration step. . titrate the homogenates of the passage of interest and select a virus dilution that produces plaques in a well. this allows identification of individual clear single plaques. . only circle the unambiguous clear single plaques. . compared to vero cells, we found that the mers-cov rna genome is more stable when propagated in huh cells (less likely to introduce genomic deletions, insertions, or point mutations). isolation of a novel coronavirus from a man with pneumonia in saudi arabia dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk person-toperson spread of the mers coronavirus-an evolving picture the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission spread of mers to south korea and china infectious diseases epidemic threats and mass gatherings: refocusing global attention on the continuing spread of the middle east respiratory syndrome coronavirus (mers-cov) comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques intratracheal exposure of common marmosets to mers-cov jordan-n / or mers-cov emc/ isolates does not result in lethal disease infection with mers-cov causes lethal pneumonia in the common marmoset mouse dipeptidyl peptidase is not a functional receptor for middle east respiratory syndrome coronavirus infection wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase -mediated entry of the middle east respiratory syndrome coronavirus domestic pig unlikely reservoir for mers-cov the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection asymptomatic middle east respiratory syndrome coronavirus infection in rabbits rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus a human dpp -knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection cd + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a mouse model for mers coronavirus-induced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease we thank chris wohlford-lenane and jennifer bartlett for careful review of the manuscript. this work is supported by the national institutes of health p ai . we also acknowledge the support of the cell morphology core and pathology core, partially supported by the center for gene therapy for cystic fibrosis (national institutes of health p dk- ) and the cystic fibrosis foundation. p.b.m. is supported by the roy j. carver charitable trust. key: cord- -pk ealu authors: hu, yi title: a farewell to the “sick man of east asia”: the irony, deconstruction, and reshaping of the metaphor date: - - journal: rural health care delivery doi: . / - - - - _ sha: doc_id: cord_uid: pk ealu susan sontag revealed how a disease could be turned into a metaphor in social evolution, from merely a disease of the body to moral judgment or even political oppression. in her article “aids and its metaphors” written in , she offers a plan to do away with the metaphor: “with this illness, one that elicits so much guilt and shame, the effort to detach it from these meanings, these metaphors, seems particularly liberating, even consoling. but the metaphors cannot be distanced just by abstaining from them. they have to be exposed, criticized, belabored, used up” (songtag ). in sontag’s terms, “metaphor” mainly refers to the symbolic social oppression of the diseases. for example, cancer is a metaphor for the defect of the sick person in personality. while diseases were a biological phenomenon, the “metaphor” was a social one. what i would like to demonstrate here was none other than the related “political metaphor” started by the “anti-germ warfare.” united states had the intention to involve china in the war after crushing the democratic people's republic of korea in one movement. from the very beginning of the war, the chinese people and the chinese government maintained to resort to peaceful methods when solving the korea problem and that warnings be given to the united states about withdrawing the armed forces from taiwan, stopping the aggression against north korea, and solving the problem of korea and the far east peacefully. however, the united states ignored these warnings. in the early winter of , the american aggressors crossed the th parallel and attacked the areas around yalu river and tumen river as well as the airspace of northeast china. many a chinese were killed in bomb attacks and property was ruined. as the national security was seriously threatened, the chinese people's volunteer army entered korea on october , , and began the great war of "aiding korea and defending the homeland." this was a war between two parties with disparity of strength in many aspects. economy: while half of the world's population was involved in world war ii, causing million deaths or injuries, the united states somewhat benefi tted from the war. it quickly grew into the largest industrial power in the world. at that time, the us industrial output value accounted for more than half of the total capitalist world industrial output value. us steel production reached . million tons in ; wheat production accounted for more than % of production of the capitalist countries; the industrial and agricultural output value reached . billion us dollars. in , the us gold reserves were valued at more than . billion usd, accounting for % of the total gold reserves of the entire capitalist world. in the aggression against korea, the direct expenses of the war of the united states reached more than billion usd. war material destined for north korea totaled million tons (peng dehuai ) . in comparison, the nascent prc from the ruins of wars did not even completely liberate all its territory. successive wars not only crippled china's modern industry but also damaged its primitive agriculture. in , new china's industrial and agricultural output value was only . billion yuan, less than a fractional amount of that of the united states if converted into us dollars. armed forces: one third of the us army, one fi fth of its air force, and most of its navy were assembled in the war in korea, in addition to the troops of the vassal countries (peng dehuai ) . by contrast, the people's liberation army was still trying to eradicate the remnant kuomintang forces in the southwest and northwest of china. only those , border guards in northeast china could be mobilized, but some of the forces must be reserved to protect the northeast industrial base. military equipment: the united states boasted atomic bombs and other weapons of mass destruction, the world's greatest number of advanced combat aircrafts, and the world's largest battle fl eet. eighteen aircraft carriers were under construction at the end of world war ii, and the number and the gross tonnage of them accounted for % of the world's total. every infantry division was equipped with more than tanks and seventy-mm-diameter canons; the fi repower of the us army was also at the top of the world. almost all the most advanced weapons (except atomic bombs) were employed in the korean war. the us superiority in navy and air force was maintained all the time. in contrast, china did not have any tanks or air force of its own. air defense weapons were very few. on the other hand, the whole volunteer army were only equipped with seventy-mm-diameter guns. basically, this army was still the so-called millet plus rifl es -even the rifl es were composed of those of different periods and different types. thanks to the support from the soviet union, and from all the chinese people in their donation campaign, an air force was created and fi repower was strengthened. the enemy's superior state was not fundamentally changed, nevertheless. command: the us commander in chief changed three times during the war: douglas macarthur (dismissed because of his defeats in the battles), matthew bunker ridgway (notorious for launching the germ warfare), and mark clark (who signed the armistice agreement). actually, they had all been prominent commanders who withstood the test of world war ii and gained unrivalled fi rsthand experience. the united nations forces under their command in the korean war had employed a variety of tactics, such as the blitzkrieg, taking advantage of the weaknesses (in operation chromite), "strangling battle" that paralyzed china's transportation line, and the inhumane germ warfare. the united states and people all over the world were dumbfounded with the result of the war between the "sick men of east asia" and the world police: after signing the armistice agreement, the united states had to admit that the korean war was "the wrong war, at the wrong place, at the wrong time, and with the wrong enemy." former us secretary of defense marshall once said, "the myth has been punctured. the united states is not such a great power as it has been imagined" (peng dehuai ) . indeed, the myth had been exploded; seemingly powerful countries are sometimes like a "paper tiger," a phrase used once by mao zedong meaning someone or something outwardly powerful or dangerous but inwardly weak or ineffectual. then, how about the "sick men of east asia"? were they still sick beyond cure and doomed to a hopeless fate? peng dehuai said, "long gone are the days when the western invaders could occupy a country if only they could shoot a couple of cannons on the oriental sea" (peng dehuai ) . historical evidence convincingly suggested that the sick men were not always sick. when the awakened sick men were organized and had a stronger will, they would become strong enough to defeat a powerful enemy and to rewrite the history of the "sick man of east asia." the confession confi rmed that "the master plan of the germ warfare in korea was ordered by the meeting of the u.s. joint chiefs of staff in october ." this plan was sent to the far east commander in chief (general ridgway) to "start the germ warfare in korea." "various kinds of military weapons, carriers, and various kinds of aircrafts" were to be experimented "in every area possible or a combination of areas" and "under extremely hot or cold weather." "depending on the results and the situation in korea, the fi eld trials might be extended to be a part of formal war operation." the plan was transferred from ridgway to the us fifth air force via the us far east air force commander lieutenant general wiranto and was implemented on a large scale on a trial basis in november . the first air force alliance participated in this experimental mission and in the formal operational task of "building a trans-korean contaminated zone" in may . meanwhile, the chinese people's committee for world peace held "exhibitions of crimes committed in the germ warfare of the u.s. government" in beijing, vienna, berlin, etc. evidence collected by various parties of the us germ warfare in fl agrant defi ance of the geneva convention (note: in , the geneva protocol or the "protocol for the prohibition of the use in war of asphyxiating, poisonous or other gases, and of bacteriological methods of warfare" was signed by various countries in geneva) was publicly displayed (fang shishan ). on february , , mark clark, the general commander in the invasion of korea, published a declaration, in which it was admitted that schwable and bligh were the us air force personnel, that they made the confessions, but it was supposed that the confessions were "fake," extorted by the china side through "torture" (li siguang ). any facts of the "germ warfare" were emphatically denied. subsequently, those air force prisoners of war were forced to make an affi davit to the united nations general assembly, and the so-called proposal relating to china's "atrocity" (li siguang ) was brought forward to the united nations, together with countries such as the uk, france, australia, and turkey, the intention being to deny crimes committed in the germ warfare categorically. in this regard, the chinese government refuted that there were other captured prisoners of war, other than schwable and bligh, who had confessed: a lieutenant inuk and a lieutenant quinn of the third bomber team of the us air force and a lieutenant o'neal and a lieutenant knits of the eighteenth fighter-bomber brigade. from the confessions made by the six men from the us air force, one could clearly see every step of the germ warfare from planning to implementation. on the other hand, north korea and china's lenient policies for prisoners could be detected from the confessions made by the prisoners of war of the us air force, from talks of those prisoners of war who had been repatriated, from the reports made by the british and american journalists, or even from us army minister stevens and the british army minister. it could be safely concluded that those confessions were none other than "blame of consciousness" rather than a result of "torture." just as schwable exclaimed, "morally, it is an irreparable crime"; "from the standpoint of dignity and loyalty, it is shameful." the united states crimes committed in waging germ warfare could be confi rmed by evidence from many other aspects. after investigating in northeast china and north korea, scientists of the international scientifi c commission collected a body of evidence (physical specimens including insects, bacteria, and other clinical evidence) for the investigation of the pacts concerning bacterial warfare in korea and china. a conclusion had been drawn that the united states had organized a large-scale, disguised germ warfare. dr. joseph needham, who was a member of the royal society of biologists and the international scientifi c commission, published an open letter saying that the truth of the germ warfare "was by no means determined by what the air force personnel confessed, nor was it determined by what they had denied in the new and different circumstances" (li siguang ). dr. samuel b. pessoa (brazil), also a member of the international scientifi c commission and who participated in the investigation, wrote after his visit to "exhibitions of crimes committed in the germ warfare of the u.s. government": "although it appeals to the broad mass of the population, the exposure process of all the facts is highly scientifi c; it is in no way exaggerated; it is with the aim to explain the truth, or to explain the real situation. what i lament on is that such good techniques of the exhibition should be used to expose such dirty evil deeds." the bloody crimes of the united states committed in the germ warfare, as well as other crimes such as the ill-treatment of the prisoners of war and the massacre of civilians, were also exposed after the investigation by some impartial bodies such as investigation group of crimes of the germ warfare made by the imperialist united states, investigation group of the international association of democratic lawyers, and the international democratic women's federation. overshadowed by the long-term hegemonic discourse of colonialism, the backward countries had not only become the exploited and the plundered but also been oppressed by various political metaphors such as the "sick man of east asia." in the metaphorical politics, the colonial countries and the developing countries were often accused of, and blamed for, being the sources of a variety of diseases, communicable diseases particularly, thus falling into both a moral and political dilemma. as revealed by guenter b. risse, the socially marginalized groups, minorities, and the poor are often accused of being the culprit during outbreaks of diseases. in europe, jews were regarded as the creators of the black death. in new york, irish people there were considered responsible for the outbreak of cholera. in brooklyn, the italians were seen as a source of poliomyelitis. in such cases, the colonial countries would assume the role of a guardian to prevent the spread of the diseases and play the role of the "benevolent" and even the "savior" through dispatching missionary doctors. the irrefutable evidence of germ warfare launched by the united states reveals another perspective of history: the controller of communicable diseases can also be the initiator of communicable diseases. the historical process confi rmed this perspective: the major diseases popular in the modern world (smallpox, syphilis, pulmonary diseases encompassing tuberculosis, pneumonia, and sars) originated in europe. it was with the footsteps of the colonizers, and sometimes as the earliest forms of chemical and biological weapons, that these diseases were spread to the colonial areas. it was from the diseases brought by european settlers that the great majority of indians in north and south america died (diamond ) . a return to the humane world was said to have begun since the renaissance and the enlightenment in western societies. the rise of rationalism directly contributed to the development of modern science and led to powerful scientism, as well as something closely related to it -the technological revolution and the industrial revolution. in line with rationalism, humanism triggered cultural reform and institutional reform, with the western democracy being one of the solid achievements. when guns and fl eets of the western countries easily forced open the door to the colonies, conquer in thought also began, forming the distinct dichotomy between the traditional and the modern, the advanced and the backward, the civilized and the barbarian. an image of the "civilized world" began to take shape and strengthened in the long colonization process. this positive image was, however, tarnished by the launching of the germ warfare of the united states and by its sophistry. after investigation, the international women's federation published "a report of the international women's investigation group on the atrocities made by the u.s. and the rhee armies." in the report, brute facts were established, such as the us army cold-bloodedly killed korean residents. in areas that had temporarily been occupied by the us army and syngman rhee's army, hundreds of thousands of civilian inhabitants, young and old alike, were tortured, burned, killed, or buried alive. the atrociousness had exceeded what the nazis and adolf hitler had made when they occupied europe (li siguang ). a canterbury dean johnson, invited to china, said after he learned the news that the united states had launched germ warfare, "a country, under the name of christianity, is shameful, connected with this matter." a representative from el salvador, dias, wrote after a visit to the exhibition, "this exhibition exposed, most conclusively, the way of the u.s. armed forces in launching germ warfare. the u.s. government, high command of the army, and scientists have committed heinous crimes against humanity and no punishment of any kind is suffi cient to ease the anger caused by such crimes." a costa rican deputy, sanz, wrote at the peace conference of asian and the pacifi c regions, "what i saw here was evidence and documents demonstrating the employment of bacteriological weapons by a self-styled civilized country. we must stop it, and expose it in every possible way." a stark historical fact was gradually ascertained in these accusations and angry words: the construction process of the "civilized world" was built in a very "uncivilized" manner. examples were many: the enclosure movement in britain where "sheep eat people," "reign of terror" of the french jacobins, the genocide waged against the native americans in the westward movement, etc. in his communist manifesto , karl marx seemed to have pointed out a more desirable attitude as to the complex interweaving of the "civilized" and the "uncivilized": he affi rmed that the development of capitalism had created unprecedented social productive forces, the results of which even exceeded the sum of any previous era. on the other hand, the actual process of capitalism was ruthlessly criticized: "sweating blood and fi lth with every pore from head to toe." that the weak and the sick men could actually defeat the strong world police, that the controller of communicable diseases should become the real source of the communicable diseases, and that the self-proclaimed civilized world was really permeated with fi lthy, uncivilized behavior were so astonishing that when history unveiled to its real image and shatter illusions surrounding it, an irony took the place of the metaphor of the "sick men of east asia" constructed on the basis of hygiene. in this dramatically ironic process, the deconstructive process of the metaphor of "the sick men" also began. in foucault's thorough analysis of the modern medical system, the complex underlying mechanism of the construction of metaphors such as the "sick man of east asia" was presented. far from being merely a process of "medical progress," the modern system of western medicine was also a social process in which the technology for social organization and social control continuously improved and intensifi ed. the medical system virtually became the origin of the "modern political system." in defi ning what is "healthy" and what is "unhealthy," what is hygienic and what is unhygienic, modern medicine also implies political or moral judgments of being "sinful" or "decadent." more importantly, foucault believed that the modern medical system, which originated from state behavior, such as controlling the spread of epidemics, was also an "aggressive system" full of "war mentality." in etiology, all diseases come from the infection of bacteria (microorganisms), to practice medicine is to fi ght with microbes, and to be a doctor is to be a warrior. diseases cannot be wiped out without a social system of well-organized, combative doctorpolice. that is why the practice of the western colonial countries or imperialistic countries was discovered to have a close connection with modern hygiene when they were rebuilding the world order. apartheid was necessary because the colonized people were thought to be the sources of communicable diseases in addition to being cheap labor resources. the metaphor of the "sick man of east asia" implied physical and moral denigration to the oppressed state and its people; in addition, the world police system is to prevent, control, and eradicate what was, in their eyes, the physical diseases as well as the social "diseases" -resistance, revolts, rebels, etc. the creation and proposal of the metaphor of the "sick man of east asia" alone did not mean, in reality, that they could be turned into a dominant and oppressive force. only when the target of the discourse had accepted and internalized the metaphor as it was found to be supported by social facts could the realistic force of the metaphor be brought out into full play. during this process, violence plays a vital role. violence had become the premise and the basic properties in the rise of the nation-states. through the writings of famous thinkers and theorists in modern times, we can discover that almost without exception, they would emphasize this feature of the state: hobbes described the state directly as a potentially violent "leviathan"; karl marx argued that the state is nothing but a machine for the oppression of one class at the hands of another; max weber articulated his celebrated defi nition of the state as a human community that "successfully claims the monopoly of the legitimate use of force within a given territory." giddens even considers "military industrialization" the defi ning moment from the traditional country to the modern nation-state. organized violence of the state not only became a space construction instrument dividing the borders and establishing boundaries but also played a crucial role in the process of colonization when the original pattern of world trade drastically changed, a new world order and market order were created, and the advantageous position of the western counties was assured. the premodern agrarian countries were defenseless when confronted with such organized, monopolized, and industrialized state violence. the fi asco of combating the western countries undermined the sense of superiority and confidence these agricultural countries originally enjoyed. more often than not, they felt hopeless and shameful in being forced to cede their territory and pay indemnities. the frequent attacks of feelings of hopelessness and shamefulness would suppress their original resistance awareness and let them accept the status quo. thus, they internalized the metaphor of the "sick man of east asia." in chinese people's refl ections upon a weak and declining china, and in their futile actions, the implications of the metaphor of the "sick man of east asia" were further broadened and more widely accepted. the repeated defeat and failure, and the growing sense of the nation experiencing a crisis especially, forced the chinese people to exert themselves to fi nd ways to save the country. "westernization movement," the "reform movement of ," the "new deal of autonomy," and "new culture movement" were all evidence of a continuous self-denial process focusing fi rst on some particular objects, then on the institutions, and then on culture. in this series of self-denial, "to resort to the other places" or "to resort to novel ideas in a foreign land" became the fi nal or the most practical choice. however, the choice was fi rst and foremost based on the premise of self-negation. the "sick men" was turned from a metaphor to a self-portrait of and a realistic oppressive discourse to the chinese people of the time. of course, it also meant space and possibility for resistance to the "oppression." the social fact of sickliness reinforced the shaping of the metaphor of the "sick man of east asia." since the han dynasty, the civil and the military had been separated and the civilians and the offi cers did not have anything to do with each other. this situation was aggravated in the song dynasty. "when it is established that the emphasis is on the civil side, the military side has faded, and the atmosphere is sort of soft. two millenniums of corruption are deep in the brain of the civilians" (liang qichao ) . opium importation, the fl ooding of opium after the opium war, especially, consumed not only china's fi nancial resources but also the nationals themselves. in addition, in the frenzied plunder and exploitation of the western countries, coupled with the infl ux of western industrial products, the country's economy rapidly slumped and the people's living standards were dramatically lowered, and the infi rmity due to malnutrition had become chronic. different from the traditional relatively static agrarian society, there tended to be more interactions among people who were involved in modern industry and commerce, which was conducive to the spread of diseases. thus, scenery of a "sick country" consisting of "sick people" emerged. the successful attempt to "clean" the country by banning prostitution and drugs after the founding of new china not only highlighted the characteristics of the nascent state as being "clean" but also brought about multiple perspectives: if the social ills and crimes that had lasted for thousands of years could be extirpated in a relatively short time in the resolute attempts of a new regime and the "patients" that had long been victimized could "turn from a ghost to a human" with the care and reconstructive attempts of the state, then what gave rise to so many sick persons in the fi rst place? why hadn't they stepped onto the highroad to health earlier? who should be responsible for the overall "sickness" of the state? besides internal inspection, the external reasons were also uncovered and questioned. the new state attributed the internal and the historical reasons to the "old exploitative system," which was entirely consistent with william mcneill's theory of the "microparasite" and "macroparasite." in mcneil's mind, the relationship between the ruler and the ruled in human history is macroparasitic, while the relationship between the human body and the pathogenic microorganisms is microparasitic (mcneill ) . the existence of the "parasites" not only produces such social ills as prostitution or drugs but also weakens the effective unity of the grassroots society during the national crisis. the bottomless pit of double "parasites" made the grassroots society miserable. perhaps this could be cited as a reason why mao zedong felt so exhilarated that he spent a sleepless night when learning the news that schistosomiasis was eradicated in yujiang. the outbreak of the war in north korea, the launching of the germ warfare in particular, revealed a more complex parasitic mode. macroparasitically, the western countries had been the occupier in the colonial world order by virtue of its military power in modern times. meanwhile, china had suffered continual defeats and the loss of sovereignty and dignity. reduced to a semicolonial country, china was in an even more miserable situation than that of a colonial country. the exploitative means of the parasite countries included, among other things, the ceding of territory, fi nancial exploitation, priorities the western countries claimed, and unfair market competition in the coastal areas and in the inland areas alike. the multiple parasites and long-time extortion made the oncerich-and-beautiful exploited countries become ugly, weak, and sick. the state and its people were both sick. after the sick men awakened and began an organized resistance, however, the western powers turned to violence (the war in north korea) as a new parasitic means. microparasitically, the natural properties of pathogenic microorganisms were separated and more social, political, and even cultural signifi cances were added. metaphors such as the "sick man of east asia," the "yellow peril," and others were used to denigrate the chinese people politically, morally, and in many other ways. next, diseases caused by microparasites, communicable diseases especially, were used as a means to extend the rights or benefi ts of the macroparasites (the plague in northeast china in part i can be referred to in this regard). in addition, advanced medicine was imported to strengthen its advantageous and civilized position so that the sentiments of resistance or rebelling of the colonial people could be broken down in a secret and artful way. furthermore, when the macroparasites went through a crisis in their survival, microparasite could be turned to as the last resort; "germ warfare" was invoked as a means to debilitate the combating force in the colonial area and to incur social panic. in reality, standing in strong contrast to the irony, and with the disintegration of the sick man metaphor, the metaphor began to be effectively dissolved and a turnaround occurred when resistance by means of armed forces turned to ideological condemnation by means of exposure of the crimes committed by the united states in bacterial warfare. while the western society used modern hygiene to construct the metaphor of the "sick men" and to establish the colonial order, those "sick men," who now had means of criticism at their hands, also used the knowledge framework for modern hygiene to create a basic narrative model and correspondent discourse system of the "virus or pathogens versus colonizers or aggressors." in a time when a variety of discourses and thoughts of "modern," "modernity," "postmodern," and even "post-postmodern" emerge, "farewell" seems to have become a popular and fashionable word in contemporary china: "a farewell to the tradition," "a farewell to revolution," "a farewell to ideology," and "a farewell to the state." however, can one so easily bid farewell? the olden times of the "sick men" are fading, but history is still unraveling itself. at this moment, when we indulge in vivid imagination and visions of modernization or modernity, we seem to have forgotten the past, which is not very far-gone. however, if we could bid farewell to the sick man metaphor because of "weapons of the weak," then how about modernization? when the modernization complex that has clung to the minds of chinese people since the modern times, when sometimes it even becomes an oppressive discourse, in what way is its basic approach different from the construction of the metaphor of the "sick men"? when the irony in between (e.g., growth without development) presents itself continuously, do we possess the basic consciousness and ability to deconstruct it? have we found the "weapons of the weak"? maybe what follows will be useful and offer some food for meditation. guns, germs, and steel: the fates of human societies suppression of u.s. germ warfare crimes. people's daily, . li siguang on new people -on the emphasis on military affairs plagues and peoples (yang yulin i think this turn is more than an "imagination"; it also evolved into realistic social action and achieved many instantaneous or far-reaching these forms need almost no prior coordination or planning. with their tacit understanding and informal networks, the peasants could help themselves without directly or symbolically fi ghting against the authority report on chinese people's voluntary army in the war to resist u.s. aggression and aid korea epidemics and history: ecological perspectives and social responses weapons of the weak: everyday forms of peasant resistance illness as metaphor social history of diseases in modern china key: cord- - vdqq authors: norrie, philip title: how disease affected the end of the bronze age date: - - journal: a history of disease in ancient times doi: . / - - - - _ sha: doc_id: cord_uid: vdqq dr. norrie provides a summary of the fifteen currently accepted causes for the end of the bronze age in the near east and then goes on to discuss the sixteenth reason—infectious disease epidemics. these are the real reason that the end of the bronze age in the near east was called either the “catastrophe” or the “collapse” due to its short time frame of years, the mass migration of the general population and the “sea peoples” plus the abandonment of cities such as hattusa, the capital of the hittite empire c. bce. the diseases most likely to cause this collapse are smallpox, bubonic plague and tularemia. in bce the ancient near east was dominated by egypt in the nile valley and into the levant, the kingdom of babylon in mesopotamia and the minoan civilization in crete. anatolia was made up of small city-states including arzawa. egypt and babylon's military tactics were based on foot soldiers and were no match for the hordes of invaders who came from the central asian plains and swept through the near east. they came in fast twowheeled horse-drawn chariots with a driver and an archer on board. this system of weaponry was developed and refi ned earlier, during years of warfare on the plains of central asia. this type of weapon or style of warfare was unknown among the classical oriental civilizations. the foot soldiers of egypt and babylonia were unable to defend against these invaders. in bce , the hyksos swept into the nile delta region, and in bce the hittites swept into mesopotamia. the middle bronze age civilizations c. bce displayed all their characteristic social traits: a low level of urbanization due to an agricultural based society; small cities centered around royal palaces; numerous temples; a strict separation of classes between an illiterate mass of peasants and craftsmen and a powerful military elite. knowledge of writing and education was reserved to a small minority of scribes and the aristocrats. developments in the ancient near east after bce include the hittite empire beginning c. bce , in c. bce the minoan civilization reached its peak and mycenae in greece was occupied with the mycenaean civilization fl ourishing until c. bce . in c. - bce the mitanni kingdom in northern mesopotamia began, minoan culture ended on crete and the kassites ruled babylonia. later the mitanni kingdom was divided in two with the western half taken over by the hittite empire and the eastern half taken over by the assyrians. at the end of the bronze age between and bce only the egyptian empire remained with all other empires, kingdoms and citystates having been destroyed in the "catastrophe". human cultural development can be measured by means of the types of materials used to make tools, utensils and weapons. first there was the stone age where hard fl int stone was sharpened by chipping away and honing the cutting edge on another fl int stone. this resulted in a sharp cutting edge, which could be used as a knife, axe or spear tip. the next stage was the copper age, the fi rst of the metal ages. copper by itself is a relatively soft metal; so copper-only metal implements were weak. so the metal workers looked for ways to make the copper stronger. through trial and error they arrived at the idea of mixing or alloying small quantities of tin with the copper to form bronze. the dates for the bronze age varied from region to region depending on the region's copper and tin ore supplies or that region's ability to trade for bronze ingots. for example, the bronze age in the near east (the region of interest in this book) went from c. - bce , while in south asia it was from - bce , in europe - bce , in china - bce and in korea - bce . by contrast the sub-saharan african region bypassed the bronze age altogether going from the stone age directly to the iron age with the use of imported iron, because the region had no copper or tin mines and no ability to trade for bronze. some cultures went straight from the stone age to the iron age, bypassing the copper and bronze ages, when their culture was infi ltrated by immigrants who brought iron age tools with them; such as when the british colonized australia, new zealand and fiji. focusing on ancient near east, the near east had the earliest bronze age because "metallurgy developed fi rst in anatolia, modern turkey. the mountains in the anatolian highland possessed rich deposits of copper and tin". copper was also found in cyprus, the negev desert and iran. tin was found in more distant places such as far off cornwall (uk) and the north west indian subcontinent, so trade in tin became a vital trade route commodity. the near eastern bronze age has been divided into the early bronze age (c. - bce ), middle bronze age (c. - bce ) and the late bronze age (c. - bce ) by scholars. a more detailed dating system is shown in table . . the next stage in human cultural development was the iron age; this is the third and fi nal stage in the usual three-stage sequence of stone age, bronze age and iron age or the fourth in the more detailed fi ve-stage sequence of stone age, copper age, bronze age, iron age and steel age. the iron age ended in each region with the beginning of what is known as the historical period, that is, the local production of suffi cient detailed written sources and records. again the dates for the iron age vary from region to region depending on that region's access to iron ore mines. again anatolia was blessed with abundant iron ore in its mountains. consequently the ancient near east had the earliest iron age from to bce followed by india - bce and europe - bce , china - bce , korea - bce and japan bce - ce . other historians give earlier dates for the iron age in anatolia saying the earliest iron ware dates from about bce and forged weapons at the latest by bce . according to the late eminent oxford historian john morris roberts when discussing the hittite empire, he stated it "enjoyed a virtual monopoly of iron in asia, this not only had great agricultural importance but, together with their mastery of fortifi cation and the chariot, gave the hittites a military superiority which was the scourge of egypt and mesopotamia". also "the use of iron weapons by the hittites was believed to have been a major factor in the rapid rise of the hittite empire". by bce , iron was in common use around the middle east but it would take a few more centuries before it supplanted bronze as the dominant metal even though it was lighter and stronger. one theory for the collapse of the bronze age was a lack of tin either due to it having been mined out or because its trade routes had been disrupted due to raiders, thus forcing metalworkers to look for an alternative metal. hence iron became the next preferred metal. another explanation for the end of the bronze age is simply that iron, once discovered, was found to be a better metal because it was much stronger. a hittite tomb in anatolia housed a dagger that had a smelted iron blade dating from bce , making it one of the earliest ever examples of smeltered iron. the late bronze age saw the slow continuous transition from bronze to iron throughout the near east region as price and availability of iron, not just the iron-making technology above, determined the rate of expansion of iron usage. thus it was a gradual transition from late bronze age to early iron age over several centuries in the near east around bce . the end of the hittite empire occurred c. bce and was part of a much larger event-the end of the bronze age. the end of the bronze age was known as the "catastrophe" because it signaled the end of all the known empires in the eastern mediterranean and near east including the hittite empire, the mycenaean kingdoms and the egyptian empire in syria and canaan, but not the central nile based egyptian empire. it was followed by the "ancient dark age" when all that was glorious about these lost empires including, not only their palaces and cities but also their history, religions, writings, art, music, administration and organization, temples, libraries, political systems and trade routes were lost. this loss lasted many centuries until the emergence of the classical greek, assyrian and persian empires and the hebrews later. the catastrophe lasted from c. -c. bce and was characterized by its short time frame of about years, mass migrations of populations and mass destruction where whole cities were destroyed and burnt, but also curiously many whole cities were just abandoned intact. it was also characterized by another mass population migration involving raiders called the "sea peoples" who invaded the near east from the mediterranean region. it was "violent, sudden and culturally disruptive". the dartmouth university archaeology department has an online lesson about the collapse of the mycenaean palatial civilization in greece around bce . it summarizes the causes of this collapse as: economic factors, climate change, internal social upheaval, invasion from outside the aegean world and changes in the nature of warfare. historian robert drews in his book the end of the bronze age has on his list of possible causes of the collapse the following: earthquakes, mass migrations, ironworking, drought, systems collapse, raiders and changes in warfare. the internet website, www.enotes.com , lists the possible causes for the bronze age collapse, which includes the following: volcanoes, earthquakes, migrations, raids, ironworking, drought, changes in warfare and general systems collapse. note that there is no mention of disease as a possible cause of the end of the bronze age in any of these three lists. by contrast this book proposes the more comprehensive list of sixteen causes for the end of the bronze age, including diseases. but the fi nal answer for the causes of the end of the bronze age will be multifactorial as some things will apply to one area, such as earthquakes in greece that do not apply to other areas. a possible major cause though, one that knows no boundaries or political borders when it spreads are infectious disease epidemics. so what is the evidence? mt. hekla is a volcano located on the southern side of iceland. according to volcanologists and egyptologists, it erupted in bce throwing an estimated " . cubic km of volcanic rock into the atmosphere, placing its volcanic explosivity index (vei) at . this would have blocked out the sun leading to cooler temperatures in the northern parts of the globe for a few years afterwards. thus plants would not grow causing famine and eventual disease in the weakened population. traces of this eruption have been identifi ed in scottish peat bogs, and in ireland a study of tree rings dating from this period has shown negligible tree ring growth for a decade". the volcanic explosivity index is a relative measure of the explosiveness of a volcanic eruption on a scale of - , with being a mega-colossal eruption where the cloud column of ejected material would climb to a height of over km. such a large volcanic eruption could have caused famine in the near east contributing to the demise of the region. the scale is logarithmic where each interval on the scale represents a ten-fold increase in the material ejected. by comparison the largest volcanic eruption in recent history was mt. krakatoa in which had a volcanic explosivity index of , and mt. vesuvius which destroyed pompeii in had a volcanic exposivity index of . mike baillie is the emeritus professor of paleoecology at the queen's university in belfast, northern ireland and is an expert of dendrochronology, which is the science of dating by means of tree rings. he has built a year by year chronology of tree ring growth reaching back "the last , years". his work has confi rmed "that the events such as those at bc, bc and ad , represented abrupt environmental downturns with profound effects on human populations". these dates correspond with dynastic changes in china, the end of the bronze age in the near east and the plague of justinian, respectively. these events were most likely volcanic in cause but he also argues that it could also be due to the impact of debris from comets blocking out the sun and causing temperatures to fall for years, hence poor growth in trees, as shown in their tree rings, and all other plants leading to famine which makes the general population more prone to disease. this has been formalized in his paper "hints that cometary debris played some role in several tree ring dated environmental downturns in the bronze age". earthquakes amos nur is the emeritus professor of geophysics at stanford university, california, usa and he: postulates that earthquakes tend to occur in "sequences" or "storms" where a major earthquake above . on the richter magnitude scale can in later months or years set off second or subsequent earthquakes along the weakened fault line. he shows that when a map of earthquake occurrence is superimposed on a map of the sites destroyed in the late bronze age, there is a very close correspondence. in his paper "the end of the bronze age by large earthquakes?" nur proposes that a large earthquake storm lasting years from c. to c. bce could have contributed to the end of the bronze age. twentieth-century geophysical data about the geography of active tectonic faults especially at geological plate boundaries, the location of earthquakes, the geography of ground motion intensity, and earthquake frequency-magnitude statistics in the eastern mediterranean show that most of the sites that collapsed at the end of the bronze age must have experienced destructive earthquakes repeatedly in their past. recent and historical evidence shows also that these massive earthquakes reoccur every few hundred years in bursts, or "storms" of large events that sweep across broad portions ( - km in length) of the eastern mediterranean and over short periods of time ( years) . this suggests that a large earthquake could have contributed to (and probably did contribute to) both the physical and political collapse of the great centers of civilization at the end of the bronze age. this probably began by an earthquake storm that unzipped the plate boundaries in the eastern mediterranean between and bce . the earthquakes in this -year long storm could have rendered many of the urban centers militarily vulnerable, thus inviting attacks, not by powerful distant sea people but by opportunistic indigenous or neighboring populations. these attacks may have led in turn to the political and social collapse of the centres, followed by a dark age of recovery and rebuilding often lasting a few hundred years. figure of nur's paper shows "superposition of the sites destroyed at the end of the bronze age in bc and the earthquake intensity vii in our century . the coincidence suggests that the end of the end of the bronze age could be related to earthquakes". (see the table of bronze age earthquake destruction in nur's paper.) drews in his book end of the bronze age dismisses the idea of earthquakes totally. he says that archaeologists only look at their own site and an earthquake may be relevant only to this site and not the whole of the eastern mediterranean. nur, on the other hand, has shown that earthquakes occurred all over the eastern mediterranean and near east. elizabeth b. french is an authority in mycenaean pottery and a former warden of ashbourne hall, manchester university and the former director of the british school of athens, from to , which is a facility established in to promote british-based greek archaeological research and study. in french wrote: archaeologists of my generation, who attended university in the immediate aftermath of schaeffer's great work in , were brought up to view earthquakes, like religion as an explanation of archaeological phenomena to be avoided if at all possible. thus, it is only recently that an earthquake at mycenae has begun to be a serious hypothesis. claude frederic-armand schaeffer ( - ) was the fi rst to conduct the excavations at the ancient city port of ugarit at ras shamra in syria from that showed that the city was destroyed c. bce by the invading sea people as told by ammurapi, the king of ugarit, in a letter to the king of alasiya. in , schaeffer fi rst suggested that an earthquake may have destroyed late bronze age sites. his idea was rejected by archaeologists because the catastrophe was spread over a -year period and could not have been the result of a single earthquake. schaeffer did not know then about -year long "earthquake storms" as proposed by nur, which have put earthquakes back on the agenda. schaeffer was a french scholar and archaeologist who was the curator of the prehistoric and gallo-roman seum in strasbourg ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the museum of national antiquities in saint-germain-en-laye . fritz schachermeyr ( schachermeyr ( - was an advocate for earthquakes playing a role in the end of the bronze age, which he proposed in his book griechische fruhgeschichte . he has a similar view to nur in advocating that catastrophic earthquakes destroyed troy in western turkey, mycenae in greece and knossos in crete about bce . fritz was a professor specializing in ancient history and more specifi cally in ancient tsunamis usually accompany an underwater earthquake or volcanic eruption. so in line with the above evidence about earthquakes and volcanoes then a destructive tsunami may also have occurred, such as when the volcano on the island of thera erupted sending a massive tsunami to crete destroying many minoan coastal settlements. during the catastrophe many new ethnic groups started to appear in the near east region. indo-european tribes such as the phrygians, thralians, proto-armenians, macedonians and dorian greeks seem to have arrived at this time-possibly from the north. there also seems to have been widespread migration of the aramaeans, possibly from the south east. the people involved in the mass migrations could have been "pushed" by drought and subsequent famine, earthquake destruction, raiders, and "sea people" invasions. they could also have been "pulled" by the prospect of better lands and food, plus safety being far away from raiders. another one of the possible "push" scenarios could be fl eeing disease, and this should be factored in by historians. the "sea peoples" is the name given to a confederacy of seafaring raiders who invaded the eastern mediterranean at the time of the catastrophe. they invaded cyprus, the hittite empire and the levant. they also tried to invade egypt but were repelled. the pharaoh merneptah (reigned c. -c. bce ) referred to them as "the foreign countries or peoples of the sea" in his great karnak inscription. he fought and defeated them at perire, in the western nile delta during the fi fth and sixth years of his reign. later the great pharaoh ramesses iii (reigned c. -c. bce ) had to deal with a later wave of invasions by the sea peoples. the following inscription from his medinet habu mortuary temple describes what happened, who they conquered and who made up their confederation force: the foreign countries (i.e. sea peoples) made a conspiracy in their islands. all at once the lands were removed and scattered in the fray. no land could stand before their arms: from hatti, qode, carchemish, arzawa and alashiya on, being cut off (i.e. destroyed) at one time. a camp was set up in amurru. they desolated its people, and its land was like that which has never come into being. they were coming forward toward egypt, while the fl ame was prepared before them. their confederation was the peleset, tjeker, shekelesh, denyen and weshesh, lands united. they laid their hands upon the land as far as the circuit of the earth, their hearts confi dent and trusting: 'our plans will succeed! ammurapi was the last bronze age king of ugarit. he wrote a letter to the king of alasiya telling how the sea peoples had invaded and destroyed his country: the sea people, like the other mass migrations, may have been "pushed" out of their lands by earthquakes, drought, and famine; and "pulled" to new lands looking for food and minerals such as iron, tin and copper. they too may have been "pushed" out of their lands by infectious disease. there are many hypotheses about who were the "sea peoples". . philistine hypothesis : the hebrews called the southern coastal plain of ancient palestine "philistia" and the people who lived there in the fi ve towns called the "philistine pentapolis" of askelan, ashdod, ekron, gath and gaza were called "philistines". there are two schools of thought as to their origin. one school says they were mycenaean because they made distinctive mycenaean iiic pottery, whereas the other school says they were an indigenous canaanite culture. either way they became a force that helped attack egypt as part of the "sea peoples". . minoan hypothesis : after the eruption of the volcano at thera (today's santorini), sometime between - bce , crete would have been devastated by fi res and ash causing a cooling climate resulting in crop failures and famine, plus the tsunamis destroying the coastline. the people of the minoan culture would have been forced to leave crete, some scholars say initially to anatolia and from there later to the levant as "sea peoples". . greek migration hypothesis : greece not only possibly supplied people for the philistines but also supplied people who migrated to sicily and sardinia as well as fought in troy and later occupied cyprus. so they would have been bands of raiders/invaders like the sea peoples. . trojan hypothesis : historian eberhard zangger proposes that the "sea peoples" may have come from troy and its allies, and that the greek literary tradition of the trojan war, as told by the poet homer, may well refl ect the greek efforts to counter those raids. zangger ( ) is a swiss german who has a phd in natural sciences from stanford university and was a senior research associate in the department of earth sciences at the university of cambridge from - . he is a writer on geoarchaeology especially in the prehistoric aegean area. mycenaean city states fought each other over decades. refugees from this fi ghting then turned to piracy for survival and that this in turn led to becoming "sea peoples" raiders. . italian peoples hypothesis : italy may have supplied some of the sea peoples from the etruscans. . anatolian famine hypothesis : in this hypothesis extensive drought in anatolia, which led to famine, in turn forced people from places in anatolia such as lydia to go to sea and become sea-going migrants. these displaced people could later become sea peoples. a famous passage from herodotus portrays the wandering and migration of lydians from anatolia because of famine: in the days of atys, the son of manes, there was a great scarcity through the whole land of lydia … so the king determined to divide the nation in half … the one to stay, the other to leave the land ... the emigrants should have his son tyrrhenus for their leader ... they went down to smyrna, and built themselves ships … after sailing past many countries they came to umbria ... and called themselves … tyrrhenians. . invader hypothesis : historian michael grant proposed that there may have been another origin for the "sea peoples" besides the aegean. he proposed that "there was a gigantic series of migratory waves, extending all the way from the danube valley to the plains of china." grant ( grant ( - ) received a doctor of literature from the university of cambridge and was an english classicist, numismatist and author of more than seventy books on ancient history and ancient coins. fellow historian sir moses i. finley has suggested that the carpatho-danubian region of europe was the original centre of this disturbance. sir michael grant and sir moses i. finley suggest that these people were invaders who destroyed sophisticated cities and built simpler and less complex settlements with plain pottery and simple tools, on top of the ruins. this demonstrates a cultural discontinuity from a more advanced culture to a less advanced culture after the invaders or raiders took over. sir moses i. finley was born in the usa as moses israel finkelstein but changed his surname to finley in . he studied at syracuse university and columbia university and taught ancient history at columbia university and rutgers university in the usa, specializing in the social and economic aspects of the ancient world. after being attacked for his left wing opinions by senator joseph mccarthy in he immigrated to britain where he taught classics at the university of cambridge from , eventually becoming professor of ancient history from to when he was also knighted. the collapse of the bronze age should also be seen as part of the bigger technological picture and changes taking place at the time, that is, the slow change from bronze making to iron working. even though the hittites in anatolia were the fi rst great power to have iron at the time of the collapse, the general regional shift from bronze to iron occurred after the collapse of the bronze age c. bce . the sea peoples' weapons for example were made of bronze, not iron. so iron confi rmed the collapse and end of the bronze age but did not cause it. bronze is made from a combination of - % copper and - % tin. in the ancient world tin was an element in short supply. without tin the only metal available for implements and weapons was the much softer plain copper of the copper age. the near east had few tin mines with anatolia being the main source of tin. the majority of the tin was imported from outside the near east, with cornwall in britain and other sources to the distant east of the near east, such as afghanistan, being the main suppliers. hence the tin trade and its trade routes were very important, like oil trade today. if the tin trade collapsed for whatever reason, such as war or disease, then the bronze age would fi nish, as people would have to look for alternative metals such as iron, also the bronze age could have ended because iron is a stronger hence better metal and would have replaced bronze as a metal of choice. herodotus (c -c bce ) was a greek historian who is regarded as the "father of history". he wrote a detailed history of the greco-persian wars called the "histories" and he "was the fi rst historian known to collect his materials systematically, test their accuracy to a certain extent a nd arrange them in a well-constructed and vivid narrative". he proposed that the bronze age ended because of a long drought that caused famine and social disruption. examples of grain shortages in the hittite empire and demands for urgent resupply of hatti in the late bronze age, just before the end, were mentioned in the last chapter. dendrochronological (tree ring) studies have shown drought years leading up to bce . "starting from bce, we see indices smaller than per cent, except for two years, and bce. in addition there is a continuous decrease in indices in the last fi ve years of the empire. examining the tree rings shows the existence of dry years which contributed to drought on the lands". peter ian kuniholm founded the aegean dendrochronology project in and has been its director ever since. this project examines tree rings all over the aegean and near east to make up a chronology of the area from neolithic time to the present. he has shown that in bce trees grew at only . % of their normal rate, in bce at . % and in bce at % indicating three consecutive years of drought, which would lead to famine. examination of the data presented in this paper shows that the occurrence of three such poor growth years in a row was rare. kuniholm is the professor of archaeology and dendrochronology at cornell university. he gained his ba at brown university in , his ma at vanderbilt university in and his phd from the university of pennsylvania in . other researchers also support the arid climate change theory at the end of the bronze age. anthropologist brandon drake from the university of new mexico studied various parameters including mediterranean sea temperatures to show the change in the climate to much drier times at the end of the bronze age and early iron age. by contrast, archaeologist jennifer moody studied the change in building structure over the late bronze age period to show that buildings had verandas, and were airy with good ventilation to keep them cool because the climate became " signifi cantly drier than previously". famine naturally what follows drought is famine. as discussed above, there was correspondence between the hittite king and the egyptian pharaoh about urgent grain shipments for hattusa. famine can drive people from their lands and could be one of the causes for the mass migrations seen at this time, and could also have pushed the sea peoples from their land in search of new territories with food. famine can also weaken people and make them more prone to disease. for example prior to the black death, which started in europe in , there was the great famine which lasted from - which greatly weakened the population of europe. the great famine was not caused by drought, as in the end of the bronze age though, but by several years of excessive rain fall. during this time crops were fl ooded or could not be planted. disease could also cause famine because the farmers or slaves necessary to work the fi elds would have died, hence crops would not have been planted or maintained, let alone harvested, which required considerable man power. robert drews "suggests that these collapses occurred as the result of a fundamental change in the nature of warfare in this period". the horse drawn chariot with an archer behind the driver was introduced in the seventeenth and eighteenth centuries bce and subsequently dominated the near eastern battlefi elds. they did not dominate in the aegean as the topography was different being more rugged terrain with fewer large fl at areas suited to chariot warfare. these chariots were manned by a socially and economically privileged warrior elite (e.g. the hittite empire, the mycenaean kingdoms, city states of coastal syria and the levant (such as ugarit), the hurrian kingdom of mitanni, the kassite kingdom of babylon etc). drews argues that these large massed chariot formations involving sometimes thousands of chariots became vulnerable when they were harassed and killed by highly mobile, lightly armed infantry who used new and better weapons with great success. these new weapons included (a) the aspis-a highly maneuverable small round shield - feet in diameter as opposed to the sakos or large immobile shield; (b) the javelin which had elliptical heads instead of a barb which allowed easy retraction hence reuse. massed javelins thrown on the run using massed swarming formations were very effective against massed chariots; (c) the new nave type ii sword which was about cm long which "optimized both for slashing as well as stabbing or thrusting" and fi nally (d) "the infantryman's corslet, which protected the trunk, arose, along with greaves for protecting below the knees". this new warfare meant that an entire elite social order of charioteers became redundant and were replaced by infantry and cavalry. this meant signifi cant disruptions of the aristocracy and consequently the royal families. unfortunately drews only provides a single answer solution to a very complex multicausal problem, hence is guilty of oversimplifi cation. also his approach does not apply to the aegean where chariots were less dominant due to the hilly topography. after the end of the bronze age mycenaean pottery continued to depict war chariots as part of the decoration thus confi rming that they had not died out. this assumes that the potters were portraying only contemporary and not historical scenes. the late bronze age palatial kingdoms all had fatal centralized, complex and top-heavy political structures, which made them vulnerable. this bureaucratic domination and top-heavy administration involved the use of a whole scribal class of record keepers and was highly specialized. it also involved the exploitation of the peasantry who funded the whole expensive top-heavy system with their taxes. so when the system was stressed by famine, war or disease for example, it was unable to cope due to lack of funding to prop up "the system". once the administration was affected then other things such as trade became affected. once trade in all important commodities such as tin, grain, olive oil, wine and timber was stopped, the end was inevitable. a weakened administration could also not handle other crises such as peasant revolts, defection of mercenaries, overpopulation and invasion by sea peoples. the general systems collapse theory was fi rst proposed by joseph tainter vast sums of money were needed to run an empire especially one with an expensive and large bureaucracy. besides their administrative expense, there was the cost of the royal family and the large armies necessary not only to defend the empire but also to help expand it. once the revenue from taxes dried up the system, lacking funding, collapsed. so this is a consequence of the the reduced income caused by peasants migrating and dying due to famine, war or disease, but not a cause of the end of the bronze age. this theory put forward variously by vermeule ( ), lakovides ( ) and betancourt ( ) suggests that the piratical activity of the "sea peoples" would cripple trade routes, hence disrupt commerce so much as to bring down the late bronze age empires. the theory refers to a consequence of the "sea peoples" activity and not to why the "sea peoples" were active in the fi rst place. so once again there is a theory which turns out to be a "consequence of" and not a "cause of" the end of the bronze age. the hittite royal families provide many examples of internal fi ghting within a royal family. murder, even of the incumbent king as a means to gaining control of the throne, was accepted practice. fighting between different branches of the royal families leading to civil war would have badly destabilized any empire. oliver dickinson from durham university had a similar view when he stated in chapter "the collapse at the end of the bronze age" in his contribution to the oxford handbook of the bronze age aegean (c - bc) that "it is a waste of effort to try to isolate a single cause or prime mover for the collapse." this is because it was most probably the result of several causes. this is what this book is arguing that the end of the bronze age in the near east, referred to as either "the catastrophe" or "the collapse" [ dickinson preferred the term collapse], was the result of a cascade of events beginning with volcanic eruptions and comets. these caused drought which in turn caused famine, which weakened the population so they (rich and poor alike, because disease does not discriminate) were prone to whatever infectious disease happened to be prevalent at the time. this is in agreement with dickinson's "natural disasters like earthquakes, localized droughts leading to famines, or epidemic diseases could have acted as catalysts for trouble or have exacerbated an already deteriorating situation". dickinson also showed that many sites were abandoned at the end of the bronze age not reoccupied for many centuries. this would be expected when an infectious disease epidemic is active. his abandoned city list included places such as gla, messenia and krisa in the aegean, besides the well documented abandonment of hattusa, the hittite capital in anatolia. guy middleton's phd, "theories of mycenaean collapse", favored the idea of infectious disease as a cause of the collapse, so it included a section on "plagues and epidemics" but stated "the main problem with the plague hypothesis is the lack of positive evidence". it also stated "much about the plague hypothesis seems attractive in the way it can explain the changes in settlement pattern, material and social cultural and long-term decline in population levels". the plague hypothesis was also good for "explaining the variability of collapse because "palatial areas may have been more densely settled and thus more seriously affected, while possibly less populous peripheral areas may have been less affected". once the central palace was adversely affected: "this lack of strong central power and instability seems to fi t with the kind of unstable and more mobile society suggested for palatial greece." emeritus robert arnott from oxford university wrote chapter "disease and the prehistory of the aegean" in health in antiquity edited by helen king, in which he listed some of the possible diseases from that time which included: thalassemia, malaria, tuberculosis, brucellosis, malnutrition, scurvy, anemia, measles, chickenpox, oral infection, pneumonia, hemolytic disease, enteropathies, cholera, typhoid, dysentery, tetanus, hookworm, mumps, whooping cough and amoebiosis. note that most diseases listed are infectious diseases. he also gave a possible reason why disease has not been considered as a cause for the end of the bronze age because "many such scholars are completely unaware of the social effects of disease and the major consequences that ensued whenever contacts across disease boundaries allowed a new infection to invade a population that lacked any acquired immunity." arnott referred to the research of j. lawrence angel (also known as the archaeological "bone man") and robert sallares who have found the lethal falciparum malaria dna in ancient skeletons, making malaria a strong infectious disease that possibly contributed to the end of the bronze age. he also supported the drought leading onto disease hypothesis stating "if widespread climatic change and drought were mainly responsible for the decline and collapse of the mycenaean world in the twelfth century bc, as has been suggested … then a natural consequence would have been widespread epidemic disease (e.g. cholera and typhoid) brought on by the shortage of clean drinking water". in conclusion, arnott paints a very bleak picture of life for the ordinary man in ancient times "the harsh reality of a society where life was hard, death and disease were everyday occurrences and the day-to-day ambition of those who lived outside the palaces was simply survival". eric watson-williams wrote an article about the end of the bronze age called "the end of an epoch" in which he championed bubonic plague as the sole cause for the catastrophe. "what seems so puzzling is the reason why these apparently strong and prosperous kingdoms should disintegrate" he questioned. he cites abandonment of cities, the adoption of the practice of cremation of the dead instead of the usual burial because so many people died, and it was necessary to destroy the decomposing bodies quickly, and the fact that bubonic plague is very deadly killing animals and birds as well as humans, affects large areas, spreads rapidly and lingers for many years as reasons for his choice of bubonic plague. unlike panagiotakopulu, he provides no physical evidence, but uses history to compare how things were during later bubonic plague epidemics such as the plague of justinian and the black death to how things were around bce . watson-williams argues that there were four epidemics of the bubonic plague that we know of in ancient times, namely the hittite epidemic of bce , the exodus from egypt in the reign of merneptah c. bce , the plague of the philistines c. bce and fi nally the plague "that which destroyed the army of sennacherib ( b.c.)". he also quotes a pestilence that killed , men in three days during the reign of king david c. bce that may have been bubonic plague. lars walloe from the university of oslo had a similar view to that of watson-williams when he wrote his article "was the disruption of the mycenaean world caused by repeated epidemics of bubonic plague?" he noted the "large movements of population"; "the population decreased in successive steps during the fi rst two or three epidemics of plague down to perhaps half or one-third of its pre-plague level", and that there was "a substantial reduction in agricultural production". this led to famine and the abandoning of settlements. he thus concluded that bubonic plague accounted for all of these observations rather than other infectious diseases such as anthrax. any historian trying to fi nd the cause of the end of the bronze age and the hittite empire must explain: the short time frame of approximately years, when it occurred between - bce ; the mass migrations not only of normal people but also of the "sea peoples"; and the fact that so many large cities, such as the hittite capital hattusa, were simply abandoned and not destroyed or occupied by raiders or invaders. disease in the form of infection epidemics provides one plausible explanation; there is nothing like a severe widespread plague to deliver the fi nal fatal blow to an empire or an era. the bubonic plague is caused by the yersinia pestis and is transmitted to man by fl eas from small rodents such as rats. it is a very lethal disease killing two out of every three people infected within four days. the usually accepted fi rst outbreak of bubonic plague was the plague of justinian in . "the fi rst recorded outbreak of bubonic plague was the world's fi rst great pandemic. called the mortalitas magna (great death) or the plague of justinian, it began in arabia … the pestilence reached constantinople by the spring of a.d. and lurked around the eastern mediterranean until the s." new research however by mark achtman has suggested that the bubonic plague began near china c. bce and spread to europe via central asia's "silk road". mark achtman's research was published in nature genetics in an article titled " yersinia pestis genome sequencing identifi es patterns of global phylogenetic diversity". his team compared seventeen whole genomes of yersinia pestis isolates from various global sources and "conducted phylogenetic analyses on this sequence variation dataset, assigned isolates to populations based on maximum parsimony and, from these results, made inferences regarding historical transmission routes. the phylogenetic analysis suggests that yersinia pestis evolved in or near china and spread through multiple radiations to europe, south america, africa and southeast asia, leading to country-specifi c lineages". mark achtman bsc, msc, phd from the university of california, berkeley, usa is a canadian microbiology researcher at the university college, cork, ireland where he specializes in the population genetics of bacterial pathogens and microbial phylogeography. prior to this he was a researcher at the max-planck institute in berlin, germany specializing in molecular genetics and infections biology. but, as shown earlier in this book, kozloff and panagiotakopulu argue that bubonic plague, coming from india [? indus valley civilization] could have occurred during the reign of amenhotep iii early in the fourteenth century bce , that is, predating achtman's chinese suggestion by over years. thus, if achtman continued his research he may fi nd that china may have contracted the plague from india, and that india was the primary source of the bubonic plague. this all leads to the plague of the philistines, also known as the plague of ashdod, which occurred c. bce and may have been caused by the bubonic plague. if egypt had bubonic plague during the reigns of amenhotep iii and his son, akhenaten (c - bce ) then it could have recurred to the north east in southern canaan fi fty years later. the philistines lived in south canaan at the end of the bronze age and ruled their fi ve city-states (pentapolis of gaza, ashkelon, ashdod, ekron and gath). they were the kingdom of israel's worst enemy and they fought each other many times. their origin is obscure though. some historians, such as carl ehrlich, believe they were "sea peoples" from either the aegean or mycenae in greece who settled in southern canaan after being defeated by pharaoh ramesses iii in c. bce , while others such as riemschneider believe they came from anatolia as refugees from the crumbling hittite empire. as stated earlier the plague of the philistines occurred c. bce while others think it may have occurred later, either bce or in the second half of the eleventh century bce . but peter kuniholm's aegean dendrochronology project has shown that our current dating scheme for ancient times maybe anywhere up to years out when compared to his tree ring dating scheme. "long standing assumptions and conventions in other egyptian and old world chronology and history will need to be re-examined" he has stated, hence the bce stated above could in fact have been about years out making it c. bce . according to author lee allyn davis in his book natural disasters , the philistine plague was caused by bubonic plague and began in bce after the philistines captured the ark of the covenant (a box that contained ancient hebrew records such as the ten commandments and the first torah scroll) from the israelites. the fi rst recorded plague is the one which beset the philistines in bce , and which is recorded in the bible in the book of samuel. the philistines in this year defeated an army of nomadic hebrews at ebenezer, captured the sacred ark of the covenant and carried it in triumph to ashdod, a city near the mediterranean sea. but their triumph was immediately tainted, according to samuel : : "the hand of the lord was against the city with a very great destruction; and he smote the men of the city, both small and great, and they had emerods (swellings) in their secret parts". the description makes it clear that bubonic plague had invaded the army of philistines, probably from a stricken ship. if it had originated in the ark of the covenant as the bible notes, it would have been mentioned in the old testament. wherever they took the ark (of the covenant), the philistines took plague too. they moved from ashdod inland to gath, then to ekron. the plague followed them. terrifi ed, they trundled the ark of the covenant into a cart pulled by two milk cows. if the cows took the ark to the hebrew border town of beth-shemesh, they reasoned that the lord of israel was responsible for the plague, and had indeed smitten them. the cows took the ark into the fi eld of beth-shemite, joshua stopping alongside a huge stone. israel rejoiced, but not for long. in samuel : , the bible chronicles the inexorable progress of the plague; "and he smote the men of beth-shemesh, because they had looked at the ark of the lord, even he smote the people fi fty thousand and three score and ten men; and the people lamented because the lord had smitten many of the people with a great slaughter". davis gets his idea of the plague coming from a stricken ship and not the ark of the covenant from a book about plague called plague an ancient disease in the twentieth century by charles t. gregg. in it gregg states that the plague of the philistines occurred in the twelfth century bce and that: the plague of the philistines probably invaded the town of ashdod from a stricken ship rather than with the ark of the covenant and then infected the crowds that conveyed the ark to the other affl icted cities. had the plague begun in israel there should have been accounts of it in the old testament, but samuel mentions the disease no more. others who also think the plague of the philistines was bubonic plague include w.j. simpson and w.w.c. topley and g.s. wilson. simpson ( ) affi rms that the pestilence was bubonic plague, and that the "emerods" were plague buboes, and his assertion has been repeated by later writers. topley and wilson ( ) , for example, assert that "in the fi fth and sixth chapters of the first book of samuel there is an unmistakable account of bubonic plague." in his book on the plague of the philistines shrewsbury goes against the conclusions of his fellow british bacteriologists simpson, and topley and wilson and instead endorses the belief of the fi rst century romano-jewish historian titus flavius josephus ( -c. ce ) who stated "categorically that it was dysentery, and he also discriminates the epidemic from the simultaneous plague of mice". it is possible that josephus had access to authentic material that has since been lost; it is certain that he was many centuries nearer the ancient jewish tradition than we are, and, though it appears to be fashionable in some quarters to decry the value of tradition as an adjunct to history, it is not wise to ignore it. with the support of josephus, i submit, therefore that the plague of the philistines was one of the forms of bacillary dysentery, either shiga or a virulent flexner dysentery, and that the "emerods" were "piles." note his point that "it is not wise to ignore it". in other words he is warning us that it is not wise to ignore the ideas of josephus. the basis for his fi ndings were the facts that he thought fi rstly the "secret part" of the body as the only part not visible when naked, that is, the anal area and that "hemerods" were in fact hemorrhoids or piles, which would be made worse with dysentery. this is in contrast to the others who thought the "secret part" was the genital area in general hence the "hemerods" were buboes or the swollen lymph nodes in the groin; because buboes are infl amed lymph nodes and there are no lymph nodes in the anal area. the communicable diseases that have scourged armies throughout historical times are typhus fever, typhoid fever, and dysentery, and there can be no doubt that the pestilence that affl icted the philistines fi rst erupted in their army, and was then disseminated by their victorious troops among the civilian population of their cities. typhus fever and typhoid fever can be dismissed, because neither disease is accompanied by the development of swellings around the anus; but bacillary dysentery is frequently associated, because of the invariable concomitant tenesmus, with the formation of external piles, those rounded protuberances from the anal margins that are at fi rst hot, red and painful, but which later often itch considerably as they resolve. it is commonly stated in medical textbooks that bacillary dysentery in hot climates is more prone to give rise to piles than is the disease in temperate ones. the usual explanation given is that the anal sphincter is generally more lax in individuals living in hot lands than in those living in temperate regions. but shrewsbury gives another reason for his conclusion that the plague of the philistines was in fact dysentery and not bubonic plague. there is still the third clue to be considered; to wit, the fact that the gethrites, after deliberate consultation, made "seats of skins" for themselves. to those who still opine that the pestilence of the philistines was bubonic plague, the question may be put: why should any sane individual make themselves seats of skins as a palliative for bubonic plague? of what conceivable use would a skin seat be to a person who is collapsed, delirious, and bed-ridden almost from the onset of that disease? if the "emerods" were plague buboes, the action of the gethrites was utterly nonsensical; but if, as i have argued, they were hemorrhoids, then anyone who has suffered from external piles will agree that the gethrites showed good sense. the difference in comfort to the sufferer from piles between reclining upon a skin seat and sitting cross-legged upon the ground must be experienced to be appreciated, but i can assure the reader that it is profound. the signifi cance of the plague cannot be underestimated. because of this plague the philistines returned the ark of the covenant back to the israelites to get rid of it, which consequently allowed them to fl ourish. whatever opinion is held about the nature of the pestilence that ravaged the philistines, the fact that it caused them to return the ark of the covenant to the defeated and demoralized israelites is indisputable. that restoration deprived the philistines of the inestimable moral advantage that the possession of the ark gave to them, and at the same time restored to the israelites the focus upon which all their national activities and aspirations converged. i therefore affi rm that this act, because it saved the israelite society from almost certain extinction, must be ranked as one of the decisive acts in human history, and i draw support for that affi rmation from the authoritative pronouncement of osterley and robinson ( ) : "yet small and insignifi cant among the nations of the world as israel was, without political infl uence or extended power, it may safely be said that no other people of antiquity holds a place of such profound importance in the history of human thought. it was israel who gave to the world a religion which has directed the spiritual life of nearly half mankind, and, not only among the jews themselves, but in the two daughter faiths of christianity and islam, has molded the beliefs of men in every continent save central and eastern asia". william oscar emil osterley and t.h. robinson were historians who wrote the book a history of israel published by oxford university press in . the other aspect of this plague is the fact that it occurred in palestine, which shrewsbury referred to as the "cock pit" ) of the ancient world. according to osterley and robinson "palestine was the commercial, military and political center of the ancient world, and on it focused all the greatest movements of the peoples" because any conqueror had to control it if they wanted free movement from europe or asia into africa or in the reverse. so controlling palestine was vital because of this movement factor between europe, asia and africa, the same factor that would also have easily spread the plague via the fl eeing masses into adjoining regions thus spreading it throughout the near east during the end of the bronze age. the following hypothesis about tularemia is included for the sake of completeness, so that no option is left out or eliminated from the list of potential infectious diseases that caused the end of the near eastern bronze age. tularemia, also known as rabbit fever, is a bacterial infection caused by francisella tularensis . it is easily spread and is highly virulent making it the perfect infective agent for an epidemic. for these reasons it, along with dysentery (already discussed), the bubonic plague (already discussed), smallpox (to be discussed as the next infective disease), and anthrax (to be discussed later), have been used as infective disease agents for biological warfare. francisella tularensis infects small mammals such as rabbits, hares and other rodents who can in turn infect humans directly or via their ticks or mosquito or fl ies. "humans can get the disease through a bite from an infected tick, horsefl y, or mosquito; breathing in infected dirt or plant material (causing pneumonia); direct contact through a break in the skin with an infected animal or its dead body (most often a rabbit, muskrat, beaver, or squirrel) or eating infected rare meat". so the disease is easily contracted either by direct contact with host rodents, ticks, mosquitoes, fl ies, ingestion of contaminated food and water or as aerosol particles being inhaled. siro trevisanato, an italian-canadian molecular biologist proposed that tularemia was used by the hittites as a germ warfare agent, caused the hittite plague of bce and later still caused the plague of the philistines. trevisanato ( -) graduated in with a ba in biology from suny purchase, usa; in with an msc in biochemistry from new york medical college, valhalla, usa and a phd in in molecular biology from the university copenhagen, denmark. avaris was a port city in the northeastern region of the nile delta. it was the major administrative hub for the nile delta during the hyksos era (fifteenth dynasty) and later became the capital of egypt under the hyksos. it was occupied from c. to bce and according to trevisanato was the port through which tularemia entered egypt c. bce . paragraph , column , lines to of the hearst papyrus tells of an infectious disease attributed to "the asiatics", that is, people from the syria-canaan-transjordan area also known as the "canaanite illness". it has also been attributed to bubonic plague or typhus. austrian archaeologist manfred bietak uncovered the ruins of avaris at tell el-dab'a in the eastern nile delta. there he also found unequivocal signs of an epidemic. more precisely, the layer dividing the so-called stratum g from stratum f of avaris contains a large number of tombs, which are characterized by a hasty job. this fact is the signature of emergency situations such as those existing during a plague. artefacts in the tombs revealed that they and, thus the epidemic, could be dated to c. bce . during the epidemic of bce the hebrew population in avaris fared a lot better than the local egyptians because the egyptians living in the city had no immunity to tularemia while the hebrews, who were exposed to sheep and other animals, had a natural immunity to tularemia and survived. if the disease had been bubonic plague or typhus then the hebrews would have died at the same rate as the egyptians. trevisanato also thinks that the hittite epidemic of bce was due to tularemia rather than bubonic plague. a deadly epidemic, also dubbed the hittite plague, affected most of the middle east towards the end of the fourteenth century bce . the present study determined that its onset was described in the egyptian royal archives, enabling to date it to the last reigning years of akhenaten, that is, just before bce , and locate the focus to an area northeast of byblos (present-day lebanon). letter ea states that "there is a pestilence in simyra". anyone from simyra was barred from entering nearby byblos, and donkeys were not to be used in caravans because of the pestilence. the measure did not work, as evidenced by letter ea stating the pestilence did reach byblos, and by letter ea stating the byblos ruler became chronically ill. the plague spread further south as attested by the ruler of amurru in present-day southern lebanon, who referred to his relationship with byblos, and mentioned he was now sick, and was going to die (ea ). still further south, along the coastal trade route from byblos, coeval letter ea reports megiddo "is consumed by pestilence". the east-west trade road going through simyra linked the mediterranean coast to the euphrates. reports from bce show that east of simyra in babylon, an aristocratic woman died from plague (ea ), and the local ruler was ill (ea ), consistent with the spread along the trade route. west of simyra, coeval letter ea from cyprus stated "the hand of nergal is in my country", killing many, in particular individuals linked to copper mining. the attribution of the plague to the mesopotamian god of pestilence nergal points to an origin from the east, that is, via canaanite harbors and indicates that the etiological agent also travelled by ship. note that the letters referred to in the above quote are amarna letters and are quoted from william moran's book the amarna letters published in by the john hopkins university press, usa. trevisonato argues that initially the epidemic was confi ned to the central area of the near east from cyprus to iraq and from israel to syria, sparing egypt and the neshite or hittite empire. later, after fi ghting between egypt and the hittites at amka on the litani river to east of byblos and simyra in c. bce , the tularemia infection spread into anatolia via the egyptian prisoners of war to cause the -year hittite plague. it would have also spread via the various trade routes that went through the levant. in c. bce , arzawa in western anatolia attacked the hittites. eventually, after a hittite counterattack, the hittites laid siege to the capital of arzawa. during this siege the hittites left sheep carrying tularemia outside the city walls. the starving inhabitants of the city brought the sheep into their city where they, unbeknown to the city inhabitants, spread the disease among the population. thus this is the fi rst documented example of biological warfare. trevisonato argues why he thinks the epidemic was caused by tularemia: the reconstruction of the dynamics of the epidemic helps identifying the etiological agent. a disease lasting - years, infecting humans and animals, causing fever, disabilities, and death, spreading via rodents aboard ships as well as donkeys, points to francisella tularensis , the etiological agent of tularemia. this disease can linger for a long time, and its longevity is incompatible with shorter-lived epidemics such as from bubonic plague, which for instance hit europe around - , and in - . furthermore, the description in neshite records, e.g. knees, debilitation, and sensation of internal burning, is also coherent with tularemia. moreover, tularemia also fi ts the onset of the infection, as it infects caravans stopping for rest, turning them into carriers for the etiological agent. the aforementioned trading route between the mediterranean sea and the euphrates would have had such contact points allowing for the spread of the pathogen. trevisonato further believes that the plague of the philistines was also due to tularemia, which has canaan, the setting for the plague of the philistines, as a natural reservoir as shown above. he does not believe it was due to bubonic plague or dysentery and puts forward a strong argument: an etiological agent for the philistine plague. the biblical data appear to center around the box as a vehicle for the disease, as well as the rodents that appear shortly thereafter, and are depicted in the "settlement" paid in gold. the hebrew word akhbar for the rodents fails to distinguish between mice and rats. rats would have carried y. pestis but bubonic plague fails to adequately explain the epidemic. mice are a better option; they can carry diseases, and fi t the other data relative to the historical text, i.e. box, idol, and settlement payment. the gold-plated wooden box measured . × . × . cubits (ex. . - ; ex. , - ), that is, . × . × . m, giving a volume of roughly l, offering a nest to mice but not rats. the former animals average g and are small enough to enter the box through a small aperture possibly hidden by the gold covering. the latter animals average g requiring a wider aperture and more internal space. mice nesting in the box would have explored their new habitat upon each transfer of the box, thus offering an explanation for the box transmitting the disease. mice also explain the otherwise odd detail of a small philistine idol falling on the fl oor. once the box was hosted in the philistine temple, the animals exiting the box from the same aperture, would have tipped over the statuette, eventually breaking the extremities after repeated falls ( sa. . - ). the fi ve replicas in gold of rodents and tumors to settle the dispute with the hebrews ( sa. . - ) also favor mice over rats. given the specifi c gravity of gold, just over kg/l, a gold mouse would be shy of g, while a rat would be shy of kg. considering - g tumors, the philistines were paying roughly - kg of gold in total. rat-like tumors would have resulted in - kg of gold, where the tumors would have only contributed marginally (additional - %) to the gold already provided by the rats, raising the question of their raison d'être. linking mice to the box and to the disease singles out tularemia as the disease portrayed by the biblical text; mice are known to carry francisella tularensis , the etiological agent for tularemia. moreover, the text calls for a disease that originated from animals, can be communicated, can form tumors, and is deadly. so now there are arguments for the three virulent infections namely bubonic plague, dysentery and now tularemia, being present in the near east at the end of the bronze age. smallpox or variole is a virulent infection caused by a virus. it was fi rst called smallpox in europe in the late fi fteenth century to distinguish it from the "great pox" or syphilis. thought to have originated in north eastern africa , bce , smallpox then spread to egypt and from there onto india. ramesses v is the fourth pharaoh of the twentieth dynasty of egypt and he died c. bce most likely from smallpox. the well-preserved mummy of ramesses v shows classical smallpox lesions on the face, neck and shoulders as verifi ed by donald r. hopkins in . this period was a time of expansion of the egyptian empire, so cases of smallpox could have been imported into egypt because of this expansion into new territories and war. if the pharaoh, who would have been protected from all harm, fi nally succumbed to smallpox; how long had it been ravaging the general population of egypt and how far had it spread in the near east? sir marc armand ruffer in described a smallpox like rash on a mummy from the same period (twentieth dynasty) as ramesses v, thus giving further primary physical evidence of the existence of smallpox in the near east at the time of its ending between and bce . sir marc armand ruffer ( ruffer ( - was an anglo-french pathologist who pioneered paleopathology. in he graduated from oxford with a ba, then in gained his mbchb from university college in london followed by his md in . in he was knighted for his services to bacteriology and hygiene and for his services to the red cross organization. normally a pharaoh is mummifi ed and buried precisely days into the reign of his successor but ramesses v was buried two years after his death-why? hopkins suggests three possible reasons for this. first the body may have deteriorated due to the infection hence it needed prolonged mummifi cation. second the embalmers feared being infected by the smallpox. finally there may have been a shortage of embalmers because they too had been killed by the smallpox epidemic. there may have also been a shortage of stone masons and stone cutters as well, because ramesses vi was also buried in the tomb of ramesses v, that is, two pharaohs in the one tomb, which is not the usual practice. tom slattery has degrees in east asian studies from the university of california, berkeley and in english from central washington university. in his book the tragic end of the bronze age. a virus makes history slattery argues that smallpox may have killed ramesses v and started the end of the bronze age. he also argues that as people died with smallpox there were not enough men to mine tin that was vital in bronze production, hence the bronze age ended because less bronze was able to be made. slattery also thinks that the hittite plague of bce was due to smallpox. the evidence for the existence of tuberculosis during the bronze age comes again from sir marc ruffer and his examination of egyptian mummies. in his book studies in the paleo pathology of egypt he identifi es the typical spinal lesions of pott's disease as shown in plate ix, fi gures and . modern science has also identifi ed the dna of mycobacterium tuberculosis in the spine of egyptian mummies, thus providing primary physical evidence for the existence of tuberculosis in the near east in the late bronze age. in the crowded living conditions in cities with malnutrition and poverty, tuberculosis would have had the perfect breeding conditions in which to fl ourish and devastate the local population in the late bronze age with its high mortality rate. infl uenza in an unprotected population had the potential to be catastrophic but there is no good evidence to support this theory. we only have its potential as a cause of an epidemic. poliomyelitis had the potential to be devastating and egyptian paintings confi rm its existence in the late bronze age, so it is a defi nite possible cause of an epidemic. anthrax is a bacterial infection caused by the bacillus anthracis and is usually fatal in both humans and animals. because it is so lethal it is used as one of the agents in biological or germ warfare today. in the late bronze age with no vaccine or antibiotics an epidemic would be catastrophic. the endospores of the bacterium anthracis can survive for decades or even centuries and when they are inhaled, ingested or come in contact with the skin they can cause the disease in animals or humans. herbivorous animals usually ingest the spores whilst grazing and carnivores usually ingest the spores when eating an infected animal. humans usually contract the disease by inhalation of spores, direct contact of spores with the skin or consumption of the fl esh of an infected animal. one of the ten plagues suffered by egypt, as described in the bible, could have been anthrax with the description of the typical anthrax sores on the animals, which confi rms that anthrax existed in the near east in the bronze age; hence this is another possible cause of an epidemic. measles in a vulnerable population has the potential to cause a lethal epidemic, but unfortunately there is no good evidence or records to show it occurred. we only have its potential as a cause of an epidemic. malaria has been infecting humans "for the entire history of the species" and existed in the near east from anatolia through the levant and into the nile in egypt in the late bronze age, as it still does today. so it had the constant potential to cause death in humans, especially the virulent falciparum strain during the end of the bronze age era. typhus is a zoonosis that is transferred to humans via lice. it was common among groups of people who lived very closely together such as soldiers in barracks, sailors in ships, prisoners in gaols and refugees in camps; hence it was known variously as "barrack fever", "ship fever", "gaol fever" or "camp fever". was the epidemic that the egyptian prisoners of war took into the hittite empire, that caused the hittite epidemic of bce , really typhus "camp fever" and not smallpox or bubonic plague ? also, as discussed earlier-typhus may have caused the plague of the philistines. in summary, what was needed was an epidemic or several epidemics that had the ability to be lethal, widespread and able to continue for long periods of time or recur frequently; this would be able to change the course of history by destroying empires such as the mycenaean and hittite empires and ending the bronze age. because the end of the bronze age is approximately , years ago, then the virgin population factor has to be taken into consideration as some of the epidemics, such as infl uenza and measles, may have been the fi rst appearance of these infections in history, hence they would be a lot more lethal than they are today in the same area, the near east. of the diseases discussed above, there is evidence that bubonic plague, smallpox and tularemia fi t these criteria and there is evidence of their existence in the near east at the time. dysentery and typhus would be more localized and shorter lived, but still very lethal in its time and place. poliomyelitis and tuberculosis would be too slow to kill and spread over a wide area but potentially lethal locally. anthrax and malaria are also possibilities. there is no good evidence or records to support the existence of infl uenza and measles. they may well have occurred and been lethal, but if they did occur they would have been seasonal and relatively shortlived epidemics. this is in agreement with itamar singer's understanding who said "plagues are already attested in anatolia in the old assyrian colony period (cecen ), and are often mentioned in late bronze age syrian documents (klengel b)". so it is possible that diseases could have contributed to the end of the hittite empire and the end of the bronze age in a major way. because of the timeframe of about years from c. - bce there was plenty of time to have several major lethal epidemics such as bubonic plague, smallpox and tularemia occur on a widespread scale, supported by more local epidemics such as dysentery, poliomyelitis, tuberculosis, measles, infl uenza, anthrax and malaria. in a time of poor hygiene, no antibiotics and no vaccines these infections (fi ve of which are so virulent that they are still used as germ warfare agents today, namely bubonic plague, smallpox, tuberculosis, anthrax and dysentery) had the potential to devastate the near east and should be factored in as potential cofactors in the future by historians. any future discussions about the end of the hittite empire and end of the bronze age should be considered as incomplete if disease is not considered and included. early bronze age metallurgy in northeast aegean the age of iron ironware piece unearthed from turkey found to be the earliest steel the babylonians ancient history from the first civilization to the renaissance from bronze to iron arms and armour of the greeks the age of iron the catastrophe " part : what the end of the bronze-age civilization means for modern times the catastrophe the collapse of mycenaean palatial civilization the end of the bronze age end of the late bronze age and other crisis periods : a volcanic cause hints that cometary debris played some role in several tree-ring dated environmental downturns in the bronze age poseidon ' s horses : plate tectonics and earthquake storms in the late bronze age aegean and eastern mediterranean the end of the bronze age by large earthquakes evidence for an earthquake at mycenae in archaeoseismology verlag, der osterreichischen akademie der wissenschaften, . wien. (vienna: greek prehistory, the austrian academy of sciences t he great karnak inscription of merneptah : grand strategy in the thirteenth century bc medinet halu inscriptions of ramesses iii's eighth year lines - as translated by the ancient mediterranean the bronze and archaic ages the bronze age -iron age transition in europe contribution of drought to the collapse of the hittite empire dendrochronological dating in anatolia : the second millenium bc ", (der anschnitt, anatolian/metal iii the infl uence of climatic change on the late bronze age collapse and the greek dark ages changes in vernacular architecture and climate at the end of the aegean bronze age the collapse of complex societies vermule . . lokovides a cultural chronology of disease from prehistory to the era of sars y ersinia pestis genome sequencing identifi es patterns of global phylogenic diversity the philistine in transition ; a history from ca die herkunft der philister the plague of the philistines , and other medical -historical essays by encyclopedia of plague and pestilence from ancient times to the present anatolian tree rings and the absolute chronology of the eastern mediterranean - bc natural disasters , (facts on file science library plague an ancient disease in the twentieth century the plague of the philistines the works of flavius josephus translated by w. whiston tularemia did an epidemic of tularemia in ancient egypt affect the course of world history hittite plague ", or epidemic of tularemia and the fi rst record of biological warfare the biblical plague of the philistines now has a name , tularemia -years-terro. epidemics of the past smallpox: years of terror whqlibdoc. who.int/smallpox/wh_ _ _p .pdf. ramesses v. earliest known victim note on an egyptian resembling that of variola in the skin of a mummy of the twentieth dynasty - bc chronology of the pharaohs the tragic end of the bronze age a virus makes history studies in the paleopathology of egypt characterization of mycobacterium tuberculosis complex dnas from egyptian mummies by spoligotyping early origin and recent expansion of plasmodium falciparum mutanuinden kultepe-texten " in (atti del congresso internazionale di hittitologia epidermien im spatbronzezeitlichen syrien -palastina " in michael. (historical, epigraphical and biblical studies in honor of michael heltzer key: cord- -k l unc authors: lu, li; lankala, srinivas; gong, yuan; feng, xuefeng; chang, briankle g. title: forum: covid- dispatches date: - - journal: cult stud crit methodol doi: . / sha: doc_id: cord_uid: k l unc covid- pandemic is the first truly global crisis in the digital age. with death count worldwide reaching , merely months after its first outbreak in china in late december and . million cases reported in countries and territories as of july , this ongoing pandemic has spread far beyond domain of world health problem to become an unprecedented challenge facing humanity at every level. in addition to causing social and economic disruptions on a scale unseen before, it has turned the world into a site of biopolitical agon where science and reason are forced to betray their impotence against cultish thinking in the planetary endgame depicted in so many dystopian science fictions. it is in this context that this forum offers a set of modest reflections on the current impacts incurred by the covid- virus. blending ethnographic observations with theory-driven reflections, the five authors address issues made manifest by the crisis across different regions, while keeping their sight on the sociopolitical problems plaguing our life both individually and collectively. taken together, they provide a grounded documentary for the archive that the covid- virus is making us to construct. the apparition of these faces in the crowd. -ezra pound, "in a station of the metro" the french word envoi is polysemic, defined as dispatch, the action of sending, something that is sent, a poetic dedication or dedication of a literary work, and the marking of the beginning of a process. this article is a dispatch from hubei, china, based on the author's -month stay in his hometown qianjiang, a small city in the middle of hubei, during the coronavirus pandemic. firsthand observations sent from the epicenter give us a clear picture of what the coronavirus has done. moreover, this article argues that the coronavirus marks a spectral moment in which a repressed trauma returns. there have been fierce debates on the origins of the coronavirus and the political, economic, and social significances of the pandemic. popular representations of the coronavirus which isolate, stigmatize, and terrify the other are symptoms of a returning trauma, which is caused by bodily memories of being victims in past disasters. a derridean reading of the envoi highlights the inherent failure of sending: what is sent can always be held up by a malfunctioning in the process of the sending or postal system, and the meaning of the trauma is lost. this traumatic failure results in a repetition in representation and the return of what is sent to the writer/sender. proposing a supplement, this article foregrounds bodily knowledge acquired through social and political trauma by virtue of fear of the coronavirus. this fear of what is familiar reminds us of the feeling of the uncanny. according to freud and derrida, the uncanny is related to the spectral working of a hidden desire that repeatedly returns as a haunting body, representation, and history. this line of thinking helps us to better understand conflicting representations of the coronavirus. the coronavirus is a ghost. this is not merely a metaphorical proposition; this is accurate in the sense that the coronavirus instantiates our phantasms, fears, and desires toward ghosts. in this regard, derrida's specters of marx provides us with a basic framework for understanding the coronavirus as a ghost. the first teaching of derrida is that ghosts do not come at just any time but in spectral moments that do not belong to time. by pointing precisely to the c scxxx . / cultural studies <span class="symbol" cstyle="symbol">↔</span> critical methodologiesli et al. present or now-time, derrida ( ) regards spectral time as "a disjointed now that always risks maintaining nothing together in the assured conjunction of some context whose border would still be determinable" (p. ). second, a ghost is a phenomenon in the game of repetition and difference. neither exclusively situated in life nor in death, neither visible nor invisible, a ghost is "the frequency of a certain visibility. but the visibility of the invisible. and visibility, by its essence, is not seen, which is why it remains epekeina tes ousias, beyond the phenomenon or beyond being" (derrida, , p. ) . third, to "make oneself fear" is essentially ineluctable in the experience of a ghost. one becomes frightened of a ghost "on the condition that one can never distinguish between the future-to-come and the comingback of a specter" (derrida, , p. ) . in other words, what one fears is not the ghost, but the fear, imagination, and one's subject inspired by the ghost. finally, "a ghost never dies, it remains always to come and to come-back" (derrida, , p. ) . whatever repression the dead may suffer, the return of the dead is anticipated, and "this being-with specters would also be, not only but also, a politics of memory, of inheritance, and of generations" (derrida, , p. xviii) . in light of derrida's framework on specters, once the coronavirus finds a host, it starts to live a ghostly life. the coronavirus pandemic irrupted during a time of turmoil. as the chinese president xi jinping has expressed, the world is experiencing profound shifts unseen in a century. while the trend of globalization is markedly receding, nationalism, popularism, and isolationism are on the rise. the eulogic discussions of "chimerica," a popular term coined by the british epidemiologist neil ferguson in , are being replaced by the theories and practices of the china-u.s. decoupling. the trade dispute between china and the united states puts an end to the chinese ideal of great harmony in the world. in addition, as his campaign slogan "america first" shows, donald trump epitomizes the idea of american exceptionalism. in traditional chinese thinking, famine, natural disasters, and plague happen when the political order or legitimacy are out of joint. during this disjointed moment, a plague was anticipated, even fabricated before it came. according to a widely circulated story in the we-media during the height of the coronavirus, wang yongyan, an academician specializing in chinese medicine at the chinese academy of engineering, predicted half a year ago that a plague would come after the dongzhi (winter solstice), one of the chinese solar periods. in addition, he predicted that the plague would last until next spring. in hindsight, rumors about a new virus were spreading right after the dongzhi. or, simply put, divination went hand in hand with the plague during a time of disjointing, disjunction, or disproportion. while scientists are still trying to track down patient zero, conspiracy theories about the origins of coronavirus have been spreading. bat soup and biological warfare are on the top of the list of suspected criminals. like ghosts, the coronavirus takes shape in the game of visibility and invisibility. in this game, ways of seeing determine how a virus can be understood. approximately made up of . microns, the coronavirus can only be seen under an electron microscope, made visible with the help of scientific equipment and representations. in contrast, a poet like ezra pound sees the invisible through his gifted imagination. his imaginative inspiration and aesthetic reflection allow his keen observation to become a line of poetic beauty and philosophical complexity. through this form of observation, an invisible apparition becomes visible in the faces of the crowd. similarly, the depiction of a fictional killer-virus called wuhan- in dean koontz's novel the eyes of darkness, re-gained popularity among those who regard it as an imaginative depiction of the coronavirus. in line with this imaginative depiction, mr. wang, well-trained in traditional chinese medicine, claimed that his prediction was based on his reading of the xiang (image) of the sky, earth, plants, animals, and human beings. visible to the naked eye, xiang functions as the visible traces from which an invisible plague becomes visible to an expert in traditional chinese medicine. in other words, with scientific support, talent, and training, people are able "to see this invisibility, to see without seeing, thus to think the body without body of this invisible visibility" (derrida, , p. ) . in this way, the ghostly nature of the coronavirus lies in the different frequencies of its visibility. however, despite our faith in being able to depict, and make distinctions between, the invisible and the visible, the way of seeing the coronavirus, especially in this time of turmoil, is politically conditioned and manipulated. when i took a night bullet train to wuhan with my family for vacation, it felt like an ordinary chinese family reunion trip during the spring festival: carriages packed with passengers, luggage, excitement, anxiety, and weariness in the air. one of the reasons for the peaceful atmosphere was that china and the united states had signed a trade agreement a few days before, sending a false message to the world that rationality and peace would return. one thing was markedly noticeable on the train: most passengers wore a facial mask for fear of an officially unidentified but unofficially sarslike virus. to my surprise, a line of masked faces was greeted at the exit by the smiling faces of relatives or friends, the indifferent faces of railroad workers, and the shrewd faces of barkers at the hankou railway station. this lack of consistency indicates that aspects of the coronavirus were kept secret. furthermore, this scenario at the station reminded me of a horrifying scene in the film the cassandra crossing, an eye-opening disaster thriller for my generation directed by george p. cosmatos. in this harrowing film, an international express carrying a virus-infected terrorist approaches a station at night. when the train reached the station, the passengers, who were kept from the truth, were confronted with members of the u.s. army in white biological hazard protective suits lined up on both sides of the platform. in both cases, the dynamic of the visibility and the invisibility of a virus was of political significance. the facial masks and the protective suits were used not only to protect people from a virus but also to make the secret of the virus both visible and invisible. in other words, political manipulation complicates the ways that a virus is seen and how the coronavirus, in particular, is seen as a political ghost. the coronavirus pandemic frightened people because it looked like the return of a specter, namely sars. because of its fatality and residua, sars remains an unresolved trauma for many chinese. at the early stages of the coronavirus pandemic, what was most frightening was its assumed high fatality rate. similarly, the short notice given for the lockdown of wuhan, a huge city of more than million residents, sent a clear message to everyone that the novel coronavirus was the grim reaper. corona, the brand of the first car i owned and of the beer i had on my first visit to a mexican restaurant, was colored by images of a fearful virus, deserted streets, calm officials on tv news channels, and panicking crowds in wuhan hospitals caught on video by the we-media. unlike the countries who proposed or actually enacted herd immunity, the chinese authorities imposed very tough immunity measures, a lesson learned from the sars pandemic, when highways, the railway station, and docks in my hometown were closed overnight. nursing homes were under quarantine; no visitors were allowed in. local authorities advised avoiding public gatherings, including public square dancing and playing majiang. the most popular forms of social activity, especially for retired people, were no longer available. after the initial panic, it was discovered that the fatality rate of the coronavirus was much lower than sars. according to the world health organization, the sars mortality rate worldwide was about %. in early february, the chinese authorities claimed that the coronavirus mortality rate in wuhan was about %. subsequently, what elicited fear in the population was the future-to-come, particularly in the form of social unrest. on one hand, stricter quarantine measures were implemented: all roads were quickly blocked with cranes or tankers or stones; vehicles' use was not allowed, unless a special permit was issued; all grocery shops, markets, restaurants, and hotels were shut down; residents were not allowed to exit their residential areas except for grocery shopping at an arranged supermarket. in addition, central and local authorities watched closely for other concerns, such as food shortages and the inflation of prices. thanks partly to its rich agricultural products in a land of fish and rice, the impact of the coronavirus on food supply and prices did not affect my hometown. however, under the restrictions put in place, my hometown looked like a ghost town, and the uncertainty of the future frightened people of all social strata. in fact, what people fear most is that the coronavirus will never die and will come back again and again, either in the form of a future-to-come or a return of the dead. regardless, despite the medical or political ambition to eradicate the coronavirus, we might have to accept the fact that the virus will co-exist with us forever. for instance, the coronavirus has been mutating, and the way the coronavirus replicates itself in the cells of other organisms is ubiquitous. this mechanism of repetition and difference functions both literally and metaphorically. on one hand, the coronavirus reproduces itself through difference. merely a collection of genetic materials that seems to think with/like a human once it infects its host, the virus induces a feeling of the uncanny, a topic to which i will return later. in addition, news sources reported that infected patients tested positive again after they had been released from the hospital. robert redfield, director of the u.s. centers for disease control and prevention, admitted that some deaths from coronavirus have been discovered posthumously (cnn, ) . in other cases, the coronavirus acted like a whimsical tyrant who inadvertently signed a death sentence. for example, the only cases of death in my residential area was an old couple who lived in an apartment very close to that of my parents. they got infected by their son and daughter who came back from wuhan. what remained a mystery was that the son and the daughter had stayed with their parents for more than days, much longer than the latent period of coronavirus. days after they were hospitalized, they died one after another. on the other hand, the coronavirus reproduces difference in its host organisms. the neighborhood my mother lives in is an acquittance community and an aging society. cadres and volunteers from the neighborhood committee have diligently attended to the needs of the old. aware of the higher fatality rate of the old, an ageist exhortation to quarantine was broadcast repeatedly through a portable loudspeaker placed at the gate of the neighborhood committee building. as stigmatized targets, senior residents were susceptible to the emotion of shame and, for this reason, chose to stay at home. the use of broadcasts and the instigation of shame illustrates how the coronavirus (re)produces, moderates, and polices the line between the public and private spheres. the coronavirus also changed the affective, moral, and power economy of the family. the spring festival is supposed to be the perfect time for a temporary family reunion of joy and harmony. when the lockdown continued longer than everyone expected, generational conflicts broke out. in extreme cases, the political infected families while they were trying to contend with the coronavirus during quarantine. for example, fang fang, a veteran chinese writer who lived in wuhan, posted her thoughts on life in quarantine on her or her friend's weibo account. those posts were later collected and published under the title wuhan diaries. public opinion on those posts varied and eventually led to a political debate between left-wing and right-wing netizens, eventually affecting family members who conflicted in their attitudes toward the wuhan diaries. along with the coronavirus, the memory of personal, generational, and political traumas returned. sars, the cultural revolution, natural disasters, and national humilities were recurring themes in representations of the coronavirus. the suffering and trauma in the epicenter deserve an envoi/dedication, and efforts have been made to achieve this goal, such as daily national and international coverage, fang fang's wuhan diaries, and we-media postings. in these kinds of representation, a rhetoric of "suffering as sublime" is usually at play. in addition, stigmatizing the suffering of others, or blaming the other for one's suffering, is another kind of dedication. both kinds of representations of the coronavirus attempt to take the moral higher ground by attempting to fix the coronavirus as a mere object awaiting to be represented. no matter what position the representation takes toward the coronavirus and its significances, the will to truth turns a dedication quickly into a testimony and even a perjury. a virus is an infectious agent that replicates only within a host organism. for the host, a coronavirus is a deadly stranger and an intimate family member at the same time. familiar, frightening, and secretive, the coronavirus reminds us of the uncanny, as discussed by freud. in his pioneering study, freud focused on the unsettling psychological state of the uncanny. distinct from the feeling of fear, the uncanny is a kind of terrifying feeling that is associated with something known and familiar. after an etymological investigation of the german words heimliche/unheimliche, and a close reading of hoffmann's story "the sand-man," freud ( ) unearthed the origins of the uncanny: "it may be true that the uncanny [unheimlich] is something which is secretly familiar [heimlich-heimisch], which has undergone repression and then returned from it, and that everything that is uncanny fulfills this condition" (p. ). he also associates the feeling of the uncanny "with the omnipotence of thoughts, with the prompt fulfillment of wishes, with secret injurious powers and with the return of the dead" (freud, , p. ) . following the lead of freud, derrida worked on the concept of the uncanny to engage with marx's concepts of repetition, specter, and fear. refuting the claims that the tenants of marxism have died, derrida emphasizes the strange familiarity of the specter of marxism in the age of advanced capitalism. as derrida insists, the specter of marxism will continue to return from the future to visit us, to live with us, and to alert us. similarly, derrida ( ) interprets the uncanny through the concept of absolute hospitality, in which "one may deem strange, strangely familiar and inhospitable at the same time (unheimlich, uncanny)" (p. ). remaining structurally open to future interpretation, the uncanny in derrida's account presupposes a materialism without substance, a messianic without messianism. derrida's understanding of the uncanny is critical to my reading of the coronavirus as a ghostwriter of envoi. as a ghostwriter, the coronavirus is a ghost who writes from the future. as a stranger and a family member, it writes with and in the place of the host. by writing an envoi, a kind of writing haunted by failure and repetition, the coronavirus makes itself visible and frightening in a spectral moment. however, the envoi is not exclusively governed by a ghostly logic that is followed by and instantiated through the coronavirus. in critiquing the tendency to unearth an ultimate truth, eve kosofsky sedgwick & frank ( ) regard affects as a possible way out of the binary opposition of truth and falsehood in representation. by invoking the power of the performativity of shame, they highlight the negative affects neglected by identity politics, dismissed and stigmatized: without positive affect, there can be no shame: only a scene that offers you enjoyment or engages your interest can make you blush. similarly, only something you thought might delight or satisfy can disgust. both these affects produce bodily knowledges. (sedgwick & frank, , p. ) in their view, shame is neither subversive nor mandatory; it works with other affects, drives, and representations to adapt the body to its situation. foregrounding bodily knowledge acquired through trauma commits us to thinking differently about representation and the envoi in question. fear of the coronavirus is not only the fear of a returning trauma as a ghostly logic in representation. more importantly, the coronavirus writes itself and writes about bodily memories of trauma in a constant play of materialization: inscribing fear in itself and on the body of the host permanently. with the end of the lockdown in hubei, the coronavirus pandemic is almost over in china. however, the coronavirus has been sending, and will keep sending, its fearful envoi. the enduring sign of the coronavirus pandemic for indians was not related to medicine or public health. it was the unprecedented exodus of migrant workers from metropolitan centers to their native rural districts, sometimes hundreds of miles away (mukhopadhyay & naik, ; petersen & chaurasia, ) . the scale of this migration was vast and is still being understood. it certainly provokes disturbing questions about urbanity and the fragility of a political compact that kept people in their place through calibrated deprivation (dahdah et al., ) . but for our purposes here, i will explore the ways in which it underscores the varying effects of the pandemic on different classes of people and the diversity of its signification. the virus in india is both a medical event to be dealt with through appropriate public health measures and a mediated discourse that has developed its own ramifications and responses. i argue that both forms of the virus have had tragic and miserable consequences, but on different classes and groups of people. like the televised persian gulf war of that jean baudrillard found to be a distinct and distorted signifier of the actual fighting on the ground, the virus itself is not the same phenomenon once it is transformed into a signifier for other meanings and purposes. the novel coronavirus later named covid- emerged in the public consciousness as a distinct problem with the rapid rise in infections in several indian states by february . in march, the government of india mandated an immediate "lockdown" of the entire country. this new term burned itself into the national consciousness and its many vernaculars almost instantly, as its meaning became physically apparent. it involved the physical arrest of people wherever they happened to be at the moment, and the prohibition of all commerce, traffic, and circulation. it was announced with a -day notice period by the prime minister, in an eerie echo of a similar announcement in of the withdrawal of paper currency. that tragic farce had laid a historical precedent for this second tragedy to come. as a deeply iniquitous society and economy were forced to a halt, the effect was expectedly unequal. metropolitan indian citizens soon learned to cope with the new hardships of "work-from-home," homeschooling, online classes and meetings, and such social-media-driven innovations as cooking and cleaning without domestic servants and entertaining themselves in their houses and apartments. the government also encouraged the adoption of derivative coping mechanisms as soon as they were observed in other countries: applauding medical workers from the safe confines of apartment balconies and terraces; singing, chanting and clanging metal plates and dishes with utensils in cacophonous, solidarity of the gated classes; lighting lamps and candles; and waving mobile phone flashlights at appointed times (krishnan, ) . however, the actual effect of the virus became inseparable from the effect of the "lockdown." the sudden impoverishment of the majority of the country's population led to starvation, medical neglect, and a national panic. while invisible to the citizens in its first few weeks, it became impossible to ignore, when workers across indian cities started to simply walk back to their native villages. their exit from cities also emphasized the fragility of urban belonging: that in a crisis, indian cities were fundamentally empty shells, drawing people not through cosmopolitan attractions or civic rewards but by rural misery. at this point, the virus was still largely a media phenomenon, while the "lockdown" was what had directly affected most indians: the sudden disappearance of work, wages, commerce, and circulation magnified the precarity of urban existence. the largely informal national economy quickly unraveled in a crisis. this crisis was exacerbated by the role of the virus in continuing the ideological and political discourses of the chaotic period immediately preceding the lockdown. the use of the virus to carry out "politics by other means" can be seen in other polities as well, but its entanglement with indian politics is particularly useful as a means to understand the virus as a set of signifying practices. the context of this political use of the virus as a signifier is also inseparable from the highly mediatized nature of indian politics and society. the virus emerged as a discursive phenomenon in india at a crucial juncture in a national conflict over changes to the country's citizenship laws. with the rise to national power of the ruling rashtriya swayamsevak sangh (rss, or the national volunteers organization-a fascist group founded in ), india's national government had been attempting since to achieve its political goal of abolishing its secular and liberal constitution through a steady dismantling of public institutions (roy, ) . this conflict worsened in with the re-election of the rss-controlled government headed by the current prime minister, and the consequent repeal of laws that had hitherto guaranteed the autonomy of the occupied territory of kashmir. this was followed by a critical change to citizenship laws to specifically exclude muslims from gaining indian citizenship and institute a new "citizens' register" to determine afresh the legal status of all residents. with reports of the parallel construction of detention camps outside major cities, the fascist inspiration and ominous intent of the new laws became clearer and more immediate. protests and political resistance to the new measures emerged across the country, and were met with violent responses from the police and rss groups. matters had reached a head when the nationwide lockdown was suddenly imposed. except in a few indian states such as kerala, with still functioning local health systems, the lockdown did not involve any public initiative to test or prevent the spread of the virus. instead, in keeping with the ruling ideology of our time, citizens were mandated to protect themselves, on pain of being brutalized by the police if they failed. in this chaotic sauve qui peut scenario, the rhetoric of basic preventive measures took on ominous ideological connotations depending on who you were and where you lived. as it became clear that only access to clean running water, adequate space, and a home to live in would guarantee the efficacy of the public health guidelines, medical advice became meaningless for much of the country's population, especially the inhabitants of vast informal urban settlements in the metropolitan cities. in effect, a dual situation emerged: a parallel virus had infected the classes who lived in gated urban communities and formal neighborhoods and who followed its progress in daily primetime news trackers. positive cases, testing ratios, death rates, and other numbers soon flew across television and website screens in macabre charts, graphs, and complex animations, as breathless studio anchors enthusiastically tracked the competitive fatalities across states, regions, cities, and countries. as the formal state and civil society response to the virus grew more and more into a media discourse, its actual effect on the population was determined by existing social conditions and ideological practices than by the ideals of public health. in the early period of its spread, the illusion of its control was maintained through the interpellation of the mass television audience as ideal national subjects. in a series of televised speeches, the prime minister exhorted citizens to planned acts of mass discipline, such as the applause, noise-making, and lamp-lighting exercises mentioned earlier. it took several costly weeks for the citizens to realize that this national son-et-lumière had only served to deafen and obscure a different and more real virus that had silently spread illness and death among urban populations who did not have houses or apartments with balconies. a starved public health system soon proved inadequate and unprepared. because this real crisis was not mediated or televised, there was no appropriate or meaningful response to it. the easy congruity of the eagerly adopted virus prevention measures with the practice of caste-based rituals of discrimination was not lost on most indians (george, ) . this fortunate coincidence enabled the easy normalization of virus prevention as a legitimization of existing hierarchical practices. the convenient prescription of social distancing appeared to keep the privileged class of wealthy and respectably middle-class white-collar workers as far away as possible from the physical contact or proximity of their social inferiors. the pandemic thus seemed tailor-made for defenders of hindu caste hierarchies, a righteous and suitably scientific legitimation of social discrimination. the fantasy of caste purity would have remained an abhorrent social remnant if it had not become part of state policy in the last few years. but in the context of the stateled legitimation of religious hierarchies and the consequent onslaught on emancipatory laws, this entanglement of the virus with caste and with the violent hate crimes against muslims acquired a dangerous dimension. it is this distrust of and disgust with a compromised public health system that drove so many indians streaming out of cities and into the relative safety of their impoverished rural communities. the alienation of muslims as a national other has been a part of the basic doctrine of india's current ruling group ever since its founders, awed by the nazi policy of extermination, adopted a similar goal for the erasure of non-hindu communities in india. the mass protests and popular uprising against the rss's attempts to irrevocably alter the basic structure of the country's republican constitution had reached a tipping point when the covid- epidemic was suddenly deemed emergent enough to impose an unprecedented "lockdown," in effect a de facto police state across the country. the imposition of the lockdown allowed police to destroy protest sites, detain protestors, and unleash a reign of terror across indian cities. caught in the initial crossfire were members of an apolitical muslim religious group, the tablighi jamaat, whose convention in delhi had been interrupted by the lockdown. jamaat members trapped in the organization's premises by the curfew were found to be infected with the virus. the consequent media narrative of the discovery of the infection among the jamaatis veered into the fantastical, with nightly news anchors debating the strategies of a "corona jihad" that was to be waged by militant muslims using the virus as a weapon (perrigo, ) . this dog-whistle narrative of muslim bodies as unclean spreaders of a foreign disease dovetails with similar narrative frames used to portray hindus from laboring and working castes as well. the manufacture of conspiracies surrounding the coronavirus can be seen across the world and is not unique to india. a disturbingly large proportion of americans, for example, appear to believe that the virus has been manufactured to enable mind-control through vaccination and g cellular signals by a ruling elite (fisher, ) . on rare occasions, these conspiracies do spiral out into real effects such as the bombing of cellular towers in britain and the anti-vaccination movement in the united states. in india, however, the covert encouragement of such theories by the state itself, to legitimize the hatred toward muslims, exacerbates and normalizes the rumors as mainstream prime time news which is then amplified and shared through an organized social media campaign (ellis- petersen & rahman, ) . the vilification of the muslim other serves two purposes, one of furthering the state's broader agenda of religious and caste purity, and the other more immediate goal of providing a scapegoat for the inescapable rise in infections and deaths due to the virus and the inability of the state and society to understand the crisis. the brutal police crackdown that accompanied the lockdown and the violence of its imposition across the country were a small reminder of the routinization of the "lockdown" as a way of life in the occupied valley of kashmir, part of the only muslim-majority state in the indian union. the effects of the police state as a normalized entity have been multiplied since the abrogation in of constitutional laws guaranteeing the region's autonomy, even if such laws were honored more in the breach in preceding decades (zia, ) . the uncanny resemblance of a public health curfew to a military occupation is not coincidental, but the result of the colonial origins of both, and of the state institutions they represent. the symbiotic existence of caste-based discrimination, the extermination of a religious minority, and the colonial occupation of an entire province within the same body politic is made possible by the continuous interpellation of the mass of people to become national citizen-subjects. this call to obedience, broadcast daily through primetime television and magnified through the near-mandatory use of mobile phones, is the only sign of a nation-state that is otherwise absent in the real world. the failure to stop the spread of the real virus is obscured as the interpellated citizen is urged, cajoled, and threatened to participate in the simulacral fight against a mediated virus in a purely semiotic realm. the washing of hands without the precious reality of running water, the maintenance of "social distance" in the absence of space, the exhortation to "work from home" for a population that is not housed, and the discourses of online socialization and commerce are all much more than signs of mere denial: they are the components of this new semiotic space, enabling the call to national belonging in a new domain, bereft of its mooring in the world. from a broader historical perspective, the coronavirus epidemic does not appear to have affected indians as much as the far greater fatalities caused by more prosaic diseases, hunger, and the increasingly toxic air and water (rukmini, ) . what has caused the greatest pain and panic is the response to the epidemic. this response has been not to the virus itself, but to a simulacral virus that appears to occupy the same space and shares the same name as covid- , but which is a mere signifier, pointing to other, older evils. like baudrillard's hyperreal war, it has surpassed the real virus itself and has come to occupy its place. it cannot be wished away or prevented with a vaccine, it needs a response in kind: of new counter-signs and counter-discourses. a catastrophic pandemic unseen in a century, the current raging of covid- around the globe has undoubtedly produced a unique symbolic site for global, regional, and national imaginations. as the earliest epicenter of this infectious illness, china has witnessed the proliferation of discourses about the evolution of the pandemic on various media platforms, through which the chinese public has the rare chance to reflect on important issues regarding identity construction, social reformation, and nation building. while much attention has been paid to the stigmatization of china in euro-american politics, media, and everyday whisper that label the natural coronavirus as a cultural and ethnic fault (fu, ) , what has been overlooked is how china has portrayed other countries in this global health crisis, especially those surrounding nation-states in the same geopolitical area. east asia, or the sinosphere in the broader sense, with the collective memory of fighting sars in , is thought to have responded to covid- more efficiently than many western countries (salmon, ) . how, then, is the east asian encounter with covid- depicted in the chinese public discourse? how does such depiction envisage china's relations with neighboring countries and its position in the area? in this essay, i discuss the ways in which the coronavirus pandemic has been appropriated by the chinese public for a (re)imagination of east asia. by exploring the evolving representations of its neighboring countries throughout the epidemic on chinese media platforms including weibo, wechat, and zhihu, i argue that the talk of the regional responses to covid- envisions a china-centered union of selected east asian countries in parallel with the historical tributary system of the sinosphere. through the expression of the nostalgia for imperial china, the discursive reconstruction of the east asian identity is a ratification of china's contemporary ambition to reclaim its geopolitical dominance. synchronized with the rapid transmission of the coronavirus in china and east asia between january and march , the chinese public in this early phase drew close attention to the unfolding of the epidemic in its nearby countries, and japan and south korea in particular. with the disease breakout involving diamond princess (japan) and shincheonji church of jesus (south korea) frequently making news headlines, the discussions of how those countries responded to covid- flourished online, which, in combination with the continuous debates over china's own pandemic threat management, contributed to the imagination of the covid- rampancy as a regional challenge that china and its neighbors faced together. central to the discursive formation of this imagined community was the celebration of the incessant interaction and cooperation between china and some east asian countries to combat the virus collectively. in the wake of the outbreak when china was threatened by the crumbling of its health care system, the countries under the spotlight-japan and south korea-were widely appraised for the sympathetic and supportive approaches they took to help china overcome the severe shortage of medical resources. the media reports of japanese and south korean governments leading the international aids to china (gong, ) were echoed by numerous warm anecdotes on social media championing the heartfelt support from their people. perhaps the most well-known story of this kind, a japanese institution wrote a chinese-language verse on the boxes of masks it donated to the province of hubei: "rivers low, mountains high; the same moon in the sky" (trans. zhao, ) ("山川异域，风 月同天"), which immediately went viral online because of its signification of the long-lasting friendship between china and japan. according to account of the expedition to the east by the great master (唐大和上东征传) written by omi no mifune (淡海三船) (see wong, ) , this sentence was from an ancient poem written on the edges of the buddhist robes japanese missions (遣唐使) brought to tang china as the tribute from prince nagaya (長屋王). given its profound roots in the history of japanese envoys to imperial china learning from the chinese culture and civilization, this verse went beyond re-fostering the traditional sino-japanese solidarity. analogizing japan's mask donation with ancient japanese envoys' gifts, it also evoked the retrospective commemoration of the hierarchy between china and japan in history which almost vanishes in the modern era. therefore, the popularity of this verse may indicate the aspiration for the reoccurrence of such bi-lateral relations. indeed, this was only one example of the ubiquitous imaginary of the pan-east asian cooperation and exchange of goods and information as a modern emulation of the tributary system through which imperial china maintained its diplomatic and trade relations to neighboring countries and consolidated its dominance in the region for over a millennium. after china started to keep the pandemic under control and resume the production of medical supplies, this metaphor was further perpetuated in an attempt to accentuate that china's supplies of medical goods and anti-epidemic lessons to nearby countries drastically outnumbered what it was initially given. on weibo, china's return of masks and respirators to its neighbors was often explicitly compared with the "vassals' gifts" chinese emperors assigned to tributary states in posts like this: tribute is both the highest form of alliance and an advanced way of investment, but (this time) it is based on masks! recently, xinwu district in wuxi, jiangsu province donated , to toyokawa, aichi prefecture in japan in return for the , masks, protective clothing and other anti-epidemic materials toyokawa donated to xinwu district in february. (weibo source, march , ) this nostalgic use of metaphor implies a crucial undertone of sinocentrism of the public imagination of the community comprising china and bordering countries fighting against the coronavirus. the tracing of the origin of east asian solidarity to the past is suggestive of the ambition of the present. the chinese public not only fantasizes about a reunion of china, japan, and south korea for covid- but more importantly yearns for the recovery of their nation's leadership and centrality in this battle. as the coronavirus expands rampantly to the rest of the world from march onward, chinese media coverage quickly catches up with the shift of the epicenters from east asia to europe and north america and reformulates the pandemic as a global health crisis. against the depiction of how covid- created chaos, helplessness, and dysfunction in western societies stands the stark contrast of east asia as a safer zone where the outbreaks have been largely contained with success. with the similar control of cases less than , , japan and south korea remain at the heart of this imagined safe zone in company with china even though the reality has seen even fewer confirmed cases in other parts of asia as well as the recent resurgence of virus spreading in all these three countries. this rhetoric is in concert with the prevalence of online deliberations about why east asia as an area has performed better than other parts of the globe in the containment of the virus. at the core of these discourses lies the construction of an east/west binary which frames the global responses to the pandemic into a competition in which "we" (the east/ east asia) have triumphed "them" (the west/euro-america). although china and neighboring countries diverge in the official approaches to handle the pandemic, their relative efficiency in virus containment in comparison with the west is considered to be guided uniformly by the cultural values they share as part of the "confucius east." in particular, collectivism-the principles of prioritizing community interests to personal interests, pursuing social harmony, compliance to authority, avoid causing inconvenience to others-has been glorified as the main drive for the people in east asia to more effectively cope with the governmental strategies in contact tracing, testing, social distancing, and mask wearing. similarly, the regional cooperation in the pandemic management is regarded as a manifestation of these values. for example, the reflections on how south korea has set a model of disease control using mass tracing and testing tend to recognize the smooth uptake of this procedure facilitated by koreans' collectivist mind-set that downplays individual privacy and complies with the data-mining measures to track and publicize their locations, activities, and close contacts. meanwhile, other popular discussions blame the religiosity of the shincheonji church members whose gatherings caused the initial covid outbreak in south korea, which is reflected from the titles of zhihu posts that describe the diffusion of the virus through "hallelujah" such as "the occupation of south korea by covid- , everything has to start from 'hallealia'" and "south korean cult hallelujah devastated the country." apparently, these titles have no intention to mask the underlying tone mocking at the role of christianity in the acceleration, not mitigation, of disease spreading, which further serves as a foil to the power of confucianism to help south korea navigate away from the disaster. in fact, satire targeting at christianity represents the broader criticism of western cultural values in hindering the efficacious enforcement of restrictive and surveilling measures against the coronavirus. the east asian identity is thus reaffirmed through the clashes between the eastern and western civilizations. however, it is worth noting that the narratives about the east asian conquest of covid- are again permeated with the metaphor of the tributary system delineating china as the leader and role model in this imagined "safe zone." not only does the attribution of the regional success to confucianism call up the historical chinese centrality in the sinosphere but the emphasis on china's ability to offer lessons and instructions from its early experience for its neighbors to benefit from also ratifies the restoration of the "teacher/student" relation between imperial china and pre-modern japan and korea. far from a total reenactment of the historical sinosphere, this chinese imaginary of east asia engages with a purposeful selective process that amplifies china's solidarity with some east asian countries but simultaneously mutes others in the same region. as remarked earlier, a majority of the online narratives about the cooperative responses to covid- in east asia revolves around china, japan, and south korea, with less frequent inclusion of singapore as well as occasional reference to such countries as mongolia and myanmar. this emphasis on forming a coalition with japan and south korea is compatible with china's longterm agenda of promoting and dominating the china-japan-south korea union (中日韩一体化), which was recently reiterated by the three governments' consensus to speed up the negotiation of the free trade zone (中日韩自贸 区) (wang, ) . in this sense, the covid- crisis has offered a discursive site for the chinese state to rebuild this trilateral bond and remodel its significant neighbors whose national images, due to the respective disputes around diaoyu islands and thaad (terminal high altitude area defense), have been negative in china for almost a decade. while the china-japan-south korea triangle is romanticized in connection with other small countries, the alienation of some confucius societies from this imagined "cooperative" east asia is quite striking, especially given the outstanding results some of them have produced in the prevention of disease transmission. the first excluded category includes taiwan, hong kong, and macau-the territories outside the mainland in the great china area. whereas macau is often forgotten by the media as it has always been, both taiwan and hong kong are widely criticized and mocked for their attempt to politicize the pandemic as a weapon to confront beijing and increase international recognition. the second group pertains to north korea and vietnam-the authoritarian states that have close political and ideological bonds with china. for instance, north korea has been constantly questioned and satirized because of the lack of transparency in the disclosure of its epidemic circumstances. vietnam's outstanding handling of the virus which led to only confirmed cases and death was nearly silenced in the mainstream media coverage. in the unusual reference to vietnam in some zhihu conversations, vietnam's success was rarely celebrated but considered as a "threat" to china's leadership in containing the pandemic in the area. the trivialization and exclusion of these countries/ regions from the chinese imagination of east asia as a collective force fighting against covid- is not unexpected. in the first place, the negative attitudes toward them (except macau) reflects a backlash against the restrictive, noncooperative methods those governments have enforced to block the virus from mainland china (e.g., full border closure; ban on exports of medical supplies), which signifies their resistance to be incorporated into the modern tributary system chinese people have aspired. yet for taiwan and hong kong, this exclusion repeats the endeavor of chinese official propaganda to erase the distinction between them and the mainland and disavow their political autonomy. instead of being completely out of the picture, their responses to the coronavirus are mainly discussed as part of the chinese experience to consolidate the national identity. for north korea and vietnam, the negative impression may partly result from china's ongoing diplomatic conflicts with them in recent years regarding the south china sea and denuclearization, respectively. nevertheless, the shaking of the "socialist brotherhood" on the matter of covid- also implies the reluctance of the chinese public to articulate a regional identity around the axis of a shared political regime. in fact, assimilating itself with ideological and political allies is likely to obscure the focus of this imaginary on china's historical and cultural alignment with japan and south korea. as covid- begins to shift both the scholarly and media focus on an international scale to reconsidering the dark sides of globalization (chan & haines, ) and mourning for the disruption of european union (trofimov & pancevski, ) , china's reversed agenda of imagining a regional union is stunningly intriguing. on one hand, the eagerness to build solidarity with east asian countries represented by japan and south korea might be a strategy to react to the racialization of covid- as a "chinese virus" and the demonization of china as a "public enemy" and "trouble maker" in the euro-american political and media agenda (viala-gaudefroy & lindaman, ). by articulating china's resemblance (and collaboration) with the bordering democratic capitalist states (rather than the "socialist brothers") in the "confucius-inspired" success of halting the virus, the public discourse strives to construct a collective identity of the east so as to brush off china's label of the other imposed by the western imagination. ironically, this consolidation of the eastern identity also serves as a repercussion to otherize the west as the loser to the coronavirus. on the other hand, the rise of this east asian imaginary centering around china's historical and cultural bonds with japan and south korea has far-reaching implications for china's geopolitical strategies beyond the covid- pandemic and the realm of public health. rested upon the trope of the imperial tributary system, this imagination reflects how the chinese public discourse echoes the state ambition to recuperate the historical dominance of china in the sinosphere, which is part of chinese communist party's long-term project of "the great revival of the chinese nation" (中华民族伟大复兴), or in xi jinping's term, the "chinese dream" (中国梦). incorporating japan and south koreathe most important american allies in east asia-into the imagined tributary network might serve the specific purpose of weakening the u.s. hegemony in the region (see ikenberry, ) , whereas the tactic exclusion of north korea and vietnam indicates the indifference of many chinese to the state's political and ideological "comrades" (whose traditional alliance with china has often proven itself unstable and delusionary in the changeable economic and political dynamics in east asia). more importantly, this selective reimagination of the eastern union expresses the chinese public's nostalgic ideal of the nation's revival, which dreams of a return to the middle kingdom, the empire that reunites and leads east asia through culture and history. during the year , which is anticipated to be the warmest year in human history, we failed to stop the rampant spread of a coronavirus called covid- and its disastrous impact on societies and individual lives. unlike its "cousin" sars, which broke out in early and vanished into thin air largely because of rising temperatures, the current respiratory epidemic has yet to show any sign of amelioration with the arrival of summer. news photos have shown audiences an incredibly bleak, bizarre, and somewhat surreal picture of life during the pandemic. streets are evacuated. stores are closed. public services are paralyzed. modernized cities have become empty and ghostly quiet. only scattered people equipped with medical face masks walk anxiously in this futurist, apocalyptic scene. to use timothy morton's concept, the covid- pandemic has become a "hyperobject," a phenomenon that possesses an ahuman time scale and an extremely diffused quality in occupying space. in such a space-time reconfiguration, or, in plain language, during this type of disaster, humankind becomes an obsolete idea, as humans no longer play a meaningful role in the space-times created by and for "hyperobjects." unfortunately, such a concept bares relevance in light of the uncontrollable proliferation of the coronavirus across the globe at this juncture. worse still, some epidemiologists warn that a new round of outbreak will likely occur soon in the coming fall. a possible scenario could repeat the conditions after the / fukushima daiichi nuclear disaster, when breathing with face masks, people eventually became accustomed to a state of emergency as the conditions for living and dying in the anthropocene. "[p]oison has become a normal feature of daily life, the second nature we have to inhabit" (berardi, , p. ) . while one can attribute the deterioration of nature to neoliberalism and its disastrous governance, this essay, rather, speculates on what foregrounds the involutional relationship between humans and the earth beyond the "nature-culture" divide. whether one is willing to admit or not, viruses are neither creation ex nihilo nor culturally and politically constructed representation. instead, they are beings that have always been part of earth's composition. in a prophetic book, the natural contract, the late philosopher michel serres ( ) describes the evolution of the earth's composition. in ancient law and modern science, nature was treated as an objective reference point, because it had no subject. existing objectively "out there," the earth was a space that did not depend on humans but only acted passively in relation to causality. yet, witnessing the ecological crisis arising in the th century, humans realized that the earth has been affected by our behavior and is now behaving like an aberrant subject! in recent scholarship, this subject has been referred to as gaia, the capricious goddess of the earth (see latour, , p. ) . the earth is full of action and so is covid- . as described in news reports, the coronavirus looks for and hijacks its hosts; it finds easy purchase on, and takes control of, human bodies; it kills many, but not all, of its hosts so as to keep moving, spreading, replicating, and surviving. it would be impossible to talk about the virus without referring to those actions. cited by the washington post, a virologist came up with a vivid analogy for viruses by comparing them with destructive burglars. "they break into your home, eat your food, use your furniture and have , babies" (kaplan et al., ) . as the word "object" refers to entities that are inanimate and subject to chains of causality, viruses, in this sense, hardly fit into this definition. for instance, covid- remains mostly enigmatic, not least because it is considered strikingly sneaky-"the virus doesn't really want to kill us. it's good for them, good for their population, if you're walking around being perfectly healthy," said another virologist in the same washington post article (kaplan et al., ) . besides doing things such as breaking-into, eating, and having-babies, the virus is further endowed with intentions-it does not want to kill us! however, the coronavirus should not be mistaken for a subject, especially a subject-agent, which is historically associated with liberal humanism since the enlightenment and which is deeply rooted in the "nature-culture" divide, an ontological regime referred to by latour as "the modern constitution" (see latour, b) . the idea of the subject as a product of euro-american modernity is indivisible from its aim to achieve individual sovereignty and autonomy. in a politico-legal sense, bounded individualism is the most evolved form of this idea in the wake of the global expansion of capitalism. faced with an unprecedentedly active earth in the late th century, nonetheless, this anthropocentric conception of the subject-agent has been confronting exponential challenges, among which the current coronavirus pandemic constitutes the latest one. to be clear, the term "subject" is a mismatch for covid- , not because it is agentless and incapable of doing the same things that humankind does. the contrary is true: the state of being of the virus-what it is-can unfold only through its actions and long after its performances. at stake for the virus and humans is that there are "no pre-constituted subjects and objects, and no single sources, unitary actors, or final ends" (haraway, , p. ) far from being a de-animated object, or an anthropomorphized subject, gaia, the increasingly "rioting" earth, is a collective of actions that distributes agency in heterogeneous and surprising ways. as a result, "we must not believe in advance that we know whether we are talking about subjects or objects, men or gods, animals, atoms, or texts" (latour, a, p. ) , and also viruses until their actions are captured, and rendered into shapeswhether the shape of a human or of a virus. the story of the human-centered history is being replaced by an explosion of narratives about the increasingly animated and animating earth. however, the dualism of the subject versus the object, unfortunately, is still perniciously conserved in the mainstream reaction to the covid- pandemic. when societies are forced to act on the pandemic, the virus is almost exclusively treated as an object subject to the chain of causality. this tendency is clearly reflected in the mobilization of wartime rhetoric and discourses in conjunction with governments' anti-epidemic measures. for instance, when visiting wuhan right after its lockdown, sun chunlan, china's vice premier, warned that the country was facing "wartime conditions." likewise, only month later, president donald trump declared a national state of emergency over the coronavirus outbreak in the united states. in this antagonistic discourse, contending with the virus, a not-yet-tamed and potentially threatening other, is framed as a relationship between humans and their enemies. for those who believe humans and only humans make history, a self-proclaimed war on the virus is unavoidable! peace, accordingly, is only imaginable to be reached, or more precisely restored, to an already existing order, established primarily for humans. mobilized to describe the relationship between covid- and humans, "war" is a terrible and even dangerous choice in terminology, due to its undertone of human exceptionalism. in fact, nearly % of the cells in a human body is "part of a vast community of companion species, particularly bacteria and viruses" (smart & smart, , kindle ) . unfortunately, most humans have yet to learn the meaning of living and becoming-with these beings who are made by and making humans at the same time. in her book staying with the trouble: making kin in the chthulucene, donna haraway ( ) invites readers to contemplate our troubling present, the chthulucene, an emerging regime of naturecultures, as opposed to the "nature-culture" divide. contrasting to the discourse of the anthropocene and the capitalocene, both of which are conceived as human-induced condition, the "chthulucene" is, first and foremost, concerned with earth beings who live in "manifold forms and manifold names in all the airs, waters, and places of earth"-they are monsters which "demonstrate and perform the material meaningfulness of earth processes" (haraway, , p. ) . the vicious coronavirus is evidently one of these monsters. despite the havoc it is creating in the present, the epidemic is a manifestation of the biotic and abiotic powers inherent in earthly actors and is part of "ongoing multispecies stories . . . in times that remain at stake" (haraway, , p. ) . as implied by the title, one of the valuable lessons of haraway's book is that, for humans in particular, there might be no better option other than to stay with troubles, of which humans are never innocent. staying with the troubles demands caring for all the threads that bind us together and make our existence possible in the first place-humans are made by countless earth beings and vice versa. it also means that we are required to weave unexpected and even dangerous connections with others, in haraway's ( ) words, making kin as oddkin "in unexpected collaborations and combinations . . . we become-with each other or not at all" (p. ). this insight is particularly useful for thinking about viruses. because viruses have "no cellular machinery of their own, they become intertwined with ours. their proteins are our proteins" (kaplan et al., ) . in this sense, the evolution of humans and viruses is inseparable from the process of involution of the two into one. in other words, becomingwith means that, by definition, a "we" always precedes an "i," a "you," or a "they." the so-called "asymptomatics" provide an excellent example of this point. asymptomatics refer to those who test positive for covid- but, confusingly, do not suffer from illness or show any symptom of the disease. asymptomatic infections or carriers are possibly greater in number than those with symptoms. at this point, it is impossible to decide which of the two types is more typical of covid- infections, because, as a researcher at the university of oxford says, "there is not a single reliable study to determine the number of asymptomatics" (shukman, ) . in the same news report, neil hall, a biomedical expert, suggests considering asymptomatic cases of the coronavirus as the "dark matter" of the epidemic, as invisible and not-yet identified dark matter is believed to make up most of the matter in the universe. despite the fact that no conclusion has been reached about the enigmatic phenomenon of asymptomatics, the differences that manifest among patients reveal that the virus, and the particular cases of infection, should be examined as specific units. in other words, between the virus and humans, the specificity of an encounter matters. unlikely to be autopoietic systems that reproduce autonomous units, the virus and an infected body constitute a collectively produced, sympoietic system that does not have self-defined spatial or temporal boundaries. in these cases, and from a non-anthropocentric, philosophical point of view, the idea of bounded individualism has to be discarded for good. beyond the divide between the subject and the object, what emerges are ontologically heterogeneous practitioners who are involved in each other's lives. besides evolution, living also relies on involution. without any intention of "romanticizing" covid- and the current pandemic, staying with the trouble, as articulated by haraway ( ) , is "to make kin in lines of inventive connection as a practice of learning to live and die well with each other in a thick present" (p. ). the coronavirus does not happen as a matter of fact, which "passively" waits to be discovered, investigated, tamed, or neutralized by "active" humans. what we call the covid- pandemic manifests itself as a differentiating and relational effect because it matters by bringing into being various relations between humans, and between humans and their oddkin. in this view, science is only one practice among many others to capture the efficacy of its mattering. in addition to biomedical measures, a more critical question for the coronavirus crisis is "what method does the matter demand" (thompson, , p. ) ? proposed by haraway for living in the chthulucene, the string figure might also serve as an appropriate method and image for the pandemic, characterized by its exceptional contagiousness and interactivity. consisting of "passing on and receiving, making and unmaking, picking up threads and dropping them," the string figure is all about "becoming-with each other in surprising relays" (haraway, , p. ) . crucial to this method is that it does not guarantee what is obtained turns out to be good in the end, because living itself has become so dangerous in this very thick present-agencies are distributed, conflicting, and entangled in a myriad of practitioners, human and non-human alike. in this pandemic, we are all playing the game of string figures with our oddkin. it is not beneficial to judge in advance who is a subject and who is an object, or which one is active and which one passive, as all participants might be capable of something that matters in one way or another. for example, one thing that the respiratory disease teaches us is that not only breathing matters but also the manner how one breathes matters to others. life and death happen inside specific connections and their mattering in mundane, and even fleeting, encounters. making covid- matter requires us to reanimate "what is coming into states of matter and mattering in bodies, stories, acts, and events" (stewart, , p. ) , in other words, in the vicissitudes of our ordinary lives. for the future of this thick present, one key is to stop imagining the crisis of the coronavirus as something wholly predicated on effective vaccines and scientific solutions. instead, humans must learn to connect and also care for threads, some of which are obvious, some elusive, some vicious and dangerous, and some fictional. we may need to discard terms such as "overcoming" or "solution," and turn to terms like "participation" concerning all that we are uncertain of but have to live and become with, together, in the "metamorphic zone" called the earth (latour, , p. ). what does it mean, the plague? it is life, that is all. the most abundant biological entities on earth, viruses are forever and everywhere. suspended between living and being dead, they are simply there, a slimy strip of ribonucleic acid (rna), as biologists tell us. poorer in life than tardigrades, incapable of movement, and having no logistic of their own, they ride on and feed off others to replicate themselves, to become the viruses that they are. as smart schoolchildren know, they are transmissible and must be so transmitted as to go viral, to become the viruses as we know them. dependent entirely on carriers, that is, exploiting others' hospitality, without which they have no life (but also no death either), viruses exemplify transmissibility. they live and thrive, as it were, only if their hosts are susceptible, in motion, and in contact and they die or die down when susceptible hosts are either unavailable or no longer hospitable. defined, that is, made finite, by transmissibility, and yet transcending its barren finitude through parasitism, viruses exist and operate like pure media, self-generating and selfgenerated by being entirely coterminous with the channel through which they flow and multiply. interpolating and encoding themselves in the metabolic cycle of others, thereby reproducing themselves passive-actively, they mediate by colonizing others and, in so doing, mediate themselves by proxy, going about so energetically and indiscriminately as to cause the demise and thus thwarting unwittingly their own propagation. if viruses communicate anything, if their shadowy occupation of host bodies sends any message, it is their very own communicability, their ability to disseminate themselves over a large population with effort less than minimal. although all over creation and in abundance, most of the viruses cause us no harm and we pay them little attention, even though they populate our body and capitalize on its resources. they become a matter of grave concern when they infect us, when they not only put themselves inside (in-ficere) our body and stain its normal functioning, but also threaten to afflict as many people as their "infectivity" attacks. more dangerous and less tamable than most microbes, viruses invade our body and compromise it at the cellular level. they do not just make us sick; they bring about plague. once seen as a cause of infection, viruses accrue significance and take on the label "pathogens." to refer to viruses as pathogens implies that they are "medicalized," that they not only enter into a relation with humans who regard them as toxic and virulent, but are also seen as a problem to be addressed in a methodical, systematic, that is, "scientific," manner. it is through this medicalization that viruses are individuated and identified as a distinct biological entity and, having been so captured and given a name-for example, h n , mers-cov, sars-cov- , covid- , and the like-by what might be called the "clinical gaze" and its taxonomic procedures, they enter into sciences and become a focus of medical research, made all the more pressing if and when they create public health crisis. more than one hundred years after martinus beijerinck gave the name contagium virum fluidum (contagious living fluid) to the incitant of tobacco mosaic first discovered by adolf mayer and dimitri ivanovsky, viruses are now actively collected, classified, and manipulated by scientists in highly restricted spaces called laboratories, most of which, like the viruses housed carefully therein, are hidden from public eyes. while slimy poisons were once thought to be sent down by god to punish us for our sins, we now see viruses not only as an object of scientific investigation but also as a medical challenge that nature poses to us as biological creatures on earth. like birds, bats, and rats, we are all equal opportunity hosts to killer germs. not all viruses are fully pathogenic, but pathogenic viruses are ever ready to go viral when the conditions are ripe. however, although viral infection may break out and spill over, it does not mean that there is a pandemic. "pandemics," as virologists tell us, "begin when a brandnew virus infects a human who also at that point is able to transmit the virus to other humans" (buettner, ) . two points should be noted without delay. first, pandemics are not created by transmission of viruses from some source to humans, but from humans to humans. breakouts of viral infections among members of a primate community deep in the amazon rainforests, for example, may be large scale and may disturb ecological balance alarming to conservationists, but they do not for all that count as pandemics in the sense that the term is properly used. viruses might infect one or more individuals, but humans are responsible for creating the conditions that transform infections to outbreaks and outbreaks into pandemics. pandemics, in other words, are not natural or biological phenomena; they name a human crisis, a contagious malady plaguing humans who are both agents and patients at the same time. contagious diseases are disastrous to all, locked, as we are, in the same bubble in which microbes live and grow, but pandemics are decidedly more pernicious in that we become, often unknowingly, the source and the cause of our own infestation. second, pandemics are "declared." as is the case with catastrophic events in history, like wars, famines, or mass cultural anomaly as bizarre as the chinese sorcery scare of , whose duration and identity result from an act of punctuation and sense-making entirely sociopolitical in nature, pandemics too begins with a performative act that announces their beginning and, having made them to begin in this way, determines when they reach their end, even though the viruses and their carriers may still be with(in) us (see kuhn, ) . naming not microbial activities in nature but a crisis for humans, pandemics are events made real, public, and urgent, as just said, by a performative-a speech act, to be exact-whose authority in pronouncing their beginning and end depends on the very force that makes the declaration authoritative and forceful in the first place. brought into being by discourse and public communications, pandemics are social constructions; they signal a state of emergency-appearing, first, as physical ailments on the part of individuals, subsequently identified and ratified by medical and scientific community as a real health problem, and finally materialized by authoritative broadcast and public acknowledgment, thereupon becoming a public policy issue to be addressed by political leadership, all these over a determinate territory. once established as such, a pandemic individualizes a collective paroxysm, making it a public enemy by giving it a face, a name, a certain life span in the social calendar, without which the havocs wreaked by the virus would not be the crisis its name designates and invokes. it is in this declarative nature of pandemics that we can see how viruses, once medicalized and publicly acknowledged, are inevitably entangled with science, history, culture, and politics. socially constructed by a decision, by a cut or break into regular time, they mark a "zone of exception," a temporal heterotopic, as it were, where we, individually and collectively, stand to one another as equal subjects to illness, unfreedom, and death in the unending drama of man against nature and its hostile elements. viruses are viruses are viruses. they have no political content; operating according to the laws of physics, chemistry, and biology, they come and go on their own rules and on their own times, as nature dictates. in sharp contrast, pandemics are biopolitical phenomena; they are moments of discontinuity or rupture in social order, shot through from start to finish with forces and factors that shape culture, history, and economy, which in turn determine what they mean and how they come about and come to pass. moreover, and importantly, a pandemic is not a single, monolithic event; it is a series of localized epidemics, each with its own point of origin, its own history, its own epidemiological pattern and impacts. further still, all these factors crisscross one another in a complex, nonlinear fashion, amassing multiple agents and stakeholders in such a critical fashion that the language of war is often used by the authority in charge to quell the infectious assault. pandemics force social changes precisely because the changes they incur invite resistance. it is for this reason, perhaps for this reason alone, that pandemics inevitably appear as a site of social contestations, politicizing and politicized by the heterogeneous constructions barely betrayed by the name of a single virus. it is for this reason too that pandemics assert themselves as a sign of generalized cultural and economic strife, a symptom of social struggle underlying the health terror that a viral breakout unfailingly induces. covid- is a novel virus, novel in that scientists do not fully understand how it afflicts the body and therefore cannot predict its epidemiological paths. to control its spread, we have no choice but to employ methods developed from past experiences, such as quarantine/isolation, social distancing, face coverings, and contact tracing, to name a few now well-known. because viruses are infectious, to control its spread is, understandably, to separate and to isolate. this means that people be kept away from one another. instead of gathering or being together, we make ourselves scare; better yet, we isolate ourselves, even if begrudgingly. more than that, the injunction of isolation leads straightaway to insulation in that the ultimate, foolproof means of isolation is to literally atomize ourselves, to turn ourselves into windowless monads. indeed, all the mitigation measures we hear about of late-quarantine, mask wearing, hand washing, and social distancing-are in reality anti-social measures. don't reach out and don't touch anyone! cover up your face! just as social distancing-a contradiction in terms of sorts-means keeping physical distance, and just as mask wearing reduces mutual recognition based on simple vision to its unnatural minimum, (self-)isolation and quarantine all but eliminate human contact of all kinds. when the plagues struck, we were all lepers; when covid- strikes, we are all windowless monads. pandemics are born of communicable diseases, yet for this reason, they force us to be incommunicable. flattening individuals and bringing to a halt exchange and commerce of every kind, they turn a society into one that is against society. if there is a history of pandemics, it is a history of anti-social history. neither alive nor dead, neither this nor that, viruses are by nature improper. never proper, that is, never being (of) themselves, they appropriate-always ready to make others their own. they are pure media, as suggested earlier. viruses are pure because they mediate unconditionally. however, inasmuch as unconditional mediation performed by viruses leads to the demise of their host, upon whom they depend for their parasitic reproduction, viruses end up annihilating themselves by their very nature; they are always already their own collateral casualties. rendering themselves nil by simply being and subsisting as themselves, pure media are no (longer) media. unconditional mediation ends all mediations. by bringing society to go against itself, viruses commit suicide, so to speak, by killing their host, by the unconditional abuse of others' hospitality. and, alas, weat least some of us-are spared. covid- is a new virus. but, unlike the known flu viruses, or h n , sars, and the like, covid- is considered "novel," not the least because, as indicated earlier, it frustrates scientists' understanding. "it has been like nothing else on earth," says an infectious-disease expert, who falls victim to the virus; "i knew i had the disease; it couldn't have been anything else," but "i don't understand what's happening in my body" (yong, ) . there are many things, inanimate or living, on earth that are like nothing we know so far, and there are many things happing in our body that we do not understand at all. covid- can justifiably be called "novel," but isn't every virus novel in its own way and at some moment in time? isn't being novel the normal course of event in life and in life sciences as well? "there is novelty here," remarks a prominent epidemiologist karl friston upon leaving a lab meeting about covid- , but he quickly adds, "so this, from my point of view, is just an average day" (kosner, ) . being novel is the very characteristic of all viruses and many other things in nature as well. the novelty of covid- may not be as novel as we think. what is possibly novel about covid- is the fact that it gives us the first pandemic in our truly globalized age. the global village, in which we now live, is so hyper-connected-not only by technology but also through affluence, commerce, and global travel-that an infectant can travel from one city to another as fast as jet streams flow. connectivity translates qualitative diversity into measurable multiplicity, reducing distance and difference for the formation of the common, which in turn strengthens connectivity. to be alive, as few would disagree, is to be connected, literally and in every other sense. but this means that we must live in and with the risks that global connectivity brings to us. to be connected brings with it the possibility of being stranded in harm's way. as covid- makes clear, "connectivity is the killer" (kosner, ) . after all, life depends on maintaining boundaries and keeping differences. deadly viruses are deadly because they breach them. as infection rate rises, so does anxiety. and bleak scenes spread as wide as the virus goes. deserted streets, boardedup stores, closed factories, shot-down public transportations; remote learning, work-from-home; stock markets crashed . . . and, worse yet, "i just lost my job." individual solation leads quickly to desolation across the board. and economy bears the brunt of a colossal coronal attack. shortly after covid- spread out of wuhan, china, to europe in january , stories about the economic plight began to top the list of topics in public forums and news media. the future we face seems to lie in one of the two choices: to die from hunger or to die from the disease (餓死 或病死), as the expressions go in chinese media. it is not for no reason that a policy brief released in june by the united nations on the impact of the pandemic is given the title "the world of work cannot and should not look the same after this crisis" (guterres, ) . the address on the launch of this brief, given by the secretary-general antónio guterres ( ) , begins as follows: the covid- pandemic has turned the world of work upside down. every worker, every business and every corner of the globe has been affected. hundreds of millions of jobs have been lost . . . many small and medium-sized enterprises-the engine of the global economy-may not survive. after painting a depressing picture of the future and explaining how difficult it will be for the world economy to return to "normal," guterres's ( ) address makes a hardly perceptible turn when he says "let's not forgot the pre-covid- world was far from normal." it seems then that, rather than shattering the world of work as we know it, the covid- pandemic simply exposes in higher resolution the "tremendous shortcomings, fragilities and fault lines" that have been eroding society and economy from the bottom-up for decades. the pre-covid- world, in which we thought we lived a normal life, is not as normal as we think (see guterres, ) . to save the economy under siege is to "return to normal" as soon as possible, so cry the bureaucrats and journalists alike. but what is "normal" in this case? what does "being normal" mean exactly? is the world, old or new, ever normal? there are norms regulating life, but has there ever been a "normal life" as such? the so-called normal life, a life before covid- , to which we pray to return, is in truth one of recollection, a romantic one at that, as the un policy brief readily admits. just as a viral infection may display more than one symptom on the part of its victims, embody more than one single illness, and create more than one single public health challenge, life, as it is actually lived, is hardly reducible to one normal life. in fact, the socalled normal life is the one that brought us the pandemic in the first place. to live is to live normally; to return to normal is what living is all about. the so-called new normal is both new and not so new, which is to say, it is neither really new nor really normal. perhaps the world has never been and will never be normal, whatever our idea of "being normal" means. if a pandemic can turn the world upside down, it is because life has been turning and turning again. and anything that returns cannot be entirely new. humans have been haunted by viruses since time immemorial. from the prehistoric pandemics in northeastern china , years ago, uncovered at sites now called hamin mangha and miaozigou, to the justinian plague ( - ad) that may have helped to bring down feudalism, or the small pox outbreak that finally toppled the aztec empire before hernán cortés returned to the region in the spring of , viral infections have tormented the lands and their people over millennia. traveling with host animals and humans, viruses had gone global long before globalization became a fact. there are known pandemics in the last years, all displaying the same pattern of spiking in seasonal waves after the initial attacks. covid- , and some of its coronal cousins, will undoubtedly expand the list. to those who are living through its assault, the impacts brought about by covid- are more or less clear and more or less measurable. but what is the meaning of covid- when the current pandemic is over? will it be remembered? if so, in what way and to what extent? if the history of pandemics has taught us anything, it is that history tends to repeat itself, that viral outbreaks are an ineliminable part of the natural history, in which humans are a part and in which no "zone of being" is free from viral infection. recall the spanish flu of , the worst pandemic during the last two centuries. it is estimated to have wiped out million people worldwide, meanwhile infecting million, a third of the world's population at the time. however, despite its short distance of mere one hundred years from us, few people today know much about it, and still fewer are able to understand or feel the impact it had at that time. its centenary a short time ago passed noiselessly, certainly not for lack of stories or records. like the many plagues before it, the spanish flu, it seems, never quite made itself into what reinhart koselleck ( ) calls the "the space of experience" (p. ). failing to make its way into collective memory, it is also helpless in figuring into our "horizon of expectation" (koselleck, , p. ) . if the spanish flu faded largely from memory, all the woes caused by covid- are, likewise, likely to dissipate in time, regardless of how we feel and say about it now. there was a pre-coronavirus world, and there will be a post-coronavirus world, but viruses, known or novel, will outlast our worlds. viruses are everywhere and forever. so, plagues will never disappear for good (camus, , p. ) . but what then does it mean, the pandemic? it is life, that is all. a troubled memory, fading, under the vast indifference of the sky. until the gate of oran closes again. assembled in this forum, the five short essays provide some modest reflections on the coronavirus pandemic and its still unfolding consequences. committed to a variety of disciplinary perspectives and interests, the authors did not set out by pursuing any preset direction or common agenda supposedly carried out collectively in our intellectual labor. rather, what unifies the diverse inquiries in these essays is the shared awareness about the confusion in the public discourse that constantly fails to distinguish a coronavirus called covid- from the covid- pandemic, or as briankle reminds us in his essay, from "a series of localized epidemics." this alertness constitutes a common ground in addressing specific issues or phenomena in these essays. this forum is anything but comprehensive. if it can contribute to the discussion of the crisis, it is most likely because all the essays refuse to bind the pandemic exclusively with the coronavirus and to position the virus and humanity in rigid opposition to each other. in her multispecies ethnography, anna tsing tells a marvelous story about matsutake mushrooms. "when hiroshima was destroyed by an atomic bomb in ," says she, "it is said, the first living thing to emerge from the blasted landscape was a matsutake mushroom" (tsing, , p. ) . when human history temporarily comes to a halt in disasters, matsutake, and also viruses in our case, may well survive and continue to thrive with their own stories. histories are being made every day by humans and non-humans alike; however, the future for those histories to converge has still yet to come. as demonstrated in the essays gathered here, while governments and the public are desperate to frame the virus in their own social and political narratives, the virus also works hard to inscribe its historicity on the earth and humans too. if a message must be dispatched out to all at this juncture, it is that for a future of collaborative survival, the stake of living together has nothing to do with harmony and conquest, but is derived from "disturbance-based ecologies" (tsing, , p. ) , that is, plagues. plagues are life, that is all. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. gong yuan https://orcid.org/ - - - xuefeng feng https://orcid.org/ - - - x notes . in a trilogy of contemporary essays, baudrillard ( ) argued that the war in the desert was a new phenomenon, because it was defined and shaped by its discursive aspect as a form of television programming, regardless of what happened on the ground. . that earlier announcement called "demonetisation" led to a vast contraction of commerce and an immiseration of a majority of the population which has still not recovered, and is now widely considered an unnecessary exercise of personal whimsy. for an analysis of the economic consequences, see ghosh et al. ( ) . . the decline of indian agriculture is not adequately discussed in the celebration of urban growth. its effects are however inescapable and directly lead to the growth of informal settlements in cities (balakrishnan, ; jaffrelot & thakker, ) . . mediatization, or the analysis of events with their mediated construction as the starting point, is a phenomenon that has grown in importance across cultural contexts, as media theorists attempt to understand the increasing influence of media forms on culture, especially with the virtualization of human interaction and the redefinition of community through the use of social media and mobile communication. for a fuller discussion, see couldry and hepp ( ) . discontents: an indian history ( ), jayal ( ) traces the current shift in the discourse of indian citizenship from an egalitarian rights-based model to a new regime predicated on religious and cultural identity, given shape through the concomitant technocratic frames of transactional welfare and biometric identity. . four short essays provide further context and narrate the response: shankar et al. ( ) . . partha chatterjee's ( chatterjee's ( , insightful categorization of indian society separates the distinct ontological domain of a small formal "civil society" that includes rights-bearing citizens, from the vast undifferentiated mass of the population that constitutes "political society" and which forms the actual locus of democratic practice. . the current indian government has dramatically increased the acquisition and use of big data in governance, including a reliance on biometric identification for access to welfare programs and the use of mobile phones for access to services: part of the government's covid- response was in the form of a mandatory mobile application that purported to use location tracking to show active virus infections in the user's vicinity. an analysis of its invasive nature can be found in a working paper by deb ( ). . societies always declared war on problems they cannot solve: war on drugs, war on poverty, and the like. it is no surprise to see donald trump refers to covid- as an "invisible enemy" and calls himself a war-time president in his speeches on the covid- pandemic. similar examples abound across the board throughout history. it is widely recognized by epidemiologists today that the model developed by john snow based on the cholera outbreaks between and in england is too linear to be of any use in contemporary pandemics. like the global climate instability or financial market volatility, pandemics are nonlinear phenomena, displaying a high degree of uncertainty that defies simple causal explanation. on this, see kosner ( ) . . it is therefore not surprising that we observe donald trump repeatedly refers to covid- as wuhan virus or kung flu in reaction to his rise and fall in poll and public opinion as he tries to find scapegoat, in this case, china, for his sorry failure in handling the crisis. a virus is always more than a virus when it enters the body politic. india's brutally uneven development patterns are mapped in routes migrant workers are taking home. scroll the gulf war did not take place bifo covid- : straight answers from top epidemiologist who predicted the pandemic the plague. vintage diseasescape: coping with coronavirus, mobility, and politics. global-e the politics of the governed: reflections on popular politics in most of the world lineages of political society: studies in postcolonial democracy cdc director says some coronavirus-related deaths have been found posthumously conceptualizing mediatization: contexts, traditions, arguments the covid- crisis in india: a nascent humanitarian tragedy. books & ideas public policy imperatives for contact tracing in india specters of marx: the state of the debt, the work of mourning and the new international coronavirus conspiracy theories targeting muslims spread in india. the guardian why coronavirus conspiracy theories flourish, and why it matters. the new york times the standard edition of the complete psychological works of sigmund freud as coronavirus spreads, so does racism and xenophobia covid- lockdown and the pandemic of caste demonetisation decoded: a critique of india's currency experiment chinese-japanese-s. korean relations evolve to meet the challenge of covid- . global times the world of work cannot and should not look the same after this crisis the companion species manifesto staying with the trouble: making kin in the chthulucene american hegemony and east asian order by revealing magnitude of migrant worker phenomenon, covid- points to rural distress. the indian express reconfiguring citizenship in contemporary india the coronavirus isn't alive. that's why it's so hard to kill sediments of time: on possible histories karl friston takes on the pandemic with the brain's arsenal high on talk, low on substance: modi's speech showed india is ill-prepared for covid. the caravan soulstealers: the chinese sorcery scare of the pasteurization of france we have never been modern agency at the time of the anthropocene migrant workers distrust a state that does not take them into account it was already dangerous to be muslim in india. then came the coronavirus india racked by greatest exodus since partition due to coronavirus. the guardian india: portents of an ending: modi, the rss and the rise of the hindu far right. the nation how covid- compares against other killer diseases in india why east beat west on covid- : east asia has handled and contained the pandemic far better than the west on nearly all metrics shame in the cybernetic fold: reading silvan tomkins the natural contract the demagogue and his labyrinth. the baffler coronavirus: the mystery of asymptomatic "silent spreaders posthumanism: anthropological insights mattering compositions labyrinth of linkages: cinema, anthropology, and the essayistic impulse coronavirus crisis threatens to split an already fractured eu the mushroom at the end of the world: on the possibility of life in capitalist ruins donald trump's "chinese virus south korea agree to continue push for free-trade deal despite ongoing tensions across region. south china morning post buddhist pilgrim-monks as agents of cultural and artistic transmission: the international buddhist art style in east asia, ca covid- can last several months the popularity of "rivers low, mountains high; the same moon in the sky" and its english translation the hindu rashtra comes of age. contending modernities li lu is an associate professor of literary theory at the school of chinese language and literature and a research associate at the center for literary theory at beijing normal university. he teaches courses on marxian aesthetics, critical theory, and translation theories.srinivas lankala teaches in the department of communication at the english and foreign languages university, hyderabad. he works in the areas of semiotics and critical theory, and engages with questions of politics, history, and nationalism. key: cord- - ef u mz authors: alsolamy, sami; arabi, yaseen m title: infection with middle east respiratory syndrome coronavirus. date: journal: can j respir ther doi: nan sha: doc_id: cord_uid: ef u mz nan t he middle east respiratory syndrome coronavirus (mers-cov) was first recognized as a new febrile respiratory illness in saudi arabia in june . as of september , , the who reported laboratory-confirmed cases, including at least related deaths. cases have been reported in countries; however, the majority of cases have occurred in saudi arabia ( %) and south korea ( %) ( ) . mers-cov infection has been documented in dromedary camels; evidence suggests that they are the most common source of animal-to-human transmission ( ) . although the exact mode of transmission from camels is unknown, the presence of high viral loads in the upper respiratory system of infected camels suggests that transmission occurs through close contact ( ) . humanto-human transmission of mers-cov has been demonstrated among close household contacts ( ). however, sustained community transmission has not been observed. in a cross-sectional serosurveillance study involving , individuals in saudi arabia, positive serology was documented in only . % of individuals sampled ( ) . transmission within health care settings has been a predominant feature of mers-cov infection, and has been attributed to breaches of infection prevention and control practices ( ) . coronaviruses are a family of single-stranded rna viruses. mers-cov is the sixth coronavirus and the first lineage c betacoronavirus known to infect humans. severe acute respiratory syndrome coronavirus is of lineage b ( ). mers-cov enters cells via a common receptor, the dipeptidyl peptidase- , and it infects type i and type ii alveolar cells ( ) . the virus has primarily been detectable in respiratory secretions, with the highest viral loads in the lower respiratory tract ( ) . the median incubation period of mers-cov infection is . days, but it can be as long as days ( ) . the most severe cases of mers-cov infection have been reported in adult patients with underlying comorbidities, including diabetes mellitus, ischemic heart disease, end-stage kidney disease or immunosuppression ( , ) . however, severe infection may also occur among younger patients, especially health care workers. the disease spectrum ranges from asymptomatic infection to rapidly progressive multiorgan failure. the most common clinical features in severe cases are fever ( %), cough ( %), dyspnea ( %) and gastrointestinal symptoms ( %) ( ) . laboratory abnormalities commonly associated with severe mers-cov infection include leukopenia, lymphocytopenia and thrombocytopenia, in addition to elevated serum levels of creatinine, lactate dehydrogenase and liver enzymes ( ) . initial chest radiographs are abnormal in the majority of symptomatic patients. findings range from minimal abnormality to extensive bilateral infiltrate consistent with acute respiratory distress syndrome patterns ( ) . respiratory samples in suspected patients should be tested using real-time reverse transcriptase-polymerase chain reaction. lower respiratory tract specimens have been found to be more sensitive than upper respiratory tract specimens for the detection of mers-cov ( ) . rapid progression to hypoxemic respiratory failure requiring mechanical ventilation usually occurs within the first week and it is notable that it has been associated with acute kidney failure ( ) . the management of patients with mers-cov infection entails early case recognition, appropriate patient isolation, strict implementation of infection prevention and control measures, and supportive treatment. the who has issued interim guidance for the management of suspected and confirmed mers-cov infection ( ) . early supportive management includes supplemental oxygen to all patients with signs of hypoxemia or respiratory distress, conservative fluid management, early endotracheal intubation in patients with laboured breathing or persistent hypoxemia, and a lung-protective ventilation strategy ( ) . other adjunctive hypoxemic rescue therapies, such as early prone positioning and neuromuscular blockade, may be considered in patients with moderate-to-severe acute respiratory distress syndrome ( ) . in addition, systematic corticosteroids should generally be avoided unless there is another indication. high-flow oxygen and noninvasive ventilation should be used with caution because of the potential to generate aerosols ( ) . a systematic review to assess the risk for transmission of respiratory pathogens to health care workers through aerosol-generating procedures found that the following procedures were associated with an increased risk for pathogen transmission: endotracheal intubation, noninvasive ventilation, tracheotomy and manual ventilation ( ) . to date, there are no clinical trials involving humans for virusspecific therapies for mers-cov infection. data regarding ribavirin, interferon and convalescent plasma are limited ( ) . other medications, such as mycophenolic acid, chloroquine, chlorpromazine, loperamide and lopinavir, have shown an inhibitory effect on mers-cov replication in vitro; however, in the absence of clinical data, these drugs are not recommended for clinical use outside clinical trials ( ) . there is ongoing work investigating monoclonal or polyclonal antibodies against mers-cov, but no clinical data to date ( ) . the overall case-fatality rate of mers-cov infection is %, but the mortality rate of mechanically ventilated patients reaches % to % ( ) . given the potential for transmission in the health care setting, compliance with infection control measures is critical. according to who guidelines, droplet precautions should be added to the standard precautions when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of mers-cov infection, and airborne precautions should be applied when performing aerosol-generating procedures with mers-cov patients ( ) . the centers for disease control and prevention (georgia, usa), on the other hand, recommends using airborne precautions with mers-cov patients at all times ( ) . hospitals should develop an infectious disease emergencies response plan. after more than three years since the first mers-cov patient was identified, this virus continues to be a significant global threat because of its high fatality rate and the gaps in our knowledge about the disease. this open-access article is distributed under the terms of the creative commons attribution non-commercial license (cc by-nc) (http:// creativecommons.org/licenses/by-nc/ . /), which permits reuse, distribution and reproduction of the article, provided that the original work is properly cited and the reuse is restricted to noncommercial purposes. for commercial reuse, contact reprints@pulsus.com middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia: disease outbreak news evidence for camelto-human transmission of mers coronavirus family cluster of middle east respiratory syndrome coronavirus infections presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study mers-cov outbreak in jeddah -a link to health care facilities genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome: knowledge to date association of higher mers-cov virus load with severe disease and death, saudi arabia an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected -interim guidance hypoxaemic rescue therapies in acute respiratory distress syndrome: why, when, what and which one? aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). cdcgov <www.cdc.gov/coronavirus/mers/infection-prevention-control.html> key: cord- -wy kj l authors: abidin, crystal; zeng, jing title: feeling asian together: coping with #covidracism on subtle asian traits date: - - journal: soc media soc doi: . / sha: doc_id: cord_uid: wy kj l since the onset of covid- , incidents of racism and xenophobia have been occurring globally, especially toward people of east asian appearance and descent. in response, this article investigates how an online asian community has utilized social media to engage in cathartic expressions, mutual care, and discursive activism amid the rise of anti-asian racism and xenophobia during covid- . specifically, we focus on the . -million-strong facebook group “subtle asian traits” (sat). throughout the covid- pandemic, the , new posts it publishes daily have swiftly pivoted to the everyday lived experiences of (diaspora) east asians around the world. in this article, we reflect on our experiences as east asian diaspora members on sat and share our observations of meaning-making, identity-making, and community-making as east asians collectively coping with covid- aggression between january and may . this quote-posted by a member of subtle asian traits (sat), a facebook group-exemplifies how the ongoing health crisis has shaped what it means and feels to be asian. as two asian diaspora scholars who have spent several years living in a string of cities where our cultural identities have been questioned, contested, and commodified, a facebook group like sat can feel like a mothership to soothe returning aliens to an online "home." while popular among east asians in general, sat caters especially to the asian diaspora, having been launched in "as a joke" by eight asian-australian teenagers to reflect on their intersectional experience. since then, sat boasts an impressive . million members, including actors, musicians, and public figures of asian descent who praise it for championing asian representation through its leadership and advocacy work online and offline. on sat, everyday experiences of "being asian" are parsed through banal or critical internet vernacular as reminders and reflections of our cultural identities: members celebrate memes, viral videos, and asian excellence alongside sincere diary entries, heart-breaking sob stories, and riveting confessions about "growing up asian." members congregate daily to share their origin stories, family cultures, food preferences and traditions, stereotypes about asian bodies, and entertainment. in internet speak, sat is tl;dr a repository of a universal (east) asian experience and a validation of one's diaspora struggles, all packaged into a massive facebook group. in the age of covid- , sat has become a congregational node for the asian diaspora community on facebook. from propagating "quarantine trends" (e.g., homemade dalgona coffees which require the effort of whipped coffee and milk but are instaworthy to simulate the café experience; and recommendations of korean dramas in every genre to soothe the soul) to joking about asian mothers' pseudo-scientific anti-covid remedies, sat's + daily posts from its s msxxx . / social media <span class="symbol" cstyle="mathematical">+</span> societyabidin and zeng research-article curtin university, australia university of zurich, switzerland members have swiftly pivoted to reflecting on what it means to be "asian" during the pandemic. however, sat is not just about memes and humor. (for the record, the authors' favorite meme was when sat members collectively agreed that all our asian mothers were graduating from the "whatsapp and wechat university of misinformation"; someone even designed a mock graduation certificate for our moms.) since knowledge of the outbreak first occurred in early , disheartening incidents from people of east asian appearance have been reported worldwide (giuffrida & willsher, ) and on sat (figure ), recounting experiences of being verbally and physically attacked. in the fight against #covidracism, social media serves as the key arena where asians can speak out about their own encounters and launch various counter campaigns (zeng, ) . in particular on sat, the pandemic has shifted the tonality of camaraderie and community to focus on sharing, resolving, and teasing out the other universal east asian experience of coping with and surviving covid- race-based aggression. in our study of sat's comments sections, we have observed various forms of coping strategies used by group members to counteract racism, most of which are expressed through lengthy threads. in response to such shared struggles, as participant-researchers we observed that sat members collectively cope with the rising prejudice against asians through catharsis, escalation, and problem solving. amid the pandemic of #covidracism, sat has become the "go to" arena for airing our grievances and for seeking resonance and support within the east asian (diaspora) community. we describe this coping strategy where users share their own feelings and experiences as catharsis. for instance, sat members often circulate self-deprecating tiktok videos of themselves holding in coughs out of fear of being ostracized, or humorous stories of how our asian appearances command spatial buffers when we are in public spaces (figure ) . "mask on vs mask off" was another hot topic in the group during the early phase of the outbreak. on one hand, for most asian people, wearing masks is a common and natural thing to do in crowded public places, especially when one is ill, when dealing with bad environmental conditions (e.g., fine dust in south korea, pollution in china), to keep warm (e.g., countries with winter seasons), or even to retain a degree of anonymity or privacy (also influenced by east asian celebrity cultures where masks are worn when makeup is off). on the other hand, as diaspora members in countries without this cultural context, we are concerned about cultural clashes, misunderstandings, and unintended consequences around mask wearing. sat members actively shared our own stories about asians wearing masks in global northern countries receiving eyerolls, being avoided, or even being attacked in public ( figure ) . escalation is the second strategy sat members have commonly utilized to cope with #covidracism and refers to the practice of members amplifying the reach and visibility of anti-asian racism incidents. in cases of assault, sat members often suggest that we make reports to the authorities, media, and other facebook support groups (e.g., crimes against asians). when confronted with racist assaults, it seems to be a cultural response for early-generation diaspora asians to take non-confrontational approaches such as "quietly enduring the pain" or "walking away"-indeed many of our comments threads referenced these notions in a variety of east asian adages and idioms. this norm of "not making a scene" has made asians the invisible victims of racism (chou & feagin, ) . but unlike our parents' generation, the younger asian diaspora on sat are more vocal and more willing to decry the unfair treatment they receive, and strategize with others on possible solutions. from providing advice on self-defense to offering tangible help to asian businesses suffering from the lack of customers, sat members also provide problem-solving solutions. providing advice and comforting messages to each other regarding our life and career struggles has always been a recurring theme of posts and comments in this group (this shared commiseration is partly in jest and partly genuine emotional support for those of us who have grown up with #highexpectationasiandads and #tigermoms). throughout the pandemic, sat's "agony aunts" have similarly offered suggestions on some measures that fellow asians can take when reacting to covid-triggered mistreatment. for example, circulated footage of an asian couple getting beaten up on a subway in philadelphia triggered widespread outrage and heated discussion, and the group's discussions called upon fellow asians to intervene and take action if we ever witnessed such a situation. the centrality of sat as a hub for the east asian diaspora to cope with racism is evident through the volume of content posted daily. however, this community is by no means a virtual utopia with only "rainbows and butterflies" (or "kpop and boba," in the vernacular of our fellow sat members). throughout the pandemic, incidents uncovering in/out-group conflicts have regularly emerged in the group. in january and february, when covid- first emerged in europe and the united states, many of the responses from non-chinese sat members were in the form of complaints that they were "not even chinese" and should not be subject to mistreatment. this form of rhetoric incurred the wrath of other commenters who suggested that unity is important as racists do not care for such subtleties. in early march, when footage of anti-asian assaults circulated on sat, the comments section on sat was also flooded with racist comments alluding to the african-american identity of the aggressor. there were yet other threads where inter-asian and inter-minority tensions broke out, in displays of "competitive grief olympics" or "competitive onedownmanship" (ask & abidin, ) where users attempted to measure and compare the extent of their pain and outrank each other. on threads where aggressive comments accumulated, trolls emerged, or conversations derailed from the ethos of sat (e.g., offering political commentary on actions by politicians and authorities, addressing racist and xenophobic language), admins and moderators would step in to lock the comments and curtain further discussion. while it was found in other studies of antagonistic comments sections that disagreements can be used as springboards for negotiations and teachable moments (abidin, ; johnston, ; uldam & askanius, ) , this was not usually the case for sat given the truncation of conversations due to admin blocks. furthermore, due to the volume of posts and pace of new updates on the page in a non-chronological fashion, it is unlikely that members would return to previously seen posts to keep track of the development of discourse on comment threads, unless they had specifically bookmarked them to do so. although these occasional incidents of "fighting racism with racism" and "out-grieving each other" are significantly outweighed by numerous voices calling for rationality and solidarity, they still reveal the limitations of sat as a space for discursive activism given that the values and ethos of all . million of us cannot be singularly aligned. as the weeks turned into months, the daily routine of scrolling through sat has become a way of equipping ourselves with vocabularies, literacies, and potential reactions for managing the fast-changing covid- situation. like many members of sat, we would sift through the pages and send each other links to "must see" posts, often closely monitoring the ones that we felt were on the cusp of "going viral" (jing's attempt at dalgona coffee was a moderate success whereas crystal's homemade bubble tea never eventuated). other times, we had tagged other diaspora friends in posts that related to recent accounts of their own experiences with anti-asian racism and xenophobia, especially if these had occurred in cities in the vicinity of their residence. although there are often encouraging advocacy and activism initiatives among sat members-such as the grassroots organization of small relief and aid for asians businesses that were the targets of racially motivated attacks, tips and hacks for educating elderly family members about covid- misinformation, and suggestions for maintaining personal safety and well-being-we do not want to overstate the singularity or utility of sat. ultimately, the imagined utopia and harmony of a space like sat is fragile and superficial and can be easily disturbed or destabilized when the tonality of topics ventures into more serious discussions. this was acknowledged by the sat admins in a formal group announcement: [. . .] however, please also recognize that sat is currently run by a group of mostly volunteer college students and generally focuses on sharing light-hearted posts-so the group might not currently be equipped to ensure the kind of nuanced, civil discussion platform around certain topics in our community [. . .] . ( march ) for the most part, what sat has really provided for members during covid- is a sense of companionship through the ups and downs: we have shared heart-wrenching anecdotes detailing experiences with racism, missing the funerals of loved ones far away, and precariousness of job security; alongside the first world problems of missing constant access to bubble tea, deteriorating beauty standards, and running out of korean dramas to watch. alongside its role as an evolving repository of "how to be (east) asian," through the sharing of family histories, traumas, material consumption, and niche asian pop cultural references, sat is also a living archive of how it feels to be (east) asian in the time of covid- . the impressions of normalcy, fun, and the semblance of unity through laughing together is a welcome albeit brief distraction, from the looming crisis outside of our screens and beyond our control. victim, rival, bully: influencers' narrative cultures around cyber-bullying my life is a mess: self-deprecating relatability and collective identities in the memification of student issues. information ?scroll=top&needaccess=true myth of the model minority: asian americans facing racism outbreaks of xenophobia in west as coronavirus spreads. the guardian subscribing to sex edutainment: sex education, online video, and the youtube star online civic cultures: debating climate change activism on youtube sensationalist media is exacerbating racist coronavirus fears. we need to combat it. the guardian the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: crystal abidin's segment of this research is supported by an australian research council discovery early career researcher award (de ). crystal abidin https://orcid.org/ - - - crystal abidin (phd, curtin university) is senior research fellow and arc decra fellow in internet studies at curtin university. her research interests include influencer cultures, online visibility, and internet pop cultures.jing zeng (phd, university of zurich) is a senior research associate at the university of zurich. her research interests include digital culture, online activism, and online misinformation. key: cord- - gs xcw authors: leist, sarah r.; cockrell, adam s. title: genetically engineering a susceptible mouse model for mers-cov-induced acute respiratory distress syndrome date: - - journal: mers coronavirus doi: . / - - - - _ sha: doc_id: cord_uid: gs xcw since , monthly cases of middle east respiratory syndrome coronavirus (mers-cov) continue to cause severe respiratory disease that is fatal in ~ % of diagnosed individuals. the ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with mers-cov in human infections. the inability of mers-cov to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. identification of human dipeptidyl peptidase iv (hdpp ) as the receptor for mers-cov infection opened the door for genetic engineering of mice. precise molecular engineering of mouse dpp (mdpp ) with clustered regularly interspaced short palindromic repeats (crispr)/cas technology maintained inherent expression profiles, and limited mers-cov susceptibility to tissues that naturally express mdpp , notably the lower respiratory tract wherein mers-cov elicits severe pulmonary pathology. here, we describe the generation of the – (+/+) mers-cov mouse model in which mice were made susceptible to mers-cov by modifying two amino acids on mdpp (a and t ), and the use of adaptive evolution to generate novel mers-cov isolates that cause fatal respiratory disease. the – (+/+) mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for mers-cov. the chapter starts with a historical perspective on the emergence of mers-cov and animal models evaluated for mers-cov pathogenesis, and then outlines the development of the – (+/+) mouse model, assays for assessing a mers-cov pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice. in february of mers-cov was listed as a priority on the r&d blueprint for the global strategy and preparedness plan outlined by the world health organization (who) [ ] . the r&d blueprint includes viruses that pose a global public health risk, and for which there are no available therapeutic countermeasures [ ] . twenty-seven countries have reported cases of mers-cov with most cases confined to the arabian peninsula. diagnosed cases of mers-cov in countries outside the arabian peninsula are primarily traveler associated. the potential for global spread of mers-cov was realized in when a single traveler returning to south korea initiated an outbreak that infected people resulting in % fatality and caused widespread fear that crippled the economy for nearly months [ ] [ ] [ ] . human-to-human transmission is often associated with close contact in the health care setting, but can also occur between family members within a household [ ] . asymptomatic individuals pose a particular risk of transmission due to their unknown carrier status as demonstrated in the health care setting [ ] . despite the high percent of fatalities associated with mers-cov outbreaks on the arabian peninsula most epidemiological studies suggest r values < , indicative of a low risk of sustainable human-to-human transmission, whereas epidemiological studies from the south korean outbreak describe r values (> ) akin to more sustainable human-to-human transmission [ ] . recurring spillover events from dromedary camels (zoonotic reservoir for mers-cov on the arabian peninsula) likely contribute to newly diagnosed cases in humans [ ] [ ] [ ] . the potential for continuous reintroduction to humans increases the risk of mers-cov adapting in humans to acquire enhanced human-tohuman transmission profiles, a scenario suspected to have initiated the sars-cov pandemic in - [ ] . effective public health measures and culling of civet cats, the zoonotic host for sars-cov, brought the sars-cov pandemic to a rapid end [ ] . eliminating mers-cov through culling of infected camel herds is not a practical solution. furthermore, detection of pre-emergent mers-cov-like, and sars-cov-like, strains circulating in bat species indicate that the natural environment is ripe for future human exposures to potentially pathogenic coronaviruses [ ] [ ] [ ] . therefore, the development of therapeutic countermeasures that can interfere with mers-cov pathogenesis is critical to break zoonotic-to-human and human-to-human transmission cycles that may instigate global spread. evaluating the toxicity and efficacy of novel mers-cov therapeutics require the availability of animal models that effectively recapitulate mers-cov pathogenesis during fatal cases of human infections. therefore, the first question in generating a mers-cov animal model would be: what are the pathological features of a human infection? limited histopathological findings from human autopsies indicate that fatal cases of mers-cov results from pneumonia initiated by infection of bronchiolar and alveolar epithelia of the lower respiratory tract (lrt) [ , ] . pneumonia in the lrt is also the prominent finding on radiographs from x-rays and cts of diagnosed human cases [ ] . high viral loads in tracheal aspirates from patients are also associated with severe pulmonary disease [ ] , which is indicative of actively replicating mers-cov in the lrt. initial evaluation of the human mers-cov emc/ isolate in rhesus macaques demonstrated replication in the lrt with mild pneumonia-like disease ( fig. ) [ ] . achieving respiratory pathology reflecting a lethal human disease proved to be more complicated in nonhuman primates. severe respiratory disease in the marmoset produced clinical endpoints consistent with fatal disease that required euthanasia ( fig. ) [ , ] . evaluation of two human isolates, jordan and emc/ , and a tissue cultureadapted mers-cov strain (mers- ) in nonhuman primates resulted in mild disease in rhesus macaques or marmosets ( fig. ) [ ] [ ] [ ] , confounding the reproducibility of near-lethal disease in nhps. nonhuman primates are central to late-stage preclinical evaluation of therapeutic countermeasures, but may be impractical for initial preclinical studies. a small animal model may be applicable if there is limited therapeutic available for toxicity and efficacy testing, especially if large animal numbers are needed to determine confidence and reproducibility. early studies in mouse, hamster, and ferret revealed that conventional small animal models were fully resistant to mers-cov infection and replication (fig. ) [ , , ] . a seminal study identifying the mers-cov receptor as human dipeptidyl peptidase iv (hdpp ) [ ] , and publication of the crystal structure of hdpp interacting with the receptor binding domain (rbd) of the mers-cov spike protein [ ] , exposed tropism determinants critical for susceptibility. dipeptidyl peptidase iv contact amino acids at the hdpp /rbd interface are highly conserved among mers-cov-susceptible mammalian species (human, camel, and specific events since the emergence of mers-cov in are emphasized above the timeline. references to mammalian models evaluated for mers-cov pathogenesis comprise hamster [ ] , ferret [ ] , rabbit [ ] [ ] [ ] [ ] , camel [ ] [ ] [ ] , nonhuman primates [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and mouse [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] bat) (fig. ) [ ] . although mouse, hamster, ferret, and guinea pig dpp orthologs exhibit high overall similarity to hdpp , specific amino acid differences at the dpp /rbd interface account for the inability of these species to support infection [ ] [ ] [ ] [ ] [ ] [ ] . overexpression of a mouse dpp (mdpp ) with changes in the contact residues at the dpp /rbd altered cellular profiles from resistant to susceptible to mers-cov infection [ , , ] . the dependence on dpp -specific contact was further substantiated by similar studies evaluating modified dpp orthologs from the hamster, ferret, and guinea pig [ ] . dipeptidyl peptidase iv was identified as the major determinant of mers-cov tropism. researchers rapidly leveraged knowledge of the dpp receptor to generate susceptible small animal models ( fig. ) [ ] . zhao et al. utilized a unique approach for producing susceptible mice that could replicate human isolates of mers-cov in the lungs by infecting mouse lungs with an adenovirus that constitutively expresses the full-length hdpp gene ( fig. ) [ ] . transient expression of hdpp supported infection and replication with human strains of mers-cov in the lungs and indicated that this technology may be an effective rapid response platform for initial (c) zoomed-in view of the human dpp structure (light gray) with highlighted mers-cov rbd contact residues (dark gray). species-specific contact residues that differ from human are highlighted in red evaluation of emergent and pre-emergent viruses. however, pathology associated with a fatal mers-cov infection was not observed in the ad-hdpp model [ ] , which limited the capacity to evaluate the efficacy of therapeutic countermeasures. genetic engineering of mice would be necessary to develop preclinical mers-cov mouse models with respiratory phenotypes that reflected clinical outcomes in patients. knock-in of full-length hdpp rendered mice susceptible to human isolates of mers-cov at low infection doses ( fig. ) [ ] [ ] [ ] . knock-in mice exhibited severe pulmonary pathology and increased mortality; however, widespread constitutive expression of full-length hdpp resulted in high levels of mers-cov infection and replication in extrapulmonary tissues [ ] [ ] [ ] . in some studies, higher viral loads could be detected in the brain compared to the lungs [ , ] . mice with infections of the central nervous system (cns) exhibited encephalitis that corresponded with the kinetics of mortality [ ] . currently, there is no evidence to support a cns component associated with mers-cov pathogenesis in humans. attempts to restrict hdpp expression to epithelial cells of the lungs using constitutive tissue specific promoters (e.g., cytokeratin k ) yielded outcomes similar to those observed with sars-cov mouse models, wherein high levels of mers-cov infection/replication were detected in the brains (fig. ) [ ] . to circumvent confounding problems associated with global bio-distribution of overexpressed hdpp receptor, researchers engineered mouse models using sophisticated molecular approaches. pascal et al. employed regeneron's velocigene technology to replace sequences encoding nearly the entire mdpp genomic region with those encoding the exons/introns from the hdpp genetic region ( fig. ) [ ] . retaining the mdpp and genetic elements that regulate expression maintained inherent expression profiles of full-length hdpp in mice [ ] . importantly, mers-cov infection/replication was readily detected in the lungs with little involvement of extrapulmonary tissues [ ] . infection with human isolates of mers-cov caused moderate respiratory pathology with mortality determined by euthanasia of mice at % weight loss [ ] . unfortunately, commercial restrictions limit the availability and use of this model to the broader scientific community. in addition to the concerns raised above, the first generation of mouse models was developed with the full-length hdpp , which may alter the inherent physiological properties of the mouse. the multifaceted involvement of dpp in maintaining immune homeostasis is of significant importance regarding susceptibility to infectious disease [ ] . dpp exists in two forms: ( ) a membrane anchored form on the surface of multiple cells types (e.g., b cells, t cells, nk cells, and epithelial cells to mention a few) and ( ) a secreted form that can be identified in human serum [ ] . dpp interacts with and modifies heterologous protein molecules involved in nociception, neuroendocrine function, metabolism, cardiovascular function, immune regulation, and infection [ ] . modification of heterologous protein function can proceed through cleavage of n-terminal amino acids through the enzymatic activity of the α/b-hydroxylase domain, or allosteric interaction/ signal transduction [ ] . the species specificity of dpp is exemplified by the interaction of hdpp with adenosine deaminase (ada), a well-recognized binding partner of hdpp , which modulates downstream t cell functions [ ] [ ] [ ] . the hdpp /ada interaction evolved in higher mammalian species (human, nhp, bovine, rabbit), but not in mouse or rat [ ] [ ] [ ] . interestingly, in one study ada was demonstrated to block infection of mers-cov in tissue culture [ ] , indicating that the binding site on hdpp for ada, and the mers-cov rbd, may overlap. consequently, introducing full-length hdpp into mice may skew innate immune mechanisms that could influence responses to therapeutic countermeasures. in the second generation of mers-cov-susceptible mouse models amino acid residues predicted to function at the mdpp / mers-cov rbd interface were modified to avoid the introduction of full-length hdpp ( fig. ) [ , ] . li et al. recently developed a mouse model wherein the mdpp genomic region encompassing exons - were replaced with the respective genomic region from hdpp , referred to as an hdpp knock-in model (hdpp -ki) [ ]. exons - encode contact amino acids at the hdpp /mers-cov rbd interface that were able to support replication of human mers-cov isolates in the lungs, but did not elicit a mortality phenotype [ ] . adaptive evolution of human mers-cov in the hdpp -ki mouse resulted in mouseadapted viruses that evoked a lethal respiratory phenotype with little involvement of extrapulmonary tissues. the lethal respiratory phenotype is a consequence of novel mutations acquired during adaptive evolution. a combination of mutations in both the s and s regions of the mers-cov spike protein facilitated a lethal respiratory phenotype [ ] . results in the hdpp -ki model substantiate an earlier mouse model referred to as the - +/+ model, which was designed with only two amino acid changes in mdpp to generate mers-cov susceptible mice. genetic engineering and implementation of the - +/+ mouse model, combined with mers-cov adaptive evolution, is the subject of this chapter. initial studies in tissue culture revealed that human and rodent cell types were resistant to mers-cov infection upon overexpression of mdpp ; however, overexpression of hdpp conferred permissivity to infection/replication [ ] . comparative structural modeling of hdpp and mdpp revealed putative contact residues in mdpp amenable to modification at the dpp /rbd interface. modification of two amino acids (a l and t r) was sufficient to endow mdpp with the capacity to mediate mers-cov infection/replication [ ] . shortly after the emergence of mers-cov into humans in , the crispr/cas genome editing technology became available for applications to modify mammalian genomes in vitro and in vivo [ ] [ ] [ ] . recognizing our unique situation, we designed crispr/cas targets to modify the mouse genome encoding amino acids a and t in exons and of the mdpp gene (fig. ) [ , ] . concomitant with mouse development, in vitro studies were initiated to adapt mers-cov to the modified mdpp [ ] . tissue culture adaption resulted in mers- virus, which contained an rmr insertion and s l mutation in the s region of the mers-cov spike protein [ ] . a mers- molecular clone exhibited enhanced replication kinetics and higher titers compared to human mers-cov isolates. additionally, the mers- virus replicated to higher levels in the lungs of - +/+ mice, compared to human and camel mers-cov isolates [ ] . based on these data the mers- virus was used to initiate passaging in mice heterozygous for mdpp with a l and t r mutations, - +/À (fig. ) . we reasoned that adaptation around one expressed copy of the mdpp with - mutations, and a wild-type mdpp expressed copy, might cultivate generation of a mouse-adapted mers-cov that could utilize wild-type mdpp as the primary receptor. after passages we obtained a mouse-adapted mers-cov (mers c ) exhibiting a lethal respiratory phenotype in the - +/+ mice [ ] . our mers-cov reverse genetic system was used to generate an infectious clone of the mouse-adapted virus, icmersma [ ] . lethal respiratory pathology with icmersma required high infectious doses (  pfu). an additional passages of icmersma in - +/À mice bore a novel mouse-adapted mers-cov that produced lethal respiratory disease at doses of  pfu, and lung pathology associated with severe respiratory disease at  to  pfu [ ] (fig. ) . this mers-cov model system ( - +/+ mice and mouseadapted mers-cov viruses) is now being employed to: ( ) understand complex virus-host interactions [ , , , [ ] [ ] [ ] [ ] , ( ) evaluate antibody-based therapeutics [ ] , ( ) evaluate drug-based therapeutic countermeasures [ ] , and ( ) evaluate anti-mers-cov vaccines [ , ] . the goal of this chapter is to provide an outline of how to rationally design a mouse with altered susceptibility to mers-cov. for additional information there are a number of detailed reviews and book chapters describing the design and utilization of the crispr/cas technology for generating mouse models [ , ] . . agarose gel separation based on size allows for discrimination between target dna, cas digested targets, and guide rnas. (b) schematic utilizing crispr/cas technology to genetically engineer mice. fertilized c bl/ j zygotes are collected and injected with rna encoding cas , dpp single guide rna, and oligos to facilitate homology-directed repair (hdr). microinjected zygotes are implanted into pseudopregnant recipient female c bl/ j mice. offspring are screened by sequencing for the intended change at positions and . mice identified as having the appropriate changes are backcrossed to c bl/ j mice to maintain the pure c bl/ j background, or may be crossed to any desired strain (e.g., balb/cj or s /svimj). (c) table describing sequences of cas guide rnas and oligos for hdr to genetically engineer amino acid changes at position (ala to leu) and (thr to arg). (d) sequencing chromatograms highlighting how the f offspring from embryo implantation can be a mosaic of insertion/ deletions (indel's) generated by random non-homologous end joining from cas cutting at the genomic alleles, and the hdr repair that incorporates the intended changes encoding amino acids at positions and . pure homozygous - +/+ lines were obtained by backcrossing onto c bl/ j mice. the highlighted mutations caa (ttg in the reverse orientation) and aga encode the novel l and r amino acids . after a predetermined number of passages the region encoding the spike protein of mers-cov is sequenced using rt-pcr to amplify the region of interest followed by standard sanger sequencing (see note ). . after passages viruses were plaque purified by diluting the heterogenous stock of virus À to À , and infecting a monolayer of vero ccl cells similar to a standard plaque assay. . single plaques are isolated using a pipet tip and the virus expanded on a freshly seeded monolayer of vero ccl cells. . virus stocks are generated, viral rna is isolated using standard trizol purification, and the region encoding the mers-cov spike protein is amplified by standard rt-pcr techniques and sequenced using standard sanger sequencing. . mutations identified by sequencing must be confirmed using a reverse genetic system to generate an infectious clone encoding the identified mutations [ ] . cockrell (fig. ) the details for generating and using the crispr/cas system to generate mutations are outlined in the materials and methods by cockrell et al. [ ] . notably, the - +/+ mice were initially generated in the animal models core facility at the university of north carolina at chapel hill. the extensive technical expertise required for genetic engineering of mice is the subject of many expert reviews and book chapters that will not be covered here. nevertheless, we provide a conceptual overview of the steps to generate the - +/+ mice. . design guide rnas to target each of the a and t alleles. cockrell et al. designed the guide rnas to direct the cas to cut as near the mutation site as possible (fig. ) (see note for helpful resources to design and genetically engineer mouse knockouts). . test guide rnas in vitro for the capacity to cut a target sequence (fig. ) . (a) generate double-stranded odns or a plasmid containing the target sequence with the correct pam site. (b) assemble ribonucleoprotein (rnp) complexes according to manufacturer's instructions (see note ). (c) subject double-stranded dna with target sequence to rnp complexes and assess digestion pattern on an agarose gel (fig. ). . two separate oligos are also designed to introduce the novel mutations on exons (a l) and (t r) of mdpp , through homology-directed repair (fig. ). . fertilized zygotes are collected from c bl/ j female mice that were superovulated and mated to male c bl/ j mice. cas endonuclease, combined with odns encoding the replacement alleles for and in mdpp , were all pronuclear injected into the fertilized zygote [ ] (fig. ) . the fertilized zygotes were from c bl/ j mice. . the injected embryos were implanted into pseudopregnant recipient females. . newly born pups were screened for the presence of the correct change at the and alleles by standard pcr amplification and sanger sequencing (fig. ) (see note ). . - +/+ mice are brought into the bsl laboratory days before start of the experiment to allow for environmental acclimation. . mice are anesthetized via intraperitoneal injection of - μl of a ketamine ( mg/kg)/xylazine ( mg/kg) mixture (see note ). level of anesthesia is assessed by pedal reflex. . measure initial weight (day weight) while waiting for mouse to be anesthetized (see note ). . once a pedal reflex is no longer triggered, mice should be immediately infected by the intranasal route. holding the animal vertically, apply μl of virus solution by pipetting onto their nostrils and allow them to inhale. to ensure that all of the μl reach the lower respiratory tract hold the mouse upright for an additional s (see note ). . note any inconsistencies during infection, including: ( ) presence of bubbles of inoculum from nasal cavity, ( ) occurrence of inoculum in mouth, or ( ) failure to inhale entire dose of inoculum. notes will help to explain potential inconsistencies in readout parameters and may be used as exclusion criteria for inefficient infections. . mice are put back into the cage and placed on their back to ensure virus solution will stay in the lungs. note: cages are returned to cage rack, but the respiration of mice is continuously monitored by observing breathing. . place mice next to each other to keep body temperature as close to normal as possible. . check cage after - min to confirm that all mice wake up from anesthesia and infection. adaptation of mers- in - +/À mice (fig. ) . mouse adaptation was initiated in heterozygous - +/À mice by infecting with μl of the mers- infectious clone (see note ). . at days post-infection the mouse is euthanized by extended exposure to isoflurane.~ ml of isoflurane is added to the bottom of a jar that can be firmly sealed (see note ). a thoracotomy is performed to expose the lungs. . lungs are removed and placed in a ml gasket sealed skirted screw cap tubes. tubes are previously prepared with ml of  pbs containing~ - mm of glass beads. lungs are homogenized for s in a bead homogenizer. . lung lysates are centrifuged in a microcentrifuge for min at max speed to pellet debris. . this is considered passage (p ) and μl of lung homogenate is used to infect a naïve - +/À mouse. . the process is repeated for a desired number of cycles. . after infection mice are monitored daily for weight loss for the entire duration of the experiment. . to record daily weights, pick up mice by the tail, identify by ear notch, and place into cup on a scale. record weight and calculate percentage of starting body weight (see note ). . mice can also be monitored to determine if they are moribund using a clinical scoring scale whereby: ¼ no clinical signs; ¼ ruffled fur; ¼ ruffled fur with hunched posture (only slight with no signs of dehydration); ¼ as defined in number with more severe signs of dehydration and some loss of body strength; ¼ pronounced dehydration and prominent loss of mobility; ¼ unresponsive to stimuli and prominent eye squinting. . it is important to note that weight loss might not always be the most appropriate parameter and animals should be euthanized at the discretion of the researcher even if animals have not reached % of their starting weight. mice that approach % of their starting body weight ( % weight loss) are euthanized via isoflurane overdose followed by a secondary euthanasia method (thoracotomy or cervical dislocation). depending on the experimental circumstances an institutionally approved exception may be implemented to allow continuation of the experiment (increasing the frequency with which the mice are monitored will likely need to be implemented) (see note ). . respiratory function can be performed every day over the entire course of infection, or on single selected days. investigating a novel respiratory virus may require the investigator to perform a time course to determine the most effective time points for measurement. the largest differences between groups typically correlate with peak viral replication. . at each time point measured the mice need to be randomized into different chambers to avoid technical artifacts (e.g., a mouse measured at day in chamber should be evaluated in a different chamber on day measurement). practical considerations dictate that - animals can be measured at any one time, and each group of - mice may take an hour for a proficient technician. therefore, experiments should be carefully planned to limit the number of mice to be evaluated. . mice that are difficult to handle can be slightly anesthetized by applying isoflurane to the chamber in order to remove them from the chamber and return to their cage (see note ). . open thorax, paying attention not to cut into lung tissue. . assess lung tissue for reddish discoloration and record severity by applying a number (no hemorrhaging) to (severe hemorrhaging in all lobes of the lungs). . harvest lung tissue and place in tubes prefilled with sample type specific solution. . put scissors and forceps first into cidecon to remove any residual blood and then into % ethanol in order to not crosscontaminate samples. . whole lung can be used for one assay or different lobes can be used for different assays. steps - from subheading . . . cut into superior vena cava and collect blood with a pipette. . blood is typically transferred to a serum/plasma separation tube that allows for separation of serum/plasma from cells. . in the event that it is necessary to harvest cells for flow cytometry analysis or vetscan hm analysis, the blood sample can be transferred to a tube containing edta. note: vetscan hm is a veterinary diagnostic machine that analyzes basic immune cell counts and additional hematological parameters within min. . all vetscan assays are performed under bsl conditions. removal of samples for downstream analysis outside of bsl conditions require specific inactivation procedures that must be pre-approved by the institutional biosafety committee. . this can also be achieved using a readily transfectable human embryonic fibroblast cell line such as hek t cells and selecting for stably transfected cells. . cells can be counted by seeding an extra well, but it is safe to assume that the cell number is approximately doubled after h. . this can also be achieved using a microscope to assess plaque size if plaques can be readily detected. . since the major determinant of mers-cov tropism is the spike protein, it would be anticipated that mutations having the most significant impact on infection might occur within the gene encoding the spike protein. . at the time that these mice were being generated in early , crispr/cas reagents were not readily available. additionally, there were few bioinformatics tools available to facilitate guide rna design and off-target potential. in the current research environment crispr/cas reagents can be sourced from multiple commercial entities and there are a number of bioinformatics tools to assist with design. addgene is a nonprofit plasmid repository where crispr reagents and resources can be readily obtained (https://www.addgene. org/). additional guidance for generating mice using crispr/cas technology can be found in more comprehensive protocols [ , ] . . all relevant reagents and protocols can now be obtained from commercial sources as readily synthesized rnas and purified proteins (e.g., integrated dna technologies). . although a number of pups may be identified to have the correct mutation, many will likely be mosaic for random mutations including insertions/deletions due to the higher efficiency of non-homologous end joining (nhej) after cas digestion compared to the desired hdr employed to mediate allele modification. . the administered dose will depend on the weight of the animal which should be predetermined the day prior to initiating the experiment. . it is not necessary to anesthetize mice for measuring daily weights. . it is important that the inoculum reaches the lower respiratory tract for a successful mers-cov infection. . mouse adaptation can be initiated with any mers-cov strain that exhibits some pulmonary replication. . mice should never come into contact with the isoflurane. to prevent direct contact, a layer of aluminum foil is placed at the bottom of the jar and this is covered with two additional layers of paper towel. . the percent body weight is typically calculated after leaving the bsl environment. therefore, the weight sheets should have the anticipated weights of each animal at % weight loss. this will provide a real-time indication of when the mice are approaching the criteria established for humane euthanasia. . institutional approval is required for animals to be placed under exception. it cannot be emphasized enough that all animal work should be pre-approved by appropriate university iacuc and ibc committees and should be in accordance with the recommendations for the care and use of animals by the office of laboratory animal welfare at nih. . assessing respiratory function using plethysmography under bsl conditions requires costly equipment and extensive training prior to use. . anesthesia should be avoided prior to measuring lung function to prevent interference with lung function measurements. the who r&d blueprint: review of emerging infectious diseases requiring urgent research and development efforts comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from economic impact of the mers outbreak on the republic of korea's tourism-related industries costly lessons from the middle east respiratory syndrome coronavirus outbreak in korea middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control mers transmission and risk factors: a systematic review high prevalence of mers-cov infection in camel workers in saudi arabia reported direct and indirect contact with dromedary camels among 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middle east respiratory syndrome coronavirus infection in rabbits enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection lack of middle east respiratory syndrome coronavirus transmission in rabbits bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (mers-cov) after experimental infection replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels pneumonia from human coronavirus in a macaque model treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset infection with mers-cov causes lethal pneumonia in the common 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organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease elevated human dipeptidyl peptidase expression reduces the susceptibility of hdpp transgenic mice to middle east respiratory syndrome coronavirus infection and disease cd + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a human dpp -knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov acute respiratory infection in human dipeptidyl peptidase -transgenic mice infected with middle east respiratory syndrome coronavirus haagmans bl ( ) dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptorbinding domain complexed with human receptor dpp coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection mouse dipeptidyl peptidase is not a functional receptor for middle east respiratory syndrome coronavirus infection glycosylation of mouse dpp plays a role in inhibiting middle east respiratory syndrome coronavirus infection permissivity of dipeptidyl peptidase orthologs to middle east respiratory syndrome coronavirus is governed by glycosylation and other complex determinants host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase cut to the chase: a review of cd /dipeptidyl peptidase- 's (dpp ) entanglement in the immune system direct association of adenosine deaminase with a t cell activation antigen revisiting an old acquaintance: cd and its molecular mechanisms in t cell function crystal structure of cd / dipeptidyl-peptidase iv in complex with adenosine deaminase reveals a highly amphiphilic interface multiplex genome engineering using crispr/cas systems rna-guided human genome engineering via cas one-step generation of mice carrying reporter and conditional alleles by crispr/cas-mediated genome engineering the human sodium iodide symporter as a reporter gene for studying middle east respiratory syndrome coronavirus pathogenesis giving the genes a shuffle: using natural variation to understand host genetic contributions to viral infections middle east respiratory syndrome coronavirus nonstructural protein is necessary for interferon resistance and viral pathogenesis mers-cov accessory orfs play key role for infection and pathogenesis broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses generating genetically modified mice: a decision guide genome editing in mouse embryos with crispr/cas reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses new metrics for evaluating viral respiratory pathogenesis key: cord- - y j j authors: adney, danielle r.; bielefeldt-ohmann, helle; hartwig, airn e.; bowen, richard a. title: infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: y j j middle east respiratory syndrome coronavirus is a recently emerged pathogen associated with severe human disease. zoonotic spillover from camels appears to play a major role in transmission. because of logistic difficulties in working with dromedaries in containment, a more manageable animal model would be desirable. we report shedding and transmission of this virus in experimentally infected alpacas (n = ) or those infected by contact (n = ). infectious virus was detected in all infected animals and in of in-contact animals. all alpacas seroconverted and were rechallenged days after the original infection. experimentally infected animals were protected against reinfection, and those infected by contact were partially protected. necropsy specimens from immunologically naive animals (n = ) obtained on day postinfection showed virus in the upper respiratory tract. these data demonstrate efficient virus replication and animal-to-animal transmission and indicate that alpacas might be useful surrogates for camels in laboratory studies. middle east respiratory syndrome coronavirus is a recently emerged pathogen associated with severe human disease. zoonotic spillover from camels appears to play a major role in transmission. because of logistic difficulties in working with dromedaries in containment, a more manageable animal model would be desirable. we report shedding and transmission of this virus in experimentally infected alpacas (n = ) or those infected by contact (n = ). infectious virus was detected in all infected animals and in of in-contact animals. all alpacas seroconverted and were rechallenged days after the original infection. experimentally infected animals were protected against reinfection, and those infected by contact were partially protected. necropsy specimens from immunologically naive animals (n = ) obtained on day postinfection showed virus in the upper respiratory tract. these data demonstrate efficient virus replication and animal-to-animal transmission and indicate that alpacas might be useful surrogates for camels in laboratory studies. cov) was first detected in samples from a man in saudi arabia who had severe respiratory disease in ( ) . since its identification, > , cases of infection have been documented, and the case-fatality rate is ≈ % ( ) . although efficient human-to-human transmission has been documented, zoonotic spillover probably plays a major role in human infection ( ) ( ) ( ) ( ) ( ) . dromedary camels were identified early after recognition of the virus as a possible reservoir host for the disease, although not all patients report contact with camels. numerous investigators have reported the presence of mers-cov rna or infectious virus in nasal swab specimens of dromedary camels in saudi arabia ( , , ( ) ( ) ( ) , qatar ( , ( ) ( ) ( ) , oman ( ) , the united arab emirates ( ), nigeria ( ) , and egypt ( ) . in some areas of the middle east and africa, nearly % of animals tested were serologically positive for mers-cov, which suggested widespread circulation among camel populations ( , , ) . historical samples contained specific antibodies against mers-cov as long ago as , which indicated that mers-cov has been circulating much longer than originally believed ( , ) . young animals appear to be at a greater risk for productive infection, and handling practices, such as weaning or shipping animals, might play a major role in animal-to-animal transmission. many dromedary camels tested had high antibody titers. these results support field data suggesting that young animals become infected, and their immune responses probably are repeatedly boosted by subsequent exposure to the virus ( ) . however, it is currently unknown whether these repeated exposures result in productive infection or whether antibodies generated from a previous infection are protective. we have previously demonstrated that dromedary camels can be experimentally infected with mers-cov and found that mild upper respiratory tract disease associated with shedding copious amounts of virus by nasal secretions develops during the first week after infection ( ) . however, because of the cost of dromedaries, their size, and the requirement for specialized facilities to conduct such studies, it would be useful to identify alternative animal models that respond similarly to infection with mers-cov. we report characterization of an alpaca model of mers-cov infection in which we evaluated virus shedding and pathology, transmission by contact, and protective immunity weeks after initial infection. results indicate that alpacas might be a useful substitute for dromedary camels in certain types of mers-cov experiments. animal experiments were approved by the animal care and use committee of colorado state university. every effort was made to minimize stress and pain of the animals. animals were infected with a low-passage human isolate of mers-cov (strain hcov-emc/ ). this strain was propagated in vero e cells cultured in dulbecco modified eagle medium as described ( ) . nine locally bred alpacas were obtained by private sale for use in this study. animals were allowed to acclimate to the facility for week before infection and were fed hay ad libitum. one day before infection, animals were subcutaneously injected with an identification and temperaturesensing transponder (lifechip; destron fearing, dallas/ fort worth airport, tx, usa), and their body temperatures were monitored throughout the study. alpacas a -a were housed together and experimentally infected by intranasal instillation of pfu of mers-cov diluted in sterile phosphate-buffered saline ( ml/nare). two days later, alpacas a -a were introduced into the same room as alpacas a -a and housed together for the duration of the study. nasal swab specimens were collected by inserting and rotating sterile swabs into both nares, immediately placed in virus transport medium, and frozen until assay. blood was collected weekly into serum-separating tubes for detection of neutralizing antibodies. animals a -a were held in the facility for days postinfection, and all animals were then reinfected intranasally with pfu of mers-cov. three additional alpacas (a -a ) were also infected to serve as infection controls and evaluate tissue distribution of virus replication. nasal swab specimens were collected daily from all animals for days, at which time animals a -a were humanely euthanized. tissues collected at necropsy for detection of infectious virus from these animals included nasal turbinates, trachea, larynx, and all lung lobes. these samples plus additional samples, including brain, kidney, liver, skeletal muscle, heart, spleen, bladder, mesenteric lymph node, submandibular lymph node, and mediastinal lymph node, were fixed in formalin for histopathologic and immunohistochemical analysis. nasal swab specimens and serum samples collected from alpacas a -a were sampled for weeks after the second infection, and then these animals were then humanely euthanized. tissues were fixed in % neutral-buffered formalin for > days and embedded in paraffin. tissue sections were stained with hematoxylin and eosin and evaluated by a veterinary pathologist (h.b.-o.). immunohistochemical analysis was preformed to detect mers-cov antigen by using a rabbit polyclonal antiserum against hcov-emc/ antigen (diluted : , ) as a primary antibody as described ( ). mers-cov was titrated from nasal swab specimens in virus transport medium and homogenized tissue by plaque assay as described for camels ( ) . a -ml volume of virus transport medium was considered a - dilution, and -fold serial dilutions were prepared in ba medium. neutralizing antibodies were detected by plaque reduction neutralization test (prnt) as described, and seropositive animals were identified by using a % neutralization cutoff ( ) . field studies and experimental infections suggest that mild respiratory disease associated with nasal discharge develops in mers-cov-infected camels ( , , ) . similar to dromedaries, none of the alpacas had any appreciable increase in body temperature during challenge or rechallenge ( figure ). unlike dromedary camels, none of the alpacas emerging infectious diseases • www.cdc.gov/eid • vol. , no. , june had any observable nasal discharge over the course of infection. all alpacas maintained consistent activity level, temperament, and food intake throughout the study. nasal swab specimens were collected from infected animals immediately before challenge, on days - postinfection, and on day postinfection. all experimentally infected animals (a -a ) had detectable infectious virus on day postinfection but had stopped shedding virus by day postinfection (figure , panels a, b) . the co-housed animals (a -a ) were placed in the room with the infected animals days after initial virus infection. nasal swab specimens were collected from co-housed animals on days - after infection of animals a -a , and then times/ week through day . infectious virus was detected from animal a during days - and from animal a only on day . we did not isolate infectious virus from animal a (figure , panel a). although infectious virus was detected in animal a on day , infectious virus was not detected in animal a until day (figure , panel a) . we speculate that animal a became infected by contact with animal a after animals a -a had cleared their infections, which suggested that transmission is linked to intimate animal contact, rather than to aerosol transmission. to test whether previous infection was protective against subsequent virus challenge, all original study animals (a -a ) were allowed to clear their infections and rechallenged by intranasal infection on day postinfection. challenge was also performed with immunologically naïve alpacas (a -a ) (infection controls). the immunologically naive animals became infected and shed virus during days - postinfection, at which time they were euthanized. the animals that became infected through contact (a -a ) shed minimal virus between days - after rechallenge, but not on days - . in contrast, animals that had been experimentally infected were completely protected against rechallenge and did not shed detectable quantities of virus ( figure , panels c, d). serum was collected weekly and tested for neutralizing antibodies against mers-cov. all experimentally infected animals (a -a ) had detectable levels of antibodies beginning on day (table) . although infectious virus was isolated only from of the co-housed animals, these animals had neutralizing antibodies detected first on day (animals a and a ) or day (animal a ) (table) . nasal turbinate, upper trachea, lower trachea, larynx, and all lung lobes were sampled at necropsy from alpacas a , a , and a and tested for infectious virus by using a plaque assay. virus was detected in the nasal turbinates, larynx, and trachea of the alpacas but not in any of the lung lobes tested (figure ). gross lesions were not observed at necropsy in any of the alpacas. however, microscopic analysis of formaldehydefixed tissue sections from animals a -a showed mild squamous metaplasia of the epithelium of the turbinates in animal a (figure , panel a) and rare foci of mucosal erosion accompanied by minimal-to-mild subepithelial infiltration of neutrophils and macrophages and fewer lymphocytes ( figure , panel c). all animals also had follicular hypertrophy and hyperplasia of the draining lymph nodes, which suggested immune activation. immunohistochemical analysis detected rare, scattered, virus antigen-positive cells in respiratory epithelium of turbinates (figure , panel b ) and in rare cells interpreted to be intraepithelial leukocytes. virus antigen was not detected in any of the other tissues examined. animals a and a had histopathologic evidence of mild encephalitis with perivascular infiltrates of lymphocytes and monocytes and mild gliosis (figure , panel d) . we did not assay brain tissue for virus, either by isolation or pcr, because of the high potential of contamination from the nasal cavity during extraction. brain tissue was negative for virus by immunohistochemical analysis, but the etiology of the encephalitis observed remains unknown and might have been unrelated to mers-cov infection. many difficulties are associated with high containment experiments involving dromedary camels. thus, additional animal models are necessary for mers-cov research. because of their greater availability in the united states and smaller size, we tested an alpaca model. we report an alpaca model of mers-cov infection in camelids and analysis of animal-to-animal transmission and reinfection dynamics. infected alpacas shed considerable quantities of infectious virus nasally, although at lower concentrations than those reported for dromedary camels ( , ) . in addition, none of the infected alpacas had a noticeable nasal discharge, which is distinctly different from what has been observed in camels and might explain the relatively low efficiency of contact transmission we observed with alpacas. , antibody titer a a a a a a < < < < < < < < < < *alpacas a -a were experimentally infected, and alpacas a -a were co-housed with infected alpacas. titers were determined by using a % cutoff. infectious virus was detected in nasal swab specimens from of alpacas co-housed with experimentally infected animals, and each of the co-housed animals had neutralizing antibodies against mers-cov, which indicated virus transmission. the antibody titers observed approximate those seen for infected dromedaries with the exception of a , whose antibodies titers remained low until after rechallenge ( ) . finally, experimentally infected alpacas were completely protected against subsequent virus rechallenge, and contact-infected alpaca were only partially protected. these results suggest that infection can easily spread among closely grouped camelids infected with mers-cov. camels are frequently moved within the middle east for grazing, camel shows, and races. such movement enables mixing and close mingling of animals and could play a major role in mers-cov transmission among animals and to handlers. khalafalla et al. reported that animals bound for slaughter were held in a livestock market for several days, transferred to an abattoir, and kept for up to hours before slaughter ( ) . our data suggest that these handling practices could promote animal-to-animal virus transmission and that at the time of slaughter virus could potentially be transmitted to slaughterhouse workers. a major question related to the pathogenesis of mers-cov infection in camels, and of great relevance to vaccination strategies, is whether animals that have been infected are resistant to reinfection and virus shedding and, if so, for how long. our experimentally infected animals were completely protected against rechallenge days later, which suggests that sterilizing immunity can be achieved. however, the animals that were infected through contact (animals a -a ) shed infectious virus after reinfection, albeit at much lower levels than infected control animals (animals a -a ). although not tested in the present study, it might be surmised that the in-contact animals would have acquired sterilizing immunity from the second (booster) infection. these results support field data that suggest that young animals become infected and probably receive booster infections; most older animals have acquired immunity and are not susceptible to infection and virus shedding ( ) . this finding also highlights the possibility that widespread vaccination of dromedary camels could result in a major decrease in virus transmission to humans. to date, neutralizing antibodies against mers-cov have not been detected in camelids outside africa or the middle east. however, if virus were to be introduced into immunologically naive camelid populations, it probably would be readily transmitted among animals. many new world camelids are valued for their fiber, and such transmission might devastate fiber-related industries ( ) . thus, as travel-associated cases of mers-cov infection continue to be documented, human-to-human virus transmission and possible human-to-animal virus transmission should be monitored. this study had several limitations. each of the experimental groups had only animals, which limited our ability to perform statistical analyses. in addition, we evaluated protective immunity weeks after the original infection, which is a relatively short period and does not fully recapitulate seasonal exposures. thus, further studies are necessary to better understand duration of immunity in camels and alpacas. note added in proof: crameri et al. also report experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus in this issue of emerging infectious diseases ( ) . isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) evidence for camel-to-human transmission of mers coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates occupational exposure to dromedaries and risk for mers-cov infection mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through wholegenome analysis and virus cultured from dromedary camels in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels isolation of mers coronavirus from a dromedary camel high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels antibodies against mers coronavirus in dromedary camels antibodies against mers coronavirus in dromedary camels mers coronavirus neutralizing antibodies in camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels infection with mers-cov causes lethal pneumonia in the common marmoset dynamics of passive immunity to west nile virus in domestic chickens an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels mers-cov in upper respiratory tract and lungs of dromedary camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries medicine and surgery of camelids experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus we thank erasmus medical center (rotterdam, the netherlands) for proving virus isolate hcov-emc/ and vincent munster and the rocky mountain laboratories for providing the rabbit antiserum against mers-cov. key: cord- -g n n authors: khudhair, ahmed; killerby, marie e.; al mulla, mariam; abou elkheir, kheir; ternanni, wassim; bandar, zyad; weber, stefan; khoury, mary; donnelly, george; al muhairi, salama; khalafalla, abdelmalik i.; trivedi, suvang; tamin, azaibi; thornburg, natalie j.; watson, john t.; gerber, susan i.; al hosani, farida; hall, aron j. title: risk factors for mers-cov seropositivity among animal market and slaughterhouse workers, abu dhabi, united arab emirates, – date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: g n n camel contact is a recognized risk factor for middle east respiratory syndrome coronavirus (mers-cov) infection. because specific camel exposures associated with mers-cov seropositivity are not fully understood, we investigated worker–camel interactions and mers-cov seroprevalence. we assessed worker seroprevalence in slaughterhouses and live-animal market in abu dhabi, united arab emirates, during – and administered an epidemiologic survey in and . across sampling rounds during – , we sampled – workers, and %– % were seropositive for mers-cov at each sampling round. one ( . %) of seronegative workers tested at multiple rounds seroconverted. on multivariable analyses, working as a camel salesman, handling live camels or their waste, and having diabetes were associated with seropositivity among all workers, whereas handling live camels and either administering medications or cleaning equipment was associated with seropositivity among market workers. characterization of high-risk exposures is critical for implementation of preventive measures. m iddle east respiratory syndrome (mers) coronavirus (mers-cov) was first identified as a cause of severe respiratory tract infections in saudi arabia in october ( ) . the clinical spectrum of mers ranges from asymptomatic infection to acute respiratory distress syndrome and death ( ) . as of april , , a total of , laboratory-confirmed cases of infection have been reported by countries to the world health organization (who); the reported case-fatality rate is % ( ) . all reported cases have an epidemiologic link to the arabian peninsula, and imported cases have been reported in europe, asia, north america, and africa. the united arab emirates has reported the third-highest number of mers cases since ( ) . mers-cov is a zoonotic virus, and dromedaries (camels) are recognized as a major virus reservoir for spillover to humans ( ) . multiple studies have isolated mers-cov or mers-cov rna from camels across the arabian peninsula and africa ( ) ( ) ( ) ( ) ( ) ( ) ( ) . serologic studies of camels in the middle east and africa have revealed mers-cov seroprevalence of > %- % ( , ( ) ( ) ( ) . in natural infection, camels have been found to shed mers-cov in respiratory secretions and to a lesser extent in stool ( , ) . evidence of virus rna has also been found in milk collected by traditional milking procedures, which involve calf suckling as a stimulus for milk letdown ( ) . epidemiologic links between infected camels and human mers-cov infections have been shown, with identical or nearly identical mers-cov genomes found in human cases and in camels with which they had direct contact ( ) ( ) ( ) . also, a case-control study identified exposure to camels as a risk factor for human mers-cov infection ( ) . human seroprevalence studies also support the association between mers-cov infection and camel contact; in saudi arabia mers-cov seroprevalence was found to be times greater in camel shepherds and times greater in slaughterhouse workers compared with the general population ( ) . further studies have also shown high seroprevalence in specific occupational groups with various camel exposures (e.g., seropositivity was detected in . % of a cohort of camel workers in qatar [ ] and in % of a cohort of camel workers in saudi arabia [ ] ). although multiple lines of evidence suggest camel exposure is associated with human mers-cov infection, the exact mechanisms of transmission are not fully understood. information on specific risk factors relating to camel interactions are needed to further understand how the virus might be transmitted from camels to humans and to guide interventions to prevent zoonotic transmission, including changes to camel management practices. because mers-cov vaccines are currently in development and have reported success in phase i clinical trials ( ), knowledge of groups at risk for mers-cov infection might also be useful when considering future vaccine use. our study aimed to identify risk factors for mers-cov seropositivity among live-animal market and slaughterhouse workers. the study sites consisted of an open-air animal market and slaughterhouses ( commercial and public). all facilities housed camels, goats, sheep, and cattle ( figure ). typically during the study period, approximately persons worked at the market, at the public slaughterhouse, and at the commercial slaughterhouse. the market investigated in this study was linked to a human mers case in ( ) . prior investigation showed a large diversity of mers-covs circulating among camels at the market; ( %) of screened camels had detectable mers-cov rna in nasal swab specimens in the spring of ( ) . we conducted rounds of worker serum sampling. the first round was conducted during may - , , and the second round during march -april and may - , . during the first rounds of sampling, all available workers at the market and public slaughterhouse were requested to provide a serum sample as part of a public health investigation. we conducted a third round of serum sampling during september -october , , and march - , . the third round of sampling included workers at the market, public slaughterhouse, and the newly opened commercial slaughterhouse. all available workers were requested to provide serum samples, although participation was voluntary. some, but not all, workers were repeatedly sampled, when feasible, during multiple rounds. we administered an epidemiologic survey to all workers only during the third round of serum sampling in and . no surveys were administered in or . the survey consisted of questions covering worker demographics; occupational history; contact with various animal species; travel history; medical history; consumption of raw camel milk, raw camel meat, and camel urine; specific tasks performed with camels; types of personal protective equipment (ppe) worn; and handwashing practices (appendix , https://wwwnc.cdc.gov/eid/article/ / / - -app .pdf). separate lists of questions covering specific camel tasks performed were asked of market and slaughterhouse workers because of the different nature of camel tasks among occupational groups. interviews were conducted in arabic by staff from the abu dhabi department of health. human serum samples were tested for mers-cov antibodies at the us centers for disease control and prevention (cdc) by using indirect elisas for nucleocapsid (n) and spike (s) proteins, followed by a confirmatory microneutralization test, as previously described ( ) . samples were initially tested by using both n and s elisas as screening assays with serum diluted to : . all serum samples with optical densities above assay cutoff were diluted serially, -fold, from : to : , , and used for endpoint titer determinations. serum samples that were positive by n or s elisa with titers at : , : , or : , , plus % of samples negative by n or s elisa at these titers, were tested by using microneutralization with live mers-cov performed in a biosafety level laboratory, as previously described ( ) . in addition, we conducted confirmatory microneutralization tests on seronegative samples from any persons who showed a change in seropositivity status over time to confirm changes in seropositivity status. samples were considered positive if positive on n and s elisa or if positive on microneutralization. specimens near the limits of detection but not consistently above or below these limits were considered indeterminate. for the epidemiologic analysis, persons with an indeterminate result were considered seronegative. we used epi info (https://www.cdc.gov/epiinfo) for data entry and r version . . (https://cran.r-project.org/ bin/windows/base/old/ . . ) for data analysis. we performed comparisons between prevalence of work practices by setting (market vs. slaughterhouse) by using the pearson χ square test. we used univariable logistic regression to estimate odds ratios, % cis, and p values (wald test) for all associations between potential risk factors and seropositivity. we assessed associations between demographics, occupational history, contact with various animal species, consumption of camel products, travel history, and medical history with seropositivity for all workers. we separately tested associations between specific interactions with camels, types of ppe worn, and handwashing practices with seropositivity for stratified subgroups of market and slaughterhouse workers because of the different nature of work setting and standard practices between these populations. we then performed additional exploratory data description by occupation on the basis of results of univariable analyses. we developed multivariable logistic models to identify associations between risk factors and seropositivity. first, we constructed a model of risk factors common to all workers and then constructed occupationally stratified models (i.e., separate models for market workers and slaughterhouse workers) to model specific interactions with camels, ppe use, and handwashing practices. we combined or eliminated highly correlated variables, which were determined by condition indices and variance decomposition proportions. we reduced categorical variables to binary options if small group size was observed. we performed initial variable selection by using least absolute shrinkage and selection operator (lasso) and then tested person-variable significance by using the likelihood ratio test with a cutoff of p< . within an ordinary logistic regression model. we then included age and number of years worked at current setting as potential confounders in all final models. we excluded persons with missing data at the lasso stage but included them for the final logistic regression model. for the stratified market worker and slaughterhouse models, we also included variables significant in the all workers model but not directly relating to camel interactions (e.g., reported underlying conditions) in the final occupationally stratified models. we did not include significant variables directly relating to camel exposures in the all workers model in the stratified models because more specific camel risk practices were assessed in the stratified models. for market and slaughterhouse models, we tested interactions between significant risk practices and select ppe use and handwashing practices for a protective effect. we sampled workers in round ( ), workers in round ( ), and workers in round ( and ); overall mers-cov seroprevalence was % for round , % for round , and % for round . twenty-one persons had specimens taken at rounds and , twenty-three at rounds and , thirteen at rounds and , and twenty-two at all rounds ( figure ). of persons who were seronegative at their first sample, only ( . %, % ci . %- . %) seroconverted: a -year-old man who was a cleaner at the public slaughterhouse tested negative at round and positive at round . of persons who were seropositive at their first sample, ( %) was later found to be seronegative: a -year old man who was an administrative supervisor at the market was resampled between rounds and . this person did not report handling camels or their waste and did not perform any tasks directly relating to camels. one additional person who had a positive serologic result at their first and second samples and an indeterminate result at their third sample was not subsequently evaluated for change in seropositive status. because some study participants might have had different medical record numbers across the sampling rounds, we could not determine all potential seroconversions or losses of seropositivity, although we also performed matching by name and age. we compiled serologic results for all participants who ever tested positive (appendix , https://wwwnc.cdc.gov/eid/ article/ / / - -app .pdf). in total, persons both completed the epidemiologic survey and were sampled during round . one additional person completed the epidemiologic survey but refused serum sampling and was not included in any analyses. all workers were men, and their median age was years (range - years). the median number of years worked at the current settings was (range . - years). we observed no significant effect of age (p = . ) or years worked (p = . ) on seropositivity on univariable analysis. worker occupations were categorized into animal handlers (n = ), camel salesmen (n = ), other animal salesmen (n = ), animal or waste transporters (n = ), butchers (n = ), cleaners (n = ), veterinarians (n = ), and other (e.g., supervisor, cashier, and tourist guide) (n = ). salesmen only worked in the market, and butchers only worked in the slaughterhouses. the remaining occupations were found in both settings, but each person could only work at a slaughterhouse or the market. none of the workers reported working at any other job outside of the market or slaughterhouses, and the only animals reported present at home were poultry and stray cats. overall, ( %) of market workers had daily contact with camels or their waste, compared with ( %) of slaughterhouse workers (p = . ). certain ppe use and handwashing were more frequently reported by slaughterhouse workers than market workers. among slaughterhouse workers, % reported wearing a dust mask (equivalent to a surgical mask), compared with % of market workers (p< . ). only % of slaughterhouse workers reported taking their work clothes home, compared with % of market workers (p< . ). eighty-one percent of slaughterhouse workers reported washing their hands before and after each animal-related task, compared with % of market workers (p< . ). ninety-three percent of slaughterhouse workers reported washing their hands at the beginning and end of the day, compared with only % of market workers (p< . ). rates of seropositivity were higher among market workers ( [ %] of ) than among slaughterhouse workers ( [ %] of ), although this difference was not statistically significant on univariable analysis (p = . ). by occupation, camel salesmen and animal or waste transporters had significantly higher odds of seropositivity than the reference group of other salesmen (table ) . univariable analyses showed that several characteristics were associated with seropositivity among all workers (table ) , including handling camels or their waste daily. not all seropositive workers reported handling camels or their waste; workers initially claimed they never handled camels or their waste, although of these later reported that they contacted either camel equipment, viscera, or waste within the slaughterhouse. for the subgroup of market workers, univariable analyses revealed multiple camel exposures to be associated with seropositivity and handwashing practices that were inversely associated with seropositivity (table ). for the subgroup of slaughterhouse workers, no individual risk factors were associated with seropositivity (table ) . because camel salesmen had the highest odds of mers-cov seropositivity, we summarized their frequency of specific camel exposures separately (figure ). direct observation of camel salesmen in the market showed that most of their time was spent in the camel pens, including while they ate and rested, and direct handling of the animals occurred frequently (data not shown). for the multivariable model evaluating risk factors associated with seropositivity in all workers, the following variables remained in the final logistic regression model: handling camels or their waste daily (adjusted odds ratio [aor] . , % ci . - . ), working as a camel salesman (aor . , % ci . - . ), and self-reported diabetes (aor . , % ci . - . ). all factors significantly increased odds of seropositivity. for market workers, multivariable analysis resulted in a final model in which the following variables were each independently associated with seropositivity: handling live camels (aor . , % ci . - . ), administering medications to camels (aor . , % ci . - . ), and self-reported diabetes (aor . , % ci . - . ). cleaning equipment was also significantly associated with seropositivity (aor . , % ci . - . ); substituted for administering medication to camels, this factor produced a model with a near-identical fit along with the other risk factors. given that administering medications to camels was highly correlated with cleaning equipment, the statistical significance of both factors was lost if both factors were included in the model because of collinearity (ρ = . ). none of the select ppe and handwashing practices evaluated as interactions with risk practices showed a significant protective effect. no individual risk factors were significantly associated with slaughterhouse workers by multivariable analysis. our study investigated risk factors for mers-cov seropositivity in animal market and slaughterhouse workers at a site previously associated with zoonotic transmission of mers-cov. given the large number of camels present, including many young camels, and the mixing of camels from multiple sources, this site probably facilitates mers-cov transmission among camels. our results demonstrated a relatively high mers-cov seroprevalence in workers at this site, ranging from % to % at each round across all occupations. because we did not record occupation and other risk factors during the first sampling rounds, we were unable to further assess reasons for the different seropositivity rates between sampling rounds. we found particularly high seroprevalence in specific occupational groups, namely camel salesmen ( %) and animal or waste transporters ( %). previous studies of workers with occupational exposure to camels have reported either lower seropositivity rates (e.g., . % of workers with occupational camel contact seropositive in qatar [ ] and . % of camel shepherds seropositive in saudi arabia [ ] ) or comparable seropositivity (e.g., % of camel workers positive in saudi arabia [ ] ). our rates of seropositivity might underestimate actual exposure to mers-cov. previous studies have demonstrated that examining mers-covspecific t cells from mers patients is more sensitive than examining serum antibodies alone ( ) . to examine t-cell responses, peripheral blood mononuclear cells must be collected, which was beyond the scope of our study. on multivariable analysis, we found that contact with camels or their waste, working as a camel salesman, and self-reported diabetes were all independently associated with seropositivity in all workers. because of small stratum size, belonging to other occupational groups could not be meaningfully explored as risk factors. diabetes has previously been shown to be a commonly reported underlying condition in mers cases ( ) , has been associated with risk for infection in a case-control study ( ) , and has been associated with increased risk for death in mers patients ( ) . we found an association between diabetes and mers-cov seropositivity in a cohort with occupational exposure to camels. although persons with diabetes might be at increased risk for mers-cov infection, the association between diabetes, mers-cov infection, and the resulting antibody response is still not fully understood. however, because persons with diabetes are considered at high risk for developing severe disease from mers-cov infection, who recommends these persons take precautions when visiting farms or markets where camels are present, including avoiding contact with camels ( ). among market workers, handling live camels and either administering medications to camels or cleaning equipment were practices associated with significantly increased risk for mers-cov seropositivity. given that administering medications to camels was highly correlated with cleaning equipment, neither factor was statistically significant if both were included in the model. the biological importance of these associations might therefore be difficult to interpret, because either or both risk factors could be statistically associated with mers-cov seropositivity and have an undefined strength of association. practices potentially associated with camel calves, such as milking or assisting with camel birth, were not associated with mers-cov seropositivity despite a higher prevalence of viral rna in camels < year of age compared with other ages ( ) and a previously reported association between milking camels frequently and seropositivity ( ) . however, these practices were not commonly reported by market workers in our study, limiting the power to detect an association with seropositivity. no specific work practices were found to be associated with seropositivity among slaughterhouse workers. compared with market workers, slaughterhouse workers had less exposure to live camels and a higher self-reported prevalence of potentially protective practices such as ppe use and frequent handwashing. although our multivariable analysis did not show a significant association between ppe use (e.g., wearing a dust mask and gloves) or handwashing practices and seropositivity, the small sample size might have restricted the power to detect interactions between ppe and camel exposures. because camel-to-human transmission of mers-cov is not fully understood, who recommends broad preventive measures for slaughterhouse and market workers, including wearing facial protection when feasible, washing hands before and after each animal-related task, and washing soiled work clothes and shoes at the work place to avoid exposing family members to soiled work clothing ( ) . where feasible, increased use of such measures could be encouraged, particularly in market workers, to decrease risk for infection. because only a single human mers case has been reported in connection with the study site, our reported rates of seroprevalence suggest unrecognized transmission (and potentially unrecognized illness) at this site. however, because the length of time mers-cov antibodies persist is unknown ( ), the time and place these infections might have occurred is unknown; transmission potential also exists in the united arab emirates outside of markets and slaughterhouses. whether infections were symptomatic is also unknown. participants were asked whether they had seen a healthcare provider for respiratory illness in the previous months, but such reported illness was not associated with seropositivity, and multiple pathogens other than mers-cov could be responsible for any reported respiratory illness. despite these limitations, mers-cov was detected in camels at the market during our study period ( ) , and an interim seroconversion was noted in worker, suggesting active zoonotic transmission. taken collectively, our findings suggest an underestimated prevalence of human mers-cov infection in settings where the virus is circulating among camels, probably resulting from camel-to-human transmission. our study had additional limitations, including the overall sample size and limited number of subjects within specific substrata. concentration of camel interactions within particular occupational groups limited our ability to differentiate risk among specific camel interactions, despite our use of multivariable analysis. furthermore, because most persons reported interactions either daily or never, determining whether increased risk was associated with increased frequency of individual tasks was not possible. also, some mers-cov infections might not result in detectable antibodies, particularly when the infections are asymptomatic or mild ( ) . persistence of detectable mers-cov antibodies after infection is not well-defined, limiting the ability of serologic testing to define previous infection. finally, because of incomplete linkage of study participants by medical record numbers across the sampling periods, not all potential seroconversions or losses of seropositivity could be determined. in summary, our study found significantly increased odds of mers-cov seropositivity in persons with exposure to camels, in particular among those who handle live camels. odds of seropositivity were also significantly higher for camel salesmen, suggesting that preventive measures such as ppe use could focus on specific occupational groups, in addition to individual work practices. determining groups at highest risk for zoonotic mers-cov infection could also inform future vaccine trials in geographic regions where mers-cov is known to circulate. middle east respiratory syndrome coronavirus (mers-cov) is a novel cov known to cause severe acute respiratory illness in humans; approximately % of confirmed cases have been fatal. human-tohuman transmission and multiple outbreaks of respiratory illness have been attributed to mers-cov, and severe respiratory illness caused by this virus continues to be identified. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) world health organization. who mers-cov global summary and assessment of risk middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction risk factors for mers coronavirus infection in dromedary camels in mers coronaviruses from camels in africa exhibit region-dependent genetic diversity middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation 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for mers-cov infection high prevalence of mers-cov infection in camel workers in saudi arabia inovio pharmaceuticals i. inovio's mers vaccine generates high levels of antibodies and induces broad-based t cell responses in phase study man in germany dies of complications stemming from mers virus diversity of middle east respiratory syndrome coronaviruses in dromedary camels based on full-genome sequencing inclusion of mers-spike protein elisa in algorithm to determine serologic evidence of mers-cov infection recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study korean society of infectious diseases. clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea acute middle east respiratory syndrome coronavirus infection in livestock dromedaries risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during - : a case-control study mers-cov antibody responses year after symptom onset we thank yassir eltahir for critical input during the conceptualization and implementation of this study. this investigation was considered a public health response by the abu dhabi department of health and cdc. dr. khudair is a senior officer in the communicable disease control and management section of the abu dhabi department of health. his research interests include mers-cov and tuberculosis. dr. killerby is an epidemiologist in the division of viral diseases, national center for immunization and respiratory diseases, cdc. her research interests include respiratory viruses such as mers-cov, human coronaviruses, and adenoviruses. key: cord- -mswb q authors: zumla, alimuddin; dar, osman; kock, richard; muturi, matthew; ntoumi, francine; kaleebu, pontiano; eusebio, macete; mfinanga, sayoki; bates, matthew; mwaba, peter; ansumana, rashid; khan, mishal; alagaili, abdulaziz n.; cotten, matthew; azhar, esam i.; maeurer, markus; ippolito, giuseppe; petersen, eskild title: taking forward a ‘one health’ approach for turning the tide against the middle east respiratory syndrome coronavirus and other zoonotic pathogens with epidemic potential date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: mswb q the appearance of novel pathogens of humans with epidemic potential and high mortality rates have threatened global health security for centuries. over the past few decades new zoonotic infectious diseases of humans caused by pathogens arising from animal reservoirs have included west nile virus, yellow fever virus, ebola virus, nipah virus, lassa fever virus, hanta virus, dengue fever virus, rift valley fever virus, crimean-congo haemorrhagic fever virus, severe acute respiratory syndrome coronavirus, highly pathogenic avian influenza viruses, middle east respiratory syndrome coronavirus, and zika virus. the recent ebola virus disease epidemic in west africa and the ongoing zika virus outbreak in south america highlight the urgent need for local, regional and international public health systems to be be more coordinated and better prepared. the one health concept focuses on the relationship and interconnectedness between humans, animals and the environment, and recognizes that the health and wellbeing of humans is intimately connected to the health of animals and their environment (and vice versa). critical to the establishment of a one health platform is the creation of a multidisciplinary team with a range of expertise including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral phylogeneticists, and researchers to co-operate, collaborate to learn more about zoonotic spread between animals, humans and the environment and to monitor, respond to and prevent major outbreaks. we discuss the unique opportunities for middle eastern and african stakeholders to take leadership in building equitable and effective partnerships with all stakeholders involved in human and health systems to take forward a ‘one health’ approach to control such zoonotic pathogens with epidemic potential. benefit the large majority of affected people. some foreign aid workers and researchers were not familiar with local cultural and medical services norms and aroused local anxieties. the evd epidemic highlighted the need for developing more comprehensive local, national, international, and global surveillance, as well as epidemic and outbreak preparedness response infrastructures. multiple animal, human, and environmental factors are obviously playing a critical role in the evolution, transmission, and pathogenesis of zoonotic pathogens, and these require urgent definition to enable appropriate interventions to be developed for optimal surveillance, detection, management, laboratory analysis, prevention, and control in both human and animal populations. an important need exists for establishing long-term, sustainable, trusting and meaningful and equitable collaborations between the animal, human, ecosystem, and environmental health sectors at the local, national, and international levels. these should include sustainable political and funder support for developing human and laboratory capacity and training that enables effective human-animal health cooperation leading to proactive surveillance, early detection of potential pandemic pathogens, and rapid initiation of public health prevention and control guidelines and interventions. whilst a long list of pathogens with epidemic potential are on the radar of the world health organization (who), ideally 'prevention is better than cure' and new pathogens should be dealt with at the animal source, tackling the drivers and triggers of pathogen evolution and emergence. this requires close cooperation between human and animal health systems and an appreciation of human impacts on the environment at all levels and easy access to adequate laboratory facilities. on december , an expert panel convened by who prioritized a list of emerging pathogens ''considered likely to cause severe outbreaks in the near future, and for which no, or insufficient, preventive and curative solutions exist''. , the list of the top includes the new viral zoonotic pathogen of humans mers-cov, , which was first isolated from a patient who died of a severe respiratory illness in a hospital in jeddah, saudi arabia in june . the emergence of mers-cov in was the second time (after sars-cov ) that a highly pathogenic coronavirus of humans emerged in the st century. a strong link between human cases of mers-cov and dromedary camels has been established through several studies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mers-cov is endemic in the camel populations of east africa and the middle east , , and presents a constant threat to human health in both regions. retrospective studies using stored serum from different geographical locations have indicated that mers-cov has been circulating for several decades. as of may , , there have been laboratoryconfirmed cases of mers reported to the who, with a mortality of % ( cases died). whilst most mers cases have been reported from the middle east (a large proportion from saudi arabia), mers cases have been reported from countries in all continents. the who has held nine meetings of the emergency committee (ec) for mers-cov. since evidence of sustained human-to-human transmission of mers-cov in the community is lacking, the who currently does not recommend travel restrictions to the middle east. however, mers-cov remains a major global public health threat with continuing reports of new human mers cases in saudi arabia, where millions of pilgrims from over countries travel throughout the year. furthermore, a more intensive farm-based camel livestock system has emerged and there is a large, wellestablished trade in camels between countries at the horn of africa and countries in the middle east. this has increased significantly, particularly following the lifting of the ban on live animal imports from somalia by saudi arabia in / . somalia now exports some five million live animals every year to the gulf arab states (including camels), making it the single biggest exporter of live animals in the world. the positive experience of reviving somalia's livestock export industry through increased investment in animal disease prevention and control strategies highlights how effective the 'one health' approach can be. most of the african countries do not have the resources, expertise, or capacity, including laboratory facilities, to have active surveillance for mers-cov in place. in light of this, the need for increased vigilance and watchful surveillance for mers-cov in sub-saharan africa has been highlighted previously. such an initiative could be supported through investments by countries that import large numbers of camels and other livestock from the region. the epidemic potential of mers-cov was recently illustrated by a large outbreak in hospitals in seoul, the republic korea, in mid- : mers-cov was imported by a traveller to the middle east (an agriculture businessman), resulting in mers cases with deaths. the first case was reported on may , and over the ensuing weeks, the number of secondary, tertiary, and perhaps quaternary cases of mers from this single patient rose rapidly, resulting in the largest mers case cluster occurring outside the middle east. the unprecedented outbreak was attributed to poor infection control measures at the hospitals. sequencing studies of the mers-cov isolate showed genetic recombination of mers-cov in the case exported from korea to china. however, recombination is a frequent event in mers-cov and the korean outbreak is unlikely to represent a special form of the virus. nonetheless, the potential evolution of mers-cov into a more virulent form needs to be monitored closely. research on sequencing seems to have stagnated and there have been no further sequences published from new human mers cases reported from the middle east. furthermore, the genetic evolution of mers-cov strains infecting humans over the past year remains unknown. there is an urgent need for more sequencing studies on mers-cov evolution in camels and humans, with the development of appropriate local capacity for these studies. the kingdom of saudi arabia has kept proactive watchful mers-cov surveillance with regular reports to the who of mers-cov cases. the who and ministries of health of middle eastern countries continue watchful surveillance of the mers-cov situation, and the watchful anticipation is that mers-cov may disappear with time like sars-cov. however, with the continuing, regular reports of community cases of mers-cov from saudi arabia, there are no signs of this happening in the near future and lessons must be learnt from the korean outbreak. whilst there is a growing camel livestock industry in the region, elimination of the virus is unlikely in the short term. several animal, human, and environmental factors are obviously playing a critical role in the repeated movement of mers-cov from camels to humans. the disease ecology remains largely unknown. urgent definition is required to enable appropriate interventions to be developed for optimal surveillance, laboratory detection, management, prevention, and control in both human and animal populations. whilst several ad hoc research studies have been conducted and findings published over the past years, more comprehensive investments in tackling mers-cov have not been forthcoming. there remain huge knowledge gaps on mers-cov. much of the information that we have about the source of mers-cov infections is based on small local studies and it is difficult to develop general country-wide policies without a clear understanding of the zoonotic problem. questions remain, for example are new local mers outbreaks in saudi arabia always seeded by the same type of human exposure to camels? are there particular regions of africa that provide infected camels to saudi arabia? or is there a general risk from all regions? is there a way to efficiently control the entry of infected camels? are animal vaccination strategies economically viable given the large number of imported animals and the frequency of the infection? a clear policy in which full virus genome sequences are generated from every outbreak in the country and in which virus from subsets of imported camels is routinely screened and sequenced after years, would provide incredibly useful information about the transmission patterns of the virus and how to stop it. certainly the resources and expertise to perform this sequence monitoring are available and only governmental support is needed to run such a survey. the cost of such a survey would be far less than the management costs and grief associated with a single hospital outbreak. numerous priority research questions regarding mers-cov (basic science, epidemiology, management, and development of new diagnostics, biomarkers, treatments, and vaccines) in both humans and camels, highlighted years ago by the who mers expert groups and by others, remain unanswered. these have again been raised recently, highlighted by calls from saudi arabian health care staff and scientists , and by yet another who mers expert group, which has defined a ''roadmap for research and product development against mers-cov''. in the who set up the global outbreak alert and response network (goarn) for better coordination of surveillance efforts across the globe. it networks institutions and partner agencies, with cooperation with other agencies such as public health england and the us centers for disease control and prevention (cdc) and consortia such as the international severe acute respiratory and emerging infection consortium (isaric). recent consortia such as glopid-r aim to bring together research funding organizations on a global scale to facilitate an effective research response within h of a significant outbreak of a new or re-emerging infectious disease with pandemic potential. the past years has seen outbreaks of ebola virus, zkv, and mers-cov, [ ] [ ] [ ] which indicate that the global community needs to seriously reflect on what is critically missing from current political, scientific, and public health agendas, and how to delineate what is required at the national, regional, and global levels to prevent future epidemics. the factors and operating conditions that promote the emergence and geographical spread of zoonoses are complex and may be related to a single event or chain of multiple events influenced by the genetic evolution of the pathogen, environmental and climate changes, anthropological and demographic changes, and movement and behaviour of humans, animals, and vectors. with animal, human, and environmental factors playing a critical role in its evolution, mers-cov requires more close collaboration between human and animal health systems and university academics to reduce the risk of pandemic spread. moreover, a better understanding of the agricultural dynamics involved in its persistence and spread in camels and studies on interactions between hosts in the environment are urgently needed. the intermittent detection and reporting of mers cases in the community and sporadic nosocomial mers-cov outbreaks will require a more coordinated response plan to study clinical cases, conduct translational basic science and clinical trials research, and perform longitudinal sequencing studies from human and camel mers-cov isolates. a more collaborative mers-cov response plan is required to better define mers-cov epidemiology, transmission dynamics, molecular evolution, laboratory capacity, optimal treatment and prevention measures, and development of vaccines for humans and camels. a better understanding of the prevailing disease ecology and investigations into the dynamics of infectious agents in wildlife could act as a better means of preventing outbreaks in livestock and people at source. the 'one health' concept is an important concept that focuses on the relationship and interconnections between humans, animals, and the environment, and recognizes that the health and wellbeing of humans is intimately linked to the health of animals and their environment (and vice versa). [ ] [ ] [ ] [ ] [ ] a balanced ecological approach improves understanding of the true threat of novel pathogens and helps to avoid costly, poor, and inappropriate responses to new diseases. in many cases, solutions can be found through altered development pathways and are not inevitably requiring of costly, unsustainable technical and pharmaceutical interventions. thus it is ideally suited to the mers-cov situation in which camels, humans, and environmental factors are central to its persistence and evolution. since the kingdom of saudi arabia is host to millions of pilgrims each year travelling from all continents, tackling the threat of mers and other infectious diseases with epidemic potential will require enhanced closer cooperation between those who provide human health, animal health, and environmental health services, locally, nationally, regionally, and internationally: the middle eastern, european, african, asian, and american governments, veterinary groups, the who, the food and agriculture organization (fao), the african union, the united nations international children's emergency fund (unicef), the world bank, office international des epizooties (oie), cdc, public health england, the newly formed africa cdc, and funding agencies among others. they should now demonstrate increased commitment towards local, national, and global multidisciplinary collaborative efforts to secure optimal health for people, animals, and the environment. global efforts need to be focused on establishing the capability for and strengthening of surveillance systems in developing countries, particularly in africa where emerging and re-emerging zoonoses are a recurrent problem. a prime emphasis should be on developing awareness and response capacity in all countries and on promoting interdisciplinary collaboration and coordination. critical to the establishment of a well-functioning 'one health' platform is the creation of a multidisciplinary team with a range of expertise, including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral geneticists, and researchers, with easy access to adequate laboratory facilities, who will collaborate in order to learn more about zoonotic spread between animals, humans, and the environment and to monitor, respond to, and prevent major outbreaks. there is an urgent and critical need to build a sustainable public health programme and rapid response capability for outbreaks of zoonotic pathogens in the middle east and in low-income countries, especially in africa. importantly there is a need for capacity development programmes designed to strengthen research training and build career pathways for the best and brightest post-doctoral researchers, including phd and masters students working at the interface of humans, animals, and environment. these should include national or regional laboratory facilities, as surveillance requires laboratory support to be meaningful. the development of human and animal health research leaders will create a critical mass of local research capacity and the development of self-funding research environments in african universities and research institutes. this capacity growth could be facilitated through the further development and support of a geographical network of equitable and enduring south-south and north-south partnerships. . need for more effective political and scientific engagement to eradicate the threat of mers-cov and other zoonotic diseases the persistence of mers-cov years since its first discovery has created major opportunities for each of the middle eastern and african countries to take leadership of the 'one health' approach with a view to bringing this under regional and global umbrellas, to tackle new emerging and re-emerging infectious diseases with epidemic potential. this will also devolve current dominance of the global health agenda by western groups and consortia and allow equitable partnerships to be established with long-term sustainability. the past year has seen some progress in research into mers-cov, but there remains a need for a more effective, coordinated, and multidisciplinary 'one health' consortium to take forward mers-cov research on priority areas already defined by saudi scientists , and the who mers committee. the establishment of regional 'one health' centres of excellence in the middle east (under the league of arab states) and at specific geographical locations in west, central, east, and southern africa could make an important difference in mitigating the risks and factors that pose a risk to both human and animal health. furthermore, any operational plan developed will contribute to strengthening the sentinel surveillance systems in sub-saharan africa in the preparedness and response to potential outbreaks. regional centres should be sufficiently empowered to manage the spectrum of 'one health' approaches to zoonotic disease control in humans and animals, from behaviour change and social interventions for prevention to surveillance of infections and antimicrobial resistance, and preparedness and response to outbreaks. a model for the major syndromes (respiratory, neurological, haemorrhagic, gastro-enteric, and sepsis-like presentations) should be developed so that clinical protocols may be adapted rapidly for any major outbreak during mass gatherings. this should include the development and introduction of innovative and smart platforms for data sourcing, sample collection, and analysis, in order to give clinicians and public health workers continuously updated information on which clinical decisions may be based. there is a pressing need to develop and strengthen the national ethics and medicines regulatory frameworks in sub-saharan africa in order to strike a balance between the public health interest, the interests of the pharmaceutical industry, and ethical values. parallel initiatives across africa and the tropics could be harmonized to create regional networks that can serve as a repository for expert 'one health' advice on agriculture, sustainable livestock, and the links to human development. there are several ongoing important initiatives on developing 'rapid response' and broader 'one health' capacity development groups in europe, asia, and the americas to assist in the surveillance and response to emerging infectious disease threats. the public health systems of west african countries failed with the ebola epidemic, and the response from the who and the international community was very slow and uncoordinated. this led to thousands of people, including over health care workers, losing their lives. the factors governing the appearance and disappearance of new coronaviruses affecting humans are complex and it has been over years since the first patient died of mers-cov. mers cases continue to be reported throughout the year from the middle east. there is a large mers-cov camel reservoir and there is no specific treatment or vaccine. the precise pathway from infected camel to the recurring mers hospital outbreaks needs to be understood in order to devise effective control measures. with million people visiting saudi arabia every year for umrah and/or hajj and the increasing importation of live animals from sub-saharan africa, the potential risk of global spread will be everpresent, especially if mutations or recombinations in mers-cov occur. a major 'one health' initiative to tackle mers-cov at source in animal populations is thus required. middle eastern and african governments should now work more closely together and increase collaborative efforts with international partners and global public health authorities if we are to prevent yet another global zoonotic pandemic. conflict of interest: all authors have a specific interest in 'one health'. the authors declare no conflicts of interest. there was no financial support. emerging and re-emerging infectious threats in the st century world health organization. ebola virus disease outbreak world health organization. zika virus be prepared: europe needs ebola outbreak consortium ethics for pandemics beyond influenza: ebola, drug-resistant tuberculosis, and anticipating future ethical challenges in pandemic preparedness and response ebola: missed opportunities for europe-africa research rapid spread of zika virus in the americas-implications for public health preparedness for mass gatherings at the brazil olympic games lessons from the ebola outbreak: action items for emerging infectious disease preparedness and response challenges in controlling the ebola outbreak in two prefectures in guinea: why did communities continue to resist? world health organization. who publishes list of top emerging diseases likely to cause major epidemics world health organization. a research and development blueprint for action to prevent epidemics middle east respiratory syndrome 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in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov) world health organization. ihr emergency committee concerning middle east respiratory syndrome coronavirus hajj: infectious disease surveillance and control middle east respiratory syndrome-need for increased vigilance and watchful surveillance for mers-cov in sub-saharan africa middle east respiratory syndrome coronavirus (mers-cov)-update. disease outbreak news origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis saudi ministry of health. weekly mers-cov monitor middle east respiratory syndromeadvancing the public health and research agenda on mers-lessons from the south korea outbreak state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans advancing priority research on the middle east respiratory syndrome coronavirus in riyadh, ksa knowledge gaps in therapeutic and non-therapeutic research on the middle east respiratory syndrome world health organization. a roadmap for research and product development against middle east respiratory syndrome-coronavirus (mers-cov) international severe acute respiratory and emerging infection consortium. the website for the international severe acute respiratory and emerging infection consortium (isaric) global research collaboration for infectious disease preparedness website emerging infectious diseases and pandemic potential: status quo and reducing risk of global spread development of medical countermeasures to middle east respiratory syndrome coronavirus what is one health? one health global network one health: a new professional imperative. one health initiative task force one world, one health. oie-world organisation for animal health sharing responsibilities and coordinating global activities to address health risks at the animal-human-ecosystems interfaces. a tripartite concept note international organization for standardization. who develops iso standards. geneva: who towards a one health approach to controlling zoonotic diseases key: cord- -vevsgkp authors: alharbi, naif khalaf; padron-regalado, eriko; thompson, craig p.; kupke, alexandra; wells, daniel; sloan, megan a.; grehan, keith; temperton, nigel; lambe, teresa; warimwe, george; becker, stephan; hill, adrian v.s.; gilbert, sarah c. title: chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: vevsgkp abstract the middle east respiratory syndrome coronavirus (mers-cov) has infected more than humans, since . the syndrome ranges from asymptomatic and mild cases to severe pneumonia and death. the virus is believed to be circulating in dromedary camels without notable symptoms since the s. therefore, dromedary camels are considered the only animal source of infection. neither antiviral drugs nor vaccines are approved for veterinary or medical use despite active research on this area. here, we developed four vaccine candidates against mers-cov based on chadox and mva viral vectors, two candidates per vector. all vaccines contained the full-length spike gene of mers-cov; chadox mers vaccines were produced with or without the leader sequence of the human tissue plasminogen activator gene (tpa) where mva mers vaccines were produced with tpa, but either the mh or f promoter driving expression of the spike gene. all vaccine candidates were evaluated in a mouse model in prime only or prime-boost regimens. chadox mers with tpa induced higher neutralising antibodies than chadox mers without tpa. a single dose of chadox mers with tpa elicited cellular immune responses as well as neutralising antibodies that were boosted to a significantly higher level by mva mers. the humoral immunogenicity of a single dose of chadox mers with tpa was equivalent to two doses of mva mers (also with tpa). mva mers with mh or f promoter induced similar antibody levels; however, f promoter enhanced the cellular immunogenicity of mva mers to significantly higher magnitudes. in conclusion, our study showed that mers-cov vaccine candidates could be optimized by utilising different viral vectors, various genetic designs of the vectors, or different regimens to increase immunogenicity. chadox and mva vectored vaccines have been safely evaluated in camels and humans and these mers vaccine candidates should now be tested in camels and in clinical trials. middle east respiratory syndrome (mers) is caused by a novel betacoronavirus (mers-cov) that was isolated in late in saudi arabia [ ] . the syndrome (mers) is described as a viral infection that causes fever, cough, and/or shortness of breath and to a lesser extent gastrointestinal symptoms such as diarrhea [ ] . severe disease from mers-cov infection can cause respiratory failure and organ failure, and cases can be fatal, especially in patients with co-morbidities such as diabetes and cardiac complications. however, the infection can be asymptomatic or mild in many cases [ ] [ ] [ ] [ ] [ ] . mers-cov has spread to countries and infected more than humans with a mortality rate of % [ ] . dromedary camels, especially juveniles, contract the infection and shed the virus, without notable symptoms of disease; this is now known to have been occurring since the early s [ ] [ ] [ ] [ ] [ ] [ ] . the mechanism of camel to human transmission is still not clear, but several primary cases have been associated with camel contact, which is considered an important risk factor [ ] [ ] [ ] . therefore, camels http [ ] [ ] [ ] [ ] [ ] [ ] . other livestock animals such as sheep, goats, cows, chicken, and horses have proved seronegative in many studies [ ] [ ] [ ] [ ] . further, these animals did not productively contract mers-cov when they were inoculated experimentally [ , ] . therefore, to date, dromedary camels are the only confirmed animal reservoir. there is currently no approved vaccine against mers-cov for camels or humans despite active vaccine research and development. a number of vaccine candidates have been developed using various platforms and regimens and have been tested in several animal models [ ] . viral vectors are potent platform technologies that have been utilised to develop vaccines against malaria, tuberculosis, influenza, hiv, hcv, ebola, and many viral pathogens. these vectors include adenoviruses, poxviruses, yellow fever viruses, and alphaviruses [ , ] , and they are preferred for their ability to induce cellular immune responses in addition to humoral immunity. here, we report development of mers-cov vaccine candidates that are based on two different viral vectors: chimpanzee adenovirus, oxford university # (chadox ) [ ] and modified vaccinia virus ankara (mva) [ , ] . each viral vector was developed by generating two alternative versions, resulting in four vaccine candidates that all encode the same complete mers-cov spike gene (s). the two chadox based vaccines were produced with or without the signal peptide of the human tissue plasminogen activator gene (tpa) at the n terminus. previous studies have shown that encoding tpa upstream of recombinant antigens enhanced immunogencity, although results differed depending on the antigens employed. the tpa encoded upstream of influenza a virus nucleoprotein, in a dna vector, enhanced both cellular and humoral immune responses in mice [ , ] , whereas the same leader sequence resulted in increased humoral sequences but decreased cellular responses to hiv gag [ ] . the two mva based vaccines were produced with either the mh or f poxviral promoter driving antigen expression, both including the tpa sequence at the n terminus of mers-cov spike protein. previously, we reported the ability of the strong early f promoter to enhance cellular immunogenicity of vaccine antigen candidates for malaria and influenza, as compared to utilising p . or mh early/late promoters which resulted in a lower level of gene expression immediately after virus infection of target cells, but higher levels at a later stage [ ] . here, we continue to assess the f promoter in enhancing cellular immunogenicity, and to investigate its ability to impact on humoral immune responses. the four vaccine candidates were evaluated in a number of different regimens in mouse models that showed a single dose of chadox mers inducing higher cellular and humoral immunogenicity than a single dose of mva mers, or equivalent to two doses of mva mers. chadox based vaccines have been tested in different animal models, including camels [ ] , and in human clinical trials and proved safe and immunogenic [ ] . therefore, based on our data, chadox mers can be readily developed for use as a mers vaccine in humans. furthermore, utilising chadox mers for camel vaccination can serve the one-health approach whereby blocking mers-cov transmission in camels is expected to prevent human infections. the spike (s) gene of mers-cov camel isolate (genbank accession number: kj . ) was synthesised by geneart gene synthesis (thermo fisher scientific). the s transgene was then cloned into four shuttle plasmid vectors following in-fusion cloning (clontech). two plasmids contained the s transgene within the e homologous region of chadox , driven by the human cytomegalovirus major immediate early promoter (ie cmv) that includes intron a. one of the chadox shuttle plasmids was designed to include the tpa signal sequence upstream of the transgene sequence while the second plasmid did not contain the tpa. the chadox shuttle plasmids contained the s transgene within gateway Ò recombination cassettes. to construct mva mers, one of the shuttle plasmids for mva was designed to have the upstream and downstream (flanks) of the f l orf as homologous sequence arms. inserting the s transgene within these arms enabled the utilisation of the endogenous f promoter, which is part of the right homologous arm, while deleting the native f l orf. this resulted in the shuttle vector for generation of f -mva mers (f shuttle vector). the mh promoter sequence was subcloned upstream of the s transgene; and this mh -s transgene was then subcloned into the f shuttle vector. this resulted in the shuttle vector for generation of mh -mva mers (f /mh shuttle vector). mh -mva mers contained the mh promoter at the f l locus, however, the endogenous f promoter is intact and located upstream of the mh promoter. the endogenous f promoter could not be replaced with the mh since it is part of the essential upstream orf. the chadox shuttle plasmid, described above, was used to validate the expression of mers-cov spike protein in vitro. an african green monkey kidney cell line (vero cells) was seeded into -well plate to % confluence. then the plasmid dna was transfected into vero cells using lipofectamine Ò (thermo fisher scientific) following manufacturer's instruction. twenty-four hours after transfection, cells were fixed, permeabilised, and immunostained using a rabbit polyclonal anti-mers-cov spike antibody, following standard protocols. dapi stain was used to label nuclei. the chadox mers vaccines were prepared by gateway Ò recombination between the chadox destination dna bac vector (described in [ ] ) and entry plasmids containing the coding sequence for mers-cov spike gene (chadox shuttle vectors explained above), according to standard protocols. chadox mers genomes were then derived in hek a cell lines (invitrogen, cat. r - ), the resultant viruses were purified by cscl gradient ultracentrifugation as previously described [ ] . the titres were determined on hek a cells using anti-hexon immunostaining assay based on the quicktiter tm adenovirus titer immunoassay kit (cell biolabs inc). for mva mers vaccines chicken embryo fibroblast cells (cefs) were infected with mva parental virus that encodes dsred marker instead of the native f l orf and transfected with mva shuttle plasmids containing mers-cov spike gene (explained above) to allow recombination with the mva genome and deletion of dsred marker whilst keeping the f promoter sequence. recombinant mva expressing mers-cov s protein was purified by plaque-picking and fluorescent selection using the sorting function of cyclone robotic module of a moflo flow cytometer (dako cytomation, denmark) as previously described [ ] . f -mva mers and mh -mva mers were confirmed to lack the native f l orf (and the dsred marker), and contain mers-cov s by pcr (identity and purity pcr screening). the sequence of the s transgene amplified from these vaccines was confirmed. the recombinant viruses (vaccines) were amplified in cm monolayers of cefs cells, partially purified over sucrose cushions and titrated in cefs cells according to standard practice, and purity and identity were again verified by pcr. female balb/c mice (harlan, uk) aged - weeks were immunised intramuscularly (i.m.) in the upper leg (total volume ml) with a total of iu of chadox mers with or without tpa or with a total of pfu of either f -mva mers or mh -mva mers. for induction of short-term anaesthesia, animals were anaesthetised using vaporised isofloh. in prime only regimens, mice were vaccinated with chadox with blood samples taken at days post immunisation (d.p.i) or d.p.i. for serum isolation; and spleens were collected at d.p.i. in heterologous prime-boost regimens, mice were vaccinated with chadox mers and boosted with mva mers at d.p.i; mice were bled at d.p.i. splenocytes were harvested for analysis by ifn-c elispot or intracellular cytokine staining (ics) and flow cytometry as previously described [ , ] , using re-stimulation with mg/ml s mers-cov s-specific peptide (vydtikyysiiphsi); for vaccine cellular immunogenicity [ ] ); or mg/ml e and f (g) mva vectorspecific peptides [ ] (for anti-mva immune responses). in the absence of peptide re-stimulation, the frequency of ifn-c + cells, which was typically . % by flow cytometry or less than sfc by elispot, was subtracted from tested re-stimulated samples. lg/ml with capturing antigen (s recombinant protein from mybiosource, ca, usa) were used to coat elisa plates, and standard endpoint elisa protocol was followed, as previously described [ ] . sera were prepared in a -fold serial dilution in pbs/t and then ll were plated in duplicate wells. serum from a naïve balb/c mouse was included as a negative control. goat anti-mouse total igg conjugated to alkaline phosphatase (sigma) and pnpp tablet ( mg p-nitrophenylphosphate, sigma) substrate were used in the assay. mers pseudotyped viral particles (merspp) were produced and titrated using huh . cell line as described previously [ ] . for the merspp neutralisation assay, serum samples were serially diluted in -well white plates (nunc). a standard concentration of the merspp were added to the wells and plates were incubated for h at °c. after incubation, huh . cells ( , cells per well) were added to the plate in duplicates. following h incubation, cells were lysed and luciferase activity was measured. ic neutralisation titres were calculated for each mouse serum sample using graphpad prism. induction of virus-neutralising antibodies was confirmed according to previously published protocols [ , ] . briefly, mouse serum samples were tested for their capacity to neutralise mers-cov (emc isolate) infections in vitro with % tissue culture infective doses (tcid ) in huh- cells. sera of non-immunised mice served as negative control. graphpad prism (graphpad software) was used for statistical analysis and to plot data. all animal procedures were performed in accordance with the terms of the uk animals (scientific procedures) act (aspa) for the project licenses / or / and were approved by the university of oxford animal care and ethical review committee. all mice were housed for at least days for settlement prior to any procedure in the university animal facility, oxford, uk under specific pathogen free (spf) conditions. the spike gene from a camel isolate (camel/qatar_ _ mers-cov isolate, genbank accession number kj . ) was cloned into four shuttle vectors that facilitate homologous recombination with the genome of chadox or mva. four recombinant viral vectors, two chadox and two mva, were derived as described in the materials and methods. chadox based vaccine candidates were generated with or without the signal peptide of the human tissue plasminogen activator gene (tpa). the spike transgene expression in chadox mers vaccine candidates is under the control of the human cytomegalovirus major immediate early promoter (cmv ie) that includes intron a. in mva mers vaccine candidates, the tpa was also inserted upstream of the spike transgene, which was under the control of either the ectopic mh promoter or the endogenous f promoter (fig. a) . all of our mers-cov vaccine candidates contain the same codonoptimized spike transgene. the expression of the newly synthesized transgene was first tested by transfection of an african green monkey kidney cell line (vero cells) with the adenovirus shuttle vector, and immunofluorescence staining of the transfected cells ( fig. b and c) . this was performed to confirm the expression of the codon optimized spike transgene in mammalian cells. the level of transgene expression from the four vaccine candidates was not evaluated in vitro. we have previously reported that differences in mva promoter activity detectable in vitro does not correlate with in vivo immunogenicity [ ] , and that only in vivo expression correlates with the in vivo immunogenicity. to evaluate humoral immune responses to chadox mers with or without tpa, balb/c mice were vaccinated with  iu of chadox intramuscularly. serum samples from and d.p.i. were collected and evaluated by elisa. both vaccine candidates induced a high level of s -specific antibodies (mean endpoint titre (log ) = . with tpa, . without tpa), unlike the control vaccine, chadox encoding enhanced green fluorescent protein (chadox -egfp, mean endpoint titre (log ) = ). these antibody levels were similar between the two candidates (with or without tpa) at day . however, at d.p.i. chadox mers with tpa induced significantly higher s -specific antibodies than chadox mers without tpa (mean endpoint titre (log ) = . with tpa, . without tpa, fig. a ). serum samples from day were selected for merspp neutralisation assay. serum antibodies induced by chadox mers with tpa showed significantly higher neutralisation activity than without tpa (mean titre ic (log ) = . with tpa, . without tpa; fig. b ). in order to confirm that the psuedotyped virus neutralisation assay was producing biologically relevant results, serum samples from mice immunised with chadox mers with tpa were also tested in a neutralisation assay utilising wildtype mers virus. this assay confirmed the neutralisation activity of mouse antibodies (nab) with a median of vnt (virus neutralization test antibody titre; fig. c ). we therefore continued to evaluate chadox mers with tpa in addition to generating mva mers vaccine candidates with tpa. having established the utility of tpa in chadox mers vaccines (referred to as chadox mers in the rest of this report) at increasing humoral responses, spleens were collected at d.p.i. from immunised balb/c mice. splenocytes were processed to evaluate cellular immune responses to chadox mers in elispot and intracellular cytokine staining (ics). peptide s , described by others [ ] , was used to re-stimulate the cells in both assays and elispot data showed a high level of ifn-c secreting splenocytes (median = sfu/ splenocytes; fig. a ). ics data confirmed the ifn-c secreting cd + splenocytes also secreted tnf-a and il- (fig. b ). to evaluate humoral immune responses to heterologous primeboost vaccination, balb/c mice were immunised with chadox were collected and evaluated by elisa and merspp neutralisation assay. at d.p.i. chadox mers induced similar levels of s -specific antibodies and nab as observed previously ( fig. a and b) . at d.p.i. s -specific antibodies were boosted to a higher level (mean endpoint titre (log ) = by chadox mers boosted to . by mh -mva mers or . by f -mva mers); fig. a ) with nab also enhanced to a statistically significant level (mean titre ic (log ) = . by chadox mers boosted to . by mh -mva mers or . by f -mva mers; fig. b ). there was no difference in antibody levels induced using either the f or mh promoter in the mva. at d.p.i. splenocytes were also processed to evaluate cellular immune responses to chadox mers mva mers prime-boost vaccination in elispot and ics as shown in fig. . the t cell responses to mers s were boosted by the mva vaccinations; in the ics experiments, f -mva and mh -mva boosted the percentage of ifn-c + splenic cd + t cells to . and . % respectively (fig. d) whereas the percentage was . % after chadox mers prime in fig. b . the percentage of tnf-a + splenic cd + t cells were also increased by mva boost (comparing fig. b and d) . utilising the f promoter resulted in a trend towards greater cell-mediated immunogenicity ( fig. c and d) . splenocytes were also re-stimulated with mva backbone-specific e and f(g) peptides and evaluated in ics. both mva based vaccines induced similar responses to e or to f(g) pep- tides, weeks after mva vaccination (fig. e and f) . this similarity confirmed the efficiency of vaccine titration, vaccination, and sample processing because responses to each of those peptides are not expected to be different unless there is variation in the doses administered or sample preparation. overall, mva mers vaccines were able to boost the humoral and cellular immune responses to cha-dox mers prime vaccination. there was no difference between the f and mh promoter in the resulting antibody titres after cha-dox prime/mva boost, but there was a trend towards increased cellular immunogenicity when the f promoter was used. to evaluate humoral immune responses to a homologous mva mers prime-boost vaccination, two groups of balb/c mice were immunised with f -mva mers or mh -mva mers and boosted with the same vaccine after three weeks. serum samples from d. antibodies (mean endpoint titre (log ) = . and . respectively; fig. a ). at d.p.i s -specific antibody levels had increased to . and . respectively (fig. a) . the titres of nab (mers pp assay) were also similar for both vaccines (mean titre ic (log ) = . (f -mva mers) and . respectively; fig. b ). utilising different promoters in mva vectors did not result in differences in the induced antibody levels. however, at d.p.i. ifn-c secreting splenocytes induced by f -mva mers were statistically significantly higher than those of mh -mva mers ((median = and sfu/ splenocytes, respectively, fig. c ). both mva vaccines induced similar vector-specific immune responses as expected ( fig. d and e) . vaccines against mers-cov have been developed and tested in a number of animal models (including non-human primates [ ] [ ] [ ] and camels [ ] ) as well as in human clinical trials [ ] . all vaccine candidates focused on the spike antigen because it contains the receptor-binding domain used for cell entry by the virus, against which neutralising antibodies may be induced, and it is conserved. therefore, the improvement of mers-cov vaccines focuses on platform and vaccination regimens rather than antigen selection and optimisation. here, we focused on using the same antigen (transgene) to develop a vaccine against mers-cov, and to assess different vectors, different versions of each vector, and different vaccination regimens. we generated a number of mers-cov vaccine candidates based on the same codon optimized spike transgene and ensured its expression in vitro before we evaluated the humoral and cellular immunogenicity in a pre-clinical balb/c mouse model. chadox based vaccine candidates were produced with or without tpa. the tpa signal peptide was predicted to enhance the humoral immunogenicity of encoded vaccine antigens, based on previous reports [ ] . our data supported this hypothesis and showed a significant increase in the s -specific antibody levels at d.p.i. the level of neutralising antibodies was also increased when tpa was utilised. however, chadox mers without tpa was still a potent vaccine candidate, inducing a high level of both s -specific binding antibodies and mers-cov neutralising antibodies. neutralisation activity of mouse serum antibodies was assayed by using mers-cov pseudotyped viral particles (merspp), an approach used by a number of researchers for other human pathogens such as hiv, influenza, and hcv to overcome the necessity of handling bsl- viruses [ ] . additionally, we confirmed the ability of serum samples from vaccinated mice to neutralise live mers virus. we therefore selected chadox mers with tpa (simply referred to chadox mers) for further evaluation. chadox mers also induced cellular responses for mers s, with polyfunctional cd + t cells detected in the spleen of immunised mice. this supports the potency of the chadox viral vector in inducing t cellular immunity, observed previously in animal models [ , , ] as well as in humans [ ] . following chadox prime/mva boost, mva significantly boosted the neutralising antibody titres to higher levels. no difference in humoral immunity was found when either the f or mh promoter was used. regarding the promoter effect on mva cellular immunogenicity, we have previously reported that utilising the f promoter enhanced malaria and influenza antigens in mva [ ] . here, we again report that f -mva mers induced higher t cell responses than mh -mva mers in a homologous prime-boost mva mers vaccination. all of our vaccine candidates induced humoral (with nab) and cellular immune (with polyfunctional cd + t cell) responses against mers-cov spike antigen. modest effects on immunogenicity of different versions of the vaccines were noted, with the use of the tpa leader sequence in chadox , and the use of the f promoter in mva producing small increases in immunogenicity compared to no leader sequence, or the mh promoter. the protective level of either antibodies or cellular immunity required to counter mers-cov infection in humans or in animal models is not yet defined, despite some efforts [ ] [ ] [ ] [ ] . the ideal vaccine would provide rapid onset of immunity and complete protective efficacy after a single dose, with a long duration of immunity. complete protective efficacy of one dose of chadox expressing the external glycoprotein of rift valley fever virus has been demonstrated in multiple species and it is already known that chadox rvf is highly immunogenic in camels [ ] . to date, the only vaccine against mers to be tested in camels is an mva vectored vaccine [ ] which was protective in hdpp transgenic mice immunised with a homologous prime/boost regimen [ ] but in camels required two doses given both intranasally and intramuscularly to provide partial protection and reduction of virus shedding [ ] . here we find that a single dose of chadox mers is as immunogenic as two doses of mva mers, suggesting that this regimen should be tested for protective efficacy in camels. however if this is not completely protective, administration of mva mers as a heterologous boost should be considered next. in our hands one dose of mva resulted in an endpoint titre of logs, two doses of mva produced . logs, one dose of chadox produced logs, and chadox /mva prime boost produced . logs. if a single dose of chadox mers is not protective and a two dose regimen is required, chadox /mva would be more likely to provide complete protection than mva/mva. chadox mers should now be evaluated for immunogenicity and efficacy in larger animal species, including both camels and humans. scg is a co-founder of, consultant to and shareholder in vaccitech plc which is developing vectored influenza and mers vaccines. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates mers-cov outbreak in jeddah-a link to health care facilities state of knowledge and data gaps of 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with middle east respiratory syndrome coronavirus infection key: cord- -w on x authors: ahmadzadeh, jamal; mobaraki, kazhal; mousavi, seyed jalil; aghazadeh-attari, javad; mirza-aghazadeh-attari, mohammad; mohebbi, iraj title: the risk factors associated with mers-cov patient fatality: a global survey date: - - journal: diagn microbiol infect dis doi: . /j.diagmicrobio. . sha: doc_id: cord_uid: w on x risk factors associated with middle east respiratory syndrome coronavirus (mers-cov) infection outcome were established by analyses of who data from september , to june . of the reported cases, cases, including mers-cov deaths, were analyzed. the case fatality rate was % ( % ci: . – . ). compared to mers patients ≤ years old, those with > years had the adjusted odds ratio estimate for death of . [ % ci: . – . ]. this index was . [ % ci: . – . ] for saudi patients in comparison to non-saudi; . [ % ci: . – . ] for patient with comorbidity in comparison to those without comorbidity; . [ % ci: . – . ] for those who had close contact to a camel in the past days and . [ % ci: . – . ] for patients with > days with onset of signs and hospital admission compared to patients with ≤ days. the middle east respiratory syndrome (mers) is a relatively new viral respiratory infection caused by a novel coronavirus, the middle east respiratory syndrome coronavirus, or mers-cov (alshahrani et al. ) . coronaviruses are single-stranded positive-sense rna viruses (zumla et al. ) , with human pathogenic strains resulting in a common cold to a severe acute respiratory syndrome (castaño-rodriguez et al. ). mers-cov, an emerging zoonotic virus (alamoudi et al. ; cong et al. ) is pathogenic in human, resulting in shortness of breath, cough, fever, diarrhea, and frequently pneumonia (cong et al. ; sherbini et al. ) . the exact animal origin of mers-cov is not fully understood, but the transmission pattern and the evidence from virologic studies suggest that it may have originated in bats and was transmitted to camels sometime in the distant past (hu et al. , wang et al. . several published studies have assessed the epidemiological status of mers-cov infection. most of these epidemiological studies were derived from specific cohorts with a small sample size, or carried out in a single medical center (alraddadi et al. ; assiri et al. ; harriman et al. ; memish et al. ; mobaraki and ahmadzadeh b; park et al. ) . however, numerous questions about the epidemiological status and associated risk factors of mers-cov at the global level remain unanswered. for this study, we used the publicly available world health organization (who) mers global epidemiologic data (world health organization ) to assess characteristics, clinical information, global distribution status, and probable risk factors associated with mers-cov patient mortality. in this worldwide comprehensive survey, were analyzed publicly available data from the who website:(http://www.who.int/csr/don/ archive/disease/coronavirus_infections/en/) related to laboratory-confirmed mers-cov cases from september , until june , . data for the analyses was downloaded on june , . this who database is periodically updated by the who. epidemiological characteristics of each patient were retrieved including: age, gender, travel history to endemic countries, nationality, country/city of origin. also retrieved was clinical data on the symptomatic mers patients including day/ month of the onset of symptom, day/month of the admission to the hospital, and comorbidities (diabetes mellitus, hypertension, and ischemic heart disease). exposure to hazardous contacts was also collected, including healthcare workers (who worked in the hospital), camels, consumption of raw camel products, and exposure to mers-cov morbid patients at home or hospital within days prior to the onset of symptomology. all the collected information was checked for missing or invalid data. of a total input of mers patient cases, with a complete data set were included in the analysis. in order to avoid measurement bias and miscategorization of cases, of the patients with incomplete data were not included. statistical analysis was conducted using the spss, version (ibm inc., armonk, ny, usa). quantitative measurement is expressed by the mean and standard deviation. qualitative variables are presented as absolute frequency and percentage. logistic regression was used to calculate the adjusted odds ratio (aor) and unadjusted odds ratio (uor) with a % confidence interval to assess the probable relationship between the risk factors and the final outcome (dead/survived) of laboratory-confirmed mers-cov cases. any p-value given was two-sided and was considered statistically significant at . . for this study, a total of sporadic/clusters patients with confirmed mers-cov infection and complete data were used in the analysis. the characteristics and the number of mers cases per year can be found in . most of the mers cases were male and male-specific % cfr was approximately % ( / ). the male vs. female ratio in the fatal and nonfatal cases was . ( / ) vs. . ( / ), respectively (see table ). table illustrates unadjusted and adjusted or for mortality in the laboratory-confirmed mers-cov cases in the survey period. the adjusted analysis showed that the age groups n years ( . ; % ci: . - . ), saudi nationality ( . ; % ci: . - . ), and comorbidity ( . ; % ci: . - . ) were independently associated with higher chances of mortality. additionally, in comparison to mers patients who had ≤ days from onset of clinical signs to hospital admission, adjusted or estimates of the mortality was . ; % ci: . - . for those who had n days from the onset of clinical signs to hospital admission. the adjusted or estimates of mortality was . ; % ci: . - . for mers patients who were exposed to a camel in the last days compared to those who were not exposed. other probable risk factors such as gender, exposure to a morbid case of mers in the last days, healthcare worker, and admission in negative pressure isolate room or icu had no significant association with higher mortality (p n . for all). ). mers-cov started in saudi arabia by sporadic infections in mid- and later its outbreak progressed to other countries (alamoudi et al. ). due to the occurrence of a large number of mers-cov cases and its high worldwide mortality rate, this infection must be considered a public health threat (lessler et al. ) . the current study focuses on the epidemiological trend of mers-cov infection and mortality rate analysis of its worldwide cases in the aforementioned dates. the findings of this study may have important implications for the infection control practice and also help to ensure global health security. based on the analysis, the overall global %cfr of mers was . % [ / ], which is substantially lower than the %cfr in the mers-cov endemic region (table ) . for example, hunter et al. found an overall cfr of % in the abu dhabi (hunter et al. ) and petersen et al. found an overall cfr of % in the kingdom of saudi arabia (petersen et al. ) however, our estimates were higher than the largest mers outbreak in south korea (cfr of %) (cowling et al. ) . our analyses of the who data was approximately similar to the cfr of % reported by memish et al. (memish et al. ) , and also cfr of . % declared by mobaraki et al. (mobaraki and ahmadzadeh a). the regional variation of cfr from previously conducted studies may be skewed due to severity of disease and smaller sample sizes than have been investigated previously. on the whole, the cfr of % related to the mers-cov infection in the present study should be considered as a major health concern at the global scale. thus, the characteristics of this disease and the potential risk factors associated with patient fatality should be studied comprehensively. our findings confirm that the mortality pattern of the mers in saudi arabia is different from the observed countries in the middle east and affected countries beyond. by far, the greatest burden of this disease in terms of mortality and morbidity rates is located in four countries including saudi arabia, south korea, united arab emirates, and jordan. in this regards, differences in the virus and the genetic background of the population affected can play a role. other reasons can include a difference in the availability or ability to implement patient isolation procedures as well as differences in overall medical technology among involved countries. consistent with the previous reports, age range n years is associated with death in cases of mers-cov infection (alzeer ; gautret et al. ; memish et al. ). this finding is in line with a saudi arabian case report series. it showed that the age range of the individuals (n years) had a greater association with mortality and per every year increase in age, the odds of mortality increased by % (majumder et al. ) . the reason for the higher fatality rates in this age range is unclear, but they may have underlying diseases and impaired immune functions that exacerbate the symptom of mers-cov infection and increase the chances of death. also, calculating the or (table ) suggested that having saudi nationality, comorbidity, the interval time of onset sign and admission to the hospital n days are other potential risk factors for the disease progression and mortality related to mers-cov infection. although camels are a suspected reservoir, this study could not find a risk relationship in the mortality of patients who had contact with camels in the days prior to clinical signs. meanwhile, global concern rests on the ability of mers-cov to cause major illnesses in direct and indirect contact with camels and its products, namely drinking unpasteurized camel milk (conzade et al. ; harrath and abu duhier ; kamau et al. ) . details as to the specific mechanism of zoonotic transmission from dromedaries to humans remain unclear, and further epidemiological studies are required in this regard. in line with the findings of alghamdi et al. (alghamdi et al. ), we observed a higher rate of mers-cov incidence in males than females (table ) . however, based on our findings (table ) this gender difference in mortality rates related to mers-cov was not statistically significant (aor = . ; % ci: . - . ), p = . . the current study suffered from some limitations. of the total worldwide cases ( laboratory-confirmed cases of mers-cov), only cases with complete data were investigated in the current study. it should be noted that from mers-cov cases in south korea, only details related to cases were published in the disease outbreak news on the who website. the lack of complete data for all mers cases potentially increases the occurrence of selection and measurement biases in the result. therefore, it might be more appropriate to conduct further large-scale epidemiological studies with complete data related to all morbid cases of mers to obtain a better understanding of mers-cov emergence in humans and also associated risk factors related of this infection. in the future, we may closely monitor the mers-cov infections globally to better understand the risks of this new infection for public health and to provide helpful recommendations for controlling and preventing it. recommendations might change and be updated as additional data becomes available. indeed, despite the above limitations, such studies might be useful to implement educational programs, and access health care for early diagnosis and prevention of modifiable factors to reduce high mortality rates associated with mers-cov. based on our analyses of the who data, years after the emergence of the mers-cov incidence; saudi arabia still has the highest rate of infection. this study estimated a global % cfr ( % ci: . - . ) for mers patients. the results demonstrated a link between mortality and some risk factors such as age n years old, saudi nationality, comorbidities, the interval time of onset sign and the admission to the hospital n days. unlinked data. all authors express their satisfaction with the publication of this paper. this project was funded by the urmia university of medical sciences (grant no. ir.umsu.rec. . ). the funding bodies had no role in study design, data collection, analysis, preparation of the manuscript, or the decision to publish. the data used for the analysis can be obtained from the study authors. we thanks to the urmia university of medical sciences for supporting this study (grant no. - - - ) . we also appreciate all reporting countries with the confirmed mers cases for investigations, data collection and sending of it to the who. the authors would like to acknowledge the miss gazhal akhavan masoumi for editing this paper. preliminary epidemiologic assessment of mers-cov outbreak in south korea no molecular evidence of mers-cov circulation in jeddah, saudi arabia between - : a single-center retrospective study the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus respiratory tract infection during hajj hospital outbreak of middle east respiratory syndrome coronavirus role of severe acute respiratory syndrome coronavirus viroporins e, a, and a in replication and pathogenesis mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj , despite a high rate of respiratory symptoms sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia hospital-associated middle east respiratory syndrome coronavirus infections bat origin of human coronaviruses transmission of middle east respiratory syndrome coronavirus infections in healthcare settings knowledge and practices regarding middle east respiratory syndrome coronavirus among camel handlers in a slaughterhouse an update to middle east respiratory syndrome coronavirus and risk of a pandemic in estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia mortality risk factors for middle east respiratory syndrome outbreak, south korea family cluster of middle east respiratory syndrome coronavirus infections etiology of severe community-acquired pneumonia during the hajj-part of the mers-cov surveillance program current epidemiological status of middle east respiratory syndrome coronavirus in the world from . . to . . : a cross-sectional study an update to middle east respiratory syndrome coronavirus and risk of a pandemic in hospital outbreaks of middle east respiratory syndrome health-care associate transmission of middle east respiratory syndrome corona virus, mers-cov, in the kingdom of saudi arabia middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data bat origins of mers-cov supported by bat coronavirus hku usage of human receptor cd world health organization. emergencies preparedness, response:mers-cov.disease outbreak news middle east respiratory syndrome the authors declare that they have no competing interests. key: cord- -wbi ird authors: ahmed, anwar e.; alshukairi, abeer n.; al‐jahdali, hamdan; alaqeel, mody; siddiq, salma s.; alsaab, hanan a.; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; aloudah, nouf m.; baharoon, salim; alsalamah, majid a.; al johani, sameera; alghamdi, mohammed g. title: development of a risk‐prediction model for middle east respiratory syndrome coronavirus infection in dialysis patients date: - - journal: hemodial int doi: . /hdi. sha: doc_id: cord_uid: wbi ird introduction the middle east respiratory syndrome coronavirus (mers‐cov) infection can cause transmission clusters and high mortality in hemodialysis facilities. we attempted to develop a risk‐prediction model to assess the early risk of mers‐cov infection in dialysis patients. methods this two‐center retrospective cohort study included dialysis patients who were suspected of mers‐cov infection and diagnosed with rrt‐pcr between september and june at king fahd general hospital in jeddah and king abdulaziz medical city in riyadh. we retrieved data on demographic, clinical, and radiological findings, and laboratory indices of each patient. findings a risk‐prediction model to assess early risk for mers‐cov in dialysis patients has been developed. independent predictors of mers‐cov infection were identified, including chest pain (or = . ; p = . ), leukopenia (or = . ; p = . ), and elevated aspartate aminotransferase (ast) (or = . ; p = . ). the adequacy of this prediction model was good (p = . ), with a high predictive utility (area under curve [auc] = . %; % ci: . % to . %). the prediction of the model had optimism‐corrected bootstrap resampling auc of . %. the youden index yielded a value of . or greater as the best cut‐off for high risk of mers infection. discussion this risk‐prediction model in dialysis patients appears to depend markedly on chest pain, leukopenia, and elevated ast. the model accurately predicts the high risk of mers‐cov infection in dialysis patients. this could be clinically useful in applying timely intervention and control measures to prevent clusters of infections in dialysis facilities or other health care settings. the predictive utility of the model warrants further validation in external samples and prospective studies. an important lesson was learned from the world's largest middle east respiratory syndrome coronavirus (mers-cov) outbreaks that occurred in saudi arabia and south korea: that health care-associated infection is a major cause of rapid pathogen spread in health care settings with a high risk of cluster infections. in particular it was discovered that they spread rapidly in hemodialysis, inpatient, emergency, and intensive care facilities. [ ] [ ] [ ] [ ] [ ] [ ] dialysis patients were associated with a high risk of mortality compared to the national mortality estimates in the mers-cov population. , assiri et al. were able to track hospitals, units, rooms, beds, symptoms onset, and diagnoses status to map a large cluster of infections between april and may , . according to the authors, the clusters developed in the hemodialysis facility, where health careassociated infected patient who underwent long-term hemodialysis transmitted the virus to dialysis patients and the transmission then continued to other hospital settings. in a recent study, assiri et al. reported a high likelihood of transmission in dialysis patients and health care workers within the outpatient dialysis facility. among laboratory-confirmed mers-cov patients in south korea, only dialysis patient was identified, but no cluster viral transmissions were identified in other patients who utilized the same hemodialysis facility. park et al. developed a guideline to control cluster infections and prevent mers outbreaks in hemodialysis facilities. earlier studies on dialysis patients focused on virus transmission and clinical outcomes, while limited by the small number of mers cases. our understanding of early diagnoses of mers and identifying patients at high risk of infection is incomplete, particularly in a hemodialysis facility. a mers-cov risk assessment tool is urgently needed to accurately identify dialysis patients at high risk of infection and apply infection control measures to prevent future cluster transmission in these patients and patients in other health care facilities. exploring an efficient screening system to detect mers-cov infection at an earlier stage may result in immediate isolation and improve clinical outcomes and economic burdens. , a valid risk-predictive model for mers-cov infection in dialysis patients may increase the likelihood of early virus detection. the authors attempt to develop an algorithm that combines demographic, clinical, radiological, and laboratory data to assess the early risk of mers-cov infection in dialysis patients who are suspected of having mers-cov infection and were diagnosed by real-time reverse transcription-pcr (rrt-pcr) between september and june . the authors hypothesized that mers-cov infection in dialysis patients could be predicted by a set of clinical, radiological, and laboratory indices. this two-center retrospective cohort study included dialysis patients who were suspected of having mers-cov, according to the saudi ministry of health guidelines, . acute respiratory illness and/or chest radiological findings of pneumonia, . hospitalized with health care associated-pneumonia, . upper or lower respiratory tract illness within days after exposure to a confirmed/probable case of mers-cov infection, and . high fever ( c), headache, body aches, nausea/ vomiting, diarrhea, or with or without respiratory symptoms, leucopenia, and thrombocytopenia. data were abstracted into potential predictors of mers, including demographic data (age and gender); clinical presentations (fever, cough, short breath, chest pain, abdominal pain, diarrhea, vomiting, diabetes); radiology findings in chest (abnormal ct scan or x-ray); baseline laboratory measurements (number of white cells) (wbc) /l in the blood, blood platelet count /l, alanine transaminase (alt) u/l, and aspartate transaminase (ast) u/l). in order to evaluate whether mers-cov infection was associated with a decrease in wbc count, a cut-off of less than ( /l) indicates leukopenia. similarly, platelet count of less than ( /l) indicates thrombocytopenia, alt greater than (u/l) indicates elevated alt, and ast greater than (u/l) indicate elevated ast. data were analyzed using stata (statacorp. . stata statistical software: release . college station, tx: stata-corp llc). overall sample summary and subgroup analysis were provided in table . p value of independent samples t test/chi-square test and unadjusted odds ratio (or) were reported to test whether specific characteristics were associated with mers-cov infection in dialysis patients ( table ). the area under the curve (auc) and % confidence interval (ci) were used to evaluate the accuracy of each predictor in identifying mers-cov infection ( table ) . we developed the mers riskprediction model in dialysis patients using the stepwise logistic regression model. fifteen potential predictors of mers-cov were evaluated at a . . the goodness-offit of the final model was evaluated using the hosmer-lemeshow test. a p value of greater than % (a > . ) indicates the model fit the data well. the discrimination of the model was evaluated by the receiver operator characteristic curve and was compared with the each of the most important predictors (figure ). the risk model was internally validated in bootstrap samples drawn with replacement from the study sample (n ). the model was presented in the form of the predictive probability of mers-cov infection in dialysis, which is a function of the important selected variables, refer to the supplement file. the youden index was used to identify optimal probability cut-off value for the mers risk stratification. of the dialysis patients studied, % had respiratory symptoms, and . % had gastrointestinal symptoms at presentation. the sample age was relatively older at . . years and . % were males (table ) the auc in table shows the predictors of mers infections. it indicates that chest pain, diabetes, abnormal radiology findings, and elevated ast (auc . ) were the most powerful predictors of discriminating mers. when controlled for potential predictors (table ) , the final risk-prediction model retained independent variables (at a . ) that increased the risk of mers-cov infection. mers dialysis patients were more likely to have chest pain (or . ; p . ), leukopenia (or . ; p . ), and elevated ast (or . ; p . ). according to the hosmer-lemeshow test, the adequacy of this prediction model was good (p . ). the model shows high potential for predicting mers (auc . %; % ci: . % to . %). the prediction of the model had optimism-corrected bootstrap resampling auc of . %. figure shows that the riskprediction model improved the accuracy of risk classification as compared to the individual predictors. the predicted probability of mers can be calculated by: [ exp ( . - . chest pain - . leukopenia - . elevated ast)] . table presents cut-off values for risk probability. this is the first study to develop a risk-prediction model in dialysis patients who screened for mers-cov infection by rrt-pcr. the study included data on dialysis stepwise selection significant at a . . patients from centers, kfgh-jed and kamc-r. mers-cov infection is common in dialysis patients, , , and is associated with increased rapid spread, which can be prevented through early detection, isolation, and monitoring individuals at risk. subsequently, a predictive model was developed for mers-cov infection in hemodialysis facilities. the model shows promising accuracy in detecting high-risk dialysis patients with an auc of . %. the model identified the most important clinical and laboratory characteristics that could help in distinguishing mers-cov infection from other respiratory illnesses. dialysis patients with chest pain were associated with a -times higher risk of mers-cov infection than dialysis patients without chest pain. earlier studies reported that chest pain was one of the most common symptoms in the mers-cov population. , in agreement with a matched case-control study, we found no differences between mers and non-mers groups in regards to fever, shortness of breath, cough, and other gastrointestinal symptoms. dialysis patients with low wbc count or leukopenia was associated with a -times higher risk of mers-cov infection as compared to dialysis patients without leukopenia. this finding is in agreement with saudi ministry of health guidelines, as they developed a tool to identify and evaluate individuals for mers-cov infection, and several other reports, , , where the wbc was found to be lower in patients with mers-cov infection. our findings support the matched case-control study which showed that mers-cov patients are more likely to have leucopenia and transaminitis. in concordance with earlier studies, , , elevated ast was found to be a feature of mers-cov infection, where dialysis patients with elevated ast were associated with -times higher risk of mers-cov infection as compared with dialysis patients with no elevated ast. according to our risk-prediction model, alt has poor predictive utility. this association was also described by ajlan et al., where normal alt levels have been frequently encountered in mers patients. a prospective study is needed to understand further the link between abnormal ast and mers-cov infection in dialysis patients. the model with the mentioned predictors can be useful in clinical decision to identify high-risk dialysis patients for further investigations and interventions. we presented a simple form of a probability prediction model to calculate the potential risk of infection. for instance, a randomly selected dialysis patient who presented with chest pain, leukopenia, and elevated ast has a probability of mers of . . another case, a randomly selected dialysis patient who did not present with chest pain, leukopenia, or elevated ast has a probability of mers of . . the cut-off values of the probabilities that discriminate between the high-risk and low-risk mers were provided in table . according to the youden index, a cut-off value (p . ) produces sensitivity and specificity of . and . , respectively was found optimal to identify high-risk mers infection. diabetes and abnormal radiology were risk factors for mers-cov infection when we presented the unadjusted analysis. however, these factors were not significant after adjustment for other confounding factors. several limitations should be reported that could influence the prediction of the risk model. the model needs to be validated in a prospective mers-cov investigation. the study included two of the largest hospitals in saudi arabia, yet the model may not be generalizable to dialysis patients in other hospitals. although this study is the largest rrt-pcr study on dialysis patients who screened for mers-cov infection, yet it is limited by the small number of cases screened. the authors were not able to include many other potential confounding factors in the analysis because they were not available. we also acknowledge that the small number of dialysis patients and unequal distribution of mers between hospitals limit our report. despite the limitations mentioned, the prediction ability of the model appears to be promising in clinical decision making to identify suspected dialysis patients with mers-cov infection at an early stage of the infection. in summary, this risk-prediction model in dialysis patients appears to depend markedly on chest pain, leukopenia, and elevated ast. the model accurately predicts high-risk of mers-cov infection in dialysis patients. this could be clinically useful in applying timely intervention and control measures to prevent clusters of infections in dialysis facilities or other hospital settings. the predictive utility of the model warrants further validation in an external sample and a prospective study. hospital outbreak of middle east respiratory syndrome coronavirus managing mers-cov in the healthcare setting middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases guidelines for the laboratory diagnosis of middle east respiratory syndrome coronavirus in korea outbreak of middle east respiratory syndrome at tertiary care hospital multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia the predictors of -and -day mortality in mers-cov patients 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observational study early identification of pneumonia patients at increased risk of mers-cov infection in saudi arabia health care associated middle east respiratory syndrome (mers): a case from iran predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea epidemiological and clinical characteristics of patients with middle east respiratory syndrome coronavirus in iran in middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings key: cord- -wfaqim d authors: modjarrad, kayvon title: mers-cov vaccine candidates in development: the current landscape date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: wfaqim d middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than people, resulting in more than deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of mers-cov have created an urgent need for effective public health countermeasures, paramount of which is an effective means of prevention through a vaccine or antibody prophylaxis. despite the relatively few number of cases to-date, research and development of mers-cov vaccine candidates is advancing quickly. this review surveys the landscape of these efforts across multiple groups in academia, government and industry. middle east respiratory syndrome (mers-cov) was first isolated in september from a patient in saudi arabia who presented two months earlier with severe acute respiratory infection and acute renal failure [ ] . retrospective testing of samples in jordan identified earlier cases from a nosocomial outbreak in april [ ] . although the majority of mers-cov cases (∼ %) have occurred in saudi arabia, other countries have confirmed imported or autochthonously transmitted cases ( fig. ) [ , ] . the most recent and largest outbreak outside of saudi arabia occurred in south korea in may [ ] , raising concern for an eruption of regional outbreaks or accelerated global spread, similar to the phylogenetically related severe acute respiratory syndrome coronavirus (sars-cov) that killed nearly a thousand people a decade earlier [ ] . although the definitive host for mers-cov has not yet been established, closely related coronaviruses have been isolated from bats across wide geographic areas [ ] [ ] [ ] . mounting evidence has strongly implicated dromedary camels as the intermediate animal reservoir, as serological surveys throughout the middle east and north africa have demonstrated them to have a high prevalence of mers-cov binding or neutralizing abs [ ] [ ] [ ] [ ] . additionally, outbreak investigations have suggested epidemiologic linkage between farm camels and human cases [ ] . mers-cov is a spherical, enveloped, single-stranded, positive sense rna beta-coronavirus [ , ] . its genome contains a replicase * tel.: + . locus at the end and codes for structural proteins toward the end. the most immunogenic of the viral proteins is spike (s), a trimeric, envelope-anchored, type i fusion glycoprotein that interfaces with its human host cognate receptor, dipeptidyl peptidase (dpp ), to mediate viral entry [ , ] . s comprises two subunits: s , which contains the receptor-binding domain and determines cell tropism; and s , the location of the cell fusion machinery. although dpp has a broad tissue distribution, most of the clinical manifestations of mers-cov can be attributed to its localization to the lower respiratory tract [ , ] . much like other coronaviruses, mers-cov can also cause significant dysfunction of the gastrointestinal, cardiovascular, renal, and neurologic systems. mers-cov is distinct, though, in its tendency to cause greatest harm to older individuals with concurrent comorbidities of one or more of these organ-systems [ , ] . despite past efforts to develop coronavirus countermeasures in response to the sars-cov pandemic, there are currently no prophylactic or therapeutic interventions of proven efficacy for mers-cov or any other coronavirus infection. although combination treatment with ribavirin and interferons were shown to improve clinical outcomes in mers-cov-infected non-human primates (nhps), treatment was initiated very soon after viral challenge (∼ h) and results have not been replicated in humans [ ] . in fact, no experimental interventions have demonstrated appreciable benefit in acutely ill patients in a consistent or controlled manner. rapidly scaled treatments based on naturally occurring neutralizing antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have demonstrated mortality reductions for other respiratory infections and may hold promise for mers-cov as well [ ] . their development, however, is limited by logistical challenges, local technical capacity, and donor supply. supportive management, adapted from guidelines developed for sars-cov, has thus far been the mainstay of mers-cov treatment. the global will to develop a coronavirus vaccine faded in the aftermath of sars-cov pandemic but has since gained renewed momentum in the face of the current mers-cov outbreak. previous approaches to coronavirus vaccine development were broad and included whole-inactivated and live-attenuated viruses, recombinant vectors and protein subunits, as well as dna and rna based platforms [ ] . most developers based their immunogen designs on the s surface glycoprotein, the primary target for neutralizing antibodies during any natural coronavirus infection. a number of preclinical and clinical studies showed that the sars-cov s protein subunit, and specifically the rbd at its core, could serve as a dominant target for neutralizing antibodies in mice, non-human primates, and humans [ ] . s , therefore, became the basis for a number of promising sars-cov vaccine candidates. the s protein subunit and rbd have also been the basis for several mers-cov vaccine candidates (fig. ) [ ] [ ] [ ] [ ] [ ] . resolution of rbd crystal structures alone or in complex with the dpp receptor [ , , ] have informed the design of immunogens that have been expressed either as recombinant protein fragments or conjugates to the fragment crystallizable (fc) region of human antibodies. both types of constructs, in formulation with aluminum salt or oil-in-water adjuvants, have elicited neutralizing antibodies of high potency across multiple viral strains. despite their demonstrated immunogenicity in animal models and anticipated safety in humans, rbd or s -subunit based vaccine candidates are limited in their epitope breadth. although the coronavirus genomes are not as variable as other rna viruses, the rbd is the most mutable region, containing mutation sites that define antibody escape variants [ , ] . thus, vaccine candidates that elicit a more diverse antibody repertoire as well as a robust cellular immune response may offer the advantage of broader and more durable protection. full-length s used as an immunogen could at least increase the breadth of the antibody response; however, it has been difficult to express, and may require additional work to produce a stable soluble trimer of the s ectodomain. investigators at the university of maryland, in collaboration with novavax, inc., have overcome this problem through the development of s rosettes that are stable and immunogenic in murine models [ ] . vaccines that mimic natural infection, such as live-attenuated viruses or recombinant viral vectors, may elicit even more robust immunity. live attenuated viruses have historically been among the most immunogenic platforms available, as they have the capacity to present multiple antigens across the viral life cycle in their native conformations. although a live-attenuated mers-cov has yet to be tested, one has been constructed and has the potential to be protective [ ] . however, manufacturing live-attenuated viruses requires containment in a biosafety level or facility. additionally, live-attenuated viruses carry the hazards of inadequate attenuation or reversion to wild type form and causing disseminated disease, particularly in immunocompromised hosts. given that moderately immunocompromised adults with co-morbidities such as diabetes mellitus and chronic kidney disease have suffered the most severe mers-cov disease, these individuals may comprise a target population for immunization, thus making a live-attenuated virus vaccine a less viable option. replication competent viral vectors could pose a similar threat for disseminated disease in the immunosuppressed. replication deficient vectors, however, avoid that risk while maintaining the advantages of native antigen presentation, elicitation of t cell immunity and the ability to express multiple antigens. to date, two recombinant vector platforms-modified although replication deficient vectors are relatively safe and immunogenic, their ability to deliver genetic material for expression could be impeded by pre-existing or developing immunity to the vector itself. one way to overcome this limitation is by administering different vectors in a so-called prime-boost immunization regimen. as this strategy has been effective for other pathogens, it is likely that the same success could be recapitulated for mers-cov. the use of more than one type of platform or antigen in a single vaccine also increases the likelihood of inducing a broad repertoire of antibodies with diverse mechanisms of viral neutralization. one vaccine regimen developed at the us national institutes of health is based on full-length s dna and a truncated s subunit glycoprotein and has elicited neutralizing antibodies in mice directed at both the s -within and outside the rbd-and s subunits. immunization with these constructs also protected nhps from severe lung disease after intra-tracheal challenge with mers-cov [ ] . a dnaonly vaccine, expressing multiple antigens, has also been developed by inovio pharmaceuticals and geneone life science inc. and has been advanced to a phase i first-in-human trial. each of the vaccine candidates that have been mentioned is being developed for prophylactic use. however, as the total number of cases (> ) [ , ] and reproductive rate (∼ . ) of mers-cov are both relatively low [ ] , it will be difficult to define the target populations for vaccination that would support the investment in manufacturing and advanced product development. also, with such low incidence and lack of robust animal models, it would be difficult to achieve a vaccine efficacy result that would be sufficient to support licensure. human mabs, on the other hand, could be used without as much discrimination in an outbreak setting for post-exposure prophylaxis and early treatment. the advantages of mabs over polyclonal antibodies (administered through convalescent plasma or hyperimmune globulin) are their higher potency, greater specificity, more extensive pre-licensing evaluation and consequently improved safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic level detail for the design of better immunogens. however, the timeline for mab development may be longer and potentially cost more than some vaccines. despite requirements for greater upfront investment, several groups have developed highly potent mabs that are currently being advanced through pre-clinical stages of testing (fig. ) . some have been isolated from immunized animals (mice/humanized mice/nhps) [ , ] , while others have been identified from either an antibody human phage library [ ] [ ] [ ] [ ] or memory b cells of infected and recovered human survivors [ ] . almost all of the mabs that have been reported target the spike rbd. it is likely that mabs directed at other sites on the spike glycoprotein have been recovered but are not as potent neutralizers. most of those that have been published bind to recombinant spike with picomolar affinity and neutralize mers-cov pseudovirus at a half maximal inhibitory concentration (ic ) of . mcg/l or less. additionally, some have demonstrated protective efficacy in pre-and post-exposure prophylaxis animal models [ , ] . the successes thus far in isolating potent and protective mabs may prove useful for therapeutic development where the target population is well defined. it may prove more challenging to advancing these products to licensure and fullscale production at affordable costs for the purpose of prophylaxis in as of yet undefined populations. the vaguely defined epidemiology of mers-cov has complicated the design and implementation of appropriate public health countermeasures. most transmission events have occurred either in the setting of household clusters or nosocomial outbreaks [ ] [ ] [ ] [ ] [ ] [ ] . it is also likely that the virus has been introduced multiple times into human populations from a large zoonotic reservoir, i.e. dromedary camels. given the broad distribution and ownership of camels in the arabian peninsula where most cases have occurred, a targeted vaccine campaign may prove difficult. as the outbreak in the republic of korea revealed, patients and workers in the same healthcare facility as an infected patient are at high risk for secondary acquisition. an optimal strategy may be to use vaccines in conjunction with stringent infection control practices in hospitals where mers-cov cases are being treated. the epidemiologic link of mers-cov between bats, camels, and humans presents an opportunity for a veterinary vaccine to interrupt the transmission cycle. a successful precedent for this so-called "onehealth" approach toward mitigating human disease with a veterinary vaccine exists in the example of equivac ® , a hendra virus vaccine developed solely for horses [ ] . although hendra virus is even more rare than mers-cov, it is highly fatal with no treatment other than intensive supportive management. in , a protein subunit vaccine was licensed and rolled-out in australia, where all outbreaks of the virus occurred. since that time, the incidence in horses has fallen precipitously and no human cases have been detected [ ] . a similar strategy may be applicable to mers-cov; however, a veterinary vaccination in this context would be deployed solely for the sake of protecting humans, as the virus causes only mild upper respiratory illness in camels. safety and reduction in viral shedding would have to be demonstrated in immunization, challenge and transmission studies of camel or camelid populations, one of which has shown efficacy [ ] . one of the primary challenges to developing countermeasures to mers-cov is the lack of an appropriate animal model that recapitulates the natural history of human disease. much of the difficulty originates from the absence of the virus's cognate dpp receptor. one group approached this problem by successfully transducing mice with an adenoviral vector expressing human dpp [ ] . although more relevant than a standard murine model, transient transduction of the desired protein may result in inconsistent tissue expression of adenovirus antigens. agrawal et al. made an important advance with the development of a transgenic mouse model that demonstrated productive, disseminated mers-cov infection [ ] . although rhesus macaques do not manifest full clinical disease, they develop a transient lower respiratory infection that can be quantified and evaluated by computed tomography. investigators at the nih rocky mountain laboratories (rml) and integrated research facility (irf) have also independently been developing potentially lethal marmoset models that could be used for the evaluation of vaccines, mabs and therapeutics [ ] . as mers-cov vaccines-both active and passive-are developed and tested, not only will more relevant animal models be required, but there will also be a need for a more detailed understanding of the epidemiology, immunology, and pathogenesis of the virus. in the aftermath of the west african ebola virus epidemic and in the face of the current zika virus outbreak, the global health community has coalesced around the realization that a multi-faceted plan is required to quickly and efficiently respond to global public health emergencies. the world health organization is currently developing a blueprint by which that preparation and response can follow, with mers-cov highlighted as a case study. although mers-cov still causes relatively few cases in a limited geographic distribution, its high case fatality and sudden outbreak in in korea have proven it to be a pathogen of public health concern. the concentration of the epidemic to saudi arabia also raises the specter of international spread every year during hajj, one of the largest mass gathering events in the world. ultimately, the development of a safe and effective vaccine for mers-cov may not yield its greatest benefit for the current epidemic but for the knowledge gained in creating a platform for combating coronaviruses as a whole. the opinions expressed herein are those of the authors and should not be construed as official or representing the views of the us department of defense or the department of the army. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological findings from a retrospective investigation east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of update: middle east respiratory syndrome coronavirus (mers-cov) probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea a decade after sars: strategies for controlling emerging coronaviruses detection of coronaviruses in bats of various 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preand postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus family cluster of middle east respiratory syndrome coronavirus infections hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections hendra virus vaccine, a one health approach to protecting horse, human, and environmental health hendra virus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection key: cord- - znb authors: omrani, a.s.; shalhoub, s. title: middle east respiratory syndrome coronavirus (mers-cov): what lessons can we learn? date: - - journal: j hosp infect doi: . /j.jhin. . . sha: doc_id: cord_uid: znb the middle east respiratory coronavirus (mers-cov) was first isolated from a patient who died with severe pneumonia in june . as of june , a total of , mers-cov infections have been notified to the world health organization (who). clinical illness associated with mers-cov ranges from mild upper respiratory symptoms to rapidly progressive pneumonia and multi-organ failure. a significant proportion of patients present with non-respiratory symptoms such as headache, myalgia, vomiting and diarrhoea. a few potential therapeutic agents have been identified but none have been conclusively shown to be clinically effective. human to human transmission is well documented, but the epidemic potential of mers-cov remains limited at present. healthcare-associated clusters of mers-cov have been responsible for the majority of reported cases. the largest outbreaks have been driven by delayed diagnosis, overcrowding and poor infection control practices. however, chains of mers-cov transmission can be readily interrupted with implementation of appropriate control measures. as with any emerging infectious disease, guidelines for mers-cov case identification and surveillance evolved as new data became available. sound clinical judgment is required to identify unusual presentations and trigger appropriate control precautions. evidence from multiple sources implicates dromedary camels as natural hosts of mers-cov. camel to human transmission has been demonstrated, but the exact mechanism of infection remains uncertain. the ubiquitously available social media have facilitated communication and networking amongst healthcare professionals and eventually proved to be important channels for presenting the public with factual material, timely updates and relevant advice. the middle east respiratory syndrome coronavirus (mers-cov) was first identified in september . as of june , a total of , mers-cov infections have been reported to the world health organization (who). despite an accumulation of clinical experience and scientific knowledge, new mers-cov infections continue to be reported almost on daily basis. what lessons can we learn after three years of clinical experience and scientific research? lesson one: no substitute for continuous vigilance a -year-old man was admitted on june th, , to a hospital in jeddah, saudi arabia, with severe pneumonia and multi-organ failure. the patient died after days of hospitalization. indirect immunofluorescence assays and real-time polymerase chain reaction (pcr) for widely occurring respiratory viruses failed to identify an infective aetiology. interestingly, cytopathic changes consistent with viral replication were noted in llc-mk and vero cell cultures of the patient's respiratory samples. slides of the infected cell cultures reacted strongly with the patient's serum but not with any of control sera stored in the same hospital. however, pancoronavirus pcr yielded positive results. the pcr fragments were sequenced at the erasmus medical centre in rotterdam, the netherlands, and phylogenetic analysis showed that the novel coronavirus belonged to lineage c of the genus betacoronavirus. , on september , an email was posted on program for monitoring emerging diseases mail (promed-mail) announcing the discovery of a novel human coronavirus. meanwhile, a critically ill -year-old qatari man was transferred by air ambulance on september th, , to a hospital in england. he had developed respiratory symptoms on september rd followed by multi-organ failure. his upper and lower respiratory tract samples were negative for influenza a/b, parainfluenza e , rsv a/b, human metapneumovirus, enterovirus, rhinovirus, adenovirus, human bocavirus, and the human coronaviruses (nl , e, oc , hku ). on september st, , one day after the above promed-mail posting, the patient's respiratory samples tested positive by pancoronavirus pcr. once sequenced, a base-pair fragment from this isolate showed . % homology with the erasmus medical centre's isolate. the third patient was a -year-old man who presented to a hospital in riyadh, saudi arabia, on october th, , with severe pneumonia and renal failure. mers-cov was detected in samples from the patient's upper and lower respiratory tract. prior to all of this, an outbreak of respiratory illness was reported in april from an intensive care unit in a hospital in zarqa, jordan. a retrospective epidemiological investigation in november identified probable cases, two of whom had died. mers-cov was detected by reverse transcription (rt)epcr in stored samples from the two deceased patients. seven more were subsequently confirmed by serological testing. a pattern began to emerge, characterized by severe pneumonia, multi-organ failure, and an epidemiological link to a country in the middle east. in may , the virus, which had been initially known as human coronaviruseerasmus medical centre (hcov-emc), was named the middle east respiratory syndrome coronavirus (mers-cov). notably, phylogenetic analysis of the first five available mers-cov sequences suggested a common ancestor dating back to mid- . furthermore, anti-mers-cov antibodies were detected in out of , serum samples [ . %; % confidence interval (ci): . e . %] obtained between december and december from provinces in saudi arabia. the authors extrapolated that just fewer than , individuals ( , ; % ci: , e , ) in saudi arabia could be seropositive for mers-cov. it is therefore reasonable to assume that human mers-cov infections had taken place in the region for some considerable time before it was identified. it is possible that the identification of the virus might have been delayed even more, had it not been for the meticulous investigation by a single virologist, dr a.m. zaki, of the first reported case of mers-cov. the first lesson one has to learn from mers-cov and its discovery is that continuous vigilance and perseverance with diagnostic investigation of undiagnosed infectious diseases are essential to identify emerging pathogens. lesson two: yet again, prevention is better than cure clinically, mers-cov infection may range from an asymptomatic or mild upper respiratory illness to a rapidly progressive and fatal disease. e the majority of hospitalized patients with mers-cov infection present with fever and respiratory symptoms including cough and shortness of breath with clinical and radiological evidence of pneumonia. , fatigue, myalgia, headache, and gastrointestinal symptoms such as vomiting and diarrhoea are also frequent. , respiratory and renal failure are frequent complications of severe mers-cov infection, in addition to acute liver injury, cardiac dysrhythmias, and coagulopathy. e overall mortality is around . %, but exceeds % in critically ill patients and in those with significant comorbidities. , e for reasons yet to be understood, mers-cov infection is rare in children. , in-vitro studies have identified numerous agents with anti-mers-cov activity including interferon, ribavirin, mycophenolate, cyclosporine and lopinavir. the combination of interferon and ribavirin showed promising results in experimentally infected macaques. however, in retrospective clinical studies the combination was not associated with significantly improved overall survival. , treatment of patients with mers-cov infections remains largely dependent on supportive measures. diagnosis is confirmed by detection of mers-cov rna in respiratory samples by real-time pcr targeting the upe and orf b genes. samples obtained from the lower respiratory tract have higher viral loads and better diagnostic yield than those obtained from the throat or nasopharynx. , , moreover, viral shedding is considerably prolonged in symptomatic and severely ill mers-cov patients compared with asymptomatic infected contacts. interestingly, detection of mers-cov in blood has been associated with worse clinical outcome. , mers-cov may also be detected in stool for up days and in urine for up to days from disease onset. under certain conditions, mers-cov can survive on plastic and steel surfaces for up to h. in the absence of appropriate precautions, the environment surrounding a symptomatic mers-cov patient can therefore become extensively contaminated with viable, potentially infectious virus. human-to-human transmission of mers-cov has been well documented in family clusters, community settings and more often in healthcare settings. e , , , common denominators in the largest hospital outbreaks have been overcrowding, especially in emergency departments, and poor adherence to infection control standards. , , , however, mers-cov continues to have relatively limited infectiousness. for example, screening identified secondary mers-cov infections in only % of close family contacts and % of healthcare contacts. , moreover, no secondary cases were identified following extensive epidemiological investigations of imported cases in the uk, germany, france, greece, the netherlands, and the usa. e it has been phylogenetically demonstrated that mers-cov transmission chains have not extended beyond two to three months and that the virus has remained genetically stable over the past three years. , given an effective reproduction number (r ) of less than one, human-to-human mers-cov could be readily interrupted with effective preventive interventions. , indeed, even the most explosive hospital outbreaks of mers-cov infection, such those that occurred in jeddah and riyadh in april to may , were brought under control through a strategy based on early case detection and implementation of appropriate infection prevention and control measures; namely contact and droplet precautions for general care in addition to airborne precautions for aerosolgenerating procedures such as intubation and respiratory tract suctioning. e the poor prognosis associated with mers-cov, especially in patients with multiple comorbidities, and the lack of effective anti-viral therapy make appropriate infection prevention and control all-important. just as is true for most infectious diseases, mers-cov reminds us again that prevention is better than cure. the initial case definitions for mers-cov case finding and reporting focused on patients who are hospitalized, had evidence of acute pulmonary disease with an epidemiological link to confirmed cases or to countries in the middle east. , as more clinical experience and epidemiological data became available, updated definitions removed the requirement for hospitalization. the reporting of several community and hospital clusters during the first half of the year , often without identifiable human or animal sources, led to speculation that individuals with no or only mild respiratory symptoms might have a role in mers-cov transmissions. , , this was reflected in the who revised interim definition published in july where patients with acute febrile illness of any severity were included; in addition to a recommendation to proactively test asymptomatic close contacts of confirmed mers-cov infections. memish et al. later showed that mers-cov was detectable for up to days in % of asymptomatic contacts. in another report, an asymptomatic healthcare worker had detectable mers-cov for more than five weeks. although mers-cov transmission from an asymptomatic individual remains a strong probability, this has never been documented. , in the meantime, clinicians were becoming increasingly aware that mers-cov infections were being diagnosed in patients whose clinical presentations did not conform to those definitions, including the absence of fever, lack of respiratory involvement and the predominance of gastrointestinal or nonspecific generalized symptoms. , , in the aftermath of the surge of mers-cov infection in jeddah and riyadh in april and may , the ministry of health in saudi arabia revised its case definition and surveillance guidance to recommend mers-cov testing in any of four patient categories: e patients with clinical or radiological evidence of community-acquired pneumonia; e patients with clinical or radiological evidence of healthcare-associated pneumonia; e patients with acute febrile illness and myalgia, headache, diarrhoea, nausea, or vomiting, and unexplained leucopenia or thrombocytopenia; e contacts of individuals with confirmed or probable mers-cov infection who develop upper or lower respiratory symptoms within two weeks of exposure. as better understanding of the epidemiology of mers-cov developed, it became obvious that a considerable proportion of cases were probably missed. , during the steep learning curve of an emerging infectious disease, regularly updated guidelines are important. such guidelines are inevitably based on incomplete evidence and hence may not be comprehensive or applicable in all situations. clinical acumen and heightened medical awareness are essential for early detection of unusual mers-cov cases and to prevent delays in diagnosis and to mitigate additional exposures. a zoonotic origin was suspected soon after the identification of mers-cov. bats are known natural hosts for several coronaviruses and hence were the initial target for investigation. , more than faecal samples were collected from wild bats in the area around where the first mers-cov patient lived. a -nucleotide fragment of mers-cov rna was detected in one faecal pellet from an egyptian tomb bat. the sequenced amplification product was genetically identical to the mers-cov sequence obtained from the index human case. more recently, a closely related coronavirus was isolated from bats in south africa, suggesting that mers-cov ancestors might exist in old world bats. , to date, no further evidence is available to confirm the role of bats as natural hosts or reservoirs for mers-cov. on the other hand, the evidence implicating dromedary camels in mers-cov epidemiology is more consistent. a role for dromedary camels is supported by the following observations: À neutralizing mers-cov antibodies are highly prevalent in dromedary camels from across the arabian peninsula, north africa, and eastern africa. e mers-cov antibodies were detected in stored camel sera dating as far back as the early s. e the prevalence of mers-cov seropositivity is significantly higher in camels aged more than two years than in juvenile camels. , , À several groups have reported the detection of mers-cov by rtepcr in nasal and faecal samples from dromedary camels in the arabian peninsula. , , e one study reported mers-cov positivity in more than % of lung tissue samples obtained from dromedary camel carcasses. rtepcr was positive in camels that had prior evidence of mers-cov seropositivity, indicating that animal reinfection is possible. interestingly, the prevalence of mers-cov rna is significantly higher in juvenile than in adult camels. , , furthermore, all mers-cov strains obtained from dromedary camels are phylogenetically clustered within human isolates, supporting possible animalehuman intertransmission. it is important to note, however, that mers-cov seroprevalence studies in individuals with close contact with camels have yielded inconsistent results. a national serosurvey in saudi arabia found prevalence of mers-cov antibodies that was times higher in camel shepherds (p ¼ . ) and times higher in slaughterhouse workers (p < . ), compared with the general population. similarly, mers-cov serology was positive in individuals who had occupational exposure to dromedary camels in qatar but not in those without such exposure. on the other hand, mers-cov antibodies were not detected in sera obtained from individuals who had close contact with camels that had documented mers-cov infection two to three months earlier. likewise, screened slaughterhouse workers and other animal workers in western and southern saudi arabia were all seronegative for mers-cov antibodies. , collectively, the available data strongly suggest that mers-cov is highly prevalent in dromedary camels in the arabian peninsula and that transmission of infection from camels to humans, although inefficient, does occur. however, the exact mechanism and route of infection it is still unclear. infections. , , , one pertinent cause for concern has been the potential global spread of mers-cov during the annual hajj pilgrimage when millions of muslims from around the world gather in mecca, saudi arabia. e though those concerns are well founded, several surveillance studies over the past three years have not identified any mers-cov infections among hajj pilgrims while they are in saudi arabia or after their return to their home countries. e the situation was entirely different in the recent outbreak in south korea where a single imported case resulted in a total of laboratory-confirmed cases of mers-cov infection, including deaths. the index patient was a -year-old man who developed respiratory symptoms seven days after returning to seoul from a two-week visit to bahrain, saudi arabia, united arab emirates, and qatar. he sought medical care in several hospitals before he was diagnosed with mers-cov infection. , a combination of late recognition, overcrowding in emergency departments and hospital wards, multiple incidents of patient movement between different healthcare facilities, and delayed implementation of adequate infection control precautions culminated in the largest single outbreak of mers-cov infection. , , e the outbreak involved patients, visitors, care-givers and healthcare workers, and spanned across six different hospitals in three south korean cities. , notably, phylogenetic analysis of mers-cov strains from south korea revealed no significant biological changes compared to previously sequenced viruses. the outbreak in south korea was eventually controlled through a series of measures including aggressive contact identification, screening and strict isolation, and rigorous infection control precautions. , , within a few weeks, south korea went from a country with no reported mers-cov cases to one that has the second largest number in the world. , with air travel becoming readily accessible and affordable, the south korean experience demonstrates vividly that in the context of an infectious respiratory illness, there is simply no room for complacency. adequate assessment of patients presenting with febrile illness must include their recent travel history to enable early application of proper control measures and to expedite laboratory confirmation and appropriate clinical management. the past decade has witnessed an exponential rise in internet-based social media sites such as facebook, twitter, and youtube. healthcare professionals are increasingly using social media applications to follow medical developments and emerging scientific literature and to share their own research findings, observations, and opinion. the general public often uses these tools as news outlets to seek and share medical and scientific information. however, in the context of mers-cov, social media have been a double-edged sword. for example, social media were at some point rife with inaccurate information that included rumours of hospitals closed due to mers-cov outbreaks and certain social events being nodes for mers-cov transmission. the authors are aware of examples of information and photos shared on social media resulting in patients losing their right to privacy and confidentiality. patients often cancelled their clinic appointments or scheduled surgical procedures for fear of acquiring mers-cov while in hospital. some avoided attending emergency departments despite having acute problems that required medical attention. some individuals posted videos and messages challenging the suggestion that camels may be a source of mers-cov infection. scepticism and mistrust in governmental agencies and the medical community were sometimes promoted and propagated. on the other hand, various government agencies, scientific organizations and healthcare professionals used social media to enhance networking and facilitate communication of epidemiological, medical and scientific developments; in addition to presenting the public with factual material, timely updates, and relevant advice. the saudi ministry of health, for example, posts daily updates on its website and through social media outlining details of current mers-cov cases. the korean ministry of health and welfare did the same during their mers-cov outbreak. such steps are important to gain the public's trust and to remove barriers to appropriate sources of information. taking on board the surging role of social media and using them effectively to disseminate appropriate information turns them into invaluable tools for controlling an emerging infectious disease such as mers-cov. mers-cov is an emerging infectious disease of probable animal origin. sustained human-to-human infection has not occurred and its potential for causing widespread epidemic remains limited. vigilance, early recognition, and institution of appropriate protective measures are the most effective control measures. none declared. middle east respiratory syndrome coronavirus (mers-cov); summary of current situation, literature update and risk assessment isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans novel coronavirus e saudi arabia: human isolate. archive number severe 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austria clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus (mers-cov) e the philippines investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in family cluster of middle east respiratory syndrome coronavirus infections laboratoryconfirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response health protection agency uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections a scenario-based evaluation of the middle east respiratory syndrome coronavirus and the hajj middle east respiratory syndrome (mers) coronavirus. what travel health advice should be given to hajj pilgrims preventive measures against mers-cov for hajj pilgrims the hajj pilgrimage and surveillance for middle east respiratory syndrome coronavirus in pilgrims from african countries potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj , despite a high rate of respiratory symptoms lack of mers coronavirus but prevalence of influenza virus in french pilgrims after prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the hajj from the hajj: it's the flu, idiot high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana circulation of respiratory viruses among pilgrims during the hajj pilgrimage saudi arabia, south korea preliminary epidemiological assessment of mers-cov outbreak in south korea lessons to learn from mers-cov outbreak in south korea spread of mers to south korea and china list of hospitals with known mers exposure preliminary data from sequencing of viruses in the republic of korea and the people's republic of china. mers-cov situation assessment world health organization. managing contacts in the mers-cov outbreak in the republic of korea geneva: who. available at middle east respiratory syndrome coronavirus (mers-cov): summary and risk assessment of current situation in the republic of korea and china e as of korean society for healthcare-associated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea dangers and opportunities for social media in medicine social media and health care professionals: benefits, risks, and best practices social media and clinical care: ethical, professional, and social implications middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd key: cord- - zjpqnqp authors: hoteit, rouba; shammaa, dina; mahfouz, rami title: use of the human coronavirus (mers) genesig kit for mers-cov detection date: - - journal: gene rep doi: . /j.genrep. . . sha: doc_id: cord_uid: zjpqnqp introduction: mortality due to mers-cov infection is common especially among immunocompromised patients. the pathogenesis and the transmission mode of this virus are still not well understood. the name of the virus is derived from the area of its appearance and the genomic sequence that was used in the development of qrt-pcr assays for mers-cov detection was retrieved from the first detected case isolate. the employed assays target various regions including the area upstream of the envelope gene (upe) that is used for screening and the open reading frames (orf) a and b used for confirmation. aim: this study assesses the use of a mers-cov specific assay for screening of respiratory samples in anticipation of the possible spread of the virus in the region. methods: respiratory specimens were tested using the qualitative one-step qrt-pcr genesig human coronavirus (mers) kit (primerdesign™). results: out of the tested samples, were negative for mers-cov and one sample was found mers-cov positive. conclusion: the genesig human coronavirus (mers) kit is very useful for the screening of suspected respiratory cases in the middle east area as well as other regions. the middle east respiratory syndrome (mers) is a novel coronavirus first discovered in september in a cell-culture from a patient after his death from pneumonia in june in saudi arabia (assiri et al., ) . mortality due to mers-cov infection is common especially among immunocompromised patients (al-abdallat et al., ) . coronaviruses are the largest positive single-stranded rna viruses with a genome that can reach up to kb long. the genome entails a ′-terminal cap structure and is polyadenylated at its ′ end, and is occupied by two large overlapping open reading frames, orfs a and b (subissi et al., ) . mers-cov is part of the c lineage of the betacoronavirus genus and it is related to the hku and hku coronavirus detected in bats (omrani et al., ) . mers-cov and highly homologous mers-like cov were isolated in dromedary camels and african neoromicia capensis bats, respectively. although mers-cov is known to have zoonotic origins the pathway of transmission from the animal reservoir to humans is still not well understood. it is, however, suggested that a bat to camel to human route could explain the mers-cov outbreak in humans, due to the emerging camel trade between equatorial africa and saudi arabia during the last years (gralinski and baric, ) . mers-cov causes severe lower respiratory tract infection that sometimes requires admission to the intensive care unit. patients usually respond well to alpha interferon or cyclosporine treatment, however, no specific antiviral or vaccine is available for mers-cov (ohnuma et al., ) . more than cases from countries worldwide have been infected and diagnosed with mers-cov since june , and almost a third of those died (saad et al., ; who, c ) . patients with mers-cov infection tend to have leukocytosis, lymphopenia and some express thrombocytopenia and coagulopathy. moreover, creatinine levels, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase levels may be elevated and thus implying a liver or kidney disease (van den brand et al., ) . males and elderly are more commonly infected with mers-cov than females and younger people, and human-to-human transmission in the community is mainly in healthcare settings where poor infection control measures are implemented especially in intensive care units, and where overcrowding of patients may occur (saad et al., ) . other reported cases of mers-cov, especially the ones reported in europe or the us are more likely associated with the travel history to the middle east. the rate transmission of the virus from infected patients to their household contacts is as low as % (drosten et al., ) . rapid detection and implementation of proper infection control measures are two essential steps to limit and control mers-cov transmission in healthcare settings. this includes applying contact and airborne precautions such as wearing gloves, gowns and surgical masks upon entry to a room of mers-cov infected patient in isolation and removing such personal protective equipment upon leaving the room (zumla and hui, ) . protective measures outside healthcare settings include avoiding contact with dromedary camels, the host of mers-cov, and restraining from drinking camel urine or raw milk or eating raw or undercooked camel meat. moreover people working with camels must wear protective clothing and facial masks, and maintain a good personal hygiene (reusken et al., ) . ideally, lower respiratory specimens such as sputum, bronchoalveolar lavage, bronchial wash and tracheal aspirates are the most representative and appropriate for mers-cov testing (cdc, june , ) . the name of the virus is derived from the area of its appearance and the genomic sequence that was used in the development of qrt-pcr assays for mers-cov detection was retrieved from the first detected case isolate (lu et al., ) . the employed assays target various regions including the area upstream of the envelope gene (upe) that is used for screening and the open reading frames (orf) a and b used for confirmation . the aim of this study was to assess the use of a mers-cov specific assay for screening of respiratory samples referred to a major tertiary care center in anticipation of the possible spread of the virus in the region. rna was extracted from respiratory specimens using the qiaamp viral rna mini kit (qiagen, hilden, germany) that does not use phenol/ chloroform extraction or alcohol precipitation protocols. briefly, a lysis buffer was used to isolate non-degraded viral rna and inactivate rnases. the sample was then transferred to the qiaamp mini spin column under optimal conditions that ensured effective binding of the rna to the qiaamp membrane. possible contaminants were removed using two different wash buffers, then, a rnase-free buffer was employed to elute the rna. mers-coronavirus qualitative one-step qrt-pcr testing was done using the genesig human coronavirus (mers) kit (primerdesign™, london, uk). the hcov primers are designed for the specific and exclusive in vitro detection of the novel coronavirus isolates based on sequences for the emc strain by targeting the orf ab and the upe regions of orf . the orf ab and orf /e primer and probe sets are designed to be specific to the mers strain regardless of the species of origin and are detected through the fam channel. an internal rna extraction control is available in the kit and was added to the lysis buffer as an external source of rna template, during the extraction step, and co-purified with the sample rna to monitor this process as well as to rule out the presence of inhibitors in the realtime pcr step. for the latter purpose, a separate mix of primers for the internal control, with limited concentrations that allow for multiplexing with the target sequence primers without affecting the ability to detect the target even when present in low concentrations, was used. the detection of the internal control was through the vic channel. moreover, a primer mix for the endogenous control actin beta (actb) gene was used to evaluate the quality of the original biological sample. the detection of actb is through the vic channel, thus a separate reaction mix than that of the target is necessary to test for actb as multiplexing for actb and hcov primers is not possible. for each sample, three reaction mixes were prepared. for the detection of orf ab and orf /e two μl reaction tubes were prepared as follows: μl of oasig™ qrt-pcr mastermix, μl of hcov_ (orf ab or orf /e) primer/probe mix, μl of internal extraction control primer/probe mix, μl of rnase/dnase free water and μl rna material. to detect the endogenous actb control, a separate μl reaction mix was prepared, containing, μl of oasig™ qrt-pcr mastermix, μl of endogenous actb primer/probe mix, μl of rnase/dnase free water and μl rna material. the amplification conditions involved a reverse transcription step at °c for min, an enzyme activation step at °c for min and cycles of °c for s followed by °c for s. a positive control for orf ab and orf /e, as well as a negative control (rnase/dnase free water) was used with each run. the test requires min hands-on time and the pcr protocol lasts two hours after which the rotorgene-q (qiagen, uk) software is used for analysis. out of the tested samples, were negative for mers-cov and one sample was found mers-cov positive. the positive result was validated by the naval medical research unit (namru) laboratory, located in cairo-egypt, in collaboration with the world health organization (who). all other results were validated with a cap accredited referral laboratory in germany and the concordance rate was %. fig. represents a graph of the amplification of the mers-cov rna and the endogenous actb in the mers-cov positive sample. positive and negative controls were used and signal detection was done on the fam channel. the graph in fig. represents the detection of the internal rna extraction control (iec) in the fluorescence channel vic, for the same positive sample. iec was only added to the sample during extraction and not to the positive or negative controls. an acceptable ct value of the iec ranges between and . the genesig human coronavirus (mers) kit is a rapid and useful kit for the screening of suspected respiratory cases in the middle east area as well as other regions. the collaboration of different molecular microbiology laboratories with the world health organization (with viral cultures done as gold standard reference testing) has always been a rewarding and teaching experience. no extensive sensitivity and specificity work-up has been performed from our side on multiple samples due to the presence of a single mers-cov positive case; however, our lower limit of detection is compatible with the copies/ml reported by the manufacturer. the clinicians have advised that this detection limit is acceptable for their management algorithms. to our knowledge, this is the first report that describes the clinical application of the genesig kit in the mers-cov epidemic era. hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide transmission of mers-coronavirus in household contacts molecular pathology of emerging coronavirus infections real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia inhibition of middle east respiratory syndrome coronavirus infection by anti-cd monoclonal antibody a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case rna and neutralising antibodies in milk collected according to local customs from dromedary camels clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities middle east respiratory syndrome coronavirus (mers-cov) c who infection control and mers-cov in health-care workers key: cord- -row mn authors: chan, jasper fuk-woo; lau, susanna kar-pui; woo, patrick chiu-yat title: the emerging novel middle east respiratory syndrome coronavirus: the “knowns” and “unknowns” date: - - journal: j formos med assoc doi: . /j.jfma. . . sha: doc_id: cord_uid: row mn a novel lineage c betacoronavirus, originally named human coronavirus emc/ (hcov-emc) and recently renamed middle east respiratory syndrome coronavirus (mers-cov), that is phylogenetically closely related to tylonycteris bat coronavirus hku and pipistrellus bat coronavirus hku , which we discovered in from bats in hong kong, has recently emerged in the middle east to cause a severe acute respiratory syndrome (sars)-like infection in humans. the first laboratory-confirmed case, which involved a -year-old man from bisha, the kingdom of saudi arabia (ksa), who died of rapidly progressive community-acquired pneumonia and acute renal failure, was announced by the world health organization (who) on september , . since then, a total of cases, including fatalities, have been reported in the middle east and europe. recent clusters involving epidemiologically-linked household contacts and hospital contacts in the middle east, europe, and africa strongly suggested possible human-to-human transmission. clinical and laboratory research data generated in the past few months have provided new insights into the possible animal reservoirs, transmissibility, and virulence of mers-cov, and the optimal laboratory diagnostic options and potential antiviral targets for mers-cov-associated infection. introduction: the "new sars"? ten years after the devastating epidemic of severe acute respiratory syndrome (sars) caused by sars coronavirus (sars-cov), which resulted in a total of deaths among more than confirmed cases in over countries, the world is facing a new challenge posted by a "sars-like" infection caused by another novel coronavirus emerging from the middle east, which was originally named human coronavirus emc/ (hcov-emc) and recently renamed by the coronavirus study group of the international committee for taxonomy of viruses as middle east respiratory syndrome coronavirus (mers-cov). e the complete genome of the virus was sequenced and released in october after the isolation of the virus from two patients with severe community-acquired pneumonia in bisha, the kingdom of saudi arabia (ksa), and doha, qatar, first announced by the world health organization (who) on september . as of may , , the total number of laboratory-confirmed cases of mers-cov infection has increased to with deaths, including two cases confirmed retrospectively from a jordanian cluster of severe respiratory disease reported by the ministry of health of jordan in april (table ) . , , e although the number of laboratory-confirmed cases remains limited, the severe clinical manifestations with an unusually high mortality rate of over %, the spread of the infection beyond the geographical confinement in the middle east, and the epidemiological evidence of human-to-human transmission arising from the recent clusters of cases in a family in the united kingdom (cases to ), and in hospitals in ksa (cases to , and ) and france (cases and ), have raised significant concerns on the possible emergence of another sars-like epidemic in the near future. in anticipation of the potential spread of this highly pathogenic virus from the middle east and europe to other parts of the world, especially the densely populated southeast asia, an updated review of the latest research findings on mers-cov and their implications on the clinical management of mers-cov infection is essential. viral genomic studies reveal the first lineage c betacoronavirus associated with human infection mers-cov belongs to the genus betacoronavirus in the family coronaviridae under the order nidovirales. coronaviruses are enveloped viruses with positive-sense singlestranded rna genomes. studies in their biodiversity, comparative genomics and phylogeny in the past years have improved our understanding of this family of viruses. e according to the most recent classification by the coronavirus study group of the international committee for taxonomy of viruses, there are four genera in coronaviridae, namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. , the genus alphacoronavirus contains two human coronaviruses, hcov- e and hcov-nl , which are associated with the common cold. no human coronavirus has been discovered in gammacoronavirus and deltacoronavirus, which mainly contain avian coronaviruses with just a few mammalian coronaviruses. the genus betacoronavirus is comprised of four lineages (a, b, c and d) which contain four coronaviruses associated with human infections: hcov-oc and hcov-hku (lineage a), sars-cov (lineage b), and mers-cov (lineage c). mers-cov is the first known lineage c betacoronavirus associated with human infection and is phylogenetically closely related to the other lineage c betacoronaviruses including tylonycteris bat cov hku (ty-batcov-hku ) and pipistrellus bat cov hku (pi-batcov-hku ), which were discovered in lesser bamboo bats (tylonycteris pachypus) and japanese pipistrelle bats (pipistrellus abramus), respectively, captured in hong kong, china. , , analysis of the genome of mers-cov revealed that it has a genome size of , bases with the rdrp and s genes having over % and around % amino acid identities with those of ty-batcov-hku and pi-batcov-hku . , molecular clock analysis using the rdrp gene showed that mers-cov might have diverged from the most recent common ancestor of lineage c betacoronaviruses in year w ad ( bc to ad). it is postulated that the emergence of mers-cov represents another series of interspecies transmission events in coronaviruses, from bats to possibly other animals and then to humans, a scenario similar to the sars epidemic. epidemiology reports and cell line susceptibility studies suggest possible animal reservoirs and human-to-human transmissions epidemiological linkage with animals, including camels, goats, sheep, and farm animals or their caretakers before symptom onset in some of the reported cases supported the hypothesis of mers-cov being a zoonotic agent (table ) . furthermore, in vitro data from cell line susceptibility studies showed that the virus had a broad species tropism and was able to replicate in bat, primate, porcine, rabbit, and civet cell lines. , as in the case of sars-cov, which likely emerged from its natural animal reservoir the horseshoe bats (rhinolophus sp.), and jumped to other mammals during their caging in the wild life markets of south china and then to humans, mers-cov might have also originated from bats to these susceptible animal species before adapting to humans. , , in addition to pipistrellus bats, which are the natural hosts of the closely related pi-batcov-hku and are also found in the middle east, rousettus, rhinolophus, myotis, and carollia bat cell lines are also susceptible to mers-cov infection in vitro. active surveillance of different bat and animal species predominantly found in the middle east would help to delineate the natural bat reservoir and the evolutionary pathway of mers-cov among other susceptible animal species. indeed, a recent study showed that a number of different bat species in ghana and europe are infected with coronaviruses, which also share close homologies with mers-cov. determination of the virus' animal hosts might in turn facilitate the control of the outbreak as in sars, where closure of the wet markets likely contributed to the cessation of the epidemic. human-to-human transmission represents a new stage in the evolution of mers-cov infection. there are, so far, six the seroprevalence and transmissibility of mers-cov remain undetermined, although animal-to-human and human-to-human transmissions both appear to be limited at this stage (fig. ) . among persons seeking medical attention in a hospital in jeddah, ksa, none had mers-covspecific igg detectable by indirect immunofluorescence assay, suggesting that the current situation is likely to be different from those of other human coronaviruses which are endemic in humans. , the lack of secondary cases among nearly contacts of cases and who did not practice optimal infection control measures before the diagnosis of mers-cov infection was confirmed, implied that this novel virus might be less efficient than sars-cov in human-tohuman transmission. , , however, the findings of these studies should be interpreted with cautions for two reasons. first, only a relatively small number of subjects ( / in case and / in case ) were tested by reliable laboratory tests. second, the timing of testing was not clearly described and might be suboptimal for the purpose of excluding the diagnosis. it has been proposed that a second test should be performed in symptomatic patients with initially negative results by reverse transcriptionpolymerase chain reaction (rt-pcr), within the first days of symptom onset, based on the observation in sars where viral load peaked at day of symptom onset. , e therefore, the apparently limited spread of mers-cov at present might be an underestimation and ongoing transmission of the virus should be cautiously monitored. unlike its close relatives in bats, mers-cov is highly pathogenic in humans. the most common clinical presentation among the laboratory-confirmed cases of mers-cov infection is acute severe community-acquired pneumonia with acute renal failure following an estimated incubation period of e days (table ) . , this is unusual among human infections caused by coronaviruses, in which severe pneumonia with respiratory failure is seldom seen, except in sars. the other human coronaviruses, namely hcov- e, hcov-nl , hcov-oc , and hcov-hku , predominantly cause acute self-limited upper respiratory tract infections, and only occasionally cause lower respiratory tract infections in the elderly and immunocompromized populations. only two of patients (cases and ) had a self-limiting, mild, influenza-like illness not requiring hospitalization. asymptomatic or mild infections have otherwise not been detected among the other contacts of confirmed cases by rt-pcr of upper respiratory tract specimens and/or serological tests, saudi residents in jeddah by indirect immunofluorescent antibody testing of archived sera, and french hajj pilgrims with upper respiratory symptoms and rt-pcr of prospectively collected nasal swabs. , , , the other ( . %) patients, many of whom had no underlying medical condition, developed rapid clinical deterioration with lower respiratory tract involvement and respiratory failure requiring ventilator support within a few days to week after initial systemic symptoms of fever, myalgia, and malaise, and upper respiratory tract symptoms such as rhinorrhea and sore throat. this correlated with the finding in our recent cell line susceptibility study, which showed that mers-cov replicated much better in the lower respiratory tract cell lines including calu- (polarized airway epithelium), a (lung adenocarcinoma), and hfl (embryonic lung fibroblasts), than the upper respiratory tract cell line hep- (laryngeal epidermoid carcinoma). another in vitro study also showed that pseudostratified human bronchial epithelium cultures are highly permissive to mers-cov infection. in ex vivo organ cultures, mers-cov productively replicated in both human bronchial and lung tissues, whereas sars-cov only productively replicated in lung tissue. recently, a macaque model showed that mers-cov caused acute, localized to widespread pneumonia, resulting in mild to moderate clinical disease resembling the illness observed in humans. radiologically, pneumonia is evident by focal consolidations involving single or multiple lobes, with progressive involvement of bilateral lung fields, especially the lower zones. in case , thoracic computed tomography scans revealed mediastinal hilar lymphadenopathies, airspace opacities with air bronchograms, scattered ground-glass opacities, interstitial septal thickenings, and nodularities in the upper lobes without significant pleural and pericardial effusions. acute renal failure was the other dominant clinical feature in mers-cov infection and is seen in at least six of the reported cases. many of the remaining severe cases probably also developed renal impairment, although their clinical details were not available. this was unusual, even when compared to sars in which . % of the patients had abnormal urinalysis and detectable viral load by quantitative rt-pcr in urine, but only . % developed acute renal failure with histological evidence of acute tubular necrosis and most did not require renal replacement therapy. this clinical presentation correlates with the in vitro finding of efficient replication of mers-cov in kidney cell lines, including hek , vero, llc-mk , and p. , , more importantly, the presence of renal involvement appeared to be a poor prognostic factor, as those with renal failure either died or required renal replacement therapy, while two cases without renal impairment survived ( table ). the lack of extrapulmonary lesions observed in the macaque model of mers-cov infection suggested that acute renal failure was more likely due to hypoxic damage than a direct viral cytopathic effect. other clinical, laboratory and microbiological findings reported in mers-cov infection included pericarditis, disseminated intravascular coagulation, leukocytosis with neutrophilia and lymphopenia, thrombocytopenia, anemia, hyponatremia, hypoalbuminemia, elevated liver enzymes, lactate dehydrogenase, c-reactive protein, and procalcitonin levels, possible secondary bacterial pneumonia caused by klebsiella pneumoniae, staphylococcus aureus, and acinetobacter sp., and coinfection with other respiratory viruses, including influenza a(h n )pdm and type parainfluenza virus. , , e , it is interesting to note the absence of watery diarrhea in the acute phase of mers-cov infection, despite the in vitro finding of viral replication in the colonic cell line caco- (colorectal adenocarcinoma), in contrast to sars in which around % of patients developed enterocolitis. , it remains to be seen in future case cohorts whether this is due to under-reporting, or a genuine difference in clinical presentations between the two diseases. together with severe acute respiratory and renal failure, these clinical features underscore the success of the innate immune evasion mechanisms of mers-cov in humans, leading to overwhelming infection and possible cytokine dysregulation, as reflected by the lack of interferon through inhibition of interferon regulatory factor family (irf- ). , the discovery of dipeptidyl peptidase (dpp ), a multifunctional -amino-acid-long type-ii transmembrane glycoprotein exopeptidase expressed on human non-ciliated bronchial, renal, enteric, hepatic and prostatic epithelial cells, which is important in the regulation of hormone and chemokine bioactivity, glucose metabolism, t-cell activation, chemotaxis modulation, cell adhesion, apoptosis and regulation of tumorigenicity, as a functional receptor for mers-cov, provides further insights into the unique pattern of organ involvement and unusually severe clinical presentation of this emerging infection. clinical utility and practical concerns of published laboratory diagnostic options several laboratory methods are available for establishing a virological diagnosis of mers-cov infection. the most definitive tests are viral culture from respiratory, fecal, urine, or tissue specimens, and/or a fourfold rise in the serum neutralizing antibody titers taken at to days apart. the first clinical isolate of mers-cov was cultured on monkey kidney cells, like vero and llc-mk , which showed cytopathic effects of syncytium formation, rounding and detachment of cells. subsequent studies have identified various human cell types, including bronchial epithelial, colonic, hepatic, renal, and neuronal cells, monocytes and histiocytes that support the replication of mers-cov. however, the use of viral culture is limited by the requirement of a biosafety level three setting, which is not available in most clinical microbiology laboratories. a serum neutralizing antibody test has the disadvantage of requiring convalescent sera and is therefore mainly used for diagnosis in the convalescent instead of acute phase. , furthermore, the sera of sars patients may contain lowtiter cross-reactive neutralizing antibodies against mers-cov, which may lead to the wrong serodiagnosis, especially in countries where the general population had previous exposure. it remains to be seen whether neutralizing antibodies against mers-cov might also crossreact with other closely related lineage c betacoronaviruses, like ty-batcov-hku and pi-batcov-hku . in most of the laboratory-confirmed cases, diagnosis was established by the detection of nucleic acid by rt-pcr of respiratory tract samples, including combined nose and throat swab, sputum, tracheal aspirate, bronchoalveolar lavage, and/or urine taken between day (cases and ) and day (case ). two highly sensitive real-time rt-pcr assays targeting regions upstream of the e gene (upe), with sensitivity of up to . rna copies per reaction and within an open reading frame (orf) b, with sensitivity of up to rna copies per reaction, have been proposed for screening and confirmation of mers-cov infection, respectively, and are available in about half of the countries in the who european region. , additional testing of other gene targets with partial or whole genome sequence analysis may also be used for confirmation. , for example, pan-coronavirus rt-pcr targeting the rdrp gene was used in case and a real-time quantitative rt-pcr assay targeting the n gene has been used in in vitro studies, and may also be useful for clinical diagnosis, although further evaluations are needed. other potential diagnostic options which might be used in areas without rt-pcr are n or s protein-based enzyme-linked immunosorbent assays, which were shown to be highly sensitive and/ or specific for the diagnosis of sars. of note, the detection of other respiratory viruses in the respiratory tract specimen does not preclude the need for specific mers-cov testing in patients with epidemiological risk factors, or unusually severe disease, despite antiviral treatment as exemplified by the presence of coinfections in cases e . the exceptionally high crude mortality rate of % in mers-cov infection is partly due to the lack of specific anticoronavirus treatment and effective vaccine. in most of the cases, intensive supportive treatment with extracorporeal membrane oxygenation, renal replacement therapy, and empirical broad-spectrum antibacterial and antiviral agents were used. case , who is still in a critical condition nearly months after symptom onset, also received a corticosteroid in the initial phase of treatment, but its efficacy is unknown. its use might be limited by serious side effects and the availability of ecmo for organ support during the critical phase. alternatively, type i interferons appear to be promising therapeutic options, as mers-cov has been shown to be much more sensitive than sars-cov to the antiviral action of interferon in vitro. the replication of mers-cov was shown to be reduced in human lung ex vivo organ cultures treated with type i interferons. a recent study showed that while both interferon-a b and ribavirin reduced the replication of mers-cov in vero and llc-mk cells, the combination of the two drugs achieved the same endpoints at a much lower concentration, which might facilitate their clinical applications. other potential specific antiviral targets include type ii transmembrane serine proteases (tmprss ) and endosomal cathepsins, which are responsible for mers-cov s protein activation required for virus-cell fusion and entry of the virus into host cells. the identification of dpp , but not angiotensinconverting enzyme (ace ), aminopeptidase n, and carcinoembryonic antigen-related cell adhesion molecule (ceacam ), as a functional receptor for mers-cov implies that in vivo manipulation of dpp levels and development of inhibitors against the s domain-dpp interface might have potential therapeutic roles in mers-cov infection, as with ace analogues in the case of sars. finally, the recognition of the predicted receptor-binding domain /critical neutralizing domain at residues to in the mers-cov s protein might facilitate vaccine development for this emerging infection. in the past few months, following the who's announcement of the first two cases of mers-cov infection on september , , clinicians and scientists worldwide have collaborated closely in an attempt to prevent a sars-like epidemic from happening again. the discovery of certain important characteristics of mers-cov including its genome arrangement, phylogenetic relatedness with other coronaviruses, in vitro tissue and species tropism, and functional receptor, enhanced our understanding of the clinical presentation, pathogenesis, epidemiology, and design of diagnostic and therapeutic options of this emerging novel human coronavirus. however, key questions concerning the definitive animal reservoirs of the virus, evolutionary process, transmissibility, and the prognostic factors and optimal treatment modalities of the infection, remain elusive. more importantly, the increasing number of laboratory-confirmed cases in the middle east, and the recent evidence of human-tohuman transmission in europe are suggestive of continuing viral adaptations in humans, which might precede a largescale epidemic. indeed, as of june , after the acceptance of the article, the total number of laboratoryconfirmed cases have increased to with fatalities. additional clusters of cases involving household and/or hospital contacts were reported in ksa, italy, and tunisia. collaborations between global and local health authorities and their sustained support for further research on mers-cov are crucial to control this "new sars". severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection coronavirus as a possible cause of severe acute respiratory syndrome the severe acute respiratory syndrome relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sars-like pandemic isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans latest outbreak news from promed-mail: novel coronavirus e middle east recovery from severe novel coronavirus infection evidence of person-to-person transmission 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tmprss and is targeted by neutralizing antibodies detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction laboratory capability for molecular detection and confirmation of novel coronavirus in europe inhibition of novel b coronavirus replication by a combination of interferon-a b and ribavirin a predicted receptor-binding and critical neutralizing domain in s protein of the novel human coronavirus hcov-emc global alert and response (gar): middle east respiratory syndrome coronavirus (mers-cov) e update key: cord- - kuh njb authors: elkholy, amgad a.; grant, rebecca; assiri, abdullah; elhakim, mohamed; malik, mamunur r.; van kerkhove, maria d. title: mers-cov infection among healthcare workers and risk factors for death: retrospective analysis of all laboratory-confirmed cases reported to who from to june date: - - journal: j infect public health doi: . /j.jiph. . . sha: doc_id: cord_uid: kuh njb background: approximately half of the reported laboratory-confirmed infections of middle east respiratory syndrome coronavirus (mers-cov) have occurred in healthcare settings, and healthcare workers constitute over one third of all secondary infections. this study aimed to describe secondary cases of mers-cov infection among healthcare workers and to identify risk factors for death. methods: a retrospective analysis was conducted on epidemiological data of laboratory-confirmed mers-cov cases reported to the world health organization from september to june . we compared all secondary cases among healthcare workers with secondary cases among non-healthcare workers. multivariable logistic regression identified risk factors for death. results: of the laboratory-confirmed mers-cov cases reported to who, were healthcare workers and were non-healthcare workers. compared with non-healthcare workers cases, healthcare workers cases were younger (p < . ), more likely to be female (p < . ), non-nationals (p < . ) and asymptomatic (p < . ), and have fewer comorbidities (p < . ) and higher rates of survival (p < . ). year of infection ( – ) and having no comorbidities were independent protective factors against death among secondary healthcare workers cases. conclusion: being able to protect healthcare workers from high threat respiratory pathogens, such as mers-cov is important for being able to reduce secondary transmission of mers-cov in healthcare-associated outbreaks. by extension, reducing infection in healthcare workers improves continuity of care for all patients within healthcare facilities. middle east respiratory syndrome coronavirus (mers-cov) was first detected in a patient living in saudi arabia in september of [ ] . subsequent cases have included human infections across the arabian peninsula, occasional importation of cases outside the arabian peninsula and associated clusters in other regions of the world. outbreaks of non-sustained, human-to-human transmission have occurred primarily in healthcare settings [ ] . while mers-cov appears to be inefficient at transmitting between humans in the general community, about half of the reported mers-cov infections have occurred in healthcare settings [ ] . healthcare-associated transmission of mers-cov has been reported in france, jordan, saudi arabia, united arab emirates, republic of korea and the united kingdom and has on occasion resulted in large outbreaks [ ] [ ] [ ] [ ] [ ] [ ] [ ] . secondary transmission has occurred between patients, from patients to healthcare workers, and from patients to visitors of the hospital. to date, there has been limited evidence of transmission documented between healthcare workers in saudi arabia [ ] and anecdotal evidence from healthcare workers to patients in saudi arabia [ ] . because hcw have not been consistently tested for mers-cov infection, nor has detailed outbreak investigations occurred within all hospital outbreaks, the role of healthcare workers in onward transmission remains unclear. given the large number of hcws infected to date, it is possible that hcw can propagate an outbreak within a healthcare facility. among reported secondary mers cases in such outbreaks, a substantial proportion have been healthcare workers [ , , [ ] [ ] [ ] [ ] [ ] . the clinical spectrum of mers-cov infection ranges from asymptomatic infection to severe pneumonia with acute respiratory distress syndrome and other life-threatening complications [ ] [ ] [ ] [ ] . mild symptoms are non-specific and can include headache, tiredness, fever, mild cough, sore throat and runny nose. some patients may present with gastrointestinal symptoms, including diarrhoea. the non-specificity of mers signs and symptoms poses several challenges, not only for the timely identification and isolation of infected patients, but also for reducing secondary transmission within the healthcare facility, particularly to healthcare workers and between patients. preventing mers-cov infection in healthcare workers is critical because of their role in the clinical management of patients and in ensuring adequate infection prevention and control measures are implemented in healthcare facilities. unnecessarily exposing healthcare workers to mers-cov affects the safety of both the healthcare workers and other patients in the healthcare facility, potentially propagating secondary transmission in healthcare-associated outbreaks. understanding mers-cov infection in healthcare workers to date and the risk factors for adverse outcomes is important for preventing future infection of healthcare workers, for informing and updating infection prevention and control measures in healthcare facilities and for reducing secondary mers-cov transmission within healthcare settings. the international health regulations [ ] require all laboratoryconfirmed cases of mers-cov to be reported to the world health organization (who) within h of laboratory confirmation [ ] . in this study, we use the epidemiological data of all mers cases reported to date to who to describe secondary cases of mers-cov infection among healthcare workers and to identify the risk factors for death among healthcare workers with secondary infection. a retrospective analysis was conducted on epidemiological data of laboratory-confirmed mers-cov cases reported to who [ ] from september to june . we looked specifically at healthcare workers involved in the delivery of health care to patients within the same healthcare facility as a confirmed or suspected mers-cov case. primary cases were defined as cases with laboratory confirmation of mers-cov infection with no epidemiological link to a suspected or confirmed human mers case. secondary cases were defined as those with laboratory confirmation of mers-cov infection and with a direct epidemiological link with a confirmed or probable mers-cov case. prior to , data from individual cases including occupation, signs/symptoms, exposures and risk factors for infection, etc. was not collected systematically. following the large mers outbreak in jeddah and riyadh in , case report forms and policies related to the investigation of cases and contacts became more systematic from onwards. given the inconsistencies in the collection and reporting of epidemiologic data from mers-cov cases to who prior to , we performed a secondary analysis with data reported only from january to june . descriptive analysis was performed for all mers-cov cases reported to who from to . for all statistical analyses, p < . was considered statistically significant and all analyses were performed using the epidemiological data display package in r, version . . . (https://cran.r-project.org/ package=epidisplay). we compared all secondary cases in healthcare workers with all secondary cases among non-healthcare workers, using the student t-tests for continuous variables and chi-squared tests for categorical variables. we also aggregated survival outcomes of healthcare workers with mers-cov infection by year of infection. to identify risk factors for adverse outcomes in healthcare workers with secondary infection, we performed multivariable logistic regression analysis on all healthcare worker secondary cases. variables considered were dichotomous (sex, residency, symptomatic clinical presentation, presence of any comorbidities), categorical (year of infection) or continuous (age). the final multivariable model, constructed using the backward, stepwise elimination method, included all variables with an adjusted p < . . our analysis considered all cases of mers-cov reported to who up to june . among these cases, were primary cases, secondary cases, cases for which the information reported was insufficient to be able to determine whether it was a primary or secondary case, and cases with missing case information. among all cases, ( . %) deaths occurred. of the cases, were reported as healthcare workers. the mean age of healthcare workers was . (interquartile range . - . ) years and . % were women. five were primary cases, were secondary cases, cases had insufficient information to be able to determine whether it was a primary or secondary case and cases had missing case information. fig. shows the epidemic curve of cases of mers-cov among healthcare workers and non-healthcare workers reported from to june . table provides further description of the healthcare worker cases, as well as the healthcare worker cases reported to who from january to . table compares all secondary healthcare worker cases and all secondary non-healthcare worker cases. compared with secondary non-healthcare worker cases, secondary healthcare worker cases were younger (p < . ), had a higher proportion of women (p < . ), were non-national residents (p < . ), and had asymptomatic infection (p < . ), fewer comorbidities (p < . ) and higher survival (p < . ). further comparison between laboratory-confirmed mers-cov cases in healthcare workers and non-healthcare workers are shown in the supplementary material. tables a and b shows the survival outcomes of all infections by year of infection. tables a shows outcomes of all infections by year of infection for healthcare workers, while table b shows outcomes of all infections by year of infection for non-healthcare workers. there have been no fatal mers infections among healthcare workers since . table shows the regression coefficients and adjusted odds ratios ( % confidence interval) for the two variables retained in the final multivariable risk model for death in secondary cases among healthcare worker. year of infection and having no comorbid conditions were found to be independent protective factors against death in healthcare workers with secondary mers-cov infection. healthcare workers include, but are not restricted to: doctors, nurses, pharmacists, physiotherapists, radiologists, rehabilitation staff, infection prevention and control staff, intensive care staff, ambulance staff, respiratory therapists, auxiliary healthcare workers, attendants, laboratory, x-ray and ultrasound technicians, and healthcare administrators. a lack of consistency in reporting specific job titles prohibited subgroup analysis by different roles of healthcare worker. healthcare workers continue to constitute a substantial proportion of secondary mers-cov infections. that is, among all cases of mers-cov reported to who as of june , healthcare workers accounted for . % of all mers-cov cases and . % of secondary mers-cov cases. this is similar to other respiratory pathogens: in the outbreak of severe acute respiratory syndrome, % of cases in hong kong, % in canada and % in singapore were healthcare workers [ ] . protecting health care workers from infectious hazards is paramount to ensuring their safety in delivering health care. in addition, being able to protect healthcare workers, constituting the front line response against high-threat respiratory pathogens, such as mers-cov, is important for reducing secondary transmission in healthcare-associated outbreaks. reducing infection in healthcare workers, even if their infection does not cause morbidity or mortality, will improve the continuity of care for all patients in the same healthcare facility. we also found that the demographic and clinical profile of secondary infections in healthcare workers was different from secondary infections among non-healthcare workers. healthcare workers infected with mers-cov were younger, more were female and non-national residents, and had fewer comorbidities, more asymptomatic infections and higher survival. importantly, no deaths have occurred among healthcare workers with secondary mers infection since the end of . these results are similar to the findings in individual outbreaks reports [ , , [ ] [ ] [ ] [ ] [ ] [ ] ] . the highest burden of mers cases to date is in saudi arabia where healthcare workers are more likely to be female, a large proportion of whom are expatriates. the younger age and fewer comorbidities may partly explain the higher survival observed among healthcare workers, as well as the possibility of earlier identification or suspicion of mers among healthcare workers. indeed, the case fatality rate among all healthcare workers was . % compared with . % among all non-healthcare workers. this is the first study to perform multivariable logistic regression to identify risk factors for death among healthcare workers with secondary infections reported to date. the analysis showed that year of infection ( - ) and having no comorbidities were independent protective factors against death. the decline in risk of death since may reflect the substantial improvements in surveillance and the infection prevention and control measures that have been introduced in affected countries in recent years. in saudi arabia, for example, the ministry of health regularly review and update national infection prevention and control guidelines, according to which, healthcare workers who had unprotected "high-risk exposure" (within . m of the patient) or have suggestive symptoms regardless of exposure type are required to stop performing their duties immediately; to have a nasopharyngeal swab tested for mers-cov; to not resume their duties until cleared by the infection control team and to delay travel until cleared by infection control team [ ] . any healthcare worker who tests positive for mers, any healthcare worker who develops mers suggestive symptoms and any healthcare worker who had unprotected high-risk exposure are considered clear and able to resume work if they meet all of the following criteria: asymptomatic for at least h, and the -day observation period is over, and they have at least one negative rt-pcr [ ] . the enhanced infection prevention and control (ipc) efforts which have been introduced since include regular training of healthcare workers on ipc, auditing of ipc in healthcare facilities, improved case notification and isolation within emergency departments, and comprehensive contact-tracing and testing of all contacts, including healthcare workers, regardless of the development of symptoms. guidelines on contact tracing were revised after to include the testing of all contacts of confirmed mers cases (including healthcare workers) for mers-cov. prior to , contacts were only tested if they developed symptoms. this has increased the detection of asymptomatic or mildly symptomatic cases, which in turn, may have decreased the case fatality rate. specific ipc measures which may have contributed to the decreased case fatality rate include more systematic use of appropriate personal protective equipment and increased testing of asymptomatic personnel which effectively increases the detection of asymptomatic healthcare workers who are less likely to die, therefore increasing the denominator of healthcare worker cases and decreasing the case fatality rate. in addition, earlier detection of cases, and more efficient contact tracing have likely identified mers-cov infections in healthcare workers at an earlier stage of infection, enabling more timely treatment and clinical management, and, as a result, a reduction in the case fatality rate. further efforts to prevent and manage emerging respiratory disease infections, including mers among healthcare workers, still need to be made. these include improving identification and rapid diagnosis of mers, further understanding of the mecha- nisms of transmission in healthcare settings, optimizing the layout of emergency departments for better triage of patients with respiratory symptoms, standardization of infection prevention and control practices and (re)training at facilities with high hospital staff turnover, and auditing of healthcare facilities for adherence to infection prevention and control measures. these will be critical to minimizing transmission in healthcare facilities, particularly until interventions are introduced to stop the virus entering the human population from the dromedary camel reservoir. the role of environmental contamination was evaluated in a number of hospitals following the mers outbreak in the republic of korea and collaborative, experimental studies were conducted to evaluate the viability and persistence of mers-cov on surfaces and in the air [ ] [ ] [ ] . the role of mild or asymptomatic cases in transmission chains, however, remains unclear [ ] [ ] [ ] [ ] . further epidemiologi- outcomes cal and environmental studies to determine route(s) of secondary transmission will also be critical. the results of our study are strengthened by the size of the study, which included all cases reported to who since the first case was notified in . our database has cases from all countries reporting cases globally. however, since , some inconsistencies have occurred in the way data have been collected and reported to who, for example in the use of a systematic data collection tool and in reporting the outcome of all cases. challenges also exist in classifying cases based on the available information at the time of reporting. for example, thorough outbreak investigations, which include full genome sequencing to clarify the transmission chains within the outbreak or separate introductions from outside, may find that cases that were initially classified as secondary case are in fact primary cases [ , ] , but this information is not systematically relayed to who. efforts are currently being made to retrospectively review and update the epidemiological data for all cases reported to who to date, particularly before . healthcare workers still make up a significant proportion of secondary mers-cov infections. understanding transmission to healthcare workers, preventing infection and improving clinical management of infected healthcare workers will all be critical to further reducing the incidence of secondary infections in healthcare settings. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. who mers-cov global summary and assessment of risk epidemiological findings from a retrospective investigation hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia response to emergence of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission epidemiological investigation of mers-cov spread in a single hospital in south korea middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control molecular epidemiology of hospital outbreak of middle east respiratory syndrome clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study middle east respiratory syndrome a cohort study of patients suspected for mers-cov in a referral hospital in saudi arabia geneva: world health organization middle east respiratory syndrome case definition for reporting to who: interim case definition severe acute respiratory syndrome (sars) and healthcare workers middle east respiratory syndrome coronavirus: guidelines for healthcare professionals -version . environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers outbreak units stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection the authors would like to thank the many individuals involved in the collection of individual case data and in the care of mers-cov infected patients in the affected countries. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jiph. . . . all authors contributed to the study design, and reviewed and edited the manuscript. aae, rg and me performed the statistical analysis. all authors were involved in the interpretation of the findings. no funding sources. none declared. the case-patient data reported to who is anonymized, thus neither informed consent, nor approval from an institutional review board were required by who or countries providing this data to who under the international health regulations ( ). key: cord- -xzfo jjq authors: todd, ewen c. d. title: foodborne disease in the middle east date: - - journal: water, energy & food sustainability in the middle east doi: . / - - - - _ sha: doc_id: cord_uid: xzfo jjq food safety is a concern worldwide and according to the world health organization, developing countries are probably more at risk of foodborne illness because many of these, including those in the middle east, have limited disease surveillance and prevention and control strategies. specifically, the middle east and north africa (mena) region has the third highest estimated burden of foodborne diseases per population, after the african and south-east asia regions. however, it is difficult to determine what the burden is since little is published in peer-reviewed journals or government reports for public access. this chapter reviews autonomous nations, namely, afghanistan, bahrain, egypt, iran, iraq, israel, palestine, kuwait, lebanon, oman, pakistan, qatar, saudi arabia (ksa), syrian arab republic (syria), united arab emirates (uae) and yemen. countries range in size from bahrain with . million inhabitants to pakistan with a population of million. agriculture and local food production is much influenced by water availability for irrigation. water shortages are most severe in the gulf countries which rely on aquifers, desalination, and recycled waste water for most of their water supplies. this means that most food is imported which is expensive if not subsidized through petrodollars. this impacts food security which is a particular concern in countries under conflict, particularly, syria, yemen and iraq. gastrointestinal infections are frequent in this region from salmonella typhi and other salmonella spp., shigella spp., campylobacter jejuni and c. coli, rotavirus, hepatitis a virus, parasites, and more rarely from aeromonas, yersinia enterocolitica, brucella spp., and middle east respiratory syndrome coronavirus (mers-cov). reports indicate that children are the most susceptible and that many isolates are multidrug resistant. chemical contamination of water supplies and crops are probably more of a concern than published reports indicate, because of widespread indiscriminate use of fertilizers, antibiotics, and pesticides, coupled with increased industrial pollution affecting the water supplies. like many other parts of the developing world, foodborne disease surveillance is limited and outbreaks are most often reported through the press but with insufficient detail to determine the etiological agents and the factors contributing to the outbreaks, leading to speculation to the cause by those interested or responsible for food prevention and control. however, there are some well investigated outbreaks in the region that have those details, and reveal where the shortcomings of both the establishments and the inspection systems have been. where the causative agents are known, the kinds of pathogens are generally similar to those found in the west, e.g., salmonella, but many outbreaks seem to have short incubation periods that point to a toxin of some kind of chemical or biological origin, but these are almost never identified. because of sectarian warfare, residents and refugees have been given food that has made them sick and solders? have been deliberately poisoned. research has been focused on microbial contamination of locally-sold foodstuffs and manager and employee knowledge of food safety and hygienic conditions in food preparation establishments. an innovative pilot project in qatar is to use seawater and sunlight for raising crops through the sahara forest project. all countries have some kind of food establishment inspection system, but they tend to be punitive if faults are found in management or employees on the premises rather than being used for their education for improving food safety. restaurants may be closed down and owners and employees fined for often unspecified infringements. however, some food control agents are moving towards employee training through seminars and courses before problems occur, which is a good disease prevention strategy. unfortunately, many of the food handlers are from asian countries with languages other than arabic and english, which makes effective food safety communication and training difficult. tourists visiting popular resorts in turkey and egypt have suffered from foodborne illnesses, usually of unknown origin but poor hygienic conditions are blamed with law suits following, and the adverse publicity affects the long-term viability of some of these resorts. food exports, important for local economies, have occasionally been contaminated resulting in recalls and sometimes illnesses and deaths, notably fenugreek seeds from egypt (e. coli o :h ), pomegranate arils from turkey (hepatitis a virus), and tahini from lebanon (salmonella). overall, in recent decades, the middle east has made strides towards improving food safety for both residents and foreign visitors or ex-pat workers. however, within the countries there are large discrepancies in the extent of effective public health oversight including food safety and food security. currently, almost all of the countries are involved to a greater or lesser extent in the civil wars in syria and yemen, or are affected through political tensions and strife in egypt, iraq, iran, israel, palestine, lebanon and turkey. in addition, the current overproduction of oil on a world-wide scale has led to a rapid decrease in revenues to most gulf states. all this points to a severe setback, and an uncertain foreseeable future for improvements in obtaining both sufficient and safe food for residents in this region. the world health organization (who) eastern mediterranean region, comprising countries in the middle east and north africa (mena), has the third highest estimated burden of foodborne diseases per population, after the african and south-east asia regions. according to the who ( a), more than million people living in this region are estimated to become ill with a foodborne disease every year and million of those affected are children under years. diarrheal diseases caused by e. coli, norovirus, campylobacter and nontyphoidal salmonella account for % of the burden of foodborne disease. an estimated people die each year from unsafe food, caused primarily by diarrheal diseases, typhoid fever, hepatitis a, and brucellosis. both typhoid fever and hepatitis a are contracted from food contaminated by the feces of an infected person and the source of brucellosis is typically unpasteurized milk or cheese from infected goats or sheep. half of the global cases of brucellosis are in people living in this region, with more than , people infected every year, causing fever, muscle pain or more severe arthritis, chronic fatigue, neurologic symptoms and depression. cholera, which after a short incubation period of - days causing severe diarrhea and dehydration, is returning to those countries with limited public health infrastructure caused by conflict, such as iraq (agence france-presse ). the list of countries covered by this chapter is similar to that of who but leaving out north african countries except egypt (which has territory in eastern asia) and adding turkey which is not always considered in the region because it is not arabic, but has interesting food safety data. therefore, the countries under review are afghanistan, bahrain, egypt, iran, iraq, israel and palestine, kuwait, lebanon, oman, pakistan, qatar, saudi arabia (ksa), syrian arab republic (syria), united arab emirates (uae) and yemen. gulf countries bahrain, kuwait, oman, qatar, ksa and the uae have similar social, political, economic, culture, religion, language and ancestry with several similarities in their food control systems and food safety programs (al-kandari and jukes ). a food and agriculture organization (fao) report covering international investments in agriculture in the near east (not identical to the countries chosen for this chapter, but many of the findings apply) states that this region is characterized by a mix of very different countries' resources and incomes (tanyeri-abur and elamin ). the wealth in the richer countries of the region is primarily dependent on oil revenues and the past economic growth has been closely linked to the oil market; about % of regional gross domestic product (gdp) is concentrated in the high income countries (qatar, kuwait, uae, saudi arabia, and bahrain) which are home to only . % of the population in the region, and many of these are expatriates working in these countries. the report indicates that food insecurity varies sharply in the region but overall the percent of the undernourished population does not exceed % in most countries of the region, except for sudan, mauritania, djibouti and yemen where the proportion of undernourished exceeds %; however, in these percentages will be totally out of date for countries like syria and iraq and in neighboring countries where refugees have reached because civil war and jihadi terrorist groups have put considerable stress on public health facilities and food availability. the countries in the region however, are largely similar when it comes to the challenges in achieving sustainable agriculture and food security. for most of these countries, the overwhelming concern is to secure adequate and stable supplies of food at the national level, making food security a concern for both rich and poor countries of the region (tanyeri-abur and elamin ). the three major problems affecting most of the countries are (i) limited water availability; (ii) population growth; and (iii) heavy dependence on food imports. water scarcity in particular, is the most critical development problem in the region and the single most important factor in limiting agricultural growth, and water availability has been declining steadily since the late s. the region as a whole has % less availability of renewable water per person in - than in - . lack of water for irrigating crops but also for potable water supplies affects many of the countries, particularly in the gulf region. it is important to note that the wealthiest countries are also those with the highest water depletion record, namely, the uae and qatar. the unprecedented growth in investment in agriculture is in large part a result of the food crisis of , which brought about a rethinking of agricultural support policies, mostly in countries of the gulf and particularly saudi arabia, which has invested heavily in the last years in large-scale agricultural production using up valuable water resources. saudi arabia announced in january that it would phase out wheat and agricultural production in the course of the next years. in july , qatar and uae took similar policy decisions (tanyeri-abur and elamin ). crops grown in the region may serve as fresh food sources for the population, but much of the food is imported with limited locally processed products, and if the policies of ksa, qatar and uae expand to other countries, more will be imported in the future (tanyeri-abur and elamin ). thus, the main foodborne disease issues are with homemade, restaurant and street food, where isolated claims of illness are followed up by inspections and possible punitive action by public health agencies responsible for food safety. those countries that rely on tourism for their main source of gdp have sometimes been damaged by adverse publicity, e.g., egypt, and to a lesser extent, turkey and lebanon. according to the food and agriculture organization, less than % of the world agricultural trade is conducted in the region. even though by tradition many of these countries relied on growing their own food, today some of these countries import almost % of their food; . % of the food in the world alone was imported to saudi arabia and united arab emirates in , and the food trade balance in food in middle east is negative, estimated at over us $ billion dollars (tajkarimi et al. ). there are specific restrictions prevalent in the arab-speaking countries related to islam and judaism with the prohibition of eating pork and blood, the drinking of alcohol, and mixing dairy foods and meat under halal and kosher food laws. therefore, parasites related to pigs, e.g., trichinella and taenia spp., are unlikely to be prevalent in these populations. however, there are many muslim and jewish feast occasions with large gatherings such as eid linked to ramadan and particularly the muslim hajj, which put a strain on food preparation, distribution and storage. good health conditions for travelers to saudi arabia for the pilgrimage to mecca (hajj) are critical and any incident that occurs has to be quickly contained to prevent extensive infectious disease outbreaks (memish and al rabeeah ) . traditional middle eastern foods are mainly related to legumes, leafy greens, fruit, dairy products and meat on special occasions; details can be found in brittin ( ) . in urban areas today, grocery stores and supermarkets can supply most of the food requirements of a family but imported foods tend to be expensive. also, some fruit and vegetable items are seasonal and are only available once or twice a year such as local plums, almonds and bananas, which tend to be cheaper than imported varieties. quality of raw produce in stores varies but they often have short shelf lives and can spoil quickly because of harvesting ripe products, bruising, and high storage temperatures. traditional rural foods include aromatic stews, stuffed vegetables, wild leaves, pulses and cracked wheat, and occasional goat or lamb meat. a typical middle eastern meal starts with a variety of cold and hot mezze (appetizers), salads and pastries, especially in greece, turkey and lebanon. many contain herbs, cheese, pickles, nuts, seeds, and parsley and lettuce are widely eaten in salads or traditional mezzes. most mezzes are vegetarian and fresh fruits and vegetables are an integral and important part of the cuisine when they are in season. tabbouleh, a salad where parsley is a major ingredient with small pieces of tomato, and some bulgur (ground wheat) in it, is often served in leaves of romaine lettuce or raw cabbage. almost as popular is fattoush, a mixed bowl of lettuce, tomatoes, cucumbers, and fried or toasted pita chips, typically seasoned with a dusting of sumac and pomegranate molasses. since leafy greens do not have a final decontamination step, they are at risk from environmental fecal contamination as reported in lebanon by faour-klingbeil et al. ( ) . hummus, a smooth chickpea paste made with tahini/tehineh, lemon juice or citric acid, garlic and salt, and often served with olive oil, is the most ubiquitous mezze. since tahini and hummus are major exported products from the region, particularly lebanon, they are prone to salmonella contamination, and are sometimes recalled from other countries, which is damaging to the local economies. dairy products are also served regularly at meals and these are locally made or imported. labneh, strained yogurt, very similar to greek yogurts, is widely used as a base for mezze which might have olive oil, pine nuts or za'atar (a mixture of thyme, sumac, and sesame seeds) added. cheeses including the popular haloumi are frequently served in restaurants. shawarma/ shwarma is frozen or refrigerated raw or marinated meat (lamb, beef or chicken) cooked on a vertical rotisserie popular throughout mena countries and now frequently seen in western nations. higher fish consumption tends to be close to where these are locally caught, either sea or river netted. one example from iraq is masquf (split large fish cooked on stakes over a fire, and eaten outdoors by a river, served with slices of tomato and onion and arab bread. crustaceans are less frequently eaten but can be obtained from imports. cosmopolitan foods are widely available in the larger cities, as are multinational fast-food chains. foodborne illnesses have been sporadically reported throughout the region over the past decades and global assessments of the kinds of problems encountered reviewed, e.g., todd ( ) and al-mazrou ( ) and more recently by tajkarimi et al. ( ) . these last authors indicate that reporting foodborne disease is functioning well in jordan, kuwait, oman, saudi arabia and uae, compared to other countries in the region. however, the foodborne outbreak surveillance systems in middle eastern developing countries are still limited with reporting of less than % of the actual outbreaks; one reason is that many foodborne illnesses occur in homes and those ill may not visit medical care facilities. in addition, available laboratory analytical support for public health agencies is often minimal or lacking, even though some research institutions may have up-to-date equipment and technical expertize. change is gradually coming and a food and drug authority has been established in both saudi arabia and jordan (al-kandari and jukes ). also, new food legislation has been initiated by egypt, lebanon and syria (tajkarimi et al. ) , but is currently stalled in last two countries. improvements in inspection service, hand held computers, customized software and improved surveillance systems are some examples of developments in food safety systems in the region. jordan, saudi arabia and bahrain have been developing unified food safety activities from farm to fork (al-kandari and jukes ). however, there is a need for substantive food safety education for all foodservice staff. increasing quality and quantity of the food safety training and human resources in governmental agencies in the region will improve the public health infrastructure. for example, the municipality of dubai has established an international annual food safety conference to improve the food safety education system of those in the region, now in its th year ( ). the following sections of the chapter focus on five aspects: gastrointestinal infections; foodborne disease outbreaks in specific countries; food safety related research and surveys; issues relating to tourism and exported food; and government oversight of the food industry, with specific examples from countries in the region. gastrointestinal diseases are frequently encountered in the middle east and many etiological agents have been identified where specific studies have been carried out to look for bacterial, viral and parasitic pathogens. the average annual incidence of culture-proven shigellosis in israel was / , from to , but each reported case was considered to represent cases indicating the high burden of the disease in the country (cohen et al. ) . orthodox jewish communities, living in highly crowded conditions and with a high number of children aged < years were the epicenter of country-wide biennial propagated epidemics of s. sonnei shigellosis. s. flexneri was the leading shigella serogroup in israeli arabs. isolates showed high rates of resistance to ampicillin and trimethoprim/sulfamethoxazole, but very low rates to quinolones and third-generation cephalosporins. there is no indication if foods or water were vehicles of these shigellosis cases. also, in israel a study of pregnancy-related listeriosis cases from to , identified cases, resulting in a yearly incidence of - cases per , births (elinav et al. ). there were fetal deaths, two neonate deaths and one maternal mortality. the incidence of israeli pregnancy-associated listeriosis has a high yearly variability and is one of the highest worldwide. the geographical distribution varied greatly between years and had a different epidemiological pattern compared with nonpregnancy-related listeriosis. the sources of the infections were not studied but all listeriosis cases have a foodborne link. this has to be further researched as to diet, and the unawareness of the israeli public of the risk for certain food products contributing to the extremely high incidence in israel, in both general and pregnancy-associated listeriosis, as occurs in other countries. a total of stool samples were collected from palestinian patients with acute diarrhea from which ( . %) yielded enteropathogenic bacteria. salmonella, campylobacter coli/ jejuni, and aeromonas hydrophilia were isolated in equal numbers from samples / ( % each), shigella boydii / ( . %), yersinia enterocolytica / ( . %) (abdelateef ) . many strains were antibiotic-resistant. children younger than years old were more susceptible to infectious diarrhea; in addition, diarrhea was more frequent in those living in crowded houses, and in houses rearing poultry, including pigeons. salmonella enterica serovar typhi continues to be an important public health problem in kuwait. analysis of the isolates from patients, collected between and , showed that the majority were from patients from the indian sub-continent, and many strains were drug resistant (dashti et al. ) . typhoid fever in kuwait is predominantly associated with those who have traveled from endemic areas to work in kuwait. the circulation of enteric viruses among the population of cairo, egypt, between march and february was studied by kamel et al. ( ) . at least one type of virus was detected in % of fecal samples, . % of which were positive for rotavirus, % for norovirus, . % for adenovirus, and . % for astrovirus. over % of infections were mixed infections. among the noroviruses, half belonged to the predominant ggii. cluster which were similar to those circulating elsewhere, but there were also new ggii. variants that were not associated with any previously known ggii. isolate. although norovirus is rarely implicated in foodborne outbreaks compared with the us and other western countries, it is clearly present in egypt. further studies are required to assess the disease burden of enteric viruses in egypt and the impact of atypical strains. the disease burden of hepatitis a and e in egypt is one of the heaviest worldwide, based on serological analysis, with hav infections occurring very early in life, with almost % seropositivity after the first years of life (kamel et al. ) . to determine the actual contamination levels in the environment, these authors conducted a survey of hav and hepatitis e virus (hev) in sewage in cairo. hav was detected by rt-pcr in of ( %) sewage samples. in addition, all the hav-positive samples were also positive for enteroviruses. that only one stool sample was hev-positive might be explained by the lower level of excretion of the virus in stools, the fragility of the virion in the environment, and technical difficulties in concentrating and amplifying the virus with standard methods. bacterial etiology was found in . % of cases of childhood diarrhea in dhahira, oman, mostly shigella sonnei and to a lesser extent salmonella (patel et al. ) . antibiotics were prescribed in . % of cases and the resistance to the common antibiotics tested was low. one reason for the low pathogen isolation rate could be that many cases had viral etiology. rotavirus was detected in stool specimens from ( %) of children, who were admitted to regional public hospitals in oman for a median of days with severe diarrhea (al awaidy et al. ) . a diverse rotavirus strain pattern in oman was identified with g ( %), g ( %), and g ( %) accounting for most of typeable strains. the authors estimated the burden for the omani government at us$ , and us $ . million annually to treat rotavirus-associated diarrhea in the outpatient and hospital settings, respectively. they recommended a rotavirus vaccination program that would substantially reduce the burden of severe diarrhea among children in the country. unlike the above countries where the health care system functions for most residents, though not always to western standards, the same cannot be said for pakistan, particularly in rural areas. poor nutrition combined with diarrheal and other foodborne diseases puts the population at risk for serious illness and death, especially among infant and children in pakistan (akhtar ) . cholera, campylobacteriosis, e. coli gastroenteritis, salmonellosis, shigellosis, typhoid, and brucellosis have been demonstrated to be the major foodborne illnesses in the country as well as infectious diseases caused by viral and parasitic agents. many fatalities have been associated with food poisoning but the actual agent has rarely been determined. many health experts believe that rapid spread of gastrointestinal diseases cannot be controlled if the public has no awareness of prevention and control measures against cholera and other forms of gastroenteritis, and that in most parts of the country, sewage is continuously contaminating streams, lakes, springs, wells, and other drinking water sources (qasim ). in may , an epidemic of diarrhea and gastroenteritis occurred in kamalia, toba tek singh, with over children and others being admitted to hospitals which had few medical supplies. apart from lack of potable drinking water, the main reason given for the rise in cases was the heat of summer when there were frequent power cuts so that food "rots" or becomes "stale" (islam ) . in remote areas of pakistan, cholera has been responsible for many outbreaks. two examples in july and august of , both in areas of conflict near afghanistan, give an idea of local but severe outbreaks. in one case authorities seemed not to want to be involved and in the other vaccinations are carried out. although water is the primary vehicle of the vibrio cholerae pathogen, it can easily contaminate prepared foods through poor hygienic practices. in july , five deaths from cholera occurred in pashtoon kot area, balochistan region of pakistan (federally administered tribal areas) along the afghan border (staff ), some km from quetta, in the absence of any emergency medical aid. the condition of an additional people suffering from the disease was said to be critical. a local tribal elder expressed the fear that outbreak of cholera might cause loss of life at large scale. he complained that the doctor and paramedics deployed at the basic health center in panjpai live in quetta and are rarely seen at the center. officials of the provincial health department appeared to be unaware about the cholera outbreak and loss of lives (or ignored these), as they sent no medical teams to the affected area. in fact, pakistani government rebuffed international media's claims, and did not respond to requests to dispatch healthcare professionals to the balochistan area. it was assumed the outbreak would continue without medical aid. in , cholera outbreaks killed hundreds of people, mostly children, in flood-hit districts of nasirabad, jaffarabad and jhal magsi where waterborne diseases were reported at a large scale because of consumption of contaminated water by local people. in august , two people died and others had fallen ill, following a cholera outbreak in kurram tribal agency near afghanistan (hussain ) . dhand and kudiad khel were the worsthit areas but vaccinations were carried out amid tight security, and tribesmen were instructed not to drink water directly from the well and boil it first instead since the wells had been contaminated from the rain water. around people were shifted to parachinar headquarters hospital, while others were discharged after medical aid. sometimes diseases kept at bay by functioning public health systems come back when these break down as is occurring in a few of the countries embroiled in internal strife and outside attacks. for instance, in iraq in october, , > cases and deaths of cholera occurred which started along the euphrates valley in september with the governorates of baghdad and babil, south of the capital, being the worst affected with more than cases each. the epidemic then spread to the northern autonomous kurdish region, which hosts hundreds of thousands of people displaced by conflict from other parts of iraq (agence france-presse ). a previous outbreak killed four people in the kurdistan region in . the united nations says the number of people displaced by conflict in iraq since the start of has topped . million which would exacerbate the spread of the disease. authorities blamed the cholera outbreak mostly on the poor quality of water caused by the low level of the euphrates. limited vaccination programs are in place in areas of conflict. in october, , two persons arriving in kuwait from iraq tested positive for cholera and both were provided proper treatment and recovered. the ministry of health recognized that further cases could be discovered among people arriving from iraq, but because kuwait has a well-structured health infrastructure with water and sewers grids, and a supply of healthy and safe food, the disease should not spread into the kuwaiti population (anonymous a) . probably there are some cases in yemen and syria, countries also with limited public health infrastructures, but have yet to be identified. in saudi arabia, a country with a well-maintained health system, the main infectious disease concern today are the infections and deaths arising from exposure to the middle east respiratory syndrome corona virus (mers-cov), which has reservoirs in camels and bats (todd and greig ) . a potential food source for this virus and other pathogens is from unpasteurized camel milk, as camel farmers drink the milk as well as being exposed through other aspects of camel contact. this brief review indicates that diarrheal diseases, caused by cholera, dysentery, hepatitis a, salmonellosis, shigellosis, typhoid fever, and other enteric diseases through water and food are major contributors to ill health in the region in agreement with the who ( b) report on global estimates of foodborne diseases. in the region, not very many outbreaks of foodborne disease tend to be investigated, or at least reported publically, and those that are tend to have fatalities or are very large. for instance, in june, , two children and one adult were brought to a hospital in dubai, uae, with suspected food poisoning (vomiting) after they ate take-away food (the father was out of town). although the mother eventually recovered, the two young children ( and years old) died, one on arrival and the other the next day. the cause was not determined (saberi and scott ) . it is not known if the family or restaurant was primarily responsible for the deadly gastrointestinal attack as bacteria can multiply quickly in the hot summer months, and the public had been recently warned to minimize eating out at this time of year, especially at smaller eateries where hygiene levels are often of lower standard. a toxin was likely involved to cause fatalities so rapidly, but it could have been an accidental contamination of the food with a chemical such as a pesticide, as much as it could have been with an enterotoxin produced by staphylococcus aureus or bacillus cereus through careless ambient temperature storage. unfortunately, this was one episode in a string of incidents, most of them with fatalities, in the county. in april, , a -year-old died of suspected food poisoning in sharjah, and in august, a -year-old girl died of food poisoning in abu dhabi. in march, , six people fell ill after eating buffet food at a restaurant in the large ibn battuta mall, dubai; in november of the same year, employees at a cement factory were hospitalized after consuming what was considered rotten food prepared at the factory kitchen in another emirate, ras al khaimah. in may, , a -year-old girl died of suspected food poisoning in sharjah. the indian family of four rushed to the hospital after series of vomiting but were too late to save the girl. dubai has been reporting foodborne outbreaks and cases through its foodborne disease investigation and surveillance system since ; in that year there were cases reported in the first nine months (saseendran ) . in , suspected cases of foodborne illnesses were reported but only cases were confirmed. no deaths were reported since the surveillance system was in place. egypt has had a particular problem with foodborne illnesses in universities and schools, mostly without a confirmed etiology, which seem to be related to poor food quality. food poisoning is not uncommon in egyptian university dormitories, where basic hygiene standards are often not observed, but the following outbreak was one of the largest. on april , hundreds of egyptian students angered by a mass outbreak of food poisoning at a cairo university stormed the offices of the country's top muslim cleric and university president, ahmed el-tayeb, because of the students who were hospitalized after a meal served at the university dormitories in the nasr city district of cairo (associated press ). the university is affiliated with al-azhar mosque, the world's foremost seat of sunni muslim learning, and awards degrees in sciences and humanities, as well as in religious studies. in the protest, thousands of al-azhar students blocked roads, broke into el-tayeb's offices by the main campus, and chanted slogans against the university's management. the causative agent was unknown, and only with the incubation period, types of symptoms and their duration would it be possible to consider the potential etiologies of this illness. because of their poor quality, campus meals were not very popular before they were being blamed for the current food poisoning outbreak. although investigators were not able to find a specific cause, the university suspended its food services director and some other staff members. within a few weeks food poisoning affected students on april , at the same university, al-azhar (masriya ) . investigations were initiated within the university and by the ministry of health, and apparently "bad tuna" had been served at the campus cafeteria; no further details were given. if tuna was the vehicle of the outbreak, scombroid poisoning was the likely cause of the illnesses. the allergic-like symptoms generally begin - minutes after ingestion and usually resolve in a few hours. scombroid fish poisoning occurs after fish, most frequently tuna, with high levels of accumulated histamine or other biogenic amines, is eaten. but "bad tuna' could equally be contaminated with bacterial or viral enteric pathogens with a longer incubation period. a month later there was another outbreak. because at least three outbreaks of food poisoning occurred at al-azhar university between april and may with over cases of food poisoning detected in the university's male dorms, the dorm's director, the university's kitchen manager and eight chefs were sentenced in november, , to years in prison with a financial bail. in a similar situation, egypt's top prosecutor ordered a swift investigation into the cases of food poisoning reported in two primary schools in october, , in suez (masriya ). an official of the ministry of education indicated that the poisoning was caused by the consumption of milk provided by the schools. the distribution of milk to all schools in the governorate was halted until the milk's validity was ensured. if milk was responsible, the etiological agent could be bacillus cereus enterotoxin if the onset time was short, or less likely an infectious disease pathogen such as salmonella or e. coli o :h . on january , , female students were diagnosed with food poisoning at al-azhar university in upper egypt's assiut/ asyut governorate, by the banks of the nile, and were briefly hospitalized in an assiut city (anonymous b) . this follows a similar incident which occurred in april when students, also in the girls' dormitories, contracted food poisoning on the university campus in luxor. this report also flags two major poisoning incidents involving at least students ill consecutively at its campuses in cairo in (probably the ones already discussed). the reason given for these repeated mass foodborne illnesses among university students is the quality of the food served them. apparently cheap, subsidized food is poorly stored, cooked and distributed to the poorer university students. in most cases the attorney general would open a criminal investigation that would be closed without knowing the microbiological cause of these outbreaks. the promed-mena editor speculated that enterotoxins of staphylococcus aureus were the most probable cause of such communal food poisoning, as a toxic dose of less than . microgram in contaminated food is sufficient to produce symptoms of staphylococcal intoxication. this toxin level is reached when s. aureus populations exceed , /g, a condition likely to be present in these university kitchens because of intense pressure on them to feed a huge number of students in a short time, taking into consideration that most of these kitchens lack basic hygienic measures with regard to safe food handling. the editor also considered shigella, with its low infective dose ( - depending on the species) as another possible agent. however, the incubation period and symptoms of s. aureus intoxication and shigellosis or dysentery are quite different. pakistan is similar to egypt in that much of the country is rural but with very large cities with high populations (total population is million in egypt and million in pakistan, the most populous of all middle eastern countries). in september, , more than of the flood victims at a relief camp in bengali boys sindhi section school in ibrahim hyderi vomited after eating cooked food and then fell unconscious; of them had to be taken to a nearby hospital (aligi ) . a local philanthropist had been providing cooked food to the flood victims but by the time the food arrived at the relief camp, the cooked rice had turned "stale". since the rice did not show any sign of spoilage, it was served to the flood victims. a similar incident had taken place days earlier at another town where more than flood affectees had fallen unconscious after consuming "stale" food and were hospitalized. none was seriously affected. during the investigation, it was noticed that the sanitary situation in and around the relief camps was very poor. even though the reason for the illness was not determined, the police took action against the donor and two caterers. in fact, based on the information of the vehicle and the symptoms, bacillus cereus enterotoxin which is known to be produced in boiled rice, was the most likely agent. in the following two outbreaks yoghurt is blamed for the serious illness and deaths though details of the symptoms are not given. rapid onset of symptoms indicates the presence of a toxin of some kind, although yoghurt is not a food known to be frequently contaminated with pathogens because of its high acidity. either the yoghurt was made under very unhygienic condition with the source of the milk perhaps being spoiled (possibly containing bacillus cereus enterotoxin), or a chemical had been added accidently such as a pesticide, or deliberately and illegally to enhance the flavor. however, it is possible other foods were involved and yoghurt was not the contaminated vehicle. in january , in lahore, a hospital employee died and two other employees became critically ill after eating contaminated yoghurt. the three employees ate rice with yoghurt at a local restaurant (ians ) . action was taken against the restaurant owner and manager. no further details are known. in early april, , a rawalpindi family of ten became seriously ill after eating a home-prepared evening meal where yoghurt was suspected to have been the contaminated food, and they were taken to a hospital, where a teenage boy and -year-old girl died (asghar ) . the surviving family members remained in critical condition for some time but eventually recovered; the cause of the illnesses was not discovered, although it was postulated by a relative who had eaten the yogurt with the meal that it was possibly poisonous or, strangely he thought a lizard might have fallen into it. in february , at least four people died and another seven were hospitalized in a critical state after eating home-cooked biryani (a dish made with spices, rice and meat or vegetables) in a suburb of karachi (mahmood ) . the owner of a grocery shop, who provided the ingredients, was arrested, and a sample taken for analysis. it is not known if any toxin was found. a month later in march, in faisalabad, more than children and women were ill after eating contaminated aalo-chanay (potatoes, chick peas, onions, tomatoes and spices) purchased from an unidentified vender (anonymous c) . as soon as the children ate the aalochaney, they felt ill and started vomiting. although they were immediately rushed to a rural health center, one boy died. a medical opinion was given that the eaters suffered from "diarrhea and cholera". however, the onset was too rapid for anything but a toxin of some kind, most likely heat-resistant since the aalo-chanay was cooked. also, in march , as many as student nurses and eight staff nurses were hospitalized with acute food poisoning at a hospital in rawalpindi after eating food at the nursing hostel, but none was critically ill (anonymous d). the nurses residing in the hostel started reporting complaints of vomiting and diarrhea along with high-grade fever at an undisclosed time after a meal. the hospital administration was criticized for failing to provide safe food and drinking water to its employees and demanded immediate inquiry into the case, but none was reported on. the illnesses are consistent with an enteric infection such as salmonella or norovirus. in april , at least constables suffered from diarrhea and were admitted to hospitals when they ate food during the sehat ka insaf program, which is a blanket method of administering the polio vaccine along with eight other vaccines, hygiene kits and vitamin a drops in order to circumvent polio-specific terrorist attacks in pakistan. local administration purchased packed food, including piece of chicken and juices from a local supporter (mayar ) . no further details are given but the chicken could have been undercooked or cross-contaminated with enteric pathogens such as salmonella and campylobacter; if the packs had been left at ambient temperatures for some time, these pathogens could have multiplied on the chicken to large numbers. over thirty children in faisalabad were hospitalized over days because of diarrhea and gastroenteritis, three seriously, and other children were expected to be ill. undetermined contaminated food was postulated as the cause, more than usual because of the extreme seasonal heat combined with frequent power outages to allow rapid bacterial growth in contaminated food. the unavailability of clean drinking water was mentioned as a contributing factor to the increasing number of gastrointestinal disease cases. hospital administrators complained that vaccines and medications were required but were not forthcoming from the health department. probably many family meals were contaminated because of the lack of potable water and any unspecified enteric bacterial pathogens present could grow rapidly in the heat. children are more vulnerable than healthy adults to infections which might explain the high proportion of sick children seeking medical help. botulism outbreaks occur periodically in iran. in a study of stool and serum specimens of patients with clinical symptoms of botulism, who were at inpatient and outpatient medical centers in tehran and other areas of iran, between april to august , specimens of patients showed the toxin and spores of c. botulinum (modarres ) . type e was the most common causative agent found in this study, being responsible for . % in all specimens; other etiologic types, in order of frequency were types a ( . %) and b ( . %). type e strains are typically associated with fish and freshwater and marine sediments. the results of this study indicate that the cases had consumed salted fish, smoked fish and canned fish, along with cans of green beans and cucumbers. a similar result over a decade later confirms that c. botulinum type e is a major pathogen in iran. in gilan province, of fish samples collected in , % of processed fish and . % of non-processed fish contained clostridium botulinum, mainly type e (tavakoli and imani fooladi ) . the processing is insufficient to kill the spores or reduce much of toxin produced because the fish tend to be partly cooked with the intestines kept intact. a total of traditional food product samples ( cheese, kashk [a type of dried yoghurt or thick cream], and salted fish) were examined using a bioassay method for detection of clostridium botulinum toxin (hosseini et al. ) . standard monovalent antitoxins were used to determine the toxin types. c. botulinum toxins were detected in . % of examined samples ( . % of cheese samples and . % of salted fish samples). none was found in kashk samples. c. botulinum types a and e were dominant in cheese and salted fish samples, respectively. consumption of these traditional foods either raw or processed may contribute to foodborne toxicity in iranian populations. in may , a quickthinking mother immediately brought her -month old boy to an israeli hospital when she saw he was suffering from vomiting, difficulty in breathing, listlessness, glassy-eyed, apathetic, and an inability to nurse or eat (bender ) . a doctor at the hospital diagnosed the child as suffering from infant botulism. he decided to treat the baby with the antitoxin stored in the emergency stocks, even before they got back the lab test results. the hospital like all israeli medical facilities keep ample supplies of biological and chemical warfare antidotes on hand in case of war or terrorist attacks, and staffers are regularly drilled in dealing with the symptoms of various chemical, neural and blister agents. the infant started recovering soon after the administration of the antidote. in the rare disease of infant botulism, spores of clostridium botulinum are ingested and the infant's flora is not mature enough to prevent germination and slow growth of the toxigenic pathogen. it is entirely possible that infant botulism occurs more frequently in the region but is not diagnosed. foodborne disease surveillance depends on an infrastructure of reporting and diagnosis in hospitals, epidemiologists, and food testing laboratories. lebanon is an example of a country where modernization in public health seems to occur at a glacial pace. however, diseases including those of foodborne and waterborne origin, are documented and published. the law of december , regarding communicable diseases in lebanon mandates all physicians, from private or public sectors, in hospitals or ambulatory services, to declare to the epidemiologic surveillance unit of the moph all diseases considered a risk to public health. the data available at the ministry of public health (moph) are compiled from different sources, and the declaration of cases remains irregular and insufficient (moph ) . in , foodborne and waterborne diseases were the most frequently reported in lebanon at a rate of . ‰ (total of cases), with the highest rate in the bekaa ( . ‰) and the lowest in the south ( . ‰). the most common infection was viral hepatitis a, which represented . % of the total food and waterborne diseases with cases. there were also cases of typhoid ( . %), cases of food poisoning (unspecified, . %), cases of dysentery ( . %), cases of brucellosis ( . %, cases of parasitic worms ( . %) and cases of hydiatic cyst ( . %). no cases of cholera and trichinosis were declared. hydiatic cyst (cystic echinococcosisis) caused by echinococcosis (typically e. granulosis) is acquired by contact with animal feces contaminated with tapeworm eggs. sources include contaminated food (meat), water, and animal fur. cysts containing tapeworm larvae may grow in the body for years before symptoms appear. when cysts become large, they may cause nausea, weakness, coughing, and belly or chest pain. occasionally, well-investigated outbreaks are published; the following two examples are from lebanon and neighboring jordan. in may , employees suffered from diarrhea, fever, and abdominal pains . - . h (mean, . h) after eating chicken noodles au gratin at a catered lunch served at a bank cafeteria (hanna et al. ). a few cases had systemic infections. salmonella enteritidis (se) was confirmed in stool and blood cultures within - h after hospital admission of the first cases, and also in leftovers of the suspect food. the same dish had been served at the bank in the past with no apparent health problems. preparation normally started in the evening prior to the day the dish is served. however, in this instance, some of the constituents had been prepared days ahead, because the dish was to be served on a monday, immediately after the week-end closure. no salmonella was found in rectal and nasal mucosal swabs taken from all kitchen workers, or in the tanker water supply (although it had high fecal coliform counts), but se was found in a frozen batch of the same raw chicken breast consignment that had been used for the chicken noodles. the batch of chicken came from a large producer of poultry and eggs in lebanon, who was advised of its potential involvement in a major foodborne outbreak. however, the investigators were refused access to the poultry-producing facility. it is highly likely that contaminated chicken carcasses had been, and would continue to be, shipped to many parts of lebanon. that the same se strain occurred in the patients, the raw chicken, and the leftover food was confirmed through random amplified polymorphic dna polymerase chain reaction (rapd-pcr). it would appear the -day delay in the chicken noodle preparation was significant in allowing the salmonella present in the ingredients not only to survive but probably to grow; undercooking, cross-contamination, inadequate storage and reheating all may have played a role in the outbreak, but no more information was available to determine which of these were the key factors in the outbreak. the bank management decided to sue the caterer and because they were aware of apparently inefficient way that public authorities were conducting the procedure, they took the initiative to call upon an independent investigative team to obtain solid evidence to win any court action. the caterers, concerned that they would be the only party blamed for the salmonella outbreak, had succeeded in concealing some raw and cooked items from destruction by the public health authorities, which was their normal practice after a complaint. these items were central to establishing contamination upstream from the caterer's kitchen. no action seems to have been taken against the poultry producer who was the source of the se, a pathogen that is invasive of flocks and difficult to eradicate. the authors complained about the obsolete lebanese laws dating back to the s that still governed what should be done following a report of "food poisoning". public health officers are mandated to stop the spread by destroying allegedly contaminated food items and closing down incriminated facilities. hanna et al. ( ) stated that this kind of action is generally lauded by the public but does not help determine the cause to develop appropriate prevention and control strategies. they also complained that because no investigation is typically done, many non-implicated foods and ingredients are wastefully discarded. the jordanian example is over two decades old, but is worth noting in detail. in september , a -case outbreak of salmonellosis occurred in a university hospital in amman after employees, patients and visitors ate in the cafeteria. the incubation period ranged from to h. symptoms included diarrhea ( %), fever ( %), abdominal pain ( %), dehydration ( %), and bloody stool ( %); were hospitalized (khuri-bulos et al. ) . cultures of eight food items were negative, but stool culture on of patients and of kitchen employees yielded salmonella enteritidis (se) group d . a cohort study revealed a foodspecific attack rate of % for the steak and potato meal and % for the rice and meat meal. stratified analysis of the steak and potato meal revealed that the potatoes were implicated most strongly. cultures were obtained from all kitchen employees, who showed no symptoms of illness, but of grew se group d . one asymptomatic, culture-positive employee had prepared the mashed potatoes on september , h before the first case presented at the hospital emergency with severe gastroenteritis symptoms. all of the food workers had negative stool cultures months earlier. the potatoes were mashed by machine, but peeled after boiling and mixed with milk by hand, using a ladle but no gloves. two different batches, the first of which was served exclusively to hospitalized patients and the second to a few remaining patients and employees, were prepared and served within to minutes of preparation. from the epidemiological data it can be assumed that the infected handler fecally contaminated only the second batch of potatoes, thus sparing most of the highly susceptible inpatients from exposure. furthermore, while potatoes clearly were implicated, individuals who ate steak only had an elevated risk of being attacked. this probably was due to surface contamination of foods being served on the same plate. kitchen employees harboring salmonella were excluded from work until they had three negative stool cultures taken week apart; it took weeks for them to return to work. stool surveillance that was routinely carried out in the hospital was ineffective in detecting infected employees to prevent this outbreak and the investigators recommended that employees adhere to proper hygienic practices including thorough washing of hands, especially when preparing food. today, salmonella is only one of many of the pathogens that can be encountered in foodborne illness. one of the newer pathogens, well established in the west is norovirus (nov), which causes more cases of foodborne disease in the u.s. than any other agent (scallan et al. ). in may , a significant increase in acute gastroenteritis (age) cases was noted in the american health clinic at incirlik air base (iab) in adana, turkey. this increased rate of age led to discussions with local turkish military public health authorities, which confirmed that the turkish military community and the residents of adana were also experiencing an anecdotal increase in age illnesses (ahmed et al. ). an epidemiologic investigation was launched to attempt to identify the cause and possible source of this age outbreak at iab from may to june with the peak incidence of cases during the week of may -june , with a total of patients seeking medical care at the clinic. of the total infected persons, patients completed the case survey, % reported diarrhea, % reported vomiting, and % reported fever. the median number of days between symptom onset and clinic visit was days. during the days prior to symptoms, % of respondents reported travelling off base, % reported eating off base, and % reported using an outdoor pool. this outbreak had a significant negative operational impact, degrading mission readiness with nearly % of the american population in a -month period affected. initiation of a clinic case-based investigation yielded stool specimens in which nov was detected in %, with % of the positive nov specimens identified without a copathogen. dna sequencing data demonstrated that several relatively rare genotypes of nov contributed to this outbreak; four different genotypes were isolated from positive specimens. two of the nov strains were previously reported in iraq and only from deployed troops, while the other two genotypes were reported in south africa and in the us. in turkey, little systematic data on circulating nov genotypes exist. however, giib/gii. strains have been frequently identified in turkish children with gastroenteritis; strains belonging to this genotype have been found in europe and mainly in children. previous reports from british troops deployed to iraq indicated that two nov strains isolated were responsible for cases of gastroenteritis there. similar mixed nov outbreaks have been previously observed and are often attributed to systematic failure of cooking/cleaning/drinking water supplies (ahmed et al. ) . one limitation of this investigation was that the survey was not used to capture data from a control group, those without recent age, preventing carrying out a risk factor analysis. another limitation was the lack of environmental samples that could be tested for nov in order to track the source of outbreak. from anecdotal information, it is likely many in the local population and the turkish military base were ill, but a formal outbreak investigation in the turkish population was never performed. from the multiple genetic types involved, one specific contaminated food or water source seems unlikely. the largest turkish nov outbreak was in keçiborlu province of isparta county between april and , , with patients seeking medical help from the healthcare centers, after suffering from nausea, vomiting and abdominal pain (more frequent than diarrhea) (s€ ozen et al. ) . because of underreporting, the number of affected people was estimated to be higher. municipal water was the suspected source but no samples tested positive. as a cautionary note, the authors suggest that nov may not be the only causative agent of gastroenteritis outbreaks, especially from an undetermined fecal source, and bacterial, viral and parasitic agents should be examined together with the nov. in saudi arabia, a national policy for reporting, notifying, and recording incidents of bacterial food poisoning was established in (al-joudy et al. ). since then salmonella food poisoning outbreaks have been reported from different regions of ksa, exhibiting seasonal and regional variations, with chicken, meat, and rice being commonly incriminated food items, and frequently reported in the saudi epidemiological bulletin. al-mazrou ( ) reviewed the history of foodborne outbreaks in ksa and saw an increase over the last few decades, especially those caused by salmonella, with the main food vehicles being chicken, meat and eggs, and s. enteritidis being the most frequent salmonella serovar responsible. according to promed editorials, restaurants and communal feasts and institutional feeding (such as in school cafeterias, hospitals, nursing homes, prisons, etc.) where large quantities of food are prepared several hours before serving are the most common settings in which foodborne illness incidents occur (http://www.promedmail.org). for instance, in , a hospital in the jizan region received suspected food poisoning cases that were ill after taking meals from a restaurant, including a woman who suffered from severe diarrhea, abdominal pain, vomiting and dizziness (fagbo ) . the restaurant was closed down and three of its workers were detained pending the results of laboratory tests. the report of an investigative committee could not find a specific cause, but noted that the restaurant had earlier been responsible for some hygienic violations. in , cases suspected of foodborne illness after eating a meal at a restaurant were admitted to various hospitals in the najran region (alhayat ) . most of the cases were not seriously ill. no report was given on the samples that were taken from the suspected restaurant, which was closed temporarily. there is an interesting observation related to variant creutzfeldt-jakob disease (vcjd); four cases have occurred in the us since the disease was first diagnosed in the united kingdom in linked to consumption of cow meat suffering from bovine spongiform encephalopathy (bse); two of these were associated with the united kingdom (where bse was first reported), but one came from saudi arabia and the most recent case in had extensive travel to the middle east and europe (cdc ) . this may indicate some source of vcjd in the middle east including saudi arabia. one of the big concerns for ksa is the annual hajj with millions of muslims from around the world converging on mecca, in saudi arabia, each year. no other mass gathering can compare with the hajj, either in scale or in regularity, and various communicable disease outbreaks of various infectious diseases have been reported repeatedly, during and following the hajj (memish ) . in , an outbreak during the hajj occurred where all the cases came from one tent occupied by soldiers located in a government camp in mina, makkah province, near mecca (al-joudi ). the camp was served by a catering company that prepared and distributed three meals daily (breakfast, lunch, and dinner). a case was defined as any individual who developed diarrhea with or without abdominal pain after eating at the camp in mina in january, . of the soldiers who were interviewed, ( %) had developed gastroenteritis, most commonly manifested by diarrhea ( %), and abdominal pains ( . %). the mean incubation period was . ae . h and the epidemic curve suggested a common point source outbreak. out of three served meals, lunch with a rice dish was found to have a statistically significant association with illness. unfortunately, no food remnants were found for sampling, and the results of stool cultures of all diarrhea patients, and rectal swabs from all food handlers were inconclusive. temperature abuse was cited as a contributory factor in this outbreak. based on the incubation period and symptomatology, bacillus cereus would be the most likely etiological agent. another example of a foodborne illnesses associated with the hajj occurred in when bangladeshi pilgrims were taken to hospitals in madina (medina) after eating a meal prepared by an unlicensed caterer (promed-mena ). they suffered from abdominal pains associated with diarrhea and vomiting. the pilgrims were all treated and discharged, except for one who remained hospitalized. samples of the food they had eaten were sent for analysis but the results are not known. considering the mass of people converging on this small part of the middle east, it is surprising there are not more foodborne disease outbreaks. this may mean excellent food control by the authorities or some illnesses are simply not recognized and reported. at least bahrainis suffered from food poisoning after eating catered sandwiches served during a wedding celebration, the biggest mass poisoning outbreak in the country's history (promed-mena ) . the wedding took place in the safala village, near the eastern island of sitra. all eventually recovered after treatment but one man who had sickle cell disease, died. teams were formed to investigate the outbreak, and blood specimens from all workers at the bakery who prepared the egg, cheese, and mayonnaise sandwiches along with leftover sandwiches and their ingredients on the caterer's premises were sent for bacteriological analysis. the bakery which supplied the sandwiches was closed by the public health directorate at the ministry pending the investigation's results. unfortunately, no final report was released to the public. the promed editor considered the etiological agent could be salmonella or staphylococcus aureus enterotoxin, depending on the length of the unstated incubation period. the region experiences some unusual type of illnesses relating to on-going hostilities. for instance, the united nations has been sending aid to reach besieged towns in syria, close to the lebanese border, but in october, , it sent hundreds of boxes of "moldy" high-energy biscuits past their 'sell-by' date in september ( of the boxes transported) to zabadani and madaya, apparently causing food poisoning (afanasieva et al. ; muhkalalati and kieke ) . officials stated these could be the only cause of an outbreak of food poisoning among almost residents who came to makeshift hospitals, mainly children who had vomiting, diarrhea and abdominal swelling almost immediately after eating the biscuits. the biscuits were described as "moldy and rotten and had been poorly stored". apparently, when the last aid order that was sent was filled, there was a shortage of food. the red crescent, who was filling the order, took some of the expired goods to complete it. however, these biscuits had only just expired and normally would not have posed any health risks to those eating them. nevertheless, the words poorly stored suggest that moisture may have encouraged microbial growth (visible mold more likely than bacteria because fungi can grow aerobically in the presence of the presumably elevated sugar content in the high energy biscuits). also, contributing to the symptoms, the residents of zabadani and madaya had been blockaded for consecutive days, and their immune systems were extremely weak. refugees are also at risk of gastrointestinal diseases from contaminated water or food. up to two million syrian migrants fleeing syria due to the civil war were living in turkey, and supplying them with safe and secure food supplies is a challenge for any host country. one incident, no doubt, one among many indicates the risk of contaminated food. in april, , five security forces were injured after syrian migrants in a tent city in turkey's southeastern province of mardin reportedly attacked guards over allegedly being poisoned from the lunch at the camp (anadolu agency ). some syrian migrants were detained after the incident; syrian migrants out of the currently residing in the temporary sheltering center in mardin's derik district applied to the center's hospital with symptoms of food poisoning, dizziness, and vomiting. after treatment they were discharged, none of them in a critical condition. although an investigation was conducted and samples from the lunch sent to the lab for analysis, no further information was available on the outbreak. promed speculated that if the lunch food was the vehicle, it would be a short incubation illness likely caused by staphylococcus aureus, bacillus cereus, clostridium perfringens, or possibly a non-biological toxin. these illnesses may cause vomiting, diarrhea, or both, and are usually short in duration (less than h), and are not associated with prominent fever. in iraq, no recent foodborne disease outbreaks have been published, but no doubt many have occurred in the last decades with so much public health infrastructure dismantled. only the most newsworthy of outbreaks are being covered by the press today. iraq and other middle eastern countries are in sectarian turmoil and on two occasions islamic state (isis/is/isil) fighters (jihadis) were likely poisoned by cooks who infiltrated their camps. in november, , a group of defected syrian soldiers (free syrian army men) who posed as cooks reportedly poisoned isis militants after they ate a contaminated lunch at the fath el-sahel camp, where of them were based (gee ). apparently about a dozen of the jihadis were killed and taken to nearby field hospitals. the 'cooks' immediately fled, along with their families, with the help of fellow revolutionaries. seven months later, in july , jihadis died after ingesting an iftar meal eaten by isis militants (akbar ; variyar ) . it remains unclear whether the jihadis, who were breaking their ramadan fast in mosul, iraq, died of accidental food poisoning or intentional poisoning, but it is likely a repeat attack of the earlier incident described above. the nature of the poison or details of the illnesses in either episode are not known. however, in both episodes, onset and severity of the attack were rapid, probably caused by a relatively tasteless chemical in lethal doses added to one or more foods. targeting the military by any means including poisoning food has always been a strategy of opposing forces. in february , a deliberate attack was foiled when afghan border police detected a significant amount of bleach in fruit and coffee stored at their main border checkpoint between afghanistan and pakistan, a likely attempt to poison the afghan security forces (tucker ) . the police decided that although none of this food had been consumed, the level of contamination was high enough to cause serious injury, and it must have been done intentionally. there had been previous incidents of intentional food poisoning aimed at afghanistan's civil defense forces, including an episode in kabul in when several people were sickened. in , in southern helmand province militants killed four afghan policemen and two civilians inside a police checkpoint by poisoning their yoghurt coordinated with an attack (anonymous a). there had been several recent poisoning incidents involving members of the afghan national police, as part of attempts by the taliban to infiltrate the security forces; three police officers were reported missing, along with their weapons and a police vehicle, following that attack in helmand province. taliban militants had first poisoned the police officers' yoghurt before launching a full scale attack on the checkpoint. similar tactics had been used by insurgents in helmand before. the same thing happened again in january when a rogue policeman collaborating with insurgents in southern province of uruzgan shot dead colleagues after first poisoning their food, but no further details are given (reuters ). turkey does have food laws that are supposed to limit food contamination and resultant foodborne illnesses. the turkish food code stipulates that all turkish food businesses have to provide food hygiene training commensurate with the work activities of their staff. to see what progress had been made in this area baş et al. ( ) evaluated knowledge, attitudes, and practices concerning food safety issues among food handlers in ankara, conducting face to face interviews and administrating questionnaires. the majority of the food handlers who responded ( . %) had not taken a basic food safety training (and probably most of non-respondents had not either). the mean food safety knowledge score was . ae . of possible points. the self-reported hygienic practices showed that only . % of those who were involved in touching or distributing unwrapped foods always used protective gloves during their working activity. of those food handlers who used gloves, only . % and . % always washed their hands before putting them on and after removing them, respectively. in addition, there was a difference handlers' scores depending on where they worked. scores were higher for food handlers in catering establishments ( . ae . ), school food services ( . ae . ) and hospital food services ( . ae . ) than restaurants ( . ae . ), hotels ( . ae . ), takeaways ( . ae . ) and kebab houses ( . ae . ). these scores may also be biased upwards since they were self-reported and not observed practices. the study demonstrated that food handlers in turkish food businesses often have lack of knowledge regarding the basic food hygiene, e.g., critical temperatures of hot or cold ready-to-eat foods, acceptable refrigerator temperature ranges, and cross-contamination. those who were trained scored better, and the authors stated there was an immediate need for education and increasing awareness among food handlers regarding safe food handling practices. in istanbul from / , thermophilic campylobacter was isolated from . %, . %, and . % of beef, mutton, and chicken samples tested, respectively (bostan et al. ). there was no significant seasonal variation in the prevalence of the pathogen. c. jejuni was the species most commonly isolated from chicken meat, while c. coli was the most common in beef ( . %) and mutton ( . %) carcasses. campylobacter isolates were most often resistant to tetracycline ( . %), followed by trimethoprim-sulfamethoxazole ( . %), nalidixic acid ( . %), erythromycin ( . %), enrofloxacin ( . %), ciprofloxacin ( . %), chloramphenicol ( . %), and gentamicin ( . %). the results of this study suggest that a high proportion of meat samples, particularly chicken carcasses, are contaminated by campylobacters, most of which are antimicrobial-resistant strains. in yemen, the prevalence of salmonella in food was determined in sana'a city from april to april by ahmed ( ) . of the different food samples collected from local markets, salmonella spp. were isolated from ( . %). the highest prevalences were in red meat ( . %), chicken ( . %), eggs ( . %), cooked foods ( . %), raw milk and milk products ( %), juices ( . %), vegetables ( . %), sandwiches ( %), and pastries ( . %). serogroups identified were b, c , c -c , d , e , and e , and some foods contained more than one isolate with different serogroups, especially red meat. because handlers in foodservice facilities play a major role in transmission of foodborne diseases (greig et al. ) , studies have been carried out to demonstrate their knowledge of practices related to food safety. in jordan, osaili et al. ( ) measured food safety knowledge of food handlers working in fast food restaurants in the cities of amman and irbid. a total of food handlers in fast food restaurants participated in this question survey study. the overall knowledge of food handlers on food safety concepts was considered to be fair ( . %). the food safety aspect with the highest percentage of correct answers was "knowledge of symptoms of foodborne illnesses" ( . %) and "personal hygiene" ( . %), while the lowest percentage of correct answers was for "safe storage, thawing, cooking and reheating of the foods" ( . %), critical practices to prevent the survival and growth of pathogens. the mean knowledge score of "personal hygiene" reported in the study was much higher than . % and . % reported by martins et al. ( ) and baş et al. ( ) , for the food handlers in portugal and turkey, respectively. also, only . % of respondents considered the duration of hand washing to be ! s. when they were asked how they check that the poultry is sufficiently cooked, only % knew "when the meat has the correct thermometer reading", although % of the respondents had thermometers in their restaurants. about % of them answered that poultry is cooked "when it has been cooked for the stated time" ( %) and "when it looks cooked" ( %). about % of them would store leftovers on the steam table ( %) and in the refrigerator ( %) while about % of the correspondents would store leftovers at room temperature in kitchen or in the oven. a low percentage of the respondents ( %) reheated leftovers to the appropriate temperature ( c). about % and % of the respondents had heard about salmonella and hepatitis a virus, respectively, but % of the respondents knew about listeria monocytogenes, staphylococcus aureus, bacillus cereus, escherichia coli o :h , clostridium perfringens, campylobacter jejuni, or shigella. food workers who had enrolled in a food safety training course had significantly higher total food safety knowledge score than those who did not take any training. there was no association between the experience or any other characteristic of food workers and total food safety knowledge score. this study suggests adopting proper food safety education training courses to food handlers, periodic evaluation of food handlers' knowledge and food safety training course materials. also, the authors considered that better pay for food handlers would improve the food safety status in foodservice institutions. similar concerns over practices that could lead to food contamination and foodborne illnesses were demonstrated in lebanon. a survey was conducted in beirut to evaluate the knowledge, attitudes and practices related to food safety issues of food handlers (n ¼ ) in foodservice establishments (n ¼ ), and to assess the influence of management type on enactment of safe practices on food premises (faour-klingbeil et al. ) . the data suggest that while respondents do have some knowledge of food safety aspects, substantial gaps in their knowledge and self-reported practices associated with critical temperature of foods and cross contamination remain, therefore posing health risks to consumer health. food handlers in corporate managed food outlets showed a significantly higher awareness on food safety practices. it is concluded that the management type is an integral element of the theory of planned behavior that influence food handlers' practices and substantiate the need for more research work on safe food handling in the context of food safety culture framework in food businesses. as in many other mena countries, there is a critical need for food safety education interventions and technical guidance fostered by synergistic participation of the private and public sector to support food handlers in smes (small and medium sized enterprises). parasites are not often looked for in middle eastern countries but they are frequent, and one of the ones of most concern for pregnant women is toxoplasma gondii which is transmitted through undercooked meat and cat feces. since stray cats are common in some localities, of fecal samples of stray cats examined in kuwait, ( . %) were found to be infected with oocysts of coccidian protozoa (abdou et al. ) . toxoplasma gondii was found in . %, and cats < months old had higher infection rate with oocyst of enteric protozoa than older cats. a serosurvey of the stray cats revealed that . % were positive to t. gondii igg. toxoplasma sero-positivity was observed in a higher number of adult cats compared to younger ones suggesting that with age the risk of exposure to t. gondii increases. thus, pregnant women handling cats and particularly kittens or cleaning out sand boxes have a chance of infecting their fetuses and eating raw meat. in pakistan, enteric pathogens are present not only in water but also foods contaminated from the environment or through human actions. mishandling of foods allows these pathogens to contaminate and multiply in them. for example, street-vended fruit salads, locally called fruit chats, offered for sale at high ambient temperatures without coverings, and khoya and burfi, two indigenous sweet dairy products, and locally produced ice cream are often heavily contaminated with enterobacter, e. coli, klebsiella, salmonella and s. aureus (akhtar ) . these contamination scenarios have led to outbreaks with cases severe enough to be hospitalized. bus and train stations where pulses (edible seeds of various crops as peas, beans, or lentils), ground meat dishes, and chickpeas are sold to passengers, and are also heavily contaminated with bacteria including clostridium perfringens. sweet dishes and home-prepared foods in small communities are commonly contaminated with s. aureus, c. perfringens, and bacillus cereus leading to rapid intoxications. one study confirmed campylobacters to be present in % of tested samples of milk and meats and . % of vegetables in three major cities of pakistan (akhtar ) . a wide array of vegetables is routinely consumed in this country and serve as a rich source of vitamins, minerals, bioactive compounds, and fiber but these can be sources of enteric infections if they are consumed contaminated. shigella spp. has been shown to develop resistance and is generally thought to be a major cause of foodborne illnesses, especially among the poor where health care facilities are minimal; shigellosis is associated with poor sanitary conditions and unsafe water for drinking and preparing foods. possible etiologies can be postulated in the following outbreaks. unfortunately, it is not only pathogens that give rise to food-associated disease. soomro et al. ( ) highlighted the indiscreet use of pesticides in agriculture and its impact on environmental pollution. despite the increased production cost associated with extensive use of pesticides, their use is common in developing countries. numerous studies have demonstrated substantial levels of pesticide residues in various foodstuffs in pakistan, and the groundwater has been observed to be considerably polluted in many parts of punjab and sindh provinces of pakistan (akhtar ) . commonly used open rural wells in the punjab were polluted with six pesticides: bifenthrin, λ-cyhalothrin, carbofuran, endosulfan, methyl parathion, and monocrotophos. in the hyderabad region % of the tested samples of eight vegetables (cauliflower, green chili, eggplant, tomato, peas, bitter gourd, spinach, and apple gourd) were found to be contaminated with pesticide residues exceeding maximum recommended limits (mrls) (tariq et al. ; anwar et al. ) . heavy metals such as cadmium (cd), copper (cu), lead (pb), and zinc (zn) arising from increased industrialization can contaminate agricultural soils and these can be found in fruits (including widely-consumed mangoes), fruit juices, vegetables directly from soil uptake or from the processing and packaging (akhtar ) . for instance, spinach, coriander, and peppermint, grown in sindh province contained . - . mg/kg of arsenic resulting in a total ingestion of arsenic . - . μg/kg body weight/day in diet (arain et al. ; khan et al. ) . aluminum concentration in branded and nonbranded biscuit samples from hyderabad were found to range . - . and . - . mg/kg, respectively (jalbani et al. ) . similarly, javed et al. ( ) detected higher concentrations of cd, cr, ni, and pb residues (mg/l) in bovine and goat milk. pakistani foods are more prone to aflatoxin contamination because of the warm and humid climate, and the situation is exacerbated by malpractices during handling and storage of edible commodities (mobeen et al. ) . samples of broken rice, wheat, maize, barley, and sorghum ranged - % with the highest aflatoxin concentration ( . μg/kg), in wheat samples (akhtar ) . chilies are widely eaten and exported, but aflatoxin levels can be eightfold higher than the eu permissible limits to pose a potential health risk to pakistani consumers; concentrations can be reduced by more appropriate care and handling of the chilies at pre-and postharvest stages. nuts and dried fruits in pakistan are cultivated and processed in the northern areas and have been shown to have aflatoxin levels above the eu limit of μg/kg in up to % of samples (ahmad et al. ; luttfullah and hussain ) . aflatoxin m in milk and milk products requires regular monitoring in pakistan since % of the total tested samples of milk were found to exceed the us tolerance limit of . μg/l (hussain and anwar ; hussain et al. ) , and buffalo milk had higher levels of aflatoxin compared with cow's milk. intentional deception of consumers by blending low cost and inferior quality ingredients to make more profit of food intended for sale is prevalent in pakistan, where families are exposed toxic dyes, sawdust, soapstone, and harmful chemicals in beverages, oil or ghee, bakery products, spices, tea, sweets, bottled water, and especially milk and milk products where more than % of samples tested have had adulterants added (akhtar ) . one of the more innovative research projects to provide more home-grown food is in qatar. the sahara forest pilot (sfp) pilot study demonstrated that there are significant comparative advantages using saltwater for the integration of food production, revegetation and renewable processes: ( ) seawater cooling system for greenhouses supports production of high-quality vegetables throughout the qatari summer, and reduces freshwater usage to less than half that of comparable greenhouses in the region; ( ) solar and desalination technologies were successfully integrated as designed into the sfp system, such as the greenhouse and evaporative hedges providing wet-cooling efficiencies without cooling towers; ( ) the external evaporative hedges provide cooling of up to c for agricultural crops and desert revegetation with vegetable and grain crops growing outdoors throughout the year; ( ) commercially interesting algae showed good tolerance to heat and high evaporation rates in the leftover salty water (miss ; clery ). the concentrated solar power plant uses mirrors in the shape of a parabolic trough to heat a fluid flowing through a pipe at its focus. the heated fluid then boils water, and the steam drives a turbine to generate power. hence, the plant has electricity to run its control systems and pumps, and can use any excess to desalinate water for irrigating the plants. in summary, sfp allows food production in all months of the year ( crops) with half the fresh water usage than in comparable greenhouses. on the basis of the pilot success, sfp is now engaged in studies aimed at building a -hectare test facility near aqaba in jordan, large enough from the -hectare operation in qatar to demonstrate a commercial enterprise. tourism is popular in several middle eastern countries, particularly beach and coastal resorts in egypt and turkey. tourism has been the major economy in egypt for many years but can be threatened not only by civil unrest and terrorism but also by foodborne illness (costa ). tourists might not stop coming to egypt due to a few reports of diarrhea; however, widespread reporting of severe cases, and lawsuits, will make tour operators much more selective, and bring pressure on the egyptian hospitality industry to improve its hygienic standards. the greater challenge is for egypt to ensure that it has the capacity to sustain a safe food supply for its own people. in doing so, it provides safe food for those who want to explore its rich history and seaside resort areas. multiple reports of illness have been reported from nile river cruises and a resort town on the coast. from september to november, , cases of hepatitis a imported from egypt were reported to the german public health authorities (bernard and frank ) . investigations pointed to a continuing common source of infection, most likely linked to nile river cruises. in addition, eight cases from france had been travelling on a nile cruise and one on a red sea diving safari (couturier et al. ). one specific cruise ship was mentioned by six of ten belgian cases (robesyn et al. ). those who took a nile cruise had typically done this in combination with a hotel stay. at least three different ships and three different hotel accommodations were mentioned in the travel histories of the french cases. the patients affected had not been vaccinated, which emphasized the need for more effective travel advice before trips to hepatitis a endemic countries (sane et al. ) . possible sources of infection might have been contaminated food obtained from a common food catering company consumed onboard, contaminated tap water supplies for the ships' bunkers, or a common exposure on shore (e.g., a restaurant where tourist groups from various ships were taken during day trips). as all of these ships continuously traveled up and down a short stretch of the river (aswan to luxor and back) with standard mustsee stops along the way, the cases possibly shared an exposure on land. both the long incubation period of hepatitis a ( - days) and long delays in collecting information on the individual cases precluded any rapid intervention on location. no specific food source was identified but it could have been juices as recognized in an earlier major outbreak. in , tourists returning from egypt included hepatitis a case-patients from european countries who were infected with a single hav strain (genotype b) (frank et al. ). the case-control study identified orange juice most likely contaminated during the manufacturing process, e.g., by an infected worker with inadequate hand hygiene or by contact of fruit or machinery with sewage-contaminated water. citrus fruit and citrus juices have occasionally been implicated as vehicles of hav and salmonella infections, with contamination typically occurring during production, or preparation just before consumption. as hav is resistant to acid, it likely can survive for prolonged periods in orange juice. it is also possible that leafy greens could contribute to foodborne illness in egypt. an international study of contamination of leafy green lettuce and spinach samples taken between and from open-field farms in belgium, brazil, egypt, norway, and spain showed that the egyptian samples were the most contaminated at . % (liu et al. ) . these authors claimed that temperature had a stronger influence than did management practices on e. coli presence and concentration. region was a variable that masked many management variables, including rainwater, surface water, manure, inorganic fertilizer, and spray irrigation. temperature, irrigation water type, fertilizer type, and irrigation method should be systematically considered in future studies of fresh produce safety. also in the spring of , a young couple was ill with vomiting and abdominal cramps after their first meal at a sharm el sheikh -star hotel in the egyptian coastal resort area, and they remained there in their bedrooms for the rest of their week (this is staffordshire ). both continued to have ongoing issues months later, with one of them suffering from reactive arthritis. other guests also complained about diarrhea. they stated that the food was disgusting; the meat was undercooked, the buffet was left out for long periods of time, with new food being piled on top of the old food, and there were flies landing on food items. in august , a family stayed at a resort hotel, also in sharm el sheikh, and all suffered severe symptoms including diarrhea, stomach cramps, and vomiting. they were put into the hotel clinic given antibiotics and intravenous drips but had not completely recovered after they returned home (galley ) . at the time other guests were also ill. they noticed that the food including chicken and beef, appeared to be undercooked a couple of times, and that one of the chefs touched raw meat and then touched cooked meat without changing gloves. the booking company confirmed that "a very small number" of guests staying at the resort in reported that they had been unwell, "with symptoms similar to a virus". the company said that guests were offered the appropriate support and advice by their overseas holiday advisors. it claimed that all of its hotels were subject to stringent monitoring and audits and this hotel achieved an extremely high score in its audit carried out in the summer of . however, high audit scores do not necessarily correlate with day-to-day safe hygienic practices (powell et al. ) . the popular beach resort of sarigerme, turkey, on the aegean sea also has had a reputation for gastroenteritis, with repeat problems of foodborne illness with british tourists on vacations organized by tour companies, although the actual hotels were different. in , an outbreak of gastric illness at this resort led to £ . m paid out in compensation, with people suffering from infections including salmonella, cryptosporidium, campylobacter and e. coli (hutchison ) . in september, , hundreds of british holidaymakers suffered from salmonellosis after returning from a hotel complex in sarigerme (disley ) . final figures may have been close to , and several were hospitalized. in october , the swannell family had booked a week's stay at the first choice holiday village resort in sarigerme, when mark swannell, , fell seriously ill a few days into the break with diarrhea, abdominal pain, nausea and lethargy (hutchison ) . he said that some of the food he was served at the hotel had been undercooked, with some chicken bloody in the middle, food was not served at the correct temperature, food was left uncovered for prolonged periods of time, and the same food had been served more than once. the family stated that cutlery, crockery and table linen used in the restaurant was not up to standard, and they saw cats in the public areas of the hotel and in the restaurant. legal action was taken. in addition to ill tourists in middle eastern countries, contaminated exported food can affect those abroad, as illustrated in the following u.s. outbreak. from march to august , of patients identified with hepatitis a in ten states, ( %) were admitted to hospital, two developed fulminant hepatitis, and one needed a liver transplant, but none died (collier et al. ) . almost all cases reported consuming pomegranate arils (seeds) from one retail chain. hepatitis a virus genotype ib, uncommon in the americas, was recovered from specimens from people with hepatitis a virus illness. pomegranate frozen arils imported from turkey were identified as the vehicle early in the investigation by combining epidemiology, genetic analysis of patient samples, and product tracing. the product was then removed from store shelves, the public warned not to eat the seeds, recalls took place, and post-exposure prophylaxis with both hepatitis a virus vaccine and immunoglobulin was provided. this investigation showed that modern public health actions can help rapidly detect and control hepatitis a virus illness caused by imported food. egyptian trade has also been adversely affected by exports. in , there were three outbreaks of hepatitis a sickening persons in -european countries. in the first report in april, persons in four scandinavian countries were infected with hepatitis a (andrews ) . epidemiological investigations traced those cases to frozen strawberries grown in egypt and morocco, though no strawberries were found to be positive for hav. the second outbreak in april was larger in extent with ill in countries, all having recently visited egypt, and the outbreak strain of the virus had the same subgenotype as the first outbreak associated with strawberries. an epidemiological investigation into the second outbreak suggested the likely source was strawberries or another fruit distributed to hotels in egypt. the third outbreak was reported in germany in may, after nine germans were infected with hepatitis a after traveling to italy. this third outbreak infected about italian residents, as well as nine germans, one dutch traveler and five polish travelers; irish residents with no travel history to italy were infected by the same strain of the virus. separate investigations in italy and ireland both implicated imported frozen mixed berries as the source, with most of those berries coming from eastern europe. it is not known if these berries came from other regions, such as egypt, or were local to eastern europe. contributing factors to the larger number ill was lack of vaccination. because hav infections were declining in europe over the last few decades, fewer people had developed antibodies to repel the virus. couple that with the fact that hepatitis a was not on the vaccination schedule for citizens of many of the countries affected, and the result was a highly susceptible population. also, most of the european travelers to egypt were not advised to get hepatitis a vaccinations when staying in all-inclusive resorts, which were attracting an increasing number of europeans traveling to egypt. further, the investigators believe contamination of the berries occurred early in the food production chain. investigators suspect that irrigation water contaminated with sewage water likely contaminated the strawberries in the two outbreaks connected to egypt. but the contamination might have also been caused by infected workers in the field or the processing facility, or by contaminated water sprayed on the berries sometime before distribution. the outbreaks indicate that fresh and frozen berries are efficient vehicles of hav infection, as previously demonstrated in the us and elsewhere (palumbo et al. ) . european authorities agreed that "the experience demonstrated the absolute necessity for extensive collaboration between countries and between the public health and food sectors to identify as quickly as possible the vehicle of infection and, ideally, to control the outbreak in a timely fashion." a more serious outbreak damaged egypt's food export trade. in july , the european union (eu) banned the import of certain egyptian seeds and beans till at least october following an official report that a single batch of egyptian fenugreek seeds probably caused two european outbreaks of e. coli infections responsible for ill persons and at least deaths. a task force of health officials set up by the european food safety authority (efsa) reported that one lot of fenugreek seeds imported from egypt was the most likely common link between the two outbreaks in northern germany and in bordeaux, france (anderson ) . both were traced back a year and a half to a shipment of , pounds ( , kg) of fenugreek seeds, that was loaded onto a ship at the egyptian port of damietta on november , . on the ship's arrival at antwerp, belgium, the seeds were barged to rotterdam to clear customs. the sealed container was trucked into germany to an unidentified importer, who resold most of the lot. an unidentified german company then resold about pounds of the seeds to the german sprouter, which is believed to be the source of the sprouts that caused the extensive german outbreak. the german importer also sold about pounds of sprout seed to the english company thompson & morgan, which repackaged the seeds into . -ounce ( grams) packages. those packages were shipped to a french distributor, who resold the seeds to about garden centers around france. investigators believe that one of those packets was the source of the second european outbreak with cases in the bordeaux area. because the seeds were likely contaminated with e. coli o :h at some point before leaving the importer, and more contaminated seeds could be in circulation, it was deemed appropriate to consider all lots of fenugreek from the egyptian exporter as suspect. soil contact or animal or human fecal contamination of the seeds likely occurred during their production or distribution in egypt. even a negative laboratory test of those seeds could not be interpreted as proof that a batch was not contaminated. trace-forward findings indicate the german importer sold seeds from the suspected lot to companies, and the shelf life of the seed can be up to years. by mid-october, , the european commission (ec) lifted import restrictions on fresh and chilled podded peas and green beans and other fresh produce from egypt, but the ban on egyptian seeds and sprouts, scheduled to expire on october , was to be extended until the end of march, , following an "unsatisfactory audit" of seed producers in egypt (news desk ). the extended ban involved arugula sprouts, leguminous vegetable sprouts (fresh or chilled), soy bean sprouts, dried (shelled) leguminous vegetables, fenugreek seeds, soy beans and mustard seeds. the ec audit showed that measures taken by the egyptian authorities to address shortcomings in the production of seeds that may be sprouted for human consumption were not sufficient "to tackle the identified risks." those shortcomings were not seen in the growing and processing sites for fresh peas and beans, and therefore those vegetables were no longer considered a food safety risk. there is no need for actual illnesses to occur to affect trade. recalls, seizures, and bans can be employed by importing countries if standards are not met, and force exporting countries like egypt to take action. for instance, in the ec suspended the import of peanuts from egypt due to the presence of aflatoxin in concentrations in excess of maximum levels specified in eu regulations (technical cooperation department ) . egypt is a major peanut exporting country and the european markets then accounted for % of its peanut exports. this decision was repealed on december and was replaced by another decision, which imposed a requirement for certification to accompany every consignment and required systematic analysis of consignments and documentation by the importing member state. under this system only egyptian exporters were allowed to ship to the eu. in august , the decision was replaced by another decision that required the competent authorities in eu member states to undertake random sampling and analysis of % only of peanut consignments from egypt for aflatoxin b and total aflatoxins. this improvement came as a result of the efforts that the egyptian government put in complying with the requirements of the eu. to this end, the egyptian ministries of agriculture and land reclamation (malr) and ministry of foreign trade (moft) issued ministerial decree no. / , which covered all stages of production, processing, sampling and exporting of peanuts. the main provisions of the decree were: exported peanuts must be produced, inspected and prepared according to set scientific procedures; and exporters who violate the rules would be suspended for year; the decree also established the legal limit for aflatoxin in peanuts in both the domestic and eu export markets. in the egyptian domestic market, the legal limit was mg/kg aflatoxin b and mg/kg total aflatoxin content. for the eu market, the legal limits were mg/kg aflatoxin b and mg/kg total aflatoxin content. in addition, the decree specified the sampling procedures that must be followed for export certification. in september of the food and veterinary office sent a mission to egypt to assess egypt's compliance with its certification system requirements. a number of recommendations on steps egypt should take to improve the control system of foodstuffs intended for export to the eu were made. in response, the egyptian authorities declared that they were taking actions to address the mission's recommendation. but to achieve that there was a need to coordinate among a number of egyptian agencies involved in the production and export of peanuts and aflatoxin control: malr, the central administration for plant quarantine (capq), the agricultural research center (arc), the ministry of foreign trade (moft), and the customs service. also a laboratory capable of testing for mycotoxins was necessary. alongside this; egypt had technical assistance from international organizations in order to build human and physical capacities necessary for achieving compliance. the action by the eu forces egypt to improve the safety of its peanut production which would be beneficial both to europeans and to all who eat products made from egyptian peanuts, including the domestic consumers. lebanon used to be a tourist haven but is less today because of a seemingly dysfunctional government following a civil war. the country produces food for both the domestic and overseas markets. unfortunately, some exported food has caused illnesses and recalls. twenty-three cases of salmonella bovismorbificans in eight states and in the district of columbia (washington, d. c.) from august to november, were linked epidemiologically to hummus eaten at three mediterranean-style restaurants in the d. c. area, all owned by the same individual (goetz ) . although samples collected from all ingredients used to make the hummus tested negative for any salmonella, the hummus was recalled and the outbreak ceased. during its investigation of the restaurants, the d.c. department of health discovered multiple food safety violations at the establishments, including inadequate food temperature control, insufficient hand washing, and the presence of pests and insects, which had to be corrected. it is not clear if any abusive temperature conditions could have allowed growth of the salmonella in the hummus. the public was not notified because by the time the hummus had been withdrawn from the market, there were no further cases. however, the contaminated ingredient in the hummus was not discovered until may, , when a traceback by the u.s. food and drug administration (fda) revealed that the tahini used to make the hummus in one of the restaurants had recently been associated with recalls in canada for contamination with s. cubana (september ) and s. senftenberg (february ). all tahini linked to these outbreaks had been imported from the same company in lebanon. the fda then mandated that all tahini products coming from this lebanese company be tested for salmonella before entering the u.s. and has recommended that u.s. and canadian officials partner to inspect the tahini manufacturing plant. this was the first time s. bovismorbificans had been implicated in a tahini outbreak in the u.s. as a result of this outbreak, the author stated it is important for public health officials and consumers to be informed that products made with imported sesame paste have been shown to be associated with salmonella outbreaks and that they should be considered as possible sources for foodborne illness in the future. in fact, contaminated sesame seed paste was in the news a few days before a cdc report on the outbreak was made public, after a supply of contaminated tahini was stolen from a california importer's warehouse, where it was being stored because a sample had tested positive for salmonella. the tahini, which had also been imported from lebanon but from a different manufacturer, was awaiting destruction, and the fda warned the public that the stolen, potentially contaminated tahini may be on the market. lebanese tahini has been implicated in several outbreaks in the past and subject to recalls (harris et al. ) . government oversight of the food industry is variable across the region with many regulations stemming back to colonial days, but modernization changes are gradually being considered or implemented. unfortunately, where some middle eastern countries are slowly moving forward to improve food safety, others are slipping back in their oversight because of conflict and lower public health priorities. there are relatively few large food processing operations except those managed by multinational companies, and most of the government oversight is on smes particularly small foodservice outlets. the states in the gulf cooperation council (gcc), each have an aggressive food safety policy but do not always follow identical approaches, some of which are well-established and some of which are innovative. the ksa has had a food inspection system in place for many years with reports of outbreaks published regularly, though no doubt it could be improved with more cooperation between the ministry of health, the municipalities and the saudi food and drug authority (sfda). the sfda was established under the council of ministers resolution no ( ) dated january , , as an independent body that directly reports to the prime minister (el sheikha ). the sfda is responsible to regulate, oversee, and control food, drug, medical devices, as well as set mandatory standard specifications thereof, whether they are imported or locally manufactured. the control and/or testing activities can be conducted at the sfda or any other agency's laboratories. moreover, the sfda is in charge of consumers' awareness on all matters related to food, drug and medical devices and associated other products and supplies. the sfda has to negotiate with the moh their mutual responsibilities following specific foodborne disease instances or consumer complaints. bahrain claims to have one of the more advanced food control systems in the region. in july , as ambient temperatures heated up, the ministry of heath urged people to make sure the food they consume is properly stored during the summer months to avoid microbial growth and risk of food poisoning, e.g., keeping meat and fish at c and to cook food thoroughly (haider ) . the ministry was aware that both visitors and locals want to eat safe food, especially as bahrain is moving towards more tourism with people are eating out more often. the ministry ordered shops to provide appropriate storage facilities, e.g., coolers and refrigerators, for food as part of its efforts to protect the public's health. inspectors were checking food stalls, ice-cream parlors and vegetable shops to ensure that customers were not being sold contaminated or rotten products. the ministry claimed to thoroughly investigate any complaints it receives, and to facilitate this a new hotline number was launched by the ministry for general public to report food contamination complaints against supermarkets, restaurants, coffee shops and hotels. specific advice for consumers included: being careful when buying salads; fruits and vegetables should be washed thoroughly before they are consumed; and dairy products such as milk, cheese and eggs, should always be refrigerated, since microorganisms grow faster in these products. the ministry claimed that bahrain has one of the best food control methods and food safety records in the region, and could even act in the future as a consultant in this field for other countries, including other gcc states. by , government oversight had stepped up. in april, the ministry of health warned people against buying food advertised on social media or sold on the street by unlicensed retailers in bahrain, either made in people's homes or by street hawkers (anonymous c) . the ministry stated that control of these home operations is difficult if someone suffers from food poisoning since inspectors are not allowed to go into homes. many homes sell food without a license and some would-be entrepreneurs even have barns where they slaughter livestock and market the meat illegally. there were , inspection visits conducted in by inspectors from the food safety and licenses group, which closed of around registered outlets. inspections cover imported food from ports right up to where it reaches restaurants and food outlets; , visits revealed around , tonnes of imported food were permitted for consumption, but tonnes were considered as non-consumable (rejected), during the same period. one of the more recent important programs is the smart inspection project launched in april . inspectors, many with masters and phd degrees, visit restaurants and coffee shops to take food samples, as well as explain to staff how to store food and ensure its safety (anonymous c) . it includes awarding food outlets that achieve a % food safety standard a blue sticker, while those meeting % of standards get a green sticker. outlets that fail to achieve basic standards are warned with a red sticker. the total number of outlets assessed between august and february was ; were presented with blue stickers, with green stickers and with red stickers. this project features daily inspections and is focused on small food outlets, some of which have caused food poisoning in the past. inspection visits depend on the hygiene of each outlet and the complaints received about them; some require two or more visits annually. high-level restaurants already have certified inspectors for evaluation and most of them require only one visit per year. the ministry's ultimate goal through this project is to decrease cases of foodborne disease, particularly important as bahrain is increasing its tourism efforts and, thus, ensuring food safety is essential. to support the ministry's initiatives, live demonstrations on food safety practices were promoted in kitchens in hypermarkets. however, if red sticker facilities fail to take advantage of educational material, they may be punished for neglecting food safety standards and guidelines though public prosecution. in a bid to improve standards of hygiene in restaurants, qatar's supreme council of health (sch) increased the number of spot checks on food outlets and has launched a hotline for residents to report food poisoning (walker ). the council is responsible for monitoring food establishments and implementing qatar's food laws along with the ministry of municipality and urban planning (mmup/baladiya). the sch embarked on an intensive inspection campaign, collecting food samples from all restaurants and food outlets in the country including suppliers. the inspection teams, which include specialized doctors from the sch's communicable diseases department and the environmental health inspection department, also medically check workers responsible for preparing food to ensure they are not carrying infections. those found to be handling food in an unhygienic way would be immediately dismissed. following a hotline complaint call, a report is filed, a team from the sch visits the affected people, then inspects the related food outlet and collects samples for laboratory examination. the latest crackdown was in response to the illness of a family of four which suffered food poisoning after eating chicken, rice and salad at a popular turkish restaurant which was closed down because a medical report prepared by the sch's environmental health section confirmed that the outlet served contaminated food and violated health regulations. tests conducted in the central food laboratory at sch found three types of bacteria causing diseases in food served by the restaurant. medical tests on the victims also showed that they were infected by the same bacteria, as well as one of the restaurant workers. another popular turkish restaurant was closed for months after it was found that several customers were treated in the hospital for food poisoning symptoms including intense nausea, vomiting and diarrhea. as part of the sch's new campaign, experts would undertake community awareness drives, and organize seminars and training sessions about food contamination to improve understanding among owners and workers in food establishments. other closures occurred because of serving food with moldy ingredients, rotten vegetables in the kitchen, insects in pasta, and generally violating the provisions of the food law. the mmup increased the number of spot-checks and naming and shaming erring establishments on its website in arabic. the amendments to the food law gave greater powers to authorities to fine and close down venues that break the law including temporarily closing down establishments if it has violated food safety and hygiene regulations, and also has the power to recommend severe penalties. a follow up to one of these closed doha turkish restaurants was after a trial when five staff were each been handed fines, jail sentences and deportation orders after they were found guilty of causing food poisoning to approximately customers ill with vomiting, nausea and diarrhea (santacruz ) . the restaurant was accused of serving spoiled and unsafe food on october, . an affected pregnant woman gave birth to her baby months prematurely. the manager of the restaurant was fined approximately $ and sentenced to spend months in jail while three other staff members were each fined approximately $ and sentenced to month in jail. during an inspection it was found that another staff member did not hold the necessary health certificate and was subsequently fined approximately $ and also sentenced to month in jail. as well as the staff members being sentenced to jail and fined, the court of environmental misdemeanours also found that the restaurant itself was guilty of causing the food poisoning outbreak, and issued the restaurant with approximately $ in fines and ordered it closed for a further months. in other parts of the world these penalties would seem unduly harsh, as it would be difficult for this restaurant ever to recover financially. coupled with education, there has been recent enforcement blitzes on food establishments such as hotels, restaurants and bakeries by oman municipalities, and a leading bakery in muscat was closed down because of rats in the premises in late december, (staff . this led food safety experts and the public to call for stricter rules and heftier fines to be imposed after surprise checks conducted by the muscat municipality, especially when it was disclosed that nearly half the restaurants in the bausher area were not following food safety standards. surprise inspections by the muscat municipality at restaurants in bausher found that around restaurants did not meet food safety standards and were violating rules formulated by the municipality. also, in the same time frame, ibri municipality officials were forced to shut down commercial shops and they destroyed more than km of outdated food in . according to the municipality's officials, health violation letters were issued throughout the year, as well as warnings were issued to different institutions operating in the wilayat of ibri. there are no easily-accessible reports on government oversight in pakistan and inspection actions are more likely to be released to the public through the press. in , the islamabad capital territory (ict), administration conducted a drive against adulterated food items with unannounced inspections of food outlets in different markets and imposed fines amounting to rs , (about us$ ) on owners for unhygienic conditions at their premises including restaurants, cafes, bakers, candy (sweet) stores, and a hotel was sealed (app ) . cleanliness conditions at the outlets' kitchens were found unsatisfactory and unhygienic while workers had not been vaccinated against viral diseases. some business owners were also paying less to their workers in contravention of the minimum wages act. business owners were directed to improve cleanliness conditions and ensure food safety standards failing which strict action would be taken against them. a cattle market was also ordered to "beef up" its security. punjab, pakistan's most populous province, has a population that is more than double that of california, and lahore, the provincial capital, has a vast array of food outlets. from the available press reports, the punjab food authority (pfa) has a mixed record of oversight of food operations. a pfa team visited the polo ground restaurant at the race ground park and found expired food, blocked sinks and unhygienic conditions in the kitchen and food storage area in contrast to the claimed high quality standards by the management of the supposedly high-class restaurant (raza ) . the team faced resistance from the management but it managed to enter the kitchen for inspection. pfa officials said the kitchen condition was similar to that of an ordinary road-side eatery, dispelling general perception that restaurants serving the elite follow higher standards of hygiene and food safety. however, the pfa in lahore had received a complaint that an assistant food safety officer had received rs , (about us$ ) bribe from the restaurant owner so he could keep his restaurant open (anonymous d) . another restaurant on peco road sealed by the pfa for poor hygiene and unsanitary conditions of its workers in the second week of march, was opened for business the very next day. typically, according to the pfa's standard operating procedure (sop), a restaurant sealed for the first time may resume business after a week. at the end of the week, the proprietor has to submit an affidavit assuring the authority that all problems pointed out by the food safety officer had been taken care of prior to reopening it for business. the pfa director general (dg) had constituted a three-member committee to probe the complaint of bribery but it was later shelved. similar situations occurred when restaurants that had reopened before the stipulated period for closure had expired. in the first week of , a restaurant was fined rs , (about us$ ) for unhygienic conditions and lack of soaps in the workers' washrooms, instead of following the pfa sops of sealing the premises. the sops regarding duration of closure and required permission from the pfa dg were stated to be flouted openly. however, a pfa spokesperson denied any wrongdoing, and the sop was being observed to the letter. she said a written permission from the dg used to be mandatory in order to de-seal restaurants, but now an operations deputy director can also issue permission for it. she also stated that the restaurant on peco road had not reopened on orders of the pfa; its owner had de-sealed it illegally. these reports indicate that there may be some illegal activities including bribery by inspectors but miscommunication on how much leeway inspection staff have on prevention and control practices may be more of the issue. in mid- ayesha mumtaz became the new operations director of the pfa, tasked with ensuring food in punjab is unadulterated and safe (reeves ) . her self-declared war on unhygienic food generated so much publicity in the last months that she became a household name in pakistan. mumtaz says many food producers know nothing about hygiene but are willing to learn. there's also a hardened mafia who are only interested in profit, she says. everyone in the street seems to know about mumtaz. storekeepers begin shooing away customers, hauling down the shutters, and heading into the shadows in the hope that mumtaz's scrutinizing eye will not fall on them. these traders would sooner lose business than risk a visit from a woman whose campaign to clean up the kitchens and food factories of pakistan has made her a national celebrity. she declared that the pfa cannot allow them to get away with their "perverse" activities and to "play havoc" with the lives of the people. consumers are unaware that the cakes and sweets that they buy over the counter are produced amid unhygienic conditions. she has found spoons encrusted with filth, fly-blown cans of gooey liquid lying around haphazardly, dirty containers, grimy rags and rusty tin cans, moldy scraps of cake, all involved in making cakes and sweets to be sold to the public. civil servants in pakistan are often accused of being lazy and corrupt. mumtaz is being feted as a rare example of a government official who actually champions the public's rights. she and her inspectors have so far raided more than , businesses, and pakistanis seem to approve. her fans call mumtaz the fearless one. hundreds of thousands have clicked like on the pfa's facebook page in appreciation of her work. there was a very famous hotel in the heart of lahore that she inspected and found the chiller where they keep all the foods together (vegetables with chicken, meat), but also a big rat; this became big news for the public. however, there are complaints that she does her raids with police and cameras to be broadcast nationally even before the owners are convicted, according to the lahore restaurant association. in , the abu dhabi food control authority (adfca) planned to check all food handlers by . the authority's emirate food safety training (efst) program, started in , provides basic training in food hygiene and safety to those who work in food outlets (olarte ) . according to the adfca, small catering businesses in most countries have the lowest standards of food safety, and most workers in abu dhabi's small restaurants are illiterate and do not speak fluent arabic or english, making it a challenge for them to understand and follow safety guidelines and regulations; % of managers and % food handlers in the capital speak south asian languages such as urdu, hindi and malayalam (pennington ) . the training is now offered in four languages -english, arabic, urdu and malayalam -which the majority of food service personnel speak, and covers basic food hygiene issues including staff hygiene, food temperature, cross-contamination, cleaning and sterilization. to help them understand and follow food-safety rules, the adfca is using photographs to teach employees how to handle food safely according to international standards. the scheme is an extension of a pilot involving small restaurants carried out in - . as part of the efforts to ensure retention of their learning, the adfca conducted spot checks at food outlets in marina and khalidiya malls, and gave guidance and advice to staff for those with violations, rather than just penalizing them, the normal practice in most middle eastern countries. the field operations manager at the adfca noted that the differing cultures, education and languages are the barriers that sometimes hinder food handlers from carrying out what they are trained to do. he recommends that supervisors should quiz them on hygienic and safety issues so that they know how to properly prepare and serve food. those who have learning difficulty or are illiterate are given assistance through illustrations, in order to make it through the lessons and pass the examination. one of the critical elements of food safety that the adfca has to monitor and ensure, is that food handlers are aware of cold ready-to-eat food being kept at c, while hot food should be kept and served very hot > c. the adfca categorizes the food premises and carries out inspections based on their risk factors -high, medium and low. restaurants and hypermarkets belong to the high-risk group; warehouses to the medium risk; while groceries, honey shops and vegetable and fruit outlets are considered low risk. recently, the establishment of the egyptian food safety authority was initiated by the minister of trade and industry, with the support of the ministry of health and the ministry of agriculture. it would be responsible for food safety and consumer protection through the provision of sound data and guidance to deal with processed or genetically modified food in accordance with food safety standards (anonymous b) . the strategic plan for the new draft law includes a revision of all egyptian laws and legislation that deal with food safety since , including around other legislations. the authority would need to apply food safety standards on imported food the same way it does for locally produced foodstuffs. adopting the draft law would in effect cancel all existing laws and create one food safety law for the country. the food safety authority plans to monitor the foods consumed by egyptians of different age groups as a basis for where to put resources. another issue to be faced is that studies in egypt based on us statistics have revealed that the cost of food spoilage costs the country million egyptian pounds annually. the chamber of food industries indicated that a unified body for food safety to apply international quality specifications and unite regulators was lacking. this reduced the competitiveness of local products, especially since most foreign countries do not recognize egyptian regulations. it was hoped that investors in food industries would bring in new investments to the sector in the upcoming period if a food safety authority were to be established, as per a ministerial decision issued in . the food safety authority has received several approvals from governments that ruled during the -year period following the revolution, but apparently nothing has been yet finalized until recently (mefreh and saeed ) . in a similar way to egypt, the lebanese government has been debating a new law on food safety for many years but unlike egypt, it has yet to make much progress. lack of agreement at the parliamentary level has resulted in different ministries (health, agriculture, industry, environment, tourism) taking action as they see fit. the latest was in november , when the minister of health conducted an extensive campaign of inspections in lebanese establishments and naming of facilities that did not meet the ministry's expectations (naylor ) . the minister personally revealed that numerous supermarkets, bakeries, butchers and restaurants had been violating food safety and sanitation standards. they shut down slaughterhouses, restaurants, supermarkets and other retailers selling contaminated food. for instance, changes needed to be made for the slaughterhouse to conform to health standards; the report said livestock must be hanged during slaughter and not laid on the ground and that the abattoir should also be equipped with refrigerators and storage units for separate types of meat and their cuts. however, discord among ministries is apparent with the tourism minister trying play down the publicity of the health minister's food safety blitzes by saying "we are in favor of full transparency, but we feel like we were 'deceived' because the food safety situation in lebanon is good and better than other countries. we apologize to tourists, but more importantly, any of the ministry of health staff is ready to apologize to the lebanese citizens for the public sector's failures throughout the years?" (yaliban ) . foodborne disease surveillance is limited in lebanon and cannot be used to indicate the actual level of foodborne illnesses in the country. lebanese food exports are also being required to conform to international standards. tahini made from sesame seed paste is a major food export to the west, but recalls of tahini manufactured in lebanon because of salmonella contamination are more frequent than they should be; one recent example was a health hazard alert for certain clic, al nakhil and al koura brand tahina products that may have contained salmonella, recall/advisory dated august , posted from canadian food inspection agency [also see tahini/hummus linked illnesses under foodborne disease in specific countries]. under the new us food and drug administration food safety modernization act, foreign companies importing foods to the us must demonstrate that they have the operational plans and facilities sufficient to produce safe food before they can ship any product to the us (fda ), which is causing some concern among lebanese tahini manufacturers and government agencies. thus, although there is knowledge about foodborne disease and other food safety issues within government, industry and academia, the political inertia means that many foodborne illnesses will continue to occur but not be properly reported or know what factors were present to cause the outbreaks. industry currently is taking the lead; apart from companies promoting food safety like boecker and gwr food safety, mena food safety associates (mefosa) (http://www.mefosa.com/), based in beirut, assists mena companies hone their competitive edge by establishing and verifying procedures and practices that ensure quality, wholesome and safe products through consulting, auditing and training services in haccp, gmps, and hygienic practices. however, lebanon's lack of a coordinated system of government oversight of the food industry pales into insignificance compared to that in syria. prior to the war, syria's healthcare system had hospital and doctor levels equivalent to other middle-income countries such as brazil, turkey and china, with life expectancy of years, and most of the disease burden being similar to that in the west with non-communicable diseases, but four years of violence have changed all of that. child vaccination levels dropped from % pre-conflict to % in march (templeton ) . as a result, outbreaks of diseases that had long been under control have spread across the land and into neighboring countries: hepatitis, measles, leishmaniasis, multi-drug-resistant tuberculosis, typhoid and even polio, which had not been seen in the middle east for years. life expectancy has dropped by two decades. medical personnel are clearly targeted because they are seen as potential enemies helping the opposite side. the majority of syria's doctors have been killed or fled the country (> medical workers have been killed since ). the situation has been called the worst humanitarian catastrophe this century, and the worst concerted attack on healthcare in living memory. at least , syrians have been killed and more than million others have been forced from their homes since the conflict began on march , , with over four million people in areas that are hard to reach for humanitarian aid, and Á million have fled mostly to neighboring turkey, lebanon, jordan, and northern iraq, while others have sought safety in europe, provoking a political crisis in the -member bloc (devi ) . another middle eastern country under stress but with less publicity is yemen. currently there is little government oversight into food as there is little to be had. the situation in yemen is characterized by large-scale displacement, civil conflict, food insecurity, high food prices, endemic poverty, diminishing resources, and movement of refugees and migrants (wfp ). the un world food programme (wfp) has been in yemen since . in , wfp conducted a comprehensive food security survey which found that % of the people ( . million) were food insecure, of which some five million were severely food insecure, meaning they were unable to buy or produce the food they need to survive. the organization's protracted relief and recovery operation (prro), aims to reach six million people between mid- and mid- with , metric tons of food and us$ . million in cash and vouchers at an overall cost of us$ million. if the conflict continues, this goal is unlikely to be met in time since both the airport and shipping port are areas being fought over. the wfp has been attempting to bring in relief supplies but cannot do so under fire, which means that only small amounts are occasionally delivered to the country (mukhashaf and miles ) . one example of this occurred in aden on july , when a ship docked after waiting a month to unload enough u.n. food aid to feed , people for a month. previous repeated attempts to send ships to aden were been blocked due to severe fighting in the port area. the prro is aligning wfp's activities with moves to increase the government's capacity to respond to the crisis and will promote recovery and resilience to enable food insecure households and communities to better withstand and recover from the effects of conflict and shocks. there are many similarities as well as substantial differences in the descriptions of issues concerning food safety and foodborne disease of each country in the region. gastrointestinal diseases are frequent throughout the middle east with some countries identifying their etiologies, such as egypt, kuwait, israel, pakistan, turkey, yemen. these include bacteria and parasites, e.g., salmonella, shigella, campylobacter, enterotoxigenic e. coli (etec), giardia, entamoeba, and occasionally enteric viruses such as hav and norovirus. however, none of the countries has a well-functioning foodborne disease surveillance system, but a few report on a regular basis like ksa, and starting recently, lebanon with pulsenet. mostly it seems that only large outbreaks or ones with fatalities that are reported on, and mainly through the press. these outbreaks are often related to point sources which are in most cases communal foods prepared for a large number of individuals as in feasts, student hostels, schools, campuses, or military camps. however, the actual etiological agents and the factors contributing to outbreaks are only rarely determined. one example is a very large outbreak in bahrain in with at least people suffering from foodborne illness after eating contaminated egg-andmayonnaise sandwiches served at a wedding party, but the etiology was not determined, even though clinical specimens and food samples were analyzed, at least in a publically-released report (promed-mena ) . based on the type of preparation including the length of time taken for preparation of the implicated food and the time from consumption to the appearance of symptoms of foodborne illness, the types of symptoms, and what has already occurred historically in foodborne disease outbreaks, possible agents can be surmised, such as bacillus cereus and staphylococcal enterotoxins, and salmonella, shigella, or norovirus infections, but promed is continually asking for more information once an outbreak is announced, and hardly ever receiving it (promed-mena ). all this indicates that even if clinical specimens or food samples are taken and analyzed, laboratories are only rarely able to determine an etiologic agent, or at least report on their results. most agents described with the little information available seem similar in all the mena countries and to those encountered in the west. however, a few pathogens are more likely to be restricted to a few nations, such polio in pakistan, cholera in iraq, mers-cov in ksa, and botulism in egypt and iran where river fish are often eaten (one case of infant botulism was diagnosed in israel but it is a rare disease anywhere); the first two are more likely transmitted though water or poor hygienic conditions, the third by camels, and only botulism exclusively through food. brucellosis is widespread in the middle east but only a few country studies indicate its link to meat or dairy products. much of the middle east is in the throes of conflict which results in unique situations in specific countries to exacerbate foodborne disease or food poisonings; these include relief agencies supplying "stale" food to those trapped and starving by the syrian civil war, almost lack of food at all in yemen, deliberate poisonings of enemies in afghanistan, syria and iraq, accidental pesticide poisonings in iran, preventing unsafe food being sold to those on the hajj in ksa, improperly prepared catered food for foreign troops in bases in afghanistan, iraq, kuwait, ksa, and turkey. countries where tourism is a major source of income can be adversely affected by bad publicity over complaints over food served in resorts, such as in egypt and turkey. also, gulf countries tend to employ workers from india and other surrounding territories, and these are typically housed in camps or separate communities from citizens and visitors, and are transported to work sites and back; conditions are not always conducive to safe food, and outbreaks are occasionally reported either from their work sites or their overnight residences where meals are prepared or catered. most food to many of these countries is imported, especially those with limited agricultural land and adequate water supplies; fruits and fresh vegetables, tend to be grown in rural or peri-urban settings for local consumption and these can be contaminated at source through polluted river or well water, such as in the bekaa valley of lebanon and mountain communities in pakistan, and the nile, tigris and euphrates fluvial plains. on one occasion, iranian watermelons were recalled and future sales banned in ksa, qatar, and uae because they were suspected of being poisoned or were injected with pesticides (nobody claimed to be ill after eating the melons), because holes were found in a few of them. however, the rationale of iranian farmers deliberately losing money seems to counter this argument, and it is more likely a sectarian economic barrier (abdullah ) . in fact, with the temporary ban the price of watermelons went up in the countries that had banned them. random tests carried out on the fruit confirmed they were free from any chemical substances, insecticides or other pollutants. the holes were most likely caused by emerging insect pupae. countries outside the gulf region reported no problems with the imported iranian melons. where some processed foods are exported, there is a risk of the importing countries recalling these if they cause foodborne illnesses or contaminants are found in them. this has happened in egypt with hepatitis a virus in strawberries and e. coli o :h in fenugreek seeds causing serious illnesses in europe and restricting further trade for an extended period. the same issue affected turkish pomegranate arils and lebanese tahini (made from imported ground sesame seeds), both containing salmonella, exported to the us. large to medium operations for broiler chickens and egg layers in ksa, kuwait, lebanon and other countries try and meet national standards or international guidelines for salmonella but are not always achieved, resulting in recalls and fines. governments are also aware of increasing concern over campylobacter in chickens, as widely-eaten poultry is a major source of this pathogen, but campylobacteriosis is not often cited as causing foodborne disease. raw milk (cow, sheep and camel) and raw milk cheese are still widely consumed in the middle east at the local level, though not usually obtained through supermarkets, and the risk of infections is high, as it is in other parts of the world, but with the added concern of brucella spp. and mers cov (the latter in the gulf countries where camels are bred and milked), both serious pathogens. yoghurt, surprisingly since it is acidic and is a source of gut beneficial lactobacilli, apparently was the foodborne vehicle to cause illnesses and deaths in afghanistan, israel, and pakistan. no agent was found in any of the samples. in the afghani example, the yoghurt was claimed to be deliberately poisoned; in the israeli one, it was apparently "stale" given to palestinian prisoners; there were two episodes in pakistan, one was from a home-prepared meal and the other from a restaurant which served rice and yoghurt. for prevention and controls strategies, most countries seem to rely on local authorities (municipalities) to do inspection of food facilities, more typically restaurants than processing plants as there are far more of them. illegal sales for unapproved products by local entrepreneurs are sometimes an issue, e.g., homeslaughtered meat in bahrain, and palestinians shipping food to israel. these illegal operations probably occur more often in porous borders within the region, and are only recognized when authorities decide to become vigilant in this area. some countries have conducted research and surveys much more than others based on the publication record, e.g., egypt, israel, palestine, ksa, turkey, and to a lesser extent, iran, lebanon, pakistan, uae, and yemen, but some research may occur without formal publication in recognized journals, making it difficult to have a true picture of how food safety problems are recognized and controlled. a few surveys have shown that home makers and food employees have limited knowledge of food safety, as in other regions. thus, some agencies or industry associations, sometimes in collaboration with outside organizations like fao or who, have attempted to train food employees in basic haccp principles, including best hand hygiene practices, and speakers give the latest food safety issues at the annual dubai international food safety conference, now in its th year. a few governments have established food safety agencies that have broad powers to inspect and control without overlapping responsibilities; these include jordan and ksa with food and drug administrations, uae with abu dhabi and dubai food control authorities, oman with its national food quality and safety centre, and pakistan with a punjab food authority. egypt and lebanon are initiating food safety authorities. israel, palestine and jordan have a cross-border agreement to collaborate on food safety issues. typical of many food control agencies in developing countries, periodic campaigns are launched to "crack down" on foodservice operations and sometimes processing plants. these are usually stimulated by complaints of the public, or the need for the responsible ministry to be seen doing something to justify its existence in compliance with regulations (if they exist). this has occurred recently in lebanon, qatar and pakistan. one issue is that poorly constructed or out-of-date regulations may be interpreted in different ways by the owners and the agencies (kullab ) . if a violation is found, the facility may be fined and/or temporarily closed down until it has satisfied the inspectors at the next visit. in one extreme instance in qatar, the owners and employees, were fined, imprisoned and deported. unfortunately, although the names of those at fault are often publicized by the media, their specific violations and how they relate to the regulations are not usually documented or at least publically released. another issue is that whether illnesses are suspected or not following a complaint, inspectors often insist that all food be discarded as soon as a sufficient violation, which may be unrelated to the complaint, has been determined; this prevents any samples being taken for outbreak investigations (hanna et al. ), as well as using the outbreak for a teaching tool for the owner and other similar operations. in conclusion, some progress has been made in the surveillance of foodborne disease in the middle east, but the disease's health and economic burden is barely being considered in many countries for future decision-making policies, an issue that is being tackled at the global level (who b). food control agencies seem to be trying to stop apparent abuses but have limited resources to do much more. this region, in particular, is severely strained because of sectarian distrust, on-going civil wars, and terrorist attacks, with refugees from iraq seeking shelter toward europe but stalled in turkey and lebanon for long periods of time. the crisis in syria is considered the greatest humanitarian disaster of the twenty first century, or even since world war ii, and it looks like the on-going fighting including outside armed forces will make food insecurity in the affected countries even worse in the foreseeable future. less public attention has been directed to yemen where food insecurity is a major concern. this coupled with gulf countries losing their wealth over low oil prices and a resultant stagnant global economy means a focus on food safety will likely become lower in priority for many of these countries. since secure food has to be safe, as illustrated by "stale" food being issued to besieged syrian residents and prisoners, it is important that relief agencies and countries themselves be aware of the risk of foodborne diseases associated with immunocompromised persons, particularly children. however, even in countries where the food supply is acceptable, inadequate hygienic practices put the local and tourist population at risk of illness and exported foods jeopardize industry profits and a poor reputation for future trade. as demonstrated by ksa, jordan and uae, single agencies or multiple agencies with clear-cut roles responsible for food safety, should be pursued by governments in consultation with industry and academia. duplication creates ambiguities for enforcement and education strategies as well as being unnecessarily costly. water supplies are also critical and some governments are weaning away farmers from depleted groundwater aquifers, and making irrigation more efficient where there are sustainable supplies. water for irrigation and processing has to be both free of pathogens and unacceptable levels of chemicals, and effectively treated waste water can substitute for groundwater. the sahara forest project in qatar is one example of a very dry country using seawater resources effectively; an even larger project is being considered from the -hectare in qatar to a -hectare test facility in jordan (clery ) . all these issues are being compounded by climate change and expected higher temperatures in already arid lands, which will make the region all the more dependent on more expensive imported foods. gulf counties have enough petro-dollars to afford these, but other countries are struggling to be self-sufficient for the near future even if the fighting ceases. the repair to destroyed infrastructure will be 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waterborne disease in developing countries -africa and the middle east. dairy food and environmental sanitation viruses of foodborne origin: a review. virus adaptation and treatment afghan cops; food poisoning at border post isis fighters die in 'iftar poisoning'; more ill after eating ramadan meals in mosul. international business times, india edition amid growing complaints, sch launches food poisoning hotline in qatar yemen: current issues and what the world food programme is doing who's first ever global estimates of foodborne diseases find children under account for almost one third of deaths. world health organization who estimates of the global burden of foodborne diseases. foodborne diseases burden epidemiology reference group - . world health organization food scandal: food poisoning rate in lebanon lowest, says tourism minister key: cord- -qdmunb l authors: zhao, yongkun; wang, chong; qiu, boning; li, chufang; wang, hualei; jin, hongli; gai, weiwei; zheng, xuexing; wang, tiecheng; sun, weiyang; yan, feihu; gao, yuwei; wang, qian; yan, jinghua; chen, ling; perlman, stanley; zhong, nanshan; zhao, jincun; yang, songtao; xia, xianzhu title: passive immunotherapy for middle east respiratory syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: qdmunb l middle east respiratory syndrome (mers) is a highly lethal pulmonary infection caused by a coronavirus (cov), mers-cov. with the continuing spread of mers-cov, prophylactic and therapeutic treatments are urgently needed. in this study, we prepared purified equine f(ab’)( ) from horses immunized with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins. both igg and f(ab’)( ) efficiently neutralized mers-cov replication in tissue culture. passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of mers-cov infected mice. our data show that horses immunized with mers-cov vlps can serve as a primary source of protective f(ab’)( ) for potential use in the prophylactic or therapeutic treatment of exposed or infected patients. middle east respiratory syndrome (mers)-cov is an emerging pathogen that causes severe pneumonia in humans in the arabian peninsula and in travelers from this region (assiri et al., a; zaki et al., b; zumla et al., ) . human-to-human spread has been documented (assiri et al., b) . while infections of immunocompetent patients generally present with only mild symptoms, the elderly and patients with pre-existing illnesses such as diabetes or renal failure are likely to develop more severe disease (assiri et al., a) . as of september , , cases with deaths ( . % mortality) had been reported to the world health organization, although the actual number of infections could be much larger since mild, asymptomatic or undiagnosed cases are likely to be common (drosten et al., ) . as yet there are neither licensed vaccines nor any prophylactic or therapeutic treatments effective against mers-cov. given the ability of coronaviruses to rapidly adapt to new hosts, a major public health concern is that mers-cov will further adapt to replication in humans, triggering a global severe acute respiratory syndrome (sars)-like pandemic (peiris et al., ; zaki et al., a) . as of now, the most promising treatment is the passive administration of anti-mers-cov neutralizing antibodies. several research groups have developed and produced anti-mers patientderived or humanized monoclonal neutralizing antibodies in vitro that were able to protect mers-cov infected mice (corti et al., ; li et al., ; zhao et al., ) . however, since these antibodies react with a single epitope on the mers-cov spike (s) protein and since coronaviruses are prone to mutate, this approach has raised concerns about possible antibody escape (corti et al., ; sabir et al., ) . recently, we showed that sera from middle east dromedary camels contained high levels of anti-mers-cov neutralizing antibodies. passive immunotherapy with sera from these animals significantly reduced virus loads and accelerated virus clearance from the lungs of mers-cov infected mice . this provides proof of concept that immune animal sera are potentially useful in the treatment of patients with mers (hayden et al., ) . passive immunotherapy with animal sera or antibodies has been successfully used to prevent rabies and to neutralize snake venom (both et al., ; gutierrez et al., ) . convalescent plasma used to treat patients with sars has been found safe and has demonstrated some efficacy in a study with a small number of patients (mair-jenkins et al., ) . however, neutralizing antibody titers in mers patients are generally low and the limited number of mers survivors makes this approach impractical (drosten et al., ) . here, we show that immunization of healthy horses with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins induces strong polyclonal neutralizing antibodies against mers-cov. since administration of whole antibodies can induce allergic responses in some humans, we further tested f(ab') fragments prepared by digestion of antibody with pepsin. prophylactic or therapeutic treatment of mers-cov infected mice with either igg or f(ab') significantly decreased the virus load in their lungs. mers-cov vlps were produced and purified as previously described . in brief, army worm sf cells were infected with a single recombinant baculoviruses co-expressing mers-cov structural protein genes s, m, and e, at a multiplicity of infection (moi) of . . culture supernatants were harvested at h post-infection and centrifuged at g for min to remove cell debris. following centrifugation of the clarified supernatants at , g for h at c the resulting vlp pellets were resuspended in pbs and loaded onto a e e % discontinuous sucrose gradient. after an additional centrifugation at , g for . h at c, bands between and % sucrose containing mers-cov vlp were collected. four -year-old healthy horses received multi-point intramuscular injections of . , . , , , and mg mers-cov vlps in ml pbs at weeks , , , , and , respectively. freund's complete adjuvant (sigma) was included in the first dose, and incomplete adjuvant in the remaining ones. sera were collected from the jugular vein weeks after each injection, and stored at À c before further analysis. mers-cov specific antibodies in the sera were measured by an indirect enzyme-linked immunosorbent assay (elisa) using purified mers-cov receptor-binding domain (rbd) protein (i.e., s protein residues e cloned into the pet- a expression vector and purified by ni-nta affinity chromatograph column). briefly, -well microtitration plates (corning costar, usa) were pre-coated with ml purified rbd antigen diluted in . mol/l carbonate sodium buffer (ph . ) to a final concentration of mg/ ml and incubated at c overnight. after blocking with skimmed milk for h at c, ml twofold serially diluted serum samples were added to the wells, and incubated at c for h. the plates were washed three times with pbs containing . % tween- (pbst), before addition of ml hrp-labeled rabbit antibody against horse igg (bioss, china; : , ) and incubation at c for h. after washing with pbst, ml , , , '-tetramethylbenzidine (tmb) (sigma, usa) as substrate was added to each well and incubated for min. the reaction was stopped with ml m h so . optical densities at nm were measured in an elisa plate reader (bio-rad, usa). horse antiserum was diluted with vol of normal saline ( . % nacl) and a half volume of saturated ammonium sulfate was then added and mixed gently at room temperature for min before centrifugation at g for min. the resulting sediment was redissolved in saline and mixed with a one-third volume of saturated ammonium sulfate. after incubation at ambient temperature for min and centrifugation at g for min, the second sediments were dissolved in normal saline and dialyzed against normal saline to remove any remaining ammonium salt. immunoaffinity resins were prepared by coupling mg rbd protein to . m sodium periodate-activated sepharose b ( g), and then incubating with ml sodium borohydride for min. after reaction with m tris (ph . ) for min, a purified igg sample was diluted -fold with pbs and incubated with the rbd resin overnight at c with constant rotation. the flowthroughs (anti-rbd depleted) were collected, and then the flowthroughs were tested against the rbd protein by elisa to ensure rbdspecific igg all bound with the rbd sepharose b. after washing with pbs, the bound antibodies (anti-rbd) were eluted in . m glycine-hcl buffer (ph . ). the eluates were neutralized with m tris buffer (ph . ), and then dialyzed against pbs. all samples were adjusted to the same protein concentration and sterilized by passage through microspin filters ( . mm pore size; millipore). neutralizing activity of the igg, rbd-specific igg, and flowthroughs were tested. the ph of the horse antiserum was adjusted to . with mol/l hcl. following incubation with pepsin ( iu/ml) at c for . h, the reaction was stopped by adjusting the ph to . with mol/l naoh. the solution was then applied to protein-a and protein-g columns sequentially to remove whole immunoglobulins. the purity of the resulting f(ab') protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by coomassie blue staining and the target fraction in the gel was analyzed in a thin layer chromatography scanner (transmission, zigzag scan, dual wavelength, swing width: mm, delta y: . mm) (cs- , shimadzu). specific pathogen-free week old balb/c mice were purchased from charles river laboratories international and maintained in the animal care facility, university of iowa. briefly, all mice were housed in thoren individually ventilated cages. caging and bedding were autoclaved. irradiated diet was fed. filtered water ( . mm filter) was provided with edstrom automatic watering system. hepa-filtered cage changing stations were used. all persons entering animal rooms worn autoclaved gowns, gloves, hair bonnets, face masks, and shoe covers. serum samples, purified igg or f(ab') were serially diluted in dmem and mixed with an equal volume of mers-cov containing pfu. following incubation at c for h, aliquots were added to cultures of vero cells in well plates and incubated at c in % co for h with gentle rocking every min. plates were then overlaid with . % agarose/dmem/ % calf serum. after further incubation for days, agarose plugs were removed using a small spatula, and the remaining plaques were visualized by staining with . % crystal violet. six-week-old female balb/c mice were lightly anesthetized with isoflurane and transduced intranasally with .  pfu of ad -hdpp in ml dmem as described elsewhere (zhao et al., ) . five days post transduction, mice were infected intranasally with mers-cov (  pfu) in a total volume of ml dmem. mice were monitored daily for morbidity (weight loss) and mortality. all work with mers-cov was conducted in the university of iowa biosafety level (bsl- ) laboratory. separate groups were injected with ml horse antiserum or mg igg or f(ab') intraperitoneally (ip) day before or after intranasal infection with  pfu mers-cov. control mice were given an equal volume of normal horse serum (sigma). to obtain virus titers, lungs were harvested from subgroups of animals at the indicated time points (see results) and homogenized into ml of phosphate buffered saline (pbs), using a manual homogenizer. lung homogenates were aliquoted into micro tubes and kept in À c. virus was titered on vero cells. cells were fixed with % formaldehyde and stained with crystal violet three days post-infection (p.i.). viral titers are expressed as pfu/g tissue for mers-cov (zhao et al., ) . due to the biosafety risk, mers-cov must be handled in a bsl- laboratory, whereas vlps can be rapidly generated under bsl- conditions as an immunogen inducing high antibody titers. in addition, the horse provides little risk to humans and produces high antibody yields, making these animals an effective source for production of hyperimmune sera (zheng et al., ) . rbd-specific igg titers in the sera were all above : , after five immunizations (fig. ) as assessed by elisa. rbd contains the major neutralizing epitopes of the s protein, as shown by the observation that absorption of sars patient convalescent sera with sars-cov rbd removes the majority of neutralizing antibodies (he et al., ) . independent research groups have also shown more directly that the mers-cov rbd sequence contains the major antigenic determinants for inducing neutralizing antibodies, and that neutralizing epitopes within mers-cov s are also localized primarily in the rbd region (du et al., ; mou et al., ) . here, we have demonstrated that anti-rbd antibodies function as major components of neutralizing antibodies. we found that rbd-specific igg neutralized mers-cov infection with half maximal inhibitory concentration of . mg/ml, and .  mg/ml for flowthroughs (fig. ) , suggesting that the rbd of s protein act as an important neutralization determinant of mers-cov. our results demonstrate that equine antibodies are polyclonal and recognize more antigen determinants in mers-cov s protein than single mabs, which could potentially prevent antibody escape. the integrity of igg and f(ab') fragments was evaluated using an sds-page gel (fig. a) . the purity of the f(ab') fragments after protein-a/g chromatography was > % after gel electrophoresis (fig. b ). passive transfer of blood products from other humans poses a safety concern, with possible contamination with agents of blood-borne diseases (e.g., hiv, hepatitis). heterologous antibody carries a potential risk of allergic reaction, but generation of f(ab') fragments, results in antibodies being less immunoreactive and safer for use in humans. while we successfully generated equine antibodies against mers-cov vlps, their protective effect against authentic mers- fig. . robust mers-cov rbd-specific antibody in immunized horse sera. horses (n ¼ ) were injected intramuscularly with mers-cov vlps and boosted every two weeks an additional times. sera were collected weeks after each immunization. rbd-specific antibodies in immunized horse sera were detected using elisa. cov infection remained untested. using a plaque reduction neutralizing assay, we confirmed that immune sera significantly neutralized mers-cov infection in vitro, with a half effective maximal dilution of : , (fig. a, b) . further, we found that equine igg and f(ab') also neutralized mers-cov infection with half effective maximal concentrations (ec ) of . mg/ml and . mg/ml for igg and f(ab') , respectively (fig. c, d) . collectively, these results show that equine antibody products exhibit highly potent neutralizing activity against mers-cov. next we asked if adoptive transfer of equine antibodies could protect mice from mers-cov infection prophylactically and therapeutically. by using a mouse model we previously generated (zhao et al., ) , we injected animals with immune serum (fig. a, b) , purified igg (fig. c, d) or f(ab') (fig. e, f) i.p. day before (fig. a , c, e) or after (fig. b, d, f) mers-cov challenge. in both prophylactic and therapeutic settings, passive transfer of equine immune antibodies resulted in a e log reduction of virus titers in the lungs of mers-cov infected mice, and accelerated virus clearance in the serum treated group (fig. a, b) . we did not observe any difference in body weight loss and pathologic changes on the exterior surface of the lungs in treated and untreated mice after fig. . neutralizing activity of the rbd-specific antibodies in igg. in vitro neutralization tests of total igg, rbd-specific igg, and flowthroughs, were determined in a series of -fold dilutions and % neutralization was calculated using graphpad prism. infeciton, since in this model, mice only develope mild lung disease. rapid virus replication and inflammatory cell infiltration in the infected lungs are the major parameters to measure (zhao et al., ) . since the half-life of f(ab') in vivo is relatively short and mers-cov is cleared within days in this model (zhao et al., ) , we did not inject f(ab') antibodies before day À or after day p.i. of note, the purified igg seemed to have lower protective potency than that of the immune serum in vivo (fig. ) . the concentration of igg in serum is > mg/ml. we used ml of immune serum (equal to mg igg) per mouse which is much higher than the immune igg we used ( mg/mice). the other reason could be we purified immune igg using saturated ammonium sulfate precipitation method, which needed to be performed under room temperature. we speculated that some iggs were degraded or misfolded, and unable to bind to mers-cov spike protein under this circumstance. while, immune sera were properly stored at À c and contained high concentration of bsa and other proteins, which made the antiserum more stable. to date, there are several anti-mers-cov antibodies developed from different origins. each antibody contains its own advantages and disadvantages. for monoclonal antibodies, mouse-derived monoclonal antibody needs to be humanized before human use (li et al., ) ; a human neutralizing antibody derived from a convalescent mers patient can be produced in large amount from cho cells (corti et al., ) . however, the single clone antibody raises the concern of viral escape mutant when applied to human. administration of transchromosomic bovine human immunoglobulins (luke et al., ) or dromedary immune serum resulted in rapidly viral clearance in infected mouse lungs. the disadvantage of these antibodies is that these animals are not readily available. compared to the antibodies described above, the administration of equine igg-derived f(ab') fragment proved to be a versatile and feasible method (lu et al., ; zhou et al., ) . it provides a useful platform to produce therapeutics against emerging infectious diseases. in summary, by immunizing healthy horses with mers-cov vlps, we have successfully developed the first equine igg-derived f(ab') fragment that neutralizes mers-cov in vitro and in vivo. both prophylactic and therapeutic treatments decreased virus loads and accelerated virus clearance in the lungs of mers-cov-infected mice. therefore, horses immunized with mers-cov vlps can serve as a useful initial source for developing protective f(ab') fragments, for the purpose of preparedness and to serve as a strategic reserve for a potential mers epidemic and other emergent pathogens. the authors declare no competing interests. epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak 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generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from ebola virus infection inhibition of infection caused by severe acute respiratory syndrome-associated coronavirus by equine neutralizing antibody in aged mice middle east respiratory syndrome ad -hdpp transduced balb/c mice ( wks, female) were injected intraperitoneally with ml horse serum key: cord- -r molh c authors: jeong, soo young; sung, se in; sung, ji-hee; ahn, so yoon; kang, eun-suk; chang, yun sil; park, won soon; kim, jong-hwa title: mers-cov infection in a pregnant woman in korea date: - - journal: j korean med sci doi: . /jkms. . . . sha: doc_id: cord_uid: r molh c middle east respiratory syndrome (mers) is a lethal respiratory disease — caused by mers-coronavirus (mers-cov) which was first identified in . especially, pregnant women can be expected as highly vulnerable candidates for this viral infection. in may , this virus was spread in korea and a pregnant woman was confirmed with positive result of mers-cov polymerase chain reaction (pcr). her condition was improved only with conservative treatment. after a full recovery of mers, the patient manifested abrupt vaginal bleeding with rupture of membrane. under an impression of placenta abruption, an emergent cesarean section was performed. our team performed many laboratory tests related to mers-cov and all results were negative. we report the first case of mers-cov infection during pregnancy occurred outside of the middle east. also, this case showed relatively benign maternal course which resulted in full recovery with subsequent healthy full-term delivery without mers-cov transmission. middle east respiratory syndrome (mers) is a lethal respiratory disease caused by mers-coronavirus (mers-cov) and occurs mostly in the middle east, initially by camel-to-human transmission, and then by human-to-human transmission. however, the disease was spread to other continents, probably by an index case, with subsequent pandemic outbreaks through human-to-human transmission through droplets and contact. during these respiratory viral outbreaks, pregnant women can be expected as highly vulnerable candidates for infection ( ) . a mers outbreak occurred in korea in with infections, including deaths ( , ) . we experienced a case of a korean pregnant woman who was confirmed for a mers-cov infection via a polymerase chain reaction (pcr) test. this is the first case of a mers-positive pregnancy reported outside the middle east and is also the first case of having been exposed and confirmed on rd trimester of pregnancy with full-recovery and successful full-term delivery. on may , , the patient's mother was exposed to the th mers patient, had a fever starting from june and was diagnosed with mers on june . while febrile, she had been in close contact with her daughter, a -year-old pregnant woman (gravida para ). on june ( weeks and days of gestational age [ga]), this pregnant woman visited the emergency room complaining of mild myalgia. based on this contact history with a mers patient and her symptoms, a mers-cov pcr test was performed and the result was found to be positive on june . starting from june , the patient developed dyspnea and sputum production. although chest auscultation was normal, the oxygen saturation (spo ) was % in room air and chest radiography showed diffuse opacity in the left lower lung area compared to a previously obtained radiographic image. the laboratory findings included a leukocyte count of , /mm (normal range , - , /mm ), with a differential of . % segmented neutrophils, . % lymphocytes, and . % monocytes; and c-reactive protein level of . mg/ dl (normal range - . mg/dl). she was given supplemental oxygen for hypoxia and conservative treatment, with hydration and pain control. the antiviral agents used in other severe mers-cov patients were not used in this patient, because her symptoms and laboratory findings were not severe. also, there was no evidence of any potential harm to the fetus and pregnant woman related to those drugs. after several days, her dyspnea and myalgia improved. the spo was % in room air and chest radiography showed interval improvement. on june and , mers-cov pcr was performed and the results were negative. she had no symptoms related to mers. on june , the patient manifested abrupt vaginal bleeding with rupture of membranes. a fist-sized blood clot was found through speculum examination and she had abdominal pain. fetal cardiotocography showed no deceleration, but a variability of fetal heart rate changed from moderate to minimal. with an impression of placental abruption, her obstetrical team decided on emergent cesarean delivery. a , g male newborn was delivered at weeks and days of gestation. apgar scores at and minutes were and , respectively. as expected, about % placental abruption was found (fig. ) . after delivery, the baby was immediately moved to the airborne infection isolation room (aiir) and received an initial care with all health care personnel (hcp) completely protected according to the centers for disease control and prevention (cdc) guidelines ( ). mers-cov pcr tests and antibody tests were performed with umbilical cord blood and placenta, and all results were negative. a systematic testing procedure for coronavirus infection, including chest radiograph and serial reverse transcription (rt)-pcr assays with peripheral blood and nasopharyngeal swab, did not demonstrate the presence of mers-cov in the newborn. mers-cov antibody tests were performed with mother and newborn sera on june and june , respectively ( ). in the mother's serum, immunoglobulin g (igg) was detected, albeit weakly, ( . ) via enzyme-linked immunosorbent assay (elisa; euroimmun ag, luebeck, germany), and via indirect immunofluorescence test (iift; euroimmun ag) with a titer of : . igm and iga were not detected through elisa and the plaque reduction neutralization test (prnt) result was below the cutoff value. however, mers antibodies for igg, igm, and iga were not detected in the newborn's blood samples ( table ) . the patient and her newborn baby were discharged in stable condition on june with no clinical abnormalities on followup at the outpatient clinic. mers-cov was first isolated from a patient who died from a severe respiratory illness in jeddah, saudi arabia in june ( ) . since then, more than , confirmed cases were reported to https://doi.org/ . /jkms. . . . the world health organization (who). clinical features of mers are variable, and infected patients can be asymptomatic or have an acute febrile illness, upper respiratory tract disease, or even multiple organ failure resulting in death ( ) ( ) ( ) ( ) ( ) . however, there are limited data about the clinical features of mers-cov infection during pregnancy and the perinatal outcome of patients diagnosed with mers-cov infection. to our best knowledge, there have been reported cases in which pregnant patients had positive laboratory results for mers-cov including this case ( ) ( ) ( ) ( ) (table ) . unlike other cases, this case is not only the first mers-cov infection during pregnancy occurred outside of the middle east, but also the first case of mers confirmed on rd trimester of pregnancy showing good outcome of both mother and baby. currently, an exposure time to this virus during pregnancy and a severity of maternal disease could be expected to affect the perinatal outcome. however, there is limited knowledge about the clinical implications of mers-cov infection on the maternal, fetal, and placental aspects of pregnancy. from the maternal aspect, there is no epidemiologic data regarding whether pregnant women are more susceptible to mers. also, it is unknown whether mers-cov infected pregnant women have a more severe disease course compared with the non-pregnant population. in our case, she showed a mild disease course. she had low level of igg antibody by elisa and iift but not detectable neutralization activity by prnt. it has been suggested that neutralizing antibodies are produced at low levels and are potentially short-lived after mild or asymptomatic mers-cov infection ( , ) . from the fetal aspect, it is unclear whether mers was a causative factor in the stillbirth or preterm birth. fetal specimen and/or placenta were not available for evaluation in the previous cases. as pregnancy alters maternal pulmonary function and consumes more oxygen, severe respiratory illness during pregnancy results in maternal hypoxemia. maternal hypoxemia can be associated with poor fetal oxygenation, which eventually could lead to preterm birth or stillbirth. also, altered immune responses during pregnancy could affect the fetal outcome ( ) . from the placental aspect, there have been no reports of mers causing pathology of the placenta including infarction, insufficiency, or villus placentitis. our case showed placenta abruption clinically, which can be caused by maternal infection. there is no evidence of a relationship between mers-cov and placenta disorder. however, the possibility that this virus may be a cause of placenta abruption should be of concern. lastly, the remaining question was whether the virus could cross the placenta causing significant infection in the fetus, and whether mers could cause vertical transmission. camel-tohuman transmission, and human-to-human transmission via contact, droplet, and possibly airborne routes are the known modes of transmission ( , ) . however, there are no data about perinatal transmission of mers-cov. moreover, if the mother mounts an appropriate immune response to produce enough neutralizing antibodies without serious conditions, passive antibodies transferred from mother to fetus may have a protective effect on the fetus. there is only one case reporting the mother's serologic data previously ( ) , in which stillbirth occurred at approximately months of gestation, although the mother had mers-cov antibody by elisa (titer : , ) , immunofluorescent antibody (ifa), and microneutralization titer assay (titer : ). in our case, although the mother had igg antibody (titer : by iift), antibody was not detected in neonatal serum. this finding may provoke different interpretations in regard to the role of maternal antibodies in the fetus or to transmission of maternal antibodies, necessitating more data in the future. to know whether prenatal transmission of mers-cov can occur, collection of samples including amniotic fluid, placenta, and umbilical cord is needed from an infected pregnant patient. further studies with a larger sample size will help in understanding of the pathophysiology and perinatal outcome of mers during pregnancy and the optimal mode of delivery. the effect of asian influenza on the outcome of pregnancy mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome coronavirus (mers-cov) nosocomial outbreak in south korea: insights from modeling interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak isolation of a novel coronavirus from a man with pneumonia in saudi arabia characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea mers outbreak in korea: hospital-to-hospital transmission better understanding on mers corona virus outbreak in korea case definition and management of patients with mers coronavirus in saudi arabia impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus infection during pregnancy: a report of cases from saudi arabia middle east respiratory syndrome coronavirus during pregnancy stillbirth during infection with middle east respiratory syndrome coronavirus transmission of mers-coronavirus in household contacts persistence of antibodies against middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control the emergence of the middle east respiratory syndrome coronavirus we thank dr. christian drosten and dr. marcel a. muller in institute of virology, university of bonn medical center for performing immunoglobulin a (iga) enzyme-linked immunosorbent assay (elisa) and plaque reduction neutralization test (prnt). we also thank prof. kyong ran peck in division of infectious disease, department of medicine, samsung medical center, sungkyunkwan university school of medicine for providing the reagent and practical help for us to obtain the antibody test results. the authors have no potential conflicts of interest to disclose. jong-hwa kim https://orcid.org/ - - - key: cord- - iyynbup authors: furuyama, taima n.; antoneli, fernando; carvalho, isabel m. v. g.; briones, marcelo r. s.; janini, luiz m. r. title: temporal data series of covid- epidemics in the usa, asia and europe suggests a selective sweep of sars-cov- spike d g variant date: - - journal: nan doi: nan sha: doc_id: cord_uid: iyynbup the covid- pandemic started in wuhan, china, and caused the worldwide spread of the rna virus sars-cov- , the causative agent of covid- . because of its mutational rate, wide geographical distribution, and host response variance this coronavirus is currently evolving into an array of strains with increasing genetic diversity. most variants apparently have neutral effects for disease spread and symptoms severity. however, in the viral spike protein, which is responsible for host cell attachment and invasion, an emergent variant, containing the amino acid substitution d to g in position (d g), was suggested to increase viral infection capability. to test whether this variant has epidemiological impact, the temporal distributions of the sars-cov- samples bearing d or g at position were compared in the usa, asia and europe. the epidemiological curves were compared at early and late epidemic stages. at early stages, where containment measures were still not fully implemented, the viral variants are supposed to be unconstrained and its growth curves might approximate the free viral dynamics. our analysis shows that the d g prevalence and the growth rates of covid- epidemic curves are correlated in the usa, asia and europe. our results suggest a selective sweep that can be explained, at least in part, by a propagation advantage of this variant, in other words, that the molecular level effects of d g have sufficient impact on population transmission dynamics as to be detected by differences in rate coefficients of epidemic growth curves. the coronavirus disease outbreak is caused by sars-cov- (severe acute respiratory syndrome coronavirus type ) and was declared by the world health organization (who) as a pandemic in march , (world health organization, c , d . as of june , , the virus has already infected more than . million people and caused more than , deaths worldwide ( . % fatality ratio) (dong et al., ; johns hopkins university, ) . this is the third coronavirus caused outbreak in less than years (world health organization, a) . from november to may , sars-cov- (severe acute respiratory syndrome caused by coronavirus type ) affected countries worldwide, accounted , confirmed cases and deaths ( . % fatality ratio) (drosten et al., ; ksiazek et al., ; lee et al., ; peiris et al., ; zhong et al., ; centers for disease control and prevention -department of health and human services, ; world health organization, ; centers for disease control and prevention, ) . mers-cov (middle east respiratory syndrome caused by coronavirus) spread to countries around the globe, totalizing , confirmed cases and deaths ( . % fatality ratio) continuously since april (zaki et al., ; hijawi et al., ; centers for disease control and prevention, ; world health organization, , b . several conditions contribute to the transmission speed of sars-cov- , such as transmission during the asymptomatic phase and wide human susceptibility to this pathogen (arons et al., ; fam et al., ; gandhi et al., ) . the central concern for governments and general population is the collapse of healthcare systems and lack of essential care. therefore, cumulative information about the sars-cov- mechanisms inside the host cell, its epidemiology and its genetic patterns are necessary to halt the virus spread, to prevent the disease and heal the infected individuals. the comparison of several sars-cov- strains with the wuhan reference genome genome (genbank accession nc_ ) reveals a g to a transition at position , that leads to a d to g amino acid substitution at position in the spike protein. molecular evidence suggests that this substitution is advantageous for viral propagation in vitro because of increased spike protein abundance and reduced shedding (zhang et al., ) . a previous study by korber and collaborators (korber et al., ) conjectures that the d g substitution in sars-cov- spike protein could be responsible for higher transmission rates observed in a global scale. the study shows that there is a higher prevalence of d in china and in the united states before march while after march the g prevalence significantly increases in europe and united states. if there is a correlation between the d g variant prevalence and higher sars-cov- transmission, then the epidemiological data might reveal a significant correlation between d g prevalence and the growth rate coefficients of epidemic curves globally. here we present evidence that the prevalence of d g is correlated with increased growth rate coefficients in temporal series of covid- epidemiological data. the relative dynamics of d and g variants observed is what would be expected in the case of a selective sweep. data on prevalence of d or g was obtained from the los alamos distribution map of d and g sars-cov- (elbe and buckland-merrett, ; shu and mccauley, ; los alamos national laboratory, ) . the data was downloaded as "data- - - .csv" file for "all" time range. the time range considered was days ( weeks). data: obtained from coronavirus covid- global cases by the center for systems science and engineering (csse) at johns hopkins university; the red cross; the census american community survey; the bureau of labor and statistics: (https://github.com/cssegisanddata/covid- ). the epidemic growth rates were obtained from coronavirus covid- global cases by the center for systems science and engineering (csse) at johns hopkins university; the red cross; the census american community survey; and the bureau of labor and statistics data (johns hopkins university, ; american red cross, ; united states census bureau, ; u.s. bureau of labor statistics, ). the time range considered was the same as the considered in the los alamos data, being the first day of time series of confirmed cases on february , and the last day of time series of confirmed cases in may , . six states and counties of the usa were considered in the analysis further grouped as east or the west coast. accordingly, west coast states data included (with number of counties in parentheses): oregon ( ), washington ( ), california ( ), being a total of the west coast. the east coast states included (number of counties in parentheses): new york ( ), connecticut ( ), virginia ( ) totaling counties of the west coast. the time range was divided into an early start period and a late start period. the early start considered counties such that the epidemics started in the range / / - / / ( counties). the late start considered counties such that the epidemics started in the range / / - / / ( counties). nine counties did not have at least days of non-zero time series. the analysis was also made for other regions of the world, considering a broader division: world (some countries have more than administrative region): western countries (europe): belgium ( ), denmark ( ), france ( ), germany ( ), italy ( ), luxembourg ( ), netherlands ( ), portugal ( ), spain ( ), united kingdom ( ); total number of administrative regions = . eastern countries (eastern asia and oceania): australia ( ), bangladesh ( ), china ( ), india ( ), japan ( ), south korea ( ), singapore ( ), taiwan ( ), thailand ( ), vietnam ( ); total number of administrative regions = . the first day of time series of confirmed cases was / / (eastern countries); / / (western countries) and the last day of time series of confirmed cases was / / . regarding the early and late epidemic periods, the time frame was: early start: countries such that the epidemics started in the range: / / - / / (western countries; regions) and / / - / / (eastern countries; regions). late start: countries such that the epidemics started in the range: / / - / / (western countries; regions) and / / - / / (eastern countries; regions). one region did not have at least days of nonzero time series and the eastern regions are all early starters, so in this case we compared the eastern regions with the early western region and the late western regions. the logistic model parameters were obtained from logistic regression (spiegelhalter, ) using python with pandas libraries (https://pandas.pydata.org/) and scikit-learn (https://scikitlearn.org/stable/about.html). plots were generated with matplotlub (https://matplotlib.org/) and seaborn (https://seaborn.pydata.org/index.html). the initial analysis consisted in the logistic models derived from us data comparing us east coast (predominantly g ) with west coast (predominantly d ), the asia-europe data in west (predominantly g ) and east (predominantly d ) in the early and late epidemic stages. figure depicts the plots of logistic models with the corresponding logistic model (blue line) and its confidence band (light blue shading). figure a shows the early start counties of usa, figure b shows the late start counties of usa, figure c shows the early start countries of the asia-europe axis and figure d shows the late start countries of asia-europe axis. the comparisons between the logistic models in early and late epidemic stages show that at the early stages the growth rates between west and east, either us or europe, are significantly different, while in late stages the west-east differences are not significant. this test therefore suggests that in the early epidemic stages the predominant variant pattern reflected a "founder effect", especially in the us, where the west coast infections derived from an asian d type whereas in the east cost the infection dynamics started with european derived ancestors, of the g type. this is observed from d g distribution data. the early epidemic stage in both us and asia-europe show significant differences in the odds ratios in the west and east portions, showing that the growth rates might be impacted by the g substitution. at the late stages the growth rates are not distinguishable. european late stages show odds ratios < and in the us the odds ratios drop from . to . . the growth curves of the d and g variants in west us, east us, west asia-europe and east asia-europe at different time periods, of days each, are shown in figure . the time series of frequency variants (d/g) reveal that irrespective of geographic region and early and late epidemic stages, the g variant increases and surpasses the d . figure a shows the usa dynamics and figure b shows the asia-europe dynamics. each curve corresponds to the variant frequency in the corresponding geographic region. the frequencies of variants in the same region are complementary to each other. in us east the g started at approximately % and d at ~ % with subsequent increase of g to % and extinction of d . in us west, g started at ~ % and d ~ % and after days g increased to more than % whereas d decreased to less than % (figure a) . this type of dynamics is highly suggestive of a selective sweep because of an increased infectivity/replication rate of g . the effect is very similar in europe west and east where irrespective of the initial frequencies of d and g , the later always predominates after days (figure b) . in supplementary table s the frequencies of d and g are compared in the east and west coast for the us while supplementary table s depicts the asia-europe frequencies of d and g . this data show that the growth rate of g is significantly higher than the d growth rate. also, it indicates that the phenomenon is global, not restricted to a geographic location of specific host population. the initial cases in the east coast are likely to have originated from european strains (predominantly g ) whereas in the west coast the initial infections were caused by asian strains d predominant in that continent at that time. the first confirmed covid- case in the united states was in the state of washington on january , (centers for disease control and prevention, ; holshue et al., ) . this would explain the similarities in transmission processes in the us west coast when compared to china, japan and taiwan. on the other hand, in the us east coast, especially new york, the likelihood of the beginning of the covid- epidemic is of european origin. in the present work we analyzed the frequencies of d and g variants in us west and east and asia-europe west and east. we have shown that irrespective of initial frequencies of these variants at early epidemic stages, g always predominates, and very quickly either becomes fixed or significantly surpasses d after days of the initial infection. in a previous study (korber et al., ) conjecture on founder effects and selection when d and g are compared. the analysis shown in figure indicates that at early epidemics the founder effect is more prominent and at later stages the selection is more significant. the populational dynamics depicted in figure , indicates a selective sweep of g over d . a selective sweep is a population genetics process in which a novel beneficial mutation increases its frequency to a point where it reaches % and is therefore, "fixed" in the population (hermisson and pennings, ) . the current covid- epidemics and the discovery of a spike protein variant with a mutation from d to g at position has given an opportunity to show such process in action due to the detailed epidemiological data and viral genome sequence availability. our analysis also shows that the founder effect and selective sweep are not specific to a country or region, which suggests that the selective advantage of g over d is global and occurs irrespective of the genetic variation and ethnic background of the host populations. although the results indicate that there is a robust difference between the d g variant and the epidemic growth rate curve it is important to point there are several mutations occurring in the viral genome, which could compose a mutation balance in the viral fitness. that is, it is unlikely that the fitness increase by g alone drive the epidemic curves. as shown by (korber et al., ) other mutations hitchhike around g by recombination and therefore the combined fitness of several mutations increase the fitness of sars-cov- to a point that explains the selective sweep observed in figure . nevertheless, we provide robust population evidence for the hypotheses raised by which combine founder effect and selection and believe that g predominance over d is an example of selective sweep in a viral population. author contributions imvgc, lmrj, mrsb, fa: data analysis planning and conceptualization. tnf, fa: performing the data analysis. all authors: writing and editing the manuscript. the authors declare no conflict of interests. shows the late start countries of asia-europe axis (black dots). the blue curve is the corresponding logistic model with its confidence band (light blue shading). in each panel, the horizontal axis is the growth rate of the initial segment of days of the corresponding time series of confirmed cases, and the vertical axis is a binary variable indicating the corresponding region where the time series is from (east/west coast county in usa, panels (a) and (b) or east/west country in asia-europe axis, panels (c) and (d)). panel (b) shows the asia-europe axis. in each panel, the horizontal axis shows time period of time (corresponding to days each) and the vertical axis show the frequency. each curve corresponds to the variant frequency in the corresponding region, according to the side legends. the frequencies of variants in the same region are complementary to each other. adapted from (https://cov.lanl.gov/apps/covid- /map/) (korber et al., ) . available at presymptomatic sars-cov- infections and transmission in a skilled nursing facility cdc -severe acute respiratory syndrome (sars) available at: cdc.gov/coronavirus/mers/about/index.html first travel-related case of novel coronavirus detected in united states cdc -severe acute respiratory syndrome -fact sheet: basic information about sars an interactive web-based dashboard to track covid- in real time identification of a novel coronavirus in patients with severe acute respiratory syndrome data, disease and diplomacy: gisaid's innovative contribution to global health: data, disease and diplomacy ace diversity in placental mammals reveals the evolutionary strategy of sars-cov- asymptomatic transmission, the achilles' heel of current strategies to control covid- soft sweeps: molecular population genetics of adaptation from standing genetic variation epidemiological findings from a retrospective investigation first case of novel coronavirus in the united states covid- dashboard by the spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov- a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong sars-cov- sequence analysis pipeline -sars-cov- map: distribution of d and g coronavirus as a possible cause of severe acute respiratory syndrome gisaid: global initiative on sharing all influenza datafrom vision to reality probabilistic prediction in patient management and clinical trials available at china's latest sars outbreak has been contained, but biosafety concerns remain -update . world health organization middle east respiratory syndrome coronavirus (mers-cov) -key facts coronavirus disease (covid- ) situation report - mers situation update who director-general's opening remarks at the media briefing on covid- - who timeline -covid- . world health organization isolation of a novel coronavirus from a man with pneumonia in saudi arabia the d g mutation in the sars-cov- spike protein reduces s shedding and increases infectivity. biorxiv epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china west east g d total g d total * data on the prevalence of variant of the virus with respect to the residue of spike protein is prevalent in the infected population (d or g). data from the site "distribution of d and g " (https://cov.lanl.gov/apps/covid- /map/) from (korber et al., ) . key: cord- - ckjl w authors: kang, hee sun; son, ye dong; chae, sun‐mi; corte, colleen title: working experiences of nurses during the middle east respiratory syndrome outbreak date: - - journal: int j nurs pract doi: . /ijn. sha: doc_id: cord_uid: ckjl w aims: to explore working experiences of nurses during middle east respiratory syndrome outbreak. background: since the first case of middle east respiratory syndrome was reported on may , in south korea, people, including health care workers, were infected, and died. design: a qualitative descriptive study. methods: seven focus groups and individual in‐depth interviews were conducted from august to december . content analysis was used. results: the following major themes emerged: “experiencing burnout owing to the heavy workload,” “relying on personal protective equipment for safety,” “being busy with catching up with the new guidelines related to middle east respiratory syndrome,” and “caring for suspected or infected patients with caution.” participants experienced burnout because of the high volume of work and expressed safety concerns about being infected. unclear and frequently changing guidelines were of the common causes of confusion. participants expressed that they need to be supported while caring for suspected or infected patients. conclusion: this study showed that creating a supportive and safe work environment is essential by ensuring adequate nurse staffing, supplying best‐quality personal protective equipment, and improving communication to provide the quality of care during infection outbreak. information on the new guidelines and job-related information via text messages using smartphones was helpful for the nurses. • creating a supportive work environment and providing adequate training for nurses is essential. the implications of this paper: • nurse managers and hospital administrators should establish strategies to prevent nurses from burnout and to ensure their safety during the outbreak of infectious diseases. • clear and consistent practice guidelines and effective communication methods among nurses should be developed. • increasing awareness of health care workers about infectious diseases to enhance emergency preparedness is essential. with mers on may , , which was days after his first visit (lee & ki, ; yang et al., ) . a mers outbreak in korea was caused by hospital-to-hospital transmission because patients were moved to other hospitals without appropriate quarantine (ki, ; kim et al., ) . it was exacerbated by overcrowding in the emergency room, delayed diagnosis, and lack of self-protection (balkhy, perl, & arabi, ; xia, zhang, xue, sun, & jin, ) . as more and more mers cases were reported, hospitals restricted the visitors and checked all visitors and employees for the presence of fever. additionally, for the temporary screening of mers-suspected cases, triage was set up to screen the infected or suspicious patients and to block the cross-transmission in and outside hospitals. furthermore, the government adopted the national safe hospital program to control mers infections within hospitals (korea centers for disease control and prevention, along with high risk of being infected, studies reported that health care personnel experienced occupational risks, distress, and the fear of contacting and transmitting the disease during epidemics of h n , severe acute respiratory syndrome (sars), and ebola virus (bukhari et al., ; chou et al., ; corley, hammond, & fraser, ; koh, hegney, & drury, ; speroni, seibert, & mallinson, ) . nurses also reported positive experiences of becoming more confident, mature, and broad-minded while caring for sars patients (liu & liehr, ) and positive feelings about their experience of caring for h n patients (honey & wang, ) . however, few studies have been conducted on nurses' working experiences during the mers outbreak. the aim of the study was to explore the working experiences of nurses during the mers outbreak. data were collected using focus group interviews and individual in-depth interviews from august to december until the data were saturated. focus group questions were developed based on a literature review (chou et al., ; corley et al., ) . each focus group was comprised of to participants. individual in-depth interviews were conducted for those who were not able to meet in focus groups because of time conflicts. prior to the interview, participants were informed about the reasons for doing this study and the goals of the study. the first author (hsk), who has experience with qualitative research, conducted the focus groups and the individual in-depth interviews. the focus group discussions and individual interviews were conducted in a private room at a site with convenient participant access. each session lasted for to hours. no one was present besides the participants and the researchers during the interviews. a semistructured interview guide was used. we conducted a pilot test with nurses caring for the patients with mers and refined the interview questions. the following questions guided the interviews: • what are your working experiences of caring for suspected or infected patients with mers during the outbreak? • what are the challenges of working during the mers outbreak? to ensure consistency and accuracy of our data, interviews were audio-taped with the participants' permission and transcribed verbatim. the researchers made field notes during and right after the • it's so sweaty and hard to breathe with it. it is hard to work and see clearly while wearing it (protective measures) and i feel dizzy when wearing it for long hours. • (we were) sweating, (find it) hard to breathe; it was difficult to work wearing personal protective equipment. being busy with catching up with the new guidelines for mers frequently changing guidelines • mers guidelines kept changing. at first, (we were told to) do thing this way and this is the guideline. we needed time to understand and practice a new guideline; however, guidelines kept changing without considering our adjustment to a new one. • the most difficult thing was that protocols were changed daily. working along with memorizing new protocols was very difficult. while workload increased, (we) were told this has been changed this way and that has been changed that way in shift change meetings. sharing the new information • we promptly communicated and shared updated information among nurses within the unit, through kakao talk (a free mobile instant messaging application for smartphones with free texting). • we had a notice note summarized about new information on mers. when changing shifts, we read the note and were also told what we have to be cautious because of what has been changed and it helped. it helped because we never had mers before and didn't know how we have to send the specimens and did not know how to cope with it. it worked as basic guidelines. lack of support • why do you have to do it, and what if you are infected? why? why does it have to be you? • when the patients' condition was bad and when we were having a hard time, no one showed appreciation of our hard work. identifying the best way to care for patients • after spending many days in the isolated room, we started using a messenger. we supported each other and shared information. it was very helpful for me to ask my colleagues when i was unsure about patient care. • we made a package for mers patients, a package for mers. at first, we brought water bottles to patients because they cannot come out a negative pressure room and we complained regarding this matter. next thing, we agreed to make a package for the patients in the isolation room. when a patient comes, we give this package that has water, sleeper, and disposable products that patients need. (continues) interviews to help understand the interviews. there were no repeat interviews carried out. the study was approved by the institutional review board ( - -hrsb- - ). all participants were informed about the purpose of this study and participants' right to withdraw from the study at any time, without penalty. confidentiality of participants was ensured, and written informed consent was obtained from each participant. the responses from the participants were analysed, using qualitative content analysis (krueger & casey, ). data collection was conducted concurrently with data analysis and continued until no new information emerged from the responses. the researchers read each verbatim transcript several times to obtain an overall understanding of the content and to gain a sense of the whole. the meaning units (words, sentence, and paragraphs) in the interviews related to nurses' work experiences were identified and coded. the codes were sorted into similar things together and grouped into categories based on similarities and differences. after assessing themes across groups, overarching themes were derived. two of the investigators independently coded each transcript. when discrepancies in coding occurred, the investigators discussed and resolved them by consensus. trustworthiness of the study was maintained following criteria by lincoln and guba ( ) . the study participants included female and male nurses. their mean age was . ( . ) years, ranging from to years, and their work experience ranged from months to years. the following major themes emerged: "experiencing burnout owing to the heavy workload," "relying on personal protective equip- participants reported discomfort in wearing ppe all day on duty. the amount of time of wearing ppe varied according to their work and the severity of the patients' condition. participants said that they preferred a mask that led to less breathing difficulties. one participant said, "i prefer the mask made by a company because it has a space that helps me to breathe easily. many nurses prefer to use it." the patterns of staying in the isolation room wearing a papr differed. nurses from one hospital stated that they stayed in the isolation room for a maximum of hours while wearing their papr and then came out; they stayed in the anteroom (a room in front of the negative pressure isolation room) and went back into the isolation room when needed. contrary to this, nurses from another hospital stayed in the isolation room for their entire shift, except for the lunch hour. meanwhile, nurses who wore a papr said that they felt like wearing a space suit. they had a backache from wearing heavy equipment. "i had put the battery of the papr on a side participants communicated with others by writing on paper. they reported that it was easy to hear the intercom sound in the room, but it was difficult to talk back because the head shield of the papr blocked out sounds. permission to use computers or smartphones in the isolation room varied across hospitals. in one hospital, nurses working in an isolated intensive unit communicated with other nurses in another isolated room using a smartphone messenger application. they said it helped them to know how others in isolation rooms were doing and to ask them when they were unsure about patient care. participants said that they gradually returned to normal life. the hospitals rewarded working with mers patients differently. these included participating in healing programs, receiving financial incentives, eating out in teams, or receiving several days off for resting. a participant said "i enjoyed participating in the healing camp. the post-trauma prevention education was also helpful. i was relieved to hear that we could seek psychiatric counselling if necessary." after completing their volunteered job with the mers patients, participants stated that they had learned on site while caring for infected patients and that it was very rewarding and worthwhile. they expressed that they felt being matured and gained a lot of confidence from these experiences. participants also said that when they returned to their work unit, they often heard "you did a good job" from their peers, and that "it felt supportive and healing." this study explored the nurses' work experience during the mers outbreak. our participants reported that their workload increased with time. this result indicates that, as part of emergency planning, nurse managers and hospital administrators should prepare for the extra workload during the emergency of an infection outbreak and to ensure quality of care. participants reported that restricting unauthorized access of visitors was one of the main issues. restricting visitors was one of the strategies used for controlling further outbreak during the norovirus outbreak (danial et al., ) . visiting hospitalized patients in a group is a part of the korean culture, as it is a way of expressing support and wishing for a quick recovery. rather than just restricting the visitors, it would be helpful to suggest alternative ways of expressing support for patients, such as sending a message through a phone or social networking service. participants expressed their concerns about the possibility of being infected. in fact, health care personnel who had close contact with mers patients were at a high risk for infection (alraddadi et al., ) . previous studies support our results. during the mers epidemic, health care workers felt fearful about being infected; however, they continued to work during the epidemic as it was their professional and ethical duty (al-dorzi et al., ; khalid, khalid, qabajah, barnard, & qushmaq, ) . emergency room nurses working during the outbreak of mers also expressed high concerns about being infected, and that they would have like to avoid caring for patients with mers if there was a choice (choi & kim, ) . these fears of nurses could be reduced by sharing the correct information about the quality of the protection devices they wear and appropriate ways to use them to prevent the transmission of infection (speroni et al., ) . in addition, hospitals experiencing mers epidemic suggested that institutional plans be made in advance to provide personal safety equipment when there is a rapid increase in its demand (al-dorzi et al., ; stirling, hatcher, & harmston, ) . however, our participants mentioned discomfort in wearing ppe. likewise, a study on a simulation exercise for health care workers wearing ppe in a hospital in the uk reported that they found the ppe uncomfortable, and even basic tasks took longer than usual while wearing it (phin et al., ) . thus, feedback from nurses on protection devices would help medical equipment companies design more comfortable medical protection equipment. our participants complained of having to continuously catch up with the frequently updated guidelines. likewise, it was reported that one of challenges for hospitals was the changing and conflicting guidelines and the overwhelming amount of information that required sifting through during the h n influenza pandemic (rebmann, regarding going back to routines, our participants felt supported when they received positive responses from peers when they went back to their unit after taking care of patients at risk. additionally, hospitals implemented various programs for health care professionals to reward or appreciate their hard work during the outbreak. the common response of participants on these was very positive. in a study, nurses who took care of h n high-risk infected patients and who worked in an isolated area in taiwan said that nurses needed counselling services (honey & wang, ) . after any outbreak, it may be important to offer a healing program for nurses, to help them share their experiences and feelings with others. a limitation of this study was that all participants were staff nurses. further research is needed to explore the mers experiences of patients, nurse managers, and other health care workers. because of variations in nurses' work schedule, the focus groups were quite small. the disadvantage of a small group is that it limits generating a rich diversity in views and the total range of experiences, although the advantage of smaller groups is that they are easier to recruit and allow everyone to have a greater opportunity to share experiences. another limitation was that this was a cross-sectional study. longitudinal studies are needed to examine the impact of any changes in hospital regulations or policies on nursing care. these study results suggest that nurse managers and administrative personnel should understand that overload of nurses' work during the outbreak may lead them burned out, which may negatively affect quality of care to patients. furthermore, establishing consistent and solid practice guidelines and efficiently disseminating them and training health care workers to deliver them could lead to less confusion during an infection outbreak. it is important to acknowledge nurses' work as valuable and to create a supportive environment in workplace of nurses. these efforts will empower nurses to work as an expert and will positively influence the quality of care. finally, it is essential to raise awareness about infection control among health care workers and people in general to strengthen emergency preparedness. the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel middle east respiratory syndrome coronavirus: current situation and travel-associated concerns preventing healthcareassociated transmission of the middle east respiratory syndrome (mers): our achilles heel middle east respiratory syndrome: a new global threat middle east respiratory syndrome coronavirus (mers-cov) outbreak perceptions of risk and stress evaluation in nurses factors influencing emergency nurses' ethical problems during the outbreak of mers-cov uniformed service nurses' experiences with the severe acute respiratory syndrome outbreak and response in taiwan the experiences of health care workers employed in an australian intensive care unit during the h n influenza pandemic of : a phenomenological study lessons learned from a prolonged and costly norovirus outbreak at a scottish medicine of the elderly hospital: case study new zealand nurses perceptions of caring for patients with influenza a (h n ) healthcare workers emotions, perceived stressors and coping strategies during a mers-cov outbreak mers outbreak in korea: hospital-to-hospital transmission the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea nurses' perceptions of risk from emerging respiratory infectious diseases: a singapore study middle east respiratory syndrome coronavirus outbreak in the republic of korea focus groups: a practical guide for applied research strengthening epidemiologic investigation of infectious diseases in korea: lessons from the middle east respiratory syndrome outbreak naturalistic inquiry instructive messages from chinese nurses' stories of caring for sars patients personal protective equipment in an influenza pandemic: a uk simulation exercise pandemic preparedness: implementation of infection prevention emergency plans worry experienced during the middle east respiratory syndrome (mers) pandemic in korea nurses' perceptions on ebola care in the united states, part : a qualitative analysis communicating the changing role of a nurse in an epidemic: the example of the mers-cov outbreak in saudi arabia consolidated criteria for reporting qualitative research (coreq): a -item checklist for interviews and focus groups modeling the transmission of middle east respirator syndrome corona virus in the republic of korea middle east respiratory syndrome in persons, south korea working experiences of nurses during the middle east respiratory syndrome outbreak none. the authors declare no conflict of interest. all listed authors meet the authorship criteria and all authors are in agreement with the content of the manuscript. sun-mi chae http://orcid.org/ - - - key: cord- - b hvorz authors: watson, john t.; hall, aron j.; erdman, dean d.; swerdlow, david l; gerber, susan i. title: unraveling the mysteries of middle east respiratory syndrome coronavirus date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: b hvorz nan cov) is a novel cov known to cause severe acute respiratory illness in humans; ≈ % of confirmed cases have been fatal. human-to-human transmission and multiple outbreaks of respiratory illness have been attributed to mers-cov, and severe respiratory illness caused by this virus continues to be identified. mers-cov was first reported in september , and subsequent investigations documented illness onsets as early as april ( ) . as of february , , the world health organization has reported laboratory-confirmed cases of mers-cov infection, including deaths, indicating an ongoing risk for transmission to humans in the arabian peninsula ( ). the median age of reported case-patients is years (range - years); most cases are in males ( ). most index casepatients have at least reported chronic comorbid condition ( ) and have resided in, or recently traveled to, jordan, qatar, united arab emirates, oman, kuwait, or saudi arabia ( ). in france, germany, italy, united kingdom, and tunisia confirmed cases of mers-cov have been identified in travelers returning from these countries ( ) . although a zoonotic reservoir of mers-cov has been speculated, very little is known about the specific exposures that result in primary human cases. mers-cov infection causes severe acute hypoxemic respiratory failure, extrapulmonary organ dysfunction, and high rates of death; however, the spectrum of illness and clinical course are not fully defined ( ) . evidence suggests that mers-cov is capable of limited human-to-human transmission, which results in outbreaks in family and health care settings ( , ) . the reported cases include multiple distinct spatiotemporal clusters and identified infections in health care workers ( ) . modeling performed to assess the extent of human infection and the transmission potential of mers-cov (as of august ) estimated that most symptomatic case-patients had not been detected but that chains of transmission were not self-sustaining when infection control was implemented ( ) . despite evidence of human-to-human transmission, the number of contacts infected by persons with confirmed infections appears to be limited; sustained transmission in the community has not been documented ( ) . the hajj, the annual religious pilgrimage to saudi arabia, involved . million pilgrims from countries in but resulted in no reports of confirmed cases in the weeks after the pilgrimage ( ). little is known about the pathogenic potential and transmission dynamics of mers-cov. although multiple health care-associated clusters have been identified ( ), further investigation is needed of the specific risk factors for transmission within health care facilities. basic information about the temporal and causal patterns of viral shedding and their relationships to clinical outcomes is critical to further understand the virus and to shape prevention and control measures needed to limit transmission. standard, contact, and airborne precautions appear to be effective in limiting transmission and are recommended by the centers for disease control and prevention to manage known or suspected mers-cov infection in hospitalized patients as a primary means of preventing and controlling transmission ( ) . potential animal reservoirs and mechanism(s) of transmission of mers-cov to humans remain unclear. of the minority of case-patients for whom information is available about exposure to animals, few have reported owning or visiting a farm with camels, goats, sheep, chickens, ducks, or other animals ( ). a zoonotic origin for mers-cov was initially suggested by its high genetic similarity to bat covs ( ) and the identification of closely related viruses in bats ( ). recent reports have described additional data from camels. these include real-time reverse transcription pcr detection and limited sequencing of mers-cov from camels from a farm in qatar linked author to infections in humans in october ( ) and, more recently, in camels in saudi arabia ( ) , and antibodies against mers-cov in camel serum from the arabian peninsula, including serum from the united arab emirates drawn in ( ) ( ) ( ) ( ) ( ) . however, more epidemiologic data linking cases to infected animals are needed to determine whether a particular animal species is a host for the virus, a source of human infection, or both. this month's issue of emerging infectious diseases presents results of a study that provides evidence of mers-cov in dromedary camels in egypt ( ) . only other reports of mers-cov detection in animals have been published: in a bat and in camels ( , , ) . however, these reports were based on limited genetic information. in contrast, on the basis of their sequence analysis of nearly the entire viral genome showing > % nt sequence identity with human mers-cov, chu et al. provide the most compelling evidence thus far of mers-cov infection in dromedary camels ( ) . although the authors also found neutralizing antibodies to mers-cov (or a mers-like cov) in most of the camels, they did not find serologic evidence of infection in the abattoir workers who had contact with the infected animals. this finding leaves key questions about zoonotic transmission unanswered. most notably, it remains unclear whether zoonotic transmission of mers-cov occurs between camels and humans and, if so, what the directionality and risk factors are for such transmission. these lingering gaps in knowledge about mers-cov emphasize the need for more epidemiologic study to determine risk factors for human infection, more population-level data on the prevalence of mers-cov in camels, risk factors for infection and shedding in camels, and continued vigilance for other possible sources of infection. also, the camels tested were in egypt and were locally reared or imported from sudan or ethiopia, countries in which no cases have been identified in humans. thus, the geographic area for surveillance should be widened beyond the arabian peninsula and include eastern africa, which is a source for importation of dromedary camels. this study emphasizes the need to further define exposure information for all mers-cov cases regarding camels and other animals, as well as exposure to ill humans who might have undetected mers-cov infections. understanding the role of dromedary camels and possibly other animals in transmission of mers-cov to humans remains a priority for future investigation to enable development of targeted control measures and prevent future cases and deaths from this emerging pathogen. dr watson is a medical officer with the division of viral diseases, centers for disease control and prevention, in atlanta, georgia, usa. his research interests include the epidemiology and control of viral respiratory diseases. latest outbreak news from promed-mail: novel coronavirus-middle east middle east respiratory syndrome coronavirus (mers-cov)-update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels mers coronaviruses in dromedary camels, egypt. emerg infect dis middle east respiratory syndrome coronavirus in bats, saudi arabia key: cord- -t jngq authors: ramshaw, rebecca e.; letourneau, ian d.; hong, amy y.; hon, julia; morgan, julia d.; osborne, joshua c. p.; shirude, shreya; van kerkhove, maria d.; hay, simon i.; pigott, david m. title: a database of geopositioned middle east respiratory syndrome coronavirus occurrences date: - - journal: sci data doi: . /s - - - sha: doc_id: cord_uid: t jngq as a world health organization research and development blueprint priority pathogen, there is a need to better understand the geographic distribution of middle east respiratory syndrome coronavirus (mers-cov) and its potential to infect mammals and humans. this database documents cases of mers-cov globally, with specific attention paid to zoonotic transmission. an initial literature search was conducted in pubmed, web of science, and scopus; after screening articles according to the inclusion/exclusion criteria, a total of sources were selected for extraction and geo-positioning. each mers-cov occurrence was assigned one of the following classifications based upon published contextual information: index, unspecified, secondary, mammal, environmental, or imported. in total, this database is comprised of unique geo-positioned mers-cov occurrences. the purpose of this article is to share a collated mers-cov database and extraction protocol that can be utilized in future mapping efforts for both mers-cov and other infectious diseases. more broadly, it may also provide useful data for the development of targeted mers-cov surveillance, which would prove invaluable in preventing future zoonotic spillover. middle east respiratory syndrome coronavirus (mers-cov) emerged as a global health concern in when the first human case was documented in saudi arabia . now listed as one of the who research and development blueprint priority pathogens, cases have been reported in countries across four continents . imported cases into non-endemic countries such as france, great britain, the united states, and south korea have caused secondary cases [ ] [ ] [ ] , thus highlighting the potential for mers-cov to spread far beyond the countries where index cases originate. reports in animals suggest that viral circulation could be far more widespread than suggested by human cases alone [ ] [ ] [ ] . to help prevent future incidence of mers-cov, public health officials can focus on mitigating zoonotic transfer; however, in order to do this effectively, additional research is needed to determine where spillover could occur between mammals and humans. previous literature reviews have looked at healthcare-associated outbreaks , importation events resulting in secondary cases , , occurrences among dromedary camels , , or to summarize current knowledge and knowledge gaps of mers-cov , . this database seeks fill gaps in literature and build upon existing notification data by enhancing the geographic resolution of mers-cov data and providing occurrences of both mammal and environmental detections in addition to human cases. this information can help inform epidemiological models and targeted disease surveillance, both of which play important roles in strengthening global health security. knowledge of the geographic extent of disease transmission allows stakeholders to develop appropriate emergency response and preparedness activities (https://www.jeealliance.org/ global-health-security-and-ihr-implementation/joint-external-evaluation-jee/), inform policy for livestock trade and quarantine, determine appropriate demand for future vaccines (http://cepi.net/mission) and decide where to deliver them. additionally, targeted disease surveillance will provide healthcare workers with updated lists of the methods and protocols summarized below have been adapted from previously published literature extraction processes [ ] [ ] [ ] [ ] [ ] , and provide additional context surrounding our systematic data collection from published reports of mers-cov. data collection. we identified published reports of mers-cov by searching pubmed, web of science, and scopus with the following terms: "middle eastern respiratory syndrome", "middle east respiratory syndrome", "merscov", and "mers". the initial search was for all articles published about mers-cov prior to april , , and was subsequently updated to february , . these searches were conducted through the university of washington libraries' institutional database subscriptions. we searched the web of science web of science core collection (the subscribed edition includes science citation index expanded, -present; social sciences citation index, -present; arts & humanities citation index, -present; emerging sources citation index, -present). we searched the standard scopus database and the standard, freely available pubmed database; these products have a single version that is consistent across institutional subscriptions or access points. in total, this search returned , related abstracts, which were collated into a database before a title-abstract screening was manually conducted (fig. . flowchart) . articles were removed if they did not contain an occurrence of mers-cov; for example, vaccine development research or coronavirus proteomic analyses. non-english articles were flagged for further review and brought into the full text screening stage. the accompanying supplementary file highlight the title and abstract screening process and the inclusion and exclusion criteria. full text review was conducted on , sources. to meet the inclusion criteria, articles must have contained both of the following items: ) a detection of mers-cov from humans, animals, or environmental sources, and ) mers-cov occurrences tagged with spatial information. additionally, extractors attempted to prospectively manually remove articles containing duplicate occurrences that were already extracted in the dataset. extractors only prospectively manually removed articles if it was clear the articles contained data we were confident had already been extracted and had high-quality data. we excluded sources based on full text review. in addition, we reviewed citations and retroactively added relevant articles to our database if they were not already included. we retroactively added and subsequently marked ten articles for extraction using this process. in total, we extracted peer-reviewed sources reporting detection of mers-cov that included geographic and relevant epidemiological metadata. geo-positioning of data. google maps or arcgis was used to manually extract location information at the highest resolution available from individual articles. we evaluated spatial information as either points or polygons. the geography was defined as a point if the location of transmission was reported to have occurred within a × km area. point data are represented by a specific latitude and longitude. a point references an area smaller than × km in order to be compatible with the typical × km resolution of satellite imagery used for global analyses. the geography was defined as a polygon if the location of transmission was less clear, but known to have occurred in a general area (e.g. a province), or the location of transmission occurred within an area greater than × km (e.g. a large city). we used contextual information to determine location in instances where the author's spelling of a location differed from google maps or arcgis. maps provided by authors were digitized using arcgis. we used three different types of polygons: known administrative boundaries, buffers, and custom polygons. relevant administrative units were sourced from the global administrative unit layers curated by the food and agricultural organization of the un for known administrative boundaries of governorates, districts, or regions, and paired with the occurrence record. buffers were created to encompass areas in cities and regions without corresponding administrative units. to ensure that buffers encompassed the entirety of the area of interest, google maps was used to determine the required radius. in areas with unspecified boundaries (e.g. table mountain national park and the border region between saudi arabia and uae) arcgis was used to generate custom polygons, which were assigned a unique code within a defined shapefile for ease of re-identification. this database is publicly available online , . each of the rows represents a unique occurrence of mers-cov. rows containing an index, unspecified, or imported case represent a single case of mers-cov. rows containing mammal and secondary cases may represent more than one case but are still unique geospatial occurrences. table shows an overview of the content available in the publicly available dataset. in addition, online-only table lists occurrences by geography, origin, shape type, and publication and online-only table provides citations of the data. index unspecified mammal import secondary absent environmental www.nature.com/scientificdata www.nature.com/scientificdata/ . pathogen: name the pathogen identified (e.g. mers-cov, bat coronaviruses, and other mers-cov-like pathogens). . pathogen_note: miscellaneous notes regarding pathogen. . patient_type: index, unspecified, na, secondary, import, or absent. • index: any human infection of mers-cov resulting after direct contact with an animal and no reported contact with a confirmed mers-cov case or healthcare setting. • unspecified: cases that lacked sufficient epidemiological evidence to classify them as any other status (e.g. serosurvey studies). • na: non-applicable field; case was not a patient (e.g. mammal) • secondary: defined as any cases resulting from contact with known human infections. cases reported after the index case can be assumed to be secondary cases unless accompanied by specific details of likely independent exposure to an animal reservoir. • import: cases that were brought into a non-endemic country after transmission occurred elsewhere. • absent: suspected case(s) ultimately confirmed negative for mers-cov. . transmission_route: zoonotic, direct, unspecified, or animal-to-animal. • zoonotic: transmission occurred from an animal to a human. • direct: only relevant for human-to-human transmission. • unspecified: lacked sufficient epidemiological evidence to classify a human case as zoonotic or direct. • animal-to-animal: transmission occurred from an animal to another animal. . clinical: describes whether the mers-cov occurrence demonstrated clinical signs of infection. denoted by yes, no, or unknown. • yes: clinical signs of infection were present/reported. clinical signs among humans may range from mild (e.g. fever, cough) to severe (e.g. pneumonia, kidney failure). clinical signs among camels include nasal discharge. • no: clinical signs of infection were not present/reported. • unknown: subject(s) may or may not have been demonstrating clinical signs of infection. for example, some authors did not explicitly mention symptoms, but individuals reportedly sought medical care. another example being when a diagnostic serosurvey was conducted during an ongoing outbreak. the term "unknown" was used when articles lacked sufficient evidence for extractors to definitively label as "yes" or "no". . diagnostic: describes the class of diagnostic method that was used. pcr, serology, or reported. . diagnostic_note: more detailed information related to the specific test used (e.g. rk , igg, or igm serology). . serosurvey: describes the context if serological testing was used. • diagnostic: testing of symptomatic patients. • exploratory: historic exposure determined among healthy asymptomatic individuals. . country: iso code for country in which the case occurred. . origin: open-ended field to provide more details on the specific in-country location of mers-cov case. . problem_geography: this field was utilized if the mers-cov case was reported in a location that could cause uncertainty when determining exact geographic occurrence (e.g. hospital, abattoir). . lat: latitude measured in decimal degrees. . long: longitude measured in decimal degrees. . latlong_source: the source from which latitude and longitude were derived. . loc_confidence: states the level of confidence that researchers had when assigning a geographic location to the mers-cov case (good or bad). an answer of 'good' meant the article stated clearly that the case occurred in a specific geographic location and no assumptions were required on part of the researcher. an answer of 'bad' meant the article did not clearly state the specific geographic location of the mers-cov case, but the researcher was able to infer the location of occurrence. the field site_notes was utilized to detail the logic behind researchers' decisions when inference was required. . shape_type: the geographic shape type assigned to the mers-cov occurrence (point or polygon). . poly_type: if the mers-cov occurrence was assigned a shape_type of polygon, was it admin (gaul), custom, or buffer? . buffer_radius: if a mers-cov occurrence was assigned a buffer, what is the radius in km? . gaul_year_or_custom_shapefile: file path used to reach the necessary shape file in arcgis. users of this dataset can find custom shapefiles created for this dataset at: https://cloud.ihme.washington.edu/index. php/s/dgoykyqnbjg f /download . poly_id: a standardized and unique identifier assigned to each gaul shapefile. . poly_field: which type of polygon was used to geo-position the occurrence? (e.g. if admin polygon was used, enter adm _code) . site_notes: miscellaneous notes regarding the site of occurrence. . month_start: month that the occurrence(s) began. if the article provided a specific month of illness onset, the month was assigned a number from - ( = january, = february, etc.). if the article did not provide a specific month of illness onset, then researchers assigned a value of 'na' . month that the occurrence(s) ended, defined as the date a patient tested negative for mers-cov. if the article provided a specific month for recovery, the month was assigned a number from - ( = january, = february, etc.). if the article did not provide a specific month of symptom onset, then researchers assigned a value of 'na' . . year_start: year that the occurrence(s) began. if the year of illness onset was not provided in the article, the ihme standard was used: (year_start = publication year - ). year that the occurrence(s) ended. if the article did not provide a specific year for recovery, the ihme standard was used: (year_end = publication year - ). . year_accuracy: if years were reported, this field was assigned a value of ' ' . if assumptions were required, this field was assigned a value of ' ' . all data extracted from the original search (october to april , ) was reviewed independently by a second individual to check for accuracy. challenging extractions from the updated search (may , to february , ) were selected for group review during bi-weekly team meetings. upon extraction completion, all data were checked to ensure they fell on land and within the correct country. while the protocol implemented above was designed to reduce the amount of subjective decisions made by extractors, total elimination was not possible. wherever a subjective decision had to be made, the extractor utilized the various notes fields in order to document the logic behind decisions. these decisions were subsequently reviewed by other extractors. the techniques described here can be applied to collect and curate datasets for other infectious diseases, as has been previously demonstrated with dengue and leishmaniasis . additionally, since these data were collected independently through published reports of mers-cov occurrence, they may be used to build upon existing notification data , . our ability to capture occurrences in this dataset is contingent on the data contained within published literature. therefore, this dataset does not represent a total count of all cases. instead, this dataset's value lies within its geo-precision. data were extracted with a focus on obtaining the highest resolution possible. these data may be merged with other datasets, such as who or oie surveillance records, and are intended to complement, not replace, these resources. together, published reports and notification data can provide a more comprehensive snapshot of current disease extent and at-risk locations. an important consideration, whether using the literature data alone, or in combination with other databases, is the potential for duplication. various pieces of metadata can be used to evaluate where potential duplicates could lie, such as common date fields (month_start, month_end, year_start, year_end) or consistent geographic details (lat, long, poly_id, shape_type) or shared epidemiological tags (patient_type). researchers may wish to consider further steps, such as fuzzy matching of geographic data (e.g. matching a point with an overlapping buffer) or temporal data (e.g. matching a precise month with an overlapping month interval). we acknowledge this duplicate-removal process will not catch all matching records, but it will likely catch several. we recommend occurrences are layered from top to bottom in the following order: index (green), unspecified (orange), mammal (yellow), import (blue), secondary (purple). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this approach because it will allow researchers to remove several duplicates without erroneously deleting any two occurrences that are truly unique (i.e. not duplicates). essentially, we recommend a sensitive approach above a more specific approach, as the latter simply risks culling too many records that aren't actually duplicates. when merging with other databases, consistency in metadata tagging is essential. for the who disease outbreak news data feed , for instance, nomenclature for case definitions is slightly different, with who definitions of "community acquired" and "not reported" comparable to "index" and "unspecified" respectively. in addition, it is important to recognize what information is beyond the scope of these additional databases. again, when comparing to the who dataset, it is important to recognize that serologically positive cases do not meet the case definition used in the who database. these adjustments need to be identified on a dataset-to-dataset basis. among cases tagged as index or unspecified. occurrences tagged as index are coloured green, those tagged as unspecified are coloured orange. points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this database can be combined with other covariates (e.g. satellite imagery) to produce environmental suitability models of mers-cov infection risk and potential spillover on both global and regional scales as achieved with other exemplar datasets [ ] [ ] [ ] [ ] . this information can be useful in resource allocation aimed at improving disease surveillance and contribute towards a better understanding of the factors facilitating continued emergence of index cases. the addition of sampling techniques and prevalence data may improve this dataset. researchers were ultimately unable to add these data due to inconsistencies in the way literature reported sampling techniques and prevalence date by geography. an attempt to extract these data using the current approach would have led to sporadic inclusion of this information and would not have been comprehensive for the entire dataset. moving forward, we recommend authors report sampling technique and prevalence data at the highest resolution geography possible, as seen in miguel et al. . we encourage continued presentation of paired epidemiological and geographic metadata that would allow for more detailed analysis in the future. this database may also be utilized in clinical settings to provide an evidence-base for diagnoses when used in conjunction with patient travel histories. additionally, it can be used to identify geographies for surveillance, particularly areas where mers-cov has been documented in animals but not humans (e.g. ethiopia and nigeria). identifying locations for surveillance will, in turn, inform global health security. while models will increase the resolution at which these questions can be addressed, datasets such as this provide an initial baseline. a major limitation of this database is the potential for sampling bias, which stems from higher frequency of disease reporting within countries where there exists strong healthcare infrastructure and reporting systems. this database does not attempt to account for such biases, which must be addressed in subsequent modelling activities where such biases are of consequence. similarly, another limitation is potential duplicate documentation of singular occurrences. this can happen when the same occurrence is assigned different geographies (e.g. point, polygon) in multiple publications. even though extractors made efforts to prospectively manually identify duplicate occurrences, this was challenging because the process relied upon papers providing sufficient details for extractors to determine a duplicate occurrence (e.g. geography, patient demographics, dates of occurrence, diagnostic methods, etc.). however, the majority of papers did not report such details for each occurrence. in those instances, it was impossible for extractors to discern whether occurrences may have been duplicates from a previous artic le. even case studies inconsistently reported patient details and demographic information. these are some examples of challenges faced by extractors when we attempted to identify duplicates. without sufficient contextual clues, extractors lacked evidence to determine duplicity and thus likely extracted some unique occurrences more than once. despite efforts to remove duplicate occurrences from the database, it is possible that some remain. geographic uncertainty is similarly problematic for analyses such as this. in some cases, polygons, as opposed to points, are utilised as a geographic frame of reference, reflecting the uncertainty in geotagging in the articles themselves. for some occurrences, there is a strong assumption that the geography listed corresponds to the site of infection. while the use of km × km as the minimum geographical unit allows for some leeway in this precision, it is possible that even with the point data (often corresponding to household clusters) these may not map directly with true infection sites. this must be considered in any subsequent geospatial analysis. finally, this database represents a time-bounded survey of the literature. while all efforts were made to be comprehensive within this period, articles, and therefore data, will continue to be published. efforts to streamline ongoing collection processes are still to be fully realized . regardless, we hope that this dataset provides a solid baseline for further iteration. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission enhanced mers coronavirus surveillance of travelers from the middle east to england control of an outbreak of 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epidemiology of hospital outbreak of middle east respiratory syndrome notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia outbreak of middle east respiratory syndrome at tertiary care hospital patient characteristics infected with middle east respiratory syndrome coronavirus infection in a tertiary hospital predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients report of middle east respiratory syndrome coronavirus (mers-cov) infection in four patients with hematological malignancies treated at king fahad medical city risk factors for middle east respiratory syndrome coronavirus infection among 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respiratory syndrome coronavirus a phylogenetically distinct middle east respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in dubai with rising seroprevalence with age acute middle east respiratory syndrome coronavirus infection in livestock dromedaries antibodies against mers coronavirus in dromedary camels clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate middle east respiratory syndrome coronavirus antibody reactors among camels in dubai middle east respiratory syndrome coronavirus during pregnancy polyphyletic origin of mers coronaviruses and isolation of a novel clade a strain from dromedary camels in the united arab emirates prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate time course of mers-cov infection and immunity in dromedary camels transmission of middle east respiratory syndrome coronavirus infections in healthcare settings response to emergence of middle east respiratory syndrome coronavirus diversity of middle east respiratory syndrome coronaviruses in dromedary camels based on full-genome sequencing identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates mers-cov in pregnancy some epidemiological studies on mers coronavirus in dromedaries in the united arab emirates -a short communication emerging and reemerging diseases in the world health organization (who) eastern mediterranean region-progress, challenges, and who initiatives melinda gates foundation opp# and s.i.h. was supported by opp curated and catalogued the database. s.s. provided managerial support. all authors participated in interpreting and summarizing the results. r.e.r. wrote the first draft of the manuscript. all other authors critically reviewed the manuscript had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis s.i.h. and d.m.p. are members of the editorial board of scientific data. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to d.m.p.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /.the creative commons public domain dedication waiver http://creativecommons.org/publicdomain/zero/ . / applies to the metadata files associated with this article. key: cord- -rhhmvnlp authors: joseph, sunitha; wernery, ulrich; teng, jade ll; wernery, renate; huang, yi; patteril, nissy ag; chan, kwok-hung; elizabeth, shyna k; fan, rachel yy; lau, susanna kp; kinne, jörg; woo, patrick cy title: first isolation of west nile virus from a dromedary camel date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: rhhmvnlp although antibodies against west nile virus (wnv) have been detected in the sera of dromedaries in the middle east, north africa and spain, no wnv has been isolated or amplified from dromedary or bactrian camels. in this study, wnv was isolated from vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in dubai. complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage a. there was no clustering of the present wnv with other wnvs isolated in other parts of the middle east. within lineage a, the dromedary wnv occupied a unique position, although it was most closely related to other wnvs of cluster . comparative analysis revealed that the putative e protein encoded by the genome possessed the original wnv e protein glycosylation motif nys at e – , which contained the n-linked glycosylation site at n- associated with increased wnv pathogenicity and neuroinvasiveness. in the putative ns protein, the a s substitution observed in other cluster wnvs and p , which has been implicated in neuroinvasiveness, were present. in addition, the foo motif in the putative ns a protein, which has been implicated in neuroinvasiveness, was detected. notably, the amino-acid residues at positions in the present dromedary wnv genome differed from those in most of the closely related wnv strains in cluster of lineage a, with the majority of these differences observed in the putative e and ns proteins. the present study is the first to demonstrate the isolation of wnv from dromedaries. this finding expands the possible reservoirs of wnv and sources of wnv infection. west nile virus (wnv) is a positive-sense single-stranded ribonucleic acid (rna) virus in the flaviviridae family. wnv is the leading cause of mosquito-borne encephalitis in humans in many parts of the world. in addition to humans, many animals are also susceptible to wnv infections. large outbreaks have occurred globally in both humans and other animals, such as horses and pigs. birds are the natural reservoirs of wnv. bird-to-bird, bird-to-mammal and bird-to-human transmissions are achieved by mosquito bites, with humans and other mammals serving as dead-end hosts because of low viral loads. camels are one of the most unique mammals on earth and have shown perfect adaptation to desert life. there are two surviving old-world camel species: camelus dromedarius (dromedary or onehumped camel), which inhabits the middle east and north and northeast africa, and camelus bactrianus (bactrian or two-humped camel), which inhabits central asia. among the million camels on earth, % are dromedaries. although wnv is known to infect some of the new world camels, such as llamas and alpacas, only antibodies against wnv have been detected in the sera of old-world camels, such as the dromedaries in the middle east, north africa and spain, with a seroprevalence of %- %. [ ] [ ] [ ] [ ] to date, no wnv has been isolated or amplified from either dromedary or bactrian camels. recently, the emergence of middle east respiratory syndrome (mers) and the isolation of the mers coronavirus (mers-cov) from dromedaries boosted interest in the search for novel viruses in dromedaries. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this article, we report the first isolation of wnv from a dromedary calf in the united arab emirates during the process of mers-cov screening and the results of the comparative genome and phylogenetic analysis. clinical samples were obtained during a necropsy of a dromedary calf at the central veterinary research laboratory in dubai, the united arab emirates, using standard procedures. the central veterinary research laboratory in dubai is the center for performing necropsies of dromedaries from sheikhs in the uae with the aim of finding the cause of death and preventing the spread of infectious diseases to other camels or herds. nasal swabs and pooled ground trachea/lung samples were inoculated onto vero cells for mers-cov screening. the pooled clinical samples were diluted -fold with viral transport medium and filtered. two hundred microliters of the filtrate was inoculated into μl of minimum essential medium (gibco, grand island, ny, usa). four hundred microliters of the mixture was added to -well tissue culture plates with vero cells by adsorption inoculation. after h of adsorption, the excess inoculum was discarded, the wells were washed twice with phosphate-buffered saline, and the medium was replaced with ml of minimum essential medium (gibco). cultures were incubated at °c with % co and inspected for cytopathic effects daily using inverted microscopy. negative-contrast electron microscopy was performed as previously described. tissue culture cell extracts infected with dromedary wnv were centrifuged at g at °c. then, the pellet was resuspended in phosphate-buffered saline and stained with % phosphotungstic acid. samples were examined with a philips em s electron microscope (philips scientifics, eindhoven, the netherlands). sample preparation for illumina sequencing rna was extracted from the isolated virus using the qiaamp viral rna mini kit (qiagen, hilden, germany). reverse transcription and polymerase chain reaction (pcr) were performed using the super-script iii reverse transcriptase (invitrogen, carlsbad, ca, usa) and a random primer containing a -base arbitrary sequence at the ′ end followed by a randomized octamer ( n) at the ′ end. a single round of priming and extension was performed using the klenow fragment polymerase (new england biolabs, ipswich, uk). pcr amplification with a primer consisting of only the -base arbitrary sequence of the random primer was performed with cycles of °c for s, °c for s and °c for min and a final extension at °c for min in an automated thermal cycler (applied biosystems, carlsbad, ca, usa). standard precautions were taken to avoid pcr contamination, and no amplified pcr product was observed in the negative control. the pcr product was purified using the minelute pcr purification kit (qiagen) following the manufacturer's protocol. the purified dna was eluted in μl of eb buffer and used as the template for library construction. library construction for illumina sequencing the metagenomics library was generated using a dna library prepared with the nextera xt dna sample preparation kit (illumina, san diego, ca, usa) according to the manufacturer's protocol. briefly, ng of input dna was tagmented by the nextera xt transposome at °c for min. this transposome simultaneously fragmented the input dna and added an adapter sequence to the ends, thereby allowing amplification by pcr in subsequent steps. the sequencing library with the tagmented dna was amplified in cycles of °c for s, °c for s and °c for s and a final extension at °c for min in an automated thermal cycler (applied biosystems). the amplified dna library was purified using . × ampure xp beads (beckman coulter, indianapolis, in, usa) to remove very short library fragments from the population. the amplified dna library was analyzed using the bioanalyzer instrument (agilent technologies, santa clara, ca, usa). the purified sequencing library was quantified using the kapa library quantification kit (kapa biosystems, wilmington, de, usa) and subsequently sequenced on the illumina hiseq with bp paired-end reads (rapid run mode). image analysis and base calling were performed with scs . /rta . (illumina). fastq file generation and the removal of failed reads were performed using casava ver. . . (illumina). genome assembly and complete-genome sequencing illumina sequence reads were quality trimmed by prinseq-lite, and de novo genome assembly was performed with mira . . the ′ and ends of the viral genome were confirmed by rapid amplification of cdna ends (race) using the ′/ ′ race kit (roche, penzberg, germany). the nucleotide sequence of the genome and the deduced amino-acid sequences of the open reading frames were compared with those of other wnv strains with complete genomes in the vipr sequence database (http://www.viprbrc.org/). n-linked glycosylation sites were predicted using the netnglyc . software (http://www.cbs.dtu.dk/ services/netnglyc/). complete polyprotein nucleotide sequences were aligned with muscle. a maximum-likelihood phylogenetic tree with bootstraps was constructed using the gtr substitution model; gamma distribution among sites was conducted in mega . the nucleotide sequence of the dromedary wnv genome isolated in this study has been lodged in the genbank sequence database under accession no ku . necropsy of the female -month-old kg fresh dromedary calf revealed pale muscles and myocard, massive lung congestion and colitis. the causes of death were white muscle disease, colisepticemia and candida enteritis. at the first passage, the vero cells inoculated with both the nasal swab and pooled trachea/lung samples showed identical cytopathic effects on day , with cell rounding, progressive degeneration and detachment ( figure a) . electron microscopy showed enveloped icosahedral virions - nm in diameter with a -to -nm electron dense core ( figure b ). both the cytopathic effects and viral morphology were inconsistent with those of mers-cov. the complete genome of the isolated virus was sequenced and assembled. the size, g+c content and genome structure were similar to other wnvs. the single polyprotein is putatively cleaved by proteases into three structural proteins (capsid (c), premembrane/ membrane (prm/m) and envelope (e)) and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ). phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage a (figure a ). the present dromedary wnv occupied a unique position within lineage a, although it was most closely related to other wnvs of cluster ( figure b ). comparative analysis based on the complete polyprotein sequence showed the highest ( . %- . %) overall amino-acid identities to wnv strains isolated from a mosquito in kenya (genbank accession number ay ), horses in italy and morocco (genbank accession numbers af , ay and ay ) and a human in italy (genbank accession number jq ). detailed annotation revealed that the putative e protein encoded by the genome possessed the original wnv e protein glycosylation motif nys at e - , which contained the n-linked glycosylation site at n- associated with increased wnv pathogenicity and neuroinvasiveness. in the putative ns protein, the a s substitution observed in other cluster wnvs and p , which was implicated in neuroinvasiveness, were also present. in addition, the foo (flavivirus overlapping orf) motif in the putative ns a protein that was implicated in neuroinvasiveness was detected. notably, amino-acid residues in the present dromedary wnv genome differed from those in most of the closely related wnv strains in cluster of lineage a (figure ) , with the majority of these differences observed in the putative e and ns proteins. we report the first isolation of wnv in a dromedary. the first evidence for the presence of hemagglutination inhibiting and complement-fixing antibodies in dromedaries from nigeria against wnv was published in . , recently, a study has shown the presence of neutralizing antibodies against wnv from dromedaries in north africa. in the united arab emirates, antibodies against wnv have been reported in % of dromedary sera and . % of tested equine sera, supporting the conclusion that wnv is present in the country. in the present study, we isolated a wnv from two independent respiratory samples from a dromedary calf using vero cells, which is one of the most widely used cell lines for the recovery of wnv. this finding was consistent with previous studies that showed that wnv could also be detected in respiratory specimens from birds, humans and other mammals. , notably, the glycosylation motif nys in the e protein, the a s substitution and p in the ns protein and the foo motif in the ns a protein, which are important for pathogenicity and neuroinvasiveness, were also detected in the present wnv isolate. [ ] [ ] [ ] however, because there were no clinical signs in the dromedary calf, the pathogenic role of wnv in dromedaries remains to be determined. viral cultures using other samples from the dromedary were not performed because the samples were initially intended for mers-cov isolation. the present dromedary wnv belongs to wnv lineage a. the whole-genome phylogenetic trees showed that the present dromedary wnv was most closely related to other wnvs in cluster of lineage a with high bootstrap support ( figure ). however, there was no clustering of the present wnv with wnvs isolated in other parts of the middle east, such as israel, egypt and cyprus (data not shown). indeed, two wnvs from israel (genbank accession number ay ) and cyprus (genbank accession number gq ) belong to lineage , another isolate from israel (genbank accession number hm ) belongs to cluster of lineage a, and two other isolates from israel (genbank accession number hm ) and egypt (genbank accession number eu ) belong to cluster of lineage a. moreover, amino-acid positions located in various regions of the genome encoding five different proteins showed marked differences with the corresponding amino acids in the genomes of the isolate's close relatives in cluster . interestingly, most of the amino-acid differences between the present dromedary wnv and the closely related wnvs from africa and europe are located in the e and ns genes. the e protein is generally the least conserved protein because it is exposed to external selection pressure, whereas the ns protein is highly conserved because it contains the viral methyltransferase and rna-dependent rna polymerase. complete-genome sequencing of more wnv strains and comparative genomic and phylogenetic studies are needed to ascertain whether dromedary wnvs form a unique cluster in lineage a. the mers epidemic and the discovery of dromedaries as the reservoir of mers-cov have boosted interest in the search for novel viruses in dromedaries. prior to , viruses from at least eight families were found to infect camels. in the last two years, we have reported the discovery of a novel dromedary camel cov (uae-hku ), a novel genotype of hepatitis e virus, a novel genus of enterovirus, a novel astrovirus, and novel picobirnaviruses and circoviruses in dromedaries. , [ ] [ ] [ ] [ ] these discoveries have remarkably widened the spectrum of viruses known to infect dromedaries. notably, after the description of the novel genotype of hepatitis e virus in dromedaries, another group found this dromedary camel hepatitis e virus as a cause of chronic hepatitis e infection in a liver transplant recipient with a habit of camel milk and meat consumption, highlighting the importance of camels as a source of infectious diseases. the present study is the first demonstration of the isolation of wnv in dromedaries and the first wnv complete-genome sequenced from a dromedary among the relatively few full-length wnv genome sequences from the middle east. further studies will help elucidate whether dromedaries can serve as another possible source of wnv infection and determine the tissue tropism of wnv in s joseph et al figure summary of amino-acid changes in the various putative proteins encoded by the dromedary wnv genome relative to those of its most closely related wnv strains in cluster of lineage a. only changes that occurred in ⩾ strains are shown. dots indicate no difference from the dromedary wnv. west nile virus in a dromedary s joseph et al viral diseases of new world camelids a transversal study on antibodies against selected pathogens in dromedary camels in the canary islands rift valley and west nile virus antibodies in camels emerging viral diseases in dromedary camels in the southern morocco seroepidemiological studies for the detection of antibodies against nine infectious diseases in dairy dromedaries (part- ) middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of a novel coronavirus from a man with pneumonia in saudi arabia a phylogenetically distinct middle east respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in dubai with rising seroprevalence with age a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease acute middle east respiratory syndrome coronavirus infection in livestock dromedaries isolation and characterization of a novel betacoronavirus subgroup. a coronavirus, rabbit coronavirus hku , from domestic rabbits metagenomic analysis of viromes of dromedary camel fecal samples reveals large number and high diversity of circoviruses and picobirnaviruses the challenge of west nile virus in europe: knowledge gaps and research priorities viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the new york strain of west nile virus loss of dimerisation of the nonstructural protein ns of kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of ns ns ' of flaviviruses in the japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness a survey for haemagglutination-inhibiting antibody to west nile virus in human and animal sera in nigeria west nile complement fixing antibodies in nigerian domestic animals and humans west nile fever in the united arab emirates west nile virus detection in kidney, cloacal, and nasopharyngeal specimens fatal hemorrhagic fever caused by west nile virus in the united states novel betacoronavirus in dromedaries of the middle east new hepatitis e virus genotype in camels, the middle east a novel astrovirus from dromedaries in the middle east a novel dromedary camel enterovirus in the family picornaviridae from dromedaries in the middle east chronic infection with camelid hepatitis e virus in a livertransplant recipient who regularly consumes camel meat and milk we are grateful to dr ismail hassab elrasoul mohammed khair (central veterinary research laboratory, dubai, uae) for sending the carcass. we also thank the members of the centre for genomic sciences, the university of hong kong, for their technical support. this work is partially supported by the theme-based research scheme (project no t / / ), the university grant committee, and the strategic research theme fund, the university development fund, the university of hong kong. dromedaries. other studies on the epidemiology, disease spectrum and ecology of wnv in this group of unique animals are also warranted. key: cord- -ajd ofc authors: hui, david s.; leung, chi‐chiu title: contemporary concise review : respiratory infections and tuberculosis date: - - journal: respirology doi: . /resp. sha: doc_id: cord_uid: ajd ofc nan respiratory tract infections (rti), such as communityacquired pneumonia (cap) and tuberculosis (tb), are important causes of morbidity and mortality. the ongoing outbreaks of middle east respiratory syndrome coronavirus (mers-cov) infection and epidemic waves of avian influenza a(h n ) since their emergence in and , respectively, have raised global concern in recent years. the huge clinical burden of common respiratory viruses, such as respiratory syncytial virus (rsv) and seasonal influenza, on healthcare resources and utilization highlights the importance for developing more effective treatment modalities in order to reduce morbidity and mortality. in this contemporary concise review, articles related to these common respiratory infections and tb published recently in respirology and selected articles published in other journals that highlight advances in knowledge are summarized. pneumonia rsv is an important cause of acute rti in early life. high bacterial loads colonizing the upper rt are often observed during rsv infections in children. brealey et al. identified an association between co-detection of rsv and streptococcus pneumoniae and more severe disease in a brisbane-based cohort of young children aged under years with acute rti, suggesting the bacteria may play a pathogenic role in these young children. their findings may potentially have major implications for the management of young children with acute rti in determining whether hospitalization is required and in directing the appropriate use of antibiotics, in addition to understanding factors involved in the development of asthma and new pathways of immunomodulation. • in tropical countries, the spectrum of pathogens with pneumonia includes diseases such as melioidosis, scrub typhus, leptospirosis, hanta virus, chikungunya, dengue and parasitic pneumonias. • up to % of patients hospitalized for cap develop cardiovascular complications. • further clinical and mechanistic studies are required to define the role of vascular endothelial growth factor (vegf)-a as a marker of resolution of lung inflammation in ventilatorassociated pneumonia (vap). • the provision of procalcitonin assay results to emergency department and hospital-based clinicians did not result in less use of antibiotics than did usual care among patients with suspected lower rti. • β-lactam and macrolide in combination reduced the -day mortality compared with β-lactam alone in patients with low drugresistant pathogen (drp) risk. • systemic corticosteroid therapy was associated with delay in mers-cov rna clearance in critically ill patients. • early treatment of patients with avian influenza a(h n ) within - days from illness onset with a neuraminidase inhibitor (nai) can reduce mortality. • baloxavir marboxil is a selective inhibitor of influenza virus cap-dependent endonuclease that is effective in the treatment of uncomplicated influenza. • the role of adjunctive immunomodulating agents for patients with severe influenza deserves further investigation. the complex interaction of climate change, socioeconomic factors and human migration patterns has led to changing patterns of respiratory infections in tropical climate but also increasingly in temperate countries. tropical and poorer countries, especially south east asia, account for almost one-third of the global burden of tb pandemic. in addition to tb, lim and siow have emphasized that in tropical countries, the spectrum of pathogens with pneumonia includes diseases such as melioidosis, scrub typhus, leptospirosis, hanta virus, chikungunya, dengue and parasitic pneumonias. clinicians must be aware of the possible aetiology of pneumonia in the locality of practice and not solely dependent on published treatment guidelines. up to % of patients hospitalized for cap develop cardiovascular complications (such as new onset or worsening cardiac failure or arrhythmias, myocardial infarctions and cerebrovascular accidents) in the acute phase and even up to years thereafter. cardiac complications result from complex interactions between co-morbid conditions, upregulation of the sympathetic system, relative ischaemia, systemic inflammation and direct pathogen-mediated damage to the cardiovascular system. these mechanisms could provide the target of future therapeutic intervention to reduce incidence of cardiovascular complications and improve outcomes in patients with cap. a significant increase in human blood vegf-a is associated with early resolution of vap; vegf-a was an independent predictor of resolution despite disease severity. experimental chronic pulmonary infection by pseudomonas aeruginosa in the mice model led to increased vegf-a concentrations in the lungs and yet a reciprocal reduction of pro-inflammatory cytokines and myeloperoxidase activity. further clinical and mechanistic studies are required to define the role of vegf-a as a marker of resolution of lung inflammation in vap. the standardized approach to treatment makes cap a target for comparative performance and outcome measures. hadfield and bennet discussed clinical-and patient-reported outcomes, including endpoints such as the time to clinical stability and patient satisfaction, and strategies to improve these outcomes including use of a risk stratification tool, local antimicrobial guidelines with antibiotic stewardship and care bundles to include early administration of antibiotics and early mobilization. interestingly, the provision of procalcitonin assay results to emergency department and hospital-based clinicians did not result in less use of antibiotics than did usual care among patients with suspected lower rti in a randomized controlled trial (rct) involving hospital sites in the usa. there are limited published data evaluating the effects of antibiotic in cap according to the drp risk. in a post hoc analysis of a prospective multicentre cohort of patients with cap, okumura et al. have shown that β-lactam and macrolide in combination reduced the -day mortality (adjusted odds ratio: . , % ci: . - . ) compared with β-lactam alone. in addition, among patients at low risk for cap-drp, independent host factors associated with mortality included high arterial carbon dioxide (paco ) level, non-ambulatory status, leucopenia, low haematocrit, older age, tachypnoea, low serum albumin level and low body temperature. these factors should be evaluated in future clinical trials in patients with low cap-drp risk for stratification to assess antibiotic effects. in an accompanying editorial, waterer emphasized the importance of starting a macrolide early followed a short time later by a third-generation cephalosporin, preferably all within h of presentation to hospital. in saudi arabia (fig. ) . nosocomial outbreaks are a major hallmark of mers-cov infection. in , the largest outbreak outside the middle east occurred in south korea following the return of a businessman to seoul from a trip to qatar, uae, saudi arabia and bahrain, resulting in a total of confirmed cases with deaths. the risk factors for primary, household and nosocomial transmission are listed in table . another businessman returned to seoul from kuwait on august with atypical presentation of mers-cov infection including diarrhoea and weakness without respiratory symptoms. with improved infection control awareness and disease surveillance after the major outbreak in , the patient was isolated by the health authority quickly without causing any outbreak. treatment of mers-cov infection is mainly supportive at present. an rct is in progress in saudi arabia comparing lopinavir/ritonavir ( mg/ mg) bd for days, recombinant interferon-β b [ . -mg/ml subcutaneous (sc) injections on alternate days for days] and standard supportive care versus placebo and standard supportive care in patients with laboratory-confirmed mers requiring hospital admission. it is difficult to forecast the timing, size and severity of influenza seasons despite advances in surveillance system. nai has been the mainstay of treatment for seasonal and avian influenza but its effectiveness is limited by delay of patient presentation for management. early treatment of patients with avian influenza a(h n ) in zhejiang, china, within - days from illness onset with an nai can reduce mortality. of the patients, ( %) patients who had received nai within days died versus of ( . %) patients who received treatment within - days versus of ( . %) patients who were treated after days (p < . ). the median durations of viral shedding from nai therapy initiation was . days (interquartile range (iqr): - days) for patients who took nai within days, which was significantly shorter than that for those who took nai within - days ( . days (iqr: . - . days)) or after days ( days (iqr: - days)) (p < . ). introduction of a bivalent h /h vaccination of poultry in mainland china in october has resulted in better control of a(h n ) outbreaks, with detection of only three human cases from october to september , and a corresponding reduction of a(h n ) viruses detected in poultry and environmental samples of a(h n ) in china. baloxavir marboxil is a selective inhibitor of influenza virus cap-dependent endonuclease, with therapeutic activity in preclinical models of influenza a and b virus infections, including strains resistant to current antiviral agents. early treatment with a single-dose baloxavir was superior to placebo in alleviating influenza symptoms (in patients - years of age (median difference: . h) and among those - years of age (median difference: . h)). in addition, baloxavir was superior to both oseltamivir and placebo in reducing the viral load day after initiation of treatment in patients aged - years with uncomplicated influenza, although there was no significant difference in the median time to alleviation of symptoms between baloxavir and oseltamivir recipients. however, there was emergence of mutant viruses with the development of decreased susceptibility to baloxavir after treatment in a small percentage of cases. the therapeutic role of baloxavir in older or immunocompromised patients with severe seasonal or avian a(h n ) influenza especially with some time delay in administration of the drug later in the clinical course of the infection or in combination with an nai requires investigation. lee et al. have found significant anti-inflammatory effects with adjunctive macrolide treatment in adults with severe influenza infections, although virus rna decline was unaffected. the role of adjunctive immunomodulating agents for patients with severe influenza deserves further investigation. according to estimates by the world health organization, million incident tb cases and . million tb deaths occurred in . the estimated tb incidence declined only by % from to , barely higher than the . % annual decline in tb mortality in the united kingdom before the availability of effective treatment. social inequities continue to hamper tb control in many parts of the world, and major gaps remain in reaching, diagnosing and effectively treating tb patients, especially in resource-limited settings. on september , heads of state and government meeting at the united nations general assembly committed to mobilize us$ billion to implement tb prevention and care, and us$ billion for research on an annual basis by . hopefully, this important political commitment and additional funding support will promote universal health coverage and social protection, facilitate access to quality tb care and accelerate the development of better tools to meet the targets of the end tb strategy by . unlike other bacterial infections, a single critical concentration that inhibits the growth of % of phenotypically wild type strains is used to classify phenotypic susceptibility or resistance to tb drugs. , however, this may not reflect the achievable serum drug level or clinical response of a mutant strain. clinical breakpoint (minimal inhibitory concentration at or below which the relevant strain is likely to respond to treatment) may be more informative for isoniazid, rifampicin, fluoroquinolones and other drugs that can be used at higher doses. the target drug coverage of commercial rapid molecular tests suitable for direct application to clinical specimens is still too limited to guide the formulation of individualized treatment regimens for mdr-/rifampicin-resistant (rr-) tb. , whole-genome sequencing holds promise for revolutionizing the predictive power of genotypic drug susceptibility test, but cost, throughput, facility requirement, background noises and replicative errors are important hurdles to overcome before its direct application to clinical specimens. bacillary resistance to tb drugs continues to emerge. about . % of new tb cases and % of previously treated cases in were mdr-/rr-tb, while . % of mdr-tb cases were extensively drug-resistant (xdr-)tb. standardized treatment regimens, while having facilitated programmatic implementation, could have inadvertently accelerated the development of mdr-tb and xdr-tb through progressive selection of resistant mutants. , the standardized -to -month shorter mdr-tb regimen showed marginally less favourable outcome ( % vs %) than the conventional -to -month regimen in the preliminary results of the stream stage trial. in some mdr-tb hotspots such as eastern europe, south east asia, pakistan and brazil, the high prevalence of resistance to one or more of the drugs used in the shorter regimen may render - % of mdr-tb patients ineligible for the regimen by the strict drug susceptibility criteria. partly based on an updated meta-analysis of observational data in the treatment of mdr-tb, the world health organization proposed reclassification of tb drugs used in the longer conventional regimen for mdr-tb into three different categories in august . group a drugs, including levofloxacin/moxifloxacin, bedaquiline and linezolid, are to be prioritized. group b drugs, including clofazimine and cycloserine/terizidone, are to be added next. group c drugs (ethambutol, delamanid, pyrazinamide, imipenemcilastatin, meropenem, amikacin/streptomycin, ethionamide/prothionamide and p-aminosalicylic acid) are included when drugs from groups a and b cannot be used. with the favourable outcomes achievable by the optimized background regimens in the treatment of mdr-tb in both the stream stage trial ( %) and delamanid trial ( %), the need for the relatively toxic drug, linezolid, and the expensive new drug, bedaquiline, with very short and incompletely established safety record, remains to be established, at least for fluoroquinolone-susceptible mdr-tb. ageing and co-morbidities yew et al., in their review of the epidemiological, clinical and mechanistic perspectives of tb in older people, highlighted the increasing clinical and public health challenge posed by tb in the ageing population in many asian countries. the complex interactions among oxidative stress, mitochondrial dysfunction and immunological dysfunction may contribute to the development of tb in the geriatric population and worsen the disease outcomes, especially in presence of co-morbid conditions such as smoking and diabetes mellitus. in a : propensity score-matched analysis of data from a longitudinal health insurance database in taiwan, lin et al. showed % reduction in tb risk among diabetic patients put on metformin as compared with non-metformin users, after adjusting for co-morbidities, diabetic complications, anti-diabetic therapy type and statin use. in another longitudinal study from the same locality, metformin users were associated with % reduction in mortality during tb treatment despite their higher haemoglobin a c level. the protective effect of metformin therefore appears independent of diabetic control. while the sizeable protective effects of metformin in the abovementioned studies suggest a potential role of the drug as host-directed therapy in the treatment of latent tb infection and active tb, randomized trials are need to delineate its exact role(s) before introduction into clinical practices. park et al. retrospectively reviewed the data of patients with chronic obstructive pulmonary disease (copd) in the korean copd subtype study cohort. the copd assessment test (cat) scores, total st george's respiratory questionnaire for copd (sgrqc) scores and exacerbation prevalence were significantly higher, and lung function was significantly poorer in copd patients with prior tb than those without, both at the baseline and on follow-up over years. poorer lung function was observed among patients with prior tb, irrespective of whether they had visible lung lesions on chest radiographs. despite the introduction of igra using more specific antigens to avoid interference by prior bcg (bacillus calmette-guerin) vaccination, all existing tests for latent tb infection and predictive biomarkers for future disease development are subject to a generic limitation imposed on their positive predictive value by the generally low absolute disease risks. shorter rifamycin-based regimens (e.g. weekly rifapentine plus isoniazid for weeks and daily rifampicin for months ) are proving safe and effective alternatives to months of isoniazid, especially in terms of lower risk of hepatotoxicity. however, the risks of other adverse effects (e.g. systemic or hypersensitivity reactions to rifamycins) [ ] [ ] [ ] remain considerable relative to the number of tb cases averted. a targeted approach is therefore necessary to optimize the benefit versus risk ratio at the expense of reduced population coverage. despite the higher risk of tb infection among household contacts, household transmission accounts only for a small portion of the overall transmission in the ongoing tb epidemic. the safety of rifapentine in pregnancy remains to be established even though no excess risks of foetal loss or congenital anomalies were observed among pregnant women inadvertently exposed to either isoniazid or isoniazid plus rifapentine in two clinical trials. novel tb vaccine candidates being developed include whole-cell vaccines, adjuvanted protein subunit vaccines, viral vector-delivered subunit vaccines, plasmid dna vaccines, rna-based vaccines etc. vaccines are useful as a first-line tool in controlling infectious diseases because they are more readily applied on a population scale. unlike preventive treatment, the protective effects of vaccines will not be nullified by reinfection after the intervention. however, with the high global burden of latent tb infection, vaccines capable of preventing pulmonary tb in infected individuals will be needed to reduce tb incidence quickly. at least novel tb vaccine candidates are now in clinical trials. a subunit vaccine, m /as e, showed % protection against active pulmonary tb in hiv-negative adults with a positive igra in a recent phase iib trial. this proof-of-concept study brings fresh hopes for new and possibly transformative tb vaccines in the near future. streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children emerging role of viral and bacterial co-infection in early childhood pneumonia in the tropics respiratory infections in the asia-pacific region: problems and cautious optimism pneumonia as a cardiovascular disease early increase of vegf-a is associated with resolution of ventilator-associated pneumonia: clinical and experimental evidence determining best outcomes from community-acquired pneumonia and how to achieve them proact investigators. procalcitonin-guided use of antibiotics for lower respiratory tract infection central japan lung study group. mortality in patients with community-onset pneumonia at low risk of drug-resistant pathogens: impact of β-lactam plus macrolide combination therapy empiric antibiotics for community-acquired pneumonia: a macrolide and a beta-lactam please middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission an atypical case of middle east respiratory syndrome in a returning traveler to korea from kuwait and the miracle trial group. treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β b (miracle trial): study protocol for a randomized controlled trial saudi critical care trial group. corticosteroid therapy for critically ill patients with middle east respiratory syndrome macrolides in critically ill patients with middle east respiratory syndrome difficulties of predicting the timing, size and severity of influenza seasons a clinical approach to the threat of emerging influenza viruses in the asia-pacific region benefit of early initiation of neuraminidase inhibitor treatment to hospitalized patients with avian influenza a(h n ) virus influenza h /h virus vaccination in poultry and reduction of zoonotic infections baloxavir marboxil investigators group. baloxavir marboxil for uncomplicated influenza in adults and adolescents a step forward in the treatment of indfluenza anti-inflammatory effects of adjunctive macrolide treatment in adults hospitalized with influenza: a randomized controlled trial the role of adjuvant immunomodulatory agents for treatment of severe influenza geneva: world health organization tuberculosis updates : innovations and developments to end tb implementing the end tb strategy in the western pacific region: translating vision into reality applying new tools to control tuberculosis drug resistance mechanisms and drug susceptibility testing for tuberculosis thoracoscopic pleural biopsy improves yield of xpert mtb/rif for diagnosis of pleural tuberculosis genexpert mtb/rif for rapid diagnosis and rifampin resistance detection of endobronchial tuberculosis xpert mtb/rif for rapid detection of tb and rifampicin resistance in the evaluation of tracheobronchial lesions: what's to stop its use? technical report on critical concentrations for tb drug susceptibility testing of medicines used in the treatment of drug-resistant tb. geneva: world health organization drug-resistant tuberculosis: an update on disease burden, diagnosis and treatment prediction of susceptibility to first-line tuberculosis drugs by dna sequencing new drugs and regimens for tuberculosis position statement on the continued use of the shorter mdr-tb regimen following an expedited review of the stream stage preliminary results treatment correlates of successful outcomes in pulmonary multidrug-resistant tuberculosis: an individual patient data meta-analysis rapid communication: key changes to treatment of multidrug-and rifampicin-resistant tuberculosis (mdr/rr-tb). geneva: world health organization epidemiological, clinical and mechanistic perspectives of tuberculosis in older people metformin is associated with a lower risk of active tuberculosis in patients with type diabetes metformin use reverses the increased mortality associated with diabetes mellitus during tuberculosis treatment repurposing metformin to prevent and treat tuberculosis history of pulmonary tuberculosis affects the severity and clinical outcomes of copd latent tuberculosis infection: opportunities and challenges update on tuberculosis biomarkers: from correlates of risk, to correlates of active disease and of cure from disease isoniazid-rifapentine for latent tuberculosis infection: a systematic review and meta-analysis four months of rifampin or nine months of isoniazid for latent tuberculosis in adults latenttb-nstm study team. short-course regimens of rifapentine plus isoniazid to treat latent tuberculosis infection in older chinese: a randomised controlled study where is tuberculosis transmission happening? insights from the literature, new tools to study transmission and implications for the elimination of tuberculosis exposure to latent tuberculosis treatment during pregnancy: the prevent tb and the iadhere trials tuberculosis vaccines: opportunities and challenges phase b controlled trial of m /as e vaccine to prevent tuberculosis middle east respiratory syndrome key: cord- -zjr csla authors: hillman, john r.; baydoun, elias title: food security in an insecure future date: - - journal: water, energy & food sustainability in the middle east doi: . / - - - - _ sha: doc_id: cord_uid: zjr csla food security in the middle east is directly affected by a challenging combination of ongoing destructive conflicts, a global economic downturn, widespread poverty, high population growth, corruption, intolerance, and the potentially damaging consequences of climate change. many arab countries demonstrate nearly all the features of those countries classified as poor, less developed, or failing to achieve the eight millennium goals. even the economies of the richer oil-exporting countries in the region have been seriously damaged by the downturn in oil and gas prices as new sources come on stream elsewhere and demand falls as a result of renewable sources of energy becoming available. in a previous article , we considered definitions of food security in the modern era of rising global populations, discussing how food security might be attained in terms of security of water and fossil-fuel-derived energy supplies, climate change, rapid urbanisation, changing dietary trends, and modification of the natural environment leading to depleted natural resources, increasing environmental pollution, and the need to introduce modern technologies. the concepts of sustainable agriculture and uncertainty were also addressed, notably in respect of fresh thinking about key components of agricultural systems. these included (babu and blom ) vertical and horizontal integration of farming-related businesses to allow adequate capitalisation for enhanced efficiency measures; (bardshaw and brook ) policy shifts to remove market-distorting subsidies, tariffs, import and export bans, and excessive bureaucracy; improved crop and livestock breeding, including entirely new species; (bolukbasi et al. ) automation in agriculture and horticulture; (breisinger et al. ) protected cropping; (cong et al. ) new-generation agrochemicals; (elasha ) new agronomic practices; (fan et al. ) novel foodstuffs; (fao ) habitat reconstruction and land renovation; (fao ) biofuels and biodiesel; (garland et al. ) periurban and urban agriculture; (grebner et al. ) industrial biotechnology; (grivetti and ogle ) farming the seas and oceans; long-term carbon storage; and (hillman and baydoun ) new ways of thinking about carbon trading. more recently, we reviewed mitigation and adaptation processes and strategies to address the impacts of climate change on food, water, and energy security in the arab middle east (hillman and baydoun ) . here, we consider potential adaptations to an insecure global future generally, and to the concerns in the arab middle east specifically, in the light of the economic realities of wide disparities in wealth, competition for resources, and widespread poverty in many parts of the globe, coupled to a relatively high population growth, on-going conflicts, attempted cultural genocides, potential conflicts, endemic corruption and nepotism, and epidemics of infectious diseases. most arab countries are classified as poor, less developed, or failing to achieve the eight millennium goals of the united nations, and these arab countries share several undesirable features (table ) . even the much richer oil-exporting arab nations are under pressure. after a decade of relatively high oil prices, these nations have accumulated more $ . trillion in sovereign assets reinforced by substantial infrastructural investments supplemented by high levels of spending on imported military hardware. now, however, oil prices are under pressure as global oil and gas prices slumped in and remain depressed, possibly for the medium to long term as new sources of oil and gas come from hydraulic fracturing (fracking) and from iranian exports as the economic blockade on iran is being relaxed, coupled to greater energy efficiency in industry and new sources of renewable energy. this price depression has exposed the degree to which the economies of these arab oil exporters are dependent on oil and their failure in most instances to diversify their economies as their populations continue to expand alongside public expectations of continuing governmental largesse. nearly all of the immediate adaptations arab countries must undertake in order to adjust to a raft of severe insecurity issues require strategic planning and value-for-money infrastructural and civil-society improvements, and any preparatory changes in rural and urban areas will differ in scale and design. longer-term adaptations will be reliant on more stable conditions and a stepwise improvement of educational standards and attitudes. at the time of writing, no arab country is deemed to have acceptable levels of budget transparency according to the latest open budget index released by the international budget partnership (see www.internationalbudget.org). the future is especially insecure because of the persistence in the region of a combination of incompatible political and economic ideologies, religious and ethnic groupings overtly intolerant of others, introvert nationalism and disrespect of others, disconnection from democratic principles, profound cultural divides, ignorance -some wanton, inability to adapt to modernity, and malevolent community and national leaders. from a noble history of toleration, hospitality, and learning, arab society is fragmenting, defiled by the actions of relatively few. arabs are killing arabs either directly or indirectly; arabs are inflicting as-yetuntold horrific crimes on other arabs either directly or indirectly. attacks on arab countries by their neighbours might be used as an excuse to divert attention from their own failing donor-dependent economies or social structures, or most often to steal resources and ensure that the neighbouring country is suppressed from developing normally. at the global level, many would say that future conflicts and insecurity in much of the world are inevitable, simply because of the impacts of expanding global populations and the obvious competition for limited resources. the intensity of this competition must be analysed in context of the alarming table fourteen features of countries classified as poor, less developed, or failing to achieve the eight united nations millennium goals. the listed features are closely interrelated . poverty common in both the urban and especially the rural poor, poverty is sometimes concentrated in regions, often in marginalised ethnic or religious groups, and may relate primarily to girls and women. many of the urban poor operate in the unofficial economy. the rural poor tend to be land-constrained, dependent on rain-fed, low-yield subsistence agriculture with little or no access to modern technology (modern cultivars and livestock breeds, fertilisers, pesticides, automation, agronomy advice, veterinary support), and usually do not own their land. the rural poor encounter barriers to trade (e.g. transport, storage) and are unable to meet quality assurance standards. with no or limited access to social benefits (primarily pensions, child support, education, training, and healthcare), the poor may be hungry, thirsty, suffer ill health and low standards of accommodation, and die early. the poor are susceptible to exploitation. . hunger and thirst access to food and potable water may involve substantial travel on foot, and the basic requirements may only be met wholly or in part by humanitarian assistance. food quality and safety are usually low, and cooking is often dependent on wood for fuel. symptoms of malnutrition are prevalent. . disease slum dwellings, insanitary conditions, poverty, and hunger lead to a vulnerability to pandemics, made worse by poor or no public health provision. high maternal and child mortality and low general life expectancy characterise poverty and there is a reliance on traditional and/or herbal medicine. crops and livestock are subject to catastrophic attacks by pests and diseases. . poor environmental management poor countries suffer a depletion of their natural resources, including freshwater supplies and native flora and fauna. mineral and fossil-fuel resources are extracted to be exploited by industries in other more-developed countries. land, water, and the atmosphere may be polluted with few or no remediation efforts. national, regional, and international environmental regulations are not properly implemented. agricultural soils tend to be subject to erosion, salination, solarisation, desertification, and nutrient depletion. even without climate-change predictions, the general anthropogenic environmental degradation currently taking place increases the vulnerability to flooding, sand storms, ill health, and displacement of peoples. most poor countries are enduring adverse climate trends, and climate-change predictions point to even harsher conditions (erratic rainfall with rising temperatures, heat waves, hot extremes, and storms; rising sea levels and acidification of seas and oceans; increased desertification with effects on agriculture as well as the natural flora and fauna; socio-economic and health implications). . poor infrastructure quantity and quality of the built infrastructure tends to be low, or even absent (e.g. roads, ports, airports, telecommunications and access to the internet, hospitals and clinics, reliable power and fuel supplies, potable water sources, sanitation systems and sewage disposal, protected natural environments, cold and pest-free storage of agricultural and horticultural produce). the cost of living is worsened by relatively high overland transport costs. facilities may be inadequate to meet demand and maintenance neglected. rapid urbanisation and conflicts exacerbate infrastructural deficiencies. . corruption political autocracy coupled to a lack of transparency in government and public services, a weak judiciary, lack of consultation on major issues, and suppressed media enable a climate of corruption and nepotism to permeate all areas of society, including schools, colleges, and universities. a low regard for human rights, democratic processes, health and safety measures, and international law, is made worse by an overburdensome bureaucracy (staffed by poorly paid civil servants and police) dependent on bribery to function. corruption allows laws to be broken with impunity, and protests ignored. in most areas of society, a lack of altruistic leadership and (continued) poor countries manufacture few value-added products, offer little or no advanced training, and lack participation in the global knowledge economy. there is little or no foreign direct investment and free trade is constrained. financial assistance from donor countries is increasingly being audited to ensure compliance with attempts to prevent unauthorised expenditure. many poor countries have not managed to have constructive relationships with potential donor countries, and some try to align themselves with one of the main international political power blocs. unrelieved debt burdens have led to high interest rates on loans and difficulty of obtaining credit. private savings are minimal and the country may be subject to periodic flights of capital. their economies are grossly imbalanced with little spent on healthcare, education, and other social benefits compared with defence and vanity projects. the economy may be damaged by previous and/or ongoing conflicts, and may in any case have limited absorptive capacity properly to manage additional resource inflows and outflows. agriculture, unofficial transactions, and remittances from those working abroad contribute disproportionately to the real economy. . poor education limited or no access to free schooling accounts for a general low level of literacy and numeracy, especially of females. this is reflected in the absence of high-grade internationally competitive universities, colleges, and research institutes, despite a high level of parental financial sacrifice to secure a supposedly good education for their children. science and technology tend to be poorly taught and there is undue influence of religions with regressive attitudes to modernity. a lack of investment in research and development (r&d) and a lack of a critical mass of scientists, engineers, and technologists impede industrial development, and prevent those that remain from joining international consortia, participate in learned societies, access essential literature and training programmes, and have the opportunity to use state-of-the-art instrumentation, software, and laboratory consumables. the best educated and the most talented and entrepreneurial usually emigrate, leading to a brain drain. intellectual property rights are often ignored and little benefit is derived from traditional knowledge and its products tend to be exploited in other countries. crucial demands and needs of less-developed countries are rarely the targets of major international r&d projects. . gender inequality low educational attainment in most girls and women is reflected in female political, economic, and social representation and participation failing to match their proportion of the general population. cultural and religious influences often lead to females being regarded as inferior to males. family planning is limited, and in the absence of social security and reasonable incomes, large families are the norm. . high population growth this is a feature of the poorest social groups, and correlates with low life expectancy and gender inequality. in some countries high population growth may exceed the economic capacity of the country to feed itself, leading to a propensity to generate refugees, displaced persons, and terrorists. . vulnerability to transnational terrorism terrorism often relates to a combination of one or more of the following: poverty, hunger, poor educational attainment, disconnection from democratic principles, susceptibility to indoctrination by intolerant religious ideologies, criminal activities, and psychiatric disorders. poor countries are usually unable to defend themselves from terrorists, and groups of terrorists may receive covert support from other countries and agencies. poor countries may form the battlefield for fighting between different groups of terrorists. recovery from terrorism and conflicts in general may take several generations, and lead to psychological and physiological after-effects in the survivors and their progeny. projected changes and options for adaptation as well as mitigation (curbing emissions) detailed in the latest fifth assessment report of the international panel on climate change (ipcc, www.ipcc.ch/). the immediacy of food security during social instability in the arab region forms the backdrop to this article, rather than the longer-term infrastructural and social transformations needed to mitigate and adapt to climate-changing emissions, transformations that demand political stability and sophistication. the global population is estimated to be around . billion at present and there is an % probability that it will increase to between . billion and . billion in (garland et al. ) , so stabilisation of the population is highly unlikely this century. moreover, human population reduction is not a quick fix for environmental problems, and even a catastrophic event that killed billions of people would have relatively little effect on the overall impact of humans on the environment . weak public sector low salaries, complex and inefficient bureaucratic processes, poor educational attainment, incompetence, widespread acceptance of corruption through bribery and political interference, and the lack of an investigative free media, account for the justifiable lack of confidence in the public sector. a low regard for human rights, legal processes, and justice undermines societal advancement. there is also poor custodianship of cultural heritage and essential infrastructure. . neighbouring countries many poor countries have reliance on and vulnerability to neighbouring countries for access to water, transport and communication networks, foodstuffs, energy, control of environmental issues (e.g. desertification, flooding, biodiversity protection, pollution etc.), and security. neighbouring countries may (a) create security problems by aiding terrorists and insurgents, (b) provide an uncontrolled source of refugees, (c) invade, (d) steal water or other natural resources, and/or (e) issue mendacious media releases or operate diplomatically to undermine the confidence of donor countries, aid agencies, and investors. the more-developed countries rarely understand the sheer difficulty of managing a poor country facing inter-ethnic conflict, and terrorism and poverty. . international agencies, non-government organisations, international media and the united nations poor countries are monitored by a plethora of international and national bodies, and extensive reports generated, but the necessary actions -political and military -to solve the main issues and problems are rarely carried out. unwarranted aggression inflicted on other countries -poor or not, or the suppression of their populations or specific parts of their populations by nation states eventually must be counteracted by military intervention. sadly, poor nations are oftentimes regarded as pawns in international power struggles, and remain either exploited for their resources, or ignored. moreover, economic downturns in donor countries reduce the level of aid and absorption of refugees, made worse by certain countries cynically failing to meet their initially well-publicised aid pledges. (bardshaw and brook ) . population increases could undermine attempts to ameliorate attempts to reduce climate-modifying emissions. climate-change predictions for the arab region are deeply concerning. most scientists support the conclusions of the latest fifth assessment report of the ipcc. formed in by the united nations environment programme (unep) and the world meteorological organization (wmo), and relating closely to the un framework convention on climate change -the main multilateral forum for addressing climate change, ar comprises three working group reports and a synthesis report with its summary for policymakers. the main issues are covered in detail: observed changes and their causes; future climate changes and their risks and impacts; future pathways for adaptation, mitigation and sustainable development; and a more detailed analysis of adaptation and mitigation including policy options, technology and finance. in the arab human development report authored by balgis osman elasha, ( ), the impacts of climate-change projections in the arab region are given in stark detail. in the coming decades, arab societies and their industries will be profoundly and adversely affected by projected temperature increases in excess of c and severely reduced rainfall, threats of increasing frequencies of impacts originating from el nino events, changes in the seasonal distribution and predictability of rainfall, depleted aquifers, reduced river flows, rising sea levels, flash floods, in addition to increased numbers of dust storms and hurricanes. as the arab region itself is a relatively small direct contributor to global greenhouse-gas emissions, although its fossil-fuel exports and its importation of goods that took energy to create are substantial contributors to them, adaptation must be a crucial factor in policy developments in more settled times that will themselves be dependent on stable and peaceful arab countries. questions arise as to whether organisations such as those overseen by the united nations, multilateral groups such as the european union, individual nations and several charities will be capable of initiating and then maintaining peaceful conditions and food security. food and other forms of aid are likely to face increasing demands at a time when the economies of many donor countries are enduring continuing austerity and recessionary conditions, and when there is growing but unjustified cynicism about the effectiveness of these aid organisations. irrespective of the many estimates that total global food production can readily meet the needs of the present global population, political reality is that all countries and people are patently not equal and are unlikely to be so for the foreseeable future. aid can assist in partially rebalancing the inequalities, not least where several countries in the arab region are currently in turmoil and others are deeply troubled. some regard most of the arab world as regressing, out of synchrony with, and lacking sympathy from, the world at large. food costs and poverty are primary concerns, exacerbated by insecurity of energy and water supplies as well as rampaging insurgents and those wishing to impose unacceptable regimes and suppression of minorities, denying their citizens proper democratic freedoms. wars and conflicts are all too easily incited in the absence of strong democratic civil society involvement and usually quickly bankrupt countries; destroy nearly all parts of their economies; ruin infrastructure including homes, businesses, transport and communication networks; disrupt family life and social interactions; generate displaced people and refugees; and attract foreign interference, including active participants. war crimes are commonplace. psychological aftereffects are noted in civilians and combatants long after fighting has died down. populations in the arab middle east have endured asymmetric warfare between countries with vastly different military capabilities, provoking guerrilla tactics, chemical warfare, civil war, and even unconventional warfare through acquiescence, capitulation, and clandestine support for long-term insurgencies. wherever they occur, warfare and conflicts are always associated with the participants having distorted views of history -some drawing on ancient history -with widely diverse concepts of ethics, and complicated by ethnic and profound religious differences. little consensus exists on triggering factors, theories, and outcomes. prodigious sums of money have been expended in the arab middle east on armaments and defence forces to the exclusion of adequate investments in social welfare, education, and research and development. political instability for whatever reason usually leads to food insecurity and conflict. during social disturbances and the onset of widespread conflict, the normal mechanisms underpinning food production, importation, storage, processing, transport and retailing are profoundly disrupted, creating conditions that promote the formation of ghettos, and further stimulate corruption, robbery, and the black market. normal policing and social order collapse, social behaviour degrades, and criminal activities dominate. as the economy collapses, the terms of trade are transformed. disease epidemics become manifest. the restoration of domestic and business normality takes years and may never be achieved within one or more generations. food insecurity itself may lead to political instability, not least in a world of global intercommunications when citizens of a poor country or region can view with understandable envy the lifestyles of those in rich countries or communities. it is therefore a basic duty of political and community leaders to ensure that food security is a foremost priority for those people within their sphere of responsibility. insecurity of food supplies can be created by adverse weather conditions; the depredations of pests, weeds, and diseases; and salt contamination that can be caused by poor irrigation and agronomic practices. around million hectares of hitherto fertile land on earth have been damaged by salt. according to economics of salt-induced land degradation and restoration (qadir et al. ) , only tree planting, deep ploughing, and growing salt-tolerant crops coupled to digging drains and dykes around the affected area can address the problem. neglecting the health of africa's soils, many of which suffer almost irreversible degradation and nutrient deficiency, will lock the continent into cycles of food insecurity for generations to come, according to the montpellier panel report. indeed, was designated the 'international year of soils' by the th un general assembly. since the s, there are particular issues relating to selection pressures on destructive pests, weeds, and diseases in the vast monocultural single-cultivar agricultural systems that are also the present-day main sources of global food aid. many of these main producing areas are experiencing irregular rainfall patterns and failing irrigation arrangements. crop failures in these areas have quick knock-on effects on volatility of the global agricultural commodity markets; this is a situation likely to get worse as the population inexorably increases and demands more food. moreover, the world has yet to experience the sort of dramatic harvest failures that occurred in the s and before. another aspect of the agriculture sector (generally accepted to include crops, livestock, fishing, and forestry) in the developing world is that it absorbs circa % of the economic impacts caused by medium-and large-scale natural hazards and disasters. between and , these events in developing countries affected more than . billion people and caused more than $ billion, but agriculture only received about . % of all humanitarian aid (fao www.fao.org/emergencies/how-we-work/resilience/en/). delegates from fao at the conference announced the launch of a facility that will focus on bringing together technical expertise and financial resources with the aim of building greater resilience of agriculture to natural extreme weather events. the combination of advanced crop and livestock breeding, agrochemicals, automation, better agronomy and livestock husbandry, increases in the land area farmed, and efficient larger-scale better-capitalised production units have collectively prevented malthusian disasters. since , food production has more than quadrupled, using less than % more cultivated land, allowing civilisation to proceed and expand. food security is no longer an issue in many countries, and the global economy, human health, and societal development have been, for the most part, positively influenced by agricultural advancement. in the period - , when the global population increased from around . billion to around billion, average agricultural commodity prices decreased by an average of . % p.a. because supplies rose faster than demand (unpublished presentation by prof. ingo pies in to biennial development meeting of pottinger). nonetheless, feeding any substantial increases in the global population will only be possible by technological innovations because of the ecological limitations on increased water and fertilizer supplies and increasing the area farmed. likewise, various climatechange predictions amplify justifiable concerns about global agricultural productivity. supply and demand market dynamics are complex and ultimately resilient to political interference, although many countries and trading blocs try to manipulate production by tariffs, export bans, subsidies, inhibiting technological developments and market processes. the spikes in prices of traded wheat, rice, maize, and soya in and were initially blamed on speculation, especially the index tracking funds and derivatives markets. yet this type of speculation does not trade physical goods but price risks, and is therefore a form of insurance market. in addition, speculation would be expected to be associated with high stocks as farmers opt for storage rather than sales. in and , however, stocks were very low and caused the price rises. even so, government policy failures, including protectionist export bans and inadequate promotion of agricultural efficiency, contributed to panic buying, as exporters reduced their offer and importers increased their demand in response. calls by civil society groups to ban speculation by index-tracking funds and derivatives markets were always and continue to be profoundly misguided. all countries should have policies to sustain and constantly review food production and supply, especially if there is a significant dependence on food imports. reasonably substantial reserve food stocks are essential cushions to prevent price bubbles and food shortages, but excessive stocks can distort markets such as when they are released in large quantities as general food aid and undermine the operation of normal agriculture markets in developing countries. some price volatility is an essential component of healthy competitive markets, driving adaptation, risk taking and innovation. debates about the environmental costs of different kinds of agriculture, not least in terms of water and energy security in respect of the arab middle east, and in terms of the destruction of natural habitats and loss of biodiversity, as well as cultural and other changes, have stimulated possible strategies to address these concerns. one approach is for each country to have a "roadmap" for its agricultural development, and these roadmaps might be aggregated into a regional roadmap. the us report: a science roadmap for agriculture -cited as task force on building a science roadmap for agriculture, national association of state universities and land-grant colleges, experiment station committee on organisation and policy, "a science roadmap for the future", november (www.nasulgc.org/comm_food. htm) http://agsci.oregonstate.edu/files/main/roadmap .pdf pioneered a way to define the needs of agriculture and help shape the future direction of the various strands of agriculturally relevant science. this impressive us-specific study followed a conceptual framework of needs to (a) be competitive in a global economy; (b) add value in future harvests; (c) adjust agriculture to a changing climate; (d) be good stewards of the natural environment and natural resources; (e) make agricultural enterprises profitable; (f) make families and communities strong; and (g) modify foods for improved health and safety. one important relevant outcome of this work is the obvious requirement to grow crops and practice livestock husbandry in the most appropriate environments, enabling different environmental zones to have a critical mass of expertise and facilities. for the water-constrained arab middle east, this would mean increased reliance on food imports that could also be regarded as a form of water importation. but this would be possible only if there were formal agreements between food-producing and food-recipient countries, and these agreements were economically and politically stress-resistant. to this caveat would be questions of how to pay for imports, and acquiring resources required to redirect the agricultural workforce into other wealth-creating activities. introduction of a logical, science-led agricultural roadmap in the region may be impossible at present but needs to be initiated. on a global level, food commodity and non-food agricultural commodities prices have declined by - % in the period mid-september to mid-september (see the economist commodity-price index ( ) in www.economist.com/indicators), reflecting relatively clement weather conditions in most of the producing areas, greater production efficiency, balance sheets strong enough to bear losses, competition to gain market share, and continuing investments. importing countries, however, are affected by the strength of their currencies amongst other factors, such as social upheaval. unlike other commodities whose prices direct reflect industrial demand, prices of foodstuffs reflect the effects of weather, pests and diseases, the demands of a rising global population, and many kinds cannot be readily stockpiled. a further factor operating at the global level is the domination of farm supplies by six international companies: monsanto (the largest seed producer), syngenta (the largest agrochemical producer), bayer, basf, dow chemical, and du pont). all of these companies have been active in acquiring other companies and patents, thus reducing competition, and potentially reducing innovation as they robustly defend their intellectual property. in this era of low commodity prices and developing resistance to older-type herbicides, farmers are constrained by input costs of fertilisers, seeds, agrochemicals and veterinary medicines that have steadily risen in over the past decade, only partially alleviated by the recent reduction in fuel and lubricant costs. pandemics -epidemics of infectious or contagious disease that have spread through populations across a large region, crossing international boundariesdrastically curtail food production and distribution, aggravating poverty in both the rural and urban poor. pandemic-causing diseases include the ever-present cholera, influenza such as the and h n outbreaks, typhus, smallpox, measles, tuberculosis, plague (yersinia pestis), leprosy, malaria, human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids), viral haemorrhagic fevers (ebola, marburg, crimean-congo, lassa, rift valley, dengue, yellow fever, etc.), and now there are new diseases such as severe acute respiratory syndrome (sars). vaccine development is necessarily slow, and treatment of bacterial diseases is hampered by the rapid development of antibiotic resistance. the propensity of diseases to mutate, acquire new vectoring capabilities, have reservoirs in wild animals, and even persist in spore form, mean that there must be constant vigilance. as one of the major global food-producing bloc of nations, and as one of the major food importers and donors of food and other forms of humanitarian aid, the european union (eu) bears crucial responsibility for the deleterious effects of its complex and highly bureaucratic common agricultural policy. its massive subsidy regimes impact adversely on global markets and its well-meaning but often poorly thought-through environmental regulatory decisions are not based on sound scientific evidence. so-called "greening" policies are being introduced that may be deemed to enhance the environment but are likely to decrease profitable production. social measures to support small-scale inefficient producers also distort the global marketplace. likewise, the series of restrictions being introduced on the use of a wide range of agrochemicals within the eu and for imported commodities, without carrying out proper impact assessments and fast-tracking alternatives, imperil production. in the medium to long term, a more serious issue is the virtual ban in genetically modified (gm)) crops, inhibiting their uptake in countries intending to export to the eu as well as suppressing state-of-the-art research and development and associated investments in eu countries. of particular relevance in this regard is the recent and largest statistically rigorous review of the agronomic and economic effects of the current range of commercially available gm crops on farming (klümper and qaim ) . in examining publications between and march , it is therefore a near-complete survey. in essence, the two main types of gm crop -resistance to insect pests and tolerance to the wide-spectrum weedkiller glyphosate -conferred considerable yield improvements and much higher profits than conventional crops. gm crops and related products in the development pipeline were not considered, and these promise great advances in nutrition enhancement, environmental clean-up, new medicines, new products for manufacturing industries, and improved crop and forest species. moreover, existing gm crops have greater impacts in poorer countries than in richer countries because their insect pests and pernicious weeds are more difficult to control. by including non-peer-reviewed papers (book chapters, working papers, conference papers etc.) as well as peer-reviewed papers for the meta-analysis, it was possible both to correct for academic bias in focusing just on the most dramatic effects, and include data for many ancillary effects, such as the effects of fertilisers. the eu ban on gm crops is therefore denying poor exporting countries from reaping the full benefits in yield, profitability, and commodity quality, in addition to reducing potential eu food-aid exports. one positive feature of the eu is the bureaucratic system to improve and monitor the quality and safety of foodstuffs, from raw ingredients through to ready-meals, and gm crops have been found to be safe. that for many years most of the feed protein in the eu comprises imported gm maize and soya bean, the issue of gm crops should not be ignored for much longer. opposition to the processes of modern genetic modification and ownership of the processes (often deeming them to be "unnatural"), and disregarding the quality, safety, and valuefor-money of the product, actually condemns conventional agricultural practice, and demonstrates ignorance of naturally occurring mutations and horizontal gene transfer. even so, in early november , the newly elected president of the european commission, j-p juncker, in what is widely regarded as a blatant act of appeasement to greenpeace and other so-called "environment" pressure groups, sacked the eu's chief scientific advisor, professor anne glover, for her support of gm technology, compounding this regressive stance with abolition of the post. all countries and trading blocs should have influential and competent teams of chief scientific advisors. research and development in gene identification, construction, insertion, editing, and expression, coupled to high-throughput phenotyping are collectively revolutionising agricultural, horticultural, and forestry sciences. gm technology is not simply the insertion of genes using various technologies from a similar or different species into a recipient organism. it includes the concept of gene silencing -the prevention the reduction of expression of certain genes -a process that can take place at either the transcription or translation cellular processes. it is not the equivalent of gene knockout but is essentially gene knockdown because the methods to silence genes do not completely eliminate the expression of a specified gene. the methods to silence genes include rna interference (rnai or posttranscriptional gene silencing), small interfering double-stranded rna (sirna), and crispr. of special interest is the crispr (clustered, regularly interspaced, short palindromic repeats) toolkit that is derived from research on prokaryotic antiviral systems and currently involving the cas and cpr endonucleases (jinek et al. ; cong et al. ; bolukbasi et al. ) . as viruses constantly evolve to escape from these antiviral systems, bacteria probably evolve new systems. crispr technology is able to recruit heterologous domains that can regulate endogenous gene expression as well as label specific genomic loci in cells, so that is feasible to engineer germ lines and thus the path of evolution. this technique is replacing methods using mutagenic agents, virus vectors, zinc-finger nucleases and transcription activator-like effector nucleases (talens). its relative simplicity and evolutionary significance for all life forms, including humans, means that internationally agreed regulatory frameworks are essential. the technology does not involve implanting genes from one organism into another, and is not therefore creating transgenic organisms; it is gene editing. there is now realistic expectation of new perennial cereals; incorporation of c- photosynthetic characteristics in existing c- crops; enhancement of nitrogen fixation by free-living soil microorganisms in the vicinity of crop roots; tolerance and resistance to biotic and abiotic stresses; and modification of lignification, texture and endogenous components (such as vitamin content, acrylamide in potatoes, antinutritionals, toxins, proteins, oils, and carbohydrates) of a wide range of existing and potential crop species (see www.isaaa.org/kc/cropbiotechupdate). besides the present-day generation of improved livestock species and new forms of husbandry, the use of balanced diets based on competitively priced synthetic amino-acid and fatty-acid products will lessen the need for large-scale soya and maize production. ancillary advances are taking place in mechanisation; diagnostics; predictive modelling and decision-support systems; remote sensing; protectedcropping systems; and weed, pest, and disease control. diminution of abiotic and biotic stresses in the field and under protective cropping is now the focus of major research initiatives. of the circa , species of angiosperms, according to the food and agriculture organisation of the united nations (fao ) , only species provide % of human energy needs and only four species (rice, wheat, maize, and potato) account for % of energy intake, and % of crop diversity was lost in the last century. around % of human calorie intake comes from crop species (grivetti and ogle ) and % comes from just three grasses -wheat, maize and rice (see www.knowledgebank.irri.org/ericeproduction/importanceofrice.htm) dependency on such a narrow genetic base is a threat to food security and is only partially alleviated by investments in in situ and ex situ plant gene/seed banks, germplasm collections, and dna libraries. just species of angiosperms and gymnosperms have been cultivated for human consumption in human history, with around , angiosperm species yet to be discovered. in theory, most angiosperms should be capable of being biotechnologically modified for food and non-food uses. will scientists in the arab middle east fully participate in these exciting developments? valuable collections in the arab region are inadequately respected for their worth under peaceful conditions but are now extremely vulnerable during this period of war-like conditions and enduring financial pressures. coming into force in , the international treaty on plant genetic resources for food and agriculture (international seed treaty) was designed to complement the convention on biological diversity (cbd) and was designed to guarantee food security by (a) conservation, exchange, and sustainable use of all types of plant genetic resources; (b) offering fair and equitable benefit-sharing; and (c) recognition of farmers' rights. critics of the international seed treaty point to great variability across countries of access to collections and interpretation and implementation of farmers' rights. moreover, in adopting the cbd's outlawing of biopiracy -the uncompensated commercialisation and profiteering of seeds, propagules, growing plants, and their products from source areas -has severely inhibited the acquisition and exchange arrangements in collections until better processes come into force. gap analyses are methods to identify gaps in ex situ collections of wild-plant relatives of agriculturally relevant species as a means to guide efficient and effective collection strategies (villegas et al. ) . a gap analysis by the international center for tropical agriculture managed by the global crop diversity trust and the millennium seed bank in kew examined priority gene pools of the globally most important food crops and their wild relatives. most at risk were eggplant, potato, apple, sunflower, carrot, sorghum, and finger millet (see www.cwrdiversity. org/conservation-gaps/). ongoing conflicts and disorder in the arab region justify an independent review of a regional red list index based on the list of threatened species released by the international union for the conservation of nature (www. iucn.org) in order to evaluate the extinction risk of species and subspecies of the natural flora and fauna. certain individual countries in the eu are important donors of humanitarian aid in their own right. according to a recent report (november , ) from the organisation for economic co-operation and development, economic stagnation especially in the eurozone portion of the eu poses a major risk to world growth. if the stagnation were to continue or even get worse, humanitarian assistance would inevitably be reduced, and countries that hitherto were willing donors would become increasingly introverted. as evidenced by growing problems of graft, corruption and authoritarian government in certain (but not all) members of the eu that were formerly dictatorships or in the sphere of influence of the former soviet union, democratic norms (human rights, respect for minorities, tolerance, free press, independent judiciary and rule of law, active civil-society groups, transparency of accounting for taxpayers' money etc.) take time to become bedded into the fabric of society. in considering the medium-to-long-term future of the eu, it is sobering to note that throughout european history, confederations between its diverse nations and subsets have rarely persisted unless full political, legal, monetary, and more profoundly, cultural fusion had taken place. all governments worthy of the title must ensure that there are relief mechanisms to enable the provision of basic food supplies and fresh water together with functioning standby electricity-generating equipment in unsettled times caused by natural or man-made disasters. surely governments have the ultimate responsibility to attend to the needs of their people and not themselves. rarely observed, governments need genuine food, nutrition and agricultural experts as an integral part of the decision-making hierarchy. such experts must have a proper understanding of the pre-conflict or pre-disaster food, fresh-water, and energy supplies and their distribution systems, and how they can be safeguarded, modified and employed to proper effect, and how alternative mechanisms can be deployed. sadly, this aspect seems to be neglected at the present time in the chaotic condition of certain countries in the middle east. much can be learned from countries in europe during the wars that raged in the nineteenth and twentieth centuries. simply standing by and watching the population adapt slowly to acquiring barely adequate water and food supplies inflicts untold misery on innocent people. all governments should have readily accessible emergency supplies (reserve stocks) and transport systems, and be willing to introduce rationing if need be. special protection measures are needed for water supplies and farms to make sure production can continue no matter the degree of impairment. in more settled times, each government should establish a group of experts to construct interactive databases as the foundation of an agriinformatics and metrics organisation. this would collate information on supply and demand changes, supply-chain details, imports, crop and livestock genetics, commodity production levels, labour-force composition, pricing, inputs, availability of decision-support systems, advisors and research bodies, grant funding, biotic and abiotic stress factors, natural resource constraints, predictive modelling of shocks to the agricultural system and disruptive events, etc. other research organisations would interact with this organisation to ensure best practice and enhance agricultural resilience, demonstrate efficient use of inputs, exploit wastes, optimise the use of mechanisation, and foster skills. arab countries still have to utilise fully the international capabilities and potential of (international centre for agricultural research in the dry areas -currently based in beirut given the conflicts in syria) and other members of the cgiar consortium (formerly the consultative group on international agricultural research). the plethora of aid agencies offering humanitarian and development aid encompass those that are organised by a single government, multilateral donors, non-governmental organisations, philanthropic and charitable organisations, businesses, and individuals. reliefweb (www.reliefweb.int) provides a relatively comprehensive directory of humanitarian organisations. fragmentation of the total aid effort is becoming a worrisome issue. the international committee of the red cross, part of the international red cross and red crescent movement along with international federation of red cross and red crescent societies and national societies, is mandated internationally to uphold the four treaties and three additional protocols of the geneva conventions. these conventions are rules that apply in times of armed conflict both within and between countries, and define the rights of civil and military prisoners and protections for wounded people and for civilians. weapons of war are dealt with by the hague conventions and the biochemical warfare geneva protocol. enforcement of the conventions is through the un security council but is rarely invoked, primarily because of profound ideological differences about democracy and human rights between the five permanent members of the security council with veto powers, so there tends to be diplomatic reliance on regional treaties and national laws. parenthetically, there are ten non-permanent members of the security council without veto powers that are elected by the general assembly of the united nations for a two-year period. on november , the un security council pledged to counter the global terrorist threat and increase cooperation to address the perils posed by foreign terrorist fighters such as those that are a notable feature of conflicts in the arab region. the un office on drugs and crime is also involved in this initiative. other international related treaties include the united nations multilateral treaty referred to as the geneva protocol or convention relating to the status of refugees as well as the declaration on the protection of women and children in emergency and armed conflict adopted by the united nations in . humanitarian aid is distinguished from humanitarian intervention, which involves armed forces protecting civilians from violence or genocide. the united nations office for the coordination of humanitarian affairs is mandated to coordinate humanitarian responses, usually in concert with the international committee of the red cross. valuable reference material can be found in (a) grebner et al. ( ) , the state of hunger in developing countries as a group has improved by % since . even so, the level of hunger is still serious with an estimate of million people continuing to go hungry. the highest levels are south of the sahara and south asia. in the ifpri global nutrition report , evidence is summarised to show that improvements in nutrition status will make large contributions to sustainable development goals, namely poverty, food, health, education, gender, and employment investment in nutrition has a highest benefit ratio. projections from the world health organization (who) and unicef demonstrate that the world is not on track to meet any of the six world health assembly (wha) nutrition targets (reducing child stunting, reducing anaemia in women of reproductive age, reducing low birth weight, reducing the number of overweight children, increasing exclusive breast feeding, and reducing child wasting), although many countries are making good progress in meeting nutrition outcomes. the manifestation of malnutrition is changing as countries are now facing complex, overlapping, and connected malnutrition burdens. three of the chapters in the ifpri publication resilience for food and nutrition security (fan et al. ) are germane to this article. breisinger et al. ( ) briefly mention the arab spring and uses egypt, somalia, sudan, and yemen as case studies of conflict-affected countries. mabiso et al. ( ) have specific reference to the syrian refugee crisis, and take a global overview of the complex relationships between refugees and host countries. babu and blom ( ) introduce a model that seeks to delineate the key capacity components of a resilient food system, considering a country's capacity to create, manage, and utilise human resources for a resilient food system. significant challenges to aid provision include (a) harnessing the necessary stream of funding when grandstanding promises by countries are often never met; (b) establishing and coordinating the basic support network; (c) ensuring the logistics arrangements are effective, including communication networks; (d) prevention of resource misappropriation; (e) protection for officials and support workers on the ground; (f) protecting the vulnerable people needing aid; (g) operating with transparency and integrity; and (h) laying the structural and procedural foundations for self-reliance. effective lines of communication with donors and international agencies and charities are pivotal so that emergency arrangements can be established without delay and hindrance. these bodies need to deal with those individuals in the recipient countries truly knowledgeable about the capacity and specific problems facing food and water security, and fast-moving internal developments. the experts in the recipient countries must have the authority to be able to (a) quantify the levels of demand, (b) direct supplies, (c) recommend the siting of depots and distribution centres, and (d) highlight points of accessibility and vulnerability. in poorly governed countries, experts must be prepared to deal directly with these donors, agencies, and charities, difficult as that might be. the complexities of globalisation extend beyond food and water security (lerche ) . when people are deliberately persecuted, and honest law enforcement collapses, then non-partisan protection must be afforded, usually with outside security forces. unfortunately, ideological differences mean that the international community has often been shown to be ineffective in bringing about rapid termination of conflicts by imposing observers or armed forces, although thanks to relatively few major international donors, humanitarian relief has been forthcoming, albeit frequently late and inadequately funded. dealing with refugees and displaced people requires expertise and sympathetic support. housing provision together with monitoring and combatting infectious diseases, usually run in parallel with the issuance of food supplies. governments that prepare for worst-case scenarios are to be commended. even the distribution of authoritative guidance for populations in stress would represent a small step in the right direction, as would reinforcing the institutions that bind civil society, such as voluntary rescue and care organisations. networks of low-temperature clean and secure depots with associated distribution centres should be set up at the outset of disasters and conflicts. even in peaceful times, a marked cut in food waste helps food security. according to m. m. rutten (rutten ) around a third of the food for humans produced annually (about . billion tonnes) is either lost or wasted, and in developing economies the situation tends to be worse, with in excess of % lost during harvesting, processing and storage (fao. . global food losses and food waste -extent, causes and prevention (see www.go. nature.com/um vga). basic needs of refugees, as recommended by the unhcr and related organisations, are modest but are directly applicable to those displaced or besieged in their own country. unhcr recommends each refugee receive more than calories per day, recognising that a lack of food variety and inadequate supply of fruit and vegetables lead to deficiencies in essential vitamins and minerals. calorific intake can be reduced if the provided foodstuffs do not conform to traditional diets, or if the rations are traded to acquire other non-food goods and services. encouragement is needed to set up temporary gardens. fresh-water provision is of primary importance, with a minimum of litres per person. a greater volume is needed, though, to prevent public-health problems of diarrhoea, cholera, and even polio. thus, clean-water sources and pumps are required along with taps within walking distance. vessels are needed for transfer and storage of water. water-purification tablets should be provided. sanitation systems are essential for hand washing and the safe disposal of urine, faeces, sanitary towels, wound dressings, infected and contaminated materials, and for the disposal of dead bodies. monitoring of faecal contamination is recommended. housing refugees and displaced people at short notice demands special expertise to avoid overcrowding and give adequate protection against inclement conditions. overlaying the fundamental needs for food, water, and shelter are meeting basic medical needs, particularly of the young, women, and the old and frail. in addition, within a short time, children require to be educated. host communities and host countries sometimes resist integration of forcibly deracinated people and can grow resentful at the costs incurred, especially if the host economy is weak. most financial assistance from donor countries is given to aid agencies rather than host countries. large-scale influxes of refugees can soon overwhelm the host country's infrastructural resources (chiefly fresh water, energy, housing, hospitals and healthcare systems, education, and waste disposal). other problems arise from combatants embedded in refugee cohorts, spreading the conflict and increasing policing costs. cultural incompatibilities between refugees and the host population create hostilities. refugees can suffer the dire consequences of being rendered stateless. in general, it is fair to say that humanitarian care is not able to sustain basic needs in the medium to long term. as a consequence of a funding crisis for humanitarian aid in the arab middle east, the world food programme was forced to suspend its desperately needed food-aid-voucher scheme for more than . million syrian refugees at the beginning of december , the onset of winter. this suspension meant that refugees were less welcome in host countries and border closures are already being implemented in the immediate area as well as in the european union. axiomatically, just as responsible governments must be alert to and prepared for civil and other forms of unrest, they should always promote food production and remove any impediments to the uptake of improved technologies so that their economies have inbuilt resilience to dreadful events. likewise, governments should have in their ranks, or instantly available for consultation, competent scientists, technologists, and engineers able to advise on food, water, and energy resource distribution and allocation. over the past few decades, public-sector agricultural research and development in virtually all countries have suffered financial reductions and financial resources have been switched to activities regarded as more exciting and with greater wealth-creating potential; history shows this to be monumentally misguided. the urban disregard for agriculture is likely to continue as urbanisation increases, until the point food security threatens social stability. active or benign neglect of food-producing, food-processing, and food-distribution industries as well as of the scientists, technologists, and engineers underpinning its productivity, improvement and efficiency reveal incompetent governance. as an aside, the dearth of scientists, technologists, and engineers in active politics accounts for numerous policy failures. graduates in the arts (such as history and politics) and social sciences dominate politics and the upper echelons of the machinery of government (civil service) worldwide, people with little understanding or appreciation of business let alone of the "hard" sciences and engineering and their essential utility (and limitations) for mankind. perhaps this explains the growing dissatisfaction with the prevailing political classes. the scientific approach is that of the quest for knowledge by constantly questioning, developing and testing hypotheses by experimentation so that opinions change as "facts" change, oftentimes undermining policies that are not evidence-based, whereas many political parties are founded on inflexible belief systems, as are almost all religions. one aspect of food security in times of conflict and community disharmony has been the remarkable resilience of researchers to continue their studies or just maintain libraries, databases, records, laboratories, and genetic resources under the most trying conditions. the pursuit of knowledge is a fundamental feature of humans, as is the search for improvement. when there is blatant disregard of national constitutions as well as united nations treaties, protocols and conventions, and universities, colleges, schools, and research institutes become targets of malevolent forces, then the rest of the world must have no other option than to intervene, regardless of diplomatic niceties, in order to restore at least the vestiges of societal normality. as a first step, food security and the provision of fresh water for the besieged people must be a priority. if and when particularly large, heavily populated countries become embroiled in conflicts and/or major natural disasters, the existing international support efforts are likely to fail. this, in turn, may lead to a series of related conflicts, as opposing ideological pressures culminate in outright wars, invasions, and suffering on a huge scale. throughout the world, history has shown that unless they are relatively rich (and that may not be enough), smaller or militarily weak larger countries are influenced, for good or ill, by their more powerful and sometimes aggressive neighbours. as recent events demonstrate, conflicts in smaller countries rarely bring about rapid corrective measures from the international community, and adverse and damaging propaganda actively promoted in donor countries can prolong the suffering. ultimately though, food and fresh-water security are a prerequisite and eventually underpin stability, peaceful and thriving economies. today, much of the arab world is poorly governed and insecure for its citizens; they urgently deserve a better life. many of the most talented arabs seek a better life elsewhere. the warfare must be ended forthwith. grossly and unfairly misunderstood by much of the rest of the world, arabs demonstrate admirable resilience and stoicism yet retain their sense of humour tempered by understandable cynicism and justifiable suspicion of conspiracies. enemies of the arabs subject them to a tirade of insults and demeaning innuendos, often designed to deny them basic rights and international support. nonetheless, arabs must not be the continuing authors of their own misfortune, and a first step would be an end to internal conflicts followed by an effective region-wide clampdown on corruption at all levels. remember -it is the victor who determines the writing and shape of history. if the level of insecurity in the region were to get worse, then not only the arabs but also the rest of the world would pay a high price, so it is in everybody's interest to help restore peace. bluntly, the solution to their problems lies in the actions of the arabs themselves. in fully grasping the opportunities available through top-quality education , high standards of integrity and tolerance can be demanded from those in leadership roles in communities, organisations, businesses, and local and national government. ignorance can be reduced, even if not eliminated. essential components of democracy can be established, including independent and diverse news media, an autonomous judiciary operating to high standards of justice and unaffected by pressure groups and politicians, freedom of speech, and dynamic humanities and artistic sectors. wealth, and security of food, water, and energy, can and must be assured through the knowledge economy. harmony can be restored to communities suffering deep-seated divisions. furthermore, countries in the arab middle east will then be in a position to interact much more effectively and comprehensively in the international arena so that, if needed, external support and assistance can be fully and timeously harnessed. despite all the odds, this transition must be accelerated from the current dangerous condition to a much more enlightened and prosperous existence. education throughout society has proved to be a slow process, and can be resisted by regressive forces and indolence, so responsible leadership is a prerequisite. arab scientists, technologists, and engineers must contribute actively to this transition, thereby securing a safe, healthy, and buoyant future for all arabs. research and development priorities must be reassessed in the light of worsening nexus of water, food, and energy insecurity, and the desperate need to return to peaceful conditions. in legal jargon: time is of the essence. finally, a buoyant growth potential for the arab middle east is dependent on the fundamentals of demography, education, access to capital, technology, careful custodianship of its natural resources and environment, and social stability; all are threatened by this insecurity nexus. building capacity for resilient food systems human population reduction is not a quick fix for environmental problems the future of universities in the arab world. arab academy of science creating and evaluating accurate crispr-cas scalpels for genomic surgery food security policies for building resilience to conflict multiplex genome engineering using cripr/cas systems mapping of climate change threats and human development impacts in the arab region ( pp.) food-and-nutrition-security food and agricultural organisation of the united nations action to build resilient livelihoods. prepared by the food and agricultural organization of the united nations (fao) for the world conference on disaster risk reduction world stabilization population unlikely this century global hunger index: the challenge of hidden hunger ( pp.) value of traditional foods in meeting macro-and micronutrient needs: the wild plant connection overview of the roles of energy and water in addressing global food security an overview of mitigation and adaptation processes and strategies to address the impacts of climate change on food, water, and energy security in the arab middle east a programmable dual-rna-guided dna endonucleases in adaptive bacterial immunity a meta-analysis of the impacts of genetically modified crops the conflicts of globalization resilience for food security in refugee-hosting communities international budget partnership. www.internationalbudget.org organisation of economic co-operation and development economics of salt-induced land degradation and restoration what economic theory tells us about the impacts of reducing food losses and/or waste: implications for research, policy and practice. agriculture and food security national association of state universities and land-grant colleges, experiment station committee on organization and policy, a science roadmap for the future. www.nasulgc.org/comm_food.htm the economist commodity-price index a gap analysis methodologhy for collecting crop genepools: a case study with phaseolus beans key: cord- - apdhf e authors: hussels, stephanie; sherman, claire; ward, damian; zurbruegg, ralf title: south and east asian insurance market growth and development date: journal: handbook of international insurance doi: . / - - - - _ sha: doc_id: cord_uid: apdhf e recent economic research, notably by king and levine ( a, b), levine and zervos ( ), levine ( ), levine, et al. ( ), and beck, et al. ( ), indicates that financial services and its various components, including insurance and banking, have substantial potential for spreading positive externalities throughout the commercial sector of an economy. such benefits can stem from improved access to capital by firms, better allocation of capital to investment projects, greater risk management, and enhanced portfolio diversification and liquidity for individual investors. while existing economic research shows the development of financial services is generally important for economic growth, a number of previous studies by outreville ( ) and ward and zurbruegg ( ) provide empirical evidence that insurance market development in its own right can promote economic development. the importance of the insurance industry to the wider economy is seen to stem from the relative size of the insurance industry to gdp in many developed economies, the transfer of risks, and the scale of insurance companies’ financial intermediary functions. recent economic research, notably by king and levine ( a, b) , levine and zervos ( ) , levine ( ) , levine, et al. ( ) , , indicates that financial services and its various components, including insurance and banking, have substantial potential for spreading positive externalities throughout the commercial sector of an economy. such benefits can stem from improved access to capital by firms, better allocation of capital to investment projects, greater risk management, and enhanced portfolio diversification and liquidity for individual investors. while existing economic research shows the development of financial services is generally important for economic growth, a number of previous studies by outreville ( ) and ward and zurbruegg ( ) provide empirical evidence that insurance market development in its own right can promote economic development. the importance of the insurance industry to the wider economy is seen to stem from the relative size of the insurance industry to gdp in many developed economies, the ^' the authors wish to thank the editors and an anonymous referee for very instructive and helpful comments in developing this chapter. transfer of risks, and the scale of insurance companies' financial intermediary functions. while the link between financial services development and economic growth is well-established, the focus of this chapter is to highlight research that has sought to identify the factors that will promote the demand and supply of the insurance sector. in particular, recent empirical research on insurance markets by beck and webb ( ) , browne, et al. ( ) , esho, et al. ( ) , and ward and zurbruegg ( ) have shown that the level of insurance demand within an economy can be influenced by a number of particular variables, including economic, legal, political, and social factors. despite these results, there has been little information presented on which specific factors should be fostered to aid financial development via the insurance market. to address this problem, the objective of this chapter is to present a synopsis of the existing literature and provide some insight on how the development of the south and east asian insurance market might be achieved. insurance companies who intend to expand their business activities abroad can also utilize this knowledge to select markets in south and east asia. in focusing upon south and east asia, the chapter is organized as follows: first, the relative size, economic importance, and likely developments of the insurance markets within the region are presented. second, empirical evidence relating to how demand and supply within the insurance sector can be developed is discussed. finally, this empirical evidence is then fiirther examined to consider the effects of economic stimulation in the insurance market on insurers operating in south and east asia. before delving into the specific demand and supply features of the regional insurance market in south and east asia, a preliminary statistical review of the countries considered within this chapter is listed in table . , including data on population, gross domestic product (gdp), inflation rates, and net written insurance premiums. net written life and non-life insurance premiums for - are included in table . . this helps to establish a comparative basis to examine insurance market forces within the region, starting below with an analysis of demand. in many of the emerging markets of asia, data by underwriting class is unfortunately difficult to obtain. therefore, in table . we only provide a breakdown of non-life underwriting classes for the developed economies of hong kong, japan, and singapore. source: various national annual statistical reports and swiss re ( a) , pp. , . note: gdp is gross domestic product. with the recent growth of the south and east asian insurance market, it is no wonder that it is attracting much attention from the global insurance market. life insurance premiums for south and east asia reached a phenomenal growth rate of . percent in and non-life insurance premiums grew by . percent in the same period (swiss re b). as figure . indicates, patterns of growth within the region's insurance industry suggest that changes in premiums are correlated with gdp growth, and with gdp predicted to grow at a real rate of . percent per annum between and , this growth is likely to continue (swiss re b) . this link between gdp and demand for insurance can be explained through the s-curve relationship (enz ) , which indicates that as gdp grows, people begin to have sufficient income and assets to warrant insurance protection. this leads to a steep increase in insurance demand greater than the growth in gdp. however, eventually, as more and more people reach a level where they have already attained insurance, growth in premiums stabilizes. this particular level tends to be different depending on the social security system in place within the particular country. re ( ), p. , , ; swiss re ( ), pp. , , ; swiss re ( a), pp. , , ; swiss re ( a), pp. , , ; swiss re ( b), p. , , ; swiss re ( a) , pp. , , for insurance premiums. note: data for excludes sri lanka and vietnam. table . provides evidence of the increase in premiums in most of the countries in south and east asia. in particular, china has seen outstanding growth in premiums for both life and non-life insurance, at . percent and . percent in , respectively (swiss re b). this is not surprising when considering the changing socio-economic structure of the chinese population. a rising middle class with a greater income to protect has opened a new market for insurers. assisted by a decline in restrictive regulations, insurers have been able to offer more insurance products to this developing middle class resulting in a higher growth rate in premiums. the threshold where gdp growth will begin to outpace premium growth has yet to be reached, and hopeful insurers in this market will continue to capitalize on this opportunity until it is attained. note: * not adjusted for inflation. in general, south and east asian countries have relatively high savings rates. the financial volatility experienced within the region's previous decade has meant that people are looking for safer options to invest their savings, and insurance has recently been seen as one way to reduce risk. by capturing a wide customer base, insurance companies have been able to diversify this risk on behalf of the investors within the region. a culture of self-reliance within many of these countries has also fueled the demand for insurance. as faith in the ability of some governments to provide for people in later life fades, people are procuring insurance policies to ensure their well-being. also, as with much of the world, an ageing population in south and east asia is driving demand for pension and investment-linked insurance products, either through direct personal investment or through governments searching for ways to fund their pension schemes. the - financial crisis in the region has had numerous effects on the insurance industry, one of which is the increased emphasis on risk awareness and risk minimization. initially, the real estate market experienced a downturn that significantly changed the security holdings of banks. additionally, bad corporate debt insfigated the collapse of many banks within the region (roubini ) . as a result, the currencies within asia started to plummet and central banks unsuccessfully tried to support their weakened position. as international funds were being funneled out of the region, and assets were declining in value, companies, governments, and the public within the region became more aware of the risks of investing and relying on established financial bodies to provide good credit assessments. for the insurance industry, this has fueled an increase in demand as a currently limited, but growing public awareness of insurance develops. another factor that has enhanced awareness was the severe acute respiratory syndrome (sars) epidemic, which first appeared in southern china in november and spread to canada, china, taiwan (china), germany, hong kong, singapore, slovenia, thailand, vietnam, and the united kingdom by march (world health organization ) . in particular, sars has increased the necessity of health-related insurance, and although initially having a marginally negative impact on demand where travel is a major component, it should stimulate further demand for insurance. furthermore, due to other events such as the indonesian riots in , the earthquake in taiwan, and the tsunami that struck several south and east asian countries in late , a need to insure against the risks of both natural and man-made disasters has become apparent within the region. there are differences in the demand for both commercial and personal non-life insurance that are often dependent on the country's stage of development. more rapidly developing countries such as china, india, and indonesia found a greater proportion of their demand came from commercial insurance in when compared to their counterparts (swiss re a). this is partly due to the lack of awareness for personal insurance and the low income of members of the general population. however, as stated previously, this is changing. additionally, the rise in total premiums may be due to a relative increase in the price of commercial premiums compared to personal insurance. in summary, the growth of gdp, changes in socio-economic structures within the region, the increased awareness of the need for insurance, and the need for risk minimization highlighted by recent damaging events have lead to an increased demand for both life and non-life insurance within the south and east asian region. nevertheless, coupled with this ongoing increase in demand, is an unlocking of the supply of insurance for this region, which is explored in following section. both singapore and hong kong have well-established insurance markets and, due to the deregulation of financial markets, insurance markets in other countries within this region have also begun to grow over the past five to ten years. changes to the structure of companies and the regulatory environment have been pivotal in opening new avenues for the supply of insurance within the region. the structure of insurance companies can be defined in several ways, namely, whether they are domestic or foreign, government-owned or private, listed or nonlisted, or by their size. data on the market share held by government and foreign owned insurance companies are listed in table . . also, broadly speaking, within south and east asia's non-life insurance market in , percent of companies were foreign (including joint ventures and branches) versus percent domestic companies (swiss re a). interestingly, this percent of companies represents only percent of the dollar share of net written premiums within the region. these divisions vary widely depending on the country of focus. for example, in singapore percent of the premiums are held with foreign companies, whereas, only . percent are held by foreign companies in south korea (swiss re a). the opening of insurance markets to foreign companies has allowed investment of much needed capital into the region, where the effects of the asian financial crisis of and sluggish world investment market continue to linger. note: market share is calculated using the total premium volume of those firms with > % ownership. > (>) highlight the % held by firms with exactly %) foreign ownership or equal ownership (greater than % ownership) for some countries. * ownership data is from . ** ownership data is from . n/a is not applicable. between and , the performance of foreign companies was more impressive than domestic companies, with growth in premiums for foreign firms amounting to . percent, whilst domestic companies experienced a slight decline (swiss re a). despite this, domestic companies have experienced greater profit margins, albeit, they have not had to incur the start up costs that foreign insurers have. foreign companies have also shown a better ability to assess risk within the region with lower loss ratios in most countries. however, this is only within the short-term as foreign firm admittance is relatively recent and long-term losses may not yet be apparent. in general, there are no discemable differences between the types of businesses that foreign and domestic insurers underwrite, with the exception of motor insurance, which is more prevalent in the domestic realm. within environments characterized by deregulation, consolidation, and a decline in government ownership, the withdrawal of underwriting restrictions and premium controls have provided an increasing opportunity for insurance companies to become progressively more commercially driven and strategically focused. large insurance companies have generally taken the initiative, now representing percent of the insurance companies in the region, but holding percent of the dollar share of premiums.^*^ as with many markets, the large consolidated firms have been able to utilize their economies of scale and breadth of expertise to become more competitive. this push for consolidation has meant that a considerable number of smaller companies have become prime merger and acquisition targets for the large insurance companies. large companies may also be able to diversify to a greater extent when compared to the smaller companies. however, they tend to follow regional market trends, which would not make them as resilient as global companies when considering shocks such as the asian economic crisis. an indication of the degree of market concentration in the non-life sector is provided in table . . ^ large companies refer to the top - firms within the country. place within government-owned insurance companies. however, now that the markets are opening to private companies, it is hkely that these matters will receive lesser consideration. interestingly, performance of government-owned insurers is surprisingly consistent with their private counterparts (swiss re b). nonetheless, this may be attributed to their greater ability to secure government business (swiss re a) and use of established distribution networks and databases. it is also worth pointing out that in islamic nations there is a general preference for mutual insurance, which is a form of risk sharing. this would be in contrast to private, profit-seeking insurance companies that profiteer fi-om speculation on life expectancies through the issue of price-related premiums, which does not conform well to sharia islamic law. the world trade organization (wto) has made a significant impact in the south and east asian region by encouraging insurance market liberalization. in particular, market deregulation is breaking down barriers for foreign insurers and creating a more market-based system in countries such as china, india, taiwan, malaysia, and thailand. in china, amendments to the insurance law, stipulate a market-based insurance system where companies have more control over their policies and premium rates (aliens arthur robinson ). the introduction of new regulation on the administration of foreign-invested insurance companies (china insurance regulatory commission ) also provides more detail for foreign companies who wish to enter the market. although restrictions still remain, there have been a number of foreign licenses granted and this step is seen as an indication of further liberalization to come. another country that has undergone substantial deregulation is thailand. it has gone from a market where a limited amount of foreign investment has been allowed to a market that encourages foreign capital and involvement. there is considerable foreign involvement in the thai insurance market with much of the foreign investment in complex share agreements and joint venture structures. the reason for this complexity is that foreign insurance companies are limited to a percent holding in any thai insurance company; and there are still restrictions on direct foreign investment, where companies with sole licenses can only operate from one location unless they are in a joint venture with a thai insurance company (aliens ). also, until , motor vehicle insurance was not compulsory in thailand. the introduction of compulsory motor insurance will obviously increase the market and, if further commitments to wto agreements are met, this should allow a greater opening of this large sector of the thai insurance industry to foreign investment. de-tariffication of the non-life sector is particularly relevant in china, taiwan, and india. tariffs are placed on most lines of insurance within india, with fire ( percent) and motor insurance ( percent) being the most prevalent insurance provided (the tariff advisory committee ), currently, information regarding claims is not complete and this means that the tariffs set by the tariff advisory committee are based on assumptions rather than calculated risk esfimates. reforms are in place to change the tariff-based system to a risk-based premium setting, which will assist in a better match of assets coming in to the risk being taken. in general, both the hfe and non-hfe insurance markets in south and east asia are becoming more liberahzed, although some countries are far more regulated than others. given the above discussion of the overall structure and developments in the insurance markets in south and east asia, there is a particular trend in the regional market that deserves particular attention, the recent rise of bancassurance. career agents are agents that are usually licensed to write insurance exclusively for one company and are employed as the most common form of distribution for insurance in south and east asia for both life and non-life insurance (see tables . and . , respectively) (swiss re b). although their prevalence as the primary means of distribution was a result of their ability to capture a dispersed customer base with a physical presence, there are other burgeoning alternative avenues for distribution such as direct marketing, insurance brokers, financial planners, and the topic in question, bancassurance, which is when a bank distributes insurance products. although only a small percentage of both the life and non-life insurance markets, bancassurance is now a fast-growing distribution channel in south and east asian markets. the bancassurance market may seem to contribute to only a small percentage of the insurance market as a whole. however, as figure . indicates, it varies greatly among individual country markets. for example, the life bancassurance market is as high as percent in singapore and the non-life bancassurance market as high as . percent in hong kong (swiss re b) . life insurance is the most common type of insurance sold through banks, which can be attributed to its similarity with other financial products sold by banks, such as mutual funds and time deposits. the main products offered in the life bancassurance market are whole-life, term-life, health/disability, retirement/pension insurance, endowments, annuifies, and policies linked to loan agreements. the benefit of tying the bank's existing financial products to insurance also influences the types of insurance products offered. the majority of bancassurance products are linked in some manner to deposits, mortgages, or other investment products. in some cases, entirely new insurance products have been created, such as depositor's insurance, which provides a payout in the case of an event (i.e. death) that is a multiple of the depositor's cash balance and over-draft insurance, which provides payment of an existing overdraft in case of a sfipulated event (i.e. death) (munich re group ). however, the similarity of some of these products to existing bank products should cause some concern, as there is a risk that substitution may take place. although a smaller secfion of the bancassurance market, non-life insurance offered by banks also includes a wide variety of products. this includes fire insurance, home insurance, and in some cases travel and accident insurance. once again these products are linked to the banks' existing products. for example, home insurance is typically linked with mortgage loans. the motor insurance market is one form of non-life insurance that has not been developed heavily by the south and east asian bancassurance merchants, as it is less related to the banks' existing products. varying degrees of regulation in different countries also dictate the use of this form of insurance distribution. countries such as singapore and hong kong have been using bancassurance for some time and have little or no restrictions as opposed to countries, such as south korea, where bancassurance is gradually being introduced through the staged relaxation of rules (asia insurance review ). where restrictions do apply, they are quite varied between countries. for example, in thailand, the restrictions are focused on particular products and licenses. for instance, a bank cannot issue non-life insurance products unless it is under a brokerage license and until recently, an insurance company was not permitted to pay commissions to banks (scor ) . alternatively, in china, restrictions focus on the parties of the arrangements, where it is often stipulated that insurers have to deal with each bank branch individually. in south korea, the introduction of bancassurance has been gradual and restrictions have focused on the types of insurance products permitted to be sold by banking institutions (asia insurance review ). overall, restrictions governing affiliations between banks and insurance companies as well as restrictions on the distribution of insurance products by non-insurers have been relaxed after the introduction of market liberalization, thus contributing to the rise of bancassurance. despite these current barriers, there has been a push for financial deregulation throughout south and east asia. as a result of this deregulation, boundaries between providers of financial products are blurring and countries that currently disallow bancassurance, such as the philippines, are expected to accommodate this distribution channel through banking subsidiaries within the philippines or through banks that are affiliated with insurance companies. south korea is also hoping to introduce bancassurance; however those with concerns about the polarization of a market that is already dominated by large banks and insurers may inhibit this. some restrictions shape the operations of the bancassurance markets in south and east asia. in particular, restrictions on foreign insurer entry have meant that the majority of bancassurance agreements are between domestic banks and foreign insurers. this form of alliance allows the foreign insurer to tap into the pertinent market without having to form a partnership with direct competitors and to gain access to the market by using existing networks. as a consequence, the level with which these partners are collaborating is mainly through distribution agreements ( percent) with the remaining operations based on the more integrated joint ventures, subsidiaries, holding companies, and financial conglomerates (swiss re b). these foreign insurers, which are involved in approximately percent of bancassurance operations in south and east asia (swiss re b), also have more experience with this type of distribution elsewhere in the world. the rise of bancassurance in south and east asia is also due to synergies between insurance products and banks' particular channel attributes. for example, this would include a bank's staff already being knowledgeable about such products, an established customer base, and customer information being used to target key segments with great accuracy. along with the increase of experienced foreign insurers in the market, synergies have fueled a major push for bancassurance within the region. the banks' incentive to decrease costs and recoup diminishing interest margins has been heightened due to the asian financial crisis. it has been recognized that the efficiency of using exisfing bank channels and various other distribution channels is greater than using the current career agents system (limra international ) . with this acknowledged, bancassurance has been difficult for governments, banks, and insurers to ignore. introducing these new higher-yield products also helps to delay the outflow of a bank's funds, which has been significant recently due to low rates on deposits. another issue highlighted by the financial crisis was the lack of stringency in the credit culture within many of the south and east asian economies. by tying in insurance products to bank loans, for example, fire insurance for a housing loan, the risk of the asset is reduced somewhat. as a rising middle class is fueled by greater economic growth, a new market has emerged. therefore, the traditional distribution systems are no longer targeted towards the entire market. as many of the financial products that are consumed by the new middle class are accessed through existing bank systems, banks provide the perfect avenue to provide their insurance needs as well. this new market is also not necessarily looking for the more traditional insurance products, but is looking for tailored products as the market becomes more financially savvy. consequently, banks and insurers are now collaborating to provide comprehensive coverage and more integrated products. with the lack of pension schemes throughout the region, likely through deficient government and employment pensions, consumers are also becoming increasingly motivated to provide for their own future. in summary, the main issues for bancassurance in this region are the restructuring of the industry with an emphasis on consolidation and integration, a shift in previously restrictive regimes, creating synergies, the need to tap into new markets through more efficient channels, product innovation, and the influx of global competition and partnerships. another fundamental change in the region's insurance industry is the deregulation and liberalization of the market. this also leads on to two final areas that need further discussion as they underlie the movement of the insurance sector in the region that has arisen from deregulation: the type of business that is underwritten in the region as well as the importance of financial intermediation. in general, commercial lines of insurance have seen greater growth than personal, with commercial growth of . percent versus . percent for personal lines of nonlife insurance in (swiss re b). as would be expected, growth in commercial net premiums written increases as the amount of business undertaken within the country increases. this explains the greater proportion of commercial lines within the more developing nations such as india and china compared to the more developed markets such as south korea and taiwan. throughout the region much of the non-life insurance growth can be attributed to motor vehicle insurance, in particular, over half the premiums in both taiwan and thailand relate to this line. other countries within the region also show a large representation with most attributing approximately a third of all premiums to motor vehicle insurance. insurers will come under further pressure to compete within the particular motor vehicle insurance markets in the region as motor vehicle premium rates in india, china, and taiwan are liberalized. the sars epidemic is also thought to have generated a rise in both personal and commercial policies. medical insurance may be affected directly through an increased awareness of health issues such as sars. to add to this, individuals are now conscious of the advantages of having coverage for the use of private medical services because public resources were put under strain during the epidemic. commercial lines of insurance should receive increases in premiums due to sars as disruptions to business and cancellation of events make businesses wary of further health epidemics, even those unrelated to humans, such as the avian flu crisis. although declining, government providers often play a big role within the insurance markets of south and east asia. in particular, governments often provide medical and unemployment insurance as well as pensions. this varies from country to country quite substantially. for instance, in india and hong kong, governments do not usually provide for any of these three lines of insurance. alternatively, singapore, south korea, and taiwan provide all three types of insurance as well as other lines such as crop insurance, workers' compensation, and motor vehicle insurance (swiss re b). some governments have even started to provide insurance against terrorism, particularly for the aviation industry. with greater competition and a more client-oriented focus. south and east asian insurance is becoming more diverse and the scope of contracts available to consumers and businesses are tailored to more specific risks. large insurers, who are now segmenting their considerable customer base to a greater extent, are also developing several innovative products. specialist firms are also continuing to service niche markets. overall, these changes show that the traditional role of insurance companies when providing policies is changing. however, so too is their role as a financial intermediary. despite the world downturn that began in late , optimism within the global financial markets in the beginning of has strengthened the financial sector. also, growth in bank lending within the region has not been as affected from to present. however, increases in both consumer and commercial lending have been met with more prudence, to avoid any future investment bubble. the continued confidence of financial intermediaries is an important facet of the recovery from the previous decade's slump. in particular, insurance companies' role in allowing risks to be spread throughout the region (and the world) and their injection of funds back into the region is pivotal. large and foreign insurers are more conservative in their investment activities focusing on more passive investments such as cash and deposits. wide ranging regulation of insurance investment is still prevalent, although changes are expected to increase flexibility and allow for higher yield investments (organization for economic co-operation and development (oecd) ). in general, the majority of insurance companies' investments are kept in cash and cash-equivalents (including other relatively risk-free assets, such as government bonds), and more so for non-life insurers than life insurers due to the different liability requirements. over the asian financial crisis, equity investments declined and bond investments increased (swiss re a). this is not surprising as the volatility of the south and east asian stock markets increased over the last decade. thus, insurers are adjusting portfolios because regulators have consequently maintained a stringent policy on investing in equity markets and because many insurers were negatively affected by their higher weighting of equity holdings during the financial crisis. moreover, despite the fact that the proportion of real estate investments remained relatively stable, there was an overall decrease in the value of insurers' investments. consequently, solvency concerns have come to light as regulators are now looking at insurers' balance sheets with more scrutiny. losses incurred on local investment assets have meant that reshuffling of investments has had to take place to secure appropriate reserves, particularly within thailand, indonesia, and south korea. finally, reinsurance has started to be used to alleviate the mismatch between the assets and liabilities of insurance companies, although restrictions to foreign company entry have hindered the entry of some of the large global reinsurers into the region. due to the long-term nature of life insurance, companies within this sector experienced a greater mismatch between assets and liabilities. in indonesia, problems arose because too few investments were denominated in u.s. dollars to offset the policies that were based in the same currency. this currency risk, which was a by-product of insurers trying to gain fi^om higher returns on domestic investment, proved to be quite detrimental once the financial crisis emerged. there was a mismatch in the maturities of assets and liabilities, where longer-term policies were matched with what proved to be shorter-term investments with diminishing value. perhaps a closer look at what is determining demand and supply within the region will help to alleviate problems such as that experienced in indonesia. this, therefore, leads us into the next section that focuses on academic research of these determinants. the insurance market growth discussed previously has been enormous across asia. but growth in economies such as hong kong, singapore, india, and china has been more pronounced. for key decision makers, such as managers and policy makers, it is essential to understand those factors that promote higher growth rates. the purpose of this section is to provide a basic insight into the drivers of insurance market growth by drawing upon current empirical studies. the discussion highlights the factors that promote the demand and supply of insurance and relate these findings to the insurance market of asia. economies flourish under economic, legal, and political stability. it is therefore of little surprise that economic sectors, such as insurance, should also do well under similar conditions. economic growth and economic stability, coupled with effective political and legal institutions, have been found to promote the development of insurance. however, for a better understanding of the differing rates in insurance market growth experienced in the markets across asia, it is necessary to explore these factors more fully. there is broad agreement that growth in gdp and/or gdp per capita leads to higher demand for insurance (beenstock, et al. ; truett and truett ; browne and kim ; outreville ; ward and zurbruegg ; beck and webb ) . as economic prosperity increases, the need and ability to purchase insurance also increases, although not at a constant rate. as consumers become increasingly wealthy, they can afford to retain risks within their current financial portfolios. therefore, the strongest link between income and insurance consumption occurs in those countries with moderate levels of gdp per capita, which ward and zurbruegg ( ) found to be the asian 'tiger' economies.^'^ however, as these economies continue to grow, the demand for insurance will wane, which must be an important consideration for insurance companies seeking to exploit growth in asia on a longterm basis. economic stability is equally important for insurance consumption. high rates of inflation devalue the net present value of insurance; and therefore reduce its attractiveness to consumers. moreover, excessive inflation can be linked to macroeconomic instability, with consequences for consumption, investment, and exports. ward and zurbruegg ( ) provide evidence that inflation is around . times more important for insurance demand in asian economies than it is for developed oecd countries. this emphasizes the role that broad macroeconomic ^' in this discussion, the 'asian tiger economies' are categorized as consisting of hong kong, south korea, malaysia, singapore, taiwan, and thailand. stability plays in the long-term development of the demand for insurance, a role that is mirrored by the accelerated growth of insurance within the relatively stable economies of singapore, hong kong, india, and china. recent debate on economic development and economic stability has been dominated by discussions of the function played by legal and political institutions. prominence, however, has usually been given to the effectiveness of legal systems in promoting commercial transactions within an economy. as a contractual exchange of risks, insurance market development is arguably very sensitive to legal and political effectiveness. ward and zurbruegg ( ) and esho, et al. ( ) find clear support for this hypothesis in both the life and non-life sectors. importantly, a direct positive correlation seems to exist between an improvement in the legal systems within a country and life insurance demand. it is, therefore, highly evident that the effectiveness of the legal environment within an economy is very important for the development of the insurance sector. the significance of legal and political variables is usually measured from constructed indices. a good example is from bcnack and keefer ( , ) , who use an index of governance constructed from five international country risk guide (icrg) variables that reflect the security of private property and the enforceability of contracts: 'corruption in government,' the 'rule of law,' 'expropriation risk,' 'repudiation of contracts by government,' and 'quality of the bureaucracy.' the higher the index the more effective the legal and political system is. in the case of indonesia where the index was low, weak functioning legal systems are associated with low gdp growth and low insurance market growth. the message for international insurance companies is, therefore, very clear. along side considerations of economic stability, legal effectiveness is also a necessary condition for economic and insurance market development, especially within the asian economies. social factors can also play a role in the development of insurance market demand. hofstede ( ) argues that the level of insurance within an economy depends on the national culture and the willingness of individuals to use insurance as a means of dealing with risk. while an appealing argument, esho, et al. ( ) and park, et al. ( ) fail to identify national culture as a significant driver of insurance demand. however, of particular interest to asia is the role of muslim dominated countries, where religious beliefs inhibit those forms of insurance that facilitate speculation of future events, thereby discouraging growth of the insurance sector. since asia has a number of important muslim based societies, including indonesia and malaysia, these maybe expected to have a lower rate of insurance market development. the supply of insurance, more so within many asian economies is composed of domestic and international supply. the amount of supply, the number of firms, and the openness of competition, are all important for an understanding of market development, pricing, and ultimately profitability. unfortunately, little is empirically known about the determinants of domestic supply in the insurance industry. however, there has been an enormous research agenda in measuring the efficiency of various insurance industries around the world, see cummins and weiss ( ) for a review. however, little has been done to develop this work into an understanding of growth rates in domestic supply. admittedly, the use of malmquist indices to measure productivity developments over time is useful, but these indices have not been linked to the dynamics of the insurance market, including such topics as growth rates in premiums, number of insurance companies, or the types of risks being traded. all of which are important supply related topics. a tradeoff is often seen to exist between increased competition and financial stability. however, this view, arguably, places too great an emphasis on regulation being supply constraining, rather than supply enhancing. regulation can restrict flawed products and facilitate the development of competition, innovation, and new beneficial products. regulation can also be an ally of the sector, rather than simply being an enforcer of rules. swiss re ( a) provides a brief review of regulatory characteristics and expected changes in the south and east asian region. it is notable that the fastest growing insurance markets. hong kong, singapore, and south korea have the most liberal regulatory environments. admittedly, prudent regulation is still important and the need to regulate foreign insurance companies in asia will only grow as globalization continues, but even globalization brings risk reduction. for example, globalization enables international insurance companies to diversify a broad range of risks, from the obvious examples of underwriting to the investment opportunities presented by different capital markets. in addition to these operational risk reductions, globalization also offers world players in the insurance market the opportunity to strategically reduce risk by broadening activities across many different international markets. as a result, financial stability can be strengthened by accessing a broad array of strategic positions in a variety of developing, emerging, and developed economies. a key development in the leading insurance markets of the world has been the emergence of alternative risk financing, characterized by cat bonds, insurance derivatives, and the securitization of insurance lines (swiss re b and cowley and cummins ) . the benefits of such developments are the ability to draw on additional capital and to diversify risk beyond the insurance companies' balance sheet, or that of its reinsurer. these capital market developments highlight to the emerging economies of the world that the development of insurance supply is intrinsically linked to the development of the entire financial system and ultimately the depth and diversity of the accessible financial markets. economies, such as singapore and hong kong with well-developed capital markets and access to the capital markets of north america and europe, should be able to bolster underwriting capacity and supply by broadening capital market access and financial innovation. the international supply of insurance services can take two broad forms, the export and import of insurance services and the provision of insurance services through foreign direct investment. in assessing the attractiveness of international markets to insurance companies ma and pope ( ) reveal that the number of domestic competitors and high levels of existing demand are most important. in essence, international insurance companies appear to seek out high growth markets, with an abundance of existing suppliers; and therefore, no dominant incumbent. this may ease entry into the market by facilitating accommodation and switching between suppliers. in asian countries, where state provision of insurance has led to the creation of a dominant incumbent, international entry into these markets maybe limited. this may then constrain future price competition and long-term growth of the market. in addition to examining the factors that attract the import of insurance services, it is necessary to understand why insurance may be supplied internationally across national borders. a key argument is that the continued and growing supply of financial services across international boundaries is suggestive of competitive advantages gained from access to superior and cheaper factors of production, particularly labor and capital. recent research conducted at the world bank also sheds light on recent liberalization commitments in financial services trade, and how there are domestic and international forces that drive this liberalization process (schuknecht, et al. ) . international insurance companies can use this analysis to benchmark their own domestic resource base and level of competitive advantage against the international markets into which they are contemplating entrance. promoting economic development stimulates both life and non-life consumption. in particular, national income seems to have a stronger impact on life insurance consumption in asia, than on the oecd markets. decision makers should, however, not overvalue these results. recent research indicates that the effect of national income on insurance demand decreases enormously after controlling for legal and political factors. the empirical resuhs generally suggest that economic stability, conditioned on legal and political stability, appears to be more crucial for long-run success than economic development alone. these findings are also reflected in the impact of income per capita on the demand for life insurance in the asian 'tiger' economies. after controlling for legal and political factors, income elasticities tend to be smaller. while economic development may occur at faster rates in asia, the link to insurance market development does not appear to be as strong. insurance companies intending to expand their business activities abroad should consider these factors when choosing which markets to enter. in the non-life sector, empirical findings highlight the fact that the link between insurance demand and the legal environment is focused on the single issue of property rights enforcement. this indicates that the effectiveness of the legal system in enforcing contracts is paramount to the development of the insurance market. however, in contrast to the life insurance industry, existing research highlights that the non-life insurance business is largely unaffected by cultural and institutional factors. in fact, esho, et al. ( ) state that the development of non-life insurance is 'technically rather than culturally located' indicating the importance of the legal and regulatory environment. on the supply side, decision makers need to be aware of how a number of key findings fi-om the literature, in particular, the factors that promote a comparative advantage in insurance, drive international insurance supply. an important determinant is the capital to labor ratio. applying this finding to the insurance sector is likely best achieved by arguing that the level and quality of technical infi-astructure is important for supply side development in the insurance sector. to price risks and manage losses, decision makers should include the quality of office space, the clustering of insurance companies, services for shared experience and technical advice, and the reliability and breadth of telecommunication and computer systems. the quality of capital and labor is also important since a comparative advantage is found to be related to the amount of schooling and the level of research and development (r&d) expenditures. again, relating these directly to the insurance sector, r&d becomes important in the development and implementation of risk management and control systems. fire systems, construction methods, motor vehicle security, and safety are further examples, along with the development of underwriting models and the ability to model financial and capital market risks. when coupled with an educated workforce and the development of professional training, the insurance sector can exploit and leverage knowledge available within its broader environment. in terms of attracting an international supply of insurance. ma and pope ( ) show that international involvement in domestic markets is promoted by increased domestic competition and increased liberalization. therefore, in order to promote supply, the emerging economies of asia need to enable greater competition among existing and potential entrants and present flexibility and growth through increased liberalization. deregulation, increased licensing of insurance companies, reduction in rate setting, and the broadening of underwriting lines and potential investment options are all suitable policies to promote. the south and east asian economies offer a variety of risks and opportunities for global and domestic insurance companies. in the relatively developed and economically stable economies of singapore, india, south korea, hong kong, and japan growing economic prosperity is strongly associated with insurance market growth. with increasing deregulation, fi*eedom of market access to foreign companies, and a willingness to integrate financial services through bancassurance, these markets are competitive growth spots for the world's leading insurers. in the more emergent economies of the region, where economic, legal, and political effectiveness are less-assured, commercial confidence and insurance market development is more open to macroeconomic shocks and potential financial crises. governments need to foster a commercial environment, which creates greater confidence for economic exchange and insurance risk transfers. legal stability and the enforcement of property rights are essential for an economy to grow and to make insurance sectors attractive and profitable for both domestic and international players. in the future, it will be interesting to see how domestic competitors rise to the challenge of international rivals; and the fight may not be one sided. singapore, in the areas of telecommunications, aviation, and shipping, has shown itself to be a dominant regional competitor; and the same is possible in financial services, and in particular insurance. international rivals should not assume that the markets of south and east asia offer effortless opportunities. perhaps most interestingly, the insurance sector stands in stark contrast to many other industries reflecting the trends of globalization. while many manufacturing industries view south and east asia as a source of low-cost inputs, global insurance companies view south and east asia as a potential source of premium growth. the empirical evidence suggests that intra-industry trade in insurance is promoted by a convergence in economic activity across economies. therefore, an increasing international supply of insurance in south and east asia will benefit from increased economic growth, and more importantly, salaried employment and rising personal disposable income. all factors which are likely, however, to impede further development of low cost manufacturing and subsequent export growth. it is without a doubt that the south and east asian economies are now arriving at an important economic crossroad, with pressures to expand into the financial services industry while also trying not to lose their cost-advantages in their already established manufacturing sector. how successfully these economies migrate from manufacturing to service-based economies may have important implications for the medium term economic development of their respective markets, and the consequential desire to purchase insurance. annuity. an annuity is a life insurance policy in reverse, whereby the purchaser gives the life insurance company a lump sum of money and the life insurance company pays the purchaser a regular income, usually monthly. bancassurance. bancassurance is the amalgamation of assurance and banking business within a financial environment, whereby banks are used as distribution channels to sell insurance products. a broker is an individual who arranges and services insurance policies on behalf of the insurance buyer. the broker is the representative of the insured, although the broker receives compensation in the form of a commission from the company. career agents. career agents work on behalf of the insurance company, selling products to potential policyholders. they are also referred to as a tied agent. cat bonds. cat bonds are risk-based securities that allow (re)insurance companies to transfer natural catastrophe insurance risk to institutional investors in the form of bonds. cat bonds help to spread peak exposures. they are also called catastrophe bonds. a claim is a demand by an insured for indemnity for loss incurred from an uninsured peril. commercial insurance. commercial insurance is sold by privately formed insurance companies with the objective of making a profit. depositor's insurance. depositor's insurance provides a payout that is a multiple of the depositor's cash balance in the occurrence of a certain event (i.e. death). endowment insurance. endowment insurance provides the insured with the face value of a policy if the insured or the beneficiary is alive on the maturity date stated in the policy. foreign direct investment reflects the objective of obtaining a lasting interest by a resident entity in one economy ('direct investor') in an entity resident in an economy other than that of the investor ('direct investment enterprise'). the lasting interest implies the existence of a long-term relationship between the direct investor and the enterprise and a significant degree of influence on the management of the enterprise. direct investment involves both the initial transaction between the two entities and all subsequent capital transactions between them and among affiliated enterprises, both incorporated and unincorporated. gdp. gross domestic product represents the total value of final goods and services produced within a country's borders during a specific time period, usually a year. gnp. gross national product is the total value of final goods and services produced by domestically owned factors of production. income elasticity. income elasticity of demand measures the responsiveness of the quantity demanded of a good to the income of the people demanding the good. it is measured as the percentage change in demand that occurs in response to a percentage change in income. insurance derivatives. insurance derivatives are investment instruments which are determined directly and solely by the loss pattems of natural catastrophes. liability insurance. liability insurance is insurance for damages that a policyholder is obliged to pay because of bodily injury or property damage caused to another person or entity based on negligence, strict liability, or contractual liability life insurance. life insurance provides for a payment of a sum of money upon the death of the insured. more specifically, in exchange for a series of premium payments or a single premium payment, upon the death of the insured, the face value minus outstanding policy loans and interest is paid to the beneficiary. malmquist index. the malmquist index is a measure of the degree of concentration in a market. it is the sum of the squares of the percentage market shares of all companies in the market. a mutual insurance company is an insurance carrier, without capital stock, that is owned by the policyholders. it may be incorporated or unincorporated. organisation for economic co-operation and development is an intemational organization of those developed countries that accept the principles of representative democracy and a free market economy. it originated as the organisation for european economy co-operation (oeec) to help administer the marshall plan for the re-construction of europe after world war ii. later, its membership was extended to non-european states, and in it was reformed into the oecd. over-draft insurance. over-draft insurance covers the repayments of an overdraft facility in the case of certain events (i.e. death). premium density. premium density is premiums per capita. a premium is the payment, or one of the periodical payments, a policyholder agrees to make for an insurance policy reflecting his/her expectation of loss or risk. premiums earned. premiums eamed are premiums an insurance company has recorded as revenues during a specific accounting period. premiums written. premiums written are premiums for all policies sold during a specific accounting period. product liability insurance. product liability insurance is insurance for the manufacturer or supplier for of goods for damage caused by their products. research and development can be defined as any project to resolve scientific or technological uncertainty aimed at achieving an advance in science or technology. advances include new or improved products, processes, and services. sars. severe acute respiratory syndrome is a viral respiratory illness caused by a coronavirus, called sars-associated coronavirus (sars-cov). sars was first reported in asia in february . over the next few months, the illness spread to more than two dozen countries in north america, south america, europe, and asia before the sars global outbreak of was contained. term life insurance. term life insurance is a life insurance policy that has a set duration limit on the coverage period. once the policy is expired, it is up to the policy owner to decide whether to renew the term life insurance policy or to let the coverage terminate. permanent life insurance. permanent life insurance, also referred to as ordinary life insurance or whole life insurance, is a life insurance policy that covers an insured for their entire lifetime, assuming premiums are paid as specified in the policy. whole life insurance provides a guaranteed fixed sum (sum assured) upon death of the life or lives assured. time deposits. time deposits are savings accounts which require notice of withdrawal. wto. world trade organization. focus: insurance and reinsurance asia interview with the regulator-bancassurance in korea: future supervisory directions finance and the sources of growth economic, demographic, and institutional determinants of life insurance consumption across countries the determination of life premiums: an international cross section analysis international property-liability insurance consumption an international analysis of life insurance demand regulations of the people's republic of china on administration of foreign-invested insurance companies securitization of life insurance assets and liabilitiqs analyzing firm performance in the insurance industry using frontier efficiency and productivity methods the changing focus in the supervision of insurance company investment the s-curve relation between per-capita income and insurance penetration law and the determinants of property-casualty insurance general insurance in japan fact book insurance as a product of national values office of the commissioner of insurance finance and growth: schumpeter might be right institutions and economic performance: crosscountry tests using altemative institutional measures law, finance, and economic growth financial intermediation and growth: causality and causes stock markets, banks, and economic growth survey of alternate distribution channels: asia determinants of intemational insurers' participation in foreign non-life markets bancassurance in practice the economic significance of insurance markets in developing countries life insurance markets in developing countries determinants of insurance pervasiveness: a cross-national analysis basic readings and references on the causes of crisis explaining liberalization commitments in financial services trade bancassurance across the globe: meets with a very mixed response world insurance in : another boom year for life insurance: retum to normal growth for non-life insurance india: transition from uniform insurance tariff system to risk-based approach the demand for life insurance in mexico and the united states: a comparative study does insurance promote economic growth? evidence from oecd countries update -severe acute respiratory syndrome (sars), www.who key: cord- -o pmuhd authors: mine, yoichi; gómez, oscar a.; muto, ako title: human security in east asia: assembling a puzzle date: - - journal: human security norms in east asia doi: . / - - - - _ sha: doc_id: cord_uid: o pmuhd this chapter describes the motivation of the research project, provides the theoretical framework of the entire book, and gives a summary of the findings of the case study chapters. in the process of diffusion of human security norms in east asia, several features have emerged. first, east asians have accepted a comprehensive definition of human security regarding the perception of threats. second, east asians tend to think that human security and state security are complementary. third, the constituent elements of the human security norms such as freedom from fear and from want, freedom to live in dignity, protection, and empowerment are already accepted by east asian nations. we need an extra effort to elevate human security to a full-fledged norm in the region. human security is an international norm concerned with global public interest, or a concept that aims to be an international norm such as human rights, the sustainable development goals (sdgs), and corporate social responsibility (csr). in general terms, norms denote codes of desirable (or undesirable) behaviors shared in a specific community. one of the strongest norms common in human society is that "homicide is evil." even when the death penalty and war are allowed, they are considered exceptions to this norm. written norms become statutes and formal regulations, while social consciousness supporting specific codes of conduct can also be called norms. normative sciences not only describe facts but also inquire into "how the object ought to be," covering logic, ethics, and aesthetics. the study of norms can be part of such an intellectual exercise: we describe and evaluate what people consider to be appropriate behaviors, nationally and internationally. let us try to answer the above question. why does human security not fade out? it is because the international community needs this concept. though not explicitly using this term, the un can be thought of as being originally organized to realize human security beyond international security. the originality of human security as an international norm lies in its attempt to shift the referent object of security from "states" to "individuals" and to urge various actors to conduct themselves accordingly. the two world wars in the twentieth century claimed large numbers of human lives and stripped as many of their dignity and property in the all-out wars between nation states. in order not to repeat such calamities, the un conferred on its security council the authority to limit the sovereignty of states threatening international peace and security and to impose military sanctions under international law. in the un, state sovereignty is not necessarily an inviolable sanctuary, even though "non-interference" remains a major norm in international society. it is often assumed that hobbes' "realism" and kant's "idealism" are poles apart. however, if the nation-state is invented to overcome the havoc caused by the war of every man against every man (hobbes (hobbes [originally , and a world federation is shaped to avoid the devastation caused by the war of every state against every other state (kant (kant [originally ), these two world views are conterminous in a single spectrum. in this light, the ultimate objective of both nation states and international organizations is to realize the security of individuals by ensuring freedom for all people. therefore, it is of pressing importance to evaluate government functions on the extent to which they serve this objective. although we cannot deny the crucial roles of nation states and national governments, the strong nation states are those that effectively serve the security of individuals living in their territories, not those that demand citizens' sacrifice for state security too easily. in a nutshell, the normative message of human security boils down to a powerful proposition that the ultimate objective of governance at all levels is to provide security (or ensure freedoms) for every individual. the core message of human security is thus very simple, but many other intentions and meanings have been subsumed in this concept along the way. if the objective is the security of individual persons, we must be able to characterize the core constitutive elements of such a secure state, as well as the principal means to achieve that goal, which can be described as norms themselves. human security is being formed as a "norm-complex" in which different existing norms are combined and nested under the umbrella of human security (kurusu ) . this hybrid nature of human security is observable in the consensus-based resolution on the definition of human security adopted by the united nations general assembly (unga) in september . that resolution stipulates that human security is "an approach to assist member states in identifying and addressing widespread and cross-cutting challenges to survival, livelihood and dignity of their people." according to the resolution, a common understanding on the notion of human security includes: "(a) the right of people to live in freedom and dignity, free from poverty and despair. all individuals, especially vulnerable people, are entitled to freedom from fear and freedom from want, with an equal opportunity to enjoy all their rights and fully develop their human potential." the resolution then enumerates certain qualifications of the concept: "(b) human security calls for people-centred, comprehensive, context-specific and preventionoriented responses that strengthen the protection and empowerment of all people and all communities," "(c) human security recognizes the interlinkages between peace, development and human rights, and equally considers civil, political, economic, social and cultural rights," "(d) the notion of human security is distinct from the responsibility to protect and its implementation," and "(e) human security does not entail the threat or the use of force or coercive measures" and "does not replace state security." human security thus makes much of "national ownership," local contexts and bottom-up initiatives, and pays respect to all generations of human rights. based on the characterization of human security in past documents, including this unga resolution as well as the commission on human security ( ) and undp ( undp ( , , we defined the practice of human security for the present research as follows: to ensure three freedoms (freedom from fear, freedom from want, and freedom to live in dignity) for individuals and communities vulnerable to large-scale and cross-border threats, by combining protection from above and empowerment from below. although this definition may still feel too complicated, with careful attention, one finds that the concept has been made dynamic by incorporating new elements into a set of established norms. let us discuss three points. first, while taking the concept of "freedoms from fear and want" as a given, human security brought in the third element, "dignity." realizing a world free from "fear and want" is the ideal of the universal declaration of human rights, and these two freedoms can be represented by civil liberties and socio-economic rights. they are embedded in the national constitutions of many nations as well as in international human rights law. on the other hand, dignity corresponds to a moral attitude when aiming at the realization of these freedoms: to express respect for humanity, recognizing that every human being has intrinsic worth (rosen ) . it is impossible to think of the human rights of the dead, even though we do think of the dignity of the dead. this is because dignity is a relational concept, and practical methods to respect the irreplaceability of others depend on local cultural contexts. second, while human security does not deny the importance of protection, it incorporates the element of "empowering" people from below as a complement to protecting them from above. empowerment is a process that enables people to become the masters of their own lives and may require the redistribution of power and resources between the powerful and the powerless. in the context of social development, friedman ( ) developed a theory of empowerment focusing on community development and livelihood support. women's empowerment has been incorporated into both the millennium development goals (mdgs) and, more recently, the sdgs. self-evaluation tools for empowerment processes have also been developed (fetterman et al. ) . if practitioners of human security want to translate empowerment into practice, it is important for them to unambiguously respect the agency of local people while avoiding their protracted dependence on assistance wherever possible. by reinforcing the power not only of individuals but also of communities and local governments, the excessive power of national governments can also be effectively checked. in human security, it is important to lower the level of the focus of empowerment from the national to the subnational and down to the community level. thus, in the human security discourse, by adding the concepts of dignity and empowerment, the elements of culture and agency have been grafted onto existing norms of human rights and humanitarian intervention. this deserves more attention as a new value that has been added to the human security idea. in east asia, where social hierarchy is relatively strongly rooted, the concept of dignity based on the premise that individuals are embedded in society can be accepted more easily than the concept of empowerment that might "disturb" public order. however, as a counterbalance to public authorities' sometimes excessively paternalistic protection, the emphasis on empowerment is undeniably of great significance in this region. the third source of power that can dynamize human security is the awareness that human society is in danger. mahbub ul haq, a pakistani economist and the first advocate of human security in the un, wrote: "a powerful, revolutionary idea, the emerging concept of human security forces a new morality on all of us through a perception of common threats to our very survival (…) while great religions often move the human spirit through the sublimeness of their messages, they also carry in their messages the fear of eventual punishment. much human change comes from a fear for human survival (haq , ) ." we cannot fully control the forces of nature or the fate of humanity. in envisioning a sustainable future for human beings and nature, the human security idea is expected to contribute to the realization of the sdgs through its emphasis on serious and pervasive threats (downside risks) and people's vulnerability to these. human security as defined in the unga resolution makes much of national ownership in organizing human security action. the implication of this approach will be discussed further in the rest of this volume. modern international norms involving many and diverse stakeholders tend to be complex, which relates to the ways a norm is established. there is a normative process of norm-making: in other words, a desirable process that is the standard way of setting a new norm. wise people may gather to put bonum commune of humankind into statutory forms and diffuse this downward. however, the actual processes of norm creation and diffusion are a little different. for an idea to be established as a norm, it must be internalized in the minds of the members of society irrespective of whether it is legally enforced or not. for this purpose, it is desirable for as many parties as possible at the center and at the periphery to actively participate in the process of norm-making instead of passively waiting for the advent of a new norm. in this process, both universal and local values tend to slot into a new norm, thereby making it hybrid, composite, or complex. international norms are said to have life cycles. at the beginning, "norm entrepreneurs" propose a new norm, which is accepted by several states (the norm emergence stage). then, after a certain "tipping point," the norm diffuses quickly and prevails throughout international society (the norm cascade stage). finally, the norm is internalized in every country and becomes "taken for granted" (the internalization stage) (finnemore and sikkink ) . however, as clarified by amitav acharya in the case of the security regime in southeast asia, foreign norms may be opposed, modified, or displaced by existing local norms in local space. norms are not simply accepted or rejected but are also localized (acharya ; . conversely, new norms that are (re)created by local actors in the periphery may eventually reach the core nations and/or challenge global powers (acharya ; towns ) . as indicated by the concept of bricolage in cultural anthropology (lévi-strauss ) , people living in communities bring together various indigenous and foreign materials to ingeniously create a new modality of life. proposed norms are to diffuse or fade out while being transformed vertically from the un headquarters to a small village, and horizontally across diverse world regions and nations. the process of initiation, diffusion, and regeneration of a norm is called "norm dynamics." as described above, the concept of human security was first advocated by a group of norm entrepreneurs at the undp, consisting of mahbub ul haq and others. after that, several countries including canada reinterpreted the human security concept, and this gave rise to an offshoot norm called responsibility to protect (r p), which defined the conditions for international society to intervene into a sovereign state with military and/or non-military measures to directly protect citizens from the horror of "genocide, war crimes, ethnic cleansing and crimes against humanity." on the other hand, countries including japan, thailand, and the philippines understood the nature of threats in broader and more comprehensive ways and tried to redefine human security to avoid confrontation between state sovereignty and humanitarian imperatives by emphasizing prevention and sensitivity to local contexts. it should be noted that the comprehensive human security initiative of the latter group, maintaining the universality of un-based messages, has passed through the process of localization in asia. a radical change of international norms is often triggered by a dispute or a grave event (sandholtz and stiles ) . the prime minister of japan, keizo obuchi, officially advocated human security for the first time in singapore in after the asian financial crisis (he was foreign minister at the time) (kurusu ) . the commission on human security, which released the final report on the comprehensive human security approach in , was co-chaired by the former united nations high commissioner for refugees (unhcr) sadako ogata and the nobel prize-winning economist amartya sen, a combination of east asian and south asian universal figures (chs ). pitsuwan and caballero-anthony ( ) relate the effects of the financial crises, as well as the multiple humanitarian crises, that have made evident the significance of human security as a "compelling normative framework." still, they argue "that as far as institutionalizing human security in its security practices, … asean still has a long way to go," particularly because of gaps in economic security, protection from disasters and of minorities and migrants, among others. in the rest of this introductory chapter, we discuss how the concept of human security has been received in east asia in terms of the perspective of norm dynamics. what do asian countries accept, reject, or remodel of the idea of human security born in the un? in this book, the so-called asean plus three countries (the member states of the association of southeast asian nations (asean) plus china, japan, and south korea) is defined as east asia. in this region that has experienced "miraculous" growth (world bank ), the nexus between economic development and human insecurities is prominent. japan is not the only country that has accepted the human security norm in asia. the late surin pitsuwan, a member of the commission on human security and distinguished fellow of the jica research institute, persevered in his effort to diffuse the concept of human security in southeast asia, serving as the minister of foreign affairs of thailand and then as secretary general of asean. as discussed in chap. , in the gov-ernment of thailand set up the first government ministry in the world bearing the name of human security: the ministry of social development and human security. in thailand, knowledge on human security had been widely diffused among academic researchers, but the practice of human security canalized by the establishment of this ministry came to focus on the social welfare of the vulnerable: persons with disabilities, the elderly, children, women, and ethnic minorities. the philippines also paid attention to human security as soon as the undp report was released, and multiple efforts of localization can be enumerated, including the design of a "human security index." there have been attempts of co-option as well. an antiterrorism law called the "human security act" was enacted in , inviting criticism from filipino civil society (chap. ). application of the concept of human security in thailand and the philippines headed in the opposite directions of benign welfare and hardline public order. the chinese government does not often mention human security, but chap. argues that china articulates a vision similar to this concept and practices it without saying so. that is partly because china, a permanent member of the un security council, is expected to promote international norms embraced by the un system. the acceptance of human security by way of participation in multilateral stages is applicable to south korea as well. in , south korea became a member of the organisation for economic co-operation and development-development assistance committee (oecd-dac). in addition, ban ki-moon promoted human security in his capacity as un secretary-general. also, the government of south korea has occasionally referred to the importance of human security in addresses by its president and foreign minister (chap. ). thus, in east asia, several countries have accepted the concept of human security to varying degrees under government initiatives. in the meantime, local scholars have also accumulated academic inquiries. in addition to two major single-authored books (howe ; nishikawa ) , a train of edited volumes on human security in the east asian contexts has been published (kassim ; peou ; teh ; tow et al. tow et al. , umegaki et al. ). moreover, with relatively limited circulation, the proceedings, commentaries, and policy recommendations based on international conferences held in bangkok, seoul, jakarta, and so on, have been published one after another (banpasirichote et al. ; hernandez and kraft ; thabchumpon ; unesco unesco , wun'gaeo ) . these publications have shared a certain feature: authors based in east asia transmit messages mainly to readers within the region. these earlier studies, especially most of the edited volumes, discuss how concrete issues can be interpreted using the concept of human security and how those issues can be addressed on the ground. however, there is little research that digs into the processes by which individual countries in the east asian region have accepted the human security norm in their own ways. the country-by-country analyses in this book are expected to fill this gap. in , when the final report of the commission on human security was published, sadako ogata returned to japan to take the helm of the japan international cooperation agency (jica). under her presidency, the human security idea became embedded in the spirit of the agency. when a part of the japan bank for international cooperation (jbic) and jica were integrated to set up the new jica in , the jica research institute was established and launched several international research projects related to human security. then, in , a research project to directly investigate the norm dynamics of human security in east asia was set up. based on a common questionnaire, researchers from east asian countries were to work on interview surveys and document research to elucidate the present status of human security in each country (see fig. . ) . the researchers participating in the project-the authors of the chapters in this book-are a combination of senior and young scholars specializing in international relations, political science, development studies and other disciplines and working for universities and think tanks in various parts of the region. the researchers agreed to ask questions about the following three topics in the interviews: first, local perceptions of threats (the ranking of human security issues that are considered important in each country and in the east asian region); second, the ways of (selective) acceptance of the concept of human security (the understanding of freedoms from fear, from want and to live in dignity, the strategy for combining protection and empowerment, and the understanding of preparedness for calamities, and so on); and third, the question of national sovereignty (whether to allow foreign actors to operate within the country in case of natural disasters and violent conflict, as well as whether to take action in territories of other countries in such a case). at the same time, respondents were allowed to change the combination of interview questions to adapt to their countries' unique circumstances. in addition, it was agreed that the researchers would welcome responses criticizing human security. the interviewees included government officials, lawmakers, researchers at universities and think tanks, nongovernmental organization (ngo) activists, religious leaders, journalists, business persons, and international organization staff. though they were not necessarily statistically representative, in-depth interviews were conducted (some of the survey activities included anthropological interviews with villagers in the countryside and focus group discussions). the interviews reached more than a hundred, and two workshops for chapter authors were organized in tokyo and manila. in the next section, we put together the research outcomes in the light of norm dynamics, including the localization processes. first, let us think about what threats to human security we face. classifying the sources of threats to human security into those derived from the physical system (the earth), from the living system (animals and plants), and from the social system (human beings), akihiko tanaka called for a clearer understanding of the mechanism in which these threats bring about human insecurities. to that end, close collaboration between different academic disciplines including the natural sciences and engineering, the biological and ecological sciences, and the social sciences and the humanities is required (tanaka ) . in our surveys of the east asian countries, local experts were asked to enumerate the threats to human security. though priority ranking varies from country to country, an integrated list of threats arranged according to the above three systems can be as follows: climate change, typhoons/ cyclones, floods, volcano eruptions, earthquakes, tsunami, infectious diseases such as severe acute respiratory syndrome (sars), avian influenza and hiv/aids, food crises, lack of basic health and education, environmental pollution, urbanization, extreme poverty, unemployment, migration, human trafficking, violent conflicts, interstate military conflicts, religious intolerance, organized crime, oppression from the government, and so forth. meanwhile, the undp's human development report listed seven main categories of human security: economic, food, health, environmental, personal, community, and political security (undp , chap. ) . in the case studies of cambodia (chap. ), thailand (chap. ), the philippines (chap. ), and vietnam (chap. ), human security challenges are classified in line with these seven categories. these areas correspond not only to the divisions of the un specialized agencies but also to government ministries, so that the classification could be accepted as familiar and practical. such a diversity of threats largely overlaps with the so-called nontraditional security (nts) issues. while military threats from foreign states are considered "traditional," many threats that simultaneously affect multiple countries are of a non-military nature and fall into the category of "non-traditional" threats. as pointed out in the cases of china (chap. ), indonesia (chap. ), malaysia (chap. ), the philippines (chap. ), south korea (chap. ), and vietnam (chap. ), there is growing interest in nts among policymakers and researchers in china, south korea and in the asean countries, which seems to have contributed to the acceptance of human security in the region (caballero-anthony et al. ; caballero-anthony and cook ; li ) . however, there is substantial difference between the nts and the human security approach: while the actors that address such diverse threats still concentrate on the national governments in the former, more emphasis is placed on peer collaboration between states and other actors in the latter. the role of national armies in coping with human security challenges should be limited. chapter presents the opinion of an indonesian military officer who argued that the term "security" should not be used until the poverty level or the impact of a disaster exceeded a certain threshold and becomes a real threat to the survival of all citizens. if every threat was considered a security challenge, the military would be overwhelmed by the resulting deluge of duties. human security is regarded as a principle of official development assistance (oda) policies in japan, and to a lesser extent, in south korea. as described in chap. , in japan, the idea to combine efforts toward development and peacebuilding has gradually taken root under this framework. as an added value of human security, japanese interviewees emphasized the importance of a "comprehensive approach" in which diverse actors (including ngos and private firms) cooperate, as well as the significance of working among grassroots people and paying more attention to real needs in the field. meanwhile, most experts pointed out that human security challenges lie on the domestic front, too. people who were familiar with the concept of human security interpreted the great east japan earthquake and the resultant fukushima disaster as a typical human security issue. in addition, the aging population and a possible collapse of social security in the future can also be serious domestic human insecurity issues. in terms of domestic human security challenges, the case of singapore as presented in chap. is also revealing. while singapore has achieved a high degree of human security as a developed country in southeast asia, this small city-state is also going through acute human insecurities such as growing inequalities, increasing psychological stresses on citizens, the survival race between small enterprises, and discrimination against migrants and minorities. here, social media cuts two ways by spreading messages virally: it can mobilize good will but may also deeply wound people. in singapore, with strong administrative control from above, empowerment is supposed to be of great significance. besides, singapore assists neighboring countries in the form of philanthropy, even though this is not officially classified as oda. it is pointed out that the philippines has also provided humanitarian assistance while receiving assistance itself (chap. ) . in the great east japan earthquake, japan, a major provider of oda, received goodwill support from many countries including recipients of japan's assistance (chap. ) . it is noteworthy that the line separating providers from recipients of oda is blurred in the case of humanitarian crises. a country that has faced a series of exceptionally acute threats to human security is cambodia (chap. ). this country is considered "a showcase of human insecurities" that started with the genocide under the pol pot regime (it is said that around million people were killed in a country with a population of million). the interviewees enumerated contemporary sources of threats in cambodia such as the government, natural disasters, diseases, political insecurity, and land issues. some respondents pinpointed the problem of the "government approach, relying on the heavy presence of security forces and legal means to threaten and detain people." it is widely perceived that cambodian society has been destabilized and that human security has been threatened despite (or due to) recent economic growth. one of the topical concerns in east asia that has wider political implications is the north korean issue (chap. ). an emergency on the korean peninsula could bring about an exodus of refugees and other situations, which may potentially give rise to grave human insecurities both regionally and globally. the risk of military conflict over maritime interests could also be a threat to human security, as voiced by several countries. the necessity to address cross-border issues such as human trafficking, air pollution, infectious diseases, food security, and cybersecurity was also pointed out by many interviewees. how far has the concept of human security permeated east asian countries so that stakeholders can jointly address the multiple threats described thus far? as pointed out in those chapters that discuss the experiences in the philippines (chap. ), malaysia (chap. ), and thailand (chap. ), east asian experts did not fully understand the difference between human security and human rights or human development, while activists in civil society tended to use the discourse of human rights more often than that of human security. however, even though the human security norm has not prevailed in east asia, the concept has been accepted at least partially, as argued in several of the case study chapters. the survey carried out in vietnam (chap. ) broke down human security into the seven security categories of the undp and found that all these elements were inscribed into the vietnamese constitution and other laws. in addition, even when interviewees were not familiar with the concept of human security, they "were able to quickly connect the abstract concepts of 'freedom from fear', 'freedom from want', and 'freedom to live in dignity' to specific examples in their lives." human security in vietnam "can be said to be a jigsaw puzzle, in which the pieces are identified, but have not been put together." the surveys in indonesia (chap. ) and south korea (chap. ) also found, by examining official documents, that the elements of human security defined in this chapter, such as the three freedoms, protection, and empowerment, were all written into these documents to varying degrees (the former in domestic policies and the latter in oda policies). in addition, people who were interviewed in cambodia pointed out that the three freedoms were closely linked to each other in substance (chap. ). what is the most important element among the components that make up human security? the study of japan (chap. ) presented the expert opinion that the third "freedom to live in dignity" could be a real added value of the human security approach, indicating that "dignity is an idea of waiting and caring." the survey in the philippines also mentioned that the concept of dignity had potential to lead human security to a higher dimension and emphasized the importance of local contexts. moreover, people in cambodia said that having dignity is associated with "having a moral character; with notions of respect, pride, and having value and independence; and of helping others and having an honest character." a rural resident made a candid remark: "dignity is most important because it is about no discrimination, having rights to do what we want, not being looked down upon by wealthy people." while the expectation of state protection was found in many interview results, empowerment was mostly referred to in general terms. however, protection and empowerment make an effective pair in reality. empowerment leads to a series of concepts that value people's agency, such as ownership, self-help support, resilience, and capacity development in the practice of development cooperation, while the same concept is expected to promote collaboration between governments and civil society in domestic policies. given that the asian approach to human security tends to give relative weight to the role of states as discussed below, the counterbalance of empowerment is needed all the more in this region. in many countries, we also asked the interviewees whether foreign support should be accepted in case their own country suffers an uncontrollable crisis due to a natural disaster or violent conflict (and whether their country should support neighboring countries in case the latter suffers the same situations). the common pattern of responses to these hypothetical situations was that foreign support was undesirable during political unrest but welcome when a natural disaster occurs. it was also preferred that the support should be provided in multilateral rather than unilateral frameworks, as mentioned in the studies on malaysia (chap. ), the philippines (chap. ), and vietnam (chap. ). these reactions illustrate that east asians tend to think that state security could be compromised in favor of humanitarian concerns in certain emergency situations, especially in case of natural disasters. it should be remembered that sadako ogata stressed that human security and state security complement one another (ogata ) . as to the role of states in realizing human security, both a loose consensus and a subtle disagreement could be found among east asian countries. the case study of china argues as follows (chap. ). on the one hand, we can establish the causal connection that state security contributes to human security. the idea that people should not be easily sacrificed for national objectives is absolutely correct because human beings are not means but ends in themselves. on the other hand, national security and personal security can be compatible. the perception that states are a "necessary evil" is not a chinese but a western idea. east asians naturally expect a great deal from their governments: people expect the governments to protect them just like parents protect their children. this represents a view of states as benevolent and "paternalistic." the relationship in which a stable state guarantees people's security is also expressed in the case study of vietnam (chap. ). on the "right" of this view of states, there is another understanding that human security is part of state security, that is, state security subordinates human security, not vice versa. in this research project, such a view was expressed by government officers from indonesia (chap. ) and malaysia (chap. ) . from the government side, however, some added that the role of the military in human security should be strictly limited. indonesian interviewees opined that military operations should be firmly placed under civilian control, even though the military effectively responded to the earthquake and tsunami in . this is because they consider that the military is essentially not trained to respond to nonmilitary threats, and it is often better to entrust the duty of maintaining public order to police forces in disaster situations. on the "left" side, there are countries with impressive traditions of civil society activism such as the philippines (chap. ) and thailand (chap. ), which have strongly influenced the trajectories of acceptance of human security. in the case studies of malaysia (chap. ) and singapore (chap. ), dynamic and strained relationships between the government and civil society are vividly depicted. the chapter on malaysia places expectations in consolidating human security through empowerment of local governments, more active dialogues between the government and civil society, and regional cooperation through the networks of asean and ngos, against the backdrop of the government repression of free speech, religious intolerance, and the surge of rohingya refugees. when severe threats to human security are actualized, the relationship between state sovereignty and human security may become extremely tense. as described in chap. , when cyclone nargis hit myanmar in , the military government refused to accept foreign aid, even while lowland residents were caught in the flooding. it is said that the dead and missing persons numbered nearly , . though western countries such as france threatened to make a r p-type humanitarian intervention, the government of myanmar rejected such operations and instead decided to accept coordinated assistance from organizations such as asean and the un. this multilateral collaboration has become a model for humanitarian operations in east asia. the case study of japan (chap. ) warns that the concept of human security could be "politicized" in the contexts of domestic debates on security and securitization. in contrast, the study of thailand (chap. ) voices concern that human security is now too "depoliticized," arguing that the concept has been reduced to the practice of social welfare and is now rarely discussed in thai diplomatic contexts. however, behind the activities of the ministry of social development and human security seeking to improve the well-being of the socially vulnerable seems to lie the buddhist concept of mercy as well as an attempt to integrate human security with the concept of sufficiency economy advocated by king rama ix. these dynamics of politicization, depoliticization, and local reinterpretation are interesting in terms of the "norm localization" discussed in this chapter. keeping in mind the urgent problems including land grabbing and king sihanouk's political legacy in cambodia, chap. emphasizes the importance of "cooperative leadership" based on the spirit of tolerance and compromise. the key to ensuring human security in cambodia is to realize voluntary collaboration among opposing political parties, between the government and civil society, and between the central and local governments, and to make the government listen to the voice of the people. different countries have different perceptions as to which state and non-state actors should be valued as against others. however, we can safely say that there is a shared understanding in the region that diverse actors, including national governments, should coordinate each other's activities to secure freedoms and development for individual persons in the face of serious and pervasive threats. just as a world where autonomous villages cease to make decisions on their own affairs is hard to imagine, it is unlikely that the governments of nation states will cease to make their own decisions. national governments are important because most of them have strong powers and the authority to ensure security for individuals by utilizing well-developed institutions, resources, and national cohesion. however, overly powerful state security mechanisms require an antidote, which can be the human security norm. as history illustrates, when pluralist thinking that endorses critical roles played by non-state actors is denied, the world as well as national politics go awry. the purpose of the association of world peoples is not only to promote the security of nations but also to ultimately promote the security of all human beings. in this sense, the security council of the un could be renamed a human security council. in the practice of human security, neither "western individualism" nor "oriental despotism" is required in their pure forms; it seems that asian versions of human security have begun walking along the middle road between the two. in east asian nations, perceptions of diverse threats as sources of insecurities largely overlap, and therefore the conditions for collective action to address common threats also seem to be maturing. even though the term human security is not officially used very often, in this research it was found that the constituent elements of human security, namely, the three freedoms as well as protection and empowerment, have been accepted more or less in all parts of east asia. if regional spaces for dialogues are provided, the human security idea may diffuse in the short term like a cascade. an international network of experts sharing the value of a specific norm and assuming key roles in its diffusion as well as policy coordination is called an epistemic community (haas ) . in the process of this research, we witnessed the emergence of a bridged community with a shared interest in human security in east asia. the process of this research endeavor itself might be part of the formation of such a community. lastly, we would like to pay notice to the fact that the outcomes of this research reflect not only east asia's potential unity but also its actual diversity. once we zoom in to the regional space of east asia, we can see a kaleidoscopic diversity of human security stakeholders and their values. this is the reason why this book is entitled human security norms rather than the human security norm. the latter is only in the making: there remain forces that resist the idea of human security, while east asian nations are developing their own human security norms with different interpretations and preferences. the country-by-country analyses in subsequent chapters are based on independent research, which seems to have succeeded in shedding light upon the diversity of the history, society, and political economy of the region. the chapters are arranged in the alphabetical order of countries, so readers can start with any chapter while referring to the comparative analysis in chap. . we return to the issue of east asia's diversity in chap. , in which we will suggest a direction to proceed with the practice of human security in this region. notes . martin and owen ( ) present a stock-taking collection of reflections on the concept and its application. bourbeau ( ) captures the multidisciplinary nature of the study of security, including human security. . in international relations, norms are defined as "shared expectations about appropriate behavior held by a community of actors" (finnemore , ) or "collective expectations for the proper behavior of actors with a criticizing the proposition that human rights make sense only when they are legally guaranteed, amartya sen argues that strong moral imperatives of what to do and not to do make up human rights. these imperatives may call for legislation, but legal provision is not a prerequisite for human rights see also the discussions of "composite norms united nations general assembly, follow-up to paragraph on human security of the world summit outcome the concept of dignity was explicitly introduced to the human security discourse in the commission on human security ( ) as one of the triad of "survival, livelihood and dignity annan ( ) has introduced "freedom to live in dignity" into the agenda of the un reform, and tadjbakhsh and chenoy ( ) have attempted to incorporate dignity fully into the human security perspective. on the other hand, while the concept of empowerment is widely diffused in social movements in the americas and south asia after a norm is 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and australia. harnham: ashgate norms and social hierarchies: understanding international policy diffusion 'from below human insecurity in east asia united nations development programme) proceedings of the international conference on human security in east asia. seoul: korean national commission for unesco the east asian miracle: economic growth and public policy human security now: strengthening policy networks in southeast asia key: cord- -arjtjy authors: reuss, annicka; litterst, annette; drosten, christian; seilmaier, michael; böhmer, merle; graf, petra; gold, hermann; wendtner, clemens-martin; zanuzdana, arina; schaade, lars; haas, walter; buchholz, udo title: contact investigation for imported case of middle east respiratory syndrome, germany date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: arjtjy on march , , a patient from united arab emirates who had severe respiratory infection was transferred to a hospital in germany, days after symptom onset. infection with middle east respiratory syndrome coronavirus (mers-cov) was suspected on march and confirmed on march ; the patient, who had contact with an ill camel shortly before symptom onset, died on march . a contact investigation was initiated to identify possible person-to-person transmission and assess infection control measures. of identified contacts, were available for follow-up. ten contacts experienced mild symptoms, but test results for respiratory and serum samples were negative for mers-cov. serologic testing was done for ( %) of nonsymptomatic contacts; all results were negative. among contacts, the use of ffp /ffp face masks during aerosol exposure was more frequent after mers-cov infection was suspected than before. infection control measures may have prevented nosocomial transmission of the virus. % also had > underlying chronic condition ( ). the median age of case-patients was years (range months to years). all cases were directly or indirectly related to countries in the middle east or on the arabian peninsula. mers-cov shows a close genetic relationship with coronaviruses found in bats ( , ( ) ( ) ( ) ( ) ( ) ( ) , but no zoonotic link has been confirmed. person-to-person transmission has been reported in the work environment, among family contacts, or to health care workers (hcws) ( ) ( ) ( ) . although situations involving consecutive human transmission events have been documented ( ) , none of the known clusters have led to sustained person-to-person transmission in the general population. in europe, single imported infections have been reported in the united kingdom, germany, france, and italy, and secondary cases have been reported in the united kingdom, france, and italy ( , , ) . because a large proportion of cases are fatal and the virus could acquire the ability to spread more efficiently (as was the case with severe acute respiratory syndrome coronavirus), who has recommended thorough contact investigations for confirmed human cases to identify, quantify, and prevent person-to-person transmission ( ) . in germany, mers-cov infection was initially reported in a person from qatar ( ) . he was in his third week of illness and was already on mechanical ventilation when he was admitted to a hospital in essen in october . a retrospective contact investigation found no indication of person-to-person transmission to contacts in germany ( ) . on march , , the institute for virology of the university of bonn reported an imported case of mers-cov infection to the department of health and environment in munich (city health department). a -year-old man from abu dhabi, united arab emirates, had been admitted to a hospital in munich and had positive test results for mers-cov infection ( figure ). clinical details and virologic findings have been reported elsewhere ( ) . briefly, the patient had underlying multiple myeloma and had received several modes of treatment, including high-dose chemotherapy and autologous stemcell transplantation in . on march , , influenzalike illness with fever and cough developed in the patient. after his symptoms worsened, he was hospitalized in his country on march with a diagnosis of pneumonia; he was intubated on march and transferred by flight ambulance services to germany on march , eleven days after illness onset, for further intensive care treatment and mechanical ventilation. general infection control guidelines of the munich hospital required that patients from areas such as the middle east, where prevalence of multidrug-resistant pathogens is high, be isolated until colonization or infection with a multidrug-resistant pathogen is ruled out. this rule is particularly enforced when patients have been previously hospitalized in the country of origin. thus, at the time of hospital admission in germany, the patient was isolated from other patients. when mers-cov infection was suspected and included in the differential diagnosis on march , standard hygiene measures for hcws were changed to infection control measures as recommended for severe acute respiratory syndrome patients, including the use of ffp face masks for usual patient care ( ) . mers-cov infection was diagnosed in the patient on march ; he died on march of multiorgan failure and acute respiratory distress syndrome. after mers-cov infection was diagnosed, the city health department, in cooperation with the state health department, the institute for virology in bonn, and the robert koch institute, initiated an investigation to ) monitor all contacts of the patient to identify possible person-to-person transmission, ) assess infection control measures, and ) explore possible sources for the patient's infection to prevent further cases. for the investigation, the city health department assessed all contact persons (contacts) retrospectively and monitored them prospectively. all contacts received a questionnaire for retrospective documentation and prospective daily self-monitoring of symptoms, exposure to the patient, and infection control measures applied. for every day from march through april , information was collected about the contacts' distance from the patient (< meters vs. > meters); type of contact with the patient (aerosol-producing procedures, non-aerosol-producing procedures, care of patient, handling of urine catheter, handling of respiratory samples in the laboratory, handling of urine samples in the laboratory); type of protection used (surgical mask, ffp mask, ffp mask, ffp mask, gown, gloves, protective glasses); and symptoms experienced by the contacts (cough, fever, temperature, sore throat, diarrhea, shortness of breath). an aerosol-producing procedure was defined as respiratory suction, bronchoalveolar lavage, intubation, or bronchoscopy. on the basis of the self-reported information in the questionnaires and personal interviews with the contacts, we divided contacts into groups. close-distance contacts had face-to-face contact with the patient (< meters from the patient) or direct contact with secretions or body fluids of the patient, irrespective of protective measures worn. all other contacts were classified as less-close-distance contacts. according to who recommendations on the duration of follow-up at that time, close-distance contacts were asked to contact the city health department daily for days after the last exposure to the patient. those who failed to do so were contacted by the city health department, supported by the occupational health service of the hospital. less-close-distance contacts were asked to report to the city health department only in case of onset of symptoms. respiratory illnesses in contacts that occurred - days after exposure to the patient were assessed through the city health department by telephone contact with the contact; a respiratory tract sample was taken from any contact with respiratory illness. in addition, attempts were made to obtain paired serologic samples from all contacts, the first taken immediately after contact and the second > days after the last exposure. because the mers-cov patient was on mechanical ventilation and could not be interviewed, family contacts who had accompanied him to germany were interviewed about the onset of his symptoms and possible exposures in the days before disease onset. for the interview, a structured questionnaire was used, and information collected was documented on paper. pcr testing and serologic testing were done as described ( , ) . serum samples from contacts were tested for mers-cov antibodies if a serum sample was taken > days after last exposure. in addition, serum samples were tested for antibodies against influenza a, b, and c; rhinovirus a, b, and c; parainfluenzavirus , , , and ; respiratory syncytial virus a and b; human metapneumovirus; coronavirus e, nl , oc , and hku ; and adenovirus. all samples were analyzed at the institute for virology of the university of bonn. data from the city health department's contact monitoring, the contacts' questionnaires, and the laboratory findings were integrated in database. results were validated and analyzed by using stata version . (statacorp, college station, tx, usa). the city health department identified contacts. of these, ( %) were classified as close-distance contacts and ( %) as less-close-distance contacts (table) . four ( %) of the contacts were members of the patient's family, ( %) §probability that the distribution as indicated occurs by chance given the column and row totals. ¶nonsymptomatic contacts are asymptomatic persons and those who were symptomatic before exposure. of other professional groups. clinical follow-up was available for ( %) contacts. a respiratory symptom or fever developed in ( %) contacts. of these, swab specimens were collected from ( %) and blood samples from ( %). all swab specimens were negative for mers-cov; ( %) was positive for cov nl- , and ( %) were positive for rhinovirus. all serum samples were negative for mers-cov antibodies. all symptomatic contacts had > sample type (respiratory swab or serum) collected for laboratory testing; results of pcr and serologic testing were available from ( %), pcr only from ( %), and serologic testing only from ( %). in addition, serologic test results were available for ( %) of the nonsymptomatic contacts; all were negative for mers-cov antibodies. overall, persons for whom serologic testing results were available were more likely to be close-distance contacts than were persons without available serologic results (p = . ; table) . the family members who accompanied the patient were his wife, daughter, son, and son-in-law. their ages were - years, and none reported symptoms. the patient's children and son-in-law had their last contact with the patient on march and his wife on march . because no protection measures had been used until after march , the family members were considered at high risk for infection. all provided respiratory swab and serum samples on march ; all samples had negative results. serum samples taken > days after last exposure to the patient were not available. mers-cov infection was added to the differential diagnosis for the patient on march . the daily numbers of hcws who had any contact with him (regardless of protection measures) and of those who had aerosol exposure were lower after that date than before ( figure ): . hcw per illness day vs. . hcw per illness day (p = . ) and . hcw per illness day vs. hcw per illness day (p = . ). among hcws with aerosol exposure, ( %) of daily exposures occurred while ffp or ffp masks were being used before march ; after that date, ( %) of daily exposures occurred while ffp or ffp masks were being used (p< . ). the patient was a -year-old married man from abu dhabi, united arab emirates; he had a medical history of multiple myeloma. at the time of his mers-cov infection, he was receiving corticosteroid therapy. his profession was camel breeding; in the weeks before his onset of illness, of his camels was reported to have had a respiratory illness. in the questionnaire, we did not differentiate between dromedary (camelus dromedaries) and bactrian (c. bactrianus) camels. his neighborhood had palm trees, and bats were known to dwell in the area. the patient had no known contact with other mers-cov patients, had no personal contacts in qatar or jordan, and had no travel history in the days before illness onset. he consumed different types of fruit juices and cooked goat meat, beef, and sheep meat, but no raw meat. he ate dates from his region, but he reportedly did not consume date or palm syrup. other than the camels on his farm, he had no contact with animals; he did not practice falconry and did not visit camel racetracks or animal markets. we describe the case and contact investigation of a confirmed case of mers-cov infection that was imported to germany. we did not identify person-to-person transmission from the patient to any of the contacts. as with the previous imported case in this country, the patient was already on mechanical ventilation when he was transferred to germany. however, whereas the previous case was in the late third week of illness, this patient was in the second week of illness. sample from this patient taken from different body locations and at different times were positive for mers-cov by pcr, and the viral load detected was several logs higher than in samples from the patient with the previous imported case ( , ) . these results indicate that this patient may have been more infectious than the previous patient. nosocomial transmission from mers patients to hcws has been documented ( , , ) . in our study, the patient was isolated during the first days of his hospital stay (before mers was suspected), although the reason for this intervention was the hospital's policy to isolate every patient from the middle east, irrespective of the assumed diagnosis, because of perceived increased risk of carrying drug-resistant pathogens, rather than any special measures taken because of the patient's respiratory illness. after mers was suspected, hcws used ffp masks significantly more frequently than they had before, and fewer hcws had daily contact with the patient. our result suggest that, in the later stages of this disease, the combination of standard protection measures (use of surgical masks for potentially aerosol-generating procedures), cautious handling of the patient (because of his potential to harbor drugresistant bacteria), and possible decreased infectiousness compared with the first week of illness may have prevented transmission to hcws. these findings also underline the importance of following who recommendations on infection prevention and control when managing a patient who may be infected with a pathogen that could lead to nosocomial transmission ( ) . regarding possible sources of infection, an extensive interview was conducted with family members because the patient could not be interviewed. the patient's illness was likely a primary case, and possible exposures that might have caused the mers-cov infection were explored. of note were the presence of bats in the neighborhood of his residence, the patient's profession as camel breeder, and his contact with a camel that was reported to have had a respiratory illness before his own illness onset. bats are a likely reservoir for mers-related cov ( , ) , and serum samples from omani racing camels have shown to have neutralizing antibodies against mers-cov ( ). these findings suggest these animals' possible relevance (e.g., as intermediate hosts) for human acquisition of mers-cov. two complementary monitoring instruments for contact persons were used: active follow-up with daily telephone contact and a self-administered monitoring questionnaire. both methods have merits, and a combination of both is likely to ensure the most thorough contact follow-up. advantages of personal interviews on the telephone are immediacy and the possibility for the interviewer to receive intangible information, such as the self-assessment of symptoms, as well as the opportunity to answer questions from the contacts. this process enables a more specific way to judge a person's health status. on the other hand, a daily monitoring questionnaire provides detail in clinical information, exposure, and protection measures that might be used for more in-depth analyses (e.g., when a few contacts have become infected). such a questionnaire could be expanded to include a section for contact persons to fill in the names of persons with whom they had face-to-face contact during each day. this information might become crucial for second-generation contact tracing when contacts under observation become infected. rapid availability of this type of information is essential for efficient investigation of clusters or outbreaks similar to those that have been reported already ( ) . in conclusion, we conducted a contact investigation of an imported case of mers-cov infection in germany. laboratory testing of symptomatic and asymptomatic contacts of the index case-patient did not indicate transmission of the virus. furthermore, we documented the change from standard hygiene to infection control measures after mers-cov was suspected, an adaptation that may have prevented nosocomial transmission. exposure to camels as a possible etiologic mechanism for human mers-cov infection requires further evidence from other studies. dr reuss is an epidemiologist at the respiratory infections unit, robert koch institute, berlin, germany. her research interests include emerging infectious respiratory diseases, pandemic preparedness, and influenza vaccination. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. novel coronavirus infection in the united kingdom middle east respiratory syndrome coronavirus (mers-cov)-update state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans human betacoronavirus c emc/ -related viruses in bats, ghana and europe coronaviruses in bats from mexico full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus close relative of human middle east respiratory syndrome coronavirus in bat genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans group c betacoronavirus in bat guano fertilizer middle east respiratory syndrome coronavirus (mers-cov)-update health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission investigation of an imported case of middle east respiratory syndrome coronavirus interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus contact investigation of a case of human novel coronavirus infection treated in a german hospital clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection empfehlungen des robert koch-institutes für die hygienemaßnahmen und infektionskontrolle bei patienten mit schwerem akutem respiratorischem syndrom (sars) assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections updated rapid risk assessment: severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus infections in health care workers infection prevention and control of epidemic-and pandemic-prone acute respiratory diseases in health care. who interim guidelines key: cord- -k m xz e authors: chertow, daniel s.; kindrachuk, jason title: influenza, measles, sars, mers, and smallpox date: - - journal: highly infectious diseases in critical care doi: . / - - - - _ sha: doc_id: cord_uid: k m xz e influenza, measles, sars, mers, and smallpox illnesses are caused by highly infectious viral pathogens that induce critical illness. these biologically diverse viruses enter and replicate within host cells triggering viral- and host-mediated damage that results in pneumonia and multiorgan failure in severe cases. early case identification and strict infection control limit healthcare transmission. vaccination allowed smallpox eradication and limits global measles and seasonal influenza mortality. while sars-coronavirus (cov) is no longer circulating, mers-cov and zoonotic influenza viruses, with pandemic potential, remain persistent threats. supportive critical care is the mainstay of treatment for severe disease due to these viral infections. measles virus is a pleomorphic, enveloped, negative-sense, single-stranded rna virus of family paramyxoviridae of approximately nm to nm in diameter [ ] . measles virus causes mild to severe illness during seasonal outbreaks in endemic areas and intermittent outbreaks in nonendemic area [ ] . measles virus codes for six structural and two nonstructural proteins (fig. . b) [ ] . hemagglutinin (h) and fusion (f) glycoproteins project from the viral surface and facilitate viral binding to cellular receptors and fusion with the host cell membrane, respectively. matrix (m) protein underlies the envelope providing structure. the inner nucleocapsid is composed of rna coated by nucleoprotein (n), bound by the polymerase complex which includes the large (l) polymerase protein, and phosphoprotein (p), a polymerase cofactor. the remaining nonstructural proteins include c and v. coronaviruses are spherical, enveloped, positive-sense, single-stranded rna viruses of family coronaviridae of approximately nm in diameter [ ] . coronaviruses are the causative agents of an estimated % of upper and lower respiratory tract infections in humans resulting in rhinitis, pharyngitis, sinusitis, bronchiolitis, and pneumonia [ ] . while coronaviruses are often associated with mild disease (e.g., hcov- e, hcov-oc , hcov-nl , hcov-hku ), severe acute respiratory syndrome coronavirus (sars-cov), a lineage b betacoronavirus, and middle east respiratory syndrome coronavirus (mers-cov), a lineage c betacoronavirus, are associated with severe and potentially fatal respiratory infection [ , ] . sars-and mers-cov transcribe and subgenomic rnas, respectively, which encode for the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural proteins (fig. . c) [ ] . s, e, and m are all integrated into the hostderived lipid envelope, and s facilitates host cell attachment to angiotensinconverting enzyme (ace)- receptors for sars-cov and dipeptidyl peptidase (dpp)- receptors for mers-cov [ , ] . the n protein encapsidates the viral genome to form the helical nucleocapsid. the viral replicase-transcriptase complex is made up of nonstructural proteins (nsp - ) including a unique proofreading exoribonuclease that reduces the accumulation of genome mutations [ ] . poxviruses are oval-to-brick-shaped double-stranded dna viruses of family poxviridae that range in size from to nm [ ] . viruses within genus orthopoxvirus that cause human disease include cowpox virus (cpxv), monkeypox virus (mpxv), vaccinia virus (vacv), and variola virus (varv), the etiologic agent of smallpox [ ] . poxviruses contain a biconcave viral core where the dna genome, dnadependent rna polymerase, and enzymes necessary for particle uncoating reside ( fig. . d ) [ ] . this nucleosome is surrounded by a core membrane that is flanked by two proteinaceous lateral bodies. a single lipid membrane surrounds the cellassociated form of the mature virion (mv). a second host-derived lipid envelope covers the extracellular virion (ev) [ , ] . poxvirus genomes are comprised of a large, linear double-stranded viral dna genome that encodes ~ genes. highly conserved structural genes are predominantly found in the middle of the genome, whereas variable virulence factor genes that function in immune evasion, virulence, and viral pathogenesis are found at the termini of the genome [ ] . wild aquatic birds are natural reservoirs for nearly all influenza a virus subtypes, which spread to domestic avian species and mammals, including humans [ ] . h n and h n subtypes are exceptions in that they have only been isolated from bats [ , ] . certain h and h subtypes are highly pathogenic to domestic poultry when transmitted from wild birds, known as highly pathogenic avian influenza (hpai) viruses [ ] . hpai viruses cause spillover infections in humans that may be severe or fatal. examples include outbreaks of h n and h n hpai viruses in asia with high case fatality among humans, although limited human-tohuman transmission [ , ] has been reported. hpai virus adaptations might lead to sustained human-to-human transmission, and so poultry outbreaks are managed by flock depopulation [ ] . influenza a subtypes isolated in swine include h to h , h , and n and n . subtypes that spillover into humans cause mild to severe illness and are known as swine "variant" viruses [ ] . currently circulating seasonal influenza a subtypes h n and h n and influenza b viruses, yamagata or victoria lineage, cause annual epidemics during fall through spring in temperate regions and infections throughout the year in the tropics [ ] . antigenic drift of h and n surface glycoproteins drives annual epidemics. from to , seasonal influenza caused approximately million illnesses, million hospitalizations, and , deaths in the united states alone [ ] . when two or more influenza a viruses infect a common host, such as a bird or pig, individual gene segments may recombine to form a novel virus, known as antigenic shift. influenza pandemics occur when novel viruses emerge into an immunologically naïve population and become adapted for sustained human-to-human spread. the "spanish" influenza pandemic was the most severe on record, resulting in an estimated million deaths [ ] . less severe pandemics occurred in , , and . in an effort to improve preparedness and response to seasonal, pandemic, and zoonotic influenza, the world health organization (who) conducts global surveillance of influenza a and b isolates (fig. . a) [ ] . measles is pathogenic for humans and nonhuman primates, although sustained transmission occurs only among humans raising potential for global elimination [ ] . historically, measles infected an estimated % of children by age years, resulting in approximately million global deaths each year [ ] . with the introduction of the measles vaccine in and advances in global vaccination programs, measles cases and mortality have drastically declined (fig. . b ). by , % of children worldwide had received at least one dose of the measles vaccine by age year, and during - , global measles mortality decreased by %, preventing an estimated million deaths [ ] . of the known measles genotypes, only five were detected in circulation during - . despite these gains, measles remains endemic in many regions of the world including africa, western pacific, south east asia, and europe, and measles has resurged in previously low-incidence areas (e.g., regions within europe and the americas) with epidemics attributable to importation of cases and suboptimal immunization coverage [ ] [ ] [ ] . an estimated % population immunity is required to prevent measles transmission within communities, a prerequisite for global elimination [ ] . chinese horseshoe bats are the putative reservoir for sars-cov, and dromedary camels are thought to be the reservoir for mers-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . animal-to-human transmission likely occurs following direct contact with intermediate hosts [ , ] . during the - sars epidemic, cases and deaths were reported from countries with no cases reported since ( fig. . c) [ ] . human-to-human transmission of sars-cov occurred primarily in healthcare settings with healthcare workers comprising % and > % of reported cases in china and canada, respectively [ ] . mers was first reported in saudi arabia in with > cases and > deaths reported from countries through [ ] . while most cases have been reported from the arabian peninsula, an imported case to south korea in resulted in a large outbreak in multiple healthcare facilities [ ] . mers transmission occurs primarily in healthcare facilities and to a lesser degree within households [ , ] . while the only known reservoir for varv is humans, it has been postulated that the virus emerged from an ancestral rodent-borne poxvirus more than , years ago [ , ] . numerous smallpox epidemics have occurred throughout recorded history including more than million fatalities during the twentieth century alone [ ] [ ] [ ] . smallpox was eventually eradicated following the implementation of the smallpox eradication program by the who from to ( fig. . d) which was facilitated by the absence of a zoonotic reservoir for varv [ ] . influenza viruses are transmitted by large respiratory droplets by coughing, sneezing, or talking or through contact with infected surfaces [ ] . influenza viruses bind to sugar moieties on the surface of airway epithelial cells where early viral replication, propagation, and shedding occur during an average - days of incubation period [ ] [ ] [ ] . peak viral replication typically occurs within days of symptom onset and resolves within - days, lasting longer in children and immunocompromised hosts [ ] [ ] [ ] . on average one person infects -one to two additional people; however, this reproductive number (r ) varies by viral strain and social and environmental factors [ ] . viral infection impairs the airway mucosal barrier and disrupts the alveolar-capillary membrane contributing to leakage of fluid and inflammatory cells into the alveolar space which impairs gas exchange resulting in hypoxemia [ , ] . bacterial coinfection often complicates severe cases contributing to respiratory failure and death, with staphylococcus aureus and streptococcus species as predominant copathogens [ ] . seasonal influenza virus infection is largely limited to the respiratory tract; however, h and h hpai viruses have a polybasic cleave site within the hemagglutinin allowing for replication outside of the respiratory tract [ , ] . infection with one strain of influenza does not confer complete immunity to other strains or subtypes [ ] . measles is among the most highly contagious respiratory infections, spread by exposure to large respiratory droplets through coughing, sneezing, or talking; by indirect contact with infected surfaces; or by small infectious droplets that can remain suspended in air for up to hours [ , ] . respiratory tract dendritic cells, lymphocytes, and alveolar macrophages are early targets of infection where during an average -to -day incubation period measles replicates and spreads to local lymphatics and respiratory epithelium and then disseminates in blood via infected lymphocytes to epithelial and endothelial cells in most organs [ ] [ ] [ ] . the infectious period begins with fever onset and extends for several days after rash appears [ ] . the estimated r of measles is - dependent upon host susceptibility and social and environmental factors [ ] . measles infects and disrupts tissues throughout the body; however, severe disease is primarily due to lower respiratory tract and neurological complications [ ] . natural measles infection confers lifelong immunity, and passive transfer of maternal antibodies protects newborns during the early postnatal period [ ] . individuals who recover from measles infection are at increased risk of secondary infection [ , ] . sars-cov is transmitted by large respiratory droplets and by contact with infected surfaces. epidemiologic data also support small droplet airborne transmission of sars-cov although the estimated r of . - . argues against this being a predominate route of spread [ , ] . sars-cov binds to angiotensin-converting enzyme (ace)- receptors on respiratory epithelial cells, pneumocytes, and alveolar macrophages resulting in diffuse alveolar damage and respiratory failure [ , ] . sars is a systemic infection with viremia detected in most cases affecting multiple cell types and organs [ , ] . acute kidney injury is multifactorial with evidence of acute tubule necrosis, vasculitis, and glomerular fibrosis, and central nervous system manifestations are at least in part attributable to direct infection of neurons resulting in edema and degeneration [ ] . mers-cov is transmitted by large respiratory droplets and by contact with infected surfaces with an estimated r of < to > outside of versus within healthcare settings, respectively [ ] . mers-cov binds dipeptidyl peptidase (dpp ) on respiratory epithelial cells and pneumocytes where it undergoes productive replication during a - days incubation period [ ] . viral shedding from the lower respiratory tract may persist for weeks [ , ] . viremia, while not documented in all cases, is associated with severe disease and productive infection of dcs, and macrophages is thought to facilitate immune dysregulation [ , ] . dpp is broadly expressed on cells outside of the lung; however, few autopsy data are available to define viral distribution [ , ] . varv is transmitted primarily by large respiratory droplets and to a lesser degree through contact with contaminated objects such as scabs, bedding, or clothing or by airborne small respiratory droplets [ , ] . varv is thought to replicate in airway epithelium and spread to regional lymph nodes [ , ] . varv replicates within lymph nodes and disseminates via the bloodstream seeding distant sights including skin, spleen, bone marrow, liver, kidney, and other organs [ ] . fever manifests following an average days incubation, and rash follows fever by - days, concurrent with high-level viral shedding from oropharyngeal secretions [ , ] . the estimated r of smallpox is between . and [ ] . high-level viremia is detected more often with hemorrhagic compared with ordinary type smallpox, although exact mechanisms of organ failure observed in fatal case are not well defined [ ] [ ] [ ] [ ] . influenza infection manifests as acute onset of fever, chills, malaise, headache, and myalgias following an average - days asymptomatic incubation period [ ] . most infections are self-limited resolving within - weeks. upper or lower airway complications include otitis media, sinusitis, bronchitis, and pneumonia with or without bacterial coinfection [ , , ] . risk factors for severe infection include age > years or < years; pregnancy; preexisting respiratory, cardiac, neurologic, or metabolic conditions; immunosuppression; and obesity. progressive lethargy and shortness of breath, typically within days of symptom onset, suggest development of lower respiratory tract complications which may rapidly progress to respiratory failure and death in severe cases [ ] . pneumonia due to influenza infection alone versus influenza and bacterial coinfection cannot be reliably distinguished by clinical or radiological grounds, and so a high index of suspicion is needed. influenza complications outside of the respiratory tract include exacerbation of underlying heart disease including ischemic heart disease and heart failure, myocarditis, encephalopathy, and encephalitis [ ] . measles infection manifests by acute onset fever, coryza, conjunctivitis, and cough [ ] . small white papules, koplik spots, appear on the buccal mucosa within days of fever onset, followed by development of diffuse maculopapular rash or days later. diarrhea commonly begins shortly following rash onset and may result in dehydration. symptoms typically resolve within days of fever onset in self-limited illness. groups at increased risk for measles complications include malnourished infants and those with vitamin a deficiency, adults > years old, and immunocompromised individuals [ ] . respiratory complications include otitis media, laryngotracheobronchitis (croup), and pneumonia. pneumonia, often complicated by bacterial coinfection, is the most common severe complication of measles contributing to respiratory failure and death [ , ] . predominant bacterial copathogens include streptococcus pneumonia, staphylococcus aureus, and haemophilus influenzae. three rare but severe neurologic complications occur [ ] . acute disseminated encephalomyelitis (adem) is a demyelinating autoimmune process that occurs within weeks of acute illness in approximately in cases. adem is characterized by fevers, seizures, and neurologic deficits. measles inclusion body encephalitis is a progressive lethal brain infection occurring within months of acute illness primarily among individuals with impaired cellular immunity. subacute sclerosing panencephalitis (sspe) occurs - years following initial infection resulting in seizures and cognitive and motor decline resulting in death. sspe affects an estimated in , infants under year of age and is attributed to host responses to defective viral particle production in the brain. following an average -day incubation period, sars-cov infection presents with fevers, chills, dry cough, headache, malaise, and dyspnea commonly followed by watery diarrhea [ ] [ ] [ ] . age > years and pregnancy are associated with severe disease manifested by progressive respiratory failure within weeks of illness onset [ , ] . common laboratory features of sars included lymphopenia, thrombocytopenia, abnormal coagulation parameters, and elevated lactate dehydrogenase, alanine aminotransferase, and creatine kinase levels [ ] [ ] [ ] . acute kidney injury and proteinuria were observed in % and % of patients, respectively [ ] . initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of days [ ] . gastrointestinal symptoms, including vomiting and diarrhea, occur in onethird of patients [ ] [ ] [ ] [ ] . the median times from symptom onset to hospitalization, icu admission, and death are , , and days, respectively [ ] . mers patients present with a rapidly progressing pneumonia requiring mechanical ventilation and additional organ support with the first week of illness [ ] . severe disease has been linked to comorbidities including diabetes mellitus ( %), chronic renal disease ( %), hypertension ( %), chronic cardiac disease ( %), chronic pulmonary disease ( %), and obesity ( %) [ ] . the median age of those with confirmed mers is years with a male-to-female ratio of . : [ ] . laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes [ , , , , ] . high lactate levels and consumptive coagulopathy have also been reported [ , ] . chest radiographic abnormalities are due to viral pneumonitis with or without secondary bacterial pneumonia, and acute kidney injury occurs in up to % of patients [ , , , [ ] [ ] [ ] . as the smallpox disease course was related to the clinical presentation of disease, rao proposed a clinical classification system [ ] that was later adopted by the who in [ ] . ordinary type smallpox was the most common clinical type of smallpox. the incubation period was - days and was followed by fever onset ( . - . °c), headaches, backaches, vomiting, and diarrhea [ ] . lesions first appeared on mucous membranes (including the tongue, palate, and pharynx) ~ day prior to macular rash development, where lesions began on the face followed by proximal regions of the extremities, the trunk, and the distal extremities. lesion development followed a centrifugal dispersion pattern, typically most dense on the face, with papules appearing within days of macular rash development. papules became vesicular ~ - days later followed by a pustular stage ( - days postrash) that peaked ~ days postrash. pustule resolution quickly followed and was accompanied by lesion flattening, fluid reabsorption, hardening, and scab formation ( - days postrash). rao proposed for ordinary type smallpox to be further subdivided based on the macular rash pattern [ ] . these included discrete ordinarytype smallpox, characterized by discrete skin lesions; confluent ordinary-type smallpox, where pustular skin lesions were confluent on the face and extremities; and semiconfluent ordinary-type smallpox, where skin lesions were confluent on the face but disparate over the rest of the body. modified-type smallpox, where lesions were less numerous than in ordinary-type smallpox, was primarily associated with vaccinated individuals and had an accelerated nonfatal disease course [ ] . flattype and hemorrhagic-type smallpox were the most lethal forms of the disease but were also very rare (~ % and % of patients, respectively) [ ] . flat-type smallpox had high cfrs in both unvaccinated and vaccinated patients ( % and %, respectively). hemorrhagic-type smallpox was nearly % fatal in both vaccinated and unvaccinated individuals, and death normally came prior to macular rash development. the clinical symptoms of flat-type smallpox were more severe during the prodromal period and did not subside. skin lesions were flat and often black or dark purple. respiratory complications were common and patients were febrile throughout disease. death typically occurred - days post-fever onset. hemorrhagic-type smallpox could be divided into early and late hemorrhagic-type smallpox. the early form was characterized by hemorrhage (primarily subconjunctival) early in the disease course. generalized erythema, petechiae, and ecchymosis within days of fever and flat matter lesions formed across the entire body surface. lesions turned purple by day with death by day as a result of cardiac and pulmonary complications. in the late form, hemorrhages occurred following rash development and death followed between and days post-fever onset. in healthcare settings, patients under evaluation for influenza should be isolated, and standard, droplet, and contact precautions should be implemented [ ] . traditional antigen-based rapid diagnostic assays (rdas) for influenza lack sensitivity and cannot be relied upon to rule out infection [ ] . newer antigen-based rdas that employ a digital scan of the test strip, and molecular assays that employ isothermal amplification technology have improved sensitivity and specificity that more closely approximates highly sensitive and specific reverse transcriptase polymerase chain reaction (rt-pcr)-based assays [ ] . acceptable sample types for influenza testing include nasopharyngeal swab or wash and bronchoalveolar lavage specimens. individuals suspected of zoonotic influenza infection should have case evaluation and specimen testing coordinated through local or state public health authorities. measles should be considered in patients without preexisting immunity and a compatible febrile rash illness. travel to a region with ongoing measles transmission or exposure to other individuals with a febrile rash illness should raise suspicion. patients under evaluation for measles require isolation and implementation of standard, airborne, and contact precautions. local or state health authorities should be contacted within hours to assist with confirmatory testing, case finding, and infection control. measles is typically confirmed by measles-specific igm serology or detection of measles rna in a nasopharyngeal, throat, or urine specimen by rt-pcr [ ] . a fourfold or greater rise in measles igg titers between acute and convalescent samples tested or more weeks apart can assist with diagnostic uncertainty. virus can also be cultured from respiratory, blood, and urine specimens in appropriate public health laboratories. while sars is no longer circulating, mers should be suspected in individuals with a compatible febrile illness and an epidemiological risk factor [ ] . risk factors include travel to the arabian peninsula or contact with a confirmed or suspected case within days of symptom onset. patients under evaluation for mers require isolation and implementation of standard, airborne, and contact precautions. confirmatory testing and infection control should be coordinated through local or state health authorities. mers may be confirmed in designated public health laboratories by rt-pcr testing of lower respiratory tract specimens [ ] . multiple other specimen types including upper respiratory tract samples, serum, and stool should also be collected for testing. serologic testing can be used to evaluate for suspected infection among individuals no longer shedding virus [ , ] . smallpox has not been observed in over years; however, concerns remain for use as a bioweapon. major and minor criteria have been established to assist clinicians in recognition of smallpox [ ] . individuals under evaluation should be isolated, and standard, airborne, and contact precautions should be implemented. local or state health authorities should be contacted to assist with confirmatory testing and public health interventions. pcr identification of variola dna or isolation of the virus from a clinical specimen is required to confirm a diagnosis in specialized highcontainment laboratories. annual seasonal influenza vaccination is recommended in the united states for all individuals aged months or older and has been associated with decreased risk of pneumonia and death, particularly among high-risk groups [ ] [ ] [ ] . seasonal influenza vaccination does not provide protection against novel strains. consequently, efforts are underway to develop a vaccine that would protect against most or all influenza strains [ ] . three classes of drugs are licensed for the treatment of influenza in the united states [ ] . adamantanes, including amantadine and rimantadine, are not currently recommended given resistance of circulating seasonal strains. baloxavir morboxil, a cap-dependent endonuclease inhibitor, was recently approved for the treatment of uncomplicated influenza [ ] . neuraminidase inhibitors (nai) include oral oseltamivir, inhaled zanamivir, and intravenous peramivir. prophylactic use of nais is recommended in unvaccinated individuals with risk factors for severe disease and during institutional outbreaks to limit spread. therapeutic use is recommended for individuals with suspected or confirmed influenza that have developed or are at high risk for influenza complications [ ] . influenza complications, including respiratory and multiorgan failure, are managed with supportive care. bacterial coinfection should be considered and empirically treated early pending results of microbiologic testing among severe cases. measles can be effectively prevented through vaccination, typically given in combination with vaccines for rubella (mr), mumps (mmr), or varicella (mmr-v). who recommends the first dose of measles vaccine be administered at or months of age in high and low prevalence settings, respectively [ ] . a second dose should be administered after a minimum of -week interval. nonimmune individuals that have been exposed to measles should receive post-exposure prophylaxis with mmr or immunoglobulin within hours or days, respectively, although not concurrently [ ] . clinical management of patients with measles consists of fluid, electrolyte, and nutritional support and early recognition and treatment of bacterial coinfection [ ] . two doses of vitamin a in children under years have been associated with reduced risk of pneumonia and death [ ] . who recommends administering , iu of vitamin a daily for days in children aged year and older, with reduced dosing in younger infants [ ] . there are currently no licensed therapeutics or vaccines for sars or mers. consequently, supportive care is the mainstay of treatment [ ] . renal replacement therapy is frequently required in severe illness [ , , ] . empiric antibiotics are often administered given potential for secondary bacterial infection. ribavirin and pegylated interferon alpha b have been administered to mers patients, although effectiveness data is lacking [ ] . aerosol-generating procedures including endotracheal intubation are associated with increased risk of healthcare worker infection necessitating strict adherence to infection control measures, including use of eye protection in addition to standard, airborne, and contact precautions [ ] . while routine smallpox vaccination ceased at the end of the smallpox eradication program, it is still employed for those at increased risk for exposure. first-generation vaccines comprise a significant proportion of both the us national and global vaccine stockpiles [ ] . however, first-generation vaccines carry high risk of adverse events due to use of replication-competent vacv and potential manufacturing contaminants. second-generation smallpox vaccines have reduced concerns for contaminants and are expected to have similar protective efficacy as first-generation vaccines. acam ® has garnered us food and drug administration licensure for vaccination of those at high risk for orthopoxvirus exposure and is part of the us strategic national stockpile [ ] . acam ® and the lister-derived vaccines rivm and elstree-bn also contribute to the global stockpile. imvamune (mva), a third-generation vaccine, is licensed in europe and canada and is part of the us national stockpile. passive immunization with vig has been employed to treat complications of vaccinations [ , ] . there has also been increasing interest in the development and licensure of small molecule antivirals for treatment of orthopoxvirus infections. cmx (brincidofovir), a dna synthesis inhibitor, has demonstrated protection against lethal varv in nonhuman primates [ ] and has been granted ophan drug designation while also being included in the us strategic national stockpile. st- (tecovirimat), which 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children middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease recovery from severe novel coronavirus infection ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review smallpox vaccine and its stockpile in clinical guidance for smallpox vaccine use in a postevent vaccination program the efficacy of vaccinial immune globulin. a -year study experience of anti-vaccinia immunoglobulin in the united kingdom efficacy of tecovirimat (st- ) in nonhuman primates infected with variola virus (smallpox) an orally bioavailable antipoxvirus compound (st- ) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge key: cord- -gtmo ixs authors: al-tawfiq, jaffar a.; rabaan, ali a.; hinedi, kareem title: influenza is more common than middle east respiratory syndrome coronavirus (mers-cov) among hospitalized adult saudi patients date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: gtmo ixs background: since the initial description of middle east respiratory syndrome coronavirus (mers-cov), we adopted a systematic process of screening patients admitted with community acquired pneumonia. here, we report the result of the surveillance activity in a general hospital in saudi arabia over a four year period. materials and methods: all admitted patients with community acquired pneumonia from to were tested for mers-cov. in addition, testing for influenza viruses was carried out starting april . results: during the study period, a total of patients were screened for mers-cov and only ( . %) tested positive. from january to december , a total of patients were tested for both mers-cov and influenza. none of the patients tested positive for mers-cov and ( . %) were positive for influenza. the detected influenza viruses were influenza a ( , . %), pandemic h n (n = , . %), and influenza b (n = , . %). pandemic h n was the most common influenza in with a peak in peaked october to december and influenza a other than h n was more common in with a peak in august and then october to december. conclusions: mers-cov was a rare cause of community acquired pneumonia and other viral causes including influenza were much more common. thus, admitted patients are potentially manageable with oseltamivir or zanamivir therapy. the emergence of the middle east respiratory syndrome coronavirus (mers-cov) in september had attracted international attention. the virus was initially isolated from a patient with a fatal community acquired pneumonia (cap) in saudi arabia [ ] . since then, multiple hospital outbreaks occurred within [ ] [ ] [ ] [ ] [ ] [ ] and outside saudi arabia [ ] [ ] [ ] [ ] . as of may st, , the world health organization reported laboratory-confirmed cases worldwide and at least related deaths [ ] . a wide-spectrum of mers-cov infection was described and ranges from mild to severe and fulminant infections leading to severe acute respiratory disease [ , [ ] [ ] [ ] . in the kingdom of saudi arabia, the number of mers-cov cases was as of may th, [ ] . since most of the cases of mers-cov in saudi arabia occurred due to intra-and inter-hospital transmissions, there was an increased amplification of the transmission [ ] [ ] [ ] [ ] [ ] [ ] ] . early detection and isolation of patients with mers-cov infection remains an important factor for the control of mers-cov transmission [ , ] . one of the goals of the surveillance of emerging respiratory viruses is the rapid and early identification and placement of control measures [ ] . following the initial description of the disease [ ] , the ministry of health in the kingdom of saudi arabia put in place a surveillance and screening program for patients admitted with respiratory illness [ ] . similarly, we adopted universal screening of admitted patients with community acquired pneumonia. here, we report the result of the surveillance activity in a general hospital in saudi arabia over a four year period. the study was conducted at a -bed general hospital, which also accepts referred patients. the hospital provides medical care for about , individuals eligible for medical care. the hospital has intensive care units (cardiac, medical, surgical, pediatric, and neonatal) [ ] . all admitted patients with community acquired pneumonia from suspected mers-cov was an acute febrile respiratory illness (fever, cough, or dyspnea) with radiographic evidence of pneumonia [ ] . we collected data for all suspected patients using a standard microsoft excel data collection sheet. both electronic and paper medical records were reviewed. we recorded the age and the date of admission and the mers-cov and influenza results. the study was approved by the johns hopkins aramco healthcare institutional review board (irb). suspected patients had either dacron-flocked nasopharyngeal swabs, or sputum testing for mers-cov. the testing was done at the saudi ministry of health mers-cov laboratory and at the main hospital. clinical samples were screened with real-time reverse-transcriptase (rt)epcr as described previously [ ] . the test amplified both the upstream e protein (upe gene) and orf a for mers-cov and if both assays were positive then the diagnosis of mers-cov was made, as described previously [ ] . the influenza test was carried out at the johns hopkins aramco healthcare centre, dhahran, using the cepheid ® xpert flu assay multiplex real-time pcr. the tested influenza viruses were pandemic h n , influenza a (other than h n ), and influenza b. the test was systematically carried out starting april . statistical analysis was done using excel and descriptive analyses were done for demographic, results of the tests and the monthly number of cases. minitab ® (minitab inc. version , pa , usa; ) was used to calculate the mean age ( ± sd) of patients with influenza. during the study period from to , a total of patients were screened for mers-cov and only ( . %) tested positive. during the first two years (april -march ), a total of patients were screened for mers-cov. only . % of them were positive for mers-cov (table ) and unfortunately these were not systematically screened for influenza. there was an increased number of tests in november -march (fig. ) . from april to december , a total of patients were tested for both mers-cov and influenza. none of the patients tested positive for mers-cov and ( . %) were positive for influenza. the detected influenza viruses were influenza a ( , . %), pandemic h n (n = , . %), and influenza b (n = , . %) ( table and fig. ). it is interesting to note the pattern of the influenza in and (fig. ) . pandemic h n was the most common influenza in and influenza a other than h n was more common in . the influenza season peaked october to december and the season had a peak in august and then october to december (fig. ). there was a significant difference in the mean age ( ± sd; % ci) of patients with h n and other influenza (fig. ) in this study, we presented the surveillance data on mers-cov over a four year period and the surveillance for influenza over a two year period. mers-cov was only detected in ( . %) from a total of patients as detailed in previous publication [ , ] . the earliest surveillance study from saudi arabia was done from october to september and tested a total of samples [ ] . in that study, the mers positivity rate was % [ ] . a second surveillance of mers-cov in saudi arabia was conducted from april , to february , and included a total of , suspected mers cases [ ] . the study showed only ( . %) mers-cov positive cases [ ] . in a study in the united states, two ( . %) imported cases were detected among patients-under investigation in - [ ] . in a surveillance study of unique persons from the united arab emirates between january , and april , , ( %) tested positive for mers-cov infection [ ] . in the south korea outbreak, ( %) had mers among , suspected cases [ ] . in a small study from saudi arabia, mers-cov was not detected in cases tested november and january (winter time) [ ] . thus, the overall positivity of mers-cov among a large cohort remains low. there is a need for a better tool to identify patients with high probability of mers-cov. however, a case control study and a large cohort study did not reveal significant predictor of mers-cov infection [ , ] . the monthly frequency of suspected mers cases that were tested showed variation with an apparent increase in the tested number during november -march . this apparent increase likely represented an increased activity of influenza during that time. there was no relation to the hajj season as it occurred during september - , (fig. ) . in addition at that time, there were no known outbreaks in the kingdom of saudi arabia to account for such an increase in the testing. the outbreaks occurred in al-hasa in may [ ] and in riyadh in august [ , , ] . previous studies had shown increased testing of patients for mers-cov during outbreaks [ ] . in the current study, the season was predominated by pandemic h n whereas influenza a was more common during . similarly, in the united states the - season was predominated by pandemic h n and h n was more common during the - season [ , ] .we found that influenza rather than mers-cov was more common among the tested patients. the findings are also consistent with other studies among travelers and pilgrims where influenza far exceeded mers [ ] [ ] [ ] [ ] [ ] . similarly, in a small study in saudi arabia, influenza viruses were detected in % of patients [ ] . similarly, among a small study of suspected mers cases in the united states of america, influenza was the most commonly ( %) identified respiratory agent [ ] and another study found influenza a and b in % of investigated patients [ ] . thus, it is important to test for common respiratory pathogens such as influenza viruses and it should be noted that identification of a respiratory pathogen should not exclude mers-cov testing [ ] . one report indicated co-infection with influenza and mers in four patients [ ] . however, epidemiologic differences between different countries should remain as an important predictor of the existence of mers-cov infection. the mean age of patients with h n was younger than the other influenza patients of at least years ( . vs. . for influenza a, . for influenza b, and . for influenza negative patients (p < . ). the inital cases of pandemic h n were also younger than the influenza negative patients [ ] . in a small study of patients, influenza b patients were younger than other influenza [ ] and in another study the mean age was lower for patients with influenza b ( . yr) than (h n ) pdm influenza infection. however, these studies included children and thus are not comparable with the present study [ ] . similar results were obtained in travelers returning from the middle east. these studies showed the lack of mers-cov among travelers and that influenza was more common among french travelers [ , ] , austrian returning pilgrims [ ] , british travelers [ ] , german travelers [ ] , and travelers to california, united states [ ] . the presence of influenza infection among those travelrs stress the need for influenza vaccination in travelers, notably tfor those going for the hajj and umrah in saudi arabia. in conclusion, mers-cov was a rare cause of community acquired pneumonia (cap) and other viral causes including influenza are much more common. the epidemiology of influenza mirrored the epidemiology of influenza worldwide. the study highlights the importance of the surveillance system to elucidate the epidemiology of respiratory infections in order to formulate appropriate control measures. interhospital and intra-hospital 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influenza not mers cov among returning hajj and umrah pilgrims with respiratory illness hajj-associated viral respiratory infections: a systematic review influenza a and b viruses but not mers-cov in hajj pilgrims laboratory testing for middle east respiratory syndrome coronavirus interim guidelines for clinical specimens from pui | cdc n the impact of coinfection of influenza a virus on the severity of middle east respiratory syndrome coronavirus pandemic influenza a ( h n ) in hospitalized patients in a saudi arabian hospital: epidemiology and clinical comparison with h n -negative patients differences in clinical features between influenza a h n , a h n , and b in adult patients clinical differences between influenza a (h n ) pdm & influenza b infections identified through active community surveillance in north india lack of mers coronavirus but prevalence of influenza virus in french pilgrims after infections in symptomatic travelers returning from the arabian peninsula to france: a retrospective cross-sectional study enhanced mers coronavirus surveillance of travelers from the middle east to england acute respiratory infections in travelers returning from mers-cov-affected areas all authors have no conflict of interest to declare. all authors have no funding. key: cord- - rz byz authors: morra, mostafa ebraheem; van thanh, le; kamel, mohamed gomaa; ghazy, ahmed abdelmotaleb; altibi, ahmed m.a.; dat, lu minh; thy, tran ngoc xuan; vuong, nguyen lam; mostafa, mostafa reda; ahmed, sarah ibrahim; elabd, sahar samy; fathima, samreen; le huy vu, tran; omrani, ali s.; memish, ziad a.; hirayama, kenji; huy, nguyen tien title: clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta‐analysis date: - - journal: rev med virol doi: . /rmv. sha: doc_id: cord_uid: rz byz middle east respiratory syndrome (mers) is a respiratory disease caused by mers coronavirus. because of lack of vaccination, various studies investigated the therapeutic efficacy of antiviral drugs and supportive remedies. a systematic literature search from databases was conducted and screened for relevant articles. studies reporting information about the treatment of mers coronavirus infection were extracted and analyzed. despite receiving treatment with ribavirin plus ifn, the case fatality rate was as high as % in the ifn‐treatment group and exactly the same in patients who received supportive treatment only. having chronic renal disease, diabetes mellitus and hypertension increased the risk of mortality (p < . ), and chronic renal disease is the best parameter to predict the mortality. the mean of survival days from onset of illness to death was . ( % ci, . ‐ . ) for the ifn group compared with . ( % ci, . ‐ . ) for the supportive‐only group (p = . ). delay in starting treatment, older age group, and preexisting comorbidities are associated with worse outcomes. in conclusion, there is no difference between ifn treatment and supportive treatment for mers patients in terms of mortality. however, ribavirin and ifn combination might have efficacious effects with timely administration and monitoring of adverse events. large‐scale prospective randomized studies are required to assess the role of antiviral drugs for the treatment of this high mortality infection. tions range from supportive to antiviral therapy. two in vitro studies suggested a possible efficacious effect of interferon alpha- b and ribavirin in the treatment of mers infection. , consequently, the investigators further examined the efficacy of these drugs in an animal study. potential benefits of these antiviral treatments in both in vitro and animal studies persuaded clinicians to question the feasibility and applicability of such an approach in humans. therefore, we aimed to recapitulate the evidence from all human published data about mers clinical management in a systematic review and metaanalysis, to summarize the efficacy and safety of current applied therapeutics and define risk factors associated with outcomes. as a result, we may provide a better approach to more compatible management of fatal consequences of mers infections and the risk imposed by recent outbreaks in densely populated areas. our study was performed according to the recommendations of the preferred reporting items for systematic reviews and meta-analyses. we developed a protocol of methods and registered it in the international prospective register of systematic reviews (pros-pero) (reference, crd ). in june , we conducted a systematic search of after removing duplicates, three trained reviewers were assigned to independently screen the titles and abstracts of all references generated from the aforementioned search strategy on the basis of the following inclusion and exclusion criteria. inclusion criteria were (a) any study that gives information about the treatment of mers-cov infection, (b) all types of study designs were included, and (c) no restriction was made with respect to language, age, and area. exclusion criteria were (a) data that could not be reliably extracted, (b) data sets considered as overlapping, (c) studies published before / / , (d) book chapter, thesis, letter, conference paper, poster, or editorial, and (e) animal or in vitro studies. three reviewers compared their screening results and discussed the differences. a consensus was reached through discussion. extracted data included publication year, year of research, country and city of the patients, year of subject recruitment, study design, participant enrollment, data collection method, baseline characteristics before treatment, diagnostic method of mers-cov, time from admission to treatment start, treatment for mers-cov, and outcome survival. this work was conducted by investigators evaluating the references independently, and all disagreements were discussed to reach a consensus from supervisors. the quality of included clinical data was assessed using (care) statement for case reports and the metrics tool for nonrandomized studies. three reviewers were assigned to assess each included reference independently. mortality rates were treated as dichotomous variables with their respective % confidential intervals (ci). statistical heterogeneity was assessed using the i statistic , and assumed to be influential when i was greater than % or p ≤ . . , a fixed-effect model was used because there was no evidence of heterogeneity between studies. meta-analysis was performed using data analysis and statistical software (stata) that was developed by statacorp. fisher exact (or chi-square, as appropriate) and mann-whitney u tests were used for the categorical and continuous variables, respectively. the classification and regression tree (cart) model was used to identify independent variables that predict mortality outcome. invasive ventilation and renal replacement therapy were also chosen as the outcomes in the cart model because of direct correlation with severity and mortality. all possible variables were extracted to build cart (table s a total of references were retrieved. upon screening them regarding inclusion and exclusion criteria from section , eleven references were included for data extraction and analysis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] additionally, references were identified through manual search. - , , so a total of studies were eligible for selection as shown in figure . of the studies including patients, were case reports, [ ] [ ] [ ] , , , , , , were case series, , and were observational studies. , , , saudi arabia was the country of origin for patients in studies, and the rest were from france, greece, and qatar. detailed characteristics of patients in our included articles are presented in table . eight studies used specific antiviral treatment , , , , [ ] [ ] [ ] while studies used supportive treatment (including invasive ventilation, prone position, renal replacement therapy, vasopressors, corticosteroids, immunoglobulins, and oseltamivir). - , , , , omrani et al used both specific antiviral treatment and supportive treatment. the specific antiviral treatments were ifn (alpha- a, alpha- b, and beta- a), ribavirin, and several others including tenofovir, emtricitabine, lopinavir, and ritonavir. among patients recruited, were reported with detailed information regarding baseline characteristics, comorbidities, treatments, outcomes, and some with survival time, which was subsequently used to perform univariable analysis and kaplan-meier survival curves (table s , found in the supporting information). hemodialysis dependency appeared in higher frequency in the ifn group than in the supportive-only group (p = . ). conversely, renal replacement therapy and vasopressors were used more often in the supportive-only group than in the ifn group (p = . ) ( table ). the quality of case reports [ ] [ ] [ ] , , , , , , and case series , was assessed using the care checklist (table s , found in the supporting information). all of them contained an introduction and described the importance of the case in the abstract, main introduction section, demographics and symptoms of the patients, significant the quality of observational studies , , , was assessed using the metrics tool (table s , found in the supporting information). three of them had a score , , of of as they described study design, characteristics of the patient population, inclusion criteria, method quality, and mers diagnosis. however, none of the studies described data collection method, assignment method of patients, exclusion criteria, and interpretation. treatment with antiviral drugs, other than oseltamivir, was reported in studies. ifns (ifn alpha- a, alpha- b, or beta- a) in combination with ribavirin were the common remedies used in all studies. ifn beta- a was used in studies (n = ), both of which used a dose of μg/wk for treatment. , ribavirin administration was started with a loading dose followed by subsequent doses in all studies. the loading dose was mg for all studies while mg in one study of alghamdi et al. the subsequent doses, however, were variable among studies and ranged between and mg/d. the frequency of administration of ribavirin was doses per day in studies , and doses per day in another studies. , , duration of treatment with ribavirin was also variable and ranged between and days. the subsequent oral ribavirin dose was adjusted according to the calculated creatinine clearance in studies. [ ] [ ] [ ] however, the duration of treatment ( - days) and the loading dose ( mg) used in the studies were similar, regardless of creatinine clearance (table s , found in the supporting information). in addition to treatment with ifn and ribavirin, the treatment regimen in shalhoub et al included treatment with tenofovir/ regarding mortality, studies with more than cases were included in the meta-analysis. the pooled proportion of mortality was . ( . - . ) from studies including mers patients (figure ). in patients received ifn treatment, the mortality rate was high ( %) in spite of receiving treatment with ribavirin plus ifn (alpha- a, alpha- b, or beta- a). likewise, the mortality rate was high in patients who received supportive treatment only ( %, n = ). there was no statistical significant mortality difference when comparing mortality of both groups (p = ) ( there was a significant difference between death and survival in patients with chronic renal disease (crd the modeling tool, cart, identified crd as the best parameter to predict mortality ( figure a ). the performance of the decision tree tool that classified mortality outcome was at an accuracy of . %, sensitivity of . %, specificity of %, ppv of %, and npv of %. in addition, treatment with inotropes was exhibited as the best parameter to predict renal replacement therapy outcome ( figure b ). the performance of the decision tree tool that classified renal replacement therapy outcome was at an accuracy of . %, sensitivity of . %, specificity of %, ppv of . %, and npv of . %. no significant results were detected regarding the invasive ventilation outcome. survival time from admission to death was compared for ifn-treated (n = ) and for non-ifn (supportive care only, n = ) patients. all cases died within days after hospital admission ( figure a ). only one female case treated with ifn was eliminated from analysis because the patient remained intubated when the original study ended. however, she had met death criteria; thus, she was included the difference between the groups was statistically significant (p = . ) ( figure b ). the longer survival period from onset to death was simply attributed to the duration between onset and admission. our systematic review highlights the significance of age and period between the illness onset and start of antiviral therapy in mers cases' prognostic assessment. our results revealed that younger age and there is no evidence of any difference between ifn treatment and supportive treatment for mers patients in terms of mortality. the ribavirin and ifn combination might have promising effects where therapy can be started promptly and adverse effects monitored carefully; a randomized controlled trial is required to assess this possibility. concerning prognostic factors, delayed treatment, older age, and accompanying comorbidities such as hypertension, dm, chronic kidney disease, and dialysis dependence are associated with worse outcomes. because of high fatality, the seriousness of this newly emerging disease, and a limited number of available cases, we believe there is an urgent need for large-scale clinical trials on the efficacy of antiviral treatment of mers-cov infections. the authors declare no competing interests. orcid ahmed abdelmotaleb ghazy http://orcid.org/ - - - nguyen tien huy http://orcid.org/ - - - infectious diseases. mers surges again, but pandemic jitters ease middle 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predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: vgpphiu background: after the middle east respiratory syndrome (mers) outbreak in korea, prediction of pneumonia development and progression to respiratory failure was emphasized in control of mers outbreak. methods: mers-cov infected patients who were managed in a tertiary care center during the korean mers outbreak were reviewed. to analyze predictive factors for pneumonia development and progression to respiratory failure, we evaluated clinical variables measured within three days from symptom onset. results: a total of patients were included in the study: patients ( . %) did not develop pneumonia, developed pneumonia without respiratory failure ( . %), and progressed to respiratory failures ( . %). the identified predictive factors for pneumonia development included age ≥ years, fever ≥ . °c, thrombocytopenia, lymphopenia, crp ≥ mg/dl, and a threshold cycle value of pcr less than . . for respiratory failure, the indicators included male, hypertension, low albumin concentration, thrombocytopenia, lymphopenia, and crp ≥ mg/dl (all p < . ). with ≥ two predictive factors for pneumonia development, % of patients developed pneumonia. patients lacking the predictive factors did not progress to respiratory failure. conclusion: for successful control of mers outbreak, mers-cov infected patients with ≥ two predictive factors should be intensively managed from the initial presentation. middle east respiratory syndrome (mers) is an emerging lethal respiratory disease caused by a novel betacoronavirus (mers-cov). from may to july , there was a hospital-associated mers outbreak in the republic of korea reporting laboratory-confirmed cases, which is the largest recorded outbreak outside the arabian peninsula. e the outbreak featured several super-spreading events with unexpectedly high human-to-human transmission rate: of cases ( . %) were transmitted from only three patients. , e as these large transmission clusters were exclusively originated from patients with pneumonia, prediction of pneumonia development has been emphasized in control of mers outbreak. in addition, pneumonia progression to respiratory failure should be anticipated in advance to avoid urgent intubation or cardiopulmonary resuscitation which might break protection of healthcare workers. although several studies analyzed prognostic factors for fatal outcome, e predictive factors for pneumonia development and progression to respiratory failure have not been reported. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated mers-cov infected patients managed in a tertiary care center during the mers outbreak in korea. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we reviewed the electronic medical records of who were diagnosed with mers-cov infection and admitted at samsung medical center, a tertiary care university hospital which managed the largest number of mers-cov infected patients as a single center during the korean mers outbreak. as it is still unclear whether initially asymptomatic patients would develop pneumonia or not, we included all the mers-cov infected patients managed at our hospital during the outbreak regardless of symptoms presence. to avoid confusion with the case definition of mers which did not included asymptomatic cases, we used the term of 'mers-cov infected patient' rather than 'mers patients' throughout the present paper. mers-cov infections were confirmed on the basis of rrt-pcr assays targeting upstream of the e gene and the open-reading frame gene a. , disease status of included patients was assessed at six weeks from their symptom onset and patients were divided into three groups depending on pneumonia development and progression to respiratory failure: patients without the development of pneumonia (group ), patients who developed pneumonia without respiratory failure (group ), and pneumonia patients who progressed to respiratory failure (group ). for practical purposes, respiratory failure was defined as the need for mechanical ventilation (mv). the institutional review board of our hospital approved the present study. we retrospectively collected data from electronic medical records and epidemiologic investigation. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated clinical variables measured within three days from symptom onset. during the korean mers outbreak, fever was defined as body temperature ! . c to increase sensitivity of screening and the same definition was used in the present analysis. thrombocytopenia was defined as a platelet count lower than  cells/mm , lymphopenia as an absolute lymphocyte count lower than , cells/mm , and hypoalbuminemia as albumin concentration lower than . g/dl. lower respiratory tract specimens including sputum and endotracheal aspirates were used for mers-cov rrt-pcr. cycle threshold (ct) values of mers-cov rrt-pcr were used as a surrogate of viral load. pneumonia development of mers-cov infected patient was defined as presence of parenchymal infiltration on chest x-ray with respiratory symptoms. test days or events were counted from the day of symptom onset for each patient: day was defined as the day of symptom onset. for asymptomatic patients, the day of diagnosis of mers-cov infection was considered as day . to identify predictive factors for pneumonia development and progression to respiratory failure, clinical variables measured within three days from symptom onset were compared. for evaluation of pneumonia development, patients who developed pneumonia (group and ) were compared to those who did not (group ). for factors for respiratory failure, patients who progressed to respiratory failure (group ) were compared to those who did not (group and ). student's t-tests or mannewhitney u tests were used to compare continuous variables, and chi-square tests or fisher's exact tests were used to compare categorical variables. statistically significant continuous variables were re-categorized into binary factors using threshold values between mean of each group, which showed lowest p value. statistically significant categorical variables and binary factors re-categorized from continuous variables were defined as predictive factors. for significant predictive factors, as a measure of association, odds ratio (or) and % confidence interval (ci) for or were calculated using the woolf procedure. multivariate analysis was not performed due to the limited sample size. all pvalues were two-tailed, and those < . were considered to be statistically significant. ibm spss statistics version . for windows (ibm, armonk, ny, usa) was used for all statistical analyses. time course of pneumonia development and progression to respiratory failure a total of mers-cov infected patients were hospitalized during the outbreak with patients in group (including asymptomatic patients), patients in group , and patients in group . the clinical course of symptomatic mers patients progressed serially: patients developed initial symptoms after a median -day incubation period (iqr . e . ), pneumonia after a median of days from symptom onset (iqr . e . ), and respiratory failure after a median of days from symptom onset (iqr . e . ). in group patients, it took a median of days from desaturation to respiratory failure (iqr e days). the development and progression of pneumonia by time sequence is depicted in fig. . no one developed pneumonia before day of symptom onset. to evaluate predictive factors for pneumonia development, demographics, underlying diseases, and clinical variables of patients in group and were compared to those of patients in group (tables and ). identified predictive factors are summarized in table with odd ratios (or). increasing age was significantly associated with pneumonia development as a continuous variable (p z . ), and age older than years was a predictive factor for the development of pneumonia (or, . ; % ci, . e . ; p z . ). although proportion of male also increased with progression of pneumonia ( . %, . %, and . % for group , , and , respectively), statistically significant association between male sex and the pneumonia development was not identified (p z . ). fever over . c by day were more frequently detected in patients with pneumonia ( . %, . %, and . % in groups , , and , respectively), and was identified as a predictive factor for the development of pneumonia (or, . ; % ci, . e . ; p z . ). thrombocytopenia (or, not applicable (na); p z . ), lymphopenia (or, . ; % ci, . e . ; p z . ), elevated c-reactive protein (crp ! mg/dl; or, na; p z . ), and high viral load (ct value < . ; or, . ; % ci, . e . ; p z . ) were distinctly observed in pneumonia patients from the initial presentation, and identified as predictive factors for pneumonia development. to evaluate predictive factors for progression to respiratory failure, patients in group were compared to those in group and (tables e ). although the mean age of patients in each group tended to increase with progression of pneumonia ( . , . , and . years in groups , , and , respectively) and increasing age was significantly associated with respiratory failure as a continuous variable (p z . ), there was no statistically significant cut-off value for prediction of respiratory failure. proportion of male also increased with progression of pneumonia, and male sex was a predictive factor for respiratory failure (or, . ; % ci, . e . ; p z . ). among underlying diseases, hypertension was identified as a predictive factor for respiratory failure (or, . ; % ci, . e . ; p z . ). initial symptoms including fever were not significantly different between patients who progressed to respiratory failure and those who did not. among initial laboratory test results, thrombocytopenia (or, . ; % ci, . e . ; p z . ), lymphopenia (or, . ; % ci, . e . ; p z . ), hypoalbuminemia (or, . ; % ci, . e . ; p z . ), and elevated crp (crp ! mg/ dl; or, . ; % ci, . e . ; p z . ) were distinctly observed in group patients, and identified as predictive factors for respiratory failure. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) by number of predictive factors were presented in table . when patients presented with ! two of the predictive factors for pneumonia development, % of these patients developed pneumonia (sensitivity . %, specificity . %, ppv %, and npv . %). patients lacking the predictive factors for respiratory failure did not progress to respiratory failure. when patients presented with ! two of these predictive factors, . % of these patients progressed to respiratory failure (sensitivity . %, specificity . %, ppv . %, and npv . %). initial rapid propagation of mers-cov during the korean mers outbreak was caused by three super-spreading events responsible for . % of all transmissions. , , in addition to these super-spreaders, transmission of mers-cov despite application of personal protective equipment (ppe) occurred from patients with progressed pneumonia at our hospital. in this regard, identifying the predictive factors for pneumonia development and progression is not only important in patient care, but also in infection control to prevent further in-hospital transmission. the present analysis of predictive factors for pneumonia development and progression to respiratory failure using variables obtained by day of symptom onset could be conducted owing to the observation of entire clinical course of the disease from the exposure to mers-cov. compared to mers outbreaks in the arabian peninsula where community-acquired infections might simultaneously occur from animals, identifying epidemiologic links, exposure date, and symptom onset were relatively clear for each case. , in our observation, the clinical course of symptomatic mers patients progressed serially and no one developed pneumonia before day of symptom onset. this is the reason why we used clinical data obtained by day of symptom onset. there is no other comparable data to which presented time interval from the symptom onset to the development of pneumonia. although there were no ideal cut-off scores of predictive factors with good sensitivity and specificity, it should be noted that % of patients with ! two predictive factors for data are expressed as the number (%) of patients or mean ae sd. as missing values were also removed from the population parameter, variables with missing values are expressed with modified population parameters. continuous variables with statistical significance were re-categorized into binary factors which are presented in italics. abbreviations: res., respiratory; wbc, white blood cell; alc, absolute lymphocyte count; ast, aspartate transaminase; alt, alanine transaminase; bun, blood urea nitrogen; crp, c-reactive protein; ld, lactate dehydrogenase; ct, threshold cycle; rrt-pcr, real-time reverse transcriptase polymerase chain reaction. a data are presented as mean value of day e ae sd. pneumonia actually progressed to pneumonia. thus, careful and intensive management should be implemented for such patients including adequate isolation of patient in an airborne infection isolation room (aiir), minimizing chance for exposure, application of ppe with hooded coverall, and consideration of experimental antiviral treatment. , e for patients with ! two predictive factors for respiratory failure, aiirs in intensive care units should be prepared for early elective intubation. although the time interval from symptom onset to mv support was much longer than in previous reports (median days versus days), we also experienced rapid progression of pneumonia from the moment of desaturation: % of group patients required mv within days from desaturation (median , iqr e days). to avoid urgent situations which might break protection of healthcare workers, elective intubation should be considered when desaturation begins to progress. in addition, sensitivities of predictive values are relatively low with cut-off value of ! two factors, clinical course of patients with any predictive factors also should be carefully monitored. of note, thrombocytopenia, lymphopenia, and increased crp level were shared predictive factor for the pneumonia development and respiratory failure. they were observed in the very early course of the illness, indicating that inflammation had already been enhanced. lymphopenia and thrombocytopenia presenting from the initial presentation of severe mers-cov infected patients were also observed in the recent report by min et al. although time of measurements were not specifically described, these laboratory abnormalities were previously observed in severe mers cases and other respiratory viral illnesses including severe acute respiratory syndrome (sars) and influenza, which are caused by intense inflammatory response to the viruses. , e this is the first report that identified these laboratory findings as predictive factors for pneumonia development and progression to respiratory failure in mers. although other predictive factors for pneumonia development and respiratory failure were different due to discordance of statistical significance, they shared the same spectrum of etiology. age increased according to pneumonia progression and was associated with both pneumonia and respiratory failure as a continuous variable (p z . and p z . , respectively). these findings correlate with previous data suggesting that old age is associated with poor prognosis. , e , , similarly, the proportion of males increased according to disease severity, though male sex was only significant for predicting respiratory failure. although the mean age of males was older than that of females ( . and . years, respectively), it was not statistically significant (p z . ). previous data also reported that overall proportion of male was higher and was associated with severe infection. , it could be meaningful observation that the same finding was observed in the republic of korea where the social activity of females is not restricted, especially among healthcare workers. on the other hand, hypoalbuminemia and hypertension were predictive factors only for respiratory failure, while high viral load was predictive factor for the development of pneumonia. these factors were related with severe disease and poor prognosis of mers in previous reports. , , , , , , our study has several strengths and limitations. due to its retrospective nature, there may be a bias regarding collecting medical information in retrospective manner. however, as all electronic medical records were standardized to record symptoms and signs in the same way from the beginning of the outbreak, bias was minimized. secondly, there were missing values when calculating the sensitivity and specificity of predictive factors, which is another limitation of retrospective study. lastly, the present study did not perform multivariate analysis due to limited sample size and need to be validated. prospective studies with sufficient number of patients are required for validation of the predictive factors identified in the present study. despite these limitations, our data would be suitable for identifying predictive factors because we could observe entire course of the disease from exposure and apply homogenous management to patients. in conclusion, based on cases from a single tertiary care hospital during the largest mers outbreak outside of the arabian peninsula, we identified six predictive factors for the development of pneumonia and 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epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease we would like to express our sincerest condolences to the patients and families who suffered from the mers outbreak. we also greatly appreciate the health care personnel and staff members at samsung medical center and all other hospitals who worked together to overcome the mers outbreak. key: cord- -jmoyy rb authors: assiri, abdullah m.; midgley, claire m.; abedi, glen r.; saeed, abdulaziz bin; almasri, malak m.; lu, xiaoyan; al-abdely, hail m.; abdalla, osman; mohammed, mutaz; algarni, homoud s.; alhakeem, raafat f.; sakthivel, senthilkumar k.; nooh, randa; alshayab, zainab; alessa, mohammad; srinivasamoorthy, ganesh; alqahtani, saeed yahya; kheyami, ali; hajomar, waleed husein; banaser, talib m.; esmaeel, ahmad; hall, aron j.; curns, aaron t.; tamin, azaibi; alsharef, ali abraheem; erdman, dean; watson, john t.; gerber, susan i. title: epidemiology of a novel recombinant middle east respiratory syndrome coronavirus in humans in saudi arabia date: - - journal: journal of infectious diseases doi: . /infdis/jiw sha: doc_id: cord_uid: jmoyy rb background: middle east respiratory syndrome coronavirus (mers-cov) causes severe respiratory illness in humans. fundamental questions about circulating viruses and transmission routes remain. methods: we assessed routinely collected epidemiologic data for mers-cov cases reported in saudi arabia during january– june and conducted a more detailed investigation of cases reported during february . available respiratory specimens were obtained for sequencing. results: during the study period, mers-cov cases were reported. full genome (n = ) or spike gene sequences (n = ) were obtained from individuals. most sequences ( of [ %]) formed a discrete, novel recombinant subclade (nrc- ), which was detected in regions and became predominant by june . no clinical differences were noted between clades. among cases reported during february , had no recognized risks for secondary acquisition; of these also denied camel contact. most viruses ( of ) from these individuals belonged to nrc- . discussions: our findings document the spread and eventual predominance of nrc- in humans in saudi arabia during the first half of . our identification of cases without recognized risk factors but with similar virus sequences indicates the need for better understanding of risk factors for mers-cov transmission. middle east respiratory syndrome coronavirus (mers-cov) is known to cause severe respiratory illness in humans, with deaths recorded in %- % of cases reported globally [ ] . since the first recognition of mers in , all cases reported to the world health organization have been linked to the arabian peninsula, with > % of cases reported from saudi arabia [ ] . camels (camelus dromedarius) have been suspected as a reservoir for mers-cov, based on case investigations [ ] , serologic studies [ ] , and the isolation of virus from camels [ ] [ ] [ ] [ ] [ ] . direct camel contact has also been identified as a risk factor for human illness [ ] . secondary human transmission has been demonstrated among close contacts of symptomatic cases, primarily following healthcare-associated exposures [ ] [ ] [ ] and, to a lesser degree, household exposures [ ] . there is no definitive evidence of sustained human-to-human transmission in the community [ ] . mers-cov infection can exhibit a wide range of clinical manifestations, including mild or limited symptoms among those identified through contact tracing [ ] . prolonged viral shedding from the respiratory tract of those without obvious symptoms has been demonstrated [ ] , and transmission related to unrecognized cases has been suggested [ , ] but not documented. mers-cov sequences obtained to date suggest periodic introductions of the virus into human populations, presumably from an animal reservoir, with subsequent limited chains of transmission in households and healthcare settings. the temporal persistence of identified viral clades appears limited, consistent with an r of < [ , ] . intervals between the beginning and end of the circulation of a clade vary, with longer intervals suggesting the existence of undetected human cases [ ] . cases and clusters continue to be reported from countries in or near the arabian peninsula, presenting an ongoing threat for broader transmission [ ] . to assess the epidemiologic and clinical features of the disease, we investigated all cases reported by the saudi arabia ministry of health (moh) during january-june , and we attempted genetic sequencing on all available specimens. this investigation was part of an emergency public health response and was determined to be nonresearch by the moh and centers for disease control and prevention (cdc) and therefore not subject to institutional review board review. at the time of this investigation, reporting in saudi arabia was required for all patients with clinical or radiologic evidence of mers-cov infection and a positive real-time reverse transcription-polymerase chain reaction (rrt-pcr) test result [ ] . all rrt-pcr-positive cases identified at non-moh facilities required confirmation at moh laboratories. we assessed the routinely collected epidemiologic information for all mers-cov cases reported by the moh during january- june , to provide a basic epidemiologic description. for this analysis, we included only individuals who met the case definition described above (ie, symptomatic cases). february was a period of increased reporting. to perform a more in-depth analysis, we collected additional information for all individuals with laboratory-confirmed mers-cov infections during february . this included all cases meeting the case definition as described above, as well as those identified as having a laboratory-confirmed case but no recognized symptoms; individuals not meeting the case definition [ ] were typically identified through contact tracing. we reviewed available moh case investigation records and data reported through the moh health electronic surveillance network. we collected demographic information, medical history, outcome information, and treatment location. we assessed the likelihood of acquisition from another person (secondary acquisition) by determining whether a patient ( ) was a healthcare professional (hcp), ( ) had been admitted to a healthcare facility - days before illness onset, ( ) had visited any healthcare facility in the days before illness onset, or ( ) had direct contact with either another documented case of mers-cov infection or with someone with an acute respiratory illness of unknown cause in the days prior to illness. when it was not possible to determine the criteria described above by using available information, we conducted telephone interviews (in arabic) to collect additional exposure information. proxies (a close friend or immediate family member who was familiar with the patient's activities during this period) were interviewed if the case was deceased, still hospitalized, or too ill to participate. among cases without any of the aforementioned risk factors for secondary acquisition (hereafter referred to as sporadic cases), we asked during telephone interviews about the history of exposure to camels [ ] . interviewees were prompted to describe examples of camel exposures, including direct contact or visiting a live market, slaughterhouse, or race where camels were present. we also assessed travel history. demographic and clinical characteristics were reported, and differences were assessed for significance by using χ , wilcoxon rank sum, and kruskal-wallis tests, where appropriate. data were analyzed using sas, version . (sas institute, cary, north carolina). molecular testing was performed on all respiratory specimens available during january-june . specimens and molecular testing at the moh respiratory specimens, including nasopharyngeal and oral pharyngeal swabs, both separate and combined, nasopharyngeal and tracheal aspirates, and sputa collected from suspected mers cases were tested at moh laboratories by upe and orf a rrt-pcr assays [ ] . available specimen aliquots (or rna extracts) that tested positive for mers-cov by both assays were shipped on dry ice to the cdc (atlanta, georgia) for sequencing. sample aliquots ( - µl, if available) were extracted on a nuclisens easymag (biomerieux), and µl of total nucleic acid elutes were recovered. the specimen extract were retested by mers-cov n and/or n rrt-pcr assays [ ] , and sequencing was attempted on confirmed positive samples. overlapping nested primer sets were used for amplification and sanger sequencing of the mers-cov spike genes and selected genomes (supplementary table ). amplicon sequencing was performed in both directions, using sequencing and internal amplification primers, with the bigdye terminator v . cycle sequencing kit on a xl genetic analyzer (thermo fisher scientific). sequencher . software (gene codes) was used for sequence assembly and editing. nucleotide sequences were aligned using clustal x, version . , implemented in bioedit, version . . . phylogenies were estimated using neighbor-joining and maximum likelihood methods implemented in molecular evolutionary genetics analysis, version . [ ] , and bayesian inference, using mrbayes v . . [ ] . the neighbor-joining method used maximum composite likelihood distance estimation and maximum likelihood used general time reversible (gtr) model of nucleotide substitution with γ-distributed rate variation and a proportion of invariant sites (gtr + g + i). mrbayes was performed under a gtr model of nucleotide substitution with categories of γ-distributed rate heterogeneity and a proportion of invariant sites (gtr + + i). putative recombination events were identified using recombination detection program software, version . (rdp ; available at: http://web.cbio.uct.ac.za/~darren/rdp.html), with the default settings [ ] . the complete genome sequence of each of the viruses in the nrc- clade was aligned with the genomes outside the clade. the multiple sequence alignment was then imported into the rdp software for detection of recombination. the software uses several algorithms, including gene-conv, bootscan, maxchi, chimaera, siscan, and seq, to detect putative recombination events. the potential minor and major parental sequences and the beginning and end breakpoints of the potential recombinant sequences were also defined by rdp software. putative recombinant events were considered significant when a p value of ≤ . was observed for the same event, using ≥ algorithms. time estimates to the most recent ancestor were calculated using the bayesian markov chain monte carlo (mcmc) method implemented in beast v . . [ ] . the coding regions (orf ab, s, orf , orf a, orf b, orf , e, m, and n) in the genomes grouping within nrc- were concatenated, and the hky+ Γ substitution model was used with independent rates for each of the positions in the codon. a lognormal relaxed molecular clock (uncorrelated) was used with gaussian markov random field bayesian skyride coalescent. bayesian mcmc analysis was run for million steps. parameters for tmrca, rate, and trees were sampled every steps, with the first % removed as burn-in. time estimate values thus obtained were also compared with strict and exponential relaxed clock models. during january- june , mers-cov cases from of the regions of saudi arabia were reported by the moh; mers-cov-positive individuals with no recognized symptoms, and who therefore did not meet the case definition, were not included. the longest period between case reports was days. among these cases, were hospitalized, and ( %) died. most patients were male ( [ %]) and of saudi nationality ( [ %]). median age was years (range, - years). of the symptomatic cases reported during the study period, had respiratory specimens available for further testing at the cdc; specimen was also available from an individual with no recognized symptoms who did not meet the case definition. of the available respiratory specimens collected during january- june , spike gene sequences were obtained from (supplementary table table ). recombination analysis on the newly available genome sequences from nrc- identified possible recombination events involving sequences from outside the clade as potential minor and major parental strains. the first event had a predicted breakpoint at nucleotide position ( % confidence interval [ci], - ), located in orf ab, and the second event had a predicted breakpoint at ( % ci, - ), located in the spike gene. recombination analysis was performed using rdp software, and events detected with a p value of ≤ . were considered evidence of true recombination (supplementary table ). to date the emergence of nrc- , mcmc analysis was performed on the concatenated coding regions of the genomes grouping within nrc- , using beast. the most recent common ancestor of the virus was approximately . years table ). among the cases reported during january- june , nrc- was first detected in a case with onset in mid-january (figure a) . during the study period, nrc- viruses were detected in regions of saudi arabia (figure ) , and the proportion of patients identified with nrc- increased steadily over time ( figure b ). nrc- was next compared to past and present subclades within clade b, using sequences available in genbank (figure ). nrc- was more widely distributed geographically than any other identified members of clade b. the duration of circulation of recognized subclades ranged from to days. at the conclusion of our investigation period, nrc- had been circulating for days, which was longer than of other identified subclades. in our analysis, the longest circulating subclade reported was riyadh_kkuh- _ , which was first detected in july and was still circulating as of may . during our investigation period, of sequenced viruses belonged to riyadh_kkuh- _ . no viruses belonging to clade a were detected. a comparison of patients infected with nrc- versus other circulating viruses revealed no significant differences in age, sex, rate of mortality, time between onset of symptoms table ). there was also no difference in mean cycle threshold values, a proxy for virus load, with respiratory specimens containing nrc- versus other clades, although these were not adjusted for timing of specimen collection (supplementary table ). for our more detailed analysis of cases reported during - february , we identified mers-cov-positive patients ( table ). of these, patients ( %) satisfied the case definition for routine reporting and required hospitalization; the remaining individuals ( %) had no recognized symptoms (and did not satisfy the case definition) but are included in this analysis. the patients were reported from different healthcare facilities across regions in saudi arabia; of these facilities reported ≥ cases within the same -day period. of these patients, sequences could be obtained from , of which ( %) were associated with nrc- . no clinical differences were apparent when comparing nrc- to other circulating viruses ( table ) . the patients with laboratory-confirmed disease reported during february were also classified according to their reported exposures during the weeks before illness onset. record review and interviews were conducted during - march . among the cases, were classifiable using information obtained by the initial case investigation. interviews were attempted for the remaining patients. of these, ( %) were interviewed; individual refused to participate, and patients were not available. proxy interviews were conducted for of interviews, including for patients who were deceased and for of patients who survived. among the patients, ( %) were determined to have had household contact with a confirmed mers-cov case, ( %) were hcps, ( %) were inpatients in a healthcare facility, ( %) were hospital visitors, and ( %) were unable to be classified owing to a lack of available information (table ) . notably, patients ( %) denied exposure to a healthcare facility or to a person with acute respiratory illness in the weeks before illness onset and were classified as sporadic cases (tables and ); among these, individual reported visiting a camel farm in the weeks before illness onset. among the sporadic cases, were available for interview, and were interviewed by proxy. among the interviewed by proxy, were deceased and were too ill to participate in the interview. sequences were obtained for sporadic cases, and ( %) were nrc- , including the individual who had visited the camel farm. in the republic of korea [ , ] , thailand (accession number kt ), and china in [ , ] . previous documentation of the duration of circulation in humans of different mers-cov clades in saudi arabia during - noted an average detection time of days [ ] . in contrast, we demonstrate that nrc- has persisted longer than most previously documented clades. nrc- was found to eventually predominate over the -month study period and attain a wide geographic distribution in a comparatively short period. while this apparent emergence and clade displacement is suggestive of greater epidemiologic fitness [ ] , we observed no clinical differences between nrc- and other clades; the implications for virus replication and transmission need further study. during preparation of this manuscript, sequences obtained from camels in oman in may [ ] and saudi arabia during july -april [ ] were reported that showed similar recombination features and phylogenetic association with nrc- . in camels, nrc- (referred to as lineage [ ] ) was first detected in july and became predominant in saudi arabia during a period that overlaps with our study, corroborating our findings of an increased prevalence in humans relative to other clades. recombination has been documented among covs [ ] and has been linked to the emergence of more-pathogenic strains of some animal covs [ ] [ ] [ ] . evidence of intraspecies recombination has also been found with the human covs hku [ ] , nl [ ] , oc [ ] , and, more recently, mers-cov [ ] . genome analysis of human mers-cov strains from saudi arabia in and the recent outbreak in south korea/china [ ] [ ] [ ] and camels as noted above [ , ] revealed a probable signature recombination event between different parental clade b viruses involving a region of the orf ab and spike genes. we confirmed this finding and documented an increasing prevalence of this virus in humans among samples collected since january from geographically distant communities in saudi arabia. similar to recent reports [ ] , we estimate that this recombinant virus emerged sometime in mid-to-late . based on recently available sequence data from camels in saudi arabia, nrc- (lineage ) was predicted to have diverged between december and june [ ] . in our study, further analysis of patients with laboratoryconfirmed mers-cov reported in february revealed individuals with no recognized risks for secondary acquisition; none of these reported direct camel contact, although individual reported visiting a camel farm. of those sequenced, most were infected with genetically very similar viruses, suggesting a potential for limited transmission from those with unrecognized mers-cov infection. these findings highlight the importance of strengthened epidemiologic and laboratory surveillance. most cases identified in saudi arabia in february had documented exposure to healthcare facilities, a well-demonstrated risk factor for mers-cov infection [ ] [ ] [ ] . seventeen of affected facilities in saudi arabia in february experienced mers-cov infection clusters. moreover, of patients in february ( %) were visitors to healthcare facilities. this is similar to the jeddah outbreak, where % of investigated cases were visitors [ ] . recommendations to limit visitation in facilities with ongoing mers-cov transmission should be reinforced to limit these exposures. our investigation, which was performed as part of an emergency public health response, is subject to several limitations. first, specimens were not available for all cases during the study period, meaning that many viruses remained untyped; however, we observed no demographic differences between cases who had specimens sequenced and those who did not. second, since its emergence in , surveillance and sequencing of mers-cov strains has been incomplete; variations in sequence availability and documentation might have influenced the extent of persistence and geographic spread that we have determined for past circulating virus strains. case definitions, testing practices, and testing locations have also changed during this period. third, although we were able to obtain full genome sequences from nrc- samples, all of which possessed the expected recombinant signal, our sequencing was mostly limited to the spike gene alone, which poses the risk of misclassifying recombinant viruses as belonging to nrc- . this is illustrated in the recent study by sabir et al [ ] , which reported multiple novel recombinant viruses in camels, including recombinants between nrc- (lineage ) and other virus clades. fourth, because of the high morbidity and mortality of mers-cov infection, interviews with cases were not always possible, necessitating the use of proxies. it is possible that, combined with issues of recall, the quality of the information collected varied. of particular consideration, of sporadic cases were classified on the basis of interviews with proxies, and pre-illness exposures might not have been accurately recognized and reported. fifth, some camel exposures may have gone unrecognized because of disincentives for reporting camel exposures, given their cultural and economic significance in saudi arabia. sixth, given the existing evidence of association between mers-cov illness and pre-illness healthcare exposure or exposure to sick individuals [ , , ] , our risk classification was hierarchical; that is, reported exposure to a setting where secondary acquisition was likely took precedence over reported exposure to camels. as such, we did not assess camel exposures in individuals with recognized risks for secondary acquisition. finally, although we have attempted to link the results of our epidemiologic investigation with mers-cov sequences obtained from investigated cases, we cannot fully assess the possible role of virus introductions from nonhuman sources. recent phylogeny of mers-cov sequences from camels in saudi arabia indicated that the novel recombinant subclade (referred to as nrc- in our manuscript) was also predominant in camels during a period overlapping with our study [ ] . as such, our detection of closely related viruses in humans might in part reflect multiple introductions from camels with similar strains. virus introductions from other currently unidentified sources might also be factor. virus transmission dynamics within and between human and nonhuman sources of mers-cov will likely influence transmission routes in ways not yet fully understood. this investigation describes the emergence, persistence, and widespread circulation of a novel recombinant mers-cov in saudi arabia. a lack of clearly defined epidemiologic links in some cases highlights the need for ongoing intensive epidemiologic and laboratory surveillance to better understand mers-cov transmission and to focus infection prevention and control efforts. supplementary materials are available at http://jid.oxfordjournals.org. consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. potential conflicts of interest. all authors: no reported conflicts. all world health organization. summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel mers coronaviruses in dromedary camels human infection with mers coronavirus after exposure to infected camels, saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities 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coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene and evidence of recombination molecular epidemiology of human coronavirus oc reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination mers-cov recombination: implications about the reservoir and potential for adaptation acknowledgments. we thank shifaq kamili, for logistics of specimen shipment; and laura wright of the centers for disease control and prevention (cdc) geospatial research analysis and services program.disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc.financial support. this work was supported by the saudi arabia ministry of health and the cdc as part of an emergency response. in the weeks before becoming ill, none had known contact with a patient with mers, a person with severe respiratory illness, and anyone mildly ill, and none reported working in or visiting a healthcare facility.abbreviations: g, full genome sequence obtained; s, spike region sequence obtained; ns, not sequenced. a genbank accession number. key: cord- -p kk up authors: horby, peter w,; pfeiffer, dirk; oshitani, hitoshi title: prospects for emerging infections in east and southeast asia years after severe acute respiratory syndrome date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: p kk up it is years since severe acute respiratory syndrome (sars) emerged, and east and southeast asia retain a reputation as a hot spot of emerging infectious diseases. the region is certainly a hot spot of socioeconomic and environmental change, and although some changes (e.g., urbanization and agricultural intensification) may reduce the probability of emerging infectious diseases, the effect of any individual emergence event may be increased by the greater concentration and connectivity of livestock, persons, and products. the region is now better able to detect and respond to emerging infectious diseases than it was a decade ago, but the tools and methods to produce sufficiently refined assessments of the risks of disease emergence are still lacking. given the continued scale and pace of change in east and southeast asia, it is vital that capabilities for predicting, identifying, and controlling biologic threats do not stagnate as the memory of sars fades. i t is a decade since severe acute respiratory syndrome (sars) emerged and in a few dramatic months redefined perceptions of global vulnerability to emerging infectious diseases. it is believed that sars originated from southern china, with the first cases identified in guangdong province, china, where sporadic cases and small outbreaks occurred between november and early january . a larger outbreak, triggered by nosocomial transmissions in hospitals, began during mid-january in guangzhou city, the capital of guangdong province ( ). on february , , the international community, including the world health organization (who), became aware of this unusual cluster of severe pneumonia cases, which included many health care workers. detailed information about the outbreak was not available to the international community, and when who issued a global alert on march , , the virus had already spread to other countries and caused outbreaks in areas outside guangdong, including hong kong, china; hanoi, vietnam; singapore; and toronto, ontario, canada. the sars epidemic provided a dramatic demonstration of the weaknesses in national and global capacities to detect and respond to emerging infectious diseases, and it was in many ways a watershed event that had a transformative effect on many of the clinical, public health, and other professionals involved. but has the response to sars had any lasting effect on the probability of new infectious agents emerging, being detected at an early stage of emergence, and being effectively controlled? more than % of the global population lives in east and southeast asia, and despite impressive improvements in health, infectious diseases remain a major problem in the region. in , % of the estimated . million deaths among children < years of age in southeast asia were attributable to infectious diseases (e.g., pneumonia and acute diarrhea) ( ) . alongside this existing pool of known human pathogens, a large and diverse population of mammalian wildlife species and domestic livestock reside in the region, acting as reservoirs or amplifying species from which new infectious diseases of humans might emerge ( , ) . the reemergence of highly pathogenic avian influenza a(h n ) virus in , the isolation of novel bat-associated reoviruses from humans in malaysia in , and the discovery of a novel tick-borne bunyavirus associated with fever and thrombocytopenia in rural farmers in china in attest to the existence of a pool of potential zoonotic pathogens in east and southeast asia (table) ( , , ) . we review how the conditions that drive the emergence of infectious diseases and the systems to detect and control them have changed in east and southeast asia in the decade since sars. over the past decade, east and southeast asia have been home to many of the top-performing world economies, and this macroeconomic success has resulted in large increases in the demand for natural resources. the demand for hardwood, firewood, wood pulp, agricultural and grazing land, living space, roads, minerals, and power has had an enormous effect on the ecosystems of the region. deforestation occurred throughout the s, but the last decade has seen net increases in forested areas of china, the philippines, and vietnam because of active afforestation (including new commercial plantations). net forest losses continue, however, in myanmar, cambodia, indonesia, and papua new guinea ( figure ) ( ) . the conversion of natural environments into agricultural or other commercially viable land (e.g., dams, mines) is usually associated with a decrease in biodiversity. a reduction in biodiversity can lead to increased disease transmission through a variety of mechanisms (e.g., reduced predation and competition) and cause an increase in the abundance of competent hosts and the loss of buffering species, leading to increased contact between amplifying host species and compatible pathogens ( ) . although a reduction in biodiversity can lead to increased disease transmission, a large diversity of mammalian wildlife species is also associated with a large diversity of microbial species, which both increase toward the equator ( , ) . therefore, tropical areas (e.g., myanmar, cambodia, and parts of indonesia) that have a rich pool of existing and potential pathogens but are experiencing ongoing ecosystem disruption and biodiversity loss may be at a particularly high risk for the emergence of zoonotic diseases. land-use changes are ongoing, but much of east and southeast asia already has very high pressures on productive land. the rate of land-use change in much of the region has probably peaked, and the region is now in an era of increasing intensification of land productivity. in fact, over the last decade, china has increased agricultural output despite a slight decrease in total agricultural land area ( figure ) ( ) . this intensification is driven largely by demographic pressures, which are predicted to result in a % increase in food production by ; the consumption of grains is expected to decrease and demand for meats, fruits, and vegetables is expected to increase ( ) . the recent high and volatile prices for food commodities are a good indicator of the current vulnerability of agricultural production systems. the environmental consequences of intensified agricultural production include the depletion and degradation of river and groundwater, reduced soil quality, and water and soil contamination with chemical fertilizers and pesticides. the loading of aquatic ecosystems with nitrogen and phosphorous (eutrophication) is a widespread environmental change with an as-yet unquantified effect on the risk for disease emergence. eutrophication can result in potentially harmful blooms of cyanobacteria, but little is known about the effect on pathogens that cause disease in animals and humans. there is, however, evidence that eutrophication can alter ecosystems in such a way as to increase the transmission of parasitic diseases of amphibians, the concentration of vibrio cholerae, and the abundance of mosquito vectors ( ) . given the trend of increasing intensification of crop and animal production in east and southeast asia, much more attention should be given to the effect of the large-scale contamination of water and soil with nitrogen, phosphorous, and other chemicals on the functioning of ecosystems and on disease dynamics. demand for livestock products in east and southeast asia has risen dramatically over the past years: the per capita consumption of meat in developing countries has more than tripled since the early s, and egg consumption has increased -fold ( ) . the increased demand for meat has been met by more intensive and geographically concentrated production of livestock, especially pigs and poultry. east and southeast asia are home to million pigs ( % of the world pig population) and . billion poultry ( % of the world poultry population) ( ) . the food and agriculture organization of the united nations (fao) has projected that pork consumption in china will increase by % between and ( ) , and rabobank, an agricultural finance group, predicts a % increase in global meat demand over the next years, with % of that occurring in asia. the demand will be particularly strong for poultry because of its relatively low cost and short production cycle, and because there are fewer cultural restrictions regarding poultry than there are that concern pork and beef. in addition, meat-producing companies will continue to consolidate at the global level. the intensification of livestock farming often results in more effective separation of domestic and wild animals, improved veterinary supervision and input, reduced movement of animals, and reduced species mixing, all of which may reduce the likelihood of disease emergence. however, higher densities of short production-cycle domestic animals, such as pigs and, in particular, poultry, introduce a vulnerability because such animals usually have limited genetic variation. higher genetic diversity within a host species is often associated with differences in susceptibility to infection, thereby limiting the potential for infections to spread rapidly ( ) . recent outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus throughout east and southeast asia, which at times co-occurred with outbreaks of streptococcus suis infections, and the detection of reston ebola virus infection in pigs in the philippines highlight the ongoing risk for disease emergence, amplification, and crossover from livestock to humans in east and southeast asia ( , ) . in east and southeast asia, antimicrobial drugs are used extensively in the livestock and aquaculture sectors to treat or prevent infections, and they are used non-therapeutically as growth promoters, which requires the prolonged administration of sub-therapeutic doses. this practice has a demonstrable effect on the emergence and prevalence of potentially clinically relevant resistant microorganisms in food animals. furthermore, the subsequent excretion of antimicrobial drugs into the environment may subject environmental bacteria to antimicrobial selection pressures ( ) . it is clear that the continued use of non-therapeutic antimicrobial drugs in livestock and aquaculture industries that are increasing in scale and intensity poses a threat to human and animal health ( ) . reducing contact between domestic and wild animals, whether the wild animals remain wild or are captive in breeding farms or markets, is a key tactic recommended by the fao for reducing risk to human health, and this reduced contact is part of the wider fao strategy for biosecurity. improving biosecurity in farms in east and southeast asia is a major challenge because a large proportion of the farming is done in backyard and small-to medium-scale commercial farms, and there is often a mix of commercial and backyard farming in any location ( ) . the longerterm vision is to restructure the livestock production sector toward a more integrated and controlled system in which controls benefit animal health and welfare, human health, and commercial profitability without adversely affecting the livelihood of poor persons. in east and southeast asia, increasing intensification of animal husbandry may lead to healthier, better isolated animals and a subsequent lowered risk for emerging disease events. however, should an emerging infectious disease event occur, this intensification may result in greater amplification of disease in large, naive monocultures, as demonstrated in the netherlands when they experienced major outbreaks of classical swine fever in - and avian influenza in . the role of civet cats in the sars pandemic and the smuggling of avian influenza a(h n ) virus-infected birds of prey into europe showed that the legal and illegal wildlife trade is an effective conduit for zoonotic pathogens to enter new niches ( , ) . wild animal products remain popular in east and southeast asia as traditional medicines, tonics, food delicacies, or symbols of wealth. although all countries in the association of southeast asian nations (asean) are signatories to the convention on international trade in endangered species of wild fauna and flora, asia continues to host the largest illegal wildlife trade in the world ( ) . the regional and global connectivity of east and southeast asia continues to increase; there is visa-free travel between asean countries, relaxation of previously restrictive international travel policies in some countries, and a proliferation of budget airlines. increased travel and trade between africa and asia is a particularly notable phenomenon: exports from africa to asia increased % from to . this africa-asia traffic is a new corridor for the exchange of potential emerging infectious pathogens. the ongoing development of a regional road transport network within east and southeast asia will also offer new opportunities for pathogen dispersal because, compared with air travel, roads offer a more egalitarian form of connectivity that includes animals as well as humans. increases in domestic travel also continue; only % of the world is now classified as remote (i.e., > hours travel time to a big city), and an estimated . billion passenger trips were made during china's lunar new year celebrationsthe greatest annual human migration on earth. increased connectivity provides greater opportunities for pathogens to disperse beyond their traditional niches and presents a formidable challenge to the tracking and containment of outbreaks ( ) . between and , the global population is expected to increase from . billion to . billion (an increase of . billion [ %]), and this increase will be concentrated in cities ( ) . most of the growth will occur in developing countries, particularly in asia, which will experience an increase in its urban population of . billion persons. in many ways, this concentration of population growth in urban areas is a positive development, and the popularity of cities is a testament to the fact that cities generally provide better economic and education opportunities, better living and sanitation conditions, better nutrition, and therefore better health than in underdeveloped rural areas. cities are, however, key in the epidemiology of many infectious diseases because they can function as "pace-makers" that drive temporal and spatial transmission dynamics of local epidemiology (e.g., dengue), hubs for national and global spread (e.g., sars and hiv), or bridges between human and animal ecosystems (e.g., influenza a subtypes h n and h n ). east and southeast asia have made considerable progress in health and social welfare improvements: during - , a total of million persons in china and india moved out of slum conditions. however, urban poverty remains a concern. in , an estimated million persons in asia lived in slums ( ), and, at the end of , there were an estimated million rural migrant workers in china, many of whom lacked residency rights and had limited access to health care and other social supports ( ) . circular migration between rural and urban settings is common and may facilitate the transfer of pathogens from wild or rural ecosystems to urban areas, with the potential for rapid amplification in settings with high concentrations of migrant workers. almost % of the emerging infectious diseases identified in asia during - represent the emergence of a new pattern of antimicrobial drug resistance ( ) . the major driver of such resistance is drug pressure. in east and southeast asia, the sequential development of resistance by malaria parasites to chloroquine, sulfadoxine-pyrimethamine, mefloquine, and now artemisinin is a measure of both the adaptive capacity of the parasite and the failure of health systems to implement effective drug combination and cycling strategies to avoid resistance. bacteria in east and southeast asia show high rates of resistance to antimicrobial agents; examples include multidrug-resistant acinetobacter baumannii, salmonella enterica, and enterobacteriaceae ( ) . a high level of antimicrobial resistance is a marker of the failure to control access to antimicrobial drugs and to influence prescribing behaviors. over-the-counter antimicrobial drugs are available without a prescription throughout much of east and southeast asia, even though antimicrobial drugs are officially prescription-only medicines in most countries. left unchecked, the supply-and demand-side incentives for inappropriate antimicrobial drug use will lead to a region awash with antimicrobial drugs and with the potential for public health disasters, such as artemisinin resistance, which now threatens global malaria control. the sars pandemic highlighted what had been apparent to some since the s: few countries possessed the necessary surveillance and response capacities to rapidly detect and control emerging infectious diseases ( ) . the deficiencies of the international health regulations at the global level had long been recognized, and attempts to revise them were ongoing before , but the sars outbreak added new urgency and momentum for change. the international health regulations were successfully revised in , and for the first time they defined a series of core capacities that each country is required to establish to detect, report, and control public health emergencies of international concern. the target for attaining these core capacities was set as june , and an assessment undertaken in found that although these core capacities had not yet been fully achieved in several countries of east and southeast asia, considerable progress had been made ( figure ( ) . an analysis of the global capacity for detecting outbreaks showed improvements in the median time from outbreak start to outbreak discovery between ( . days) and ( . days) and from start to public communication ( days in to days in ); the who western pacific region was the only who region that showed a statistically significant improvement in both areas ( ) . many of these improvements were facilitated by a large increase in political and financial support for emerging infectious disease surveillance and response from national governments and donor agencies after the outbreaks of sars and influenza a(h n ). although data on the total expenditure for emerging infectious disease surveillance, preparedness, and response in east and southeast asia are not available, examples of international support include the first and second asian development bank greater mekong subregion regional communicable diseases control projects ($ . million and $ million, respectively), the canada-asia regional emerging infectious disease project ($ . million), the us government foreign assistance for disease control, research, and training (>$ million in asia during - ), and the us agency for international development's pandemic and emerging threats program. as a consequence, pandemic and epidemic preparedness planning has improved in most countries of east and southeast asia, but gaps between the plans and the ability to operationalize them remain in many countries ( , ) . since , international and national authorities have increasingly recognized the importance of more effective animal health surveillance. however, limited resources in most countries have meant that investments into improved surveillance capacity have occurred largely in those countries affected by major outbreaks, such as the case with an outbreak of nipah virus infection in malaysia and of influenza a(h n ) virus infection in thailand, china, vietnam, and indonesia. where these types of investments did occur, they were often species-and threat-specific, rather than to facilitate strategic enhancement of generic surveillance efforts for dealing with emerging disease threats. not too dissimilar from the situation in high income countries, institutional and administrative boundaries between the human and animal health sectors have largely prevented the development of integrated surveillance systems. it has been said that there have been more international meetings about influenza a(h n ) virus than human cases of the disease. whether this is true or not, a benefit of the many meetings held after the outbreaks of sars and influenza a(h n ) virus has been a strengthening of regional and international professional partnerships. clinicians, epidemiologists, virologists, veterinarians, and public health officials in east and southeast asia are now better connected and familiar with their colleagues than they were before ( , ) . networks of trusted colleagues are a powerful force for sharing expertise, clearing confusion, and bridging divides, and these newly formed partnerships are perhaps one of the greatest unquantified achievements of the last decade. to coordinate and harmonize the diversity of initiatives spawned by sars, the who south east asia office and western pacific office jointly developed the asia pacific strategy for emerging diseases in . this plan provides a common framework for strengthening national and regional surveillance and response capacity for emerging infectious diseases in the countries of the asia pacific region; it was revised and re-endorsed by the who regional committees in . a more troublesome dimension of international partnerships since has been the dispute over the sovereignty and sharing of pathogen samples. although these disputes have not benefited disease surveillance in the short term, they do have a legitimate basis, and it must be hoped that in the medium term, an airing and resolution of these issues will result in greater trust, improved surveillance, and a more equitable distribution of benefits. in this context, the ratification in of who's pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits is a major step forward. a major shift, from west to east, is underway in the global center of gravity: east and southeast asia are becoming the dominant force of economic, social, and environmental change. while rapid development has brought east and southeast asia many benefits, it has also resulted in widening health inequalities, environmental degradation, increased migration and urbanization, and a concentration of persons, food production, and economic activity. these changes might facilitate the emergence and transmission of new pathogens, but it would be simplistic and disingenuous to present the extensive changes in east and southeast asia as inevitably increasing the risk of emerging infectious diseases. it seems likely that the probability of new emerging infections may be reduced by many of these socioeconomic changes, such as urbanization and the industrialization and commercialization of agriculture and food production; however, the scale and effect of any individual emergence event may increase because of a greater concentration and connectivity of livestock and persons. surveillance and response capacities have improved in the last decade, and east and southeast asia are far better prepared to detect and respond to emerging infectious diseases. however, we are still lacking the tools and methodologies to produce a sufficiently refined assessment of the distribution and profile of disease emergence risks that encompasses geographic heterogeneity; the interaction of different drivers of pathogen evolution, crossover, and dispersion; and dynamic systems and the uncertainty inherent in such assessments. given the continued scale and pace of change in east and southeast asia, it is vital that the capacity to predict and identify biologic threats and to protect the public's health does not stagnate as the memory of sars fades. epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china global, regional, and national causes of child mortality: an updated systematic analysis for with time trends since ecology drives the worldwide distribution of human diseases global trends in emerging infectious diseases avian influenza a (h n ) in patients in vietnam streptococcus suis and porcine reproductive and respiratory syndrome streptococcal toxic shock syndrome caused by streptococcus suis serotype a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new 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impacts on human health the transfer of antibiotic resistance from food to humans: facts, implications and future directions livestock production: recent trends, future prospects bats, civets and the emergence of sars highly pathogenic h n influenza virus in smuggled thai eagles summarizing the evidence on the international trade in illegal wildlife environmental change and infectious disease: how new roads affect the transmission of diarrheal pathogens in rural ecuador united nations habitat. state of the world's cities: / -cities for all: bridging the urban divide united nations, department of economic and social affairs, population division. population distribution, urbanization, internal migration and development: an international perspective antimicrobial resistance among pathogenic bacteria in southeast asia hot spots in a wired world: who surveillance of emerging and re-emerging infectious diseases summary of states parties report on ihr core capacity implementation national institute for viral disease control and prevention, chinese center for disease control and prevention. influenza weekly report. overview of influenza surveillance in china strengthening indonesia's field epidemiology training programme to address international health regulations requirements global capacity for emerging infectious disease detection pandemic influenza preparedness and health systems challenges in asia: results from rapid analyses in asian countries health system resource gaps and associated mortality from pandemic influenza across six asian territories laboratory capacity building in asia for infectious disease research: experiences from the south east asia infectious disease clinical research network (seaicrn) the evolution and expansion of regional disease surveillance networks and their role in mitigating the threat of infectious disease outbreaks we thank nguyen thi thanh thuy for preparation of figure .dr horby is a senior clinical research fellow at the oxford university clinical research unit in vietnam and an adjunct associate professor in the department of infectious diseases, yong loo lin school of medicine, national university of singapore. his research interests include the emergence and control of infectious diseases, including avian and seasonal influenza; community-acquired pneumonia; dengue; and hand, foot and mouth disease. key: cord- -pdhjl authors: park, wan beom; kwon, nak-jung; choe, pyoeng gyun; choi, su-jin; oh, hong sang; lee, sang min; chong, hyonyong; kim, jong-il; song, kyoung-ho; bang, ji hwan; kim, eu suk; kim, hong-bin; park, sang won; kim, nam joong; oh, myoung-don title: isolation of middle east respiratory syndrome coronavirus from a patient of the korean outbreak date: - - journal: j korean med sci doi: . /jkms. . . . sha: doc_id: cord_uid: pdhjl during the outbreak of middle east respiratory syndrome coronavirus (mers-cov) in korea, persons were infected, resulting in fatalities. we isolated mers-cov from the oropharyngeal sample obtained from a patient of the outbreak. cytopathic effects showing detachment and rounding of cells were observed in vero cell cultures days after inoculation of the sample. spherical virus particles were observed by transmission electron microscopy. full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade b of mers-cov. middle east respiratory syndrome coronavirus (mers-cov) is a betacoronavirus causing a severe acute respiratory infection ( , ) . it was first isolated from the sputum of a patient with severe pneumonia in saudi arabia in ( ) . since then, countries have reported , laboratory-confirmed cases of infection with mers-cov to the world health organization (who), including fatalities ( ) . the korean outbreak of mers-cov was initiated in may by a business man returning from the middle east ( ) . the transmission of mers-cov continued until early july, resulting in cases with deaths. one of the most important characteristics of the korean outbreak was large clusters of cases due to superspreading event at hospitals, accounting for > % of the total cases. another characteristic was that many cases of second-and third-generation of transmission occurred ( , ) . this finding is quite contrast to the previous studies suggesting limited person-to-person transmissibility of mers-cov ( , ) . to better understand transmissibility and assess epidemic risk, characterization of mers-cov of the korean outbreak would be of paramount importance ( ) . here, we report the mers-cov isolated from a patient of the korean outbreak. a -year-old healthcare worker was admitted to the hospital because of fever and cough. on may , , he was unknowingly exposed to the index case (designated as patient number by korea ministry of health and welfare) of the hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) at emergency department of a hospital ( ) . two days later, he developed fever and dry cough. on june , he was diagnosed with mers-cov infection as sputum sample was positive on real-time reverse transcriptase polymerase chain reaction (rt-pcr) assay, and admitted to the isolation unit of the mers-designated hospital by the government. he had a history of cough variant asthma, but did not take any regular medication, and otherwise healthy. on admission (june , ), the physical examination revealed a body temperature of . °c, a respiratory rate of breaths per minute, a pulse of per minute, and a blood pressure of / mmhg. chest radiography showed patchy consolidation in the upper zone of the left lung. his pneumonia progressed, and on june , he developed shortness of breath, his arterial oxygen saturation decreased below %, requiring oxygen supplementation, and chest radiography showed multiple con- solidations in the both lungs. on june , he was intubated and mechanical ventilation was started. his hypoxemia worsened rapidly, and veno-venous extr acorporeal membrane oxygenation support was started since june . on july (day of his illness), real-time rt-pcr for mers-cov turned negative, and was removed from the isolation unit. he recovered gradually. the patient's oropharyngeal samples were obtained by using utm tm kit containing ml of viral transport media (copan di-agnostics inc., murrieta, ca, usa). the samples were stored at - °c until assays. we inoculated m onolayers of vero cells with the samples and cultured the cells at °c in a % carbon dioxide atmosphere. cytopathic effects consisting of rounding and detachment of cells were observed days after the inoculation of the sample taken on day of his illness ( fig. a and b) . the rna titer in the sample was . × copies/ml for upe gene and . × copies/ml for orf a gene. in order to observe virus particles, vero cell monolayer showing the cytopathic effects was fixed as previously described ( ) . it was cut on ultramicrotome (rmc mt-xl) at nm. ultrathin sections were stained with saturated % uranyl acetate and % lead citrate before examination with a transmission electron microscope (jem- ; jeol usa inc., peabody, ma, usa) at kv. spherical particles ranging to nm in diameter were observed within the cytoplasm of infected cells (fig. c and d) . for full-length genome sequencing of the virus isolate (mers-cov hu/kor/snu _ / ), vero cell monolayer showing cytopathic effects was harvested and used for rna extraction. rna was extracted by using qiaamp viral rna mini kit (qiagen, valencia, ca), according to the manufacturer's instructions. the rna was used for cdna synthesis using su-perscript iii reverse transcriptase (invitrogen, ma, usa) by each specific rt primer as described previously ( ) . finally, about . kb pcr products were amplified by each primer pair (table ) , and the amplicons were sheared by covaris s according to the bp target bp condition (covaris, ma, usa). to generate the next generation sequencing (ngs) library, the fragments were ligated with adapter and index (barcode) using truseq nano dna ht library prep kit (illumina, ca, usa), and the library was sequenced by miseq (illumina, ca, usa). the ngs data were aligned to mers-cov, nc_ , used for binary sequence alignment/map (bam) file generation, and genome assembly. in order to evaluate genetic relationship between this isolate and homo sapiens and camelus dromedaries mers-cov sequences reported from other countries, phylogenetic analyses were conducted using the whole genome, the s gene and the ofr a gene. the full-length genome sequence ( , bp) of the virus isolate was obtained and deposited in the genbank (accession no. ku ). the genome sequence of the virus had high level of nucleotide identity ( . %- . %) to those of mers-cov reported previously ( fig. a) . of note, the closest ones were korea/seoul/ - - and - - (genbank accession no. , that were directly sequenced from sputum of the same patient as ours ( ) . a previous study about s gene of mers-cov reported from korea showed that a culture isolate from patient number contained two nonsynonymous variants (s r and v l) ( ) . these variants were not found in our isolate and there was no difference in amino acids of s protein between our isolate and directly sequenced ones (kt - ). this difference can be explained by cell culture-adaptation in that our culture isolate was obtained before passage whereas one with nonsysnonymous variant was from the third passage in vero cells. phylogenetic analyses of the whole genome showed that this virus closely clustered with those reported from korea (gen-bank accession nos. kt , kt -kt ), china (genbank accession no. kt . ) and saudi arabia in (genbank accession no. kt - ). phylogenetic analyses based on orf ab genes revealed that this virus fell into the group , but those based on s genes showed that this virus belongs to the group along with other viruses reported from korea ( fig. b and c) . these findings are compatible to a previous study ( ) . in summary, we isolated mers-cov from a patient with severe pneumonia who had been infected during the korean outbreak in . we also obtained full-length sequence of the the evolutionary history was inferred by using the maximum likelihood method based on the tamura-nei model ( ) . evolutionary analyses were conducted in mega ( middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. middle east respiratory syndrome corona saudi arabia: disease outbreak news middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission characteristics of mers and sars in the healthcare setting: a comparative study interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study what needs to be done to control the spread of middle east respiratory syndrome coronavirus? middle east respiratory syndrome coronavirus superspreading event involving persons, korea processing tissue and cells for transmission electron microscopy in diagnostic pathology and research full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus microevolution of outbreak-associated middle east respiratory syndrome coronavirus variations in spike glycoprotein gene of mers-cov origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees mega : molecular evolutionary genetics analysis version . we thank ms. myoung im shin (department of pathology, seoul national university hospital) for her technical assistance in electron microscopy. the authors have no potential conflicts of interest to disclose. key: cord- -zy qjaai authors: gong, shu‐ran; bao, lin‐lin title: the battle against sars and mers coronaviruses: reservoirs and animal models date: - - journal: animal model exp med doi: . /ame . sha: doc_id: cord_uid: zy qjaai in humans, infection with the coronavirus, especially the severe acute respiratory syndrome coronavirus (sars‐cov) and the emerging middle east respiratory syndrome coronavirus (mers‐cov), induces acute respiratory failure, resulting in high mortality. irregular coronavirus related epidemics indicate that the evolutionary origins of these two pathogens need to be identified urgently and there are still questions related to suitable laboratory animal models. thus, in this review we aim to highlight key discoveries concerning the animal origin of the virus and summarize and compare current animal models. in the aftermath of the sars outbreak, its high morbidity and mortality made the identification of natural reservoirs and an appropriate animal model necessary in order to ascertain the interspecies transmission chain, to develop procedures for protecting public health, to promote research on the sars-cov mechanism, and to establish animal models for use in developing antivirals and vaccines. although the sars outbreak occurred over years ago, another member of the coronaviridae family of viruses has since caused illness in the middle east. this illness has been named middle east respiratory syndrome and the pathogen (mers-cov) has been shown to be a type of coronavirus that is highly related to sars-cov. infection with mers-cov results in higher mortality and new symptoms such as renal failure. thus, failure to fully resolve the initial sars pathogen has been followed by a more virulent infection with a related virus, mers-cov. therefore, we need urgently to identify the origins of the viruses and find ways to deal with them. to make a useful comparison, this review will investigate the yuan ky et al assayed different species of bats, different species of rodents and rhesus macaques. the results proved that sars-cov in bats was the most closely related to that in humans. in this assay, the sars-like cov in chinese horseshoe bats has %- % sequence homology with human sars-cov. moreover, the s m motif in its ʹutrs and the phylogenetic analyses of four fully characterized genomes of sars-like cov indicated that the horseshoe bats have great potential to be one of the natural reservoirs. , given that, the same research suggested that, even though there are no available migration patterns, it is known that bats can migrate approximately miles for hibernation, and the distance between their wild habitats and markets in shenzheng and hong kong is only miles, which means that geographically widespread infections of sars-like cov can be explained by transmission via bats. recently, a five year study provided futher evidence that bats in the yunnan province of china are more likely to be the prime reservoir than those detected elsewhere. the study compared the nonstructural protein genes orf a and b of the virus and concluded that sarsr-cov strains from a cave in yunnan village were more closely related to human sars-cov and cell entry studies demonstrated that three newly identified sarsr-covs with different s protein sequences are all able to use human ace as the receptor. just as horseshoe bats were postulated to be the primary sars host, van boheeman et al. indicated that two kinds of bats carry similar mers coronaviruses. then, susanna k. p. lau et al. used sequences of rna polymerase (rdrp), spike (s), and nucleocapsid (n) genes to determine that human mers-cov rdrp is more closely related to the pipistrelle bat cov hku ( . %- . % amino acid identity) and the s and n genes are more closely related to the tylonycteris bat cov hku (respectively, . %- . % and . %- . % amino acid identity), indicating that these three viruses may share the same ancestor. however, these results did not definitively prove bat cov to be the ancestor of the human cov. subsequently, victor max corman et al isolated a virus named "neo cov" from south african neoromicia capensis bats, and this virus has % similarity with mers-cov, with even higher rates for some specific viral rna segments. additionally, mers-cov has been found to grow better in bat cells than in human, bovine, cat, and swine cells. interestingly, within this article, it is noted that the great horn of africa, where neo cov was discovered, is also a place where camels are transported and traded. therefore, the original transmission from bats to camels may have occurred in sub-saharan africa. based on the above evidence, there is a strong possibility that bats are the initial mers-cov host. a paper by hana fakhoury et al analyzed the possible viral distribution route that mers-cov traveled in the spring of , leading the authors to speculate that hibernation might be the reason for seasonal outbreaks. after hibernation, bats wake up in the warm, food-abundant spring weather and eat palm or other seeds. those seeds may carry bat feces and then drop to ground, where they might be eaten by camels. if mers-cov is found in cereal plantations near where bats congregate, this view will be confirmed. during the sars outbreak, masked palm civet cats (paguma larvata) and raccoon dogs (nyctereutes procyonoides) were found to carry sars-like viruses, even before the virus was discovered in lesser bamboo bats (tylonycteris pachypusa and pipistrellus). at the same time, wild animal sellers were found to possess higher than average relevant neutralizing antibodies. initial phylogenetic analyses reveal that the sars-covs in civets and humans actually come from two distant branches, but the sars-cov from civets during the incipient phase of the epidemic had . % sequence similarity to the human sars-cov. until recently, it was thought that civets were the immediate zoonotic source of sars-cov in the guangdong sars outbreak. regarding how the civets gain the cov, janies et al used dynamic homology phylogenetic analyses to investigate other species besides civets and the results further supported the idea of civets as the immediate reservoir. it is worth mentioning that the "other species" even included humans. however, some phylogenetic analyses including humans have shown a limited numbers of infectious civets in the relevant areas. , based on these data, it is likely that civets may be only a "bypass" reservoir that adapted transiently before the epidemic. whatever the truth is, we can at least be sure that the civet cat is one of the intermediate hosts. in ruminants, almost all evidence indicates that camels are the most additionally, an investigation in saudi arabia revealed that younger camels are prone to infection by mers-cov. with regard to the transmission route, there are three possible ways for the virus to be conveyed to humans from camels: organs, flesh, and discharges such as feces and camel milk. mers-cov rna was found in a camel lymph node in qatar. this finding indicates that mers-cov may be maintained in camel organs or muscles. if that speculation proves true, the convention in the middle east of cooking and trading camel meat and organs might be the interspecies transmission route. mers-cov can also survive longer in camel milk than in other ruminant milk. in addition to the above two routes, mers-cov rna can be detected in % of nasal discharge and % of feces in camels. thus, there is supportive evidence for the three postulated transmission routes, but further verification is needed to confirm them. it should also be noted that mers is a type of infectious respiratory disease, and therefore, in addition to the above three possible transmission routes, infection via aerosols produced by camels should be considered. research in qatar in showed that mers-cov was found in alpacas centrally housed with camels. although mers-cov has so far not been found in camelids other than dromedaries outside of the arabian peninsula, the increasing export of alpacas increases the risk of an outbreak, since they have the potential to be the interspecies host. while it is not clear that alpacas have the same function as the camels in the outbreak, their ability to spread the virus exists. currently it is very popular to trade and keep ornamental alpacas, so strict quarantine is necessary. reusken et al infected swine and chickens with sars-cov and determined that they are not susceptible to becoming hosts. in contrast, dromedary camels have been found to be capable of sars-cov infection. a similar capability to transmit mers-cov makes camels a focal link in the coronavirus transmission chain. as for other mers-cov domestic reservoirs, between and , some investigators tested serum samples from sheep, goat, cattle and chickens, which pervade saudi arabia and europe. on the other hand, another study that found mers-cov rna, but no neutralizing antibodies, in lambs. to some extent, in the context of methodical investigations, sheep could be another virus carrier in addition to camels. thus, it would be prudent to conduct field research and take necessary precautionary measures to preclude the possibility of transmission of coronaviruses from sheep. coronaviruses can be found in many kinds of birds. within hong kong alone, cov-hku has been found in nightingales, cov-hku in thrushes, cov-hku in munias, cov-hku in whiteeyes, cov-hku in sparrows, and cov-hku in magpies. fortunately, avian coronaviruses are not that closely related to sars-cov. additionally, these are not migratory birds and therefore do not expand the range of the pathogen. moreover, unlike bats, these birds were accessible enough to sterilize. however, in light of the findings above, randomly hunting them for food or for pets is unwise. compared to other symptom-limited models, non-human primary models are better-established models that have close psychological and physical similarities with humans (table ). the animal's age is the key factor affecting these results, but this is hard to identify in wild-caught monkeys. another suitable and well-established model is the common marmoset (saguinus mystax), which can show more severe clinical signs than rhesus macaques when infected with mers-cov. [ ] [ ] [ ] when administered through a combination of intraoral, intranasal and intravascular inoculation, with doses ranging from tcid to pfu, mild to moderate respiratory disease was observed, and interstitial pneumonia was observed clinically and microscopically. when infected with sars-cov, common marmosets exhibit fever, diarrhea, multifocal pneumonitis and hepatis. research using this model is progressing. the common marmoset is a potential nonhuman primate model for sars-cov infection and deserves more attention. to date, mers-cov only has two mature models. this section will deal with additional non-human prime models for sars-cov. rhesus, cynomolgus (macaca fasicularis), and african green (chlorocebus aethiops sabaeus or cercopithecu aethiops sabaeus) monkeys have been used to investigate vaccine immunogenicity or efficacy against sars-cov. squirrel monkeys (saimiri sciureus) and mustached tamarins (saguinus mystax) have been shown to be incapable of being infected. in the case of cynomolgus monkeys, clinical evidence, such as lethargy, temporary skin rash or respiratory distress, has not been reported. however, to days post-inoculation (dpi), there was diffuse alveolar damage and extensive loss of epithelium from alveolar and bronchiolar walls. , regarding african green monkeys, clearance of the virus takes approximately dpi, and the infection niduses are patchy. respiratory secretions cannot accurately reflect the viral titers. however, there is a report that showed that the titer is higher and the residence time is longer in african green monkeys than in two other kinds of old world monkeys (cynomolgus and rhesus). in contrast, young balb/c mice aged - months and -to month-old balb/c mice displayed clinical syndromes such as weight loss, dehydration, and ruffled fur at - dpi, along with interstitial pneumonia, cov, reaching the viral replication peak at or dpi and viral shedding by dpi. there was histochemical evidence of pneumonia but no clear clinical symptoms. in addition, recovered hamsters produced high levels of neutralizing antibodies to resist subsequent infection. unlike mice, the hamsters developed temporary viremia and viral replication in the liver and spleen. however, no inflammation was observed in those organs. this experiment proved that hamsters develop a more severe syndrome than mice. martine be et al three alpacas were experimentally intranasally inoculated with mers-cov. all of them shed viruses and antibodies were found in their serum. additionally, infected alpacas spread the virus to two other healthy alpacas housed in the same space, indicating the alpacas' own natural transmission capability and their potential as natural hosts. the authors also noted that mers-cov may be able to infect alpaca kidney cells. as with camels, alpacas never showed symptoms such as fever. however, unlike camels, alpacas did not demonstrate observable nasal secretion. hagamans bl et al regarding susceptibility to infection, goats are more susceptible than sheep. though the mers-cov neutralizing antibody has not been found in horses, three of the four experimentally infected horses had detectable viral replication in their nasal secretions starting at dpi. determining the evolutionary pathway leading to the ability to transfer between species may be helpful in predicting the next epidemic outbreak. regarding models for mers-cov, camels do not develop the same clinical signs as humans, although they are natural hosts. in addition, they require too much space to house and thus are not the first choice for a laboratory model. rabbits can be infected by mers-cov, but the histology is unstable, which would limit routine observation of disease. compared to those animal models, other than dpp transgenic mice, rhesus macaques and marmosets are currently the best mers animal models. since their immune system is like that of humans in terms of physiology and anatomy, they can be used to study the pathogenesis mechanism and the efficacy of vaccines and antivirals. however, research using rhesus macaques requires bsl- laboratories and high investments. moreover, they are smart, fast, and strong, which means precautions must be taken against them escaping. therefore, the need for more user-friendly models still exists, but to date non-human primate models are still the best option. however, there is still a possibility of establishing new models. chi wai yip et al phylogenetically analyzed the dpp receptor in various species and determined that humans, rhesus macaques, horses and rabbits belong to one family. cattle and swine are not in the same group, but their receptor is close to the human dpp receptor. small animals (including ferrets and mice) have dpp receptors far more distantly related to that of humans. this analysis could provide a reference point for potential animal models. in summary, non-human primate models are still the best choice of model. comparatively speaking, there is a greater variety of sars-cov animal models than mers-cov animal models. regarding priorities for research on vaccines and antivirals for both coronaviruses, suitable mers-cov models should be considered first. m- ), the chinese national major s & t project wild animal surveillance for coronavirus hku and potential variants of other coronaviruses prevalence and genetic diversity of coronaviruses in bats from china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats discovery of a rich gene pool of bat sars related coronaviruses provides new insights into the origin of sars coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syn rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat re-emerging middle east respiratory syndrome coronavirus: the hibernating bat hypothesis possible role of an animal vector in the sars outbreak at amoy gardens prevalence of igg antibody to sars-associated coronavirus in animal traders civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates comments to the predecessor of human sars coronavirus in - epidemic cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques pneumonia from human coronavirus in a macaque model treatment with interferon-a b and ribavirin improves outcome in mers-cov-infected rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus macaque model for severe acute respiratory syndrome an animal model of sars produced by infection of macaca mulatta with sars coronavirus treatment with lopinavir/ritonavir or interferon-b b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset infection with mers-cov causes lethal pneumonia in the common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n / or mers-cov emc/ isolates does not result in lethal disease pneumonitis and multiorgan system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans glycosylation of mouse dpp plays a role in inhibiting middle east respiratory syndrome coronavirus infection severe acute respiratory syndrome coronavirus infection of golden syrian hamsters therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters pathology of experimental sars coronavirus infection in cats and ferrets animal models for sars studies of severe acute respiratory syndrome coronavirus pathology in human cases and animal models infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas replicative capacity of mers coronavirus in livestock cell lines asymptomatic middle east respiratory syndrome coronavirus infection in rabbits inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding bats as animal reservoirs for the sars coronavirus: hypothesis proved after years of virus hunting phylogenetic perspectives on the epidemiology and origins of sars and sars-like coronaviruses how to cite this article: gong sr, bao ll. the battle against sars and mers coronaviruses: reservoirs and animal models key: cord- -o m xdra authors: spanakis, nikolaos; tsiodras, sotirios; haagmans, bart l.; raj, v. stalin; pontikis, kostantinos; koutsoukou, antonia; koulouris, nikolaos g.; osterhaus, albert d.m.e.; koopmans, marion p.g.; tsakris, athanassios title: virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen date: - - journal: international journal of antimicrobial agents doi: . /j.ijantimicag. . . sha: doc_id: cord_uid: o m xdra abstract serological, molecular and phylogenetic analyses of a recently imported case of middle east respiratory syndrome coronavirus (mers-cov) in greece are reported. although mers-cov remained detectable in the respiratory tract secretions of the patient until the fourth week of illness, viraemia was last detected days after initiation of triple combination therapy with pegylated interferon, ribavirin and lopinavir/ritonavir, administered from day of illness. phylogenetic analysis of the virus showed close similarity with other human mers-covs from the recent jeddah outbreak in saudi arabia. immunoglobulin g (igg) titres peaked weeks after the onset of illness, whilst igm levels remained constantly elevated during the follow-up period (second to fifth week of illness). serological testing confirmed by virus neutralisation assay detected an additional case that was a close contact of the patient. an upsurge of middle east respiratory syndrome coronavirus (mers-cov) infection has been recently described in countries of the arabian peninsula resulting in exported cases from these countries to the european union [ ] . cases of mers-cov infection are associated with a high case fatality rate since there is no available treatment. there is a scarcity of data on specific therapeutic interventions for the disease. published reports propose the use of known antivirals based on extrapolation of data from: (i) the severe acute respiratory syndrome (sars) epidemic that was also associated with the circulation of a novel coronavirus; (ii) in vitro data; (iii) animal experimental infections and therapy data; and (iv) limited clinical data for actual mers-cov infections [ ] [ ] [ ] . however, no clear-cut recommended therapeutic regimen exists and the evidence for grading such interventions is generally low, with the exception of the use of convalescent serum that based on biological effects is given the highest grade [ ] . moreover, little is known about the viral kinetics of mers-cov-associated infection, especially when a specific antiviral or other therapeutic intervention is attempted. a case of mers-cov has recently been described in greece in a traveller who had extensive contact with the healthcare environment in jeddah (saudi arabia) [ ] . here we describe molecular, serological and phylogenetic analyses of this case as well as evidence for a second case that was a close contact of the first patient. furthermore, we provide evidence of the kinetics and the pattern of viral excretion in biological specimens obtained from the first greek case while the patient was on a triple antiviral regimen. a preliminary report of the first imported, laboratory-confirmed mers-cov case in greece has been described elsewhere [ ] . a full description of the course of illness and treatment regimen in http relation to kinetics of virus shedding and immune response was prepared by review of the patient records. in the course of the outbreak investigation, of patient's contacts, including the patient's wife, provided an oropharyngeal sample for pcr testing week after contact with the positive case; additional contacts were included in the serology examination group. all were submitted to personal clinical monitoring for fever and upper respiratory infection symptoms and were advised to call the hellenic centre for disease control and prevention (cdc) command centre immediately in such an instance. in addition, all were offered the chance to provide serum samples on a voluntary basis for specific anti-mers antibody testing at baseline (same time as the oropharyngeal pcr testing) and weeks after exposure. during the patient's stay in the intensive care unit (icu), samples from the oropharynx, trachea, urine and faeces were tested for diagnostic evaluation and to monitor viral shedding. a real-time reverse transcription pcr (rt-pcr) method based on amplification of the upstream envelope gene (upe), the nucleocapsid (n) gene and the open reading frame (orf) a of the virus was used for detection of mers-cov according to previously described methodology [ , ] . immunoglobulin g (igg) and igm antibody titres in serum samples were determined using an anti-mers-cov indirect immunofluorescence assay (euroimmun ag, lübeck, germany). confirmation of the serological findings was performed with a virus neutralisation assay as described previously [ ] . samples from the patient's upper respiratory tract underwent conventional or molecular testing for the presence of other respiratory pathogens: thus, cultures applied for bacterial testing, whilst real-time rt-pcr was performed for several respiratory viruses including influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza, adenovirus, enterovirus, bocavirus and human metapneumovirus (hmpv) (m.w.s. r-gene; biomérieux, marcy-l'Étoile, france). specific urine antigen testing of urine samples was utilised for legionella pneumophila and streptococcus pneumoniae (binaxnow ® ; alere, orlando, fl). a stool culture was performed due to a history of possible typhoid fever, diagnosed by treating physicians in saudi arabia [ ] . nucleotide sequences of -kb concatenated sequences of representative mers-covs were analysed and a phylogenetic tree was constructed by the phyml method as described previously [ ] . a -year-old patient of greek origin who was a permanent resident of jeddah presented to a tertiary care centre a few hours after arriving in athens (greece) on april . his chief complaints included fever since april and diarrhoea since april . the most likely source of exposure was the hospital environment in jeddah. the patient had no known co-morbidities. at the time of initial evaluation, a fever of . • c was noted together with low oxygen saturation ( %), although the patient exhibited minimal respiratory symptoms. a chest radiograph depicted bilateral lung infiltrates consistent with viral pneumonia. the patient was immediately placed under isolation because of suspicion of mers-cov infection, and an antimicrobial regimen targeting communityacquired pneumonia was initiated. on april , mers-cov infection was confirmed by means of viral rna detection in a pharyngeal swab at the department of microbiology, university of athens medical school (athens, greece). after laboratory confirmation of mers-cov, the patient was transferred to a specialised respiratory disease unit in the 'sotiria' chest diseases hospital of athens where he was treated in a negative pressure regular room in isolation until april when, due to deterioration of his respiratory function and development of acute respiratory disease syndrome (ards), he was intubated, ventilated and transferred to a negative pressure room in the icu of the same hospital. an empirical antiviral regimen was initiated on day of illness consisting of oral (p.o.) lopinavir/ritonavir ( / mg twice daily), pegylated interferon ( g subcutaneously once per week for days) and ribavirin ( mg p.o. loading dose, followed by mg p.o. every h for days) based on available evidence [ ] [ ] [ ] , ] (fig. ) . the patient remained intubated exhibiting hypoxia and occasionally hypercapnia while breathing inspired oxygen in the range of . - . . he remained febrile with a plateau temperature of > • c and a maximum value of . • c on day of illness. fever started subsiding below • c on day . acute kidney injury was diagnosed on day of illness and rapidly progressed to non-oliguric renal failure that reverted to rifle injury level (i.e. two-fold increase in the serum creatinine, or glomerular filtration rate decrease by %, or urine output < . ml/kg/h for h) on day . the patient's diarrhoea resolved gradually starting on day and he developed constipation thereafter with normalisation of his bowel movements and gastrointestinal function on day . owing to development of jaundice and hyperbilirubinaemia attributed to ribavirin [ ] , the drug was discontinued on day . during the course of his hospitalisation, the patient was diagnosed with adenocarcinoma of the colon and eventually died from septic shock months and days after the initial diagnosis. cultures and antigen detection were negative for l. pneumophila and s. pneumoniae. virological testing was negative for the presence of any other respiratory virus. no relevant enteric pathogens were identified as a cause of the patient's diarrhoea. rna was detected in several consecutive patient samples from different sites that included faecal material and serum (fig. ) . shedding of mers-cov in the respiratory secretions of the patient was noted until the fourth week of illness, whereas viraemia was last detected days after onset of illness and days after initiation of the triple combination antiviral regimen. consecutive urine testing did not reveal the presence of mers-cov rna (fig. ) . serological testing showed a peak igg titre during the third week of illness, whilst during the fourth and fifth weeks igg titres were substantially declining. igm titres were persistently elevated during the whole survey period (day until day of illness) (fig. ). viral neutralisation assays performed at erasmus medical center (rotterdam, the netherlands) confirmed the immunofluorescence testing results. initial and follow-up serological testing were performed on serum samples from patient's contacts. seroconversion was revealed in one of them who developed an igg titre of / and an igm titre of / at days after making contact with the patient. this was a -year-old man with a past medical history of coronary artery heart disease and diabetes. the presence of specific mers-cov antibodies was confirmed by the virus neutralisation assay. without other symptoms from any other system. no nasopharyngeal pcr testing was performed at the time since he was outside the incubation period of days. he has been well since then and during the time that he was symptomatic he only had contact with his family members (four persons). the initial patient's wife had a brief episode of fever on may . oropharyngeal pcr testing was negative for mers-cov, and all contacts remained seronegative on repeat testing. partial genomic sequencing [ ] revealed the close phylogenetic relationship with clinical mers-cov strains associated with severe respiratory infection from patients in jeddah (fig. ) . in this report, we further characterised serological and virological parameters of the first mers-cov case in greece. rising titres of igg were demonstrated in sequential serum samples, with the peak titres approximately weeks into the course of the disease. this is in accordance with serological testing guidance from the world health organization (who) recommending baseline testing from initial contact with an affected case and repeated serological testing on day [ ] . on the other hand, igm titres of the patient remained constantly elevated above the threshold of detection, albeit at a lower level than igg antibodies, for a prolonged period of ≥ month of follow-up. thus, isolated use of igm testing without concomitant igg determination appears not to be sufficient to reveal a recent infection. it should be noted, however, that in the absence of detailed studies, use of serological testing for mers-cov detection in humans needs to be further evaluated. prolonged shedding of the virus was noted from the respiratory tract of the patient. this finding is consistent with data regarding the sars coronavirus. in a report dealing with patients affected by sars, prolonged shedding of the virus was noted in stool (up to days) and respiratory specimens (up to days) [ ] . data regarding the length of mers-cov excretion from different body sites are scarce [ ] . excretion of the virus probably depends on the amplitude of replication in different body sites, the underlying immune status and co-morbidities, and appropriate antiviral therapy. the non-detectable viral rna in serum by day after initiation of the antiviral treatment could be explained either by viral clearance in an otherwise immunocompetent person or by effectiveness of the instituted antiviral regimen. literature on appropriate antiviral intervention for mers-cov is very limited and currently no evidence-based therapy exists. the regimen chosen was based on the best available literature as well as evidence from animal and patient data that have been described elsewhere [ ] [ ] [ ] . the role of interferon therapy for mers-cov infection needs to be further elucidated. an attenuated interferon-␤ (ifn-␤) response has been described as a result of mers-cov infection [ ] and extensive use of interferon-based regimens alone or in combination with ribavirin has been described for sars [ ] . however, interferons appear to have a better antiviral effect on mers-cov compared with sars-cov in in vitro experiments [ ] . in vitro, ifn-␤ appears to exhibit the best anti-mers-cov effect [ ] . interferon activity has been enhanced by the addition of ribavirin in in vitro experiments [ ] . furthermore, this combination has shown promising clinical and radiological effects in rhesus macaques experimentally infected with mers-cov [ ] . thus, the clinical team elected to use this combination despite the fact that a prestigious public health agency ranks ribavirin use as not supported by high-quality evidence [ ] . in a more recent update published by the same public health agency, the use of interferons and lopinavir is ranked under the recommendation of benefit is likely to exceed risk, whereas the combination of interferon and ribavirin is ranked as data are inadequate for assessment [ ] . the frequent side effects of ribavirin limit its use in combination regimens for actual mers-cov-infected patients, as was the experience with the current patient where liver toxicity, although not definitively associated, was mainly attributed to this medication. the renal function deterioration of the patient described here was considered multifactorial and probably also a complication of the virus infection [ ] . the possibility that drug toxicity might have contributed in the renal dysfunction could not be excluded, however. no drug levels were measured since the patient was under continuous renal replacement therapy at that time and drug levels would be unreliable. in the actual clinical human setting, the combination of ribavirin and interferon has been tried, with no successful outcome reported among any of the mers-cov-infected recipients [ ] . nevertheless, the group studied consisted of severely ill patients who received this combination quite late in the course of their disease [ ] . lastly, we added the protease inhibitors lopinavir/ritonavir based on experience accumulated from the sars epidemic where the addition of this agent to ribavirin improved the outcome of infection [ ] . expanding the knowledge regarding viral kinetics and the pattern of shedding especially in association with specific therapeutic interventions has important implications for infection control in the healthcare environment, especially as it relates to potential transmission to other patients and healthcare workers [ ] . phylogenetic analysis of the greek mers-cov strain showed close similarity with circulating patient viral strains from the recent jeddah outbreak as well as with a strain isolated from a dromedary camel in qatar. this is in accordance with previous genetic studies that have shown identical viral strains between infected humans and dromedary camels and generated the hypothesis that dromedary camels are among the reservoirs of the virus in nature [ , ] . also, the presence of mers-cov-specific antibodies in camels across a wide geographic area in africa and the arabian peninsula signifies the possibility for zoonotic transmission between camels and humans [ ] [ ] [ ] . the potential for transmission across different individual strains should be further explored. in this patient, the most likely source of exposure was the hospital environment in an endemic area, as his wife was hospitalised in a local hospital in jeddah [ ] . it appears that healthcare-associated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic in the recent upsurge [ ] . in conclusion, we describe the genetic stability of the mers-cov in a strain from the recent jeddah outbreak. although reassuring, this finding should not limit the level of awareness regarding the increased number of cases in the arabian peninsula reported recently and the potential evolution and more efficient transmission of the virus. a who committee recently concluded that the conditions for a public health emergency of international concern have not yet been met. nevertheless, important gaps in current knowledge about mers-cov exist. more investigations to clarify the natural reservoir and modes of transmission are necessary. persistence of virus shedding in patients' secretions and the effect of immune status and antiviral therapy together with the implementation of appropriate infection control measures are of paramount importance in limiting further spread of this potentially lethal virus. funding: this work was funded by a grant from the netherlands organisation for scientific research (nwo) [no. - - - ]. competing interests: none declared. ethical approval: not required. european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov). ecdc what are our pharmacotherapeutic options for mers-cov? ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy clinical decision making tool for treatment of mers-cov v a case of imported middle east respiratory syndrome coronavirus infection and public health response assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections detection of a novel human coronavirus by 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through whole-genome analysis and virus cultured from dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of the authors would like to acknowledge the department of epidemiological surveillance and response of the hellenic centre for disease control and prevention (cdc), and especially dr georgia spala, theano georgakopoulou and agoritsa baka, for mers-related activities, as well as dr spyros sapounas for contacting the case investigation of the second case. key: cord- -cog nma authors: watkins, kevin title: emerging infectious diseases: a review date: - - journal: curr emerg hosp med rep doi: . /s - - - sha: doc_id: cord_uid: cog nma purpose of review: this review highlights some of the recent concerning emerging infectious diseases, a number of them specifically that the world health organization has categorized as priorities for research. recent findings: emerging and reemerging infectious diseases account for significant losses in not only human life, but also financially. there are a number of contributing factors, most commonly surrounding human behavior, that lead to disease emergence. zoonoses are the most common type of infection, specifically from viral pathogens. the most recent emerging diseases in the usa are emergomyces canadensis, the heartland virus, and the bourbon virus. summary: in addition to the aforementioned pathogens, the severe acute respiratory syndrome, middle east respiratory syndrome, nipah virus, new delhi metallo-ß-lactamase- enterobacteriaceae, rift valley fever virus, and crimean-congo hemorrhagic fever virus are reviewed. these pathogens are very concerning with a high risk for potential epidemic, ultimately causing both significant mortality and financial costs. research should be focused on monitoring, prevention, and treatment of these diseases. in , an expert committee that produced the institute of medicine report on emerging infections defined them as "new, reemerging, or drug-resistant infections whose incidence in humans has increased within the past two decades or whose incidence threatens to increase in the near future." additionally, six major contributors to these diseases were presented and included changes in human demographics and behavior, advances in technology and changes in industry practices, economic development and changes in land-use patterns, dramatic increases in volume and speed of international travel and commerce, microbial adaptation and change, and breakdown of public health capacity [ ] . a common theme to recent emerging infectious diseases is that the majority are of animal origin [ ] . in fact, some vector-borne pathogens that are projected to be introduced into new regions include rift valley fever and japanese encephalitis viruses in the americas and crimean-congo hemorrhagic fever virus in eurasia [ ] . additionally, the majority of them have been viral. in addition to the cost of human life, emerging or reemerging infectious diseases can be very costly financially. the recent zika outbreak is estimated to have cost $ . billion in economic losses in [ ] . the world health organization has prioritized a number of infectious diseases as requiring urgent need for research and development given the concern for potential of severe outbreaks. this article reviews the majority of emerging infections on this list, a few others that have emerged in the usa as well, and those that have not recently emerged but that the who has prioritized as having a high likelihood of causing outbreaks in the future. in , an outbreak in southern china of atypical pneumonia of undetermined etiology spread to neighboring countries and eventually across the world. the coronavirus that caused the severe acute respiratory syndrome (sars) was then discovered [ ] . hong kong and beijing were the most severely affected cities with estimated costs in the far east of this article is part of the topical collection on infectious disease * kevin watkins kwatkin @gmail.com approximately $ billion by may alone [ ] . over probable cases were reported in countries with a mortality rate of about % by the end of the epidemic in july . sars reemerged in small scales from the end of to . it is a positive-sense rna coronavirus whose mean incubation period is days [ ] . the clinical presentation varied by patient age: children developed typical mild upper respiratory tract infection while teenagers and adults developed a more severe but predictable course [ , ] . phase i of the course was associated with increasing viral load with flu-like symptoms. phase ii was characterized by recurrence of fever with hypoxemia and progression of pneumonia. about % would require supplementary oxygen and approximately % would require intubation due to the development of acute respiratory distress syndrome (ards). watery diarrhea was a prominent extrapulmonary manifestation. additionally, hepatitis was a common complication and patients occasionally developed neurologic manifestations including status epilepticus. the clinical worsening in phase ii is thought to be immune-mediated. laboratory findings include lymphopenia, elevated aminotransferases, elevated lactate dehydrogenase (ldh), and creatine kinase (ck), with results consistent with disseminated intravascular coagulopathy. progression from unilateral opacities to bilateral involvement was found on imaging. sars is detected in respiratory secretions, urine, and feces [ ] . however, the most notable finding is diffuse alveolar damage [ ] . advanced age, severe hepatitis, high initial ldh, high neutrophilia on presentation, diabetes mellitus, low cd and cd counts, and high initial viral load are all prognostic factors for severe disease. some survivors have shown persistent lung function abnormalities. it is spread by close contact via droplet transmission, though there is some evidence that it might follow airborne transmission [ ] . the virus has a high rate of infecting healthcare workers, accounting for approximately % of cases [ ] . the original outbreak was thought to have started from the handling of wild animals, particularly the civet cat and raccoon dog [ , ] . if suspected, a patient should be placed in respiratory isolation. a single test result, either positive or negative, is insufficient to make conclusions about a patient's diagnosis as both false positives and false negatives occur. it is initially tested for via serology or reverse-transcriptase-polymerase chain reaction (rt-pcr). however, two specimens obtained from either different sources or from the same source on different occasions confirm the diagnosis. specimens can be obtained from a nasopharyngeal swab or stool. additionally, seroconversion is another diagnostic method. it is vital to identify patients with sars, as isolation within days of illness significantly reduces secondary transmission [ ] . although ribavirin was used in the initial outbreak, it has not been shown to have in vitro activity against the virus. corticosteroids should be used only in the late-phase for rescue purposes as it may prolong viremia if given early [ ] . most of the other agents that have shown promise, including monoclonal antibodies, nitric oxide, protease inhibitors, and interferons have not yet shown activity in humans and there have been very few animal studies [ , ] . part of the concern about the potential for future epidemics lies in the fact that the family of viruses from which sars comes is notorious for frequent mutations [ ] . additionally, there has been decreased worldwide attention, leading to decreased funding for research, as there have been no reported cases of sars since [ ] . the middle east respiratory syndrome coronavirus (mers-cov) was first identified in . it is a ß coronavirus that is enveloped with a positive-sense rna genome. the clinical features may include flu-like symptoms, gastrointestinal symptoms, severe pneumonia with acute respiratory distress syndrome, septic shock, disseminated intravascular coagulopathy, and multi-organ failure [ ] . lab findings may include lymphopenia, thrombocytopenia, elevated ldh levels, and elevated aminotransferases [ , ] . however, approximately % of cases had no or mild symptoms while almost % had severe symptoms [ ••] . the incubation period is - days. children and younger adults seem to be less susceptible [ ] . a large proportion of the confirmed pediatric cases have been asymptomatic and have been acquired via household contacts [ ] . primary infection results from contact with a dromedary camel or its products, though human-to-human transmission is possible. cases acquired via human transmission tend to be more mild and the number of infected contacts tends to be limited. in fact, the majority of secondary cases have been associated with healthcare settings, where the majority of outbreaks have occurred and about % of cases have been healthcare workers [ ] . with the exception of south korea, outbreaks have been limited to the middle east where dromedary camels are found [ ] . in the south korean outbreak, there were cases and deaths [ ••] . cases have been imported to europe, asia, africa, and the usa [ table ]. the cases in the usa were detected promptly, so no secondary cases occurred. both cases occurred in may and were healthcare workers in saudi arabia [ ] . as of july , there have been laboratory-confirmed cases, with the majority of these in saudi arabia. the reproduction number is less than with significant heterogeneity [ ••, ] . in the south korean outbreak, "spreaders" had significantly higher rates of underlying pulmonary disease. most transmissions occurred early in the disease course, during days - after symptom onset and were associated with patients going to multiple healthcare facilities before diagnosis [ ] . human-to-human transmission seems to require close contact, though has not been sustained. most cases requiring hospitalization are those with chronic comorbidities, such as obesity, diabetes, end-stage renal disease, or chronic lung disease [ ] . the mortality rate is about % [ ••] . among those who were critically ill, patients frequently had underlying chronic disease, and extrapulmonary manifestations were common [ ] . mers can be detected in respiratory tract secretions with bronchoalveolar lavage specimens providing a better yield than nasopharyngeal swabs; however, serum samples may also be obtained [ ] . the centers for disease control and prevention (cdc) recommends evaluation for patients with respiratory symptoms and travel to an affected country within days or for those who are in a cluster of patients with severe acute respiratory disease in which mers-cov is being evaluated [ ] . testing should not be performed before h of exposure [ ] . healthcare workers with exposure during aerosol-generating procedures are at high risk for transmission; because of this, the cdc recommends airborne and contact precautions for suspected or known cases [ ] . patients suspected to have mers should be admitted if they have shortness of breath, pneumonia, or hypoxemia. otherwise, they may be cared for at home in isolation; however, these cases should be carefully selected for potential of spread [ ] . treatment is primarily supportive with mechanical ventilation and renal replacement therapy as needed as there are no specific antivirals that have been developed [ ] . a number of studies have been performed with a variety of medications, including interferons (ifn), ribavirin, protease inhibitors, mycophenolic acid, cyclosporin a, chloroquine, nitazoxanide, antibiotics, fusion inhibitors, and steroids. perhaps the most promising human studies have involved a combination of ribavirin and interferons, though overall results have been mixed. some have shown no benefit, but have been critiqued that treatment was too late. other studies have shown improved -day survival but not at days, and others have shown improved survival at both [ , , ] . a small retrospective study had all eight patients who received combination therapy with ifn-ß and mycophenolic acid survive, though they had lower acute physiology and chronic health evaluation ii scores compared to patients who received other agents [ , ] . two new viruses that have recently been discovered in the usa are the heartland and bourbon viruses. the heartland virus is a member of the bunyaviridae family in the genus phlebovirus with single-stranded, negative-sense rna [ ] . the lone star tick (amblyomma americanum) transmits the disease [ ] . it was first discovered in from a farmer in missouri. clinical features include flu-like symptoms, leukopenia, thrombocytopenia, and mild transaminitis and develop within days of tick bite. six additional cases were identified during - ; four of these patients were hospitalized and one patient with multiple underlying diseases died. five patients were in missouri and one was in tennessee [ , ] . the deceased patient had developed hypoxemic respiratory failure with severe thrombocytopenia, acute kidney injury, and upper gastrointestinal bleeding and his family ultimately opted to transition to comfort measures. his clinical course was consistent with severe infection from ehrlichia chaffeensis except that he did not improve with doxycycline [ ] . diagnosis should be suspected when a patient does not improve with doxycycline therapy and can be made with detection of viral rna by rt-pcr [ •]. the bourbon virus is in the genus thogotovirus within the family orthomyxoviridae and is pleomorphic with an rna genome. the case patient was from bourbon county, kansas, in . within several days of tick bite, he developed flu-like symptoms. later findings included leukopenia, thrombocytopenia, hyponatremia, and mild transaminitis; the case patient then developed congestive heart failure and later died after withdrawal of care [ ] . a study of tick populations in missouri revealed that amblyomma americanum ticks were carriers of the virus, though transmission from these ticks is not certain [ ] . little is known otherwise about the disease as confirmed cases have been scarce. nipah encephalitis was first noted in an outbreak of fatal encephalitis starting in in malaysia. it is an rna virus from the family paramyxoviridae, genus henipavirus. the outbreak initially involved pig-farming villages but spread to other areas [ ] . since discovery, outbreaks have been recognized almost yearly in bangladesh and occasionally in india [ ] . it is characterized by a high attack rate with neurologic features at presentation. the strain in malaysia rarely displays respiratory symptoms, though the more concerning bangladeshi strain often has a respiratory component [ , ] . prodromal symptoms consisting of sore throat, myalgias, fever, headache, and vomiting are common with the ultimate development of altered mental status and potentially seizures and coma. the majority of patients show brainstem dysfunction with pinpoint pupils, dysautonomia, and abnormal vestibulo-ocular reflex. myoclonus may also be present and characteristically involves the diaphragm and anterior neck muscles. patients commonly develop thrombocytopenia and abnormal liver function tests. cerebral spinal fluid analysis yields normal glucose with elevated white cell counts and protein typical of viral infections. diagnosis can be made by rt-pcr. most patients with encephalitis had abnormal electroencephalograms and magnetic resonance imaging studies (mri). mris commonly show small discrete hyperintense lesions in the subcortical and deep white matter [ ] . pathophysiologically, nipah virus causes a widespread vasculitis most commonly involving the brain and lungs [ ] . mortality rates have been quoted to be between and %. the majority of people infected develop severe disease. neuropsychiatric sequelae are not uncommon (one third of survivors) with persistent cognitive impairment, cerebellar signs, and peripheral nerve lesions [ , ] . relapse and late-onset encephalitis may also occur months or years after the acute illness, though rates are rare (about %). additionally, relapse or late-onset disease has a lower mortality rate of about % with minimal brainstem involvement. transmission can consist of consumption of food contaminated by bat saliva (raw sap is common), contact with infected animals, or human-to-human spread [ , ] . sustained human-to-human transmission beyond generations has not been recognized and the reproductive number has averaged . . those that are at greatest risk of infection are providers of fatally infected patients with prominent respiratory secretions. however, most patients do not transmit infection to anyone [ ] . bats of the family pteropodidae are the natural reservoirs, though numerous mammals can become infected. treatment consists of supportive care. treatment with ribavirin was tried during a malaysian outbreak with % reduction in mortality; however, this was not a controlled clinical trial and did not reach statistical significance [ ] . the concern about a potential for pandemic arises from the fact that it is an rna virus and the bangladeshi strains have high genetic variability with potential for an increase in the reproductive number [ ] . an experimental human monoclonal antibody (m . ) has shown promise in nonhuman studies [ ] . another emerging infectious disease is the dimorphic fungus, emergomyces canadensis. although other emergomyces species have been discovered throughout europe and africa, e. canadensis was only recently discovered in north america [ ] . emergomyces was previously named "emmonsia-like" pathogens. there are numerous species of emergomyces, with the most common being e. africanus that causes infections in those with hiv in south africa. no animal infections have been documented [ ] . there have been four cases noted so far in immunocompromised patients that developed systemic disease with fungemia. patients commonly had pneumonia as well as skin lesions. it is suspected that inhalational infection occurs and that geographic range involves central and western north america [ ] . the diagnosis is made via histopathology or culture of affected tissue [ ] . limited susceptibility testing has shown susceptibility to itraconazole and amphotericin b. treatment should follow typical guidelines for dimorphic fungal infections in immunocompromised hosts with amphotericin b for - weeks followed by itraconazole for months [ ] . one of the most concerning emerging infectious diseases is the n e w d e l h i m e t a l l o -ß -l a c t a m a s e - ( n d m - ) enterobacteriaceae. these "superbugs" seemed to have originated in the indian subcontinent and cases were first reported in , though retrospective analysis shows its presence as early as . the ndm- gene is carried on a plasmid, bla ndm- , that is easily spread among bacteria [ ] . although carbapenamases are not new, the level of promiscuity of ndm- is higher than others [ ] . additionally, studies have shown both a high level of inter-lineage and inter-species gene transfer though the dominant mechanism seems to be the latter. it is most commonly found among escherichia coli and klebsiella pneumoniae, but has also been found within all species of enterobacteriaceae as well as acinetobacter baumanii and pseudomonas species [ ] . although gram-negative bacteria have typically been treated with ß-lactams, fluoroquinolones, and aminoglycosides, ndm-positive pathogens contain up to antibiotic resistance genes and are only susceptible to tigecycline and colistin, with the exception of a few strains sensitive to aztreonam and certain aminoglycosides [ ] . multiple variants have been reported (i.e., ndm- ). ndm- is widely found throughout both hospital strains as well as community strains in india, which is consistent with the rates of extendedspectrum ß-lactamase (esbl) of % in both the community and hospital setting [ , ] . it is likely that the overuse of antibiotics led to excretion into the waste water systems, ultimately resulting in selection-pressure for multidrug-resistant organisms [ , ] . in fact, some estimate that at least million indian residents have gut flora consisting of ndm- containing bacteria [ ] . ndm- has spread to numerous other countries; it is believed that the medical tourism industry propagated its dissemination. in fact, seven out of eight tourists to india were colonized with esbl-positive bacteria upon return, which they did not have upon departure [ ] . the balkans have been another area with high rates of ndm-positive bacteria, which has now spread to all continents except south america and antarctica [ fig. from ref- erence ] ]. ndm-positive bacteria have even now been found in companion animals in the usa [ ] . adding to the concern is that there are few antimicrobials in the research pipeline with activity against gram-negative pathogens. rift valley fever virus (rvfv) is a single-stranded rna virus of the genus phlebovirus, family bunyaviridae. the majority of infected individuals are asymptomatic. most patients who are symptomatic have a self-limiting febrile illness; however, a small minority develop severe disease with acute hepatitis, renal failure, and hemorrhagic manifestations. those who survive this phase often develop neurologic symptoms consisting of vision loss and encephalitis. long-term sequelae may develop with blindness, hemiparesis, quadriparesis, incontinence, hallucinations, or coma. the fatality rate is - %, though patients with hemorrhagic disease have a fatality rate as high as %. diagnosis is made by detection of viral rna and immunoglobulin serology, particularly during the acute febrile stage [ ] . it is often transmitted by mosquitoes, typically of the aedes genus, though exposure to infected animal tissue or consumption of their products can also cause disease [ ] . rvfv is endemic in sub-saharan africa with periodic outbreaks after heavy rainfall and flooding. it has spread to the middle east and the french island of mayotte. outbreaks often have significant effects on both humans and livestock. characteristics of an rvfv outbreak include the sudden increase in abortions and mortality of young animals with the appearance of disease in humans. models have been developed with successful predictions of outbreaks using satellite imagery of sea surface temperatures, rainfall, and vegetation. given this spread out of africa, there is concern about it reaching the mediterranean basin and europe. a number of competent vectors for rvfv have been identified in europe. it has additionally been isolated from other hematophagous arthropods including ticks and sand flies, though their significance is not currently understood. there is currently no specific treatment, though a vaccine, tsi-gsd- , has provided protection following primary inoculation and single boost schedule [ ] . crimean-congo hemorrhagic fever virus (cchfv) produced its first major known outbreak from to in the crimean peninsula, though it has likely been causing outbreaks for centuries. it was not identified until . as such, it has not recently emerged but is listed as a priority agent for research and development by who due to its potential for spreading and causing more severe outbreaks. cchfv is a member of the bunyaviridae family, genus nairovirus. it has many aliases, including asian ebola, karakhalak (black death), khunymuny (nose bleeding), and khungribta (blood taking). cchfv derives its name from outbreaks both in crimea and the belgian congo and has been reported throughout many parts of africa, the middle east, europe, and asia [ fig. from reference ]. crimean-congo hemorrhagic fever (cchf) is the most widespread tick-borne viral infection of humans [ ] . it is a negative-sense rna virus transmitted by numerous tick species; because of this, agricultural workers are most commonly affected [ , ] . additionally, human-to-human transmission may occur via contact with skin, mucous membranes, or body fluids of infected patients [ ] . standard barrier precautions are recommended for patients with suspected disease [ ] . domestic livestock are the main reservoirs. outbreaks have been increasing in recent years. likely targets of the virus are endothelial cells, hepatic cells, and kupffer cells. the clinical course of cchf follows phases consisting of incubation, prehemorrhagic, hemorrhagic, and convalescence [ ] . there is a spectrum of severity from a mild, febrile illness to hemorrhagic shock with multiorgan failure [ ] . the incubation period varies by method of acquisition; it typically takes - days by tick bite and - days by exposure to infected patients. the prehemorrhagic stage is nonspecific with flulike symptoms. the hemorrhagic stage follows and develops within - days of symptoms in severe disease [ ] . one differentiating feature is that severely ill patients often develop a pattern of large ecchymosis not seen in other hemorrhagic fevers [ ] . laboratory findings include leukopenia, thrombocytopenia, elevated aminotransferases, elevated ck and ldh, fig. distribution of crimean-congo hemorrhagic fever virus and prolonged prothrombin time and activated partial thromboplastin time. convalescence usually occurs - days after onset of illness [ ] . diagnosis is commonly made via detection of viral rna with rt-pcr or serology. there has traditionally been no specific treatment for cchf, though ribavirin has demonstrated both in vitro and in vivo activity [ ] . furthermore, both the cdc and who recommend use of ribavirin [ •] . a report from turkey claimed that a combination of methylprednisolone, intravenous immunoglobulin (ivig), and freshfrozen plasma was beneficial, but there was no control group for comparison [ ] . ivig and methylprednisolone may be beneficial by improving thrombocytopenia [ •, ] . two vaccines have been developed, though they have not been through randomized clinical trials [ ] . cchf has never been demonstrated in vaccinated individuals and the bulgarian ministry of health has reported a fourfold decrease in cases since vaccine implementation [ , ] . ribavirin might be beneficial for post-exposure prophylaxis [ ] . mortality rates have varied between and %, with worsening prognosis if the disease is acquired nosocomially, with higher levels of aminotransferases, higher viremia, or with coagulopathy [ , ] . long-term sequelae have been documented but are rarely permanent and include impaired vision and other neurologic symptoms [ •] . cchf has epidemic potential with high rates of mortality, nosocomial infection, and difficulty with prevention and treatment [ ] . additionally, it is the most genetically diverse of the arboviruses [ ] . attempts to control cchf via eradication of tick vectors have proven inefficient and domestic animals are asymptomatic even when highly viremic [ • ]. infectious diseases promise to be one of the biggest challenges in the coming decades, with new ones emerging unpredictably. successful clinical outcomes require a proactive approach with research and development, as human behavior contributes to new infections emerging, particularly with zoonoses. although this strategy would prove costly, this article has demonstrated a few examples of how much an emerging infectious disease can cost during an outbreak. this article has reviewed a number of emerging infections that the who has categorized as priorities for research and development due to their potential for epidemics or even a pandemic. additionally, it has reviewed a concerning drug-resistance mechanism that threatens to provide bacteria with resistance to all antibiotics. finally, a few infectious diseases that have emerged in the usa have been covered, though little is known about them, with a paucity of cases to date. conflict of interest the author declares that he has no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. emerging infectious diseases: a cdc perspective emerging infectious diseases in : years after the institute of medicine report emerging infectious disease: a proactive approach a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome (sars): a review of the history, epidemiology, prevention, and concerns for the future severe acute respiratory syndrome and coronavirus severe acute respiratory syndrome (sars) severe acute respiratory syndrome and influenza: virus incursions from southern china who guidelines for the global surveillance of severe acute respiratory syndrome (sars) anti-sars coronavirus agents: a patent review ( -present) middle east respiratory syndrome coronavirus: a comprehensive review middle east respiratory syndrome: what clinicians need to know middle east respiratory syndrome coronavirus: update for clinicians this article gives the majority of a summary of perhaps most significant recent emerging infectious disease in terms of both mortality and number of cases middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus a review of treatment modalities for middle east respiratory syndrome treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia a new phlebovirus associated with severe febrile illness in missouri notes from the field: heartland virus disease-united states heartland virus associated death in tennessee update and commentary on four emerging tick-borne infections novel thogotovirus associated with febrile illness and death bourbon virus in field-collected ticks nipah encephalitis-an update the pandemic potential of nipah virus therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody emergomyces canadensis, a dimorphic fungus causing fatal systemic human disease in north america emergomyces: a new genus of dimorphic fungal pathogens causing disseminated disease among immunocompromised persons globally global spread of antibiotic resistance: the example of new delhi metallo-ß-lactamase (ndm)-mediated carbapenem resistance the new medical challenge: why ndm- ? why indian? emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study the emergence of pan-resistant gramnegative pathogens merits a rapid global political response rift valley fever virus: a review of diagnosis and vaccination, and implications for emergence in europe risk factors associated with human rift valley fever virus infection: systematic review and meta-analysis crimean-congo hemorrhagic fever crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity crimean-congo haemorrhagic fever virus: past, present and future insights for animal modelling and medical countermeasures recent advances in research on crimean-congo hemorrhagic fever key: cord- -mfyff ne authors: modjarrad, kayvon title: treatment strategies for middle east respiratory syndrome coronavirus date: - - journal: journal of virus eradication doi: nan sha: doc_id: cord_uid: mfyff ne middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than people, resulting in almost deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of this novel pathogen have created an urgent need for effective public health countermeasures, including safe and effective treatment strategies. despite the relatively few numbers of cases to date, research and development of mers-cov therapeutic candidates is advancing quickly. this review surveys the landscape of these efforts and assesses their potential for use in affected populations. respiratory tract infections are the leading cause of mortality in resource-limited settings, accounting for more than million deaths each year globally [ ] . epidemic-and pandemic-prone respiratory viruses are the aetiological pathogens in many cases, and have caused several of the most prominent infectious disease outbreaks of the past two decades: these include h n influenza in , severe acute respiratory syndrome (sars) in and pandemic h n influenza in . most recently, middle east respiratory syndrome coronavirus (mers-cov) has emerged as a novel cause of severe acute respiratory illness after first being identified in a saudi arabian patient in [ ] . although initially restricted to the arabian peninsula, this emerging pathogen has respectively infected and killed more than and people on four continents across countries [ , ] . phylogenetically related to sars-cov [ ] , mers-cov has a similar clinical presentation [ ] [ ] [ ] [ ] , albeit with a higher case fatality rate (~ % versus %) [ ] [ ] [ ] . dromedary camels serve as the principal animal reservoir for this virus; and zoonotic spillover from dromedaries to humans has, thus far, driven the course of the epidemic [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although humanto-human transmission has been documented -particularly in the context of nosocomial outbreaks [ ] [ ] [ ] [ ] [ ] [ ] -the spread of mers-cov is inefficient and unsustained, as reflected in an estimated reproduction rate of no higher than . [ , ] . mers-cov is an enveloped, single-stranded, positive-sense rna virus that comprises a -kilobase genome that codes for four structural proteins and an rna polymerase [ ] , typical of the coronaviridae family ( figure ). the most immunogenic of these proteins is the virus' only surface glycoprotein, spike (s) [ ] [ ] [ ] that mediates viral attachment and fusion via the host cognate receptor, dipeptidyl peptidase (dpp ) [ ] . although the broad principles of the virus' life cycle and its mechanisms of pathogenesis are beginning to be understood, this knowledge has not yet translated to a licensed therapy or vaccine. much of the work to develop safe and effective mers-cov countermeasures has centred on vaccines, but the relatively low prevalence of the disease, the sporadic nature of the case clusters and the dearth of detailed knowledge on chains of transmission highlight the need for greater investments into the discovery of effective therapeutic and secondary prophylactic regimens for infected and exposed individuals. efforts to research and develop treatment strategies for mers-cov are accelerating but remain limited in their scope and stage of advancement. there are few novel compounds being studied that are specific for mers-cov molecular targets, as most treatment options, investigational and licensed, are being repurposed from their use for other rna viruses or other non-infectious diseases. the current landscape of mers-cov therapies, therefore, is dominated by an armamentarium of repositioned drugs with in vitro activity against mers-cov replication, but is also speckled with agents that are directed towards and derived from host immunity. the current review surveys the landscape of therapeutic products in each category and assesses their potential for advanced testing and development. respiratory and circulatory support, preservation of renal, hepatic and neurological function, and prevention of secondary infections. beyond implementing basic principles of critical care medicine, immune-based therapies have been used most commonly during both the sars-cov pandemic of and the current mers-cov epidemic, each time yielding equivocal results. there have been some promising animal data where combination treatment with ribavirin and interferon (ifn)-α b improved clinical outcomes in mers-cov-infected non-human primates (nhps). however, treatment was initiated very soon after viral challenge (~ hours), a window that is unlikely to be replicated in a real-world clinical setting [ ] . various ifn regimens, in combination with ribavirin, have been intermittently administered to severely ill patients, although typically in an ad hoc manner and in the absence of systematic evaluation [ ] [ ] [ ] [ ] [ ] . individual case reports and uncontrolled case series not only limit determination of whether an intervention works but if it is safe as well. ribavirin, for example, is a potent nucleoside analogue that has been used with varying measures of success against a range of rna viruses [ ] . however, patients can experience significant toxicities when given the drug alone or in combination with an interferon, including but not limited to haemolytic anaemia and metabolic abnormalities. interferons also can elicit systemic adverse effects, psychiatric disturbances and neutropenia [ ] . thus, without the benefit of randomised controlled trial data, it becomes difficult to assess whether the treatment is worse than the disease. certain strategies, however, have been shown to worsen clinical outcomes in the setting of a coronavirus infection. for example, studies during the sars pandemic showed that corticosteroids, when used early on sars-cov infected patients, significantly increased viral load, icu admission and mortality [ , ] . the role for interferon therapies has been less clear in the current mers-cov epidemic, as some data show a positive impact on proximate outcomes, such as oxygenation and inflammation, but no effect on more significant outcomes like hospital stay and long-term survival [ , , ] . rapidly scaled treatments based on naturally occurring neutralising antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have been shown to be relatively safe and potentially effective for reducing mortality from several infections such as sars-cov and influenza [ ] [ ] [ ] , and may hold promise for mers-cov as well. this strategy, however, relies on the rapid identification of cases and contacts and immediate deployment of products to have maximal impact. one study found that convalescent plasma decreased mortality in sars-cov patients only if administered within days of illness [ ] . a network for the use of convalescent plasma for case clusters of mers-cov is currently being assembled [ ] to test its safety, efficacy and feasibility. however, actualisation of this plan is limited by logistical challenges, local technical capacity and donor supply. unfortunately, no host-derived experimental interventions have yet demonstrated appreciable benefit in acutely ill, mers-covinfected patients in a consistent or controlled manner. this reality, although, has not slowed down the discovery and advancement of passive prophylactic products derived from vaccinated and infected animals and humans. despite intensive efforts to develop a mers-cov vaccine, the prevalence and transmissibility of this emerging pathogen are both relatively low [ , ] , making it difficult to define a target population for vaccination. mabs, on the other hand, can be administered in the setting of an outbreak without the need to discriminate who might be at greatest risk for infection. they can be used to treat cases early in their natural history and for post-exposure prophylaxis of case contacts. mabs also carry the benefits of higher potency, greater specificity, more extensive pre-licensing evaluation and consequently a more vetted safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic-level detail for the design of better immunogens. they also have been proven as effective therapies in the areas of cancer treatment and autoimmune disease management. although there is only one pathogen, respiratory syncytial virus, for which a mab is licensed for use, there are a number of other infectious disease indications-such as ebola virus disease treatment and human immunodeficiency virus primary and secondary prevention-for which mabs are being tested in advanced phase clinical trials (www.clinicaltrials.gov). despite all of these advantages, the timelines and costs of mab research and development (r&d) are respectively longer and higher than that for polyclonal antibody preparations. in spite of the requirements for greater upfront investments and a more rigorous testing and approval process, several groups have identified highly potent mers-cov mabs and are advancing them through preclinical stages of development (table ) . some have been isolated from immunised animals (mice/humanised mice/nhps) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , while others have been identified from either an antibody human phage library [ ] or memory b cells of infected and recovered human survivors [ ] . almost all of the published mabs and all of those in development target the s receptor-binding domain (rbd), which contains the most immunogenic epitopes on the virus. many bind to the rbd, expressed both on a recombinant s and on the surface of live virus, with picomolar affinity and neutralise mers-cov at a half maximal inhibitory concentration (ic ) of ng/μl or less. additionally, several groups have demonstrated new york blood center mersmab s imunised mouse rbd in vitro [ ] nih national cancer institute m , m , m human antibody library rbd in vitro [ ] nih niaid d , f , g , g s/s immunised mouse rbd, s , s nhp efficacy [ ] regeneron regn /regn humanised mouse rbd mouse/nhp efficacy [ ] tsinghua university mers- , mers- human antibody library rbd in vitro [ ] rbd: receptor binding domain; s: spike glycoprotein; s : spike domain containing rbd; s : spike domain containing fusion machinery. journal of virus eradication ; : - protective efficacy in pre-and post-exposure prophylaxis animal models. because most of the antibodies target the rbd, there is a potential for viral escape from any one mab. thus, there may be a need to develop antibodies against other vulnerable sites on s or to investigate the use of combination mabs to overcome the potential emergence of therapeutic resistance. it is likely that mabs directed at other sites on the s glycoprotein have already been recovered but are not as potent neutralisers, as is the case in one report [ ] . a more efficient search for potent neutralising antibodies that target epitopes outside the rbd could be facilitated by a more detailed understanding of the atomic-level structure of the entire s glycoprotein, as has already been resolved for the rbd. the successes thus far in isolating potent and protective mabs, although significant, are likely to be tempered by the challenges of advancing these products to licensing and full-scale production at affordable costs for as of yet undefined populations in a relatively short timeframe. thus, mabs should be advanced along a development pipeline in parallel with a program of rational drug design and discovery. although intensive, supportive care still serves as the primary treatment option for mers-cov and mabs are the focus of the most advanced r&d efforts, antiviral therapies are being actively investigated for use in severely ill patients. there are two main pathways for drug discovery: de novo development and repurposing licensed medications. there are few new antivirals for mers-cov; however, the ebola epidemic has had an unanticipated consequence of facilitating their development. one in particular, gs- developed by gilead sciences, is an adenine analogue that is incorporated into viral rna to disrupt replication [ ] . it has shown survival benefit in nhps inoculated with ebola virus and is now advancing through a phase i dose escalation trial. it has been claimed to have in vitro activity against mers-cov as well, but publication of these data is pending. similarly, bcx is a nucleoside analogue that is being developed by biocryst pharmaceuticals for potential treatment of filoviruses, coronaviruses and other rna viruses [ ] . additionally, small interfering rna molecules and peptide inhibitors are being investigated for their ability to disrupt mers-cov replication, although these products are still in very early phases of investigation [ , ] . as the life cycle and genetic sequence of this new coronavirus has become better elucidated, the rational design and development of novel and approved agents with potent antiviral activity have become possible. the advent of high-throughput screens of licensed compounds and small molecules has also allowed researchers to efficiently evaluate large libraries of drugs for their in vitro antiviral activity against novel targets [ ] [ ] [ ] [ ] [ ] [ ] . to date, several dozen licensed drugs have been reported to inhibit mers-cov replication. using slightly different screening technologies, different groups have converged on some common classes of compounds, including nucleoside analogues, antibacterial protein synthesis inhibitors, kinase signalling modifiers, antimetabolites and antiprotozoal agents. mycophenolic acid, an inhibitor of both t an b lymphocytes, has also been found to have strong activity against mers-cov, as it does against other rna viruses such as west nile, hepatitis c and dengue [ ] . only one of the drugs to show in vitro activity against mers-cov, lopinavir, however, has been tested in an animal model. mers-cov-challenged marmosets that were treated with this protease inhibitor had better clinical, pathological, virological and radiological outcomes than controls or those treated with mycophenolate mofetil [ ] . additionally, two peptides, hr p and hr p are being developed as potential fusion inhibitors [ ] . by acting on the six-helix bundle core of the mers-cov s protein to prevent protein-mediated cell-to-cell fusion, this class of compounds may hold potential beyond mers-cov towards a long-term objective of a pan-coronavirus antiviral. given some of the common life cycles and pathways of pathogenesis for rna viruses and homologies in protein structures across different coronaviruses, there may be economies of effort and investment in developing antivirals that have activity against more than one virus or family of viruses. irrespective of the breadth of these novel or repurposed compounds, treatment studies should be carried out prospectively according to protocols that plan for the collection of quality controlled data and serial biological sampling to assess viral evolution and biomarkers of favourable clinical outcomes. recent infectious disease outbreaks such as the h n influenza pandemic, the h n influenza epidemic in china, the ebola crisis in west africa and now the mers-cov outbreak have highlighted the need for better r&d preparedness and improved coordination of clinical testing in the face of the accelerating number of emerging and re-emerging infectious diseases. the ability to have an armamentarium of countermeasures and clinical trial infrastructure in the early phases of an outbreak is critical for mounting an effective public health campaign. for example, the sars-cov pandemic caused more than cases of severe acute respiratory illness and nearly deaths but few prospective, controlled studies were undertaken to determine the optimal management of the disease. consequently, treatment options for sars-cov were never defined clearly and thus difficult to adapt for mers-cov. although global coordination has resulted in the advancement of some urgently needed, novel countermeasures for mers-cov, they will have to be developed along faster timelines than before, with greater investments earlier in the preclinical development pipeline that can generate products for more timely efficacy testing in affected populations. as the global community takes lessons from the most recent outbreak and prepares for the potential of another regional epidemic or broader pandemic, stakeholders in mers-cov r&d must set out a sound strategy now for where to best target their investments in anticipation of the changing dynamics of the current and future outbreaks. global and regional mortality from causes of death for age groups in and : a systematic analysis for the global burden of disease study isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) summary and literature update -as of european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov) from sars to mers: years of research on highly pathogenic human coronaviruses clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient evidence for camel-to-human transmission of mers coronavirus epidemiological investigation of mers-cov spread in a single hospital in south korea transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea transmission among healthcare worker contacts with a middle east respiratory syndrome patient in a single korean centre transmission characteristics of mers and sars in the healthcare setting: a comparative study health-care associate transmission of middle east respiratory syndrome corona virus, mers-cov, in the kingdom of saudi arabia assessment of the middle east respiratory syndrome coronavirus (mers-cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach estimation of mers-coronavirus reproductive number and case fatality rate for the spring saudi arabia outbreak: insights from publicly available data genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a -residue region in the spike protein that efficiently elicits neutralizing antibodies a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc treatment with interferon-alpha b and ribavirin improves outcome in mers-cov-infected rhesus macaques ifn-alpha a or ifn-beta a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome: a case report ribavirin and interferon-alpha b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review interferon in oncological practice: review of interferon biology, clinical applications, and toxicities the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis the use of convalescent plasma to treat emerging infectious diseases: focus on ebola virus disease a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for middle east respiratory syndrome coronavirus (mers-cov) evaluation of candidate vaccine approaches for mers-cov exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections structural basis for the neutralization of mers-cov by a human monoclonal antibody mers- identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus nucleotide prodrug gs- is a broad-spectrum filovirus inhibitor that providescomplete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx in a non-human primate model of filovirus infection structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus design of potential rnai (mirna and sirna) molecules for middle east respiratory syndrome coronavirus (mers-cov) gene silencing by computational method antiviral drugs specific for coronaviruses in preclinical development a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors alternative screening approaches for discovery of middle east respiratory syndrome coronavirus inhibitors treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset the opinions expressed herein are those of the authors and should not be construed as official or representing the views of the us department of defense or the department of the army. key: cord- -umuiovrd authors: bindayna, khalid mubarak; crinion, shane title: variant analysis of sars-cov- genomes in the middle east date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: umuiovrd background coronavirus (covid- ) was introduced into society in late and has now reached over million cases and , deaths. the middle east has a death toll of ∼ , and over , of these are in iran, which has over , confirmed cases. we expect that iranian cases caused outbreaks in the neighbouring countries and that variant mapping and phylogenetic analysis can be used to prove this. we also aim to analyse the variants of severe acute respiratory syndrome coronavirus- (sars-cov- ) to characterise the common genome variants and provide useful data in the global effort to prevent further spread of covid- . methods the approach uses bioinformatics approaches including multiple sequence alignment, variant calling and annotation and phylogenetic analysis to identify the genomic variants found in the region. the approach uses samples from the countries of the middle east sourced from the global initiative on sharing all influenza data (gisaid). findings we identified distinct genome variants including downstream gene variants, frame shift variants, missense variants, start lost, start gained, stop lost, synonymous variants and upstream gene variants. the most common, high impact variants were deltinsg, delcinsc, delcinsc and delainsa. variant alignment and phylogenetic tree generation indicates that samples from iran likely introduced covid- to the rest of the middle east. interpretation the phylogenetic and variant analysis provides unique insight into mutation types in genomes. initial introduction of covid- was most likely due to iranian transmission. some countries show evidence of novel mutations and unique strains. increased time in small populations is likely to contribute to more unique genomes. this study provides more in depth analysis of the variants affecting in the region than any other study. funding none on january h , the china centre for disease control reported that of suspected cases of pneumonia were due to a novel human coronavirus (cov), now known as severe acute respiratory syndrome cov (sars-cov- ) . the genome for this novel virus was then made publicly available on the global initiative on sharing all influenza data (gisaid) the next day. sars-cov- is an easily spreadable virus which would evolve into a global pandemic of at least million cases and , deaths . one of the first countries to experience a significant outbreak was iran. the country reported its first confirmed case on th february from a merchant in qom who travelled from china . . many of the first countries with infections in the middle east were linked to travellers from iran including lebanon, kuwait, bahrain, iraq, oman and uae. covid- continued to spread to the remaining middle eastern countries with a death toll of over , people according to health authorities. this number is expected to be an underestimation due to countries effected by war including libya, syria and yemen. needless to say, there have been devastating effects to the region and the real effects are expected to be unreported . researchers are racing to develop a vaccine that can provide viral immunity and avoid additional deaths. sar-cov- is transmitted using the spike protein which binds to human angiotensin-converting enzyme (ace ) receptor; the virus is easily transmittable due to mutations in the receptor-binding (s ) and fusion (s ) domain of the strain . transmission could be made even easier if more mutations accumulate. although mutations are rare, they can create new strains and it is not guaranteed that the current leading vaccine trials will be effective as sars-cov- continues to mutate . by categorizing variants, we can identify any new strains and how the mutations are likely to affect spread. as the middle east is often under reported, it is important to characterise the variants of strains that are commonly present. analysis of the common variants in the middle east is essential to develop a vaccine that treats the strains in the region. this analysis helps understand the viral genome landscape and identify clades of the region. • our hypothesis is that variants found in sars-cov- genomes from middle eastern samples will indicate delivery from iran. we will use bioinformatics tools and publicly available samples to explore the composition of strains within each country. we expect that many strains will show evidence of iranian origin. • the aim is to explore the structure of middle eastern genome strains using multiple sequence alignment, tree generation and variant prediction (and others). if we explore the structure and common variants of sars-cov- strains in these populations, we expect to learn more about how the virus spread. sample source: we obtained the publicly available data from the global initiative on sharing all influenza data (gisaid) . samples were also filtered to high quality when possible. samples was selected as the optimum number to cover all possible countries and remain within alignment file limit of size mb (maximum size for clustal omega tool). in countries with samples, to prevent sample sourcing from same outbreak, the earliest and most recent samples were taken. all samples were downloaded from gisaid and then concatenated into a single multi-sample file and saved in fasta format. multiple sequence alignment: using the collected samples, multiple sequence alignment (msa) was performed using clustal omega (https://www.ebi.ac.uk/tools/ msa/clustalo/) . the clustal omega online tool was used to perform the alignment (found at: https://www.ebi.ac.uk/tools/msa/clustalo/). the online tools allows up to sequences or a maximum file size of mb, therefore the maximum number of samples was used. the concatenated file of samples was uploaded to the online tool. for step , the output parameters selected were pearson/fasta. all other options were kept at the default option. the output file generated is an alignment file; the file consists of all sequences with gaps denoted by '-'. the output file format is also a fasta file. variant identification: variant calling was performed using the alignment fasta file and the snp extraction tool snp-sites (https://github.com/sanger-pathogens/snp-sites). these tools identify the snp sites by taking a multi-sample fasta file as input. the program then restructures the data as a variant call format (vcf) file. the vcf file provides a clear mapping of snps from the aligned sequences -this allows us to easily identify the snp location and the genotype for each sample at a given locus. in the outputted vcf file, the rows correspond with each unique variant and the column provides the genotype at the given site. we used snp-eff to perform the variant annotation information such as the variant definition and the overlapping gene (found at: (https://pcingola.github.io/snpeff/snpeff.html). snpeff also predicts the effect of the variants. snpeff is integrated into the galaxy web-based tool for bioinformatics analysis (found at: usegalaxy.org) . we utilised the galaxy platform and uploaded our vcf file to galaxy using their online upload tool. to annotate variants, you must first build a database from the reference genome. this is performed using the "snpeff build" tool on galaxy. to create the snpeff database, we downloaded data from ncbi for wuhan reference nc_ . and uploaded to galaxy. to build the database, we directed to ncbi and searched for nc_ . we then downloaded the corresponding gff file, which contains the annotations, and the fasta file, which contains the entire genome. we then selected the build database option. once the database was built, we selected the "snpeff eff" tool to annotate variants. galaxy populates the fields for vcf with the uploaded file from the previous step. the output format is selected as vcf and csv report was also selected for additional useful information for downstream analysis. for genome source parameter, the option "custom" was selected to use the newly created database. other default parameters were used including bases for upstream / downstream length and bases as set size for splice sites (donors and acceptor) in bases. all filter output and additional annotation options were deselected and analysis was ran. once the analysis was executed, the annotation data is outputted as an annotated vcf and a html report file. data visualisation: once the annotated vcf was generated, the vcf was imported to r for extraction of the variant annotation information. the annotated data was imported, manipulated and plotted using r v . . . the dplyr v . . package was used to summarise and align the data . the ggplot package was used to align the identified variants and visualise the types of mutations that re-occur . the x-axis in the plots indicates the variant position along the sars-cov- genome; the left y-axis indicates the sample name and the right y-axis represents the country of origin for each sample. this plot is used to compare the genome in different populations. the data was then ordered by date of first reported case, meaning that wuhan is followed by united arab emirates and the final country is cyprus. phylogenetic analysis: phylogenetic analysis was performed using beast (bayesian evolutionary analysis sample trees) v . . , to perform bayesian analysis of molecular sequences using monte carlo markov chains (mcmc) . the analysis followed the approach recommended to reconstruct the evolutionary dynamics of an epidemic. the aim of this is to obtain an estimate of the origin of the epidemic in the region and understand how it spread through the middle east. to undertake the analysis, we opened beauti, the graphical application used to analyse the control file. although it requests a nexus file, the fasta file can also be used. the data was uploaded using the import data option and appeared under the partitions section. beauti confirmed that sites are present in the uploaded data. the default options are selected for site model and clock model. next, we specified the individual virus dates by selecting the "tips" panel and selecting the "use tip dates" option. a tab delimited file was uploaded which specified the upload date. this information was extracted from the names as they were downloaded from gisaid. next, we set the substitution model by selecting the "sites" tab and selected the default options of hky model, the default estimated base frequencies and select gamma as site heterogeneity model. next, the molecular clock was selected under the "clock" tab as a strict clock since we know that the frequency of mutation is low. the tree options are elected under the "tree prior" tab as "random starting tree" for the tree model and "coalescent: exponential growth", a model that assumes a finite but constant population size and predicts that all alleles will be removed from the population individually. this provides additional predictions on the reproductive rate. in the "priors" tab, select the scale as for prior distribution which models the expected growth for a pandemic. the operators require no changes from the default. the mcmc option for chain length is set as , and sampling frequency to . tree visualisation: finally, we summarised the tree using the treeannotator tool, an additional package as part of beast. we first select the file generated using beast and outputted the tree file. then the output nexus file was imported to figtree program to display. once we opened figtree to display, we re-ordered the order by increasing value and then switched on branch labels. we switched on node bars and selected the % highest posterior density (hpd) credible intervals for the node heights. we plotted a time scale by turning on the scale axis and then setting the time scale section for offset as . , the latest date of collection for our samples. sequence alignment and variant calling were completed successfully. once these were complete, variation annotation was performed. we identified distinct genome variants which are recorded in table . the most common, high impact variants were deltinsg, delcinsc, delcinsc and delainsa. the frequency of each unique variant type can be found in table , which outlines the locus of all snps with over instances. frame shift variants start lost stop lost upstream gene variant these results were then used to generate the dotplot of variant alignment (figure ). the dotplot successfully indicated a pattern in variants that could not be easily identified from the alignment or annotation files. the alignment includes samples in facets based on their country. the alignment shows a pattern in variants that occur between each country. for example, this is prominent in qatar, jordan and oman where the pattern makes the country distinctive from the variants plotted for other countries. in addition, the phylogenetic tree generated branching indicative of an iranian origin ( figure ) the aim of this study was to identify whether covid- was introduced to the middle east from iran and also to explore the genomic composition in the region. our study performs sequence alignment to compare all sequences against the reference genome. once this is complete, the annotated variants were extracted to generate a plot mapping variants, grouping samples by country. the plot as seen in figure , shows clear distinctive patterns within countries that are not obvious from the generated alignment and annotation files. this may indicate a diverse, new strain is circulating in the country. cyprus has little diversity in the variant mapping which is surprising given its late date for first reported cases. another interesting point is that time-varied samples were taken for countries with samples. we see not indication that there are distinct groups within countries. this further indicates the the mutation frequency is low. it also indicates that there is more variation in the genomic composition in samples from different countries than differences found in samples from different collection times. smaller populations can cause greater accumulation of variants through genetic drift. this may occur given local lockdowns and travel restrictions that have been enforced worldwide. it is possible that these genomic strains with new mutations may create a situation where the countries develop a deathly strain that is not prominent in other parts of the world. this could result in a situation where a country is disproportionately affected by accumulating deaths or an inefficient vaccine. phylogenetic trees help in understanding the evolutionary relationships between groups. in the present context, we use them to identify the earliest strains and to track the spread of covid- across the middle east. the tree shows that uae samples are distinguished and form one clade. this correlates with their early intervention and lockdown and subsequently appears to have resulted in a unique genome. samples from qatar also form the majority of clade, with many of the wuhan samples, indicating that they are similar to the wuhan samples and show little distinction. egypt also becomes a distinct branch earlier than most samples. these examples are indicative of the global response -the lockdown of each country and prevention of spreading has resulted in sars-cov- strains of great similarity within each country. if lockdowns were not enforced, it is likely that these clades would be less distinguisable as mutations are spread between countries. as we expected, of the highest branches points attach to iranian samples, further implementing iran in the initial spread across the middle east. the phylogenetic tree therefore indicates what we suggest in our hypothesis -most samples originate from the iranian sample. this is not surprising given the vast number of cases and early crisis state of the country. however, it is useful to see that the variant analysis shows what we suspect at the genome level. a related study also came to this conclusion by using contact tracing from cases related to religious events in the city of qom, iran . risk assessment: outbreak of acute respiratory syndrome associated with a novel coronavirus covid- situation reports mapping the incidence of the covid- hotspot in iran -implications for travellers challenges to testing covid- in conflict zones: yemen as an example phylogenetic analysis and structural modeling of sars-cov- spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop on the origin and continuing evolution of sars-cov- data, disease and diplomacy: gisaid's innovative contribution to global health fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega snp-sites: rapid efficient extraction of snps from multi-fasta alignments a program for annotating and predicting the effects of single nucleotide polymorphisms, snpeff: snps in the genome of drosophila melanogaster strain w the galaxy platform for accessible, reproducible and collaborative biomedical analyses: update r: a language and environment for statistical computing. r foundation for statistical computing dplyr: a grammar of data manipulation ggplot : elegant graphics for data analysis bayesian phylogenetic and phylodynamic data integration using beast . virus evolution is visiting qom spread covid- epidemic in the middle east? the authors are grateful for the timely sequencing and release of genomes to make this study possible and to dr. anusha c p for her comments. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. iran epi_isl_ iran epi_isl_ iran epi_isl_ iran epi_isl_ iran epi_isl_ iran epi_isl_ iran epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ israel epi_isl_ jordan epi_isl_ jordan epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ saudiarabia epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ turkey epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ unitedarabemirates epi_isl_ wuhan epi_isl_ wuhan epi_isl_ wuhan epi_isl_ wuhan epi_isl_ wuhan epi_isl_ wuhan epi_isl_ table : catalogue of sample accession id by country. key: cord- -ay cnzn authors: chan, jasper f.w.; chan, kwok-hung; kao, richard y.t.; to, kelvin k.w.; zheng, bo-jian; li, clara p.y.; li, patrick t.w.; dai, jun; mok, florence k.y.; chen, honglin; hayden, frederick g.; yuen, kwok-yung title: broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: ay cnzn objectives: middle east respiratory syndrome coronavirus (mers-cov) has emerged to cause fatal infections in patients in the middle east and traveler-associated secondary cases in europe and africa. person-to-person transmission is evident in outbreaks involving household and hospital contacts. effective antivirals are urgently needed. methods: we used small compound-based forward chemical genetics to screen a chemical library of known drugs against influenza a virus in biosafety level- laboratory. we then assessed the anti-mers-cov activities of the identified compounds and of interferons, nelfinavir, and lopinavir because of their reported anti-coronavirus activities in terms of cytopathic effect inhibition, viral yield reduction, and plaque reduction assays in biosafety level- laboratory. results: ten compounds were identified as primary hits in high-throughput screening. only mycophenolic acid exhibited low ec( ) and high selectivity index. additionally, ribavirin and interferons also exhibited in-vitro anti-mers-cov activity. the serum concentrations achievable at therapeutic doses of mycophenolic acid and interferon-β b were – and – times higher than the concentrations at which in-vitro anti-mers-cov activities were demonstrated, whereas that of ribavirin was ∼ times lower. combination of mycophenolic acid and interferon-β b lowered the ec( ) of each drug by – times. conclusions: interferon-β b with mycophenolic acid should be considered in treatment trials of mers. a novel lineage c betacoronavirus, previously known as human coronavirus emc/ and later renamed as middle east respiratory syndrome coronavirus (mers-cov), has emerged in the arabian peninsula since april to cause a "severe acute respiratory syndrome (sars)-like" disease in laboratory-confirmed cases with fatalities in countries in the middle east, europe, and north africa as of october . e animal-to-human transmission has been suspected in view of mers-cov's close phylogenetic relatedness to other lineage c betacoronaviruses found in bats in hong kong, mexico, europe, and africa, e and its broad species tropism in various animal cell lines including those of bats, primates, pigs, civets, and rabbits. , recently, a serological study of major livestock suggested dromedary camels to be a possible host based on the high prevalence of mers-cov neutralizing antibodies in dromedary camels from oman. however, targeted studies are needed to confirm this finding and its possible relevance to human cases of mers-cov infection as most cases did not have contact with camels and the virus has not been isolated in animals yet. the epidemic continues to evolve with recent outbreaks occurring among epidemiologically-linked household contacts in the kingdom of saudi arabia, the united kingdom, italy, and tunisia, and hospital contacts in the kingdom of saudi arabia, jordan, the united kingdom, and france providing evidence for mers-cov's potential for person-to-person transmission. e unlike most other human coronavirus infections which are generally mild, most patients with mers have suffered from rapidly progressive pneumonia with some also developing acute renal failure, hepatic dysfunction, gastrointestinal upset, pericarditis, disseminated intravascular coagulation, and/or cytopenias. , the resulting crude mortality rate of nearly % in documented cases far exceeded those seen in all other human coronavirus infections including sars despite aggressive supportive treatment including extracorporeal membrane oxygenation in some of the mers cases. while mild and asymptomatic cases have been recognized, , , these recent case clusters signify a global health threat especially in view of the unusual clinical severity of mers, travel of infected persons to other countries and influx of religious pilgrims to the kingdom of saudi arabia, and the lack of proven effective specific antiviral treatment. after our initial success in applying chemical genetics in probing novel targets and compounds for antiviral development, we started looking for broad-spectrum antiviral compounds that may be active against both influenza a viruses and coronaviruses, the two viral pathogens responsible for causing the recent pandemic and largescale epidemics. while neuraminidase inhibitors such as oseltamivir and zanamivir remain effective against most seasonal and avian influenza a viruses, e proven antiviral therapeutic options for coronavirus infections is lacking. given the limited time available to develop novel anti-mers-cov agents in this evolving epidemic, we attempted to provide an alternative solution by identifying potential broad-spectrum antiviral agents against mers-cov and influenza a viruses by a small compound-based forward chemical genetics approach using chemical libraries consisting of drug compounds already marketed or having reached clinical trials in the united states, europe, or asia (microsource discovery systems, usa). we then assessed the anti-mers-cov activities of the identified drug compounds in cell culture by cytopathic effect (cpe) inhibition, viral yield reduction, and plaque reduction assay (pra) assays, as well as drug cytotoxicity. a clinical isolate of mers-cov was kindly provided by r. fouchier, a. zaki, and colleagues. the isolate was amplified by one additional passage in vero cells to make working stocks of the virus (  tcid /ml). all experimental protocol involving live mers-cov isolate followed the standard operating procedures of the approved biosafety level- facility as we previously described. the influenza a/ wsn/ (h n ) virus was expanded in chick embryo as we previously described. chemical reagents and high-throughput screening (hts) a total of pre-existing drug compounds (microsource discovery systems) were screened against influenza a/ wsn/ (h n ) virus. high-throughput screening (hts) was carried out in a fully automated beckman coulter core system (beckman coulter, usa) integrated with a kendro robotics co incubator (thermo fisher scientific) at chemical genetics unit, department of microbiology, research center of infection and immunology, li kashing faculty of medicine, the university of hong kong as we previously described with modifications. briefly, compounds were added in -well microtitre plates (tpp) in duplicate with a final concentration of mm or mm and , madinedarby canine kidney (mdck) cells per well in ml complete eagle's minimal essential medium (emem) supplemented with % heat-inactivated fbs. cells were then inoculated at an moi of . with influenza a/ wsn/ (h n ) virus for detection of broad-spectrum antivirals. after infection, the plates were incubated at c with % co and monitored daily using a leica dm inverted light microscope for virus-induced cpe. drugs that gave full protection of mdck cells (no cpe) were selected for further evaluation with mers-cov in a biosafety level- laboratory. the cytotoxicity of selected drug (ribavirin: e . mg/ml; introna , e . iu/ml; avonex: , e . iu/ml; rebif: , e . iu/ml; betaferon: , e . iu/ml; mmf: e . mg/ml) was determined by thiazolyl blue tetrazolium bromide (mtt) assay according to manufacturer's instructions. the endpoint was the % effective cytotoxic concentration (tc ). the drug compounds identified as primary hits showing a ec of less than or equal to um and a selectivity index of more than were diluted with serum free mem and added to confluent vero cells in -well culture plates in triplicate for h at c. after incubation, the drugcontaining media was removed, and mers-cov at . moi was added together with fresh drug-compound media to each well containing approximately , cells. following h adsorption at c, the virus-compound mixture was removed and the cells were washed times with mem to remove unbound virus. subsequently, media with antiviral compounds were added to the cells for further incubation for h at c in a % co humidified environment. cpe was examined by inverted light microscopy, and ml of supernatant was collected for virus quantification, as we previously described with modifications. thereafter, ml of serum free mem and ml of mg/ml mtt solution (prepared in  pbs, filtered) were added to the wells. the monolayers were incubated as above for h (away from light). finally, ml of % sds with . m hcl was added and further incubated at c with % co overnight. the activity was read at od with reference wavelength at od . the interferon and non-interferon drug compound with the lowest % effective inhibitory concentration (ec ) and highest selectivity index were selected for combination studies using the cpe inhibition assay. for the drug compounds with antiviral activity in the mtt assay, further evaluation by quantitative virus yield reduction and plaque reduction assays (pra) was performed. virus yield quantification was performed by quantitative rt-pcr using total nucleic acid extracted from culture supernatants of the vero cells infected by mers-cov on day post-infection as we previously described. pra was performed as we previously described with modifications. briefly, it was performed in duplicate in well tissue culture plates (tpp). the vero cells were seeded at  cells/well in mem (invitrogen) with % fbs on the day before carrying out the assay. after e h incubation, e plaque-forming units (pfu) of mers-cov virus were added to the cell monolayer with or without the addition of drug compounds and the plates further incubated for h at c in % co atmosphere before removal of unbound viral particles by aspiration of the media and washing once with mem. monolayers were then overlaid with media containing % low melting agarose (cambrex) in mem and appropriate concentrations of drug compounds and incubated as above for h. next, the wells were fixed with % formaldehyde (bdh) overnight. after removal of the agarose plugs, the monolayers were stained with . % crystal violet (bdh) and the plaques counted. the percentage of plaque inhibition relative to the control (without the addition of compound) plates was determined for each drug compound concentration. the ec and the % cellular cytotoxicity concentration (cc ) were calculated using sigma plot (spss) in an excel add-in ed v . the pra were carried out in triplicate and repeated twice for confirmation. high-throughput screening (hts) ten drugs compounds, namely mycophenolic acid, flufenamic acid, tolfenamic acid, meclofenamate sodium, mefenamic acid, ribavirin, mercaptopurine, pyrimethamine, emetine, and estradiol were identified as primary hits with protective results in chemical library screening against influenza a/wsn/ (h n ) virus (table ) . neuraminidase inhibitors were not identified because they were not included in the chemical library. amantadine was not identified because the virus strain had an m gene mutation (s n) conferring drug resistance. using both ec and tc as the hit selection criteria, only mycophenolic acid exhibited a low ec of < mm with a high selectivity index of > . mercaptopurine, which is a competitive, selective, and reversible inhibitor of the sars-cov papain-like protease, in addition to mycophenolic acid (sigmaealdrich, usa), ribavirin (tianxin pharmaceutical, china), intron a (recombinant interferon-a b, schering-plough, usa), avonex (recombinant interferon-b a, biogen idec, denmark), rebif (recombinant interferon-b a, merck serono, italy), betaferon (recombinant interferon-b b, bayer schering pharma, germany), imukin (recombinant interferon-g b, boehringer ingelheim, germany), nelfinavir mesylate hydrate (agouron pharmaceuticals, usa), and lopinavir (abbott, usa) were also tested in the mtt assays because of their documented in vitro anti-sars-cov activities in previous reports. e among them, only mycophenolic acid, ribavirin, intron a, avonex, rebif, and betaferon showed anti-mers-cov activity at the tested concentrations ( table ) . cpe was completely absent in vero cells infected with mers-cov on day post-infection at concentrations of ! . mg/ml for mycophenolic acid and ! mg/ml for ribavirin, and was decreased but not absent in the tested concentrations of intron a, avonex, rebif, or betaferon (table ) . combination studies showed that the ec of mycophenolic acid was lowered by . e . times in the presence of . e . iu/ml of betaferon, and that the ec of betaferon was lowered by . e . times in the presence of . e . mg/ml of mycophenolic acid (table ) . the mean baseline viral load in the cell culture supernatants without drugs was . ae . log copies/ml. there was a % reduction in viral load as compared to the baseline in cell culture supernatants inoculated with each of the six drugs (fig. ). there was a > -log reduction in viral load in cell culture supernatants inoculated with mycophenolic acid, ribavirin, rebif, and betaferon. there was > -log reduction in the viral load in cell culture supernatants at iu/ml of betaferon and > -log reduction at the highest concentration of , iu/ml (fig. c) . the largest reduction in viral load at clinically relevant drug levels was a nearly -log reduction at mg/ml of mycophenolic acid. mycophenolic acid, ribavirin, and rebif achieved % plaque reduction at concentrations of . mg/ml, mg/ml, and , iu/ml respectively (figs. and ) . the maximum percentages of plaque reduction achieved by intron a, avonex, and betaferon were . % at , iu/ml, . % at iu/ml, and . % at iu/ml respectively (fig. ) . in pra, betaferon achieved e % plaque reduction at iu/ml (fig. c ). novel antiviral targets for sars coronavirus and influenza a virus have been identified previously using small compoundbased forward chemical genetics approaches similar to ours. , , in this study, we identified ten compounds among approved drugs with as primary hits in chemical library screening that possess antiviral activities. some may offer potential therapies in the evolving mers-cov epidemic. influenza a/wsn/ (h n ) virus, instead of mers-cov, was used for initial screening because its manipulation did not require a biosafetly level iii laboratory. other human betacoronaviruses such as hcov-oc and hcov-hku were not used because of their slow replication and low viral titres in cell culture. among the identified drug compounds, only mycophenolic acid exhibited an ec of < mm, which is a common cut-off value for lead compound detection, and a high selective index of > . additionally, we tested other agents reported to have in vitro activities against sars-cov and/or mers-cov. , , imukin (interferon-g b) and the hiv protease inhibitors, nelfinavir mesylate hydrate and lopinavir, showed suboptimal ec in the initial cpe inhibition assay and were therefore not further evaluated. together with mycophenolic acid, four other drug compounds in five preparations, namely ribavirin, intron a, avonex, rebif, and betaferon, showed in vitro anti-mers-cov activity of varying magnitude across four assays. mycophenolic acid is a selective, non-competitive, and reversible inhibitor of inosine- -monophosphate dehydrogenase (impdh). it inhibits the proliferation of t and b lymphocytes and production of immunoglobulins by depletion of the lymphocyte guansine and deoxyguanosine nucleotide pools. its major clinical indication is prevention of graft rejection in solid organ and haematopoeitic stem cell transplantations. in addition to potent immunosuppressive activity, mycophenolic acid also has broad activity in vitro and/or in animal models against different viruses including west nile, japanese encephalitis, yellow fever, dengue, chikungunya, and possibly hepatitis b viruses. furthermore, it inhibited the in vitro and in vivo replication of hepatitis c virus by augmentation of interferon-stimulated gene expression and depletion of guanosine. , combination treatment with interferon-a showed additive effects on interferon-stimulated gene expression and enhanced interferon-induced luciferase reporter activity. as for coronaviruses, mycophenolic acid test test test , . t t t , . t t t remarks: -, negative; þ is defined as %e % involvement; þ is defined as > %e % involvement; þ is defined as > %e % involvement; þ is defined as > % involvement; t, druginduced toxic effects in vero cells. was found to be ineffective against sars-cov in an animal model, although it did not significantly increase the viral load in the lungs of sars-infected balb/c mice as ribavirin did. we are unaware of data on its activity against other human coronaviruses. our study is the first to demonstrate the anti-coronavirus activity of mycophenolic acid against the novel mers-cov. in addition to mycophenolic acid, our in vitro findings indicated that ribavirin, interferon-a, and interferon-b had anti-mers-cov activities in vitro. in the case of sars-cov, their antiviral activities in in vitro susceptibility tests had been conflicting. none of them were tested systemically in large-scale randomized controlled trials and the results from clinical trials involving their use in sars were often confounded with the concomitant use of corticosteroids. , although their clinical use in mers-cov infection has not been described, a recent study found that ribavirin had in vitro anti-mers-cov activity at very high concentrations which was potentiated when given together with interferon-a b. another study showed that mers-cov is e times more sensitive to pegylated interferon-a than sars-cov in vero cells, which is possibly related to the lineage-specific genetic differences between the two coronaviruses with mers-cov lacking the homolog of the sars-cov orf protein responsible for the blockade of interferon-induced nuclear translocation of phosphorylated transcription factor stat . furthermore, the delayed and aberrant induction of inflammatory cytokines and chemokines by mers-cov might support the use of adjunctive immuno-modulatory treatment combined with antivirals in patients with mers. , among the four preparations of interferons tested, betaferon exhibited the lowest ec of . iu/ml, which was below the mean peak serum concentration of iu/ml after a subcutaneous dose of million iu or an intravenous dose of . million to million iu. although the other preparations of interferons also demonstrated in vitro anti-mers-cov activities, their ec were generally above the peak serum concentrations achievable with usual therapeutic dosing. combination treatment consisting of mycophenolic acid and betaferon resulted in a . e . -fold reduction in the ec of mycophenolic acid in vero cells with . e . iu/ml of betaferon, and . e . -fold reduction in the ec of betaferon in vero cells with . e . mg/ml of mycophenolic acid. our finding may provide the basis for combinational mycophenolic acid and betaferon in future clinical trials. compared with ribavirin and interferons, mycophenolic acid exhibits a number of attributes that support its practical use in mers-cov infection. it is commonly available in two forms, the prodrug mycophenolate mofetil and the salt mycophenolate sodium, and could be given orally or parenterally. the serum concentration of mycophenolic acid peaks at around e mg/ml after a mg oral dose of mycophenolate mofetil or . mg/ml after a mg oral dose of mycophenolate sodium. these far exceeds its ec of . mg/ml and is e times higher than the concentrations at which the replication of mers-cov is inhibited in cell culture and pra. with average plasma elimination half-lives of . h and . h after a mg oral dose and mg intravenous dose of mycophenolate mofetil respectively, the usual regimens consisting of mg twice daily oral or mg twice daily intravenous mycophenolate mofetil would be sufficient to achieve levels well above the ec throughout the dosing interval. in contrast, the ec of ribavirin for mers-cov between . and . mg/ml is just marginally effective in some cell lines and greatly exceeds the drug's serum concentration with usual oral doses. peak concentrations with high intravenous doses may reach approximately mg/ml in humans, but steady-state requires at least weeks to achieve. , furthermore, the use of ribavirin, and hence also its combination with interferon-a b, may be limited in the clinical setting, because a significant proportion of patients with mers-cov infection have developed acute renal failure often requiring renal replacement therapy. , it has been suggested that systemic ribavirin should best be avoided in patients with a creatinine clearance of < ml/min because of the increased risk of haemolytic anaemia. although mycophenolic acid may also be associated with acute renal impairment, the dosage adjustment in such a setting is generally well established. the potent in vitro anti-mers-cov activity of mycophenolic acid may allow it to be used as a monotherapy if concomitant interferon is not available or tolerated by the patient. finally, drug level monitoring for mycophenolate mofetil is generally available in most tertiary hospitals which are the usual referral centers for cases of severe mers-cov infections requiring intensive care facilities such as extracorporeal membrane oxygenation. however, the risk of immunosuppression at the onset of adaptive immune responses or polarization towards a deleterious th response by mycophenolic acid needs to be considered. one possible approach is a short course of mycophenolate mofetil combined with an interferon, particularly interferon-b b, to provide synergistic antiviral and immune-enhancing effects against mers-cov. these options should be considered for study in randomized control clinical trials for this highly fatal disease. there are a number of limitations in our study. firstly, the cytotoxicity assay likely underestimated more subtle effects of candidate compounds on host cell growth and metabolism. for example, ribavirin inhibits replication of uninfected mdck cells at concentrations of mg/ml and above but does not cause overt cytotoxicity until much higher concentrations are reached. , secondly, we used vero cells alone to study the antiviral activity of ribavirin. vero cells have been described as being comparatively resistant to ribavirin due to their inefficient conversion of the drug into its mono-and tri-phosphate forms. however, we decided not to perform the experiment using another cell line as this has been done in another recent report using vero and llc-mk cell lines which also demonstrated anti-mers-cov activity of high ribavirin concentrations similar to our findings. it would be important to extend these in vitro studies to human respiratory epithelial cell systems and explants. to optimize treatment options for mers-cov infection, further studies on the anti-mers-cov activities of other potential anti-coronavirus agents which have been previously identified for sars-cov should be undertaken. replication of many coronaviruses including sars-cov and mers-cov requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease ( clpro) and a papain-like protease (plpro). however, the protease inhibitors such as nelfinavir and lopinavir were not found to be active in our in vitro study. helicase inhibitors are another group of agents with in vitro anti-sars-cov activities but their anti-mers-cov activities remain undetermined. , inhalational nitric oxide was used as rescue therapy for sars and might be useful for treating mers-cov infection if organic nitric oxide donors such as s-nitro-n-acetylpenicillamine also show anti-mers-cov activity. , antiviral peptides or neutralizing antibodies designed against heptad repeat region of s which may inhibit membrane fusion and cell entry of sars-cov could theoretically be harnessed for mers-cov since the s region shared significant homology amongst betacoronaviruses. e other agents with in vitro anti-sars-cov activities such as glycyrrhizin, baicalin, reserpine, aescin, valinomycin, niclosamide, aurintricarboxylic acid, mizoribine, indomethacin, chloroquine, and experimental agents like small interfering rna (sirna) and inhibitors targeting the binding interface between the s domian and receptor in vivo, should also be evaluated. , , we did not test these agents in this study because most of them have the problems of either not being commercially available or having therapeutic levels that are not easily achievable clinically. recently, cyclophilin inhibitors, such as cyclosporine which is available commercially, have also been reported to exhibit anti-mers-cov and anti-coronavirus activity in cell culture and viral load studies. , further evaluation of its potential therapeutic effects of these commercially available 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macaque model the authors have no financial or any other conflicts of interest regarding the contents of the investigations. key: cord- -v hw cdi authors: ejima, keisuke; aihara, kazuyuki; nishiura, hiroshi title: probabilistic differential diagnosis of middle east respiratory syndrome (mers) using the time from immigration to illness onset among imported cases date: - - journal: journal of theoretical biology doi: . /j.jtbi. . . sha: doc_id: cord_uid: v hw cdi abstract middle east respiratory syndrome (mers) has spread worldwide since . as the clinical symptoms of mers tend to be non-specific, the incubation period has been shown to complement differential diagnosis, especially to rule out influenza. however, because an infection event is seldom directly observable, the present study aims to construct a diagnostic model that predicts the probability of mers diagnosis given the time from immigration to illness onset among imported cases which are suspected of mers. addressing censoring by considering the transmission dynamics in an exporting country, we demonstrate that the illness onset within days from immigration is suggestive of influenza. two exceptions to suspect mers even for those with illness onset within days since immigration are (i) when we observe substantial community transmissions of mers and (ii) when the cases are at high risk of mers (e.g. cases with close contact in hospital or household). it is vital to collect the information of the incubation period upon emergence of a novel infectious disease, and moreover, in our model, the fundamental transmission dynamics including the initial growth rate has to be explored to differentiate the disease diagnoses with non-specific symptoms. middle east respiratory syndrome (mers), caused by the novel mers coronavirus (mers-cov), has been reported since march (cauchemez et al., ) . mers-cov has not caused substantial human-to-human transmissions yet, but the extent of geographic distribution of this disease has gradually expanded from middle east countries (e.g. jordan and saudi arabia) to other countries, including europe. recent studies have suggested that mildly symptomatic cases are common (cauchemez et al., ; fisman and tuite, ; assiri et al., a) , while hospitalized cases tended to exhibit severe respiratory symptoms (assiri et al., b; guery et al., ) . in october , a saudi boy was suspected of mers in hong kong and was admitted to a hospital which is equipped with an isolation ward (south china morning post, ) . two days in advance, his father had developed fever and coughing, and the boy followed similar symptoms to his father. one day after admission to the hospital in hong kong, the boy was tested positive to influenza a (h n ) virus, while testing negative to mers-cov. the similar suspected cases caused by influenza have been also contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yjtbi reported from other countries (nishiura et al., ) . not only influenza but also many other respiratory viruses induce only nonspecific clinical signs and symptoms. thus, it is difficult to selectively detect and differentially diagnose only mers among imported cases with upper respiratory symptoms (e.g. by screening febrile individuals at an international border (nishiura and kamiya, ) ). laboratory diagnosis such as pcr takes time and cost, and there would be a substantial number of suspected imported cases to be tested and isolated in hospital if we intend to test and intervene all suspected febrile individuals arriving from affected countries. as complimentary information to partly resolve this problem, a probabilistic model has been proposed to help differential diagnosis based on a known incubation period (nishiura et al., ) . employing a bayesian approach, and assuming that the incubation period distribution and the prior probability (or the population risks) of all suspected respiratory viruses are known, the model has permitted us to calculate the probability of mers given a certain length of the incubation period. nevertheless, it has been recognized as a core issue of infectious disease epidemiology that an infection event of non-sexual directly transmitted diseases is seldom directly observable (clancy and o'neill, ) . thus, the exact length of the incubation period is seldom known for each individual case. nevertheless, it is frequently the case that the time of illness onset is remembered among cases, and the time from immigration to illness onset among imported cases is readily available and can be useful for demonstrating the practical usefulness of the probabilistic model to assist clinical diagnosis. the present study aims to construct a statistical model that predicts the probability of mers diagnosis given a certain length of the time from immigration to illness onset among imported cases. through this exercise, we also aim to assess practical and theoretical importance of the proposed model and identify associated data gaps in epidemiological observations. further to the present study, nishiura and inaba ( ) proposed an estimation framework of the incubation period based on the time from immigration to illness onset among imported cases of influenza a (h n - ) in japan. while that study aimed to estimate the incubation period distribution, the present study extends the model structure in the earlier study to predict the probability of mers diagnosis. in particular, the present study focuses on the distinction between influenza and mers. during the early stages of the epidemic, the epidemiological parameters of mers had yet to be estimated based on the empirical data. thus, we also examine the corresponding estimates of the severe acute respiratory syndrome (sars) as a substitute for mers during the early stages. let t be the time from immigration to illness onset in an imported case which developed a disease in country b at time tz (where t¼ stands for the time of immigration; fig. ). suppose that he or she traveled to country a (where mers has spread) for k days. in the case of resident of country a, we may drop the data or assume that k- . we consider two different patterns of spread in country a: (i) an endemic state (i.e. the risk of infection is in a stationary state) and (ii) an epidemic state. in the case of the latter, we assume that the epidemic of novel coronavirus is in an early stage with an approximately exponential growth of infections. the frequency of exposure among imported cases during their travel is assumed as proportional to the incidence in country a. the exponential growth rate of incidence is known to be characterized by the basic reproduction number r and the mean generation time t g (wallinga and lipsitch, ) . if the generation time is exponentially distributed, the growth rate r is calculated as r ¼ (r À )/t g , while we have r ¼ln(r )/t g for a constant generation time (wallinga and lipsitch, ) . given identical values of r and t g , the exponential distribution is known to yield the largest value of r among all the possible distributions of the generation time, while a constant t g gives the smallest r (roberts and heesterbeek, ) . here we briefly describe the time from immigration to illness onset among imported cases (nishiura and inaba, ) . let i(t,τ) be the number of incubating individuals at time t after immigration and at infection-age τ (i.e. the time since infection). supposing that the rate of illness onset at infection-age τ is γ(τ), the dynamics of the density of incubating individuals are described by ∂iðt; τÞ ∂t þ ∂iðt; τÞ ∂τ ¼ ÀγðτÞiðt; τÞ; ð Þ with boundary conditions iðt; Þ ¼ for t ; ið ; τÞ ¼ jðτÞ: here j(τ)¼i( ,τ) represents the density of incubating population at an infection-age τ at the time of immigration t ¼ (i.e. the initial age distribution). i(t,τ) can be integrated as for τ Àt , where l(τ) represents the survival probability of incubating individuals at infection-age τ. the survival probability is calculated by using the rate of illness onset at infection-age τ, γ (τ) as follows: and, based on survival analysis, the probability density of the incubation period f(τ) is given as note that l(τ) is also written as À f(τ) where f(τ) is the cumulative distribution of the incubation period. let c(t) be the number of new symptomatic cases (illness onsets) at time t after immigration. supposing that the duration of travel is k days, then the mechanism and timing of importation: spending k days for travel, an exposure occurs in country a with a risk proportional to the incidence. entering country b, the exposed individual develops illness at t days since immigration. since an infection event is not directly observable, the exact length of the incubation period has to be inferred by addressing censoring and using an explicitly infection-age structured model. which can be rearranged as the frequency of illness onset is obtained by normalizing c(t) over t. hereafter, we focus on differential diagnosis of two specific diseases, influenza and mers. suppose that both diseases are growing in a similar manner (i.e., both in an endemic state or both in an exponential growth phase with different growth rates due to different r and t g ). as was discussed by nishiura and inaba ( ) , the frequency of exposure in an epidemic case is written as for τ k. in the endemic case, we have r ¼ , and thus, ( ) is simplified as let i represent a label for disease i and θ i be the population parameter of the incubation period of disease i. the probability density of observing the illness onset at t days from immigration, g i (t), is written as the function g i (t) is the normalized version of c(t) in eq. ( ). let q i be the prior probability of disease i in country a that may be derived from cause-specific prevalence such as those based on viral etiological study. as was discussed by nishiura et al. ( ) , a bayesian approach is employed, and the present study uses the following formula for the differential diagnosis: where i¼ denotes mers. in practical instances, the exact time of illness onset may not be precisely known for all suspected cases due to coarsely recorded data, and we may only know that the illness onset occurred at time t from immigration where rt rt m , where t m is the possible maximum incubation period. in such an instance, we use a doubly interval censored likelihood (reich et al., ) which we use to compute the following crude model for prediction: it should be noted that eq. ( ) investigates the cumulative probability of mers from time to t m (and thus, the interpretation is different from ( )), but the consistent discrete version of ( ) can be obtained by alternatively integrating time from (t m À ) to t m in ( ). for illustration of the proposed predictive model, we consider the differential diagnosis between two diseases, using influenza and mers as the case study. in addition, we consider sars and compare it against influenza, because sars share many clinical, virological and epidemiological features in common with mers and the empirical data of the incubation period and epidemiological parameters were available during the early stages of mers outbreaks (wallinga and lipsitch, ; donnelly et al., ) . let r and t g of influenza be . and . days, respectively. according to recent studies, r and t g of mers are assumed as . and . days, respectively (cauchemez et al., ; breban et al., ) . similarly, r and t g of sars are assumed as . and . days, respectively (donnelly et al., ) . the incubation period was assumed to follow a lognormal distribution with the scale parameter (or the median incubation period), exp(μ) ¼ . , . and . days and shape parameter s ¼ . , . and . for influenza, mers and sars, respectively (cauchemez et al., ; nishiura and inaba, ; reich et al., ; donnelly et al., ) . for illustration, we assumed that the length of travel was k ¼ days (which is consistent with the empirical best estimate (cauchemez et al., ) ) and also that a prior probability for each disease was . . to compare against influenza, a common disease, the equal prior yields a conservative result when the diagnosis involves a novel infectious disease without known q i (nishiura et al., ) . for the exposition of our proposed method, we first compare mers against influenza, using both continuous and discrete models (i.e. eqs. ( ) and ( )), and examine the possible time from immigration to illness onset as ranging from to days. as mentioned above, as an alternative to mers during the early stages of pandemic, we also compare influenza against sars. in both comparisons, we assume endemic and epidemic scenarios, and the latter is restricted to the early exponential growth phase. in the epidemic scenario, we use two different exponential growth rates, i.e., one assuming that the generation time is a constant, and the other assumes that the generation time follows an exponential distribution. as the sensitivity analysis, we compute the probability of influenza (in comparison with mers) given the time from immigration to illness onset, by varying the length of travel (from to days), prior probability of influenza (from . to . ), and r of mers (from . to . ) (cauchemez et al., ) . fig. a shows the probability of influenza given the exact time from immigration to illness onset (computed by eq. ( )). one minus the probability of influenza gives the posterior probability of mers. since the median incubation of influenza is . days shorter than that of the mers, the probability of influenza is high if the cases develop the disease shortly after immigration. however, as time goes by since immigration, the probability of influenza lowers % at day , and the probability of mers exceeds that of influenza thereafter. exponential growth of cases during the travel yielded higher probability of influenza than the case of uniformly distributed risk, although the difference was hardly visible on day since immigration and later. if the growth rate was much greater, the probability of infection shortly before immigration would be elevated, increasing the probability of influenza. a similar qualitative pattern was seen in the comparison between influenza and sars (fig. b) . in this comparison, the posterior probability of influenza again appeared to be greater than % given that a case develops illness within days since immigration. fig. c and d shows the average probability of influenza, as compared with mers and sars, respectively, given that the case developed a disease between day and day t since immigration (computed by eq. ( )). although the prediction becomes crude as compared to those based on the exact length of time in fig. a and b, it clearly indicates that the average posterior probability of influenza is greater than % as long as the case develops the disease within days since immigration. the probability is gradually lowered thereafter eventually reaching to %, i.e., the prior probability. again, comparison between influenza and sars yielded very similar results to that between influenza and mers. fig. a -c examines the sensitivity of the posterior probability of influenza to the duration of travel, the prior probability of influenza and r of mers, respectively. fig. d -f shows the corresponding results of the average probability of influenza using eq. ( ). as the duration of travel was extended, it appeared that the posterior probability of influenza decreased for the early days from immigration ( fig. a and d) . since the incubation period of influenza is short (on the order of a few days at most), the long travel indirectly increased the likelihood of mers. short travel (e.g. day) led us to have the high probability of influenza for those developing illness within days since immigration, because the distribution of time from immigration to illness onset becomes closer to the incubation period distribution. the posterior probability of influenza appeared to be very sensitive to prior probability of influenza ( fig. b and e) . especially, if the prior probability of mers was high (e.g., % due to widespread community transmission of mers or if we have to examine cases at high risk such as those following close contact), the posterior probability of influenza was apparently smaller than a and b) the posterior probability of influenza given illness onset at t days since immigration (as compared to middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars)), calculated by using eq. ( ). we assumed that the travelers stay in country a for days with two different rates of exponential growth (where exponential corresponds to exponentially distributed generation time, while exponential corresponds a constant generation time) or the uniformly distributed risk over time. the prior probability of influenza was assumed as . . r of mers was assumed to be . . (c and d) the average probability of influenza given illness onset from to t days since immigration (as compared to middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars)), calculated by using eq. ( ). only the results that assumed exponential growth with exponentially distributed generation time are shown, but other assumption yielded quantitatively similar estimates. other parameters are identical to those adopted in panels a and b. % for those developing illness within days since immigration. in the realistic situation with much greater endemicity of influenza than mers for general travelers, the illness onset within days from immigration was clearly suggestive of influenza. r of mers had little impact on the prediction of influenza diagnosis (fig. c and f), which is in line with the limited sensitivity of the posterior probability to the growth rate of infection in fig. . the findings are summarized in table . it appeared that the posterior probability of influenza for those developing illness within days since immigration is strongly influenced by the prior probability of influenza. this indicates that the high probability of influenza would not be the case even for those developing illness in days if mers transmission was more widespread than influenza or if the cases were at high risk of mers (e.g. with suspected exposure to camels or other mers cases in hospital) (assiri et al., b; reusken et al., a reusken et al., , b . the present study proposed a probabilistic model that permits us to estimate the posterior probability of a specific infectious disease given the time from immigration to illness onset among imported cases. the estimation requires us to assume that we know not only the incubation period and the prior probability but also the length of travel and the transmission dynamics of exporting country. we have shown that the illness onset within days from immigration is suggestive of influenza rather than unless varied in the corresponding univariate analysis, the duration of travel was days, the prior probability of influenza was assumed as . , and r of mers was assumed to be . . only the results that assumed exponential growth with exponentially distributed generation time are shown, but other assumption yielded quantitatively similar estimates. sensitivity of differential diagnosis to four different variables. baseline assumption sensitivity of diagnosis to the increase in assumed value how sensitive within assumed parameter range? growth rate of infection exponential growth or endemic steady state mers or sars, which is consistent with a simpler model based on the known exact length of the incubation period (nishiura et al., ) . the results of using sars data were similar to those obtained using mers data, which were consistent with similar viral etiology and common clinical characteristics between two diseases (assiri et al., a; guery et al., ; cotten et al., ; de wit et al., ) . moreover, we have demonstrated that our approach to doubly interval censored data can correspond to common practical settings in which the exact length from immigration to illness onset is not known. assuming that the risk of mers is likely much smaller than . , the illness onset within days can be said to be strongly suggestive of influenza as compared to novel coronavirus infection. nevertheless, this predictive statement is not applicable to suspected cases at high risk of mers-cov infection, such as those previously exposed to camels or other confirmed cases in household or hospital (assiri et al., b; reusken et al., a reusken et al., , b ); a high weight should be given to the prior probability of mers among these cases. to the best of our knowledge, the present study is the first to explicitly relate the observable time length (i.e. the time from immigration to illness onset) to the differential diagnosis of infectious diseases, examining the sensitivity of the prediction model to key model variables. by doing so, we have theoretically shown that accounting for such delay mechanism is critical not only for quantifying the natural history (nishiura and inaba, ) , but also for utilizing and interpreting the observable information among imported cases. moreover, the applicability of the model is not limited to imported cases. the proposed statistical framework is widely applicable to other settings in interpreting the observable epidemiological data: the most typical application may be the distinction between nosocomial and community infections in hospitals by using the time from hospitalization to illness onset among hospitalized cases with an infectious disease (lessler et al., (lessler et al., , ejima et al., ) . the formulation on this subject is our ongoing research. in practical terms, our proposed model not only predicts the posterior probability of influenza but also suggests that various data gaps have to be filled in empirical observation. as discussed in an earlier study (nishiura et al., ) , it is vital to collect the information of the incubation period upon emergence of a novel infectious disease. it should also be emphasized that the viral etiological study is valuable to directly quantify q i based on empirical data, although such data may be only applicable to general travelers (and not the travelers with close contact with other mers cases or animals (nishiura et al., ) ). in addition, the present study identified that addressing censored information of exposure at an exporting country requires us to understand the transmission dynamics at a global scale (and not at the country level) (lam et al., ) . namely, at least, either the exponential growth rate of infection, or a combination of the estimates of r and t g has to be derived from epidemiological data nishiura, ) . if we have a disease with similar etiology and characteristics (e.g. sars as a substitute of mers), r , t g and the incubation period of the substitute disease could complement the uncertainty by the time these estimates become available for the novel disease. two specific limitations have to be noted. first, our assumed exposure rests on a homogeneous population model, and the heterogeneous transmissions as well as the heterogeneous incubation period have yet to be explored extensively. second, if the transmission dynamics is dependent on the illness onset mechanism klinkenberg and nishiura, ) , the dependence structure has to be addressed within the model system, requiring us to account for this matter explicitly in the process of model building. since the proposed clinical prediction solely relied on the incubation period and assumed transmission dynamics, improvements have to be made with a broader scope. our ongoing future study includes an explicit assessment of the diagnostic performance of the proposed prediction system (i.e. assessment of validity and reliability), and also an inclusion of additional exposure variables other than the incubation period in the model (e.g. the presence of risky contact behavior during travel). despite the need to drastically improve the model structure to fully achieve the practical modeling exercise, we believe that 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infectious disease: implications for sampling to appropriately quantify transmission potential estimation of the incubation period of influenza a (h n - ) among imported cases: addressing censoring using outbreak data at the origin of importation incubation period as part of the case definition of severe respiratory illness caused by a novel coronavirus how to interpret the transmissibility of novel influenza a(h n ): an analysis of initial epidemiological data of human cases from china missing information in animal surveillance of mers-cov estimating incubation period distributions with coarse data middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study model-consistent estimation of the basic reproduction number from the incidence of an emerging infection saudi boy in hong kong has flu, not sars-like virus: south china morning post how generation intervals shape the relationship between growth rates and reproductive numbers hn received funding support from takahashi industrial and economic research foundation and the japan science and technology agency (jst) presto program. ke received scholarship support from the japan society for promotion of science (jsps). ka received funding support from the aihara project, the first program from jsps, initiated by cstp. the funding bodies were not involved in the collection, analysis and interpretation of data, the writing of the manuscript or the decision to submit for publication. key: cord- - rec xg authors: haagmans, bart l; al dhahiry, said h s; reusken, chantal b e m; raj, v stalin; galiano, monica; myers, richard; godeke, gert-jan; jonges, marcel; farag, elmoubasher; diab, ayman; ghobashy, hazem; alhajri, farhoud; al-thani, mohamed; al-marri, salih a; al romaihi, hamad e; al khal, abdullatif; bermingham, alison; osterhaus, albert d m e; alhajri, mohd m; koopmans, marion p g title: middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation date: - - journal: lancet infect dis doi: . /s - ( ) -x sha: doc_id: cord_uid: rec xg background: middle east respiratory syndrome coronavirus (mers-cov) causes severe lower respiratory tract infection in people. previous studies suggested dromedary camels were a reservoir for this virus. we tested for the presence of mers-cov in dromedary camels from a farm in qatar linked to two human cases of the infection in october, . methods: we took nose swabs, rectal swabs, and blood samples from all camels on the qatari farm. we tested swabs with rt-pcr, with amplification targeting the e gene (upe), nucleocapsid (n) gene, and open reading frame (orf) a. pcr positive samples were tested by different mers-cov specific pcrs and obtained sequences were used for phylogentic analysis together with sequences from the linked human cases and other human cases. we tested serum samples from the camels for igg immunofluorescence assay, protein microarray, and virus neutralisation assay. findings: we obtained samples from camels on oct , . we detected mers-cov in nose swabs from three camels by three independent rt-pcrs and sequencing. the nucleotide sequence of an orf a fragment ( nucleotides) and a · kb concatenated fragment were very similar to the mers-cov from two human cases on the same farm and a mers-cov isolate from hafr-al-batin. eight additional camel nose swabs were positive on one or more rt-pcrs, but could not be confirmed by sequencing. all camels had mers-cov spike-binding antibodies that correlated well with the presence of neutralising antibodies to mers-cov. interpretation: our study provides virological confirmation of mers-cov in camels and suggests a recent outbreak affecting both human beings and camels. we cannot conclude whether the people on the farm were infected by the camels or vice versa, or if a third source was responsible. funding: european union projects emperie (contract number ), antigone (contract number ), and the virgo consortium. an emerging betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-causes illness characterised predominantly by mild-to-severe respiratory complaints, with most patients requiring admission to hospital because of pneumonitis or acute respiratory distress syndrome. old age and the presence of comorbidities or immunosuppression seem to increase the risk of infection and are associated with severe forms of the disease. however, some patients can remain asymptomatic or mildly symptomatic and atypical presentations such as gastroenteritis have occurred. as of dec , , laboratory-confi rmed cases, including deaths, have been reported to who. all of these cases were directly or indirectly linked to the middle east region, including saudi arabia, qatar, united arab emirates, kuwait, oman, and jordan. coronaviruses reside in many animal hosts and can adapt to diff erent species, including people. mers-cov belongs to the lineage c betacoronaviruses, which are associated with bats and the closest relatives of mers-cov have been identifi ed in vesper bats from europe, asia, and south africa. [ ] [ ] [ ] notably, this coronovirus is able to replicate in various bat cell lines. molecular clock dating of epidemiologically unlinked human mers-cov isolates estimated their divergence from a common ancestor in mid- , with a cluster of isolates from the eastern parts of the arabian peninsula diverging in late . these fi ndings suggest that the reported mers-cov diversity in human beings is the result of several independent, geographically structured, zoonotic events from an unknown reservoir in the middle east. , human-to-human transmission has been noted, especially in health-care settings and households, but is thought to be relatively ineffi cient. to determine how circulation of mers-cov in human beings is maintained and to mitigate the chain of transmission, identifi cation of possible animal reservoirs and modes of transmission is needed. limited available exposure history of patients suggest that contact with livestock, including dromedary camels, might have a role. , , , in addition, our recent investigations have provided evidence for the circulation of mers-cov or a related virus in dromedary camels. both mers-cov spike protein binding antibodies and virus neutralising antibodies were reported in dromedary camels from diff erent regions, including oman and egypt, but no virus shedding could be detected and, therefore, the signifi cance of these observations remained an issue of debate. , in this study we aimed to assess presence of mers-cov in dromedary camels from a farm in qatar that was linked to two human cases of mers-cov. in october, , the qatar supreme council of health recorded two cases of laboratory confi rmed mers-cov. case involved a -year-old man who was the owner of a farm that he visited regularly. the patient had substantial contact with animals including camels, sheep, pigeons, and hens, and no history of travel outside of qatar in the weeks before becoming ill. mers-cov infection was diagnosed by e gene (upe) pcr on a sputum sample collected on oct , , in the national virology laboratory of qatar. case involved a -year-old male employee of the farm owned by case and was identifi ed in the close-contact investigation of that case. he was reported on oct , , and had no underlying medical conditions and no recent travel history, but had regular contact with the animals on the farm. mers-cov infection was diagnosed in a throat swab taken on oct , , by the national virology laboratory of qatar. diagnosis for both cases was confi rmed (orf b and n gene pcr) by public health england. , qatari authorities, with support from who, did an epidemiological investigation at the farm within week of diagnosis of the fi rst case, which included the collection of various clinical samples from the farm's dromedary camels. full details of the outbreak investigation will be described elsewhere. we collected serum, rectal swabs, and nasal swabs from all camels present on the premises where the two men had been in contact with the animals. after an experienced animal handler fi xated the camel by nose pinching, samples were obtained through swabbing with fl ocked swabs (floqswabs, copan improve diagnostics, brescia, italy) that were inserted deep into one of the nostrils of the camel. after sampling, swabs were put into tubes containing viral transport medium-utm (copan diagnostics, brescia, italy). in addition, we obtained fi ve stool samples from three diff erent cages. the sample collection was done jointly by the communicable disease control outbreaks rapid response team of the public health department and the animal health team, which used full personal-protective equipment including n masks, goggles, disposable gowns, gloves, and head covers during the handling of the animals, and during the environmental and human sampling. serum samples were stored at - °c and all other samples were stored at - °c and were shipped to the netherlands on dry ice. we tested nose swabs with two independent rt-pcr targets specifi c for mers-cov (upe and nucleocapsid [n gene]) in one laboratory (department of viroscience, erasmus medical center, rotterdam, netherlands), and did rt-pcr testing with a third target (orf a) in another laboratory (national institute of public health and the environment, bilthoven, netherlands). we inoculated vero and huh- cells for h with cellculture medium containing samples from camel nose or rectal swabs, or with mers-cov (emc isolate) as a positive control. after washing, the cells were incubated with medium containing % fetal bovine serum at °c. we stained formaldehyde-fi xed huh- cells infected with mers-cov by use of heat-inactivated (at °c for min) camel sera or a mers-cov patient's serum according to standard protocols with fl uorescein isothiocyanateconjugated antibodies as a second step. we isolated rna from μl of swab medium or culture supernatant with the qiaamp viral rna minikit (qiagen, hilden, germany) and eluted it in μl. camel mers-cov rna was quantifi ed on the abi prism with the taqman fast virus -step master mix (applied biosystems, bleiswijk, netherlands), with μl isolated rna, one taqman mix, · u of uracil-n-glycosylase, primer, and probes targeting the n gene, upe, or orf a as described elsewhere. , we used rna dilutions isolated from a mers-cov isolate emc stock as a standard control. μl of rna was reverse transcribed with the superscript iii fi rst strand synthesis system (invitrogen, bleiswijk, netherlands) with random hexamers. cdna was used to amplify six diff erent camel mers-cov fragments, including a partial spike gene by use of pfu ultra ii fusion hs dna polymerase (agilent technologies, santa clara, ca, usa). we did the pcr as follows: one initial denaturation step of °c for min, followed by cycles of °c for s, °c for s, °c for min, and a fi nal extension of °c for min. the amplifi ed camel mers-cov fragments were sequenced directly on both strands with the bigdye terminator version . cycle sequencing kit on an abi prism genetic analyser (applied biosystems). we generated phyml phylogenetic trees with seaview software with the approximate likelihood-ratio test based on a shimodaira-hasegawa-like procedure, which used a general time reversible as substitution model. we used nearest neighbour interchange and subtree pruning and regrafting-based tree search algorithms to estimate the tree topologies. rna from human samples (sputum for qatar case and throat swab for qatar case) was extracted with the nuclisens easymag system (biomérieux, basingstoke, uk). viral sequences from the two human cases were isolated (with the same techniques as for camel cases) in the reference department, public health england, london, uk. we used primers designed against sequences of mers-cov jx _emc/ and bat coronaviruses hku and hku to amplify the entire genomes from human cases for sequencing on an illumina miseq sequencer, subsequently indicated as qatar_ _ (case ) and qatar_ _ (case ). the sequences obtained in this study were deposited in genbank under the following accession numbers: camel_mers-cov/qatar_ _ _orf a_ _ partial, kf ; camel_mers-cov/qatar_ _ _ orf a_ _partial, kf ; camel_mers-cov/ qatar_ _ _orf b_partial, kf ; camel_ mers-cov/qatar_ _ _spike_partial, kf ; camel_mers-cov/qatar_ _ _orf b_full, kf ; camel_mers-cov/qatar_ _ _orf a_ _ partial, kf ; qatar_ _ , kf ; and qatar_ _ , kf . we tested all sera for the presence of igg antibodies reactive with mers-cov (residues - ), severe acute respiratory syndrome coronavirus (sars-cov; residues - ), and human coronavirus oc (residues - ) s antigens as described previously. , we report results as the relative mean fl uorescent intensity (rfu) for each set of quadruplicate spots per antigen. we did igg antibody testing only because anti-camel igm conjugates were not available. for virus neutralisation, serum samples were heatinactivated by incubation for min at °c. we prepared twofold serial dilutions with well plates, starting dilution at / . mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with penicillin, streptomycin, and % fetal bovine serum, to a dilution of tcid per ml. we then added μl of virus suspension to the plates and the plates were incubated at °c for h. next, we incubated virus-serum mixtures on well plates containing vero cells for h, and then washed them with phosphate buff er saline and incubated them with imdm and % fetal bovine serum for days. the sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. samples from camels were obtained on oct , (table, fi gure ). nose swab specimens from fi ve camels were positive for all three assays (upe, n gene, and orf a), one sample reacted in two assays, and fi ve camels tested positive in only one rt-pcr assay. subsequently, all samples were subjected to a pcr assay targeting the spike gene of mers-cov (nucleotides - ) to obtain a fragment for sequencing as defi nitive confi rmation. such sequencing confi rmed the presence of sequences specifi c to mers-cov in three camels. alignment of these partial spike gene sequences with known human mers-cov sequences, including those determined from the two related human cases and some other human mers-cov isolates such as hafr-al-batin_ _ (kf ) and riyadh_ _ (kf ), showed % identity, but the sequences diff ered from emc mers-cov, which is used routinely in the erasmus mc laboratory, by one nucleotide. the three camel mers-cov sequences obtained by this approach were identical. virus culture attempts were not successful, although one sample tested for camel yielded a positive rt-pcr of the supernatant (table) and staining of the fi xed virus-infected cells (data not shown). further passage of the virus on vero cells, however, was not successful. in addition, serum from camel showed low levels of mers-cov as determined by rt-pcr, and all other camel sera tested negative (data not shown). rectal swabs and faecal materials were negative for mers-cov when tested for reactivity in the upe and n gene rt-pcrs. further sequencing of the virus from camel with primers specifi c to mers-cov rendered six diff erent fragments covering · kb across the mers-cov genome (fi gure ). fragments (nucleotides - ), ( - ), and ( - ) were located in orf a, fragment ( - ) in orf b, fragment ( - ) in the spike gene, and fragment ( - ) in orf b. the sequences obtained from the human cases from the same farm diff ered by one nucleotide diff erence in orf a and one in orf b. the mers-cov emc isolate diff ers by eight nucleotides from the camel mers-cov in orf a. alignment of the divergent nucleotide orf a fragment (data not shown) and a · kb concatenated sequence fragment was used for phyml phylogenetic analysis. the camel mers-cov clustered with viral sequences obtained from the two human cases related to the farm and with a sequence from hafr-al-batin as the next closest relative (fi gure ). less stringent methods of analysis do show limited bootstrap support (data not shown), in line with relatively limited sequence variation within this fragment. all camel sera were positive in the immunofl uorescence assay when tested in a / dilution (fi gure ). when serum samples from camels were tested at diff erent dilutions on the microarray, we noted reactivity with mers-cov antigen, but not the sars-cov antigen. reactivity to human coronavirus oc antigen, as a proxy to betacoronavirus, varied between negative (less than the cutoff value of ) and saturating signals (data not shown). consistent with the array data, all serum samples had mers-cov neutralising capacity, with titres varying between and (fi gure ). we also noted a strong correlation between microarray titres and virus neutralising antibody titres (fi gure ). the two camels with the highest mers-cov load in the swabs as shown by taqman assay (camels and ) had a relatively low serum neutralisation titre of . we present, to our knowledge, the fi rst virological confi rmation of mers-cov in dromedary camels (panel). we and others previously reported mers-cov neutralising antibodies in dromedary camels from the canary islands, oman, and egypt, suggesting circulation of mers-cov or a mers-cov-like virus in camels. , high prevalences and antibody titres were reported in the camels from oman and egypt suggesting widespread circulation. however, virological testing was unable to detect mers-cov viral sequences in camels, probably because only faecal and serum samples were analysed. in addition, shedding kinetics of mers-cov in camels (and human beings) are unknown, and therefore, inter pretation of negative rt-pcr results from screening is diffi cult because positive and negative predictive values remain to be determined. in the outbreak reported, we showed evidence for presence of the virus in six camels according to internationally recommended criteria of two independent rt-pcr targets. furthermore, we used sequence confi rmation as an additional test to increase the level of certainty-during an emerging disease outbreak such as this, options for validation of assays for clinical testing are limited. discrepancies between assays can be explained by diff erences in detection limits, or by diff erences in specifi city of the primer sets. in our study, n gene rt-pcr yielded four additional weak positive results that could not be confi rmed by any other method, and an individual orf a rt-pcr identifi cation was made for only one animal. these cases could have resulted from mispriming, or represent presence of levels of virus around the detection limit, as can be expected at the end of the infection cycle. nevertheless, our results suggest a widespread and recent outbreak in this herd, coinciding with infection in two people. our data should not be taken as evidence for infection of people from the camels. comparison of sequence data from the orf a gene and a concatenated · kb sequence from the human and camel viruses showed that these viruses are very similar, but distinct. notably, samples from camels and people were analysed in diff erent laboratories in the netherlands and the uk. the data provided show proof that camels can be infected with mers-cov. the sequence diff erence between human and animal viruses from the farm, based on the available overlapping sequence, is so small that we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels were infected from a third as yet unknown source. although additional sequencing might provide improved resolution, it probably will not provide conclusive evidence. the most important unknown is the exact timing of infections, both in the infected people and camels. if the virus entered the farm through either the people or the camels, the infections observed would probably have resulted from a chain of transmissions that introduced mutations. because we did not identify any seronegative animals that were pcr positive, and because all animals had mers-cov neutralising antibodies, our sampling probably took place relatively late in the outbreak on this farm. a more detailed analysis of the outbreak, including testing of additional animals and environmental samples is ongoing, as are attempts to obtain full mers-cov genomes of the human and animal specimens. we cannot exclude the possibility that other common livestock species including cattle, sheep, and goats, or other animals including wild species were involved in the spread of mers-cov. while confi rmation of the source is awaited, we recommend that a detailed case history is taken of any cases of mers-cov, including review of any animal exposures (including animal products), and targeted (prospective) serosurveys to determine what risk factors are associated with human infection. we searched pubmed for articles published in english up to dec , , with the search terms "novel coronavirus emc" or "mers-cov". we identifi ed reports linked to the middle east respiratory syndrome coronavirus (mers-cov). one report described the detection in one bat specimen of a single bp fragment in a highly conserved area of the coronavirus genome, identical to the human mers-cov laboratory isolate emc. our report describes the fi rst detection of mers-cov in dromedary camels on a farm in qatar that had been linked to human cases of the disease. our study provides proof that mers-cov has infected dromedary camels in qatar. our results suggest the simultaneous occurrence of a mers-cov outbreak in people and camels. on the basis of the present data, we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels could have been infected from a third as yet unknown source. we recommend that detailed case histories be taken, including review of any animal exposures including animal products, and targeted (prospective) serosurveys to determine what risk factors-other than contact with an individual with the infection-are associated with human infection. blh wrote the report, drew the fi gures, and generated, analysed, and interpreted data. shsad, mma, and mpgk coordinated the study, wrote the report, and interpreted data. cbemr wrote the report, drew the fi gures, did the literature search, and analysed and interpreted data. vsr drew the fi gures, and generated, analysed, and interpreted data. mg, rm, and ab generated and interpreted data. gjg generated, analysed, and interpreted data. mj generated and analysed data. ef led the fi eld investigation, obtained samples, and collected and interpreted data. ad supervised fi eld outbreak management and collected and interpreted data. hg and fa supervised animal health fi eld investigations. ma-t coordinated the study and interpreted data. saa-m coordinated the study. hear led outbreak management and collected and interpreted data. aak coordinated the study and interpreted data. admeo wrote the report and interpreted data. blh, admeo, and vsr hold a pending patent for mers-cov. admeo is scientifi c adviser of viroclinics biosciences. all other authors declare that they have no confl icts of interest. doi: . / currents.outbreaks. bf e e f ad fa ddb . who. global alert and response (gar): novel coronavirus infection-update ecology, evolution and classifi cation of bat coronaviruses in the aftermath of sars close relative of human middle east respiratory syndrome coronavirus in bat human betacoronavirus c emc/ -related viruses in bats, ghana and europe genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt alert and response (gar): novel coronavirus infection-update alert and response (gar): novel coronavirus infection-update detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections seaview version : a multiplatform graphical user interface for sequence alignment and phylogenetic tree building specifi c serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus: data gaps for laboratory preparedness we thank who and the food and agriculture organization of the united nations for their generous help in organisation of the study, theo bestebroer for providing middle east respiratory syndrome coronavirus specifi c primer sets, berend jan bosch for providing antigens for the microarray, and jeroen cremer for technical support. contributions to the study were funded through the european union fp projects emperie (contract number ; to blh and admeo) and antigone (contract number ; to mpgk and admeo). key: cord- -dmiplvt authors: almekhlafi, ghaleb a.; albarrak, mohammed m.; mandourah, yasser; hassan, sahar; alwan, abid; abudayah, abdullah; altayyar, sultan; mustafa, mohamed; aldaghestani, tareef; alghamedi, adnan; talag, ali; malik, muhammad k.; omrani, ali s.; sakr, yasser title: presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: dmiplvt background: middle east respiratory syndrome coronavirus infection is associated with high mortality rates but limited clinical data have been reported. we describe the clinical features and outcomes of patients admitted to an intensive care unit (icu) with middle east respiratory syndrome coronavirus (mers-cov) infection. methods: retrospective analysis of data from all adult (> years old) patients admitted to our -bed mixed icu with middle east respiratory syndrome coronavirus infection between october , and may , . diagnosis was confirmed in all patients using real-time reverse transcription polymerase chain reaction on respiratory samples. results: during the observation period, patients were admitted with mers-cov infection (mean age ± years, [ %] males). cough and tachypnea were reported in all patients; ( . %) patients had bilateral pulmonary infiltrates. invasive mechanical ventilation was applied in ( . %) and vasopressor therapy in ( . %) patients during the intensive care unit stay. twenty-three ( . %) patients died in the icu. nonsurvivors were older, had greater apache ii and sofa scores on admission, and were more likely to have received invasive mechanical ventilation and vasopressor therapy. after adjustment for the severity of illness and the degree of organ dysfunction, the need for vasopressors was an independent risk factor for death in the icu (odds ratio = . , % confidence interval: . – . , p = . ). conclusions: mers-cov infection requiring admission to the icu is associated with high morbidity and mortality. the need for vasopressor therapy is the main risk factor for death in these patients. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. middle east respiratory syndrome coronavirus (mers-cov) is a novel betacoronavirus that was first reported in september [ ] . by january , , a total of laboratory-confirmed cases of infection with mers-cov, including at least related deaths, had been reported to the world health organization [ ] . although mers-cov infections have been reported from countries around the world, the majority of cases have originated in saudi arabia, south korea, the united arab emirates, jordan and qatar [ ] . interhuman mers-cov transmission occurs in community and healthcare settings [ ] [ ] [ ] [ ] [ ] . the exact source and mode of transmission of mers-cov to humans remains uncertain. however, mers-cov circulates among dromedary camels in africa and the middle east with occasions of documented camel-human inter-transmission [ ] . there have been several reports outlining the clinical features and outcomes of patients with mers-cov infection [ , [ ] [ ] [ ] [ ] [ ] [ ] . however, very few have focused on critically ill patients in intensive care units (icu) [ ] [ ] [ ] . there is therefore a need for more data to understand the various clinical and prognostic aspects of this potentially lethal disease, particularly for the most severe cases that require admission to the icu. we performed a retrospective study to describe the clinical features and outcomes of patients admitted to our icu with laboratory-confirmed mers-cov infection. the study was approved by the institutional review board of prince sultan military medical city ( riyadh, saudi arabia), a large tertiary-care referral center in riyadh, saudi arabia. informed consent was waived due to the retrospective, anonymous nature of data collection. we included all patients aged years or more with confirmed mers-cov infection who were admitted to our -bed mixed medico-surgical icu between october , and may , . all icu patients with a confirmed diagnosis of mers-cov were registered in a special logbook. for the purpose of the current study, all patients' records were reviewed by a senior intensivist (s. hussain, a. alwan, a. abudayah, s. altayyar, m. mustafa, t. aldaghestani, a. alghamedi, a. talag or m. malik). clinical data and laboratory parameters from confirmed cases of mers-cov were transcribed onto specially developed case record forms. these included the initial manifestations of respiratory infection, the clinical picture on admission to the icu, laboratory indices of organ failure, radiographic findings, interventions during the icu stay, treatment modalities, and final outcome. the acute physiology and chronic health evaluation ii (apache ii) score was calculated from the data obtained within hours of admission to the icu [ ] . the sequential organ failure assessment (sofa), score, calculated daily by the physician in charge of the patient, was also noted [ ] . since the first reported cases of mers-cov in saudi arabia in september , all suspected cases in our institution are strictly isolated and nasopharyngeal swabs are obtained for initial screening. deep respiratory samples (tracheal aspirates or bronchoalveolar lavage fluid) are obtained from all patients admitted to the icu with suspected respiratory infections, in addition to blood samples to perform cultures and polymerase chain reaction for common respiratory viruses and atypical microorganisms. urinary samples were obtained to detect legionella antigens in only two patients (legionella infections are not common in saudi patients). cultures of tracheal aspirates are analyzed quantitatively and bacterial counts of at least colony-forming units are considered positive. these investigations are repeated in the icu whenever secondary infections are suspected. clinical specimens aimed at detecting possible mers-cov infection are processed and analyzed at the national reference laboratory of the saudi ministry of health. mers-cov infections are identified using real-time, reverse transcription polymerase chain reaction (rt-pcr). the standard assays target amplifications of the upstream e protein (upe gene) and open reading frame (orf) a; both need to be positive to confirm infection, otherwise another sample is required to confirm the diagnosis [ ] . the sample requires days of processing for the final results to be available. routine laboratory testing in our icu includes complete blood counts, coagulation profile, electrolytes, renal function, liver profile and arterial blood gases. these parameters are measured on admission to the icu and at least once daily thereafter (at : am) throughout the icu stay. all patients with suspected or confirmed mers-cov infection were isolated in single rooms, either on the hospital floor or in the icu. patients were admitted to the icu according to the guidelines of the society of critical care medicine for icu admission, discharge, and triage [ ] . patients were classified into four categories according to their icu admission priority: priority one comprised critically ill patients who were unstable and need intensive treatment and monitoring, with significant likelihood of recovery; priority two were stable patients who required intensive monitoring because of the possibility of decompensation; priority three were unstable patients who had a low likelihood of recovery because of the severity of acute disease or because of comorbidities; priority four were those who had little or no anticipated benefit from icu admission. patients classified as priority one and two and most of those classified as priority three were admitted to our icu or full critical care services were mobilized and provided for in the isolation ward until a bed was available in the icu. priority four patients were not admitted to the icu and remained in the isolation ward. general ward patients with mers-cov infection were transferred to the icu if their condition deteriorated or organ failure developed. the infection control precautions recommended by the saudi ministry of health guidelines were strictly implemented to prevent possible transmission of mers-cov to other patients or to the healthcare staff [ ] . supportive treatment was provided according to our standard operating procedures and in accordance with the surviving sepsis campaign guidelines [ , ] . antiviral therapies, such as oseltamivir, and ribavirin/interferon alfa- a, were prescribed at the discretion of the attending physician. protective lung ventilation was applied in mechanically ventilated patients. prone positioning was considered in some patients with severe refractory hypoxemia. extracorporeal membrane oxygenation (ecmo) and high-frequency oscillation were also available as a last resort, when considered necessary by the attending physician. statistical analyses were performed using spss statistics for windows (ibm corp., armonk, ny, usa). the kolmogorov-smirnov test was used to verify whether there were significant deviations from the normality assumption of continuous variables. nonparametric tests of comparison were used for variables evaluated as not normally distributed. difference testing between groups was performed using student's t test, mann-whitney test, chi-square test and fisher's exact test, as appropriate. friedman's test was used to assess the time course of organ function. to identify the risk factors for death in the icu, we performed multivariable logistic regression analyses. due to the relatively small number of deaths in our study, we adjusted only for the severity of illness on admission to the icu (apache ii score) and the degree of organ dysfunction as assessed by admission sofa score. potential risk factors for icu mortality were selected among the demographic characteristics, comorbidities, mode of acquisition of mers-cov, initial manifestations, procedures and therapies, and superimposing infections. variables yielding p < . in the univariate analysis, apa-che ii score and sofa score were included in a multivariable logistic regression analysis. these variables were introduced separately into multivariable models including apache ii and sofa scores on admission to the icu. adjusted odds ratios (or) and % confidence of interval (ci) were computed. none of the covariates simultaneously introduced in a multivariable model were collinear. data are presented as mean ± standard deviation (sd), median value ( th- th interquartile range [iqr]) or number (%), as appropriate. all statistics were two-tailed and a p < . was considered statistically significant. during the observation period, cases with confirmed mers-cov infections were diagnosed in our institution [ ] (fig. ) ; patients were managed in the hospital ward, patients were admitted to other icus or received critical care service in the ward, and patients were admitted to our icu ( between october , and december , and between january and may , ). patients were admitted to our icu because of respiratory failure (pao /fio < mmhg). the mean age of the patients admitted to our icu (n = ) was (sd ) years and ( %) were males. the characteristics of these patients on admission to the icu are shown in table . eighteen ( . %) patients had community-acquired mers-cov infection, while for ( . %), including two healthcare staff, infection was acquired in the hospital. twenty-seven patients ( . %) had at least one comorbidity. the median number of concomitant comorbid conditions was three (iqr: - ). initial clinical manifestations had occurred at a median of days (iqr: - ) prior to hospital admission. patients had been treated for a median of days (iqr: - ) in general hospital wards before their admission to the icu. only four patients ( . %) were admitted to the icu on arrival at the hospital. cough and tachypnea were reported in all patients. other common initial symptoms were fever ( . %), abdominal pain ( %), sore throat ( . %), and fatigue ( . %) (additional file ). crackles ( . %), tachycardia ( . %), and rhonchi ( . %) were the most commonly identified initial physical signs. bilateral pulmonary infiltrates were present in the chest x-rays of ( . %) patients and lobar infiltrates in six ( . %). only one patient had a normal chest x-ray at the time of admission to the icu. on admission to the icu, no patients had microbiologically proven co-existing bacterial pneumonia. secondary infections, as evident from positive quantitative cultures of deep tracheal aspirates, occurred in ( . ) patients within a median of days (iqr: - ) after admission to the icu. the most commonly isolated microorganisms were acinetobacter baumannii ( . %), only four ( . %) patients had positive blood cultures; acinetobacter baumannii (n = ), escherichia coli (n = ), methicillin-resistant staphylococcus aureus (n = ), and vancomycin-resistant enterococcus species (n = ). invasive mechanical ventilation was applied in ( . %) patients during the icu stay; ( . %) within hours of admission to the icu, and ( . %) patients received noninvasive ventilation (table ) . eleven ( . %) patients were treated with high-frequency oscillation and five ( . %) with prone positioning. only one patient received ecmo. the ventilatory parameters are presented in additional file . vasopressor therapy using norepinephrine was initiated in ( . %) patients (table ) . oseltamivir was administered to ( . %) patients for a median of days (iqr: - ). combined ribavirin plus interferon alfa- a therapy was used in ( . %) patients ( table ). all patients received at least one antimicrobial agent during the icu stay (additional file ). antifungal therapy was only used in four of the five patients with positive cultures for candida but the necessity of this therapy is uncertain. the overall icu mortality rate was . % (n = ). the median icu and hospital lengths of stay were (iqr: - ) and (iqr: - ) days, respectively. the major causes of death were hypoxemic respiratory failure ( . %) and refractory septic shock ( . %). one patient died from sudden cardiac arrest after icu discharge but while still in the hospital. furthermore, one patient died within year after discharge from the icu because of septic shock related to an infected wound. only one patient was lost to follow-up after hospital discharge. the sofa score and glasgow coma scale (gcs) increased markedly over the first weeks in the icu in the whole cohort, while other parameters of organ function remained largely unchanged (additional file ). compared with those who were discharged alive from the icu, nonsurvivors were older, had higher apache ii and sofa scores on admission to the icu, and were more likely to require invasive mechanical ventilation and vasopressor therapy and to have been ventilated using highfrequency oscillation (tables and , and additional files and ). nonsurvivors had a persistently low pao /fio throughout the first weeks in the icu, whereas survivors showed a slight increase over time (fig. ) . after adjustment for the severity of illness and the degree of organ dysfunction, the need for vasopressors was the only independent risk factor for death in the icu (or . , % confidence interval . - . , p . ) (additional file ). the critically ill patients with confirmed mers-cov infection in our cohort frequently had organ failure with an overall mortality rate greater than %. comorbidities were common in this cohort of patients. not surprisingly, mortality in the icu was associated with older age, severe disease and organ failure. the need for vasopressor therapy was an independent risk factor of death in the icu. since the first reported case of mers-cov infection in , several authors have described various cohorts of patients with this serious infection [ , - , , , ] . [ , ] . our facility is a large tertiarycare medical center in riyadh, central saudi arabia. we herein provide a detailed account of the largest single cohort of critically ill mers-cov infected patients reported thus far. in agreement with previous reports from saudi arabia, comorbid conditions were common in our patients with mers-cov infections with a median of three comorbidities per patient [ , , , ] . in contrast, only . % of the individuals involved in the recent mers-cov outbreak in south korea had any preexisting chronic medical conditions [ ] . however, only . % of patients in the korean outbreak were aged years or older and nearly half ( . %) were caregivers or healthcare personnel [ ] . the differences in the demographic characteristics of our cohorts and the mode of acquisition of mers-cov infection may explain, at least in part, the discrepancy in the patterns of associated comorbidities between the saudi and korean cohorts. the respiratory manifestations of mers-cov infection in our cohort were similar to those observed in previous reports from saudi patients [ , , , , ] . cough and tachypnea occurred in all patients and % of cases had bilateral pulmonary infiltrates, denoting severe respiratory illness, which required a median of days to reach the peak of clinical deterioration such that icu admission and organ support therapy were required. gastrointestinal manifestations, such as abdominal pain, diarrhea, vomiting, and abdominal tenderness, were relatively common in our cohort. this was also a common finding in the previous literature in patients with mers-cov infection as well as those with severe acute respiratory syndrome (sars) [ , , , , , ] . our data confirm previous studies that reported a high prevalence of nonrespiratory organ failure in critically ill patients with mers-cov [ , ] . the mechanisms of organ dysfunction and failure in these patients are yet to be determined. cytokine dysregulation has been suggested to be involved in the pathophysiology of mers-cov-related organ failure. direct viral invasion may also occur as the virus was recovered from urine and stool in one patient [ ] . in agreement with the results of the previous reports on critically ill patients with mers-cov infection [ ] [ ] [ ] , more than % of our patients received vasopressor support, underscoring the high prevalence of cardiovascular dysfunction in these patients, and suggesting that disturbances in tissue perfusion may also have been involved in the pathophysiology of the organ failure. lower rates of vasopressor support have been reported in patients with sars [ , , , , , ] with, as a result, lower mortality rates than those reported in patients with mers-cov infections. even though overall mortality rate was high in our cohort, it is still comparable with rates reported in previous studies ( . - . %) [ ] [ ] [ ] . in all studies, almost all patients had significant comorbidities and median apache ii scores of or higher. we observed significantly higher apache ii and sofa scores in icu nonsurvivors compared to those who survived severe mers-cov infection, underscoring the strong association between mortality and the severity of disease. epidemiological analyses have suggested that mers-cov is unlikely to trigger sustained human epidemics at present [ , ] . nevertheless, nosocomial outbreaks have resulted in considerable morbidity and mortality, in addition to disruption of medical services and substantial economic losses [ , , ] . the most severe infections usually require icu admission, necessitate major resource utilization and result in high fatality rates. identifying possible risk factors for poor prognosis in patients with mers-cov infection is therefore crucial to enable appropriate allocation of healthcare resources and early transfer of high-risk patients to the appropriate medical facilities. our data show that the need for vasopressor therapy was an independent risk factor for death in the icu. indeed, the major causes of death in our study were hypoxemic respiratory failure and refractory septic shock, which confirm the role of respiratory and cardiovascular system failures as determinants of outcome in this population. this was also evident from the persistent hypoxemia observed in the nonsurvivors. to date, published data on the risk factors for poor prognosis specific to critically ill patients with mers-cov infection are lacking. in cohort studies of patients with any degree of severity of mers-cov infection, older age, diabetes, chronic renal failure, chronic respiratory disease, high viral load in lower respiratory tract samples, shorter incubation period and mers-cov viremia have all identified as independent predictors of mortality [ , , [ ] [ ] [ ] . secondary respiratory infections occurred commonly in this cohort, predominantly with gram-negative bacteria. although candida species were frequently isolated, these are probably not relevant as respiratory pathogens and the necessity of antifungal therapy is uncertain. interestingly, acinetobacter baumannii, which is an emerging fatal infection in icu patients worldwide, was isolated from deep tracheal aspirates in one in four patients. this may explain, at least in part, the relatively high mortality rates in this cohort. specific therapeutic options for mer-cov infections are limited and their efficacy is not well established [ ] . all patients in this report received antiviral treatment with either oseltamivir or combined ribavirin/interferon alfa- a therapy; two patients received both. although a previous study from the same institution showed that combined ribavirin/interferon alfa- a therapy was associated with significant improvement in survival at days, this benefit was not maintained at days after the onset of the disease [ ] . the retrospective and observational nature of this study does not allow precise assessment of the efficacy of these therapies. in the absence of a vaccine or a specific treatment, prevention of viral transmission through adequate infection control methods is the mainstay in the management of mers-cov outbreaks. appropriate isolation of patients with suspected or proven infections is crucial. in view of the high fatality rates of these patients in the icu, it may be reasonable to closely monitor patients with suspected infections in the general wards for early signs of organ dysfunction to prevent unnecessary delay in the provision of intensive care services and reduce mortality rates in these patients. our study has some limitations. we included patients with confirmed mers-cov infection from one icu of a large medical center. possible variations in the geographic distribution of the disease and in local practice may hinder extrapolation of these data to other cohorts in saudi arabia and other countries. the relatively low number of patients may have biased the statistical comparisons presented in this report and overestimated mortality rates. multivariable adjustment was also limited to the variables included in the models. collaborative efforts are needed to provide an insight into the risk factors for poor prognosis in these patients. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus ?id= &list= db c-fe d- c- - ff cb b &source=http% a% f% fecdc% eeuropa% eeu% fen% fpress% fepidemiological% fupdates% fpages% fepidemiological% f updates% easpx. accessed a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link 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(mers-cov) infection surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd middle east respiratory syndrome in the shadow of ebola middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart spread of mers to south korea and china mers coronavirus: diagnostics, epidemiology and transmission mortality risk factors for middle east respiratory syndrome outbreak, south korea association of higher mers-cov virus load with severe disease and death, saudi arabia association between severity of mers-cov infection and incubation period therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) infection: how close are we? curr treat options infect dis ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study we would like to thank dr. hassane nijimi (free university of brussels) for the statistical revision of this study and dr. karen pickett for editorial assistance with the manuscript. the study was supported only by institutional funds. mers-cov infections requiring admission to the icu are associated with high morbidity and mortality rates. the need for vasopressor therapy is the main risk factor for death in these patients. this report describes the clinical features and outcomes of critically ill patients with confirmed middle east respiratory syndrome coronavirus (mers-cov) infection. patients with mers-cov infections frequently had organ failure, and mortality rates were greater than %. the need for vasopressor therapy was an independent risk factor for death in the icu. the authors declare that they have no competing interests.authors' contributions gaa, ym, aso, mma, and ys conceived the study. sh, aal, aab, sa, mm, ta, aalg, at, and mkm participated in data collection. ys processed the data and performed the statistical analyses. ys and gaa drafted the manuscript. all authors read, revised, and approved the final manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - t hg wi authors: bernard-stoecklin, sibylle; nikolay, birgit; assiri, abdullah; bin saeed, abdul aziz; ben embarek, peter karim; el bushra, hassan; ki, moran; malik, mamunur rahman; fontanet, arnaud; cauchemez, simon; van kerkhove, maria d. title: comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from to date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: t hg wi since its emergence in , , cases and deaths due to middle east respiratory syndrome coronavirus (mers-cov) have been reported to the world health organization. most cases were due to transmission in healthcare settings, sometimes causing large outbreaks. we analyzed epidemiologic and clinical data of laboratory-confirmed mers-cov cases from eleven healthcare-associated outbreaks in the kingdom of saudi arabia and the republic of korea between – . we quantified key epidemiological differences between outbreaks. twenty-five percent (n = / ) of mers cases who acquired infection in a hospital setting were healthcare personnel. in multivariate analyses, age ≥ (or . , %ci: . – . ) and the presence of underlying comorbidities (or: . , % ci: . – . ) were associated with increased mortality whereas working as healthcare personnel was protective (or . , % ci: . – . ). at the start of these outbreaks, the reproduction number ranged from . to . ; it dropped below within to weeks. this study provides a comprehensive characterization of mers hca-outbreaks. our results highlight heterogeneities in the epidemiological profile of healthcare-associated outbreaks. the limitations of our study stress the urgent need for standardized data collection for high-threat respiratory pathogens, such as mers-cov. such large healthcare-associated (hca) outbreaks have mainly been limited to the kingdom of saudi arabia (ksa) and the united arabian emirates (uae) until the spring , when a single imported case of mers returning from the middle east initiated a cluster of cases in the republic of korea (rok) across at least hospitals and much of the country . super spreading events in healthcare settings has been described for several previous mers outbreaks, including an outbreak in al-hasa governorate in and during the outbreak in rok, where approximately % of the transmission events were epidemiologically linked to five mers cases , , . superspreading events in health care facilities have been observed in similar high threat respiratory disease pathogens, such as severe acute respiratory syndrome (sars) in canada, china, singapore [ ] [ ] [ ] . while more than half of the laboratory confirmed mers-cov infections reported globally to date are associated with human-to-human transmission in healthcare settings , there has been little human-to-human transmission reported in household settings . outbreak investigations and scientific studies conducted during or after mers hospital outbreaks have identified that aerosol-generating medical procedures with improper or inadequate personal protective equipment place medical personnel and patients sharing wards with mers patients and family visitors at higher risk for mers-cov infection , , with exposure to infectious droplets being the likely source of contamination. although close unprotected contact with a mers patient is generally considered necessary for human-to-human transmission , several studies have revealed that mers-cov particles can persist on surfaces as long as several days, raising the possibility of a role of fomites in transmission , . fomite transmission is further supported by observed viral spreading between rooms that were clearly separated , and outbreaks that occurred in hemodialysis units , . factors leading to healthcare-associated outbreaks include overcrowding in emergency departments, slow triage and isolation of suspected patients and inadequate compliance to infection prevention and control procedures , , . however, few studies have described or compared the characteristics of hca-outbreaks as a whole in terms of their size, epidemiologic factors , , or the role of interventions to stop transmission , . here, we provide the largest comprehensive study of eleven healthcare-associated outbreaks that occurred between and june . we carried out a comparative analysis of these outbreaks in terms of epidemiological profiles, demographic characteristics and clinical outcome. study design. we analyzed epidemiological datasets of laboratory-confirmed mers patients and focused our study on eleven healthcare-associated outbreaks that were reported in ksa and rok since , when policies and procedures for case identification and comprehensive contact identification and follow up became systematic and were implemented by affected countries. the data used documented mers-cov infections reported to who under the international health regulations ( ). we only included clusters of cases/outbreaks that were linked to healthcare facilities. supplemental rok case-based data were provided as a detailed line list of the korean mers cases included in a published study . we defined laboratory-confirmed mers-cov infection as following who guidelines , . we defined a hca-outbreak as the occurrence of or more laboratory-confirmed mers-cov infections with reported epidemiologic links between cases and during which the human-to-human transmission events were documented within a single healthcare facility, with no more than days apart between cases symptom onset. the mers outbreak in the republic of korea in is treated as a single outbreak. individual-level variables included information on age, sex, nationality, occupation (healthcare personnel (hcp) yes/no), dates of symptom onset, date of notification to who, presence of any pre-existing co-morbid conditions, and clinical outcome. in case of missing or conflicting information and when information from the country was not available, we considered the data as missing. statistical analysis. descriptive analysis was performed by hca-outbreak (outbreak-level analysis) using aggregated data, and for all cases (individual-level analysis). all analyses were conducted using stata, version (college station, tx: statacorp lp), microsoft excel (version . , jones, chicago usa) and r. outbreak-level analysis. we calculated the duration, size and case fatality ratio for each outbreak. the duration of an outbreak was calculated as the number of days between the date of symptom onset of the first reported case to the date of symptom onset of the last reported case. we obtained weekly smoothed estimates of the case reproduction number based on the approach developed by wallinga and teunis , using the r package. we assumed that the serial interval of mers-cov had a gamma distribution with a mean of . days and a standard deviation of . days, as described elsewhere . individual-level analysis. we summarized case characteristics as frequencies and proportions for categorical variables, as median and interquartile ranges (iqr) for continuous variables. chi-square tests were used to compare subgroups of cases when appropriate. a p value of less than . was used to indicate statistical significance. univariate analysis identified variables significantly associated with fatal outcome, which were included in a multivariable model. model selection was performed using a multilevel mixed-effects logistic regression with backwards selection taking into account clustering of individuals by outbreak. for the variable "age", the cut-off was fixed at , based on the results of the univariate analysis. variables with p-values < . were retained in the final model. all data used in these secondary analyses were de-identified data obtained from who or datasets from peer-reviewed literature. as such, these data were deemed exempt from institutional review board assessment. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ general characteristics of hca-outbreaks. since january to october , , laboratory-confirmed mers-cov infections have been reported to who. figure a illustrates the global epidemic curve since mers was first identified in humans. each peak is associated with a health care associated outbreak (colored lines, fig. a ). from , affected countries implemented systematic contact tracing and follow up (including laboratory testing), investigation and data collection of mers suspect cases , . in our analysis, a total of laboratory-confirmed mers cases from eleven distinct hca-outbreaks during - were included ( table ). the eleven hca-outbreaks varied in terms of duration, size and epidemiological profile ( table , fig. b) . the median number of total reported cases per outbreak was (interquartile range, [iqr] - ), ranging from to cases. the median duration was days (iqr - ), ranging from to days. three outbreaks began with sporadic cases during the first two to five weeks of the outbreak, while the other eight displayed a rapid increase to the peak. the median time between onset of symptoms of the first reported case and the peak of incidence was weeks (iqr - . ), ranging from to weeks. the case fatality ratio (cfr) in outbreaks was % ( reported deaths among cases), compared with the global overall cfr of . % ( reported deaths among , cases reported as of october (table ) . during hca outbreaks, cfr ranged from to % (p < . ) and cfr was significantly lower among hcp mers-cov infections compared to non-hcp mers-cov infections ( % vs. % p < . ). demographic and clinical characteristics. the demographic and clinical characteristics of the cases from hca outbreaks included in our analyses are summarized in table . the median age was (iqr, - ), and significantly varied by outbreak (p < . ). five outbreaks had a median age < and the other outbreaks had a median age ≥ . the majority of cases were male ( %, n = / ), and the sex ratio among cases differed significantly between outbreaks (p < . ). the overall proportion of hcp was % (n = / ). this proportion varied significantly by outbreak (p < . ), from % to % (table ) . median age was significantly lower among hcp than non-hcp cases ( iqr - vs iqr - , p < . ) and the proportion of females was higher among hcp than non-hcp ( % vs %, n = , p < . ). more than half ( %, n = / ) of cases had at least one underlying co-morbid condition (table ) , and this was significantly lower among females compared to males ( % vs %, respectively, n = , p < . ) and among hcp compared to non-hcp ( % vs %, n = , p < . ). sixteen percent (n = / ) of cases were asymptomatic at time of reporting (table ). this proportion varied significantly between outbreaks ranging from % to % (n = , p < . , fig. c ). median age of asymptomatic cases was (iqr, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the majority of whom were females ( %, n = / ) and had no underlying co-morbid conditions ( %, n = / ). the proportion of hcp among asymptomatic infections was high ( %, n = / ), and the cfr was null. the median duration between symptom onset and case notification to who was days (iqr - ). risk factors associated with fatal outcome. in univariate analysis, fatal outcome was significantly associated with age (p < . ), presence of underlying comorbidities (p < . ), non-hcp status (p < . ), and male sex (p < . , estimation of time-varying reproduction number. at the start of each hca outbreaks, the case reproduction number r (t) ranged from . ( % ci . - . ) to . ( % ci . - . ) ( table and fig. ). estimates of r (t) dropped below within to weeks from the first reported case in the outbreak (n = outbreaks, median weeks, iqr - ). year of outbreak period of time* www.nature.com/scientificreports www.nature.com/scientificreports/ we provide here a comparative characterization of mers hca-outbreaks and report substantial heterogeneity between hca-outbreaks illustrating the complexity of the factors contributing to the emergence of a cluster of cases associated with nosocomial transmission. the duration and epidemic profiles of outbreaks varied; some started with an apparent sharp increase in incidence while others began more slowly with isolated cases emerging intermittently for a few weeks before a cluster of cases appeared in a healthcare facility. some outbreaks had a sharp decline in cases, while others experienced a long tail lasting several weeks after the peak. the median estimates of the reproduction number r(t) in the early stages of outbreaks included in our analyses reached as high as . in the republic of korea, as has been found by others , likely facilitated by multiple superspreading events at two hospitals . what is perhaps most informative from a public health perspective is the length of time it took to bring the outbreaks under control. all of outbreaks reached r t values below within to weeks after the first cases were identified, highlighting that the time frame in which hospital and ministry officials can implement control measures to stop nosocomial outbreaks. factors explaining differences in hca outbreak size and duration might include variations in the speed in which cases were suspected and timing of interventions implemented in healthcare settings, including contact identification, management and isolation of patients, improved infection prevention and control measures and in some cases, the requirement to close departments [ ] [ ] [ ] [ ] [ ] . in this study, we were not able to evaluate the impact of interventions in these outbreaks. prevention of large hca outbreaks since (fig. a) , may be, in part, explained by improvements in contact tracing policies implemented in . in , contact tracing became more systematic with the identification and follow up of high (close, unprotected contact) and low risk contacts (protected hcw). in affected countries, national ministries of health and hospital staff comprehensively list all contacts of known mers patients, including healthcare workers at all facilities/departments the patient visited, patients who shared wards/ rooms with mers patients, family and visitors and occupational contacts. follow up of contacts includes the testing of all high-risk contacts, regardless of the development of symptoms. recommendations stated that positive contacts are placed in quarantine (home or hospital isolation for asymptomatic or symptomatic secondary cases, respectively) until they test negative , [ ] [ ] [ ] . additionally, affected countries enhanced infection prevention and control procedures education, and training, and implemented visual triage systems to reduce delays in testing, isolation and care of suspected mers-cov patients. this has again been recently illustrated by the lack of secondary cases following the identification of a confirmed case of mers in korea in september was due to the rapid and comprehensive isolation, treatment and management of contacts of the patient. the variation in outbreak size and duration is also affected by superspreading events early in some outbreaks, during which a limited number of cases generated a disproportionately large proportion of the secondary cases under specific conditions in hospitals, occurring in some outbreaks [ ] [ ] [ ] . two super spreading events have been documented in ksa and in the republic of korea. in the republic of korea, the practice of "doctor shopping", extended stays in overcrowded emergency departments, cultural practices of large numbers of family members visiting sick relatives, and environmental contamination amplified transmission from some patients to others , , , . during the outbreak in ksa in at the ministry of the national guard hospital, a high number of secondary cases were among hcp very quickly after the hospitalization and a surgical procedure of the index case . these events triggered comprehensive review ipc in hospitals, emergency department layout, movements of patients, triage of respiratory visits, duration of emergency department stay, training of hospital staff and disinfection of healthcare facilities. our study confirmed that age and presence of comorbidities are linked to increased risk of death, similar to previously published results , whereas being hcp was protective. the protective effect of hcp could be explained by the fact that hcp are more likely to be younger (< years old) and have fewer underlying medical conditions than hospitalized patients, but also that they are likely to be identified earlier or seek medical care soon following contact with a confirmed patient. the proportion of asymptomatic secondary cases identified during outbreaks has increased since . there is no evidence to suggest that this represents changes in virus pathogenicity, epidemiology or transmission www.nature.com/scientificreports www.nature.com/scientificreports/ patterns of mers in recent years. however, the increase in the number of reported asymptomatic cases is hypothesized to be due to earlier detection efforts from more aggressive contact identification and testing during hca-outbreaks since as testing policies adopted and implemented by ksa and other countries have changed following the large outbreaks in jeddah/riyadh in , , . in , - % of the laboratory confirmed hcp secondary cases experienced no symptoms and were detected as part of a policy to test all contacts irrespective of symptoms (table ) . we believe that the identification of hcp asymptomatic cases, and their subsequent isolation, has had a strong impact on prevent further human to human transmission in health care settings. this is visually demonstrated in fig. c by the outbreak labelled sau _ , which included (of www.nature.com/scientificreports www.nature.com/scientificreports/ reported cases) asymptomatic cases. while this is a large number of secondary cases, we argue that the early identification, isolation and recovery of these asymptomatic/mildly symptomatic cases effectively stopped human to human transmission. our study has several limitations due to variability in the completeness and quality of case-based data provided to who since and also due to the lack of detailed information on the timing specific interventions were implemented in relation to each outbreak. without detailed information on the timing of interventions in each health care facility it was not possible in our analyses to determine which intervention or combination of interventions had the greatest impact on stopping the mers outbreaks. moreover, prior to , contacts without symptoms were not tested for mers-cov infection, thus the rate of identification of secondary cases was drastically different prior to , which complicates the comparison of data collected before and after . the improvements in data reporting on cases (e.g., more systematic reporting of underlying conditions, reported exposures, contacts between patients) from allowed us to perform better analyses with less missing values. we continue to encourage the policy of identifying, following and testing of all high risk contacts of mers patients in hca-outbreaks , , . the natural history of asymptomatic infection and role of asymptomatic or mildly symptomatic hcp in transmission of the virus between patients, requires detailed scientific studies to better understand their potential role in transmission . the sharing of outbreak experiences between affected hospitals within and between countries and a detailed evaluation of the impact of non-therapeutic interventions is critical to our understanding and for the prevention of nosocomial outbreaks of respiratory pathogens. health care professionals and hospitals currently have tools to limit the extent and impact of such events, which include early identification and isolation of suspect patients and strict adherence to standard infection prevention and control measures. these are the hallmark of effective mers-cov control. a combination of interventions including the efficient triage of patients with respiratory symptoms at hospital entry; limiting wait times and overcrowding in waiting areas; isolation of suspected and confirmed cases; appropriate use of droplet personal protection equipment by hcp; basic hand hygiene; increased protective aerosol precautions for hcp during aerosol-generating medical procedures; efficient surface and environmental decontamination of areas with mers patients, and extensive contact tracing, can prevent human to human transmission in 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dynamics in south korea transmission characteristics of mers and sars in the healthcare setting: a comparative study risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea middle east respiratory syndrome risks of death and severe disease in patients with middle east respiratory syndrome coronavirus asymptomatic infection of middle east respiratory syndrome coronavirus using serologic survey in korea world health organization. disease outbreak news the authors would like to thank the many individuals involved in the collection of case-based data and in the care competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -rtmsrh authors: zumla, alimuddin; rustomjee, roxana; ntoumi, francine; mwaba, peter; bates, matthew; maeurer, markus; hui, david s.; petersen, eskild title: middle east respiratory syndrome - need for increased vigilance and watchful surveillance for mers-cov in sub-saharan africa date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: rtmsrh nan the past two decades have witnessed the emergence of several new and old respiratory tract infectious diseases, which threaten global health security due to their epidemic potential. , these include multi-drug resistant tb, severe acute respiratory syndrome (sars), avian and swine influenza and more recently the middle east respiratory syndrome (mers). mers is a new zoonotic disease of humans caused by a coronavirus (mers-cov) which was first isolated in september, from a patient who died from a severe respiratory disease in jeddah saudi arabia. since then mers has attracted global media attention because it is associated with a high mortality ( %) in individuals who have co-morbidities such as diabetes, chronic renal, liver or lung illnesses or in those who are immunocompromised. , the recent unprecedented outbreak of the mers , in south korea which arose consequential to the importation of mers-cov by a south korean traveler to the middle east, alarmed global public health authorities and highlights the potential of mers-cov to spread across the globe and cause local outbreaks. the who director general convened the ninth meeting of the emergency committee (ec) under the international health regulations regarding mers-cov on june to discuss the korean outbreak. as of rd june the total number of mers cases reported from the republic of south korea now stands at ( currently receiving treatment, recovered, deaths. , of cases, patients and hospital staff had contracted the virus nosocomially, friends, colleagues and relatives had come in contact within healthcare facilities while visiting family members with mers. virological and serological studies from several middle eastern, west and east african countries indicate that bats and dromedary camels are likely reservoirs of mers-cov. [ ] [ ] [ ] [ ] however, human mers-cov infections appear to be endemic only to countries in the middle east where sporadic cases continue to occur in the community throughout the year. the outbreak in seoul, republic of korea, has been linked to a single individual who had travelled to saudi arabia. the first mers case in thailand was reported last week and the patient had a history of travel from the sultanate of oman. of note is the striking absence of any mers cases (primary or travel related) reported from sub-saharan african (ssa) countries. , the reasons mers-cov predominantly affects humans in the middle east and is not endemic in africa where mers-covinfected camels and bats are present requires further study. a likely explanation may be that this may simply reflect the lack of clinical awareness of exposure risk, diagnosis and treatment of respiratory tract infections largely remains clinically based and empiric in most ssa countries coupled with absence of surveillance. every year an estimated million pilgrims from over countries travel to the kingdom of saudi arabia to participate in hajj pilgrimage, the mini-pilgrimage umrah (which is performed at any time of the year), or for the month of ramadaan. of these, an estimated million pilgrims come from sub-saharan african countries. there were no cases of mers reported during the , and hajj pilgrimages or the ramadaan period. [ ] [ ] [ ] however, the risk of mers-cov spreading globally remains due to the continuous influx of pilgrims and the persistent low levels of endemic mers-cov transmission to humans in saudi arabia. there is also the possibility that mers-cov may mutate into a form more adaptable for human to human transmission over time. the potential risk of mers-cov infection to pilgrims who visit saudi arabia from different regions of the world was estimated by coker and colleagues based on overall incidence of mers cases in saudi arabia since its first discovery in . their estimates based on the most likely scenario using recent pilgrim numbers for sub-saharan africa are that there will be at most ten returning pilgrims each year with mers-cov infections. national surveillance systems should be on alert for the low but long-lasting risk of mers-cov infected pilgrims returning from the umrah throughout the year, and also for the large numbers of refugees at several conflict zones in the middle east (those migrating from syria to turkey and from the yemen border into saudi arabia and beyond). the recent mers outbreak in the republic of korea was associated with secondary, tertiary, quarternary and quinary cases of mers-cov transmission, though fortunately there has been no sustained community transmission. , the republic of korea mers-cov outbreak has many similarities with that of previously reported mers-cov outbreaks which occurred at healthcare facilities in several cities in saudi arabia and from jordan - which were all associated with breaches and gaps in infection prevention and control protocols. these lapses in korean hospitals enabled mers-cov infected and uninfected patients, staff and visitors to mix freely in busy and crowded accident and emergency departments, within wards and multi-bed hospital rooms, with no isolation or quarantine of suspected cases. public health measures such as enhanced contact tracing and isolation and quarantine put in place by the korean government to control the outbreak eventually led to the decline in the numbers of mers cases and the outbreak is being brought under control. the importance of infection controls measures was also illustrated by the saudi arabian hospital mers outbreaks, where well-trained health care and workforce brought the hospital outbreaks under control quickly. , the who ec meeting noted that the who ec referred to the outbreak as a 'wake-up' call and state that in a highly mobile world, all countries should always be prepared for the unanticipated possibility of outbreaks of mers-cov and other serious infectious diseases. the korean mers outbreak is the largest recorded from outside the middle east and the largest imported from a returning traveller to the middle east, raising several important issues for global surveillance and control. it illustrates that mers-cov, three years after its first discovery remains an important global public health risk with many unanswered questions. further international spread should be anticipated and countries with weaker health systems and lack of laboratory facilities to accurately screen for mers-cov need to be vigilant. this will pose major challenges. there are important lessons here for sub-saharan african and other developing countries from where mers-cov cases have not yet been detected. as the recent ebola virus disease epidemic illustrates, african countries may be very vulnerable to a korea-like mers-cov outbreak, which may arise from returning pilgrims or other travellers from saudi arabia or from traders between saudi arabia and the horn of africa. mers-cov is transmitted through mers-cov-infected respiratory secretions for which contact and droplet precautions are recommended. [ ] [ ] [ ] [ ] the korean mers outbreak highlights that hospitals provide ideal conditions for amplifying mers-cov transmission arising from close contact between patients, healthcare and ancillary staff, relatives and other visitors, which enables spread of mers-cov. , it is critical that every country should maintain a high level of vigilance and perform mers-cov surveillance according to widely available expert recommendations, - whether or not mers cases have been detected in their countries, it ensuring infection prevention and control protocols are in place at all health-care facilities. those who travel must be educated to follow basic hygiene measures and those develop ill health during their trip to the middle east, or soon after their return should seek medical care and volunteer the history of travel to their healthcare provider. sub-saharan african governments must pay serious attention to strengthening infection control and public health surveillance systems. all healthcare workers and travellers from africa to the middle east should be aware of the threat to global health security posed by mers-cov. considering a diagnosis of mers at first presentation may be difficult due to non-specific symptoms at clinical presentation. however it is important that prevention and control measures are instituted at first consideration of mers as a diagnosis to prevent spread of mers-cov. hospitals and clinics providing care for patients infected with suspected or confirmed mers-cov infection should take appropriate measures to decrease the risk of mers-cov transmission from the infected patient to other patients, doctors, nurses, allied health-care workers, relatives and visitors. health-care workers should be educated and trained in infection prevention and control and should have continuing professional development on these issues. over the past decade, several surveillance systems have been introduced to monitor the emergence of new infectious pathogens. as the ebola virus epidemic in west africa showed, surveillance systems in african countries for infectious diseases with epidemic potential require strengthening. more effective national, regional, and international surveillance systems are required to enable rapid identification of emerging respiratory epidemics, diseases with epidemic potential, their specific microbial cause, origin, mode of acquisition, and transmission dynamics. in light of the republic of korea mers outbreak increased vigilance and surveillance for mers-cov should be carried out by health services in african countries using current clinical and public health guidelines for mers-cov. although resources may not allow for making an accurate diagnosis of mers, a high degree of awareness of the possibility of mers-cov infection in all returning pilgrims will allow early, isolation of patients and putting in place infection control measures, avoiding a repeat of the korea outbreak. sub-saharan african countries need to protect themselves against the possible outbreaks akin to the korean one. mers-cov should be included in list of pathogens by the african network of influenza surveillance and epidemiology (anise) and mers-cov should be made part of the strengthening influenza sentinel surveillance in africa (sisa)' with national, regional and international reporting mechanisms in liaison with other stakeholders involved in global infectious diseases surveillance. new, low cost, rapid, sensitive and specific diagnostic tests that can be used at all points of healthcare are require for all infectious diseases which threaten global health security. the exact mode of transmission and pathogenesis of mers-cov and other novel respiratory tract viruses such as h n influenza a virus require definition so that more effective prevention and management measures can be developed and introduced. a united and coordinated global response is needed to tackle emerging respiratory tract infections and to fill major 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middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndromeadvancing the public health and research agenda on mers-lessons from the south korea outbreak coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers infection prevention and control of epidemic-and pandemicprone acute respiratory infections in health care -who guidelines. geneva, world health organization infection prevention and control during health care for probable or confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection. update th infection control advice -middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov): prevention in travelers surveillance for emerging respiratory viruses idsr as a platform for implementing ihr in african countries establishing a national influenza sentinel surveillance system in a limited resource setting, experience of sierra leone rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects emerging novel and antimicrobial-resistant respiratory tract infections: new drug development and therapeutic options key: cord- -w l h g authors: okba, nisreen ma; raj, v stalin; haagmans, bart l title: middle east respiratory syndrome coronavirus vaccines: current status and novel approaches date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: w l h g middle east respiratory syndrome coronavirus (mers-cov) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. the re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. ongoing efforts to develop mers-cov vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. extending on our current knowledge of mers, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish mers-cov transmission to humans. in addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next cov outbreak. nisreen ma okba, v stalin raj and bart l haagmans middle east respiratory syndrome coronavirus (mers-cov) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. the re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. ongoing efforts to develop mers-cov vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. extending on our current knowledge of mers, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish mers-cov transmission to humans. in addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next cov outbreak. coronaviruses are the largest positive sense single stranded rna viruses. there are six human coronaviruses (hcov) to date; hcov- e, hcov-oc , hcov-nl , hcov-hku , severe acute respiratory syndrome (sars)-cov, and middle east respiratory syndrome (mers)-cov. prior to the sars-cov epidemic in - , covs were known to cause mild respiratory infections in humans. sars-cov, on the other hand, infected around cases causing severe respiratory disease with a % fatality rate [ ] . ten years later, mers-cov emerged in the human population also to cause severe respiratory infection [ ] . in contrast to the sars-cov epidemic, which was contained within one year, mers-cov still continues to cause outbreaks with increasing geographical distribution, four years after its first identification. as of march nd , cases in countries have been reported to the who with deaths accounting for a % case fatality rate (http://www.who.int/emergencies/mers-cov/en/). like sars-cov, mers-cov emerged as a result of zoonotic introduction to the human population. despite its close genome similarity with bat coronavirus hku and hku [ ] , accumulating serological and molecular evidence pointed to dromedary camels as the most probable reservoir for mers-cov [ ] [ ] [ ] . this poses a continuous risk of virus spill-over to people in contact with camels, such as those working in slaughter houses and animal farms, evidenced by the presence of mers-cov antibodies in sera of those individuals [ , ] . nosocomial transmission, however, accounts for the majority of mers-cov cases reported in outbreaks [ ] [ ] [ ] , although a substantial part of infections that occur result in unrecognized asymptomatic or mild illnesses [ ] . thus, in addition to camel contacts, other highly-at-risk groups are healthcare workers and patient household contacts [ , , ] . considering the ongoing mers-cov outbreaks, it is crucial to develop intervention measures among which vaccines play an important role. despite the fact that the emergence of mers-cov and sars-cov has dramatically changed the way we view covs, there is no licensed cov vaccine or therapeutic drug available to date [ , ] . a cornerstone for rational vaccine design is defining the determinants of immune protection. accumulating data from studies done so far on mers-cov and other coronaviruses revealed that a combination of both virusspecific humoral and cellular immune responses is required for full protection against coronaviruses. especially neutralizing antibodies are considered key players in the protective immunity against covs. neutralizing monoclonal antibodies (mabs) reduced viral loads in mers-cov receptor-transduced mice, rabbits and macaques [ ] [ ] [ ] [ ] . similarly, convalescent camel sera increased virus clearance and decreased lung pathological outcomes in mice with an efficacy directly proportional to anti-mers-cov-neutralizing antibody (nab) titers [ ] . also polyclonal sera produced in transchromosomic bovines protected mice against mers-cov challenge [ ] . evidence for the protective role of antibodies also comes from recent studies analyzing immune responses in patients that survived or succumbed to mers-cov. although neutralizing antibodies were only weakly inversely correlated to viral loads, serum antibody responses were higher in survivors compared to fatal cases but viral rna was not eliminated from the lungs [ , ] . administration of convalescent sera, however, did not lead to significant reduction in viral loads [ , ] . the presence of mucosal iga abs, on the other hand, was found to influence infectious virus isolation [ ] . besides humoral immunity, cellular immune responses are also considered to play a crucial role in protection against coronaviruses. while b-cell deficient mice were able to clear mers-cov, those lacking t-cells failed to eliminate the virus, pointing out the crucial role of t-cells in viral clearance [ ] . this is supported by the observation that t-cells were able to protect aged mice against sars-cov infection [ ] and the fact that a reduced tcell count was associated with enhanced disease severity in sars patients [ ] . along with other studies, these data highlight the importance of t-cells for virus clearance and protection against mers-cov [ , ] and sars-cov [ , ] . it is also noteworthy to mention that while neither antibodies nor memory b cells were detectable -years post-infection [ ] , sars-cov-specific memory t-cells, despite being low in frequency, persisted up to years post-recovery [ ] . nonetheless, the protective capacity of such memory response is not known. hence, taking into account the waning of virus-specific humoral responses, generating a long-lived memory t cell response through vaccination could be favorable, but as proper b-and t-cell immune responses are required for efficient protection, vaccination should target the induction of both. at the moment we lack information concerning the longevity of anamnestic immune responses following mers-cov infection, except for a recent study showing that antibody responses, albeit reduced, persisted up to months post-infection [ ] . the role of immune responses in protection is also in line with the observed increased fatality among the aged population following mers-cov infection. retrospective studies on mers-cov patients from saudia arabia and south korea have found a significant correlation between old age and mortality [ , , [ ] [ ] [ ] , a pattern that has been also reported for other respiratory viruses such as sars-cov [ ] and influenza virus [ ] . this is most likely caused by immunosenescence; a failure to produce protective immune response to new pathogens in elderly due to impaired antigen presentation, altered function of tlrs, and a reduced naïve b and t cell repertoire [ ] . this agerelated increase in mortality was also reported in sars-cov laboratory-infected animals, that is, mice and nonhuman primates (nhps) [ , ] , and was associated with low neutralizing antibodies and poor t-cell responses [ , , ] . several factors that play a role in t-cell activation were also found to be dysregulated in an age-related manner. age-related increase in phospholipase a group iid (pla g d), and prostaglan-dind in the lungs contributed to a diminished t-cell response and severe lung damage through diminishing respiratory dendritic cell (dc) migration [ , ] . likewise, adoptive transfer of t-cells to mice enhanced viral clearance and survival [ ] , highlighting the contribution of a reduced t-cell response in severe disease outcome. these observations also highlight the need for more effective preventive measures for the elderly. in this sense, induction of a potent airway t-cell response may be crucial to protect against covs [ ] . thus, a promising approach to protect against mers-covinduced fatality is to enhance virus-specific tissue (airway) resident memory t-cell responses through intranasal vaccination. although the mers-cov genome encodes for nonstructural proteins (nsp - ) and four structural proteins, the spike (s), envelope (e), membrane (m), and nucleocapsid (n) [ ] , the viral structural proteins, s and n, show the highest immunogenicity [ ] . while both s and n proteins can induce t-cell responses, neutralizing antibodies are almost solely directed against the s protein, with the receptor binding domain (rbd) being the major immunodominant region [ ] . thus, current mers-cov vaccine candidates mainly employ the spike protein or (parts of) the gene coding for this glycoprotein. these mers-cov vaccines candidates were developed using a wide variety of platforms, including whole virus vaccines, vectored-virus vaccines, dna vaccines, (table ) and protein-based vaccines ( table ) . although live attenuated vaccines produce the most robust immune responses, they pose a risk from reversion to virulence. inactivated virus vaccines may cause harm due to incomplete attenuation or the capacity to induce lung immunopathology [ ] . viral-vector-based vaccines, on the other hand, provide a safer alternative and have been developed using modified vaccinia virus ankara (mva) [ , , ] , adenovirus (adv) [ , ] , measles virus (mev) [ ] , rabies virus (rabv) [ ] , and venezuelan equine encephalitis replicons (vrp) [ , ] , all expressing mers-s/s proteins. additionally, vrp expressing the n protein have also been developed [ ] . a major hurdle facing these viral-vector-based platforms is preexisting immunity in the host which potentially can impair the vaccine efficacy. however, this can be prevented by using virus strains not circulating in the targeted population or immunization strategies involving heterologous prime-boost immunization, for example, mva and adv. although plasmid dna vaccines are considered to be of low immunogenicty in humans, current versions developed seem to induce potent immune responses. dna-based vaccines directed at inducing anti s responses were also shown to exert protection in nhps [ , ] . noteworthy to mention is middle east respiratory syndrome coronavirus vaccines: current status and novel approaches okba, raj and haagmans ad/hdpp -mice, mice transduced with hdpp in an adenoviral vector; alum, aluminum hydroxide; e, envelope protein; hdpp , human dipeptidyl peptidase ; i.m., intramuscular; i.n., intranasal; m, matrix protein; mers-cov, middle east respiratory syndrome coronavirus; nab, neutralizing antibodies; nd, not done; nhp, non-human primate; rntd, recombinant n-terminal domain; rbd, receptor-binding domain; rrbd, recombinant rbd; rbd-fc, rbd fused to the antibody crystallizable fragment of human igg; s, spike protein; s , s domain of spike protein; s - , amino acid residues - of the s protein; s - -klh, peptide s - coupled to keyhole limpet haemocyanin; s.c., subcutaneous; vlps, virus-like particles; a i.m.;alum/cpg odn produced higher neutralizing antibody responses than s.c.; ifa/cpg odn. b s - -fc, s - -fc, s - -fc, s - -fc, and s - -fc were tested and s - -fc had the highest nab titers although some produced equal s igg response [ ] . c mf produced the highest immunogenicity at low doses of antigen compared to s - -fc only, or with freund's/alum/mpla-sm/isa /mf . [ ] mg of antigen with mf was sufficient to produce humoral and cellular immune responses similar to higher doses ( mg or mg) [ ] . d i.n. + poly(i:c) vaccination induced stronger systemic cellular responses and higher local immune responses in mice lungs (iga and neutralizing antibody titers) than s.c. + montanide isa vaccination. that a combination of dna (s) and recombinant protein (s ) in a heterologous prime-boost immunization strategy induced higher immune response (nab) compared to each component alone [ ] . additionally, protein-based vaccines were developed in various platforms as virus-like particles (vlps) [ ] , nanoparticles [ ] , peptide-based [ ] , and subunit vaccines directed against various regions of the spike protein s [ ] , the n-terminal domain [ ] , and the rbd [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . those vaccines have the highest safety profile among vaccine platforms but confer variable degrees of immunogenicity which need adjustment for the dose, adjuvants, and site of administration to get optimal protective responses. adjuvants influence the type and magnitude of immune response produced by vaccines, and thus the doses used [ , , ] . additionally, the route of administration is a determining factor for the type of vaccine-induced immune response produced in the host. while intranasal (i.n.) vaccination with sars-n produced a protective airway t-cell response against sars-cov in mice, subcutaneous (s.c.) vaccination, inducing systemic t-cell responses, did not [ ] . likewise, i.n. vaccination with mers-rbd induced a significantly higher neutralizing and iga antibody responses in the mice airways compared to s.c. vaccination [ ] . this is important because mucosal immunity and airway memory t-cell responses are crucial players in protection against respiratory viruses, since these areas are the first to encounter the virus. therefore, along with selecting antigens for a vaccine, the route of vaccination and adjuvants are key players that cannot be neglected in vaccine design. because the spike protein and more specifically the s domain, is highly divergent among different covs, neutralizing antibodies only provide homotypic protection. thus far, the variability in the amino acid sequence of the spike protein observed among mers-cov strains is low [ ] , and circulating mers-cov strains did not show any significant variation in the serological reactivity [ , ] , implying that the development of a vaccine that is effective against one strain is likely to be protective against all circulating strains. another risk posed from the development of antibody escape variants is still present [ , ] , although this is not likely to happen as mutations in two epitopes may be required, and mutants that develop may have reduced viral fitness [ , ] . while the rbd is considered an ideal vaccine candidate for mers-cov, the spike s domain and n protein are more conserved, and thus adaptive immune response directed against these proteins can potentially lay the basis for a more broadly acting coronavirus vaccine. however, evidence for cross reactive immune responses against different covs is limited to a few studies. convalescent sars-cov patient sera weakly neutralized mers-cov [ ] and sars-s reactive antisera showed low level neutralization of mers-cov [ ] . extra-rbd s or s epitopes could be responsible for this effect, as some neutralizing epitopes have been identified in these regions of the s protein [ , ] . these may not be as immunodominant as the rbd epitopes but could provide a rationale for the development of a cross protective cov epitope-focused vaccine. a recent study also demonstrated the potential role of adaptive response against n protein in protection against mers-cov infection as this vaccine candidate produced a protective t-cell response against mers-cov challenge which was also partially protective against sars-cov [ ] . moreover, infection of mice with sars-cov reduced mers-cov titers days p.i. upon challenge suggesting the development of a cross reactive t-cell response [ ] . thus, mapping and focusing the immune response towards these critical neutralizing and t-cell epitopes, which could be subdominant, may provide a way to induce immune responses with a broader activity against different covs. immune focusing may also be beneficial for the generation of a robust virus-specific immune response. as during vaccine preparation, some epitopes which are normally hidden in the full length protein structure get exposed. some epitopes could be immunodominant and have a negative contribution on the overall neutralization capacity produced by the vaccine [ ] . this also holds true for some non-neutralizing immunodominant epitopes, as s -based vaccines induced slightly higher neutralization than whole s ectodomain-based ones [ , ] . additionally, the rbd induced higher neutralizing antibodies compared to an s subunit vaccine [ ] , and shorter regions of rbd induced even higher neutralization responses [ ] , indicating that additional regions inducing non-neutralizing antibodies may contribute negatively to the overall neutralization response produced. additionally, antibody-dependent enhancement of the viral infection by non-neutralizing antibodies [ ] [ ] [ ] , despite not being reported so far for mers-cov, needs also to be taken into consideration when developing a coronavirus vaccine. one approach to enhance the efficacy of subunit vaccines is to mask those negativelycontributing epitopes through glycosylation [ ] . other approaches are immunefocusing and epitope-based vaccines, all aiming at narrowing the immune response to target only critical or beneficial epitopes to produce a stronger protective response. a prerequisite to reach that goal is to map epitopes targeted by the immune system and identify their biological role as being neutralizing, non-neutralizing, infection enhancing, containing a tcell epitope, and so on. this can be achieved by analyzing the activity and fine specificity of convalescent patient sera, infected animal polyclonal sera, monoclonal antibodies, animal and human pbmcs. subsequently the predicted epitopes can also provide a basis for potential vaccine candidates when produced as nanoparticles or vlps. further characterization of the immune responses induced by these vaccine candidates when evaluated in an animal model may be utilized to optimize the vaccines for efficacy (figure ). this epitope-focused vaccine approach may allow for targeting less immunodominant b-and t-cell epitopes having broader protection, avoid eliciting immune responses against epitopes playing no role in protection or having a negative or harmful role. in addition to better targeting of protective immunodominant epitopes, a combination of those epitopes, b-and tcell epitopes targeting different viral proteins, could be used to produce a broader and stronger protective immune response for both strain-specific and universal cov vaccines. next to the choice of the mers-cov vaccine candidate, it is also important to take into account the target population that needs to be protected through vaccination. populations at risk of mers-cov infection include camel contacts, healthcare workers and patient contacts. the latter two groups could benefit from the rapid onset of immunity though passive immunization using mabs or convalescent sera, provided that it is given in time. another alternative strategy for short-term protection is the use of vaccines capable of rapidly inducing high titers of neutralizing antibodies. this will provide a short-term immunity beneficial to protect those highly-at-risk groups when a new case is identified, to prevent outbreaks. to prevent virus infection of primary cases, vaccination of the dromedary camels may also be considered. so far, among the available vaccine candidates, only two have been tested in dromedary camels, pvax -s and mva-s. pvax -s, a dna-based vaccine, induced neutralizing antibodies in two of three camels tested so far, but has not been tested for efficacy [ ] . the other candidate mva-s, a viral-vector-based vaccine, induced systemic neutralizing antibodies and mucosal immunity which conferred protection against mers-cov challenge and reduced virus shedding in vaccinated camels [ ] therefore, this vaccine candidate may provide a means to prevent zoonotic transmission of the virus to the human population. for camel contacts and healthcare workers in endemic areas, being at a continuous risk of mers-cov infection either from infected camels or patients, respectively, it would be beneficial to induce a longer-term (mucosal) protection. while these could be rewarding approaches to stop mers-cov outbreaks, it is still worthwhile to develop platforms and vaccines that aim to induce more broad protection against different related covs, that could potentially cause future outbreaks. learning from previous epidemics, the who issued a list of priority pathogens posing a risk to the human population and requiring urgent research and development (r&d) for intervention measures, among which mers-cov and highly pathogenic covs are of high priority. the lack of intervention measures along with the increase in geographical area and ongoing mers- epitope-based vaccine design. following a virus infection, potential protective b-and t-cell epitopes are mapped. peptides or proteins containing promising epitopes are produced and formulated using a suitable platform, for example, nanoparticles and tested for immunogenicity and efficacy in animals. follwing several cycles of testing and optimization, a final vaccine suitable for human use may be produced. cov cases, raise worries for the future occurrence of larger epidemics as a result of virus adaptation in the human population and more efficient human-to human transmission. further development of mers-cov and other cov vaccines thus needs proactive collaborative efforts from researchers filling knowledge gaps, and market stakeholders providing funding for this costly process. the latter can be insufficient and/or unsustainable, therefore hindering development of even some promising candidates. in an initiative aiming at accelerating vaccine r&d process by providing sustained funding to be prepared for future epidemics, the world economic forum launched the coalition for epidemic preparedness innovations (cepi) [ ] . cepi is an international non-profit association aiming at removing barriers facing vaccine development for epidemic infections and getting ready for future epidemics, including mers-cov. however, we still face a number of challenges despite the fact that various promising mers-cov vaccine candidates are currently available. there is a lack of animal models mimicking the disease in humans in which vaccine platforms can be tested prior to human use. we need to take into account the populations to target with vaccination, with camels and camel handlers being the most relevant ones. the lack of full understanding of the pathogenesis and immune responses to the virus in humans and camels, which is crucial for vaccine development, also needs further investments. in addition, the longevity of immune responses post-vaccination has not been evaluated for vaccine candidates, which is important for the vaccination scheme development and for the choice of the best candidates for further development. lastly, most of the vaccine candidates are developed against the highly variable spike protein and thus may not be able to provide protection against cov strains evolving in the future. a more targeted vaccination approach aiming at conserved epitopes should be considered for the development of a more broadly-acting cov vaccine. given the propensity of covs to jump the species barrier, current efforts to develop a mers-cov vaccine may also be of benefit to prepare for potential novel covs that may emerge in the future. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest the immunobiology of sars* isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study occupational exposure to dromedaries and risk for mers-cov infection presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission characteristics of mers and sars in the healthcare setting: a comparative study unraveling the drivers of mers-cov transmission risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel transmission of mers-coronavirus in household contacts clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia animal models of middle east respiratory syndrome coronavirus infection a comparative review of animal models of middle east respiratory syndrome coronavirus infection b -n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n / . virology prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome airway memory cd (+) t cells mediate protective immunity against emerging respiratory coronaviruses this study shows the efficacy of an anti-nucleocapsid vaccine for mers-cov, t-cell mediated protection and potential cross protection, and the role of route of administration in protection haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study memory t cell responses targeting the sars coronavirus persist up to years post-infection persistence of antibodies against middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility mortality risk factors for middle east respiratory syndrome outbreak, south korea association of higher mers-cov virus load with severe disease and death mortality associated with influenza and respiratory syncytial virus in the united states grubeck-loebenstein b: vaccines for the elderly mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice exacerbated innate host response to sars-cov in aged non-human primates evasion by stealth: inefficient immune activation underlies poor t cell response and severe disease in sars-cov-infected mice successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus t cell-mediated immune response to respiratory coronaviruses review on the role of t-cells in protection against respiratory coronaviruses age-related increases in pgd( ) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice critical role of phospholipase a group iid in age-related susceptibility to severe acute respiratory syndrome-cov 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orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type or vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform one-health: a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus a mouse model for mers coronavirus-induced acute respiratory distress syndrome evaluation of candidate vaccine approaches for mers-cov a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice the amino acids - of the mers-cov spike protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents the recombinant nterminal domain of spike proteins is a potential vaccine against middle east respiratory syndrome coronavirus (mers-cov) infection recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase (hdpp ) transgenic mice from mers-cov infection intranasal vaccination with recombinant receptorbinding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution recombinant receptor-binding domains of multiple mers-coronaviruses induce cross-neutralizing antibodies against divergent human and camel mers-coronaviruses and antibody-escape mutants cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc ( ) by both immunofluorescent and neutralizing antibody tests introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus sars cov subunit vaccine: antibody-mediated neutralisation and enhancement antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins vaccine initiative marks bold resolution engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate this work was supported by the zoonoses anticipation and preparedness initiative (zapi project; imi grant agreement no. ), with the assistance and financial support of imi and the european commission, inkind contributions from efpia partners. key: cord- -vkjcheaz authors: hao, xin‐yan; lv, qi; li, feng‐di; xu, yan‐feng; gao, hong title: the characteristics of hdpp transgenic mice subjected to aerosol mers coronavirus infection via an animal nose‐only exposure device date: - - journal: animal model exp med doi: . /ame . sha: doc_id: cord_uid: vkjcheaz background: middle east respiratory syndrome coronavirus (mers‐cov), which is not fully understood in regard to certain transmission routes and pathogenesis and lacks specific therapeutics and vaccines, poses a global threat to public health. methods: to simulate the clinical aerosol transmission route, hdpp transgenic mice were infected with mers‐cov by an animal nose‐only exposure device and compared with instillation‐inoculated mice. the challenged mice were observed for consecutive days and necropsied on days , , , and to analyze viral load, histopathology, viral antigen distribution, and cytokines in tissues. results: mers‐cov aerosol‐infected mice with an incubation period of ‐ days showed weight loss on days ‐ , obvious lung lesions on day , high viral loads in the lungs on days ‐ and in the brain on days ‐ , and % survival. mers‐cov instillation‐inoculated mice exhibited clinical signs on day , obvious lung lesions on days ‐ , continuous weight loss, % survival by day , and high viral loads in the lungs and brain on days ‐ . viral antigen and high levels of proinflammatory cytokines and chemokines were detected in the aerosol and instillation groups. disease, lung lesion, and viral replication progressions were slower in the mers‐cov aerosol‐infected mice than in the mers‐cov instillation‐inoculated mice. conclusion: hdpp transgenic mice were successfully infected with mers‐cov aerosols via an animal nose‐only exposure device, and aerosol‐ and instillation‐infected mice simulated the clinical symptoms of moderate diffuse interstitial pneumonia. however, the transgenic mice exposed to aerosol mers‐cov developed disease and lung pathology progressions that more closely resembled those observed in humans. middle east respiratory syndrome coronavirus (mers-cov), which was first identified in saudi arabia in and causes acute respiratory illness, multiorgan failure, shock and even death, is an important highly pathogenic coronavirus that is similar to severe acute respiratory syndrome coronavirus (sars-cov) and produces severe infections with a high mortality rate. [ ] [ ] [ ] at the end of may , there were a total of laboratory-confirmed cases of mers with associated deaths (case-fatality rate: . %, which is higher than the fatality rate of sars) worldwide according to world health organization (who) statistics. mers cases have been reported in countries, including countries in the middle east, africa, europe, asia, and north america as well as australia, and case numbers continue to increase, posing a global threat to public health. in china, the first patient infected with mers-cov from south korea was diagnosed in may , and it will be extremely important to prevent, control, and treat mers-cov infections during any future outbreaks. hence, effective small animal models are needed to investigate viral pathogenesis and evaluate mers-cov therapeutics and vaccines. nonhuman primate animal models of mers-cov in both rhesus macaques and common marmosets were established in previous reports, , however, these models are limited by restricted availability, high costs, expert husbandry requirements, and ethical concerns. , traditional small animals such as mice, hamsters, and ferrets cannot be infected with mers-cov owing to absence of the necessary dipeptidyl peptidase (dpp ) receptor that interacts with the receptor binding domain of the mers-cov spike protein (s protein) [ ] [ ] [ ] mers-cov fails to replicate in mice, which are readily available, have a defined genetic background and low cost and are frequently used in infectious disease research, due to variations in the dpp receptor. previous studies showed that transgenic mice expressing the human dpp (hdpp ) receptor could be infected intranasally with mers-cov and developed acute pneumonia. [ ] [ ] [ ] therefore, hdpp transgenic mice were selected for exposure to mers-cov-containing aerosols using an animal nose-only exposure device. there are two modes of mers-cov infection, animal-to-human and human-to-human transmission. some reports have found that airborne transmission via the coughing and sneezing of infected dromedary camels or contact with respiratory secretions and consumption of unsterilized milk from infected camels can significantly increase the risk of mers-cov infection in humans. , kim et al discovered extensive viable mers-cov contamination in the air and surrounding environment in mers isolation wards. according to the who, it has been suggested that human-to-human transmission, to a very limited extent, is caused by inhalation of droplets or airborne virus and close contact with patients. the above studies have demonstrated that mers-cov has a risk of aerosol transmission. in addition, aerosol inhalation is the main clinical route of infection for viral respiratory illnesses. there are different clinical presentations in animal models established by different infection routes. comparative studies using approaches with different perspectives will contribute to a deeper understanding of mers. in this work, to simulate the aerosol transmission route for comparison with the instillation route, hdpp transgenic mice were exposed to mers-cov aerosols by an animal nose-only exposure device. after infection, we analyzed the mouse characteristics of weight loss, survival, viral replication, tissue pathology, viral antigen distribution, and cytokine and chemokine profiles, which provide additional data to investigate the pathogenesis of mers-cov-induced disease and evaluate relevant therapeutics and vaccines. specific pathogen-free transgenic c bl/ mice expressing hdpp all animals were fed under absl- conditions for days before the start of the study. on two consecutive days prior to infection, each mouse was trained in an animal nose-only aerosol device. infected mice were kept in the absl- laboratory throughout the study and observed daily to ensure that they had enough water and food. mers-cov (human betacoronavirus cemc/ , complete genome genbank: jx . ) was provided by the ilas, cams. the virus was propagated and expanded in vero e cells (american type culture collection, usa) cultured and passaged at °c and % co by routine methods. purified and concentrated progeny viruses were titrated using vero e cell-based infectivity assays, and viral titers are expressed in units of % tissue culture infectious dose per microliters (tcid / μl). mers-cov stocks at a concentration of . tcid / μl were stored at − °c. an animal nose-only aerosol exposure device (in-tox products) was located in an absl- laboratory and comprised a nose-only exposure chamber and nebulizer inside a class Ⅱ biological safety cabinet (bsc Ⅱ), a control box, mouse restraint tubes, a clean compressed air tank and a vacuum pump ( figure ). the exposure device, which exposed only the mouse nose, generated mers-cov aerosol particles of . ± . μm to infect transgenic mice expressing hdpp and simulated the natural route of infection. as shown in table , transgenic mice were randomly assigned to an aerosol group, an instillation group, an aerosol control group, and an instillation control group, and the body weight of each mouse was measured on the day of infection (day ). each group contained mice; five mice in each group were used to analyze clinical symptoms, weight loss and survival, and three mice in each group were randomly chosen for necropsy on days , , , and postinfection. mers-cov virus suspensions ( . tcid ) and serum-free dulbecco's modified eagle medium (dmem) were separately added to the nebulizer reservoir to infect exposed mice in the aerosol and control aerosol groups, respectively, for minutes. according to the instructions of the exposure device and mouse respiratory rate ( ml/min per mouse), the nebulizer flow rate was set to . l/ min, the diluter flow rate was set to . l/min, and the nebulizer pressure was set to psi. mice were anesthetized with . % tribromoethanol ( . ml/ g of body weight, intramuscular (im)) for intranasal inoculation with . tcid of mers-cov in the instillation group and serum-free dmem in the instillation control group. infected mice were observed for consecutive days to analyze the clinical symptoms of disease, weight change, and survival. the mice were euthanized with . % tribromoethanol ( . ml/ g of body weight, im) when they reached % weight loss. aerosol control dmem aerosol to analyze clinical signs, weight loss, and survival a instillation control dmem suspension to analyze clinical signs, weight loss, and survival on days , , , and postinfection, three animals randomly selected from each group underwent necropsy to obtain tissue specimens for assessing viral distribution, associated histopathology, and cytokine levels using quantitative reverse transcription-pcr (qrt-pcr), hematoxylin and eosin (h&e) staining, immunohistochemistry (ihc), and enzyme-linked immunosorbent assay (elisa). total viral rna was extracted from tissues (lungs, brain, kidneys, spleen, liver, heart, and intestine) homogenized using the rneasy control. a standard curve was generated for pcr using - copies of a qualified standard plasmid to calculate copy numbers for each reaction. formalin-fixed lung, brain, and kidney samples were embedded in paraffin wax and sectioned at an approximately -μm thickness. deparaffinized and hydrated tissue sections were routinely stained with h&e to examine histopathological changes. immunohistochemical staining was performed to assess the expression of a viral antigen using a rabbit two-step detection kit (zhongshan golden bridge biotechnology co., ltd) with a rabbit polyclonal anti-mers-cov nucleoprotein (np) antibody (sino biological inc). visualization was then performed by dab staining and hematoxylin counterstaining. supernatants of tissue homogenates from infected mice ( µl) were added to the bottom of an antibody-coated plate. the levels of interleukin (il)- β, il- , il- , il- , tumor necrosis factor (tnf)-α, interferon (ifn)-γ, and ifn-β were assayed using elisa kits (kete biotechnology co., ltd). chemokine and cytokine concentrations were recorded as pg/ml of homogenate or ng/l of homogenate. data were analyzed using spss or graphpad prism . software. the experimental results are presented as the mean plus standard deviation (sd). one-way anova was used to assess differences in body weight, viral load, and cytokine levels among different groups. student's t test was performed for two-group comparisons. p < . was considered statistically significant. the infected mice in both the aerosol and instillation groups displayed significant clinical symptoms, such as huddling, hunching, ruffled fur, weight loss, and death. there were significant differences in weight change (p < . ) and survival (p < . ) between the mers-cov infection groups and the control groups. the incubation period, however, was - days after aerosol infection and day after instillation inoculation. after mers-cov aerosol exposure, hdpp transgenic mice showed profound clinical signs on days - , rapid weight loss on days - and % survival by day (acute death or euthanasia at % weight loss). the intranasally infected transgenic mice displayed rapid weight loss on days - and % survival by day (acute death or euthanasia at % weight loss). there were significant differences in disease progression (p < . ) after challenge between the aerosol group and the instillation group. transgenic hdpp mice infected with mers-cov aerosols exhibited milder disease and slower disease progression than did those inoculated intranasally (figure a,b) . no obvious abnormalities, including weight loss or signs of clinical illness, were detected in the aerosol control and instillation control groups. there were no significant differences in weight change or survival rates between mice inoculated with dmem in the above two control groups (p > . ; figure c ). based on qrt-pcr analyses of tissue rna contents, we identified high viral loads in the lungs and brain in mice and a small amount of viral rna in other tissues after mers-cov infection via the aerosol or instillation route ( figure a ,b). however, there were significant differences in the tissue viral loads of infected mice between the two groups (p < . ). after mers-cov aerosol infection, high viral loads were detected in the lungs at - days and in the brain at - days. viral loads were high in the lungs and brain of intranasally infected mice at days and . there were significant differences (p < . ) in the viral loads in the lungs and brain between the two groups at days and . the viral loads in the lungs and brains of the mice in the aerosol group were significantly lower than those of the mice in the instillation group. high levels of viral rna accumulated more slowly in the tissues of the mers-cov aerosol-exposed mice than in those of the mice infected intranasally ( figure ). as shown in figure the data are presented as the mean change ± sd for each group (n = ). mice in the instillation group died acutely or were euthanized when they reached % weight loss; these mice had a % survival rate by day , which produced no results for weight loss on days and . a key indicating the color coding for the groups is provided in the figure. *p < . , **p < . , ***p < . , and ****p < . mice in the instillation group died acutely or were euthanized when they researched % weight loss; these mice had a % survival rate by day , so no tissue lesion results were available on days and mice infected with mers-cov via the aerosol inhalation or intranasal instillation route, but no obvious lesions were found in other tissues. there were no abnormalities in the tissues of the normal control group. it was clear that the appearance of the lungs exhibited obvious congestion and dark brown regions on days - in the aerosol group. the mers-cov-intranasal mice showed gross lung lesions on day and more severe lung lesions on day . gross lung lesions developed more slowly and were milder in the aerosol group than in the instillation group ( figure a ). microscopically, challenged mice developed moderate acute interstitial pneumonia and brain pathology, but no pathological changes were detected in other tissues in the mice. in the aerosol group, the lungs of the exposed mice showed alveolar septal widening, inflammatory cell infiltration, and vessel dilatation and congestion at - days, gradual development of severe pathological changes and inflammatory cell infiltration in perivascular regions at - days, focal hemorrhages at - days, and an expanded pathology range at day ( figure b ). dilatation and congestion of the cerebral vessels were not clearly observed until day , and few areas of neuron deformation necrosis were found in the cerebral cortex, hippocampus, and thalamus before day ( figure c ). on days and after intranasal infection, we found moderate acute interstitial pneumonia and brain lesions ( figure b ,c). tissue lesions, however, were milder in the aerosol group than in the instillation group. furthermore, there were significant differences in the progression of lung and brain lesions in the two infected groups. tissue lesion progression was slower in the aerosol-infected mice than in the instillation-infected mice ( table ). the expression of a mers-cov antigen was primarily evaluated using ihc assays and was found in endothelial cells and alveolar pneumocytes in the lungs and in cerebral cortical neurons, dendrites, axons, microglia and the hippocampus in the brains of aerosol-and instillation-challenged mice but not in control mice ( figure a,b) . prominent mers-cov expression was also observed in renal tubular epithelial cells ( figure c ). however, there were significant differences in the timing of virus expression in the tissues of the mice postinfection. after mers-cov infection, ihc assays with a rabbit polyclonal anti-mers-cov np antibody found that viral antigens predominantly appeared in tracheal endothelial cells at day postinfection in the lungs of the aerosol-infected mice and in both tracheal endothelial cells and pneumocytes in the lungs of the aerosol-infected mice at - days; these changes were observed in the lungs of the instillation-infected mice at and days, respectively. in addition, the mers-cov antigen was discovered in the brain and kidneys in the aerosol group at - days and in the instillation group at and days. based on these results, we concluded that the distribution of the mers-cov antigen in the lungs, brain and kidneys after infection was slower in the aerosol group than in the instillation group. there were significant differences in the level of related proinflammatory cytokine and chemokine profiles, including il- β, il- , il- , il- , tnf-α, and ifn-γ, between infectious groups (the aerosol group and instillation group) and the control group (p < . ). significantly elevated levels of il- β, il- , il- , il- , tnf-α, and ifn-γ were discovered in the lungs and brains of mice in the aerosol group with increased cxcl- at - days (p < . ) postchallenge and in those of mice the instillation group at and days postchallenge ( figure ). in the aerosol group, the exposed mice showed peak il- and concentration in the lungs and il- and cxcl- concentrations in the brain at - days, and peak tnf-α and ifn-γ levels in the lungs and brains with peak il- level at a -, no apparent changes; +, mild alveolar septum widening; ++, moderate alveolar septum widening; and +++, severe alveolar septum widening. b -, no apparent changes; +, infiltration of a few interstitial inflammatory cells; and ++, some interstitial inflammatory cell infiltration. c -, no apparent changes; and +, a small amount of exudate in alveoli. d -, no apparent changes; +, mild dilatation and congestion of vessels; and ++, moderate dilatation and congestion of vessels. e -, no apparent changes; and +, mild hemorrhage. f nd, not done. mice in the instillation group died acutely or were euthanized when they reached % weight loss, which occurred by day . f i g u r e immunohistochemical staining of mouse tissue samples after infection. a, immunohistochemical staining of the lungs of infected mice. b, immunohistochemical staining of the brains of infected mice. c, immunohistochemical staining of the kidneys of infected mice. mice in the instillation group died acutely or were euthanized when they researched % weight loss; these mice had a % survival rate by day , so no tissue lesion results were available on days and - days. after intranasal infection, however, the levels of il- β, il- , il- , tnf-α, and ifn-γ in the lungs and il- , il- , il- , and ifn-γ in the brains peaked at - days. the secretion of some cytokines and chemokines in the aerosol group was slower than that in the intranasal group (p < . ). the mice in the instillation group died acutely or were euthanized when they researched % weight loss; these mice had a % survival rate by day , so no results were available on days and . the results represent the mean ± sd for each group (n = ). *p < . , **p < . , ***p < . , and ****p < . distribution in tissues between the aerosol-and instillation-challenged mice (table ) . after mers-cov infection, the disease progression in the mice in the aerosol group was slower than that in the mice in the instillation group. sanders et al showed that virus droplets were deposited and concentrated in the lungs through the respiratory tract of mice inoculated intranasally, resulting in fast disease onset. , correspondingly, after instillation infection with mers-cov, we found that mice with a short airway and high concentration of virus deposited in the lungs displayed weight loss at day and lung lesions at day , consistent with intranasal mouse models established by adam, agrawal and li et al [ ] [ ] [ ] ; these mice also exhibited % survival by day . previous studies reported that aerosol particles ≤ μm penetrated the respiratory tract to reach the alveoli and were diffusely distributed in the lungs. , in addition, virus aerosols entered the blood circulation through the alveoli, and other viruses slowly replicated in the lungs after mice inhaled mers-cov-containing aerosols (particle size: . ± . μm). compared with instillation-inoculated mice with virus deposition in the lungs, aerosol-exposed mice displayed slower disease progression with an incubation period of - days, lung lesions on day , continuous weight loss on days - , milder clinical signs, and % survival on day . we found that the progressions of virus replication and lung lesions in challenged mice were slower in the aerosol group than in the instillation group. based on high viral loads in the lungs and brain of challenged mice, which was consistent with previous reports, and acute renal failure in mers patients, we carried out h&e staining to assess histopathological changes and immunohistochemical staining with a specific antibody to further characterize mers-cov expression in the lungs, brain, and kidneys. a relatively high viral load in the lower respiratory tract is associated with severe illness in viral respiratory diseases. , at - days postinfection, mice in the intranasal group, which had high viral loads in the lungs and brain at - days, exhibited acute interstitial pneumonia and pathological brain changes. in the aerosol group, mice developed acute interstitial pneumonia at - days and pathological brain changes at - days, which were caused by high levels of virus rna in the lungs at - days and in the brain at - days, respectively. higher virus rna levels in the instillation group might contribute to the more severe high level on days and high level on days to high level on days and histopathology lungs moderate acute interstitial pneumonia on days to moderate acute interstitial pneumonia on days to brain relatively mild brain lesion on days and brain lesions on days and lungs in bronchial endothelial cells on day in both tracheal endothelial cells and alveolar pneumocytes in the lungs on days to in both tracheal endothelial cells and alveolar pneumocytes in the lungs on days and in cerebral cortical neurons, dendrites, axons, glial cells, and the hippocampus on days to in cerebral cortical neurons, dendrites, axons, glial cells, and the hippocampus on days and in renal tubular epithelial cells on days to in renal tubular epithelial cells on days to cytokines and chemokines b high levels on days to , including cxcl- high levels on days to middle east respiratory syndrome patients exhibit a median incubation period of - days, with a range of - days. we discovered that instillation-inoculated mice exhibited clinical signs within day but that the incubation period of aerosol-exposed mice was - days, which more closely resembled the period observed in humans. mers-cov binds to hdpp receptors that are primarily expressed in the lower respiratory tract and alveoli, resulting in a wide range of disease symptoms in patients, from no symptoms to mild respiratory illness or severe acute pneumonia, which rapidly progresses to acute lung damage, multiorgan failure and even death. , clinically, chest radiography and chest computed tomography (ct) show no lung lesions in patients in the early stages of illness, but pneumonia is identified during the course of the disease and includes patchy densities, extensive diffuse and focal alveolar space opacities, interstitial infiltrates, and consolidation. pulmonary pathological changes in aerosol-infected mice were similar to those noted in patients with respiratory tract infection. additionally, immunohistochemical staining revealed that a mers-cov antigen was expressed in alveolar pneumocytes and endothelial cells, the brain, and the kidneys in challenged transgenic mice. studies of a fatal case of mers-cov infection evidenced that the expression of a mers-cov antigen was predominantly localized in pneumocytes and endothelial cells, resulting in cell necrosis and pneumocyte damage; however, no viral antigens were detected in other tissues in the fatal case. as demonstrated in previous studies, we also discovered high viral loads, pathological changes and the expression of a mers-cov antigen in the brain of challenged mice; and no brain lesions, but multiorgan damage, were observed in mers patients. , , zhou et al demonstrated that human dendritic cells and macrophages were permissive to mers-cov replication, indicating that the multiorgan injury induced by mers-cov may be associated with the distribution of the hdpp receptor in many cell types that are spread throughout multiple organs. some studies have indicated that mers-cov has cell and tissue tropisms, especially tropisms for pneumocytes and neurons, and synapses may be one of the structures by which viruses diffuse through the brain after mers-cov infection. , the mechanisms underlying the brain lesions and death induced by mers-cov infection in hdpp transgenic mice remain complex and complicated and need to be further investigated. hdpp transgenic mice were successfully infected with mers-cov aerosols by an animal nose-only exposure device, and aerosol-and instillation-infected mice all simulated the clinical symptoms of moderate diffuse interstitial pneumonia. compared to instillation-infected mice, aerosol-infected mice more closely resembled infected humans in terms of the progression of disease and pathology in the lungs, which provided additional data for studying pathogenesis and evaluating the efficacy of preventive and therapeutic agents for mers-cov. the current work was supported by the national science and technology major projects of infectious disease (grant number zx - ). none. hg was the principal investigator, designed and supervised the study, and wrote the grant application. xyh performed the main experiments. xyh and ql performed the cell experiments. xyh and fdl conducted the animal experiments. yfx completed the pathology experiments. xyh and hg conceived the experiments, analyzed the data and wrote the paper. all authors read and approved the final manuscript. xin-yan hao https://orcid.org/ - - - middle east respiratory syndrome: emergence of a pathogenic human coronavirus nurses' experiences of care for patients with middle east respiratory syndrome-coronavirus in south korea middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome coronavirus (mers-cov). mers monthly summary discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory 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ghorbani, saied; khatami, alireza; farzi, rana; baradaran, behzad; turner, diana l.; turner, raymond j.; bahr, nathan c.; idrovo, juan‐pablo title: comparison of confirmed covid‐ with sars and mers cases ‐ clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta‐analysis date: - - journal: rev med virol doi: . /rmv. sha: doc_id: cord_uid: fmg scx introduction: within this large‐scale study, we compared clinical symptoms, laboratory findings, radiographic signs, and outcomes of covid‐ , sars, and mers to find unique features. method: we searched all relevant literature published up to february , . depending on the heterogeneity test, we used either random or fixed‐effect models to analyze the appropriateness of the pooled results. study has been registered in the prospero database (id ). result: overall articles included in this study; covid‐ confirmed patients ( studies), sars ( studies), and mers patients ( studies) were included. the most common symptom was fever; covid‐ ( . %, p < . ), sars ( %, p < . ), and mers ( %, p < . ), respectively. analysis showed that % of covid‐ patients, % of sars patients, and . % of mers patients had an abnormal chest x‐ray. the mortality rate in covid‐ ( . %, p < . ) was lower than sars ( %, p < . ) and mers ( %, p < . ) between all confirmed patients. conclusions: at the time of submission, the mortality rate in covid‐ confirmed cases is lower than in sars‐ and mers‐infected patients. clinical outcomes and findings would be biased by reporting only confirmed cases, and this should be considered when interpreting the data. during the last two decades, coronaviruses have been recognized as one of the most critical human pathogenic viruses that affect global health and cause concern in the world health system. coronavirus is classified into four genera: alpha, beta, delta, and gamma. major (about % mortality). there is no vaccine or targeted treatment currently available for covid- infection. treatment is mostly supportive, although multiple experimental antiviral medications are being evaluated. , thus, prevention and rapid diagnosis of infected patients are crucial. the trigger for rapid screening and treatment of covid- patients is based on clinical symptoms, laboratory, and radiographic findings that are similar to sars and mers infections. in this study, we attempted to distinguish the clinical symptoms, laboratory findings, radiographic signs, and outcomes of confirmed covid- , sars, and mers patients. all findings are compared to determine unique features among each of them. these data could be helpful in the early diagnosis and prevention of infection as well as providing more reliable epidemiological data on a large-scale for health care policies and future studies. this study was conducted according to the preferred reporting items for systematic reviews and meta-analyses statement (prisma) guidelines, and it has been registered in the prospero database (id ). we searched all studies published up to february , from the following databases: embase, scopus, pubmed, web of science, and the cochrane library. search medical subject headings (mesh) terms used were: "covid- ," "coronavirus," "severe acute respiratory syndrome," "sars virus," "severe acute respiratory syndrome coronavirus ", "coronavirus infections," "middle east respiratory syndrome coronavirus," and all their synonyms like "wuhan coronavirus," "sars-cov- ," and "covid- ," " -ncov" and mers. moreover, we searched for unpublished and grey literature with google scholar, centre for disease controls (cdc) and who databases. we also examined references of included articles to find additional relevant studies. there was no language restriction, and all included studies were written in english or chinese languages; the latter was translated by https://translate.google.com/. additional search strategy details are provided in table s . duplicate studies were removed using endnote x (thomson reuters, new york, ny, usa). records were initially screened by title and abstract by independently four authors (ap, sg, ak, and rf). the full-text of potentially eligible records was retrieved and examined, and any discrepancies were resolved by consensus. studies had to fulfill the following predetermined criteria to be eligible for inclusion in our meta-analysis. all case-control, cross-sectional, cohort studies, case reports, and case series peer-reviewed studies were included if they reported the number of confirmed cases of patients with demographic data, [and] [or] clinical data, [and] [or] laboratory data, [and] [or] risk factor data. studies were excluded if they did not report the number of confirmed cases. letters to the editor, individual case reports, review articles, and news reports were also excluded. duplicate information from the same patient was combined and counted as a single case when the data was reported twice. all covid- included publications were published in , and all patients were from china. the following items were extracted from each article: first author, center and study location in china, countries, sample collection time, patient follow-up time, the reference standard for infection confirmation, number of confirmed cases, study type, and all demographic, clinical, laboratory data, and risk factor data. three of our authors (sg, ak, and rf) independently extracted data, and all extracted data were checked randomly by another author (ap); differences were resolved by consensus. quality assessments of studies were performed by two reviewers independently according to the critical appraisal checklist recommended by the joanna briggs institute, and disagreements were resolved by consensus. the checklist is composed of nine questions that reviewers addressed for each study. the "yes" answer to each question received one point. thus, the final scores for each study could range from zero to nine (table s ). data cleaning and preparation were done in microsoft excel (microsoft©, redmond, wa, usa), and further analyses were carried out via comprehensive meta-analysis software version . (biostat, englewood, nj). determination of heterogeneity among the studies was undertaken using the chi-squared test (cochran's q) to assess the appropriateness of pooling data. depending on the heterogeneity test, we used either random or fixed-effect models for pooled results. in the case of high heterogeneity (i > %), a random effect model (m-h heterogeneity) was applied, while in low heterogeneity cases (i < %), a fixed-effect model was used. percentages and means ± sds were calculated to describe the distributions of categorical and continuous variables, respectively. p values reflect study heterogeneity with <. being significant. we also used the funnel plot, begg's, and egger's tests based on the symmetry assumption to detect publication bias ( figure s ). the process of study selection is displayed in figure quality assessment of included studies was performed based on the critical appraisal checklist, and the final quality scores of the included studies are represented in table s . in brief, studies by chen et al, wang et al, huang et al, guan et al, zhang et al, cheng et al, li et al, xu et al, figure s ). cough was the second most common symptom presenting in covid- % ( % ci . - , p < . ), sars . % ( % ci - , p < . ), and mers % ( % ci - , p < . ) of patients ( figure s ). age is an exception, presented in mean age in years. ( % ci . - , p < . ), or runny nose % ( % ci - , p < . ). more detail information about demographics and clinical characterization of covid- (table s ) , sars (table s ) , and mers patients (table s ) demonstrated in the supplementary material. the greatest risk for covid- patients . % ( % ci . - , p < . ) up to february , is a history of recent travel to wuhan, contact with people from wuhan, or were wuhan residents, and . % ( % ci . - , p < . ) had exposure at the seafood market(s (table s ) , sars (table s ) , and mers patients (table s ) is demonstrated in the supplementary material. most covid- confirmed patients required hospitalization . % ( % ci - , p < . ) and . % ( % ci . - , p < . ) were deemed to be in critical condition. the mortality rate of covid- confirmed cases was . % ( % ci . - . , p < . ), sars % ( % - , p < . ), and mers % ( % ci - , p < . ) (figure ). the laboratory findings showed that among a subset of patients . % increased the percentage value. the actual mortality rate from covid- is almost certainly much lower than that found in this study. as more data emerges from screening asymptomatic or mildly symptomatic individuals in china and around the world, the exact mortality rate will be better understood. among covid- , sars, and mers patients, leukocytosis was found in . %, %, and %, respectively, and leukopenia in %, %, and %, respectively. most of the patients with coronavirus had abnormal chest radiological findings. on the other hand, runny nose and rhinorrhea are less common symptoms in coronavirus-infected patients, and that it is typically expressed on pulmonary alveolar epithelial cells. another study reported that following covid- infection deregulated cytokine/chemokine response and higher virus titer causes an inflammatory cytokine storm with lung immunopathological injury. inflammation related to the cytokine storm in the lungs may then spread throughout the body via the circulation system. covid- patients have been reported to have increased plasma concentration of inflammation-related cytokines, including interleukin (il)- , , , , tumor necrosis factor-α (tnf-α), and monocyte chemoattractant protein i (mcp-i) especially in moribund patients. several limitations of this study exist. publication bias and study heterogeneity are unavoidable in this type of study. therefore, it should be considered when interpreting the outcomes of the reports and our final data set. furthermore, this study likely overestimates disease severity due to a lack of screening for asymptomatic or mildly symptomatic individuals and subsequent publication bias related to these factors. likely, many infected persons have not been detected, thus falsely elevating the rates of hospitalization, critical condition, and mortality. the lower quality analysis and reporting in some of the included publications is another limitation of the study. to prevent language bias, we included reports in languages other than english. additionally, we searched for a variety of sites and databases to prevent internet platform bias. using egger's regression test, we did not find significant publication bias. journal bias is an issue facing those who carry out a meta-analysis, yet it does not usually affect the general conclusions. however, we cannot reject the occurrence of other biases in this study, such as choice bias, since several journals are not indexed in embase, scopus, pubmed, web of science, and the cochrane library and unpublished data from some regions of the world. fever and cough are the most common symptoms of covid- -, sars-, and mers-infected patients. the mortality rate in covid- confirmed cases was lower than sars-and mers-infected patients. clinical outcomes and findings may be biased by reporting only confirmed cases, and it should be considered when interpreting the data. a novel coronavirus outbreak of global health concern three emerging coronaviruses in two decades: the story of sars, mers, 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declares pandemic because of "alarming levels" of spread, severity, and inaction comparison of severe and non-severe covid- pneumonia: review and meta-analysis. medrxiv single-cell rna expression profiling of ace , the putative receptor of wuhan immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid- patients angiotensin-converting enzyme in acute respiratory distress syndrome empirical assessment of effect of publication bias on meta-analyses supporting information section at the end of this article. how to cite this article comparison of confirmed covid- with sars and mers cases -clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta-analysis none. the authors have declared that no conflict of interests. key: cord- -pqtem t authors: mcfee, r.b. title: middle east respiratory syndrome (mers) coronavirus date: - - journal: dis mon doi: . /j.disamonth. . sha: doc_id: cord_uid: pqtem t nan ) ( ) was isolated from a patient who died from severe pneumonia and multi-organ failure in saudi arabia ( , ) . this newly identified respiratory viral illness was caused by a novel coronavirus, which was initially designated as human betacoronavirus ( ) ( ) ( ) ( ) , but was eventually named middle east respiratory syndrome coronavirus (mers cov). reminiscent of, and worth considering as a caution for greater vigilance towards emerging pathogens, the suddenness that sars cov emerged as a new cause of severe pulmonary illness, has been replicated in this new aggressive respiratory illness mers cov. unlike sars cov, it has not caused the thousands of cases over a short period of time. but it also differs from sars cov in that it carries a much higher case fatality rate, and causes more severe illness ( , ) . unlike sars which seems to have gone quiescent, new mers cases have been reported well after the initial outbreak, including december ( b) . since september there have been cases reported in countries across continents, with most human cases occurring in saudi arabia (map and ) ( ) . while the greatest spike in cases occurred near , new cases to date continue to be reported. consider by the end of / the number of mers cases , with deaths, resulting in a case fatality rate of . % ( b, c) . earlier data from world health organization (who) as of / / there have been laboratory confirmed cases of mers cov since / , reported from countries (maps and ), resulting in deaths, yielding a significant case fatality rate (~ %) ( ); a fatality rate for a coronavirus that is significantly greater than that associated with sars cov delta (~ %), at least among confirmed cases ( ) ( ) ( ) . these data demonstrate mers continues to cause illness, though thankfully not at pandemic rates in the same manner as covid- . as with other cov, including sars cov and covid- , the exact epidemiology, including the magnitude of mild or asymptomatic illness, remains unknown. that said, among those presenting with mers, males over yrs of age seem to be at higher risk of severe disease symptoms and death among those infected. the risk associated with age seems consistent with covid- findings as well. abnormalities associated with mers cov include thrombocytopenia, lymphopenia, leukopenia, elevated serum lactate dehydrogenase (ldh), elevated aspartate aminotransferase (ast), elevated alanine aminotransferase (alt), along with abnormal renal function tests ( , , ) . superinfection from viral or bacterial causes has been found in some patients and may complicate the course of the disease. severe cases of mers may require intensive care and mechanical ventilation, with a relatively large number of patients progressing to respiratory or renal failure. a retrospective radiology review of ct findings in laboratory confirmed cases of mers cov noted that cough, fever, and dyspnea were the most common presenting symptoms. three of the seven subjects died ( ) . the high case fatality rate associated with mers cov has sparked interest in the role of virus on the immune system. one area of interest is the potential for cytokine storm, which is also found in covid- patients, and currently being studied extensively ( b) . some studies have shown mers cov is associated with an attenuated interferon (ifn) response, and does not induce inflammatory cytokines ( , , chest xrays should be obtained early in the course of progressive pulmonary illness ( figure ) ( ). radiographic findings may include unilateral or bilateral patchy densities or opacities, interstitial infiltrates, consolidation, and pleural effusions. rapid progression to acute respiratory failure, acute respiratory distress syndrome (ards), refractory hypoxemia, and extrapulmonary complications (acute kidney injury requiring renal replacement therapy, hypotension requiring vasopressors, hepatic inflammation, septic shock) has been reported. key to appropriate testing is a thorough history and physical. it is important to consider multisystem function as well as pulmonary status in patients with severe respiratory illness, including suspected mers cov, especially those returning from regions where aggressive pathogens are noted. involving infectious disease and pulmonologist specialists early on, as well as good critical care management are essential. as noted earlier, laboratory findings at admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels (ldh). renal and hepatic function tests, at least for baseline information are worth obtaining. in the ajlan study ( ) , all seven patients had lymphopenia. elevated creatinine developed after admission in all patients. ast also were elevated in all patients. of note, co-infection with other respiratory viruses and a few cases of co-infection with communityacquired bacteria at admission has been reported in mers cov patients. not surprisingly, nosocomial bacterial and fungal infections have also been reported in mechanically-ventilated patients. according to clinical experience reported to cdc, mers-cov virus can be detected with higher viral load and longer duration in the lower respiratory tract compared to the upper respiratory tract, and has been detected in feces, serum, and urine. however, very limited data are available on the duration of respiratory and extrapulmonary mers-cov shedding. as mentioned earlier, aggressive testing, isolation, and management are critical in treating highly pathogenic coronaviruses, especially mers, which among patients who present for medical care, has a high case fatality rate, extrapulmonary involvement in some cases, and rapid deterioration. this includes early radiographic studies ( b). according to the aljan radiology study ( ) , chest xray findings were most notable for airspace and interstitial opacities with mers cov. radiographic findings such as airspace opacities are nonspecific, whereas the interstitial changes were described as reticular or reticulonodular. total lung opacification and thickening of the bronchovascular markings have been reported as well. previous chest ct findings with mers have been reported as bilateral patchy or extensive opacities ( , b) . not unexpected, imaging that had features consistent with acute respiratory distress syndrome were typically identified in sicker patients, and can be found in covid- patients as well (table ). in aljan ( ) mers cov the most common findings on ct were shows that airspace opacities on ct are common in patients hospitalized with mers-cov infection. airspace opacities are suggestive of an organizing pneumonia pattern. it was also described in h n influenza a viral infections. in most of our patients, ground-glass opacities were more extensive than consolidation. septal thickening and pleural effusions also were demonstrated. importantly, tree-in-bud pattern, cavitation, and lymph node enlargement were not seen in our cohort. chest xrays and ct scanning ( figure ) are recommended depending upon the presentation, and clinical course. in the aljan study ( ) the most common ct finding in hospitalized patients with mers -cov infection reveals predominantly subpleural and basilar airspace changes, with more ground glass opacities than consolidation. it is important to note this is a small study. nevertheless this pattern is consistent with organizing pneumonia. patients recently returning from the middle east, presenting with significant respiratory illness, with ct findings of peribronchial region abnormalities, organizing pneumonia, should be considered for mers cov infection, and if possible, queried about international travel and occupational exposures. -year-old man with middle east respiratory syndrome patient was a smoker who was healthy otherwise. ct was performed days after admission, and days after onset of symptoms. patient was eventually discharged. lower lung ct image show large right lower lobe and small focal left lower lobe subpleural consolidations the cdc provides case definitions. the reader is requested to review periodically this site for updates as the mers cov situation can change ( , ) . although emergency testing can be possible for suspected asymptomatic cases, the cdc guidelines for sending samples for laboratory confirmation are predicated upon a combination of clinical and epidemiological factors to identify persons under investigation (pui) - table . the other two categories per cdc are: a confirmed case is a person with laboratory confirmation of mers-cov infection. confirmatory laboratory testing requires a positive pcr on at least two specific genomic targets or a single positive target with sequencing on a second. laboratory confirmation of mers cov can be obtained by real time polymerase chain reaction (rt pcr) a probable case is a pui with absent or inconclusive laboratory results for mers-cov infection who is a close contact of a laboratory-confirmed mers-cov case. examples of laboratory results that may be considered inconclusive include a positive test on a single pcr target, a positive test with an assay that has limited performance data available, or a negative test on an inadequate specimen. nb the reader is advised to check for updated testing procedures with the cdc ( ) . this is an updated version of the interim guidance document issued by the centers for disease control and prevention (cdc) january based on input from public health partners, healthcare providers, professional organizations, and others. cdc will continue to update the document as necessary to incorporate new information that increases our understanding of mers-cov. updates: minor changes were made to clarify specimen type and collection procedures. of note -respiratory specimens should be collected as soon as possible after symptoms beginideally within days. however, if more than a week has passed since symptom onset and the patient is still symptomatic, respiratory samples should still be collected, especially lower respiratory specimens since respiratory viruses can still be detected by rrt-pcr. for example -if symptom onset for a pui with respiratory symptoms was less than days ago, a single serum specimen, an np/op specimen and lower respiratory specimen should be collected for cdc mers rrt-pcr testing. . if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. . if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. for short periods (≤ hours), most specimens should be held at - °c rather than frozen. for delays exceeding hours, freeze specimens at - °c as soon as possible after collection (with exceptions noted below). label each specimen container with the patient's id number, specimen type and the date the sample was collected. broncheoalveolar lavage, tracheal aspirate, pleural fluid collect - ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at - °c up to hours; if exceeding hours, freeze at - °c and ship on dry ice. have the patient rinse the mouth with water and then expectorate deep cough sputum directly into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at - °c up to hours; if exceeding hours, freeze at - °c and ship on dry ice. nasopharyngeal swab and oropharyngeal swab (np/op swab) use only synthetic fiber swabs with plastic shafts. do not use calcium alginate swabs or swabs with wooden shafts, as they may contain substances that inactivate some viruses and inhibit pcr testing. place swabs immediately into sterile tubes containing - ml of viral transport media. np/op specimens can be combined, placing both swabs in the same vial. refrigerate specimen at - °c up to hours; if exceeding hours, freeze at - °c and ship on dry ice. nasopharyngeal swab -insert a swab into the nostril parallel to the palate. leave the swab in place for a few seconds to absorb secretions. swab both nasopharyngeal areas. oropharyngeal swab (e.g., throat swab) -swab the posterior pharynx, avoiding the tongue. nasopharyngeal wash/aspirate or nasal aspirate collect - ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at - °c up to hours; if exceeding hours, freeze at - °c and ship on dry ice. for serum antibody testing: because we do not want to delay detection of mers infection and since the prevalence of mers in the us is low, serologic testing on a single serum sample collected or more days after symptom onset may be beneficial. this is in contrast to serologic testing for many other respiratory pathogens which require collection and testing of acute and convalescent serum specimens. serologic testing is currently available at cdc upon request and approval. please be aware that the mers-cov serologic test is for research/surveillance purposes and not for diagnostic purposes -it is a tool developed in response to the mers-cov outbreak. contact cdc's emergency operations center (eoc) ( - - ) for consultation and approval if serologic testing is being considered . a single serum specimen collected optimally during the first - days after symptom onset is recommended. note: the kinetics of mers-cov are not well understood. once additional data become available, these recommendations will be updated as needed. the minimum amount of serum required for mers-cov testing (either serologic or rrt-pcr) is µl. if both mers-cov serology and rrt-pcr tests are planned, the minimum amount of serum required is µl ( µl for each test). serum separator tubes should be stored upright for at least minutes, and then centrifuged at - relative centrifugal force (rcf) for minutes before removing the serum and placing it in a separate sterile tube for shipping (such as a cryovial). refrigerate the serum specimen at - °c and ship on ice-pack; freezing and shipment of serum on dry ice is permissible. children and adults: collect tube ( - ml) of whole blood in a serum separator tube. a minimum of ml of whole blood is needed for testing pediatric patients. if possible, collect ml in a serum separator tube. specimens from suspected mers cases must be packaged, shipped, and transported according to the current edition of the international air transport association (iata) dangerous goods regulations. shipments from outside of the united states may require an importation permit that can be obtained from cdc. specimens should be stored and shipped at the temperatures indicated above. if samples are unable to be shipped within hours of collection, they should be stored at - °c and shipped on dry ice. when shipping frozen specimen from long distances or from international locations, it is best to use a combination of dry ice and frozen gel ice-packs. the gel ice-packs will remain frozen for a day or two after the dry ice has dissipated. all specimens must be pre-packed to prevent breakage and spillage. specimen containers should be sealed with parafilm® and placed in ziplock bags. place enough absorbent material to absorb the entire contents of the secondary container (containing primary container) and separate the primary containers (containing specimen) to prevent breakage. send specimens with cold packs or other refrigerant blocks that are self-contained, not actual wet ice. this prevents leaking and the appearance of a spill. when large numbers of specimens are being shipped, they should be organized in a sequential manner in boxes with separate compartments for each specimen. saturday delivery is planned, special arrangements must be made with the shipping company. there are no commercially available or fda approved therapeutics specifically designated as mers cov antivirals. as of there remain a variety of candidate therapeutics in development against covid specifically, highly pathogenic coronaviruses such as sars and mers, and coronaviruses in general, including those that cause "common cold-like" symptoms, but none have emerged as fully capable of treating these viruses with the same consistency as neuraminidase antivirals have against influenza. and even in that context there are some clinical considerations, and limitations. there continues to be ongoing research into repurposing antivirals that are approved for other clinical indications such as hiv, influenza, and other illnesses, as possible treatments -monotherapy or in combination, against covid- and other coronaviruses. brief updates are provided below. covid- has reenergized research into greater study of the pathogenicity of coronaviruses, and newly or better described characteristics of highly pathogenic coronaviruses has led to increased insights into additional antiviral and vaccine targets. significant research into a wide array of therapeutic approaches is undergoing at unprecedented levels internationally. as will be seen in the covid- therapeutics section of this article, multiple options are being clinically tested that warrant consideration for the clinician faced with treating covid- patients. the mainstay of therapy for mers cov remains supportive care, especially respiratory care, while managing circulatory, renal, hepatic and neurological function, as well as protecting against secondary infections. although attempted in both the sars cov and early mers cov outbreaks, immune based interventions -interferon -resulting in equivocal outcomes. non human primate studies using ifn a b and ribavirin against mers cov demonstrated improved outcomes but it is worth noting treatment was initiated soon after viral infection initiated; this is unlikely going to be the case with human infection, where a delay in presentation to health care, or delayed diagnosis will likely preclude the same rapidity of treatment that occurred in the study ( - , , ) . the role for interferon in mers cov remains unclear; clinical trials are needed to better characterize successful strategies. but the limited number of cases make such studies difficult. in addition to various interferon treatment protocols, ribavirin -a potent nucleoside analog -has been utilized against rna viruses with varying degrees of success ( , , , b, c) . unfortunately ribavirin presents a risk for adverse effects including hemolytic anemia. interferon is not without side effect risk either. the early use of corticosteroids in sars cov infected patients resulted in increased viral load, critical care/intensive care unit admission, and death ( , c). other approaches to cov include neutralizing antibodies from convalescent plasma or hyperimmune globulin; these may hold some promise in reducing the cfr ( c, [ ] [ ] [ ] . convalescent plasma was shown to decrease mortality in sars cov patients if provided to patients within days of illness ( ) . however in order for this to be effective, rapid diagnosis of patients, and survivors is required in order to obtain, and utilize these interventions. towards that end, a network for convalescent plasma is being assembled, allowing safety, effectiveness and logistic feasibility testing. ( ) . that said, to date no host derived interventions have been shown to consistently confer significant benefit to severely ill mers cov patients via controlled study. while to date there are no antivirals currently licensed for use against mers cov, some candidate drugs are in development. two categories of candidates showed early promise. an adenine analogue that can be incorporated into viral rna, which can disrupt virus replication, and has shown in vitro activity against mers cov, as well as benefit against ebola in non human primates (nhps). also there is a nucleoside analogue for treatment of filoviruses, cov, and other rna viruses. researchers are working on other molecular approaches to treat cov ( , , ) . ribavirin is a guanine, oral nucleoside analogue, which inhibits viral rna-dependent rna polymerase. in trials exploring the use of ribavirin against mers, it was often in combination with other therapeutics, including interferon. evidence revealed it did not confer significant clinical benefit on outcomes or viral clearance ( c, , ) . it has shown some in vitro activity against sars, where high concentrations/high doses were required to inhibit viral replication ( . g to . g po every hours), and combination therapy via intravenous or enteral administration ( b, c, ) . studies on the use of ribavirin as a treatment for sars were either inconclusive in terms of clinical benefit, or suggested possible harm referable to adverse events, which included hepatic and hematologic deleterious effects ( c, ). ribaviran as a potent nucleoside analogue has been, and continues to be used with varying degrees of success against rna viruses, but there is the potential for adverse side effects including hemolytic anemia, metabolic derangements. interferon can also elicit adverse effects, although they have demonstrated value against viral infection ( ) ( ) ( ) ( ) . another repurposing of antivirals involved lopinavir-ritonavir combination therapy trialed against mers. the medication is an oral protease inhibitor ( b, c). early success with treating sars in , using lopinavir/ritonavir and ribavirin was suggested ( b, ) . mortality and need for intensive respiratory support was noted ( b, ) . in an animal study involving marmosets, lopinavir-ritonavir or interferon beta - b reduced viral load and improved lung pathology ( b, ) . combination antiviral therapy for patients hospitalized with severe influenza was noted in some cases to confer greater results, for those with high viral loads at presentation ( b, , ) . it is worth noting that studies have shown both sars coronavirus and mers coronavirus their viral loads peak at ~ - days after symptoms begin compared to covid- coronavirus where viral loads seem to peak at the time of clinical presentation ( b, , ) . this adds another level of clinical challenge in terms of managing this new, highly pathogenic coronavirus, and underscores the importance of time sensitivity in the administration of potentially lifesaving medications. another promising approach includes monoclonal antibodies (mabs). mabs have already demonstrated benefit against certain cancers and autoimmune disorder management ( , ) . to date the only pathogen for which there is a licensed mab is respiratory syncytial virus (rsv). who recommends standard precautions with all patients, especially those displaying early signs of respiratory illness, as the diagnosis -whether common cold, or mers cov or rsv or pertusis or other pathogen related infection. also droplet precautions should be added to standard precautions when providing care to such patients, including those with acute respiratory illness. although eye protection is recommended for probable or confirmed cases of mers cov, and airborne precautions for aerosol generating procedures, this would seem prudent even in cases clearly not mers cov or sars cov. from an infection control perspective, among the known human coronaviruses they are capable of surviving on environmental surfaces for up to hours ( ) . the sars and mers experiences, as well as previous hcov, underscore the threat of transmissibility; coronaviruses may be transmitted from person-to-person by droplets, hand contamination, fomites, and small particle aerosols. avoiding camels, especially dromedary camels, farms, as well as not consuming raw milk, urine, or meat are also worth considering. camels infected with mers cov may not appear ill. if contact with animals, especially dromedary camels cannot be avoided, strict hand washing is recommended. personal protection, including face/eye if working with camels or other animals. patients who must travel to the region are encouraged to present to a health care facility especially if fever and respiratory symptoms develop. according to a who update, multiple cases from the united arab emirates, qatar, oman, kuwait, and bahrain have links to dromedary camels. these patients were thought to be infected via contact with infected dromedaries or close primary cases (uae). a good practice for anyone who works with potential contaminants -whether in the us or middle east farm -remove work clothes that may carry materials from farms and animal contact (poster ) ( ). to date few cases have occurred in europe or the us. however with the emergence of medical tourism -cosmetic and advanced surgeries -patients who reside in the middle east may bring with them diseases endemic to the region, including mers cov. the astute clinician will be aware of potential importation of illness. as of there are sporadic cases of mers-cov, which continues to cause illness since its emergence in , and with a mortality rate estimated at greater than %. mers still remains a potential outbreak threat, and is a dangerous disease causing pathogen. as with sars cov and other viral threats of public health concern, there is a great need for both prophylactic measuresvaccines, and more targeted therapies. there is significant research effort underway internationally to develop an effective vaccine. as with the sars cov, there are a variety of approaches. one attempt which shows early animal efficacy is by a vaccine candidate consisting of chimeric virus-like particles (vlp) expressing the receptor binding domain (rbd) of mers-cov. the researchers have fused canine parvovirus (cpv) vp structural protein gene with the rbd of mers-cov; it self-assembles into chimeric, spherical vlp (svlp). this svlp retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to cpv virions. immunization with svlp induced rbd-specific humoral and cellular immune responses in mice. svlp-specific antisera from these animals were able to prevent pseudotyped mers-cov entry into susceptible cells, with neutralizing antibody titers reaching : . of note, interferon (ifn) -ifn-γ, il- and il- secreting cells induced by the rbd were detected in the splenocytes of vaccinated mice by elispot. mice given svlp or an adjuvanted svlp vaccine elicited t-helper (th ) and t-helper (th ) cell-mediated immunity. although early stage, resarch svlp displaying the rbd of mers-cov may become, or lead to an effective vaccine against mers-cov. human research is needed ( ) . of note, several of these approaches will be applied to developing vaccines against covid- , which will be discussed in the next section ( c). -or -a member of a cluster of patients with severe acute respiratory illness (e.g., fever and pneumonia requiring hospitalization) of unknown etiology in which mers-cov is being evaluated, in consultation with state and local health departments in the us. and close contact with a confirmed mers case while the case was ill. fever may not be present in some patients, such as those who are very young, elderly, immunosuppressed, or taking certain medications. clinical judgement should be used to guide testing of patients in such situations countries considered in the arabian peninsula and neighboring include: bahrain; iraq; iran saudi arabia; syria; the united arab emirates meters), or within the room or care area, of a confirmed mers case for a prolonged period of time (such as caring for, living with, visiting, or sharing a healthcare waiting area or room with, a confirmed mers case) while not wearing recommended personal protective equipment or ppe (e.g., gowns, gloves, nioshcertified disposable n respirator, eye protection); or b) having direct contact with infectious secretions of a confirmed mers case (e.g., being coughed on) while not wearing recommended personal protective equipment. see cdc's interim infection prevention and control recommendations for hospitalized patients with mers for detailed information regarding healthcare personnel (hcp) please review cdc interim u.s. guidance for monitoring and movement of persons with potential middle east respiratory syndrome (mers-cov) exposure for more information: call -cdc-info ( - ) or visit www.cdc.gov/travel poster ( ) mers references . image of middle east respiratory syndrome (mers) coronavirus as isolated by the national institute of allergy and infectious diseases isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus in a patient transferred to the united kingdom from the middle east middle east respiratory syndrome coronavirus (mers -cov) announcement of the cornoavirus study group epidemiological, demographic, and clnical characteristics of cases of midle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study world health organization (who) mers update and global summary treatment strategies for middle east respiratory syndrome coronavirus radiology perspective of coronavirus disease (covid- ): lessons from severe acute respiratory syndrome and middle east respiratory syndrome ajr middle east respiratory syndrome coronavirus (mers-cov) differential cell line susceptibility to the emerging novel human betacoronavirus c emc/ : implications for disease pathogenesis and clinical manifestation world health organization middle east respiratory syndrome coronavirus emergency preparedness response survival of human coronaviruses e and oc in suspension after drying on surfaces: a possible source of hospital-acquired infections middle east respiratory syndrome coronavirus (mers cov): what lessons can we learn? assessment of the middle east respiratory syndrome coronavirus (mers -cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach estimation of mers -coronavirus reproductive number and case fatality rate for the spring saud arabia outbreak insights from publicly available data hadi mohamed rae. outbreak of middle east respiratory syndrome coronavirus in saudia arabia: a retrospective study middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia 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response by the novel middle easst respiratory syndrome coronavirus: implications for pathogenesis and treatment middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd middle east respiratory syndrome coronavirus (mers cov) infection family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middlle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers cov): evidence and speculation middle east respiratory syndrome coronavirus the pathogenesis and treatment of the 'cytokine storm' in covid- treatment with interferon-alpha b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha a or ifn beta in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study triple combination of interferon beta- b, lopinavirritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open label randomized, phase trial pharmacologic treatments for coronavirus disease (covid- ); a review centers for disease control (cdc) mers clinical laboratory testing recommendations the effectiveness of vonvalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis feasibility, safety, clinical and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol a conformation dependent neutralizing monoclonal antibody specifically targeting receptor binding domain in middle east respiratory syndrome coronavirus spike protein nucloeotide prodrug gs- is a broad spectrum filovirus inhibitor that provides complete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx in a non-human primate model of filovirus infecton clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and metaaanalysis ribavirin and interferon therapy for critically ill patients with mers: a multicenter observational study sars: systematic review of treatment effects modjarrad k treatment strategies for middle east respiratory syndrome coronavirus treatment with interferon-alpha b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha a or ifn beta in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment withlopinavir/ritonavir or interferon b b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset antiviral combinations for severe influenza efficacy of clarithromycin-naproxen-oseltamivir combination in the treatment of patients hospitalized for influenza a (h n ) infection: an open-label randomized, controlled, phase iib/iii trial temporal profiles of viral load inposterior oropharyngeal saliva sample and serum antibody responses during infection by sars cov- : an observational cohort study medical treatment of viral pneumonmia including sars in immunocompetent adult novel chimeric virus-like particles vaccine displaying mers-cov antiviral res key: cord- -sqxlnk e authors: park, jiyeon; yoo, seung yeon; ko, jae-hoon; lee, sangmin m.; chung, yoon joo; lee, jong-hwan; peck, kyong ran; min, jeong jin title: infection prevention measures for surgical procedures during a middle east respiratory syndrome outbreak in a tertiary care hospital in south korea date: - - journal: sci rep doi: . /s - - -x sha: doc_id: cord_uid: sqxlnk e in , we experienced the largest in-hospital middle east respiratory syndrome (mers) outbreak outside the arabian peninsula. we share the infection prevention measures for surgical procedures during the unexpected outbreak at our hospital. we reviewed all forms of related documents and collected information through interviews with healthcare workers of our hospital. after the onset of outbreak, a multidisciplinary team devised institutional mers-control guidelines. two standard operating rooms were converted to temporary negative-pressure rooms by physically decreasing the inflow air volume (− . pa in the main room and − . pa in the anteroom). healthcare workers were equipped with standard or enhanced personal protective equipment according to the mers-related patient’s profile and symptoms. six mers-related patients underwent emergency surgery, including four mers-exposed and two mers-confirmed patients. negative conversion of mers-cov polymerase chain reaction tests was noticed for mers-confirmed patients before surgery. mers-exposed patients were also tested twice preoperatively, all of which were negative. all operative procedures in mers-related patients were performed without specific adverse events or perioperative mers transmission. our experience with setting up a temporary negative-pressure operation room and our conservative approach for managing mers-related patients can be referred in cases of future unexpected mers outbreaks in non-endemic countries. high-volume healthcare facility, which is how the previous south korea outbreak occurred . moreover, there may be very few hospitals that are prepared to provide perioperative care for mers patients. therefore, herein, we share our experience of providing infection prevention and control measures for surgeries for mers-related patients in our hospital. during the mers outbreak in our hospital, six mers-related patients underwent surgery including three possibly exposed patients, one directly exposed patient, and two mers-confirmed patients who recovered from the disease. all patients were negative during two preoperative mers screenings using real-time reverse transcription polymerase chain reaction (rrt-pcr) . figure shows the total number of surgeries performed during the outbreak period at our hospital and the distribution of mers-related patients undergoing surgery. temporary set-up of a negative-pressure operating room. the operating rooms in our hospital were generally positive-pressure environments, and we had no permanent negative-pressure operating rooms. because a negative-pressure operating room is the optimal environment to prevent airborne virus spreading to adjacent areas , two of our operating rooms in the main operating suite of the hospital were temporarily converted into negative-pressure operating rooms to perform surgical procedures on mers-related patients. operating rooms no. and were selected because they were connected to each other, but each room had separate atmospheric air inlets and exhaust systems. they also had separate air-conditioning and humidification systems. of the two connected rooms, one was used as the main operating room and the other was used as the anteroom where healthcare workers (hcws) applied and removed the personal protective equipment (ppe). in each room, fresh air was supplied from an inlet duct and discharged outside through the exhaust duct (fig. ) . because a constant exhausting air volume was maintained through the outlet duct, negative pressure in the operating room was achieved by decreasing the inflow air volume that entered through the inlet duct. first, the blades of the air volume control damper in the inlet duct were closed as much as possible (fig. ) . however, because the damper was not intended to be air-tight, the inflow volume to the operating room did not decrease sufficiently. second, as an additional measure to decrease the inflow volume, we opened the access hole in the inlet duct, which was originally used for duct inspection purposes (fig. ) . finally, a smoke test was carried out to ensure negative pressure. the room pressure was maintained at − . pa in the main operating room and at − . pa in the anteroom (fig. ) ; − . pa is below the negative pressure room standard of − . pa . www.nature.com/scientificreports www.nature.com/scientificreports/ airflow in both rooms reached - air exchanges per hour, according to airflow velocity measurements with an anemometer (ebt balometer; tsi alnor ® , minnesota, united states). in this environment, removing airborne contaminants requires minutes for % efficiency and minutes for . % efficiency . therefore, minutes of room ventilation was required after aerosol forming high-risk procedures, such as endotracheal intubation or extubation , . the cleanliness level of each room was also measured using a particle counter (tsi ; tsi, united states): main operating room = , and the anteroom = , , which were much lower than the institutional target level of < , for general surgery (fig. ) . cleanliness level was defined as the number of particles smaller than . µm in . m . equipment preparation and disinfection. all built-in instruments such as computers, telephones, and ventilators were covered with plastic paper. sufficient amounts of drugs, fluids, and other equipment were prepared in the operating room before surgery, and other unnecessary equipment was moved out. additionally, we used disposable equipment, when possible. high efficiency particulate air (hepa) filters were installed in the breathing circuits, both on the inspiratory and expiratory limbs of the ventilators and at the patient's site that connected to endotracheal tube. after operations with mers-exposed patients, minutes of room ventilation was followed by surface disinfection with diluted chlorine bleach ( ppm) , . cleaners wore standard ppe while disinfecting surfaces. for mers-confirmed patients, surface disinfection was performed twice. institutional guidelines for perioperative management of mers-related patients. during the mers outbreak, we set the following principles for perioperative management of mers-related patients: all elective surgeries for mers-confirmed patients were postponed to reduce the risk of potential in-hospital transmission. for mers-exposed patients, surgical procedures were delayed until after the potential incubation period of days . when a mers-related patient required an urgent or emergency operation, mers-cov pcr tests were performed twice with distinct specimens preoperatively, to account for asymptomatic mers patients or delayed positive conversion in symptomatic mers-exposed patients. for patients with ambiguous pcr results or without a pcr test, operations were performed according to the management guidelines for mers-confirmed patients. all the surgical procedures for mers-related patients were performed in the last order of the day as possible. perioperative protection level for hcws. when an operation for a mers-related patient was scheduled, the division of infectious diseases and infection control department confirmed the protection level of the hcws, according to institutional guidelines (table ). in principle, standard ppe was applied to hcws who cared for asymptomatic mers-exposed patients. standard ppe includes surgical gloves, surgical gowns, eye shields, and n respirators. while managing mers-confirmed or mers-exposed patients with mers-associated symptoms including fever, myalgia, respiratory symptoms, or diarrhea, hcws implemented enhanced ppe, which included coverall clothes with head cover, shoe covers, goggles, two pairs of surgical gloves, and powered air purifying respirator (papr) or n respirators. although we performed preoperative mers-cov pcr screening, enhanced ppe was still recommended when managing symptomatic mers-exposed patients regardless of their pcr results. anesthesiologists were recommended to apply enhanced ppe (including papr from the middle of the outbreak) when managing all mers-related patients because they were most directly exposed to the aerosol-producing high-risk procedures, such as endotracheal intubation and extubation. only minimal numbers of hcws were present in the operating room. institutional education regarding the precise use of ppe was provided to the all associated hcws and they were assisted by skilled nurses in the operating room during the ppe donning and doffing processes. patient transfer for operation. mers-related patients were transferred directly to the negative-pressure main operating room through an exclusive path and elevator by a physician wearing proper ppe. the walls and the floor of the passageways and the elevator were covered with plastic paper. mers-related patients wore a www.nature.com/scientificreports www.nature.com/scientificreports/ surgical mask during transfer. because only anesthesiologists wore enhanced ppe when in proximity to asymptomatic mers-exposed patients, minutes of room ventilation was performed after anesthetic induction, including endotracheal intubation. the surgical team then entered the main operating room through the anteroom. in the cases of symptomatic mers-exposed patients or mers-confirmed cases, all hcws wore enhanced ppe and the -minute ventilation time was not required. after completion of operation procedures, patients who were moved to the general ward recovered in the main operating room without going through the post-anesthesia care unit. thirty minutes of room ventilation was performed after tracheal extubation. a physician in the main operating room sent the patient into the corridor, while the other physician outside the main operating room wearing ppe took over and transferred the patient to the general ward directly through the exclusive pathway (fig. ) . patients moving to the intensive care unit (icu) were transferred while remaining intubated. before transfers, we injected patients with a sufficient amount of intravenous muscle relaxant and sedative drugs to prevent coughing or movement and we applied a portable ventilator or bag-valve mask with a hepa filter to the patient. operations for six mers-related patients. the details of the six mers-related patients undergoing surgery are presented in table . two patients had operations during phase and four patients during phase of the outbreak (fig. ) . the negative-pressure operating room was set up to be used from phase . regarding ppe levels for the hcws attending these six patients, standard ppe was applied during management of patient a (asymptomatic mers-exposed patient), while anesthesiologists wore enhanced ppe for high-risk procedures (tracheal intubation). enhanced ppe was applied to hcws for patient b because the patient was symptomatic and still within the two-week incubation period, even though both pcr results were negative. enhanced ppe, including papr to reduce risk of mers-transmission, was applied for patient c who underwent surgery in the middle of the outbreak (phase ). papr provides more perfect sealing and protection of the head surface. patients d and e had documented mers-cov infection and their recovery was confirmed with symptom resolution and two negative mers-cov pcr tests. however, enhanced ppe with papr was applied to hcws because the infection risk could not be eliminated during exposure to a large amount of body fluid, especially during cesarean section (patient d). after spinal anesthesia, she recovered in the main operating room and was transferred directly to the general ward. patient f had a history of exposure to a mers patient in the emergency room and was isolated due to a fever. enhanced ppe was applied to the hcws for patient f and she underwent surgery with only one set of negative pcr results because of her emergency condition. mers, as well as sars, is associated with coronaviruses, both of which have high affinity for the lower repiratory tract and easily produce severe pneumonia [ ] [ ] [ ] [ ] [ ] . although mers has lower human-to-human transmission potential and has resulted in fewer large outbreaks than sars, there may be occasional amplification of clusters in healthcare settings [ ] [ ] [ ] . moreover, mers case fatalities are reported to be much higher than sars ( - % for mers and - % for sars) , , , , . unlike sars, ongoing small and large mers outbreaks in the arabian peninsula foster potential future mers outbreaks in non-endemic countries. however, there is likely to be a very limited number of hospitals that are prepared with negative-pressure operating rooms, except for a few hospitals in hong kong that experienced the sars outbreak . almost all hospitals generally have positive-pressure operating rooms and they may experience an outbreak without facilities that are prepared for perioperative management of mers patients, as our hospital did in . one of the highlights of our experience during the outbreak was the temporary set-up of a negative-pressure operation room with an adequate pressure gradient (≥ . pa) by modifying two connected operating rooms according to us centers for disease control and prevention (cdc) guidelines . continuous negative pressure was maintained in the main operating room (− . pa) and the anteroom (− . pa). this temporary setting was possible because the two adjacent rooms had separate atmospheric air inlets and exhaust systems. although we could not measure the airflow pattern or dispersion of infectious particles directly , the cleanliness levels in both operating rooms were , particles, well below the institutional target cleanliness for general surgery (< , particles). although the precise route of mers-virus transmission is currently not clearly understood , mers, as well as sars, is known to spread by direct contact with infectious material, such as large respiratory droplets, and also by airborne routes , . touching contaminated objects may also be a source of transmission; this is different from tuberculosis, which is transmitted by airborne routes , . therefore, when performing procedures that generate aerosols, such as endotracheal intubation, in patients with mers or sars, hcws must wear enhanced ppe, including gloves, a gown, either a face-shield that fully covers the front and sides of the face or goggles, and respiratory protection at least as protective as an n filtering face piece respirator , , . when removing ppe, care should also be taken not to contact contaminated materials. considering potential aerosol generation in operating rooms and the transmission risk of mers-cov while changing ppe, the temporary modification of an operating room to a negative-pressure room with an anteroom should provide suitable protection for hcws participating in operations on mers-related patients . a second highlight of our experience is the highly conservative application of ppe to hcws. at the time of the outbreak, there were no specific guidelines for perioperative management . therefore, we used a conservative approach based on our experience and previous reports. first, although the previous guidelines recommended that asymptomatic mers-exposed patients be managed as general patients undergoing surgery, we applied standard ppe to hcws and we performed mers-cov pcr screening twice. although mers progressed gradually after symptom onset , we could not exclude the possibility that asymptomatic mers-exposed patients had the potential to develop symptomatic disease perioperatively. moreover, we observed development of mers after the known incubation period of days in an immunocompromised host , ; thus, the possibility of exceptional cases could be considered. furthermore, a certain proportion of asymptomatic mers-exposed patients could actually be asymptomatic mers-infected patients. approximately % of laboratory confirmed mers patients have been classified as asymptomatic or having nonspecific mild symptoms at the time of testing . the potential for transmission from asymptomatic mers-cov pcr-positive person is currently unknown, but there are reports about prolonged viral rna detection in the upper respiratory tract in asymptomatic pcr-positive person . considering these points, it would be reasonable to prepare more conservatively than the existing guidelines call for. www.nature.com/scientificreports www.nature.com/scientificreports/ another point on which our preparations differed from the guidelines was the application of enhanced ppe, which emphasizes full protection of the body surface with a hooded coverall. during the outbreak in our hospital, mers transmission events occurred among hcws who were equipped with standard ppe, including n masks. transmission may have occurred after possible contamination of uncovered head or face surfaces . therefore, if a patient with a mers contact history had mers-associated symptoms, applying enhanced ppe (either a n respirator or papr) during surgery would be appropriate for hcws because numerous droplets and aerosols may be produced during airway interventions. because the n may fit inadequately if worn for a long time or after movement during surgery, wearing papr will be more beneficial. however, unlike hcws dealing with ebola virus, impermeable and fluid-resistant gowns are not required because body fluids are not infectious as with ebola virus diseases , , , . our experience was limited in that, as a mers outbreak outside the endemic country, we did not have an opportunity to perform surgical procedures in actively virus-shedding mers-infected patients. additionally, our infection-prevention protocols would be too conservative to apply in mers-endemic situations. however, considering the potential risk of infected hcws, preventing mers transmission is extremely important in the management of a mers outbreak. importantly, our experience can be generalized to other non-endemic countries for managing potential outbreaks of emerging respiratory diseases. in the era of globalization, a mers outbreak can occur in any country outside the middle east. a very limited number of hospitals are equipped with negative-pressure operating rooms, and therefore, most hospitals are likely to experience a mers or other outbreak in an unprepared circumstance. we hope that this report will help other hospitals in preparing for future mers outbreaks and infection control in unexpected conditions. this study was based on all available data at the samsung medical center from the mers outbreak and on interviews with hcws associated with the outbreak. the study was approved by samsung medical center institutional review board. the documents for review included electronic medical records of mers-related patients who underwent operative procedures and institutional guidelines for perioperative management of mers-related surgical patients. the mers guidelines were prepared through multidisciplinary team discussions that were held by our hospital's infection control department during and after the mers outbreak. the records about the temporary set-up of a negative-pressure operating room were also reviewed. we also collected data through interviews with hcws who participated in surgery and anesthesia for mers-related patients. we defined mers-related patients as those who were possibly or directly exposed to mers or who had a previously confirmed mers diagnosis . in brief, patients who had a potential but unconfirmed close contact history with a mers patient were defined as possibly exposed patients, and they were allowed to continue their normal activities until mers-like symptoms developed. directly exposed patients included those who had close contact with a known mers patient and who did not wear proper ppe; these patients were isolated in their homes or in private negative-pressure rooms at our hospital. because the number of mers-infected patients continuously increased at our hospital, we partially closed the hospital on june , at which point outpatient-care clinics were closed and the emergency department was only available for life-threatening emergencies. all elective surgeries were postponed if possible . we defined the early phase of the outbreak (before june ) as phase and the middle phase of the outbreak (from june ) as phase . for mers-cov pcr tests, either sputum or nasopharyngeal swab samples were collected and sputum samples were preferred if available . sputum was collected directly into a sterile, leak-proof, screw-capped sputum collection sterile container and nasopharyngeal swab was collected with an eswab ( c, copan diagnostics inc., murrieta, ca, usa). clinical samples were screened by rrt-pcr testing with amplification targeting the upstream e region (upe) and confirmed by subsequent amplification of the open reading frame (orf) a using powerchek ™ mers real-time pcr kits (kogene biotech, seoul, korea). all rrt-pcr reactions were performed using the fast real-time pcr system (applied biosystems, foster city, ca, usa). the pcr reaction was performed in a total volume of μl ( μl pcr reaction mixture and μl template rna). thermocycling conditions included a step at °c for min, followed by °c for min and then cycles of s at °c and s at °c. positive viral template control and no-template control were included in each run. the glyceraldehyde- -phosphate dehydrogenase (gapdh) gene was amplified simultaneously as a heterologous internal control to monitor pcr inhibition. a positive test result was defined as a well-defined exponential fluorescence curve that crossed the cycle threshold (ct) < cycles for both upe and orf a. a sample was considered "equivocal" if the upe result was positive but the ct value for orf a was > and < . we interpreted the result as "indeterminate" if ( ) the upe result was positive but the ct value for orf a was undetected or if ( ) the ct value for upe was > and < . institutional review board statement. the study was performed in accordance with the declaration of helsinki and experimental protocols were revised and approved by irb at samsung medical center. 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hospital-associated outbreak surgical protocol for possible or confirmed ebola cases anesthetic implications of ebola patient management: a review of the literature and policies j.p. this author helped with data collection, data analysis, writing of the first draft and revision of the manuscript, and archiving of the study files. s.y.y. this author helped with data analysis, discussion of results, writing and revision of the manuscript, archiving of the study files and approval of final version. j. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.r.p. or j.j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -o i hy authors: holliday, ian; tam, wai-keung title: e-health in the east asian tigers date: - - journal: int j med inform doi: . /j.ijmedinf. . . sha: doc_id: cord_uid: o i hy objective: the article analyzes e-health progress in east asia's leading tiger economies: japan, hong kong, singapore, south korea and taiwan. it describes five main dimensions of e-health provision in the tigers: policymaking, regulation, provision, funding and physician-patient relations. methods: we conducted a series of fieldwork interviews and analyzed key healthcare websites. results and conclusion: our main finding is that the development of e-health in the region is less advanced than might be expected. our explanation focuses on institutional, cultural and financial factors. the application of information technology (it) to public sector operations sometimes captured in the notion of e-government is starting to have an impact on developed healthcare systems the world over. as time goes by, that impact is expected to become even more pronounced. ''the consensus seems to be that new information technologies will significantly affect almost every aspect of health care,'' wrote blumenthal [ ] . in this article, we examine the progress of e-health in the five leading economies of east asia: japan, hong kong, singapore, south korea and taiwan. each seeks to place itself at the forefront of the information revolution and has high levels of internet access and usage. each also has a sophisticated healthcare system dedicated to securing maximum healthcare benefit at minimal cost. by standard outcome indicators, these systems all have very good records. the tigers, therefore, form a cluster in which e-health might be expected to be notably advanced. however, our finding is that although some progress is being made, it remains limited. it is also variable across the five societies. the article begins by reviewing some of the literature on e-health taking from it a series of critical dimensions and issues. it then briefly analyzes the two relevant contextual aspects of the east asian tigers: their participation in the information age and the nature of their healthcare systems. on these bases, it examines their e-health progress, focusing on the major themes unearthed in the contemporary literature. finding limitations and variations, it concludes by thinking through possible explanatory factors, focusing on institutional, cultural and financial issues. much of the existing e-health literature has been developed in the context of the united states, reflecting both us leadership in the information age and the continuing search for solutions to us healthcare problems. five main themes are prominent. four of the five address distinct dimensions of the broad policy and management framework for healthcare, examining internet impacts on policymaking, regulation, provision and funding. the fifth theme looks inside the healthcare sector and inside the surgery, at the implications of the internet for physician-patient relations. eventually, this may have policy significance, but for now, it is best treated separately. the major argument made about healthcare policymaking is that the us government has been slow to engage with the numerous issues generated by the it revolution [ ] . the core features of that revolution, notably enhanced information flows, increased networking possibilities and novel commercial opportunities, are now well documented [ ] . however, it is said that in us healthcare, most policy actors in both congress and the executive branch continue to focus on pre-information age agendas. although, the bush administration has started to address these concerns, the result remains something of a lack of internet-related policy activity and only a limited number of perspectives on the internet's potential to transform the us healthcare system. clearly structural features of the us system, including fragmentation both of government and of the healthcare sector, play key roles. looking at the narrower sphere of regulation, concerns are expressed about the failure of regulatory agencies to keep pace with internet-related developments. goldsmith notes that the internet generates many potential regulatory problems, ranging from licensing e-health practitioners to monitoring information quality in a virtual world with no boundaries [ ] . fried et al. detail some of the obstacles placed in the way of e-health by existing regulations, holding that individuals and organizations must navigate a maze of rules and codes, old and new, if they wish to implement fresh ideas and approaches [ ] . kassirer's prediction is that the courts will play a role when substandard medical advice provided through web sites or e-mail yields poor medical outcomes. he believes that courts will be especially important when professional advice is given without a direct patient encounter, or when state lines are crossed [ ] . some regulatory issues are us-specific, but many have much wider relevance. partly building on the regulatory theme, analysts have also debated the limitations currently imposed on healthcare provision through the internet. kleinke argues that the internet will not contribute to a solution to the administrative redundancies, economic inefficiencies, and quality problems that have long plagued the us healthcare system. instead, it will exacerbate the cost and utilization problems of a system in which patients demand more, physicians are legally and economically motivated to supply more, and public and private purchasers are expected to pay the bills [ ] . goldsmith holds that the challenges of standardizing coding and formats for clinical information, and protecting patient privacy, will hinder the realization of network computing potentials in healthcare [ ] . the problems to which these and other authors point are structural. economic, organizational, legal, regulatory, and cultural conflicts rooted in the us healthcare system are all barriers to e-healthcare provision. further problems are found in the sphere of healthcare funding. shortliffe criticizes congress for focusing on short-term benefits, arguing that research investment for e-health must be balanced between basic and applied analyses [ ] . robinson examines the effect of distinct forms of capital on the development of the healthcare internet. in the late s, venture capital flooded into the ehealth sector, rising dramatically from us$ million in early to us$ million in late . in the same period, e-health firms went public, raising us$ . billion at their initial public offerings. however, the technology-sector crash in late hit the e-health sector especially hard, prompting an extended period of consolidation between e-health and more conventional firms [ ] . us funding problems thus relate to both the public and the private sectors. finally, analysts have looked inside the surgery at physician-patient relations. existing survey data show that citizens make considerable use of the internet for healthcare information and services, mostly of a generic kind [ ] . indeed, anderson reports that in , % of us adults with internet access did so [ ] . as more patients go online, increasing numbers will turn up in surgeries with internet-fueled questions and concerns. meeting the growing expectations of these individuals will be a significant challenge for physicians [ ] . assessing the likely impact, kassirer argues that the internet will change the physician-patient relationship in unpredictable ways, with some aspects of electronic communication strengthening the bond, and others undermining it [ ] . goldsmith believes patients have most to gain from the emergence of the internet, arguing that it will rebalance the steeply asymmetrical medical knowledge held by patients and physicians [ ] . using information gained through internet searches, patients can now open their dialogues with physicians at a much higher level than before, and thereby gain leverage in the care process. ball and lillis also discuss the potential challenges the internet presents to physicians. as internet searches often generate as many questions as answers, physicians are likely to find themselves under increased workload pressures [ ] . the variable quality of healthcare information accessed through online searches [ ] , a matter that is being actively addressed by bodies such as the health on the net foundation (www.hon.ch) and the internet healthcare coalition [ ] , can only reinforce those pressures. zupko and toth hold that physicians sometimes encounter a form of cultural shock when confronted by well-informed patients [ ] . it is therefore perhaps not surprising that an april survey found that physicians are much more reluctant than patients to use the internet for healthcare interactions. while % of patients wanted to exchange e-mail with their doctors, only about % of doctors actually did so. physician-patient confidentiality, time concerns and increased exposure to malpractice liability were cited as primary reasons for doctors' wariness [ ] . in the face of this mounting speculation and evidence, lumpkin is sanguine, however, contending that the physician-patient encounter is little changed, despite widespread internet usage in healthcare [ ] . though focused on the us, the existing e-health literature generates key themes for an analysis of progress in other parts of the world, including the east asian tigers. however, before exploring those themes, we first present some basic contextual information about our five societies. two features of the east asian tigers are particularly relevant to this analysis: their participation in the information age and the nature of the healthcare systems in which their application of it needs to be assessed. in this section, we examine both features. looking at broad social participation, it was exploited more rapidly in the five east asian tigers than in any other global cluster. for many years, the nielsen//netratings global internet index has ranked all five societies in the top worldwide for personal computer (pc) connections and internet access and usage. the four smaller societies, hong kong, singapore, south korea and taiwan, are particularly advanced. furthermore, the severe acute respiratory syndrome (sars) crisis that hit the region in spring gave a major boost both to internet usage in general, and to e-health in particular [ ] . in hong kong, for instance, the number of active internet users increased by % from february to april , before falling back by % from april to june . the overall increase was %. also at the start of , the time spent online by hong kong people first increased by % and then fell back by %, registering an overall increase of % [ ] . consistent with the image of economic and social dynamism, they have projected for many years now, the east asian tigers are among the most advanced it societies on earth. to some extent, this strong it orientation is the product of developmental state strategies. with the partial exception of hong kong, the east asian tigers have long placed considerable faith in stateled growth strategies. furthermore, for many years they have frequently focused those strategies on it and it-related sectors. in japan, in the s, the fabled ministry of international trade and industry targeted supercomputers and the fifth generation as a major development project [ ] . despite a long period of economic stagnation in the s, the japanese it industry remains a significant global force. in singapore in the s, the state took the lead in nurturing wafer fabrication, the most sophisticated ''front end'' of the semiconductor industry. chartered semiconductor manufacturing, established by the government in , is now the third largest silicon foundry in the world. in south korea, in the early s, the state reorganized the public-sector telecommunications system by closing inefficient firms and allocating profitable segments to major chaebol like samsung and goldstar, enabling them to establish specialized chip businesses. by the early s, samsung had become the world's number one producer of dynamic random access memories for pcs and workstations. in taiwan since the s, the ministry of economic affairs and the state-controlled electronics research service organization have played crucial roles in developing the semiconductor industry. today, it is the fourth largest in the world, and firms within it have entered into strategic alliances with leading industry players in the west [ ] . even in hong kong, where a developmental state took longer to emerge, the government is currently overseeing the construction of a flagship cyberport, intended to host a strategic cluster of companies and professional talents, specializing in it applications, information services and multimedia content creation, and designed to project a hi-tech international digital city image. the east asian tigers are also leading players in the development of e-government. the un/aspa benchmarking survey of all un member states placed singapore at number (a long way behind the us, but only fractionally behind australia and new zealand), south korea at number and japan at number . all three states featured in the top category of high e-government capacity. the survey did not assess hong kong and taiwan, neither of which is a un member state. the report noted that singapore ''demonstrated a balanced and citizen-centric e-government program, while possessing the benefits of a high technological infrastructure and human capital measures''. it held that south korea ''made perhaps the most dramatic advances in its e-government program by successfully implementing several new online transaction features''. it was more critical of japan, arguing that it had ''yet to live up to its rather significant potential''. ''japan's e-government program has not yet reached a comparable level of sophistication as that of the regional leaders due primarily to achieving only a limited interactive presence among national government websites'' [ ] . a january analysis of e-government in east and southeast asia reached similar conclusions, identifying the five tigers as regional leaders [ ] . accenture's survey looked at only two of the five east asian jurisdictions analyzed here. it ranked singapore number in the world after canada, and japan number [ ] . looked at from many different perspectives, then, the east asian tigers are leading participants in the emergent information age. healthcare systems in the tigers share a basic orientation, but are otherwise quite varied. the orientation is best termed productivist, in that in each society social policy has usually been subordinate to economic objectives. while the governments of all five tigers certainly get involved in social policy, they usually do so either for economic reasons or after they have made provision for their various economic goals. the main stimuli to this strong focus on economic development were, in all cases, the devastation brought by the second world war, and the uncertainties of the international order constructed thereafter [ ] . this shared orientation has fed into healthcare policy in three main ways [ ] . in japan and its two former colonies, south korea and taiwan, healthcare was initially left chiefly to the market. only once economic policy was on track and a measure of growth had already been attained, did these societies turn their attention to planning their healthcare systems. in doing so, they concerned themselves chiefly with healthcare finance, creating social insurance systems by gradualist means. now, in all of these societies, the health insurance scheme is universal in aspiration and near universal in fact. across all three societies, healthcare provision remains privatesector-driven, with the state performing a chiefly regulatory role. traditional medicines are significant in all three societies and covered by national health insurance schemes [ ] . however, they are not consistently brought within the planning frame. in hong kong, until the early s, the colonial government took a strictly reactive and incremental approach to healthcare. its major interventions focused on subventing charitable organizations in the healthcare business, though in time, it also built hospitals and delivered care directly through them. throughout, government activity was funded out of general government revenue. the major and to date, only step change came in , with the formation of the hong kong hospital authority (hkha). this imposed state control and state funding on the secondary sector and gave hong kong a miniature version of the british national health service. however, there has never been any attempt to bring primary care within the planned healthcare system. only in , was traditional chinese medicine subjected to anything more than minimal government regulation [ ] . in singapore, the early post-war experience was similar to that of hong kong. here, however, separate initiatives were taken in the spheres of provision and funding. in , much provision was integrated at the secondary care level through creation of the state-run hospital corporation of singapore. this body subsequently sought to drive private-sector disciplines into state provision through ''corporatization''. in , in an attempt to generate integrated pathways of care, it was broken into two territorial clusters focused on the secondary sector but also having primary and tertiary elements. however, as most of the primary sector remained outside the state sector, the extent of integration was limited; in singapore, the state provides % of secondary care but only % of primary care. on the funding side, singapore in created a compulsory savings system, medisave, within the wider central provident fund scheme. it added the insurance schemes medishield and medishield plus in and and created a basic social safety net, medifund, in . these various schemes partially fund secondary care provision. there is also some direct state subsidy. funding of primary care takes place mainly through out-of-pocket expenses. the traditional sector stands outside all state planning and, as in hong kong, has only recently been brought within the regulatory framework. these healthcare systems have enviable records. not only did they make a rapid post-war transition from the contagious disease characteristic of thirdworld countries to the chronic disease characteristic of first-world societies, but also they register very favorable health outcomes as measured by standard input and outcome indicators (table ) . healthcare systems in the east asian tigers thus share a productivist orientation and strong performance. they exhibit varied state roles, with much healthcare activity lying outside the public sector and some of it falling beyond the planning horizon. in japan, south korea and taiwan, state involvement is extensive in finance but limited in provision. in hong kong, the government both funds and directly provides care in the secondary sector, but not elsewhere. in singapore, the state provides a large amount of secondary and some primary care. the funding regime is complex, comprising direct state subsidy, forced individual saving, state-run and private-sector insurance, and out-of-pocket expense. in all five tigers, both the public and private sectors play important roles and face clear incentives to take an interest in harnessing the internet for healthcare gain. note: taiwan data are from . sources: [ ] . against the dual backdrop of sophisticated it societies that make extensive use of the internet and cost-effective healthcare systems driven in variable ways by actors from the public and private sectors, we now turn to a survey of e-health in the east asian tigers. to frame the survey, we begin by providing a brief descriptive overview of the major state-run healthcare websites in the region. we then structure our analysis using the five main analytical spheres that dominate the existing e-health literature: policymaking, regulation, provision, funding and physician-patient relations. all ministries or departments of health in the east asian tigers have their own website. throughout the region, the major quasi-autonomous state agencies, such as the national health insurance agencies in japan, south korea and taiwan, the hkha in hong kong and the two big healthcare clusters in singapore, also have sites. here, we look only at the main government healthcare sites ( table ) . the overall quality is high. all have clickable links to organizational objectives and tasks. most also offer detailed information about subsidiary divisions. all contain links to the government homepage and related healthcare sites so that visitors can conduct further searches and collect additional information. all provide feedback channels. in singapore, the ministry of health (moh) offers online feedback opportunities. in taiwan, citizens can make online appointments with the director of the department of health (doh). in japan, the ministry of health, labour and welfare (mhlw) uses e-mail to solicit however, within this generally strong showing, there are also significant differences, with japan's mhlw and to a lesser extent, hong kong's hwfb lagging behind their regional counterparts in key respects. firstly, the mhlw fails to provide contact details for named officials on its website. this is standard practice in the other four tigers. singapore's moh, for example, gives address, telephone number and e-mail details for key officials. secondly, while healthcare professionals and officials in singapore, south korea and taiwan can communicate with each other through the internet, their counterparts in japan and hong kong cannot. thirdly, the range of options available to users is more restricted in japan and hong kong than in the other three tigers. in south korea, for instance, it has played a role in the surveillance system for communicable disease since . through electronic data interchange and regional database management systems, notifying and reporting systems have been computerized, and an electronic record of all notified and reported cases is kept. using the super-highway communication network, physicians and public health centers can access the notifying and reporting system, disweb, anywhere and anytime through the internet (http://dis.mohw.go.kr). in singapore, the moh site within the government's ecitizen portal enables healthcare professionals to download application forms for license renewal, approval to perform a pregnancy termination, and so on. the policymaking strand of the e-health literature castigates us policymakers for being slow to grasp the potential of the internet. such a charge is less easy to sustain in the east asian tigers, though again experience is variable. singapore and taiwan are the regional leaders. singapore's ecitizen portal addresses many aspects of citizen interaction with government, with healthcare being a prominent theme. the internet is used to reinforce the public health messages that have been disseminated by the singaporean government through other media for many years. behind the scenes, e-mail links pervade the healthcare system and enhance the cohesiveness of policy networks. in a controlled city state, those networks are in any case very tight. in taiwan, the doh in launched an ambitious e-health project, with a timeline stretching to . the health information network that is central to this initiative has a backbone funded by central government and permits local users in both the public and private sectors to participate on a self-paying basis. drawing on us experience, it seeks to promote electronic medical records, based on a smart card system, so that information can flow to all parts of the healthcare sector. a healthcare certification authority, created in , oversees promotion of this initiative. in the other three tigers, progress is less impressive. japan launched an e-japan strategy in january , designed to make it ''the world's most advanced it nation within years'' [ ] . the strategy had an explicit e-government strand. in september , the mhlw followed up by issuing a ''grand design'' for promotion of it in the healthcare sector. the aim was to computerize the entire sector by and to introduce an electronic medical record system covering % of clinics and % of hospitals with plus beds by . progress towards targets appears to be on track. however, japanese performance in the e-health domain is poor by regional standards. hong kong is also quite slow to place healthcare online. the hwfb site contains standard bureaucratic information, such as current policy initiatives and recent speeches, plus public health information that has been developed particularly since the sars crisis. here, the major networking initiative is being taken by the dominant public-sector delivery agency, the hkha. while its primary focus is provision, the networking links being created among hospitals are likely to have policy consequences. as in singapore, e-mail links also bolster ties within policy networks that are already quite cohesive. south korea is making an aggressive attempt to exploit the internet across all areas of government, but in the healthcare sphere, currently remains an average performer. turning to regulation, three main issues are raised in the literature. the first is that e-health generates a number of regulatory problems. the second is that excessive regulation may impede e-health progress. the third is that the courts are likely to have to step in when administrative regulation fails. in the east asian tigers, regulation is clearly a major concern and an evident constraint on ehealth development, often for good reason. one instance is limitations placed on consultations, which in all five tigers quite properly mandate face-toface physician-patient contact before any specific healthcare information or advice can be given. for the foreseeable future, online consultation, though technically feasible, is likely to be restricted by professional concerns. another instance is limitations placed on information sharing and exchange, which in all the tigers are again very properly re- stricted by privacy considerations. however, there is some variation in regional regulatory practice. in singapore, patients requiring repeat prescriptions can place an order online and have the medications delivered to their homes. only after months, do they have to return to the healthcare system to consult a physician. elsewhere, this practice is illegal. in japan, physicians are prohibited from answering specific questions about healthcare or disease by e-mail or telephone. regarding provision, assessments in the us literature are mainly negative. on the one hand, the argument is made that it cannot be expected to solve structural problems in healthcare systems. on the other, barriers even to less ambitious networking initiatives are held to be substantial. these are fair points, but they should not be allowed to obscure the real progress being made by healthcare systems around the world, and in our case in east asia. among the five tigers, taiwan's healthcare websites, both public and private, provide the most comprehensive services to patients. singapore ranks second, and hong kong third. japan and south korea are somewhat behind the regional pace. an overview is given in table . in taiwan, the doh operates a taiwan e-hospital site to provide free online medical advice to patients (http://taiwanedoctor.doh.gov.tw/). currently, medical practitioners and nutritionists from public hospitals form a consulting team to answer questions about specialties. patients seeking general medical advice can send questions to a particular practitioner and receive feedback online or by e-mail. in the private sector, a number of hospitals, such as the chang gung memorial hospital, have online question-and-answer services for patients. the kingnet second opinion webhospital (www.webhospital.org.tw) and the taiwan physician's net (www.doctor.com.tw) are two prominent sites providing free online medical advice to patients. established by kingnet entertainment (www.kingnet.com.tw) in , the webhospital has some voluntary physicians answering questions from the public. the taiwan physician's net brings together about physicians, whose information and advice are posted on the web. apart from getting online medical advice, patients can search for a particular physician and visit his or her office for treatment. in taiwan, patients can also make medical appointments online with many public and private hospitals. looking to the future, the doh is planning to develop a medical information exchange center to promote information sharing and enhance treatment quality. in singapore, health is one of a number of cluster points within the ecitizen site. to date, the internet is mainly used to provide general healthcare information, with the healthcare portal containing comprehensive information about healthcare providers, the healthcare establishment, healthy lifestyles and public health issues such as sars. many searches are possible. the site also allows individuals to submit complaints and feedback. only a few transactions can be undertaken online. as in taiwan, appointments can be made and altered online. through singapore's e-pharmacy services, recurrent prescription items can be ordered online and delivered throughout the island. in one of its two main healthcare clusters, patients can register online and access summary medical records. inside the healthcare system, information flows are starting to change as polyclinics and gps gain access to hospital records online. the likelihood is that enhanced integration of the public and private sectors will result. in hong kong, the hkha, which oversees almost the entire secondary sector, is currently introducing online networking in hospitals. its clinical management system is an integrated clinical workstation giving clinicians access to departmental information and patient records. it will soon develop into a longitudinal electronic patient record within the public hospital system, enabling records to be accessed by many parties simultaneously anywhere, anytime. the system will also actively support clinical decisions by offering alerts, reminders, links to medical knowledge and other aids. it is expected to play an important role in reducing medical errors and improving the quality of patient care. over the next years, the hkha is planning to create a hong kong health information infrastructure, with the aim of networking all healthcare providers in the public, private and social welfare sectors. it also intends to build an electronic medical record for every hong kong resident and provide citizens with an electronic gateway to healthcare information and evidence-based medicine [ ] . these initia-tives are likely to enhance information flows within the public healthcare system. compared with taiwan and singapore, however, hong kong lags behind in developing internet services for patients. individuals cannot register and access summary medical records online. lacking an e-pharmacy service, the hong kong system does not allow recurrent prescription items to be ordered online. japan and south korea are falling behind their regional counterparts in providing online health services to patients. their official health websites do not deliver any electronic service to individual patients. with the exception of initiatives taken by a small number of private hospitals in south korea, like the yonsei eye and ent hospital, neither public nor private hospitals in these two tigers allow patients to register online. however, in , japan's mhlw established telemedicine networks to provide specialized care to people in remote areas. the government will provide us$ million a year to form networks consisting of one large hospital and three clinics working together to supervise patients. each patient will be equipped at home with a computer that can monitor heart rate, blood pressure and other indicators, as well as a phone capable of transmitting video. they will be linked to physicians through an isdn digital phone connection, thus enabling physicians to diagnose illness by electronically transmitted data. from june , the mhlw started to establish such networks a year, so that all districts will have at least one by [ ] . in , south korea's semipublic seoul national university hospital founded ezhospital, which is business-oriented instead of patient-oriented. with three main business elements, education (content services), e-trading and system integration, ezhospital is starting to alter purchasing arrangements for both medical and non-medical supplies. as the south korean system is highly fragmented, the purchasing consortia that can be built through the internet could one day become significant. at present, however, e-purchasing is at an early stage of development. analyses of e-health funding focus on one main issue, the short-termism of us initiatives. in this domain, it is difficult to reach an overall assessment of the tigers' performance. on the one hand, their developmental state orientations make the general climate for it industrial emergence very different from the climate found in the us. in this regard, the tigers look to the long term in a systematic fashion that has no us equivalent. on the other hand, it is hard to find evidence that the tigers are investing heavily in e-health applications. moreover, because the private sector plays such a large regional role in healthcare, as it does in the us, many of the relevant initiatives fall outside the state sector and are hard to capture. there are undoubtedly many small commercial initiatives in east asia as, again, there are in the us. furthermore, like other commercial websites, private healthcare sites throughout the region rely heavily on advertising and sale of products for income. to take just two taiwanese instances, the kingnet webhospital and the taiwan physician's net offer online sales not only of healthrelated products, but also of cinema tickets. the very fragmented nature of private-sector healthcare operations throughout the tigers means that few summary assessments can be made. looking finally at physician-patient relations, the existing literature contains variable forecasts of unpredictable change, little change, and so on. however, there is a clear belief that patients have most to gain from e-health and physicians correspondingly have most to lose. in general, physicians in the tigers have tended to be wary of exploiting the internet for patient interactions. this partly reflects the tight regulatory climate in which many find themselves, with many modes of physician-patient contact outlawed. it may also reflect a certain reluctance on the part of both physicians and patients to engage in the informalities of online contact. until recently, then, the emergence of virtual physician-patient relations was highly limited. since the spring sars crisis, however, the pattern may have started to change. although it is too early to register the longterm impact of the crisis, it is clear that during the sars outbreak, many individuals sought to shift to online interactions with healthcare professionals. the fear of visiting surgeries and, in particular, hospitals that gripped the region in has certainly not disappeared and seems likely to provide a lasting stimulus to virtual delivery of healthcare. furthermore, the generic healthcare information found in great abundance on english-language websites is paralleled on regional websites operating in chinese, japanese and korean. there is also some official encouragement for patients to migrate to e-health. in may , hong-jen chang, ceo and president of taiwan's bureau of national health insurance, argued at an oecd forum that e-health could make a major contribution in informing patients. as evidence, he cited taiwanese experience in confronting hiv/aids and the role of the internet in educating patients about the disease. in the long run, he contended, patients equipped with information gained from online searches ''will translate into quality improvement and efficiency gains for the system'' [ ] . overall, east asian societies retain many traditional features, which generate some resistance to change in established modes of physician-patient contact. nevertheless, there are also factors operating in the opposite direction. one long-term impact of the sars crisis seems likely to be heightened caution about visiting healthcare facilities, for fear of contracting infectious disease, and a consequent boost for e-health. the east asian tigers form the most wired cluster of societies found anywhere in the world. moreover, they have long had a developmentalist orientation that has seen their states become involved in many aspects of economic and social development. in the sphere of e-health, however, their performance is strong at the level of basic web provision, but otherwise not particularly advanced. on the whole, their health ministries or departments have good sites covering all the fundamentals of online provision. outside central government agencies, they often have a wealth of additional sites in the public and private sectors. beyond that, they do not make pioneering use of the internet in healthcare. there are many possible reasons for this slightly disappointing performance, some of which apply to all of the tigers and others which are specific to a particular society. in japan, the structural problems that mired the economy in stagnation for more than a decade from the early s also form part of the explanation for its sluggish e-health performance. a notable feature of the japanese healthcare system is the considerable power of the japan medical association and its extensive links to the liberal democratic party that has governed the country for almost all of the post-war period. in hong kong, the sovereignty transfer was quite disruptive, and only several years on is the political system taking a settled shape on the developmental state model. looking beyond the specific circumstances of individual tigers, however, the major explanatory factors appear to be institutional, cultural and financial. institutionally, east asian healthcare systems tend to be highly fragmented, notably in japan, south korea and taiwan. in consequence, policymakers in healthcare ministries and departments have rather few levers that they can use to direct change. in the e-health sphere, they can quite easily construct official government websites, but generating reform in the wider healthcare system is more difficult and depends on their success in building consortia of interest among many private-sector actors. in part, they seek to do this by offering ring-fenced seed money for specified development projects. in part, they resort to exhortation, calling on all members of society to engage in the project of securing and maintaining regional and/or global leadership in the information age. in these many respects, the east asian tigers have a great deal in common with the us. in the additional domain of culture, they differ from the us. while capitalism is certainly a dynamic force in east asia as in north america, it also co-exists with still vibrant cultural underpinnings. the confucian heritage that characterizes all five east asian tigers has many complex strands. among them is considerable respect for authority, hierarchy, status and so on. in the medical sphere, one consequence is that doctors tend still to be accorded considerable professional status. this may make it difficult for full commercialization to take place and for the market drive that characterizes ehealth in the us to work its way through the system. finally, the financial dimensions of healthcare in the east asian tigers should not be overlooked. these are healthcare systems that deliver the excellent outcomes already mentioned at a fraction of the cost registered in the us and, indeed, in most developed societies. as a proportion of gdp, east asian tigers spend between and % on healthcare, with most coming in at around %. this is far below the us figure of - %, and also below the highincome country standard of almost %. one result of the tigers' success in holding down healthcare costs is that the incentive to experiment with new initiatives is reduced. clearly, there still are some incentives, but they are not as strong as in the us. e-health in the east asian tigers remains at an early stage of development. all have attained a good basic standard, but few are engaged in pathbreaking initiatives. alongside institutional factors that are similar to those found in the us, cultural and financial factors help to explain this rather unsatisfactory level of performance. doctors in a wired world: can professionalism survive connectivity? milbank q the internet promise, the policy reality the information age: economy, society and culture. vol. ii. the power of identity the information age: economy, society and culture. vol. i. the rise of the network society the information age: economy, society and culture. vol. iii. end of millennium how will the internet change our health system? ehealth: technologic revolution meets regulatory constraint patients, physicians and the internet com: the failed promise of the healthcare internet networking health: learning from others, taking the lead financing the healthcare internet rethinking communication in the e-health era consumers of e-health: patterns of use and barriers the impact of cyberhealthcare on the physician-patient relationship e-health: transforming the physician/patient relationship health care web sites: are they reliable? internet healthcare coalition, e-health quality partners named exclusive education and outreach affiliate of the internet healthcare coalition physicians get on line, aspen publishers doctor-patient e-mail slow to develop, international herald tribune nielsen//netratings, internet provided vital information and alternative access to shopping, banking and education for people in hong kong as sars took hold online shopping and banking sites soared in popularity as people in hong kong shunned the crowds sars stimulates ongoing growth in internet usage in hong kong divided sun: miti and the breakdown of japanese high-tech industrial policy tiger technology: the creation of a semiconductor industry in east asia united nations/american society for public administration building e-government in east and southeast asia: regional rhetoric and national inaction high performance, maximum value productivist welfare capitalism: social policy in east asia welfare capitalism in east asia: social policy in the tiger economies traditional medicines in modern societies: an exploration of integrationist options through east asian experience agenda-setting for the regulation of traditional chinese medicine in hong kong networking health: dawning of the e-health era. paper presented to apami-mic conference at the hong kong convention and exhibition centre e-health and the informed patient, paper presented to oecd forum taiwan council of economic planning and development, taiwan statistical data book world development indicators the work described in this article was substantially supported by a grant from the research grants council of the hong kong special administrative region, china [project no. cityu / h]. initial seed funding was provided by the governance in asia research centre, city university of hong kong.we are grateful for the research support we received. we thank academics, officials and practitioners in east asia for talking to us about e-health. the usual disclaimer applies. key: cord- -ewerquin authors: sauerhering, lucie; kupke, alexandra; meier, lars; dietzel, erik; hoppe, judith; gruber, achim d.; gattenloehner, stefan; witte, biruta; fink, ludger; hofmann, nina; zimmermann, tobias; goesmann, alexander; nist, andrea; stiewe, thorsten; becker, stephan; herold, susanne; peteranderl, christin title: cyclophilin inhibitors restrict middle east respiratory syndrome coronavirus via interferon λ in vitro and in mice date: - - journal: eur respir j doi: . / . - sha: doc_id: cord_uid: ewerquin rationale: while severe coronavirus infections, including middle east respiratory syndrome coronavirus (mers-cov) cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use. objectives: we elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin a (csa) and alisporivir (alv) restrict mers-cov to validate their suitability as readily-available therapy in mers-cov infection. methods: calu- cells and primary human alveolar epithelial cells (haec) were infected with mers-cov and treated with csa or alv or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nfat, or map kinases. novel csa-induced pathways were identified by rna sequencing and manipulated by gene knockdown or neutralising antibodies. viral replication was quantified by qrt-pcr and tcid( ). data were validated in a murine mers-cov infection model. results: csa and alv both reduced mers-cov titers and viral rna replication in calu- and haec improving epithelial integrity. while neither calcineurin nor nfat inhibition reduced mers-cov propagation, blockade of c-jun n-terminal kinase diminished infectious viral particle release but not rna accumulation. importantly, csa induced interferon regulatory factor (irf ), a pronounced type-iii-interferon (ifnλ) response and expression of antiviral genes. down-regulation of irf or ifnλ increased mers-cov propagation in presence of csa. importantly, oral application of csa reduced mers-cov replication in vivo, correlating with elevated lung ifnλ levels and improved outcome. conclusions: we provide evidence that cyclophilin inhibitors efficiently decrease mers-cov replication in vitro and in vivo via upregulation of inflammatory, antiviral cell responses, in particular ifnλ. csa might therefore represent a promising candidate to treat mers-cov infection. saudi arabia [ ] and led to recurring human infections with more than , laboratory-confirmed cases and high case fatality rates of about % [ ] . in ex vivo infection of human lung tissue, mers-cov targets bronchial and alveolar epithelial cells (aec) and leads to a detachment and apoptosis of aec [ ] . recent reports analyzing autopsy material of deceased mers-cov-infected patients showed mers-cov antigen in aec and epithelial multinucleated syncytial cell conglomerates in vivo [ , ] . accordingly, severe human infection presents as pneumonia with progression to acute respiratory distress syndrome [ , ] . to date, no vaccine or specific treatment for mers-cov -or the recently ongoing pandemic caused by the novel severe acute respiratory syndrome cov (sars-cov- ) -is approved and therapy relies on supportive measures only [ , ] . while in vitro studies and experiments in non-human primates demonstrated benefits of a combination of type-i-interferon and antiviral compounds including ribavirin against mers-cov [ ] [ ] [ ] , results from retrospective patient cohorts applying similar treatment regimens remain controversial [ ] [ ] [ ] . cyclosporin a (csa) has been found to inhibit several human-pathogenic cov in cell lines originating from kidney or liver epithelia [ ] [ ] [ ] [ ] . however, the molecular mechanisms by which csa affects cov, including mers-cov, particularly in its primary target cells, the pulmonary epithelium, remain elusive. moreover, preclinical studies addressing the effect of csa or related compounds on mers-cov replication in vivo have been lacking to date. csa is known to block the peptidyl-prolyl cis-trans isomerase (ppi) activity of cyclophilins that is involved in diverse cellular processes (e.g. protein folding, ). additionally, csa forms together with cyclophilin a (cypa) and calcineurin (cna) a ternary complex which blocks the cna-dependent activation of nfat (nuclear factor of activated t-cells), a process which accounts for the immunosuppressive effect of csa [ ] . in addition, csa has also been shown to inhibit the map kinases jnk (c-jun n-terminal kinase) and p [ , ] . here, we aimed to elucidate the distinct signaling pathways by which csa affects mers-cov in clinically relevant models such as primary human aec and a murine mers-cov infection model [ , ] . we demonstrate that csa blocks mers-cov infectious particle egress, which is dependent on jnk. moreover, we for the first time provide evidence that csa triggered the activation of an antiviral defense state in lung epithelial cells. we show that csa is a potent inducer of interferon regulatory factor (irf ), type-iii-ifn (ifnλ) and multiple interferon-stimulated genes (isgs). additionally, we demonstrate that oral application of csa induced a robust ifnλ response in vivo and, importantly, significantly reduced mers-cov replication and improved disease progression in infected mice. experiments with mers-cov were performed under biosafety level conditions at the institute of virology, philipps-university of marburg, germany. human alveolar epithelial cells (haec) were isolated and cultured as previously described [ ] . human lung tissue was obtained from patients who underwent lobectomy after informed written consent (departments of pathology and surgery, university of giessen, approved by the university of giessen ethics committee; az. / ). calu- or haec were infected at a multiplicity of infection (moi) of . diluted in dmem/f without fcs at °c for h. cells were washed with dmem/f with % fcs and supplemented with stimulatory/ inhibitory reagents as indicated. h after infection (pi) cells were processed for quantitative pcr (maxima-sybr/rox qpcr-mastermix, thermo fisher) and supernatant was harvested for virus titration as described previously [ ] . all animal experiments were performed in accordance with the german animal protection laws and were authorized by the regional authorities (g / ). c bl/ mice were purchased from charles river laboratories and housed under pathogenfree conditions. mice were intratracheally inoculated with adenovirus-hdpp -mcherry (cloned at viraquest inc.) as described [ , ] . five days post transduction, mice were infected intranasally with . x tcid /ml mers-cov (emc/ ). mg/kg/day csa diluted in dmso or dmso alone were mixed with a nut/chocolate-creme, and offered to the mice for voluntary uptake. uptake was controlled daily. csa feeding started days before mers-cov challenge. mice were sacrificed or days post mers-cov infection. all data are given as mean ± sem. statistical significance was analyzed by unpaired two-tailed student's t-test or by -way anova and post-hoc multicomparison tests as indicated in the respective figures. a p value of less than . was considered significant. *p < . ; **p < . ; ***p < . . further experimental details can be found in the online supplement. to address the previously proposed antiviral activity of csa in clinically relevant cells, we infected the human bronchial epithelial cell line calu- and primary human alveolar epithelial cells (haec) with mers-cov and analyzed intracellular viral rna and infectious particle release in presence of dmso or csa ( figure ). in both calu- and haec, csa treatment led to a > % decrease of viral rna ( figure a ) and a reduction of viral titers in the supernatant by . - . log , respectively ( figure b) . interestingly, and in accordance with reports from autopsy material of mers-cov patients [ ] , mers-cov-infected calu- and primary haec both showed apoptotic cell loss and formation of multinucleated cell foci ( figure c ). addition of csa reduced cell foci formation and significantly reduced apoptosis induction ( figure c , d). in line, both cftr (cystic fibrosis transmembrane conductance regulator; figure e ) and enacβ (epithelial sodium channel beta; supplement fig.e ) protein expression was improved after addition of csa to mers-cov-infected calu- . moreover, epithelial structural integrity and ability for vectorial water transport were reduced in mers-cov-infected control cells and significantly improved to normal levels in mers-cov-infected, csa-treated cells ( figure f , g). csa is known to act via multiple signaling pathways including cyclophilin ppiase activity, the cna-nfat axis as well as map kinase signaling [ ] [ ] [ ] [ ] . using specific inhibitors, we aimed to interfere with csa-affected pathways to identify relevant molecular signaling events involved in the csa-mediated reduction of mers-cov infection. inhibition of cna by its specific inhibitor calcineurin inhibitory peptide (cip), as well as inhibition of the downstream transcription factor nfat resulted in minor, statistically non-significant changes in mers-cov viral titers in both calu- and haec (figure a , b). the non-immunosuppressive derivate of csa, alisporivir (alv), that binds the ppiase but does not induce ternary complex formation of cypa with cna, reduced viral titers to a similar extent as csa, suggesting that the cypa-ppiase activity elicits the restrictive effect on mers-cov replication rather than ternary complex-mediated signaling events. moreover, alv reduced cell foci formation and loss of epithelial integrity to a similar extent as csa (supplement fig. e ). applying specific mapk inhibitors against jnk and p , we revealed that inhibition of the map kinase jnk, but not of p reduced mers-cov titers in both calu- and haec ( figure a our data suggest that, as opposed to its well-known cna/nfat- figure c ). these data indicate that csa treatment mounts a distinct interferon-driven antiviral response in lung epithelial cells. to better understand the transcriptional programs leading to ifnλ induction in csa-treated cells, we analyzed the regulation of interferon regulatory factors (irfs). our data reveal significant upregulation of irf mrna levels upon csa treatment, but not of irf , irf or irf ( figure a ). irf is known to be a specific activator of ifnl gene expression [ ] . accordingly, we identified a significantly increased number of irf -expressing cells in csa-stimulated calu- cells by immunofluorescence ( figure b ). in line, irf sirna knockdown significantly reduced ifnl mrna levels in csa treated calu- cells ( figure c ). accordingly, irf knockdown inhibited ifnλ release by more than % as compared to control ( figure d ). to understand the extent to which the inhibition of mers-cov propagation in csa treated cells was mediated by irf -mediated production of ifnλ, we performed knockdown of irf or neutralized cell-free ifnλ, respectively. our data demonstrated that silencing of irf but not treatment by control sirna lead to a significant increase in mers-cov released viral particles in csa-treated cells ( figure a , b). moreover, neutralizing antibodies directed against ifnλ , ifnλ and ifnλ or against the less induced ifnβ were applied ( figure b ). neutralization of ifnβ had no significant impact on mers-cov replication after csa treatment, whereas application of anti-ifnλ / / treatment significantly increased mers-cov viral titers by . log level ( figure b ). these data indicate that the antiviral effects of csa were at least partially mediated by an irf -ifnλ signaling axis, and independent of type-i-ifn. as no specific treatment is approved for mers-cov or sars-cov(- ), current treatment strategies are supportive [ , ] . treatments including recombinant type-i-ifn and antivirals (e.g. lopinavir/ritonavir) have been applied off-label to treat mers-cov and yielded only moderate efficacy with controversial results in retrospective studies, and data from prospective studies or randomized controlled trials are lacking [ , [ ] [ ] [ ] . due to its receptor specificity to the human dpp , only few animal models to study mers-cov pathogenesis and mers-cov-directed antiviral compounds have been accessible to date. for this study, mers-cov infection in the mouse was facilitated via intratracheal delivery of a human dpp encoding adenovirus, that might cause low-level inflammation itself and inhomogeneous receptor distribution within the lung, present for a limited time frame. however, even if this model might not fully recapitulate the native cellular distribution or density of the receptor as seen in the human lung, high transduction efficiencies (≥ %, data not shown) allow efficient viral spread in the upper and lower respiratory airways with quick progression to severe lung injury [ ] and with moderate changes in morbidity [ ] . thus, model-specific neurotropism as seen in some of the transgenic hdpp mice [ ] or the necessity to adapt virus isolates via multiple passages, which might potentially affect its susceptibility to interventional strategies, are circumvented. while prior exposure to adenovirus evokes moderate histological changes including perivascular and bronchiolar lymphocytic infiltration (data not shown), mers-cov infection led to a clearly distinguishable granulocytic, necrotizing interstitial pneumonia with alveolar edema formation as described previously [ ] . moreover, we demonstrate that inhibition of cypa via csa or alv, which both potently block the cypa ppiase activity at the used concentrations [ ] , results in a pronounced upregulation of type-iii-ifn on both mrna and protein level, which was mediated via irf and was accompanied by expression of antiviral isgs. among those, especially ifit (interferon-induced protein with tetratricopeptide repeats ), has been reported to influence the pathogenesis of mers-cov, highlighting the relevance of our findings [ ] . of note, type-iii-ifns have recently emerged as key antiviral players in the innate immune response to viral infections at mucosal and epithelial surfaces [ ] [ ] [ ] [ ] . they efficiently restrict different respiratory viruses, and act e.g. by limiting spread from the upper to the lower airways [ , [ ] [ ] [ ] . as opposed to type-i-ifn, type-iii-ifn do not trigger detrimental immune responses that contribute to immunopathology in influenza infection [ , , , ] . this might prove to be pivotal in the context of csa-dependent stimulation of ifnλ during cov, as severe human cov infections, like mers-cov and-while data are still limitedalso sars-cov- , are characterized by an immunopathology with a strong cytokine induction [ , , ] . in addition to defining a novel pro-inflammatory, antiviral expression profile induced by csa on lung epithelial cells, this study also demonstrated for the first time gen. virol. . statistical significance was calculated using unpaired two-way student's t-test (a, b, c, d, g) with *p < . . onestep rt-pcr system (invitrogen life technologies) as described previously [ , ] . intracellular localization of endogenous irf protein was analyzed and hour post csa stimulation. dmso-treated cells were used as negative control. stimulated cells were fixed with % pfa and permeabilized with methanol/acetone for min. cells were incubated with a rabbit monoclonal-anti-irf ( : ; cell signaling) and an alexafluor -conjugated secondary antibody ( : ; dianova). cell nuclei were counterstained with dapi. the samples were mounted in fluoprep (biomérieux) and images were recorded with a confocal laser scanning microscope (leica sp ). calu cells were seeded in . µm pore size transwell cell culture dishes (corning) and cultured until achieving electrochemical resistances (ter) of ≥ Ω /cm as measured by millicell-ers device. cells were infected apically with mers-cov at ; as reported previously [ ] . for quantification of mers-cov induced apoptosis a caspase / gloassay® sds-page and western blot was analyzed as described previously [ ] . calu- cells were infected with mers-cov using a moi of . and stimulated with csa hour after virus adsorption. h pi cells were scratched off with µl pbs supplemented with protease-inhibitor mix (calbiochem) and centrifuged for min at , rpm. cell pellets were resuspended in sample buffer [ ] containing % sds and boiled at °c for min. after discharge of the probes out of the bsl laboratory another min boiling step was performed before the samples were separated using an , % sds-gel. after blotting on a nitrocellulose membrane and blocking using pbsdef with % milk powder first antibodies (anti-cftr antibody, clone mm - and mouse monoclonal anti-vinculin antibody both sigma-aldrich; enacβ antibody (e- ), sc- ; santa cruz biotechnology) diluted in pbsdef with % milk powder were incubated overnight followed by secondary antibody-incubation for h (goat anti-mouse/hrp and swine anti-rabbit/hrp; both dako). for visualization of the signals image lab software was used. all animal experiments were performed in accordance with the regulations of german animal protection laws and as authorized by the regional authorities for histopathological analyses of formalin-fixed, paraffin-embedded murine lung tissues, sections of µm thickness were cut from four to six evenly distributed planes throughout the entire lungs and mounted on adhesive glass slides. the slides were stained with hematoxylin and eosin and coverslipped. histopathological evaluation was performed using an established four grade scoring scheme [ ] including the following parameters: affected area, severity and distribution of interstitial inflammation, infiltration of macrophages, lymphocytes and granulocytes, necrosis, alveolar hemorrhage and edema as well as formation of bronchus-associated lymphoid tissue (balt) and perivascular, lymphocytic cuffing. figure onestep rt-pcr kit as described previously [ , ] . quantification was carried out using a standard curve based on -fold serial dilutions of appropriately cloned rna ranging from to copies. bar graphs in represent mean ± sem of n = - isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study middle east respiratory syndrome treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset 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access ifn-λ prevents influenza virus spread from the upper airways to the lungs and limits virus transmission interferon lambda signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses impact and regulation of lambda interferon response in human metapneumovirus infection interferon-λ mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology activation signals for interleukin- production involve activation of mkk -p and mkk signaling pathways sensitive to cyclosporin a variability of interferon-λ induction and antiviral activity in nipah virus infected differentiated human bronchial epithelial cells of two human donors a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform. perlman s, editor protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein. perlman s, editor macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection serine-arginine protein kinase regulates ebola virus transcription multivesicular bodies as a platform for formation of the marburg virus envelope spectrum of pathogen-and model-specific histopathologies in mouse models of acute pneumonia work with live mers-cov was performed in the bsl- facility of the philipps university, marburg, germany. we thank julia spengler, larissa hamann, stefanie jarmer, dirk becker and marc ringel for excellent technical and experimental assistance. we thank ralf bartenschlager for providing alisporivir. key: cord- -z sz msj authors: fanoy, ewout b; van der sande, marianne ab; kraaij-dirkzwager, marleen; dirksen, kees; jonges, marcel; van der hoek, wim; koopmans, marion pg; van der werf, douwe; sonder, gerard; van der weijden, charlie; van der heuvel, jet; gelinck, luc; bouwhuis, jolande w; van gageldonk-lafeber, arianne b title: travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia, may date: - - journal: emerg themes epidemiol doi: . / - - - sha: doc_id: cord_uid: z sz msj background: in may , middle east respiratory syndrome coronavirus (mers-cov) infection, with closely related viral genomes, was diagnosed in two dutch residents, returning from a pilgrimage to medina and mecca, kingdom of saudi arabia (ksa). these patients travelled with a group of other dutch travellers. we conducted an epidemiological assessment of the travel group to identify likely source(s) of infection and presence of potential risk factors. methods: all travellers, including the two cases, completed a questionnaire focussing on potential human, animal and food exposures to mers-cov. the questionnaire was modified from the who mers-cov questionnaire, taking into account the specific route and activities of the travel group. results: twelve non-cases drank unpasteurized camel milk and had contact with camels. most travellers, including one of the two patients (case ), visited local markets, where six of them consumed fruits. two travellers, including case , were exposed to coughing patients when visiting a hospital in medina. four travellers, including case , visited two hospitals in mecca. all travellers had been in contact with case while he was sick, with initially non-respiratory complaints. the cases were found to be older than the other travellers and both had co-morbidities. conclusions: this epidemiological study revealed the complexity of mers-cov outbreak investigations with multiple potential exposures to mers-cov reported such as healthcare visits, camel exposure, and exposure to untreated food products. exposure to mers-cov during a hospital visit is considered a likely source of infection for case but not for case . for case , the most likely source could not be determined. exposure to mers-cov via direct contact with animals or dairy products seems unlikely for the two dutch cases. furthermore, exposure to a common but still unidentified source cannot be ruled out. more comprehensive research into sources of infection in the arabian peninsula is needed to strengthen and specify the prevention of mers-cov infections. in , the middle east respiratory syndrome coronavirus (mers-cov) was isolated for the first time from the sputum of a -year-old man who presented with an acute pneumonia in kingdom of saudi arabia (ksa) [ ] . as of june , laboratory-confirmed cases of mers-cov infection have been reported to the who including ( %) fatal cases [ ] . following the first upsurge of cases in spring , a second wave occurred in , mainly in ksa [ ] . most cases so far either resided in or travelled to the arabian peninsula and neighbouring countries or were close contacts of these cases. furthermore, travel related cases have been reported from the united states of america (usa), the united kingdom (uk), germany, france, tunisia, italy, greece, algeria, egypt, the philippines and malaysia [ ] [ ] [ ] [ ] [ ] . so far, mainly sporadic cases are reported as well as a few hospital outbreaks, which is in line with a low reproduction rate (r ) of mers-cov, estimated to range from . to . [ ] [ ] [ ] . the median incubation period is estimated to be . days ( % ci . - . days) [ ] . for most non healthcare associated cases no clear source of infection could be identified, although camels are considered a reservoir as the virus and antibodies against mers-cov have been identified in camels (camelus dromedarius) and in their milk [ , ] . the exact modes of transmission of the virus to humans remain unclear [ ] [ ] [ ] [ ] , leaving the possibility for other yet unidentified sources of infection. to stop or reduce transmission of mers-cov and prevent new human infections, it is important to identify all potential sources of infections as well as risk factors and route(s) of transmission. in may , mers-cov infection was diagnosed in two dutch residents. these cases travelled with a group of other people, returning to the netherlands from pilgrimage to medina and mecca, ksa [ ] . we conducted a comprehensive epidemiological assessment of the travel group aiming to identify the likely source(s) of infection and presence of potential risk factors for these two cases. we compiled a questionnaire based on sample questionnaires originating from two who-protocols aimed to asses risk factors and investigate contacts of patients with mers-cov infection [ , ] . we adapted the questionnaire to the specific route and activities of the dutch travellers. our questionnaire covered human, animal and food exposures to mers-cov during the whole trip to ksa. the questionnaires consisted of open and closed questions, and it took approximately minutes to complete the face-to-face interview with the travellers (additional file ). nurses of the public health services administered the questionnaires approximately weeks after return to the netherlands. for the two cases the questionnaire was completed with data on the travel route and stay in ksa collected by the national coordination centre for communicable disease control. the patients were a -year-old man (case ) and his -year-old sister (case ), both having underlying cardiovascular co-morbidities and diabetes mellitus. the remaining travel group included persons ( % male) aged between and years (median age: years). seventeen ( %) of the non-cases did not report any comorbidities (table ). upon identification of case , throat swabs of all contacts were tested by pcr to assess other mers-cov cases. apart from case , who had reported mild respiratory complaints starting on the th of may (t = day ), all other travellers tested negative and did not develop symptoms. the group made a pilgrimage to medina and mecca, ksa ( figure ). they arrived on the th of april (day ) in medina, left by private bus to mecca on the th of may (day ) and returned to amsterdam on the th of may (day ). case started to feel feverish and had diarrhoea on the st of may (day ). respiratory complaints started on the th of may (day ). mers-cov was identified in case on the th of may (day ) and in case on the th of may (day ). detailed case reports of the two patients are described by kraaij et al. [ ] . during their pilgrimage, the whole group stayed in the same hotels and had breakfasts together. there was no fixed collective travel programme, but they had some joint visits to several mosques. the travellers also spent time alone or in smaller subgroups, visiting different mosques, markets and restaurants. the exposures for both the cases and asymptomatic travellers during their visit to saudi arabia are summarized in table . on the rd of may (day ), travellers (excluding the two cases) made a trip to the touristic valley wadi-e-jinn, and stopped when they came across a herd of camels laying behind an improvised razor fence. four travellers reported direct contact with the camels. eight travellers mentioned indirect contact, for example via the fences surrounding the animals. eleven travellers consumed unpasteurized camel milk, offered to them by the caretakers of the camels. besides contact with camels, two travellers reported that they spotted many pigeons in the cities and fed them. no other direct contact with animals was mentioned. page of http://www.ete-online.com/content/ / / as mentioned above, eleven travellers, excluding the two cases, consumed unpasteurized camel milk. furthermore, travellers, including case , visited one or more local markets in medina. seven of them, excluding case , bought and consumed a range of fruits such as prunes and dates. furthermore, travellers bought souvenirs, such as dates, perfume and clothing on these markets. case and his son visited two hospitals in medina on the th of april (day ) because of eye complaints of the son. as mentioned before, they spent minutes in a crowded waiting room of a general hospital where they were exposed to coughing patients. subsequently, the son was referred to a specialized eye clinic where they spent no time in a waiting room and did not notice any coughing patients. on the th of may (day ), case , accompanied by his son, visited an emergency department of a general hospital in mecca because of acute malaise, weakness, nausea and diarrhoea (without respiratory complaints). these symptoms started on may (day ). case and his son spend - minutes in the waiting room, though they had no specific exposure to coughing people. on the th of may (day ) case returned to another hospital in mecca, accompanied by his son, where he only briefly stayed in the waiting room, received antibiotic treatment and was observed for approximately three hours. four other travellers, including his son, accompanied him. two of them did not notice any coughing persons, while the other two did not answer this question. case did not visit a hospital during the pilgrimage or prior to her diagnosis in the netherlands. two travellers (case and his son) waited minutes in a crowded waiting room of a general hospital in medina ( th of april, day ) where they were exposed to coughing patients. one other traveller, who did not enter a hospital, reported coughing persons outdoors. the duration and intensity of this contact was not clarified in more detail. case and had been in close contact with each other during the entire trip, as they shared hotel rooms. all travellers had been in contact with both cases during the trip. seventeen travellers had minimal social contact with the two cases, for example only brief eye contact in the hotel lobby or in the bus. twelve travellers reported daily contact, such as visits to the hotel room of case while he was sick, or accompanying him while shopping. the son of case had the most intensive contact sharing a hotel room with both cases. nevertheless, his throat swabs tested negative to mers-cov by pcr during the follow-up after the identification of case . we found no indications that the two cases were infected via contact with specific animals or dairy products. infection during a hospital visit could be a source for case . sequence analysis of parts of the viral genome collected from both patients had shown that the strains were nearly identical, suggesting that the cases have infected each other, or that they were exposed to a common source. in the latter case, exposure during a hospital visit is unlikely because case did not visit a hospital before onset of disease. the possibility of asymptomatic infections and transmission among the accompanying travellers cannot be ruled out, but seems unlikely in view of the negative pcr results. subsequently, although considered unlikely, we cannot exclude the possibility of asymptomatic travellers being a source of infection for case and/or . no other common sources were identified although we cannot exclude the possibility of lack of accurate recall or indirect exposure to animal excreta. it might be possible that one or both cases were infected during non-specific contacts with the local population or the environment. if initial zoonotic introductions of mers-cov from camels have been followed by low level presence among the (asymptomatic) population or in the environment at large, this might result in an ongoing community-based outbreak, which, unlike sars, cannot be controlled by rigorous hospital hygiene alone. both patients were of older age and had comorbidities. in general, older age and comorbidity are considered risk factors for symptomatic mers-cov infections, although this was not the case in all outbreaks [ , ] . in this study, the distribution of symptoms towards higher age and comorbidity fits this assumption. ongoing serological analysis upon travellers and people exposed to the case in the netherlands may reveal to what extent asymptomatic infection has occurred. the remainder of the travellers did not develop symptomatic mers-cov and did not have positive pcr-test results despite exposure to camels, dairy products and the two cases. this could point to a low transmissibility of the virus, which is also illustrated by the limited number of secondary infections among contacts of import cases described elsewhere, with the reservation that asymptomatic infections among the remaining travellers cannot be excluded fully as final serologic results are not available [ , ] . currently, there is only limited epidemiological information published considering potential sources and transmission dynamics of mers-cov infection within the arabian peninsula. descriptive studies among a well-defined group of travellers can therefore provide relevant epidemiologic information, even when the number of travellers is too small to draw definite conclusions. more in depth epidemiological investigation of comparable travel groups will add to the body of evidence, although the diversity of activities during a relatively short trip makes accurate recall of potential exposure challenging. alternatively, prospective cohort studies among travellers to affected areas, whereby travellers are asked to keep a daily diary, are likely to be more informative and complete. due to the uncertainty about the source, the detailed questions of the who-questionnaire were a very useful guide to decide on the most relevant exposures to be explored, adapted to the specific route of travel. the exact source of infection remains difficult to identify. case could have been infected during his visit to a page of http://www.ete-online.com/content/ / / hospital and subsequently have infected case . however, exposure to a common source for both case and cannot be ruled out. besides, case may have been exposed to a yet unidentified source and subsequently have infected case , or vice versa. more research to relevant sources in the arabian peninsula is needed. the suggested role of underlying disease to develop mers-cov infection is in line with the age and comorbidity of the two dutch cases and the absence of symptomatic mers-cov infections among the younger and healthier travellers. additional file : the questionnaire. isolation of a novel coronavirus from a man with pneumonia in saudi arabia organization wh: coronavirus infections global alert and repsonse (gar) first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in a case of imported middle east respiratory syndrome coronavirus infection and public health response clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands world health organization: seroepidemiological investigation of contacts of middle east respiratory syndrome coronavirus (mers-cov) patients world health organization: case-control study to assess potential risk factors related to human illness caused by middle east respiratory syndrome coronavirus (mers-cov) al raiy b: clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution medical centre haaglanden, the hague, the netherlands. isalaklinieken, zwolle, the netherlands. national institute for public health and the environment, epidemiology and surveillance unit, antonie van leeuwenhoeklaan , bilthoven, ma , the netherlands. the authors declare that they have no competing interests. all readers have read and approved the manuscript.authors' contributions ef compiled the questionnaires, analysed the data and drafted the manuscript. ms is in charge of the epidemiology department; she designed and coordinated the study and reviewed the manuscript. mk assessed travel data and reviewed the manuscript. kd coordinated interviews with travellers and reviewed the manuscript. mj was responsible for laboratory testing and reviewed the manuscript. wh is in charge of respiratory epidemiology department and reviewed the manuscript. mpgk is involved in laboratory testing and reviewed the manuscript. dw coordinated interviews with travellers and reviewed the manuscript. gs coordinated interviews with travellers and reviewed the manuscript. cw coordinated interviews with travellers and reviewed the manuscript. jh coordinated interviews with travellers and reviewed the manuscript. lg was involved in clinical care and interviewed a case. jb was involved in clinical care and interviewed a case. ag was leading the study, including the study design and analysis and she reviewed the manuscript. all authors read and approved the final manuscript. key: cord- - isp wj authors: al-tawfiq, jaffar a; kattan, rana f; memish, ziad a title: middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia date: - - journal: world j clin pediatr doi: . /wjcp.v .i . sha: doc_id: cord_uid: isp wj aim: to summarize the reported middle east respiratory syndrome-coronavirus (mers-cov) cases, the associated clinical presentations and the outcomes. methods: we searched the saudi ministry of health website, the world health organization website, and the flutracker website. we also searched medline and pubmed for the keywords: middle east respiratory syndrome-coronavirus, mers-cov in combination with pediatric, children, childhood, infancy and pregnancy from the initial discovery of the virus in to . the retrieved articles were also read to further find other articles. relevant data were placed into an excel sheet and analyzed accordingly. descriptive analytic statistics were used in the final analysis as deemed necessary. results: from june to april , , there were a total of pediatric mers-cov cases. of these cases ( %) were asymptomatic and the male to female ratio was . : . the mean age of patients was . ± . years. twenty-five ( . %) of the cases were reported from the kingdom of saudi arabia. the most common source of infection was household contact ( of with reported source) and patients acquired infection within a health care facility. using real time reverse transcriptase polymerase chain reaction of pediatric patients revealed that out of ( . %) was positive in the kingdom of saudi arabia. conclusion: utilizing serology for mers-cov infection in jordan and saudi arabia did not reveal any positive patients. thus, the number of the pediatric mers-cov is low; the exact reason for the low prevalence of the disease in children is not known. middle east respiratory syndrome-coronavirus (mers-cov) was first isolated in from a patient in the kingdom of saudi arabia (ksa) [ ] . as more cases were reported, the case fatality rate changed to % from % [ ] [ ] [ ] [ ] . in addition, initially there was a predominance of males; later this ratio decreased [ , ] . mers-cov is characterized by three different patterns of disease: sporadic cases, intra-familial transmission [ ] [ ] [ ] and health care associated infection [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite the increased number overtime and the multiple health care associated outbreaks [ ] , the number of pediatric cases remained low during the study period [ ] . the initial description of cases included only a -year-old child [ ] . the first pediatric case was a -year-old child reported from jeddah, ksa on june , [ ] . later an additional three asymptomatic children were reported [ ] . the largest report of childhood mers-cov cases included eleven, of which two patients were symptomatic and nine were asymptomatic [ ] . the exact reason for this low prevalence of the disease in children is not known. in this study, we summarize the reported mers-cov cases and the associated clinical presentation and the outcome. we searched the saudi ministry of health website [ ] , the world health organization website [ ] , the flutracker website [ ] , the medical literature and the retrieved published studies for any childhood mers-cov infections. we searched medline and pubmed for the keywords middle east respiratory syndrome-coronavirus, mers-cov, in combination with pediatric, children, childhood, infancy and pregnancy from the initial discovery of the virus in june until april , . the retrieved articles were also read to find other relevant articles. relevant data were placed into an excel sheet and analyzed accordingly. descriptive analytic statistics were used in the final analysis as deemed necessary, including mean and standard deviation when applicable and frequency. the statistical review of the study was performed by a biomedical statistician. statistical review is performed before the submission of the manuscript. from june , to april , , there were a total of pediatric mers-cov cases as shown in table . of all the cases, thirteen ( ) or % were asymptomatic, and there were males, females and unreported (a male to female ratio of . : ). the mean age of patients was . + . ( . - ) years. twenty-five cases ( . %) were reported from ksa; the other patients were in jordan, united arab emirates and the republic of korea (table ). the most common source of the infection was household contact ( of with reported source), and patients acquired the infection within a health care facility. about one half of the cases were reported in , and % were reported in and . % in ( table ) . screening of pediatric patients for mers-cov infection using real time reverse transcriptase polymerase chain reaction showed that only out of ( . %) were positive in ksa [ ] . however, serologic testing of pediatric patients admitted with lower respiratory tract infection in jordan and saudi arabia revealed no positive tests [ , ] (table ) . the effect of mers-cov infection on the fetus was described in eight cases [ ] [ ] [ ] [ ] as summarized in table . the mean age of the mothers was . + . years, and the mean gestational age was . + . wk. death of the fetus was observed in ( . %) of the fetuses. despite the total number of mers cases increasing, especially in ksa, the number of pediatric cases remained low during the study period. initially, the testing in ksa was directed towards hospitalized patients with severe pneumonia. in , the saudi ministry of health added a specific case definition for mers-cov infection in children [ ] . the definition includes those ≤ years, meets the adult case definition and has either a history of exposure to a confirmed or suspected mers-cov in the proceeding d or a history of contact with camels or camel products in the proceeding d [ ] . the case definition also includes children with unexplained severe pneumonia [ ] . the change in the case definition does not account for the low rate of childhood mers-cov infection as % of the cases were reported in before the case definition was changed. one of the reasons for an increased number of cases in during the jeddah outbreak was increased testing of asymptomatic and mildly symptomatic patients [ ] . the pattern of mers-cov pediatric cases was similar to the sars outbreak. children were less affected than adults and children less than years of age had milder disease [ ] . in the largest screening of contacts, the rate of mers-cov positive children ( . %, / ) compared to . % ( / ) in adults (p = . ) [ ] . thus, in this study utilizing mers-cov pcr the positivity rate did not differ in children and adults. in adults with mers-cov infections, three patterns of transmissions were observed: sporadic (primary) cases presumed to be due to animal exposure (mainly camels), household contacts or health care associated infections [ ] . in ksa, the majority ( %) of cases were health care-associated infections, % were primary cases, and % were household contacts [ ] . in contrast, in the majority of pediatric cases that reported source of acquisition ( . % of the with reported source), the disease was acquired through household contact. this pattern indicates a low exposure of children to animals and a higher rate of health care associated infections in adult wards. the male to female ratio ( . : and . : ) was initially high [ , ] . this apparent male predominance could be explained by the nature of hospital outbreaks [ ] . eventually the male to female ratio was reduced to . : to . : [ , ] . consistent with these studies, the male to female ratio in children with mers-cov was . : and may indicate similar exposure of children to index cases in the household settings and differential host factors. possible explanations for the lower number of pediatric cases compared to adults include differential testing of adult patients and milder diseases in children; although, serologic testing of pediatric patients in ksa and jordan did not reveal any positive cases [ , ] . in the largest sero-epidemiologic survey in ksa, the study did not include children and thus it is difficult to establish the rate of sero-positivity in children [ ] . the mers-cov infection rate in children remains low and possible explanations include: a milder disease in children, asymptomatic infection, or the presence of yet to be identified factors. the development of a shorter duration of mers in children is another possible explanation. if this is the case, it may limit the development of a positive serology. in one study, delayed antibody responses as measured with the neutralization test was associated with severe diseases [ ] . the longevity of antibodies in mers-cov cases might be limited as was the case with sars [ , ] . the only study of serology among children was done among hospitalized pediatric cases who presented with lower respiratory tract infections [ ] . there is no systematic screening of exposed children using serologic testing; this limited the interpretation of available serologic studies. little data also exist regarding the effect and the likelihood of mers-cov in pregnancy. eight cases were reported [ , , ] . the outcome was favorable in the majority of cases. the exact prevalence of mers-cov antibodies and exposure of pregnant women to mers-cov is not known. in conclusion, the number of mers-cov infections in pediatric patients remains low. possible explanations include low exposure, presence of asymptomatic, mildly symptomatic patients or the presence of yet to be identified factors. the middle east respiratory syndrome-coronavirus (mers-cov) was first isolated in from a patient in the kingdom of saudi arabia (ksa). despite the increased number of mers-cov cases overtime, the number of pediatric cases remained low. the exact reason for this low prevalence of the disease in children is not known. the aim of this study is to summarize the reported mers-cov cases and the associated clinical presentation and the outcome. the first pediatric case was a two-year-old child reported from jeddah, ksa on june , . later an additional three asymptomatic children were reported. the largest report of childhood mers-cov cases included eleven, including nine asymptomatic cases. the number of mers-cov infections in pediatric patients remains low. possible explanations include low exposure, presence of asymptomatic, mildly symptomatic patients or the presence of yet to be identified factors. the immune system predisposing to severe disease and to fatal outcome remains unknown. an exploration of the virus-host interaction may add to the understanding of the low prevalence in this age group. despite the low number of pediatric mers-cov cases, it is important to continue to monitor the development of this disease in this age group and to understand the risk factors. mers-cov is a new emerging virus that was first isolated in . this complication of all known pediatric cases is a useful contribution to the medical literature, and knowing it is possible but rare is important. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: epidemiology and disease control 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middle east respiratory syndrome: years later middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution epidemiological findings from a retrospective investigation the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea microevolution of outbreak-associated middle east respiratory syndrome coronavirus managing mers-cov in the healthcare setting middle east respiratory syndrome coronavirus disease in children mers-cov summary and literature middle east respiratory syndrome coronavirus (mers-cov) case list of moh/who novel coronavirus mers ncov announced cases screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia al abdallat mm. stillbirth during infection with middle east respiratory syndrome coronavirus impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus during pregnancy middle east respiratory syndrome coronavirus infection during pregnancy: a report of cases from saudi arabia infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update drivers of mers-cov transmission: what do we know? kinetics of serologic responses to mers coronavirus infection in humans disappearance of antibodies to sars-associated coronavirus after recovery middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus (mers-cov) update. disease outbreak news key: cord- -fl nt ak authors: kleine-weber, hannah; pöhlmann, stefan; hoffmann, markus title: spike proteins of novel mers-coronavirus isolates from north- and west-african dromedary camels mediate robust viral entry into human target cells date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: fl nt ak the highly pathogenic middle east respiratory syndrome (mers)-related coronavirus (cov) is transmitted from dromedary camels, the natural reservoir, to humans. for at present unclear reasons, mers cases have so far only been observed in the arabian peninsula, although mers-cov also circulates in african dromedary camels. a recent study showed that mers-cov found in north/west- (morocco) and west-african (burkina faso and nigeria) dromedary camels are genetically distinct from arabian viruses and have reduced replicative capacity in human cells, potentially due to amino acid changes in one or more viral proteins. here, we show that the spike (s) proteins of the prototypic arabian mers-cov strain, human betacoronavirus c emc/ , and the above stated african mers-cov variants do not appreciably differ in expression, dpp binding and ability to drive entry into target cells. thus, virus-host-interactions at the entry stage may not limit spread of north- and west-african mers-cov in human cells. the middle east respiratory syndrome-related coronavirus (mers-cov) causes the severe lung disease mers (zaki et al., ) , which takes a fatal course in roughly~ % of infected patients (who, ) . mers-cov is endemic in the middle east, where the virus is transmitted from dromedary camels, the natural reservoir, to humans (perera et al., ; reusken et al., ) . human-to-human transmission is inefficient but resulted in several hospital outbreaks of mers harriman et al., ; memish et al., ) , and there is concern that the virus may adapt to humans and cause a pandemic. infection of dromedary camels with mers-cov is not limited to the middle east. african camels are frequently infected with mers-cov (ali et al., a (ali et al., , b chu et al., chu et al., , chu et al., , corman et al., ; deem et al., ; kiambi et al., ; miguel et al., ; ommeh et al., ; perera et al., ; reusken et al., reusken et al., , van doremalen et al., ) and the responsible viruses are genetically distinct from those circulating in the middle east kiambi et al., ; ommeh et al., ) . moreover, viruses isolated from animals in morocco, nigeria and burkina faso form a distinct phylogenetic subclade, c , and exhibit reduced ability to replicate in human respiratory cells . in addition, mers-cov transmission from camels to humans has not been observed in northand west-africa (munyua et al., ; so et al., ) , although two livestock handlers in kenya were shown to harbor antibodies against mers-cov (liljander et al., ) , moreover, no mers cases were documented in africa. at present, the barrier(s) impeding efficient spread of african mers-cov in human cells and camel-human transmission of these viruses remain to be identified. the mers-cov spike protein (s) is incorporated into the viral envelope and facilitates viral entry into target cells (li, ) . for this, the s protein binds to the cellular receptor dipeptidyl peptidase (dpp , cd ) via its surface unit, s , and fuses the viral membrane with a target cell membrane via its transmembrane unit, s (li, ) . binding of mers-s to dpp is essential for mers-cov infection of cells and dpp expression and the s protein/dpp interface are major determinants of mers-cov cell and species tropism van doremalen et al., ) . the s proteins of north-and west-african mers-cov of the c clade harbor - amino acid substitutions relative to mers-cov (fig. a , table ) and these substitutions might reduce s protein-driven entry into target cells. however, this possibility has not been examined so far. we employed a previously described vesicular stomatitis virus (vsv)-based pseudotyping system to study mers-s-driven host cell entry (kleine-weber et al., known to adequately model key aspects of the coronavirus entry process. in order to study host cell entry driven by s proteins from the c subclade, we employed pcr-based mutagenesis to generate expression constructs for the s proteins of mers-cov from morocco (camel/morocco/cirad-hku / , mo), nigeria (camel/nigeria/nv / , ni) and burkina faso (camel/burkina faso/cirad-hku / , bf), using a published expression construct for mers-cov emc s protein as template (kleine-weber et al., . moreover, expression constructs for all s proteins were generated that encoded a cterminal v antigenic tag. western blot analysis of cells transfected to express the s proteins under study revealed that mers-s emc, mo, ni and bf were expressed and proteolytically processed to comparable levels ( fig. b) . moreover, these s proteins were incorporated into vsv particles with similar efficiency (fig. c) . these results suggest that mutations present in north-and west-african mers-s of the c subclade do not reduce s protein expression and proteolytic processing in human cells. we next asked whether dpp binding of north-and west-african mers-s was altered. for this, t cells transfected to express the s proteins under study were incubated with soluble dpp fused to the fc portion of human immunoglobulin and binding was quantified by flow cytometry, as described previously (kleine-weber et al., ). the results showed that mers-s emc, mo, ni, and bf bound to dpp robustly and with comparable efficiency while dpp binding to cells expressing no s protein was within the background range (fig. ). finally, we tested whether the robust binding to dpp translated into efficient s protein-driven entry. for this, cell lines were selected that were shown to express low levels ( t), intermediate levels (vero ) or high levels of dpp (caco- , t + dpp ) (kleine-weber et al., ). mers-s mo, ni and bf mediated entry into all cell lines with at least the same efficiency as mers-s emc (fig. ) . moreover, under conditions of low or medium dpp expression, entry mediated by mers-s mo and bf was even more efficient than entry mediated by mers-s emc (fig. ), although these differences were not statistically significant. our results show that amino acid substitutions present in north-and west-african mers-s proteins relative to mers-s emc do not compromise s protein expression in human cells, at least when transfected cells are examined. similarly, proteolytic processing of the s proteins in the constitutive secretory pathway, which is known to be carried out by furin (gierer et al., ; millet and whittaker, ) , was not the indicated s proteins were transiently expressed in t cells, whole cell lysates (wcl) were prepared at h posttransfection and s protein expression was analyzed via western blot, using an antibody targeting the c-terminal v -tag. cells expressing no s protein were used as negative control and detection of β-actin (actb) served as loading control. similar results were obtained in two separate experiments. (c) rhabdoviral transduction vectors (vsvpp) harboring the indicated s proteins were concentrated by centrifugation and, following lysis, analyzed by western blot for s protein incorporation, using an antibody targeting the c-terminal v -tag. transduction vectors harboring no s protein were used as negative controls and detection of vesicular stomatitis virus matrix protein (vsv-m) served as loading control. similar results were obtained in a separate experiment. numbers on the left side of each blot indicate the molecular weight in kilodalton (kda). further, bands representing the precursor s protein (s , black circle) and the s subunit of proteolytically processed s protein (grey circle) are indicated. appreciably altered. moreover, binding of north-and west-african s proteins to dpp was not diminished as compared to mers-s emc, despite the presence of at least one substitution in the receptor binding domain (rbd) in each s protein tested. this finding might not be unexpected since the substituted amino acid residues do not make direct contact with residues in dpp (lu et al., ) . in keeping with these observations, all african s proteins mediated robust viral entry into non-human primate (vero ) and human cell lines ( t, caco- ) expressing different levels of dpp (kleine-weber et al., ) . in fact, mers-s mo-and bf-driven entry into cell lines expressing low or intermediate levels of dpp was augmented as compared to mers-s emc, in keeping with these s proteins showing slightly enhanced dpp binding as compared to mers-s emc. finally, it is noteworthy that mers-s activation in caco- cells mainly depends on the cellular serine protease tmprss while activation in t and vero cells is mediated by the cellular cysteine protease cathepsin l (kleine-weber et al., , ). thus, north-and west-african mers-s proteins seem to be able to use both pathways available for s protein activation in human cells. confirmation of our findings with authentic viruses is pending and we cannot exclude that, for instance, the s protein modulates recognition of the virus by sensors of the interferon system, which cannot be measured with the assays available to us. moreover, we note that a recent study examining two mers-s sequences (c subclade) from camels in ethiopia demonstrated that these sequences, when inserted into mers-cov emc, reduced viral entry and replication and increased sensitivity to antibody-mediated neutralization (shirato et al., ) . the reduction in entry was observed for vero and to a lesser degree for vero-tmprss cells and was generally modest. nevertheless, these results suggest that s proteins from viruses circulating in ethiopia might harbor mutations that diminish entry into human cells and that are not present in the mers-s proteins studied here. amino acid residues i , l , e and s in the spike protein are unique to ethiopian mers-cov and warrant further analysis. collectively, our results suggest that amino acid substitutions present in the s proteins of north-and west-african mers-cov do not compromise the ability of these viruses to enter human cells. thus, future efforts to understand why north-and west-african mers-cov isolates show reduced replicative potential in human cells should be focused on other aspects of the mers-cov lifecycle than s proteinmediated host cell entry. expression plasmids, based on the vector pcaggs, for vsv-g and mers-s emc were previously described (kleine-weber et al., . the mers-s emc plasmid was used as template for pcr-based mutagenesis to introduce the mutations found in mers-s mo (morocco, camel/morocco/cirad-hku / , genbank: mg . ), ni (nigeria, camel/nigeria/nv / , genbank: mg . ) and bf (burkina faso, camel/burkina faso/cirad-hku / , gen-bank: mg . ) ( table ). in addition, pcr-based mutagenesis was used to equip the constructs with a c-terminal v antigenic tag. the integrity of all sequences was verified using automated sequence analysis. t (human embryonal kidney) and vero (african green monkey kidney) cells were cultivated in dulbecco's modified eagle's medium (dmem; pan biotech). the human colorectal adenocarcinoma cell line caco- was grown in minimum essential media (mem, life technologies). all media were supplemented with % fetal bovine serum (fbs, pan biotech) and x penicillin and streptomycin from a x stock solution (pan biotech). the cells were incubated under humid conditions at °c and % co . for transfection of t cells the calcium-phosphate precipitation method was used. genbank: mg . v a s / n/a a s s / n/a t i s / rbd s y s / n/a r l s / ps(s ') a s s / n/a v l s / n/a mers-s ni camel/nigeria/nv / genbank: mg . v a s / n/a h y s / n/a h y s / n/a l f s / rbd l f s / rbd s y s / n/a a l s / n/a l f s / n/a mers-s bf camel/burkina faso/cirad-hku / genbank: mg . v a s / n/a a s s / n/a h y s / n/a t i s / rbd s y s / n/a a s s / n/a a amino acid position (numbering according to mers-s emc). b subunit / functional domain (if applicable); abbreviations: s = s subunit; s = s subunit; rbd = receptor binding domain, ps(s ') = priming site at the s ' position ( -rsar- ), n/a = not applicable. fig. . s proteins of north/west-and west-african mers-cov isolates from dromedary camels efficiently bind to dpp . t cells expressing the indicated s proteins or no s protein at all (control) were successively incubated with soluble dpp containing a c-terminal fc tag (sol-dpp -fc) and alexafluor conjugated anti-human antibody, before dpp binding to the respective s protein was analyzed by flow cytometry. presented are the combined data of three independent experiments for which sol-dpp -fc binding to mers-s emc was set as %. error bars indicate the standard error of the mean (sem). statistical significance was tested by one-way analysis of variance with sidak's posttest (p > . , not significant, ns; p ≤ . , **). for western blot analysis, anti-v (mouse, : , ; thermofisher scientific), anti-β-actin (mouse, : , ; sigma-aldrich), anti-vsv-m (mouse, : , ; kerafast) were used as primary antibodies and antimouse hrp (horse radish peroxidase) conjugated antibody (goat, : , ; dianova) was used as secondary antibody. antibodies were diluted in phosphate buffered saline [pbs] containing . % tween [pbs-t] supplemented with % skim milk powder. for flow cytometry, a recombinant fusion protein of the ectodomain of dpp fused to the fc fragment of human immunoglobulin (sol-dpp -fc, : , acrobiosystems) and an alexaflour -conjugated anti-human antibody (goat, : ; thermofisher scientific) were used (ligand and antibody were diluted in pbs containing % bovine serum albumin). for analysis of s protein expression, t cells were transfected with expression plasmid for mers-s proteins harboring a c-terminal v tag, as described (kleine-weber et al., , ). to investigate mers-s incorporation into vsvpp, equal volumes of supernatants containing vsvpp bearing s proteins with v tag were centrifuged through a % sucrose cushion at . g for min. subsequently, cells and vsvpp pellets were lysed and analyzed by immunoblot, following an established protocol (kleine-weber et al., . dpp binding was analyzed as described (kleine-weber et al., ) . in brief, t cells were transfected with expression plasmids for mers-s proteins and empty plasmid as negative control. at h posttransfection, the cells were washed with pbs, pelleted and resuspended in pbs containing % bsa and soluble human dpp -fc fusion protein at a final dilution of : . after incubation for h at °c, the cells were washed and incubated with alexafluor -conjugated anti-mouse antibody at a dilution of : . finally, the cells were fixed with % paraformaldehyde and analyzed by flow cytometry using an lsr ii flow cytometer and the facs diva software (both bd biosciences). fig. . host cell entry driven by the s proteins of north/west-and west-african mers-cov isolates from dromedary camels is robust. t, t transfected to express dpp , vero and caco- cells were inoculated with equal volumes of rhabdoviral transduction vectors harboring the indicated s proteins or no s protein (control). at h posttransduction, the activity of the virus-encoded luciferase, which served as an indicator for transduction efficiency, was measured in cell lysates. presented are the combined data of three independent experiments for which transduction mediated by mers-s emc was set as %. error bars indicate sem. statistical significance was tested by one-way analysis of variance (anova) with sidak's posttest (p > . , ns; p ≤ . , **; p ≤ . , ***). transduction vectors based on a replication-deficient vsv (berger rentsch and zimmer, ) and pseudotyped with the indicated viral glycoproteins (vsvpp) were generated according to a published protocol (kleine-weber et al., . target cells were transduced with equal volumes of supernatants containing vsvpp and transduction efficiency was quantified at h posttransduction by measuring the activity of virus-encoded firefly luciferase in cell lysates as previously described (kleine-weber et al., . cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt hospital outbreak of middle east respiratory syndrome coronavirus a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels mers coronaviruses from camels in africa exhibit region-dependent genetic diversity antibodies against mers coronavirus in dromedary camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity hospital-associated middle east respiratory syndrome coronavirus infections detection of distinct mers-coronavirus strains in dromedary camels from kenya mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization structure, function, and evolution of coronavirus spike proteins mers-cov antibodies in humans molecular basis of binding between novel human coronavirus mers-cov and its receptor cd hospital-associated middle east respiratory syndrome coronavirus infections risk factors for mers coronavirus infection in dromedary host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study genetic evidence of middle east respiratory syndrome coronavirus (mers-cov) and widespread seroprevalence among camels in kenya seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase middle east respiratory syndrome coronavirus (mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors thank gert zimmer and andrea maisner for providing the replication-deficient vsv vector for pseudotyping and the vero cell line, respectively. this work was supported, including the efforts of stefan pöhlmann, by the bundesministerium für bildung und forschung within the network project rapid (risikobewertung bei präpandemischen respiratorischen infektionserkrankungen; ki d). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord- -xf ukinp authors: al-abdallat, mohammad mousa; payne, daniel c.; alqasrawi, sultan; rha, brian; tohme, rania a.; abedi, glen r.; nsour, mohannad al; iblan, ibrahim; jarour, najwa; farag, noha h.; haddadin, aktham; al-sanouri, tarek; tamin, azaibi; harcourt, jennifer l.; kuhar, david t.; swerdlow, david l.; erdman, dean d.; pallansch, mark a.; haynes, lia m.; gerber, susan i. title: hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description date: - - journal: clinical infectious diseases doi: . /cid/ciu sha: doc_id: cord_uid: xf ukinp background: in april , the jordan ministry of health investigated an outbreak of lower respiratory illnesses at a hospital in jordan; fatal cases were retrospectively confirmed by real-time reverse transcription polymerase chain reaction (rrt-pcr) to be the first detected cases of middle east respiratory syndrome (mers-cov). methods: epidemiologic and clinical characteristics of selected potential cases were assessed through serum blood specimens, medical record reviews, and interviews with surviving outbreak members, household contacts, and healthcare personnel. cases of mers-cov infection were identified using us centers for disease control and prevention serologic tests for detection of anti–mers-cov antibodies. results: specimens and interviews were obtained from subjects. seven previously unconfirmed individuals tested positive for anti–mers-cov antibodies by at least of serologic tests, in addition to fatal cases identified by rrt-pcr. the case-fatality rate among the total cases was %. six subjects were healthcare workers at the outbreak hospital, yielding an attack rate of % among potentially exposed outbreak hospital personnel. there was no evidence of mers-cov transmission at transfer hospitals having acceptable infection control practices. conclusions: novel serologic tests allowed for the detection of otherwise unrecognized cases of mers-cov infection among contacts in a jordanian hospital-associated respiratory illness outbreak in april , resulting in a total of test-positive cases. serologic results suggest that further spread of this outbreak to transfer hospitals did not occur. most subjects had no major, underlying medical conditions; none were on hemodialysis. our observed case-fatality rate was lower than has been reported from outbreaks elsewhere. in april , the jordan ministry of health (jmoh) investigated a cluster of suspected pneumonia cases among healthcare personnel, of which were fatal, at a hospital in the city of zarqa [ ] . despite testing for multiple potential pathogens, the investigation did not identify a known etiology for these infections. following the discovery of middle east respiratory syndrome coronavirus (mers-cov) in september [ ] , specimens from the fatal cases in jordan were retrospectively tested and both yielded positive results for mers-cov by real-time reverse transcription polymerase chain reaction (rrt-pcr), and were reported to the world health organization (who). these were the first confirmed human cases of infection with this emergent virus, which continues to appear as sporadic cases and clusters internationally, and which is now the focus of worldwide public health investigation and response [ , ] . using newly developed serologic assays to determine mers-cov antibody responses among case contacts in this outbreak, epidemiologists from the jmoh, us centers for disease control and prevention (cdc), and regional partners conducted a retrospective seroepidemiologic investigation to ( ) confirm whether surviving outbreak members had presence of antibodies to mers-cov, ( ) ascertain whether viral transmission occurred among household contacts or to other healthcare personnel, and ( ) describe the clinical features of all detected mers-cov infections in jordan. we interviewed and collected serum specimens from available members of the initial outbreak (who were admitted to the focal outbreak hospital during the period from march to april with fever and dry cough, and with radiological evidence of pneumonia), their household contacts (who reported usually sleeping under the same roof as a defined outbreak member during february-april ), a sample of healthcare personnel from medical institutions that admitted outbreak subjects (nonsystematic enrollment, with preference toward those reporting close contact with outbreak members), and field investigators from the jmoh. hospitalized subjects meeting the initial outbreak case definition were subsequently transferred from the focal outbreak hospital to other hospitals in amman. participating healthcare personnel were employed at one of these hospitals or at jmoh during february-april . epidemiologic data were obtained through medical record reviews and personal interviews during our may investigation. interviews were conducted in arabic, and documented contact history (with outbreak members, household members, visiting travelers, and animals) and occupational exposures. we conducted medical record reviews and key informant interviews with clinicians who provided medical care to patients with suspected infection and heads of infection control units at each medical institution and at the jmoh. informed consent was obtained prior to serum collection and interviews. as a public health response to a disease outbreak, this investigation did not require institutional review board review. all work with live mers-cov was done in cdc biosafety level (bsl- ) containment facilities in atlanta, georgia. serum samples were inactivated using × rads γ-irradiation and stored at − °c until use. to maximize specificity, we defined mers-cov antibody positivity as subjects having correlated, positive laboratory results from the hku . n screening enzyme-linked immunosorbent assay (elisa), as well as confirmed positive results by either the mers-cov immunofluorescence assay (ifa) or the mers-cov microneutralization assay (mnt) (supplementary table ). an initial indeterminate test result was recorded for those subjects having only a single, uncorrelated positive test result. antibody detection by hku . nucleocapsid elisa and mers-cov ifa and mnt genetic sequencing data indicate that mers-cov is a β-coronavirus (subgroup c) similar to the bat covs hku and hku . the recombinant bthku . nucleocapsid proteinbased elisa was developed by the cdc to detect the presence of antibodies that cross-react with the hku . n protein in serum samples from possible mers cases. if cross-reactive antibodies were detected in serum samples, then confirmation of mers-specific antibodies was determined by either mers-cov mnt or ifa. pi-batcov hku . nucleocapsid (n) gene in pet- b (+) plasmid was provided by dr susanna lau, university of hong kong. his-tagged recombinant protein was expressed in escherichia coli and purified by metal affinity chromatography. recombinant hku . n protein indirect elisa was developed using a modified version of the severe acute respiratory syndrome (sars) cov n elisa described by haynes et al [ ] . sera were considered positive when the optical density (od) values were at or above the . cutoff value (mean absorbance at nm of sera from us blood donors plus standard deviations). the overall specificity of the assay was determined after screening serum samples from donors in the united states and the middle east and persons with other non-mers respiratory infections (eg, human coronavirus [hcov] oc , hcov- e, sars-cov, hcov-nl , rhinovirus, human metapneumovirus, h n ). the assay specificity was . % ( / ). serum from hku human serum was not available for evaluation; however, hku mouse hyperimmune serum did not cross-react with the hku . n protein. at a screening dilution of : , sera with od values at or near the cutoff were titered with serial -fold dilutions ( : - : ) and further evaluated using mers-cov (hu/jordan-n / ) (genbank kc . ) ifa and mnt. indirect immunofluorescence was performed by screening sera at a dilution of : or : on paraformaldehyde-fixed, acetone-methanol-permeabilized, mers-cov-infected oruninfected control vero cells. the source of the positive control for this assay was a serum sample from a patient infected with mers-cov hu/england-n / ( provided by m. zambon, public health england). antihuman immunoglobulin (ig) g, igm, and iga fluorescein isothiocyanate conjugate was used and specific fluorescence was detected under an immunofluorescence microscope. a positive result was scored when fluorescent intensity equaled or was higher than that of the positive control. a weakly positive result was scored when fluorescent intensity was lower than that of the positive control. serum samples were tested for the presence of neutralizing antibodies to mers-cov using a modified mnt method described for sars-cov [ ] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell monolayer lysis in at least of the triplicate wells. controls were included for each mnt assay performed, including the input virus back titration and mock-infected cells. all assay results were confirmed in separate assays, and representative data are presented. tests of statistical significance were performed between the mers-cov antibody-positive and -negative subjects, including fisher exact test and χ tests for categorical variables using sas software version . (sas institute, cary, north carolina). serologic specimens and interviews were obtained from subjects. we obtained serologic specimens and data from of the ( %) surviving members meeting the initial outbreak case definition; the remaining subjects were unable to be interviewed ( member was lost to follow-up and did not consent) ( figure ). we also enrolled household contacts and subjects who did not meet the initial outbreak case definition who worked in healthcare and allied professions. among the healthcare personnel interviewed, % were nurses, % were physicians, and the remaining were allied health professionals; approximately half were employed at the focal outbreak hospital. seven of the subjects tested positive for anti-mers-cov antibodies by both hku . elisa and ifa (table and supplementary figure ) , and all but also had detectable neutralizing antibody titers as determined by mnt. the subject who did not have detectable neutralizing antibodies was testpositive both by hku . n elisa and by a confirmative ifa. demographic and epidemiologic comparisons of seropositive and seronegative subjects are provided in supplementary table . sera from the fatal cases (designated outbreak subjects and ) having positive rrt-pcr tests were also tested by the described serology tests. a serum sample from outbreak subject (taken days after onset of respiratory symptoms) was positive by hku . n elisa and ifa and had detectable mers-cov neutralizing antibodies. two serum specimens from outbreak subject (collected and days after onset) were negative for anti-mers-cov antibodies. of the subjects found to be positive for anti-mers-cov antibodies during this investigation, were surviving members of the initial outbreak group and was previously unrecognized. thus, including the fatal cases previously detected and reported, a total of individuals in this outbreak had evidence of mers-cov infections by acute rrt-pcr tests (n = ) or convalescent antibody tests (n = ). the case-fatality rate among all test-positive subjects was % ( of ). we documented that each serologic test-positive subject had unprotected mers-cov exposure(s) to at least rrt-pcr test-positive subject. an additional subjects had single positive test results by either hku . n elisa or ifa, but their mers-cov antibody status was considered indeterminate because both tests were not positive (table ) . we obtained specimens and data from a total of healthcare personnel who worked during february-april, , representing a majority of intensive care (intensive care unit [icu] and coronary care unit [ccu]) personnel at the outbreak hospital as well as other personnel having close contact with initial outbreak investigation members ( figure ). these included surviving outbreak members who were healthcare personnel at the focal outbreak hospital and were not lost to follow-up, other personnel at the focal outbreak hospital, personnel at transfer hospital a, personnel at transfer hospital b, and jmoh's outbreak investigators. of the healthcare personnel at the focal outbreak hospital who survived and the who died, ( %) had cases of mers-cov. our investigation provided no evidence of mers-cov infections or transmission events among personnel at the receiving transfer hospitals, even though some patients were transferred temporally close to their symptom onset dates. interviews with surviving subjects and family members revealed that transmission opportunities among healthcare personnel were not restricted to the workplace. we obtained serologic specimens from members of households, including those from the initial outbreak group and another subjects who had resided in those outbreak member abbreviations: elisa, enzyme-linked immunosorbent assay; ifa, immunofluorescence assay; mers-cov, middle east respiratory syndrome coronavirus; mnt, microneutralization titer. a outbreak member was lost to follow-up, and outbreak member did not consent. outbreak members and were previously laboratory-confirmed positive by real-time reverse transcription polymerase chain reaction (rrt-pcr) and died. serum samples from outbreak members and were collected prior to death and stored. b serum specimens with optical density (od) values ≥ . at a : dilution against hku . n elisa were considered to be positive. specimens were further titered against hku . n at : , : , : , and : dilutions. the antibody titer was taken to be the highest antibody dilution above the cutoff od that yielded a ratio of the absorbance of the positive serum and negative serum (p/n) > . the value is the reciprocal of the dilution. c serum specimens that were positive by hku . n elisa were screened at either : or : by indirect ifa using mers-cov_jordan-infected vero cells. h outbreak members conformed to the original outbreak definition; however, some were retrospectively determined to be mers-cov test negative. they were part of the original, defined outbreak that our investigation used to trace a priori contacts and exposures, so this descriptive title is retained. i hku . n elisa od values for serum specimens from outbreak members , , and and from healthcare personnel were near the assay cutoff od value and rescreened by serial dilution. these serum samples were initially weakly positive by ifa and considered initially indeterminate. upon rescreen by ifa, the samples were determined to be negative for the presence of mers-cov antibodies. j although outbreak member was positive for mers-cov by rrt-pcr, his sera were antibody negative. presumably, this subject died before an antibody response was detectable. this case is considered to be confirmed by current who mers-cov diagnostic guidelines. households during the outbreak period. one household was lost to follow-up, and did not consent for participation. from one of these households was the symptomatic wife of an initial outbreak investigation member who tested positive for mers-cov antibodies. twelve household subjects were children < years old, all of whom were serologically test negative. a summary of underlying conditions for test-positive subjects, including the fatal cases initially identified by rrt-pcr (outbreak members and ), is presented in table . of the testpositive subjects, % were male, with a median age of years (range, - years) at illness onset. we found no evidence of underlying immunodeficiency or immunosuppressant medications/therapies among any of these subjects. one subject had an atrial septal defect, had a history of hypertension, were smokers at the time of illness, and reported a pregnancy of months' gestation. although diabetes mellitus has been observed as a potential risk factor for mers-cov [ ] , none of the subjects reported here had a prior diagnosis of diabetes mellitus and, based on serum glucose values taken during their hospitalizations, none had indications of undiagnosed diabetes mellitus. the most common presenting symptoms, as documented in medical charts, included fever ( %), cough ( %), dyspnea ( %), chest pain ( %), and malaise ( %). eight subjects presented for hospital care a median of days after symptom onset (range, - days). of these patients, ( %) had cough, ( %) had documented fever (temperature (≥ . °c), ( %) had dyspnea, ( %) had chest pain, and ( %) had malaise at some point during their disease course. less common symptoms included chills ( %), wheezing ( %), and diarrhea, vomiting, sore throat, palpitations, and confusion ( % each). seven subjects had abnormal chest radiographic findings reported within days of presentation, and of those had bilateral findings. of the remaining subjects with initial unilateral findings, went on to develop bilateral infiltrates later in their hospitalization, documented either by chest radiography or computed tomography (ct). one subject (outbreak member ) received an initial diagnosis of pericarditis, and a ct scan with abnormal pulmonary findings was reported days later ( table ) . seven of the subjects ( %) who presented to medical care were admitted; refused admission. six subjects ( %) required respiratory support with at least supplemental oxygen, and subjects ( %) received intensive care (in ccu or icu), but only the ( %) patients who died required mechanical ventilation, of which patient also required pressor support (dopamine and norepinephrine) for cardiorespiratory failure. complications among hospitalized subjects were also limited to the patients who died, of whom had hyperkalemia with associated ventricular tachycardia, disseminated intravascular coagulation, and eventual cardiac arrest; the other had pericarditis, pericardial and pleural effusions, and supraventricular tachycardia late in the course of illness. although leukopenia (< . × /l) was observed in subjects, lymphopenia (< . × /l) was observed in of the subjects who had documented complete blood counts with differentials ( %). elevated leukocyte counts (> × /l) were observed during the course for subjects ( %), both of whom died. these subjects also had laboratory abnormalities consistent with multiorgan system failure late in the course of disease. these included evidence of elevated alanine aminotransferase and aspartate aminotransferase (> u/l) and significant coagulopathy with an international normalized ratio of > . , as well as thrombocytopenia (< × /l). in addition, the subjects who died had elevated serum creatinine measurements (≥ µmol/l) on the day of their deaths. a third case had an isolated elevated creatinine measurement, but had a subsequent normal value the following day. no patient received hemodialysis (table ) . outbreak member died days after onset of symptoms (on day of hospitalization) and outbreak member died days after onset of symptoms (on day of hospitalization). the remaining subjects survived, and the who were hospitalized were discharged following a median of days (range, - days). despite having respiratory symptoms, the pregnant household subject did not seek medical care due to concerns regarding receiving chest radiography and medications. this pregnancy resulted in stillbirth during the course of her illness [ ] . surviving subjects and the family members of deceased patients reported that contact with animals was rare in this urbanized area, and no contact with camels was identified among subjects having early symptom onsets. furthermore, none of the subjects had traveled to, or had received visitors from, the arabian peninsula shortly prior to symptom onset. at the focal outbreak hospital, there were no physical barriers between ccu and icu beds, spaced approximately meters, with the exception of cloth drapes in the ccu. isolation or negativepressure rooms were not present, and infection control compliance issues were reported during the outbreak. infection control insufficiencies were not noted at the receiving transfer hospitals. we used novel serologic assays to determine antibody responses of subjects from a mers-cov outbreak investigation in jordan, including the earliest cases of this emerging virus yet discovered. in addition to fatal cases confirmed by rrt-pcr and reported to who, we discovered previously unconfirmed and unreported mers-cov infections. detection of these additional antibody-positive subjects, including healthcare personnel from the focal outbreak hospital and a family contact of antibody-positive subject, and the establishment of contacts with mers-cov infected subjects when potentially infectious, suggests that human-to-human transmission of mers-cov occurred. although community exposures were possible, healthcare-associated transmission was a plausible explanation for healthcare personnel infections. mers-cov infections were not detected among healthcare personnel at a transfer hospital having better adherence to infection control measures. compared with published descriptions of saudi arabian and french cases [ ] [ ] [ ] [ ] , among the total jordanian cases identified through our collaborative investigation, subjects were younger and had fewer underlying medical conditions, and there was a lower case-fatality rate. although all subjects with mers-cov infection in our investigation had acute respiratory illnesses during the outbreak period, % of those who were infected survived. most subjects had no underlying medical conditions and none were on hemodialysis or had indications of diabetes mellitus. one newly detected subject, who was a household contact, did not seek medical care. our data support the probability that, in outbreak settings, infections may remain undetected among subjects who have mild symptoms, lack predisposing conditions, or have barriers to accessing appropriate diagnostic care. therefore, the true mers-cov case-fatality rate may be lower than that based on symptomatic, hospitalized cases alone. the presenting symptoms we observed were largely consistent with those of previously described mers-cov cases [ ] [ ] [ ] [ ] and included fever with respiratory symptoms such as cough and dyspnea, and associated infiltrates on chest radiography. on initial presentation, many subjects did not have evidence of bilateral pneumonia. although gastrointestinal symptoms such as vomiting and diarrhea were documented for subjects, we did not observe these as presenting symptoms, as they were in saudi arabian and french cases. once hospitalized, lymphopenia, a prominent laboratory feature among previously described cases, was observed in the majority of our subjects. however, other laboratory abnormalities observed in previous reports, such as thrombocytopenia, were limited mostly to the fatal cases late in the course of illness, consistent with multiorgan system failure. also, unlike previously reported cases, renal failure was not a prominent clinical feature among our subjects, as renal dysfunction was observed only in the fatal cases on the day of death. rapid isolation of patients with suspected mers-cov and rigorous infection control practices at the receiving transfer hospitals may have been important in preventing transmission at these locations. hospitals should have established policies and procedures for the rapid identification of suspected or known mers-cov cases and implementation of appropriate infection prevention measures. the cdc recommends standard, contact, and airborne precautions for the management of hospitalized patients with known or suspected mers-cov infection [ ] . one jordanian patient was initially hospitalized with pericarditis, a manifestation similar to mers-cov case occurring in the kingdom of saudi arabia [ ] . although this jordanian patient's serologic specimens tested negative for mers-cov antibodies at periods throughout his hospital stay, acute specimen collected several days before death was confirmed positive for the virus by rrt-pcr. these laboratory findings and the patient's exposure in the ccu, where he was situated in the bed directly next to another patient with rrt-pcr-confirmed mers-cov, collectively suggest the likelihood that the patient was nosocomially infected with mers-cov and died before an antibody response was detectable. based on the knowledge of sars-cov antibody responses, igg and neutralizing antibodies to sars-cov peaked months following a patient's recovery from acute infection [ ] . antibody levels did decline over time, but detectable sars-cov neutralizing antibodies persisted up to years after onset of sars-cov symptoms [ , ] . approximately months had passed between our may investigation and the april outbreak. although this was sufficient time for infected subjects to produce an antibody response to mers-cov, the role of waning immunity on the antibody response [ ] and whether persistence of these antibodies is important for protection from reinfection remain unclear. we implemented a rigorous case definition based on an elisa-positive result plus at least correlating assay result to maximize specificity. infections with sars-cov triggered humoral and cellular immune responses in all studied humans [ ] , and high titers of neutralizing antibodies were observed in response to sars-cov infections, but such characteristics of the mers-cov immunologic response remain unknown. as for those indeterminate laboratory findings among subjects with documented mers-cov exposure(s) but having only an elisa-positive result and mild or absent respiratory symptoms, it is possible that the viral exposure to these subjects did not trigger a long-lasting ifa-or mnt-recognizable immune response. because obtaining appropriate lower respiratory specimens from subjects having mild or asymptomatic infections is challenging, the use of serologic assays to identify otherwise undetected cases of mers-cov has been demonstrated to be a useful tool. serological surveys have been conducted in retrospective case investigations around instances of mers-cov importations in europe [ ] , as well as for establishing estimates of mers-cov seroprevalence among populations at risk [ ] . further validation of serologic assays and assessments of how they complement rrt-pcr testing is needed. our investigation was unable to find evidence of any exposure (either zoonotic contacts, human contacts from the arabian peninsula, or among hospitalized contacts preceding the earliest symptomatic cases) that might explain the origin of the virus. the precise route(s) of mers-cov transmission remains unclear overall, but several mers-cov sequences have been identified in dromedary camel nasal secretions, including one that is indistinguishable from that found in infected humans [ ] . in conclusion, the jordan respiratory illness outbreak in april resulted in a total of test-positive mers-cov subjects. the source of the virus in these earliest known mers-cov cases remains unknown. compared with other reports, the improved survivability we observed is perhaps related to the youth and relative lack of underlying illnesses among the subjects we investigated. infection control practices at both transfer receiving hospitals may have been important in preventing mers-cov transmission in those facilities. since the discovery of the mers-cov, enhanced surveillance for severe acute respiratory illnesses in jordan has been implemented. international severe acute respiratory infection surveillance, collaborative investigations, and vigilance among healthcare providers are necessary components for addressing and preventing the further spread of mers-cov worldwide. supplementary materials are available at clinical infectious diseases online (http://cid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. epidemiological findings from a retrospective investigation 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patients and their clinical significance neutralizing antibodies in patients with severe acute respiratory syndrome associated coronavirus infection sars immunity and vaccination contact investigation for imported case of middle east respiratory syndrome lack of mers coronavirus neutralizing antibodies in humans middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia author contributions. d. c. p. had full access to all the data in the study and had final responsibility for the decision to submit for publication.disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc.financial support. this work was supported by the us global disease detection operations center outbreak response contingency fund.potential conflicts of interest. all authors: no potential conflicts of interest.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -hfgmmuy authors: alenazi, thamer h.; arabi, yaseen m. title: severe middle east respiratory syndrome (mers) pneumonia date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: hfgmmuy middle east respiratory syndrome (mers) is a viral respiratory infection, which ranges from asymptomatic infection to severe pneumonia and multiorgan failure, caused by a novel coronavirus named middle east respiratory syndrome coronavirus (mers-cov). majority of cases have been reported from saudi arabia. mers cases occur as sporadic cases or as clusters or hospital outbreaks. dromedary camels are thought to be a host for mers-cov. direct contact with dromedary camels within days prior to infection was identified as an independent risk factor for mers. diagnosis of mers is based on a positive real-time reverse transcriptase polymerase chain reaction (rrt-pcr), obtained from a respiratory specimen. the mainstay of management of mers-cov infection is supportive care. there is no specific antiviral therapy for mers-cov infection at present, although several modalities of treatment options have been examined or are under investigation. middle east respiratory syndrome (mers) is a viral respiratory infection, which ranges from asymptomatic infection to severe pneumonia, caused by a novel coronavirus named middle east respiratory syndrome coronavirus (mers-cov). coronaviruses are a family of viral pathogens that could cause animal and human disease. mers-cov is closely related to the severe acute respiratory syndrome coronavirus (sars-cov) but from a different lineage. the objective of this chapter is to describe the epidemiology, virology, clinical manifestations, management and prevention of mers. between september , until the end of may , the world health organization (who) has been notified of laboratoryconfirmed case of mers-cov infection from countries with associated deaths resulting in a case fatality rate of %. saudi arabia has been the major reporting country with a total number of cases and deaths (a case fatality rate of . %) ( table ) (world health organization, , mar ) . the virus was first isolated in june from respiratory specimens of a saudi patient from jeddah, saudi arabia, who presented with severe pneumonia that progressed to acute respiratory distress syndrome (ards), renal failure, multi-organ failure and eventually lead to death (zaki et al., ) . in the same week, a patient with a recent history of travel to the united kingdom (uk) in august , with cases across three hospitals, and seven deaths ( . % case fatality rate) (payne et al., ) . smaller outbreaks continued to occur in - although the number of patients and the magnitude of the outbreaks were less compared to earlier years, presumably due to better infection control practices and earlier identification of cases. transmission of mers-cov in humans occurs through animal-to-human transmission or, human-to-human transmission in the community. additionally, nosocomial transmission of mers-cov occurs frequently. all transmission described up-to-date, occurred in residents in or travelers to the arabian peninsula, or are traced to contact with patients with a history of recent travel to the arabian peninsula. animals seem to play an important role in the transmission of the mers-cov. earlier studies have suggested that bats might be the potential reservoir of mers-cov. this hypothesis that was based on the close proximity of mers-cov-phylogenetically-to tylonycteris bat coronavirus hku (ty-batcov hku ) and pipistrellus bat coronavirus hku (pi-batcov hku ) (woo et al., ) . a study from saudi arabia, a phylogenetically mers-cov identical short gene segment, was detected in a fecal sample of one of the captured bats near the home of a laboratory-confirmed mers-cov patient . however, live mers-cov has never been recovered from bats. further studies are needed to further establish the role of bats in transmission to humans including larger surveillance studies with full viral genome sequencing. epidemiologically, it seems unlikely that bats are the direct source of human cases, since none of the community-acquired laboratory-confirmed mers cases had clear bat exposure. dromedary camels are thought to be a host for mers-cov. direct contact with dromedary camels within days prior to infection was identified as an independent risk factor for mers (gossner et al., ) . camel-human transmission was also suggested in a -year-old, previously healthy man from jeddah, saudi arabia who was admitted to the intensive care unit (icu) with severe mers pneumonia, and died days after admission. the patient had owned a herd of camels and used to visit them daily until days prior to his admission. four out of the nine camels were sick with nasal discharge, week prior to the patient's onset of symptoms. the patient had significant contact with camels' excretions. respiratory specimens from the patient and one of his camels showed identical mers-cov full genome sequencing. moreover, serum antibodies for mers-cov were positive in both the patient and the camel, with the camel seropositivity preceded the patient's seropositivity suggesting that direction of transmission was from the camel to the patient. a large cross-sectional study from saudi arabia identified mers-cov infected patients who had a history of camel contact. the investigators obtained nasal swabs and serum samples from dromedary camels and found that . % of the studied camels were mers-cov polymerase chain reaction (pcr) positive, and . % of them were mers-cov antibodies positive. furthermore, of the full genome sequences of the camel mers-cov were identical to their contacted patients (kasem et al., ) . this data suggests an important role for camels in the transmission of mers-cov. however, in a cohort of patients with laboratory-confirmed mers, camel contact was reported only in patients ( . %), denied by patients ( . %), and not reported in the other patients ( . %) (conzade et al., ) . hospital-based outbreaks and community-based clusters described above suggest strongly that human-human transmission does occur. the transmission was more commonly observed in healthcare-based outbreaks, compared to community clusters. the number of close contacts who got infected by patients with confirmed mers-cov appears to be low, although, it was evident that some patients were spreading the infection to a disproportionally large number of individuals (super spreaders) (hui, ) . this phenomenon was clearly described in more than one outbreak. the first outbreak which identified the super spreader phenomena was the korean outbreak, in which a single imported index case resulted in a total of cases. it was thought that % of transmission in the korean outbreak was linked epidemiologically to five super spreaders (korea centers for disease and prevention, ) the same phenomenon was also described in a large outbreak in riyadh, saudi arabia, where out of the cases, contributed to . % of the transmission (alenazi et al., ) . however, it remains unclear if an asymptomatic individual who carries mers-cov can transmit the virus to others. the first family cluster was reported from riyadh, saudi arabia, where three laboratory-confirmed cases and one probable case were diagnosed, and two out of the four patients died . in a study that investigated index cases of mers and their household contacts, the secondary transmission rate was found to be % ([ % ci, to ] . as described above, transmission was more commonly seen in hospital-based outbreaks compared to family community transmission, particularly in emergency department (ed). this was clearly illustrated in the korean outbreak, where a single imported case had led to a total of cases, of which were nosocomial transmission (kim et al., ) the main identified reasons for hospitals-based transmission were over-crowdedness of ed, late recognition of suspected mers cases and inadequate infection control measures and proper isolation of suspected cases (stone et al., ) . environmental surfaces in hospitals is a potential source of transmission. in one study, a viable mers-cov was detected in out of surface swabs collected from patient's rooms, restrooms and common corridors (kim et al., ) . mers-cov is the sixth coronavirus that affects humans. it lies within the lineage c of the genus betacoronavirus (cov) in the family coronaviridae under the order nidovirales. it has close phylogenetic proximity to two bat coronaviruses, tylonycteris bat cov hku (ty-batcov-hku ) and pipistrellus bat cov hku (pi-batcov-hku ). like the other coronaviruses, it is an enveloped single-stranded rna virus which replicates in the host-cell cytoplasm. the size of its rna genome is approximately kb. it has structural proteins, called the e, m, and n proteins, and membrane protein called the spike (s) protein, which plays an important role in the virus attachment and entry into the host cells. due to the large increase in the number of diagnosed cases in april , there was a concern that mers-cov could have undergone mutation that led to increased virulence or transmissibility of the virus; however, this assumption was proven unlikely (drosten et al., ) . the pathogenesis and histopathology of mers-cov is poorly understood and understudied. post-mortem autopsies were rarely performed on mers patients due to cultural reasons in the arabian peninsula. most of the knowledge we have about the histopathology of mers-cov comes from in vitro, ex vivo, animal experiments and limited post-mortem reports. in a -year-old male, who died of mers-cov infection, post-mortem analysis of histopathology finding of pulmonary and extrapulmonary tissue were examined under transmission electron microscopy which showed necrotizing pneumonia, pulmonary diffuse alveolar damage, acute kidney injury, portal and lobular hepatitis and myositis with muscle atrophic changes. the brain and heart were histologically unremarkable. ultra-structurally, viral particles were localized in the pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells and macrophages infiltrating the skeletal muscles (alsaad et al., ) . in the beginning of the outbreak, the who had proposed a case definition for mers-cov infection for epidemiological purposes, that was last updated on july , . the united states (us) center of disease control and prevention (cdc) and the saudi ministry of health (moh), each had developed a case definition for suspected, confirmed and probable mers-cov infection ( table ) . in one of the earlier outbreaks in saudi arabia, the median incubation period for mers-cov infection was . days ( % ci . - . days) (assiri et al., b) . similarly, in the south korean outbreak, in , the median incubation period was . days ( th percentile . days) (korea centers for disease and prevention, ) . therefore, mers should be suspected in patients presenting with respiratory infection, and residence in or travel to the arabian peninsula within the last days prior to onset of symptoms. most of reported mers patients have been in the adult age group. only pediatric cases were reported, most of which were detected on contact tracing screening ( % were asymptomatic), and among symptomatic cases, presence of comorbidities like congenital disease were commonly present (al-tawfiq et al., ) . the mean age in one of study was . years . in another study, that described the epidemiological, clinical characteristics and demographics of mers-cov infected patients, . % of laboratory-confirmed cases were more than years of age with a median age of years. the male: female ratio was . : . eighty nine percent of patients required icu admission, and the median time to death was days (ranging from to days) (assiri et al., a) . one study from saudi arabia, have compared critically ill mers-cov patients with critically ill non-mers-cov patients, and had found that mers-cov patients tend to be younger, more likely to require mechanical ventilation and had higher mortality . there were eight reported mers-cov infection during pregnancy, from jordan, united arab emirates and saudi arabia, three of them ended with maternal death . in the beginning of the epidemic, the typical presentation of reported mers was severe pneumonia, with acute respiratory distress syndrome (ards) with or without acute kidney injury, but as the surveillance and testing had increased, milder or even asymptomatic cases have been described. in a cohort of patients, with mers-cov infection, the clinical presentation were fever ( %), cough ( %), shortness of breath ( %), myalgia ( %), diarrhea ( %), sore throat ( %), vomiting ( %), abdominal pain ( %) and hemoptysis ( %) (assiri et al., a) . a study that compared critically ill mers-cov infected patients with critically ill patients with non-mers severe acute respiratory infection (sari) found that mers patients were younger than non-mers sari patients (median [q , q ] [ , ] vs [ , ] and were more likely to be males ( . % vs . %) and to be healthcare workers ( . % vs . %). chronic comorbidities were prevalent (any comorbidity, . % in mers sari, . % in non-mers sari). after onset of symptoms, mers-cov patients presented to er with a median of days and admitted to icu after days, which was days longer compared to non-mers-cov sari patients. mechanical ventilation was required for . % of patients with mers-cov patients. at the time of icu admission, patients with mers-cov were more likely to be hypoxemic, compared with non-mers sari patients (ratio of arterial oxygen partial pressure to fractional inspired oxygen-pao /fio : . [ . , ] vs [ , ] ) . respiratory distress syndrome) and direct epidemiologic link with a laboratory-confirmed mers-cov case and testing for mers-cov is unavailable, negative on a single inadequate specimen or inconclusive. a febrile acute respiratory illness with clinical, radiological, or histopathological evidence of pulmonary parenchymal disease (e.g. pneumonia or acute respiratory distress syndrome) that cannot be explained fully by any other etiology and the person resides or traveled in the middle east, or in countries where mers-cov is known to be circulating in dromedary camels or where human infections have recently occurred and testing for mers-cov is inconclusive. an acute febrile respiratory illness of any severity and direct epidemiologic link ( ) many cases of mers present with gastrointestinal manifestations with or without respiratory symptoms. among the critically ill patients, the most described extra-pulmonary manifestations were acute kidney injury and shock (arabi et al., ) . very few patients were reported to have neurological symptoms, in addition to the pneumonia (arabi et al., ) . primary infections were more likely to be severe, as opposed to secondary cases. secondary mers infection tends to cause a milder or asymptomatic disease, however severe disease has been described. secondary cases are more likely to be younger with no comorbidities. asymptomatic infections have bee also described in patients with dromedary camel's contacts who were identified during surveillance (al hammadi et al., ) . mortality rates were reported to be higher in older age group, male gender and patients with comorbidities (assiri et al., a) . numerous cases of mers occurred among healthcare-workers; leading in some of them to severe illness resulting in admission to icu. in a study that examined critically ill healthcare-workers with mers, . % were nurses and % were physicians. . % were having comorbidities, mainly chronic kidney disease ( . %). fever at presentation, was found in / ( . %), cough in / ( . %), and gastrointestinal symptoms in / ( . %). eight out of the ( %) healthcare workers died (shalhoub et al., ) . among all hospitalized patients with severe mers pneumonia, the most commonly observed laboratory abnormalities were lymphopenia ( %), thrombocytopenia ( %) and raised lactate dehydrogenase (ldh) ( %). other abnormalities like leukopenia ( %), lymphocytosis ( %), raised aspartate aminotransferase (ast) ( %), raised alanine aminotransferase (alt) ( %), and raised lactate dehydrogenase ( %) were also observed (assiri et al., a) . in a cohort of critically ill mers-cov patients, leukopenia was observed in . %, thrombocytopenia in . %, raised alt in . %, and raised ast in . % . most of the reported symptomatic cases with severe mers pneumonia had abnormal chest-x-ray. abnormalities ranged from mild to extensive changes. peripheral ground-glass opacities were the most frequently found abnormality on cxr, in studied case (das et al., ) other findings include, unilateral or bilateral airspace opacities, increased broncho-vascular markings, patchy infiltrates, interstitial changes, nodular opacities, reticular opacities, reticulo-nodular shadowing, pleural effusions, and ards pattern. among inpatients who had chest computed tomography scan (ct scan), the most frequent findings were peripheral and bibasilar opacities bilaterally. in patient presenting with severe pneumonia, mers should be suspected based on the presence epidemiologic links (residence or travel from the arabian peninsula especially if there is history of contact with camels, contact with a person infected with mers or working or being treated in a hospital where mers patients are managed). such links should lead to application of appropriate infection control measures (see below) and to initiate diagnostic work up for mers. diagnosis of mers is based on a positive real-time reverse transcriptase polymerase chain reaction (rrt-pcr), obtained from a respiratory specimen. nasopharyngeal or oropharyngeal swab of the upper respiratory tract are often used in patients who are unable to produce lower respiratory samples. however, lower respiratory samples (sputum, endotracheal aspirate, or bronchoalveolar lavage) are preferred as they generally have better yield. in patients with suspected mers, it is recommended to send more than one specimen since a negative test does not exclude the diagnosis. in a cohort of critically ill patients with mers pneumonia, the diagnosis of mers was based on samples from the nasopharynx in of ( %) and from the lower respiratory tract (sputum, endotracheal aspirates, or broncho-alveolar lavage) in of ( %). the diagnosis was established from the first sample in % of patients, from the second sample in % of patients and from to repeat samples in % of the patients. initial negative samples collected before positive ones were predominantly from the upper respiratory tract ( . %) . several serological assays have been used including enzyme-linked immune sorbent assay (elisa) and immunofluorescence assay (ifa), which are typically used for screening, and neutralization techniques which are used for confirmation. a three different, indirect elisa have been developed and validated based on mers-cov nucleocapsid protein (n), spike (s) ectodomain (amino acids - ) and s subunit (amino acids - ) (hashem et al., ) . a single positive serological test, in the absence of positive pcr is considered a probable case, in the setting of suspected mers-cov. however, a four-fold increase in mers-cov antibody titer by neutralization tests is considered a confirmed case. viral pathogens were identified in % of critically ill patients with mers pneumonia which included other coronaviruses, respiratory syncytial virus, and influenza a virus. bacterial co-infections are described in % of critically ill patients with mers pneumonia, with acinetobacter species, pseudomonas species, klebsiella pneumoniae and staphylococcus aureus being the most frequent isolates . there is no specific antiviral therapy for mers-cov infection up to date, although several modalities of treatment options have been tried or are under investigation. the mainstay of management of mers-cov infection is supportive care. patients with suspected severe mers pneumonia-cov infection might have other respiratory pathogens as a cause of their symptoms. therefore, the who recommends starting appropriate empirical antimicrobial therapy as soon as possible, to cover community acquired or nosocomial associated pathogens, based on the presentation from the community or the hospital and based on local epidemiology and guidelines, until the microbiological diagnosis is confirmed. supportive therapy is the mainstay of management of severe mers pneumonia, which includes mechanical ventilation, vasopressor support, and renal replacement therapy if needed. oxygen rescue therapy like extracorporeal membrane oxygenation (ecmo) has been used in patients with refractory hypoxemia. in one case-control study of patients with mers, the rescue use of ecmo compared to a matched control with no-ecmo was associated with reduced in-hospital-mortality ( % compared %) (alshahrani et al., ) . another retrospective study, found that critically ill healthcare workers who died because of mers were more likely to have received ecmo than not, probably because the severity of pneumonia that led to use of rescue therapy, rather than use of ecmo itself (shalhoub et al., ) . corticosteroids have been used frequently in mers patients. a study that accounted for time-varying confounding demonstrated that corticosteroid use was not associated with difference on mortality although it was associated with prolongation of viral rna shedding (arabi et al., b) . data on other human coronaviruses, and in vitro activity of specific therapies were used to identify potential new therapy for mers-cov. examples of those include: combination of ribavirin and interferon, lopinavir-ritonavir, mycophenolate mofetil, convalescent -plasma, and, monoclonal and polyclonal antibodies ( table ) . the efficacy of ribavirin/interferon combination was suggested to be promising in vitro and animal experiments and cell culture. in a study where two cell viral cultures lines grew mers-cov, high concentrations of ribavirin or interferon alpha b were needed to inhibit viral replication, when each of the drugs was used alone, however, comparable inhibition was observed when combing them at a lower concentration (falzarano et al., a) . similar findings were observed in rhesus macaques model of mers-cov infection. among animals who received combination of ribavirin and interferon alpha b hours after inoculation did not develop respiratory symptoms and had no or very minimal chest x-ray findings of infiltrate compared to the control group. also, the treated group had a moderately lower viral genome copies and fewer severe lung histopathological changes (falzarano et al., b) . data on humans are based on retrospective studies. in retrospective cohort of patients with severe mers-cov pneumonia, ribavirin and interferon combination therapy started at median day three after diagnosis, showed improved -day survival, compared to patients who received only supportive therapy, however -day survival was not different between the groups (omrani et al., ) . other retrospective studies showed no difference in mortality between patients treated with ribavirin interferon combination, and patients who received supportive therapy shalhoub et al., ) . the largest cohort study which adjusted for time-varying confounders showed that ribavirin with interferons (alpha a and b and beta a) was not associated with difference in mortality or viral shedding. none of the patients received interferon beta b (arabi et al., ) . lopinavir-ritonavir efficacy was studied in-vitro in animals with severe mers-cov infection, in which it showed favorable outcome (chan et al., ) . there is an ongoing randomized placebo controlled trial evaluating oral lopinavir-ritonavir in combination with subcutaneous interferon beta- b in hospitalized patients with mers (nct ) (arabi et al., a) . the use of passive immune therapy with convalescent plasma was suggested as a potential therapeutic option. a study that examined the feasibility of convalescent plasma therapy for mers was limited by the small pool of donors with sufficient titers of mers-cov antibodies which may be related to the short-lasting immune response . several monoclonal antibody preparations have been developed. humanized bovine transchromosomal polyclonal antibodies against mers-cov have been developed and undergone testing in a phase i trial (beigel et al., ) . a phase ii trial in humans is being planned. mycophenolate mofetil efficacy against mers-cov was suggested in vitro studies. however, it was associated with harm in a marmoset model (chan et al., ) . remdesivir (gs- ) which is the monophosphoramidate prodrug of the c-adenosine nucleoside analog gs- , has recently been reported to inhibit sars-cov, mers-cov and bat-cov, in vitro. it has also been found to be therapeutic and prophylactic in sars-cov infected mouse modules (agostini et al., ) . most of the reported hospital-based outbreaks were attributed to lack of adherence to proper infection control practice, delayed suspected cases identification, and to overcrowded emergency room and inappropriate triage. addressing issues related to infection control practice and proper triaging of patients with suspected mers-cov, had resulted in a decline in the number and the magnitude of hospital outbreaks . the who and us cdc have published guidance for mers prevention in healthcare institutes. as per the who recommendations, patients who have probable or confirmed mers should be under contact and droplet precautions with eye protection. the patient should be under airborne precaution, when performing an aerosol generating procedure (agp) like tracheal intubation or bronchoscopy. the us cdc, on the other hand, recommends contact and airborne precautions for all suspected or confirmed mers-cov patients. viral shedding from respiratory secretions has been found to be at least weeks from onset of symptoms. therefore isolation precaution should not be discontinued until a negative pcr is obtained. patients with suspected or confirmed mers-cov, who does not require admission, can be isolated at home. it is recommended to avoid contact with camels, both direct or indirect contact like consuming raw camel's milk or meat. this is particularly for high risk individuals, such as patients with heart failure, chronic lung disease and immunosuppression. people who have to be in contact with camels should observe infection control precautions, including washing hand before and after contact, and use of appropriate personal protective equipment's (ppe) when dealing of a suspected or confirmed infected camels. it is important to note that the infected camels may not be symptomatic or might only have mild symptoms. the saudi authorities had made certain measures to reduce camel-human transmission, like banning camels in the holy areas, and moving the camels markets outside the cities. the who did not place any travel restriction to any country that have reported mers-cov cases. saudi arabia, where most of the laboratory-confirmed cases have been reported, annually hosts millions of muslims to perform hajj and omrah (pilgrimage), with no documented related cases of mers to date. there were dutch patients who developed mers after returning from hajj, but the two cases were thought to be acquired from a camel market and raw milk consumption rather than human-human transmission during hajj. table summary of treatment options for mers-cov. ribavirin/interferon combination ribavirin/interferon showed efficacy in and in vitro and in a rhesus macaques model. data in humans are based on retrospective studies. the largest cohort that accounted for time-varying confounders did not demonstrate efficacy. there may be differences in efficacy among different interferons, as interferon beta- b has the lowest inhibitory concentrations in vitro falzarano et al. ( a) and falzarano et al. ( b) lopinavir-ritonavir lopinavir-ritonavir showed efficacy in in vitro and in a marmoset model. it is being tested in combination with interferon beta- b in a randomized controlled trial (mers-cov infection treated with a combination of lopinavir /ritonavir and interferon beta- b (miracle), nct ) chan et al. ( ) convalescent plasma the feasibility of the option is limited due to the paucity of donors arabi et al. ( ) monoclonal antibodies several monoclonal antibodies exist with promising efficacy in in vitro and in animal studies polycolonal antibodies demonstrated promising efficacy in in vitro and in animal studies. phase i trial has been completed. plans for phase ii trial are undergoing beigel et al. ( ) mycophenolate mofetil efficacy has been suggested in vitro but harm in a marmoset model chan et al. ( ) remdesivir this new drug has promising efficacy in in-vitro and in animal studies. phase i trial has been completed. phase ii trial is ongoing in ebola survivors agostini et al. ( ) there is no licensed human vaccine for mers-cov till now, however, many experimental candidate vaccines are under development. another approach is to vaccinate camels, as the source of infection for many human cases, and good progress has been made in this area (alharbi, ) . as of end of december , the global case fatality rate for mers-cov infection was reported as . % ( / ). it is thought this number overestimates the case fatality rate of the disease, because milder and asymptomatic cases are likely to be underrepresented in the reported cases. this was suggested in a study that estimated the number of undetected human symptomatic cases to be % (cauchemez et al., ) . in a cohort of mers-cov infected patients, case fatality rate was higher with increasing age (assiri et al., a) . in another study that studied mers-cov infected patients, independent risk factors for mortality were, age more years, underlying cardiac comorbidity or cancer, and healthcare acquisition of the virus 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coronavirus from a man with pneumonia in saudi arabia key: cord- -decr wrd authors: kayali, ghazi; peiris, malik title: a more detailed picture of the epidemiology of middle east respiratory syndrome coronavirus date: - - journal: the lancet infectious diseases doi: . /s - ( ) - sha: doc_id: cord_uid: decr wrd nan the diff erential diagnosis of sepsis or meningitis is broad. third, although the path to commercialisation is underway, it might be challenging. inexpensive, straightforward assays are excellent from public health and global health perspectives, but disruptive new technologies are not always adopted. irrespective of how successful the commercialisation process is, the authors have developed a novel diagnostic test for neisseria meningitidis that is easy to use and provides results within min. in such a timeframe, results can lead to actionable decisionmaking, most likely to halt further diagnostic testing. the niche for lamp in meningococcal disease remains to be established, but the fact that the technique allows diagnostic confi rmation at the treating hospital rather than a reference laboratory is a good start. most cases were in countries in the arabian peninsula, with sporadic travel-related infections occurring elsewhere. although clusters of human-to-human transmission of mers-cov are well documented, transmission originates from zoonotic events, and therefore identifi cation of the animal sources of mers-cov is a priority. genetically, mers-cov is related to bat coronaviruses from china, saudi arabia, europe, and africa. , - on the basis of the evidence so far, none of the bats from these countries are likely to be the reservoir for the virus. evidence is accumulating that dromedary camels are a natural host of mers-cov. serological fi ndings suggest that more than % of adult dromedaries in the middle east and africa are seropositive for mers-cov. the virus isolated from dromedaries has spike proteins with conserved receptor-binding domains for the human dipeptidyl peptidase- receptor, , and mers-cov has been detected in camels that were in close contact with people with middle east respiratory syndrome. , the role of camels as a source of human infection with middle east respiratory syndrome is controversial because many people who have the disease have had no obvious association with camels. case-control studies that aim to defi ne risk exposures of index patients have not yet been done, and an investigation of mers-cov seroprevalence in people in contact with camels yielded negative results (ie, no association was noted). , [ ] [ ] [ ] the study by muller and colleagues is the fi rst population-based, seroepidemiological investigation of mers-cov infection in an area where zoonotic transmission is sustained. the investigators used a cross-sectional design that tested serum samples from about people in saudi arabia whose age and sex distribution was similar to that of the general population. by use of a rigorous serological testing algorithm, the investigators identifi ed mers-cov antibodies in ( · %, % ci · - · ) of samples from the general population. men had a signifi cantly higher proportion of infections ( [ · %] of ) than did women (two [ · %] of ; p= · ), and more infections were noted in central rural areas than in coastal provinces ( [ · %] of vs one [ · %] of ; p= · ). the investigators also obtained samples from camel shepherds and slaughterhouse workers, and showed that seroprevalence of mers-cov was - times higher in camel-exposed individuals than in the general population. the fi ndings from this study suggest that young men in saudi arabia who have contact with camels in cultural or occupational settings are becoming infected with mers-cov, often without being diagnosed, and might proceed to introduce the virus to the general population in which more severe illness triggers testing for the virus and disease recognition. this hypothesis could account for cases of middle east respiratory syndrome without previous animal exposure. when the data from muller and colleagues are put into context with those from studies in camels, a clearer picture of the epidemiology of mers-cov emerges (fi gure). camels seem to be a natural host for mers-cov and transmission within camel herds is well established. however, several questions need to be resolved. the route for camel-to-human transmission is unclear, and could be from one or more of the following: direct contact with infected animals, or consumption of milk, urine, or uncooked meat-all practices that are common in aff ected countries in the middle east. an intermediate host could also transmit mers-cov between camels and human beings. finally, whether dromedaries are the natural reservoir or an amplifi er host is a hypothesis that is open to further investigation. muller and colleagues' study is the fi rst step along the way to addressing these questions. human papillomavirus (hpv) vaccination programmes have been in use in many countries since following licensing of the bivalent and quadrivalent hpv vaccines. clinical trials have shown that hpv vaccines have more than % effi cacy in preventing high-grade cervical lesions caused by human papillomavirus types and , , which are the two hpv types known to cause - % of cervical cancers and large proportions of other anogenital and oropharyngeal cancers. the quadrivalent vaccine has shown similar effi cacy in the prevention of anogenital warts caused by hpv types and . furthermore, both vaccines have shown lower-but still substantial-effi cacy against related non-vaccine oncogenic human papillomavirus types. , therefore, since rollout of these vaccination programmes began, there has been great anticipation to see whether these promising trial results will translate into substantial reductions in hpv-related disease at the population level. in the lancet infectious diseases, mélanie drolet and colleagues present the fi ndings of a timely systematic review and metaanalysis assessing the population-level and herd eff ects of hpv vaccination programmes so far. the key fi ndings of the study suggest that in the preto-post-vaccination period covered by the included studies, hpv type and infections and anogenital warts have decreased by more than % in girls (< years of age) when high female vaccination coverage (> %) was reported. the authors also report signifi cant reductions in hpv types , , and infections in this same group (relative risk [rr] · [ % ci · - · ]) and in anogenital warts in boys younger than years of age ( · [ · - · ]) and in women - years of age ( · [ · - · ]), which provides strong evidence of both cross-protection and herd eff ects. smaller, but still signifi cant, reductions in hpv types and infection (rr · [ % ci · - · ]) and anogenital warts ( · [ · - · ]) also occurred in girls and young women in countries with lower vaccination coverage (< %), but there was no evidence to suggest either cross-protection or herd immunity in these countries. drolet and colleagues aimed to report the populationlevel eff ect of vaccination and, although the results are very encouraging, we need to keep in mind that their study included both clinic-based and population-based studies, of which the clinic-based studies might not be representative of the underlying population. additionally, vaccine coverage for the outcome of hpv prevalence was based on vaccine uptake in the study, which in some cases was substantially larger than that in the underlying population. although the authors did several subanalyses to investigate heterogeneity, we think it would have been helpful to do an additional subanalysis stratifying the data by clinic-based versus population-based study. disappointingly, all the included studies were from high-income countries (nine countries in total) and we are therefore none the wiser regarding the eff ect of vaccination in low-income countries in which the burden of hpv-associated disease is highest. this report shows that the maximum population-level eff ect of vaccination is achieved through delivery at a young age with high vaccination uptake rate. a result we found striking is that the greatest eff ect of vaccination, in both male and female individuals, was recorded for countries such as australia, new zealand, and the uk that have school-based vaccination programmes. this fi nding is corroborated by the results of another recent systematic review that assessed the eff ect of the quadrivalent vaccine on anogenital warts. in this study, mariani and colleagues reported that the greatest reductions in anogenital warts were achieved in countries with high vaccination uptake rates, mostly through school-based vaccination programmes, although some non-school-based programmes also achieved high vaccination uptake rates. one concern with school-based programmes is the presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional serological study isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers-cov): summary of current situation, literature update and risk assessment human betacoronavirus c emc/ -related viruses in bats, ghana and europe close relative of human middle east respiratory syndrome coronavirus in bat mers-related betacoronavirus in vespertilio superans bats middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd mers coronaviruses in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during lack of middle east respiratory syndrome coronavirus transmission from infected camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries key: cord- - plpi i authors: lassandro, giuseppe; palladino, valentina; amoruso, anna; palmieri, viviana valeria; russo, giovanna; giordano, paola title: children in coronaviruses’ wonderland: what clinicians need to know date: - - journal: mediterr j hematol infect dis doi: . /mjhid. . sha: doc_id: cord_uid: plpi i human coronaviruses (hcovs) commonly cause mild upper-respiratory tract illnesses but can lead to more severe and diffusive diseases. a variety of signs and symptoms may be present, and infections can range in severity from the common cold and sore throat to more serious laryngeal or tracheal infections, bronchitis, and pneumonia. among the seven coronaviruses that affect humans (sars)-cov, the middle east respiratory syndrome (mers)-cov, and the most recent coronavirus disease (covid- ) represent potential life-threatening diseases worldwide. in adults, they may cause severe pneumonia that evolves in respiratory distress syndrome and multiorgan failure with a high mortality rate. children appear to be less susceptible to develop severe clinical disease and present usually with mild and aspecific symptoms similar to other respiratory infections typical of childhood. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. available data on pediatric coronavirus infections are rare and scattered in the literature. the purpose of this review is to provide to clinicians a complete and updated panel useful to recognize and characterize the broad spectrum of clinical manifestations of coronavirus infections in the pediatric age. structural proteins, including the spike (s), envelope (e), membrane (m), nucleocapsid (n), and sometimes a hemagglutinin-esterase protein (he). the he protein binds to specific receptors and guides membrane fusion; the s protein is responsible for cell entry, the m and e proteins mediate viral assembly process, the inner n protein develops ribonucleoprotein complexes binding to viral rna. [ ] [ ] [ ] [ ] [ ] to date, seven coronaviruses affect humans: in s hcov- e and hcov-oc were firstly reported; , hcov-nl and hcov-hku were discovered subsequently in and , respectively. , additionally, three hcovs responsible for outbreaks involving high case fatality rates have been detected in humans in the last two decades: the severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the new coronavirus disease (covid- ) ( table ) . table . principal features of severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the most recent coronavirus disease (covid- ) . classification beta-cov beta-cov beta-cov in several studies a similar prevalence in the detection of hcovs in patients with respiratory symptoms compared to healthy children has been found. [ ] [ ] [ ] [ ] moreover, patients with other underlying medical conditions or immunocompromised appear more susceptible to developing severe infections than healthy patients. [ ] [ ] [ ] [ ] additionally, human coronaviruses are responsible for other common childhood diseases such as acute otitis media [ ] [ ] [ ] [ ] , asthma exacerbations , and conjunctivitis . they have also been involved in nosocomial infections, especially in the neonatal intensive care units (nicu). gagneur et al. in a prospective study determined the incidence of hcovrelated respiratory infections in newborns hospitalized in a nicu. among neonates, seven positive nasal samples for hcovs ( %) were detected. all children were symptomatic. oxygen and ventilatory support were frequently needed. sizun et al. evaluated the clinical role of coronaviruses respiratory infections in premature newborns. all premature infants infected had severe respiratory symptoms, including bradycardia, apnea, and hypoxemia, while chest x-ray revealed diffuse infiltrates. it has also been shown that coronavirus infections are not only responsible for respiratory symptoms but can also affect other organs and systems in children. several studies have also reported that respiratory symptoms caused by coronavirus infection may be associated with central nervous system (cns) involvement. hcovs have an intrinsic capacity to affect neurons and diffuse centrifugally from cns via the transneuronal route. , among neurological symptoms, febrile seizures, convulsions, loss of consciousness, encephalomyelitis, and encephalitis have been reported. [ ] [ ] [ ] primarily in , the viral genome was detected post-mortem in the cerebrospinal fluid of two patients with multiple sclerosis (ms). subsequently, the hcovs neuroinvasion capacity was confirmed in a large panel of human brain autopsy samples affected by ms and other neurological diseases. in , a prospective study on children with febrile seizures demonstrated that coronaviruses were frequently detected. additionally, hcovs have been implicated as possible causes of many gastrointestinal disorders in children, and gastrointestinal symptoms have been reported in several studies in more than % of pediatric patients , , . firstly, hcovs could be associated with neonatal necrotizing enterocolitis all hcovs can also be detected in stool samples of patients affected by gastroenteritis. , moreover, most of the hcovs found were coinfections with well-known gastroenteric viruses, including norovirus and rotavirus. hcovs may also be found occasionally in healthy children's stool samples. although hcovs have always been associated with respiratory symptoms, these findings suggest that other systems may also be involved in children. the absence of serious symptoms may not be coupled with serological negativity. therefore, these viruses should be considered in the differential diagnoses of most of the common diseases of childhood. the - severe acute respiratory syndrome outbreak was a viral respiratory illness caused by sars-cov. the outbreak firstly emerged in the southern chinese province of guangdong in november and then spread to countries with , people infected and died. the sars global outbreak was contained in july . since , there have not been any known cases of sars reported anywhere in the world. probably, civet cats or bats could be the initial step of the transmission to humans. humans to humans infection occurs by respiratory droplets or direct contact. healthcare or household contacts are critical routes of transmission. , sars-cov infection cases were classified by the world health organization (who) into suspected, probable, and confirmed ( table ) . the median incubation period ranges between - days. sars causes atypical pneumonia, which may progress to respiratory failure. symptoms include fever, malaise, myalgia, headache, diarrhea, and rigors. adults are more likely to develop severe illness characterized by dyspnea, lymphopenia, acute respiratory distress syndrome (ards), and a fatal clinical course in % of cases. the exact number of children affected by sars worldwide is unknown. however, children appear to be less susceptible to sars with a lower incidence of the disease and no reported mortality. the majority of children had documented exposure to adults with sars, usually a family member. most infected children had previously attended school, but the spread of the infection in the school environment has not been demonstrated, and this could probably be linked to lower infectiousness of the virus among children. , children have less severe symptoms than adults, and they rarely need intensive care. however, subclinical and asymptomatic infections appear uncommon. most children reported worldwide were healthy, previously and underlying conditions were infrequently reported. [ ] [ ] [ ] usually, children require hospitalization after - days the onset of symptoms: fever ( - %), dry cough ( - %), sore throat ( - %), rhinorrhea ( - %), malaise and myalgia ( - %), headache ( - %) are common. dyspnea, tachypnea, and febrile seizures are infrequent. aspecific gastrointestinal symptoms, including abdominal pain, appetite lack, vomiting, and diarrhea, have been reported. physical examination at presentation is negative in the majority of children, and chest auscultation does not reveal significant findings. moreover, sometimes crackles or signs of lung consolidation can be detected. as well as the clinical examination, laboratory findings are not specific in children with sars and can be confused with those of other respiratory infections typical of childhood. commonly lymphopenia, the elevation of transaminases, lactic dehydrogenase, and creatine phosphokinase are detected. other hematological abnormalities such as leukopenia, thrombocytopenia, the elevation of d-dimer levels and mildly prolonged activated partial thromboplastin times are also observed. [ ] [ ] [ ] circulating interleukin (il)- β levels might be increased, resulting in caspase- -dependent pathway activation responsible for an exaggerated and persistent inflammatory response and the consequent respiratory failure in severe cases. in children, radiological findings are nonspecific and similar to other viral respiratory abnormalities. high fever (> °c) and cough or breathing difficulty and one or more of the following exposures during the days prior to onset of symptoms: close contact with a person who is a suspect or probable case of sars cough or breathing difficulty history of travel, to an area with recent local transmission of sars residing in an area with recent local transmission of sars ) a suspect case with radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome (rds) on chest x-ray (cxr). ) a suspect case of sars that is positive for sars coronavirus by one or more assays. see use of laboratory methods for sars diagnosis. ) a suspect case with autopsy findings consistent with the pathology of rds without an identifiable cause. www.mjhid.org mediterr j hematol infect dis ; ; e commonly, the chest x-ray shows ground-glass opacity or focal consolidation. linear atelectasis and peribronchial thickening have also been reported. computed tomography (ct) shows more extensive airspace consolidation and ground-glass attenuation than chest x-ray, but it is performed in selective cases in pediatric age. [ ] [ ] [ ] usually, the clinical course is less severe in children compared to adults, and few patients require oxygen supplementation and assisted ventilation but preterm newborns, children younger than one year and older than years of age have more severe symptoms and are likely to develop respiratory distress. [ ] [ ] [ ] in pediatric age, sars infection commonly has a "biphasic" pattern. the first stage of the disease is characterized by virus replication and clinically by the onset of symptoms. the second phase is characterized by pulmonary involvement, which is typically less severe in children than in adults. most children will become afebrile within seven days, and they usually do not progress to respiratory distress, the adult third phase, that is only reported in a minimal number of cases, commonly among teenagers. , in pregnant women, sars infection is associated with a high incidence of spontaneous miscarriage, prematurity, and intrauterine growth retardation (iugr). the increased morbidities during pregnancy are likely to be due to the hypoxic state and circulatory insufficiency that worsen placental blood flow and cause miscarriage or iugr. significantly, among pregnant women, mortality is %. however, perinatal sars infections have not been documented. in none infants born from pregnant women affected, real-time pcr (rt-pcr) assays and viral cultures conducted on neonatal blood, body secretions and amniotic fluid were positive for sars. in infants, no congenital malformations have been reported. however, in premature newborns, severe gastrointestinal complications such as jejunal perforation and necrotizing enterocolitis have been described . however, it is not known if these neonatal morbidities are related to prematurity or if maternal infection is a factor that increases their incidence. it is unclear why children develop a less serious disease than adults. recurrent viral respiratory infections typical of the pediatric age could be helpful to the immune system in promptly recognizing and defeating new viral pathogens. furthermore, the immaturity of the immune system could be protective because the inflammatory cascade that causes respiratory failure in adults is more difficult to activate. additionally, children generally have fewer comorbidities than adults. children recovered quickly from sars. li et al. assessed the radiological and clinical outcomes of fortyseven children with sars after months from diagnosis. all children were asymptomatic while mild pulmonary abnormalities including ground-glass opacities and air trappings were found at ct in sixteen patients. although clinical and laboratory findings of sars are aspecific in children, certain features can be useful to distinguish sars from other respiratory viral infections. children with sars have a lower incidence of rhinorrhea and productive cough and higher incidence of monocytopenia than children with influenza. additionally, serum lactate dehydrogenase in the presence of a low neutrophil count and low serum creatine phosphokinase could be suggestive of sars infection. sars infections in children appear to be a relatively mild and aspecific disease, and the diagnosis should be accompanied by laboratory assessment. although infants and teenagers are more likely to have a worse clinical course, usually, all pediatric patients recover entirely without significant long-term sequelae. the middle east respiratory syndrome (mers) is a viral respiratory infection caused by the mers-coronavirus (mers-cov). the first identified case occurred in in saudi arabia. , subsequently, a total of confirmed cases of mers, including associated deaths with a case-fatality rate of % were reported globally; the majority of these cases were reported from arabian peninsula, and in the middle east. currently, mers is an extremely rare disease: in the last year mers was signaled only in saudi arabia. mers-cov is a zoonotic virus: dromedary camels are the primary reservoir hosts. humans are infected through contact with infected dromedary camels, animal products, or humans, especially among close contact between family members and health care workers. mers-cov infection cases were classified by the who into suspected, probable, and confirmed ( table ) . usually, the mean incubation period ranges from to days. clinical severity of the disease varies from asymptomatic to fatal forms, and the impact of asymptomatic spread is unclear. the infection can cause severe pneumonia, which may progress to ards, respiratory failure, and death, particularly in older people, immunocompromised patients, and those with chronic diseases. common symptoms include fever, cough, and shortness of breath. gastrointestinal symptoms (including diarrhea, vomiting, abdominal pain), pericarditis, septic shock and disseminated intravascular coagulation have been reported. [ ] [ ] [ ] [ ] children appear to be less susceptible to mers-cov infection, and pediatric cases described in the literature are rare with a low proportion ( . %- %) of infected children. age hospitalized with acute respiratory symptoms and/or fever. among these, none of children tested resulted positive for mers-cov. in pediatric age, few cases of mers cov infection have been described. most of the children were asymptomatic and positive during routine screening of mers-cov. al-tawfiq et al. reported a total of pediatric mers-cov cases with a mean age of years. overall, % were asymptomatic, while in symptomatic cases, fever and mild respiratory symptoms were common. subsequently, alfaraj et al. reported a total of pediatric mers-cov cases with a mean age of years. in this case series, common symptoms were fever ( %), cough ( %), shortness of breath ( %), and gastrointestinal symptoms ( %). two ( . %) patients had abnormal chest radiographic findings with bilateral infiltration, one ( . %) required ventilatory support, and two ( . %) required supplemental oxygen. four with underlying conditions (cystic fibrosis, nephrotic syndrome, craniopharyngioma, and a right ventricular tumor) had a fatal outcome. these children developed a critical form of mers infection complicated by respiratory and multiorgan failure. frequently, clinical examination revealed bilateral rhonchi and crackles while chest x-ray showed diffuse bilateral infiltrates, ground-glass opacification and pleural effusion. [ ] [ ] [ ] [ ] [ ] thrombocytopenia, leukopenia, increased creatinine and prolonged prothrombin time were the only laboratory findings reported in literature. , , mers-cov in children is less frequent than adults and appears to be associated with low mortality unless the patients have underlying comorbidities. few cases of mers-cov have been reported during pregnancy. a pregnant woman, aged years, had a stillbirth at approximately five months of gestation and another woman gave birth to a healthy term baby, but she died after delivery. in conclusion, although mers-cov represents a clinical concern for the adult population with a high fatality rate, it remains a sporadic disease in childhood. clinicians should learn to recognize and suspect mers-cov infection, as the symptoms and signs are nonspecific, based on epidemiological criteria to avoid the spread of the disease in patients at higher risk of worse clinical course. the outbreak of covid- infection (coronavirus disease ; previously -ncov) began in wuhan, hubei, china, in december , which then spread rapidly to other provinces of china and around the world. on january , , the who declared the outbreak of a public health emergency of international concern and, on march , , a pandemic. as of june , , other countries and regions, with more than . . confirmed cases, are declared. among the confirmed cases, . . are recovered, and . died. recent genetic analysis suggests the covid- emerged from an animal source. the full genome sequences showed high homology between covid- , bat coronavirus, and pangolin coronavirus, but further genetic study is required. moreover, according to current evidence, the principal route of transmission of covid- is from human to human. , covid- spread between people through respiratory droplets and contact routes. droplet transmission occurs when there is close contact with a person with respiratory symptoms such as coughing or sneezing, who may spread potentially infectious droplets. transmission may also occur by direct contact with infected persons and indirect contact with infected surfaces or objects. covid- can persist on inanimate objects for days but can be efficiently inactivated by common disinfectants. airborne transmission may be possible when a high risk of aerosolization procedures are performed, such as endotracheal intubation and bronchoscopy. the virus is also detected in stool specimens, and consequently, the feco-oral transmission is also hypothesized. [ ] [ ] [ ] [ ] the high transmissibility of covid- may be explained by its demonstrated presence in the upper respiratory tract of asymptomatic or presymptomatic subjects with viral loads comparable to those detected from symptomatic patients. the real proportion of asymptomatic cases is unclear, ranging from % to % in different studies. transmission from asymptomatic patients infected with covid- most likely contributed to the rapid and extensive spread of pandemic but further studies are needed to more accurately estimate the proportion of genuinely asymptomatic cases and their risk of transmission. [ ] [ ] [ ] [ ] [ ] [ ] [ ] covid- has been reported among all age groups. the median incubation period of covid- infection is - days with a range up to days. , covid- infection case is classified by the who into suspected, probable, and confirmed ( table ) . clinical severity of the infection varies, ranging from asymptomatic forms to critical diseases. common symptoms are fever, dry cough, malaise, lethargy, shortness of breath, sore throat, and myalgia. headache, conjunctivitis, productive cough, and diarrhea are also described. mild forms present as a common cold, and severe cases may worsen in pneumonia that may evolve to ards, shock, and multiple organ dysfunction. more severe clinical pictures are associated with stronger immune response and with the production of proinflammatory cytokines, including il- , il- , il- , and tumor necrosis factor-α (tnf-α). adverse outcomes are common in elderly patients and those with underlying diseases. the need for intensive care admission is in - % of patients. the fatality rate is estimated to range between and %. [ ] [ ] [ ] [ ] [ ] about % of covid- confirmed cases are children. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] generally, children appear to be less likely to develop a severe form of covid- infection, and commonly they have a mild clinical course with a good prognosis. few children may evolve into lower respiratory infections. probable reasons include having an immune system still immature, healthier respiratory tract, and less underlying conditions than adults. most of them have an infected contact history with family members. moreover, children, especially those with asymptomatic or milder form, may represent significant spreaders. pediatric patients appear to be likely as adults to become infected but are less likely to develop symptoms. however, future studies are needed to understand the role of children in the transmission of the virus. [ ] [ ] [ ] current researches show that the median age of infection in pediatric cases is - years. in symptomatic cases, symptoms are typical of acute respiratory infections and frequently included fever ( %) and cough ( %), which may be accompanied by nasal congestion, runny nose, conjunctivitis, pharyngitis, wheezing, myalgia, and expectoration. few children have an atypical presentation with gastrointestinal manifestations, including nausea, vomiting, and diarrhea. low oxygen saturation of less than %, dyspnea, cyanosis, and poor feeding, are less common than adults. among infants, symptoms such as irritability, reduced response, and poor feeding could be the main signs of infection. family clustering occurred for all infected infants. rarely infants require intensive care or mechanical ventilation or have any severe complications. common symptoms of pediatric age are summarized in figure . the majority of children recovers - weeks after the onset of the disease. regarding biochemical results, leukopenia and lymphopenia are frequent in children. elevation of transaminases, myoglobin, muscle enzymes, and d-dimers might be seen in severe cases. [ ] [ ] [ ] [ ] [ ] [ ] [ ] dong et al. reported that % of pediatric patients affected by covid- developed an asymptomatic, mild, or moderate form of infection. a severe disease characterized by dyspnea, central cyanosis, and oxygen saturation of less than % was reported in % of cases. a critical disease characterized by ards and multiple organs failure was reported in less than % of cases. the prevalence of severe and critical disease appears higher in younger children, particularly in children aged < -year-old and in children with underlying diseases. to date, death was an uncommon event reported in one month-old infant with intussusception and multiorgan failure and in one -year-old boy. , other systemic symptoms appear to be related to the infection, but their link has not yet been demonstrated. since the outbreak of the pandemic, a large number of rashes, urticaria, and vasculitis affecting hands and feet of healthy children and adolescents have been reported as well as itching, burning, difficulty in joint movements and pain. recently, the relationship between covid- infection and the development of cardiac diseases in children has been hypothesized. belhadjer et al. have reported a large number of febrile children resulted positive for covid- admitted in intensive care units for acute heart failure associated with a multisystem inflammatory state. in most of the children, clinical features appeared similar to those of kawasaki syndrome: lasting fever, cutaneous rash, lymphadenopathy, persistent activation of systemic inflammation and positive response to intravenous immunoglobulin. similar clinical features have subsequently been reported in children with covid- positive serology. , as in covid- infection, kawasaki syndrome is triggered by proinflammatory cascade activated primarily by innate immunity response. however, further studies are needed to establish the real pathogenetic relationship between emerging covid- and kawasaki-like syndromes. dufort et al. have recently reported the emergence of a multisystem inflammatory syndrome in children in new york state coincidental with widespread sars-cov- transmission, which can better clarify the relationship between kawasaki disease and covid- . among children admitted to the new york hospitals for multisystem inflammatory syndrome in children (mis-c), patients had a laboratory-confirmed acute or recent severe acute respiratory syndrome coronavirus [sars-cov- ] infection. this hyperinflammatory syndrome manifested with dermatologic, mucocutaneous, and gastrointestinal features associated with cardiac dysfunction. of these patients, a total of patients ( %) received a diagnosis of kawasaki's disease or atypical (or incomplete) kawasaki's disease; of the patients with coronary-artery aneurysms also received a diagnosis of kawasaki's disease. covid- infection may also trigger the onset of other immune-mediated diseases such as immune thrombocytopenia, [ ] [ ] [ ] [ ] evans syndrome, and autoimmune hemolytic anemia. among radiological findings, ground-glass opacity, mono or bilateral infiltrates, mesh shadows, and tiny nodules are frequently detected. in severe cases, radiological alterations are diffused, presenting as a "white lung." however, radiologic evidence of pneumonia might be absent in - % of children. , , [ ] [ ] [ ] [ ] [ ] in selected cases, lung ultrasound might be useful in the managing and follow-up of covid- infection. this radiological technique can precociously identify abnormalities including small pleural effusion and subpleural consolidation and appear more available then x-ray and ct. [ ] [ ] clinical examination appears mostly negative for pulmonary signs, and in rare cases, rales and thoracic retractions have been reported. whether pregnant women and children born to affected mothers are more likely to have a worse outcome is currently unclear. maternal-infant vertical transmission has not been documented. amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples from newborns delivered by infected women were tested for covid- , and all samples tested negative. data on the maternal and perinatal outcomes of pregnant women infected with covid- is limited. most pregnant women with covid- present with fever and coughing. severe and critical maternal symptomatology have also been reported, but no women died. the most common adverse pregnancy outcome is preterm birth, occurring in % of cases while the rate of perinatal death is %, including one case of stillbirth and one neonatal death. there is no data on miscarriage for covid- occurring during the first trimester. in more than a third of cases, fetal distress and frequent admission neonatal intensive care units have been reported. , rarely, cases of covid- positivity in newborns have been reported. common symptoms are fever, cough, lethargy, and vomiting milk. mottled skin and moderate respiratory distress presented with tachycardia, tachypnoea, subcostal retractions, and low oxygen saturation are also described in newborn babies. [ ] [ ] [ ] [ ] [ ] although it can be severe in some cases, compared with sars-cov and mers-cov, covid- causes less severe disease in children. a recent meta-analysis shows that children infected with covid- have less fever than that other epidemic hcovs. despite the rapid worldwide spread of covid- infection, additional data are needed to define the severity of the disease in children. the severity of the symptoms and the mortality rate will be better assessed in the future. differential diagnosis with common viral respiratory infections of childhood, such as influenza virus, adenovirus, respiratory syncytial virus, and metapneumovirus, should be considered. in the diagnosis of suspected cases, epidemiological and clinical criteria must be assessed. , , rt-pcr represents the gold standard to confirm the diagnosis of hcovs infections performed on samples of respiratory secretions. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the viral load is higher in lower respiratory tract secretion samples than in upper respiratory tract samples. therefore, suspected cases resulted in firstly negative could be re-tested with a second swab, better if with a low respiratory sampling is performed as proved for sars and mers infection. , currently, few data have been published about the sensitivity and specificity of rt-pcr nasopharyngeal swabs for covid- . in vitro analyses suggest that the rt-pcr test is highly specific and sensitive. in vivo, sensitivity is estimated to be higher than % but seems to be lower for "mild" cases while specificity is close to %. , accuracy of rt-pcr swabs in clinical practice differs depending on the site and quality of the sample. taking swabs from children may be more difficult given the intrusive nature of the procedure and further reduce the specificity and sensitivity of the test. rt-pcr of bronchoalveolar lavage fluid appears the most accurate technique of virologic confirmation, but it may not always be easily collected in all patients, especially in pediatric age. although a negative test cannot currently rule out the disease, further studies are needed to define the exact specificity and sensitivity of rt-pcr nasopharyngeal swabs. , moreover, rt-pcr appears to be useful in virus detection on stool samples. to date, serology is not considered a diagnostic method. although most patients with covid- appear positive for immunoglobulin-g (igg) within days while igm reaches a peak - days after symptom onset, the serological response is not useful for early individuation of positive patients. additionally, numerous cross-reactions occur between covid- and common hcovs , and protective immunity against covid- is not proved. despite its potential role in supporting rt-pcr in the diagnosis of covid- , the clinical and immunological meaning of serology is still unclear. the spread of the infection can be prevented if children and family members were educated about proper hygienic practices and infection control measures, including regular hand washing, cover the mouth with napkin or towel when coughing or sneezing, avoid crowded places and contact with sick people. children with hcovs should receive early supportive therapy and continuous monitoring. additional oxygen, caloric, and hydro electrolytic support should be performed if necessary. frequent checks of oxygen saturation and hematological, urinary, and biochemical parameters, including liver, kidney, myocardial enzymes, and coagulation parameters should be analyzed. finally, blood gas analysis and radiological diagnostics of the chest should be done when necessary. this strategy could be useful in the prevention of ards, multiorgan failure, and other nosocomial infections possibly treated, if bacterial, with appropriate antibiotics. in critical cases, mechanical ventilation with endotracheal intubation and other more invasive interventions, such as blood purification and extracorporeal membrane oxygenation (emco), should be adopted. additionally, the use of antiviral drugs in children with severe hcovs infections may help to reduce viral load and the duration of symptoms. however, their safety and real effectiveness have not yet been proven. interferon alfa and beta, corticosteroids, lopinavir/ritonavir, and ribavirin, were used in the treatment of sars-cov and mers-cov in adults and children. , , , however, ribavirin can cause hemolytic anemia and liver dysfunction, as well as corticosteroids, increase the risk of iatrogenic immune immunosuppression. to date, there is no evidence regarding the management and treatment of covid- infection in children. in addition to supportive therapy, the use of nebulized interferonalpha- b and oral lopinavir/ritonavir together with corticosteroids for complications and hydroxychloroquine or intravenous immunoglobulin for severe cases have been suggested. , , recently, a position paper of the italian society of pediatric infectious disease on the treatment of children with covid- infection has been published. in asymptomatic or mild cases, only antipyretic therapy is recommended. in severe or critical cases, the use of hydroxychloroquine ± azithromycin or lopinavir/ritonavir must be considered. immunomodulating therapy with methylprednisolone or tocilizumab or anakinra must be considered in case of the simultaneous presence of ards or progressive deterioration of respiratory function, the elevation of proinflammatory biomarkers and an interval of at least seven days from symptoms onset. supportive therapy should include antipyretic therapy, inhalation therapy with topical steroids and/or bronchodilators and venous thromboembolism prophylaxis therapy. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] discharge from the hospital is recommended when the patient is without fever for almost three days, respiratory symptoms have improved, and rt-pcr samples are negative. conclusions. most cases of hcovs infection in children have clinically mild symptoms and a relatively short time to resolution. children seem to have a better prognosis compared to adults, and death is a sporadic event. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. learning to recognize pediatric clinical presentations often indefinite or similar to other typical infections of this age, allows clinicians to perform a correct and early diagnosis and prevent the spread of infections in the general population. furthermore, the psychological and social impact of the pandemic outbreak should be considered, especially in the pediatric age. moreover, we think it is necessary to implement innovative clinical tools, such as narrative medicine, to recognize the burden of disease in children and caregivers. 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children's safety medication; global pediatric pulmonology alliance. diagnosis, treatment, and prevention of novel coronavirus infection in children: experts' consensus statement treatment of children with covid- : position paper of the italian society of pediatric infectious disease covid- : ibuprofen should not be used for managing symptoms, say doctors and scientists ema gives advice on the use of nonsteroidal anti-inflammatories for covid- recommendations for inhaled asthma controller medications anticoagulant treatment is associated with decreased mortality in severe coronavirus disease patients with coagulopathy discovering drugs to treat coronavirus disease (covid- ) delle fave a. perceived well-being and mental health in haemophilia a narrative approach to describe qol in children with chronic itp. front pediatr itp-qol questionnaire for children with immune thrombocytopenia: italian version validation's key: cord- -f ny ur authors: kim, seung woo; park, jung wan; jung, hee-dong; yang, jeong-sun; park, yong-shik; lee, changhwan; kim, kyung min; lee, keon-joo; kwon, donghyok; hur, young joo; choi, boyoul; ki, moran title: risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea date: - - journal: clin infect dis doi: . /cid/ciw sha: doc_id: cord_uid: f ny ur background. transmission heterogeneity was observed during the korean outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection. only of cases transmitted the infection, and super-spreading events caused transmissions. we investigated the risk factors for mers-cov transmission. methods. epidemiological reports were used to classify patients as nonspreaders, spreaders, or those associated with a super-spreading event ( or more transmissions). logistic regression analyses were used to evaluate the factors for mers-cov transmission. results. compared to nonspreaders, spreaders exhibited a longer interval from symptom onset to isolation ( days vs days) and more frequent pre-isolation pneumonia diagnoses ( . % vs . %). spreaders also exhibited higher values for pre-isolation contacts ( vs . ), pre-isolation hospitalization ( . % vs . %), and emergency room (er) visits ( % vs . %). spreaders exhibited lower cycle thresholds for the upe and orf a genes ( . vs . and . vs . , respectively). in multivariate analysis, transmission was independently associated with the cycle threshold (odds ratio [or], . ; % confidence interval [ci], . – . ) and pre-isolation hospitalization or er visits (or, . ; % ci, . – . ). the super-spreading events exhibited higher values for pre-isolation contacts ( vs ), pre-isolation er visits ( % vs . %), and doctor shopping ( % vs . %) compared to non-super-spreading events. conclusions. these findings indicate that transmission is determined by host infectivity and the number of contacts, whereas super-spreading events were determined by the number of contacts and hospital visits. these relationships highlight the importance of rapidly enforcing infection control measures to prevent outbreaks. transmission heterogeneity was a significant characteristic of the south korean outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection [ ] . transmission heterogeneity describes a state in which most transmissions are related to a few patients and most patients do not transmit the disease. numerous other infectious diseases exhibit transmission heterogeneity [ ] , and this concept is important for understanding epidemics. the course of an epidemic is influenced by the basic reproduction number (r = the average number of cases that case produces in a susceptible population) and transmission heterogeneity [ ] . as r represents an average quantity, it is often insufficient to explain individual variation, and as transmission heterogeneity reflects individual variation, it can help predict the likelihood of super-spreading events. even in instances with a low r , a disease with high transmission heterogeneity (eg, severe acute respiratory syndrome [sars]) can cause super-spreading events [ ] , such as the super-spreading that occurred during the sars outbreak [ , ] . transmission heterogeneity was observed during early mers-cov outbreaks [ ] and became prominent during the south korean outbreak. among the confirmed korean cases of mers-cov infection, > % of the transmissions were epidemiologically associated with patients [ ] , and almost % of the cases caused no transmission. furthermore, a recent study revealed that mers has greater transmission heterogeneity compared to sars [ ] . therefore, to successfully control mers-cov infection, it is essential to identify high-risk patients and perform targeted infection control [ ] . however, these patients are difficult to identify, as an individual's infectiousness is affected by complex interactions between the pathogen, host, and environment. several researchers have attempted to identify risk factors for super-spreading events during the sars outbreak [ , , ] , although there is little information regarding the high-risk group(s) from the mers-cov outbreak. the recent south korean mers-cov outbreak was triggered by a single imported case, and epidemiological tracing was performed for all laboratory-confirmed cases and their close contacts [ , [ ] [ ] [ ] [ ] [ ] [ ] . thus, it is possible to precisely reconstruct the transmission chain and identify patients who transmitted mers-cov infection. therefore, we analyzed the epidemiological characteristics that were associated with mers-cov transmission and super-spreading events. cases of mers-cov infection were confirmed using real-time reverse-transcription polymerase chain reaction (rt-pcr) assays, regardless of their clinical manifestations. the epidemiological reports were analyzed by epidemic intelligence service officers who participated in the mers-cov outbreak investigation. when a case was exposed to multiple confirmed cases, the transmission was attributed to the case with the highest likelihood of transmission, and any conflicts were resolved through the consensus of the epidemic intelligence service officers. spreaders were defined as confirmed cases of mers-cov infection that were epidemiologically suspected of transmitting mers-cov to or more persons. super-spreading events were arbitrarily defined as transmission of mers-cov infection to or more cases. the patient who triggered the outbreak was defined as patient zero. cases that were infected by patient zero were defined as first-generation cases; cases that were infected by first-generation cases were defined as second-generation cases; and cases that were infected by second-generation cases were defined as third-generation cases [ ] . isolation was defined as separating symptomatic patients from others to prevent spreading, and quarantine was defined as separating or restricting the movement of healthy individuals who may have been exposed to the infection within the maximum incubation period. the transmission date was defined as the date of contact between the spreader and suspected secondary case during the spreader's infectious period. in cases with an exposure duration of longer than day, the transmission date was defined as the day with the highest likelihood of transmission or as the median day during the exposure period in cases with consistent contact throughout the exposure. the date of sampling was the day on which the first positive respiratory specimen was collected. close contacts were defined using the guidelines on middle east respiratory syndrome [ ] , which include persons who stayed in a room or ward with a confirmed case, who directly contacted respiratory secretions from confirmed cases, or who stayed within m from the confirmed cases without wearing appropriate personal protective equipment. pre-isolation pneumonia diagnoses were based on radiographic evidence. doctor shopping was defined as visiting multiple healthcare facilities without an official interhospital transfer after developing mers-cov symptoms [ ] . epidemiological reports from the outbreak were evaluated to collect data regarding basic demographic characteristics, medical history, mers-cov exposure, symptoms and their onset date(s), sampling date(s), contact history, and post-exposure infection control. the reports were drafted during the outbreak based on direct interviews with the confirmed cases and follow-up epidemiological investigations that were performed to identify the exposure route and close contacts. hospital information systems were reviewed to identify patients who stayed in the hospital during the exposure period and healthcare providers who contacted the patient(s). persons who contacted confirmed cases outside healthcare facilities were also traced. data from closed circuit television, credit card transactions, and health insurance services were also reviewed [ ] . the numbers of close contacts were calculated based on the number of quarantines during the outbreak. all data were collected as part of the public health response and in accordance with the infectious disease control and prevention act [ ] . clinical specimens were collected in sterile containers and immediately transferred to qualified facilities. sputum samples were mixed with - × phosphate-buffered saline and vortexed vigorously to reduce their viscosity. viral rna was extracted from the clinical specimens using a qiagen viral rna mini kit (qiagen, hilden, germany). all laboratory diagnoses of mers-cov were confirmed using the world health organization guidelines [ ] and results from real-time rt-pcr assays that target upstream of the mers-cov envelope protein gene (upe) and the open reading frame a gene (orf a) [ ] . cycle threshold (ct) values for the upe and orf a genes were obtained during testing. we analyzed the ct value from the first positive mers-cov specimen (or the specimen obtained immediately after a positive screening test). categorical variables were compared using the χ test and fisher exact test, and the mann-whitney test was used for continuous variables. the variables' associations with mers-cov transmission were evaluated using multiple logistic regression analyses, and covariates were selected based on a p value of < . in the univariate analyses. a p value of < . was considered statistically significant. all analyses were performed using r software (version . . ; r foundation, vienna, austria). we identified cases of confirmed mers-cov infection. patient zero infected first-generation cases. among the cases, were responsible for transmission to second-generation cases. among the cases, infected third-generation cases. one patient with an unclear source of infection (case ) transmitted the infection to another patient. four patients exhibited unclear sources of transmission (cases , , , and ). each confirmed case transmitted the infection to - secondary cases ( figure ). there were nonspreaders and spreaders ( or more transmission). of the spreaders, cases transmitted the infection to or more cases (super-spreading event). after excluding the cases with unclear infection sources, we identified transmissions generated by spreaders. a total of transmission events ( . %) were epidemiologically linked to the super-spreading events. twenty-five transmission events ( . %) occurred within days after symptom onset, transmissions ( . %) occurred - days after symptom onset, and transmissions ( . %) occurred > days after symptom onset. a total of transmission events ( . %) occurred on the day of or after a radiographically confirmed diagnosis of pneumonia. a total of transmissions ( . %) occurred before appropriate in-hospital isolation. seven transmissions ( . %) occurred between confirmed cases and healthcare personnel after in-hospital isolation: (cases , , , and ) were doctors or nurses who managed confirmed cases, (case ) participated in cardiopulmonary resuscitation of a confirmed case, (case ) involved portable radiography for a confirmed case, and (case ) rode in an ambulance with a confirmed case during a hospital transfer. ; p = . ). the intervals from symptom onset to diagnosis or obtaining a respiratory specimen were also significantly longer among spreaders (to diagnosis: [ . - ] days vs [ ] [ ] [ ] [ ] [ ] [ ] days; p = . and to sampling: [ - . ] days vs [ ] [ ] [ ] [ ] [ ] days; p < . ). furthermore, the interval from symptom onset to isolation was longer among spreaders ( [ . - ] days vs [ ] [ ] [ ] [ ] [ ] [ ] days; p = . ). spreaders exhibited a significantly higher proportion of pre-isolation pneumonia diagnoses ( . % vs . %; p < . ) and a longer interval from the pneumonia diagnosis to isolation ( [ ] [ ] [ ] [ ] [ ] days vs [ - ] days; p = . ). the overall number of contacts was significantly larger among spreaders compared to nonspreaders ( [ . - . ] vs . [ - . ]; p = . ). compared to nonspreaders, spreaders exhibited a significantly higher proportion for pre-isolation hospitalization ( . % vs . %; p < . ), visiting outpatient clinics ( . % vs . %; p = . ), and visiting emergency rooms (ers; % vs . %; p < . ). we used logistic regression analyses to evaluate the risk factors for transmission (table ). in the univariate analyses, transmission was associated with underlying respiratory disease, ct value, interval from symptom onset to diagnosis, number of contacts, and pre-isolation hospitalization or er visits. in the multivariate analyses, transmission was independently associated with a low ct value for upe (odds ratio [or], . ; % confidence interval [ci], . - . ) and pre-isolation hospitalization or er visits (or, . ; % ci, . - . ). we compared the epidemiological characteristics of the spreaders with or more transmissions and the spreaders with or fewer transmissions (table ) . both groups exhibited similar host factors and contact durations. however, spreaders with or more transmissions exhibited higher values for we evaluated the epidemiological characteristics of patients who transmitted mers-cov during the recent south korean outbreak. among the confirmed mers-cov cases, only cases transmitted the infection to other individuals. these spreaders had higher host infectivity and wider and prolonged contacts compared to nonspreaders. the risk factors for super-spreading events included a larger number of contacts and a pre-isolation er visit. doctor shopping was marginally associated with a super-spreading event. however, both spreaders with or more transmissions and spreaders with or fewer transmissions exhibited similar levels of host infectivity. it appears that both host infectivity and the number of contacts influenced mers-cov transmission, whereas super-spreading events were mostly associated with a greater likelihood of encountering other people under diverse environmental conditions. during the outbreak, approximately % of the transmissions occurred during days - after symptom onset, and this may be a period when the risk of transmission is particularly high. furthermore, this high-risk period was temporally associated with other epidemiological factors. first, the period overlapped with the confirmed cases' visits to healthcare facilities, as hospitalization and er visits peaked during days - after symptom onset. it is well known that mers-cov outbreaks generally occur in the healthcare setting [ , , , ] , and the high-risk period may be associated with healthcare-seeking behaviors. second, the high-risk period was several days ( - days) after the radiographic diagnoses of pneumonia, which generally occurred on days - after symptom onset. although the significance of pre-isolation pneumonia has not been discussed previously, a radiographic diagnosis of pneumonia may influence transmission in ways. first, it may directly increase the chance of transmission by actively generating lower respiratory tract secretions and a productive cough. second, it may be an indirect index of disease severity and hospital visiting status. in our study, cases with pre-isolation pneumonia had lower ct values and more frequent pre-isolation hospital visits. the epidemiological significance of the high-risk period could also be observed when we compared the spreaders and nonspreaders. the spreaders were typically isolated after the high-risk period (median, days after symptom onset and days after a diagnosis of pneumonia), whereas nonspreaders were typically isolated before this period (median, days after symptom onset and day after a diagnosis of pneumonia). similar results were observed in a study of the sars outbreak, which revealed that late admission to healthcare facilities (especially > days after symptom onset) was associated with super-spreading events [ ] . thus, infection prevention measures should target isolation before this critical period (ie, within - days after symptom onset and within day after the detection of pneumonia). interestingly, the average duration from symptom onset to isolation dropped to < days during the first week of june , and reports of new cases have rapidly decreased since that time. among the host factors that were associated with transmission, only the ct value was statistically significant in the multivariate analyses. the ct value is a semiquantitative continuous variable that is inversely proportional to the viral load. ct values are associated with the severity of mers-cov infection [ ] , although its relationship with transmission has rarely been studied. in the present study, spreaders had significantly lower ct values compared to nonspreaders, which suggests that ct values might reliably predict transmission. moreover, the cases with very low ct values (ct < ) tended to transmit the infection in uncommon circumstances. in both the present study and previous studies, mers-cov transmission usually occurred in the hospital setting [ , , , ] . in contrast, cases with very low ct values transmitted the infection in more diverse settings in the present outbreak (eg, their household, in an ambulance, in an outpatient clinic, or to healthcare personnel after in-hospital isolation). these findings suggest that cases with very low ct values can potentially transmit the infection in unexpected conditions. however, our data regarding the ct values have several limitations. first, various amounts of phosphate-buffered saline were added to dilute the respiratory specimens, and this there was no multicollinearity between the independent variables (all variables: r score of < . ). abbreviations: ci, confidence interval; or, odds ratio. may have affected the ct values. second, the ct value is influenced by the specimen type and the interval between symptom onset and sample collection [ , ] , but various different types of specimens were collected at different time points in the present study. however, we only evaluated nonsputum specimens, and there was no linear correlation between the ct values and the interval from onset to sampling. our comparison of the spreaders with or more transmissions and spreaders with or fewer transmissions revealed that the spreaders with or more transmissions had an approximately -fold higher number of contacts. furthermore, there were no significant differences in host infectivity. these findings may suggest that the underlying likelihood of transmission has the greatest influence on super-spreading events rather than an intrinsic difference in host infectivity. a similar finding was observed in a previous study of the sars super-spreading event [ ] , with those super-spreaders having - contacts compared to - contacts for the spreaders with - transmissions. our study also revealed that a pre-isolation er visit and doctor shopping were associated with super-spreading events. in addition, super-spreading events were associated with the number of healthcare facilities that each patient visited for hospitalization or er treatment but not with the number of hospitals visited for outpatient treatment. in south korea, patients who seek hospitalization without prior arrangements tend to visit the er, and a history of or more er visits strongly suggests that the patient had been doctor shopping during hospitalization. specific environmental conditions have been suggested to increase the likelihood of a super-spreading event [ ] , and doctor shopping may increase the likelihood of encountering these conditions. for example, when a confirmed case changes hospital during hospitalization without an official interhospital transfer, multiple environments are exposed to the infected case (an ambulance, an er, and a ward). thus, doctor shopping can greatly increase the likelihood of encountering conditions that are suitable for a super-spreading event. in the present outbreak, of the super spreaders (cases , , , and ) transmitted the infection at or more hospitals, as they had visited multiple healthcare facilities. therefore, it is very important to have an early suspicion of mers-cov infection and minimize doctor shopping during the early stage of an outbreak. our study has several limitations. first, some of the confirmed cases had multiple potential sources of infection, and we attributed the transmission to the case with the highest epidemiological probability. the source of infection was clear in > % of the transmissions, and we excluded cases that had contact with multiple cases and an unclear source of transmission. however, as the analyses of the epidemiological data are ongoing, the list of spreaders may change if new epidemiological evidence is uncovered. second, we did not have access to genomic sequencing data, which might have provided information regarding the relatedness of transmitted strains. third, transmission may be affected by other epidemiological factors, including aerosol-generating procedures, differences in environmental conditions, and variations in crowdedness [ , , ] . however, these factors were not included in the present analysis. fourth, serological testing was not performed for every close contact, and additional asymptomatic cases may have been present. however, the seropositive rate was . % in a recent serological study of close contacts [ ] . thus, the absence of serological testing likely did not significantly influence our results. we evaluated the epidemiological risk factors for mers-cov transmission during the recent south korean outbreak. superspreading events were not related to intrinsic host characteristics and were attributable to the likelihood of transmission. therefore, strict er triage and minimizing doctor shopping during an outbreak's early stage may help prevent super-spreading events. hospital outbreak of middle east respiratory syndrome coronavirus superspreading and the effect of individual variation on disease emergence why did outbreaks of severe acute 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shopping: a phenomenon of many themes korea ministry of government legislation infectious disease control and prevention act world health organization. laboratory testing for middle east respiratory syndrome coronavirus. interim recommendations complete genome sequence of middle east respiratory syndrome coronavirus kor/knih/ _ _ , isolated in south korea mers-cov outbreak in jeddah-a link to health care facilities predicting super spreading events during the severe acute respiratory syndrome epidemics in hong kong and singapore association of higher mers-cov virus load with severe disease and death, saudi arabia an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity acknowledgements. the authors thank sung soon kim and jeong-gu nam (division of respiratory viruses, center of infectious disease, korea centers for disease control and prevention) for laboratory assistance and technical support in the production of the manuscript. in addition, we thank all administrative and laboratory staff of the korea centers for disease control and prevention who participated in the mers-cov outbreak control effort.author contributions. s. w. k. performed the literature search, study design, data collection, analysis, interpretation, and writing. m. k. contributed to the study design, data interpretation, and writing. j. w. p., y. s. p., c. l., k. m. k., k. j. l., and d. k. contributed to the data collection and interpretation. h. d. j. and j. s. y. contributed to the data collection, mers pcr testing, data interpretation, and analysis. y. j. h. and b. y. c. contributed to the study design, data interpretation, and revising the manuscript. s. w. k. and m. k. revised the manuscript. all authors contributed to writing and approved the final manuscript.potential conflicts of interest. authors certify no potential conflicts of interest. the authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- - qgx i authors: aly, mahmoud; elrobh, mohamed; alzayer, maha; aljuhani, sameera; balkhy, hanan title: occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: qgx i the emergence of the middle east respiratory syndrome coronavirus (mers-cov) infections has become a global issue of dire concerns. mers-cov infections have been identified in many countries all over the world whereas high level occurrences have been documented in the middle east and korea. mers-cov is mainly spreading across the geographical region of the middle east, especially in the arabian peninsula, while some imported sporadic cases were reported from the europe, north america, africa, and lately asia. the prevalence of mers-cov infections across the gulf corporation council (gcc) countries still remains unclear. therefore, the objective of the current study was to report the prevalence of mers-cov in the gcc countries and to also elucidate on its demographics in the arabian peninsula. to date, the world health organization (who) has reported , laboratory-confirmed cases of mers-cov infection since june , involving deaths in different countries worldwide. within a time span of years from june to july , we collect samples form mers-cov infected individuals from national guard hospital, riyadh, and ministry of health saudi arabia and other gcc countries. our data comprise a total of cases ( . % male and . % female). the age-specific prevalence and distribution of mers-cov was as follow: < yrs ( cases: . %), – yrs ( cases: . %), – yrs ( cases: . %), and the highest-risk elderly group aged ≥ yrs ( cases: . %). the case distribution among gcc countries was as follows: saudi arabia ( cases: %), kuwait ( cases: . %), bahrain ( case: . %), oman ( cases: . %), qatar ( cases: . %), and united arab emirates ( cases: . %). thus, mers-cov was found to be more prevalent in saudi arabia especially in riyadh, where cases ( . %) were the worst hit area of the country identified, followed by the western region makkah where cases ( . %) were recorded. this prevalence update indicates that the arabian peninsula, particularly saudi arabia, is the hardest hit region regarding the emerging mers-cov infections worldwide. gcc countries including saudi arabia now have the infrastructure in place that allows physicians and scientific community to identify and immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. the emergence of mers-cov dates back to july when an elderly patient of age years died from an acute pneumonia in saudi arabia, and a new coronavirus strain was isolated from his lung tissue [ ] . another case of acute respiratory disease was diagnosed in a -year old male in london who was from qatar and a new strain of coronavirus was isolated from this patient as well [ ] . shortly after, the entire genome of the new coronavirus was sequenced and deposited in the genebank database under the number jx , kc . . the phylogenetic analysis of the new virus genome revealed that homology of the nucleotide sequence between two cases was . % and the isolates were closely related to bat coronavirus (bat-cov) which belongs to group c of β-coronavirus [ ] . according to the recommendations by the international committee on taxonomy of viruses (ictv), the new coronavirus was named as 'middle east respiratory syndrome coronavirus' (mers-cov) [ ] . although, initially reported from the middle east, mers-cov exported cases have also been observed worldwide. with regard to viral origin and transmission, the first case of mers-cov infection did not relate it to any particular contact with animals before the disease onset; however, other studies did link it to dromedary camels [ ] [ ] [ ] [ ] . beta-coronaviruses are strongly associated with bats serving as reservoir host, particularly, the african neoromicia bats were speculated to be the natural reservoir of mers-cov [ ] [ ] [ ] [ ] . notably, serological evidence suggests that mers-cov has been in the circulation for at least - decades in dromedary camels [ ] . given that, the ancestral origin of mers-cov links it to african bats whereas, dromedary camels have been functioning as an intermediate host for this virus for a significantly long period of time [ ] [ ] [ ] . health facilities, hospitals and households with mers patients are considered to be the epidemic centers of mers-cov outbreaks. mers-cov is mainly spreading across the geographical region of the middle east while only sporadic cases are reported in the europe, north america, africa, and lately asia. this may be because of the widespread population of dromedary camels in the middle east; however, the scientific proof of evidence that camel farms are a potential source of mers-cov infections still remains to be established. besides, the typical seasonality pattern is not seen in case of mers-cov infections and only one report links it to camel breeding season. the modes of mers-cov transmission by droplet, contact, or airborne are not yet confirmed as well and thus its transmission among animals and from animals to human and human to human remains unclear. there is also no documentation available regarding mers-cov transmission during airplane flights. therefore, standard infection prevention and control procedures are followed including droplet and airborne precautions. the viral incubation period is from days to weeks. the viral cytopathic effects clearly show prominent syncytium formation in humans as well as non-human primates. mers-cov targets directly the lower respiratory tract (pneumocytes) in dromedary camels and continues to replicate preferentially in the airway cells of the upper respiratory tract. the clinical manifestations of mers-cov infections represent a wide spectrum ranging from asymptomatic cases to the ones with severe respiratory indexes. according to the who, mers-cov infection is an acute respiratory infection involving pyrexia of ˚c or more, cough with radiologic pulmonary presentation and also the history of the patients originating from or travel to the arabian peninsula and its neighboring countries within days of symptoms. mers-cov cases have been identified as both community-and hospital-acquired, mainly among the aged population and in patients with multiple comorbidities such as acute pneumonia, upper respiratory tract infections, influenza-like illness, or asymptomatic infection(s) in children and immunocompromised hosts. moreover, the common extra-pulmonary symptoms include diarrhea and acute renal failure. the early clinical diagnostic changes include the impaired liver and renal functions, lymphopenia, leukopenia and thrombocytopenia whereas leukocytosis, and neutrophilia are linked to progressive infections. the gold standard for diagnosis is detection of viral rna by rt-pcr in compliance with the who guidelines for positive case criteria. the virus is found to be present in different diagnostic specimens such as the lower respiratory tract, sputum, endotracheal aspirate, bronchoalveolar lavage; upper respiratory tract, nasal or nasopharyngeal swabs, urine, feces, and blood. nevertheless, positive biopsy and autopsy tissue specimens still remain to be reported. direct or indirect contact seems to explain a part of the transmission kinetics observed between dromedary camels and humans. in the general population, transmission is rather inefficient (r < . ) and it was reported that mers-cov mortality rate is % [ ] . however, once the virus is introduced into hospital setting with large numbers of susceptible patients at risk, the virus appears to be transmitted very efficiently among such vulnerable host populations. regarding viral evolution and natural reservoir, dromedary camels are the natural reservoir for mers-cov and given the multitude of different clades found in both dromedary camels and human outbreaks, it appears that virus evolution takes place in the reservoir host rather than in humans. the study objective was to report the prevalence of mers-cov infections in the gcc countries and to also investigate its demographics in the arabian peninsula. the data for the last years were collected form the king abdulaziz medical city, riyadh, ksa. further data were collected from who s table and also from ministry of health portals of the gcc countries as follows: bahrain http://www.moh.gov.bh/, kuwait www.moh.gov.kw, oman www.moh.gov.om, qatar www.moph.gov.qa, united arab emirates http://www.moh. gov.ae/, and saudi arabia http://www.moh.gov.sa/. we also consulted the gcc countries' reports and websites for the incidence of mers-cov infections between june and july . furthermore, we searched pubmed database for articles form gcc countries reporting mers-cov infections. epidemiological data including age, sex, symptoms, date of onset, and date of sampling were collected and entered into excel worksheets. descriptive analysis, frequencies and percentages were calculated using spss vr. statistical software. between june and july , a total of confirmed mers-cov cases were reported worldwide with a mortality rate of . % (n = ). the regional distributions of mers-cov were as follows: middle east had the highest number cases ( . %), followed by asia ( . %), europe ( . %) and usa with only cases officially reported ( . %) the data are summarized in table and fig a. one hundred fifty five patients out of the total confirmed cases ( . %), reported their exposure to animals, of which, out of cases ( . %) were exposed to camels, while out of ( . %) stated exposure to other animals including sheep, cows and poultry ( table ). despite the fact that out of mers-cov cases ( . %) were health careassociated infections and out of cases ( . %) involved contact with an infected family member, cases ( . %) were still reported with no exposure to any of the above. the exposure data were found missing for the remaining ( . %) patients. the distributions of mers-cov infections among gulf countries are illustrated in table and fig b. the majority of mers-cov infections ( %) reported between the time period from june to july , were from saudi arabia. while, the remaining gcc countries contributed only % of the cases with the distributions as follows: united arab emirates ( . %), qatar ( . %), oman ( . %), kuwait ( . %) and bahrain ( . %). gender analysis shown in table reveals that % of the patients were male and % were female. moreover, saudi arabia. next, we sought out the detailed demographic distributions of reported cases among governorates in saudi arabia. as shown in table and fig c, most of the cases ( %) were reported in al riyadh region, making it the worst hit area in the country followed by makkah ( %), ash sharqiyah ( %), al madinah ( %) and najran ( %). the remaining regions together contributed to % of the cases. to date, cases are still logged from ksa and newly-diagnosed positive cases are on the rise. bahrain. to date, there was only one case reported from bahrain in manama region. herein, a -year-old saudi male was admitted on the th of march, to a health care facility in bahrain for an unrelated medical condition. this person was later on tested as positive for mers-cov ( table ) . state of kuwait. according to kuwait ministry of health, a total of cases were confirmed as mers-cov infections. the first case was reported form the capital (kuwait city) on and an -year-old female from abu dhabi were traced to another confirmed mers-cov patient with no history of exposure to camels or other risk factors. finally, a -year-old expat male from abu dhabi developed symptoms on the th of june, and was later tested positive for mers-cov. finally, we searched for the pattern of mers-cov infections over the months in order to identify seasonality relationship. as shown in fig , the average of the reported cases during this years period shows that - cases are reported in the period between february and may, while about - are reported in august and september. in addition, a low point of infection occurs in the period of october-january and another one in june. the highest number of cases were reported overall during the summer time. over the past years or so, increasing numbers of mers-cov infections have been reported from the middle eastern region [ ] . herein, we present a prevalence update on the current status of mers-cov infections in the gcc countries. the data collected over a time period from june and july show that the highest number of cases ( ) were reported from saudi arabia ( %) among a total of cases reported worldwide. overall, a total of cases were reported only from the gcc region. the saudi arabian capital city of riyadh with / ( . %) cases remained the hardest hit area for mers-cov outbreaks. the incidence of mers-cov infections was found to be highest among the elderly population aged yrs or above. moreover, the gender analysis showed that there were twice more number of males infected ( / ) than females ( / ). there is no evidence that mers-cov has gender predisposition [ ] . the observed gender-related rates could be simply due to the higher probability of male exposure to camel population than females in the region [ , ] . furthermore, over one third ( . %) of mers-cov patients received intensive care among all hospitalized cases [ ] . one could argue that the hot climate shared by arabian peninsula and sub-saharan african region could contribute to the spread of mers-cov infections across these geographical regions. although lesser in number, there are still numerous mers-cov infections recorded in saudi arabia during the winter time as compared with summer. the seasonality pattern analysis identifies a -phase annual cycle wherein the outbreaks occur during the winter and summer months. altogether, the summer time represents the peak season for mers-cov infections and transmission. the evolutionarily related bat virus might have undergone modifications and adaptation in order to be able to successfully infect and multiply in the camel as an intermediate host before transmission to the human host [ ] . although, camel is a well-known animal that is widely colonized in the gulf region, it is also reared and maintained in other parts of the world. we speculate that there might be certain conditions or factors involved with regard to camel herding and shepherding exclusively in saudi arabia that would have facilitated and contributed to the survival of pathogen and fast spread of mers-cov infections from camels to humans across all over the country. there are some reports showed that human consumption of unpasteurized camel milk and or other camel products maybe a reason for the zoonotic transmission of mers-cov in the region [ ] [ ] [ ] . on the other hand, there is strong evidence that mers-cov has been circulating in the dromedary camel population for more than decades [ ] . yet, the reason that first human infected case was identified in remains unclear. we found that mortality rates were higher among the elderly group for both genders which was also concordant with a previous study [ ] . the possible explanation for the enhanced mortality in aged patients could be the presence of senescence-associated immune vulnerability in these individuals and suboptimal immune reactivity following a systemic challenge by exposure to mers-cov natural infection. other gulf countries show a few sporadic cases which may be due to the missing data that still have to be set straight or it could possibly be due to small size of camel populations is wide spread across vast geographical region. there may be still other factors involved that remain hidden at present but contribute significantly to the survival, transmission and pathogenesis of this relatively newly identified pathogen in this region of the world. notably, it appears as if coronaviruses are able to cause serious viral infections when transferred from their reservoir (wild bat) host to the human host as observed previously for ebola virus as well which was transmitted from wild bats to humans in africa. actually, many of mers-cov cases are initiated in rural areas and following hospitalization, further cases were reported. the majority of the mers-cov outbreak cases took place at health facilities; index cases are very crucial and they raise the question of the route of transmission of this zoonotic virus. this warrants caution that strict healthcare protocols and guidelines need to be followed and practiced by health care personnel in order to prevent the new outbreaks of mers-cov. in conclusion, mers-cov infections were reported to occur in saudi arabia during the whole year whereas the incidence of human outbreaks peaked in winter and summer months. the disease incidence was also highest among the elderly population aged yrs and above. besides, the fact that majority of these cases were due to human to human interaction i.e. especially among the hospitalized icu patients and not due to camel to human transmission, the local health sectors need to be made aware to mandate implementation of effective control strategies and stringent compliance with better standards of health and hygiene nationwide. despite all whistle blowing efforts aimed at raising awareness of the magnitude of the problem at home, further efforts are still needed for proper treatment and care of mers-cov-infected patients in this country. last but not least, the availability detailed reports of each and every case of mers-cov infection globally, and the gcc region particularly will provide valuable information to the scientific community that may be used to track, contain, and eradicate this disease more effectively. supporting information s table. isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate infection control and mers-cov in health-care workers mers coronovirus has probably been present in bats for many years, research shows link to mers virus underscores bats' puzzling threat genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus neutralizing antibodies in camels animal models of middle east respiratory syndrome coronavirus infection mers-cov an emerging viral zoonotic disease: three years after and counting. recent pat antiinfect drug discov middle east respiratory syndrome prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis middle east respiratory syndrome coronavirus: review of the current situation in the world mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir human infection with mers coronavirus after exposure to infected camels, saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels mers coronaviruses in dromedary camels a lesson learned from middle east respiratory syndrome (mers) in saudi arabia the authors thank the kaimrc members and infectious disease research unit for their help and support. this study was approved by the irb and supported by funds from king abdullah international medical research center kaimrc (grant #rc / ). conceptualization: mahmoud aly. key: cord- - zyv h authors: jonsdottir, hulda r.; dijkman, ronald title: coronaviruses and the human airway: a universal system for virus-host interaction studies date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: zyv h human coronaviruses (hcovs) are large rna viruses that infect the human respiratory tract. the emergence of both severe acute respiratory syndrome and middle east respiratory syndrome covs as well as the yearly circulation of four common covs highlights the importance of elucidating the different mechanisms employed by these viruses to evade the host immune response, determine their tropism and identify antiviral compounds. various animal models have been established to investigate hcov infection, including mice and non-human primates. to establish a link between the research conducted in animal models and humans, an organotypic human airway culture system, that recapitulates the human airway epithelium, has been developed. currently, different cell culture systems are available to recapitulate the human airways, including the air-liquid interface (ali) human airway epithelium (hae) model. tracheobronchial hae cultures recapitulate the primary entry point of human respiratory viruses while the alveolar model allows for elucidation of mechanisms involved in viral infection and pathogenesis in the alveoli. these organotypic human airway cultures represent a universal platform to study respiratory virus-host interaction by offering more detailed insights compared to cell lines. additionally, the epidemic potential of this virus family highlights the need for both vaccines and antivirals. no commercial vaccine is available but various effective antivirals have been identified, some with potential for human treatment. these morphological airway cultures are also well suited for the identification of antivirals, evaluation of compound toxicity and viral inhibition. respiratory diseases caused by human coronavirus infection are of both medical and socio-economic importance. currently, they are studied in various model systems, ranging from cell lines to animal models. originally, the importance of hcovs in the burden of human disease was underestimated and as a result, no general therapy exists to treat coronavirus induced disease in humans. furthermore, no commercial vaccine is available leaving the human population vulnerable to emerging coronavirus infections. both the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses have recently crossed the species barrier and entered the human population to cause severe disease. in this review, we summarize the current knowledge on human coronavirus infection emphasizing the usefulness of organotypic human airway cultures as a model system. coronaviruses (covs), a subfamily of the coronaviridae family, are positive strand rna viruses with the largest genome of all known rna viruses (≥ kb). the genomic rna is capped, polyadenylated and associated with nucleocapsid proteins within an enveloped virion. the envelope is covered by the characteristic surface glycoprotein that gives the virus particles their characteristic crown-like (latin: corona) appearance [ ] . all covs share a common genome organization where the replicase gene encompasses the ′-two thirds of the genome and is comprised of two overlapping open reading frames (orfs), orf a and orf b that encode for up to non-structural proteins. the structural gene region, which covers the ′-third of the genome, encodes the canonical set of structural protein genes in the order ′ -spike (s) -envelope (e) -membrane (m) and nucleocapsid (n) - ′. the structural gene region also harbors several orfs that are interspersed along the structural protein coding genes. the number and location of these accessory orfs vary between the cov species [ , ] . in animals, cov infections are mainly associated with respiratory and enteric disease and can have large economical impact on the veterinary industry, e.g. porcine epidemic diarrhea virus (pedv) causes gastrointestinal disease in pigs [ ] , infectious bronchitis virus (ibv) causes severe kidney and respiratory disease in chicken [ ] and bovine coronavirus (bcov) causes both respiratory disease and diarrhea in cattle [ ] . additionally, cov infections can have other disease manifestations, such as central nervous system (cns) involvement, hepatitis and peritonitis [ ] [ ] [ ] [ ] . in humans, cov infections are mainly associated with respiratory diseases that are considered to have a large impact on the economy due to reduced productivity of the working population. currently, coronaviruses that cause disease in humans have been discovered. four of those are commonly circulating and two have caused epidemics of severe acute respiratory disease. the first human coronavirus (hcov), b , was described in . in the following years, over additional strains were characterized. ten of those strains could only be isolated from primary embryonic tracheal organ culture. others were readily isolated from monolayer cultures and were antigenically related to the prototype strain hcov- e. hcov-oc , for organ culture , was isolated and found to be distinct from the e prototype strain [ , ] . in the subsequent decades, research on hcovs would center on these two distinct viruses. however, in , an unknown respiratory illness, termed severe acute respiratory syndrome (sars), surfaced in asia. research determined it to be caused by a novel coronavirus [ , ] . at the end of the epidemic, this virus had infected over people, most in china, and caused deaths [ ] . following the discovery of this virus, two additional covs causing human disease were identified. hcov-nl was isolated in the netherlands in from an infant with bronchiolitis [ ] and hcov-hku in from a patient with pneumonia in hong kong [ ] . in , another respiratory hcov, middle east respiratory (mers)-cov, was isolated from a patient with pneumonia in saudi-arabia [ ] . unlike sars-cov, this virus is still intermittently present in the human population and most recently caused a large outbreak in south-korea [ ] . to date, there have been over cases and almost deaths related to mers-cov infection [ ] . out of the known human coronaviruses, hcov- e, hcov-oc , hcov-nl and hcov-hku are commonly circulating in the human population and usually cause general respiratory illness and cold symptoms in healthy individuals [ ] [ ] [ ] . like influenza, these viruses are capable of causing more severe disease in the immunocompromised and the elderly [ ] . they infect the human airway from the luminal side and progeny viruses are released from the same side facilitating spread through coughing and sneezing [ , ] . these coronaviruses are responsible for approximately - % of all upper and lower respiratory tract infections [ ] [ ] [ ] but the interactions between them and their natural host cells are poorly understood. currently, it is hypothesized that most of the human coronaviruses may have originated from bats [ , ] . for example, hcov- e is believed to originate from african hipposiderid bats possibly using camelids as intermediate hosts [ ] . in the last years, two coronaviruses have crossed the species barrier and caused severe and fatal disease in humans. sars-cov surfaced in and mers-cov in [ , , ] . as opposed to the commonly circulating viruses, which generally only cause mild respiratory symptoms, these viruses presented with higher case fatality ratios, around and - % respectively [ , ] . currently, there is abundant phylogenetic evidence for the bat origin of sars-cov, based on sequences of sars-like viruses found among bats in the recent years [ ] [ ] [ ] . the initial transmissions of sars-cov from animals to humans were traced back to the live animal wet markets and it was hypothesized that the virus made its way into the human population using the civet cat as an intermediate host. however, successful isolation of sars-like viruses from bats [ ] and the fact that a contemporary bat sars-like virus can infect human airway cultures [ ] suggest that an intermediate host between humans and bat might not have been needed for the transmission of sars-cov. the evolutionary origin of mers-cov is less clear but it has been speculated to be bats as well. characterization of an african bat virus closely related to mers-cov shows that both the human and camel strains belong to the same viral species and phylogenetic analysis suggests that mers-cov infection in camels predates that in humans, suggesting that camels infect humans and not the other way around. furthermore, the bat virus roots the phylogenetic tree providing further evidence for the bat origin of mers-cov [ ] . additionally, human-tohuman transmission, although not robust, seems to happen simultaneously as camel-to-human transmission. therefore, any further adaptation of mers-cov to the human host must be monitored carefully and intermediate hosts identified [ ] . many bat coronaviruses have been identified in the recent years further highlighting the zoonotic potential of this family of viruses [ ] . given the documented history of coronaviruses overcoming the species barrier and causing severe disease in humans, it is important to investigate the zoonotic potential of close evolutionary relatives of common hcovs in a culture model that recapitulates the aspects of the human airway, e.g. morphology and receptor distribution. it's important to study the mechanisms of pathogenesis and the evolution of zoonotic viruses in detail in order to identify molecular determinants that affect either transmission or pathogenesis. it's also important to elucidate whether coronaviruses currently circulating in animals are a potential danger to the human population. all of the known cellular receptors of hcovs belong to the same protein family, the membrane ectopeptidases. interestingly, the catalytic activity of these peptidases is not required for viral entry but rather the co-expression of other host peptidases activates the hcov spike proteins [ , ] . it has been established that the human transmembrane serine proteases tmprssii and hat cleave and activate the hcov- e, sars-and mers-cov spike proteins during viral entry [ , ] . out of the four commonly circulating coronaviruses, hcov- e is the only one that infects non-ciliated cells using the human aminopeptidase n (hapn) as its receptor [ ] . this peptidase is predominantly expressed on non-ciliated cells in the human bronchus [ ] . sars-cov and hcov-nl both utilize the angiotensin converting enzyme (ace ) for cellular binding [ , ] . ace is expressed on ciliated bronchial cells along with endothelial cells and both type i and ii alveolar cells [ ] . mers-cov was found to use a different receptor than sars-cov, namely the dipeptyl-peptidase (dpp ) [ ] . dpp is widely expressed in endothelial cells and various epithelial tissues in the human body [ ] . in ex vivo human lung organ cultures, different tropism of sars-and mers-covs was observed. mers-cov can actively replicate in both bronchial and alveolar tissue while sars-cov primarily replicates in alveolar tissue [ ] . the wide cellular tropism of mers-cov might contribute to its associated disease severity and high mortality rate whereas the alveolar replication of sars-cov would explain why it generally presents with pneumonia. the cellular surface receptors for hcov-oc and hcov-hku are currently unknown but receptor determinants for these two viruses have been identified as n-acetyl- -o-acetylneuraminic acid and o-acetylated sialic acid, respectively [ , ] . all of these viruses can be successfully cultured and investigated in hae cultures [ , ] . the discovery of hcovs, their receptor usage, cell tropism and receptor binding domain (rbd) is summarized in table . furthermore, established reverse genetic systems for hcov- e [ ] , hcov-oc [ ] and hcov-nl [ ] allow for controlled virus mutation and fluorescent transgene insertion to better understand the interaction of these viruses with their pulmonary host cells. traditionally, respiratory viruses are studied in animal models, usually mice and ferrets [ , ] . however, it is not always possible to correctly recapitulate human infection and disease in animal models. the establishment of transgenic animal models for human disease is attainable when either the virus receptor has been identified, which is not the case for all hcovs, or when viruses can be adapted to a different host. an adapted human virus may not share the same properties as the original human virus. sars-cov was found to replicate naturally in various strains of inbred mice but to enhance clinical signs of disease the hace was introduced into these mice. this resulted in murine models with varying degree of human disease similarity. since sars-cov already replicated in mouse cells, adapting it to the murine host was quite successful. this resulted in three mouse adapted strains that caused disease in mice similar to severe sars-cov cases in humans [ ] . in an effort to establish a mouse model for hcov- e infection transgenic hapn mice were created. however, the insertion of the hapn into mouse cells is not enough to establish robust hcov- e infection in vivo. nevertheless, cells isolated from these transgenic animals could be infected in vitro [ , ] . the emergence of both sars-and mers-covs emphasized the importance of establishing animal models for human coronaviruses. currently, a few animal models for mers-cov have been established. mice carry their own variant of the viral receptor ddp that differs from the human in regions important for mers-cov spike interaction and by replacing this receptor with the human one, mers-cov can infect mouse cells but the method of hdpp insertion has an effect on the degree of pathogenesis observed in these mice [ , ] . various non-human primates (nhps) can be naturally infected with both sars-and mers-covs. however, disease presentation and pathogenesis differs between the different subspecies and nhp models are expensive, although ideal to study human infection due to their genetic similarity [ ] . to establish a link between the research conducted in animal models and humans, an organotypic airway culture system resembling the human airway epithelium has been developed. this model is a universal platform to study human respiratory viruses [ ] [ ] [ ] [ ] . they have been used successfully for infection studies with all known human coronaviruses [ , ] . furthermore, the cultures can be inoculated with a low infectious dosage to mimic natural infection in the human airway. whereas, animal models often require both high dosage and artificial inoculation routes. organotypic cell cultures are becoming increasingly common. different cell culture models exist to depict different epithelial tissues [ ] . these cultures closely resemble their tissue of origin and contain various different cell types with distinctive roles in the polarized tissue. currently, various organotypic cell culture models exist to represent the different areas of the human airways. the human lungs span a long anatomical distance and carry out different functions depending on anatomical location [ , ] . the structure of the epithelium also differs the further you descend into the airways. tracheal and bronchial epithelium is columnar and pseudostratified, with every cell in contact with the basement membrane, while the epithelium in the alveoli is comprised of a single cell layer to facilitate air-exchange [ ] . tracheobronchial cells are one of the first targets of human respiratory viruses and can be cultured in airliquid interface (ali) where the apical side of the cell layer is exposed to air while the basolateral side is submerged in medium. tracheobronchial cells cultured in that way form a pseudostratified epithelial layer that both morphologically and functionally resembles the human upper conducting airway (fig. a) [ , ] . after differentiation, these cultures contain many different cell types such as basal, ciliated and goblet cells. they also produce protective mucus, much like in vivo epithelium. when compared to primary bronchial cells in submerged two-dimensional culture, the gene expression of primary ali cultures differs significantly. however, the expression pattern of primary human bronchial ali cultures is comparable to that of in vivo epithelium. the human bronchial cell line calu- has been used as a culture model for respiratory epithelium but its gene expression in ali cultures is more similar to submerged bronchial cell cultures than differentiated epithelium [ ] . disruption of the cell layer [ ] . therefore, the primary tracheobronchial ali culture model is especially fitting for human respiratory virus research since it accurately recapitulates the primary entry point for these viruses. by using these cultures, virus replication and host interactions can be studied in natural target cells. further establishing the usefulness of this system hcov-hku was propagated for the first time in ciliated cells of bronchial hae cultures in after culturing it in conventional cell lines had failed [ ] . alveolar epithelial ali cultures (fig. b) can also be used for virus-host interaction studies and are especially applicable when a viral infection causes pneumonia and alveolar damage [ ] . hcov-hku has also been propagated in alveolar hae cultures and exhibits a strong tropism for alveolar type ii cells and causes large syncytia formation upon infection [ ] . when compared to traditional two dimensional cell cultures, the hae cultures are more cumbersome and their preparation is time consuming but they do have an advantage over traditional monolayer cell cultures when it comes to virus-host interaction studies. different types of ali cultures used for virus research are summarized in table . within the respiratory epithelium the innate immune system has a major protective role as the first line of defense against respiratory pathogens. in particular, the interferon (ifn) system orchestrates hundreds of different cellular effector proteins that (i) protect the epithelial barrier by altering the physiological and cellular environment, (ii) impair virus propagation, spread and transmission, and (iii) shape the host's adaptive immune response. recent publications have demonstrated that the innate immune system is functional in the hae cell culture system and that most pathogen recognition receptors are expressed and up-regulated upon treatment with exogenous stimuli [ , ] . in general, hcovs do not elicit a strong innate immune response in primary target cells of the human airway early during infection. despite the presence of all major pathogen recognition receptors, no elevated expression of ifn beta, pro-inflammatory cytokines or interferon stimulated genes can be observed up to h post-infection in haes infected with hcov- e, mers-or sars-covs [ ] . this is most likely due to the intrinsic cov properties harbored in the replicative non-structural proteins that actively aid in avoiding recognition by the host innate immune system. for example, the ′ termini of the viral mrna are capped making them indistinguishable from the host cellular mrnas and no longer detectable by cellular sensors. furthermore, cov replication is associated with the appearance of double membrane vesicles (dmvs) in the host cell cytoplasm, which might serve as a protective shield for viral rna to prevent recognition by cytoplasmic rna sensors [ ] [ ] [ ] [ ] . in addition to the non-structural proteins, various cov accessory proteins have been discovered to inhibit interferon signaling at different stages of the host innate immune response. for example, mers-cov accessory protein a inhibits innate antiviral signaling by suppressing the activation of mda and rigi [ , ] whereas b inhibits the induction of the ifn-beta promoter [ ] . while orf a and b are ifn antagonists in the genome of mers-cov, sars-cov orf b antagonizes ifn signaling through mavs/rigi [ ] . whereas sars-cov orf disrupts ifn signaling by blocking the nuclear translocation of stat [ , ] . these discoveries highlight that hcovs employ similar yet different strategies despite that respiratory infections with hcovs can result in severe respiratory disease there are currently no effective prophylactic or therapeutic treatment options available. however, the emergence of novel coronaviruses has emphasized the need to develop effective treatment options. for example, vaccines using the spike proteins of both sars-and mers-covs have proven protective in animal models [ , ] suggesting that a vaccine against hcovs for human use might be achievable. additionally, various drugs that inhibit hcov infection at different stages of the replication cycle have been reported and some could potentially serve as treatment options for hcov associated severe respiratory disease. for example, patients presenting with severe respiratory disease, caused by sars-or mers-covs, are generally treated with steroids and interferon, sometimes in combination with the antiviral drug ribavirin [ ] [ ] [ ] [ ] . this treatment, however, is not especially effective highlighting the need for hcov specific antivirals. many different compounds have been determined to have anti-hcov activity. for example, protease inhibitors which suppress hcov entry [ ] [ ] [ ] , cyclosporin a (csa) treatment blocks the replication of coronaviruses from all subgroups [ ] and non-immunosuppressive derivatives of csa represent a possible therapeutic option for both human and animal cov infections. hcov infection can also be inhibited by pre-treating hae cultures with either recombinant ifn alpha or lambda [ ] . similar effect has also been shown for recombinant ifn alpha and beta which could inhibit mers-cov in ex vivo lung cultures [ ] . as previously described, ifn treatment of active hcov infection is not particularly effective in vivo. therefore, the use of ifn in humans might be limited to prophylactic treatment of exposed persons and/or health care workers treating infected patients. screenings of compound libraries have also resulted in the identification of some hcov specific antivirals. for example, a novel small compound inhibitor (k ) has been identified, and showed to be effective against a broad spectrum of covs and could inhibit both hcov- e and mers-cov in hae cultures [ ] . additionally, hcov-nl has been inhibited in hae cultures with polymer-based compounds [ ] . to date, most treatment and inhibitor studies have been conducted in hcov susceptible cell lines. however, the hae cultures represent an ideal system to test the application and efficacy of those already identified, and new, antiviral compounds against hcovs in cells that represent the primary site of replication. furthermore, the hae cultures are heterogenous, containing many different cellular sub-populations, and would allow for the evaluation of compound toxicity and effect in a differentiated layer similar to human airway epithelium. compounds already shown to inhibit hcovs in cell lines should be applied to hae cultures as well before any animal or human trials. hcov induced respiratory diseases are of both medical and socio-economic importance. the emergence of sars-and mers-cov and the yearly circulation of the four common hcovs highlight the importance of elucidating the different mechanisms employed by hcovs to evade the host immune system as well as identifying antiviral compounds and human vaccine candidates. the hae culture system is based on primary human cells offering a unique platform to study respiratory viruses in cells representing the primary entry point of these viruses, bronchial epithelial cells, or investigate the interaction of hcovs and the distal airways, in type i and ii alveolar cells. additionally, the inclusion of airway epithelial cultures for other species enables the study of zoonosis and animal-to-human transmission. currently, many aspects of hcov infection and pathogenesis remain to be determined. the hae culture system, both tracheobronchial and alveolar, represents a unique platform to study virus-host interaction in natural target cells at the molecular level. these cultures are becoming more common and more relevant to hcov research. especially, for those viruses for which there is no animal model, as they provide an organotypic substitute for virushost interaction studies. the authors declare no competing interests. authors' contribution hrj wrote the review, designed tables and figures. rd revised the text, tables and figures. both authors read and approved the final manuscript. this work was supported by the r foundation, switzerland (project - ) and the university of bern initiator grant. received: december accepted: january coronaviruses: an overview of their replication and pathogenesis coronavirus genome structure and replication the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses a new coronavirus-like particle associated with diarrhea in swine a nomenclature for 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neutralizing antibodies sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus dipeptidyl peptidase distribution in the human respiratory tract: implications for the middle east respiratory syndrome we would like to thank prof. dr. volker thiel for his careful review of the manuscript and dr. eveline kindler for providing components for figures. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - w itin authors: memish, ziad a.; al-tawfiq, jaffar a.; alhakeem, rafat f.; assiri, abdullah; alharby, khalid d.; almahallawi, maher s.; alkhallawi, mohammed title: middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: w itin since the initial description of the middle east respiratory syndrome (mers) in september , a total of cases of mers-cov including deaths have been reported from saudi arabia. from august , to september , , a total of patients and contacts were tested for mers-cov. of those tested, there were ( . %) mers-cov cases reported in al-madinah al-munawwarah with one large cluster. in this report, we describe the outcome, epidemiology and clinical characteristics of this cluster of which cases involved healthcare workers. fourteen cases appeared to be linked to one cluster involving healthcare workers (hcws), family and patient contacts. of the cases, five (including hcws) were community acquired, two were household contacts, and were healthcare associated (including hcws). all except cases were symptomatic and the case fatality rate was % ( of ). the outbreak resulted in human to human transmission of an estimated cases. contact screening showed positive test in of ( . %) household contacts, and of ( . %) hcws. summary since the initial description of the middle east respiratory syndrome (mers) in september , a total of cases of mers-cov including deaths have been reported from saudi arabia. from august , to september , , a total of patients and contacts were tested for mers-cov. of those tested, there were ( . %) mers-cov cases reported in al-madinah al-munawwarah with one large cluster. in this report, we describe the outcome, epidemiology and clinical characteristics of this cluster of which cases involved healthcare workers. fourteen cases appeared to be linked to one cluster involving healthcare workers (hcws), family and patient contacts. of the cases, five (including hcws) were community acquired, two were household contacts, and were healthcare associated (including hcws). all except cases were symptomatic and the case fatality rate was % ( of ). the outbreak resulted in human to human transmission of an estimated cases. contact screening showed positive test in of ( . %) household contacts, and of ( . %) hcws. ª elsevier ltd. all rights reserved. since middle east respiratory syndrome (mers) was described in september , a total of cases of mers-cov including deaths have been reported from saudi arabia [ ] . the current case fatality rate is lower than the initial rate of % [ ] . mers-cov is known to cause three patterns of transmissions [ e ]: sporadic cases, community-transmission [ ] and healthcare associated transmissions such as the case in the zarqa, jordan [ , ] , al-hasa, saudi arabia [ ] and jeddah, saudi arabia [ , ] . the exact source of the infection for most patients remains unknown. in this report, we describe the outcome, epidemiology and clinical characteristics of this cluster of mers-cov in al-madinah al-munawwarah of which cases involved healthcare workers. all samples were tested in jeddah regional lab. we included all mers-cov cases reported from al-madinah al-munawwarah between august , and september , . a confirmed case of mers cov is defined as an isolation of mers cov from a nasopharyngeal or a respiratory sample by real time reverse transcriptase pcr, as described previously [ , ] . clinical information included demographic data, clinical symptoms and signs, co-morbidities, and contact with animals. during the study period, a total of patients and contacts were tested for mers-cov. there were ( . %) mers-cov positive cases reported in al-madinah al-munawwarah with one large cluster. of those cases, ( %) were male and ( %) were females. twelve of the cases were saudis ( %) and were non-saudis %. there were two possible clusters and two cases were sporadic in nature. the largest cluster included cases and was thought to be initiated by a year-old male resident. he was in the same hospital ward of a year-old saudi male who was thought to acquire the infection in the healthcare setting. transmission then occurred in an additional cases as illustrated in fig. . another case was from qatar, the son of a patient sharing a room with the second case although the father tested negative for mers-cov. the second cluster was from the city of hanakia located km from madina, and involved a year-old male healthcare worker (hcw), who then infected another year-old hcw. there was one sporadic case, a year-old hcw, who had no contacts with other cases. the majority of the cases ( . %) were healthcare associated infections and primary cases constituted . % and intra-familial transmission was only . %. the case fatality rate was % ( of cases). of the symptomatic cases, ( . %) had at least two of the following chronic diseases: diabetes mellitus, hypertension, end stage renal disease, cardiac disease, sickle cell anemia, obesity, or smoking. only one patient had contact with animals, he was a healthcare worker and was asymptomatic. all of the symptomatic cases had fever ( %), % had shortness of breath, % had cough, % had nausea, % headache, and % had sore throat. a total of hcws were screened and ( . %) were positive. in addition, family contacts were screened and ( . %) was positive. the current report illustrates the pattern of transmission of mers-cov. our data harmonizes with the previously described pattern of transmission of mers-cov [ e ]. the majority of the patients ( . %) were healthcare associated infections and primary cases constituted . % and intrafamilial transmission was only . %. the recent jeddah outbreak in was documented to be secondary to intrahospital and inter-hospital transmissions [ , ] . fig. shows major mers-cov outbreaks in kingdom of saudi arabia. the rate of community infections seem to be low with expansion of the infection in the healthcare setting [ ] . animal contact, especially with camels is uncommon among primary cases, and in our series only one patient had camel contact [ , ] . the case fatality of these cases was %, compared to the overall case fatality in ksa of % [ ] of the symptomatic cases, . % had at least two of underlying chronic diseases. the presence of comorbidities predisposes to increased risk of mers-cov and was shown to also correlate with case fatality rates [ ] . screening of contacts yielded less than % positivity among hcws and family contacts. in a large screening of contacts, mers-cov was detected in . % of hcws contacts and in . % of family contacts [ ] . however, the majority of the cases were acquired within healthcare facilities similar to the al-hasa and jeddah outbreak [ e ]. an interesting observation in this report is the link of one of the mers cases from qatar to this healthcare associated cluster. travel associated mers cases were reported from: turkey, austria, united kingdom, germany, france, greece, the netherlands, tunisia, algeria, malaysia, philippines, china, and the united states of america [ ] . the recent occurrence of an outbreak in the republic of korea was started with a returning traveler [ e ]. the patient traveled to bahrain ( e april), the united arab emirates ( e april), bahrain ( aprile may), saudi arabia ( e may), bahrain ( may) and qatar ( e may) [ , ] . the outbreak spanned healthcare facilities which have treated patients and six healthcare facilities have documented nosocomial transmission [ ] . as of june , , this outbreak had caused cases including deaths [ ] . the outbreak highlights the importance of infection control and early recognition and isolation of suspected cases [ ] . the kingdom of saudi arabia also hosts one of the largest mass gathering in the world hosting millions of pilgrims during the annual hajj where pilgrims visit the holly cities of makkah and al-madinah [ ] . the occurrence of mers-cov transmission during the annual hajj and subsequent development of a global epidemic is of a great concern. respiratory samples were obtained from all mers suspected cases during hajj season and all samples tested negative for mers-cov [ ] . a cohort of french hajj pilgrims were systematically sampled in with screened for mers-cov using nasal swabs prior to returning to france [ ] . although, the majority ( . ) had respiratory symptoms, none was tested positive for mers-cov [ ] . in and hajj season, a total of million pilgrims from countries visited makkah and al-madinah and no cases of mers-cov were detected during or after the hajj [ ] . screening of adult pilgrims from countries in showed no positive mers cases using nasopharyngeal swabs [ ] . although only rare cases have been associated with the umrah pilgrimage so far, there is a need for continuing surveillance among travelers, pilgrims and hcw attending pilgrims [ ] . middle east respiratory syndrome coronavirus: epidemiology and disease control measures coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers an update on middle east respiratory syndrome: years later middle east respiratory syndrome coronavirus infection control: the missing piece? middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution travel implications of emerging coronaviruses: sars and mers-cov middle east respiratory syndrome coronavirus (mers-cov) in healthcare setting family cluster of middle east respiratory syndrome coronavirus infections epidemiological findings from a retrospective investigation hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia mers-cov outbreak in jeddahea link to health care facilities screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov)- th update middle east respiratory syndrome coronavirus (mers-cov): summary and risk assessment of current situation in the republic of korea and china etiology of severe community-acquired pneumonia during hajjdpart of the mers-cov surveillance program lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj , despite a high rate of respiratory symptoms the hajj pilgrimage and surveillance for middle east respiratory syndrome coronavirus in pilgrims from african countries prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the hajj imported cases of middle east respiratory syndrome: an update we are grateful to the staff of the ministry of health in al-madinah area and the staff of the regional laboratory, kingdom of saudi arabia. middle east respiratory syndrome coronavirus none. key: cord- -shlhreve authors: sweileh, waleed m. title: global research trends of world health organization’s top eight emerging pathogens date: - - journal: global health doi: . /s - - - sha: doc_id: cord_uid: shlhreve background: on december (th), , world health organization published a priority list of eight pathogens expected to cause severe outbreaks in the near future. to better understand global research trends and characteristics of publications on these emerging pathogens, we carried out this bibliometric study hoping to contribute to global awareness and preparedness toward this topic. method: scopus database was searched for the following pathogens/infectious diseases: ebola, marburg, lassa, rift valley, crimean-congo, nipah, middle eastern respiratory syndrome (mers), and severe respiratory acute syndrome (sars). retrieved articles were analyzed to obtain standard bibliometric indicators. results: a total of journal articles were retrieved. authors from different countries contributed to publishing these articles. two peaks of publications, an early one for sars and a late one for ebola, were observed. retrieved articles received a total of , citations with a mean ± standard deviation of . ± . citations per article and an h-index of . international collaboration was as high as . %. the centers for disease control and prevention had the highest share ( ; . %) followed by the university of hong kong with ( . %). the top leading journal was journal of virology with ( . %) articles while feldmann, heinz r. was the most productive researcher with ( . %) articles. china ranked first on sars, turkey ranked first on crimean-congo fever, while the united states of america ranked first on the remaining six diseases. of retrieved articles, ( . %) were on vaccine – related research with ebola vaccine being most studied. conclusion: number of publications on studied pathogens showed sudden dramatic rise in the past two decades representing severe global outbreaks. contribution of a large number of different countries and the relatively high h-index are indicative of how international collaboration can create common health agenda among distant different countries. on december th , , world health organization (who) led a meeting of experts and health consultants in geneva to discuss and publish a priority list of pathogens likely to cause serious outbreaks in the near future bearing in mind that the suggested pathogens had limited or no available effective therapies or preventive measures [ ] . the meeting came up with a list of top eight emerging serious pathogens that are of great harmful health consequences. according to who, the list is not an ultimate one and is supposed to be reviewed annually to include any new emerging pathogens. the who list aims to lay the basis and background for national and international health planning to combat and control any potential outbreaks of these pathogens. furthermore, the who wanted countries, researchers, clinicians, and policy makers to talk about these pathogens and corresponding infectious diseases as part of global awareness and preventive policies which might include developing new and inexpensive diagnostics, therapies, vaccines, and behavioral health measures. according to who, the list of pathogens, which required urgent attention for research and development pertaining to preparedness, included "crimean congo haemorrhagic fever, ebola virus, marburg, lassa fever, middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) coronavirus diseases, nipah, and rift valley fever" [ ] . these infectious diseases are caused by viruses and some of them, such as crimean-congo and ebola, are associated with high fatality rate [ ] [ ] [ ] [ ] [ ] [ ] [ ] . marburg virus is transmitted to people from fruit bats and spreads among humans through human-to-human transmission [ ] [ ] [ ] [ ] [ ] while lassa fever is transmitted to humans through food contaminated with rodent feces or urine [ , ] . middle east respiratory syndrome is caused by a coronavirus that was first identified in saudi arabia in [ ] [ ] [ ] while sars, another coronavirus respiratory disease, was recognized on february [ , ] . nipah virus, identified in , is emerging zoonosis that affects both animals and humans [ , [ ] [ ] [ ] [ ] . rift valley fever is a viral zoonosis that was first identified among sheep on a farm in the rift valley of kenya [ ] [ ] [ ] [ ] [ ] . the who committee listed another three pathogens/infectious diseases and considered them as serious and require an action as soon as possible. these three serious diseases include chikungunya, severe fever with thrombocytopenia syndrome, and zika. literature review using pubmed, google scholar and scopus showed that bibliometric studies on sars or ebola or nipah virus have been carried out, but as a single disease and not as a group of diseases with potential future severe epidemics [ ] [ ] [ ] [ ] [ ] . the collective analysis of literature on top eight pathogens will give a more comprehensive view on these infectious diseases and will help identify which one needs to be given top priority for funding and research. it has been reported that mapping literature with certain statistical methods could help in detection of emerging infectious disease outbreaks particularly in the presence of internet with thousands of reports being easily communicated among public health specialists and healthcare providers [ , ] . based on all of the above, we carried out this bibliometric study to analyze literature on top eight emerging pathogens suggested by who. specifically, information regarding number of publications over time, contribution of various countries, international collaboration, active authors and institutions, journals that are actively publishing articles, citations analysis, geographical distribution of publications, visualization of inter-country collaboration, and top cited articles will be presented. this kind of analysis will be of value to virologists, pharmacist, medicinal chemist, and clinicians who are interested in infectious viral diseases and in developing effective preventive and curative pharmaceutical products. young researchers need to direct their research efforts toward emerging diseases because they are considered top priority and a bulk of financial support will be invested in these diseases. healthcare workers in the field of travel medicine need to be aware of the map of infectious diseases that quickly cross borders from one country to another leading to spread of diseases with potential negative impact on public health and tourism industry. for this study, scopus search engine was chosen to retrieve required literature. scopus was used because of its advantages over other databases such as web of science (wos), google scholar or pubmed [ ] . according to falagas et al. study , no database is perfect and each has certain merits over the other. for example, pubmed and google scholar are free to use in contrast to scopus and wos. pubmed lacks citation analysis in contrast to other databases. scopus offers about % more coverage than web of science and % of medline database is covered by scopus. google scholar is the largest in terms of coverage but results obtained by google scholar have inconsistent accuracy. although scopus covers a wider journal range, it is currently limited to articles published after when compared with wos [ ] . in the current study, we preferred the use of scopus because of its wider coverage since we are interested in global research activity in the eight emerging pathogens. many of the journals published from developing countries, where these infectious diseases were found, are indexed in scopus. this is reflected in the number of journals covered by scopus versus those covered by wos [ ] . in the current study, keywords used were the names of diseases that appeared in the who top eight list. to avoid errors, the names of diseases were followed by conditional keywords such as "virus or viral or fever or hemorrhagic or haemorrhagic or corona* or coronavirus or infection or infectious). fig. illustrates the steps followed along with keywords and search query used in scopus to retrieve required data. the data obtained were refined using the side functions in scopus. such functions include: ) time limitation which was set for this study from to , ) source type of data which was set in this study to be journal articles while books and book chapters were excluded, and finally ) type of documents and for the purpose of this study all types of documents were included except errata (correction). analysis of data was carried out using the "analyze" function in scopus menu bar. analysis included annual number of published documents, productivity of each country, author, preferred journals for publishing research on top eight emerging pathogens, geographical distribution, network visualization, and institution/ organization. scopus allows for citation analysis such as total number of citations, hirsch index (h-index), and top cited articles. the h-index is a parameter used to measure productivity and scientific impact of an author, institution, or country, or even a subject area [ ] . scopus can also give analysis about active journals in publishing articles on studied diseases. active journals were presented along with impact factor (if) which was obtained from the journal citation report published by thomson reuters. an important feature in scopus is that it allows exclusion or limitation which allow researchers to identify articles published by a single author or a single country. based on this, we divided articles into two types: ( ) single country publications (scp) in which all authors have the same country affiliation and such publications represent an intra-country collaboration, and ( ) multiple country publications (mcp) in which authors have different country affiliation and such publications represent inter-country collaboration. in bibliometric studies, not all data can be presented. in most bibliometric studies, active or most productive countries, authors, institutions/organizations, and journals are usually presented. in this study, with large number of retrieved documents, only countries, authors, institutions, and journals with a minimum productivity of documents were presented and ranked. the cutoff point of publications have been previously used in other bibliometric studies [ ] . for analysis pertaining to each infectious disease, only the top productive countries were presented. an important preventive aspect of most serious infectious diseases is the development of vaccines for prevention of spread. in this study, publications pertaining to vaccine development against any one of the top eight emerging pathogens were sought and presented. the search query used to search for vaccine development was the same search query used to retrieve publications on the top eight pathogens plus the keyword "vaccin*" with an asterisk to retrieve words such as vaccine or vaccination. the complete search query for vaccine data was presented in fig. . limit to journal articles: exclude errata documents: (title("crimean-congo" or ebola or "middle east respiratory syndrome" or "severe acute respiratory syndrome" or lassa or nipah or "rift valley" or marburg or mers or mers-cov or sars or ebolavirus or crimean) and title-abs(virus or viral or fever or hemorrhagic or haemorrhagic or corona* or coronavirus or infection or infectious)) and pubyear > and pubyear < and (limit-to(srctype,"j" ) ) and ( exclude(doctype,"er" ) ) search query for vaccine related documents: ( title ( "crimean-congo" or ebola or "middle east respiratory syndrome" or "severe acute respiratory syndrome" or lassa or nipah or "rift valley" or marburg or mers or merscov or sars or ebolavirus or crimean ) and title-abs ( virus or viral or fever or hemorrhagic or haemorrhagic or corona* or coronavirus or infection or infectious ) and title ( vaccin* ) ) and pubyear > and pubyear < and ( limit-to ( srctype , "j" ) ) and ( exclude ( doctype , "er" ) ) n = statistical package for social sciences (spss - ) was used to create graphs pertaining to growth of publications for each disease. mean ± standard deviation (sd) and median (q -q ) were used for descriptive statistics. finally, bibliometric studies do not involve human or animal subjects and therefore, no ethical approval by institutional review board was required. figure shows the annual growth of publications during the study period. a total of different languages were encountered in retrieved articles. english (n = , ; . %) was the most common followed by chinese (n = ; . %), french ( ; . %), and russian (n = ; . %). the majority of retrieved articles were research articles (n = , ; . %). other types of retrieved documents are shown in table . the majority of articles (n = , ; . %) were published in peer reviewed journals in the subject area of "medicine" while ( . %) were published in peer reviewed journals in the subject area of "immunology and microbiology". the subject areas with a minimum of articles are shown in table . since some journals fit into more than one subject area, the total percentages in table exceeded %. retrieved documents received a total of , citations. the mean ± sd was . ± . citations per documents while the median (q -q ) was . the h-index was . a total of ( . %) articles were cited at least once while ( . %) articles were not cited at all. a total of ( . %) publications received a minimum of citations per article. the article that received the highest number of citations was "a novel coronavirus associated with severe acute respiratory syndrome" [ ] published in new england journal of medicine (nejm) in . it received a total of citations. table shows the top cited articles. content analysis of top cited articles showed that articles were about sars, one about nipah virus and one about ebola virus. five of top cited articles were published in nejm, three in lancet, six in science, and three in nature. researchers from different countries participated in publishing retrieved articles. table shows a list of countries with a minimum contribution of articles. the list included different countries in north america, middle east, europe, asia, australia, and africa. the total number of articles produced by the list of active countries was ( . %). the united states of america (usa) ranked first in productivity with a identification of a novel coronavirus in patients with severe acute respiratory syndrome [ ] new england journal of medicine coronavirus as a possible cause of severe acute respiratory syndrome [ ] lancet characterization of a novel coronavirus associated with severe acute respiratory syndrome [ ] science the genome sequence of the sars-associated coronavirus [ ] science a major outbreak of severe acute respiratory syndrome in hong kong [ ] new england journal of medicine angiotensin-converting enzyme is a functional receptor for the sars coronavirus [ ] nature clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study [ ] lancet isolation and characterization of viruses related to the sars coronavirus from animals in southern china [ ] science identification of severe acute respiratory syndrome in canada [ ] new england journal of medicine bats are natural reservoirs of sars-like coronaviruses [ ] science koch's postulates fulfilled for sars virus [ ] nature transmission dynamics and control of severe acute respiratory syndrome [ ] science epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong [ ] lancet a pnas proceedings of the national academy of sciences total of ( . %) followed by china (n = , ; . %), hong kong (n = ; . %), and germany (n = ; . %). geographical distribution of worldwide publications on the top eight emerging pathogens was mapped using arcgis . with darker colors indicative of higher productivity (fig. ) . international collaboration ranged from . to . %. turkey had the lowest percentage ( . %) of articles with international authors while switzerland had the highest percentage ( . %) of articles with international authors. only two countries (turkey and iran) had less than % international collaboration. there was a significant correlation (pearson correlation r = . ; p = . ) between percentage of international collaboration and number of citation per article but not with h-index. visualization of international collaboration was created using vosviewer technique. in the network visualization map, the strength of collaboration between countries is expressed by the thickness of the line between any two countries. figure shows inter-country collaboration between various developed and developing countries. the thickness of the connecting lines represents the extent of collaboration between any two countries. sixteen intuitions/organizations made a contribution of a minimum of publications ( table ). the total number of documents published by these active institutions was ( . %). eight active intuitions are in northern america (usa and canada), three are in hong kong/china, two in germany, one in france, one in japan, and one is an international organization (who). the centers for disease control and prevention (cdc) had the highest productivity of ( . when productivity of each country was calculated alone the total number exceeds the number of retrieved articles. however, when productivity of all countries was dealt with collectively, the total number will be lesser than that presented in the table. the collaboration between countries created some percentage of overlap and therefore certain number of similar countries were counted twice for collaborating countries had the highest ( ) h-index followed by u.s. army medical research institute of infectious diseases ( ) and the university of hong kong ( ). five journals made a contribution of at least articles to studied diseases. figure is a visualization map of author collaboration. the map had clusters of names of authors. each cluster represents a research group working on particular pathogen(s). table shows the number of retrieved articles for each type of disease. due to the presence of articles that might have discussed more than one pathogen/ infectious disease at the same time, the total percentages exceeded %. publications on sars ( ; . %) ranked first in quantity followed by those on ebola ( ; . %) and crimean-congo ( ; . %). geographical distribution of research publications on sars, ebola, crimean -congo, and mers were mapped and presented in figs. , , and . the annual growth of publications showed that publications on sars exhibited a sharp peak in , publications on ebola exhibited a sharp peak in , and publications on mers exhibited a clear rise starting from ( fig. a and b) . country analysis of publications on each disease is shown in table . the usa ranked first in productivity in research pertaining to mraburg, ebola, rift valley four hundred seventy-two publications were related to vaccine development. research activity on vaccine development showed similar trend to overall research activity on the top eight emerging disease (fig. this study was carried out to assess worldwide research activity on emerging pathogens expected to cause serious fatal outbreaks in the near future. several bibliometric studies were carried out and published on infectious diseases in general or on a specific disease such as ebola [ ] , sars [ , ] , and nipah [ , ] . however, no bibliometric study was carried out on research activity on a group of viruses suspected of potential outbreaks in the near future. these emerging pathogens need to be looked at as one unit since most of them have similar pathogenic and epidemiologic characteristics. our study showed that research activity on emerging pathogens showed an uprising peak in due to the outbreak of sars at that time, particularly in asian countries. another uprising peak of publications was seen in due to outbreak of ebola virus and to a lesser extent the outbreak of mers-cov. between the two peaks of sars and ebola, there was a high plateau international collaboration in research on emerging diseases was high possibly due to spread of these viral infectious outbreaks across borders. furthermore, the relatively high h-index of indicates that research on these diseases is receiving a high number of citations suggestive of importance and large number of readers. a study concluded that the h-index can be used to estimate the potential impact of a pathogen and to rank individual pathogens or types of pathogens [ ] . in our study, ebola and sars had the highest h-indices which necessitate prioritizing these two pathogens in planning for the future preventive policies. the finding that professor feldmann, r. was the most prolific researcher was confirmed by other bibliometric studies [ ] . infectious diseases like acquired immune deficiency syndrome (aids), malaria, and tuberculosis are major infectious diseases affecting millions of people and draining billions of us dollars of research funds [ , ] . research activity on malaria, tuberculosis, and aids have made some success in controlling the spread of such diseases and in developing potent and effective therapies. for example, the discovery of the effective drug artemisinin has greatly changed the therapeutic approach of malaria and enhanced control and eradication of malaria [ ] [ ] [ ] . actually, the chinese scientist tu youyou, who discovered the drug artemisinin, was awarded nobel prize in medicine in [ , ] . in case of the top eight emerging pathogens which are expected to cause serious outbreaks in the near future, no effective therapy is available so far and no preventive measures are being developed to face a sudden worldwide outbreak of these infectious diseases. calls for strengthening preparedness for crimean-congo [ ] and mers-coronavirus [ ] [ ] [ ] have been published. the who stated that research remains the cornerstone for reversing trends of serious outbreaks of certain viral diseases and that research will improve methods for surveillance, prevention, and control. unfortunately, the increased funding for aids created a shortage of funding for other infectious diseases [ ] . a study that compared research output and citations among three infectious diseases indicated that funding has a positive influence on research output and citations for a particular disease [ ] . in most bibliometric studies, the usa, the uk, germany, and other european countries appeared in the most active list of publications. however, in this study, additional countries in asia and africa, and middle east did appear in the top active list for each disease emphasizing the global threat of such infectious diseases. a bibliometric analysis on infectious diseases reported that usa ranked as top productive country but china is increasing its place among the top five countries [ ] . actually, many countries start to focus their research efforts on infectious diseases as a national health burden [ ] . the participation of asian, african, and middle eastern countries in research activity pertaining to top eight emerging infectious diseases was clear and prominent. outbreaks of emerging viral infectious diseases have been commonly reported from many countries in africa, asia, and africa [ , [ ] [ ] [ ] [ ] [ ] [ ] . for example, mers-cov and crimean-congo fever have been reported in more than countries, mostly in asia, africa, and middle east [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the outbreaks of sars in hong kong and china had a great economic and public health impact [ ] [ ] [ ] . many of these infectious diseases were initially reported in africa, such as ebola, lassa fever, and rift valley fever [ ] [ ] [ ] [ ] [ ] [ ] . the marburg virus was initially reported in germany and spread to other neighboring countries and that is why china and hong kong did not show in the top productive countries on marburg disease. our study has few limitations that need to be stated. scopus is a large and comprehensive database but not all journals are indexed in scopus and therefore, some articles about the studied diseases published in un-indexed journals might be missed. furthermore, the keywords used might not be % accurate although the validity of the search query was tested by manual review of % of retrieved articles, false positive and false negative results remain a possibility. the ranking of countries and institutions based on citations did not take into account self-citations which affects the validity of results. [ ] nature accelerated vaccination for ebola virus haemorrhagic fever in non-human primates [ ] nature live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses [ ] nature medicine a dna vaccine induces sars coronavirus neutralization and protective immunity in mice [ ] nature severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice [ ] pnas marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates [ ] virology effects of a sars-associated coronavirus vaccine in monkeys [ ] lancet ebola virus: from discovery to vaccine [ ] nature reviews immunology evaluation in nonhuman primates of vaccines against ebola virus [ ] emerging infectious diseases severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus [ ] avian pathology ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenge [ ] journal of infectious diseases efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial [ ] the lancet dna vaccines expressing either the gp or np genes of ebola virus protect mice from lethal challenge [ ] virology a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial [ ] clinical and vaccine immunology single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus [ ] journal of virology development of a new vaccine for the prevention of lassa fever [ ] plos medicine these limitations and others are found in most bibliometric studies [ , [ ] [ ] [ ] [ ] [ ] . this study focused only on the top eight emerging infectious diseases expected to cause severe outbreaks in the near future. however, the other three serious infectious diseases which in include zaika were not included in the analysis. finally, we should always bear in mind that no database is perfect and even might have some bias by over-representing journals with english language. therefore, bibliometric results should always be considered with caution [ ] . the number of publications on diseases expected to cause severe outbreaks in the near future showed two clear peaks in the past two decades; one for sars and one for ebola. the clear increase in number of publication on the studied diseases during relatively short period of time is an indication of how science and health information flows rapidly across borders to create similar concerns among different countries. bibliometric methods can be used to prioritize efforts and direct research funds to help control emerging diseases [ ] . although the usa is leading the research on these diseases, the share of asian, african, and middle eastern countries was apparent. international collaboration in 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induces protective neutralizing antibodies primarily targeting the receptor binding region the author would like to acknowledge professors saed zyoud and adham abu-taha for their technical and professional editing. availability of data and materials all data present in this article can be retrieved from scopus using keywords listed in the methodology. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -peqkcix authors: khan, raymond m.; al-dorzi, hasan m.; al johani, sameera; balkhy, hanan h.; alenazi, thamer h.; baharoon, salim; arabi, yaseen m. title: middle east respiratory syndrome coronavirus on inanimate surfaces: a risk for health care transmission date: - - journal: american journal of infection control doi: . /j.ajic. . . sha: doc_id: cord_uid: peqkcix the middle east respiratory syndrome coronavirus (mers-cov) has been responsible for multiple health care–associated outbreaks. we investigated whether high-touch surfaces in rooms of laboratory-confirmed mers-cov patients were contaminated with mers-cov rna. we found out of surfaces were contaminated with mers-cov viral genetic material. hence, environmental contamination may be a potential source of health care transmission and outbreaks. meticulous environmental cleaning may be important in preventing transmission within the health care setting. the middle east respiratory syndrome coronavirus (mers-cov) has been responsible for multiple health care-associated outbreaks. we investigated whether high-touch surfaces in rooms of laboratoryconfirmed mers-cov patients were contaminated with mers-cov rna. we found out of surfaces were contaminated with mers-cov viral genetic material. hence, environmental contamination may be a potential source of health care transmission and outbreaks. meticulous environmental cleaning may be important in preventing transmission within the health care setting. © association for professionals in infection control and epidemiology, inc. published by elsevier inc. all rights reserved. in september , the middle east respiratory syndrome coronavirus (mers-cov) was identified from a patient in saudi arabia. as of march , , the world health organization reported , laboratory-confirmed mers cases in countries, with deaths ( %). in its most recent report, the centers for disease control and prevention has stressed the great importance of personal protective equipment (ppe), source control, and environmental infection control measures to help eliminate the threat of health care-associated outbreaks. most health care-associated mers-cov outbreaks has occurred in saudi arabia. although the precise mechanism of humanto-human transmission has not been elucidated, mers-cov can be recovered from plastic surfaces after hours at °c and % relative humidity (rh), and the virus is viable for hours at °c and % rh and for hours at °c and % rh. further, data from the south korean outbreak (may ) demonstrated that several environmental surfaces frequently touched by laboratory-confirmed mers patients and health care workers were contaminated by mers-cov. additionally, viral sheading was detected by viral cultures from respiratory secretions up to days postdisease onset. although mers-cov was isolated from numerous high-touch surfaces in korean hospitals affected by mers outbreak, such data are lacking in the middle east. therefore, the objective of this study was to examine the extent of environmental contamination with mers-cov during an outbreak in a saudi hospital. the study was performed in the intensive care unit (icu) at king abdul-aziz medical city, riyadh, during a mers-cov outbreak from september -october , . the icu had strict environmental cleaning policies, which included cleaning the rooms at least twice daily using ammonium-based disinfectant and chlorine solution : or , ppm, having a checklist, and frequent inspection using fluorescent light or culturing of high-touch areas. three negative-pressure rooms of laboratory-confirmed mers patients (a, b, and c) were selected for this study ( table ) . the room temperature was . °c- . °c, and rh was %- %. the air exchange rate was per hour, and the pressure gradient between the room and its anteroom ranged from . - . pa. sixteen high-touch surfaces were evaluated (table ) : in the patients' room (bedrails, mechanical ventilator, ventilator tubing, sink, garbage bin, monitor, intravenous poles, intravenous pumps, telephone, door knobs, floor, drapes-blinds, air conditioning vent, and shelf of the surgical boom) and outside (computer and medical chart). environmental samples were collected as described by julian et al. briefly, a sterile swab premoistened with viral transport media was used to swab each surface (at least cm ) horizontally, vertically, and diagonally for seconds. this procedure was repeated using eluents: / lactated ringer solution and phosphate buffer solution (pbs). virus detection was performed using specific real-time reverse-transcription polymerase chain reaction (pcr) assays for the upstream of the envelope gene and the open reading frame a. positive tests were reported as the cycle threshold value for both upstream of the envelope gene (e) and open reading (o) frame a. the demographic for the patients are summarized in table . all laboratory-confirmed mers patients were on mechanical ventilators, with an average pao /fio ratio of . the mean icu length of stay and time from last positive tracheal aspirate for mers-cov rna to environmental sampling were . days and hours, respectively. sixteen surfaces were evaluated in each of the icu rooms, with environmental samples processed ( table ) our study revealed that mers-cov viral rna was isolated from the environmental surfaces of mers patients. currently, much remains uncertain about the transmission mechanism responsible for mers nosocomial outbreaks. it was postulated from the outbreak in al-hasa, saudi arabia, in may-june that respiratory droplet and airborne transmission during aerosolgenerating procedures were the most likely transmission modes. however, genetic data from a cluster in hafr al-batin, saudi arabia, showed that direct person-to-person contact could not account for all of their cases, therefore raising the likelihood of an alternate transmission mechanism. studies on kinetics and patterns of viral excretion indicate that mers-cov rna was isolated from urine and feces and days, respectively, after initial symptoms. viral shedding from respiratory aspirates may persist up to days after illness onset. prolonged viral shedding , and survival on surfaces for hours make it difficult to ignore contaminated environmental surfaces as a potential etiology of hospital outbreaks. the rate of detecting mers-cov in our environmental samples was low ( . %) compared with recently published data (pcr positive = . %; culture positive = . %), but the current methods for isolating viruses from the environmental surfaces are not optimal. based on reported methodologies, we used a polyester swab, / lactated ringer solution, pbs and viral transport media because they seem to give the best yield for isolating viruses from fomites. however, we did screening at the tail-end of our outbreak when the patients' viral load might have been low and our infection control practices might have been optimal. additionally, mers patients were managed in our icu since and were usually cohorted in unit where the staff became very meticulous about ppe use and environmental cleaning. moreover, fairly weak disinfectants, such as povidone iodine, have a rapid virucidal activity (reduction in virus titer by ≥ log ) against mers-cov, with an exposure time of just seconds. further, leclercq et al demonstrated that at relatively low temperatures of °c, only minutes was needed to reduce the initial titer by log , while at °c virucidy dropped significantly to minute. this sensitivity to weak disinfectants could explain why our stringent environmental cleaning policies may have attenuated the recovery of viral genetic material on fomites within the patients' rooms. our finding of mers-cov rna on environmental samples within our icu shows that the viral material may contaminate fomites and can be a theoretical cause of nosocomial infections. however, we did not use viral cultures; therefore, we do not know if the positive pcrs correlate with live viruses or infectivity. despite this, we believe that in addition to proper hand hygiene and correct ppe donning and doffing, meticulous environmental cleaning is of paramount importance to eliminate health care outbreaks. middle east respiratory syndrome coronavirus (mers-cov) cdc's early response to a novel viral disease, middle east respiratory syndrome coronavirus (mers-cov) stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea comparison of surface sampling methods for virus recovery from fomites hospital outbreak of middle east respiratory syndrome coronavirus community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases rapid and effective virucidal activity of povidoneiodine products against middle east respiratory syndrome coronavirus (mers-cov) and modified vaccinia virus ankara (mva) heat inactivation of the middle east respiratory syndrome coronavirus key: cord- - okuomi authors: baloch, zulqarnain; ma, zhongren; ji, yunpeng; ghanbari, mohsen; pan, qiuwei; aljabr, waleed title: unique challenges to control the spread of covid- in the middle east date: - - journal: j infect public health doi: . /j.jiph. . . sha: doc_id: cord_uid: okuomi the covid- pandemic is spreading at unprecedented pace among the middle east and neighboring countries. this region is geographically, economically, politically, culturally and religiously a very sensitive area, which impose unique challenges for effective control of this epidemic. these challenges include compromised healthcare systems, prolonged regional conflicts and humanitarian crises, suboptimal levels of transparency and cooperation, and frequent religious gatherings. these factors are interrelated and collectively determine the response to the pandemic in this region. here, we in-depth emphasize these challenges and take a glimpse of possible solutions towards mitigating the spread of covid- . politically, culturally and religiously a very sensitive area, which impose unique challenges for effective control of this epidemic. these challenges include compromised healthcare systems, prolonged regional conflicts and humanitarian crises, suboptimal levels of transparency and cooperation, and frequent religious gatherings. these factors are interrelated and collectively determine the response to the pandemic in this region. here, we in-depth emphasize these challenges and take a glimpse of possible solutions towards mitigating the spread of covid- . having not forgotten the panic of the middle east respiratory syndrome coronavirus (mers-cov), its new counterpart severe acute respiratory syndrome coronavirus (sars-cov- ), the causative agent of coronavirus disease (covid- ) , has landed in the middle east. at june , , there were cases and deaths of covid- recorded across the middle east. although the case number was low cases in the early days of the outbreak, it has speeded at unprecedented pace later on and affecting all the middle east countries (figure ). this could reflect the levels of preparedness and response plans implemented locally in middle east countries. the middle east lies at the juncture of eurasia and africa, and constitutes an important pillar in shaping modern civilizations and religions. it is geographically, economically, politically, culturally and religiously a very sensitive area. unfortunately, the whole region has been the center for wars and conflicts over the past years. these complexities have immense impact on breading, responding and mitigating epidemics [ ] . of note, iran is the early epicenter with the highest number of confirmed covid- cases and deaths in the middle east region, followed by turkey, j o u r n a l p r e -p r o o f saudi arab, qatar and egypt (figure ) . given the rapid spread of covid- , we aim to emphasize the unique challenges of containing the epidemic in this region and to take a glimpse of possible solutions. healthcare systems within the middle east constitute a high degree of variability with regards to accessibility, capacity, and quality among different countries. the persian gulf region acquires significant wealth from the oil industry and has developed their healthcare systems [ ] . within these countries, there is a two-tiered healthcare system structure with public and private streams of financing and service delivery. who has highlighted the crucial role of early detection of covid- cases to interrupt the spread, whereas the required expertise and diagnostic capacity could differ tremendously among different healthcare providers [ ] . thus, it is essential for the authorities to ensure equal and rapid access to diagnosis and treatment of covid- regardless of nationalities, ethnicities, religions and beliefs. the healthcare systems in non-oil-producing countries are largely underdeveloped and were further weakened due to prolonged conflicts and wars. violent attacks on healthcare facilities and health workers during conflicts destroy essential health services that are most needed during the time [ ] . the syrian conflict has been marked by frequent and intense attacks against healthcare facilities [ ] . the ongoing yemen war has led to only % of the total healthcare facilities to be functional. a lesson learnt from the conflicts in the democratic republic of the congo is that frequently attacking health care workers and ebola treatment centers has gravely exacerbated the ebola outbreaks [ ] . the geopolitical context in palestine results in limited freedom of movement and economic stability causing major challenges for healthcare system maintenance [ ] . in iran, although the healthcare system has been developed, economic sanctions jeopardize social determinants of health and access to medication and healthcare [ ] . the rapid growing number of covid- cases in iran is threating the overwhelmed healthcare system but sanctions further limit the access to emergency medical supplies. rapid and adequate access to healthcare is essential for controlling the epidemic but also for minimizing severe patient outcomes [ ] . besides weakening healthcare systems, wars and conflicts in the middle east have also led to large-scale humanitarian crises. because of the syrian civil war, . out of million syrian citizens require humanitarian assistance. of these, five million have been placed in refugee camps established in turkey, lebanon, jordan, egypt and other countries [ ] . the conditions in yemen have continuously deteriorated since violence broke out in early . over four million people have been forced to flee their homes, and more than % of the population ( million) is in need of humanitarian assistance [ ] . since , yemen has experienced a large-scale cholera epidemic, affecting more than . million cases and over , deaths [ ] . emerging evidence indicates that sars-cov- is very contagious [ ] . conflicts-driven population migration will likely increase the risk of sars-cov- transmission. human-to-human transmission mainly occurs via droplets or close contacts [ ] . thus the high population density in refugee camps and a lack of j o u r n a l p r e -p r o o f sanitation and hygiene are prone to widespread of the virus [ ] . a subset of covid- patients develop diarrhea and shed viruses into feces [ , ] . this has major implications pointing to possible fecal-oral transmission [ ] . thus, it is recommended to closely monitor whether this route of transmission may occur in refugee camps where have poor water, sanitation and hygiene services. efforts must be made to minimize the risks of provoking large-scale covid- epidemics in the settings of humanitarian crisis. recalling the important communication lessons from the previous sars outbreak, transparent communication is key to an effective response to covid- outbreak. because of cultural, ethnical and religious differences, non-inclusive governments and humanitarian crises, the levels of transparent communication are clearly suboptimal in this region [ ] . two factors are of concern as obstacles for transparent communication and cooperation, including religion and ethnicity oriented diversification and conflicts. mass gatherings defined by who are planned or spontaneous events that gather substantial numbers of attendees, and these events are associated with major public health challenges [ ] . this is particularly relevant to the potential superspreading of sars-cov- . the outbreak in china coincided with a massive population movement, because of the chinese lunar new year holiday [ ] . a massive annual potluck banquet for , families held in wuhan in the middle of january was suspected as an exacerbation of the outbreak in the epicenter. analysis of nine gatherings for meal or holiday visit has indicated that superspreading events tend to occur during these close contacts [ ] . during a religious gathering, a superspreader event that a church attendee infected nearly people has occurred in korea and substantially triggered public panic. in islam, there are the five-times-daily ritual prayers and a congregational prayer on every friday in mosques, where large number of worshipers gathers. of more concerns are the large-scale pilgrimages. saudi arabia continuously receives many millions of umrah pilgrims and - million hajj pilgrims at a special season of each year from over countries (fig. a) . respiratory infections are the most common illness among pilgrims, and the associated mortality rate during hajj has been reported as . % [ ] . in iran, the early covid- cases were recorded in qom (fig. b) , a city that attracts millions of pilgrims from countries including lebanon, as pilgrims concentrating on religious rituals, there are close contacts among worshipers and insufficient self-protective measures, and therefore amplify the risk of transmission and potential super spreading of sars-cov- [ ] . the governments of saudi arabia, iran and iraq are taking heavy measures on regulating the attendance of congregational prayers and pilgrimages to mitigate the risks. in this respect, we recommend the authorities to actively communicate with religion scholars and leaders of the religious communities to facilitate decision-making and policy implementation. facing the spread of covid- in the middle east, there are major challenges to contain this epidemic. these include compromised healthcare systems, prolonged regional conflicts and humanitarian crises, suboptimal levels of transparency and cooperation, and frequent religious gatherings. these factors are interrelated and collectively determine the response to the epidemic in this region. there is no simple solution, but to leverage the communication and cooperation between political leaders, healthcare authorities, religion scholars and the general public is certainly important. immediate efforts should be dedicated to possibly lift economic sanctions and to end violence and conflict. for more effective response to the epidemic, we call to establish an independent and neutral international working group specifically dedicating to covid- , with particular focus on areas with conflicts and j o u r n a l p r e -p r o o f humanitarian crises. this new organization should work closely with all united nations agencies, all the humanitarian organizations, and the local healthcare authorities with support of the international community. finally, we shall unite and ensure commitment from all parties to contain the epidemic, as there are no physical or virtual borders for sars-cov- to cross. all authors declare no competing interests. the work was supported by the ministry of education of china for an innovative research team in university grant (no. irt_ r ; to z.m.). armed conflicts have an impact on the spread of tuberculosis: the case of the somali regional state of ethiopia healthcare for the ageing populations of countries of middle east and north africa laboratory readiness and response for novel coronavirus ( -ncov) in expert laboratories in eu/eea countries determining the scope of attacks on health in four governorates of syria in : results of a field surveillance program attacks on healthcare facilities as an indicator of violence against civilians in syria: an exploratory analysis of open-source data the exacerbation of ebola outbreaks by conflict in the democratic republic of the congo barriers preventing palestinian women from having a mammogram: a qualitative study economic sanctions threaten population health: the case of iran potential association between covid- mortality and health-care resource availability assessment of the health needs of syrian refugees in lebanon and syria's neighboring countries a call for urgent, organised medical missions in yemen the cholera outbreak in yemen: lessons learned and way forward a mathematical model for simulating the phase-based transmissibility of a novel coronavirus potential of large "first generation" human-to-human transmission of -ncov transmission routes of -ncov and controls in dental practice clinical characteristics of coronavirus disease in china epidemiologic features and clinical course of patients infected with sars-cov- in singapore enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov- possible? governance and health in the arab world mass gatherings medicine: public health issues arising from mass gathering religious and sporting events covid- control in china during mass population movements at new year secondary attack rate and superspreading events for sars-cov- mass gathering medicine (hajj pilgrimage in saudi arabia): the clinical pattern of pneumonia among pilgrims during hajj covid- : preparing for superspreader potential among umrah pilgrims to saudi arabia key: cord- -jtv jj z authors: cho, sun young; kang, ji-man; ha, young eun; park, ga eun; lee, ji yeon; ko, jae-hoon; lee, ji yong; kim, jong min; kang, cheol-in; jo, ik joon; ryu, jae geum; choi, jong rim; kim, seonwoo; huh, hee jae; ki, chang-seok; kang, eun-suk; peck, kyong ran; dhong, hun-jong; song, jae-hoon; chung, doo ryeon; kim, yae-jean title: mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: jtv jj z background: in , a large outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection occurred following a single patient exposure in an emergency room at the samsung medical center, a tertiary-care hospital in seoul, south korea. we aimed to investigate the epidemiology of mers-cov outbreak in our hospital. methods: we identified all patients and health-care workers who had been in the emergency room with the index case between may and may , . patients were categorised on the basis of their exposure in the emergency room: in the same zone as the index case (group a), in different zones except for overlap at the registration area or the radiology suite (group b), and in different zones (group c). we documented cases of mers-cov infection, confirmed by real-time pcr testing of sputum samples. we analysed attack rates, incubation periods of the virus, and risk factors for transmission. findings: patients and health-care workers were identified as contacts. mers-cov infection was confirmed in individuals ( patients, eight health-care workers, and visitors). the attack rate was highest in group a ( % [ / ] vs % [ / ] in group b vs % [ / ] in group c; p< · ), and was % ( / ) in health-care workers. after excluding nine cases (because of inability to determine the date of symptom onset in six cases and lack of data from three visitors), the median incubation period was days (range – , iqr – ). the median incubation period was significantly shorter in group a than in group c ( days [iqr – ] vs days [ – ]; p< · ). there were no confirmed cases in patients and visitors who visited the emergency room on may and who were exposed only to potentially contaminated environment without direct contact with the index case. the main risk factor for transmission of mers-cov was the location of exposure. interpretation: our results showed increased transmission potential of mers-cov from a single patient in an overcrowded emergency room and provide compelling evidence that health-care facilities worldwide need to be prepared for emerging infectious diseases. funding: none. since the fi rst identifi cation of middle east respiratory syndrome coronavirus (mers-cov) infection in , most patients infected with the virus have been exposed in the middle east. as of march , , laboratoryconfi rmed cases have been reported to who. on the basis of previous epidemiological fi ndings, the potential of mers-cov to spread to large numbers of people has been considered low, by contrast with severe acute respiratory syndrome coronavirus (sars-cov). the basic reproductive number of mers-cov was estimated to be less than · , suggesting low transmissibility. , however, a outbreak of mers-cov infection in al hasa, saudi arabia, where one patient infected seven other patients in dialysis and intensive care units, raised concerns about potential so-called super-spreaders that were reported during the sars epidemic. , from may to july, , a large outbreak of mers-cov infection occurred in south korea from a traveller returning from the middle east, which led to confi rmed cases (patient to patient ) in the country. patient was diagnosed at our hospital (samsung medical center, seoul, south korea) after transmitting the virus at several health-care facilities before he came to our hospital. patient was exposed to patient outside the hospital and sought additional care at our hospital without knowing he was infected with mers-cov. therefore, we experienced both south korea's fi rst mers-cov case and the case of highest transmission of mers-cov following a single patient exposure in an emergency room. we aimed to investigate the epidemiology of mers-cov infection in a crowded emergency room outside of the middle east and the presence of multiple super-spreaders. in may, , two patients with mers-cov infection (patient and patient ) sought care in our emergency room at the samsung medical center without knowing they were infected with mers-cov. while these patients were in the emergency room, a large number of patients, visitors, and health-care workers were exposed during both events. when mers-cov infection was suspected in patient and patient , contact investigation was immediately initiated. since no one developed mers among contacts who were exposed to patient , only contacts of patient are reported here. we identifi ed, from electronic medical record review and security video footage, all patients who had been in the emergency room with patient as contacts, regardless of the location and duration of exposure. we categorised patient contacts into three groups on the basis of their maximum exposure: patients who were in the same zone in the emergency room (group a; considered close contacts), those who were in diff erent zones but had time overlap with patient in the registration area or radiology suite ( min before and h after; group b), and those who were in diff erent zones (group c). patients who were admitted to hospital for treatment of their primary illness after exposure in the emergency room were quarantined in private rooms for days from the last exposure or discharged home after treatment was fi nished and continued isolation at home. patients and their family members who were already discharged home were reached by telephone, informed about possible mers-cov exposure, and provided with hotline numbers for any inquiries. health-care workers who were exposed were identifi ed through interviews and review of employees' duty schedules, electronic signature on medical records of patient and patient contacts, security video footage, and self-report. health-care workers who provided direct care to patient were initially considered close contacts and were placed into quarantine at home for days from the last day of exposure. other health-care workers who worked in the emergency room during the same time period continued to work with monitoring and were removed immediately from duty if symptoms developed. a confi rmed case was defi ned as a person with laboratory confi rmation of mers-cov infection from sputum samples, initially by real-time rt-pcr testing with amplifi cation targeting the upstream e region (upe) and then confi rmed by subsequent amplifi cation of open reading frame a (orf a) using powerchek mers realtime pcr kits (kogene biotech, seoul, korea). patients' demographic information, underlying disease, dates of emergency room visit, duration of stay with exact arrival and departure times, and location within the emergency room were collected. if radiographic examinations were done, the time of examination was collected. for healthcare workers, age, sex, occupation, history of patient assignments and working or visiting zone, and dates and time of duty or emergency room visits were collected. the attack rate was calculated by dividing the number of confi rmed cases by the total number of exposed individuals in the emergency room in each group. because the total list of visitors was unavailable, we estimated the number of visitors who were in the emergency room by assuming that one patient had at least one visitor during their stay; we also simulated the scenarios of two and four visitors per patient. to avoid underestimation, we chose the assumption of one visitor per patient, which would give the highest attack rate among the scenarios. the incubation period was defi ned as the time of fi rst exposure to the onset of clinical symptoms of mers-cov infections. categorical variables were presented with frequency (percentage) and continuous variables were summarised with median (range, iqr). we calculated overall comparison of attack rates across the groups with χ² test and across zones with fisher's exact test. incubation evidence before this study little information on nosocomial outbreaks caused by middle east respiratory syndrome coronavirus (mers-cov) outside the middle east had been available before the large mers-cov outbreak in south korea in , for which global alert was issued. we searched pubmed for reports published in english from may , , to dec , , using the terms "mers-cov" and "korea". we identifi ed reports, none of which provided detailed description for the contact investigation of massive transmission of mers-cov from a super-spreader in an overcrowded emergency room setting. to our knowledge, this study is the fi rst to categorise exposed patients into groups according to the type of exposure and to document group-specifi c incubation periods and attack rates. furthermore, this study provides detailed epidemiological data, including a fl oor plan of the emergency room, to understand how mers-cov spread by a single super-spreader through several modes of transmission. results from our contact investigation showed increased transmission potential of mers-cov from a single spreader, as has been documented in the severe acute respiratory syndrome epidemic. the potential for similar outbreaks anywhere in the world should be noted, as long as mers-cov transmission continues in the middle east. our study provides evidence that hospitals, laboratories, and governmental agencies should be prepared for mers-cov infection. period and exposure time were compared among groups with kruskal-wallis test, followed by tukey's test using ranks for multiple comparisons. to assess the risk factors for mers-cov infection among all patient contacts, we did a multiple logistic regression analysis based on likelihood ratio, by regressing on age, sex, underlying disease, and groups. in a subgroup analysis of patients in group a, the length of stay in the same zone and location were included. for these analyses, odds ratios and % cis were reported. p values and % cis were adjusted with bonferroni's correction for multiple comparisons if necessary. two-sided p values of less than · were considered signifi cant. we used sas version . and graphpad prism version . for statistical analyses. samsung medical center is a modern -bed university-affi liated tertiary hospital providing referral care in south korea (total population roughly million), with roughly staff , including more than physicians and nurses. the emergency room entrance is located on the ground fl oor near the south gate of the main hospital building. more than patients are seen in the emergency room each day; the average duration of stay in the emergency room was h before the mers-cov outbreak (see appendix p for details on emergency room overcrowding index). the emergency room has seven patient care areas, including zones i to iv for see online for appendix adults, a trauma zone, a resuscitation room, and a paediatric zone (fi gure ). the paediatric zone and zone iv are separated from the rest of the main areas. two negative-pressure rooms are located in the paediatric zone and two are in zone iv. the emergency room has its own radiology suite for emergency room patients only. the sizes of each zone were as follows: zone i · m², zone ii · m², zone iii · m², and zone iv · m². zones i and ii included seating areas ( chairs in zone i and chairs in zone ii), where stable patients received treatment and waited for test results. seriously ill patients, who required close observation and needed a designated bed, were moved to zone iii ( beds) or zone iv ( beds). zone iv was used for patients being admitted. beds in zones iii and iv were spaced roughly · m apart, with curtains in between. nurses were assigned to work in designated zones, whereas physicians and transfer agents took care of patients in several zones. all zones in the emergency room were covered by the same air handling units. there was no funding source for this study. the corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. upon arrival at our emergency room, he denied having travel history to the middle east and any possible exposure to people infected with mers-cov. he was treated for possible bacterial pneumonia on the basis of partial improvement from previous antibiotic treatment and increased c-reactive protein concentration of · mg/dl (normal < · mg/dl; appendix p ). during his stay in the emergency room, he was provided with a mask but frequently could not hold it because of severe respiratory symptoms. he was not isolated in a separate room; a negative-pressure room was not considered at that time. as his dyspnoea aggravated on may , supplemental oxygen was administered at l per min via a nasal cannula (up to l per min). however, no aerosol-producing procedures, including nebuliser treatments, were given. on the night of may , he received a notifi cation call from the health authorities about possible exposure to patient , notifi ed our hospital, and was immediately transferred from the emergency room to isolation in a negativepressure isolation room. mers-cov infection was confi rmed on may , and he was transferred to the nationally designated health-care facility. from may to may , he stayed in three zones in our emergency room: zone ii for roughly h on may , zone iii for h from may to may , and zone iv for h from may to may (fi gure ). additionally, from may to may , he went to the radiology suites four times. on may , he walked around and outside the emergency room and went to the toilet several times because of diarrhoea. between may and may , , the average ventilation rate in the emergency room was maintained at three air changes per h, taking h to remove airborne contaminant with a · % effi ciency. the median temperature was · °c (range · - · ), and the median relative humidity was · % (range · - · ). patients ( in group a, in group b, and in group c), an estimated visitors, and health-care workers were identifi ed as contacts of patient (table ). we assumed that each patient had one visitor and added eight extra visitors (fi ve to group a and three to group c) the epidemic curve of this emergency room-associated outbreak is shown in fi gure . the incubation period was determined from confi rmed mers-cov cases: six cases were excluded because we could not determine the date of symptom onset, and data were not available from three visitors. the median incubation period was days (range - , iqr - ). among patients and visitors in groups a-c (excluding six who were not initially identifi ed as contacts), the median incubation period was signifi cantly shorter in group a than in group c (fi gure ). excluding three patients with confi rmed mers-cov infection who were not identifi ed in the initial patient contact investigation (appendix p ), the overall attack rate for patients in the emergency room was % ( of ). patients in group a had the highest attack rate ( % [ of ]), compared with % (three of ) in group b and % (four of ) in group c (fi gure ). after adjusting for age, sex, underlying disease, and groups, patients in group a had the highest risk for mers-cov infection (table ). in group b, all three patients who had mers-cov infection had time overlap in the radiology suite with patient . the median exposure time for patients in group a to patient was · h in zone ii (range · - · , iqr · - · ), · h in zone iii ( · - · , · - · ), and · h in zone iv ( · - · , · - · ). the attack rates were % ( of ) in zone ii, % (seven of ) in zone iii, and % (three of ) in zone iv (fi gure ). after adjusting for age, sex, underlying disease, and exposure time, staying in zone ii was associated with a signifi cantly higher risk for mers-cov infection than staying in zone iv (table ) . mers-cov transmission occurred in zone iii, despite the fact that the distance from patient 's bed to the beds of other patients were as far as m (fi gure ). in zone iv, patient moved from bed to bed , and six additional cases were documented in patients and visitors occupying beds in the middle of this zone, which were not adjacent to patient 's bed. no mers-cov infection was reported in patients and visitors who had been in the emergency room on may during the time period when they were exposed only to zones ii (n= ) or iii (n= ), while patient was confi ned to zone iv. these patients were exposed to areas that were potentially environmentally contaminated but not to patient himself (fi gure ). under the assumption of one visitor per patient and excluding three visitors with confi rmed mers-cov infection who were not identifi ed in the initial visitor contact investigation (appendix p ), the overall attack rate for visitors was % ( of ). all patient contacts (n= ) any underlying disease · ( · - · ) · error bars represent % ci. mers-cov=middle east respiratory syndrome coronavirus. the attack rates for patients and visitors were % ( of ) in group a, % (six of ) in group b, and % ( of ) in group c. under the assumptions of two visitors per patient and four visitors per patient, the overall attack rates for visitors were % and %, respectively. health-care worker contacts were identifi ed, and fi ve ( %) developed mers-cov infection. three healthcare workers who were not initially identifi ed as contacts (one security guard, one physician, and one patient transfer agent) developed mers-cov infection. although they were not involved in the direct care for patient , they visited the emergency room between may and may . only close contacts were furloughed and other health-care workers were isolated when they developed symptoms. there were no secondary cases from health-care workers among contacts during the their duty hours. we did a contact investigation of the mers outbreak at the samsung medical center by grouping exposed individuals on the basis of the extent of exposure to patients and . to our knowledge, we are the fi rst to document group-specifi c incubation periods and attack rates. our results showed the increased transmission potential of mers-cov from a single patient in an overcrowded emergency room setting. overcrowding is an important issue for this outbreak and is also a common feature of modern medicine. this study is unique because the index exposure occurred in a large emergency room in a tertiary-care centre, with electronic medical record information available to track the location and duration of exposure, thus enabling near-complete tracing of exposed contacts. the classic defi nitions of close contact as being within roughly feet ( · m) or within the same room or care area for a prolonged period of time were diffi cult to apply to an emergency room setting with high patient volumes, ongoing traffi c within the emergency room and to and from the radiology suite, and large numbers of visitors and family members. we considered all patients who visited the emergency room during the stay of patients and as exposed contacts, developed criteria for close contacts by expanding on the defi nitions of the us centers for disease control and prevention (cdc), and categorised patients into diff erent groups. therefore, we could establish group-specifi c viral incubation periods and attack rates during the outbreak. in close contacts who stayed in the same zone, the incubation period was shorter and attack rate was higher than patients who stayed in diff erent zones. additionally, in zones iii and iv, patients were infected even when they were separated by curtains most of the time and were apart as far as m (for beds on either side of the nurse's station; fi gure ). similar to the sars outbreak, we observed so-called superspreaders among patients with mers-cov infection, and these super-spreaders can cause large outbreaks through several modes of transmission similar to those in the sars outbreak. among patients who stayed in various locations, those who overlapped with patient at the radiology suite or registration area had higher attack rates ( %) than the rest of the patients ( %), suggesting that transmission might occur by even brief exposures to recently contaminated objects or encounters with individuals carrying a super-spreader. comparisons of environmental exposure and patient exposure also revealed unique fi ndings. no patient developed mers-cov infection after exposure on may only to the environment that had been potentially contaminated on may (zone ii) and may (zone iii) while patient was confi ned to zone iv on may . it is plausible that even if the environment was heavily contaminated by a super-spreader, the virus might not persist long enough in the environment to be capable of causing any new infection. although patient exposure is clearly the most important factor in the spread of mers-cov, more research is needed to address the potential of environmental spread. increased viral load and larger amounts of respiratory secretions have been suggested as the factors for sars-cov super-spreaders. , in this mers outbreak, frequent ambulation of the index case could be considered as one factor related to high levels of viral transmission, in addition to large amounts of respiratory secretions and high viral load (cycle thresholds · for upe and · for orf a from patient 's sputum, and · for upe and · for orf a from patient 's sputum). of note, patient infected patients in another hospital but caused no confi rmed secondary cases in our hospital, whereas patient caused an additional cases in our hospital. the diff erence of transmissibility between these two individuals could be caused by a combination of factors such as the time from onset of disease, clinical symptoms, duration of contact exposure, pattern of behaviour inside and near the emergency room, and kinetics of viral shedding. we showed that obtaining a travel history from patients is an important element of history taking by all physicians, and not only those specialising in infectious diseases or those working in infection control. suspicion for unusual infections should be maintained if patients do not or cannot report accurate histories. readiness of laboratory support is essential for initial investigation and for control of outbreaks, and overly rigorous requirements for laboratory testing have the potential to delay diagnosis and further spread disease. hospital leadership needs to lead in preparedness for disaster management of high-risk communicable infectious diseases. emergency preparedness at a national level and communication and support from government agencies are imperative to prevent and control any serious outbreak. the results of this study need to be interpreted with caution because the study was not suffi ciently powered to study risk factors for transmission. some of our data were collected retrospectively. analysis on visitors was limited because we did not have detailed data. serological tests were not done simultaneously and attack rates were calculated on the basis of results from real-time rt-pcr of mainly symptomatic individuals. the potential transmission of mers-cov by asymptomatic carriers is under investigation. in conclusion, we report a large nosocomial mers outbreak that occurred outside the middle east. the potential for similar outbreaks anywhere in the world from a single traveller should be noted, as long as mers-cov transmission continues in the middle east. emergency preparedness and vigilance are crucial to the prevention of further large outbreaks in the future. our report serves as an international alarm that preparedness in hospitals, laboratories, and governmental agencies is the key not only for mers-cov infections but also for other new emerging infectious diseases. syc and j-mk designed the study and data collection methods, did the initial data analyses, drafted the manuscript, and approved the fi nal manuscript as submitted. yeh, who suspected and diagnosed the fi rst case of mers-cov infection in south korea, engaged in the management of mers-cov outbreak control at the samsung medical center as an infectious disease specialist and reviewed the manuscript. gep, jyel, j-hk, jyol, jmk, jgr, and jrc coordinated data collection, engaged in the management of mers-cov outbreak control at the samsung medical center, and reviewed the manuscript. sk supervised data collection and analysis, analysed the data, and reviewed the manuscript. hjh, c-sk, and e-sk did laboratory tests for mers-cov detection, coordinated laboratory data collection, and reviewed the manuscript. c-ik, ijj, krp, h-jd, and j-hs supervised data collection and reviewed the manuscript. y-jk and drc conceptualised and designed the study, and critically reviewed and revised the manuscript. all authors approved the fi nal submitted manuscript. we declare no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia. geneva: world health organization middle east respiratory syndrome coronavirus: quantifi cation of the extent of the epidemic, surveillance biases, and transmissibility interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk synthesizing data and models for the spread of mers-cov, : key role of index cases and hospital transmission hospital outbreak of middle east respiratory syndrome coronavirus superspreading and the eff ect of individual variation on disease emergence transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions superspreading sars events middle east respiratory syndrome coronavirus outbreak in the republic of korea guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) middle east respiratory syndrome coronavirus (mers) evidence of airborne transmission of the severe acute respiratory syndrome virus stability of middle east respiratory syndrome coronavirus (mers-cov) under diff erent environmental conditions severe acute respiratory syndrome-singapore severe acute respiratory syndrome middle east respiratory syndrome in persons, south korea we express our sincere consolation for the patients and their families who had mers-cov infection. we greatly appreciate the eff orts of all the hospital employees and their families at the samsung medical center, who worked tirelessly during this outbreak. we also appreciate the cooperation of all other hospitals in south korea that worked together to overcome the nationwide outbreak. we acknowledge the consultation and support of the korea centers for disease control and prevention, the mers rapid response team of the public-private joint mers task force, who, and the us centers for disease control and prevention. we also sincerely appreciate the discussion and critical feedback from michael t osterholm (university of minnesota, minneapolis, mn, usa) and janet a englund (seattle children's hospital, seattle, wa, usa). key: cord- -sn rswab authors: khan, gulfaraz; sheek-hussein, mohamud title: chapter the middle east respiratory syndrome coronavirus: an emerging virus of global threat date: - - journal: emerging and reemerging viral pathogens doi: . /b - - - - . - sha: doc_id: cord_uid: sn rswab abstract middle east respiratory syndrome (mers) is a viral respiratory illness caused by a coronavirus (cov), first identified in saudi arabia in . since then, almost cases have been reported from countries, with saudi arabia being the epicenter. this newly emerging virus is highly pathogenic and has a case mortality rate of %. it is similar to the cov causing severe acute respiratory syndrome cov (sars-cov) in that both belong to the genus beta covs that are of zoonotic origin and cause lower respiratory infection. the natural reservoir for mers-cov remains unknown. serological studies indicate that most dromedary camels in the middle east have been infected with this virus, and they maybe the potential intermediate host. however, the mode of transmission from camels to humans is poorly understood. the majority of confirmed human cases have resulted from human-to-human transmission, most probably via respiratory route. patients most at risk of developing severe mers-cov infection appear to be those with underlying conditions such as diabetes, hypertension, obesity, cardiac diseases, chronic respiratory diseases, and cancer. unlike sars-cov, mers-cov is considered an ongoing public health problem, particularly for the middle east region. in this chapter, we outline the prevailing information regarding the emergence and epidemiology of this virus, its mode of transmission and pathogenicity, its clinical features, and the potential strategies for prevention. renal failure (zaki et al., ; khan, ) . from to the end of , the world health organization reported that a total of laboratories confirmed the cases of mers-cov infection and at least deaths in countries (case fatality rate %). although sizable outbreaks have been noted in several countries, the latest being in south korea ( cases and deaths) (arabi et al., ) , the vast number of cases ( . %) have been reported from saudi arabia (fig. . ) (who, ) . this newly emerging, highly pathogenic respiratory virus is closely related to the virus that caused an outbreak of severe acute respiratory syndrome (sars) in À . both viruses are beta covs of zoonotic origin and cause similar clinical presentations. although the natural reservoir of mers-cov infection and mode of transmission to humans is not known, one factor appears to be common to all primary cases; they are epidemiologically linked to the middle east region. most secondary cases, on the other hand, have occurred as a result of human-to-human transmission. indeed, several well-documented outbreaks have occurred in healthcare settings, often in elderly men with comorbidities (arabi et al., ; chafekar and fielding, ) . unlike sars-cov, mers-cov is an ongoing public health threat, particularly for the middle east. the fact that there is no effective antiviral drug or approved vaccine available against mers-cov makes the threat even more worrisome (zumla et al., ) . mers-cov is an enveloped, single-strand, and positive-sense rna virus, which belongs to the coronaviridae family. although covs are very common and can infect a variety of different animals, including cats, pigs, and bats, they rarely jump species barrier and infect humans. human covs (hcovs) were first isolated in mid- s, and until , only two viruses, namely, hcov- e and hcov-oc , were known to infect humans (forni et al., ) . currently, six covs have been shown to infect humans. except for mers-cov and sars-cov, all others are associated with mild illnesses resembling common cold. covs are grouped into four genera, α, β, γ, and δ. the β-covs are further subgrouped in four lineages or clades, aÀd (forni et al., ; milne-price et al., ) . although mers-cov and sars-cov belong to the same genus and both cause severe lower respiratory tract infection in humans, phylogenetic and sequencing data suggests that mers-cov is in fact more closely related to several bat covs (btcovs) than to sars-cov ( fig. . ) (forni et al., ; milne-price et al., ) . these findings suggest that mers-cov probably is originated from a btcov ancestor (omrani et al., ; chan et al., a,b) . the fact that covs are rna viruses exhibiting high rates of mutation and recombination, and a propensity to cross species barrier, increases the risk of new variants emerging with higher virulence and transmission sabir et al., ) . the replication cycle of mers-cov consists of a number of important steps: attachment and entry into host cell, uncoating and release of viral rna, transcription and translation of viral specific genes, replication of viral genomic rna, and assembly and release of progeny virions from the infected cell. as is typical of most rna viruses, all of these steps take place in the cytoplasm of the host cell (de wit et al., ) . the initial attachment of mers-cov to its susceptible host cells is mediated by the viral envelop spike glycoprotein s binding to its cellular receptor, cd (also known as dipeptidyl peptidase , dpp ) (lu et al., ; raj et al., ) . a number of different cell types express dpp and hence are susceptible to mers-cov infection including pneumocytes, alveolar macrophages, bronchial epithelia, vascular endothelium, as well as a subset of mononuclear cells (meyerholz et al., ; yu et al., ) . following attachment, the virus enters the susceptible cell by fusion of its envelope with the plasma membrane and/or via receptor-mediated endocytosis (de wit et al., ) . once in the cytoplasm of the target cell, the virus particle uncoats and the positive-sense viral rna binds to ribosomes, and the viral rna-dependent rna polymerase is translated. this enzyme in turn transcribes full-length negative-sense rna that forms the template for the production of positive-sense viral genome. the viral polymerase also generates various individual mrnas that are translated into viral proteins. viral structural proteins and viral genomic rna are assembled into new virus particles in the rough endoplasmic reticulum-golgi intermediate compartment and eventually released out of the cell by exocytosis. from the infected host, it appears that the virus is shed in nasal secretions (adney et al., ) . interestingly, in bats, a recent study revealed that dpp receptor is rarely expressed in epithelial cells of respiratory tract, but highly expressed in epithelial cells of intestinal tract, indicating that fecalÀoral is probably the main mode of transmission in bats (widagdo et al., ) . of all the documented cases to date, there is no evidence for the transmission of the virus from bats or their droppings directly to humans. we also have limited data on the survival of the virus outside its host. when the virus was added to milk from dromedary camels, goats, or cow and stored at c or c, the virus could be recovered up to and hours, respectively (van doremalen et al., ) . pasteurization of the milk, however, completely destroyed mers-cov infectivity (van doremalen et al., ) (table . ). the current prevalent view is that mers-cov is a zoonotic virus that entered the human population in the arabian peninsula, via direct or indirect contact with infected dromedary camels. studies indicate that the virus had been circulating in the camel population for decades, and only recently "jumped" the species barrier to infect humans. what are the factors that precipitated the virus to cross the species barrier are unknown. most of the confirmed cases of mers-cov infection in humans have been via person-to-person transmission. the epidemiological elements in the transmission of mers-cov appear to be factors related to the virus, the host, and the environment. cases have occurred as sporadic infections, family clusters, or outbreaks in healthcare settings (kim et al., ; oboho et al., ) . although the infection is limited and nonsustained, outbreaks in healthcare settings have been particularly extensive and worrisome. the nonspecific initial symptoms, late diagnosis, and inadequate infection control measures have all contributed to the outbreaks in healthcare settings (oboho et al., ; hunter et al., ; kim et al., ) . although mers-cov cases have been detected in many countries around the world, almost all have been directly or indirectly linked to the middle east region (table . ). one of the most notable outbreaks outside the middle east occurred in south korea in may (kim et al., ; lee and wong, ) . a single infected man returning from the middle east caused a hospital outbreak in which individuals were infected (kim et al., ) . the epidemiological pattern observed in the korean outbreak was similar to that observed in the middle east; more males than females were affected, most of the patients who died had underlying conditions such as respiratory disorders, cancer, hypertension, cardiovascular problems, or diabetes (kim et al., ) . it is noteworthy that the death rate was lower in the cases from south korea compared to those reported from saudi arabia ( % vs %) (virlogeux et al., ) . the reason for this is not clear. although more than % of mers-cov cases have occurred in saudi arabia, the virus clearly has the potential of spreading to other countries. thus there is an obvious need to detect, respond, and contain any outbreak of mers-cov cases if we want to prevent the global spread of the virus. unfortunately, this is easier said than done. there are a number of risk factors prevalent in some of the countries of the middle east which support the emergence and reemergence of infectious diseases (buliva et al., ) . these risk factors include political instability, famine and war, less developed healthcare infrastructure, weak public health and surveillance systems, increased population growth and mobility, climate change, and urbanization (buliva et al., ) . in order to prevent the emergence and spread of infectious diseases such as mers-cov, it is essential to address the underlying causes and risk factors. needless to say, these are major challenges for any country, let alone the eastern mediterranean region. to successfully address these challenges, it will require not only funding, establishment of robust and effective surveillance systems, and national and international corporations but also above all, peace and security in the region. infection with mers-cov, in its initial description, resembled "sars-like" illness (chan et al., a,b) . further analysis of the epidemiological, virological, and clinical aspects of mers-cov and sars-cov revealed important differences between the two viruses. identifying unique aspects of mers-cov helped to explain how the epidemic evolved and the steps that could be taken to prevent its spread (chan et al., a,b) . serological studies have indicated that most dromedary camels in africa and the middle east, but not other animals such as sheep, goats, and cows, were seropositive for mers-cov . moreover, seroprevalence in dromedary camels appears to vary, with high rates reported in animals from egypt, ethiopia, nigeria, and sudan and lower rates in animals from tunisia (ali et al., ) . intriguingly, dromedaries from australia, canada, the united states, germany, netherlands, and japan have been reported to be seronegative for mers-cov (omrani et al., ) . importantly, population-based seroepidemiologic studies indicated that the seroprevalence of the virus was several folds higher in people who have been exposed to camels compared to those in the general population ( accumulating serologic and molecular evidence indicates that the virus in dromedaries is genetically similar to mers-cov in humans, supporting the notion that dromedary camels could be the potential source of infection to human memish et al., a,b,c; sabir et al., ) . indeed, mers-cov antibodies have been isolated in dromedary camels across the arabian peninsula, north africa, and eastern africa dating from as far back as the s (milne-price et al., ; omrani et al., ) . this finding suggests that mers-cov may have been circulating in dromedaries for over years before it was first recognized as a cause of human infection (aly et al., ) . in a recent study, a fatal case of mers-cov infection was reported in an individual who had direct contact with a dromedary camel (azhar et al., ) . sequence analysis of the virus isolated from the case and the camel was identical, clearly indicating that mers-cov can indeed be transmitted from camels to human (azhar et al., ) . it appears that active infection with release of the virus in nasal secretions, particularly during the incubation period, is important for the transmission of the virus to humans . where and how the camels acquired the infection remains unknown. it has been hypothesized that bats could be the potential source (fig. . ) (anthony et al., ; mohd et al., ; omrani et al., ) . indeed, mers-cov-like viruses have been identified in certain species of bats (anthony et al., ; woo et al., ) . the bats are present in most parts of the world and often infected with various zoonotic viruses. thus it is plausible that at some point in the past, camels acquired the infection from bats, leading to a sustained infection in the camel population (fig. . ) . mers-cov rna has been identified in the milk, nasal secretion, and feces of dromedary camels (omrani et al., ) . since camels and humans are often in close contact, particularly in the arab gulf states, humans would be at increased risk of contracting the virus from actively infected animals (mackay and arden, ; reusken et al., ) . indeed, mers-cov seropositivity in shepherds and those working in slaughterhouses in saudi arabia has been reported to be an order of magnitude higher than in the general population (arabi et al., ) . although possible, no evidence currently exists to support the transmission of mers-cov from bats to humans directly. what is certain is that transmission of the virus can occur from camels to humans, but the process is still not fully understood (al hammadi et al., ; memish et al., a,b,c) . one possibility is that some species of covs from camels and humans could recombine leading to the emergence of a new virus that can infect both, camels and human (sabir et al., ) . most mers-cov infections in humans occur through human-tohuman contact (arabi et al., ; zumla et al., ) . available data on epidemiologic observations suggest that human-to-human transmission occurs primarily through close contact with an infected individual. the mode of transmission is presumed to be via respiratory droplets or aerosols, with higher risk in situations where aerosols are generated, and inadequate personal protection or proper room ventilation is not present (kutter et al., ) . in the south korean outbreak a total of individuals were infected; of whom were directly infected by just cases, the so-called super spreaders. late diagnosis, lack of infection control measures, poor communication and healthcare management procedures, and failure to quarantine the "super spreaders" were identified as major factors contributing to this large nosocomial outbreak (kim et al., ) . cov is a common cause of mild respiratory tract infection manifesting as common cold. it is estimated that approximately one-third of all upper respiratory tract infections in adults are due to covs. sars-cov and mers-cov are the exceptions. both of these viruses have a high propensity to infect the lower respiratory track and lead to severe disease and death (de wit et al., ) . the finding that both of these viruses, but in particular, mers-cov, are able to evade the body's immune responses and infect a broad range of cells, explaining the widespread infection and development of severe disease (mackay and arden, ) . it is noteworthy that, even in the absence of viral shedding in the upper respiratory tract, most symptomatic patients have abnormal chest radiographs (fig. . ) (assiri et al., ; de wit et al., ) . the incubation period for mers-cov infection is about À days with most patients showing symptoms within days of exposure (de wit et al., ; virlogeux et al., ) . the initial clinical symptoms of mers-cov infection can range from asymptomatic to low-grade fever, cough, sore throat, myalgia, and less frequently diarrhea and vomiting. progression to more severe disease is characterized by the symptoms of shortness of breath, severe pneumonia, respiratory distress syndrome, multiorgan failure, and death (arabi et al., ; de wit et al., ; guery et al., ) . the severity of the infection appears to vary depending on the age of the patient and any underlying conditions. adults over the age of years and with comorbidities such as diabetes, hypertension, chronic renal or lung disease, cancer, and heart disease are at increased risk of developing severe disease and death (badawi and ryoo, ) . although the vast majority of confirmed cases have been in male adults, children are also susceptible to infection, albeit at lower rate and with milder disease . based on limited data, mers-cov infection in pregnancy can also lead to maternal and perinatal disease and death assiri et al., ) . not surprisingly, the severity of infection and the risk of transmission of mers-cov are significantly increased in environments such as hospitals (cho et al., ; hastings et al., ) . the clinical symptoms of mers-cov infection, especially in early stages of the infection, are typically nonspecific and can resemble a number of acute respiratory tract infections. however, acute febrile respiratory illness in a patient with a recent travel history to the middle east or direct/indirect contract with a confirmed case of mers-cov should be enough suspicion to request laboratory testing for mers-cov. studies indicate that viral replication and shedding is higher in lower compared to upper respiratory tract (de wit et al., ; memish et al., a,b,c) . hence, for laboratory diagnosis, lower respiratory tract specimens such as tracheal aspirate, bronchoalveolar lavage, or pleural fluid are preferred over upper respiratory tract specimens such as nasopharyngeal swab (mackay and arden, ; memish, et al., a,b,c) . it is essential that appropriate personal protective equipment (ppe) and infection control measures are implemented when dealing with suspected cases. the assay of choice for the laboratory diagnosis of mers-cov is reverse transcriptase real-time polymerase chain reaction (rt-pcr) on respiratory samples. this assay was established soon after the identification of mers-cov back in (zaki et al., ) . rt-pcr is not only very sensitive but also importantly a fairly rapid technique, which is essential for early diagnosis and quarantine implementation. the virus can also be cultured. a number of different cell lines are susceptible for in vitro infection, including vero and llc-mk cells (zaki et al., ) . however, cell culture approach is very slow and not easily adaptable to every diagnostic laboratory, hence the preference of rt-pcr. for determining past infection or for surveillance studies, the detection of antibodies to mers-cov using serological assay, such as enzyme linked immunosorbent assay (elisa), can be performed (mackay and arden, ) . currently, no specific antiviral therapy or vaccine is available for the treatment and prevention of mers-cov infection. supportive care and prevention of complications are the main management options that are available. however, efforts are underway for the development of therapeutic and vaccine candidates. in a marmoset model of mers-cov infection, several compounds, including ribavirin, lopinavir/ritonavir, interferon-β b, and interferon-α b, alone or in combinations, have shown varying degree of success (chan et al., a,b; falzarano et al., ) . similarly, passive immunotherapy with neutralizing antibodies against mers-cov has also shown some therapeutic value in inhibiting viral replication (luke et al., ) . in terms of vaccines, several potential candidates have been developed and are in different stages of clinical testing (du et al., ) . the possibility of developing an effective vaccine based on mers-cov spike protein is promising . in the absence of licensed antiviral or vaccine, current strategies of combating mers-cov infection are aimed at reducing the risk of animal-to-human and human-to-human transmissions. strategies recommended include avoidance of drinking unpasteurized camel milk, limiting direct contact with a sick animal, avoidance of close contact, and sharing of utensils with an infected individual, using ppe when in direct contact with an infected person and proper hand hygiene. in addition, early recognition and laboratory confirmation of infected cases, segregation/isolation of infected cases, and contact tracing and strict implementation of infection control measures in healthcare settings are all essential for controlling and preventing of mers-cov infection and spread. replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from to asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia middle east respiratory syndrome coronavirus 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coronaviruses in bats comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to sars-coronavirus coronaviruses-drug discovery and therapeutic options key: cord- -m miwtha authors: vergara‐alert, j.; raj, v. s.; muñoz, m.; abad, f. x.; cordón, i.; haagmans, b. l.; bensaid, a.; segalés, j. title: middle east respiratory syndrome coronavirus experimental transmission using a pig model date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: m miwtha dromedary camels are the main reservoir of middle east respiratory syndrome coronavirus (mers‐cov), but other livestock species (i.e., alpacas, llamas, and pigs) are also susceptible to infection with mers‐cov. animal‐to‐animal transmission in alpacas was reported, but evidence for transmission in other species has not been proved. this study explored pig‐to‐pig mers‐cov transmission experimentally. virus was present in nasal swabs of infected animals, and limited amounts of viral rna, but no infectious virus were detected in the direct contact pigs. no virus was detected in the indirect contact group. furthermore, direct and indirect contact pigs did not develop specific antibodies against mers‐cov. therefore, the role of pigs as reservoir is probably negligible, although it deserves further confirmation. middle east respiratory syndrome coronavirus (mers-cov) was first detected in in saudi arabia, and it causes severe acute respiratory illness with fever, cough and shortness of breath (zaki, van boheemen, bestebroer, osterhaus, & fouchier, ) . up to date, it has caused human infections, including related deaths (world health organization (who), ). dromedaries are the natural reservoir of mers-cov (sabir et al., ) . however, other animal species such as non-human primates (rhesus macaques and common marmosets), members of the family camelidae (alpacas and llamas), rabbits and pigs have been demonstrated to be susceptible to mers-cov infection (crameri et al., ; falzarano et al., ; haagmans et al., ; vergara-alert, van den brand, et al., ; de wit et al., de wit et al., , . the finding that pigs can be infected with mers-cov would suggest that other suidae might be susceptible to the virus. indeed, common warthogs (phacochoerus africanus), bushpig (potamochoerus larvatus) and wild boars are commonly found in the greater horn of africa or the middle east, sharing the same habitats and water sources with dromedaries (cumming, ; vergara-alert, vidal, bensaid, & segal es, ) . a recent study in alpacas demonstrated efficient animal-to-animal transmission (adney, bielefeldt-ohmann, hartwig, & bowen, ) but, to our knowledge, evidence for transmission between animals from other species has not been reported. to study whether mers-cov might be transmitted between pigs, an experimental transmission study in this animal model was designed and performed under direct and indirect contact settings. . | experimental design (bsl- ) animal facilities (irta-cresa, barcelona, spain), and divided into three groups: g , mers-cov-inoculated pigs (p -p ); g , direct contacts (p -p ); g , indirect contacts (p -p ). three extra animals were used as negative controls (g , p -p ). animals from g , g and g were housed in the same experimental box unit but placed in two different pens. the pens were separated by two fences with a cm distance among them ( figure ). tarpaulin, from the ceiling to the floor, was used to avoid contact between pen and pen . tarpaulin was also placed in the front doors of both pens. at the beginning of the experiment, g was housed in pen , and g and g in pen . g was inoculated with tcid ( % tissue culture infectious dose) mers-cov (passage human isolate hcov-emc/ ) in ml saline solution via intranasal route ( . ml in each nostril). two days later, all tarpaulins were removed and g pigs were moved from pen to pen until the end of the study. all animals were monitored daily for clinical signs (sneezing, coughing, nasal discharge and/or dyspnoea), as well as rectal temperatures until day post-inoculation (pi). nasal swabs (ns) were obtained on days , , , , pi from all animals and at days , and pi from g . animals from g and g were also sampled at , , and days pi, corresponding to days , , and after direct (g ) and indirect (g ) contact with g . two independent ns were collected and placed in pbs (for pcr analysis) and dmem containing antimicrobial drugs (for detection of infectious virus); swabbing was performed deep in both sides of the nasal cavity. sera were obtained before challenge and at , and days pi, and they were subsequently used to detect the presence of mers-cov-specific antibodies. negative control pigs were sampled (ns and sera) and euthanized before the start of the experiment. daily environmental samples (es) between day and pi were obtained from air sampling and wall surface swabbing (figure ). briefly, swabs pre-moistened with transport medium (copan universal transport medium utm-rt system) were collected from walls in pen and (es and es ). air sampling was performed using an air sampler (airport md sartorius device) located between pens, which suctioned l/min air volume for min through a gelatin membrane filter (es ). air from the box unit was sampled with cm dry membrane filters located in the air extraction of the box (es ). es were tested for the presence of viral rna. viral rna from ns and es was extracted with nucleospin â rna virus kit (macherey-nagel, germany) following the manufacturer's f i g u r e schematic representation of the experimental animal box. boxes in the animal facility of the biosafety level at irta-cresa are behind two sets of doors (with a shower in between) following the standards of a negative pressure room. animals were distributed into two pens separated by two fences with a cm distance between them. g (p -p ) was allocated in pen and g (p -p ) and g (p -p ), in pen . two days after inoculation of g with mers-cov, g was cohoused with g until the end of the experiment. tarpaulin was used to prevent contact between g and the other two groups during the firsts days after inoculation. environmental samples (es) were obtained from different locations, as represented in the scheme [colour figure can be viewed at wileyonlinelibrary.com] vergara-alert et al. instructions. the rna extracts were tested by the upe pcr (raj et al., ) , and the techniques were carried on as previously (vergara-alert, van den brand, et al., ) . ns were also evaluated for the presence of infectious virus by titration in vero cells, following previous protocol (vergara-alert, van den brand, et al., ). serum samples from days , , and pi were tested to determine the specific s -antibodies by a mers-cov s -elisa, and by a specific virus neutralization assay, as previously described (haagmans et al., ) . similar to a previous experiment (vergara-alert, van den brand, et al., ) , none of the pigs had appreciable rise in rectal temperature upon challenge, nor any clinical signs (data not shown). all mers-cov-experimentally infected animals (p -p ) shed viral rna at least from to days pi, and three of five pigs had detectable viral rna until days pi (figure a ). most importantly, all five animals shed infectious virus during the first days pi (figure b ). viral rna was detected in four of five cohoused, direct contact animals (g ) at least one time pi. the mers-cov rna load of g pigs, however, was lower than those of g (figure a) . no viral rna or infectious virus was detected in swabs from g (p -p ) and control pigs (p -p ).to test whether seroconversion occurred, serum samples were tested with a specific recombinant mers-cov s -elisa and for neutralizing antibodies against mers-cov. all five mers-cov infected animals (p -p ) had detectable levels of s -antibodies -and -weeks after the infection (figure c ). the specificity of the response was confirmed by virus neutralization assay. in p -p (but not in p ), serum neutralizing mers-cov-specific titres ( : - : ) were detected at -and -week pi (figure d) . however, at week pi, the virus neutralizing antibodies decreased ( : - : ). no mers-cov-specific antibodies were detected in serum of g , g and control pigs. in environmental samples, very low levels of viral rna were detected at different time points, with a peak at day pi (table ) . other livestock besides dromedaries are susceptible to mers-cov infection (crameri et al., ; falzarano et al., ; haagmans et al., ; munster et al., ; vergara-alert, van den brand, et al., ; de wit et al., de wit et al., , ; thus, they might be potential intermediate we thank all animal caretakers from the irta-cresa biosecurity level laboratories and animal facilities for technical assistance. this work was performed as part of the zoonotic anticipation and preparedness initiative (zapi project) [innovative medicines initiative (imi) grant ] with assistance and financial support from imi and the european commission and contributions from efpia partners. the funding from cerca programme/generalitat de catalunya to irta is also acknowledged. infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus phacochoerus africanus. the iucn red list of threatened species infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels pneumonia from human coronavirus in a macaque model dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia livestock susceptibility to infection with middle east respiratory syndrome coronavirus. emerging infectious diseases searching for animal models and potential target species for emerging pathogens: experience gained from middle east respiratory syndrome (mers) coronavirus. one health domestic pig unlikely reservoir for mers-cov middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques middle east respiratory syndrome coronavirus (mers-cov): infection, prevention and control measures are critical isolation of a novel coronavirus from a man with pneumonia in saudi arabia how to cite this article: vergara-alert j, raj t a b l e viral rna from air sampling and wall surface swabbing at different times after mers-cov infection. swabs were collected from the walls in pen and pen (es and es ), air sampling was performed with an air device (es ), and circulating air from the box was sampled with filters located in the ceiling air extraction of the room (es ) key: cord- -cob hf q authors: otter, j. a. title: the inaugural healthcare infection society middle east summit: ‘no action today. no cure tomorrow.’ date: - - journal: journal of hospital infection doi: . /j.jhin. . . sha: doc_id: cord_uid: cob hf q nan the healthcare infection society (his) decided to run its spring meeting in dubai this year as the inaugural his middle east summit. the conference was well attended, with delegates from all over the world. most of the presentations can be viewed on the his website. the conference opened with professor tawfik khoja outlining the challenges to infection prevention and control in the middle east. professor khoja focused his thoughts on the impressive joint gulf plan for infection prevention ( e ). this strategy document aims to raise the standards of infection prevention and control in the region, and is already yielding success. dr tim boswell then followed with a presentation describing the challenges in europe. among the challenges he covered were public reporting and external scrutiny, hand hygiene, antibiotic resistance, the healthcare environment, surveillance and outbreaks, an increasingly elderly population, new threats [such as ebola and middle east respiratory syndrome coronavirus (mers-cov)], meticillinresistant staphylococcus aureus (mrsa), c. difficile, and invasive devices and new complex equipment. to these i would add the increasingly cost-constrained financial environment that we face in europe. how can we invest in infection control when some hospitals can't afford to buy new pens? the next session covered viruses with pandemic potential: mers-cov, influenza and ebola (although only the first two really have pandemic potential!). dr ali omrani gave an extremely current overview of mers-cov, tracking the outbreak in south korea with up-to-the-minute slides. this illustrated how quickly the picture can change with a pandemic virus such as mers-cov. one striking aspect of dr omrani's talk was findings from saudi arabia that . % of the population are likely to have encountered mers-cov, since this is the proportion of a large community sample who were seropositive for mers-cov antibodies. this suggests a sizeable and previously unrecognized burden of asymptomatic exposure e and possibly shedding. furthermore, animal handlers are more likely to have mers-cov seropositivity, reinforcing the link between animals and mers-cov. dr omrani then discussed sharp spikes of mers-cov in jeddah, saudi arabia in , and the recent outbreak in south korea, attributing both to breakdowns in simple infection control. dr nick phin from public health england (phe) then discussed influenza, and carole fry preparedness for ebola. dr phin highlighted a useful cdc toolkit providing advice on respiratory protection for healthcare workers, and also a recent bmj review concluding that facemasks may help to prevent the spread of respiratory viruses in the community. , carole fry added some cultural perspective to ebola considerations: there are still some 'ebola deniers' in west africa! in terms of containing ebola, the british approach of using trexler isolators is safe for staff, but pretty miserable for patients. is this an appropriate trade-off? also, our careful use of personal protective equipment (ppe) for ebola has highlighted our careless use of ppe for other organisms! there are some parallels in preparing for all three of these unlikely but potentially very serious threats (mers-cov, ebola, and influenza). one of the most important challenges is dealing with the paranoia that always seems to engulf these preparations! valerie harmon delivered a useful lecture on achieving hand hygiene compliance. self-reported hand hygiene compliance rates are usually reported to be > %, but who would believe this with such a huge conflict of interest? the problem with using human beings to monitor hand hygiene compliance is that the moment another person is there, compliance improves! so, automation of hand hygiene compliance monitoring seems the best way forward. valerie's colleagues demonstrated a stateof-the-art automated hand hygiene compliance monitoring approach based on google glass. although the technology was rather prototype, the principle is there, and it seems likely that automated hand hygiene monitoring systems will come into play over the next few years. but this will not alter a fundamental problem: self-protection is a large and rather unhelpful driver for hand hygiene compliance. accurate monitoring of hand hygiene compliance using technology is important, but it must be combined with effective education to help staff to overcome themselves to comply. tim boswell's talk on the environment summarized the evidence that contaminated surfaces make an important contribution to the transmission of key pathogens, including: mrsa, c. difficile, vancomycin-resistant enterococci, norovirus, and acinetobacter baumannii. this is demonstrated most convincingly by the 'prior room occupancy' studies, showing that admission to a room previously occupied by a patient with these pathogens is a risk factor for acquisition for the incoming occupant. underpinning this are the poor levels of conventional cleaning and disinfection, illuminated by techniques such as fluorescent marking of surfaces to evaluate the cleaning process. a failure to eliminate key pathogens from hospital surfaces by cleaning and disinfection processes designed to do just this at the time of patient discharge has been demonstrated for multiple pathogens. automated room disinfection systems (principally hydrogen peroxide and ultraviolet systems) offer the potential to reduce or remove reliance on the operator to assure adequate distribution and contact time of a disinfectant e and can help to reduce the transmission of hospital pathogens. professor tibor pal provided an overview of the concerning epidemiology of multidrug-resistant gram-negative rods (mdr-gnr) in the arabian peninsula: lots of overseas healthcare and travel, and huge antibiotic usage is a toxic mix in this regard. professor pal's view is that all carbapenemases are not equal: oxa- is weedy and klebsiella pneumoniae carbapenemase (kpc)/new delhi metallo-beta-lactamase (ndm) are scary! e not least due to the emergence of resistance to last-line agents such as colistin. it does seem from the limited available data that the rate of carbapenem-resistant enterobacteriaceae (cre) in the middle east is considerably higher than in europe, and increasing fast! switching to the non-fermenters, professor pal highlighted the remarkable environmental survival properties of acinetobacter, combined with a propensity towards antibiotic resistance. although data are limited, it seems that around % of acinetobacter in the arabian peninsula are carbapenem resistant. i was next to speak, and i outlined approaches to mdr-gnr control in europe based on current guidelines. cre is a big deal in europe, especially in the uk, and has prompted unprecedented action on a national level. one key question is: do we go universal or targeted? there has been much discussion recently about abandoning traditional targeted (also known as vertical) approaches in favour of universal (horizontal). interestingly, all guidelines that i reviewed favoured a targeted approach for mdr-gnr, centred around screening and isolation of carriers. we are hamstrung by the lack of high quality studies telling us with any certainty what works to control mdr-gnr. also, how do you go about producing good guidelines? plus, importantly, how do good guidelines translate through a good policy into good practice? as to which interventions we should use for each organism, this depends on organism and setting, although screening, isolation, stewardship, hand hygiene, and cleaning/ disinfection are the pillars of infection control. but what do we do about the more controversial areas: decolonization, screening of staff, cohorting staff and patients, environment screening, and education? dr muhammad halwani then gave an overview of infection control in the middle east, focusing on acinetobacter and pseudomonas. his comprehensive review set out prevalence and resistance rates across the region, highlighting limited surveillance data e but high rates where data are available. dr halwani then reviewed the guidelines available in the region, most importantly the guidelines for isolation precautions, and the gcc strategic plan for combatting antimicrobial resistance (which has a most apt tagline: 'no action today. no cure tomorrow.'). professor david leaper gave an entertaining critique of the evidence base for infection prevention. he began by highlighting the drying of the antibiotic pipeline: the 'variations on a theme' are losing efficacy due to resistance. there is strong evidence for some interventions to prevent infection, for example, the timing of antibiotic prophylaxis prior to surgery, but other areas are more controversial. professor leaper cited the example of nasal decolonization for meticillin-susceptible s. aureus, based on this study being less convincing than you may think. but can we expect a randomized controlled trial for everything? even for parachutes when jumping out of aeroplanes? finally, carole fry and martin kiernan gave an engaging overview with uk perspectives on reductions in mrsa and clostridium difficile infection. carole described the political situation in the early s, with daily stories in the papers about the mrsa 'scandal'. this led to huge scrutiny and political drive for improvement, which has been successful contrary to the predictions of many (most?) experts! but what has caused the rate of mrsa to fall so dramatically? with many interventions during the same period, it is impossible to tell. but the high degree of scrutiny almost certainly played a key role. martin gave some useful guidance about performing an effective root cause analysis (to get to the root of the problem!): make sure the right stakeholders are around the table (junior doctors won't do); be a toddler (ask why? why? why?), document and evaluate improvement. on a personal note, the conference was very enjoyable. it had a good mixture of delegates from the middle east and elsewhere, and the talks prompted much interesting discussion! presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study hospital respiratory protection program toolkit facemasks for the prevention of infection in healthcare and community settings self-protection as a driver for hand hygiene among healthcare workers preventing surgicalsite infections in nasal carriers of staphylococcus aureus parachute use to prevent death and major trauma related to gravitational challenge: systematic review of randomised controlled trials author is a consultant to gama. key: cord- - w epdht authors: reusken, chantal bem; haagmans, bart l; müller, marcel a; gutierrez, carlos; godeke, gert-jan; meyer, benjamin; muth, doreen; raj, v stalin; vries, laura smits-de; corman, victor m; drexler, jan-felix; smits, saskia l; el tahir, yasmin e; de sousa, rita; van beek, janko; nowotny, norbert; van maanen, kees; hidalgo-hermoso, ezequiel; bosch, berend-jan; rottier, peter; osterhaus, albert; gortázar-schmidt, christian; drosten, christian; koopmans, marion pg title: middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: w epdht background: a new betacoronavirus—middle east respiratory syndrome coronavirus (mers-cov)—has been identified in patients with severe acute respiratory infection. although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of mers-cov. anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. we investigated possible animal reservoirs of mers-cov by assessing specific serum antibodies in livestock. methods: we took sera from animals in the middle east (oman) and from elsewhere (spain, netherlands, chile). cattle (n= ), sheep (n= ), goats (n= ), dromedary camels (n= ), and various other camelid species (n= ) were tested for specific serum igg by protein microarray using the receptor-binding s subunits of spike proteins of mers-cov, severe acute respiratory syndrome coronavirus, and human coronavirus oc . results were confirmed by virus neutralisation tests for mers-cov and bovine coronavirus. findings: of ( %) sera from omani camels and of ( %) from spanish camels had protein-specific antibodies against mers-cov spike. sera from european sheep, goats, cattle, and other camelids had no such antibodies. mers-cov neutralising antibody titres varied between / and / for the omani camel sera and between / and / for the spanish camel sera. there was no evidence for cross-neutralisation by bovine coronavirus antibodies. interpretation: mers-cov or a related virus has infected camel populations. both titres and seroprevalences in sera from different locations in oman suggest widespread infection. funding: european union, european centre for disease prevention and control, deutsche forschungsgemeinschaft. in , a new betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-was identifi ed in patients with severe respiratory disease in the middle east. as of aug , , laboratory-confi rmed cases, including deaths, have been reported to who. illness associated with mers-cov infection is characterised primarily by mild-to-severe respiratory complaints, most requiring hospital admission for acute respiratory distress syndrome. comorbidities and immunosuppression seem to predispose for infection and severe disease, [ ] [ ] [ ] [ ] [ ] and unpublished serological studies suggest that asymptomatic infections occur. all cases reported so far have been linked to jordan, qatar, saudi arabia, and united arab emirates. humanto-human transmission has been reported, particularly in health-care settings, but on the basis of available evidence the basic reproduction number (r ) is thought to be low, suggesting that the virus is not transmitted readily. , therefore, the primary reservoir of mers-cov is probably animals. diff erent coronaviruses have various hosts including wildlife, livestock, poultry, pets, and human beings. coronaviruses can adapt to new host species, as shown by the zoonotic origin of several human coronaviruses. human coronavirus oc has recent common ancestry with bovine coronaviruses. rhinolophid bats were identifi ed as a likely reservoir for severe acute respiratory syndrome coronavirus (sars-cov), which emerged in people in - , through intermediate carnivorous hosts. molecular clock analysis showed that bat and civet strains of viruses closely related to sars-cov only diverged a few years before the outbreak. human coronavirus e has a common ancestor with coronaviruses found in ghanaian hipposideros spp bats. mers-cov is able to replicate in various bat cell lines and phylogenetic analyses show that it is closely related to betacoronavirus lineage c viruses from pipistrellus spp bats in europe and asia. [ ] [ ] [ ] [ ] molecular clock dating of epidemiologically unlinked isolates of human mers-cov estimated their divergence from a common ancestor in mid- , , with a cluster of isolates from the eastern arabian peninsula diverging in late . this fi nding could suggest that the diversity of mers-cov in people is the result of multiple independent, geographically structured, zoonotic events in the middle east. , possible animal reservoirs need to be identifi ed to determine how circulation of mers-cov is maintained and to break the chain of transmission. mers-cov can infect cells of several species, including human beings and bats. the functional receptor is conserved between species, suggesting that receptor use is not an important barrier to cross-species transmission. data for exposure history of patients are scarce, but suggest contact with livestock, including dromedary camels and goats. , , food and agriculture organization data from show that cows, goats, sheep, and dromedary camels are the main sources of meat and milk in jordan, saudi arabia, and united arab emirates. serological studies are best suited to screen animal populations, but have not yet been reported for mers-cov in animals, although several methods have been described for testing antibodies of people. , for specifi city, who recommends use of a combination of screening assays with recombinant spike protein, and confi rmatory testing by neutralisation assays. here, we describe antibody profi ling of serum samples from major livestock species that might be relevant to the epidemiology of mers-cov in the middle east, using samples collected from herds inside and outside the region. we sampled a cohort of dromedary camels (camelus dromedarius) from two herds on the canary islands. were male, were female, were adults, nine were age - years, seven were age years, and one was age months. both herds had the same owner, with frequent exchange of animals between the herds. one herd is from a coastal dune habitat with no other livestock, while the other herd is in an inland valley close to a tropical fruit farm, in particular mango and papaya-which could attract fruit bats-and nearby (roughly m) to horse and goat farms with and animals, respectively. the camels were born in the canary islands except for three adults, which were imported from morocco. the camels are used in the tourist industry. sera were taken april-june, , nine in may, , and paired sera were taken in these months in , and , all for routine veterinary purposes. samples were obtained by jugular puncture. female dromedary camels from oman were sampled in march, . the camels were aged - years and belonged to diff erent owners from separate locations. the camels are retired racing camels now used for breeding, and blood was taken by jugular puncture for routine screening for brucellosis. omani dromedaries sera were collected for veterinary purposes from two llamas (lama glama), six alpacas (vicugna pacos), and two bactrian camels (camelus bactrianus) in the netherlands. sera were collected for veterinary purposes from two bactrian camels, alpacas, fi ve llamas, and two guanaco (lama guanicoe) in buin zoo in chile. sera from cattle (n= ), domestic goats (n= ), and sheep (n= ) were from routine submission to the dutch animal health service. sera from spanish domestic goats (n= ) were provided by the instituto de investigación en recursos cinegéticos (ciudad real, spain) from submissions for tuberculosis control in . all sera were obtained in agreement with local regulations and dutch import regulations with regard to animal disease legislation. positive human control sera for the three antigens used on the microarray were taken as described previously. all samples were stored at - °c until testing. we tested the sera for the presence of igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc s antigens in a protein microarray. the receptor-binding domains, which contain the s subunit of spike proteins of mers-cov (residues - ), sars-cov (residues - ), and human coronavirus oc (residues - ) were expressed, purifi ed, and spotted on glass slides. slides were incubated with serum and species-specifi c conjugates, as previously described. goat sera were incubated with alexa fluor -conjugated rabbit anti-goat igg fc fragment (jackson immuno research, west grove, pa, usa); combinations of the mean fl uorescent intensities of reactions of sera with mers-cov and human coronavirus oc antigens from spanish dromedary camels (a). plaque reduction neutralisation tests for bovine coronavirus and mers-cov (b): two representative sera are shown (numbers and , corresponding to camel id numbers in table ) in dilutions of / , / , and / as well as the virus input control. all samples were tested in duplicates (only one well shown) and titres were expressed as the serum dilution resulting in a plaque reduction of at least %. igg reactivity of both camel sera to mers-cov antigen and human coronavirus oc antigen in a two-step dilution series in the microarray (c). igg reactivity of two two-step serially diluted omani dromedary camel sera with human coronavirus emc antigen and human coronavirus oc antigen in the microarray (d). rfu=relative fl uorescence units. mers-cov=middle east respiratory coronavirus. cow sera with alexa fluor -conjugated goat antibovine igg (h+l; jackson immuno research); sheep sera with alexa fluor -conjugated donkey anti-sheep igg (h+l; millipore, temecula, ca, usa); and camelid sera with dylight -conjugated goat anti-llama igg (h+l; agrisera, vännas, sweden). fluorescence signals were quantifi ed as described previously. we tested the sera for igg reactivity at a dilution of / and set an arbitrary cutoff at an average signal intensity of relative fl uorescence units (rfu). this high cutoff was chosen to reduce cross-reactive false positives. we present results as rfu for each set of quadruplicate spots per antigen. negative fl uorescent intensities (caused by background correction) were assigned to . analyses were done with graphpad prism (version . ). sera were heat-inactivated before virus neutralisation by incubation for min at °c. two-fold serial dilutions of sera were prepared using -well plates, starting dilution / . mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with clemizole penicillin (penicillin g), streptomycin, and % fetal bovine serum, to a dilution of tissue culture infective dose per ml. μl virus suspension was added to the plates and the plates were incubated at °c for h. the mixtures of virus and serum were then incubated on -well plates containing vero cells for h followed by washing with phosphate buff ered saline and incubation with imdm and % fetal bovine serum for - days, after which endpoint titres were measured. all tests were repeated twice independently. we tested neutralisation activity of sera against mers-cov (erasmus mc isolate) and bovine coronavirus (nebraska strain) by plaque reduction neutralisation test ( % plaque reduction) with african green monkey kidney cells (cell line vero b ; dsmz acc ) or bovine kidney cells (cell line pt; cclv-rie ) in a -well plate format. virus ( - plaqueforming units) and heat-inactivated sera (diluted from / to / ) were pre-incubated in μl of serumfree optipro medium (life technologies, karlsruhe, germany) at °c for h. virus adsorption was done at °c for h. supernatants were removed and overlaid with avicel resin (fcm biopolymer, brussels, belgium). assays were stopped after days by fi xation with % paraformaldehyde for min. all samples were tested in duplicate and titres were expressed as the serum dilution resulting in a plaque reduction of at least %. the sponsors had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. sera were tested for igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc s antigens in a protein microarray (fi gure ). human coronavirus oc is serologically closely related to bovine coronavirus, diverging at the end of the th century. bovine coronavirus circulates in cows, sheep, goats, and old and new world camelids. [ ] [ ] [ ] [ ] because bovine coronavirus s was not available, human coronavirus oc s antigen was used as a proxy. sera from three llamas, four alpacas, one guanaco, and two bactrian camels reacted with human coronavirus oc antigen. one cow and one goat serum reacted with human coronavirus oc antigen as did sera from of ( %) spanish dromedary camels. all sera from cattle, sheep, and goats tested negative for mers-cov antigen, but sera from spanish camels ( %) did react with mers-cov antigen (fi gure ). the reactivity was highly specifi c-the same sera did not bind to sars-cov antigen but a positive control specimen did. no correlation existed between the reactivity of sera with mers-cov antigen and human coronavirus oc antigen (fi gure ). all but one serum sample that reacted with mers-cov antigen were from adult animals. one reactive serum was from a -year-old animal. to confi rm the presence of mers-cov specifi c igg in the spanish camel sera, we used a mers-cov neutralisation assay to test a subset of camel sera with diff erent degrees of reactivity with mers-cov and human coronavirus oc antigen according to microarray. nine spanish camels had mers-cov neutralising antibodies with titres varying between / and / (table ). three of the sera reacted with mers-cov spike antigen but did not neutralise mers-cov, most likely because of recognition of nonneutralising epitopes. all mers-cov neutralising sera had (almost) saturating reactivity with mers-cov antigen on the microarray, whereas reactivity with human coronavirus oc antigen varied from negative to % of saturating reactivity (table ). the variable human coronavirus oc signals suggest that mers-cov did not generally cross-react with human coronavirus oc or bovine coronavirus antigens. all nine camels with mers-cov neutralising antibodies were born and raised on the canary islands; seven were female, two were male. eight camels were adults, one was years old. to show that the reactivity of the camel sera with human coronavirus oc antigen according to the microarray was caused by the presence of bovine coronavirus igg and to further exclude mers-cov neutralising activity caused by cross-neutralisation by the bovine coronavirus antibodies, we tested camels that had suffi cient serum left (n= ) in a comparative mers-cov and bovine coronavirus plaque reduction neutralisation test (fi gure , table ). all camel sera neutralised bovine coronavirus, but with varying titres, suggesting a lower cutoff than rfu for oc in the microarray (fi gure ). five camels had high neutralising antibody titres against bovine coronavirus (and a mean signal intensity of greater than rfu for human coronavirus oc antigen on microarray) but were negative for mers-cov neutralisation, suggesting that cross-neutralisation in this direction did not occur and that the mers-cov neutralising activity was not caused by the presence of bovine coronavirus neutralising antibodies. a serum sample from a patient who had mers, neutralised mers-cov with a high titre ( / ) but neutralised bovine coronavirus less effi ciently (titre / ). the latter fi nding was most probably caused by previous infection with human coronavirus oc -this patient had a high titre ( /> ) in a human coronavirus oc recombinant spike immuno fl uorescence assay and a saturating signal with human coronavirus oc antigen in the microarray. two human serum samples positive for human coronavirus oc did not neutralise mers-cov, one of which neutralised bovine coronavirus at a titre of / (table ) . we tested sera from dromedary camels in oman at a dilution of / by microarray and mers-cov neutralisation test. all the sera showed saturating reactivity with mers-cov antigen on the microarray, no sars-cov antigen reactivity, and human coronavirus oc antigen reactivity varying between negative (below the cutoff of rfu) and saturating signals (fi gure , table ). serial dilution of two sera with saturating reactivity for both antigens at the initial dilution of / showed that mers-cov antigen reactivity was still above the cutoff at / , whereas human coronavirus oc antigen reactivity fell below the cutoff at dilutions of / to / (fi gure d). consistent with the microarray data, all sera had high mers-cov neutralising capacity, with titres varying between / (seven of samples) and / or more ( or samples). in this study we describe the presence of mers-cov neutralising antibodies in dromedary camels both in a mers-cov linked (oman) and unlinked regions (canary islands). all the sera from dromedary camels from oman and some from spain had specifi c igg reactivity with the mers-cov receptor binding domain s . we confi rmed our expectation that another betacoronavirus-bovine coronavirus-circulated in these camelids. spanish camels ( %) had specifi c neutralising antibodies against mers-cov that were clearly not caused by cross-neutralisation by bovine coronavirus antibodies. our study is the fi rst in which animals have been tested for the presence of antibodies specifi c to mers-cov (panel). animal screening is necessary to understand the epidemiology of mers-cov. at present, bats are thought to be the ultimate reservoirs for several established human coronaviruses as well as sars-cov. accordingly, phylogenetic analysis has shown that mers-cov is related to betacoronavirus lineage c viruses found in pipistrellus spp bats. , however, direct transmission of mers-cov to people from bats seems unlikely. , the identifi cation of possible intermediate hosts that are probably in closer contact with people (eg, livestock) is urgently needed. common livestock species in the middle east include dromedary camels but also cattle, sheep, and goats. based on the available data, we cannot rule out circulation of a mers-related coronavirus in these species-sera were not available from epi demi ologically linked regions. the high prevalence of mers-cov neutralising antibodies in dromedary camels from oman suggests circulation of mers-cov or a closely related virus in this population. however, attempts to identify viral sequences in spanish camel sera and faecal samples using pancoronavirus and specifi c betacoronavirus c pcr methods , , were unsuccessful (unpublished data), as was untargeted amplifi cation followed by deep sequencing of faecal samples (unpublished data). these results imply that the camels were not actively shedding the virus at the time of sampling. less than % of the animals in the canary islands had mers-cov neutralising sera with titres up to / . this low seroprevalence means either that exposure of the animals to other putative reservoirs is rare or that the virus is absent in this closed-off population of roughly animals. we cannot rule out that the population might have once had an outbreak but that by the time of sampling, antibody titres had waned and no new introductions of the virus had occurred. the camels have contact with wild rodents, rabbits, pigeons, and doves and possibly also with bats. seven insectivorous bat species, including three pipistrellus spp, are native to the canary islands, while egyptian fruit bats (rousettus aegyptiacus) have been introduced. the % seroprevalence with high titres in omani camels from diff erent owners and locations suggests a diff erent situation in the middle east, with widespread circulation of mers-cov or a closely related virus. this diff erence of epidemiology might be because the virus circulating in the middle east is diff erent to that circulating in spain, with increased animal transmissibility and human infections. in addition, the omani camels were once racing camels now held for breeding and might be kept in circumstances that favour virus transmission. for cattle, a relation has been established between the incidence and eff ects of respiratory diseases, management practices, and animal transport. , to our knowledge, the camel populations in oman and the canary islands are not connected. camels on the canary islands were originally imported in the th century from the horn of africa for labour and transport. nowadays, import of animals from africa is banned because of the risk of foot-and-mouth disease. only three camels in our study were originally imported from morocco, more than years ago. because the closest relatives of mers-cov were identifi ed very recently in neoromicia zuluensis bats from africa, the introduction of mers-cov or related viruses into some african camel populations could have occurred decades ago, giving a possible explanation for mers-cov antibodies in camels from the canary islands. in the middle east, huge numbers of camels are imported from africa to meet the demand for meat. the top fi ve camel breeding countries are all african, and saudi arabia and united arab emirates are in the top fi ve camel meat producing countries. this increased turnover of animals in the middle east we searched pubmed for "novel coronavirus emc" or "mers-cov", we identifi ed reports in english linked to the middle east respiratory syndrome coronavirus (mers-cov) published before july , . none of these reports described a serological study for mers-cov-specifi c antibodies in animals. our report describes the fi rst mers-cov serological study of major livestock relevant to the middle east. our study shows that mers-cov or a related virus has infected dromedary camel populations. both titre levels and seroprevalences in sera from diff erent locations in oman suggest widespread infection of camelids with mers-cov or a closely related virus. targeted studies are needed to confi rm our fi ndings and their possible relevance to human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels from diff erent regions for which epidemiological background information is available, as well as virological assessment of samples from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime, we recommend a detailed case history of confi rmed mers-cov cases, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. compared with the canary islands could also aff ect the epidemiology of a virus, through more frequent infl ux of immunologically naive animals. targeted studies are needed to confi rm our fi ndings and their possible relevance in relation to the human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels for which epidemiological background information is available, as well as virological assess ment of specimens from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime we recommend a detailed case history of people with mers-cov, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. who. global alert and response (gar): novel coronavirus infection recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov)-update interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk coronavirus diversity, phylogeny and interspecies jumping evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats ecoepidemiology and complete genome comparison of diff erent strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus e in bats human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human betacoronavirus c emc/ -related viruses in bats, ghana and europe genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus circulation of group coronaviruses in a bat species common to urban areas in western europe comparative analysis of twelve genomes of three novel group c and group d coronaviruses reveals unique group and subgroup features full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections specifi c serology for emerging human coronaviruses by protein microarray camel trypanosomosis in the canary islands: assessment of seroprevalence and infection rates using the card agglutination test (catt/t. evansi) and parasite detection tests antigenic and biological relationships between human coronavirus oc and neonatal calf diarrhoea coronavirus serum antibodies to bovine coronavirus in swedish sheep serosurveillance of viral diseases in korean native goats (capra hircus) enteric coronavirus infection in a juvenile dromedary analysis of the genome sequence of an alpaca coronavirus the diff erential clinical impact of human coronavirus species in children with cystic fi brosis generic detection of coronaviruses and diff erentiation at the prototype strain level by reverse transcription-pcr and nonfl uorescent low-density microarray forecast and control of epidemics in a globalized world la fauna de quiropteros del archipelgo canario coronavirus genomics and bioinformatics analysis risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds associations between the distance traveled from sale barns to commercial feedlots in the united states and overall performance, risk of respiratory disease, and cumulative mortality in feeder cattle during to close relative of human middle east respiratory syndrome coronavirus in bat we thank prof mc horzinek for helpful suggestions. rds was funded by the european public health training program (euphem), ecdc, stockholm, sweden. contributions to the study were funded through the european union fp projects emperie (contract number ; to blh, sls, ao, cd) and antigone (contract number ; to cg, cd, mpgk, ao). work in bonn was also funded by deutsche forschungsgemeinschaft (dfg grant dr / - to cd). key: cord- -w quh fx authors: hindawi, salwa i.; hashem, anwar m.; damanhouri, ghazi a.; el‐kafrawy, sherif a.; tolah, ahmed m.; hassan, ahmed m.; azhar , esam i. title: inactivation of middle east respiratory syndrome‐coronavirus in human plasma using amotosalen and ultraviolet a light date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: w quh fx background: middle east respiratory syndrome‐coronavirus (mers‐cov) is a novel zoonotic pathogen. although the potential for mers‐cov transmission through blood transfusion is not clear, mers‐cov was recognized as a pathogen of concern for the safety of the blood supply especially after its detection in whole blood, serum, and plasma of infected individuals. here we investigated the efficacy of amotosalen and ultraviolet a light (uva) to inactivate mers‐cov in fresh‐frozen plasma (ffp). study design and methods: pooled ffp units were spiked with a recent clinical mers‐cov isolate. infectious and genomic viral titers were determined in plasma before and after inactivation with amotosalen/uva treatment by plaque assay and reverse transcription–quantitative polymerase chain reaction, respectively. in addition, residual replicating or live virus after inactivation was examined by passaging in the permissive vero e cells. results: the mean mers‐cov infectious titer in pretreatment samples was . ± . log plaque‐forming units (pfu)/ml, which was reduced to undetectable levels after inactivation with amotosalen/uva demonstrating a mean log reduction of more than . ± . pfu/ml. furthermore, inoculation of inactivated plasma on vero e cells did not result in any cytopathic effect (cpe) even after days of incubation and three consecutive passages, nor the detection of mers rna compared to pretreatment samples which showed complete cpe within to days postinoculation and log viral rna titer ranging from . to . copies/ml in all three passages. conclusion: our data show that amotosalen/uva treatment is a potent and effective way to inactivate mers‐cov infectious particles in ffp to undetectable levels and to minimize the risk of any possible transfusion‐related mers‐cov transmission. the mean mers-cov infectious titer in pretreatment samples was . . log plaqueforming units (pfu)/ml, which was reduced to undetectable levels after inactivation with amotosalen/ uva demonstrating a mean log reduction of more than . . pfu/ml. furthermore, inoculation of inactivated plasma on vero e cells did not result in any cytopathic effect (cpe) even after days of incubation and three consecutive passages, nor the detection of mers rna compared to pretreatment samples which showed complete cpe within to days postinoculation and log viral rna titer ranging from . to . copies/ ml in all three passages. conclusion: our data show that amotosalen/uva treatment is a potent and effective way to inactivate mers-cov infectious particles in ffp to undetectable levels and to minimize the risk of any possible transfusion-related mers-cov transmission. t ransfusion of blood components saves millions of lives by controlling bleeding due to accidents, surgeries, or other disease complications. however, transmission of pathogens is one of the biggest risks of transfusion of labile blood components. therefore, a key mission of blood transfusion services is to provide safe blood and blood products. screening of blood products has reduced the spread of known blood-borne pathogens such as hepatitis b and c viruses (hbv and hcv), human immunodeficiency virus (hiv), and human t-lymphotropic virus (htlv). , however, other known or unknown pathogens pose a threat to the blood supply with regional differences that is difficult to address. the number of pathogens screened in blood banks is limited by the number of blood screening assays that are commercially available. therefore, pathogen inactivation offers an appealing alternative to blood screening (serology or nucleic acid testing [nat] ) because of its proactive nature and the broad spectrum of protection it offers without a priori characterization of unknown pathogens. indeed, pathogen inactivation technologies have been developed to provide safe blood products while reducing the need for the implementation of additional screening assays. the middle east respiratory syndrome-coronavirus (mers-cov) is a zoonotic pathogen that is endemic in saudi arabia and other countries in the arabian peninsula since . as of february , , mers-cov has been responsible for confirmed infections including deaths (approx. . % mortality rate) in countries. most cases ( confirmed infections) were reported in saudi arabia with a mortality rate of approximately . %. mers-cov causes respiratory illness in humans varying from asymptomatic or mild to severe pneumonia. , the elderly and patients suffering from comorbidities are the most at risk for death outcome after development of severe clinical symptoms such as severe pneumonia and extrapulmonary manifestations. the major route of transmission for mesr-cov is via droplets, fomites, and person-to-person contact through the respiratory system as well as via direct or indirect contact with zoonotic sources. so far, most mers cases are linked to residence in or travel to saudi arabia. several hospital and household outbreaks have been reported in saudi arabia as well as south korea due to nosocomial transmission and close contact with patients. , however, the number of asymptomatic cases is estimated to be much higher than the reported cases, which together with mild cases may increase the spread of the virus and pose a significant risk for blood safety. indeed, presymptomatic and asymptomatic donors may be allowed to donate blood while their donation has the potential to carry the virus, although there is no report on mers-cov detection in blood from presymptomatic and asymptomatic patients. while most of the reported mers-cov cases were due to human-to-human transmissions especially under inadequate infection control measures, dromedary camels are believed to be the reservoir host and an important source of human infection. , [ ] [ ] [ ] [ ] [ ] [ ] specifically, primary or index cases that had no contact history with infected individuals are more likely to have had direct or indirect contact with dromedaries. interestingly, current regulations of blood donation do not consider recent history of contact with dromedaries as a reason for deferral of blood donation. mers-cov has been detected in a variety of samples from infected patients including respiratory samples, serum, plasma, urine, and stool. [ ] [ ] [ ] [ ] [ ] while detection of mers-cov rna is most frequent and persistent in respiratory secretions with high viral loads, detection of viral rna in plasma or serum was documented in up to onethird of the patients and showed association with disease severity. [ ] [ ] [ ] this is reminiscent of the severe acute respiratory syndrome-coronavirus (sars-cov) outbreak which started in china in and led to global human-tohuman spread with more than confirmed cases and approximately % death rate. during the sars outbreak, viral rna was detected in serum from several patients but no transfusion-associated transmission was reported. , while sars-cov disappeared quickly, mers-cov continues to be endemic in the arabian peninsula for more than years now. similar to sars-cov, there is no proven evidence so far of transfusion-transmitted mers-cov infections, but the presence of viral rna in plasma and serum of acute patients raises this concern especially in endemic areas like saudi arabia. in fact, it is not known whether presence of viral rna in blood products could lead to transmission of mers-cov or not. therefore, it is important to find a method to mitigate the risk associated with mers-cov rna presence in blood components to proactively prevent any possible transmission of mers-cov through contaminated blood products in endemic regions and elsewhere. the intercept blood system (ibs) inactivates pathogens by forming crosslinks or monoadducts on nucleic acids using amotosalen, a photoactive psoralen, after illumination with low-energy ultraviolet a (uva) light. during this highly specific targeted process, an adduct is formed at high frequency in the nucleic acids, thus inhibiting transcription and replication. the ibs is a food and drug administration-approved pathogen reduction system that has been shown to inactivate a broad spectrum of viruses, bacteria, and parasites in plasma as well as other blood products [ ] [ ] [ ] [ ] without affecting the plasma efficacy and patient safety as demonstrated in clinical evaluation and by hemovigilance data from multiple countries. , until now there has been no data available on the inactivation of mers-cov with ibs. however, the inactivation of sars-cov with ibs, which is related to mers-cov, was successfully shown, raising the expectation of a sufficient inactivation efficacy. therefore, the aim of this study was to evaluate the efficacy of amotosalen/uva treatment to inactivate mers-cov in human apheresis plasma concentrates. african green monkey kidney-derived vero e cells (atcc # ) were grown and maintained as previously described in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs). the human mers-cov isolate used in this study was mers-cov/hu/taif/sa/ , which was characterized previously. all experiments involving live virus were conducted in a biosafety level facility at the special infectious agents unit at king fahd medical research center, king abdulaziz university, jeddah, kingdom of saudi arabia, following the recommended safety precautions and measures. mers-cov amplification and culture was done as previously described. virus was inoculated on % to % confluent vero e cells in a t tissue culture flask at multiplicity of infection of and incubated in a humidified % co incubator at c for hour with gentle shaking every to minutes. subsequently, the inoculum was replaced by ml of viral inoculation medium (dmem with % fbs, % penicillin/streptomycin, and mmol/l hepes [ph . ]) and the cells were incubated in a humidified % co incubator at c for hours or until % to % of cells showed a cytopathic effect (cpe). supernatant was collected and centrifuged to remove cellular debris for minutes at g at room temperature. virus was then aliquoted and stored at - c, and the titer was determined by plaque assay. whole blood units ( ml %) were collected and prepared at king abdulaziz university hospital, transfusion services, jeddah, kingdom of saudi arabia, from voluntary donors. briefly, blood units were centrifuged at g for minutes to separate the platelet (plt)-rich plasma. the plt rich plasma was then centrifuged at g at c for minutes. the plasma was then separated into the plasma bag and kept at not more than - c. all blood units were screened routinely for hcv antibody or antigen, hbsag, hbc antibody, hiv ( / ) antibody, htlv ( / ) antibody, and syphilis as well as hcv, hbv, and hiv by nat. two units of fresh-frozen plasma ( ml each) were pooled for each experiment. the count of red blood cells was less than /ml in the pooled plasma. in total, four pools (n ) were used in this study. pooled plasma units were then inoculated with approximately ml mers-cov stock ( : dilution). plasma units were inactivated with the intercept processing set for plasma and the intercept illuminator according to the manufacturer's instructions. residual amotosalen and the free photoproducts were removed with an intercept compound adsorption device according to the manufacturer's instructions. samples collected for testing were as follows ( fig. ) : a positive control sample from the virus stock, a negative control sample from the plasma pool before inoculation, a -to -ml pretreatment sample from the inoculated plasma pool, and an inactivated sample from the treated pooled plasma. all samples were stored at - c until testing by plaque and reverse transcriptionquantitative polymerase chain reaction (rt-qpcr) assays. for the detection of mers-cov replication, all samples were diluted at : dilution in dmem with % fbs, inoculated on % confluent vero e cells in six-well plates, and incubated for to days at c. supernatants were collected, diluted : in dmem with % fbs, and blindly passaged for two more times. each passage was observed for cpe and all collected supernatants were used for viral rna quantification by rt-qpcr. vero e cells were prepared at /ml in dmem growth medium and ml were seeded in each well in sixwell plates and incubated overnight at c. samples was then serially diluted in inoculation dmem starting from , and ll of each dilution were applied to the confluent vero e cell monolayers in each well and incubated for hour at c with gentle shaking every to minutes. after hour, inoculum was removed and replaced with overlay medium (dmem with . % agarose) and incubated for to days at c. after incubation, the overlay was removed and cells were fixed with % paraformaldehyde for minutes at room temperature and stained with crystal violet stain. plaques were examined and counted to calculate titer as plaque-forming units (pfu)/ml as previously described. in some experiments, to ml of ibs-treated plasma was tested in plaque assay to detect any residual infectious virus and to increase the dynamic range of the assay in which ll of neat plasma were inoculated in each well. for all samples (positive, negative, pretreatment, and inactivated samples) and cell supernatants, rna was extracted as previously described using viral rna mini kit (qiagen) according to the manufacturer's instructions. real-time rt-qpcr was performed using primers and probe targeting mers-cov n gene as previously described. specifically, the following forward primer -caaaaccttccctaagaaggaaaag- , reverse primer -gctcctttggaggttcagacat- , and probe -fam-acaaaaggcaccaaaagaagaatcaacagacc-bhq - were used. real-time rt-qpcr was performed in well plates on a fast real-time pcr system (model , applied biosystems) using an rt-pcr kit (quantitect probe, qiagen) according to the manufacturer's instructions in a total reaction volume of ll. rna transcript corresponding to the same target region was generated using a cdna synthesis kit (superscript rt iii, invitrogen) from a plasmid containing the mers-cov n gene according to the manufacturer's instructions and used to generate a standard curve to estimate the viral rna copy number in each sample as previously described. , each run included a positive viral template control and no-template negative control. each sample was tested in duplicate and the mean is reported as log copies/ml. samples were reported as negative if the cycle threshold values were higher than cycles. the study was approved by the biomedical ethics committee unit of king abdulaziz university hospital (approval - ). to evaluate the potential of the ibs in inactivating mers-cov in pooled plasma, mers-cov virus was spiked into pooled plasma units and treated with amotosalen and uva light. the viral titer in pretreatment samples ranged from . to . log pfu/ml with mean titer of . . log pfu/ml ( table ). the treatment resulted in no detection of viable viruses in inactivated samples by plaque assay with a mean reduction of . . log pfu/ml in viral infectivity titer from four independent experiments (experiments a-d) and fig. shows representative results from the plaque assay. therefore, larger volumes from inactivated samples were tested to increase the sensitivity and the dynamic range of the assay as shown in table . nonetheless, no viable virus was detected post pathogen inactivation treatment even when to ml of plasma was tested. the viral genomic titer was not reduced post treatment as expected (table ) . together, these data show that amotosalen/uva light pathogen inactivation technology inactivated all mers-cov infectious particles in the spiked plasma units. next, we analyzed the presence of any potential low level of replicating mers-cov in inactivated plasma units taking into consideration that genomic viral titer did not change before and after treatment ( table ) . to this end, vero e cells were inoculated with plasma samples of pathogen-inactivated units and incubated for days, followed by two additional passages with days of incubation. no cpe was observed from the negative control or inactivated samples even on day postinoculation or after passaging for three times compared to the positive or pretreatment samples, which completely destroyed all cells by day (data not shown). to further confirm these results, the viral genomic rna titer was measured by rt-qpcr from supernatant collected on day from all samples in the three consecutive passages. as shown in table , viral genomic titer in positive control and pretreatment samples ranged from . to . and . to . log rna copies/ml, respectively, in the three passages indicating presence of replicating viruses in these samples. on the other hand, no viral rna was detected in the supernatant collected from any of the three passages from the negative control or inactivated samples, confirming the complete inactivation of the infectious mers-cov in plasma. currently, there are more than known pathogens that have been recognized by the aabb as pathogens of concern for blood transfusion; however, blood donations are only screened for a small number of pathogens on a regular basis. although such specific pathogen screening and testing of blood products have significantly reduced transfusion-related infections, transmission of wellrecognized pathogens such as hiv, hbv, and hcv is still being reported in many parts of the world. , furthermore, emerging and/or reemerging pathogens which are usually of zoonotic nature pose a high risk for blood safety. the unpredictable nature of such outbreaks and the erratic dynamics of their spread represent a challenging hurdle to preparedness plans and implementation of safety measures. a recent example is the emergence of zika virus, a mosquito-borne flavivirus that is transmissible through blood transfusion. outbreaks of coronaviruses with high mortality in humans have been described for sars-cov in china and southeast asia , and mers-cov in the middle east and south korea. the detection of viral rna in the blood of patients infected with sars-cov and mers-cov suggests a potential risk for their transmission through transfusion. [ ] [ ] [ ] [ ] during a mers-cov outbreak in south korea, levels of viral load ranged from . to . log genomic copies/ml in whole blood and from . to . log genomic copies/ml in serum, while another study reported up to logs of rna copies/ml in serum from infected individuals. that corresponds to an approximate infectious titer of less than . log in whole blood and less than logs in serum according to our data which shows -log difference between genomic and infectious titers (tables and ). using ibs, we were able to show a mean reduction of . . log in mers-cov infectious titers (ranging from . to . ) in plasma (table ) which is approximately . logs higher than the minimum reduction titer recommended by the european committee of blood transfusion, suggesting an inactivation capacity of mers-cov in plasma with a high safety margin. in contrast to the infectious titer, the genomic titer was only affected slightly by the pathogen inactivation treatment ( table ). the amotosalen/uva process effectively crosslinks nucleic acids, but does not break the strands. therefore, to ensure that there are no remaining amounts of infectious virus particles, the collected supernatants were passaged three times after inactivation for days. the absence of cpe even after days of incubation in cultures inoculated with the posttreatment samples suggests that there is no infectious mers-cov remaining in these samples. no viral genomes were detected in the supernatant of cell cultures inoculated with posttreatment samples even after multiple passages mostly due to nonreplicating viral genome and degradation of viral rna during the incubation period. this is in contrast to the pretreatment and the positive control samples that showed a high level of viral rna after incubation in all three passages. this demonstrates the loss of infectious virus through passaging and the successful inactivation of infectious particles by the ibs treatment. so far, there have been no reports of any transmission of mers-cov or sars-cov via blood transfusion despite the detection of their viral rna in serum from infected individuals. nonetheless, the aabb still lists both of these viruses as pathogens of concern for blood safety. mers-cov rna detection persisted in patient serum up to days after diagnosis, and there is evidence that mers-cov could infect and replicate in macrophages, dendritic cells, and t cells from the peripheral blood. , , therefore, it is not really clear whether presence of mers-cov rna in blood, serum, or plasma could have any consequences on the transmission risks of mers-cov during transfusion. apart from that, a recent large blood virome study found that blood from % of the study population ( healthy blood donors) contained genetic material from many of these viruses are of great importance in transfusion medicine although their detection in blood does not necessarily imply infectivity since only genetic material was detected. furthermore, other nonhuman viral species have also been detected in blood from many individuals, which were attributed to contamination from commercial reagents or the environment. nonetheless, identification of other viruses such as the sewage-associated gemycircularvirus was suggested to be due to contamination during phlebotomy or plasma pooling processing. therefore, targeted testing of blood products might not be the best economic or logistic option to ensure blood safety. here, we were able to show efficient inactivation of mers-cov in therapeutic human plasma units of . logs, far above the expected viral load of mers-cov in human blood and serum described until now. a recent study investigated the inactivation of mers-cov in plasma using riboflavin and uv light. the study showed a reduction of at least . and at least . log in viral titers for pooled and individual donor plasma, respectively. however, passaging experiments after pathogen reduction were not conducted, so complete inactivation of all infectious particles cannot be taken for granted. several previous studies have also proved the efficiency of ibs in inactivating a large number of pathogens (viruses, bacteria, and parasites). the study described here adds mers-cov to the list of pathogens that can be inactivated by ibs. taken together with previously published work, our data show that ibs could represent an economically and logistically efficient protective solution to reduce the risk associated with the circulation of mers-cov in the arabian peninsula and the potential transmission of not only mers-cov but also other known or unknown pathogens for which there is no commercially available screening assays. comparison of elisa and rapid screening tests for the diagnosis of hiv, hepatitis b and hepatitis c among healthy blood donors in a tertiary care hospital in mumbai seroprevalence of hiv, hbv, hcv and syphilis infections among blood donors at gondar university teaching hospital, northwest ethiopia: declining trends over a period of 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prepared with amotosalen and uva photochemical treatment independent evaluation of tolerance of therapeutic plasma inactivated by amotosalen-hcl-uva (intercept tm ) over a -year period of extensive delivery immunogenicity of candidate mers-cov dna vaccines based on the spike protein growth and quantification of mers-cov infection active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections hiv transmission through transfusion-missouri and colorado blood transfusion safety in africa: a literature review of infectious disease and organizational challenges emerging pathogens: the epidemiology and evolution of species jumps evidence for transmission of zika virus by platelet transfusion european directorate for the quality of medicines and healthcare. guide to the preparation, use, and quality assurance of blood components productive replication of middle east respiratory syndrome coronavirus in monocytederived dendritic cells modulates innate immune response middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways the blood dna virome in , humans inactivation of middle east respiratory syndrome coronavirus (mers-cov) in plasma products using a riboflavin-based and ultraviolet light-based photochemical treatment we thank edwin raj from the blood transfusion services, king abdulaziz university hospital, king abdulaziz university, jeddah, saudi arabia, for the technical help with plasma collection and screening. the authors have disclosed no conflicts of interest. key: cord- -bgcqkz p authors: yamamoto, naoki; bauer, georg title: apparent difference in fatalities between central europe and east asia due to sars-cov- and covid- : four hypotheses for possible explanation date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: bgcqkz p the comparison of the numbers of cases and deaths due to sars-cov- / covid- shows that people in central europe are much more affected than people in east asia where the disease originally occurred. trying to explain this difference, this communication presents four hypotheses that propose the following reasons for the observed findings: ) differences in social behaviors and cultures of people in the two regions; ) possible outbreak of virulent viruses in central europe due to multiple viral infection, and the involvement of immuno-virological factors associated with it, ) possibility of corona resistance gene mutation occurring among east asians as a result of long-term co-evolution of virus and host, and ) possible involvement of hygienic factors. direct or indirect supportive evidences for each one of our hypotheses are presented and experimental approaches for their evaluation are discussed. finally, we suggest that the dynamics of the pandemic also shows that the problems of the new coronavirus can be overcome due to people's awareness of the epidemics, rational viral diagnostics and a high level of medical care. the new coronavirus sars-cov- , causing an infectious disease named covid- has caused a major pandemic with more than million people infected and more than , already dead [ , ] . its momentum is disrupting the economy, society, and healthcare systems, filling hospitals with patients, emptying public spaces, and drawing people away from work and friends. due to the test strategy used in nearly all countries, "people infected with sars-cov- " or simply named "covid- cases" are usually people with clinical symptoms or contacts to infected people. the number of infected people without clinical symptoms is therefore unknown. further testing, including antibody tests, will most likely reveal that much more people around the world have been infected than the number of defined cases of covid- . this may upset modern societies on a scale never seen before. on the other side, it may be assumed that these infected people might contribute to herd immunity and slow down further spread of the virus, provided their antibody response is longer lasting and has neutralizing potential. these aspects need further experimentation and proof. in the st century, information (including scientific and technological knowhow) circulates around the world instantly. the world has entered an era where the traffic of people, economic goods, money, but also viruses and other microbes seems to be nearly unlimited. therefore, after the outbreak of the new coronavirus epidemics in china, the transport of the virus to other parts of the world, particularly to europe, was fast and efficient. of particular note is the striking difference in the extent of medical impact caused by sars-cov- in eastern asia versus central europe [ , ] . this difference is prominent, when i) the number of infected persons with clinical symptoms, ii) the ratio between infected people with clinical symptoms and the total population of the countries, iii) the number of deaths, or iv) the death ratios of infected people with symptoms are compared between eastern asia and central europe. in this communication, we compared selected countries from central europe and east asia with respect to the medical impact of sars-cov- and covid- . based on this analysis, we propose four hypotheses that might explain the observed differences. for each one of these hypotheses, we present direct or indirect evidences to support them. none of these hypotheses by itself can explain the observed differences. rather, the mechanisms defined in individual hypotheses seem to interact and thus cause the overall effect observed. we are aware that additional mechanisms, not covered by our analysis, may also contribute to the pandemics. for the analysis of the difference regarding the number of infected people and the death tolls due to covid- between central european and east asian countries, we have chosen italy, spain, france, germany and uk from central europe and china, south korea, japan, and taiwan from south east asia. mongolia and north korea were excluded, since not sufficient information was available from both countries. for comparison, the united states, which have the highest number of infected and dead people due to covid- , were included into our analysis. table first shows the populations and gdps, in order to help estimating the scale, medical level, and affluence scale of the selected countries. it is obvious that east asia is one of the most densely populated areas in the world. it can also be seen that there is not much difference with respect of the gdps, except for china. however, china's gdp has grown rather rapidly in recent years, and it is certain that it will reach the economic level of the other countries sooner or later. european countries ranged from to per million people, whereas those of east asian countries were in the range of ~ . the ratio between the two was about . these numbers depend on the number of tests performed in individual countries, as well as on differences in the test strategy, resulting in a certain degree of uncertainty. to get a more precise picture of comparison, covid- -related deaths per nation's total population (per million people) were calculated based on the total population of each country. as seen in table , the numbers of central european countries were in the range of - deaths/million people, with the only exception being germany's lower number of . in contrast, deaths/million people in east asian countries were in the range of . - , i. e. they were nearly times lower than the values found for central european countries. importantly, also the death rate of infected people seems to be much lower in east asian countries, compared to the central european countries (except germany). the relatively lower number of infected people as well as covid- -associated deaths in germany seems to be due to strong differences in the percentage of infected people with clinical symptoms between the south and north of germany (table ) . it seems that measures taken after the first cases in bavaria (a large state in the south east of germany, which was first hit by the pandemics) were successful to prevent a strong spreading on the long run. germany had the advantage to be affected by covid- later than china and italy. getting valuable information from other countries was essential for slowing down and partially restricting the pandemics in germany. furthermore, the relatively high availability of equipment for intensive care reduced the death rate of infected people. importantly, the picture of inhomogenous distribution of the disease and associated deaths as seen for the whole of germany is also seen when the spreading of infections and deaths is analyzed within bavaria, the state being affected the most ( table ). the detailed analysis of cases and deaths in districts points to hotspots of infection and death, from which the neighbouring areas seem to be reached out. this results in an overall picture with a relatively high number of cases and deaths in the hotspot (tirschenreuth) that far exceeds the average number of cases / population and deaths / population of total germany. the immediate neighbouring districts in the north and south show about half the cases / population and less than half the deaths / population than the hotspot, whereas districts distant of the hotspot are characterized much lower numbers. this picture points to strong initial effects from local infection events, but, importantly, it also indicates that further spreading had obviously been prevented through adequate measures. since the data on the number of covid- -caused deaths in each country gave reliable data that showed strong differences between east asian and central european countries, we tried to explain these differences by four intercalated hypotheses. given the host-virus relationship, it is necessary to consider factors affecting both virulence of the pathogens and the resistance of the hosts to virus infection. several aspects might be involved in the difference. for example, lippi et al. investigated potential reasons for the noticeable difference in covid- -associated mortality rates between asian and european populations from clinical and demographic aspects in italy versus china [ ] . these authors confirmed that a higher burden of comorbidities, male sex, and older age may be considered substantial determinants of enhanced risk of death in italy compared to china. similarly, in new york city where the highest number of deaths is recorded compared to other states in the united states, factors such as high population density, the possible super spreaders of virus, the degree of poverty between races, and the lack of insurance have become important issues [ ] . hypothesis # is based on the difference in social behavior and customs of people in each area, i.e. in europe and south east asia. this might explain the difference to some extent. when comparing the lifestyles of european countries with that of asia, the first noticeable difference is the closeness of direct human contact. in asia, bowing is the mainstream greeting, and even with the spread of western culture, shaking hands is still not very common. of course, besides handshaking, there can be no hugs or kisses that are popular in western culture (if any, those are performed only after building a very close relationship). it is easy to imagine that these common actions in western societies help to spread respiratory viruses like sars-cov- very efficiently. these differences between europeans and asians, considered from a perspective of the social science might be very important in the progress of the pandemic. there is also a marked difference in obesity between westerners and asians, mainly due to differences in food culture. in addition, people wear shoes indoors in western societies, while it is strictly forbidden to do so in east asia. although the weight of this habit in preventing virus infection is not clear at this moment, this difference indicates that asians very clearly distinguish between outdoor and indoor in daily life. the habit of wearing a mask in case of a cold or fear of it also seems quite asian-specific. thus, it is very likely that differences with respect to these actions play an important role in the spread of infection. social distancing has been proven to avoiding new corona infections, and is a drug-free means of controlling de novo infections. shaking hands, kissing, and hugging are not allowed during social distancing. in favour of our hypothesis # , the switch from european style of close contact to the asian style during social distancing is regarded as an essential part of successful slowing down and preventing further infections with sars-cov- . potential effects of repeated infection on the severity of the clinical outcome, contributions of antibody-mediated enhancement of infection to the severity of disease, as well as genetic changes of the virus due to mutation of its rna genome might be underlying the observed effects. in italy, the death toll from the new coronavirus was , by the april , the third highest in the world after the united states and spain. of these, more than , people were confirmed to be infected among medical staff. this finding shows that inadequate protection of medical staff from infection is a major issue. many of the doctors who died were practitioners. they were fighting the new coronavirus without adequate protective equipment (sometimes due to unavailability of material) or underestimation of the contagiousity of the new virus, driven by the strong wish to help their patients. thus, it is highly likely, especially during the medical collapse, that these doctors will have got infections repeatedly from the infected patients one after another. so the question is what would be severe disease [ ] . mice developed higher viral loads, more severe lung pathology, and greater inflammatory responses and generated only limited influenza a virus-specific b and t cell responses. although several pathways could contribute to higher viral loads in mice that received influenza a virus repeatedly, the authors suggested the possibility that the inflammatory response elicited by the first dose of influenza a virus damaged the mucosal barrier, thus allowing the virus given as a second or third dose to penetrate more deeply into the lungs. higher viral loads will lead to the induction of stronger cytokine storms. these data are in agreement with clinical findings in humans, where high-risk individuals were exposed to multiple small doses of hbv, as well as in the woodchuck model, in which animals were repeatedly exposed to small quantities of infectious hbv [ ] . in both cases, viral infection was molecularly evident and there were detectable virus-specific cd -t cell responses but undetectable virus-specific antibody responses. as to repeated infections, another possibility is also considered that is involvement of the antibody-dependent enhancement (ade), which is a well-known phenomenon in the field of viral immunology. first, let's assume the situation of the individual who received a coronavirus infection followed by a second one. one week or so after initial infection, antibodies against the virus are elicited, and the second virus reacts with this antibody causing more serious effects. this phenomenon to increase viral infectivity and virulence has been observed with many viruses including several coronaviruses, hiv, zika virus and mosquito-borne flaviviruses (dengue virus and yellow fever virus). indeed, olsen and colleagues have shown antibody enhancement of infection with one of the animal coronaviruses, feline infectious peritonitis virus by a subset of specific monoclonal antibodies, especially those directed against specific sites on the spike protein [ ] . similarly, wang et al. generated monoclonal antibodies against sars-cov spike proteins and found that these antibodies promoted sars-cov infection [ ] . ade these aspects may be particulary relevant for a limited number of people such as medical staff. therefore, in addition to that, the emergence of more virulent virus due to mutation of the virus during the course of superinfection might also be assumed. rna viruses are, in general, highly susceptible to mutations, and the base substitution rate, which indicates the degree of change, is estimated to be + x - nucleotide substitutions per site and per year on average in case of transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus. this rate falls in the range reported for other rna viruses [ ] . in addition, it is also known that coronavirus changes considerably easily due to gene recombination or part of gene loss (deletion). for example, the new strain of canine respiratory coronavirus identified in is reported to be a recombinant of the existing canine and bovine coronaviruses [ ] . in addition, it has been known that the deletion of part of the spike gene has caused the emergence of a prcv from the ancestral porcine gastroenteritis virus [ ] . it is possible that the doctor who actually examined many infected patients could be exposed to the coronavirus repeatedly, and among the multiple virus strains infected, more virulent virus strains suitable for spread may be selected and prevailed, at least initially. according to the classical models of virulence evolution of the virus, multiple infections select for raised virulence. to explain why so many people are dying of covid- in new york city, sequence analysis of the viral genome has been performed. according to zimmer, the virus mainly came from asia through a small number of infected individuals in california, while in new york, more than people initially brought the virus mainly from europe, suggesting as if the european type viruses are more virulent in its pathogenicity and infectivity than the asian type [ ] . a number of studies have supported that through collective actions leading to common good production and immune system impairment, viral cooperation can lead to increased levels of virulence. researchers around the world have already infecting vero-e cells [ ] . based on more molecular virological studies, tan et al. proposed that sars-cov- can be divided into two major lineages (l and s). intriguingly, the s and l lineages can be clearly defined by just two tightly linked snps at positions , (orf ab: t c, synonymous) and , (orf : c t, s l). orf ab encoding replicase/transcriptase is essential for viral genome replication and might also be important for viral pathogenesis [ ] . however, there was no evidence so far that this mutation produced a more virulent form of the virus. moreover, the data examined up to now are still very limited, and follow-up analyses of a larger set of data are needed to have a better understanding of the evolution and epidemiology of sars-cov- . thus, further virological studies must focus on the relationship between differences in nucleotide sequences and infectivity/ pathogenicity of viruses since there is no firm evidence, so far, of the existence of european strains of the coronavirus or its pathogenicity being more virulent than the asian strains. human hosts and their virus have co-evolved for millions of years, during which viruses have adapted to defense system of its host by regulating pathogenic mechanisms. therefore, the possible genetic change and resulted selection of people living in east asia should also be considered from an evolutional perspective. thus, the difference in viral susceptibility and mortality of east asian people to sars-cov- could also be explained if people living in east asia may have evolved to be more resistant to viral infections, including those of novel corona viruses. in east asia, especially in china, agriculture started about , years ago, maybe , years ahead of europe. this led to an explosive increase in population, urbanization, and population density with the supply of abundant food. as a matter of course, acute viral infections such as measles, rubella, mumps, which could not be established until then, are believed to have taken roots in the human population (in the case of measles, it requires a population more than , to settle). unlike today, asia had long been much richer than europe before the industrial revolution. under the over-crowded and chaotic conditions, east asians must have experienced overwhelmingly with many plagues including several zoonoses due to the encounter with strange animal species. it is natural to consider that such epidemics are related to the change, choice, and evolution of the people who live there. east asians may have evolved to become more resistant against infectious agents in general including coronavirus. it is possible that difference of the past plagues could contribute to a difference in the susceptibility (and thus, pathogenicity) between europeans and asians against present new corona. present covid- is apparently derived from bats directly or via vector animals, and its appearance is closely related to chinese food culture. given this, it is not strange to consider the possibility that this area had been hit by coronavirus infections similar to this time before long ago. in fact, the country experienced similar endemics, sars and mers only and years ago, respectively. this suggests that coronavirus infection itself is one of the most likely candidates for east asian selection and evolution among the past plagues. although humans are a fairly homogeneous group of species as viewed from the genome, the diversity of the genome is well maintained. it avoids all human species from suffering the same disease and is a means of survival as a species, even if some disease prevails. although plague and people have been closely linked, one of the causes of human diversity is infectious disease. many genetic diseases are unfavorable to survival, but in some cases they are also advantageous for survival, and in some cases mutations have given the power to survive from the diseases that have hit the ground in the past. in east asia, where agriculture was established early on and urbanization has been achieved, plagues have been rushing to people in a messy environment since ancient times. we believe that it should be worth considering that individuals with advantageous gene mutations have been selected in relation to various epidemics, and have reached the present day. several genes may be involved in the genetic predisposition to covid- , and the combination of multiple genes may be important for the severity of the infection. among them, human leukocyte antigen (hla) polymorphisms are associated with susceptibility to various diseases such as autoimmune diseases and infectious diseases. the composition ratio of hla types varies greatly depending on the country and ethnic group. since hla is a protein of the immune system that is responsible for antigen presentation, hla has been attracting attention in relation to disease susceptibility. ellinghaus and colleagues performed gwas on patients in italy and spain [ ] . but, it was found that certain hla types are unlikely to be associated with exacerbation of the novel coronavirus at least in italy and spain. nevertheless, since hla types, which are present only in japan and other asian countries, may show resistance to the novel coronavirus, further analysis is necessary. the human angiotensin-converting enzyme (ace ) gene on chromosome has an insertion (i) or deletion (d) of a base pair (bp) alu repeat sequence in intron [ ] . therefore, in the i/d polymorphism, there are three different genotypes, ii, id and dd. ace is a metalloproteinase, which is a type i transmembrane glycoprotein. this protein plays an important regulatory function in the renin-angiotensin-aldosterone system (raas) and can convert angiotensin i (angi) vasoconstrictor, which is inactive, to angiotensin ii (angii). angii is the core product of the raas system, and causes various biological reactions through the angiotensin receptors (at and at ) while ace (angiotensin invertase ) is a homologue of ace and is well known as a receptor for sars-cov- . however, the original role of ace is to digest angii into ang - polypeptides, and protect the heart, vasodilate, resist growth, and resist proliferation. also, the activity of bradykinin can be enhanced by ace . very recently, we showed a strong negative correlation that the numbers of sars-cov- infected cases and deaths due to viral infection decreased with increasing ace ii genotype frequency [ ] . the serum ace level was significantly higher in those with the dd genotype compared with those with either the id or the ii genotype [ ] , and viral infection may lead to suppression of ace function and causes ace /ace imbalance responsible for ras over-activation and pulmonary shut-down [ ] . this can further reduce the effects of ace , which counteracts the pathophysiological effects of ang ii produced by ace , and may worsen the pathology. in patients with the d allele, especially those with the dd genotype, higher risk of morbidity and mortality from sepsis, acute respiratory distress syndrome (ards) and certain heart, lung and kidney conditions probably due to inflammation, vasculopathy and coagulopathy induced by angii [ ] is reported. thus, the ace /ace imbalance predicts that covid- patients with the d allele of ace , especially the dd genotype, have a higher severity and prevalence of covid- . further evidence for our hypothesis is provided by results obtained through the study of other viral systems. there are a few lucky people who have escaped infection while being exposed to hiv- . individuals homozygous for ccr -Δ show perfect resistance to hiv- [ ] . infection with yersinia pestis or smallpox virus were suggested as potential selective pressures favoring ccr -Δ [ ] . only european or central asians have this characteristic and in norway, ccr -Δ allele frequencies reaches nearly %. since aids, smallpox and plague are, by nature, independent infectious diseases that should be completely unrelated, it is very intriguing if these infectious diseases are linked by this mutant gene. indeed, in , lalani et al. showed that poxviruses, can exploit chemokine receptors, especially ccr to infect some cell types, notably migratory leukocytes [ ] . this further suggests a need searching for polymorphism and virological studies to see if there is a difference in the function of the ace gene as a sars-cov- entry receptor between asians and europeans. besides, several other examples are available in the resistance to certain type infections and genetic mutations. relationship between sickle cell anemia and malaria is well known [ ] . although less dramatic, it is speculated that the relationship between typhoid fever and cystic fibrosis, which is a recessive genetic disease commonly found in europeans, is similar. there is also information regarding the relationship between human abo blood type and disease, while ab type is strong against cholera and o type is resistant to tuberculosis [ ] . however, our hypothesis suffers from the lack of analogous data available in other viral infections. in other words, if this theory is relevant, we should realize earlier that there is a difference in susceptibility and pathogenicity between western and asian people even in the case of other viral infections before this corona pandemic. still, it is not impossible to explain with a claim that there was no report from the past because there was no appropriate viral pandemic involving europe and east asia in the past. if there is, it may be the spanish flu occurred at the beginning of the last century or some influenzas after that. however, in the case of spanish flu, it was before the identification of the influenza virus, and it was a special event taking place towards the end of world war i in europe, so it is quite difficult to compare the difference in the pathogenicity of the influenza virus in both areas. or is the new coronavirus indeed the first case to show such ethnic differences in terms of pathogenicity? it is hoped that this point will be answered when data in the comparative studies on new coronavirus infections in the united states, where many european and asian immigrants live, will be accumulated in the very near future. in relation to this natural selection of human traits about viral resistance, there are also some records on the relationship between hygienic condition and resistance to infectious diseases. it is generally accepted that asia used to have a denser human population and to have lower hygienic conditions than europe. is corona's low pathogenicity for east asians not related to the situation of asia being less-hygienic than in europe? a similar paradoxical seeming suggestion has been raised for childhood leukemia: the more often a child is infected during the first year of life, the less likely it seems to develop leukemia. epidemiological studies performed by kinlen [ ] , and greaves and wiemels [ ] separately showed that high socioeconomic status and lack of contact with multiple infections early in life could be a risk factor. based on these observations, zur hausen and de villiers made the following proposal by assuming the existence of a fairly universal lesser-known tumor virus such as ttv. many viral infections during pregnancy and perinatal have a negative effect on the persistence and increase of this oncovirus. intermittent viral infections, by inducing interferon, significantly reduce tumor virus load and reduce the risk of viral chromosomal alterations, prerequisites for malignant transformation [ ] . very recently, katoh et al. reported a possible association between lower rate of fatality induced by coronavirus and immunization rate against encephalitis [ ] . artificial vaccines against japanese encephalitis (je) may result in relatively lower mortality rate in some countries. je immunization is widespread or included in national programs in many asian countries including china, south korea, japan, and taiwan. in all of these countries, the fatality rate due to covid- is very low when compared with countries that don't immunize against je. also, a similar hypothesis has been proposed that the inoculation of bcg vaccine may be effective against infectious diseases caused by the new coronavirus [ ] . it is thus very intriguing to find out the reason why recipients are cross-protected against covid- conferring lower fatality rate in the countries where immunization is performed against some infectious diseases such as encephalitis or tuberculosis. it will be important to explore the underlying mechanism, such as whether this can be explained by mere nonspecific activation of immunity. as the aspect of nonspecific activation of immunity is considered as one possible mechanism that counteracts sars-cov- infection as well as the death rate of infected patients, it might seem worthwhile to ask the question whether the lower rate of infection and mortality due to sars-cov- in germany, compared to other european countries, might be connected to differences in vaccination coverages. unfortunately, such an analysis is difficult and has severe inherent limitations, as there is a very high degree of variability of vaccination coverage in germany with respect to age, sex and geography [ ] . this does not allow to consider germany as one entity, but rather would require to focus on many different regions and groups, for which it seems difficult to obtain the required complete set of data. however, when the influence vaccination coverage for risk groups is compared between european countries [ ] germany is only on place among -certainly not in favour for a more advanced immunization profile of the country. furthermore, the analysis of the national vaccination calendars of france, united kingdom, italy, spain, sweden, portugal and germany [ ] shows a large overlap of essential immunizations in these countries -a finding that is not in favour of assuming a specific aspect of immunization in germany that might have a particular negative impact on sars-cov- infections and disease. there is no doubt that central europe is much more affected by sars-cov- and covid- than east asia. the strong difference between east asia and central europe cannot be explained by eventual differences in the frequency of testing, as this would only affect the number of detected cases per inhabitants, but not the number of deaths. we are convinced that the behavioral difference in human contact in both areas of the world can be considered to have a very important influence on the spread of the sars-cov- , as seen by the impressive positive effect of social distancing on the control of covid- in europe. this shows that hypothesis # seems to be relevant to a significant degree for the differences between east asia and central europe. however, hypothesis # cannot explain the complete picture observed, as it would only have an impact on the number of cases in relation to the population, but not on the death rate of cases. as the death rates per cases are also lower in east asia compared to central europe, mechanisms suggested in hypothesis # - might also contribute to the overall effect. in addition, mechanisms not included into our hypothesis might play essential roles and await to be defined in the future. essential parts of our hypotheses for which we have no direct supportive information so far can be experimentally verified or falsified in the future. these so far unresolved aspects are i) the possible existence of more virulent strains of sars-cov- in europe, ii) the effects of repeated infections, possibly in combination with iii) ade, iv) polymorphism of ace or some other genes such as tmprss [ ] and ace [ , ] , and their relation to the function of viral receptor. covid- positive cases are already over . million even at this point in total while number of people infected with sars coronavirus- was only about , . considering its near-future expansion in developing areas such as africa and south america, the new coronavirus may reach an even stronger impact than sars-cov- . moreover, this coronavirus is very easy to mutate due to its original properties. taking these facts into account, this sars-cov- has the potential to persist in every corner of the world, has a great possibility of finding and adapting to the best environment in various climates and people's lives, and becoming established in human society. however, the pandemics have taught us some essentials for counteracting in the future. at the beginning of the outbreak of covid- in europe, the initial response, especially the delay in response to outbreaks (clusters), demographics, social behavior and lower testing capacity, etc. were sometimes very problematic in response to covid- . these experiences allowed states that were hit later by the pandemics, like germany, to adjust countermeasures. in germany, the federal and local governments have been involved in the fight against covid- from an early stage, and especially with an emphasis on looking for signs of early onset, pcr testing of very large numbers of samples for free, and isolation of defined cases. the medical system had time to be prepared and intensive care beds equipped with artificial respirators were reserved for covid- and increased in number. the needed specialized staff was trained. social distancing guidelines were introduced and widely followed. this resulted in slow-down of the pandemic. therefore, we can be confident, that even if european corona strains were more virulent than asian strains, or if europeans were more susceptible to coronaviruses, the authors declare no conflict of interest. world health organization. naming the coronavirus disease (covid- ) and the virus that causes it covid- : what is next for public health? who scientific and technical advisory group for infectious hazards clinical and demographic characteristics of patients dying from covid- in italy versus china why are so many people dying of covid- repeated low-dose influenza virus infection causes severe disease in mice: a model for vaccine evaluation repeated exposure to trace amounts of woodchuck hepadnavirus induces molecularly evident infection and virus-specific t cell response in the absence of serological infection markers and hepatitis monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins genetic evolution and tropism of transmissible gastroenteritis coronaviruses discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event most new york coronavirus cases came from europe, genomes show. the new york times patient-derived mutations impact pathogenicity of sars-cov- on the origin and continuing evolution of sars-cov- . national science review genomewide association study of severe covid- with respiratory failure online ahead of print sequence variation in the human angiotensin converting enzyme sars-cov- infections and covid- mortalities strongly correlate with ace i/d genotype an insertion/deletion polymorphism in the angiotensin i-converting enzyme gene accounting for half the variance of serum enzyme levels covid- and individual genetic susceptibility/receptivity: role of ace /ace might the double x-chromosome in females be protective against sars-cov- compared to the single x-chromosome in male? implications of the angiotensin converting enzyme gene insertion/deletion polymorphism in health and disease: a snapshot review homozygous defect in hiv- coreceptor accounts for resistance of some multiply-exposed individuals to hiv- infection evaluating plague and smallpox as historical selective pressures for the ccr -delta hiv resistance allele use of chemokine receptors by poxviruses sickle cell disease and malaria morbidity: a tale with two tails blood type biochemistry and human disease epidemiological evidence of an infectious basis for childhood leukaemia origins of chromosome translocations in childhood leukaemias virus target cell conditioning model to explain some epidemiologic characteristics of childhood leukemias and lymphomas cross-protection induced by encephalitis vaccines against covid- might be a reason for relatively lower mortality rate in some countries is bcg vaccination causally related to reduced covid- mortality? online ahead of print vaccination coverage in german adults influenza vaccination coverage among high risk groups of european countries how much money is spent on vaccination across western european countries sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven potease inhibitor covid- infections are also affected by human ace d/i polymorphism the data show that the two most southern states, bavaria and baden-württemberg have much more cases of covid- and covid- -related deaths (both in absolute numbers and per people) than the two northern states within bavaria, there are very strong differences between tirschenreuth (one of the hotspots of covid- in germany, located in the north east of bavaria on the border to the czech republic) and its directly neighbouring districts wunsiedel compared to districts that are more distant to the hotspot (oberallgäu in the north west corner, main-spessart in the south west corner and regensburg in the center of bavaria). taken together, these data show the high efficiency of sars-cov- for spreading from a site of initial infection, but also the efficiency of adequate countermeasures taken the authors state no conflict of interests. key: cord- -d r nji authors: harrath, rafik; abu duhier, faisel m. title: sero‐prevalence of middle east respiratory syndrome coronavirus (mers‐cov) specific antibodies in dromedary camels in tabuk, saudi arabia date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: d r nji the middle east respiratory syndrome coronavirus (mers‐cov) is a novel coronavirus which was responsible of the first case of human acute respiratory syndrome in the kingdom of saudi arabia (ksa), . dromedary camels are considered as potential reservoirs for the virus and seem to be the only animal host which may transmit the infection to human. further studies are required to better understand the animal sources of zoonotic transmission route and the risks of this infection. a primary sero‐prevalence study of mers‐cov preexisting neutralizing antibodies in dromedary camel serum was conducted in tabuk, western north region of ksa, in order to assess the seopositivity of these animals and to explain their possible role in the transmission of the infection to human. one hundred seventy one ( ) serum samples were collected from healthy dromedary camels with different ages and genders in tabuk city and tested for specific serum igg by elisa using the receptor‐binding s subunits of spike proteins of mers‐cov. ( , %) of the total camel sera shown the presence of protein‐specific antibodies against mers‐cov. these results may provide evidence that mers‐cov has previously infected dromedary camels in tabuk and may support the possible role of camels in the human infection. blood sera were separated, diluted at : and analyzed for mers-cov specific antibodies using the anti-mers-cov elisa camel (igg) kit manufactured by euroimmun ag (lübeck, germany). this test is based on the recombinant mers-cov spike protein subunit- and has successfully been used by other authors evaluating mers-cov in camels. optical density (od) was measured at nm using a mindray mr- elisa reader. statistical analysis was performed on spss v. . software (spss inc., chicago, il). data were expressed as percentage for continuous variables, which were normally distributed, or as percentages of total for categorical variables. pearson χ test was used to assess intergroup significance. this research was ethically approved by the research ethic committee at the university of tabuk. one hundred seventy one serum samples were collected from three areas in tabuk a primary study was conducted in tabuk instructions. this test has been successfully used and evaluated by many authors for mers-cov detection in camels. , , results have shown that a high number ( %) of dromedary camels from the different farms of tabuk riyadh and screened by elisa test showed that % of the animals were found to have antibodies to mers-cov. in the same study, archived serum samples collected from dromedary camels from to in riyadh and kharj were also analyzed by elisa and showed a high seroprevalence ( %) of mers-cov neutralizing antibodies. our data agree also with previous studies reporting wide antibody prevalence in camels in many countries, including egypt and oman. , in another study conducted in oman, all serum samples from dromedary camels were positive for mers-cov specific antibodies. similar results were also reached from a larger study conducted in united arab emirates (uae), where dromedary camels' sera screened in revealed % seropositivity. in africa, an outbreak investigating serum samples for mers-cov assessment in camels has also shown similar results in nigeria ( %), tunisia ( . %), and ethiopia ( . %). likewise, in a study conducted on archived camel sera samples collected in from egypt and between and from sudan and somalia, % were found to have neutralizing antibodies to mers-cov. similar results were also obtained in a study from kenya. while camels in the middle east and africa were highly exposed to mers-cov infection, camels from europe and australia which are geographically isolated were not exposed to the virus. however, serum samples collected from dromedary camels living in the archipelago of canary islands, between and showed that % have antibodies against mers-cov. in another study carried out in the same islands, dromedary camel sera were analyzed and only . % were seropositive for mers-cov. data showed that all the seropositive dromedary camels for mers-cov were all imported from africa. in addition, dromedary camels' sera collected from different regions between and in the islands were all tested negative for specific mers-cov antibodies. all these findings indicate that active mers-cov infection did not occur in the canary islands. the same author explained the increase of the seropositivity in adult camels by the age range of the animals which were exposed for long time to the virus infection. for young camels, specific mers-cov antibodies could be acquired from their mothers as it was reported by kamber who suggests that maternal igg antibodies in camels are acquired through the colostrum intake during the first h post-parturition and not via the trans-placental route. after h, antibody levels in the dam's milk decrease rapidly, and igg levels in serum cease to rise. we also suggest that maternal antibodies might not have been sufficient to mediate protective immunity for them. animals would then be exposed to new mers-cov infection and specific antibodies would consequently increase in - years after, which can explain the higher seroprevalence in juveniles then in adults. the predominance of viral antibodies in young camels could also be explained by animal exposure to the virus few times postparturition and not by mother immunity, which will stimulate antibody secretion in the blood. in the two last possibilities, young camels could contract viral infection after , period of epidemic mers circulation in ksa, and production of specific antibodies would consequently increase in some months after birth. respiratory specimens, as nasal swabs, and rectal swabs will be required for analysis to know whether anti-mers antibodies are issued from recent viral infection. in relation to sex, our results have shown a high seropositivity in both males ( , %) and females ( . %) without a significant difference between the two genders. this information was not reported in previous studies and indicates that the sex cannot be a limiting factor in camel's infection by the virus. in conclusion, our findings in dromedary camel's sera, even if limited to serology, constitute a strong argument suggesting that mers-cov has previously infected dromedary camels in tabuk region. we speculate that dromedary camels may support the role of these animals as potential reservoirs for human infection but we cannot confirm this relationship from the current data alone. further studies including more animals and respiratory samples are needed for a larger evaluation of camel population in this region. in addition, a whole genome sequencing of camel mers-cov is required to confirm its incrimination in human infection. the work was financially supported by the deanship of scientific research (dsr) at the university of tabuk. http://orcid.org/ - - - middle east respiratory syndrome coronavirus (mers-cov) update severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update human betacoronavirus c emc/ -related viruses in bats, ghana and europe close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study disappearance of antibodies to sars-associated coronavirus after recovery assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections specific serology for emerging human coronaviruses by protein microarray antibodies against mers coronavirus in dromedary camels antibodies against mers coronavirus in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt geographic distribution of mers coronavirus among dromedary camels absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands time course of mers-cov infection and immunity in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia studies on the supply of immunoglobulin g to newborn camel calves (camelus dromedarius) how to cite this article: harrath r, abu duhier fm. seroprevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in key: cord- - lso w o authors: adney, danielle r.; brown, vienna r.; porter, stephanie m.; bielefeldt-ohmann, helle; hartwig, airn e.; bowen, richard a. title: inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lso w o the middle east respiratory syndrome coronavirus (mers-cov) was first recognized in and can cause severe disease in infected humans. dromedary camels are the reservoir for the virus, although, other than nasal discharge, these animals do not display any overt clinical disease. data from in vitro experiments suggest that other livestock such as sheep, goats, and horses might also contribute to viral transmission, although field data has not identified any seropositive animals. in order to understand if these animals could be infected, we challenged young goats and horses and adult sheep with mers-cov by intranasal inoculation. minimal or no virus shedding was detected in all of the animals. during the four weeks following inoculation, neutralizing antibodies were detected in the young goats, but not in sheep or horses. the middle east respiratory syndrome coronavirus (mers-cov) is an emerging pathogen first described from saudi arabia in [ ] that can cause severe respiratory disease and death in roughly % of infected humans [ ] . there is considerable field and experimental evidence that dromedary camels serve as an important reservoir host involved in transmission to humans [ ] [ ] [ ] [ ] [ ] [ ] , but whether other livestock such as goats, sheep, and horses play a role in transmission has only been assessed indirectly. the virus is phylogenetically similar to betacoronaviruses previously detected in bats and there has been speculation that this disease originated through a cross-species transmission from bats to camels or humans [ ] [ ] [ ] . serologic testing of sheep, goats, and cattle from jordan [ ] and saudi arabia [ ] failed to identify animals with neutralizing antibodies to mers-cov. similarly, horses tested in the united arab emirates lacked antibodies to mers-cov [ ] . direct contact with dromedaries in saudi arabia was found to be independently associated with mers-cov illness, while contact with goats, sheep, or horses was not associated with human illness [ ] . in vitro studies in which replication of mers-cov in cultured cells was assessed have yielded mixed results with respect to species susceptibility. cells from goats, but not sheep or cattle, supported replication of mers-cov [ ] , and primary equine kidney cells supported virus replication, albeit at lower levels than observed with vero cells [ ] . transfection of the dpp receptor from goats, sheep, and horses into non-permissive mouse or hamster cells allowed replication of mers-cov [ , ] . collectively, these in vitro studies suggest the possibility that some livestock are susceptible to infection, but demonstration of infection in live animals is required to better assess their potential as reservoir hosts. the objective of this study was to determine if goats, sheep, and horses can be infected with mers-cov and assess their potential importance in viral transmission. goats (n = ) were evaluated for viral shedding, organ burden, and seroconversion and transmission to co-housed goats (n = ). limited viral shedding was observed without demonstration of viral transmission. due to the lack of transmission, only viral shedding and serology were evaluated in horses (n = ) and sheep (n = ). these animals did not become productively infected or seroconvert, indicating that such livestock are unlikely to serve as reservoirs for mers-cov and are unimportant in viral transmission. these studies were approved by the animal care and use committee of colorado state university (approval number - a) and were conducted in an association for the assessment and accreditation of laboratory animal care, international (aaalac) approved facility. two goats, three sheep, and four horses were purchased in colorado, usa. both of the goats were bred on site and gave birth to either two (doe ) or three kids (doe ). all animals were fed a complete pelleted feed supplemented with hay, and were observed at least once daily for nasal discharge, demeanor, food consumption, and clinical status. sheep, goat kids and horses were each inoculated intranasally with . × to . × plaque-forming units (pfu) of a low passage human isolate of mers-cov (strain hcov-emc/ ) propagated in vero e cells as described previously [ ] . the goat kids were maintained at all times in a room with their mothers, who served as in-contact controls to test for virus transmission. rectal temperature and nasal swabs were taken daily for seven days. one goat kid from each doe was euthanized days post-inoculation (dpi) and the remaining kids and mother goats were euthanized on day post-inoculation. the horses and sheep were monitored for viral shedding and seroconversion, and were euthanized on day post-inoculation, with the exception of horse , which was euthanized on day due to an injury. samples of nasal secretions were collected by inserting and rotating a swab into each nare and were immediately placed in viral transport medium and frozen until plaque assay was performed. plaques originating from all animals having low titers of virus were confirmed to be mers-cov by immunofluorescence using a rabbit polyclonal antiserum against hcov-emc- antigen as a primary antibody. nasal turbinates, trachea, larynx, and lung samples were collected from two kids (goat c and a) on day post-infection and frozen for virus titration or fixed in % neutral-buffered formalin for greater than days prior to being embedded in paraffin. tissue sections (hematoxylin/eosin and immunohistochemistry) were prepared and evaluated by a veterinary pathologist as previously described [ ] . in order to detect mers-cov antigen immunohistochemical analysis was performed with a previously described rabbit polyclonal antiserum against hcov-emc- antigen [ , ] . serum was collected immediately prior to inoculation and weekly thereafter until necropsy. neutralizing antibodies in sera were assayed using a plaque reduction neutralization test (prnt) with a % neutralization cutoff as described previously [ ] . goats were assessed for clinical disease, viral shedding, seroconversion, and viral transmission to their mothers. fevers were not detected in any of the goats, and no nasal discharge was observed. low levels of infectious virus were detected in two of the inoculated goat kids from doe (figure ), but not from either of the adult goats that had intimate contact or the kids from doe . serum was collected immediately prior to inoculation and weekly thereafter until necropsy. neutralizing antibodies in sera were assayed using a plaque reduction neutralization test (prnt) with a % neutralization cutoff as described previously [ ] . goats were assessed for clinical disease, viral shedding, seroconversion, and viral transmission to their mothers. fevers were not detected in any of the goats, and no nasal discharge was observed. low levels of infectious virus were detected in two of the inoculated goat kids from doe (figure ), but not from either of the adult goats that had intimate contact or the kids from doe . in order to study acute pathology and determine organ burden, two goats were euthanized on day -post infection and nasal turbinates, trachea, and lung were collected. very small but confirmed quantities of virus were isolated from the turbinates of both goats euthanized days post-infection (dpi) (figure ), which may reflect input virus or very low level virus replication. goat kid c was histologically unremarkable, however, the turbinates of goat kid a had multifocal areas of loss of goblet cells, epithelial necrosis or squamous metaplasia and attenuation and/or erosion of the epithelium, accompanied by mild to moderate neutrophil and monocyte/macrophage infiltration and occasional minimal hemorrhage. small amounts of cellular debris, leukocytes and mucus (exudate) were present in the nasal cavity, mainly associated with the aforementioned affected areas. these tissues were negative for viral antigen by immunohistochemistry (ihc) and the histopathologic lesions were very likely the result of trauma from daily swabbing rather than due to virus replication. in order to study acute pathology and determine organ burden, two goats were euthanized on day -post infection and nasal turbinates, trachea, and lung were collected. very small but confirmed quantities of virus were isolated from the turbinates of both goats euthanized days post-infection (dpi) (figure ) , which may reflect input virus or very low level virus replication. goat kid c was histologically unremarkable, however, the turbinates of goat kid a had multifocal areas of loss of goblet cells, epithelial necrosis or squamous metaplasia and attenuation and/or erosion of the epithelium, accompanied by mild to moderate neutrophil and monocyte/macrophage infiltration and occasional minimal hemorrhage. small amounts of cellular debris, leukocytes and mucus (exudate) were present in the nasal cavity, mainly associated with the aforementioned affected areas. these tissues were negative for viral antigen by immunohistochemistry (ihc) and the histopathologic lesions were very likely the result of trauma from daily swabbing rather than due to virus replication. the remaining goats were euthanized on day post-infection and the serological status of the experimentally infected kids and their cohoused dams were assessed. each of three kid goats held past day seroconverted, however, neutralizing antibodies were not detected in either of the dams (table ) . table . neutralizing antibody titers in goats experimentally infected or exposed by contact to mers-cov. mother goats are indicated as or , and their corresponded kids are indicated as a, b, c (doe ), a, or b (doe ). titers represent dilutions of serum which neutralized ≥ % of input virus. nd = not done. three sheep were experimentally infected and evaluated for clinical disease, viral shedding, and seroconversion. no nasal discharge or clinical disease was observed and all three sheep maintained consistent food intake and activity levels. a small quantity of virus was detected in nasal swabs from sheep on days , , , , and and from sheep on day ( figure ). the remaining goats were euthanized on day post-infection and the serological status of the experimentally infected kids and their cohoused dams were assessed. each of three kid goats held past day seroconverted, however, neutralizing antibodies were not detected in either of the dams (table ) . table . neutralizing antibody titers in goats experimentally infected or exposed by contact to mers-cov. mother goats are indicated as or , and their corresponded kids are indicated as a, b, c (doe ), a, or b (doe ). titers represent dilutions of serum which neutralized ≥ % of input virus. nd = not done. three sheep were experimentally infected and evaluated for clinical disease, viral shedding, and seroconversion. no nasal discharge or clinical disease was observed and all three sheep maintained consistent food intake and activity levels. a small quantity of virus was detected in nasal swabs from sheep on days , , , , and and from sheep on day ( figure ) . unlike the goats, acute pathology in sheep was not evaluated in this study. the sheep were euthanized on day post-infection, and serum samples from each week were assessed for the presence of mers-cov neutralizing antibodies. sheep developed a low titer of neutralizing antibody on day ( ), but neutralizing antibodies were not detected in either of the other two sheep at any time-point tested ( table ). unlike the goats, acute pathology in sheep was not evaluated in this study. the sheep were euthanized on day post-infection, and serum samples from each week were assessed for the presence of mers-cov neutralizing antibodies. sheep developed a low titer of neutralizing antibody on day ( ), but neutralizing antibodies were not detected in either of the other two sheep at any time-point tested ( table ) . table . neutralizing antibody titers in sheep experimentally infected with mers-cov. titers were determined by plaque reduction neutralization test (prnt) using a % cutoff. < despite no detectable rise in rectal temperature or change in appetite and activity, horses and showed mild intermittent nasal discharge prior to inoculation and throughout the days experiment. low levels of virus were detected in nasal swab samples from three of the four inoculated horses. virus was detected on day in horse , day in horse , and day in horse ( figure ) . virus was not detected in any of the nasal swab specimens tested from horse . virus isolation was performed by plaque assay from nasal swab specimens obtained from sheep experimentally infected with mers-cov. the limit of detection for this assay was . log pfu/ml, indicated by the dashed line. table . neutralizing antibody titers in sheep experimentally infected with mers-cov. titers were determined by plaque reduction neutralization test (prnt) using a % cutoff. despite no detectable rise in rectal temperature or change in appetite and activity, horses and showed mild intermittent nasal discharge prior to inoculation and throughout the days experiment. low levels of virus were detected in nasal swab samples from three of the four inoculated horses. virus was detected on day in horse , day in horse , and day in horse ( figure ). virus was not detected in any of the nasal swab specimens tested from horse . serum was collected weekly until day (with the exception of horse , which was euthanized early due to an injury unrelated to the experiment), and evaluated for the presence of mers-cov neutralizing antibodies. unlike the inoculated goat kids, none of the infected horses seroconverted (table ). serum was collected weekly until day (with the exception of horse , which was euthanized early due to an injury unrelated to the experiment), and evaluated for the presence of mers-cov neutralizing antibodies. unlike the inoculated goat kids, none of the infected horses seroconverted (table ) . the objective of this study was to determine if goats, sheep, or horses could be experimentally infected with mers-cov. very limited amounts of infectious virus were detected in nasal swab specimens of some of the experimentally infected animals, but not in uninfected, co-housed goats. it is possible that the infectious virus detected was residual from the input virus, at least on day post-infection. however, a previous study with alpacas re-challenged days after an initial infection was not able to detect infectious virus upon re-challenge indicating that input challenge virus is not detected one day after infection, although the role of secretory antibodies was not addressed in that study [ ] . similarly, another study with alpacas re-challenged on day post-infection was unable to detect viral rna upon re-challenge [ ] . in comparison to experimentally infected dromedaries, significantly less virus was isolated from the livestock in this study. since the main objective of this study was to determine the role these animals might play in transmission of the virus, we chose to test these samples by plaque assay in order to determine the amount of infectious virus present, rather than by rt-pcr which only reveals the presence of viral rna regardless of infectivity. previous studies of naturally or experimentally infected camels and experimentally infected alpacas showed variable levels of nasal discharge. studies in camels demonstrated that infected camels have nasal discharge while infected alpacas did not have any observable discharge [ , , , ] . the goats and sheep in this study did not have any observable discharge; in contrast horses and had discharge throughout the entire study. all animals were examined and healthy prior to the study, but due to the dust associated with the housing of horses we believe this discharge was unrelated to infection and instead an effect of the environment. current evidence suggests that dromedary camels are the primary reservoir of mers-cov. however, elucidating the role that other livestock such as goats, sheep, or horses could play in transmission is important for designing field studies and biosecurity strategies, and in assessing individuals at risk for viral transmission. while in vitro studies suggested that these animals could be naturally or experimental infected, the lack of support from field data coupled with the experimental data presented here suggest that these animals are unlikely to be infected and are not important in viral transmission of mers-cov. the authors declare no conflict of interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-to-human transmission of mers coronavirus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia serologic assessment of possibility for mers-cov infection in equids risk factors for primary middle east respiratory syndrome coronavirus illness in humans replicative capacity of mers coronavirus in livestock cell lines host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas infection with mers-cov causes lethal pneumonia in the common marmoset experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus key: cord- -ikm famj authors: lan, bowen; lu, puxuan; zeng, yujing; li, xin; ou, xiaxing; li, jingjing; li, hongjun title: clinical imaging research of the first middle east respiratory syndrome in china date: - - journal: radiol infect dis doi: . /j.jrid. . . sha: doc_id: cord_uid: ikm famj middle east respiratory syndrome is a viral respiratory illness caused by a novel human beta-coronavirus. based on the first case of middle east respiratory syndrome found in china, a clinical research in combination with radiological findings was studied. fever was the main clinical manifestation of this patient, and the primary imaging findings were basically the same as viral pneumonia. differential imaging diagnosis on the basis of epidemiological and experimental pathogen detection is helpful for clinical diagnosis of mers, even in distinguishing from sars and pneumonia caused by h n avian influenza. middle east respiratory syndrome (mers), also known as camel flu, is a viral respiratory illness caused by a novel human beta-coronavirus (cov) [ e ] . since it was firstly reported by saudi arabia in september , till june , more than patients have been detected worldwide, with at least cases of patients died [ , ] . until june , most of the cases of mers-cov infectors occurred in the middle east, and recently, it is reported that south korea has been sufferring from mers. symptoms may range from mild to severe, including fever, cough, diarrhea, and shortness of breath. although the exact route of transmission is still unclear, the respiratory droplet route is currently most likely [ ] . although a few cases were reported in other countries, one was found in china in may . a clinical research in combination with radiological findings was studied. a male korean patient, born in , had a close contact with his father who was diagnosed as the middle east respiratory syndrome. he began to appear back pain on may , , without fever, cough and sputum. no special treatment was given. he had a fever on may , , with body temperature up to . c, no chills, no cough, sputum, no shortness of breath, no abdominal pain, no diarrhea, and no sore throat. cold medications were ineffective. on june of , he arrived in hong kong from south korea by flight at : , and then arrived in huizhou city from shenzhen. at : pm, may of , isolation and treatment was given for him in huizhou designated hospital. during his whole journey in china, there were about close contactors, who were all confirmed to be healthy after the isolated ward. body temperature was . c, blood pressure was / mm hg, heart rate was beats/min, and breathing was times/min. breath sounds were ruder for the two lungs without coarse rales, heart rate was regular, abdomen was soft, and bowel sounded normal. the blood gas analysis, biochemistry, and blood routine examination after hospitalization were shown in table and table . pathogenic examination (sputum virological detection) showed that mers-cov was positive on june of , and the result changed to negative the second day. mobile dr was implemented by employing the chest semirecumbent on may of after hospitalization, followed by may th, june st, june rd, june th, june th and june th. obviously, pneumonia of the lower lungs was the primary finding ( fig. ). patients was in fever after may , hospitalized, lasting about a week between . c and . c (fig. ) . then with tamiflu and ribavirin antiviral therapy, broadspectrum antibiotic anti-infective therapy, oxygen therapy and improve immune function with gamma globulin, the virus was negative on june . low white blood cell count had been gradually increased to normal in two days after the virus was negative (fig. ). according to 'cases of diagnosis and treatment of middle east respiratory syndrome ( edition)' by the national health development planning commission, he was discharged on june , . it is well-known that incubation period of mers is e days. clinically, acute respiratory infection is the primary performance of mers, accompanying with high fever (even reach to e c), and sometimes with chills, shiver, cough, chest pain, headache, muscle and joint aches, fatigue, loss of appetite and so on. on the basis of pneumonia, the mers rapidly developed into respiratory failure, acute respiratory distress syndrome (ards) or acute renal failure. diarrhea and other atypical clinical manifestations might occur to individual cases (such as immunodeficiency cases). in this study, the patient suffered from fever (up to . c) and back pain firstly. after hospitalization for one week, his body temperature returned to normal (fig. ) , but still being in cough with a small amount of yellow phlegm and a little bloodshot; and there is no shortness of breath at rest and oxygen therapy. on the sixth day after his hospitalization, mers-cov was negative via the virological detection of sputum, and his body temperature had decreased to be normal, which indicated that the virus has a direct relationship with the fever. laboratory tests found that the leukocytes count in the peripheral blood had decreased obviously since his hospitalization, and the count was about . Â /l lasting for nine days, followed by increasing and till the th days it recovered to the normal level. this indicated that mers-cov mainly attacked human immune system, resulting in a significant reduce of leukocytes count; after the virus was cleared, the recovery may be a relatively slow process due to the leukocytes count recovered to the normal range for about two weeks after the virus return to be negative. the camel and bat are always thought to be the main infection source, but the animal to human infection process was not so clear till now. generally, human to human infection should be paid more attention, for mers-cov would spread table the blood gas analysis after hospitalization. results items results on the th day after hospitalization, the chest radiograph revealed the little patchy increased density shadow in the two lower lungs near the edge of heart. b. on the th day after hospitalization, the chest radiograph revealed two obviously increased patchy shadows in the two lower lungs, the degree of lower right lung was more serious. c. on the th day, the patchy lesions progressed to be large patchy consolidation shadows. d. on the nd day, chest radiograph showed two obvious absorptions in the lower lung lesions, only small pieces of the grid shadow could be observed. by direct contact of patients' secretions, or by aerosol and droplets. as is reported, there is an evidence of limited personto-person transmission of mers [ , ] . in this study, people who had more or less contact with the patient were without any further infection. this limited transmissibility is consistent with the data available to date [ ] . radiological manifestations of mers are lungs consolidation and ground glass, due to the fact that the mers-cov primary lead to viral pneumonia [ ] . imaging findings in this case were characteristic by the following three stages. ) small pieces of high density shadows in the two lower lungs near the heart edge were observed during the early period via chest x-ray examination, suggesting that it firstly progressed to pneumonia (about one week). ) subsequently, the lesions gradually expanded. further chest x-ray examination showed large pieces of high density shadows in the middle right lower lung, oval pieces of high density shadows in the left lower lung field, and clear boundaries of the two upper lungs. ) after the active antiretroviral therapy, the virus turned to be negative, the body temperature decreased to normal level (fig. ) , and leukocyte count also began to rise (fig. ) . chest x-ray of deferred examination showed the gradually foci absorbed in both lungs, being consistence with clinical changes. differential diagnosis of pneumonia imaging result from mers, sars or h n avian influenza is necessary. due to the fact that both sars and mers belong to the coronavirus family, their nucleotide homologies for one same pcr fragment are %e %, their radiographic manifestations have in common. both showed ground glass shadows and pulmonary opacities in the middle and lower lung lobes, accompanying with a rapid disease progression. via high-resolution ct scans, septal thickening and bronchiectasis could be both observed. however, ground glass opacities and consolidation of sars were relatively mild, with the lesions of a migratory change [ e ]. as for mers and h n avian influenza, their common characteristics were ground glass shadows and pulmonary opacities in both lower lung lobes, except for that disease progression of h n avian influenza infection might be more rapid [ , ] . summarily, differential imaging diagnosis on the basis of epidemiological and experimental pathogen detection is helpful for clinical diagnosis of mers, even in distinguishing from sars or pneumonia caused by h n avian influenza. epidemiological findings from a retrospective investigation what can we learn from mers outbreak in south korea? isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mersdcov): an nouncement of the coronavirus study group middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings contact investigation of a case of human novel coronavirus infection treated in a german hospital chest x-ray imaging of patients with sars health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections characteristics of imaging manifestations and dynamic changes in patients with severe pneumonia caused by h n avian influenza virus imaging and pathological analysis of severe pneumonia caused by human infections with avian influenza a (h n ) relationship of peripheral blood leukocyte counts and viral negative key: cord- - a oin authors: maltezou, helena c.; tsiodras, sotirios title: middle east respiratory syndrome coronavirus: implications for health care facilities date: - - journal: american journal of infection control doi: . /j.ajic. . . sha: doc_id: cord_uid: a oin background middle east respiratory syndrome coronavirus (mers-cov) is a novel coronavirus that causes a severe respiratory disease with high case fatality rate. starting in march , a dramatic increase of cases has occurred in the arabian peninsula, many of which were acquired in health care settings. as of may , , laboratory-confirmed cases and deaths have been reported globally. methods review of publicly available data about mers-cov health care–associated transmission. results we identified events of possible or confirmed health care–associated transmission with high morbidity and mortality, mainly among patients with comorbidities. health care workers are also frequently affected; however, they tend to have milder symptoms and better prognosis. gaps in infection control were noted in all events. currently, health care–associated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic in countries in the arabian peninsula. conclusion there is a need to increase infection control capacity in affected areas and areas at increased risk of being affected to prevent transmission in health care settings. vaccines and antiviral agents are urgently needed. overall, our knowledge about the epidemiologic characteristics of mers-cov that impact health care transmission is very limited. as the mers-cov epidemic continues to evolve, issues concerning best infection control measures will arise, and studies to better define their effectiveness in real life are needed. middle east respiratory syndrome coronavirus (mers-cov) is a novel betacoronavirus of the coronaviridae family that causes a severe respiratory disease with a high case fatality rate. [ ] [ ] [ ] [ ] the virus was isolated for the first time in september from a -year-old patient with fatal pneumonia in saudi arabia. however, the earliest identified human cases were traced back to march , to a cluster of severe respiratory infections in a hospital in jordan. up until now, all mers-cov infected cases are directly or indirectly linked to the middle east; therefore, the name mers-cov was established. over the first years after the emergence of mers-cov, the world health organization (who) has been notified of laboratory-confirmed cases, of which were fatal. however, starting in mid-to late march , a dramatic increase of cases has been recorded, many which were acquired in health care settings and concerned health care workers (hcws). as of may , , laboratory-confirmed cases and deaths have been reported to the who globally. as a result, concerns have been expressed about the possibility of a virus genetic change conferring increased transmissibility, and the novel virus received media attention globally. in the context of uncertainties about its epidemiology, the high case fatality rate, the urgent need for a specific antiviral treatment, and the unavailability of a vaccine, mers-cov has been a major public health concern of global dimensions. given the current local epidemiologic trends of mers-cov and the large numbers of travelers that fly out of the arabian peninsula, it is almost certain that an increasing number of cases will be exported to other countries; these cases, especially when that patient is seriously ill, will require medical attention and hospitalization. herein, we review publicly available data about mers-cov focusing on health careeassociated transmission. aspects relevant to infection control are also discussed. we searched pubmed from september through june , , using the terms middle east respiratory syndrome, mers, and novel coronavirus. the abstracts of articles identified through the first pubmed search were screened, and articles presenting original data on health careeassociated infections and outbreaks were included. the reference lists of these articles were also reviewed as were any relevant review articles. in addition, we searched the web sites of the who, united states centers for disease control and prevention (cdc), and european centre for disease prevention and control (ecdc). in total, we reviewed articles on mers-cov and identified articles presenting original data about possible or confirmed health careeassociated transmission events. details about the health careeassociated transmission ranged widely among these articles. to the best of our efforts, we avoided presenting duplicated data. in addition, we selected original and review articles. all articles were studied by both authors independently. mers-cov infection so far has been described in countries in the middle east (saudi arabia, united arab emirates, qatar, jordan, oman, kuwait, egypt, yemen, lebanon, iran), countries in europe (united kingdom, germany, france, italy, greece, the netherlands), country in africa (tunisia, algeria), countries in asia (malaysia, the philippines), and county in the americas (united states). molecular analyses of mers-cov or similar viruses from bats and camels suggest that these species are the natural reservoirs of the virus. , whole genome sequencing showed that human and camel viruses from saudi arabia are indistinguishable. multiple transmission routes are suspected; however, their exact contribution has not been elucidated so far. a phylogenetic study of mers-cov genomes from saudi arabia suggested that both humanto-human transmission and sporadic zoonotic events occur. the stability of the virus for prolonged periods in camel milk suggests the potential of excretion of the virus into camel milk and spread through consuming raw milk. the upsurge of cases since mid-march in the arabian peninsula (mainly in saudi arabia) is possibly attributed to an increase in the number of primary cases and hospital-acquired cases, some as a part of mainly small ( - cases), but in a few instances large, outbreaks. a change in the transmissibility pattern of the virus and increased efficacy for sustained transmission could facilitate inhospital transmission; however, epidemiologic and molecular data to this effect do not exist. family clusters of mers-cov have been recorded. the secondary attack rate in families was . % in saudi arabia in . among imported travel-associated cases, very few instances of person-to-person transmission have been verified. [ ] [ ] [ ] recent phylogenetic analysis using human sequences from jeddah suggests that the virus has not changed from previous strains. overall, it seems unlikely that the virus has increased its transmissibility or patterns of transmission. the basic reproductive number has been estimated to be < using real-time data until june and august , respectively, , even though the upper range of estimates exceeded in a scenario where infection control was not implemented. these findings indicate no pandemic potential for mers-cov so far. recently, a committee appointed by the who concluded that the conditions for a public health emergency of international concern have not yet been met. moreover, increased testing rates of less ill or asymptomatic cases may have contributed to the upsurge of detected cases. regarding characteristics of affected patients, most are men (male-to-female ratio: : ), with a median age of years (range, months- years). the spectrum of mers-cov infections ranges from asymptomatic infection to very severe pneumonia with acute respiratory distress syndrome, septic shock, and multiorgan failure resulting in death. in an analysis of confirmed and possible cases, symptomatic patients typically had fever and cough, chills, sore throat, myalgia, and arthralgia, whereas vomiting and diarrhea were present in at least one third of patients. in the same study, . % of patients developed severe respiratory disease. it appears that severe disease predominantly occurs in patients with comorbidities; % of the patients in this report had at least . the overall case fatality with the latest who figures is %. from the very first events of the mers-cov epidemic, the virus showed its health careeassociated dynamic. apart of sporadic community cases and family clusters, health careeassociated transmission has been reported on several occasions during the last years, indicating human-to-human, although inconsistent, transmission (table ) . , , , , [ ] [ ] [ ] [ ] [ ] [ ] gaps in infection control were the common denominator in the events of health care associatede transmission. , , , , , during the largest so farepublished outbreak of mers-cov that occurred in al-hasa, saudi arabia, in , health care facilities were affected through transfer of patients but also possibly because of repeated introductions of cases from the community. the outbreak extended for almost months and involved cases, including hcws. most cases were confined in the hemodialysis unit with rapid transmission and high attack rates. this outbreak gave the opportunity to elucidate several epidemiologic parameters of secondary mers-cov infection, such as the incubation period ( . days; % confidence interval, . - . days), serial interval ( . days; % confidence interval, . - . days), and heterogeneity in transmission, with many infected patients not transmitting the infection at all and infected patient transmitting the infection to others. moreover, this outbreak raised the possibility of transmission through direct or indirect contact and between rooms in the same ward. a recent study showed that mers-cov remained viable for up to hours under specific environmental conditions, which mimic the hospital environment ( c with % relative humidity), whereas its stability was not reduced during aerosolization. these data show that mers-cov has the potential to spread through contact or fomites caused by prolonged survival. a model-based study found that the virus structural characteristics render it very likely to remain viable in the environment for a long period and support fecal-oral transmission. vomiting and diarrhea are common in patients with mers-cov , , and may contribute to transmission. the mers-cov case imported in france shared his bathroom with the secondary hospital-acquired case, which raises the possibility of spread through stools. mers-cov is predominantly shed through respiratory secretions during cough. mers-cov has been detected through polymerase chain reaction for up to days in respiratory specimens and stools and up to days in urine. , , our knowledge about virus shedding and viral load kinetics throughout the clinical course of ill patients is scarce and therefore can provide limited guidance about the duration of implementation of infection control measures. the possibility of prolonged shedding under an immunocompromised status should also be investigated and considered for infection control purposes. health careeassociated mers-cov infections and outbreaks have been associated with high morbidity, high rates and prolonged use of mechanical ventilation, and fatality rates up to %. , , given the fact that health care services are often used by older people with comorbidities and in association with the severe course in the description of demographics of secondary mers-cov cases, a drop of the median age from to years old compared with primary cases has been reported. this depends on the conditions of each outbreak and may be affected by the preponderance of affected hcws in each instance. for example, in the most recent who report, the hcws who tested positive for mers-cov in the jeddah outbreaks were more likely to be younger, women, and to exhibit mild or no symptoms compared with primary cases. however, % of hcws developed a severe disease, which resulted in admission to an intensive care unit or death. unsuspected cases are the main source for the introduction of mers-cov virus from the community or another health care facility. although such patients may present with compatible symptoms, the diagnosis may not be considered early or symptoms may be mild. , , in the hospital outbreak that occurred in saudi arabia in , patients exhibited no fever during initial presentation. hcws may acquire mers-cov infection either in the community or through occupational exposure. , , nurses are mostly affected, which is attributed to their prolonged, repeated, and closer physical contact with patients. hcws may continue working despite being symptomatic. an asymptomatic or mildly symptomatic course has been described in hcws, , , which raises the possibility of transmission of the infection to their vulnerable patients during an asymptomatic phase or early incubation. patient-to-patient transmission has been noted as well. , currently, health careeassociated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic. in the recent mission report by the who authorities evaluating data on laboratory-confirmed cases in hospitals in jeddah, saudi arabia, with onset of symptoms between february and april , , one-third of the cases were considered to be primary cases (some of the investigations are still ongoing), whereas > % of the cases (including hcws) were classified as hospital acquired. in the rest of saudi arabia, out of ( . %) recent cases were identified in hcws. overall, of the ( . %) cases reported from saudi arabia from march to may , , were hcws. in mecca, another large outbreak in a hospital was described with laboratory-confirmed cases, including hcws. both outbreaks were larger than the originally described outbreak in saudi arabia. in the united arab emirates, hcws accounted for more than twothirds of cases reported during the same period. although the who points to infection control gaps for the recent propagation of mers-cov within health care facilities in saudi arabia and the united arab emirates, we do not know if this concerns the use of personal protective equipment, hand hygiene, procedures, environmental cleaning, or triage. given that no vaccines or specific antiviral prophylaxis against mers-cov are available, , the prevention and control of transmission of mers-cov within health care facilities relies solely on early detection, isolation, and strict implementation of infection control measures. rapid and accurate diagnosis is crucial to trigger contact tracing in the hospital and the community and should be ordered as soon as possible in the context of a relevant epidemiologic profile but also in the event of a health careeassociated cluster of severe respiratory illness cases. patients with confirmed or suspected mers-cov infection should be cared under contact and droplet precautions until testing results. in accordance with who guidelines, a high protection mask (eg, n respirator) along with eye goggles, gowns, and gloves should be used during aerosol-generating procedures; the latter should be performed in an adequately ventilated room (minimum of - air changes per hour) (airborne infection isolation room). for consistency with the recommendations during the h n pandemic, the united states cdc recommends the use of n respirators in all contacts with a laboratory-confirmed or suspected mers-cov infected case. the rationale for this recommendation relies on the gaps of knowledge about the potential for airborne transmission of the novel coronavirus. however, n respirators are less tolerated by hcws and are more expensive. the united states cdc also recommends that patients with confirmed or suspected mers-cov infection are placed in an airborne infection isolation room. hcws with mers-cov infection should be strictly excluded from patient care, even with mild symptoms. the role of asymptomatic hcws is under question. overall, there is a need to increase infection control capacity in affected areas and areas at increased risk of being affected to prevent transmission in health care settings. our knowledge about the epidemiologic characteristics of mers-cov that impact health care transmission is very limited. to interrupt in-hospital transmission, routes of efficient exposure and virus shedding should be well studied. the contribution of primary cases to the so-called hospital-acquired cases in the recent upsurge of detected cases in the arabian peninsula is still unclear, and further epidemiologic data and analyses are necessary. in analysis, of ( . %) cases with evidence of secondary transmission acquired the infection in the hospital environment; nevertheless, of them had additionally reported exposure to animals, therefore not eliminating an alternative source of infection. the stability of the proportion of asymptomatic versus symptomatic cases is an argument against increasing testing as a possible explanation for either primary or secondary cases. on the other hand, a reverse scenario could be that additional cases are missed because cases at the early incubation period or with low viral loads may be missed with molecular testing. a transmission event under similar circumstances has been described in the community for the first imported mers-cov case in the united states that tested negative by molecular assays but subsequently tested positive by serology. , research for the future active surveillance and testing are of outmost importance to provide answers about the epidemiology of mers-cov and evolution of the current epidemic. case-control, serologic studies in exposed hcws are needed to better define the effectiveness of infection control measures. transmission of the virus via asymptomatic shedding in feces or other routes (eg, fomites, environment) is another topic for investigation. studies of viral kinetics in affected patients with molecular analyses of samples from various body sites will provide answers for infection control as well. a vaccine against mers-cov should be developed along with specific antiviral agents. there is no doubt that mers-cov remains a serious threat and has exhibited a significant public health impact in the affected countries. currently, health careeassociated transmission plays a pivotal role in the evolution of the mers-cov epidemic in countries in the arabian peninsula. a significant cost has been encountered in terms of personnel and time required for contact tracing and means of implementing infection control and prevention measures in health care settings. so far, there is no evidence of sustained humanto-human transmission. however, significant concerns exist in terms of the increased number of health careeassociated cases, gaps in knowledge regarding transmission routes, and limited infection control capacity in affected countries. as the mers-cov epidemic continues to evolve, vaccine and specific antiviral agents against mers-cov are urgently needed. studies about the effectiveness of infection control measures will provide answers and eventually promote safety in health care facilities both for patients and hcws. epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group 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transmission a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case middle east respiratory syndrome coronavirus infections in health care workers first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description a case of imported middle east respiratory syndrome coronavirus infection and public health response stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions prediction of intrinsic disorder in mers-cov/ hcov-emc supports a high oral-fecal transmission severe respiratory illness caused by a novel coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study contact investigation of a case of human novel coronavirus infection treated in a german hospital current advancements and potential strategies in the development of mers-cov vaccines broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) preventing the spread of influenza a h n to health-care workers illinois resident who had contact with indiana mers patient tests positive for mers coronavirus key: cord- -amv los authors: widagdo, w.; sooksawasdi na ayudhya, syriam; hundie, gadissa b.; haagmans, bart l. title: host determinants of mers-cov transmission and pathogenesis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: amv los middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. in dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. differences in the behavior of the virus observed between individuals, as well as between humans and dromedary camels, highlight the role of host factors in mers-cov pathogenesis and transmission. one of these host factors, the mers-cov receptor dipeptidyl peptidase- (dpp ), may be a critical determinant because it is variably expressed in mers-cov-susceptible species as well as in humans. this could partially explain inter- and intraspecies differences in the tropism, pathogenesis, and transmissibility of mers-cov. in this review, we explore the role of dpp and other host factors in mers-cov transmission and pathogenesis—such as sialic acids, host proteases, and interferons. further characterization of these host determinants may potentially offer novel insights to develop intervention strategies to tackle ongoing outbreaks. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that was isolated in late [ ] . since then, the virus has caused multiple outbreaks and infected more than individuals, [ ] who then develop a respiratory infection ranging in severity from asymptomatic to fatal [ , ] . severe-to-fatal mers-cov patients have a higher chance of transmitting this virus since they shed a higher amount of virus progeny in comparison to the asymptomatic-to-mild ones [ ] [ ] [ ] [ ] . identifying and quarantining these patients in healthcare facilities where outbreaks have occurred, together with implementing proper infection control, has been effective in reducing transmission and containing these outbreaks [ , ] . however, new mers-cov cases are still being reported, especially in the arabian peninsula [ , ] . this is partly due to the continuous zoonotic introduction of this virus to the human population in this region by dromedaries [ ] . the dromedary camel is the only animal species that has been reported to transmit this virus to humans [ ] [ ] [ ] [ ] . mers-cov infection in these animals merely causes mild upper respiratory tract infection [ , ] , but seroepidemiological studies showed that this virus has been circulating in dromedary camels for decades, suggesting the efficient transmission of mers-cov in this species [ ] [ ] [ ] [ ] . although the clinical manifestations, as well as transmission, are remarkably different in mers-cov-infected humans and dromedary camels, the viruses isolated from these two species are highly similar, if not indistinguishable [ , ] . this indicates that host factors play a significant role in mers-cov pathogenesis and transmission. however, the identity of these host factors and how they affect the pathogenesis and transmission of mers-cov are generally not well understood. dipeptidyl peptidase- (dpp )-the mers-cov receptor, sialic acids, proteases, and interferons are ; a cartoon representation of mers-cov s protein binding to dpp (pdb code l ) (b). the s protein consists of the s and s subunits. the α/β hydrolase domain of dpp is indicated in red, β-propeller domain in green, while part of the mers-cov s protein is shown in blue. other mers-cov-interacting host factors besides dpp are less extensively studied and have mostly been investigated in vitro. glycotopes of α , -sialic acids coupled with -n-acetylated neuraminic acid are recognized by the s protein of mers-cov during attachment [ ] . in the absence of these glycotopes, mers-cov entry is reduced but not abolished, indicating their function as an attachment factor rather than a receptor [ ] . besides α , -sialic acids, ceacam and grp have also been suggested to be attachment factors for mers-cov, but their roles in vivo during mers-cov infection are not clear at this moment [ , ] . post attachment, mers-cov uses the c-terminal part of its s protein-known as s ( figure a )-to interact with host proteases, such as furin, tmprss , and cathepsins [ ] [ ] [ ] [ ] . these proteases cleave the s protein and induce conformational changes, allowing fusion between viral and host cellular membranes, resulting in the release of viral rna into the cell cytoplasm [ ] . tmprss and dpp are held in one complex at the cell surface by a scaffolding protein, the tetraspanin cd , leading to a rapid and efficient entry of mers-cov into the susceptible cells [ ] . once fusion with host cell membranes has occurred, mers-cov subsequently replicates its genetic material and produces viral proteins in the cell cytoplasm to generate new virus progeny. during this stage, mers-cov uses its nsp - polyproteins to build its replication organelles as well as its accessory proteins such as the a and b proteins to inhibit host anti-viral defense mechanisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the capacity of mers-cov accessory proteins to impede several pathways of host immune response in the lungs may be limited. mers-cov inoculation of macaques and genetically modified mice generally results in limited clinical manifestations; thus, adapting this virus through serial passaging or defecting the type i interferon pathway may be needed to enhance viral replication and pathogenesis in these animals [ , [ ] [ ] [ ] [ ] . these observations, together with studies showing type i interferon capacity to inhibit mers-cov infection in vitro [ , ] , highlight the importance of the innate immune response, especially type i interferon, as an inhibiting factor for mers-cov. so far mers-cov has been isolated from dromedary camels and humans [ , ] . both species are not only susceptible to mers-cov infection, but also capable of transmitting this virus [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] ]. however, current data indicate that virus spread is more efficient in dromedary camels than in humans [ , , [ ] [ ] [ ] ] . this difference in transmissibility could be partially due to the different tropism of mers-cov in these two species. in dromedaries, mers-cov has been shown to replicate in the nasal epithelium upon experimental in vivo infection [ ] , while in humans, mers-cov mainly replicates in the lower respiratory tract, particularly in the bronchiolar and alveolar epithelia [ , [ ] [ ] [ ] [ ] . higher viral rna levels in the sputum and lavage samples of mers-cov patients compared to nasal and throat swabs are consistent with the tropism of mers-cov in humans [ ] [ ] [ ] . this different mers-cov tropism in dromedary camels and humans is in line with the localization of dpp in the respiratory tract tissues of these two species. in humans, dpp is absent in the nasal epithelium but present in the lower respiratory tract epithelium, mainly in type ii pneumocytes [ , ] . in contrast, dpp is expressed in the nasal epithelium of dromedary camels [ ] . this difference in dpp localization between humans and dromedary camels therefore explains mers-cov tropism in these two species and highlights dpp as an essential determinant of mers-cov tropism. dpp localization has also been investigated in many other mers-cov-susceptible species. in gambian and egyptian fruit bats, dpp is expressed in the respiratory tract and intestinal epithelium, suggesting that mers-cov can target both tissues [ ] . in line with this finding, mers-cov inoculation via intranasal and intraperitoneal routes in the jamaican fruit bat led to viral rna shedding both in the respiratory tract and the intestinal tract [ ] . in contrast to frugivorous bats, dpp is limitedly expressed in the respiratory tract epithelium of two insectivorous bats, i.e., common pipistrelle and common serotine bats, but abundant in their intestinal epithelium [ ] . accordingly, sequences of mers-like-covs were mainly obtained from rectal swabs and fecal samples of insectivorous bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these findings not only support insectivorous bats as the origin host of mers-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but also indicate the importance of intestinal tropism and fecal-oral transmission of mers-like-cov in these insectivorous bats. besides bats, humans, and dromedary camels, other animal species have also been proposed as potential hosts of mers-cov. remarkably, dpp of horses, llamas, alpacas, pigs, bovines, goats, sheep, and rabbits has been demonstrated to recognize the s protein of mers-cov [ , ] . in most of these species, there is a preferential upper respiratory tract expression of dpp observed. rabbits express dpp in the upper and lower respiratory tract epithelium, and thus may allow mers-cov to replicate in both compartments [ , ] . horses, llamas, and pigs mainly express dpp in the upper respiratory tract-particularly the nasal epithelium [ ] . upon intranasal mers-cov inoculation, llamas, alpacas, and pigs developed upper respiratory tract infection, while horses did not seroconvert and only shed infectious virus in a limited amount [ ] [ ] [ ] [ ] [ ] . the reason why horses seem to be less permissive to mers-cov remains to be investigated, but a chronic co-infection in the guttural pouch, a common disease among horses, might be one of the explanations. this guttural pouch infection results in excessive mucus production that might hinder mers-cov from attaching and entering the nasal epithelium [ , , ] . sheep, on the other hand, did not seem to express significant levels of dpp in their respiratory tract, and thus did not seroconvert nor shed infectious virus upon experimental mers-cov inoculation [ , ] . comparable to sheep, goats limitedly shed infectious virus upon experimental infection and did not transmit this virus to other naïve goats upon direct contact [ ] . the results of experimental mers-cov infection in livestock animals are in line with data from epidemiological studies. mers-cov seropositive llamas and alpacas are present in the field, while horses, goats, and sheep are generally found to be seronegative [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . given the fact that experimental in vivo infection studies and dpp expression analysis in different animal species revealed that dromedary camels are not the only animals in which mers-cov has an upper respiratory tract tropism [ , , , ] , it is then relevant to question whether other animals can potentially spread mers-cov as well. new world camelids, i.e., alpacas and llamas, are able to transmit the virus to respective naïve animals upon contact [ ] . pigs and rabbits, on the other hand, hardly transmit the virus-neither by contact nor airborne routes [ , ] . most likely, this is caused by the fact that pigs and rabbits, unlike dromedary camels, shed low levels of infectious virus upon mers-cov inoculation ( figure ) . this difference indicates that other host factors besides dpp could cause interspecies variation in mers-cov infection. indeed, several glycotopes of α , -sialic acids that function as attachment factors of mers-cov are present in the nasal epithelium of dromedary camels but absent in that of rabbits and pigs ( figure ) [ , ] . the lack of these glycotopes in pigs and rabbits might limit the susceptibility and transmission of mers-cov in these animals. although the role of these glycotopes in mers-cov transmission still requires further investigation, it remains plausible that an efficient transmission of this virus might require the presence of both dpp and mers-cov-recognized glycotopes of α , -sialic acids (figure ). . it has also been reported that the lysosomal proteases from bat cells support coronavirus spike-mediated virus entry more efficiently than their counterparts from human cells [ ] . these observations suggest that host proteases from different host species may determine the species and tissue tropism of mers-cov. because mers-cov has been circulating in dromedary camels for decades before emerging in the human population [ ] [ ] [ ] [ ] , it is plausible that this virus inhibits the immune response of dromedary camels more efficiently than that of other species, including pigs and rabbits. the difference in immune response among mers-cov-susceptible species is therefore another factor that might yield interspecies variation in permissiveness to mers-cov. characterizing the difference in host proteases and immune responses among mers-cov-susceptible species, as performed for dpp and mers-cov-recognized α , -sialic acid glycotopes (figure ) , has not yet been investigated. these data, however, may further explain interspecies variation in mers-cov infection and transmission. mers-cov causes respiratory infection in humans ranging from asymptomatic to severe pneumonia [ , ] . however, it is currently unclear what causes this intraspecies variation. epidemiology data indicate that individuals with certain risk factors are at higher risk of developing severe mers-cov infection [ , ] . this implies that some host factors may dictate the outcome of mers-cov infection, thus rendering intraspecies variation. two of the risk factors, i.e., smoking and chronic obstructive pulmonary disease (copd), have been shown to upregulate dpp expression in the lungs [ , [ ] [ ] [ ] , suggesting dpp as a possible reason for intraspecies variation observed among mers-cov patients. in healthy human lungs, dpp is almost exclusively expressed in type ii pneumocytes [ , ] . type ii pneumocytes are small cuboidal cells that can regenerate alveolar epithelium upon injury, and roughly cover % of the alveolar surface area. meanwhile, around % of the surface area of the alveolus is occupied by type i pneumocytes that are morphologically flat and responsible for gas exchange [ , ] . in the lungs of smokers and copd patients, unlike in healthy human lungs, dpp is prominently expressed in both type i and ii pneumocytes, indicating upregulated expression on type i pneumocytes [ ] . autopsy reports from fatal mers-cov patients showed that both type i and ii pneumocytes expressed dpp and became infected by mers-cov, proposing a role of dpp -expressing type i pneumocytes in mers-cov pathogenesis [ , ] . damage to type i cells in the lung alveoli during viral infection may lead to diffuse alveolar damage [ ] . in line with observations made in human mers cases, common marmosets that express dpp in both type i and ii pneumocytes have been reported to produce more infectious virus upon experimental mers-cov infection, compared to rhesus and cynomolgus macaques that merely expressed dpp in type ii pneumocytes [ , [ ] [ ] [ ] [ ] . accordingly, these common marmosets developed moderate-to-severe infection, while macaques generally developed mild transient pneumonia [ , , [ ] [ ] [ ] [ ] . similarly, in genetically modified mice that displayed mers-cov tropism for type ii pneumocytes, only mild clinical manifestations were observed upon mers-cov infection [ , ] . adapting mers-cov through serial passaging or upregulating dpp expression throughout the airway epithelium in mice, however, will induce severe clinical disease [ , ] . these data altogether support the role of dpp -expressing type i pneumocytes in the pathogenesis of severe mers-cov infection. the differential expression of host factors that limits the infection should also be taken into account. dpp in soluble form has been demonstrated to protect against mers-cov infection in vitro and in a mouse model [ , ] ; however, its presence in the lungs and role in mers-cov pathogenesis remain to be investigated. the host immune response also has the capacity to inhibit mers-cov infection. mers-cov has been shown to replicate to higher levels in immunocompromised rhesus macaques [ ] , consistent with the observation that immunocompromised individuals have difficulties clearing mers-cov upon infection [ , , ] . the survivors of mers-cov infection have been shown to develop virus-specific cd + and cd + t cell responses, implying the role of t cells in virus clearance [ ] . however, the depletion of t cells in mice can either lead to failure in mers-cov clearance or improvement in clinical outcome, depending on the type of mouse model used [ , ] . therefore, the role of adaptive immune response in mers-cov pathogenesis is currently unclear. on the other hand, one of the main components of the host innate immune response, type i interferon, inhibits mers-cov replication in susceptible cells, partly by inhibiting double membrane vesicles (dmv) formation [ , , , , ] . the absence of type i interferon signaling in mice also resulted in more severe clinical manifestations and histopathological lesions upon mers-cov infection [ ] . advance age, which can cause delayed type i interferon response upon viral infection, is a well-known risk factor for fatal mers-cov infection [ , , [ ] [ ] [ ] . collectively, these data highlight the role of host innate immune response as a potent inhibitor for mers-cov infection. it is indubitable that severe mers-cov infection is not solely driven by the pathogen. additional underlying conditions increase mers-cov replication and induce severe-to-fatal clinical manifestations [ , , , , ] . it is plausible that more than one underlying condition is needed to yield a fatal outcome. dpp upregulation in type i pneumocytes and insufficient type i interferon response might be crucial determinants for severe mers-cov infection (figure ). further investigation of the host determinants of mers-cov pathogenesis may offer insights for developing novel therapeutic measures. although mers-cov has been reported to undergo some genotypic changes since it emerged in the human population [ , [ ] [ ] [ ] [ ] , this has not resulted in distinct phenotypic changes so far [ , ] . therefore, host factors remain the most significant determinant in explaining inter-and intraspecies variations observed in mers-cov pathogenesis and transmission. dpp and mers-cov-recognized α , -sialic acids might partially explain these variations, since their localization has been demonstrated to be variable between mers-cov-susceptible species [ , , , ] . dpp expression in human lungs has also been shown to vary due to certain comorbidities [ , , ] . nevertheless, it is undoubtable that the inter-and intraspecies variation in mers-cov pathogenesis and transmission is a complex phenomenon influenced by more than one host factor. current data suggest proteases and interferons as other critical host factors, but how they instigate inter-and intraspecies variations, as well as their role in mers-cov pathogenesis and transmission, still remain to be further elucidated. characterization of the host determinants of mers-cov pathogenesis and transmission could potentially offer insight into this virus epidemiology and guide novel therapeutic development. it may also help to identify the most vulnerable individuals to protect against mers-cov infection-for example, by using vaccination. author contributions: all authors contributed to the writing of the manuscript and carefully evaluated the manuscript before submission. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers-coronavirus: from discovery to intervention state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study transmission of mers-coronavirus in household contacts risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 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and mda in the innate antiviral response the endonucleolytic rna cleavage function of nsp of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum middle east respiratory syndrome coronavirus ns b protein inhibits host rnase l activation mers-cov accessory orfs play key role for infection and pathogenesis expression and cleavage of middle east respiratory syndrome coronavirus nsp - polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice a mouse model for mers coronavirus-induced acute respiratory distress syndrome rapid generation of a mouse model for middle east respiratory syndrome an animal model of mers produced by infection of rhesus macaques with mers 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coronavirus infection clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels dipeptidyl peptidase distribution in the human respiratory tract: implications for the middle east respiratory syndrome replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) human betacoronavirus c emc/ -related viruses in bats mers-related betacoronavirus in vespertilio superans bats group c betacoronavirus in bat guano fertilizer detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat close relative of human middle east respiratory syndrome coronavirus in bat further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase domestic pig unlikely reservoir for mers-cov infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding genome specialization and decay of the strangles pathogen, streptococcus equi, is driven by persistent infection strangles: a pathogenic legacy of the war horse middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis serologic assessment of possibility for mers-cov infection in equids coronavirus infections in horses in saudi arabia and oman cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels middle east respiratory syndrome coronavirus experimental transmission using a pig model receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea risk factors for primary middle east respiratory syndrome coronavirus illness in humans dpp , the middle east respiratory syndrome coronavirus receptor, is upregulated in lungs of smokers and chronic obstructive pulmonary disease patients identification of genes differentially expressed in rat alveolar type i cells renewal of alveolar epithelium in the rat following exposure to no histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset infection with mers-cov causes lethal pneumonia in the common marmoset middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase elevated human dipeptidyl peptidase expression reduces the susceptibility of hdpp transgenic mice to middle east respiratory syndrome coronavirus infection and disease pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques atypical presentations of mers-cov infection in immunocompromised hosts recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses cd + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus ifn production ability and healthy ageing: mixed model analysis of a year longitudinal study in japan age-associated failure to adjust type i ifn receptor signaling thresholds after t cell activation dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice middle east respiratory syndrome the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus mers coronaviruses from camels in africa exhibit region-dependent genetic diversity increase in middle east respiratory syndrome-coronavirus cases in saudi arabia linked to hospital outbreak with continued circulation of recombinant virus multihospital outbreak of a middle east respiratory syndrome coronavirus deletion variant deletion variants of middle east respiratory syndrome coronavirus from humans key: cord- - jj xpr authors: yu, pin; xu, yanfeng; deng, wei; bao, linlin; huang, lan; xu, yuhuan; yao, yanfeng; qin, chuan title: comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jj xpr middle east respiratory syndrome (mers), which is caused by a newly discovered coronavirus (cov), has recently emerged. it causes severe viral pneumonia and is associated with a high fatality rate. however, the pathogenesis, comparative pathology and inflammatory cell response of rhesus macaques and common marmosets experimentally infected with mers-cov are unknown. we describe the histopathological, immunohistochemical, and ultrastructural findings from rhesus macaque and common marmoset animal models of mers-cov infection. the main histopathological findings in the lungs of rhesus macaques and common marmosets were varying degrees of pulmonary lesions, including pneumonia, pulmonary oedema, haemorrhage, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. the characteristic inflammatory cells in the lungs of rhesus macaques and common marmosets were eosinophils and neutrophils, respectively. based on these observations, the lungs of rhesus macaques and common marmosets appeared to develop chronic and acute pneumonia, respectively. mers-cov antigens and viral rna were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, and ultrastructural observations showed that viral protein was found in type ii pneumocytes and inflammatory cells in both species. correspondingly, the entry receptor ddp was found in type i and ii pneumocytes, bronchial epithelial cells, and alveolar macrophages. the rhesus macaque and common marmoset animal models of mers-cov can be used as a tool to mimic the oncome of mers-cov infections in humans. these models can help to provide a better understanding of the pathogenic process of this virus and to develop effective medications and prophylactic treatments. a a a a a the middle east respiratory syndrome coronavirus (mers-cov) was identified in in a cell culture taken from a patient who died of pneumonia in saudi arabia [ ] . more men than women have become infected with this virus, and the median age of those affected is years old (range: months- years), with most of the fatalities occurring in patients over years old [ , ] . the respiratory symptoms of this infection are primarily related to severe lower respiratory tract complications (e.g., dyspnoea and coughing associated with a fever) that may become fatal, while there is generally little involvement of the upper respiratory tract. a large proportion of severely ill patients require mechanical ventilation [ , ] . complications that have been described in fatal cases include hyperkalaemia with associated ventricular tachycardia, disseminated intravascular coagulation leading to cardiac arrest, pericarditis and multiorgan failure [ ] . mers-cov seems to be widely present in dromedary camels in the middle east and in some parts of africa [ , ] . zoonotic transmission is likely to have originated from this species and is expected to continue indefinitely in these regions. the entry receptor for mers-cov, dipeptidyl peptidase (ddp ), also named cd , shows a high similarity in both humans and dromedary camels. most mers patients acquire the infection in the middle east, which subsequently leads to limited human-to-human transmission in local groups and healthcare workers and eventually to travel-related cases outside the region, all of which can result in a mild to severe or even fatal respiratory disease [ ] . finding a suitable animal model is a major challenge for understanding the pathogenesis of mers-cov infection, evaluating the safety and efficacy of mers-cov vaccine candidates and developing therapeutic interventions. experimental infections with mers-cov in rhesus macaques (macaca mulatta) [ ] , common marmosets (callithrix jacchus) [ ] , rabbits (oryctolagus cuniculus) [ ] and mice (mus musculus) [ , ] have been reported in studies on the pathological changes that occur as a result of this viral infection. however, little is known about the pathological changes in the lungs of humans infected with mers-cov, which makes it difficult to interpret data from experimental mers-cov animal models. overall, based on the known clinical aspects of mers-cov infection in humans, useful experimental animal models of mers-cov infection should exhibit a life-threatening lower respiratory tract disease. although there have been several studies in animal models on the pathogenic mechanisms of mers-cov infection, little is known about the comparative pathology and inflammatory cell response in rhesus macaques or common marmosets infected with this virus. therefore, it is vital to study comparative pathology on the association of the mers-cov antigen with its receptor, ddp , or the histopathological changes in nonhuman primate (nhp) models of mers-cov infection. here, we comprehensively describe the histopathological features of the disease and the distribution of the mers-cov antigen and ddp in rhesus macaque and common marmoset models. our findings may contribute to a better understanding of the pathogenic process of mers-cov infection and help in evaluating the suitability and efficacy of the animal models used in the development of effective medications and prophylactic treatments for this disease. this research on the mers-cov virus was discussed among the staff members of the department of pathogen biology at the institute of laboratory animal science (ilas) of the chinese academy of medical sciences and peking union medical college (pumc). the experiments and protocols for this nhp models of mers-cov infection were discussed explicitly and extensively among the staff members of the department of pathogen biology. these discussions were followed by consultations with biosafety officers and facility managers at the ilas of pumc, as well as with numerous specialists in the fields of sars-cov and general infectious disease research throughout china. all research procedures were approved by the ilas institutional animal care and use committee and the laboratory safety committee (lsc). the committee recommended that the number of animals be reduced to comply with the r (reduction, replacement, refinement) principles. therefore, our experiment was designed to include three rhesus macaques and three common marmosets to test their effectiveness as animal models of mers-cov infection. two rhesus macaques and two common marmosets were infected with the virus and one individual of each species was left uninfected to serve as a control. the animals were planned to be euthaniazed when they were suffering from fatal respiratory symptom, impending death or % of body weight loss, which included fatal dyspnea and infectious shock. the approved registration number for this study is ilas-pc- - . all experiments were conducted within an animal biosafety level (absl- ) facility, which was constructed and accredited based on national standard gb at the ilas of pumc, beijing, china. rhesus macaques and common marmosets were housed in accordance with chinese national standards, which are consistent with the standard set forth in the th edition of the nrc guide for the care and use of laboratory animals. because of the infectious nature of this study, nhps were housed individually instead of the generally recommended group or social housing. stainless steel cages measuring . - . m and . - . m depending on the weight of the individual animal, consisted of wire flooring and resting boards or perches. rooms have natural lighting and the photoperiod is supplemented during the winter months with an artificial lighting source to provide a : light cycle. temperature and humidity in animal holding rooms are maintained in accordance with recommendations in the chinese national standards for animal care. drinking potable water is obtained from the city of beijing and delivered to the animals via automated watering system (aws). the aws is checked daily to ensure proper operation i.e., water pressure, free flowing exits and absence of leakages. pans were cleaned daily and cages were washed every week by hand. all animals have individual cage id cards which contain the following basic information: study no., sex, weight, principal investigator's name and study protocol number. nhps were fed a measured amount of a commercially available nhp diet (beijing hfk bioscience co., ltd) offered twice daily. fresh fruit (apples, bananas and oranges) are supplemented on alternating days. additional environmental enrichment consists of toys, stainless steel mirrors and heavy-duty dog chew toys (nyla bones or similar), which are provided on a rotating basis. toys are left inside the cages when these are transported out of the room for washing and are sanitized at this time. damaged toys are removed from circulation. soft background music, plants, as well as pictures and photos hung on the animal room walls are provided for relaxation. opportunities for limited social interaction with compatible nhps are also provided at every other cage change when cages of compatible animals are placed in close proximity to each other while avoiding direct physical contact between animals. two rhesus macaques, - years old, were anesthetised with ketamine hydrochloride ( mg/kg, i.m) prior to the procedures and intratracheally inoculated with ml of hcov-emc ( . × tcid / ml) diluted in dmem. one mock-infected rhesus macaque was intratracheally inoculated with tissue culture media dmem for use as control. the rhesus macaques were observed twice daily, and clinical signs were recorded. the infected and mock-infected rhesus macaques were anesthetized with pentobarbital sodium ( mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. two common marmosets, - years old, were anesthetised with ketamine hydrochloride ( mg/kg, i.p) prior to the procedures and intratracheally inoculated with ml of hco-v-emc ( × tcid / . ml) diluted in dmem. one mock-infected common marmoset was intratracheally inoculated with tissue culture media dmem for use as control. the common marmosets were observed twice daily, and clinical signs were recorded. the infected and mock-infected common marmosets were anesthetized with pentobarbital sodium ( mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. none of the infected animals were euthanized or died without euthanasia prior to their sacrifice at days post-infection. the fixed samples were dehydrated and dewaxed according to conventional procedures, and -μm sections were prepared with a microtome. some sections were stained with haematoxylin-eosin (he) using routine methods. two independent pathologists observed all slides and were blinded to the experimental design. lungs were fixed in glutaraldehyde and prepared for ultrastructural observations. transmission electron microscopy was performed essentially as previously described [ ] . briefly, serial sections were dewaxed and rehydrated in graded ethanol, and a standard avidinbiotin immunoperoxidase technique was performed [ ] . table lists the primary antibodies used for ihc. optimal antibody dilutions were determined in experiments on positive control tissues. negative control sections were prepared using the same steps as described above, but the primary antibodies were derived from an irrelevant sera. sections were dewaxed and rehydrated in a graded ethanol series. ish was carried out using the enhanced sensitive ish detection kit i (boster, china) according to the manufacturer's instructions. endogenous peroxidase activity was quenched with . % hydrogen peroxide in methanol at room temperature for minutes. proteinase k digestion was performed at ˚c for min. then, pre-hybridization was performed at ˚c for hours. after removing excess pre-hybridization buffer, μg/ml digoxin (dig)-modified oligo-nucleotide antisense probes (table ) in the hybridization solution were applied to the sections, followed by incubation at ˚c overnight. after washing the slides in × saline-sodium citrate (ssc), . ×ssc, and . ×ssc buffer, the sections were incubated in a blocking buffer at ˚c for min. the sections were then incubated with biotinylated mouse anti-dig at ˚c for min and with streptavidin biotin peroxidase and biotinylated peroxidase for an additional min, with each incubation followed by three washes in phosphate-buffered saline (pbs). the sections were treated with , -diaminobenzidine for min, counterstained in haematoxylin for min, dehydrated, and mounted with neutral gum. sections for the negative controls were prepared using the same steps described above, but the antisense or sense probes were replaced with pbs at ph . . rhesus macaques were observed twice daily for clinical signs. the rectal temperature of the infected rhesus macaques increased to . ˚c at - days post-infection, and thereafter turned to normal. the infected common marmosets showed manifest symtoms of viral infection, including severe respiratory symtoms, drastical water intake decrease and overt weight loss, and the maximum body weight loss were about %. none of the mock infected nhps showed abnormal clinical signs or died during the expriment. pathological findings in the rhesus macaque tissues he stained tissues from rhesus macaques experimentally infected with mers-cov demonstrate that mers-cov induces lesions that are primarily observed in the lungs, with varying degrees of inflammation, interstitial pneumonia (fig a) , pulmonary oedema (fig b) , haemorrhaging, degeneration and necrosis of pneumocytes and bronchial epithelial cells (fig c) , and the infiltration of inflammatory cells. focal interstitial pneumonia and pulmonary oedema were observed in different parts of the pulmonary lobes, as was mild haemorrhaging. the most prominent pathological effect observed in the lungs of rhesus macaques was diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control rhesus macaque (s a fig) . pathological findings in common marmoset tissues a histopathological analysis detected numerous lesions in the lungs of the infected marmosets. exudative pathological changes were found, exhibiting haemorrhage, widespread pulmonary oedema and a large number of inflammatory cells. fibrinous exudates were observed in the oedematous alveolar cavities (fig d) . diffuse and focal neutrophil infiltration was found in the oedematous alveolar cavities (fig e) , bronchial lumen, and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells (fig f) . no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control common marmoset (s b fig) . to investigate the infiltration of specific inflammatory cells, ihc was carried out to identify cd + macrophages, cd + neutrophils, cd + natural killer cells, cd + b lymphocytes, cd + plasma cells, and cd +, cd +, cd + t lymphocytes. in the lungs of both species, the diffuse infiltration of numerous macrophages (fig b and d ) was observed in the expanded alveolar septum and the oedematous alveolar cavities. however, in the lungs of rhesus macaques, a large number of diffusely and focally infiltrating eosinophils (fig a) were found in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. however, in the lungs of common marmosets, numerous neutrophils ( fig c) infiltrated into the oedematous alveolar cavities. in both of the nhp models, other types of inflammatory cells were rarely observed. using immunohistochemical techniques and an ish analysis, we confirmed that mers-cov protein and viral rna were distributed in the lungs of rhesus macaques and common marmosets and that they were primarily located in the pneumocytes and inflammatory cells. in the lungs of rhesus macaques, mers-cov antigens were extensively distributed in type i and ii pneumocytes, alveolar macrophages (fig a) , eosinophils and bronchial epithelial cells ( fig b) . from the microscopic characteristics, the cuboidal type ii pneumocytes are located on the alveolar cavities, and smaller than macrophages. viral rna was also distributed in pneumocytes and inflammatory cells in the lungs of rhesus macaques (fig c) . in the lungs of common marmosets, a moderate level of mers-cov-positive antigens were detected in pneumocytes, and antigens were found more extensively in alveolar macrophages (fig d) , especially in the inflammatory cells around the bronchus (fig e) . viral rna was massively distributed in pneumocytes and inflammatory cells in the lungs of common marmosets (fig f) . no mers--cov-positive antigens or viral rna was detected in the lungs of the control nhps (data not shown). pathological lesions and virus distribution in rhesus macaque and common marmoset animal models are summarized and shown in table . to further determine the effects of mers-cov infection and replication in the lungs of common marmosets, ultrastructural observations were performed on lesions in infected lung samples and on mock-infected samples. virus particles were found in type ii pneumocytes ( fig a- c ) and in inflammatory cells (fig d- f) . under the electron microscope, the characteristic of type ii pneumocytes is lamellar bodies (s fig). no viral particles were observed in the lungs of mock-infected common marmosets (data not shown). to elucidate the relationship between mers-cov and its entry receptor, ddp , we determined the expression pattern of ddp in the lungs of rhesus macaques and common marmosets using immunohistochemical techniques. we found that in the lungs of rhesus macaques, ddp was strongly expressed in type i and ii pneumocytes, bronchial epithelial cells (fig a) , and inflammatory cells, primarily alveolar macrophages (fig a) . similarly, in the lungs of common marmosets, ddp was widely expressed in type i and ii pneumocytes and alveolar macrophages (fig b) . however, ddp was only weakly expressed in the bronchial epithelial cells, mainly in basal and ciliated cells (fig b) . in the present study, we analysed the histopathological features of mers-cov infection in rhesus macaques and common marmosets. moreover, we compared the distribution of mers-cov antigens, viral rna and ddp expression in the infected lungs of these species. we found that the lungs of both species exhibited varying degrees of lesions, including pneumonia, pulmonary oedema, haemorrhaging, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. comparing the different trends in the two nhp models, it can be seen that varying degrees of inflammation, especially interstitial pneumonia, were found in the lungs of rhesus macaques, indicating mild disease and trend of chronic pneumonia; however, in the lungs of common marmosets, exudative pathological changes were found, exhibiting pulmonary oedema, inflammatory cell infiltration and fibrinous exudates, suggesting acute pneumonia. similar to our results, previous study have also reported that rhesus macaques developed mild disease, and common marmoset exhibited potentially lethal disease [ ] . however, in our study we found that the prominent inflammatory cells in the two nhp models were different, which may be the causality of process in mers-cov infection. in our study, the diffuse infiltration of numerous macrophages was observed in the expanded alveolar septa and oedematous alveolar cavities of both species. however, the most prominent pathological effect observed in the lungs of rhesus macaques was a diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. in contrast, in the lungs of common marmosets, diffuse and focal neutrophil infiltration occurred in the oedematous alveolar cavities, bronchial lumen and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells. these differences in inflammatory cell infiltration suggest that inflammatory cells may function in the development of mers-cov infection. additionally, it is worth noting that eosinophils and neutrophils play important roles in rhesus macaques and common marmosets, respectively, in the development of pulmonary lesions and the pathogenesis of mers-cov infection. in the lungs of common marmoset, pulmonary oedema exhibited much more severe than that in the lungs of rhesus macaques, which may be due to the difference of inflammatory cells in the lungs of nhp models. similar to our results, previous studies have also reported that common marmosets infected with mers-cov exhibit acute bronchointerstitial pneumonia centred at doi: . /journal.pone. .g table . pathological lesions and virus distribution in rhesus macaque and common marmoset animal models. pathology of mers-cov infection the terminal bronchioles, with an influx of inflammatory cells, a thickening of alveolar septa, oedema, haemorrhaging and fibrosis in lung tissues [ ] . previous studies on rhesus macaques have shown that histological lesions induced by mers-cov infection were limited to the lungs, which exhibited interstitial pneumonia with a thickening of alveolar septa caused by oedema and fibrin accumulation, and small to moderate numbers of macrophages and even fewer neutrophils. in addition, alveoli tissue samples contained moderate numbers of pulmonary macrophages and neutrophils [ ] . previous studies on common marmosets infected with mers also showed that marmosets developed a progressive severe pneumonia or interstitial lymphohistiocytic pneumonia, exhibiting hypoproteinemia consistent with high protein pulmonary effusions resulting from alveolar oedema and interstitial lymphohistiocytic pneumonia with type ii pneumocyte and bronchial associated lymphoid tissue hyperplasia [ , ] . however, rhesus macaque model did not develop breathing abnormalities and showed no-tovery mild radiographic evidence of pneumonia [ ] . similar to our study, the common marmoset model of mers-cov infection mimics the acute and severe pathological process, yet the rhesus macaque model represents the mild infection of mers-cov. thus, the nhp models meet different needs of mers-cov researches. fatal human cases of mers-cov infection cause upper respiratory tract illness, severe pneumonia and multiorgan failure. these cases also include exudative-phase diffuse alveolar damage with the denuding of bronchiolar epithelia, the prominent formation of hyaline membranes, alveolar fibrin deposits, type pneumocyte hyperplasia, the occurrence of rare multinucleated syncytial cells, changes in alveolar septa related to oedema and increases in lymphocytes, with fewer plasma cells, neutrophils, and macrophages [ ] . these findings provide evidence for the pulmonary tropism of mers-cov infection. the pathological features of mers-cov are shared by other similar respiratory illnesses, such as severe acute respiratory syndrome (sars)-cov [ ] . previous studies that have evaluated fatal human cases of sars--cov have described diffuse alveolar damage at various stages as the most characteristic feature pathology of mers-cov infection of the disease, with sars-cov antigens primarily found in alveolar epithelial cells. in this study, we examined the histopathological features and the inflammatory cell response that occurs in the lungs of rhesus macaques and common marmosets experimentally infected with mers-cov. our findings indicate that inflammatory cells may play a crucial role in fatal mers-cov infections and that the progression of this disease in our animal models may mimic the infection in humans, making these models useful for further study of the pathogenesis, prevention and treatment of mers-cov infections. in this study, we analysed the distribution of mers-cov protein and viral rna in the lungs of rhesus macaques and common marmosets. we found that pneumocytes and inflammatory cells were positive for mers-cov antigens and viral rna. similarly, in the lungs of both species, mers-cov antigens were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, viral rna was distributed in pneumocytes and inflammatory cells, and viral protein were found in type ii pneumocytes and inflammatory cells. based on our observations, we therefore propose that mers-cov may proliferate and spread from the lungs through an inflammatory cell migration pathway. it has been reported that in fatal human cases of mers-cov infection, viral antigens were identified in both unremarkable and necrotic bronchial submucosal glands and in pneumocytes and epithelial syncytial cells. however, macrophages showed no localization with mers-cov antigens in these cases [ ] . findings of pulmonary consolidation, diffuse alveolar damage, and pleural effusion are consistent with the clinical features with mers-cov infection [ , , ] . in this study, we also detected mers-cov protein and viral rna in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells. therefore, our models of mers-cov infection using rhesus macaques and common marmosets may be suitable for use in the development of effective medications and prophylactic treatment and may serve as a tool to improve our understanding of the pathogenic process of mers-cov infection. dpp is a single-pass type ii transmembrane glycoprotein with a short n-terminal cytoplasmic tail. the structural residues comprising the receptor-binding domain have been defined via the co-crystallization of the mers-cov spike glycoprotein and dpp . dpp is typically found in type i and ii cells and alveolar macrophages. it has only rarely been detected in the surface epithelia of the nasal cavity and has also been found in a subset of mononuclear leukocytes and serous cells from submucosal glands [ ] . in fatal human cases, dpp has been observed in scattered pneumocytes and syncytial cells, bronchiolar epithelia and endothelia, and alveolar macrophages [ , , ] . in this study, we found that in the lungs of the nhps infected with mers-cov, ddp was expressed in type i and ii pneumocytes, bronchial epithelial cells, and inflammatory cells, primarily alveolar macrophages. in conclusion, we established animal models of mers-cov infection using rhesus macaques and common marmosets, which mimic the oncome of mers-cov infection in humans and provide a tool that may help in better understanding the pathogenic process of this disease. they may also be suitable models for aiding in the development of effective medications and prophylactic treatments for mers-cov infections. fouchier ra isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics 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middle east respiratory syndrome coronavirus infection in the united arab emirates time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia dipeptidyl peptidase distribution in the human respiratory tract: implications for the middle east respiratory syndrome emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study key: cord- - hadssmh authors: sherbini, nahid; iskandrani, ayman; kharaba, ayman; khalid, ghalilah; abduljawad, mohammed; al-jahdali, hamdan title: middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data date: - - journal: j epidemiol glob health doi: . /j.jegh. . . sha: doc_id: cord_uid: hadssmh background: middle east respiratory syndrome coronavirus (mers-cov), is an emerging virus respiratory infection. it has a high mortality rate and a wide spectrum of clinical features. this study describes the clinical characteristics and outcome of mers infected patients. methods: a retrospective study was conducted of all confirmed mers-cov infections from march to may at two tertiary care hospitals in al-madinah region (saudi arabia). we gathered data about demographic, clinical presentation, and factors associated with severity and mortality. results: a total of cases were identified; males ( %) and nine females ( %), age ± years. the death rate was higher for men ( %) than for women ( %). initial presentation was fever in ( %) cases, cough in ( %) cases, and shortness of breath in ( %) cases. associated comorbidities were diabetes mellitus in nine ( %) patients and chronic kidney disease (ckd) in eight ( %) patients. duration of symptoms before hospitalization ranged from . days to days. elevated liver enzymes were present in ( %) patients and impaired renal profile present in eight ( %) patients. we also describe in this study radiological patterns and factors associated with mortality. conclusion: mers-cov infection transmission continues to occur as clusters in healthcare facilities. the frequency of cases and deaths is higher among men than women and among patients with comorbidities. in saudi arabia, a beta new coronavirus was isolated for the first time at the end of from a patient who presented with acute community acquired pneumonia [ ] . he died days later from progressive severe respiratory failure and acute renal failure (arf) and his sputum sample was negative for respiratory viruses commonly tested. epidemiology of middle east respiratory syndrome coronavirus (mers-cov) was expanded after exploring the large hospital outbreak in al-hasa, saudi arabia [ ] . subsequently, the virus was designated as mers-cov [ ] . the geographic distribution of the cases has been mainly linked to the arabian peninsula particularly from saudi arabia where most of the cases were reported [ ] [ ] [ ] [ ] . however, in some countries in north america, europe, africa, and asia, the disease has been detected in some travelers from endemic countries [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the initial occurrence of mers-cov was thought to have particular predominance for male patients and those with comorbid diseases. the male-tofemale ratio was between . : and . : [ , ] ; this male predominance might have been related to the nature of the outbreak. initial cases were reported among elderly patients with a median age of years. mers-cov has a very high mortality rate, and complications arising from infection can result in severe respiratory and renal failure [ ] . symptoms of mers-cov range from mild upper respiratory symptoms to rapidly progressive severe pneumonia requiring intubation, and multiorgan failure. a significant number of patients may present with nonrespiratory symptoms such as headache, myalgia, and gastrointestinal symptoms of nausea, diarrhea, or vomiting [ , ] . this study describes the demographic, clinical characteristics, and outcome of mers-cov in al-madinah region, saudi arabia. a retrospective chart review study of all confirmed mers-cov cases recorded by two tertiary hospitals from the madinah region from march to may . institutional review board approval was obtained for our study from authorities of both hospitals. a case was confirmed as having infection if mers-cov real-time polymerase chain reaction was positive, using the recommended sampling technique (nasopharyngeal swab and tracheal aspirates or bronchoalveolar lavage in intubated patients). extraction of rna was performed with roche magna pure lc (rna viral isolation kit). samples were pretreated with lysis according to the manufacturer's instructions [ ] . we obtained data about demographic characteristics, clinical presentation, laboratory results, diagnosis, incubation period, smoking history, comorbidities, and history of contact with camels or mers-cov positive patients in regions within the madinah area. we recorded the duration of the patient's illness, microbiological test results, and reviewed imaging and treatments received. we also recorded the following outcomes: duration of mechanical ventilation, intensive care unit (icu) length of stay, and survival during hospitalization until the patient is discharged from hospital. data were analyzed using ibm spss for windows, version . . the frequency of cases of mers-cov infection and percentage of resulting deaths were calculated. statistical analyses of demographics, clinical, and laboratory descriptive data are tabulated. descriptive statistics such as means and standard deviation mean (±sd) were used to describe the age of the patients, laboratory test results, and duration of illness. frequencies and percentages n (%) were used to describe demographic and mer-cov outcomes. we also did a correlation with the outcome using the t test and fisher's exact test as appropriate with a significant value at p . . the total number of cases with confirmed mers-cov infection reported from april to may was . the majority of patients ( %) were men, and the median age was years. the most common symptoms were fever ( . %) and cough ( %), shortness of breath ( %), and vomiting and diarrhea ( %). the average duration of symptoms prior to hospitalization was days (range, - days). demographic and clinical characteristics of patients with confirmed mers-cov are shown in table . mortality rate among patients with confirmed mer-cov was %. mortality from mers-cov was significant (p < . ) and associated with older age, the presence of gastrointestinal symptoms, longer duration of symptoms prior to hospitalization ( ± . days), diabetes mellitus, chronic kidney disease (ckd), smokers, and lower blood pressure, as shown in table . most of the patients were coming from hanakia ( %) and all patients had contact with camels table . close contact with confirmed index mers-cov was documented in five patients, all of whom were healthcare professionals; three staff nurses and two clinicians. most of the patients were mildly hypoxic; oxygen saturation was . ± . at presentation with a picture of respiratory acidosis (ph . ± . and pco . ± . ). the basic liver functions show elevated alanine transaminase ( . ± . u/l), aspartate aminotransferase ( . ± . u/l), and creatinine ( . ± . mmol/l). as shown in fig. , which were collected during the patient's initial assessment. all patients had abnormal initial chest radiographs. the predominant finding was bilateral basal consolidations with ground-glass opacities, nodular or/and reticular pattern, and total diffuse multilobar involvement. all patients had appropriate supportive management and received a broad spectrum antibiotic and readjusted based on sputum cultures. among patients who required icu, the mean time of icu stay ranged from days to days ( . ± . days), and mechanical ventilation support was used in nine ( %) patients. mechanical ventilation support and longer stay in icu were significantly associated with death (p < . ) (see table ). emerging viral respiratory infections are causing a significant burden on public health and causing significant morbidity and mortality. over the past decades, many viral infection outbreaks have been reported including influenza h n such as h n , sars-cov, and the most recent mers-cov infection. the world health organization reported laboratory-confirmed cases of human infec- tion with mers-cov including at least deaths between and july [ ] . they reported that % of cases were male (n = ) and the median age was years (n = ) which is similar to our study. similar to a study reported by assiri et al. [ ] , our study showed more cases among older patients, but our study showed an association of death with older age. since , countries have been affected, including countries in the middle east, africa, europe, asia, and north america as reported from the world health organization. the majority of cases ( %) have been reported from saudi arabia. in saudi arabia, mortality secondary to mers-cov was % [ ] and in our study it was % (al-madinah). camels have been confirmed as a reservoir for mers-cov, and many hypotheses are behind this zoonotic (camels) transmission [ , ] . in our study, only five healthcare employees acquired infection from documented contact with an infected patient, but another patients were coming from areas around al-madinah; the largest number of infected patients was from the alhenakia area, where camels are prevalent. the second important mode of transmission is person-toperson transmission (travelers returning from the middle east and close contacts with mers-cov cases) [ ] . this type of transmission was confirmed by genome sequencing of mers-cov [ ] and isolates from the al-hasa healthcare-associated outbreak [ ] . in the uk, mers-cov was transmitted to a family member who visited a patient with confirmed infection and another report from france described patient-to-patient nosocomial transmission of mers-cov. in our study, five ( %) patients had transmission through close contact. approximately % of mers-cov patients in our study reported diabetes and ckd, which is similar to those from other observational epidemiological studies in saudi arabia [ , ] . the high mortalities reported early were probably due to a delay in the diagnosis and presence of comorbidities [ ] . however, the large number of mers-cov cases and ckd might have been based on the al-hasa hospital outbreak, which mainly happened in the dialysis center [ ] . we may consider the existence of chronic illnesses such as diabetes, hypertension, and ckd increasing the risk of acquiring this infection and categorize them as a high risk group for more complications and worse outcome as was revealed [ ] also in our study. we and others have found that the severity of illness associated with mers-cov infection ranges from mild to fulminant [ ] . the severity of the respiratory infections caused by mers-cov can progress to hypoxemic respiratory failure which requires the use of mechanical ventilation and death [ ] . all of our patients had significant respiratory manifestations requiring admission to the icu but % only required mechanical ventilation and died [ ] . mers-cov is known to infect cell lines of the intestinal tract [ ] , but it is not yet known what proportion of ill patients shed the virus in their stools, which is why some patients presented with gastrointestinal symptoms. identification of the full range of clinical presentations is important so that the mild cases are not missed. mers-cov is detected by reverse transcription polymerase chain reaction. to date, laboratory testing for mers-cov remains not very accurate; the sensitivity and negative predictive values are unknown. development of rapid and accurate diagnostic tests is needed urgently. results of throat swabs were occasionally negative and repeat testing for mers-cov is recommended. it seems difficult to conclude that one negative sample is enough to rule out mers-cov disease when a patient presents with respiratory symptoms and history of exposure. it is also not clear whether nasopharyngeal samples might be superior to throat samples or whether virus is shed more abundantly later in the course of the illness as it is in sars. there is evidence that repeat testing and tests on sputum or bronchoalveolar lavage fluid are of value in improving diagnostic accuracy. microbiological investigations were done routinely to exclude bacterial copathogens with community acquired pneumonia (cap). we had seven patients with methicillin-resistant staphylococcus aureus (mrsa) coinfection, two with streptococcus and one with methicillin-sensitive staphylococcus aureus (mssa) in our study population. assiri et al. [ ] stated that none of the samples screened was positive. other investigators found that one patient was coinfected with mssa and influenza b and another with streptococcus pneumonia [ ] . there might have been a selection bias because we were only screening critically ill cases, which in turn will lead to detection of more severe cases of mers-cov infection; mild cases may not come to hospital or may not be screened for mers-cov and could lead to false high case-fatality rates. clinical symptoms, laboratory investigations, and imaging findings of mers-cov are similar to those noted in other community-acquired respiratory tract infections. radiological findings in mers-patients tended to range from unilateral focal air-space opacities to multifocal or bilateral lower lobe involvement was seen with a picture of organizing pneumonia which was noted in our patients and other reports [ , ] . on the basis of findings until now, the clinical features of mers-cov infection have similarities to those seen in patients with sars-cov infection. the initial phase of nonspecific fever, cough, and shortness of breath are the major symptoms in those admitted to hospital; other common symptoms include chills, rigor, headache, myalgia, and malaise which may last for several days before progressing to pneumonia [ , ] . a significant number of patients had gi symptoms, another important similarity to sars. we found patients with mers-cov who had gi manifestation tend to progress to severe illness and this may be considered one of the poor prognostic factors. the disease may progress rapidly to a critical respiratory failure, requiring mechanical ventilation and lead to death in the icu [ ] . in our observations, all of the patients started with symptoms of fever, cough for - days. our study design has several limitations including that it is a retrospective chart review study with known inherited problems; these include missing data regarding contact with camels and documentation of all comorbidities and availability of follow up data after discharge. despite these limitations, we have been able to highlight some features in the epidemiological, demographic, and clinical characteristics of patients with mers-cov infection in al-madinah regions. the epidemiology and the transmission pattern of mers-cov to date indicate that the majority of cases occur in the healthcare setting. strengthening the infection control measures in the healthcare setting is of great importance. since about % of cases are community based, there is a real need to further prevent the animal-to-human transmission of mers-cov. the frequency of cases and deaths is higher among men than women and those around years of age are the most affected patients. the disease had higher mortality in older patients with comorbidities. also, the presence of gastrointestinal manifestations, high liver enzymes, and need for mechanical ventilation or longer stay in icu are all associated with high mortality. there are gaps in our knowledge of the epidemiology, prevalence, clinical characteristics, prognostic factors, and nature of the disease. it is also important to further delineate the transmission routes and the presence of any other animal or intermediate hosts. the influence of geographical distribution and comorbidities on the incidence and outcome of mers-cov patients should be studied further. the authors report no conflicts of interest in this work. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group managing mers-cov in the healthcare setting epidemiological and clinical aspects of coronavirus infection mers-cov middle east respiratory syndrome novel corona mers-cov infection. epidemiology and outcome update middle east respiratory syndrome coronavirus (mers-cov), summary of current situation, literature update and risk assessment. who/mers/ra/ . geneva: who lessons to learn from mers-cov outbreak in south korea epidemiological investigation of mers-cov spread in a single hospital in south korea public health response to two incidents of confirmed mers-cov cases travelling on flights through london heathrow airport in . lessons learnt travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia eds on heightened alert for mers-cov as first cases reach the us middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands middle east respiratory syndrome coronavirus (mers-cov): what lessons can we learn? screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study hospital outbreak of middle east respiratory syndrome coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during family cluster of middle east respiratory syndrome coronavirus infections prevalence of diabetes mellitus in a saudi community severe acute respiratory syndrome and coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection a major outbreak of severe acute respiratory syndrome in hong kong clinical and laboratory features in the early stage of severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings this study did not receive funding. key: cord- -qk ruj q authors: hall, aron j.; tokars, jerome i.; badreddine, samar a.; saad, ziad bin; furukawa, elaine; al masri, malak; haynes, lia m.; gerber, susan i.; kuhar, david t.; miao, congrong; trivedi, suvang u.; pallansch, mark a.; hajjeh, rana; memish, ziad a. title: health care worker contact with mers patient, saudi arabia date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: qk ruj q to investigate potential transmission of middle east respiratory syndrome coronavirus (mers-cov) to health care workers in a hospital, we serologically tested hospital contacts of the index case-patient in saudi arabia, months after his death. none of the contacts showed evidence of mers-cov infection. to investigate potential transmission of middle east respiratory syndrome coronavirus (mers-cov) to health care workers in a hospital, we serologically tested hospital contacts of the index case-patient in saudi arabia, months after his death. none of the contacts showed evidence of mers-cov infection. cov) was initially isolated in september from a -year-old man from bisha, saudi arabia ( ). in june , this index case-patient was hospitalized for severe respiratory disease in jeddah at dr. soliman fakeeh hospital and subsequently died ( ) . as of november , , a total of laboratory-confirmed cases of mers-cov infection, including deaths, had been reported to the world health organization; > % of reported mers-cov cases and deaths have occurred in saudi arabia ( ) . the clinical syndrome among hospitalized mers-cov patients includes severe acute respiratory illness, sometimes associated with hypoxemic respiratory failure and extrapulmonary organ dysfunction ( ); however, milder illness and asymptomatic infections have been identified through contact investigations ( - ). transmission of mers-cov to health care workers (hcws) has been reported ( , ), although no sustained community transmission has been identified. a zoonotic origin of mers-cov has been hypothesized; camels potentially play a role in transmission ( ), although the specific types of exposure associated with primary cases remain unknown. there remains a dearth of information on how mers-cov is spread and on transmission risks to hcw or other close contacts. our objectives were to evaluate the degree and nature of hcw contact with the mers-cov index case-patient and to serologically assess hcws for mers-cov infection. the field investigation was performed in october ; we awaited development and validation of mers-cov serologic assays before completing the study. the index case-patient was hospitalized on june , , with a -day history of fever, cough, sputum expectoration, and shortness of breath. precautions to prevent airborne transmission were taken by placing the patient in a private room with negative pressure for the first days of hospitalization. after an infectious disease consultation on day , airborne-transmission precautions were replaced with droplet-transmission precautions; after a multidrug-resistant organism was isolated on day , contact-transmission precautions were implemented ( ) . the case-patient remained in a private room, under standard and contact-transmission precautions, throughout hospitalization until he died on day . using dates and units in which the case-patient received care, hospital staff initially identified hcws who had had contact with the case-patient (came within meters of the case-patient or his bedding, equipment, or body fluids). a comparison group of approximately equal numbers of hcws (preferentially with similar job responsibilities) was selected from hcws who had had no known contact with the case-patient. hospital infection control staff administered a brief, standardized questionnaire to both groups of hcws. information was collected on hcw demographics, job duties, and symptoms of respiratory disease during june -july , , which corresponds to the period when the case-patient was hospitalized and an incubation period of - days, based on mers-cov natural history information available at the time of investigation. specific information about circumstances of case-patient contact and potential for mers-cov exposure was collected from hcws who had had contact with the case-patient. in october ( months after the case-patient's death), a blood specimen (< ml) was collected from each hcw and transported first to the ministry of health western regional laboratory in saudi arabia and then to the us centers for disease control and prevention for mers-cov testing. upon receipt, specimens were gamma-irradiated on dry ice and stored at - °c. all specimens were tested by hku . n nucleocapsid enzyme immunoassay (eia) ( ); incubations and substrate development were conducted at °c. mers-cov antibody positivity was defined as positive hku . n screening eia results and confirmed by mers-cov immunofluorescence or microneutralization assay ( , ); specimens with negative eia results were considered antibody negative. of hcws identified as having had contact with the mers-cov case-patient, were unavailable for interview and refused serum collection, leaving for the final analysis. among hcws who had had case-patient contact, median age was . (range - ) years; ( %) were female; ( %) were nurses; ( %) were physicians; ( %) were respiratory technicians; and each was a housekeeping, radiology, or infection control staff member. six ( %) hcws reported having a chronic medical condition (e.g., asthma, diabetes, hypertension), and ( %) reported smoking tobacco. according to body mass index (bmi) calculations from self-reported height and weight, nearly half of hcws were overweight (bmi . - . , n = [ %]) or obese (bmi ≥ . , n = [ %]). most of the hcws had reportedly come within meter of the case-patient ( %); touched the case-patient ( %); or touched the case-patient's bedding, equipment, or body fluids ( %) (table) . during a single shift, most ( %) hcws reported < hour of case-patient contact, but % reported > hours of contact. hcws reported having been present during airway suction ( %), nebulizer treatment ( %), sputum induction ( %), bronchoscopy ( %), and intubation ( %). infection control precautions reportedly used by hcws during contact with the case-patient included hand hygiene ( %) and/or wearing of gloves ( %), surgical mask ( %), and gown ( %); however, among those reporting use of these precautions, some admitted to < % compliance and none used eye protection. respiratory disease symptoms during june -july were reported by ( %) hcws who had had case-patient contact. among these, symptoms included cough ( %), sore throat ( %), myalgia ( %), rhinorrhea ( %), self-reported fever ( %), diarrhea ( %), and sneezing ( %); ( %) of these hcws sought medical care and received a diagnosis of pharyngitis. for comparison, respiratory symptoms were reported during the same period by ( %) hcws who had not had case-patient contact. results of eia testing of serum collected during october - , , from all hcws who had and all who had not had case-patient contact were negative for mers-cov. immunofluorescence assay of randomly selected serum specimens also gave negative mers-cov results, supporting interpretation of eia-negative specimens as antibody negative. this investigation provides indicators of transmission potential during the initial emergence of mers-cov. the lack of mers-cov transmission among hcws in this study is similar to results of some published contact investigations ( , ) but different from others that reported transmission to hcw contacts ( , ) . recovery of cultivable virus from the sputum of the case-patient reported here ( ) and the severity of illness suggest some potential for virus transmission. we did not assess use of recommended personal protective equipment during each episode of casepatient contact; however, the reported lack of use during every episode of patient contact suggests that some hcws might have been exposed to mers-cov. nonetheless, the infection control precautions that were used might have contributed to the demonstrated lack of transmission. serologic assays to determine past infections are useful for assessing the risk for mers-cov transmission ( ) . the assays used in this study have previously detected mers-cov-specific antibodies < weeks and > months after illness onset with > % specificity ( , ) . although no severely ill hcws were identified during this investigation, real-time reverse transcription pcr detection of mers-cov in respiratory tract specimens from asymptomatic or mildly ill hcws during other contact investigations has been described ( , ) . the timing of this investigation months after hospitalization of the case-patient precluded use of real-time reverse transcription pcr and potentially introduced recall bias during hcw interviews. despite numerous case-patient contact episodes among hcws, we found no serologic evidence suggesting health care-associated transmission of mers-cov from the index case-patient. rapid identification of potentially infected patients and implementation of infection control precautions can help protect hcws. us centers for disease control and prevention recommendations for management of hospitalized patients with known or suspected mers-cov infection include implementing standard, contact-and airborne transmission precautions ( ) . isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-update. disease outbreak news epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study evidence of person-to-person transmission within a family cluster of novel coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infections in health care workers human infection with mers coronavirus after exposure to infected camels, saudi arabia guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description stillbirth during infection with middle east respiratory syndrome coronavirus the united kingdom public health response to an imported laboratory first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission consortium for the standardization of influenza seroepidemiology. novel coronavirus (mers-cov) clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus (mers-cov) into the united states first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities we are extremely grateful for the collaboration of the participating hcw in this investigation. additionally, we thank idris sulemann for assistance with data entry and management and jennifer l. harcourt, hayat caidi, and azaibi tamin for performing the serologic evaluations. key: cord- - q efrqk authors: al-tawfiq, jaffar a.; omrani, ali s.; memish, ziad a. title: middle east respiratory syndrome coronavirus: current situation and travel-associated concerns date: - - journal: front med doi: . /s - - -y sha: doc_id: cord_uid: q efrqk the emergence of middle east respiratory syndrome coronavirus (mers-cov) in brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (sars-cov) in . more than mers-cov cases were recorded in months with a case fatality rate (cfr) of %. meanwhile, cases of sars-cov were confirmed in six months with a cfr of %. the clinical presentation of mers-cov ranges from mild and non-specific presentation to progressive and severe pneumonia. no predictive signs or symptoms exist to differentiate mers-cov from community-acquired pneumonia in hospitalized patients. an apparent heterogeneity was observed in transmission. most mers-cov cases were secondary to large outbreaks in healthcare settings. these cases were secondary to community-acquired cases, which may also cause family outbreaks. travel-associated mers infection remains low. however, the virus exhibited a clear tendency to cause large outbreaks outside the arabian peninsula as exemplified by the outbreak in the republic of korea. in this review, we summarize the current knowledge about mers-cov and highlight travel-related issues. the emergence of middle east respiratory syndrome coronavirus (mers-cov) in brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (sars-cov) in [ , ] . as of march , , mers-cov cases were confirmed with a case fatality rate (cfr) of approximately %, whereas sars-cov cases were recorded in six months with a % cfr [ ] [ ] [ ] . an upsurge in the number of cases was observed in march-may because of the outbreak in large healthcare facilities in jeddah, kingdom of saudi arabia (ksa) [ , ] . more recently, a larger outbreak occurred in riyadh, ksa and in multiple hospitals in south korea [ ] [ ] [ ] [ ] . wolfe et al. [ ] described the five stages of pathogen evolution that lead to diseases confined to humans. in stage , the pathogen is confined to an animal host. humans become infected by animals only in stage . stage is limited human-to-human transmission. in stage , long outbreaks with numerous cycles of human-to-human transmission occur. in stage , the pathogen exclusively infects humans [ ] . mers-cov has not yet reached stage . the interest and concern of the public and the health community in emerging infectious diseases stem from the ability of the emerging pathogens to cause pandemics with high fatality rates and the associated economic effects on affected countries. for example, sars-cov caused $ - billions of economic losses in mainland and hong kong of china, singapore, and canada, and the pandemic influenza h n in resulted in $ - billion losses worldwide [ ] . in this review, we focus on the important aspects of the recently emerged mers-cov, its effects on humans, and the transmission patterns of the disease based on available scientific data. year-old male from bisha [ , ] . the patient was hospitalized with community-acquired pneumonia; the disease rapidly progressed, resulting in acute renal failure, respiratory failure, and death [ ] . a summary of the major events in the development of the mers-cov epidemic is shown in fig. . since the first case of mers-cov, a detailed investigation was conducted to determine the environmental and animal source of the virus. extensive contact investigation was carried out on the first case that originated from bisha but was eventually hospitalized in a private hospital in jeddah [ , ] . the contact investigation started in the patient's hometown of bisha in southern ksa. the investigation included his immediate household contacts ( wives, sons, daughters, grandchildren, and house maid), as well as the community and healthcare facility in bisha, which included shepherds who took care of his five camels and healthcare workers (hcws) ( nurses and three physicians) [ ] . in total, contacts in bisha were screened and all of them tested negative for mers-cov by polymerase chain reaction (pcr) [ ] . an extensive investigation of the out of hcw who had significant contact with the same patient during his -day stay in the private hospital in jeddah tested negative [ ] . during the october investigation on the source of the virus, a team representing three agencies, namely, saudi ministry of health, center for infection and immunity of columbia university, and ecohealth alliance, interviewed the family of the index case-patient from bisha. the team also collected samples from bats within km from his home, as well as an abandoned date palm orchard, the area within km from his place of employment, a hardware store that fronted his garden, and a date palm orchard. although none of his family members nor employees recalled seeing bats, the investigating team observed the roosting bats and guano in abandoned wells and ruins within km of his home and insectivorous bats at dusk in the garden behind his store. a sample from a taphozous perforatus bat (egyptian tomb bat) captured in bisha showed % identity to the human β-cov c emc/ cloned from the index case-patient [ ] . the largest data set on the contact investigation revealed that the percentage of positive cases was . %, . %, . %, and . % among hospital patients, hcw contacts, family and contacts, and overall [ ] . with the expansion of testing to identify the full disease spectrum and the inclusion by the world health organization (who) of the positive cases by serology in july [ ] , the cfr gradually decreased from % to % as more asymptomatic and mildly symptomatic cases were included [ ] . a nationwide serosurvey for mers-cov conducted in the ksa between december and december suggested that around infected individuals were not aware of their infection, which confirmed an extremely high incidence of asymptomatic to mildly symptomatic disease [ ] . a good example of a family cluster of mers-cov infections was published in the new england journal of medicine [ ] . once the second case in that cluster was identified, an epidemiologic investigation identified the index case who was the father of the second case who was admitted earlier with congestive heart failure and community-acquired pneumonia. subsequently, the third and fourth cases where recognized [ ] . in that cluster, persons lived in the same extended household, including nine children ( < years of age) [ ] . aside from the four patients, no other family members had major respiratory symptoms and none of the hcws who managed the index case before the mers-cov diagnosis exhibited symptoms [ ] . a second family cluster was also described by omrani et al. [ ] . three patients lived in one large house in urban riyadh. none of the other family contacts had positive pcr tests. community outbreak was also described [ ] . the first patient infected his cousin, and each of the patients infected their parents. genome analysis showed multiple introduction of the virus and determined three distinct genotypes, which confirm very low patient-to-patient transmission [ ] [ ] [ ] [ ] . a number of healthcare-associated outbreaks were reported previously [ , , , ] . a cluster of acute respiratory illnesses was reported in the intensive care unit (icu) in zarqa, jordan in april [ ] . thirteen hcw cases were detected (intensivists and icu nurses) with two mortalities. an initial investigation revealed no etiology. after the announcement of the first case of mers-cov in september in ksa, samples from these patients were retested for mers-cov and confirmed to be positive in two patients by pcr and eight contacts by serology [ , ] . thus, this case demonstrated mers-cov as a healthcare outbreak. the second large outbreak occurred in al-hasa, ksa in april [ ] . a total of confirmed cases and probable cases were recorded. a detailed transmission map was drawn based on the best available epidemiological data linking these patients epidemiologically [ ] . subsequent genotyping showed multiple introduction of the virus leading to the outbreak rather than a single introduction [ ] . four of the cases did not match the transmission, which indicates that the disease was not likely transmitted between the cases but had a different genome, thus indicating multiple community introductions [ ] . the outbreak was controlled with simple infection control measures in three weeks. mers-cov was shown to have minor clades, and the most recent common ancestor of mers-cov was introduced into humans at the end of [ ] . mers-cov is closely related to pipistrellus bat cov hku (pi-batcov hku ) in bats from hong kong and these bats diverged from a common ancestor several centuries ago [ ] . in , one of the largest healthcare-associated outbreaks of mers-cov infection took place between february and april in jeddah, ksa [ ] . a total of mers-cov patients were treated in hospitals [ , ] . the number of cases in each hospital varied from to cases [ , ] . in all the cases, % were primary cases and % (including hcw) were healthcare-associated infections [ ] . the rapid increase in the cases was attributed to more sensitive case detection, active case determination, and contact tracing with changes in testing algorithms [ ] . a breakdown in infection control measures was observed with no change in the virus [ ] . a near full genome sequence of the three viruses from the early phase of the jeddah outbreak showed a highly similar virus with no genome insertions or deletions [ ] . the genetic marker influencing transmissibility was % identical to known mers-cov genomes [ ] . in , the largest outbreak outside the middle east took place in south korea [ ] . the index case was a year-old male who visited bahrain, ksa, united arab emirates, and qatar [ ] . he developed symptoms on may , and visited multiple hospitals in south korea [ ] . he caused an outbreak involving five health care facilities and cases [ ] , with cases in hospital b, cases in hospital d, cases in hospital e, and three cases in hospital f [ ] . as of june , the outbreak in the republic of korea involved health care facilities, and six facilities exhibited nosocomial transmission [ ] . the total number of cases as of july , was cases with deaths [ , ] . based on epidemiological data monitoring over the last three years, the potential seasonality of mers-cov from march to may and from september to november was observed. in april and may , the number of cases increased [ ] . one of the reasons for this increase in the number of cases is the parallel surge in mers-cov tests in jeddah [ ] . this increase is also facilitated by an intensified intra-hospital and inter-hospital transmission of mers-cov with no change in the virus genetic composition or ability to cause disease [ , ] . thus, seasonality is difficult to establish because sporadic cases were documented with amplifications mainly occurring during nosocomial outbreaks. available data to date show that mers-cov behaves differently in various conditions and in different population of patients. isolated sporadic cases, small family clusters, as well as large healthcare-acquired infections and clusters, were recorded. most cases present with respiratory symptoms and about one-third had gastrointestinal symptoms (table ) [ , [ ] [ ] [ ] [ ] [ ] . early symptoms are mild and non-specific, which last several days prior to progressing to severe pneumonia. no predictive signs or symptoms exist to differentiate mers-cov from community-acquired pneumonia in hospitalized patients [ ] . an apparent heterogeneity in transmission was observed. the severity of the disease is usually seen in primary or index cases, immune-compromised individuals, and people with underlying comorbidities. mild or asymptomatic disease usually occurs in secondary cases and was initially thought to infect the young and previously healthy individuals. however, mortalities and severe cases were observed among primary cases and young individuals [ ] . however, person-to-person transmission as a definite route of transmission is still unclear. the median incubation period was . days ( % ci, . to . ), and the serial interval was . days ( % ci, . to . ) [ ] . cfr is directly related to the number of comorbidities, the increasing detection of asymptomatic to mildly symptomatic cases over the last three years [ ] . however, mortalities were reported among healthy individuals. the median time to hospitalization was days, icu admission was days, mechanical ventilation was days, and death was . days [ , ] . mers-cov pcr was standardized, and it works extremely well with lower respiratory samples in experienced laboratories. confirmatory testing in national or regional reference laboratories with experience and load of samples will avoid reporting false positive cases. on november , , a spanish case from hajj was initially tested positive for mers-cov but was eventually sent to an outside reference laboratory for confirmatory testing; all tests were negative [ ] . if mers-cov infection is suspected and initial testing is negative, repeat testing is recommended and lower respiratory tract samples would yield higher positivity [ ] . in july , a key change in mers-cov case definition is the inclusion of a confirmed case based on serology [ ] . serologic mers-cov confirmation requires sero-conversion in two samples taken at least days apart by a screening (elisa, ifa) and a neutralization assay [ ] . since the emergence of mers-cov and till october , cases were reported in different countries [ ] . the cases included cases in the middle east, cases in europe, in asia, and cases in other countries [ ] . the who international health regulations (ihr) emergency committee convened a mers-cov emergency committee meeting on multiple occasions; after extensive deliberations and reviews of available data, the diseases weakness did not fulfill the ihr requirements to be defined as a public health emergency of international concern (pheic) and mainly sustained human-to-human transmission [ , ] (fig. ) . using a mathematical model, the risk of mers-cov was estimated to be one to seven cases per hajj and three to ten umrah pilgrims per year [ ] . in another model, . pilgrims were estimated to develop mers-cov symptoms during the hajj, and . foreign pilgrims will be infected but return home before developing symptoms [ ] . travelrelated mers-cov occurred infrequently among pilgrims performing the umrah [ ] . however, millions of pilgrims who performed the annual hajj did not exhibit mers-cov symptoms [ ] . a cross sectional study of african hajj pilgrims returning to ghana, west africa in showed that none of the pilgrims was positive for mers-cov [ ] . a cohort of french pilgrims exhibited no mers-cov infection [ ] . no mers-cov was detected by pcr among adult pilgrims from countries [ ] . although the risk of travel-associated mers-cov remains low, the potential for healthcareassociated infections in relation with an imported mers-cov is a real concern. this event took place in the republic of korea [ ] [ ] [ ] [ ] . thus, all hcws should be vigilant to the importation of mers from returning travelers and healthcare organizations should implement a strategy to screen, isolate, and diagnose these patients. serologic testing allowed the detection of eight of the contacts in the jordan cluster [ ] . the positive results were confirmed in six of nine outbreak members, one in household contacts, and one in hcws [ ] . interest-ingly, one hcw who tested positive did not recall having respiratory symptoms at the time of the outbreak [ ] . a few published serology studies did not present any background on mers-cov. in one study, eight out of abattoir workers and blood donors had weak positive tests by ifa, and none of these individuals tested positive by nt in jeddah and makkah in [ ] . in a second study, none of children with respiratory tract disease and blood donors showed neutralizing antibodies in dammam, ksa in - [ ] . an initial study of samples that were tested for mers-cov antibodies in the eastern saudi arabia revealed no positive samples [ ] . a serologic evaluation of household contacts of index patients showed probable cases of secondary transmission ( %; % confidence interval, two to seven) [ ] . a large-scale study of more than samples demonstrated that the overall seroprevalence in ksa was . % and detected an increase of -and -fold of antibody detection rate among camel shepherds and abattoir workers compared to that in the general population [ ] . the full genome sequences from mers patients with known dates and locations can help answer these questions: how fast does the virus change? when did the virus begin circulating in its current form? is the virus adapting to humans? can the geographical patterns help locate an animal source? what are the transmission patterns? a previous study showed that sequence success is as function of viral load, which is inversely proportional to the threshold cycle value (ct) in real-time pcr assays [ ] . ct values below are associated with good sequencing success rates. thus, ct values are sufficient predictors of the success that is independent on sample type or source [ , ] . mers-cov was found to be stable in the environment. the virus can survive on plastic and steel for up to h at lower temperatures and humidity; once temperature and humidity increase, the virus becomes less viable [ ] . the virus is viable at °c and % humidity for hours, at °c and % humidity for hours and at °c and % humidity for hours only [ ] . in another study, increasing the temperature from °c to °c adversely affects viral infectivity [ ] . the who advocates contact and droplet precautions with airborne isolation in hospital settings when dealing with an aerosol-generating procedure [ ] ; the united states centers for disease control and prevention (cdc) and the european centre for disease prevention and control (ecdc) call for airborne infection isolation precautions [ ] [ ] [ ] . multiple hospital outbreaks of mers-cov infection in ksa were controlled effectively using infection control precautions recommended by the who and the saudi ministry of health [ , , , ] . there are no proven therapeutic agents for the treatment of mers-cov patients. existing antiviral agents can be repurposed against mers-cov [ , ] . interferon and ribavirin were suggested to be possible therapeutic options based on the sars data [ ] . these two agents were used to treat five patients with mers-cov infection [ ] . the median time to therapy was days, and no improvement was observed [ ] . ribavirin and interferon were used in patients with mers-cov at a median of three days [ ] . the -day survival was % ( of patients) in the treatment group vs. % ( of of patients) in a historical group (p = . ) with no survival improvement at days ( % vs. %; p = . ) [ ] . in an observational study, interferon-α a with ribavirin and interferon-β a with ribavirin showed similar results in treating mers-cov [ ] . the potential repurposed drugs for mers therapy include ribavirin with or without interferon, hiv protease inhibitors (lopinavir and nelfinavir), cyclophilin inhibitors (cyclosporin and alisporivir), chloroquine, mycophenolic acid, and nitazoxanide [ ] . the use of interferon-α b and ribavirin decreased viral replication in the rhesus macaques model within h of mers-cov infection [ ] . in vitro, ribavirin inhibits mers-cov; however, the required doses are extremely high to obtain in vivo [ , ] . in vitro, nelfinavir and lopinavir achieved inhibitory concentrations against mers-cov [ ] . in a primate model, the mortality rate at h post-inoculation was % in untreated versus %- % in the lopinavir-or ritonavirtreated and interferon-β b-treated animals [ ] ; the combination was used in another case [ ] . to understand the mers-cov disease further, resolving the issues related to the specific host and the specific transmission mode and determining the factors that increase transmission within healthcare environments are crucial. the viral kinetics of mers-cov within different body compartments is another aspect that needs further examination. the optimal therapeutic options and strategies to predict the occurrence and severity of the disease require further analysis. jaffar a. al-tawfiq, ali s. omrani, and ziad a. memish declare that they have no conflict of interest. this manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. travel implications of emerging coronaviruses: sars 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interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-α for middle east respiratory syndrome: a case report key: cord- -e uaw fy authors: zhao, guangyu; jiang, yuting; qiu, hongjie; gao, tongtong; zeng, yang; guo, yan; yu, hong; li, junfeng; kou, zhihua; du, lanying; tan, wenjie; jiang, shibo; sun, shihui; zhou, yusen title: multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: e uaw fy the middle east respiratory syndrome coronavirus (mers-cov) causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. for the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of mers-cov infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against mers-cov infection. in this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase (hdpp ), the receptor for mers-cov. after intranasal inoculation with mers-cov, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day and in the lungs, kidneys and brains on day post-infection. in addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day . taken together, this study characterizes the tropism of mers-cov upon infection. importantly, this hdpp -expressing transgenic mouse model will be applicable for studying the pathogenesis of mers-cov infection and investigating the efficacy of vaccines and antiviral agents designed to combat mers-cov infection. all procedures involving animals were approved by the laboratory animal center, state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology iacuc's (the permit number is bime - ). animal studies were carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals. all experimental operations to mice were performed under sodium pentobarbital anesthesia, and mice were euthanized with overdose inhalational carbon dioxide. all efforts were made to minimize suffering of animals. considering the species differences, the hdpp -coding sequence was codon-optimized and synthesized (genscript, nanjing, china) according to the mrna sequence (accession number nm_ . ) and cloned into the multiple cloning site (mcs) of a pcaggs plasmid, leading to strong expression of the gene. the recombinant plasmid pcaggs-hdpp contained the hcmv ie enhancer (hcmviee), the cag promoter (chicken β-actin promoter/intron), a codon-optimized open reading frame (orf) of hdpp and rabbit β-globin polya ( fig a) . expression of the codon-optimized hdpp was achieved by transfection of the pcaggs-hdpp plasmid into cos- cells, followed by confirmation of expression using anti-human cd monoclonal antibody (r&d systems, minneapolis, mn, usa). to confirm the binding of hdpp to the mers-cov receptor-binding domain (rbd), transfected cos- cells were incubated with a recombinant protein expressing the rbd of mers-cov fused to human igg fc (rbd-fc) [ ] , followed by addition of dylight -conjugated goat anti-human igg to detect binding. the purified bp fragment generated from apal digestion of pcaggs-hdpp was used as the transgene for injection into the pronuclei of fertilized f (c bl/ × c bl/ ) mouse eggs to generate transgenic embryos. the mice used in this studywere backcrossed two to three times onto a c bl/ background. all animal studies were performed in strict accordance with the recommendations outlined in the guide for the care and use of laboratory animals. genomic dna from each transgenic founder line and from wild-type c bl/ mice was isolated from the liver tissue using dnazol reagent (qiagen, valencia, ca, usa) according to the manufacturer`s instructions. the hdpp dna copy numbers were determined by quantitative pcr using power sybr green pcr master mix (life technologies, carlsbad, ca, usa). total hdpp dna was normalized using a single-copy reference gene (pkd ) as an endogenous control. the primers specific for hdpp were forward ' ctacagctcctggg caacgtgctg ' and reverse ' agatgtcgtaactggcggtgtaag '. the primers specific for mpkd were forward ' ggctgctgagcgtctggta ' and reverse ' ccaggtcctgcgtgtctga '. to detect the expression of optimized hdpp in the tissues of our transgenic mice model, samples were harvested and stored immediately in rnalater rna stabilization reagent (qiagen, valencia, ca, usa) until total rna was extracted and purified using an rneasy extraction kit (qiagen, valencia, ca, usa). for each sample, μg of total rna was used as template for first-strand cdna synthesis. the resulting cdna was subjected to quantitative pcr using power sybr green pcr master mix (life technologies, carlsbad, ca, usa) to determine the relative abundances of hdpp in the tissues. the forward and reverse primers for hdpp amplicons were as the same as those noted above. the relative amount of hdpp in different tissues was obtained by normalizing mrna expression to that of the endogenous control gene gapdh [ ] . mers-cov (hcov-emc/ strain) was propagated and titered on vero cells in an approved biosafety level laboratory. following intraperitoneal anesthetization with sodium pentobarbital ( mg/kg of body weight), mice under virus infection were intranasally inoculated with mers-cov ( . tcid ) in μl dulbecco's modified eagle's medium (dmem), and mice in sham group were treated with the same volume of dmem. mice were monitored for weight loss and survival for days. we have taken special precautions and followed standard guidelines outlined in the guide for the care and use of laboratory animals to ensure that ample food and water as well as sanitary cage conditions with enrichment devices were present in order to maximize the animal's comfort. humane endpoints were used during the survival experiments. after virus infection, mice were closely monitored by daily observation and weighing of trained animal caretaker, and the monitoring will raise to twice per day once mice lost more than % of their initial body weight, or exhibited lethargy, ruffled hair coat, or hunched posture. animals were deemed gravely ill and were euthanized by overdose inhalation of carbon dioxide if they lost more than % of their weight, or lost ability to ambulate and access food or water. sera were collected on day and inactivated at °c for min before the levels of cytokines and chemokines were measured. to assess viral replication and histopathologic damage following mers-cov infection, mice were euthanized with overdose inhalational carbon dioxide, and tissues included lungs, kidneys, livers, spleens, intestines and brains were harvested on indicated time points. the viral rna copies in tissues were determined by qrt-pcr according to the protocol described elsewhere [ ] . briefly, total rna was extracted from mg of collected tissues using rneasy extraction kits (qiagen, valencia, ca, usa) following the manufacturer's instructions. mers-cov rna was quantified in a μl mixture containing μl rna using the invitrogen superscript™ iii one-step rt-pcr system with platinum taq (life technologies, carlsbad, ca, usa). the primers and probes specific for the upe envelope gene of the mers-cov virus were as follows: forward ' gcaacgcgcgattcagtt '; reverse ' gcctctac acgggacccata '; fluorescence probe / -fam/ ctcttcacataatcgccccgagctcg cg/ -tamsp/ [ ] . mouse gapdh was used as a housekeeping gene control. a standard curve was generated for pcr reaction using - copies of quantified rna transcripts to calculate copy numbers for each reaction. the results were considered positive at c t values below for primer and probe set, and were calculated as viral rna copies per gram of tissues. the tissues of infected mice were harvested aseptically at indicated time points and homogenized in minimal essential medium (mem) plus antibiotics to produce % (w/v) suspensions. tissue homogenates were centrifuged and titered on the monolayer of vero cells. the cytopathic effects (cpe) were daily observed under phase-contrast microscropy for days. the viral titer was determined as tcid by cpe-based assay, and calculated using the reed and muench method. the viral titer in tissue was expressed as log tcid /g of tissue. collected tissue samples were immediately fixed in % neutral buffered formalin, sectioned at μm thickness, and stained with hematoxylin and eosin (h&e) to examine histopathology as described previously [ ] . the expression of virus antigens and infiltration of neutrophils and macrophages in organs after mers-cov infection was detected by immunohistochemistry. briefly, formalin-fixed, paraffin-embedded lung sections were deparaffinized and hydrated using graded alcohols. expression of virus antigen and inflammatory cell infiltration was assessed using rabbit polyclonal antibody to mers-cov nucleoprotein (np) (sino biological inc., beijing, china), neutrophil marker nimp- , (santa cruz biotechnology, paso robles, ca, usa) and cd (abcam, cambridge, ma, usa). the reaction was detected using a standard streptavidin-biotin detection system (beijing zhongshan biotechnology co., ltd., beijing, china) and visualized using dab with hematoxylin counterstaining. cytokines and chemokines in mouse sera were quantified using a commercial milliplex mouse cytokine/chemokine magnetic panel kit (merck millipore, usa.). a panel of inflammatory cytokines and chemokines (ifn-γ, ip- , il- β, il- , il- , tnf-α, gm-csf, kc, mcp- , mip- ɑ, mip- β and rantes) was measured according to the manufacturer's protocols. statistical analyses were performed using graphpad prism version . . to compare the groups in terms of hdpp copies, inflammatory cytokine/chemokine levels, and tissue viral rna copies, student's t test with welch's correction was used. the significance between survival curves was analyzed by kaplan-meier survival analysis with log-rank test. p values lower than . were considered statistically significant. dpp is constitutively expressed in human parenchyma cells in tissues including the liver, intestines and kidneys as well as in t and b cells [ ] . we developed transgenic mice in which hdpp expression was driven by the chicken β-actin promotor in the pcaggs plasmid ( fig a) . expression of hdpp in vitro was achieved by transfection of the pcaggs-hdpp plasmid into cos- cells and detected by immunofluorescence analysis. the results showed that hdpp was expressed in cos- cells and that it effectively bound mers-cov (fig b- e ). after microinjection of linearized dna, founder lines that transferred the hdpp gene to their progeny were established. the levels of transgenic dna in the founder lines ranged from to copies per genome, as determined by qpcr (fig f) . in addition, the mrna levels of hdpp in the trachea, lung, liver, spleen, kidney, intestine and brain in two of the founder lines were measured by qrt-pcr. the data showed that hdpp expression was higher in the trachea, lung, spleen, kidney and brain in both lines, and the expressions were relative higher in trachea and kidney in line compared with that in line ( fig g) . in order to determine the effective infection of mers-cov in different lines of transgenic mice, mice in each line were infected with mers-cov and the changes of body weight were monitored. the results showed that all mice in line , and survived with no significant changes in body weight but all mice in line died by day following mers-cov inoculation (fig h) . in order to confirm the virus replications in transgenic mice, three mice of each line were sacrificed on day after mers-cov infection for the detection of viral titer and viral antigen expression in lungs. as expected, compared with high level of lung viral titers in line mice, no viral titer were detected (fig i) in lungs of line , and mice, which also confirmed by the negative results of viral antigen expression detected by immunohistochemistry in lung (data not shown). wild-type mice, such as balb/c, /svev and stat mice, are not permissive to mers-cov infection, and syrian hamsters do not support replication of mers-cov [ ] [ ] [ ] [ ] . macaques have been confirmed to develop mild to marked interstitial pneumonia upon mers-cov infection [ ] , but fail to progress to the extent observed in human subjects. marmoset model, which has the same interaction characteristic between its dpp and mers-cov spike protein, was partially lethal and developed a progressive severe pneumonia after mers-cov infection [ ] . in this study, we found that hdpp transgenic mice infected with mers-cov exhibited decreased activity and significant weight loss from day after infection, and all of the infected mice died by day (fig a and b) . importantly, symptoms of neural defects with paralysis were observed on day in the infected transgenic mice. compared to the sham-infected group (fig a, d, g and j) , the histopathological analysis of the tissues in transgenic mice on days and after mers-cov infection showed the presence of inflammatory tissue damage in the kidney, liver and spleen, with mild inflammatory responses in the lungs but no significant changes in the intestines. on day after inoculation, the hdpp transgenic mice exhibited mild inflammation in the lungs with focal exudation and hemorrhage (fig b) , and by day , the damage was more severe, with evidence of diffused alveolar damage, alveolar septal thickening, hemorrhage and activated macrophage infiltration (fig c) . in the kidneys, mild interstitial inflammation with inflammatory cell infiltration was observed in the interstitium and exudates of renal tubules on day (fig e) . by day , more renal tubular epithelial cells were injured, with evidence of focal hemorrhage in the renal interstitium (fig f) . in the liver, mild to moderate liver damage was seen in the transgenic mice infected with mers-cov on day , including scattered hepatocyte necrosis and numerous activated kupffer cells and macrophage infiltrates in hepatic sinusoid (fig h) , and on day , fatty change in hepatocytes with less hepatocyte necrosis was observed (fig i) . in spleens, necrosis of splenic cells and increased reticulum cells in red pulp with significant hemosiderin deposition was observed on both day and day (fig k and l) . typical characteristics of viral encephalitis were observed in the brain at day , with perivascular cuffs ( fig m) and neuronal cell necrosis in the cerebral cortex (fig n) , including hippocampal neurons (fig o) . there was no apparent damage in the intestines after mers-cov infection on day or day (data not shown). to evaluate mers-cov infection in the hdpp transgenic mice, distribution of the viral antigen np protein was assessed in mouse tissues by immunohistochemical staining. the results showed that type i and type ii pneumocytes in the lungs and renal tubular epithelial cells in the kidneys expressed viral antigen on day after mers-cov infection and that the expression was more abundant on day , and no viral antigen was detected in sham-infected group ( fig a- f) . strong viral antigen expression was evident not only in neuronal cell bodies, but also in dendrites, axons and some microglia in the cerebral cortex (fig h) including the hippocampus (fig i) . no viral antigen expression was observed in the brains of mice in the shaminfected group (fig g) . viral rna copies in lung and brain were detectable on day postinfection and increased to a higher level on day (fig j) . lower level of viral rna copies can be also detected in kidney collected on day . in order to further confirm the effective replication of mers-cov, the dynamic changes of viral titer in organs were detected. the results showed that on day after infection, only lower viral titer was detected in lung and not detectable in other organs. however, on day , viral titer was also detected in brain. on day and day , increased viral titers were detected in both lung and brain, and lower level viral titer in kidney was detectable (fig k) . these results indicate that the transgenic mice had been successfully infected and that mers-cov exhibited cell and tissue tropism especially for pneumocytes and neurons. furthermore, synapses may be one of the structures by which viruses diffuse through the brain after mers-cov infection. to date, only limited data on the immune response of mers patients are available [ ] . because of the clinical similarity between the symptoms of mers-cov and those of patients infected with sars-cov [ ] , the aberrant immune response may be related to the pathogenesis of mers-cov infection. in our mouse model, an aberrant inflammatory response was confirmed by infiltration of neutrophils in the lung, liver and spleen (fig a- f ) and macrophages in the lung, liver and kidney (fig g- l ). in addition, deposition of hemosiderin in the spleen (fig k and l ) indicated an increase in activated macrophages, and a systemic inflammatory response after virus infection was demonstrated by a significant increase in cytokines such as ifn-γ, ip- , and il- in the serum on day after mers-cov infection (fig m) . although several species, such as rhesus macaques and common marmosets are reported to be sensitive to mers-cov infection and to develop mild to marked broncho-interstitial pneumonia, small animal models are urgently needed to study the pathogenesis of this disease and evaluate the effects of vaccines and antiviral agents. a mers-cov-infection mouse model was generated by transducing mice with a recombinant non-replicating adenovirus expressing hdpp ; however, while the mice exhibit viral antigen expression and progress to interstitial pneumonia, there is no mortality after virus infection [ ] . although a transgenic mouse model expressing human dpp was also established, and its immune response was studied after infection with mers-cov [ ] , the transgenic mice in the study died on day with only progressive pneumonia and mild perivascular cuffing in brain, and no neurological disorder or other multi-organ damage was observed. different from the aforementioned transgenic mice, our hdpp transgenic mice experienced a longer incubation period post-infection and developed progressive pneumonia and neurological disorders accompanied by histological damage to the lungs, brain, spleen and liver, which more closely resembles the clinical cases. the results from this study indicate that mers-cov infection was lethal in hdpp transgenic mice with higher transgene copy number, while lines with lower levels of hdpp expressed especially in trachea had no significant clinical symptoms (data not shown), suggesting that the copy number of the transgene was closely related to the efficiency of mers-cov infection. although the expression level of optimized hdpp was not highest in the brains, the tissues had higher viral titers following mers-cov infection, revealing the tropism of mers-cov for both lungs and brains, tissues in which pneumocytes and neurons may be the main target cells (fig ) . however, mers-cov infection in brain via blood or olfactory nerves needs to be further studied due to the strong expression of viral antigens in dendrites and axons. although few autopsy reports exist on fatal mers-cov cases and atypical pneumonia and respiratory failure are suspected the causes of death [ ] , other types of extrapulmonary organ dysfunction have also been documented in mers critically ill patients with abnormal clinical manifestations [ ] [ ] [ ] . for example, acute renal failure was described in a number of mers cases [ ] [ ] [ ] [ ] [ ] . in addition, renal epithelial cells may produce almost -fold more infectious mers-cov progeny than bronchial epithelial cells [ ] . more recently, severe neurologic syndrome including altered level of consciousness from confusion to coma, ataxia and focal motor deficit was also reported in three critically cases by balkhy h et al [ ] . according to the neurologic manifestations and distinct imaging patterns, an important question was raised on the pathogenic mechanisms that underlie the occurrence of neurologic injury in patients. it has been studied that corona virus such as sars-cov are generally known for causing respiratory illness, and the strong tropism of sars-cov to cns and causing neuronal injury had also been demonstrated both in clinical and experimental studies [ ] [ ] [ ] [ ] [ ] , which suggested the possible relations of mers-cov infection with brain injury. as to the extensive expression of hdpp in the lung, kidney, placenta, liver, skeletal muscle, brain, endothelium, pancreasour and t cell in human, our globally expressed hdpp transgenic mouse manifested with multiorgan damage infected with mers-cov could be a suitable model for the pathogenic mechanisms study. systemic inflammation is believed to be a primary reason for the severe outcome in mers--cov infections [ , ] . although the transgenic mice model died after mers-cov infection with multi-organ damages, it is still uncertain whether these mice are mainly dying from lung damage. in our transgenic mice, aberrant immune response such as elevated ip- , ifn-γ and il- , which are closely related to acute virally-mediated lung injury [ ] [ ] [ ] [ ] , was also a specific manifestation after mers-cov infection. so, as to the mechanism of lethal which was due to the virus replication directly or induced by the aberrant inflammatory response need to be further studied. the mechanism of mers-cov infection caused death is complex and complicated. aberrant immune response after mers-cov infection may be one of main reasons which need to be further studied. we have added more discussions on this issue in the revised manuscript. in summary, the transgenic mouse model developed in this study was efficiently infected by mers-cov and exhibited severe acute respiratory injury and considerable extrapulmonary organ damage. this model will be useful for studying the pathogenesis of mers-cov infection and evaluating the efficacy of mers vaccines and therapeutic agents. molecular pathology of emerging coronavirus infections state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans hospital outbreak of middle east respiratory syndrome coronavirus severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) host species restriction of middle east respiratory syndrome 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syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h n virus infection treatment with interferon-alpha b and ribavirin improves outcome in mers-cov-infected rhesus macaques pathogenesis of middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection latest outbreak news from promed-mail: novel coronavirus-middle east clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection possible central nervous system infection by sars coronavirus detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis multiple organ infection and the pathogenesis of sars pathology and pathogenesis of severe acute respiratory syndrome susceptibility of human and rat neural cell lines to infection by sars-coronavirus early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus severe acute respiratory syndrome in children interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome therapy of h n pneumonia: current perspectives key: cord- -iuzfexig authors: rasmussen, sonja a.; gerber, susan i.; swerdlow, david l. title: middle east respiratory syndrome coronavirus: update for clinicians date: - - journal: clinical infectious diseases doi: . /cid/civ sha: doc_id: cord_uid: iuzfexig although much recent focus has been on the recognition of ebola virus disease among travelers from west africa, cases of middle east respiratory syndrome coronavirus (mers-cov), including travel-associated cases, continue to be reported. us clinicians need to be familiar with recommendations regarding when to suspect mers-cov, how to make a diagnosis, and what infection control measures need to be instituted when a case is suspected. infection control is especially critical, given that most cases have been healthcare-associated. two cases of mers-cov were identified in the united states in may ; because these cases were detected promptly and appropriate control measures were put in place quickly, no secondary cases occurred. this paper summarizes information that us clinicians need to know to prevent secondary cases of mers-cov from occurring in the united states. although much recent focus has been appropriately placed on the recognition of ebola virus disease in travelers returning from west africa, the recent increase in cases of middle east respiratory syndrome coronavirus (mers-cov) infection (including travelassociated cases) is also of concern [ , ] . mers-cov is a novel virus first reported in saudi arabia in . as of february , a total of laboratoryconfirmed cases of mers-cov infection, including at least deaths ( . %), have been confirmed by the world health organization (who) (figure ) [ ] . between august and february , there have been cases (and deaths) confirmed by who, with an additional cases and deaths pending who confirmation. all cases reported thus far have had a direct or indirect link through travel or residence to countries in or near the arabian peninsula (saudi arabia, united arab emirates, qatar, oman, jordan, kuwait, iran, lebanon, and yemen); at least countries (including the united states) have had travel-associated cases ( figure ). most mers-cov patients have had fever, cough, and shortness of breath, often leading to severe respiratory complications (eg, pneumonia or acute respiratory distress syndrome) [ , ] ; some cases with mild or no symptoms (often tested as part of a contact investigation) have also been reported [ , ] . other reported symptoms include chills/rigors, headache, cough, dyspnea, myalgia, sore throat, coryza, dizziness, nausea/vomiting, diarrhea, and abdominal pain. laboratory findings seen in some patients include leukopenia, lymphopenia, thrombocytopenia, thrombocytosis, and elevated levels of lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase [ , ] . in some mers-cov patients, coinfection with other respiratory pathogens has been reported [ , ] ; thus, identification of an alternative respiratory diagnosis should not rule out the possibility of mers-cov. the median age of persons with laboratory-confirmed mers-cov infection is years, approximately % have been males, and approximately % have been healthcare workers [ ] . early on in the outbreak, a predominance of male patients and those with comorbidities was noted, and most cases had severe illness. however, as additional data are collected, the full spectrum of illness is becoming more clear, with a decrease in the percentage of severe cases and increase in asymptomatic cases, as well as a decline in the male-to-female ratio, median age, and case-fatality rate [ ] . mers-cov transmission is not fully understood; however, many cases have had exposure(s) to healthcare settings during the days before symptom onset (the median incubation period is approximately days, with a range of - days). recent data highlight the likely role of camels as a source of human infection in some cases (eg, mers-cov has been found in camels in the region; mers-cov gene sequences in camels are similar to those in humans; camels have had antibodies to mers-cov or a mers-cov-like virus, suggesting previous mers-cov infection; and full gene sequencing has linked mers-cov identified in a camel to a patient who died of mers-cov) [ ] [ ] [ ] [ ] [ ] [ ] . on may and may , cases of mers-cov infection were identified in indiana and florida, respectively [ , ] . both us cases were healthcare workers in saudi arabia and presented to hospitals in the united states with mild to moderate, nonspecific findings, where they were identified as having mers-cov by astute clinicians sensitized to the need to identify and test returning travelers with respiratory illnesses. both patients were appropriately isolated and no secondary cases were identified despite extensive follow-up. these cases emphasize the importance of us clinicians' becoming familiar with the clinical and epidemiologic features and actions needed to detect and manage mers-cov patients. the number of mers cases has continued to increase; therefore, the risk of exportation of cases to the united states mandates that clinicians need to continue to be vigilant. the centers for disease control and prevention (cdc) recommends evaluation for mers-cov infection for the following patients [ ] : . patients with fever and clinical or radiographic evidence of pneumonia or acute respiratory distress syndrome and either a history of travel from countries in or near the arabian peninsula (bahrain; iraq; iran; israel, the west bank, and gaza; jordan; kuwait; lebanon; oman; qatar; saudi arabia; syria; the united arab emirates; and yemen) within days before symptom onset, or close contact with a symptomatic traveler who developed fever and acute respiratory illness within days after traveling from countries in the region. . patients who are a member of a cluster of patients with severe acute respiratory illness (eg, fever and pneumonia requiring hospitalization) of unknown etiology in which mers-cov is being evaluated, in consultation with state and local health departments. . patients with fever and symptoms of respiratory illness (eg, cough, shortness of breath) who have a history of being in a healthcare facility (as a patient, worker, or visitor) within days before symptom onset in a country or territory in or near the arabian peninsula in which recent healthcare-associated cases of mers-cov have been identified. laboratory testing for suspected mers-cov cases in the united states can be performed using the cdc's real-time reverse transcription polymerase chain reaction assay [ ] . testing of multiple specimens from different sites (ie, nasopharyngeal swab, oropharyngeal swab, sputum, serum, and stool/rectal swab) at different times after symptom onset is recommended to maximize the probability of mers-cov detection. lower respiratory tract specimens (eg, sputum or bronchoalveolar lavage) and serum should be tested when possible because mers-cov infection has sometimes been detected in lower respiratory specimens or serum when upper respiratory specimens had tested negative, including in the us cases [ , ] . most state health department laboratories in the united states are approved for mers-cov testing; clinicians should contact their state or local health department when evaluating a patient for mers-cov infection. if testing is not available through coordination with state and local health departments, samples can be sent to the cdc for testing [ ] . serologic testing, which is useful for making a retrospective diagnosis, is only available in the united states at the cdc. healthcare-associated infections have played a major role in the transmission of mers-cov [ , , [ ] [ ] [ ] [ ] . based on the experience with severe acute respiratory syndrome (sars) coronavirus patients and early data from mers-cov, strict infection control practices prevent spread of infection [ ] . thus, the cdc recommends implementation of screening and triage procedures for early recognition of potentially infected patients and prompt institution of infection control measures, including standard, contact, and airborne precautions (airborne precautions include care in an airborne infection isolation room, eye protection with goggles or face shield, and respiratory protection at least as protective as a fit-tested n filtering facepiece respirator that has been national institute for occupational safety and health certified), to prevent secondary cases in healthcare workers or other patients [ ] . healthcare workers exposed to aerosol-generating procedures (eg, sputum induction, intubation, and airway suctioning) appear to be at particularly high risk [ ] . a high degree of respiratory protection (ie, including airborne precautions) is currently recommended by the cdc because data on mers-cov transmission are limited; these guidelines are similar to what was recommended during the sars response and will be updated as additional information becomes available. no specific vaccine or treatment is currently available for mers-cov. many hospitalized patients become severely ill, with respiratory failure followed by multiorgan failure [ ] . clinical management focuses on supportive care [ , , ] . in a small retrospective cohort study, severely ill patients with mers-cov were treated with a combination of ribavirin and interferon alfa- a, vs patients in a comparison group. improved survival in treated patients was noted at days, but the results at days were not statistically significant. a randomized controlled trial will be needed to determine if this treatment is of benefit [ ] . no restrictions on travel to or from the arabian peninsula are recommended [ ] . however, the cdc recommends that travelers protect themselves from mers-cov exposure by washing their hands frequently and avoiding close contact with ill persons. given the emerging data on camels as a reservoir, the world health organization and the cdc recommend general hygiene measures (including regular handwashing before and after touching animals and avoiding contact with sick animals) for persons visiting farms, markets, or other places where animal contact is possible [ ] . consumption of raw or undercooked animal products, including camel milk, should also be avoided. additional precautions that are recommended for persons at high risk for severe mers ( people with diabetes, kidney failure, chronic lung disease, or weakened immune systems) include avoiding contact with camels as well as consumption of raw camel milk and urine. in addition, people who are traveling to the region to provide healthcare services should review the cdc's infection control guidelines for mers [ ] . travelers from the region with onset of fever or respiratory symptoms during their trip or within days of leaving the region should seek medical care. before presenting for care, patients should inform the healthcare facility of their recent travel so appropriate isolation measures can be implemented. to prevent further introduction of cases of mers-cov into the united states, the cdc has developed guidance for us travelers to countries in or near the arabian peninsula about mers-cov, including information aimed at those attending mass gatherings, such as the hajj and umrah [ ] , and public health messages about mers have been posted at us airports. the cdc has also conducted mers outreach with community-based organizations, travel agencies, institutes of higher learning, and businesses for travelers to the arabian peninsula. the cdc has also shared guidance to rapidly identify mers-cov cases among travelers to the united states with airlines, us customs and border protection, and the us transportation security administration. the cdc is working with international partners to conduct studies aimed at better understanding mers-cov transmission and risk factors for illness in community and hospital settings. as new information becomes available, the cdc will update its recommendations [ ] . mers-cov and ebola virus disease remind us all of the interconnectedness of the united states to the rest of the world. with the increase in global travel, we must remain alert for emerging infectious diseases to protect us residents and the world from these public health threats. disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention (cdc). financial support. all authors completed this work as part of their official duties at the cdc. potential conflicts of interest. all authors: no potential conflicts of interest. update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities world health organization. coronavirus infections-disease oubreak news epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome coronavirus disease in children state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans enhanced mers coronavirus surveillance of travelers from the middle east to england clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus: epidemiology and disease control measures isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-to-human transmission of mers coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome (mers): information for health providers real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus an observational, laboratorybased study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia middle east respiratory syndrome coronavirus: implications for health care facilities hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infections in health care workers hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study acknowledgments. we acknowledge huong pham, jessica rudd, aaron curns, and rebecca dahl for their assistance with collection and compilation of epidemiologic data on middle east respiratory syndrome cases. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -mr authors: oh, myoung-don; park, wan beom; park, sang-won; choe, pyoeng gyun; bang, ji hwan; song, kyoung-ho; kim, eu suk; kim, hong bin; kim, nam joong title: middle east respiratory syndrome: what we learned from the outbreak in the republic of korea date: - - journal: korean j intern med doi: . /kjim. . sha: doc_id: cord_uid: mr middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in . the korea outbreak of merscov involved cases, including fatalities. a total of % of transmission events were due to five superspreaders, and % of the mers cases were the patients who had been exposed in nosocomial transmission at hospitals. the epidemic lasted for months and the government quarantined , individuals for days to control the outbreak. this outbreak provides a unique opportunity to fill the gap in our knowledge of mers-cov infection. therefore, in this paper, we review the literature on epidemiology, virology, clinical features, and prevention of mers-cov, which were acquired from the korea outbreak of merscov. middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in september [ ] . since then, as of january , the world health organization (who) has been notified of , laboratory-confirmed cases of mers-cov infection, including at least deaths, from countries (fig. ) [ ] . most of mers-cov human infections have occurred in the arabian peninsula. saudi arabia reported , cases, including deaths (fatality rate, . %) [ ] . a recent article summarized the current knowledge on the epidemiology, virology, human pathogenesis, clinical management, and prevention of mers-cov infection [ ] . another comprehensive review article on risk factors and determinants of transmission of mers-cov from human to human in the community and hospital settings has been published recently [ ] . public health england also published an extensive review of literature for clinical decision-making support for treatment of mers-cov patients [ ] . the korea outbreak of mers-cov is the largest outside the arabian peninsula and provides a unique opportunity to fill the gap in our knowledge about mers-cov infection. since it has now been almost years since the mers outbreak in the republic of korea, we reviewed the literature to summarize the lessons we have learned from the korea outbreak. the korean journal of internal medicine vol. , no. , march fields. as of january , a total of articles were retrieved, and of them were written in english. the full texts of the english articles were reviewed and critically assessed. the korea outbreak of mers-cov resulted in cases including fatalities [ ] . the index case was a returning traveler from the middle east. the infection had spread within the hospital, and subsequently to other hospitals because of patient movement, resulting in nosocomial transmission at clinics and hospitals. the epidemic lasted for months, with the government declaring a "virtual" end to the epidemic on july , . in order to control the outbreak, the government quarantined , individuals for days, and the economic loss was estimated at . trillion korean won ( . billion us dollars) [ ] . the first patient (index case) with mers-cov infection was a -year-old korean man returning from the middle east. he ran a commercial greenhouse business in bahrain. he flew from seoul to bahrain on april , , and traveled through the united arab emirate and the kingdom of saudi arabia, and came back to seoul on may , . during his -day business trip, he did not visit a camel farm or a hospital. he did not eat camel meat or any other camel products. he did not remember meeting any persons with respiratory symptoms [ ] [ ] [ ] [ ] . seven days after returning home, on may , he developed fever and myalgia. he visited a local clinic on may , , and . his symptoms, however, did not improve and a new non-productive cough developed on th may. he visited pyeongtaek st. mary's (ptsm) hospital ( km south from seoul), a secondary hospital in gyeonggi province. his chest x-ray showed a consolidation in the upper zone of right lung, and he was admitted to the hospital for pneumonia on may . ceftriaxone and amikacin were started, but his pneumonia progressed despite the antibiotic treatment. he was transferred to , reported to who as of november , republic of korea other countries saudi arabia the chain of transmission was identified in all mers patients [ , ] . of the healthcare institutions that had taken in mers-cov-infected patients, nosocomial transmission occurred in hospitals and four clinics (plus two ambulances) [ , , ] . the epidemiological characteristics are summarized in table [ ] . the epidemic curve is shown in fig. . during his admission from may to at ptsm hospital, the first patient was unknowingly spreading mers-cov. patients, visitors, and healthcare workers (hcws) were exposed to mers-cov. therefore, by may , when the korea cdc noticed the importation of mers-cov into the republic of korea, individuals staying on the same floor as the first case had already been exposed to mers-cov. as a result, a total of mers cases occurred at ptsm hospital. of them, patients developed symptoms within days after exposure, suggesting they were first-generation cases; the remaining patients may be second-generation cases [ , ] . the mode of transmission of mers-cov at ptsm hospital remains uncertain. in addition to family members of the first patient, only one patient shared the same room (# ) with him; all the other cases stayed in other rooms, which were located > m apart from room # . all patient rooms were equipped with an air ventilating system except for room # . a recent study using a multi-agent modeling framework suggested the transmission occurred via a long-range airborne route or via a combination of a long-range airborne route and direct contact transmission [ ] . a chest computed tomography (ct) scan taken in the early stages (day or ) of illness showed very small, ground glass nodules in the periphery of the lungs, suggesting inhalation of airborne particles [ ] . many individuals had been exposed to the superspreader- at the er, and contact tracing identified patients, an estimated visitors, and hcws as contacts of superspreader- . of them, were quarantined and were under active monitoring. as a result of exposure to superspreader- , had laboratory-confirmed mers-cov infection, and an additional were secondarily infected from them [ , ] . the index case (superspreader- ) for the daejeon ( km south from pyeontaek city) outbreak was infected at ptsm hospital. he developed symptoms on may and was admitted to daechung hospital on may . as his pneumonia progressed despite the treatments, he was transferred to geonyang hospital on may . a total of secondary cases ( in daechung hospital and in geonyang hospital) were detected. although superspreader- caused the two hospital outbreaks, the me- dian time of incubation ( . days vs. . days), secondary attack rates ( . % vs. . %), and case fatality rate ( . % vs. . %) were different between the two outbreaks. the high fatality rate was associated with pre-existing pneumonia or poor underlying pulmonary function [ ] [ ] [ ] . a -year-old male, who had been exposed to the first index case on may , developed back pain on may . he is a son of the third mers case. his sister had also been exposed to mers- epidemiological characteristics have been reported by several groups (table ) [ , , , , , [ ] [ ] [ ] [ ] . the defining epidemiological characteristics were superspreading events at hospitals [ ] [ ] [ ] . early superspreading events generated a disproportionately large number of second-ary infections, and the transmission potential decreased sharply in subsequent generations [ , ] . a total of % of transmission events were due to five superspreaders, and % of the mers cases were in-patients who had been exposed within the hospitals [ ] . the spreaders transmitted mers-cov from days to of their illness (median, days), and the number of patients infected by each spreader ranged from to (interquartile range, to ) [ ] . the median incubation period and serial interval was days and . days, respectively [ , ] . r ranged from . to . , higher than the previous estimate of < [ ] . it was estimated at . for the outbreak cluster in ptsm hospital, and . for the outbreak in smc [ ] . a mathematical modeling study demonstrated that although r < overall, cluster sizes of over cases are not unexpected for mers-cov infection [ ] . compared with the non-spreaders, the spreaders had a higher frequency of fever (≥ . °c) and chest infiltrates in more than three lung zones, and longer non-isolated in-hospital days [ ] . the spreaders had higher viral load in sputum samples with the cycle thresholds for the upe and orf a genes of . vs. . and . vs. . , respectively. the spreaders with more than three transmissions had higher numbers of contacts and er visits [ ] . a brief exposure of a -minute stay and minutes of talking was enough for the transmission of mers-cov [ ] . the whole genome sequences of mers-cov isolated from the korea outbreak were reported [ , [ ] [ ] [ ] [ ] . the virus most similar to the korean isolates was a camel virus (camel/riyadh/ry / ) that belonged to lineage , a recombinant of lineage and [ , ] . viruses from lineage had been predominant in saudi arabian camels since november , but human viruses of this lineage were reported from february [ ] . a phylogenetic study of spike glycoprotein genes also showed the korean strains were closely related to the viral strains from riyadh, saudi arabia [ ] . in a molecular study, of mers-cov genome had an i t mutation and one d g mutation in the receptor binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, cd [ ] . heterogeneity analysis revealed the combined frequency of i t and d g was high ( . %), although the frequency of both mutations varied greatly among specimens [ ] . another genome sequence study also reported deletions of and nucleotides between orf and the e protein, suggesting that the virus might be defective in its ability to package mers cov [ ] . a microevolution study found no evidence of changes in the evolutionary rate [ ] . however, whether the transmissibility and virulence of the korean isolates are different from those of the previous isolates from the kingdom of saudi arabia remains to be determined by phenotypic assays. a viral shedding study showed that the copies of mers-cov rna detected by real-time reverse transcription polymerase chain reaction (rrt-pcr) in respiratory samples peaked during week , and the median value was . log in the severe group and . log in mild cases [ ] . the study also showed that viral titers were higher in throat samples than in nasopharyngeal samples. another study also demonstrated higher viral loads were associated with severity and mortality [ ] . after mers-cov infection, antibody responses developed by the third week of illness in most patients [ ] . seroconversion rates increased with the increase of dis-ease severity. in a serologic study of mers patients, the seroconversion rate was % in asymptomatic infection, . % in symptomatic infection without pneumonia, . % in pneumonia without respiratory failure, and % in pneumonia with respiratory failure [ ] . the serological response remained detectable for > months in all survivors ( / ) who had severe disease. antibody titers were not detectable in four of six patients who had mild pneumonia, suggesting that mers-cov seroepidemiological studies may underestimate the extent of mild and asymptomatic infection [ ] . the korean society of infectious diseases compiled the clinical data of the mers patients [ ] . the median age was years (range, to ). the male to female ratio was : . the most common coexisting medical conditions were hypertension ( . %), diabetes ( . %), solid organ malignancy ( . %), and chronic lung disease ( . %). patients with mers-cov infection developed a spectrum of illnesses, ranging from asymptomatic to fulminant pneumonia with fatal outcome. the respiratory symptoms were similar to other acute viral respiratory infections. at the time of presentation, fever had developed in . %, cough in . %, and sputum in . % of patients. symptoms of upper respiratory-tract infections were infrequent: sore throat was present in . % and rhinorrhea in . % of patients. gastrointestinal symptoms were also observed: diarrhea was present in . %, nausea or vomiting in . %, and abdominal pain in . % of patients. diarrhea may be due to the side effect of lopinavir/ritonavir, as % of the patients received antiviral drugs for mers-cov treatment [ ] . in the early stages of illness, cough and sputum were not prominent even in patients who later developed overt pneumonia [ ] . at admission, % ( / ) of patients had abnormalities on chest radiographs, while . % ( / ) of them developed the abnormalities during the course of the disease [ ] . sudden progression of pneumonia occurred around day of illness [ , ] . predictive factors for development of pneumonia included older age, high fever, thrombocytopenia, lymphopenia, c-reactive protein (crp) ≥ mg/dl, and a high viral load www.kjim.org https://doi.org/ . /kjim. . in sputum (threshold cycle value of rrt-pcr < . ) [ ] . forty-five patients ( . %) were treated with mechanical ventilation and patients ( . %) needed extracorporeal membrane oxygenation [ ] . the typical course of pneumonia is shown in fig. . acute kidney injury (aki) developed in . % of the patients, . % ( / ) had proteinuria, and . % ( / ) had hematuria. fifteen patients were treated with renal replacement therapy [ ] . old age is an independent risk factor for the occurrence of aki [ ] . neuromuscular manifestations were not uncommon; hypersomnolence, weakness and tingling in the extremities were reported during the treatment of mers, suggesting guillain-barré syndrome or virus-related sensory neuropathy [ ] . serial changes in chest radiographs was reported in five patients [ ] . chest ct scans revealed rapidly developed multifocal nodular consolidations with ground-glass opacity halo and mixed consolidation, mainly in the dependent and peripheral areas [ ] . there are few laboratory findings specific to mers-cov infection, although monocytosis with normal white blood cell count and low crp level were more common in mers patients at initial presentation [ , ] . the guidelines for the molecular diagnosis of mers-cov infection have been published by the korean society for laboratory medicine [ , ] . specimen type and quality is important in the laboratory diagnosis of mers-cov infection. lower respiratory-tract samples, such as sputum and tracheal aspirates, have higher viral loads than upper respiratory-tract samples [ ] . however, mers patients may not shed the virus during the early stage of their illness. therefore, initial negative results should not rule out the possibility of mers [ ] , and patients suspected of having mers-cov infection should be retested using a lower respiratory-tract sample [ , ] . when sputum cannot be obtained, throat swabs may be an alternative source of diagnostic samples [ ] . special attention should be given to diagnosing mers-cov infection in immunocompromised patients, as they may present with atypical features, such as a longer incubation period, a longer period from initial pcr positivity to symptom onset, and persistent viral shedding [ ] . an antiviral treatment guideline has been published by the writing committee with support from the korean society of infectious diseases and the korean society for chemotherapy [ ] . the guideline recommended a tri- ple combination regimen of type interferon, ribavirin, and lopinavir/ritonavir for to days. however, the guideline was mostly based on expert opinions and further study for the optimal antiviral treatment in mers is warranted. the median duration of fever was days. the median time to negative conversion of mers-cov in sputum samples by rrt-pcr was days (range, to ) [ ] . the median time from symptom onset to death was days. the case fatality rate was . % ( / ) (fig. ) . the in-hospital mortality, -day mortality, and -day (from symptom onset) mortality were . % ( / ), . % ( / ), and . % ( / ), respectively [ ] . host factors associated with mortality were old age (> years), smoking history, pre-existing pneumonia, abnormal renal function, and comorbid conditions [ , [ ] [ ] [ ] . low albumin, altered mentality, and high pneumonia severity index score at admission were risk factors for mortality [ ] . mers-cov rna in blood samples was detected in % ( / ) of patients at presentation, and it was associated with a worse clinical outcome [ ] . a shorter incubation period was also associated with an increased risk of death [ , ] . a pregnant woman infected with mers-cov in her weeks of gestational age developed dyspnea and her chest radiograph showed diffuse infiltrates in the left lower lung zone. she recovered from the infection and delivered a healthy full-term baby without vertical mers-cov transmission [ ] . a patient developed organizing pneumonia days after the resolution of mers-cov infection. he was successfully treated with corticosteroids [ ] . in a mental health study, . % of , patients who were quarantined showed symptoms of anxiety, and . % reported feelings of anger during the weeks of quarantine [ ] . mental health support, accurate information, and appropriate supplies, including food, clothes, and accommodation, should be provided to isolated persons [ ] . a case of possible transfusion-related acute lung injury following transfusion of convalescent plasma was also reported [ ] . a comprehensive "mers infection prevention and control guideline for healthcare facilities" was drafted by the guideline development committee with generous support from korean academic societies [ ] . the guideline included practical aspects of infection control and prevention based on the experience from the korea outbreak, such as the composition of members for the mers emergency committee, a space plan for an anteroom for donning and doffing, isolation of in-patients and hcws in a hospital affected by an outbreak, and management of the deceased and autopsy [ ] . the hemodialysis unit may become an epicenter for mers-cov outbreak because hemodialysis patients must receive renal replacement therapy even during the outbreak, and the risk of exposure to mers-cov continues. during the korea outbreak, a hemodialysis unit was found to have a mers patient, and a total of patients and hcws were exposed to mers-cov. with support from the korean society of nephrology, the dialysis unit could continue to operate while the exposed patients were isolated in the unit. fortunately, no further transmission occurred at the unit [ ] . after this successful experience, the society published a clinical practice guideline for hemodialysis facilities dealing with mers patients [ ] . the korea outbreak was driven by the superspreaders who visited multiple healthcare facilities; thus, generating a large number of secondary cases. limiting unnecessary contacts with patients with respiratory symptoms in healthcare settings, especially in emergency departments, is of critical importance [ ] . mers-cov could survive for longer than hours at °c and % of relative humidity, suggesting contact or fomite transmission might occur in healthcare settings [ ] . mers-cov was detected by rrt-pcr in specimens taken from the medical equipment [ ] [ ] [ ] . mers-cov was also isolated by cell culture of the air and swab samples taken from a mers isolation unit, suggesting extensive contamination of the isolation unit [ , ] . of the cases, % were infected by undocumented contact between cases (i.e., indirect transmission of mers-cov via environmental contact) [ ] . therefore, fomites with possible mers-cov contamination should be sanitized, and a minimum room ventilation rate of six air changes per hour should be implemented to minimize recirculation of pathogen-bearing droplets. meticulous environmental cleaning may be important for preventing transmission in healthcare settings. there was a case of a -year-old female nurse who was infected with mers-cov during a cardiopulmonary resuscitation lasting hour for a mers patient who had pneumonia and hemoptysis [ ] . serological surveillance conducted after the mers outbreak for asymptomatic infection among hcws involved in the direct care of mers patients showed that . % ( / ) of them were mers-cov igg positive by an indirect immunofluorescent assay. among the hcws who did not use appropriate personal protective equipment (ppe), seropositivity was . % ( / ) compared with % ( / ) in hcws with appropriate ppe use [ ] . another serological survey also showed that none of the hcws were positive for mers-cov immunoglobulin g, although . % ( / ) of the hcws reported experiencing mers-like symptoms while caring for the mers patients [ ] . in a third study, of hcws showed seroconversion by a plaque reduction neutralization test, although % to % of hcws reported mers-like symptoms [ ] . during the outbreak in smc, all hcws assigned to mers patients were screened for mers-cov, regardless of the presence or absence of symptoms. of the hcws, three ( . %) asymptomatic hcws (two nurses and one physician) were found to be mers-cov (+), but they did not transmit the virus to others [ ] . in another study, an asymptomatic nurse without ppe contacted hcws, including close contacts who were exposed within m from the index nurse and were not using ppe. none of the exposed hcws were infected [ ] . however, the potential for transmission from asymptomatic rrt-pcr positive individuals is still unknown. therefore, asymptomatic hcws who are rrt-pcr-positive for mers-cov should be isolated and should not return to work until two consecutive respiratory-tract samples test negative on rrt-pcr [ ] . the government requested the korean society of infectious diseases to participate in the control of the mers outbreak. on june , , the society organized the rapid response team, consisting of infectious-disease doctors and two infection-control professionals [ ] . critical suggestions to prevent future epidemics were made regarding a rapid alerting system for index cases, provision of a sufficient airborne infection isola- the korean journal of internal medicine vol. , no. , march tion facility, education and training of hcws for important infectious diseases, and overcrowding and visitor control in the hospital [ ] . the korea outbreak was the largest outbreak outside of the middle east. researchers had a unique opportunity to compare the nature of mers-cov infection with the middle east experience. the outbreak unveiled the weak points of infrastructure in our medical system, especially of preparedness for emerging global infectious diseases. nosocomial transmission was one of the core features of mers-cov infection. the introspection for loose medical referral systems, overcrowding at the er, a lack of expert resources and infection control infrastructure, and lack of organized preparedness for medical crises prompted the government to reform the healthcare system, and healthcare sectors to invest further in infectious diseases and infection control. although the improvement is still ongoing, the speed and content are still currently insufficient. international cooperation to prepare for and defeat emerging infectious diseases should also be emphasized. lessons we learned from the outbreak are summarized in table . a single, missed case may trigger a huge, nationwide outbreak the first line of defense is not the thermal scanner at the airport. it is doctors in the community clinics/hospitals. due to sudden deterioration of pneumonia around day of illness, patients may visit emergency department, or be admitted to intensive care unit. superspreading events may occur in healthcare settings, especially at the emergency department. patients may transmit mers-cov as early as days after symptom onset. early detection and isolation is of critical importance. aggressive strategy for quarantine maybe necessary, especially when large number of individuals are exposed in the healthcare settings. huge socioeconomic impact with an estimated . billion us dollars isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) [internet]. geneva: world health organization ministry of health kingdom of saudi arabia. mers-cov daily update ministry of health kingdom of saudi arabia, c middle east respiratory syndrome middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients uk): public health england middle east respiratory syndrome coronavirus outbreak in the republic of korea the korean middle east respiratory syndrome coronavirus outbreak and our responsibility to the global scientific community the clinical and virological features of the first imported case causing mers-cov outbreak in south korea epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital middle east respiratory syndrome in persons, south korea the first case of the korean middle east respiratory syndrome outbreak south korea coronavirus mers case list: including imported and exported cases. ministry of health & who confirmed data only fl): flutrackers, c mers-cov infection: current status-interactive and infographics probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, : epidemiology, characteristics and public health implications ministry of health and welfare. the mers outbreak in the republic of korea: learning from mers (the white paper) a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of middle east respiratory syndrome coronavirus superspreading event involving persons, korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon hospital outbreaks of middle east respiratory syndrome high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea imported case of mers-cov infection identified in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis clinical and epidemiologic characteristics of spreaders of middle east 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interventions in korea management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance [internet]. geneva: world health organization, c collaborative intervention of middle east respiratory syndrome: rapid response team institutional preparedness to prevent future middle east respiratory syndrome coronaviruslike outbreaks in republic of korea no potential conflict of interest relevant to this article was reported. key: cord- -eujbxdqi authors: ahmed, anwar e.; al‐jahdali, hamdan; alaqeel, mody; siddiq, salma s.; alsaab, hanan a.; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; ali, yosra z.; al otaibi, hazza; aloudah, nouf m.; baharoon, salim; al johani, sameera; alghamdi, mohammed g. title: factors associated with recovery delay in a sample of patients diagnosed by mers‐cov rrt‐pcr: a saudi arabian multicenter retrospective study date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: eujbxdqi background: research evidence exists that poor prognosis is common in middle east respiratory syndrome coronavirus (mers‐cov) patients. objectives: this study estimates recovery delay intervals and identifies associated factors in a sample of saudi arabian patients admitted for suspected mers‐cov and diagnosed by rrt‐pcr assay. methods: a multicenter retrospective study was conducted on patients admitted between september and june and diagnosed by rrt‐pcr procedures to have mers‐cov and non‐mers‐cov infection in which achieved recovery. detailed medical charts were reviewed for each patient who achieved recovery. time intervals in days were calculated from presentation to the initial rrt‐pcr diagnosis (diagnosis delay) and from the initial rrt‐pcr diagnosis to recovery (recovery delay). results: the median recovery delay in our sample was days. according to the multivariate negative binomial model, elderly (age ≥ ), mers‐cov infection, icu admission, and abnormal radiology findings were associated with longer recovery delay (adjusted relative risk (arr): . , . , . , and . , respectively). camel contact and the presence of respiratory symptoms at presentation were associated with a shorter recovery delay (expedited recovery) (arr: . and . , respectively). diagnosis delay is a positive predictor for recovery delay (r = . ; p = . ). conclusions: the study evidence supports that longer recovery delay was seen in patients of older age, mers‐cov infection, icu admission, and abnormal radiology findings. shorter recovery delay was found in patients who had camel contact and respiratory symptoms at presentation. these findings may help us understand clinical decision making on directing hospital resources toward prompt screening, monitoring, and implementing clinical recovery and treatment strategies. laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) has been documented in more than cases worldwide, causing related deaths from september through september . much research evidence is available on factors associated with a poor prognosis in laboratory-confirmed mers-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and non-mers-cov - patients. a high mortality rate was systematically recognized in mers-cov patients of old age, , - severe illness, , - underlying condition, , - and respiratory/gastrointestinal symptoms. however, successful management of mers-cov such as clinical recovery and its predictors has not been given sufficient attention despite the virus having been in circulation since . as per the authors' knowledge, two studies have so far addressed clinical improvement on laboratory-confirmed mers-cov patients. , the first study, shalhoub et al, was based on a case report in which their observations may not be generalized to a wider mers-cov population. the second study, al-turaiki et al, utilized publicly available data from the saudi ministry of health. the major shortcomings in their study were several potential confounding factors such as underlying medical conditions and a primary or secondary mode of mers-cov transmission that had not been included in the analysis. in addition, the recovery delay was not reported in their study, and thus, factors related to the recovery delay were not examined. as of october , , there was no available detailed data on recovery delay of laboratory-confirmed mers-cov and non-mers-cov patients. data on the time intervals between a patient's presentation or admission to a healthcare facility and the first specimen sample have been limited in patients suspected and screened for mers-cov by a real-time reverse-transcription polymerase chain reaction (rrt-pcr) test, as it might correlate with recovery delay intervals. early screening and diagnosis of mers-cov could greatly promote proper control and clinical management of cases, which may reduce the risk of transmission and increase the chance of successful outcomes. [ ] [ ] [ ] this study provides more understanding of how long a period (in days) it may take to recover from mers-cov infection. the authors have studied, retrospectively, a cohort of survivorslaboratory-confirmed mers-cov and non-mers-cov patients-to estimate recovery delay intervals and identify possible associated factors in saudi arabia. the authors assessed whether the time interval between presentation and the initial rrt-pcr diagnosis (diagnosis delay) correlates with the time interval between initial rrt-pcr diagnosis and recovery (recovery delay). we hypothesized that older age, mers-cov infection, icu admission, and abnormal radiology findings might be associated with longer recovery delay. we hypothesized that diagnosis delay might positively correlate with a recovery delay. a multicenter retrospective study reviewed medical records of patients from september to june who were admitted to the hospital and had been diagnosed by rrt-pcr assay for suspected mers-cov to have mers-cov and non-mers-cov infection. the study included patients who were admitted through emergency departments (pediatrics and adults) or patients who were admitted through outpatient clinics. screening referrals for mers-cov was made in accordance with the guidelines set by the saudi ministry of health in standard risk assessment algorithms for identifying and managing mers-cov infection. in the study population, the rrt-pcr was used to detect mers-cov in multiple and/or different clinical specimens, including combined nasopharyngeal and throat swabs, sputum, blood, stool, and endotracheal aspirate (eta). the study gathered data from the two largest hospitals in saudi from patient charts, we collected demographic data: age and gender. elderly age was defined by classifying age into two groups using a cutoff of years (≥ years). the reason behind this classification was to assess the recovery delay for this vulnerable age group, as a previous study reported a high mortality rate in this group. we collected data on route of transmission: camel contact and patient contact. the study authors collected various clinical data: fever (temperature ≥ °c); presence of any of the following respiratory symptoms: cough, bloody cough, shortness of breath, or chest pain; presence any of the following gastrointestinal symptoms: diarrhea, vomiting, or nausea; mers-cov infection; intensive care unit (icu) admission; hospital: kamc-riyadh or kfgh-jeddah; abnormal radiology findings; diabetes; renal disease; and hypertension. recovery delay was calculated as the number of days from the initial rrt-pcr diagnosis (±), which was the date found on the pathology report of the first specimen, to the clinical recovery (recovery delay), based on the date of hospital discharge or date of mers-cov or non-mers-cov infection was ruled out. in some cases, the clinical recovery was verified by taking a sample from different types of specimens at varying times. in all patients with initial rrt-pcr result, the medical records were reviewed from the date of presentation/hospital admission until days after the initial rrt-pcr diagnosis. only patients who achieved recovery were analyzed. the study excluded patients with no available clinical recovery records and no discharge records within days after the initial rrt-pcr diagnosis, as well as patients who had died. the final sample included laboratory-confirmed mers-cov and non-mers-cov patients who had recovered and were identified by reviewing patient charts, hospital discharge records, and medical practitioner notes. the analysis was conducted using ibm statistical package for social sciences (spss) ( ; spss, chicago, il). patients' characteristics were described by count and percent, and mean (± standard deviation) or median where appropriate. time intervals in days from presentation to initial rrt-pcr diagnosis (diagnosis delay) and from initial rrt-pcr diagnosis to recovery (recovery delay) were analyzed by spearman's correlation coefficient. the poisson and negative binomial models were used to model the frequency of recovery delay in days and identify unadjusted and adjusted factors associated with longer recovery delay. goodnessof-fit measures were used to compare and identify the best model. the model with the smaller deviance, larger log likelihood, smaller akaike information criterion, and smaller bayesian information criterion was considered the better model. in all analyses, a p-value of less than % was considered significant. relative risk (rr) and % confidence intervals (ci) were used to assess the strength of association between patients' characteristics and longer/shorter recovery delay. a total of patients, suspected and screened for mers-cov by an rrt-pcr test, were analyzed. the average age was years with age ranges between and years. the median recovery delay in our sample was days. of the sample, . % were male and . % were admitted to icu. fever and respiratory symptoms were common presentations, occurring in . % and . % of the patients, respectively. the chest x-ray and/or ct scan were abnormal in almost half of the samples ( . %). refer to table for other sample parameters. the longer delays in diagnosis were positively correlated with longer recovery delay (r = . ; p = . ). according to univariate negative binomial regression analysis ( a multivariate negative binomial regression analysis (table ) revealed six independent factors that affect the recovery delay. and necessitate mechanical ventilation, which is a risk factor for hospital mortality. patients admitted to icu admission were at higher risk for longer recovery delay. a previous report showing similar findings, longer icu stay, and high mortality rate was reported in mers-cov patients who were admitted to the icu. such patients would benefit from monitoring their responses to medical support and recognizing potential complications at an early stage. we found that camel contact was associated with shorter recovery delay. studies on recovery delay in patients with camel contact as compared to close contact exposure of a confirmed case or other exposure are lacking; however, camel contact has also been linked to lower -and -day mortality rates in mers-cov patients. this important association requires more studies to identify whether camel contact is an independent protective factor of shorter recovery delay. in our study, patients with respiratory symptoms are more likely to experience shorter recovery delay than patients without respiratory symptoms. this finding is probably related to the shorter lag time between symptom onset and diagnosis, in which patients with presence of symptoms could be positively affected by an early diagnosis and thus prompt medical support is deemed necessary. the time interval from presentation to initial rrt-pcr diagnosis (diagnosis delay) was positively correlated with the time interval from initial rrt-pcr diagnosis to recovery (recovery delay). early diagnosis is likely to improve clinical outcomes and reduce the economic and physical burden of a disease. , early diagnosis requires full utilization of hospital resources. individuals at high risk of mers-cov infection should be promptly screened after arrival at the healthcare facility, monitored for progression, and then having a prompt decision made for whether further rrt-pcr testing is needed. the authors noticed the following limitations. first, the study was based on chart reviews, and findings should be interpreted with caution. second, we did not collect information on the type of antiviral treatment or other supportive treatments given after diagnosis which may have affected clinical outcomes. , third, despite this being the first investigation in this population, including a number of potential predictors for recovery delay, additional relevant predictors should be explored, such as the level of camel exposure, for example, hospital-acquired infections. fourth, patients with clinical recovery were identified by reviewing the medical records of the study sample within days after the initial rrt-pcr diagnosis. studies with longer periods of follow-up in a larger population recovering from mers-cov are warranted to assess the long-term successful clinical outcomes. despite the mentioned limitations, data were aggregated directly from medical charts rather than public source databases. this chart review study was based on information from multicenters and a large sample size, and it provides valuable information on factors associated with prolonged or shorter recovery delay of patients suspected and screened for mers-cov by the rrt-pcr test. it is essential to develop interventional programs or guidelines to ensure early diagnosis, as this may reduce recovery delay intervals as well as improve patients' clinical outcomes. this research may enable identification of patients who require receiving appropriate medical support and care according to their illness progression. this also may prevent spread and transmission of the infection as individuals who are still severely ill can be appropriately isolated and managed apart from others who are responding to medical care. the study evidence supports that longer recovery delay was seen in patients with older age, mers-cov infection, icu admission, and abnormal radiology findings in a sample of patients diagnosed by rrt-pcr. recovery delay was significantly shorter in patients who had camel contact and respiratory symptoms at presentation. a prospective study is needed to evaluate the impact of camel exposure on recovery. evidence was found of an increasing recovery delay with a longer diagnosis delay. the findings may help understand clinical decision making as it directs hospital resources toward prompt screening, monitoring, and implementing clinical recovery and treatment strategies. there are no conflict of interests. middle east respiratory syndrome coronavirus (mers-cov): 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survivors recovery from the middle east respiratory syndrome is associated with antibody and t cell responses factors associated with recovery delay in a sample of patients diagnosed by mers-cov rrt-pcr: a saudi arabian multicenter retrospective study http://orcid.org/ - - - key: cord- -xxc vdnt authors: ahmed, anwar e.; al-jahdali, hamdan; alshukairi, abeer n.; alaqeel, mody; siddiq, salma s.; alsaab, hanan; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; aloudah, nouf m.; baharoon, salim; alsalamah, majid a.; al johani, sameera; alghamdi, mohammed g. title: early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: xxc vdnt background: the rapid and accurate identification of individuals who are at high risk of middle east respiratory syndrome coronavirus (mers-cov) infection remains a major challenge for the medical and scientific communities. the aim of this study was to develop and validate a risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. methods: a two-center, retrospective case–control study was performed. a total of patients with confirmed pneumonia who were evaluated for mers-cov infection by real-time reverse transcription polymerase chain reaction (rrt-pcr) between september , and june , at king abdulaziz medical city in riyadh and king fahad general hospital in jeddah, were included. according to the rrt-pcr results, patients were positive for mers-cov and were negative. demographic characteristics, clinical presentations, and radiological and laboratory findings were collected for each subject. results: a risk prediction model to identify pneumonia patients at increased risk of mers-cov was developed. the model included male sex, contact with a sick patient or camel, diabetes, severe illness, low white blood cell (wbc) count, low alanine aminotransferase (alt), and high aspartate aminotransferase (ast). the model performed well in predicting mers-cov infection (area under the receiver operating characteristics curves (auc) . ), on internal validation (auc . ), and on a goodness-of-fit test (p = . ). the risk prediction model, which produced an optimal probability cut-off of . , had a sensitivity of . and specificity of . . conclusions: this study provides a simple, practical, and valid algorithm to identify pneumonia patients at increased risk of mers-cov infection. this risk prediction model could be useful for the early identification of patients at the highest risk of mers-cov infection. further validation of the prediction model on a large prospective cohort of representative patients with pneumonia is necessary. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in . the diagnosis of this infection remains complex (al johani and hajeer, ; sung et al., ; ahmed, a) and it has a high fatality rate (ahmed, b, c; aleanizy et al., ; sherbini et al., ; kim et al., ) . the early detection and identification of individuals at high risk of a disease is the most effective strategy to improve patient clinical outcomes (ahmed, a) and reduce the costs of testing, both physical and economic (ahmed et al., (ahmed et al., , . the real-time reverse transcription polymerase chain reaction (rrt-pcr) has been found to be valid and accurate for the evaluation of respiratory swabs, sputum, and other endotracheal aspirate material (corman et al., a, b) . however, although rrt-pcr is the most accurate and sensitive test available at the authors' centers, absolute identification of mers-cov may require multiple clinical specimens from different sources and take days (corman et al., a, b; anon, a) . the saudi ministry of health (moh) has developed mers-cov visual triage guidelines to identify suspected cases (anon, b) . the current guidelines include fever, respiratory symptoms, gastrointestinal symptoms, chronic diseases, and risk of exposure to mers-cov. in clinical practice, identifying high-risk individuals can be challenging, since most laboratory-confirmed mers-cov patients report common clinical risk indices to patients with other respiratory conditions. for instance, respiratory and gastrointestinal symptoms are common for both mers-cov and non-mers-cov patients (mohd et al., ) . thus, further exploration must take place to reduce the risk of mers-cov infection. a risk prediction model is urgently needed to stratify patients with suspected mers-cov. this may decrease the risk of virus transmission to others who are in close contact, for example healthcare workers, patients, and hospital visitors, by allowing the careful management of those who are potentially infected at an early stage of infection. developing a mers-cov prediction model may more efficiently aid physicians in identifying individuals at high risk and selecting the necessary test(s) to improve prevention and control measures. several methodological studies have shown that combining demographic characteristics with clinical, radiological, and laboratory information can improve risk assessment and diagnostic accuracy (ahmed et al., (ahmed et al., , sidransky, ; etzioni et al., ) . these previous studies used a linear combination to develop an algorithm that combines demographic characteristics, symptoms, and clinical, radiological, and laboratory findings to identify the highly suspected mers-cov cases. mers-cov was initially identified in a patient being treated for pneumonia in (zaki et al., ) , and since then, pneumonia and its symptoms have remained common characteristics in mers-cov patients (saad et al., ; choi et al., ) . the aim of this study was to develop and validate a reliable risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. a two-center, retrospective case-control study was conducted utilizing data from king abdulaziz medical city in riyadh (kamc-r) and king fahad general hospital in jeddah (kfgh-jed), saudi arabia. the data were obtained between september , and june , . kfgh-jed experienced a mers outbreak between march and may (oboho et al., ) , and kamc-r experienced a large mers outbreak between june and august (al-dorzi et al., ) . both study centers applied the saudi moh case definitions (anon, b) to identify suspected individuals in the population studied, and rrt-pcr was used as the gold standard test to diagnose mers-cov in multiple and different clinical specimens when necessary. mers-cov-related symptoms were common at both centers. the project received ethical approval from two independent ethics committees: the saudi moh (irb log number - e) and king abdullah international medical research center (study number rc / ), riyadh saudi arabia. during the study, the medical records of subjects who were being assessed by rrt-pcr for suspected mers-cov were reviewed. the suspicion of mers-cov infection at both kamc-r and kfgh-jed was determined based on the saudi moh guidelines (anon, b) . two groups were compared: patients who were positive and patients who were negative for mers-cov infection. in an effort to reduce heterogeneity between the study groups, only subjects with a lung infiltrate on chest x-ray and/or computed tomography (ct) scan of the chest were included in the analysis. thus, subjects who had no available results of a chest x-ray or ct scan of the chest were excluded. the initial screening for suspected mers-cov patients includes pneumonia (anon, b) . most of the patients studied were evaluated for pneumonia immediately after presentation. the study excluded subjects who were less than years of age (pediatrics/children), as defined in the saudi moh guidelines (anon, b) , and excluded subjects who had upper respiratory tract infections (respiratory symptoms, positive or negative mers-cov rrt-pcr, and normal chest x-ray and ct scan of the chest). the final sample comprised a cohort of subjects who had a lung infiltrate on chest x-ray and/or a ct scan of the chest, who were classified according to the results of mers-cov rrt-pcr. the case group consisted of pneumonia patients who were positive for mers-cov infection, and the control group consisted of pneumonia patients who were negative for mers-cov infection. cases were defined as patients with mers-cov pneumonia who had positive mers-cov rrt-pcr on nasopharyngeal aspirate and/ or bronchoalveolar lavage in addition to a lung infiltrate on chest xray and/or ct scan of the chest. controls were defined as patients with respiratory symptoms, a lung infiltrate on chest x-ray and/or ct scan of the chest, pneumonia, and negative mers-cov rrt-pcr result of nasopharyngeal aspirate and/or bronchoalveolar lavage. nineteen predictive variables were included: age, sex, fever (temperature ! c), one composite respiratory symptom (the presence of cough, bloody cough, shortness of breath, or chest pain), one composite gastrointestinal symptoms (the presence of diarrhea, vomiting, or nausea), seven mers-cov potential risk factors (contact with sick patients or camels, severe illness (defined according to the patient's clinical status, 'yes/no', which is based on clinical judgment), diabetes, lung disease, liver disease, renal disease, and heart disease), and seven laboratory measurements (white blood cell (wbc) count (Â /l), platelets (Â /l), creatinine (mmol/l), bilirubin (mmol/l), alanine aminotransferase (alt; u/l), aspartate aminotransferase (ast; u/l), and albumin (g/ l)). the reference ranges for the laboratory measures were as follows: wbc count, - Â /l; platelets, - Â /l; creatinine, - mmol/l; bilirubin, . - . mmol/l; alt, - u/l; ast, - u/l; albumin, - g/l. no serological tests were available at the centers for these patients. stata statistical software release , (statacorp. llc, college station, tx, usa) was used for the data analysis. the sample characteristics were recorded as the frequency and percentage, or as the mean ae standard deviation (sd). laboratory measurements were summarized as the median and th- th percentiles. a subgroup analysis (chi-square test, independent samples t-test, or mann-whitney u-test) was used to identify unadjusted associations between demographic, clinical, and laboratory measurements according to mers-cov status. the performance of each bivariate predictor was further assessed by unbiased estimate, the area under the curve (auc), and its % confidence interval (ci). stepwise binary logistic regression was employed to develop a mers-cov risk prediction model and identify factors that could be used to estimate the probabilities of mers-cov infection. the goodness-of-fit of the final model was tested by hosmer-lemeshow procedure: a large p-value indicates that a model has a good fit. the discrimination ability between high and minimal risk of mers-cov infection of the final model was assessed by the auc and its % ci. a receiver operating characteristics (roc) curve was generated for the risk prediction model. two hundred random samples were drawn with replacements from the original study sample (n = ). the model internal validity was assessed in these samples by the auc and its % ci. optimal cut-off values of the probabilities for each model were determined using a generalized youden index (youden, ) . at these thresholds, it was possible to achieve the best balance between specificity and sensitivity. a total of pneumonia patients were included in the analysis: . % were confirmed mers-cov-positive and . % were confirmed mers-cov-negative. the mean age at presentation was . years, with a standard deviation of . years; age ranged between and years. of the total sample, . % had been in contact with a sick patient or camel, % had fever, . % had at least one respiratory symptom, and . % had at least one gastrointestinal symptom. the two groups were similar in the distribution of age (p = . ) and sex (p = . ). the characteristics of the sample can be found in table . subgroup analyses are presented in tables and . the chisquare test or the independent samples t-test revealed that sex (p = . ) and age (p = . ) were similar in the two groups. the risk of mers-cov infection was similar in patients with and without fever (p = . ), respiratory symptoms (p = . ), or gastrointestinal symptoms (p = . ). severe illness (p = . ), contact with a sick patient or camel (p = . ), diabetes (p = . ), heart disease (p = . ), and renal disease (p = . ) were significantly associated with an increased risk of mers-cov infection. the independent samples mann-whitney u-test revealed that the wbc count (p = . ) and platelet count (p = . ) were significantly lower in patients who were positive for mers-cov than in those who were negative for mers-cov infection. in contrast, creatinine (p = . ), bilirubin (p = . ), ast (p = . ), and albumin (p = . ) were significantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. alt (p = . ) was insignificantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. according to the individual roc curve analysis (table ) , severe illness, diabetes, wbc count, creatinine, bilirubin, albumin, and ast were the most important predictors of mers-cov infection. a risk prediction model was developed using stepwise binary logistic regression (p . ). the model retained seven independent variables that were associated with increased odds of mers-cov (table ). male sex (adjusted odds ratio (aor) . , p = . ), contact with a sick patient or camel (aor . , p = . ), diabetes (aor . , p = . ), severe illness (aor . , p = . ), low wbc count (aor . , p = . ), high ast (aor . , p = . ), and low alt (aor . , p = . ) were found to have a significant impact on the prediction of mers-cov. the hosmer-lemeshow test indicated that this model fitted the data well (p = . ). this model showed substantial discrimination, with an auc of . and a % ci of . - . ( figure ). the prediction model was validated using the bootstrap procedure. a total of random samples were drawn with replacements from the original sample, and the model showed a substantial ability to assess mers-cov infection in this study population (auc . , % ci . - . ). the findings in table were used to create a risk-probability model. the risk prediction for the model can be expressed by the following equation: predicted probability = [ + exp( . -( . Â male) À ( . Â sick patient or camel contact) À ( . Â diabetes) À ( . Â severe illness) + ( . Â wbc count) À ( . Â ast) + ( . Â alt))] À . a calculator was developed to calculate the potential risk of mers-cov infection in pneumonia patients. we determined the optimal cut-off or threshold values of the probabilities to mark the differences between the high-risk and low-risk groups. when an equal weight was given for sensitivity and specificity (m = ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. when more weight was given for sensitivity than specificity (m = . ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. when more weight was given for specificity than sensitivity (m = . ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. based on data from the two largest hospitals in saudi arabia, a risk prediction model was developed for mers-cov infection in pneumonia patients. this model was generated from a retrospective study and should be assessed prospectively for external validation. seven variables were identified as having a great impact on the mers-cov risk assessment prediction. the risk prediction model highlights the strong potential impact of male sex, contact with a sick patient or camel, severe illness, diabetes, low wbc count, high ast, and low alt on mers-cov infection. these few important parameters could be part of routine medical examinations to be performed (for the purpose of identifying highly suspected individuals) in daily clinical practice in order to make a prompt and timely clinical decision. according to the model, high ast was associated with an increased risk of being infected with mers-cov. this finding is similar to that of mohd et al., who noted high ast levels in mers-cov patients (mohd et al., ) . unlike their findings, it was noted in the present study that the impact of alt became significantly negative after controlling for several confounders. however, this type of association should be evaluated further in a prospective study in the presence of other unmeasured confounders. although sex was found to have no impact on mers-cov infection in the unadjusted analysis, the multivariate analysis revealed that the risk of mers-cov infection was . % times higher in males than in females. this may be because other factors are playing a role in the development of mers-cov in males, such as camel contact, since males are more likely than females to have contact with camels. in agreement with the recent saudi moh mers-cov visual triage guidelines for the identification of suspected cases (anon, b) , it was found that the odds of being infected with mers-cov were higher in patients with diabetes as compared to those with no diabetes. this also supports the findings of previous studies (badawi and ryoo, ; assiri et al., ; al-tawfiq et al., ; alraddadi et al., ) in which researchers systematically recognized that diabetes is a risk factor for mers-cov infection. these findings indicate that more attention should be given to assessing the risk of mers-cov infection in diabetic patients and whether the risk depends on a specific diabetes type or condition in these patients. as asserted in the saudi moh mers-cov visual triage guidelines and many other studies (muhairi et al., ; younan et al., ; reeves et al., ; sabir et al., ; azhar et al., a, b) , contact with a sick patient or camel was identified as an independent predictor of mers-cov infection, according to the risk prediction model. it must be noted that the finding in the present study could have been limited by combining camel contact and sick patient contact due to the small sample size of each category. this study shows the importance of incorporating various types of information to improve the performance of the risk prediction. according to the linear combination model, it was found that several of the parameters highlighted in the saudi moh mers-cov visual triage guidelines were not able to distinguish between 'highrisk' and 'low-risk' groups, nor did they help in predicting mers-cov infection. for instance, fever, respiratory symptoms, gastrointestinal symptoms, heart disease, and renal disease were noted to have an insignificant impact on mers-cov infection. however, in agreement with the saudi moh mers-cov guidelines and two other reports (mohd et al., ; arabi et al., ) , the odds of being infected with mers-cov were associated with a significant risk reduction of . % for each unit increase in wbc count. these results suggest that demographic, clinical, radiological, and laboratory data should be used in routine practice to identify suspected mers-cov cases, as such data could serve as the first line of prevention strategies. it was found that the accuracy of prediction (figure ) was further improved when combining various medical and patient variables as opposed to relying on a single factor (table ). this has been proven theoretically and in application (ahmed et al., (ahmed et al., , (ahmed et al., , etzioni et al., ; shen, ; pepe and thompson, ; su and liu, ; thompson, ) , where a linear combination has been used to maximize the diagnostic accuracy of a disease of interest. the strength of this study lies in the fact that a simple and applicable predictive model was developed that incorporates demographic, clinical, radiological, and laboratory data, where these were functionally associated and contributed greatly to stratifying and distinguishing between a high and a minimal risk of mers-cov infection. this simple evaluation of suspected mers-cov cases appears promising and could be implemented easily in routine clinical practice. this model could be used as a risk stratification method or a triage tool to help physicians in making an informed decision and planning the next step when deciding whether an rrt-pcr or further investigation is necessary. it was possible to derive a risk probability algorithm (range - ), a generalized youden index (choi et al., ) was used to determine an optimal cut-off to stratify the risk, and a risk probability of ! . was identified as being the optimal cut-off, with a sensitivity of . and specificity of . . several limitations to the proposed risk prediction model were identified. the study findings were based on a retrospective design; therefore this prediction model should be interpreted with caution. limited information was available on patient variables, clinical variables, and transmission routes. for example, information on primary cases and secondary cases may be supplemented by the results of clinical, radiological, and laboratory data. in this study, 'contact with a sick patient' and 'contact with a sick camel' were combined into one variable due to the small number in each category. severe illness was based on a subjective judgment. an additional potential limitation was that the duration of symptoms was not available for these patients. this study investigated a very specific population (pneumonia) at only two centers, which could compromise the applicability and generalizability of the risk prediction model. moreover, the prediction model may not be generalizable to patients who do not fulfill the moh guidelines. further validation of the prediction model on an external sample and prospective cohort of representative patients with pneumonia is necessary, specifically in relevant settings: emergency, outpatient, inpatient, and community. despite these limitations, the model developed shows promise for the identification of suspected mers-cov cases in clinical practice. this model could be applicable in various healthcare settingsinpatient, outpatient, and emergency departmentsbecause no extensive laboratory testing is required and samples may be available within short turnaround times. this may allow rapid evaluation and improve clinical decision-making. in conclusion, this study provides a simple, practical, and valid algorithm to identify individuals at increased risk of mers-cov infection among patients who have developed pneumonia. this risk model is not only useful for risk stratification and decisionmaking in clinical practice, but it could also be useful in preventing and managing the possible spread of mers-cov. the usefulness of this newly developed tool most be validated in an external prospective study. the project received ethical approval from two independent ethics committees: the saudi ministry of health (irb log number - e) and king abdullah international medical research center (study number rc / ), riyadh saudi arabia. none. none declared. accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach believe the extreme (be) strategy at the optimal point: what strategy will it become diagnostic delays in symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia 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combinations of multiple diagnostic markers comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription pcr assessing the diagnostic accuracy of a sequence of tests index for rating diagnostic tests mers and the dromedary camel trade between africa and the middle east isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors acknowledge the saudi ministry of health and king abdullah international medical research center for approving this research project. the authors would like to thank the leaders of king abdulaziz medical city in riyadh and king fahad general hospital in jeddah for their support and understanding. supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.ijid. . . . key: cord- - au nctt authors: lin, panpan; wang, manni; wei, yuquan; kim, taewan; wei, xiawei title: coronavirus in human diseases: mechanisms and advances in clinical treatment date: - - journal: medcomm (beijing) doi: . /mco . sha: doc_id: cord_uid: au nctt coronaviruses (covs), a subfamily of coronavirinae, are a panel of single‐stranded rna virus. human coronavirus (hcov) strains (hcov‐ e, hcov‐oc , hcov‐hku , hcov‐nl ) usually cause mild upper respiratory diseases and are believed to be harmless. however, other hcovs, associated with severe acute respiratory syndrome, middle east respiratory syndrome, and covid‐ , have been identified as important pathogens due to their potent infectivity and lethality worldwide. moreover, currently, no effective antiviral drugs treatments are available so far. in this review, we summarize the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. f i g u r e genome organization and structure of hcovs human covs generally cause only mild upper respiratory diseases, which is similar to common flu. , a novel cov, named sars-cov- by the world health organization (who), has emerged again at the end of , causing more infections and deaths worldwide than ever before. the absence of effective antiviral treatments and serious consequences of these three covs have highlighted the urgent need for novel drug development to prevent the spread of covs. herein, this review mainly focuses on the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. covs possess a nonsegmented, positive, single-stranded rna genome of - kb. , , all covs have a similar genome arrangement with a ′-methylated cap structure along with ′-polyadenylated tail. the replicase gene, occupying about kb, two-thirds of the genome and comprising two open reading frames (orfs), orf a and orf b, is located at the ′ end. it encodes two large polyproteins (pp) a and ab that can be cleaved by papain-like cysteine protease (plpro) and c-like serine protease ( clpro) into nonstructure proteins, involving some proteases, several rna modification enzymes, as well as rna-dependent rna polymerase (rdrp) and helicase (hel) required for virus replication. additionally, an untranslated region (utr) can also be identified at the ′-end as same as the ′terminal. structure proteins, encompassing the ′-terminal one-third of the genome, are arranged in a certain order of hemagglutinin esterase (he) protein that is present in some beta-covs, spike protein (s), small membrane protein (e), membrane protein (m), and nucleocapsid protein (n). in brief, the arrangement of the cov genome can be shown as ′-utr-replicase gene (orf a and orf b)-he protein (if have)-s protein-e protein-m protein-n protein- ′ utr-poly (a) ( figure ). cov is named for the club-shaped projections eradiating from the envelope, which forms its corona or crow-like morphology. the shape of the viral particles is roughly spherical with approximately - nm in diameters. [ ] [ ] [ ] the nucleocapsid protein and the genome rna intertwine to form a helical structure located inside the envelope. for some covs, the spikes on the surface are not only formed by trimers of s protein, but also he proteins. m protein and e protein, two transmembrane proteins, also participate in the composition of the virus (figure ). s protein, a transmembrane protein, mediates the initiation of cov infection by attaching to the specific receptors on the target cells. [ ] [ ] [ ] for a prototypical cov, s protein is usually cleaved into an extracellular receptor binding subunit (s ) and a membrane-anchored subunit (s ), responsible for virus binding and membrane fusion, respectively. , two heptad repeats (hr and hr ) and enriched alpha-helices are contained in the s domain, a feature typical of fusion protein. [ ] [ ] [ ] the receptor-binding domain (rbd) of s protein specifically binds to target receptors, leading to the conformation change of s /s complex that mediates virus entry. furthermore, the rbd also induces potent neutralizing ab response, which turns s protein into an important antigenic determinant capable of protective immunity induction and provides a vital approach for the development of immunotherapies. [ ] [ ] [ ] [ ] [ ] cov e protein is an integral membrane protein of - amino acids [ ] [ ] [ ] and is present in small amount in virions. [ ] [ ] [ ] it contains a short hydrophilic n-terminal followed by a single predicted hydrophobic domain and a hydrophilic c-terminal. although its membrane topology remains unclear, cov e protein is commonly referred to as a transmembrane protein with one transmembrane domain. [ ] [ ] [ ] accordingly, the e protein mainly targets er and golgi-complex and participates in part of the life cycle of the virus, including virus assembly, budding, particles release, envelope formation, and viral pathogenesis. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in addition, cov e protein may have a crucial role in virus production and maturation, because recombinant covs lacking the e protein exhibit significantly reduced viral titers and toxicity. [ ] [ ] [ ] [ ] [ ] the m protein is a small transmembrane protein ( ) ( ) ( ) ( ) ( ) ( ) with three transmembrane segments, an n-terminal ectodomain and a c terminal-endodomain, determining the shape of the virion. , it is considered as the most abundant structural protein and plays a pivotal role in virion assembly via interacting with other structural proteins. [ ] [ ] [ ] binding of s protein and m protein is essential to the virus assembly and the maintenance of s protein in the er-golgi intermediate compartment (ergic)/golgi complex. [ ] [ ] [ ] virus-like particles (vlps) are assembled when the combination of m and e proteins occurs, which suggests that they are required for the formation of the envelope. , [ ] [ ] [ ] additionally, when expressed alone, it can become a homomultimeric complex, the primary driving force of envelope formation. , , furthermore, as to n protein, a stable nucleocapsid and internal core of virions can be achieved when combined with m protein. , the n protein, combined with viral genomic rna to form a helical nucleocapsid inside the viral envelope, is a multifunctional protein. it contains three highly conserved domains: the n-terminal domain (ntd), responsible for rna binding; a c-terminal domain (ctd) that is a hydrophobic and helix-rich terminal, capable of dimerization and oligomerization; and an intrinsically disordered region (rna-binding domain/domain ) that is a serine/arginine-rich domain (sr-domain) with a significant phosphorylation potential. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] phosphorylation of the n protein can initiate structural modification leading to an increased rna-binding affinity. , , , the n protein binds to the genomic rna through a beads-on-astring form. likewise, except for the interaction between n protein and nucleic, the ability of complex oligomerization is another pivotal activity required for the formation of the ribonucleoprotein complexes for viral assembly. in addition to the role of the n protein in viral core formation and assembly, , [ ] [ ] [ ] it is also involved in other critical processes of viral life cycle such as virus budding and envelope formation, , - genomic mrna replication ,and genomic rna synthesis. , although the pathogenic mechanisms of human covs have not yet been fully understood, the investigation of their unique characteristics of each cov enables to distinguish the various human covs including sars, mers, and sars-cov- . the crucial early step of the infection of human covs into susceptible host cells is the interaction between viral s protein and cellular target receptors. one of the subunits of s protein, s , containing rbd, is responsible for specific recognition and binding of the target receptors. the other subunit, s , is in charge of the membrane fusion. , [ ] [ ] [ ] [ ] [ ] the tissue tropism, as well as the susceptible host species, is mainly determined by the binding of s protein to target receptors. based on lines of known evidence, human covs utilize multiple and different types of cellular receptors rather than use a common receptor. therefore, multiple cellular receptors have been identified as target receptors for the various human covs to date (table ) . angiotensin-converting enzyme (ace ), the first known human homolog of angiotensin-converting enzyme and the receptor for hcov-nl , sars-cov, and sars-cov- , is a vital component of the reninangiotensin system (ras). [ ] [ ] [ ] [ ] [ ] [ ] [ ] it is a secreted enzyme with a transmembrane domain, a single metalloprotease active site and a signal peptide, and predominantly expressed in heart, vascular endothelia, epithelia of the small intestine, kidney, and testis; alveolar macrophage and monocytes of the respiratory tract; and epithelial cells of the trachea, bronchi, and alveoli. , [ ] [ ] [ ] in contrast to its homolog ace that contributes to the promotion of lung failure pathogenesis, induction of lung edemas, and impairment of lung function, ace plays a protective role in severe acute lung injury (ali). imai et al revealed that the deficiency of ace in the murine models of acute respiratory distress syndrome (ards) deteriorated the symptom in lung function, which could be recovered by hcov-nl hcov-hku the recombinant ace . thus, downregulation of ace expression in sars patients could be used as an indicator of severe clinical outcome. besides lung damage caused by sars-cov infection could be attenuated by blocking the renin-angiotensin pathway. overall, ace could serve as a novel therapeutic target for severe respiratory diseases. dipeptidyl peptidase iv (dpp ), a type ii transmembrane protein also known as cd , is identified as a target receptor for mers. it is a prolyl oligopeptidase expressed in various cells including endothelial cells, epithelial cells, and inflammatory cells in the lung and smooth muscle. [ ] [ ] [ ] the multifunctional protein ddp is implicated in the activation of t-cell, the activity regulation of chemokines and growth factors, and the regulation of glucose metabolism. [ ] [ ] [ ] [ ] not only can it be embedded in the plasma membrane in the form of a homodimer, but it also presents in extracellular fluid like plasma as a soluble form. , although ddp is expressed in epithelial cells of the upper respiratory tract in camels, it is principally found in alveoli but rarely in the nasal cavity or conducting airway. , accordingly, in a murine mers model, though monocyte infiltration, alveolar edema and microvascular thrombosis were observed in the mers-cov-infected lungs, any symptoms were seldom found in the airways. aminopeptidase n (apn), a type of ii metalloprotease also called cd , is a ubiquitous enzyme expressed in various organs (lung, intestine, and kidney) and cells (epithelial cells, endothelial cells, leukocyte, and fibroblast). , it serves as a target receptor for hcov- e, but not for hcov-oc . hcov-oc shares the same specific target with hcov-hku , namely -o-acetylated sialic acid. , virus entry is a finely regulated process requiring a series of interactions between the virion and host cell. [ ] [ ] [ ] following the conjunction with the target receptor, cov fuse its envelope with the membrane of the host cell. these processes are forced by the conformational change of s protein, which is triggered by not only the target receptor binding but also ph acidification and proteolytic cleavage led by cell surface or endosomal proteases such as transmembrane protease serine (tmprss ), furin, cathepsin l, elastase, and trypsin. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cleavages of s protein are facilitated at two sites: the boundary between the s and s subunit (s /s ) and the conserved site upstream of the fusion peptide (s '). , the former one is aimed at releasing rbd from the membrane fusion subunit, and the latter one is important for the exposure of the fusion peptide, hydrophobic in general, which acts as an anchor to target membrane. , , then the fusion domain adopts two heptad repeats (hr /hr ) to form a compact coiled-coil conformation called -helix bundle or hb. , through direct interactions with lipid bilayers, the fusion domain disrupts two apposed membrane layers and fuses the viral envelope to host cell membrane. ultimately, the viral genome is successfully released into the cytosol of the target cell. in addition to the viral infection through the plasma membrane, the entry of covs into cells can be accomplished by the endocytic pathway, depending on the virus strains and host cells. , sars-cov, whose intermediary host is the palm civet, is a highly pathogenic respiratory virus that emerged in , leading to global pandemic that affected more than people in countries. , patients infected with sars-cov initially present "flu-like" syndrome commonly showing high fever, headache, sore throat, myalgia, and dry cough. [ ] [ ] [ ] during the disease progression, ali or ards is developed in a number of patients. pathological manifestations can be described as three phases. the first step is the disturbance of gas exchange in the first week, owing to the extensive edema, shedding of ciliated epithelial cells, and deposition of hyaline-rich substances on the alveolar membrane. in the next step, pulmonary fibrosis occurs that is characterized as the deposition of fibrin at epithelial cells and the alveolar spaces, as well as the infiltration of inflammatory and fibroblasts. finally, fibrosis of lung tissue, collagen deposition, and proliferation of alveolar and interstitial cells represent the final step of disease, about - weeks. [ ] [ ] [ ] [ ] [ ] diffuse alveolar damage (dad) accompanied by hyaline membrane formation as well as interstitial thickening is common characteristics of sars-cov inducing pulmonary damage. although many investigators have devoted to inquiring into the pathogenesis of such virus, it has not yet been fully understood until now. the immune response is the earliest alert system of the host cells warning virus attacks. ironically, it also aids viral pathogenesis. pattern recognition receptors (prrs) that are retinoic acid-inducible gene i protein (rig-i, member of rlrs family) and melanoma differentiation-associated protein (mda ) recognize viral pathogen-associated molecular patterns (pamps), such as viral components and replication intermediates, to initiate signaling cascades against virus infection. , once the pamps from invaded viruses are detected, rig-i and mda interact with the mitochondrial antiviral signaling protein (mavs) that is a mitochondrial membrane-bound f i g u r e escape mechanisms of innate immune response of sars-cov and mers-cov adaptor molecule, followed by the activation of several kinase complexes and multiple subsequent transcription factors (irf , irf , and nf-κb). activation of nf-κb induces the production of proinflammatory cytokines and chemokines in the nucleus that is a substantial cause of the ards. , phosphorylation of irf and irf by the kinase complexes results in homo-or heterodimerization of irf and irf . the dimerization initiates the transcription of type i interferons (ifns, ifn-α and ifn-β), activating the signal transducer and activator of transcription proteins (stats) to mediate antiviral response ( figure ). [ ] [ ] [ ] several defensive approaches are used by sars-cov to avoid the prrs defense system and, ultimately, host innate immune response. one approach is to replicate themselves within the double membrane vesicles (dmvs) that are protected from the prrs. , the eukaryotic mrnas contain a ′ cap that usually lacks in the viral mrnas. however, some viruses such as sars-cov are capable of building the rna cap through nsp and nsp /nap complex, helping them bypass the recognition by prrs. [ ] [ ] [ ] [ ] in addition, a couple of more proteins encoded by the viruses participate in the suppression of the innate immune response by disrupting the ifn response. among the nonstructural proteins, nsp mainly involves in the degradation of host mrnas, inactivation of host translational machinery as well as the inhibition of stat phosphorylating. [ ] [ ] [ ] sars-cov plpro interferes phosphorylation of irf and disrupts nf-κb signaling probably via interacting with sting. [ ] [ ] [ ] the nsp and nsp are also potential ifn antagonists though the mechanism is not clear. structure proteins such as the n protein and m protein are likely to suppress the type i ifn path-ways. because it was known that n protein inhibits the ifn transcription, the n protein could have a strong potential influence on the viral rna. m protein blocks the formation of signaling kinase complexes, and suppresses the irf and irf activities, suggesting the potential role of the n protein in the viral infection as well. such accessory proteins as orf b protein are able to inhibit the rig-i activity and irf phosphorylation in addition to the transcription of ifn-stimulated genes (isgs) via the isre promoter, while orf blocks the nuclear translocation of stat . , in spite of the number of research findings in vitro, they have not been validated in vivo. therefore, it is urgent to examine the findings in vivo for a clear and solid understanding of the infectious process. besides the immune response of the host, ace also plays an essential role in the pathogenesis of sars-cov. as a negative regulator on the ras, ace has been closely linked to the pathogenesis of pulmonary diseases and considered as a protective factor for ali. consequently, the downregulation of ace mediated by sars-cov binding might give an explanation for the progression of severe lung damage occurred on some sars patients. a decade after sars, another novel cov was identified as the pathogen of mers that caused a higher mortality rate ( %- %) compared with sars (around %). , sars-cov and mers-cov are both emerged from bats, and are disseminated to human through palm civets and dromedary camels, respectively. [ ] [ ] [ ] [ ] mers-cov and sars-cov share some common clinical manifestations ranging from asymptomatic to severe pneumonia in multiple organs, , , and pathological features including inflammatory cells infiltration and dad. similar to sars-cov, mers-cov is also capable of causing immune dysregulation by attenuating the innate immune response ( figure ). , type i interferon is important for the inhibition of mers-cov replication in host cells probably via the suppression of the dmvs formation. , capping viral mrna by nsp and nsp /nap complex protects mers-cov, as well as sars-cov, from the prrs recognition since the structure of nsp /nap complex in both viruses are analogous. besides, several proteins of mers-cov are involved in immune escape mechanism by involving in the signaling cascades. it was reported that irf nuclear translocation and ifn promoter activation are inhibited by m protein, orf a, orf b, and orf , former three of which also restrained the expression from an the isre promoter. inhibition of the phosphorylation of irf might be the mechanism for ifr translocation inhibiting. moreover, mers-cov orf a can interact with ifn-inducible double-stranded dna (dsdna)dependent protein kinase activator a (prkra) and subsequently control the function of rig-i and mda , resulting in the disruption of the ifn response. , orf a and orf b are thought to participate in the nf-κb signaling by downregulating the gene stimulated by nf-κb and affecting the kinase complexes, respectively. functions of plpro and nsp of mers-cov are analogous to the functions of those in sars-cov. , like ace , the entry receptor dpp for mers-cov has also a pivotal role in the disease pathogenesis and is considered as a key factor for the intraspecies variation shown in mers infection. , it is usually expressed in type ii pneumocytes that cover % of the alveolar surface. , approximately % of the surface area is occupied by the type i pneumocytes that are responsible for gas exchange. , but the autopsy reports indicated that both type i and ii pneumocytes in patients died from mers-harbored dpp expression and these pneumocytes were infected by the virus. , mers-cov infection of type i pneumocytes might lead to the damage of the cells in the alveoli subsequently causing the dad. it suggests that type i pneumocytes expressing dpp might be included in the pathogenesis of the disease. this might explain why chronic obstructive pulmonary disease (copd) patients attacked by mers-cov had poor outcomes, since the expression of dpp was predominantly upregulated on type i pneumocytes in such patients. , besides, owing to high expression of dpp shown in the kidney, the renal dysfunction might be caused by either the direct infection or the hypoxic damage. evidence of tubular injury, such as cell debris and tubular dilation, could be observed in the late stage of the infection in mers-cov-infected mice. however, no virus could be detected in such animals in the early stage after infection, meaning such pathologic changes might be related to the hypoxia. the outbreak of covid- , whose pathogen is sars-cov- , now poses a serious threat to the global public health. , since the emergence of the virus, sars-cov- has affected more than million people with more than thousand deaths worldwide as of july . next-generation sequencing of the novel virus has been developed soon after the outbreak, indicating that sars-cov- is closely related to the bat-derived sarslike covs. , it is now believed that bats are likely to be the natural reservoir, , and pangolins are regarded as intermediary host according to the later studies. typical clinical presentations of sars-cov- -infected patients include fever, dyspnea, dry cough fatigue, myalgia, pneumonia, and ards symptoms, similar to those of sars and mers patients. [ ] [ ] [ ] however, intestinal disorders such as diarrhea are less frequent in covid- patients than sars. , , furthermore, in spite of the variation of amino acid at some residues, homology modeling informed that sars-cov- and sars-cov have an analogous rbd structure, and share the same target cell receptor, ace , to mediate the viral entry. , , , [ ] [ ] [ ] it is speculated that ace is involved in the pathogenesis of sars-cov- . owing to the current sparsity of data on the pathological characters of sars-cov- , it is poorly understood. a case report from an infected patient died of this disease showed that dad with hyaline membrane formation, infiltration of inflammatory cells, and pulmonary edema could be found in the samples taken from their lungs, which is notably corresponding with the symptoms of sars and mers patients. additionally, lymphopenia is a common manifestation in covid- patients and might be an effective indicator to estimate the severity of hospitalized covid- patients. lymphopenia is also supposed to be a vital factor related to the pathogenesis that has not been elucidated so far. moreover, the concentration of some cytokines and chemokines detected in the plasma was higher in covid- patients compared with healthy individuals. moreover, higher plasma levels of gscf, il- , il- , il- , mcp , mip a, ip , and tnf-α were linked to the more severe disease. all of the data reveal that immunopathology may occupy a crucial place in the development of the disease, and further researches about the pathogenesis of sars-cov- are urgently needed in the future. owing to the fact that no effective specific antiviral therapies are currently available for sars, mers, and covid- , isolation and symptomatic support cares are the major management strategies for suspected and confirmed cases requiring hospital treatment including oxygen inhalation, fluid management, and rational use of antibiotics, to prevent organ failure and secondary bacterial infection and alleviate the symptoms. , [ ] [ ] [ ] thus, the identification of effective agents against human covs is urgently needed in the response to the current covid- outbreak. all of sars-cov, mers-cov, and sars-cov- encode structure proteins (like s protein), nonstructure proteins (eg, plpro, clpro, rdrp, and helicase), and accessory proteins that are essential for the viral life cycle and that are considered as important targets for the development of antiviral agents ( figure ). , analyses of genomic sequences and protein structure indicated that the catalytic sites of four crucial enzymes and the key drug-binding pockets in viral enzymes were conserved across sars-cov, mers-cov, and sars-cov- . therefore, the therapeutic experience based on sars and mers is capable to guide the treatment of covid- . the idea to disturb the normal life cycle of the virus provides significant insights into the clinical treatment strategy. the s protein is important for the discovery of antiviral agents due to its multifunction in virus infection. rbd located on the s subunit can bind to the host cell receptors (ace for sars-cov and sars-cov- , dpp for mers-cov) initiating the conformational changes in s subunit to get viral and cell membranes closer and trigger membrane fusion. consequently, the interaction between rbd and the host cell receptors serves as a key target for the production of neutralizing antibody followed by the vaccine development. , monoclonal antibodies (mabs) and fusion inhibitors against s and s subunit, respectively, are potential antivirus drugs to conquer the viral infection, and the agents targeting the host receptors also play a similar role. [ ] [ ] [ ] [ ] [ ] likewise, cleavage at the protease site of the s /s complex by host proteases such as tmprss and furin is necessary for the membrane fusion and syncytium formation. , the endosomal cysteine protease cathepsins promote the entry of covs into the host cell via the endosomal pathway. inhibitors of these host proteases can potently block the cell entry, which has been proved in vitro and require further validation on animals studies. once entering the host cells, covs release the nucleocapsid and genomic rna into the cytoplasm and start the translation of the replicase gene. the large replicase pp a and pp ab are cleaved by plpro and clpro to produce nonstructural proteins like rdrp and helicase, forming the replication-transcription complex. numerous agents inhibiting these proteins have shown anti-cov effects in vitro. combination of the hydrophobic domains of the replication-transcription complex to the endoplasmic reticulum membrane can form the typical cov replication structures such as dmvs and convoluted membranes, protecting covs from the detection of prrs. , viral rna synthesis produces genomic and subgenomic rnas. then the subgenomic rnas are translated to generate the structural and accessory proteins, participating in the assembly of the virion that is released into the extracellular compartment via exocytosis. small interfering rnas (sirnas) disturbing these processes could be used in the treatment of covs infections. although covs are capable of disturbing the ifn response, they are still sensitive to the ifn treatment in vitro, indicating that augmented host innate ifn response can be an effective strategy to control the viral infection. , [ ] [ ] [ ] in addition to the enhancement of inf response, several other cell signaling pathways are also regarded as potential anti-cov targets. these include calcineurin-nuclear factor of activated t cells (nfat) pathway and kinase signaling pathways such as erk/mapk and pi k/akt/mtor pathways, the inhibitors of which have exhibited anti-cov activities as well. [ ] [ ] [ ] since the discovery of new interventions may take months or even years, a more efficient approach is to repurpose existing antiviral agents approved for treating related viral infections. the followings are approved drugs or preclinical compounds that have potential antiviral abilities against sars, mers, and covid- . virus-targeted therapeutic strategies all of the major proteases of the virus are attractive druggable targets since they are essential for viral transcription and replication ( table ) . as a key part of replication-transcription complex, rdrp participates in the production of genomic rna and subgenomic rna. nucleoside analogues targeting rdrp is capable of inhibiting viral rna synthesis in a great variety of rna viruses including covs. [ ] [ ] [ ] [ ] [ ] favipiravir (t- ), a guanine analogue approved in japan for influenza treatment, has been proven to effectively interfere the rna synthesis of rna viruses such as influenza virus, ebola virus, and other hemorrhagic fever viruses at rdrp level. [ ] [ ] [ ] [ ] [ ] [ ] several studies concluded that favipiravir could inhibit avian influenza a (h n ) virus and ebola virus infection in mice. , also, favipiravir has been proved with a notable effect increasing the survival rate of ebolainfected patients from . % to . % in sierra leone. a recent study ended with the statement that favipiravir owns the ability against sars-cov- (ec = . um, cc > um, si > . ). covid- patients were enrolled in randomized trials for the evaluation of the efficacy of favipiravir plus inf-α or baloxavir marboxil (chictr and chictr ). another guanosine derivative with broad-spectrum antiviral activity, ribavirin, has been authorized for hcv and respiratory syncytial virus (rsv) treatment. accurate mechanism of ribavirin against virus infection is not clear, but inhibition of mrna capping and viral rna synthesis could be pivotal to its antiviral activity. although high dose of ribavirin has been applied to sars treatment, the anti-mers-cov effects were moderate at such dose in rhesus macaques infected by mers-cov and no obvious survival benefit has been observed in mers patients. , [ ] [ ] [ ] [ ] [ ] [ ] recently, an open-label, randomized phase ii clinical trial (nct ) has revealed that triple combination of ribavirin, interferon, and lopinavirritonavir in covid- treatment was safe and superior to lopinavir-ritonavir therapy alone in remission of symptoms, shortening virus shedding and promoting discharge of mild to moderate covid- patients. remdesivir (gs- ) is a small-molecule monophosphoramidate prodrug of an adenosine analog with the ability to interfere with the rna polymerase and the proofreading exoribonuclease and terminate the nonobligate chain. on day and mg once-daily maintenance for days. however, the first clinical trial (nct ) has been suspended so far and the second trial (nct ) with covid- patients enrolled finally indicated that remdesivir hardly shown any statistically significant clinical benefits. conversely, a research found that of ( %) hospitalized patients suffered from severe covid- and treated with compassionate-use remdesivir could achieve clinical improvement. in addition, a phase iii, randomized, double-blind, placebo-controlled trial (nct ) conducted by beigel et al uncovered the fact that remdesivir prevailed over placebo in shortening the time to recovery in adults patients. though remdesivir has been approved by the food and drug administration (fda) to treat severe covid- patients, further researches are urgently required to determine the efficacy and the indication of remdesivir therapy. a novel synthesized nucleoside analogue, bcx (galidesivir), is designed to inhibit viral rna polymerase activity via terminating nonobligate rna chain. bcx exhibits a promising antiviral capability against a wide array of . it is currently tested in phase i clinical trial (nct ) for marburg virus and can be a potential countermeasure against viral diseases that threaten the public health in the world. a recent study also concluded that penciclovir, another rdrp inhibitor that is approved for hsv, showed effects on reducing sars-cov- infection by high-dose administration (ec = . µm, cc > µm, si > . ). although resistance to nucleoside analogs has rarely been reported, it is worth noting that mutation in rdrp is probably responsible for the acquired resistance and should be monitored for the possible resistance. covs plpro enzymes display proteolytic, deubiquitylating, and deisgylating activities. [ ] [ ] [ ] plpro was first regarded as a druggable target for sars-cov, and then several compounds targeting sars-cov plpro were also found to be effective against mers-cov plpro, recently. , though numerous plpro inhibitors have been identified, many of them only inhibit enzymatic activities and do not affect on the viral replication. , a study from lin et al suggested that an fda-approved alcoholaversive drug, disulfiram could inhibit sars-cov and mers-cov plpro via different mechanisms. and the synergistic inhibition between disulfiram and known plpro inhibitors, like -thioguanine and mycophenolic acid, to mers-cov might offer the potential combination treatments against covs in clinical. another essential protease that cleaves the viral polyproteins during viral replication is clpro. similar to plpro, a great many of inhibitors have been identified with the ability to target covs clpro. among the clpro inhibitors, the human immunodeficiency virus (hiv) protease inhibitors are the most comprehensively studied such as lopinavir, ritonavir, asc f, as well as darunavir and cobicistat. lopinavir and ritonavir, applied in combination to treat hiv infection, have shown improvement in the outcome of sars patients in nonrandomized trials. , though lopinavir alone hardly displayed antiviral activity against mers-cov in vitro, the combination of lopinavir and ritonavir ameliorated the outcome in mers-cov-infected nonhuman primates. , therefore, the efficacy of the lopinavir-ritonavir combination in mers patients should be reappraised (nct ). however, no benefit was observed in lopinavir-ritonavir treatment compared to standard care in a randomized, controlled, open-label clinical trial (chictr ) involving severe covid- patients. further trials are still needed to confirm the therapeutic efficacy. in addition, several other clinical trials have been developed to confirm the efficacy of clpro inhibitors on covid- (nct , nct , nct , chictr , nct , nct , and nct ), as well as darunavir and cobicistat (nct ), asc f combined with oseltamivir (nct ). helicase acts on the duplex oligonucleotides and turns them into single strands in an atp-dependent manner in the cov replication cycle. that helicases in different covs are highly homologous making them potentially strong targets for the covs therapeutic options. based on the mechanism actions, the helicase inhibitors can be approximately classified into two groups. one is able to inhibit both the atpase and helicase activities represented by bananins derivatives, -hydroxychromone derivatives, and triazole derivatives (compound ). , the other group including ssya - and adks has the ability to inhibit the helicase activity with no or little effects on the atpase activity. however, the toxicity of helicase inhibitors needs to be examined before being applied to human patients. the transmembrane glycoprotein, s protein, is also a promising target for antiviral agents' development ( table ). one class of antiviral drugs targeting s protein mostly blocks the spike-mediated membrane fusion. a potent mers-cov inhibitor, nafamostat, has demonstrated to be inhibitive against the sars-cov- infection (ec = . µm, cc > µm, si > . ). griffithsin is a red-alga-derived lectin, which specially binds to oligosaccharides located on the surface of viral glycoproteins, for example, s glycoprotein of sars-cov and hiv glycoprotein . a wide range of human covs infection could be inhibited by griffithsin, comprising hcov- e, hcov-nl , hcov-oc , and sars-cov. , but the value of griffithsin in the treatment or prevention of covid- is needed to be evaluated urgently. s subunit of s protein contains two heptad repeat (hr and hr ). antiviral peptides analogous derived from these regions exhibited inhibition to the spike protein-mediated cell-cell fusion and viral entry in viruses such as sars-cov, mers-cov, as well as hcov- e. , , hr p, a peptide spanning hr sequences of mers-cov s protein, was capable of interacting with hr region to form a -hb complex, resulting in potent inhibition of viral fusion and replication. its analog hr p-m exhibited an obvious improvement in stability, solubility, and antiviral activity after being modified with hydrophilic residues. additionally, hr p-m intranasal administration effectively prevented experimental mice transduced by adenoviral vectors conveying human dpp from mers-cov infection with a > -fold decrease in viral titers in the lung, and this protection was intensified via the combination of hr p-m and ifn-β. another newly designed fusion inhibitor from mers-cov called mers-five-helix bundle (mers- hb), which is derived from the -hb, also displayed a potent suppression on s protein-mediated syncytial formation. compared with mers- hb, mers- hb lacks one hr , which endows its capability to interact with viral hr to interrupt the membrane fusion. besides, mers- hb could effectively inhibit the entry of pseudotyped mers-cov with % inhibitory concentration (ic ) about µm. altogether, the resistance of these drugs can be overcome by combining antiviral peptides aiming at various domains of s subunit, which may also attain synergistic effects in vitro. as for sirnas, which displayed antiviral activities in vitro as well as in sars-cov-infected rhesus macaques, are still under preclinical development and demand further studies to seek out the reliable drug delivery methods in a human before the clinical application. [ ] [ ] [ ] m, n, and e proteins and some accessory proteins are not only vital to the virion assembly but also involved in viral pathogenesis in which they function in the interruption of host innate immune response to assist viral infection. for instance, m protein as well as accessory proteins a, b, and of mers-cov act as ifn antagonist, and n protein of sars-cov serves as an inhibitor of viral rna. researches carried out by he et al and akerstrom et al emphasized that sirnas silencing m, n, e, orf a, and orf a/ b play an important role in the inhibition of viral replication of the sars-cov. , but the delivery methods still limit their application in human being. nevertheless, various agents related to these proteins are discovered via functional analysis. one example is resveratrol, a natural stilbene derivative demonstrated to reduce inflammation and exert antiviral activity against multiple viruses. [ ] [ ] [ ] [ ] [ ] [ ] in addition, it exhibited significant inhibition of mers-cov infection and prolonged cellular survival after virus challenge in vitro via deregulating the expression of n protein and the apoptosis induced by mers-cov. alternatively, hexamethylene amiloride, a viroporin inhibitor, is capable to suppress the ion channel activity of e protein of covs such as hcov- e and sars-cov. identified as dna metabolism inhibitor, gemcitabine hydrochloride is a deoxycytidine analog inhibiting both sars-cov and mers-cov with micromolar ec and low toxicity, which suggests its potential antiviral capacity for other human covs. lj and jl , two novel lipophilic thiazolidine derivatives, could induce several changes in membrane properties including the decrease in membrane fluidity, contributing to inhibition of membrane fusion, which made them become broad-spectrum enveloped virus entry inhibitors and potential anti-cov agents. host-cell-targeted therapies the host innate immune response is vital to the interruption of viral replication. recombinant interferons have been applicated in treating various viruses as well as many covs (table ) . though the host interferon response can be inhibited by the covs, they are still proved effective against covs infection such as sars-cov and mers-cov in vitro and several animal models. , , , , recombinant interferons are usually combined with other antiviral agents including ribavirin or lopinavir/ritonavir to treat sars-cov and mers-cov infections, , and the anti-covs efficacy of interferons is enhanced when added with ribavirin. a combination of interferon-α b with ribavirin reduced virus replication and improves clinical outcomes in a rhesus macaque model of mers. however, the effectiveness of combination treatments comprising interferons and ribavirin is still controversial in clinical researches. a study of five patients received interferon-α b and ribavirin showed no survival, but the finding might be not reliable owing to the delayed administration. contrarily, in another study (n = ), mortality rates of individuals receiving interferon-α b and ribavirin exhibited a significant reduction in day , compared with the individuals received standard supportive care, but no significant difference was observed in day . moreover, no significant difference in mortality rates between interferon-α b and ribavirin treatment group and interferon-β a and ribavirin ta b l e host-targeted agents for hcovs treating group was observed in a retrospective study. on the other hand, interferon-β b displayed stronger inhibition to the mers-cov replication in vitro compared with other interferons, and combined use of interferon-β b with other antiviral compounds should be evaluated in further research. , nitazoxanide, originally identified as an antiprotozoal agent, was later considered as a broad-spectrum antiviral agent able to inhibit the replication of numerous dna and rna viruses such as rsv, parainfluenza, rotavirus, hbv, hcv, hiv, yellow fever, as well as covs in vitro. several clinical trials have confirmed its potential value in treating influenza, chronic hbv, and hcv. [ ] [ ] [ ] moreover, recent research indicated that nitazoxanide was capable of inhibiting sars-cov- infection at a low micromolar concentration (ec = . µm; cc > . µm; si > . ) , and the in vivo evaluation of this efficacy is demanded. another type i interferon inducer, polyinosinic:polycytidylic acid (poly(i:c)), is an analog of dsdna with a powerful ability to reduce mers-cov load in animal models. and phase ii clinical trials demonstrated that it was well tolerated by malignant gliomas patients when injected intramuscularly. , overall, the use of interferons or interferon inducers may be a valuable strategy against covs infection and should be furtherly accessed in animal models. several host factors are considered essential to covs entry, such as the host receptors that bind to covs s protein, and host proteases that facilitated covs entry through the cell surface or endosomal pathway. thus, these factors become attractive targets for anti-cov agents' development (table ) . antibodies, peptides, and some functional inhibitors targeting the host receptors can effectively interrupt the binding between s protein and the host cells. one example is that the n-( -aminoethyl)- -aziridineethanamine (naae), a small molecular inhibitor, is able to target ace leading to the block of s protein-mediated membrane fusion, so does the synthetic peptides analogous, p and p . , similar agents were also found in mers treatment, one of which, ys , was confirmed to be well tolerated in patients with advance solid tumors. owing to their specificity to appointed receptors, they were regarded as narrow-spectrum drugs. however, the efficacy of these receptor-targeted agents have never been evaluated in any covs-infected patients and the safeties of these agents also remain unclear. host protease such as cathepsins and tmprss play a key role in the cleavage of s protein and the suppression of these proteases with potent inhibitors can obstruct the virus entry through either endosomal pathway or cell surface pathway. k is a cathepsin inhibitor with broad spectrum against enveloped rna virus including sars-cov and mers-cov. , , however, the camostat mesylate, applied in chronic pancreatitis treating, is a tmprss inhibitor that interrupts the tmprss mediated cell surface entry. , the combination of cathepsin inhibitors and tmprss inhibitors is crucial to the complete suppression of mers-cov in vitro. inhibition of another host protease, furin, which is vital to furinmediated s cleavage for covs, also can block membrane fusion and the viral entry like mers-cov. notably, inhibition of host proteases may result in some side effects and need further evaluation. some approved antipsychotic agents (chlorpromazine, triflupromazine, and fluphenazine) and cardiotonic steroids (ouabain and bufalin) can inhibit clathrinmediated endocytosis via affecting the assembly of clathrin-coated pits at the plasma membrane and binding sodium/potassium-transporting atpase subunit α (atp a ), respectively. , , thus, they are considered as clathrin-mediated endocytosis inhibitors. alternatively, endosomal acidification also has a profound impact on endocytosis. increase of endosomal ph shows an inhibitive effect on virus infection, which has been confirmed by chloroquine, a widely used autoimmune disease and antimalarial agents. chloroquine proves to be active against a wide range of rna viruses including hcov- e, hcov-oc , sars-cov, and mers-cov. [ ] [ ] [ ] [ ] [ ] recently, chloroquine is identified to function at both entry and postentry phase of the sars-cov- infection with the ec value of . µm in vero e cells. additionally, as an immunoregulator, its antiviral activity may be synergistically intensified in vivo. therefore, chloroquine is suggested as a potential option against the emerging sars-cov- . significantly, higher dose of chloroquine should not be recommended for severe covid- patients owing to the its drug safety, particularly while simultaneously accepting azithromycin and oseltamivir treatment, which was presented by a randomized clinical trial (nct ). hydroxychloroquine, a chloroquine analog with lower toxicity, was listed as a potential anti-sars-cov- agent and recommended to be applied in covid- treatment by chinese national guideline and fda. however, evidence of the benefits and harms of hydroxychloroquine therapy is still insufficient and conflicting. some small studies show that hydroxychloroquine was capable of decreasing sars-cov- shedding that could be enhanced by the combination of azithromycin and achieving a shorter time to clinical recovery. , but almost no clinical benefit was observed in other studies. , therefore, therapeutic efficacy of hydroxychloroquine is still needed to be reconfirmed. moreover, there are several factors required to be reconsidered before efficacy evaluation, such as the severity of illness, definition of the endpoints, and effects of the comorbidities. except for the innate immune response, host receptors, and other factors affecting the endocytosis, some signaling pathways have also been suggested as useful approaches for discovering anti-cov reagents (table ) . cyclophilins are peptidyl-prolyl isomerases with physiological functions showing as modulating the calcineurin/nfat pathway via reacting with covs nsp , which is important for the immune cell activation. in addition, they are also required for the replication of numerous viruses including hiv, hcv, as well as covs. consequently, inhibition of cyclophilins by cyclosporines, such as cyclosporin a (csa) and its derivatives, has shown to block the replication of a wide range of covs. , , , however, the obvious immune suppressive properties of csa limit its application in antiviral therapy. but alisporivir, a nonimmunosuppressive cyclosporin a-analog, also displayed the inhibition to the covs replication at a lowmicromolar concentration. additionally, the combined use of cyclosporine and interferon or ribavirin in vitro was beneficial to inhibit sars-cov or mers-cov infection, which needed to be furtherly evaluated in animal models. , furthermore, some antiviral agents inhibiting the cellular signaling pathways, in particular, the erk pathway and pi k/akt pathway, also interrupt the replication of covs. , , however, the efficacy and safety against covs infection of these agents still need to be reconsidered. corticosteroids, which were used in sars and mers treatment, have been linked to several complications such as psychosis, diabetes, and avascular necrosis. , they also were pointed out to be associated with viral replication prolongation in mers patients. however, an update on the efficacy of dexamethasone based on a press release publicized recently indicated that severe covid- patients given mg dexamethasone once daily shown a lower mortality (about - %) compared to patients with standard care. , besides, another agent, methylprednisolone, also exhibited potential capacity in reducing the mortality rate and achieving better clinical outcomes in severe covid- patients. , thus, it is wise to apply corticosteroids to the right patients at the right time. but physicians also need to monitor the corticosteroid-related complications. clinical trials of corticosteroid treatments are shown in table . potential immunotherapeutic options s protein of sars-cov proves to be highly immunogenic during infection and responsible for eliciting a humoral immune response in the host. antivirus antibodies could be detected in the plasma of convalescent patients' infected sars-cov and mers-cov. , convalescent plasma therapy has been applicated in treating patients infected by numerous viruses involving ebola virus, junin virus, machupo virus, and lassa fever. [ ] [ ] [ ] [ ] as for sars-cov, higher day- discharge rate and lower mortality rate have been observed among sars patients who received convalescent plasma transfusion before day of the illness. , this is consistent with another small cohorts research concluding infected patients with severe conditions who failed to respond to current therapies but finally survived after transfused with convalescent plasma, with no obvious side effects. similar results were found in mers patients. additionally, plasma adoptive therapy with anti-mers-cov antibodies could block the virus adhesion and accelerate the viral elimination from mers-cov-infected animal models. but the efficacy and safety of convalescent plasma therapy in covid- patients still need to be reevaluated. although convalescent plasma therapy proves to be a potentially effective therapeutic option for emerging covs, several factors still limit its extensive use in clinical, one of which is enough titers of serum neutralizing antibodies. the development of mabs targeting the s protein of covs is regarded as a remedial strategy (table ). potent mabs against s protein of human covs can be attained via transgenic mice immunization, convalescent b cells immortalization, and cloning of small chain variable regions from naïve and convalescent patients. the majority of mabs interact with the rbd of s protein leading to the interruption of rbd-receptor binding and block the viral attachment. a few mabs react with other regions of s protein besides the rbd. although binding to different epitopes, these mabs exhibit capacity to reduce the viral titers. two rbd-specific neutralizing mabs, mers- and mers- , which were derived from single-chain variable regions, revealed suppressive effects against both mers-cov and pseudotyped mers-cov infection at nanomolar concentrations and were recommended as promising candidates for therapeutic interventions to mers. based on similar mechanisms, other human mabs for mers-cov were also capable of competing with dpp for rbd binding and neutralizing the virus. , , [ ] [ ] [ ] [ ] when administrated to individuals at risk, some of the mabs were capable of preventing viral replication and contributing to block the transmission of mers-cov among human. thus, such antibodies could be served as prophylactic strategies in clinical and valuable tools to guild the development of effective anti-covs vaccines. , from sars-cov to sars-cov- , the emergence of severe human covs have taught us many lessons about the importance of rapid diagnostics and effective vaccines to control the outbreak caused by these viruses. due to the persistence of zoonotic sources in endemic areas, lethal covs remain existing in human society and may lead to the epidemic at any time. thus, a priority is to develop vaccines targeting conserved alleles and providing broad-spectrum protection against varied viral strains. since the emergence of sars-cov and mers-cov, several strategies were applicated in vaccine design, including inactivated virus vaccines, live-attenuated virus vaccines, viral vector vaccines, nanoparticles, recombinant protein subunits vaccines, and dna vaccines (table ) . , and clinical trials have also been developed to test the efficacy of the novel vaccines (table ) . effective vaccines are pivotal in blocking the virus spread from animals' reservoirs to human hosts. inactivated virus vaccines, preserving the viral structure and antigenicity but eliminating the infectious ability, could elicit neutralizing antibodies in animal models of sars-cov and show protection against viral replication when administrated with or without adjuvants. which may be related to the disseminated infection observed in immunocompromised patients. in addition, live-attenuated virus vaccines can induce an innate and adaptive immune response and the protective value can last for a long time. besides, other strategies for vaccines development are also evaluated in animal models. based on the experience of sars-cov and mers-cov, the development of novel sars-cov- vaccine is currently underway and requires more research. however, several concerns should be addressed about the vaccination. the first is the disease deterioration caused by vaccination. although this situation only appears in a small subset of sars vaccine studies, it is still a significant problem that needs to be properly solved. second, the variability of s protein can mediate covs escape from neutralization, suggesting that recombinant protein subunits vaccines based on s protein may demand multivalent approaches. last but not least, how to define ta b l e important clinical trials with vaccines for sars-cov, mers-cov, and sars-cov- the emergence and prevalence of highly pathogenic cov severely threaten public health. a task of top priority is to make clear the viral structural and epidemiological characteristics and block the viral dissemination as well as the progression of the disease, at the first case. to date, further understanding of the life cycle and the pathogenesis of emerging human covs makes current therapeutic strategies of antiviral infection more rational. repurposing existing antiviral drugs is an effective short-term strategy to deal with emerging cov such as the ongoing sars-cov- . various agents with different targets have been evaluated in vitro and in vivo. but not all antiviral agents are capable of achieving better efficacy than in vitro, and in vivo studies are needed to select optimal agents. suitable animal models are particularly significant. however, there are only a few effective animal models available for the studies of covs treatment, which may postpone the clinical evaluation of drugs. besides, due to the diversity of viruses and the capacity of rapidly mutating, some antiviral reagents available for existing covs may become invalid. accordingly, sequencing more natural specimens and combination therapy with two or more synergistic agents have 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identification of a peptide derived from the heptad repeat region of the porcine epidemic diarrhea virus (pedv) spike glycoprotein that is capable of suppressing pedv entry and inducing neutralizing antibodies a novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses antiviral activity of k against members of the order nidovirales purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice key: cord- - cxyogor authors: widagdo, w.; begeman, lineke; schipper, debby; run, peter r. van; cunningham, andrew a.; kley, nils; reusken, chantal b.; haagmans, bart l.; van den brand, judith m. a. title: tissue distribution of the mers-coronavirus receptor in bats date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: cxyogor middle east respiratory syndrome coronavirus (mers-cov) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase- (dpp ) as its receptor. the distribution of dpp in the respiratory tract tissues of humans and camels reflects mers-cov tropism. apart from dromedary camels, insectivorous bats are suggested as another natural reservoir for mers-like-covs. in order to gain insight on the tropism of these viruses in bats, we studied the dpp distribution in the respiratory and extra-respiratory tissues of two frugivorous bat species (epomophorus gambianus and rousettus aegyptiacus) and two insectivorous bat species (pipistrellus pipistrellus and eptesicus serotinus). in the frugivorous bats, dpp was present in epithelial cells of both the respiratory and the intestinal tract, similar to what has been reported for camels and humans. in the insectivorous bats, however, dpp expression in epithelial cells of the respiratory tract was almost absent. the preferential expression of dpp in the intestinal tract of insectivorous bats, suggests that transmission of mers-like-covs mainly occurs via the fecal-oral route. our results highlight differences in the distribution of dpp expression among mers-cov susceptible species, which might influence variability in virus tropism, pathogenesis and transmission route. immunohistochemistry to detect dpp was performed on nose, lung, intestine, kidney, salivary gland, and liver tissues of different bat species: common pipistrelle bat, serotine bat, gambian epauletted fruit bat (further referred as gambian fruit bat), and egyptian fruit bat. the assay was replicated two-three times for each tissue. we have used the same technique to map dpp localization in the respiratory tract tissues of human, dromedary camel, sheep, horse, pig, and llama , . the antibody used in this study recognizes bat dpp as was demonstrated in transfection experiments using cloned pipistrelle bat dpp . hematoxylin and eosin staining on subsequent slides of the same tissues from the bats did not show significant histological changes. dpp was not found in the nasal olfactory epithelial cells of common pipistrelle bat, serotine bat, gambian fruit bat, or egyptian fruit bat (fig. ). in the nasal tissues of common pipistrelle bat, dpp was not detected in the respiratory epithelial cells lining the nasal cavity, but was detected in the epithelial cells lining the ducts of the submucosal glands in this species. in the serotine bat and gambian fruit bat, multifocal dpp expression was detected in a limited number of nasal respiratory epithelial cells. in contrast, in the nasal tissues of the egyptian fruit bat, dpp was prominently detected at the apical surface of the respiratory epithelial cells lining the nasal cavity as well as in glandular and ductular epithelial cells of the submucosal glands. in the lungs of the common pipistrelle and serotine bat, dpp was found in the endothelial cells of the capillaries but not in the bronchial, bronchiolar or alveolar epithelial cells. in the gambian and egyptian fruit bat, dpp was detected in the bronchial, bronchiolar and alveolar epithelial cells as well as in endothelial cells of small blood vessels (fig. ). in the intestinal tissues of all four bat species, dpp was prominently expressed on the apical surface of both small and large intestinal epithelial cells (fig. ). in the kidney of all four bat species, dpp was found in glomerular cells, parietal squamous epithelial cells of the bowman's capsule, and in the proximal tubular epithelial cells. in the salivary gland of common pipistrelle bat, dpp was only detected in the ductular epithelial cells, while in the serotine bat, it was detected in a limited number of glandular epithelial cells. in the gambian and egyptian fruit bat, it was detected in both the glandular and ductular epithelial cells of the salivary gland. in the liver of the common pipistrelle bat and serotine bat, dpp was present in a limited number of endothelial cells lining the sinusoids. in contrast, in the liver of the gambian and egyptian fruit bat, dpp was detected in the bile duct epithelial cells, in the endothelial cells of the hepatic arteries, and in the endothelial cells of the sinusoids (fig. ) . variation in dpp signal and localization were occasionally observed between animals within the same species. in one common pipistrelle bat, the paranasal sinus and pharynx were examined and showed a limited number of dpp positive epithelial cells. the results of the dpp immunohistochemistry staining were scored qualitatively and summarized in table . in general, our results showed that dpp was prominently expressed in the intestine and the respiratory tract tissues of the frugivorous bats, i.e. the gambian and the egyptian fruit bat. however, it is limitedly expressed in the respiratory tract tissues of the insectivorous bats, i.e. the common pipistrelle bat and the serotine bat. in the common pipistrelle bat, dpp was not detected in the nasal respiratory, nasal olfactory, bronchiolar, or alveolar epithelium, but was abundant on the apical surface of the epithelium lining the small and large intestine. we compared these findings to our previous results on dromedary camel and human tissues . in dromedary camels, dpp is strongly detected in the nasal respiratory, tracheal, and bronchial epithelium, while there is limited expression in the alveolar epithelium (fig. ). in humans, it is not found in the nasal epithelium and is present mainly in the alveolar epithelium. additionally, we performed dpp staining on intestinal tissues of dromedary camels obtained from a previous study . we found that dpp was expressed mainly on the apical surface of the small intestinal epithelium (data not shown), similar to what has been reported for humans [ ] [ ] [ ] [ ] (fig. ). the tissue distribution of the mers-cov receptor, dpp , has previously been studied in humans, dromedary camels, and other livestock animals , . here, we show that dpp is differentially expressed among bat species, especially between insectivorous and frugivorous bats. it is strongly detected in the intestine of the common pipistrelle bat, the serotine bat, the gambian fruit bat and the egyptian fruit bat. it is also prominent in the respiratory tract epithelium of the gambian and egyptian fruit bat, but expression is limited in that of the common pipistrelle and serotine bat. given the essential role of dpp in the entry of mers-cov into cells, these results suggest that mers-like-covs are not likely able to replicate in the respiratory tract in these two insectivorous bats. this is in line with our previous report on mers-cov infection experiment in sheep, showing that the lack of dpp in the respiratory tract of the sheep was associated with restricted mers-cov replication in these animals upon intranasal inoculation . rather, in these two insectivorous bats, mers-like-covs may preferentially replicate in the gastrointestinal tract. this is partly supported by the fact that viral genomes of mers-like-covs were mainly obtained from faecal and intestinal tissue samples of insectivorous bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this intestinal tropism indicates that these viruses transmit mainly through the fecal-oral route. therefore, future screening of mers-like-covs from insectivorous bats, particularly the common pipistrelle bat, might focus on fecal material, rectal swabs, or intestinal tissues, rather than throat or nasal swabs. prominent dpp expression in both respiratory tract and intestinal epithelium of the gambian fruit bat and the egyptian fruit bat suggests that mers-cov is able to replicate in both the respiratory tract and intestine of the fruit bats. these results are in line with the fact that mers-cov was able to replicate in the lungs of jamaican fruit bat (artibeus jamaicensis) upon intranasal and intraperitoneal inoculation be detected in the rectal swabs of these animals up to day p.i. and infectious virus was also isolated in the duodenum of one of the bats at day p.i. . these data suggest that mers-cov infects and replicates in the intestine of these bats, not only in the respiratory tract. mers-cov infection in these bats is likely mediated by dpp , since hamster bhk cells, a non-susceptible cell line, could be infected by mers-cov when modified to express jamaican fruit bat's dpp . dpp expression in the intestine and respiratory tract of these jamaican fruit bats, however, was not investigated. since the jamaican fruit bat is a new world fruit bat, unlike the gambian fruit bat and the egyptian fruit bat, which are old world fruit bats, their genetic difference might influence the variation in dpp expression among these species. in contrast to the fruit bats, where dpp is expressed throughout the respiratory tract, dpp is rarely detected in the respiratory tract tissues of insectivorous bats. this limited dpp expression in insectivorous bats might significantly restrict the replication of mers-like-covs in these tissues and minimize the possibility of transmission of these viruses from the respiratory tract. the limited dpp expression in the respiratory tract of the two insectivorous bat species, particularly the common pipistrelle bat, is different from what has been reported for dromedary camels and humans. in humans, dpp is merely expressed in the lower respiratory tract, while in the dromedary camels, it is detected in the upper respiratory tract epithelium . this renders humans to develop pneumonia upon mers-cov infection, while camels develop upper respiratory tract infection , , . in the intestine of both dromedary camels and humans, dpp is mainly present in the apical surface of the small intestine epithelium [ ] [ ] [ ] [ ] . mers-cov has been isolated from faecal samples of a naturally infected dromedary camel, which suggests that this virus is able to replicate in the intestinal tract of this species . however, in dromedary camels, the chance of detecting mers-cov rna in faecal samples is much lower than from nasal swabs . we also observed that low amounts of viral rna are detectable in rectal swabs taken from mers-cov-inoculated dromedary camels . while mers-cov has not yet been isolated from human faecal samples, low amounts of viral rna could be detected in stool samples of mers patients , and several mers patients have also been reported to suffer from diarrhoea [ ] [ ] [ ] . these observations suggest that mers-cov replicates in the intestine of both dromedary camels and humans although only to a limited extent. it is currently unclear what factors restrain mers-cov replication in the intestinal tract of dromedary camels and humans. the human intestinal tract is protected by a mucus layer, commensal microorganisms, multiple innate and adaptive immune cells . also, adenosine deaminase (ada), a natural antagonist of dpp that can inhibit mers-cov infection in vitro , has also been found in the human intestine. the amount of ada in the human intestine is four times higher compared to that in the lung . the presence of dpp in the intestinal tract of bats suggests an intestinal tropism of mers-like-covs. we also detected dpp in the salivary glands and kidneys in all of the bats. in vitro, mers-cov has also been shown to replicate in primary kidney cell culture derived from common pipistrelle bat . however, there has been no further evidence supporting the susceptibility of these two tissues in vivo, nor have there been any reports of mers-like-covs isolated from these two tissues or from bat urine samples. whether these viruses are transmitted through bat saliva or urine, therefore, is currently unclear. in general, our study describes the variation in dpp distribution among four bat species, with notable differences between insectivorous and frugivorous bats. more importantly, the tissue distribution of dpp in insectivorous bats, believed to be one of the natural hosts for mers-like-covs, is different to that in dromedary camels and humans. our results indicate intestinal tropism of mers-like-covs in the insectivorous bats we examined. the existence of a co-receptor that might influence mers-like-covs tropism and replication in these bats, however, could not be disregarded. ceacam is recently reported as an attachment factor that facilitates entry of mers-cov in-vitro . whether ceacam plays an important role in-vivo, particularly in bats, remains to be investigated. in-vivo infection experiments are necessary to confirm our findings, but such studies are ethically and technically challenging. nevertheless, our data are relevant for future monitoring and surveillance of mers-like-covs in insectivorous bats, particularly in the common pipistrelle bat , , as well as for future efforts to better understand the pathogenesis and transmission of mers-like-covs in their natural host. common pipistrelle and serotine bats were found stranded and severely wounded on different occasions, and admitted to an official local bat shelter in the netherlands. the animals were euthanized by veterinarians due to ethical reasons using officially approved methods. the gambian fruit bats and three of four egyptian fruit bats used in this study originated from free-ranging populations in ghana. the bats were sampled for an unrelated study and this study was approved by the ethics committee of the zoological society of london (ref. wle ) and the council for scientific and industrial research in accra, ghana. one of the egyptian fruit bats was obtained from the captive colony at the friederich loeffler institute, riems, germany. it had been euthanized due to reasons not related to this study. all methods were performed in accordance with the relevant guidelines and regulations. after euthanasia, the bats were necropsied and tissues were collected. parts of the lung, intestine, salivary gland, liver, and kidney were obtained from nine common pipistrelle bats, seven serotine bats, three gambian fruit bats, and four egyptian fruit bats. parts of the noses were obtained from five common pipistrelle bats, six serotine bats, three gambian fruit bats, and three egyptian fruit bats. these tissues were all fixed in % formalin and embedded in paraffin. the noses were decalcified in % edta for days before being embedded in paraffin. the formalin fixed paraffin embedded tissues were sectioned ( μm), deparaffinized, and subsequently hydrated. citric acid buffer mm ph was used to retrieve antigens. blocking with normal rabbit serum % was performed prior to staining with polyclonal goat igg anti-dpp (r&d systems, abingdon, uk) in µg/ml concentration. normal goat serum (mp biomedicals, santa ana, ca, usa) in equal concentration was used as negative control. dpp staining was performed at °c overnight. secondary antibody rabbit anti-goat igg labeled with peroxidase were applied subsequently in : dilution for hour at room temperature (dako, glostrup, denmark). the red signal was revealed with -amino- -ethyl-carbazole (sigma-aldrich, st. louis, missouri, usa) before counterstaining with hematoxylin. dromedary camel intestinal tissues were obtained from three different animals sacrificed at day post infection with mers-cov in a previous mers-cov vaccination experiment . two of these animals were vaccinated beforehand, while one was not. mers-cov was not detected in the intestinal tissues of these animals using pcr, virus titration or immunohistochemistry detecting nucleoprotein of mers-cov. information on dpp expression in human respiratory and intestinal tissues was derived from the previous studies , [ ] [ ] [ ] . middle east respiratory syndrome an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan antibodies against mers coronavirus in dromedary 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knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans eptesicus serotinus pipistrellus pipistrellus proteomics. tissue-based map of the human proteome expression of sucrase-isomaltase and dipeptidylpeptidase iv in human small intestine and colon expression and different polarity of aminopeptidase n in normal human colonic mucosa and colonic tumors regional expression of epithelial dipeptidyl peptidase iv in the human intestines rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs mers coronavirus in dromedary camel herd, saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection a case of imported middle east respiratory syndrome coronavirus infection and public health response epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission intestinal epithelial cells: regulators of barrier function and immune homeostasis adenosine deaminase isozymes in human tissues carcinoembryonic antigen-related cell adhesion molecule is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus this study is supported by a top project grant ( ) and by the zoonoses in the night project ( o- - - ) both funded by zonmw. the e. gambianus and three of four r. aegyptiacus used in this study originated from a study in ghana in collaboration with richard suu-ire, from the forestry commission, accra. we would like to thank anne buschmann-balkema for her assistance in preparing the r. aegyptiacus tissues that come from the friederich loeffler institute, riems, germany. we would like to thank stichting vleermuisopvang oss for providing the tissues of p. pipistrellus. we thank brigitta m laksono for her advice on the schematic figure. competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -izn hb authors: de wit, emmie; van doremalen, neeltje; falzarano, darryl; munster, vincent j. title: sars and mers: recent insights into emerging coronaviruses date: - - journal: nat rev microbiol doi: . /nrmicro. . sha: doc_id: cord_uid: izn hb the emergence of middle east respiratory syndrome coronavirus (mers-cov) in marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. the continuing introductions of mers-cov from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. scientific advancements since the – severe acute respiratory syndrome coronavirus (sars-cov) pandemic allowed for rapid progress in our understanding of the epidemiology and pathogenesis of mers-cov and the development of therapeutics. in this review, we detail our present understanding of the transmission and pathogenesis of sars-cov and mers-cov, and discuss the current state of development of measures to combat emerging coronaviruses. supplementary information: the online version of this article (doi: . /nrmicro. . ) contains supplementary material, which is available to authorized users. mers , and a cluster of three cases of mers in the uk was identified in september (ref. ). mers-cov continued to emerge and spread to countries outside of the arabian peninsula as a result of travel of infected persons; often, these imported mers cases resulted in nosocomial transmission. in may , a single person returning from the middle east started a nosocomial outbreak of mers in south korea that involved hospitals and patients . as of april , there have been , confirmed cases of mers, including deaths in countries . this review highlights the pandemic and epidemic potential of emerging coronaviruses and discusses our current knowledge of the biology of sars-cov and mers-cov, including their transmission, their pathogenesis and the development of medical countermeasures. key features of these viruses are the dominance of nosocomial transmission, and pathogenesis that is driven by a combination of viral replication in the lower respiratory tract and an aberrant host immune response. several potential treatments for sars and mers have been identified in animal and in vitro models, including small-molecule protease inhibitors, neutralizing antibodies and inhibitors of the host immune response. however, efficacy data from human clinical trials are lacking but are needed to move these potential countermeasures forward. respectively (fig. a) . similarly to all viruses in the order nidovirales, sars-cov and mers-cov have a unique coding strategy: two-thirds of the viral rna is translated into two large polyproteins, and the remainder of the viral genome is transcribed into a nested set of subgenomic mrnas , (fig. b) . the two polyproteins, pp a and pp ab, encode non-structural proteins (nsp -nsp ) that make up the viral replicase-transcriptase complex. the polyproteins are cleaved by two proteases, papainlike protease (plpro; corresponding to nsp ) and a main protease, c-like protease ( clpro; corresponding to nsp ). the nsps rearrange membranes that are derived from the rough endoplasmic reticulum (rer) into double-membrane vesicles, in which viral replication and transcription occur . one unique feature of coronaviruses is the exoribonuclease (exon) function of nsp (ref. ) , which provides the proofreading capability required to maintain a large rna genome without the accumulation of detrimental mutations , . sars-cov and mers-cov transcribe and subgenomic rnas, er-golgi intermediate compartment (ergic) . a cellular compartment that facilitates transport between the endoplasmic reticulum (er) and the golgi complex. infected individuals who each infect a disproportionately large number of secondary cases. respectively, and these encode the four structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as several accessory proteins that are not involved in viral replication but interfere with the host innate immune response or are of unknown or poorly understood function. the envelope spike glycoprotein binds to its cellular receptor, angiotensin-converting enzyme (ace ) for sars-cov and dipeptidyl peptidase (dpp ) for mers-cov . after membrane fusion, either directly with the host cell membrane or with the endosome membrane, the viral rna genome is released into the cytoplasm, and the rna is uncoated to allow translation of the two polyproteins, transcription of the subgenomic rnas and replication of the viral genome (fig. b) . newly formed envelope glycoproteins are inserted in the rer or golgi membranes; genomic rna and nucleocapsid proteins combine to form nucleocapsids, and the viral particles bud into the er-golgi intermediate compartment (ergic). virion-containing vesicles subsequently fuse with the plasma membrane to release the virus . the first indication of the source of sars-cov was the detection of the virus in masked palm civets and a raccoon dog and the detection of antibodies against the virus in chinese ferret badgers in a live-animal market in shenzhen, china . however, these animals were only incidental hosts, as there was no evidence for the circulation of sars-cov-like viruses in palm civets in the wild or in breeding facilities . rather, bats are the reservoir of a wide variety of coronaviruses, including sars-cov-like and mers-cov-like viruses (fig. ) . thus, the search for the reservoir of mers-cov initially focused on bats, but a serological survey in dromedary camels from oman and the canary islands showed a high prevalence of mers-cov-neutralizing antibodies in these animals . in addition, mers-cov rna was detected in swabs that were collected from dromedary camels at a farm in qatar that was linked to two human cases of mers, and infectious virus was isolated from dromedary camels in saudi arabia and qatar [ ] [ ] [ ] [ ] . serological evidence for the circulation of a mers-cov-like virus in dromedary camels has been obtained in the middle east, eastern africa and northern africa, dating back as far as (ref. ). dromedary camels in saudi arabia harbour several viral genetic lineages , including those that have caused human outbreaks. taken together, these data strongly point to the role of dromedary camels as a reservoir for mers-cov. the ubiquity of infected dromedary camels close to humans and the resulting continuing zoonotic transmission may explain why mers-cov continues to cause infections in humans, whereas sars-cov, without the continuing presence of an infected intermediate host and with relatively infrequent human-bat interactions, has caused no more infections in humans. human-to-human transmission of sars-cov and mers-cov occurs mainly through nosocomial transmission; . - % of mers cases in individual outbreaks were linked to hospitals, and very similar observations were made for some of the sars clusters , . transmission between family members occurred in only - % of mers cases and - % of sars cases. transmission of mers-cov between patients was the most common route of infection ( - % of cases), whereas for sars-cov, infection of health care workers by infected patients was very frequent ( - %) . the predominance of nosocomial transmission is probably due to the fact that substantial virus shedding occurs only after the onset of symptoms , , when most patients are already seeking medical care . an analysis of hospital surfaces after the treatment of patients with mers showed the ubiquitous presence of viral rna in the environment for several days after patients no longer tested positive . moreover, many patients with sars or mers were infected through super spreaders , , , [ ] [ ] [ ] . the clinical courses of sars and mers are remarkably similar, although there are subtle differences (box ) . owing to the current sparsity of data on human mers-cov infections , the pathogenesis of this virus is poorly understood; however, similar mechanisms may underlie the pathogenesis of both mers and sars. the binding of spike protein to ace and the subsequent downregulation of this receptor contribute to lung injury during sars . although it seems counterintuitive that receptor downregulation would increase pathology, it has been shown that ace can protect against acute lung injury. the downregulation of ace results in the excessive production of angiotensin ii by the related enzyme ace, and it has been suggested that the stimulation of type a angiotensin ii receptor and middle east respiratory syndrome coronavirus (mers-cov) encode two large polyproteins, pp a and pp ab, which are proteolytically cleaved into non-structural proteins (nsps), including papain-like protease (plpro), c-like protease ( clpro), rna-dependent rna polymerase (rdrp), helicase (hel) and exonuclease (exon). an additional - orfs are encoded through the transcription of a nested set of subgenomic rnas. sars-cov and mers-cov form spherical particles that consist of four structural proteins. the envelope glycoprotein spike (s) forms a layer of glycoproteins that protrude from the envelope. two additional transmembrane glycoproteins are incorporated in the virion: envelope (e) and membrane (m). inside the viral envelope resides the helical nucleocapsid, which consists of the viral positive-sense rna ((+)rna) genome encapsidated by protein nucleocapsid (n). b | following entry of the virus into the host cell, the viral rna is uncoated in the cytoplasm. orf a and orf ab are translated to produce pp a and pp ab, which are cleaved by the proteases that are encoded by orf a to yield nsps that form the rna replicase-transcriptase complex. this complex localizes to modified intracellular membranes that are derived from the rough endoplasmic reticulum (er) in the perinuclear region, and it drives the production of negative-sense rnas ((-)rnas) through both replication and transcription. during replication, full-length (-)rna copies of the genome are produced and used as templates for full-length (+)rna genomes. during transcription, a subset of - subgenomic rnas, including those encoding all structural proteins, is produced through discontinuous transcription. in this process, subgenomic (-)rnas are synthesized by combining varying lengths of the ′end of the genome with the ′ leader sequence necessary for translation. these subgenomic (-)rnas are then transcribed into subgenomic (+)mrnas. although the different subgenomic mrnas may contain several orfs, only the first orf (that closest to the ′end) is translated. the resulting structural proteins are assembled into the nucleocapsid and viral envelope at the er-golgi intermediate compartment (ergic), followed by release of the nascent virion from the infected cell. one of a panel of recombinant inbred mouse strains derived from a genetically diverse set of founder strains and designed for the analysis of complex traits. the aggregation of leukocytes around blood vessels. (agtr a) increases pulmonary vascular permeability, thus potentially explaining the increased lung pathology when the expression of ace is decreased . immunopathology. the immune response is essential for the resolution of an infection, but it can also result in immunopathogenesis. one indication that immunopathogenesis may contribute to sars was the observation that viral loads were found to be decreasing while disease severity increased , . it is unclear whether a similar trend applies to mers , . moreover, progression to acute respiratory distress syndrome (ards) is associated with the upregulation of pro-inflammatory cytokines and chemokines, particularly interleukin- β (il- β), il- , il- , cxc-chemokine ligand (cxcl ) and cc-chemokine ligand (ccl ) , ; increased plasma levels of these molecules have been detected in patients with sars [ ] [ ] [ ] [ ] . retrospective longitudinal studies in patients who recovered from sars versus those who succumbed to the disease have shown an early expression of interferon-α (ifnα), ifnγ, cxcl , ccl and proteins that are encoded by ifn-stimulated genes (isgs) in all patients, but only patients who survived then had gene expression profiles that are indicative of the development of an adaptive immune response. by contrast, patients who succumbed maintained high levels of cxcl , ccl and isg-encoded proteins, whereas spike-specific antibodies were present at low levels or were absent , which suggests that severe disease is related to the lack of a switch from an innate immune response to an adaptive immune response. experiments using collaborative cross mouse lines and mouse-adapted sars-cov identified one host gene, trim , as important for sars pathogenesis. although there was no difference in clinical signs or viral replication in trim −/− mice compared with wild-type mice, perivascular cuffing and the number of inflammatory cells in the lungs were reduced in the trim −/− mice . a biological process in which small rna molecules induce the degradation of specific mrna molecules, thereby inhibiting gene expression. systems in which a dna molecule is produced that contains the viral leader and trailer sequences, with an assayable reporter replacing the viral orfs. when combined with the expression of viral proteins in trans, this system can be used to model the viral life cycle without the necessity of using infectious virus. the involvement of the host immune response in the pathogenesis of sars, and most likely also that of mers, suggests that drugs which inhibit viral replication will need to be combined with treatments that control detrimental immune responses. immune evasion. sars-cov and mers-cov use several strategies to avoid the innate immune response. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna (dsrna) or uncapped mrna, are detected by pattern recognition receptors (prrs), such as retinoic acid-inducible gene i protein (rig-i; also known as ddx ) or melanoma differentiation-associated protein (mda ; also known as ifih ) . this triggers complex signalling cascades involving myd that lead to the production of type i ifns and the activation of the transcription factor nuclear factor-κb (nf-κb). in turn, active nf-κb induces the transcription of pro-inflammatory cytokines (fig. a) . type i ifns signal through ifnα/β receptor (ifnar) and downstream molecules such as signal transducer and activator of transcription (stat) proteins to stimulate the production of antiviral proteins that are encoded by isgs, such as ifn-induced protein with tetratricopeptide repeats (ifit ; fig. b ). collectively, this establishes an antiviral immune response that limits viral replication in infected and in neighbouring cells (fig. ) . infection of knockout mice revealed the importance of innate immunity. infection of myd −/− and stat −/− mice, but not mice that were deficient in ifn receptors, with a mouse-adapted strain of sars-cov resulted in more severe disease than infection with a non-adapted sars-cov strain , . moreover, mers-cov infection of wild-type mice that were transduced with human dpp caused mild disease, but symptoms were more severe in myd −/− mice and ifnar −/− mice . sars-cov and mers-cov avoid host detection of their dsrna by replicating in virus-induced doublemembrane vesicles that lack prrs , , . moreover, the recognition of sars-cov mrnas, for example, by mda and ifit is prevented by capping of the viral mrnas by nsp and the nsp -nsp complex . recombinant sars-cov that lacks the methylation activity of nsp is attenuated and exhibits increased sensitivity to type i ifns. this effect is dependent on ifit or mda , as the same virus is not attenuated in mice that are deficient in either molecule . although mrna capping has not yet been shown for mers-cov, structural similarity between the mers-cov nsp -nsp complex and the sars-cov nsp -nsp complex suggests that a similar mechanism exists to avoid host recognition of mers-cov mrnas by cytosolic prrs . sars-cov encodes at least eight proteins that interact with the signalling cascades downstream of prrs; in mers-cov, several proteins have been identified with similar functions (fig. ) . the nucleocapsid protein of sars-cov has been associated with the suppression of rnai in mammalian cells . furthermore, this protein antagonizes ifn induction, probably early in the signalling cascade, as downstream signalling molecules relieve the inhibition . mers-cov orf a has a similar ifn-antagonistic function, involving the binding of dsrna and subsequent inhibition of mda activation , potentially through interaction with ifn-inducible dsrna-dependent protein kinase activator a (prkra; also known as pact), which interacts with mda and rig-i . moreover, mers-cov orf a, orf b, orf and membrane protein inhibit the nuclear trafficking of ifn-regulatory factor (irf ) and activation of the ifnb promoter . these viral proteins, except for orf , also inhibit the expression of genes that are under the control of an ifn-stimulated response element (isre), and orf a reduces the expression of genes that are stimulated by nf-κb . finally, mers-cov orf b interacts with tbk and inhibitor of nf-κb kinase-ε (ikkε), thereby suppressing the interaction between ikkε and mitochondrial antiviral-signalling protein (mavs) and inhibiting the phosphorylation of irf (ref. ). the membrane protein of sars-cov inhibits the formation of a signalling complex that contains ikkε, thus repressing the activation of irf and irf and their induction of type i ifn expression. the membrane protein of mers-cov inhibits irf function and the expression of genes that are regulated by an isre, including ifnβ , but whether this occurs through a mechanism similar to that of sars-cov is unclear. sars-cov plpro disrupts nf-κb signalling and blocks the phosphorylation of irf indirectly , . furthermore, sars-cov plpro inhibits the induction of type i ifns, potentially through the deubiquitylation of phosphorylated irf (refs , ) . similar functions have been described for mers-cov plpro . experiments involving recombinantly expressed proteins, in vitro translation, protein overexpression and minireplicon systems have shown that nsp of sars-cov blocks the ifn response through the inhibition of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have an incubation period of ~ days, and % of patients develop disease within days of exposure , , [ ] [ ] [ ] . common early symptoms are fever, chills, coughing, malaise, myalgia and headache, and less common symptoms include diarrhoea, vomiting and nausea , , , , , , , , [ ] [ ] [ ] . upper respiratory tract symptoms and viral shedding are rare, which explains difficulties in obtaining a laboratory diagnosis from nasal or nasopharyngeal swabs . abnormal chest x-rays are more common in patients with mers ( - %) , than in those with sars ( - %) , . accordingly, only - % of patients with sars require intensive care and subsequent mechanical ventilation, whereas - % of patients with mers require intensive care , , , , , , , . the higher incidence of acute respiratory distress syndrome (ards) in individuals with mers is reflected in the case fatality rate: this is ~ % for mers compared with ~ % for sars , . comorbidities have an important role in both sars and mers. several risk factors are associated with poor disease outcome, especially advanced age and male sex , , , , , , , . for mers, additional risk factors for a poor outcome include diabetes mellitus, hypertension, cancer, renal and lung disease, and co-infections , , , , , . health care settings seem to increase the risk of viral transmission owing to aerosol-generating procedures such as intubation and bronchoscopy. appropriate hospital hygiene practices and awareness are crucial to limit future nosocomial outbreaks. both nsp and nsp from sars-cov were also suggested to be ifn antagonists, but the underlying mechanism is unknown . nsp is an inhibitor of mavsinduced apoptosis; however, this occurs through an ifn-independent mechanism . finally, transcriptomic and proteomic analysis of human airway cell cultures showed that mers-cov but not sars-cov induces repressive histone modifications that downregulate the expression of certain isgs . it should be noted that most of the interactions of sars-cov and mers-cov proteins with innate immune pathways were established in in vitro systems, which rely on the overexpression of viral and, . following prr-mediated detection of a pamp, the resulting interaction of prrs with mitochondrial antiviral-signalling protein (mavs) activates nuclear factor-κb (nf-κb) through a signalling cascade involving several kinases. activated nf-κb translocates to the nucleus, where it induces the transcription of pro-inflammatory cytokines. the kinases also phosphorylate (p) ifn-regulatory factor (irf ) and irf , which form homodimers and heterodimers and enter the nucleus to initiate the transcription of type i interferons (type i ifns). both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have developed mechanisms to interfere with these signalling pathways, as shown; these subversion strategies involve both structural proteins (membrane (m) and nucleocapsid (n)) and non-structural proteins (nsp , nsp b, nsp a, nsp b, nsp , nsp and papain-like protease (plpro); indicated in the figure by just their nsp numbers and letters). b | binding of type i ifns to their dimeric receptor, ifnα/β receptor (ifnar), activates the janus kinase (jak)-signal transducer and activator of transcription (stat) signalling pathway, in which jak and tyk kinases phosphorylate stat and stat , which form complexes with irf . these complexes move into the nucleus to initiate the transcription of ifn-stimulated genes (isgs) under the control of promoters that contain an ifn-stimulated response element (isre). collectively, the expression of cytokines, ifns and isgs establishes an antiviral innate immune response that limits viral replication in infected and in neighbouring cells. again, viral proteins have been shown to inhibit these host signalling pathways to evade this immune response. iκbα, nf-κb inhibitor-α. a broadly active antiviral nucleoside analogue with several direct and indirect mechanisms of action; mainly used for the treatment of hepatitis c, in combination with interferon. having polyethylene glycol (peg) attached, to a drug for example; this moiety improves the solubility, decreases the immunogenicity and increases the stability, of the drug of interest, thereby allowing a reduced dosing frequency to be used. (table ) and sars-cov, including the use of antibodies, ifns, inhibitors of viral and host proteases, and host-directed therapies. in the absence of a clinically proven effective antiviral therapy against sars-cov and mers-cov, patients mainly receive supportive care, which is often supplemented by different combinations of drugs. ribavirin and various types of ifn have been given to patients with mers in saudi arabia and china , typically in combination with a broad-spectrum antibiotic and oxygen. the efficacy of treatments for sars-cov and mers-cov infection currently remains unclear. in addition, treatment for mers is typically started only in a late disease stage, when immunopathology predominates and antiviral drugs are likely to provide little benefit. ribavirin was used most frequently during the sars outbreak, often in combination with corticosteroids, which have an anti-inflammatory effect , - . ifnα was also given, usually in combination with immunoglobulins or thymosins, which stimulate the development of t cells, and in a small number of cases in combination with ribavirin , . none of these treatments was tested in a clinical trial, which makes it difficult to assess their efficacy. in fact, retrospective analysis did not yield a treatment combination that was clearly effective. moreover, the data from patients are contradictory about whether ribavirin, when used alone, provided a benefit or was possibly even detrimental , , , . in vitro coronaviruses have a lower sensitivity to ribavirin than other viruses. deletion of the nsp -encoding sequence increases the sensitivity of coronaviruses to ribavirin; however, the underlying mechanism is unclear and is not related to the proofreading function of nsp (ref. ). therefore, ribavirin should be considered only in combination with other antiviral treatments. although ifns are effective against mers-cov in vitro [ ] [ ] [ ] , their effect in humans has yet to be proved. the effectiveness of ifn is increased in vitro if ribavirin is added , , and a combined use of the two drugs reduces disease severity in a rhesus macaque model of mers . the potential side effects of these treatments, such as fatigue, depression and anaemia, have inhibited their use as a first-line treatment for mers, and they are generally administered only after a patient's condition starts to deteriorate. for example, one study of five patients who were infected with mers-cov indicated no survival following ribavirin and ifnα b therapy; however, therapy was not started until days after admission . a separate study found an improvement in survival days after mers diagnosis and the start of treatment, but not days after . in a third study, a combination of ifnα a and ribavirin or ifnβ a and ribavirin did not improve survival; however, some of the patients were more than years old and had preexisting renal failure . in a single case in which ribavirin and ifnα b were started shortly after admission to hospital, the patient started to improve on day after admission and made a complete recovery . ifnβ b is a more potent inhibitor of mers-cov replication in vitro than other types of ifn , , and an improved outcome of disease was observed in common marmosets after challenge with mers-cov . thus, the type of ifn that is used for treatment in humans should be reconsidered (usually, ifnα is used). furthermore, ribavirin and/or ifns should be tested in clinical trials to determine their efficacy in mers treatment and to establish treatment protocols. additional antiviral treatments. the protease inhibitors lopinavir and ritonavir, which are used in combination to treat infection with hiv, improved the outcome of patients with sars when combined with ribavirin, compared with patients who were treated with ribavirin alone , . lopinavir showed no clear antiviral activity against mers-cov in vitro , and it is thus rarely used in patients with mers. however, lopinavir and ritonavir improve the outcome in common marmosets when treatment is initiated hours after infection with mers-cov . thus, the testing of lopinavir and ritonavir in clinical trials in patients with mers should be reconsidered. one patient who received pegylated ifnα, ribavirin, lopinavir and ritonavir in combination had undetectable levels of mers-cov in the blood days after the initiation of therapy; however, this patient did not survive . the combination of ifnα, ribavirin, lopinavir and ritonavir was also used for mers treatment in south korea, but efficacy data are not yet available. however, three case reports indicate recovery in five out of seven patients who were treated with this combination [ ] [ ] [ ] . as clpro and plpro are essential for cleavage of the viral polyproteins and are distinct from cellular proteases, they are ideal drug targets, in particular plpro, compounds that mimic biologically active peptides or proteins. a complement-activated molecule that is important for the recruitment to and activation of inflammatory cells in the lungs. which is involved in both viral replication and ifn antagonism. indeed, most antiviral drug-like molecules have been developed against clpro and plpro, which was aided by the rapid report of crystal structures of these proteases . plpro was initially identified as a drugable target for sars-cov; recently, it has been noted that some of the compounds that target plpro from sars-cov are also active against plpro from mers-cov. for example, both -mercaptopurine and -thioguanine inhibit mers-cov and sars-cov in vitro ; however, the efficacy of these molecules has not yet been tested in vivo. mycophenolic acid also inhibits the replication of mers-cov in vitro , through the inhibition of plpro , but it had no effect in marmosets . as new coronaviruses are likely to emerge from bats, protease inhibitors were designed against bat coronaviruses with the goal of developing a universal antiviral compound against emerging zoonotic coronaviruses. this approach yielded an inhibitor of tylonycteris bat coronavirus hku (hku -cov), which is closely related to mers-cov . this inhibitor, named sg , indeed inhibits mers-cov replication in vitro . similarly, peptidomimetics that target and inhibit clpro of mers-cov, hku -cov and pipistrellus bat coronavirus hku (hku -cov) have also been identified, but have not yet progressed beyond the in vitro stage . several other drugs that were approved for use in humans were shown to inhibit the replication of mers-cov in vitro, notably chloroquine, chlorpromazine, loperamide and cyclosporine a , , , , although their mechanisms of action are unknown, and the benefit of cyclosporine a in patients is debatable owing to the immunosuppressive effect of the drug. although cyclophilin inhibitors that do not result in immunosuppression are available, their activity against mers-cov has not yet been tested. antibody and plasma therapy. plasma from convalescent patients and/or antibody therapies have been the leading proposed treatment for mers so far . there are several potential advantages to this approach. for example, as case numbers increase, the pool of survivors becomes larger; provided these individuals have sufficiently high antibody titres and are willing and able to donate plasma, this is a low-tech, reasonably safe treatment option. furthermore, generation of monoclonal antibodies for use in humans is well established, with a fairly straightforward path to safety and efficacy testing. however, to date, there are very few reports on the use of convalescent plasma and none on the use of monoclonal antibodies as treatments for acute, severe respiratory disease in humans. a post hoc meta-analysis of studies of either sars or severe influenza found a significant reduction in the pooled odds of mortality when convalescent plasma was used . however, study design was rated as low or very low quality, as there were generally a lack of control groups and a moderate-to-high risk of bias, which suggests that a properly designed clinical trial of convalescent plasma use in severe respiratory infections is needed . potent monoclonal antibodies that neutralize the mers-cov spike protein in vitro have been developed [ ] [ ] [ ] [ ] . however, with a few exceptions, in vivo data relating to the use of convalescent plasma or monoclonal antibodies in the treatment of mers are currently lacking. serum from high-titre dromedary camels decreased mers-cov loads in the lungs of mice that had been transduced with human dpp (ref. ). human polyclonal antibodies against the spike protein were generated by vaccinating transchromosomic bovines, and treatment with these antibodies reduced viral titres in the lungs of dpp -transduced mice when treatment was administered or hours after challenge with mers-cov . dpp -transduced mice were also treated with humanized neutralizing monoclonal antibody c h, which is directed against the receptor-binding domain of the mers-cov spike protein, day after mers-cov challenge, and this treatment also decreased viral titres in the lungs , as did the neutralizing antibody lca , which was obtained from a convalescent patient and produced recombinantly . human neutralizing monoclonal antibodies regn and regn also provided a benefit in mice that expressed human dpp and were challenged with mers-cov . the human neutralizing monoclonal antibody m reduced mers-cov replication in the lungs of rabbits following prophylactic, but not therapeutic, treatment . treatment of rhesus macaques with the human monoclonal antibody b-n day after challenge resulted in reduced lung pathology . in all of these studies, viral replication was not completely inhibited, and there were some pathological alterations to the lungs, despite the therapy. furthermore, none of the studies addressed the potential emergence of escape mutants in vivo. alternatively, antibodies that target the region of dpp that binds to the spike protein could be used to prevent entry of mers-cov; this approach was successful in vitro . however, whether such a treatment would be feasible and would not have substantial adverse effects in humans remains to be determined. host-directed strategies can also limit viral replication. for example, the spike protein of sars-cov is cleaved by cathepsin b and cathepsin l, transmembrane protease serine (tmprss ) and possibly other host proteases , . inhibition of host serine proteases by camostat reduced the entry of sars-cov and increased survival in a mouse model . however, the targeting of host proteases is more likely to result in undesirable side effects than the targeting of viral proteases. another underappreciated strategy is attenuation of detrimental host responses. the development of such treatments would require a thorough understanding of the host responses that are involved in acute lung injury and ards, processes that are unfortunately poorly understood at the moment. nonetheless, in vitro studies and limited studies in animal models with other respiratory viruses have shown that anaphylatoxin c a is important for the development of acute lung injury, and blocking anaphylatoxin c a can reduce lung pathology . vaccines that contain immunogenic parts of a pathogen rather than the entire pathogen. vaccines based on the direct introduction of a plasmid encoding an antigen; following in situ production of this antigen, an immune response is mounted against it. changes in gene expression during in vitro mers-cov infection were used to predict potential effective drugs. one of the drugs with predicted efficacy, the kinase inhibitor sb , modestly inhibited sars-cov and mers-cov replication following the treatment of cells prior to infection, but treatment after infection inhibited replication of only sars-cov and not mers-cov . vaccines. vaccination could be used to prevent infection or to reduce disease severity, viral shedding and thereby transmission, thus helping to control mers outbreaks. several vaccination strategies were developed against sars-cov and tested in animals, such as an inactivated virus, a live-attenuated virus, viral vectors, subunit vaccines, recombinant proteins and dna vaccines , . similar approaches have been used for the development of experimental mers-cov vaccines . to date, three mers-cov vaccines have been evaluated in non-human primates. in one study, rhesus macaques were primed with dna encoding the spike protein, followed by boosts with spike dna and with recombinant protein consisting of the spike subunit containing the receptor-binding domain, or primed and boosted once with the subunit protein. both approaches reduced pathological changes in lung function in animals that were infected with mers-cov weeks after the last vaccination . moreover, three vaccinations with a recombinantly expressed protein that contains the receptor-binding domain of the spike protein reduced viral loads and lung pathology in rhesus macaques that were infected weeks after the last vaccination . three dna vaccinations with a construct encoding the full-length spike sequence reduced viral loads and pathology in the lungs after challenge with mers-cov weeks after the last vaccination . one concern of vaccination in humans is vaccinemediated enhancement of disease, a process in which the disease following infection is more severe in vaccinated individuals than in unvaccinated individuals. although this was observed in only a small subset of vaccine studies that were carried out for sars-cov and has not yet been observed in any of the published mers-cov vaccine studies, it is an important concern. moreover, it is unclear who to vaccinate against mers-cov, as healthy individuals seem to be at little risk of severe disease. older patients or patients with underlying disease, who have the highest risk of severe mers, would be important target populations. however, vaccination in such patients can be problematic owing to their poor immune responses, as has been established for influenza virus . in addition, vaccination of people with a high risk of exposure to mers-cov, such as health care workers, slaughterhouse workers and camel herders, is advisable . as our understanding of the pathogenesis of emerging coronaviruses increases, so will the opportunities for the rational design of therapeutics that target viral replication or immunopathology. the rational design of new drugs and the repurposing of existing compounds have already resulted in the development of plpro inhibitors and the identification of kinase inhibitors that inhibit the replication of sars-cov and mers-cov in vitro. however, only a few potential treatments have progressed past the identification of an effect in vitro, and in vivo studies to select the most promising treatment options are required. the development of several mouse models of mers is thus an important step forward (box ) . owing to the acute nature of mers and the important role of immunopathology, combination therapies aimed at simultaneously inhibiting viral replication, limiting viral dissemination and dampening the host response are likely to yield the best results. furthermore, treatment should be started as early as possible, rather than waiting until the patient has already developed extensive lung damage. the development of therapies against sars and mers needs to focus on testing in humans, in properly controlled clinical trials. the current non-standardized, uncontrolled approach to treatment is not informative and may not be beneficial to the patient. the recent ebola outbreak has demonstrated that rapid clinical trial design and approval are possible and that exceptional situations call for deviations from normal procedures . while treatments are being developed and evaluated, the prevention of viral transmission is key to reducing the burden of mers. the large proportion of nosocomial mers-cov infections indicates that preventive measures in hospitals are currently either not fully implemented or insufficient. prevention of zoonotic transmission from dromedary camels is another possibility to decrease the number of mers cases. the most of our understanding of the pathogenesis of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) comes from animal studies. ideally, these models recapitulate all or specific aspects of human disease. several mouse models have been established, for example by using mouse-adapted sars coronavirus (sars-cov) or expressing human receptors in mice . although it has been recognized that mice might poorly mimic specific human responses to infection, the availability of knockout and transgenic mice enables the targeted study of virus-host interactions. several non-human primate models have been developed for sars-cov and mers coronavirus (mers-cov) . the close relationship of non-human primates to humans often allows faithful recapitulation of a disease and the host response. however, these benefits are countered by the need for specialized husbandry, the sometimes limited availability of the animals and reagents, and high costs. the pathogenesis of sars-cov and mers-cov in their respective reservoir hosts is not nearly as well studied as that in humans. currently, only one experimental-infection study has been carried out in bats with mers-cov , and none has been carried out for other coronaviruses. thus, data are mostly limited to the detection of coronaviruses in naturally infected bats. the detection of coronaviruses mainly in faecal samples from bats and not in oral swabs suggests that replication in bats occurs predominantly in the gastrointestinal tract , , . by contrast, a combination of intratracheal and intranasal inoculation of masked palm civets with sars-cov resulted in interstitial pneumonia, with oral and rectal viral shedding . the pathogenesis of mers-cov in dromedary camels has been studied experimentally in a limited number of animals. these animals developed transient mild disease; however, large quantities of mers-cov were shed from the upper respiratory tract, in line with the predominant replication of mers-cov in the nasal turbinates and larynx in these animals, which explains the frequent zoonotic transmission . first vaccines against mers-cov have been tested in dromedary camels , ; indeed, when camels were vaccinated with a modified vaccinia virus that expresses the mers-cov spike protein, subsequent challenge of these animals with mers-cov resulted in less viral shedding than in unvaccinated animals , thereby potentially limiting the transmission to naive animals or to humans. certainly, there has been progress in the development of vaccines and therapies against emerging coronaviruses, but more research and rigorous testing is required if we are to successfully combat these novel pathogens. when the severe acute respiratory syndrome (sars) outbreak developed into the first pandemic of the twenty-first century, it became clear that the medical and scientific communities were not adequately prepared for the emergence of highly pathogenic viruses. whereas several months elapsed and several thousand cases of sars were observed before the causative agent was identified as sars coronavirus (sars-cov) - , subsequent advances in molecular diagnostic tools, such as next generation sequencing, meant that middle east respiratory syndrome coronavirus (mers-cov) was identified before it caused a large outbreak of mers . the availability of the full-length genome of mers-cov enabled the rapid development and distribution of diagnostic assays. the first animal models of disease, several treatment efficacy studies and the identification of the reservoir followed soon after. unfortunately, the sars pandemic did not yield solid clinical data on the efficacy of treatment regimens. these data are urgently needed for the treatment of mers, as well as to prepare for novel coronaviruses that may emerge. several studies have used synthetic biology to study the zoonotic transmission potential of sars-cov-like viruses from bats , , , . the ebola virus outbreak in west africa has highlighted the need for fast-tracking of potential treatments, as several clinical trials were started only towards the end of the outbreak. the combined experiences of the outbreaks of sars, mers and ebola provide a blueprint for the response to emerging pathogens: after the identification of the causative agent, diagnostic assays need to be developed and distributed rapidly, and simultaneously, awareness of the new syndrome and reporting of (suspected) cases must be increased. in addition, infection control measures in health care facilities are essential. research needs to focus on understanding the epidemiology, including pathogen transmission and identification of the reservoir and/or intermediate hosts. animal models need to be developed, as well as therapeutic and prophylactic measures. finally, promising treatments need to 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interests. key: cord- -jv w authors: iwata-yoshikawa, naoko; okamura, tadashi; shimizu, yukiko; kotani, osamu; sato, hironori; sekimukai, hanako; fukushi, shuetsu; suzuki, tadaki; sato, yuko; takeda, makoto; tashiro, masato; hasegawa, hideki; nagata, noriyo title: acute respiratory infection in human dipeptidyl peptidase -transgenic mice infected with middle east respiratory syndrome coronavirus date: - - journal: journal of virology doi: . /jvi. - sha: doc_id: cord_uid: jv w middle east respiratory syndrome coronavirus (mers-cov) infection can manifest as a mild illness, acute respiratory distress, organ failure, or death. several animal models have been established to study disease pathogenesis and to develop vaccines and therapeutic agents. here, we developed transgenic (tg) mice on a c bl/ background; these mice expressed human cd /dipeptidyl peptidase (hdpp ), a functional receptor for mers-cov, under the control of an endogenous hdpp promoter. we then characterized this mouse model of mers-cov. the expression profile of hdpp in these mice was almost equivalent to that in human tissues, including kidney and lung; however, hdpp was overexpressed in murine cd -positive cells within peripheral blood and lymphoid tissues. intranasal inoculation of young and adult tg mice with mers-cov led to infection of the lower respiratory tract and pathological evidence of acute multifocal interstitial pneumonia within days, with only transient loss of body weight. however, the immunopathology in young and adult tg mice was different. on day or postinoculation, lungs of adult tg mice contained higher levels of proinflammatory cytokines and chemokines associated with migration of macrophages. these results suggest that the immunopathology of mers-cov infection in the tg mouse is age dependent. the mouse model described here will increase our understanding of disease pathogenesis and host mediators that protect against mers-cov infection. importance middle east respiratory syndrome coronavirus (mers-cov) infections are endemic in the middle east and a threat to public health worldwide. rodents are not susceptible to the virus because they do not express functional receptors; therefore, we generated a new animal model of mers-cov infection based on transgenic mice expressing human dpp (hdpp ). the pattern of hdpp expression in this model was similar to that in human tissues (except lymphoid tissue). in addition, mers-cov was limited to the respiratory tract. here, we focused on host factors involved in immunopathology in mers-cov infection and clarified differences in antiviral immune responses between young and adult transgenic mice. this new small-animal model could contribute to more in-depth study of the pathology of mers-cov infection and aid development of suitable treatments. m iddle east respiratory syndrome coronavirus (mers-cov) was originally isolated as a novel coronavirus from a fatal case of acute respiratory distress syndrome and renal failure in ( ). a human receptor for the virus, called human cd /dipeptidyl peptidase (hdpp ), was identified subsequently ( ) . many epidemiological and virological investigations have been undertaken since then; however, information about the pathogenesis of mers-cov is limited. in addition, because mers-cov is endemic in the middle east, the development of effective prophylactic and therapeutic treatment strategies remains a high priority. therefore, appropriate animal models are needed to better understand the pathogenesis of mers-cov and facilitate development of effective vaccines and drugs. some research groups experimentally infected nonhuman primates and small experimental animals with mers-cov ( ) ( ) ( ) ( ) ( ) . rhesus macaques appear to develop a transient lower respiratory tract infection after a combination of intratracheal, ocular, oral, and intranasal inoculation with mers-cov ( ), whereas the common marmoset develops progressive and severe pneumonia, which can be lethal ( ) . however, animal models based on nonhuman primates present both ethical and economic problems. thus, establishing a small-animal model of mers-cov infection is desirable. unfortunately, mers-cov does not infect or replicate in small rodents such as syrian hamsters ( ) , mice ( ) , or rats ( ) because they lack a functional mers-cov receptor. zhao et al. described lung infection in a mouse model transduced with an adenovirus expressing hdpp ( ) ; thus, a transgenic (tg) mouse carrying hdpp should be suitable for mers-cov studies ( ) . some research groups developed tg mice overexpressing the hdpp receptor under the control of cag or cytokeratin promoters ( ) ( ) ( ) . these mice developed severe lung disease, along with infection of the brain. autopsy data are available from only one mers patient; therefore, it is unclear whether mers-cov causes a systemic infection although there is no evidence that mers-cov infects the human brain. other studies describe development of an hdpp knock-in mouse ( ) ( ) ( ) . although the tissue distribution and expression levels of hdpp in these models are largely equivalent to those of dpp in wild-type mice, the phenotype that determines mers-cov susceptibility varies from model to model. the hdpp knock-in mouse model described by coleman et al. ( ) succumbed to infection with wild-type mers-cov. in contrast, model mice described by cockrell et al. ( ) and li et al. ( ) are susceptible to infection by serially passaged mers-cov, which induces severe lung pathology and diffuse alveolar damage (dad). these mice would be good models for studying pathogenesis of mers. here, we developed a new tg mouse model expressing hdpp under the control of its endogenous promoter to better mimic physiological expression of hdpp . these tg mice were then backcrossed onto th prone c bl/ mice. after evaluating susceptibility to mers-cov infection, we investigated age-dependent differences in disease pathogenesis because older age is one of the common factors related to mers severity and mortality ( ) ( ) ( ) ( ) ( ) ( ) . both young and adult tg mice infected with mers-cov showed transient weight loss along with moderate pneumonia and mers-cov replication in the lung; however, they did not recapitulate the severe disease and lethal infection seen in humans. young and adult tg mice infected with mers-cov did, however, show different immunopathologies. adult tg mice showed higher levels of proinflammatory cytokine-and chemokinemediated macrophage infiltration of the lungs than young tg mice. taken together, these results suggest that age affects the immunopathology of mers-cov infection in tg mice. the data suggest that other factors are required to recapitulate severe human disease in these tg mice; however, this mouse model will be useful for identifying host mediators that protect against mers-cov infection. this animal model will provide new insight into factors that cause severe mers-cov infection. expression of hdpp in tg mouse tissues. to generate tg mice showing tissueor cell-type-specific hdpp expression mimicking that in humans, we first looked at research involving tg mice harboring human enterovirus receptors (such as the human poliovirus receptor) and scarb receptor-driven endogenous promoters ( , ) . promoter sequences, which normally include a transcriptional start site, are usually isolated from the upstream regions of endogenous mammalian genes ( ) . therefore, we used a bacterial artificial chromosome (bac) clone (rp - j ) containing the complete hdpp gene and an endogenous promoter to generate tg mice harboring hdpp (fig. a) . to screen the tg mice generated, we confirmed presence of the transgene by pcr genotyping using two primers sets specific for hdpp (table , exons and ) . hdpp is a protease expressed on the surface of cells in various organs, including t cells ( , ) . the enzyme is expressed by approximately % of resting t cells isolated from blood ( ) . since handling of peripheral blood in a laboratory is relatively simple, we conducted flow cytometry analysis using a fluorescein isothiocyanate (fitc)-conjugated anti-human cd /hdpp monoclonal antibody that does no react with murine dpp to detect expression of hdpp in mice. cd -positive lymphocytes from / tested pups were positive for hdpp . these mice were then crossed with c bl/ mice to establish two independent tg lines (tg and tg ), which were maintained as hemizygotes carrying the hdpp gene. the tg animals were born at the the open and filled circles denote the centromere (cen) and telomere (tel) of human chromosome , respectively. the gray arrows indicate the genes located downstream of the hdpp gene (white arrow). the cloned region in the bac construct is denoted by a black line. gcg, glucagon gene; fap, fibroblast activation protein; ifih , interferon induced with helicase c domain . (b) expression of hdpp on peripheral blood cd -positive t lymphocytes from the transgenic (tg) mice. tg and tg , hdpp ϩ/Ϫ transgenic mouse lines and ; non-tg, hdpp Ϫ/Ϫ mouse. (c) genomic dna was extracted from tg mice, and human dpp exons to were subjected to pcr using specific primers. expected mendelian ratio and were outwardly indistinguishable from control littermates. because cd -positive lymphocytes from peripheral blood of line tg showed higher hdpp expression than those from line tg (fig. b) , tg was used for further analyses. pcr genotyping using primer sets specific for hdpp revealed that the complete hdpp gene had integrated into the genome of tg mice ( fig. c and table ) . to examine hdpp expression in human and tg tissues, we first performed western blot analysis with a goat anti-cd /hdpp polyclonal antibody (af ; r&d systems), which detected hdpp but cross-reacted weakly with mouse dpp . bands of about kda (hdpp ) were detected in all tested human tissues (liver, spleen, kidney, heart, lung, stomach, small intestine, large intestine, pancreas, brain, spinal cord, and skeletal muscle), except brain ( fig. a) . all of the tissues from hdpp tg mice expressed hdpp , including liver, spleen, kidney, heart, lung, stomach, small intestine, large intestine, pancreas, brain, spinal cord, and skeletal muscle ( fig. a) . these results suggest that the human transgene was expressed in the majority of organs/tissues in tg mice. to further determine hdpp distribution in tissues, immunohistochemistry (ihc) was performed (fig. b ). ihc using a goat anti-cd /hdpp antibody detected hdpp antigens in pneumocytes in the lung, in bile capillaries in the liver, in renal tubular epithelium, on the surface of epithelial cells lining the small intestine, in pancreatic islets, in lymphocytes in the lymph nodes, and in several types of endothelial cell and serous membranes (fig. b , left column). while hdpp expression was undetectable in brain tissue by western blot analysis, ihc revealed that endothelial cells lining blood vessels and leptomeninges of the human brain were positive for hdpp although neurons and glia were negative. in tg mice, pneumocytes and bronchial epithelial cells in the lungs, bile capillaries in the liver, the renal tubular epithelium, and the surface of epithelial cells in the small intestine were positive for hdpp (fig. b ). in addition, several types of endothelial cells and serous membranes in all tested tissues, including the central nervous system, from tg mice were positive for hdpp . notably, most lymphocytes in the t cell zones of the spleen and lymph nodes from tg mice were positive for hdpp . staining of tissues from non-tg mice was very weak or absent (except for the small intestine) (fig. b) . these data suggest that the pattern of hdpp expression in tg mice is similar to that in humans (except for pancreas and lymphoid tissues). expression of hdpp was higher in lymphocytes from tg mice than in those from humans ( fig. c and fig. b ). therefore, we investigated the immune response profile in tg mice. to assess innate immune responses in the lungs of tg , non-tg, and c bl/ mice, all animals received intranasal administration of pbs with or without (b) immunohistochemical analysis of hdpp expression in human, tg , and non-tg mouse tissues stained with an anti-hdpp polyclonal antibody. sections were counterstained with hematoxylin. scale bars, m (large images of liver, kidney, small intestine, pancreas, spleen, and lymph node), m (large images of lung and brain), and m (insets). poly(i·c), a synthetic analog of double-stranded rna (fig. ) . there was no statistically significant difference in cytokine expression levels between tg , non-tg, and c bl/ mice at h after inoculation with poly(i·c) or phosphate-buffered saline (pbs) (fig. ) . however, when we set expression levels after pbs treatment as , we noted that expression of macrophage inflammatory protein ␣ (mip- ␣), granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin- ␤ (il- ␤), il- , and il- in poly(i·c)treated tg mice was . -to -fold higher than in the other two strains. these results suggest that hdpp expression in mice does not have a marked effect on basal innate immune responses in the three mouse strains; however, tg mice show slightly stronger or earlier innate immune responses than c bl/ mice and non-tg mice. thus, when we investigated immune responses in this animal model, we made comparisons between mers-cov-infected and noninfected tg mice. susceptibility of hdpp -tg mice to mers-cov infection. in this experiment, c bl/ mice were used instead of non-tg mice because we were unable to prepare a sufficient number of non-tg mice from littermate mice for this experiment. after intranasal inoculation of -week-old tg and c bl/ mice with % tissue culture infectious doses (tcid ) of mers-cov, neither group was lethargic; however, tg mice showed mild but transient weight loss from days to postinoculation (p.i.) (tg mice, n ϭ [four females and four males]; c bl/ mice, n ϭ [five females and five males]) (fig. a ). tg mice showed seroconversion at days p.i., whereas c bl/ mice did not (tg mice, n ϭ [three females and two males]; c bl/ mice, n ϭ [three females and three males]) (fig. b ), suggesting that tg mice are susceptible to infection by mers-cov. we next examined viral replication kinetics and sites of viral replication in -weekold tg and c bl/ mice (n ϭ - [ females and to males per time point]). tg mice and c bl/ mice were inoculated intranasally with tcid of mers-cov, and tissue specimens (the maxilla [including the nostril], nasal wash fluid, lung, and lung wash fluid) were collected at h p.i. and on days , , , and p.i. the nasal wash fluid from three out of four tg mice contained barely detectable levels of infectious virus at days and p.i. (fig. c ). supernatants from maxilla tissue homogenates ( %) from tg mice contained . tcid /g at day p.i. although the titer fell by days p.i. the viral titers in lung wash fluid and supernatants of lung tissue homogenates ( %) from tg mice contained . tcid /ml and . tcid /g, respectively, at day p.i.; these values were significantly higher than those at h p.i. (p Ͻ . and p Ͻ . , respectively; one-way analysis of variance [anova]). the virus titers in the lungs were detectable up until days p.i. virus was undetectable in the respiratory tract at days p.i. although ihc revealed that various organs from tg mice were positive for the hdpp antigen (fig. b ), no infectious virus was detected in the liver, spleen, kidney, heart, intestines, and brain up to days p.i. (table ). in contrast, virus was detected in the respiratory tract of c bl/ mice at h p.i. only. these observations suggest that mers-cov infects and replicates mainly in the lower respiratory tract of tg mice and is eliminated within days of infection. several research groups have developed tg mouse models of mers-cov infection; however, in these models, viral replication and mers-cov rna were detected in the brain ( - , ). thus, we next measured the amount of viral rna in the brain of tg mice at h and at , , , and days p.i. by real-time reverse transcription-pcr (rt-pcr) using primers specific for mers-cov; however, no mers-cov rna was detected in the brain of tg mice. furthermore, another study showed that experimental infection of common marmosets with mers-cov resulted in viremia ( ) . quantitative examination of viral rna levels in tg mice revealed very low copy numbers of viral rna in the blood at and days p.i., while sera from tg mice were negative for the virus ( table ). these results suggest that although intranasal inoculation of tg mice with mers-cov causes no neuroinvasion, it may induce viremia. several animal studies have identified mers-cov rna in the lymphoid organs of infected animals ( , , ) , but no infectious virus was detected in the spleen from tg mice up to days p.i. (table ). to investigate whether lymphoid organs in tg mice are susceptible to mers-cov infection, we harvested splenocytes from tg and c bl/ mice and estimated infectivity and mers-cov rna levels (fig. d) . although the amount of infectious mers-cov in splenocytes was below the detection limit, viral rna was detected in splenocytes from tg mice (peaking at days p.i.). thus, lymphoid organs of tg mice were as susceptible to mers-cov infection as those reported in other animal studies although lymphoid organs were not a major site of mers-cov replication in tg mice after intranasal inoculation. acute inflammatory changes in the lungs of hdpp -tg mice after mers-cov inoculation. mers-cov infection of the nasal cavity, lungs, brain, spinal cord, liver, spleen, kidney, heart, and gastrointestinal tract of tg mice was examined histopathologically on days , , , , , and p.i (n ϭ ; one or two females and one or two males per time point). histopathological investigations revealed that tg mice showed progressive pulmonary inflammation associated with acute virus infection, from which they recovered (fig. ). on day p.i., there was no obvious infiltration of the lungs in tg mice; however, mild cellular degeneration and viral antigen-positive cells were seen in the bronchioles and a few alveolar areas ( fig. a to c). double-immunofluorescence staining revealed that mers-cov antigen colocalized with hdpp -positive bronchioles and alveolar cells on day p.i. (fig. ). on days and p.i., inflammatory reactions, including partial and/or mild perivascular and peribronchiolar infiltration by mononuclear cells and eosinophils in response to viral antigens, were observed in alveolar areas of lung tissue from tg mice (fig. d to i). from day p.i. onwards, the alveolar area was the main site of inflammatory responses to viral replication (fig. f , i, and l). on day p.i., the point at which tg mice showed weight loss, there was evidence of severe lung inflammation, including perivascular and alveolar septal thickening, caused by infiltrating mononuclear cells; some alveoli were positive for viral antigens (fig. k ). at day p.i., focal cellular infiltration was still evident in the peribronchioles and alveolar septa although viral antigens and inflammatory responses had cleared from the lungs by day p.i. (fig. m to p). there was no evidence of diffuse alveolar damage in the lungs up to day p.i. these findings suggest that progressive immunopathology occurred uniformly throughout the lungs but resolved within days after infection. neither histopathology nor ihc detected inflammatory infiltration or viral antigens in the nasal cavity through days p.i. in addition, there was no inflammation or viral antigen expression in the brain through days p.i. i a g i a g i a g i a g i a g i a g i a (fig. ) . in contrast, c bl/ mice showed no histopathological changes or viral antigen in any organ, including the lung. these results indicate that tg mice suffer acute pneumonia after infection of the lungs with mers-cov, which is related to expression of hdpp in the bronchiolar epithelium and pneumocytes. splenocytes from tg mice (fig. d) ; however, the spleen and other lymphoid tissues did not harbor viral antigens. furthermore, mers-cov induced t cell apoptosis upon infection in vitro ( ) , whereas immunohistochemical staining detected no evidence of apoptosis in lymphoid tissues, including spleen and lymph nodes, of mers-cov-infected tg mice. similar to findings in the other mouse models in which mouse dpp was replaced with hdpp ( ), mers-cov replication and pathology were localized in the lungs in tg mice. differences in the immunopathology of mers-cov infection between young and adult hdpp -tg mice. according to an epidemiological study ( ) , age (Ͼ years) is considered to be one of the risk factors for mers-cov infection in humans. therefore, we infected -week-old mice with mers-cov. tg mice showed significant weight loss from days and p.i. before recovering by day p.i. (tg mice, n ϭ [one female and five males]; non-tg mice, n ϭ [three females and four males]) (fig. a) . however, -week-old tg mice showed no obvious clinical signs (such as respiratory illness and mortality). the -week-old tg mice had seroconverted by days p.i., whereas the c bl/ mice had not (fig. b) . next, we examined viral replication kinetics and sites of viral replication in -week-old tg and non-tg mice (n ϭ ; two females and two males per time point). the nasal wash fluid from one out of four tg mice contained barely detectable levels of infectious virus at , , and days p.i. (fig. c) . supernatants from maxilla tissue homogenates ( %) from tg mice contained . and . tcid /g at and days p.i., respectively, although the titer was undetectable at days p.i. the viral titers in lung wash fluid from tg mice were detectable up until days p.i., while the supernatants from lung tissue homogenates ( %) from tg mice showed a high viral load from to days p.i.; infectious virus was detectable up until days p.i. viral loads in the respiratory tract peaked at days p.i. although no virus was detectable in the respiratory tract of -week-old tg mice at days p.i., infectious virus was detected in the lungs of -week-old tg mice up until days p.i. in addition, infectious virus was detected in the lungs of one of four -week-old tg mice ( . tcid /g) even at days p.i. we also found that viral titers in the nasal wash fluid, maxilla (including nostril), lung wash fluid, and lungs of -week-old tg mice on day p.i. ( . tcid /ml, . tcid /ml, . tcid /ml, and . tcid /ml, respectively) were slightly higher than those in -week-old tg mice ( . tcid /ml, . tcid / ml, . tcid /ml, and . tcid /ml, respectively) (p ϭ . , student's t test with welch's correction). histopathological analysis revealed that -week-old tg mice showed delayed and prolonged inflammatory responses in the lung compared with those in -week-old tg mice (fig. ) . interestingly, viral antigen-positive cells were seen in the alveolar area on day p.i. and then in the bronchi on day p.i., along with a sparse cellular infiltrate (fig. a to f) . cellular infiltration (which included mononuclear cells and polynuclear cells) was observed in the alveoli from days p.i.; this expanded on day p.i. (fig. g to l). focal cell infiltration was seen in the alveoli on day p.i., and lymphoid cell aggregates were seen around bronchioles and blood vessels on day p.i. (fig. m to r). next, we compared inflammation of the alveoli in -week-old tg mice and week-old tg mice on day p.i. ionized calcium binding adaptor molecule (iba- ) is expressed specifically by monocytes/macrophages and is upregulated when these cells are activated. the predominant inflammatory infiltrate within the lungs of both week-old and -week-old tg mice comprised iba- -positive large cells and cd positive mononuclear cells (fig. ) . phagocytic vacuoles were prominent in large iba- -positive cells from -week-old tg mice. ( ) . therefore, we measured the levels of cytokines and chemokines in lung samples from both -week-old and -week-old tg mice inoculated with either mers-cov or minimal essential medium (mem). measurements were made at h and at , , , and days p.i. (n ϭ to [ females and to males per time point] and n ϭ [ females and males per time point]) ( fig. a and b, for -week-old and -week-old mice, respectively). on day p.i., we observed early expression of gamma interferon (ifn-␥)-induced protein (ip- ) in the lungs of both -and -week-old tg mice, a level which was significantly higher than that in control mice; this increase lasted through day . this was followed by a transient increase in expression of interleukin- (il- ), il- , and monocyte chemotactic protein- (mcp- ) in lungs from both -and -week-old tg mice at day p.i. high levels of macrophage inflammatory protein ␣ (mip- ␣) and monokine induced by ifn-␥ (mig) were detected in the lungs of both groups of tg mice from days or to p.i., whereas il- levels increased at day p.i. ifn-␥ production in the lungs of -week-old tg mice peaked significantly on day p.i. while high values were seen in both infected and noninfected -week-old mice during the observation period. in contrast, expression of ip- , il- , and il- ␤ in -week-old tg mice was higher than that in -week-old tg mice. interestingly, il- ␣ and il- were detected only in -week-old tg mice infected with mers-cov. il- ␣, a potent proinflammatory cytokine associated with inflammation and fever, was detected from days p.i. and remained significantly elevated until days p.i.; il- (a proinflammatory cytokine that recruits monocytes and neutrophils) was detected from days p.i. and peaked at days p.i. before falling at days p.i. these results indicate that mers-cov infection induces production of acute inflammatory chemokines and cytokines in the lungs. in addition, aging causes more severe immunopathology; this means that young and adult tg mice show different pathologies in the lung after mers-cov infection. furthermore, we measured expression of mrna encoding ifn-␣ and ifn-␤ (type i ifn with antiviral activity) in the lungs of -and -week-old mice at h and at , , , and days p.i. by real-time reverse transcription-pcr (rt-pcr) ( ). we did not find any evidence of ifn-␣ or ifn-␤ in the lungs from -week-old tg mice at early times postinfection; however, we observed a transient increase in ifn-␣ expression in the lungs of two out of four tg mice at days p.i.; this level fell by days p.i. (fig. c) . day p.i. was the time point at which the amount of virus in the lungs of tg mice began to fall. no ifn-␤ mrna was detectable in lung samples from either group. on the other hand, the lungs of -week-old tg mice showed a transient increase in ifn-␣ and ifn-␤ mrna expression at days p.i. (fig. c ). in addition, ifn-␣ and ifn-␤ mrna expression was higher than that in -week-old tg mice. taken together, these results indicate that both type i and type ii ifn contribute to the immunopathology of the lungs of -week-old tg mice infected with mers-cov. here, we describe a new hdpp -tg mouse expressing the human gene under the control of an endogenous human promoter. this mouse model shows a pattern of hdpp expression that closely mimics that in human tissues and is similar to that in other recent models ( ) ( ) ( ) . this mouse model also shows susceptibility to infection by mers-cov; this mimics the nonlethal observations in other mouse models ( , ) . after intranasal inoculation with a human isolate of mers-cov, the tg mice developed acute and mild interstitial pneumonia; however, the infection was nonlethal and so did not mimic severe cases seen in human mers-cov patients. mers-cov infection can cause severe illness, resulting in acute respiratory distress syndrome, although a large number of mers-cov infections follow a mild or asymptomatic course in healthy individuals ( ) ( ) ( ) ( ) . thus, this tg mouse model reflects the natural course of a mild mers-cov infection. the majority of severe mers-cov cases occur in middle-aged and older males ( , ) . therefore, we infected tg mice aged weeks with mers-cov. the mice showed prominent proinflammatory responses and prolonged pulmonary inflammation compared with responses in tg mice aged weeks. however, none of the infected tg mice had a severe outcome, such as respiratory distress, that led to death. epidemiologically, patients with diabetes, kidney failure, or chronic lung disease, all of which might weaken the immune system, tend to have a poor outcome after infection by mers-cov (http://www.who.int/csr/don/ -september- -mers-kuwait/en/). thus, it is presumed that a combination of older age and underlying disease might also increase mortality in this animal model. this hdpp -tg mouse model, which lacks the clinical signs and mortality associated with severe mers-cov infection, is likely to be less advantageous than other lethal mouse models with respect to development of novel vaccines or antiviral agents ( ) ( ) ( ) . when we asked why the tg mice showed nonlethal responses to infection, we could not ignore the fact that virus levels in lungs were lower than those reported for other mers mouse models ( , , , , ) . one reason for this is that the transgene used in this study is a hemizygote, meaning that the copy number or expression level may be lower than that in mice homozygous for hdpp . in addition, the dpp protein is active as a dimer ( ) , but the tg mice harbor both murine and hdpp . we presume that the viral yield in the lungs of tg mice was low because of heterodimers formed by hdpp and murine dpp . to address this, we constructed structural models of homo-and heterodimers comprising human and mouse dpp . notably, the estimated interaction energy of the dpp heterodimers was greater than that of murine and hdpp homodimers (Ϫ . , Ϫ . , and Ϫ . kcal/mol, respectively). these results suggest that dpp heterodimers are as stable as dpp homodimers. cockrell et al. reported that mouse dpp does not support mers-cov entry ( ) . thus, the presence of stable dpp heterodimers may be a reason for the lower levels of infection in our mouse model. further study is necessary to clarify this. some research groups generated a mouse-adapted mers-cov for use in severe/ lethal mers-cov infection mouse models ( , ) . this may be one way to establish severe mers infection in our tg mice. while the tg mice expressed hdpp protein in the liver, spleen, kidney, heart, lung, gastrointestinal tract, pancreas, and brain, viral infection and replication were limited (mainly) to the lower respiratory tract, with little upper respiratory tract involvement, after intranasal inoculation of mers-cov. tg mice developed interstitial pneumonia, and mers-cov antigens were detected in the lungs. virus yields in the lung were up to -fold higher than those in the upper respiratory tract. most mers patients exhibit a severe lower respiratory tract infection, with little involvement of the upper respiratory tract ( ) . this suggests that mers-cov infection in tg mice mimics mild infection in humans. in vitro analysis of mers-cov suggests that the virus also infects human t cells and macrophages ( , , ) . we detected mers-cov rna in serum and spleen cells from tg mice. these data are similar to those generated from another tg mouse model in which mouse dpp was replaced with hdpp under the control of the endogenous mouse dpp promoter ( ) . mers-cov infection of t cells might affect immunopathology or induction of apoptosis in tg mice, but we found no clear evidence of this. although tg mice showed systemic viremia, infection of organs (except lung) did not lead to secondary complications. the disease phenotype (including clinical symptoms, viral titer in the lung, and acute pneumonia) appeared to be driven by infiltration by macrophages and lymphocytes; this is similar to the phenotype observed in another tg mouse model harboring hdpp under the control of the endogenous mouse dpp promoter ( ) . the tg mice described herein did not show any brain or renal lesions after mers-cov infection. other tg mouse models in which hdpp is expressed under a strong ubiquitous promoter show high levels of viral rna and inflammation in the lungs, which are accompanied by brain lesions ( , , ) . a fatal case of human mers-cov infection published by ng et al. showed no sign of mers-cov infection in the brain ( ) . to date, no reports suggest that mers-cov shows tropism for brain tissue. the primary target of mers-cov is the lower respiratory tract; however, patients with mers often show signs of acute kidney failure ( , ). in addition, mers-cov was identified in the urine of mers patients ( , ) . data from the first autopsy case did find pathological signs in the patient's kidneys although ihc revealed no evidence of mers-cov replication in the kidneys ( ) . in addition, acute renal failure in this patient was not caused by mers-cov directly; rather, it was caused by hypotension ( ) and/or acute respiratory distress syndrome ( ) . histopathological analysis identified cd -positive t cells and iba- -positive macrophages in the lungs of tg mice on day p.i., which correlated with expression of inflammatory cytokines and inflammatory infiltrates in the lung. tg mice aged and weeks showed increased expression of cytokines and chemokines associated with migration of t cells and activation of macrophages, including ip- , il- , il- , mcp- , ifn-␥, mip- ␣, mig, and il- , in the lungs at day and/or p.i. this result is the same as that observed in a hdpp knock-in mouse model reported by coleman et al. ( ) . in this hdpp knock-in mouse model, cd ϩ t cells and macrophages affected the course of mers-cov-induced disease ( ) . in addition, tg mice expressed mrna encoding the type i ifn, ifn-␣ , during the early phase of the mers-cov infection. importantly, the pathogenic and immune response data from the knock-in mouse model and our own model are similar. thus, an acute inflammatory reaction (including production of type i and type ii ifns) and infiltration by macrophages might clear the virus from the lung, thereby preventing progression to mers. interestingly, we detected il- ␣ and il- in the lungs of -week-old tg mice, but not those of -week-old tg mice, after mers-cov infection. both il- ␣ and il- are proinflammatory cytokines that attract monocytes and neutrophils; therefore, they may exacerbate immunopathology after infection. these findings support the notion that the severity of mers-cov infection is age dependent. age is one of the most common factors related to severity and mortality of mers infection; however, the underlying pathology is unclear ( ) . many studies have examined immune responses of mers patients ( , ( ) ( ) ( ) ( ) ( ) . indeed, elevated serum levels of il- , il- , ip- , and ifn-␥ are observed in patients during the early period after severe infection ( ) ( ) ( ) ( ) . in addition, a prominent proinflammatory th and th response, including production of ifn-␥, tumor necrosis factor alpha (tnf-␣), il- , and il- , is seen in patients during the acute phase of mers-cov infection ( ) . in contrast, we found that administration of poly(i·c) to tg mice induced a mild increase (or earlier induction) in innate immune responses compared with those in c bl/ mice and non-tg mice. this suggests that overexpression of hdpp might influence immune responses in tg mice. thus, we must exercise caution when assessing the relationship between immunopathology and outcome in patients and animal models of mers-cov; however, proinflammatory responses seem to contribute to immunopathology during the acute phase of mers-cov infection in both patients and mouse models. in summary, we generated an hdpp -tg mouse model showing mild respiratory infection by mers-cov. while this tg mouse has limitations as a model for human mers (i.e., lower virus titer in the lungs and mild disease), the immunopathology seems to resemble a mild and early stage of infection in humans. even though it has limitations, this tg mouse model will increase our understanding of the mechanisms underlying mers-cov infection. indeed, we recently used this mouse model to confirm a role for transmembrane protease serine type (tmprss ) during mers-cov infection ( ) . this animal model may provide new insight into disease pathogenesis and guide development of therapeutic interventions that mitigate mers. ethics statements. experiments using recombinant dna and pathogens were approved by the committee for experiments using recombinant dna and pathogens at the national institute of infectious diseases, tokyo, japan. all animal experiments were approved by the animal care and use committee of the national institute of infectious diseases and the national center for global health and medicine (ncgm) research institute and were conducted in accordance with institutional guidelines for the care and use of animals. all animals were housed in a japan health sciences foundation-certified facility. all human samples used in this study were obtained from us biomax, inc., genetex, inc., alpha diagnostics international, or protein biotechnologies. the protocols were approved by the health insurance portability and accountability act (hipaa) or institutional review board (irb). cells and viruses. mers-cov, hcov-emc strain, was kindly provided by bart haagmans and ron fochier (erasmus medical center, rotterdam, the netherlands) and was used throughout the study. vero e cells, purchased from the american type cell collection (manassas, va), were cultured in eagle's mem containing % fetal bovine serum (fbs), iu/ml penicillin g, and g/ml streptomycin ( % fbs-mem). stocks of mers-cov were propagated and titrated on vero e cells and cryopreserved at Ϫ °c. viral infectivity titers are expressed as the tcid /milliliter on vero e cells and were calculated according to the behrens-kärber method. work with infectious mers-cov was performed under biosafety level conditions. virus isolation and titration. lung wash fluids and liver, kidney, heart, spleen, intestine, and brain tissue samples from tg , non-tg, and c bl/ mice were collected at the time of postmortem examination and stored at Ϫ °c. tissue homogenates ( %, wt/vol) were prepared in % fbs-mem, and samples were inoculated onto vero e cell cultures, which were then examined for cytopathic effects (cpe) for days. blind passage was performed after freezing and thawing cells from the first-or second-round passages. if mers-cov-specific cpe were not observed in the first-, second-, or third-round cultures, the samples were deemed negative for infectious virus. viral infectivity titers were determined in vero e cell cultures using the microtitration assay described above. mers-cov neutralizing assay. blood was obtained from each mouse and allowed to clot. sera were then obtained by centrifugation and inactivated by incubation at °c for min. one hundred tcid aliquots of mers-cov were incubated for h in the presence or absence of mouse serum (serially diluted -fold) and then added to confluent vero e cell cultures in -well microtiter plates. the presence of viral cpe was determined on day , and the titers of neutralizing antibody were determined as the reciprocal of the highest dilution at which no cpe were observed. the lowest and highest serum dilutions tested were : and : , respectively. generation of hdpp -tg mice. to generate tg mice expressing hdpp , a bac vector carrying the hdpp gene (clone rp - j ) was purchased from advanced genotechs co., japan. the bac dna was purified using a large-construct kit (qiagen) according to the manufacturer's instructions and suspended in te buffer ( mm tris-hcl and . mm edta, ph . ) at a concentration of ng/l. tg mice were generated using standard procedures ( ). the purified bac clones were microinjected into the pronuclei of fertilized eggs from bdf ϫc bl/ ncr mice (slc, inc., hamamatsu, japan) and then transplanted into pseudopregnant icr mice (slc inc.). expression of the transgene was assessed by fluorescence-activated cell sorter (facs) analysis as described below. the tg mice were then backcrossed onto c bl/ ncr for five generations. after weaning, the mice were tested for tg integration by pcr and facs analysis. briefly, genomic dna isolated from ear punch tissues was subjected to pcr using hdpp -specific primers ( table ) , and lymphocytes were isolated from blood taken from the tail vein. lymphocytes were screened for hdpp protein expression by flow cytometry analysis. tg mice and their non-tg littermates were used for the mers-cov infection study. flow cytometry analysis. blood was collected from the retro-orbital venous plexus under isoflurane anesthesia using heparinized capillary tubes. the samples were then treated with red blood cell lysis buffer (sigma-aldrich, st. louis, mo) to remove erythroid cells. for immunofluorescence staining, cells kit (thermo fisher scientific). a panel of inflammatory cytokines and chemokines (basic fibroblast growth factor [bfgf], granulocyte-macrophage colony-stimulating factor [gm-csf], ifn-␥, il- ␣, il- ␤, il- , il- , il- , il- , il- , il- p /p , il- , il- , ip- , keratinocyte chemoattractant [kc] , mcp- , mig, mip- ␣, tnf-␣, and vascular endothelial growth factor) was measured according to the manufacturer's protocols. isolation of splenocytes and infection with mers-cov. spleens were removed aseptically from tg and c bl/ mice (n ϭ each), dissociated in rpmi medium, and pressed gently through a -m-pore-size nylon mesh filter. the cell suspension was centrifuged at ϫ g for min, and red blood cells were lysed with blood cell lysis buffer (final concentrations of mm nh cl, mm khco , and . mm edta, ph . ) at room temperature for min. the cells were washed twice with rpmi medium and centrifuged at , ϫ g for min. hdpp -expressing cd ϩ t cells within the splenocyte population were detected by flow cytometry analysis. the percentage of cd ϩ t cells was . % Ϯ . %, and that of hdpp -expressing cd ϩ t cells was . % Ϯ . %. the cells were resuspended in the medium and infected with mers-cov (multiplicity of infection [moi] of ). viral replication was determined after and days of culture. viral infectivity titers were measured in vero e cell cultures using a microtitration assay. to detect the mers-cov genome in splenocytes, rna from splenocytes infected with mers-cov was extracted at and days p.i. and subjected to quantitative real-time rt-pcr ( ) . molecular modeling of dpp homo-and heterodimers. dpp dimer models were constructed using the molecular operating environment (moe) (chemical computing group, inc., montreal, qc, canada) based on the crystal structure of hdpp at a resolution of . Å (pdb accession number onc). stereochemical quality was assessed using the ramachandran plot and atom clashes applications in moe. interaction energy, which is an indicator of the affinity of the dimer, was calculated using the potential energy application in moe. statistical analysis. data are expressed as the means and standard errors of the means. statistical analyses were performed using graphpad prism, version , software (graphpad software, inc., la jolla, ca). intergroup comparisons were performed using one-way and two-way anova or student's t test with welch's correction. a p value of Ͻ . was considered statistically significant. % nan ), and fc receptors were blocked by incubation for min on ice with an unlabeled anti-mouse cd /cd monoclonal antibody (clone . g ; bay bioscience co., ltd ca) and an allophycocyanin (apc)-labeled anti-human cd antibody human skeletal muscle lysates were purchased from protein biotechnologies (ramona, ca) and used under irb-approved protocols. to prepare protein samples from the tg and non-tg mouse organs, tissues were homogenized in . ml of radioimmunoprecipitation assay (ripa) buffer ( mm tris/hcl and the protein concentrations were measured using a pierce bicinchoninic acid (bca) protein assay kit after being blocked, the membranes were incubated for h with a goat anti-hdpp antibody ( . g/ml) (af followed by incubation with a donkey anti-goat horseradish peroxidase (hrp)-conjugated antibody (abcam) and an anti-rabbit hrp-conjugated antibody (abcam). the bands were detected by an immobilon western chemiluminescent hrp substrate (millipore) and an las- apparatus inflammatory cytokine profiles in % (wt/vol) lung homogenates were detected using a commercial mouse cytokine -plex antibody bead kit (thermo fisher scientific), as described by the manufacturer. inoculation of mice with mers-cov. tg and non-tg mice ( to weeks or weeks old) and tg -balb mice ( to weeks old) were anesthetized and inoculated intranasally with ϫ tcid ( l) of mers-cov. body weight was measured daily for days (n ϭ to per group), and animals were sacrificed at h and at , , , , , and days p.i. to analyze virus replication, hematological parameters, cytokine expression, and disease pathology (n ϭ to per group) for double staining of cd (t cells) and iba- (macrophages) antigen, we used a rabbit anti-human cd antibody tucson, az) and a rabbit anti-human iba- antibody diaminobenzidine (dab ) for min at °c. the second staining was performed for iba- with vina green. nuclei were counterstained with hematoxylin for s. to detect apoptosis, terminal deoxynucleotidyltransferase-mediated dutp-biotin nick end labeling (tunel) was performed using an in situ cell death detection kit (roche) dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the 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after coronavirus infection synthetic double-stranded rna poly(i:c) combined with mucosal vaccine protects against influenza virus infection we thank bart haagmans and ron fouchier for providing mers-cov (isolate hcov-emc/ ) and satoshi koike, ken fujii (tokyo metropolitan institute of medical science), and shutoku matsuyama (national institute of infectious diseases) for helpful discussions. we also thank ayako harashima and midori ozaki for technical assistance.this work was supported by the following: a grant-in-aid for research (h -shinko-wakate- ) from the ministry of health, labor, and welfare, japan; the research program on emerging and reemerging infectious diseases (grants jp fk , jp fk , and jp fk ) from the japan agency for medical research and development; grants-in-aid for scientific research from the ministry of education, culture, sports, science and technology, japan ( k and h ), and a grant from the national center for global health and medicine ( a ). key: cord- - wrsuoy authors: yang, jeong-sun; park, sunghan; kim, you-jin; kang, hae ji; kim, hak; han, young woo; lee, han saem; kim, dae-won; kim, a-reum; heo, deok rim; kim, joo ae; kim, su jin; nam, jeong-gu; jung, hee-dong; cheong, hyang-min; kim, kisoon; lee, joo-shil; kim, sung soon title: middle east respiratory syndrome in persons, south korea, date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: wrsuoy in may , middle east respiratory syndrome coronavirus infection was laboratory confirmed in south korea. patients were a man who had visited the middle east, his wife, and a man who shared a hospital room with the index patient. rapid laboratory confirmation will facilitate subsequent prevention and control for imported cases. . a nasopharyngeal aspiration specimen was collected for mers-cov laboratory testing on may . sputum samples from persons who had been in contact with the index patient were also tested for mers-cov. patient was the -year-old wife of the index patient; she had had physical contact with him while caring for him during the days of hospitalization. fever ( °c) and slight oliguria developed in patient on may . the other contact, patient , was a -year-old man who had chronic obstructive pulmonary disease, asthma, and cholangiocarcinoma and who had shared a hospital room with the index patient and had been within meters from him for hours on may . fever ( . °c) and respiratory symptoms developed in patient on may . in the hospital room, the index patient did not undergo any aerosol-generating procedures, but a severe cough developed. the same health care workers cared for the index patient and patient . laboratory diagnostic methods were performed according to world health organization guidelines for molecular detection of mers-cov ( ) ( ) ( ) . to check for contamination derived from the positive control, we designed and synthesized the mers-cov real-time reverse transcription pcr (rrt-pcr)-positive transcripts for an upstream mers-cov envelope protein gene (upe) and the open reading frame a (orf a) gene containing bp of a foreign gene (centipede). initially, nasopharyngeal samples from the index patient were positive for mers-cov by multiplex rrt-pcr. sputum samples from patients and were also positive, supporting a diagnosis of mers-cov infection. multiplex rrt-pcr results for upe and orf a were positive (table ) . according to rrt-pcr, the respiratory samples from the patients were negative for other human coronaviruses (sars-cov and human cov- e, -oc , -nl , and -hku ) and viruses that cause acute respiratory infection (influenza virus a and b; human adenovirus; bocavirus; human parainfluenza virus types , , and ; respiratory syncytial virus a and b; human rhinovirus; human metapneumovirus). for the index patient, mers-cov rna was detectable in sputum, throat swab, and serum samples but not in a urine sample collected days after symptom onset ( table ) . the viral load, indicated by cycle threshold values, was high in the lower respiratory tract sample but almost undetectable in the throat swab and serum samples. after sequential sampling repeated every - days, mers-cov rna was detected in sputum until days after symptom onset, although viral rna was inconsistently detected and patterns of viral load fluctuated ( ). other than initial fever (> °c), clinical features differed for all patients. the index patient had respiratory symptoms with cough, dyspnea, and myalgia. patient did not have a relevant medical history and showed mild symptoms. patient had underlying concurrent conditions and died days after confirmation of mers-cov infection ( figure ). virus isolation on vero cells was attempted for each respiratory specimen from the patients. the culture supernatant after inoculation was serially assessed for virus growth by using rrt-pcr and was used for blind passages every - days after inoculation. after blind passages, cytopathic effect was observed, and we isolated the mers-cov strain from south korea (kor/knih/ _ _ ) from the vero cells after inoculation by using the sputum from patient . we constructed a phylogenetic tree by using the general time reversible plus gamma model of the raxml version . . software ( ) and figtree version . . (http://tree.bio.ed.ac.uk/software/figtree) and by using complete genomes of the mers-cov isolate from south korea (genbank accession no. kt ) and reference mers-covs (figure ). because, to our knowledge, cases of mers-cov infection in south korea have not been reported, we had to establish laboratory testing protocols to overcome vulnerabilities in the absence of appropriate epidemiologic support (i.e., generate positive controls to check for contamination and repeat testing). positive controls containing foreign genes have been generated to check for laboratory contamination. for patients with an unclear exposure history (such as the index patient) and for patients with short exposure durations and unusual clinical symptoms (such as patients and ), it would be useful if the positive results of rrt-pcr could be confirmed through agarose gel electrophoresis to exclude contamination from the positive control ( , ) . the index patient had no history of potential exposure to camels, bats, or their excreta; to symptomatic persons; or to health care workers during his trip to the middle east, including saudi arabia. although the source of infection for the index patient is unclear, phylogenetic analysis of the whole viral genome showed that the isolate from south korea was closely related to the mers-cov strains isolated in saudi arabia in . ( , ) . ksa, kingdom of saudi arabia; uae, united arab emirates. because the index patient initially concealed his travel history to saudi arabia, united arab emirates, and qatar, mers-cov infection was not considered and the patient was not isolated until mers-cov infection was suspected days after symptom onset. meanwhile, other patients and health care workers had multiple opportunities for exposure to the index patient ( ) ( ) ( ) . the contacts reported here had each been exposed to the index patient. patient was probably infected via droplet transmission in the hospital room. the hospital room, originally built for persons, had been divided into rooms and lacked ventilation. furthermore, an air conditioning unit cycled the air in the room with the door and window closed. thus, poor ventilation might have played a major role in droplet transmission. detection of mers-cov rna in the respiratory tract varies up to day ( ) ( ) ( ) . in this study, virus was detected in the respiratory tract, inconsistently, for up to days. development of effective preventive measures for the mers-cov prevention will require systemic and prospective studies associated with viral shedding and use of specimens in addition to those obtained from the respiratory tract to define the kinetics of mers-cov. rapid detection of mers-cov, using multiplex rrt-pcr to detect upe and orf a genes, would be helpful for countries outside the arabian peninsula. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. emergencies preparedness, response: middle east respiratory syndrome coronavirus (mers-cov) maps and epicurves korean society for healthcare-associated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea preliminary epidemiological assessment of mers-cov outbreak in south korea probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea south korea): the centers world health organization. laboratory testing for middle east respiratory syndrome coronavirus. interim recommendations (revised) detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases address for correspondence: sung soon kim, kcdc, division of respiratory viruses phylogenetic tree comparing complete genome nucleotide sequences of middle east respiratory syndrome coronavirus (mers-cov) isolate from south korea (kor/ knih/ _ _ ) with those of reference mers-covs (genbank database). the tree was constructed by using the general time reversible plus gamma model of raxml version . . software ( ) and visualized by using figtree version we thank the division of epidemic intelligence service and national medical center for assistance with identifying patients, abstracting medical records, and collecting specimens. the public health image library (phil), centers for disease control and prevention, contains thousands of public healthrelated images, including high-resolution (print quality) photographs, illustrations, and videos. key: cord- -w eitw authors: mobaraki, kazhal; ahmadzadeh, jamal title: current epidemiological status of middle east respiratory syndrome coronavirus in the world from . . to . . : a cross-sectional study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: w eitw background: middle east respiratory syndrome coronavirus (mers-cov) is considered to be responsible for a new viral epidemic and an emergent threat to global health security. this study describes the current epidemiological status of mers-cov in the world. methods: epidemiological analysis was performed on data derived from all mers-cov cases recorded in the disease outbreak news on who website between . . and . . . demographic and clinical information as well as potential contacts and probable risk factors for mortality were extracted based on laboratory-confirmed mers-cov cases. results: a total of mers-cov cases, including deaths ( . %), were recorded in the disease outbreak news on world health organization website over the study period. based on available details in this study, the case fatality rate in both genders was . % ( / ) [ . % ( / ) for males and . % ( / ) for females]. the disease occurrence was higher among men [ cases ( . %)] than women [ cases ( . %)]. variables such as comorbidities and exposure to mers-cov cases were significantly associated with mortality in people affected with mers-cov infections, and adjusted odds ratio estimates were . ( % ci: . , . ) and . ( % ci: . , . ), respectively. all age groups had an equal chance of mortality. conclusions: in today’s “global village”, there is probability of mers-cov epidemic at any time and in any place without prior notice. thus, health systems in all countries should implement better triage systems for potentially imported cases of mers-cov to prevent large epidemics. middle east respiratory syndrome coronavirus (mers-cov) infection is considered to cause a new viral epidemic [ ] , and was first reported in a patient who died from a severe respiratory illness in a hospital in jeddah, saudi arabia, in june [ , ] . from . . to . . , world health organization (who) has notified a total of laboratory-confirmed cases of mers-cov, including at least deaths related to this infection from countries around the world [ ] . the origin of mers-cov has been widely discussed. initially, a bat reservoir was posited based on phylogenetic similarity of certain bat coronaviruses with mers-cov. however, there has been no clear bat source of infection or a consistent history of contact with bats in known cases of mers-cov to date [ , ] . another source such as dromedary was later introduced as a possible reservoir in some studies [ ] [ ] [ ] [ ] . some studies have declared that all cases of mers-cov were directly or indirectly linked to residence or travel to countries: saudi arabia, uae, jordan, qatar, kuwait, oman, yemen, egypt, iran, and lebanon [ , ] . the mers-cov infection has high mortality rates, especially in patients with comorbidities such as diabetes and renal failure, evoking global concern and intensive discussion in the media along with respiratory droplet route of its transmission [ ] . laboratory-confirmed mers-cov cases have been reported during hospital-based cluster outbreaks between . . to . . , and cases are still detected throughout the year [ ] . the occurrence of a large number of mers-cov cases and their associated deaths in the world indicate that this disease must be considered as a severe threat to public health [ ] because millions of pilgrims from countries converge in saudi arabia each year to perform hajj and umrah ceremony. upon their return to home, pilgrims hold a ceremony attended by family members and friends. oriental etiquette to share hospitality with others increases the transmission of probable mers-cov cases to others [ , ] . worldwide awareness of mers-cov is low, the disease has high intensity and lethality with unknown mode of transmission and source of mers-cov infection (i.e. whether zoonotic or human disease) [ ] . therefore, it is necessary to design and implement a research to identify some unknown epidemiological aspects and also determine the current epidemiological situation of mers-cov and its mortality risk factors in order to prevent, control and anticipate effective interventions. permission was obtained from who to conduct this analytical-descriptive epidemiological study. using census method, data related to laboratory-confirmed mers-cov cases between . . to . . were extracted from disease outbreak news on mers-cov from who website as follows. demographic information such as age, gender, reporting country, city, health care worker; clinical data and exposure status of mers-cov cases including comorbidities, exposure to camels, camel milk consumption, exposure to mers-cov cases, day/month of symptom onset, day/month of first hospitalization, day/month of laboratory confirmation, final outcome (dead or survived) of mers-cov cases were recorded. all statistical analyses were conducted using spss, version (ibm inc., armonk, ny, usa). quantitative measurement was expressed by medians and qualitative variables were presented as absolute frequency and percentage. logistic regression was used to calculate the odds ratio (or) with a % confidence interval in order to assess the probable relationship between risk factors and final outcome (dead/survived) of laboratory-confirmed mers-cov cases. p values of less than . were regarded as statistically significant. a total of mers-cov cases, including deaths ( . %), were recorded in the disease outbreak news on who website from . the median age of subjects was . years (range: - years). to assess the effect of several potential risk factors on death in morbid cases related to mers-cov infection, we used or index in order to better understand the mechanism of this relationship, and we reported both crude and adjusted or. based on this indicator, variables such as comorbidities and exposure to mers-cov cases were significantly associated with mortality in affected people with mers-cov infections ( table ) . six countries were affected with mers during the period of this study. the majority of cases (approximately . %) with highest mortality ( . %) as well as % of female cases have been reported from saudi arabia ( table ). the epidemic curve of laboratory-confirmed cases of mers between . . and . . is shown in fig. . it can easily be seen that two peaks are evident in this period: the first at the beginning of april and the second at the beginning of july . our results indicate that the number of mers-cov cases remained constant from the beginning of september to the end of january . the findings have important implications for infection control practice. especially, we found evidence that was contrary to many studies declaring that the high mortality rates are related to mers infection with increasing age [ ] [ ] [ ] . our results on mers-cov cases in global level showed that all age groups are somewhat at risk of death from this infection. the chance of mortality in mers-cov cases in all age groups is fairly equal. therefore, in the care and treatment of mers-cov cases, our results suggest that this important point is better to be considered on behalf of health care staff. in this study, we observed a higher disease occurrence and death of (table ) . a possible explanation for a higher disease occurrence and mortality of mers-cov among men is that men are likely to spend more time outdoors and hence have a higher risk of exposure to a source of infection. the evidence linking mers-cov transmission between camels and humans cannot be ignored. several studies have shown that persons with direct and indirect contact with dromedary camels had a significantly higher risk of mers-cov infection. our finding was inconsistent with other studies that did not mention such evidence (table ) . random error may be one of the reasons for obtaining this result since there were not details of exposure to camels and camel milk consumption for laboratory-confirmed mers-cov cases. our research is consistent with many studies that provided evidence of human-to-human transmission for mers-cov infection [ , , ] . figure shows two peaks during june until september, which coincides with the largest mass gathering of muslims around the world in saudi arabia to perform hajj and umrah ceremony. this finding highlights the effect of congregation in the spread of mers-cov infection. our findings in table and fig. show that most cases are reported from saudi arabia after about years since the start of mers-cov pandemic (june to january , ). so, it seems necessary that epidemiologic investigations are conducted by ministry of health in saudi arabia and international partners to better understand the transmission patterns of mers-cov. this study had a number of limitations. assessment of the relationship between mortality related to mers-cov infection and some potential risk factor requires reliable sources of mortality data. we used the data recorded in the disease outbreak news on mers-cov from who website. the quality and accuracy of this data depend primarily on quality of the recorded data reported by national ihr focal point from different countries to who. in this study, the researcher was unable to verify the accuracy of the data, which potentially results in information bias. in addition, information for some of the variables was not available and the number of missing data was high, which might introduce a negligible selection bias in results. another limitation of this research was that possible misclassification of cases may occur due to the respondent's declarations such as exposure to camels, camel milk consumption, and exposure to mers-cov cases, which potentially occurs as a result of measurement bias. despite the above limitations, the current analytical-descriptive epidemiological study may have a number of implications for health care policy by using the global data. it also reminds us that effective national and international preparedness plans should be in place as well as measures to prevent, control and predict such viral outbreaks, improve patient management, and ensure global health security. the results of this analytical-descriptive epidemiological study revealed and confirmed some potential risk factors for mers-cov cases, which were reported as a possible risk factor in previous research studies. in fact, it reminds us that there is probability of mers-cov epidemic at any time and in any place without prior notice in today's "global village". the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea the clinical and virological features of the first imported case causing mers-cov outbreak in south korea middle east respiratory syndrome coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china middle east respiratory syndrome coronavirus: review of the current situation in the world middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae world organization health middle east respiratory syndrome estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia hadj ritual and risk of a pandemic transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome novel corona (mers-cov) infection. epidemiology and outcome update epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission the authors take this opportunity to thank all the who personnel as well as reporting countries with confirmed mers cases for data collection and sending the data to who. we thank rana sidani senior communication officer in who regional office for the eastern mediterranean (cairo, egypt) for their guidance, help and permission to extract the data. this paper is dedicated to arsam ahmadzadeh and anil ahmadzadeh. as a epidemiological analysis on who data, this study did not need financial support. the dataset used and/or analysed during the current study are available from the corresponding author on reasonable request.authors' contributions km and ja designed the study and performed the search and data extraction. ja analyzed the data. km and ja wrote the manuscript. ja edited the draft. both authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - e zjaz authors: park, ji-eun; jung, soyoung; kim, aeran; park, ji-eun title: mers transmission and risk factors: a systematic review date: - - journal: bmc public health doi: . /s - - - sha: doc_id: cord_uid: e zjaz background: since middle east respiratory syndrome (mers) infection was first reported in , many studies have analysed its transmissibility and severity. however, the methodology and results of these studies have varied, and there has been no systematic review of mers. this study reviews the characteristics and associated risk factors of mers. method: we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) for english- or korean-language articles using the terms “mers” and “middle east respiratory syndrome”. only human studies with > participants were analysed to exclude studies with low representation. epidemiologic studies with information on transmissibility and severity of mers as well as studies containing mers risk factors were included. result: a total of studies were included. most studies from saudi arabia reported higher mortality ( – . %) than those from south korea ( . %). while the r( ) value in saudi arabia was < in all but one study, in south korea, the r( ) value was . – . in the early stage and decreased to < in the later stage. the incubation period was . – . days in saudi arabia and – . days in south korea. duration from onset was – days to confirmation, . – . days to hospitalization, – days to death, and – days to discharge. older age and concomitant disease were the most common factors related to mers infection, severity, and mortality. conclusion: the transmissibility and severity of mers differed by outbreak region and patient characteristics. further studies assessing the risk of mers should consider these factors. middle east respiratory syndrome (mers) was first reported in in saudi arabia [ ] . although most patients are linked to the arabian peninsula geographically, mers has been detected in many other parts of the world [ ] . a large mers cluster was also observed in in south korea [ ] . mers causes sporadic infection and intrafamilial and healthcare-associated infection. its symptoms can vary from asymptomatic infection to death. despite the infection's association with high mortality, specified antiviral therapy is lacking, especially for patients with concomitant diseases [ ] . many previous studies have assessed the risks of mers, such as factors dictating severity or an infection risk, yet the indices they present vary. for example, the case fatality rate was found to be . % in the middle east area, but . % in south korea [ ] . the incubation period was reported to be . - days in south korea [ , ] , but . in a study using data from multiple areas [ ] and . in saudi arabia [ ] . accurate assessment of the risk of mers is essential for predicting and preventing infection. a systematic review of the risk of mers, as covered in previous studies, is potentially helpful for predicting this spread, and its future impact. this study aimed at reviewing the risk of mers, focusing on indices related to infectivity and severity. we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) using the term "mers" or "middle east respiratory syndrome", encompassing articles published after . the search process was conducted in october . we also manually searched the reference lists of the included studies. human studies were included, while animal studies and reviews were excluded. only articles in english or korean were included. even if a study collected data on humans, such as collecting specimens from religious pilgrims, it was excluded if there were no mers patients in the study sample. additionally, case studies including fewer than mers patients were excluded as they were considered as having insufficient mers patient numbers and representative information. the included studies were classified as epidemiologic studies and those covering risk factors of mers. in the epidemiologic category, indices related to the risk of mers were divided into two categories; related to infectivity and related to severity. the index related to infectivity included the reproduction number (r), attack rate, incubation period, serial interval, and days from onset to confirmation. the index related to severity included the case fatality rate (cfr), days from onset to hospitalization, days from onset to discharge, days from onset to death, and days from hospitalization to death. in the risk factor category, factors related to infection, transmission, severity, and mortality of mers were analysed. even if the included studies investigated factors that were related to mortality, when they did not analyse risk factors of severity or mortality using appropriate statistical methods (e.g., regression analysis, cox proportional hazards model) or only compared prevalence factors, we excluded them from the risk factor category. in all categories, we extracted the study period, number of participants, and geographical region where the data were collected using a data extraction form confirmed after pilot assessment. a total of studies were searched, and were reviewed, excluding duplicate studies. after the title and abstract review, a further and were excluded, respectively. another four studies were included via a manual search, which left a total of studies for analysis ( fig. ). the of total included studies were classified as epidemiologic studies (table ) . r value, representing the reproduction number, indicates the average number of secondary cases generated by infectious individuals. thirteen studies reported r value of mers. four studies that used data from multiple areas had r < . [ , [ ] [ ] [ ] . studies using saudi arabia or middle east area data reported r < , at . - . [ ] [ ] [ ] [ ] , though one reported . - . [ ] . studies using south korea data showed higher values, at . - . [ ] [ ] [ ] [ ] , in the early stage, and < in the later period [ ] or with control intervention [ ] . a total of eight studies reported the attack rate. four reported the overall or secondary attack rate, and the other four reported the attack rate of specific participant groups. two studies conducted in saudi arabia showed . % [ ] and % [ ] secondary attack rates. studies in south korea showed secondary attack rates of . % in one study [ ] and . - . % in another [ ] . two studies reported the attack rate among healthcare workers (hcws). one study in south korea reported a mers incidence of . % among hcws [ ] , and another study using multiple area data reported a . - . % infection rate among hcws [ ] . the attack rate among hospital patients was % in one study [ ] and % in the early and % in the later period in another [ ] . the incubation period is the period between infection and appearance of signs of a disease. a total of studies reported the incubation period of mers. nine used data from south korea and showed a - . day incubation period [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one study using data from saudi arabia reported a . day incubation period [ ] , and another using data from multiple areas reported a . day incubation period [ ] . sha et al. compared the incubation periods between the middle east area and south korea and reported . - and days, respectively [ ] . the serial interval of an infectious disease represents the duration between symptom onset of a primary case and of its secondary cases. two studies used south korea data, reporting serial intervals of mers of . and . days, respectively [ , ] . among five studies reporting days from onset to confirmation, three studies used data from south korea. one study analysing all south korea cases reported days from onset to confirmation [ ] . park et al. reported . days for all cases, for second generation and for third generation [ ] . one study from taiwan reported days for hcws and for non-hcws [ ] . a study from saudi arabia reported days from onset to confirmation [ ] . sha et al. compared the data from middle east and south korea areas and reported - and - days, respectively [ ] . two studies from saudi arabia reported days from onset to hospitalization. one reported . - days [ ] , and the other reported . days [ ] . twenty-six studies reported on mers-related mortality. ten reported the mortality rate in south korea as . - . % [ , , - , , , , ] ; one of which, including all mers patients in south korea, reported a mortality rate of . % [ ] . ten studies analysing data from saudi arabia reported higher mortality rates, of - . % [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , although others reported mortality rates % [ ] and . % [ ] . a taiwanese study reported a mortality rate of . % [ ] . studies using data from multiple areas reported mortality rates ranging from . % [ ] to . % [ , ] . three studies reported days from mers onset to discharge. sha et al. reported days in the middle east area and in south korea [ ] . one study from saudi arabia reported days [ ] , and another in south korea reported [ ] . two korean studies reported similar periods of - days from onset to death: - . in park et al. [ ] and in ki et al. [ ] . although one study from saudi arabia reported longer than days from onset to death [ ] , sha et al., comparing data between the middle east and south korea, reported similar periods of . and days, respectively [ ] . one taiwanese study also reported a similar period of - days [ ] . two studies reported a similar length of hospitalization: [ ] and . days [ ] . of the studies included in the risk factor category, four were duplicates of studies in the epidemiologic category as they had information regarding the epidemiologic index and risk factors ( table ) . two studies reported on the risk factors of mers infection. alraddadi et al. [ ] analysed the effect of nonhuman contact, including travel history, animal-related exposure, food exposure, health condition, and behaviour and reported direct dromedary exposure, diabetes or heart disease, and smoking as risk factors of mers infection. another study reported older age, outbreak week, and nationality as risk factors [ ] . three studies analysed factors associated with spreaders. non-isolated in-hospital days, hospitalization or emergency room visits before isolation, deceased patients, and clinical symptoms, including fever, chest x-ray abnormality in more than three lung zones, and the cycle threshold value, were related to spreaders [ , , ] . four studies reported risk factors of mers severity. the included studies showed that the prnt and cd t cell response [ ] as well as a high mers virus load [ ] were associated with the severity of mers. additionally, male sex; older age; concomitant disease, including hypertension; and symptoms, including fever, thrombocytopenia, lymphopenia, and low albumin concentration, were related to mers severity or secondary disease [ ] [ ] [ ] . fifteen studies reported risk factors of mortality in mers patients. older age [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and comorbidity [ , [ ] [ ] [ ] ] , including diabetes [ , ] , chronic kidney disease [ ] , respiratory disease [ , ] , pneumonia [ ] , cardiac disease, and cancer [ ] , were the most prevalent in the included studies. male sex was reported as a risk factor in one study [ ] . smoking [ , ] and location of acquisition [ , ] were also reported. while one study noted that hcw, as a profession, was associated with mortality [ ] , non-hcws were reported to be related to mortality in two other studies [ , ] . additionally, a shorter incubation period [ , ] , longer duration of symptoms [ ] , more days from onset to confirmation [ ] , later epidemic period [ ] , and longer hospitalized days [ ] were reported as mortality risk factors. symptoms at diagnosis, including abnormal renal function [ ] , respiratory symptoms [ ] , gastrointestinal symptoms [ ] , lower blood pressure [ , ] , and leucocytosis [ , ] , were also found to be associated with mortality in mers patients. severity of illness, [ , ] such as need for vasopressors [ ] , chest radiographic score [ ] , health condition [ ] , use of mechanical ventilation [ ] , and occurrence of dyspnoea [ ] were also found to increase the mortality risk. the characteristics of mers differ between south korea and the middle east area. the r value of mers was reported to be below in the middle east area, except in one study [ ] , but was from . - . in south korea [ ] [ ] [ ] [ ] [ ] . although studies using data from the middle east area reported . - % secondary attack rates, studies in south korea reported - % secondary attack rates for patients or hospital visitors [ ] , and . - . % for the overall attack rate [ , ] . the mers incubation period was reported to be . - . days in the middle east area [ , ] , but this period was found to be slightly longer in south korea [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the severity of mers also differed between the middle east area and south korea. mortality of mers patients was found to be . % in south korea based on a report including all cases [ ] , but most studies from saudi arabia reported higher rates, from to . % [ , , , [ ] [ ] [ ] . days from onset to confirmation were similar, - days in the middle east area [ , ] and - . days in south korea [ , , ] . days from onset to discharge were slightly longer in south korea, - days in the middle east area [ , ] and - days in south korea [ , ] (table ). the transmissibility and severity of mers were different by outbreak countries, especially between the middle east area and south korea. the virus, host, and environmental factors may be the causes of the mers outbreakrelated differences between the two regions. from the standpoint of viral factors, there was a mutation of the mers coronavirus (mers-cov) in the south korea outbreak. kim et al. [ ] reported a point mutation in the receptor-binding domain of the viral spike protein in mers-cov, and another study showed that mers-cov in south korea had higher genetic variability and mutation rates [ ] . individual characteristics can also affect mers transmission. as previous studies showed, there is an association between older age and mers infection [ ] , severity [ ] , and mortality [ , ] , and the population structure may be related to transmission and severity. additionally, individuals aware of mers were found to be more likely to practice preventive behaviour [ ] , which differed by demographic characteristics [ , ] . the transmission environment may also contribute to the difference. while many mers cases were contracted through exposure to camels in saudi arabia [ ] , the south korea outbreak involved multiple generations of secondary infections caused by intra-hospital and hospital-tohospital transmission [ , ] . strategies considering various factors are therefore needed to assess the impact of mers and to better control its spread. although several studies have reported the overall r value [ , , , ] , others have shown that this value this can be variable based on the generation or a control intervention [ , , ] . especially in the south korea epidemic, the r value was particularly high in the early stage or first generation, at . - . , though it later decreased to . - . [ , ] . further studies should consider and analyse the variation of the r value depending on the period or control intervention. while earlier studies on infectious diseases assumed a homogeneous infection ability of a population, recent studies have shown the existence of so-called super spreaders, individuals with a high potential to infect others in many infectious diseases, including ebola and severe acute respiratory syndrome (sars) [ ] . the role of the super spreader is also important in the spread of mers. in south korea, . % of mers patients were associated with five super-spreading events [ ] . stein et al. [ ] asserted that super spreaders were related with the host, pathogen, and environmental factors, and wong et al. [ ] reported that individual behaviours could also contribute to disease spread. there are variations in the mortality and attack rates among studies using south korea data. for example, park et al. [ ] reported a . % mers mortality, while reports from the korean ministry of health and welfare showed . % mers mortality. this disparity may, in part, be due to small sample sizes. park et al. [ ] included only patients because the study was conducted in an early phase of a mers outbreak. we excluded studies that included cases with < subjects, which were mostly case series, to reduce those types of biases. the present review found that older age and concomitant disease were risk factors of mers infection and mortality. these results are consistent with a recent systematic review that reported older age, male, and an underlying medical condition as predictors of death related to mers [ ] ; therefore, these factors should be prioritized in protection and treatment procedures. one limitation of this study was the possibility of subject duplication. especially in south korea, the korean government publishes mers reports that include all patients. the epidemiologic index in other studies might be biased since they included partial korean patients and were analysed in the middle of an outbreak. however, we included those studies because they showed the characteristics of mers in different situations and different stages. we did not conduct a meta-analysis because of the small number of studies for each index, which might be another limitation of this study. although this study reviewed the risk factors of mers and their impact, assessing the effect size of each risk factor is important. more studies investigating the effect of risk factors on mers need to be constantly conducted. most studies on the transmissibility and severity of mers have originated from saudi arabia and south korea. even though the r value in south korea was higher than that in saudi arabia, mortality was higher in saudi arabia. the most common factors behind mers infection and mortality were older age and concomitant disease. future studies should consider the risk of mers based on the outbreak region and patient characteristics. the results of the present study are valuable for informing further studies and health policy in preparation for mers outbreaks. isolation of a novel coronavirus 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respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea the role of super-spreaders in infectious disease super-spreaders in infectious diseases clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis authors' contributions jep (corresponding author) designed the study, and conducted the data search and the analysis with jep ( st author). syj and ark participated in the data review. jep (corresponding) drafted the manuscript, and jep ( st), syj, and ark revised it. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. the authors declare that they have no competing interests. key: cord- - f sl bp authors: plipat, tanarak; buathong, rome; wacharapluesadee, supaporn; siriarayapon, potjaman; pittayawonganon, chakrarat; sangsajja, chariya; kaewpom, thongchai; petcharat, sininat; ponpinit, teerada; jumpasri, jaruphan; joyjinda, yutthana; rodpan, apaporn; ghai, siriporn; jittmittraphap, akanitt; khongwichit, sarawut; smith, duncan r; corman, victor m; drosten, christian; hemachudha, thiravat title: imported case of middle east respiratory syndrome coronavirus (mers-cov) infection from oman to thailand, june date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: f sl bp thailand reported the first middle east respiratory syndrome (mers) case on june (day ) in an omani patient with heart condition who was diagnosed with pneumonia on hospital admission on june (day ). two false negative rt-pcr on upper respiratory tract samples on days and led to a -hour diagnosis delay and a decision to transfer the patient out of the negative pressure unit (npu). subsequent examination of sputum later on day confirmed mers coronavirus (mers-cov) infection. the patient was immediately moved back into the npu and then transferred to bamrasnaradura infectious disease institute. over contacts were traced; were quarantined and self-monitored for symptoms. high-risk close contacts exhibiting no symptoms, and whose laboratory testing on the th day after exposure was negative, were released on the th day. the omani ministry of health (moh) was immediately notified using the international health regulation (ihr) mechanism. outbreak investigation was conducted in oman, and was both published on the world health organization (who) intranet and shared with thailand’s ihr focal point. the key to successful infection control, with no secondary transmission, were the collaborative efforts among hospitals, laboratories and mohs of both countries. from to july , there have been , reported laboratory-confirmed cases and deaths from middle east respiratory syndrome coronavirus (mers-cov) infection in countries [ ] . a single imported case of middle east respiratory syndrome (mers) in south korea, identified on may , resulted in laboratory-confirmed cases, amplified by infection in hospitals and the transfer of patients within and between hospitals, and caused deaths within days, mainly among patients, visitors and healthcare personnel [ ] . this highlighted the need for vigilant surveillance and the importance of swift and thorough contact tracing. the thai ministry of public health (moph) launched mers surveillance and made mers a notifiable disease in , particularly targeting people travelling into thailand from affected countries. it also initiated a nationwide public education campaign [ ] . in , mers-cov infection was classified as a dangerous communicable disease in thailand according to the communicable act b.e. (ad ) . being added to this act required all probable and confirmed cases and their close contacts to be quarantined in a designated area for the duration of the maximum incubation period of days [ ] . there have been increasing numbers of incoming travellers from the middle east seeking medical care in thailand in the past decade. more than . million medical tourists travelled to thailand in , of which . % were from united arab emirates (uae) and oman [ ] . despite that, only three cases of mers have been confirmed as of july [ ] . this study shows that, in addition to needing collaboration among different organisations during an outbreak, diagnosis cannot rely only on laboratory examination alone, especially when the specimen was not suitable. a negative laboratory result in a patient from an endemic region with mers-like clinical signs still demands cautious infection control measures in an isolation unit. on june (day ), a -year old omani man travelled to thailand, seeking treatment for his heart condition. upon arriving at the airport, the patient took a taxi to a hotel and checked in before leaving to a private hospital in another taxi. upon presentation at the emergency room at the private hospital, the patient was promptly diagnosed with heart failure and possible pneumonia. as the patient had travelled from the middle east that day, mers was suspected and he was isolated in a negative pressure room (npu). on day , the private hospital notified the bureau of epidemiology, under the thai moph, of the omani patient. rt-pcr for mers-cov on upper respiratory tract samples (nasopharyngeal swabs) that were sent on days and resulted in false negatives, leading to a -hour delay in diagnosis and a decision to transfer the patient out of the negative pressure unit (npu) on day . subsequent examination of a sputum sample later on day confirmed mers-cov infection in the patient. the patient was immediately transferred back into the npu. on day , thailand's moph officially reported the first imported mers case and the over individuals, including with high-risk of exposure were traced. thirty-six high-risk close contacts were quarantined in thailand and low-risk contacts were monitored in oman. another high-risk close contacts (airline crew members) were quarantined in the country they were situated when traced. medical records from the private hospital and bidi under the department of disease control, under the thai moph, were reviewed [ ] . further, the patient and their family members were asked to elaborate on the clinical presentations and previous medical care in oman by thai investigators. information from the omani moh was obtained via the ihr mechanism during investigation. in accordance with who interim guidelines for laboratory testing for mers-cov [ ] , mers-cov rna was tested in sputum (pre-treated with n-acetylcysteine) and via nasopharyngeal swab (when sputum was not available), using qiaamp viral rna mini kit (qiagen, hilden, germany) for extraction. two real-time rt-pcr assays targeting upstream of envelope (upe) and open reading frame a (orf- a) genes [ , ] , and one rt-pcr assay for generating amplicons for sequencing, targeting the betacoronavirus rna-dependent rna polymerase (rdrp) gene (rdrpseq assay) [ ] , were performed simultaneously by who collaborating centre for research and training on viral zoonoses, faculty of medicine, chulalongkorn university (whocc) to increase efficiency and allow reporting of results within hours of receiving the samples. respiratory specimens (sputum, nasopharyngeal and throat swabs) were collected daily from the index case from the time of patient isolation on day through to day . the respiratory samples were sent to three centres, the bidi, the thai national institute of health (nih) and whocc, for parallel real-time pcr testing of mers-cov. additional molecular sequencing was performed by whocc. the whole genome amplification of mers-cov was carried out from extracted viral rna from collected sputum of the index case. seventy sets of specific primer pairs were used to amplify the complete genome as previously described [ ] , followed by sanger sequencing. for the analysis, all mers-cov genomes with complete coding sequences available in genbank as of december (n = ), were compared with the mers-cov genome obtained in this study. sequences showing less than divergent nt positions and two representatives of the five lineages defined by sabir et al. [ ] , were selected and used for phylogenetic analysis. a phylogenetic tree was constructed using the maximum likelihood method based on the general time reversible model and , bootstrap replicates in mega [ ] . contact tracing was immediately implemented by the thai moph. contacts were divided into two categories; high-risk and low-risk. a high-risk close-contact was defined as any person who was within m of contact with the index case while the patient was symptomatic, regardless of duration of contact. airline passengers seated in the two rows surrounding the index case's seat were also considered high-risk close contacts as per who guidelines [ ] . a low-risk contact was any person who had been in contact with the patient while the patient was symptomatic, but from a distance of more than m. people were considered non-contacts if there was no evidence of direct contact with the patient or if they were not likely to be in contact with respiratory droplets, the means of transmission for mers-cov. several methods of contact tracing and active case finding were applied depending on the nature of contact, contact location, degree of symptoms at the time of contact, etc. at the hospital, attending physicians' and nurses' contact status was determined via airline passengers who sat in the two rows around the patient a quarantined. laboratory testing results for mers-cov were negative. airline crew members identified, but left thailand for their return flight on june (day ). self-quarantined and self-monitored for symptoms, being off-service for the duration of the quarantine period. none reported to have developed any illness. healthcare workers at the private hospital all were immediately quarantined in the hospital. their laboratory testing were negative for mers-cov. taxi drivers quarantined. laboratory testing for mers-cov were negative. airline passengers self-monitored for symptoms, with social distancing. hotel staff healthcare workers close relatives assigned for two weeks of follow-up from the last date of exposure. one close-contact was assigned to be tested for mers-cov infection, but refused to cooperate. healthcare contacts at the first hospital healthcare contacts at the second hospital mers: middle east respiratory syndrome; mers-cov: middle east respiratory syndrome coronavirus. a only one of whom was a thai national. interview and the hospital surveillance camera. at the hotel, potential contacts were identified by interviewing the personnel on-duty when the patient checked in and by using the hotel's surveillance camera. the investigation team from the department of disease control (ddc) at the thai moph identified the airport-to-hotel taxi driver using the airport taxi booking slip and the hotel-to-hospital taxi driver by looking at the surveillance camera from the traffic control department. the airline provided the investigation team with the passenger manifest and the thai authorities identified passengers' local address using immigration arrival cards. local health authorities in relevant provinces were informed and asked by the investigation team to locate and contact the identified passengers in their jurisdiction. some passengers voluntarily reported to a hospital or health authority in response to the moph's announcement of first imported mers case in thailand. central authorities were responsible for locating all high-risk close-contacts, while local authorities were responsible for low-risk contacts. the time lapse between the affirmed diagnosis and each contact-tracing varied. contacts at the hospital were identified within a day, while other high-risk close contacts, such as passengers on the flight, were identified within days. other low-risk contacts were traced within days. patients who were in the same npu ward as the index case at the private hospital on day were monitored despite being considered non-contacts because: i) the room for each patient was separated, ii) they had no direct contact with the index case and iii) the known mode of transmission of mers-cov is respiratory droplet. further, patients in the icu, which is where the index case was moved after being taken out of the npu hours before diagnosis on day , were monitored, despite being non-contacts. in the event they developed a new episode of fever or respiratory symptoms, samples were collected and sent for testing to rule out mers-cov infection. another concern was icu healthcare workers' simultaneous care of several patients. prompt quarantine and monitoring of patients in the icu was to be implemented if any icu healthcare worker developed any symptoms or was diagnosed with mers-cov infection. most high-risk close contacts were quarantined and all were continuously monitored for days. nasopharyngeal and throat swabs, stored in single viral transport media, from high-risk close contacts were collected on two occasions as per the bureau of epidemiology guidelines [ ]: first upon identification as being a high-risk close contact and second on day during the quarantine period. specimens were duplicated and sent to any two of three laboratories (bidi, nih and whocc) for parallel real-time pcr testing of mers-cov, using both who and commercial primers for any given sample. in line with the thai communicable disease act, high-risk close contacts were only released after completing days of quarantine and if laboratory testing on the th day of quarantine was negative. sera from three high-risk close contacts (the close relatives who travelled with the patient to thailand) were sent to the institute of virology, university of bonn medical centre, bonn, germany to test for anti-mers igg and igm using mers-cov infected vero cells for immunofluorescence assay (anti-mers-cov iift, euroimmun, lübeck, germany). the real-time rt-pcr results on the patient's nasopharyngeal swabs were negative for upe and orf- a gene targets on days and (table ) , and the patient was thus transferred to a non-npu in the icu. however, the third sample from sputum that was taken later on day as the patient's condition deteriorated and that underwent three simultaneous rt-pcr assays at the whocc, was positive for upe, orf- a and rdrp gene targets. when whocc confirmed sputum was positive for mers-cov infection, the patient was immediately transferred back to the npu that night. mers-cov was confirmed via sequencing within hours by whocc. the patient was referred to and isolated at bidi on the morning day . the patient's clinical presentation at that time was diffused bilateral pneumonia with pending acute respiratory distress syndrome [ ] . he did not report any previous illnesses pertaining to these symptoms. later the same day, sputum was collected from the patient for reconfirmation, which tested positive for upe and orf- a genes by four different laboratories: the nih, ramathibodi hospital, bidi and whocc. the thai moph proceeded to publicly announce the first confirmed imported mers case in the evening of june (day ). the patient was monitored for mers until july (day ) and was discharged on july (day ). upon laboratory confirmation on day , the case was immediately notified to the who. in order to support local handling of the outbreak, an epidemiologist and a risk communication expert were deployed from the who south-east asia regional office and who headquarters, respectively. the epidemiological investigation revealed retrospectively that on june , days before the admission to the private hospital in thailand, the patient was admitted to a regional hospital in oman, with retrosternal and left-sided chest pain, which was radiating to his left arm (figure ). the condition was associated with shortness of breath on exertion and was considered typical cardiac pain. the patient was diagnosed with acute coronary syndrome. three days later, on june , his condition had improved and he was discharged. a close relative of the patient observed a dry cough of mild degree in the patient since june . on june , he was admitted to a second hospital, displaying signs of somnolence, fatigue and elevated blood sugar level; he was diagnosed with diabetes mellitus on june . there was also decreased air entry in the right lung with fine crepitations. chest x-ray showed opacity in middle and lower zones of right lung. both hospitals' medical records confirmed the patient did not exhibit any fever or cough symptoms. the patient was discharged that day with a follow-up appointment at a regional hospital scheduled for june ; however, the patient wished to seek medical care in thailand and flew there on june . the virus sequence (tha/cu/ ) obtained from the patient was submitted to genbank (genbank accession number kt ). tha/cu/ showed closest relations ( . % nt identity) to three human mers-cov strains isolated in saudi arabia in (genbank accession numbers kt , kt and kt ). figure shows the phylogenetic tree constructed from the mers-cov whole genome obtained from the patient (tha/cu/ , , bp), among the closest relatives and representatives for each mers-cov lineage defined by sabir et al. [ ] . correspondences with a close relative of the patient and the omani moh revealed that the patient was a fisherman from ghasil village, south sharqiya governorate, but spent june-august in al mintrib village of bidiya wilayat, north sharqiyah governorate. the patient neither had any history of travel outside oman nor contact with anyone with a history of travel outside oman within the days preceding his travel to thailand. further, the patient had no contact with any person with acute respiratory infection or confirmed mers before developing symptoms. the family used to own one camel but had not had contact with that camel for few months. a close relative who lived near the patient, but did not travel to thailand with the patient, owned and cared for three camels. samples collected from these camels tested negative in oman for mers-cov by rt-pcr on day . it is also noteworthy that the patient and the patient's close relatives never consumed raw camel milk or camel urine. a total of contacts of the index case were identified after the patient was confirmed to have mers. in thailand, contacts (excluding the healthcare personnel at bidi, investigated separately in the report by wiboonchutikul et al. [ ] ) were identified. of these , were high-risk close contacts and were low-risk contacts ( table ). all patients treated in the same ward (icu) at the private hospital of first admission of index case before mers diagnosis were identified as non-contact, however they were fully monitored and followed up for days, as per the thai moph's protocol, as a precautionary measure. in oman, the outbreak investigation determined there to be lowrisk contacts and this information was published on the dedicated who system. fortunately, there was no secondary transmission associated with this case. this study demonstrates the challenges faced by physicians and the cross-border threats that exist with increasing international medical tourism. although precautions such as thermoscan are in place at airports in light of the mers-cov outbreak in the middle east and south korea, cases can slip through checkpoints due to atypical presentations. the patient flew on a commercial airline despite his sickness and was not detected by the thermoscan at the immigration checkpoint in bangkok as he was afebrile. he only had a mild, non-productive cough. this case report also provides important lessons regarding clinical case identification. the clinical diagnosis was complicated due to the existence of congestive heart failure, a condition that predisposes to either community-acquired or nosocomial pneumonia of various aetiologies. furthermore, initial chest radiographs did not show clear signs of interstitial pneumonia as expected with mers-cov infection. various contact tracing methods involving the cooperation of several authorities and business institutes, such as the border control, airline, hotel management, traffic control, local authorities and hospitals, were used to track-down all potential contacts in order to prevent an outbreak. phone calls, passenger manifests, surveillance videos and immigration cards were essential tools for the successful contact tracing. upper respiratory tract samples, such as nasopharyngeal and oropharyngeal, are often used to detect upper respiratory tract illnesses during the acute phase. in mers-cov infection, however, higher viral loads have been found in specimens from the lower respiratory tract [ ] , with sputum or endotracheal secretion samples yielding better results [ ] . this aligns with the who interim guidelines, which encourage using lower respiratory tract samples if available [ ] . physicians, surveillance staff and laboratory personnel must be well-informed about the procedures, reliability and limitations of diagnostic tests, and should be able to recognise signs of mismatch between laboratory results and clinical presentations. in this case, the initial diagnostic testing of upper respiratory tract samples on days and caused a delay in diagnosis that could have facilitated onward transmission. fortunately, the patient was isolated in a npu upon initial admission; however, he could have exposed other patients and hospital workers during the hours he spent in regular icu after the initial false negative test results. the decision to conduct repeated laboratory testing, as well as to test lower respiratory tract samples, was driven by clinical assessment and knowledge of the virus excretion pattern reported in earlier cases. the algorithm for diagnosing mers in the current who interim guidelines for laboratory testing indicates that two positive real-time pcr tests are sufficient in diagnosing mers, i.e. orf- a and upe. however, we found that performing three simultaneous assays and sequencing for upe, orf- a and rdrp genes in parallel, allowed for swift in-country confirmation of the presence of mers-cov and reporting within hours, facilitating prompt outbreak control measures. there was no secondary transmission, not even to close relatives or the healthcare workers at bidi where the patient was transferred after the diagnosis was confirmed, despite contacts, including high-risk close contacts [ ] . despite the successful outcome of infection control measures, the case provides an example of the risk of mers-cov infection importation. aside from providing technical support through the moph emergency operations centre, deployments of experts from the who south-east asian regional office also greatly facilitated the exchange of information on possible modalities of mers-cov infection with the omani moh through the ihr ( ) mechanism [ ] . this study therefore emphasises how important hospital and organisation collaboration, as well as cross-border cooperation, is to successful infection control in the event of an outbreak. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome (mers) in the republic of collaboration preparedness plea announcement nonthaburi: royal thai government gazette medical tourism: falling oil prices & middle east tourism. bangkok post middle east respiratory syndrome coronavirus (mers-cov) -thailand. geneva: who; lack of transmission among healthcare workers in contact with a case of middle east respiratory syndrome coronavirus infection in thailand laboratory testing for middle east respiratory syndrome coronavirus (mers-cov) detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia mega : molecular evolutionary genetics analysis version . for bigger datasets emergencies preparedness, response: technical guidance on contact tracing, middle east respiratory syndrome coronavirus (mers-cov) department of medical services. [guidelines for diagnosing and treating mers patients, as well as protocols to prevent an outbreak in the hospital clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus: update for clinicians international health regulations none declared. tpl, rb, ps and cp were responsible for the case investigation, sample handling and coordination of sample tests. they were also responsible for contact tracing and quarantine. cs was responsible for the clinical case management and hospital quarantine authority. sw, tk, sp, tpo, jj, yj, aj, sk and drs were responsible for the laboratory testing and sequencing of mers-cov. ar and sg analysed the sequence and submitted it to genbank. rb, sw, sg and th wrote the original manuscript. cd, vmc, rb, sw, sg and th critically reviewed the original manuscript. sg, rb, sw and th critically reviewed the revised manuscript. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indicate if changes were made. key: cord- - pnqpljt authors: munster, vincent j.; adney, danielle r.; van doremalen, neeltje; brown, vienna r.; miazgowicz, kerri l.; milne-price, shauna; bushmaker, trenton; rosenke, rebecca; scott, dana; hawkinson, ann; de wit, emmie; schountz, tony; bowen, richard a. title: replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: pnqpljt the emergence of middle east respiratory syndrome coronavirus (mers-cov) highlights the zoonotic potential of betacoronaviruses. investigations into the origin of mers-cov have focused on two potential reservoirs: bats and camels. here, we investigated the role of bats as a potential reservoir for mers-cov. in vitro, the mers-cov spike glycoprotein interacted with jamaican fruit bat (artibeus jamaicensis) dipeptidyl peptidase (dpp ) receptor and mers-cov replicated efficiently in jamaican fruit bat cells, suggesting there is no restriction at the receptor or cellular level for mers-cov. to shed light on the intrinsic host-virus relationship, we inoculated jamaican fruit bats with mers-cov. although all bats showed evidence of infection, none of the bats showed clinical signs of disease. virus shedding was detected in the respiratory and intestinal tract for up to days. mers-cov replicated transiently in the respiratory and, to a lesser extent, the intestinal tracts and internal organs; with limited histopathological changes observed only in the lungs. analysis of the innate gene expression in the lungs showed a moderate, transient induction of expression. our results indicate that mers-cov maintains the ability to replicate in bats without clinical signs of disease, supporting the general hypothesis of bats as ancestral reservoirs for mers-cov. scientific reports | : | doi: . /srep arab emirates and egypt , [ ] [ ] [ ] . mers-cov sequences and virus isolates obtained from dromedary camels in qatar and the kingdom of saudi arabia showed high sequence identity with those obtained from epidemiologically linked human cases , . together, these data suggest that rather than direct zoonotic transmission from a bat reservoir, dromedary camels are involved as the primary reservoir host for mers-cov. phylogenetic and epidemiological data suggest that rather than a single introduction in the human population, mers-cov appears to continue to be transmitted by multiple independent spillover events from dromedary camels , . after the emergence of severe acute respiratory syndrome coronavirus (sars-cov) in , the targeted focus on reservoir studies in bats has resulted in a vast increase of our knowledge on the genetic diversity of bat coronaviruses. despite the increase in genetic data on coronavirus diversity in their natural reservoirs, only very limited data are available on the impact of these viruses on the reservoir host and controlled infection experiments with coronaviruses in their reservoir hosts have not been performed. to understand the drivers of mers-cov emergence, a more comprehensive understanding of the interaction between the virus and its natural and intermediate reservoir hosts is needed. here we present data on the first experimental infection of bats with mers-cov to model the infection kinetics in a coronavirus host species, the jamaican fruit bat (artibeus jamaicensis). artibeus jamaicensis dpp receptor and cell susceptibility. the mers-cov receptor dpp is the main host restriction factor ; therefore, we first studied the interaction between mers-cov and jamaican fruit bat dpp . the paj-dpp plasmid expressing the dpp coding sequence of jamaican fruit bat under control of a cmv promoter was transfected into bhk cells, which are not permissive to mers-cov . the expression of aj-dpp in transfected cells was confirmed by flow cytometry, showing the presence of bat dpp on the surface of transfected bhk cells by determining the increase over untransfected cells ( figure s ). transient expression of bat dpp in bhk cells supported mers-cov replication, whereas transient expression of hamster dpp in bhk cells did not (fig. a) . subsequently, the replication kinetics of mers-cov were compared in llc-mk cells (macaca mulatta) and jamaican fruit bat primary kidney cells. mers-cov replicated efficiently to high titers in both cell lines (fig. b) , indicating that there is no restriction at the receptor or cellular level for mers-cov replication in jamaican fruit bat cells. clinical signs in bats inoculated with mers-cov. ten adult jamaican fruit bats were inoculated via the intranasal and intraperitoneal routes with tcid of mers-cov strain emc/ ; mock inoculated jamaican fruit bats, housed in a separate cage, were used as controls, the mock inoculated animals were inoculated with tissue culture medium via the same routes and volumes. the bats were observed at least once daily for signs of disease. bodyweight and temperature were measured throughout the experiment for a maximum of days post inoculation (dpi) for bat and (mers-cov inoculated) and bat and (mock inoculated controls). none of the bats showed signs of disease, weight loss or increased body temperature throughout the experiment ( figure s ). to examine mers-cov shedding in inoculated bats, oral and rectal swabs were collected for the duration of the experiment. mers-cov shedding was first detected on dpi, as indicated by the presence of viral rna in throat and rectal swabs and continued for a maximum duration of days. all animals, except bat , shed mers-cov from the respiratory tract ( fig. a) ; all bats except and , shed mers-cov from the intestinal tract (fig. b ). viral loads in swabs collected from the respiratory tract were higher than viral loads in swabs from the intestinal tract. tissues collected at the sequential necropsy dates of , , , and dpi were analyzed for the presence of viral rna, infectious virus, and evaluated by histopathology and immunohistochemistry. mers-cov viral rna was detected in various tissues of all inoculated bats, except bat on dpi and bat on dpi. the highest viral loads were detected in the lower respiratory tract (fig. ) . mers-cov viral rna was detected at dpi in trachea, lung, liver, spleen, bladder and nasal turbinates; at dpi in lung, spleen, duodenum, colon, bladder, turbinates and brain; at dpi in lung, liver, turbinates and brain; at dpi in heart, lung, liver, spleen and duodenum. no mers-cov viral rna was detected at dpi (fig. ) . in additon, mers-cov viral rna was detected in blood on and dpi, in bats - , indicative of viremia ( figure s ). mers-cov mrna was detected in tissues of bats to , confirming mers-cov replication on the transcriptional level (table s ) . infectious mers-cov was isolated from the lungs of bats ( dpi) and ( dpi), the bladder and nasal turbinates of bat ( dpi), and the duodenum of bat ( dpi), indicating active virus replication, mainly in the respiratory tract. only two of ten bats (bat and bat ) exhibited histopathology associated with mers-cov infection, which was mild. all mers-cov associated lesions were detected in the respiratory tract of the infected bats (fig. , table s ). bat and bat ( dpi) displayed a mild acute rhinitis, but mers-cov replication was not detected by immunohistochemistry (table s and s ). bat and displayed a multifocal interstitial pneumonia that was characterized by minimal alveolar interstitial thickening by small numbers of macrophages and neutrophils (fig. , table s ). the adjacent alveolar spaces contained small numbers of alveolar macrophages. mers-cov antigen and rna was detected by immunothroughout the lungs of bat ( dpi), but no associated pulmonary pathology was detected (fig. , table s ). cytokeratin and anti-mers-cov co-staining demonstrated mers-cov antigen in type i pneumocytes of the lungs of bat (fig. ). scientific reports | : | doi: . /srep innate immune response to mers-cov. mers-cov was most consistently detected in the lower respiratory tract of the bats. the mx , isg and rantes gene expression in the lungs of jamaican fruit bats was analyzed as an indicator of the induction of an innate immune response to mers-cov infection. a -fold increase in expression of mx was observed in the lungs of the infected jamaican fruit bats at dpi. a statistically significant upregulation of mx gene expression was detected when comparing the lungs of bats collected on dpi and dpi (two-tailed unpaired t-tests, p < . ). the maximum isg expression of . -fold occurred at dpi. statistically significant differences were observed between the dpi and dpi, dpi and dpi animals (two-tailed unpaired t-tests, p < . , p < . and p < . respectively. in addition significant differences were observed between the dpi and the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ). the rantes expression at its peak at dpi was increased . fold. statistically significant differences were observed between the versus the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ), the versus the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ), and the dpi versus the and dpi animals (two-tailed unpaired t-tests, p < . and p < . ) (fig. ). antibody response to mers-cov. sera were collected prior to inoculation and at the scheduled necropsy dates. each of the bats was seronegative for mers-cov prior to inoculation. only bat developed a mers-cov specific antibody response, both by elisa and virus neutralization assay. the sera obtained from bat had a neutralizing titer of at days post inoculation. the high sequence similarity of mers-cov to coronavirus sequences detected in bats suggests that mers-cov or its immediate ancestor originated in bats . direct contact between bats and humans is uncommon, and a domestic or peridomestic intermediate species often plays a role in the emergence of zoonotic viruses from natural reservoirs to humans [ ] [ ] [ ] [ ] . similar to the emergence of sars-cov in from the masked palm civet (paguma larvata) as an intermediate host , the dromedary camel appears to have initially served as the intermediate host for mers-cov . several aspects of the emergence of mers-cov are currently still unknown, including the role of the natural reservoir and the relationship between the natural and intermediate reservoirs. with their ability to support efficient replication of mers-cov, the availability of an annotated transcriptome , and the relative easy housing and husbandry practices of jamaican fruit bats suggest that this bat species can become an important model system to investigate the relationship between coronaviruses and their bat hosts . although the jamaican fruit bat is not the direct ancestral reservoir for mers-cov, as it is a new world bat species, generalized responses towards viruses of bat-origin rather than a direct host-pathogen relationships can be modelled. the ability of mers-cov to use dpp of multiple species as a receptor, including dpp of human, dromedary camel, and bat origin , , suggests that no prior adaptation was needed on the dpp receptor level for cross-species and zoonotic transmission to occur. with batcov-hku , a closely related coronavirus, it was shown that replication in human cells required two mutations in the spike protein . these amino acid residues, which are conserved in mers-cov, results in the activation of the batcov-hku spike protein by human cellular proteases. this suggests that batcov-hku needs these residues for replication in humans. interestingly, our data show that mers-cov replicates efficiently in jamaican fruit bat cells, suggesting that the mers-cov spike can efficiently be processed by jamaican fruit bat cellular proteases and that there is no host restriction on the post-translational modification level of the mers-cov spike in dromedary camels, humans and bats. bat coronaviruses have been primarily detected in fecal samples in field studies suggesting that these viruses have a intestinal tract tropism , , . mers-cov was able to replicate to higher titers in the respiratory tract in comparison with the intestinal tract of the jamaican fruit bats. the tissue tropism of mers-cov in jamaican fruit bats is comparable to the respiratory tract tropism observed in dromedary camels and humans , . this might suggest that mers-cov, upon cross-species transmission from bats into dromedary camels evolved from a gastrointestinal tract virus into a respiratory tract virus, similar to influenza a viruses . the ability for mers-cov to antagonize the innate immune response appears to correlate with its pathogenic potential in humans. mers-cov and related batcov-hku can inhibit innate immune signaling in a variety of human cell lines in vitro via the orf b-encoded accessory proteins lungs of jamaican fruit bat were stained with α -cytokeratin as an epithelial marker (purple) and with a polyclonal α -coronavirus antibody (brown-red) to demonstrate that viral antigen was located along the basement membrane of alveolar pneumocytes of bat at dpi (indicated by black arrows). original magnification: × . with % fetal calf serum (hyclone, logan), mm l-glutamine (lonza), u/ml penicillin and μ g/ml streptomycin (gibco). vero e , llc-mk , bhk and jamaican fruit bat primary kidney cells were maintained in dulbecco's modified eagle's media (dmem) supplemented with % fetal calf serum, u/ml penicillin and μ g/ml of streptomycin. sequencing and cloning of the jamaican fruit bat dpp sequence. total rna was extracted from primary kidney cells using the rneasy mini kit (qiagen) and cdna was synthesized using random hexamer primers and superscript iii reverse transcriptase (applied biosystems). dpp was then amplified using iproof high-fidelity dna polymerase (biorad) and primers dpp unvf and dpp unvr (primer sequences are available upon request). the obtained dpp gene sequence was synthesized in expression plasmid pcdna . (+ ) cate with mers-cov with a multiplicity of infection (moi) of . (cell lines) or (transfected cell lines) % tissue culture infectious dose (tcid ) per cell. one hour after inoculation, cells were washed once with dmem and culture medium replaced. supernatants were sampled at , , and h after inoculation. mers-cov was titrated by end-point titration in quadruplicate in vero e cells cultured in dmem supplemented with % fetal calf serum, mm l-glutamine (lonza), u/ml penicillin and μ g/ml streptomycin. cells were inoculated with ten-fold serial dilutions of virus, and scored for cytopathic effect days later. the tcid was calculated by the method of spearman-karber. animal experiments. twelve captive-bred jamaican fruit bats were used for this work , . ten bats were inoculated with tcid emc/ via a combination of intranasal ( μ l each nostril) and intraperitoneal ( μ l) routes. two mock inoculated bats were included as controls for histopathology and gene expression analyses. mock inoculated bats were inoculated with standard tissue culture media via the same routes and volumes. bats were injected with an iptt- temperature transponder (bmds) to monitor body temperature daily. animals were weighed daily and observed for signs of disease. oropharyngeal and rectal swabs were obtained on , , , , , , , and dpi and analyzed for the presence of viral rna. on , , , and days post inoculation (dpi), two bats were euthanized and trachea, heart, lung, liver, spleen, kidney, duodenum, colon, bladder, nasal turbinates and brain were collected for virological and histopathological analysis. histopathology. histopathology was performed on bat tissues. after fixation for at least days in % neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (h & e) staining. immunohistochemistry was performed using a mers-cov emc/ polyclonal rabbit antibody at a : dilution and in situ hybridization was performed using probes directed against the mers-cov emc/ n gene as described previously . rna extraction. rna was extracted from swab samples using the qiaamp viral rna kit (qiagen). rna was eluted in μ l. tissues ( mg) were homogenized in rlt buffer and rna was extracted using the rneasy kit (qiagen). rna was eluted in μ l. quantitative pcr. for detection of viral rna in samples, μ l rna was used in a one-step real-time rt-pcr upe assay using the rotor-gene tm probe kit (qiagen) according to the manufacturer instructions. in each run, standard dilutions of a titered mers-cov stock were run in parallel, to calculate tcid equivalents in the samples. for the detection of viral mrna, μ l rna was used in a one-step real-time rt-pcr using the mers-cov m mrna assay in the rotor-gene tm probe kit . artibeus jamaicensis orthomyxovirus resistance gene (mx ) gene expression was determined by qrt-pcr using mx , isg and rantes specific primers (derived from transcriptome sequencing ). the fold-change of each gene was calculated by normalizing the change in ct (cycle threshold) value of mx (Δ ct) to the ct values for hypoxanthine phosphoribosyltransferase (hprt) as an internal reference gene for each sample and comparing this to the ct values of mock inoculated bats and ( ∧ (−Δ Δ ct). mx specific primers: ′ -ccagacctgaccctgataga- ′ , ′ -tggatgtacttcctgaatgagttg- ′ and ′ -fam-atctagtgtccgatgtcagctggc-iabkfq- ′ . isg specific primers: ′ -gctgtctatcgtctgaatggg- ′ , ′ -ttcttgtccgatgtcctgaag- ′ and ′ -hex-cgatgaggc/zen/attttgtctgcaaaccc-iabkfq- ′ . rantes specific primers: ′ -agttgtcctaatcacccgaaag- ′ , ′ -cagagtgttgatgtagtcccg- ′ and ′ -fam-tgtgccga c/zen/ccggagaagaaat-iabkfq- ′ . hprt specific primers: ′ -agatggtgaaggtcgcaag- ′ , ′ -cctgaagtattcattatagtcaaggg- ′ and ′ -fam-actttgttggatttgaaattccag acaagtttg-bhq . virus isolation. tissue samples were homogenized in a tissuelyzer ii (qiagen) after addition of ml dmem. homogenates were centrifuged to pellet cellular debris and subsequently inoculated onto veroe and llc-mk cells. after hr adsorption, cells were washed once with dmem and media was replaced. elisa. antibody responses were measured in an enzyme-linked immunosorbent assay (elisa) using hcov-emc/ as described previously . briefly, emc/ containing cell culture supernatant was used to coat immuno microwell maxisorp plates (nunc) at °c overnight and diluted serum samples were added. bound antibodies were detected using a secondary protein a/g conjugated with horseradish peroxidase (hrp; pierce). sera were considered positive when absorbance was higher than three standard deviations above the mean of negative control sera. sera obtained from rabbits immunized with emc/ were used as a positive control. virus neutralization assay. two-fold serial dilutions of heat-inactivated sera were prepared in a microwell tissue culture plate and tcid of mers-cov was added and incubated for hour at °c. after incubation the virus-sera mixture was transferred to a microwell tissue culture plate with a % confluent scientific reports | : | doi: . /srep monolayer of veroe cells. the 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syndrome practical considerations for high-throughput influenza a virus surveillance studies of wild birds by use of molecular diagnostic tests the orf b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling comparison of serological assays in human middle east respiratory syndrome (mers)-coronavirus infection presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study experimental nipah virus infection in pteropid bats (pteropus poliocephalus) pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission tacaribe virus causes fatal infection of an ostensible reservoir host, the jamaican fruit bat experimental inoculation of plants and animals with ebola virus seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection bats and lyssaviruses experimental rabies virus infection in artibeus jamaicensis bats with cvs- variants middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction. euro surveillance : bulletin européen sur les maladies transmissibles growth and quantification of mers-cov infection the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the authors would like to thank drs. bart key: cord- -qtlevnbt authors: al hosani, farida ismail; kim, lindsay; khudhair, ahmed; pham, huong; al mulla, mariam; al bandar, zyad; pradeep, krishna; elkheir, kheir abou; weber, stefan; khoury, mary; donnelly, george; younis, naima; el saleh, feda; abdalla, muna; imambaccus, hala; haynes, lia m; thornburg, natalie j; harcourt, jennifer l; miao, congrong; tamin, azaibi; hall, aron j; russell, elizabeth s; harris, aaron m; kiebler, craig; mir, roger a; pringle, kimberly; alami, negar n; abedi, glen r; gerber, susan i title: serologic follow-up of middle east respiratory syndrome coronavirus cases and contacts—abu dhabi, united arab emirates date: - - journal: clin infect dis doi: . /cid/ciy sha: doc_id: cord_uid: qtlevnbt background: although there is evidence of person-to-person transmission of middle east respiratory syndrome coronavirus (mers-cov) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. methods: a seroepidemiological investigation was conducted among mers-cov case patients (cases) and their household contacts to investigate transmission risk in abu dhabi, united arab emirates. cases diagnosed between january and may and their household contacts were approached for enrollment. demographic, clinical, and exposure history data were collected. sera were screened by mers-cov nucleocapsid protein enzyme-linked immunosorbent assay and indirect immunofluorescence, with results confirmed by microneutralization assay. results: thirty-one of ( %) case patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. mers-cov antibodies were detected in of ( %) case patients with available sera, including severely symptomatic, mildly symptomatic, and asymptomatic case patients. no serologic evidence of mers-cov transmission was found among household contacts with available sera. conclusions: transmission of mers-cov was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. these results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission. although there is evidence of person-to-person transmission in household and healthcare settings [ ] [ ] [ ] [ ] [ ] , more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. these data would be of importance to the public health response given that approximately % of confirmed mers-cov cases reported to the world health organization have been described as mildly symptomatic or asymptomatic [ ] . during january - may , the department of health-abu dhabi (doh) investigated laboratory-confirmed cases and conducted extensive contact investigations in both household and healthcare settings [ ] . through these investigations, % of the laboratory-confirmed cases reported no symptoms or mild illness [ ] . contacts of case patients were tested by diagnostic pcr assays; however, results could include false negatives due to the -day incubation period. in this investigation, we use serological detection of mers-cov antibodies to evaluate if asymptomatic or mildly ill case patients had detectable mers-cov antibodies, estimate transmission rates from known cases to their household contacts, and identify potential risk factors. this investigation occurred in the emirate of abu dhabi, which occupies > % of the uae's total area [ ] and is comprised of regions: abu dhabi (capital city), al ain region, and al dhafra. the emirate of abu dhabi has a population of . million ( estimate) [ ] . the al ain region borders oman and saudi arabia and houses the second largest city in the emirate, al ain city. while al ain city is an oasis, the rest of the region primarily consists of desert and mountains. the al dhafra region is mainly desert and rural with approximately residents and a population density of residents/km [ ] . all laboratory-confirmed mers-cov cases (n = ) in the emirate of abu dhabi diagnosed between january and may and their household contacts (n = ) were eligible for the investigation. these cases were a convenience sample during the ongoing mers-cov outbreak. two of the ( . %) household contacts tested for mers-cov during initial contact investigations were pcr positive and eligible to be enrolled as cases for our investigation (figure ). the enrolled case was a healthcare worker who might have been exposed by another coworker, who also lived in the case's household; therefore, the enrolled case was a result of either household or healthcare transmission prior to this investigation's initiation. the case not enrolled in this investigation was exposed in the household. household contacts were defined as any person who stayed at least night at the same location as the case patient during the days prior to the case patient's symptom onset or the date of first positive specimen if the case patient was asymptomatic. excluded cases included palace workers and other high-level officials; their associated household contacts were also excluded. for each mers-cov case identified in the investigation, clinical information, including symptoms, was collected using the international severe acute respiratory and emerging infection consortium form, which was filled out in real time by healthcare providers and subsequently verified by retrospective chart review. in abu dhabi during this time period, all individuals who tested positive for mers-cov were admitted to a healthcare facility for observation and infection control regardless of symptom status. the same definitions for case severity were used as in al hosani et al [ ] including the following: asymptomatic cases reported no symptoms at the time of a positive test as recorded by a healthcare provider in the medical chart; mildly symptomatic cases reported symptoms, such as pharyngitis, rhinorrhea, or cough, and did not require oxygen during their hospitalization; and severely symptomatic cases required supplemental oxygenation during their hospitalization, ranging from nasal cannula to mechanical ventilation. using data collected from doh's surveillance of mers-cov cases, households with mers-cov case patients were approached. household contacts who were eligible for the investigation included those that had been identified through contact investigations associated with the case patient performed by doh officials within hours' notification. three attempts were made to contact each household. if no response was received after attempts, the household was not enrolled. households that agreed to be enrolled were given an appointment at the local disease prevention and screening center for questionnaire administration and serum collection. questionnaires were administered in english, arabic, or, if an interpreter was available, the participant's native language. data collected included demographics; residence/household description; exposure history to other mers-cov cases, healthcare settings, and animals; travel history; and medical history, including any long-term effects reported by case patients. for deceased case patients, a proxy completed the case patient questionnaire using recall. the real-time reverse-transcription pcr (rrt-pcr) results were obtained from the doh surveillance data. upper (ie, nasopharyngeal, oropharyngeal) and lower respiratory tract specimens (ie, sputum, bronchoalveolar lavage fluid, tracheal aspirates) were analyzed using rrt-pcr in the sheikh khalifa medical center laboratory. additional laboratory result verifications were performed in a random sample of specimens using nucleocapsid-based rrt-pcr [ ] . serum samples were inactivated using × rads gamma irradiation and stored at ≤ - °c until use. screening of serum specimens by mers-cov nucleocapsid enzyme-linked immunosorbent assay (elisa) was performed at the sheik khalifa medical city in abu dhabi, uae and the centers for disease control and prevention (cdc), atlanta, georgia. titers of ≥ : were reported as positive. recombinant full length mers-cov nucleocapsid protein indirect elisa was used to screen serum specimens as described by al-abdallat et al [ ] . serum samples were tested for the presence of neutralizing antibodies to mers-cov using a microneutralization assay (mnt) [ ] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell lysis in at least of the triplicate wells. positive and negative controls were included for each mnt performed and included back-titration and mock-infected cells. titers of ≥ : were reported as positive. all work with live mers-cov was done in biosafety level containment at the cdc. immunofluorescence assays (ifas) were performed by screening sera at a dilution of : and : on paraformaldehyde-fixed, acetone-methanol permeabilized mers-cov (strain mers-cov hu/england-n / ) infected or uninfected control vero cells. antihuman immunoglobulin g, m, and a fluorescein isothiocyanate conjugate was used to detect anti-mers-cov antibodies in human serum, and nuclei were counterstained with ′, -diamidino- -phenylindole to allow identification of individual mers-cov-infected cells. fluorescence was detected using a zeiss axioimager fluorescence microscope. the positive control for the assay is a serum sample from a patient infected with mers-cov hu/ england-n / . a positive result was scored when these conditions were met: cells were evenly stained (instead of punctate staining); fluorescence intensity was higher than that of the negative controls; and signal intensity declined with serial dilution. a minimum of negative controls were included with each ifa. approximately % of specimens negative by nucleocapsid elisa were screened by both ifa and mnt to confirm the negative result. mers-cov antibody positivity was defined as one of the following: ( ) of tests (ie, mers-cov nucleocapsid elisa, mers-cov mnt, and ifa) were positive; or ( ) mers co-v mnt was the only positive test. household survey data were entered into electronic forms in epi info version . (cdc). quality control and assurance were performed through epi info intelligent codes programmed into the forms. household survey data were merged with the laboratory results, and descriptive analysis was completed. differences in proportions were compared using the mantel-haenszel χ test, while differences in continuous variables were compared using the student t test. p < . was considered statistically significant. data analysis of the merged dataset was conducted with sas version . software (sas institute, cary, north carolina). following local customs, informed consent was obtained from the head of the household, who provided consent for all members of a household; however, each individual was still able to decline participation. this investigation was determined by doh and cdc to be part of a public health response, not research, and therefore not subject to institutional review board review. thirty-four case patients' households were included (supplementary table ). household residences ranged in size from m to m (interquartile range [iqr], - m ). a median of individuals (range, - ) lived in the households days prior to the diagnosis of a mers-cov household case patient. more than half of mers-cov case patients shared a bathroom with others in the household. all households reported having air conditioning. thirty-four cases of ( %) and household contacts of ( %) participated (table ) . females comprised a higher proportion of case patients compared with household contacts ( . % vs . %), and case patients were older compared with household contacts (median, years vs years). most case patients and contacts were from the al ain region of the abu dhabi emirate. seventy-one percent (n = ) of case patients reported working in a healthcare setting days prior to diagnosis, with nurses being most represented ( %, n = ) ( table ) ; only % (n = ) of household contacts worked in a healthcare setting days prior to a case patient's diagnosis. compared with household contacts, case patients less frequently reported visiting or owning a farm ( % vs %), but reported camel exposure more frequently ( % vs %). household contacts reported the following frequent exposures to mers-cov case patients: hugging ( %, n = ), using the same bathroom ( %, n = ), sharing meals ( %, n = ), and kissing or nose-kissing (ie, rubbing tips of noses against one another) ( %, n = ). case patients reported a higher proportion of underlying medical conditions than household contacts, including diabetes ( % vs %, p = . ), hypertension ( % vs %, p < . ), kidney failure ( % vs %, p = . ), and heart failure ( % vs %, p < . ) ( table ). case patients also reported taking medications for any illness more frequently than contacts ( % vs %). three case patients ( %) reported limitation to activities due to mers-cov with a median duration of days (iqr, - days) ( table ) . normal activities were resumed at a median of days (iqr, - days). of case patients, ( %) reported being asymptomatic, ( %) reported being mildly symptomatic, and ( %) were severely symptomatic. age and proportion having underlying medical conditions increased with symptom severity ( table ) . symptom duration did not have any noticeable trend with symptom severity (data not shown). all severe case patients were treated in the intensive care unit, as well as asymptomatic case, who had underlying diabetes, hypertension, and kidney disease. the median days hospitalized increased with symptom severity ( table ) . sera were obtained from of ( %) case patients and of ( %) household contacts. among the case patients with available sera (table ) among the case patients with detectable antibodies against mers-cov, all of them were aged < years, with a median age of years, compared to a median of years for case patients without detectable antibodies (table , p = . ). number of days of pcr positivity was notably higher among those who had detectable antibodies compared to those who did not (median, days vs days, p = . ). we describe the results of follow-up of mers-cov case patients and of their household contacts from the emirate of abu dhabi during - . notably, serologic testing did not find any evidence of mers-cov transmission in the households of mers-cov case patients in our investigation, suggesting that viral transmission from asymptomatic or mildly symptomatic individuals to household contacts does not readily occur. sera were tested with a combination of different laboratory assays (nucleocapsid elisa, ifa, and mnt); we feel confident that individuals identified as "negative" did not seroconvert. although there was clear evidence of household transmission in household not enrolled in this investigation, our investigation's results did not show evidence of additional household transmission. overall, our findings support current recommendations that home isolation may be appropriate for asymptomatic cases and close contacts who are ill and do not require hospitalization in consultation with local public health departments [ , ] . because this investigation occurred during may-june , many case patients were recruited from the april healthcare-associated outbreak at an al ain region hospital [ ] . a kingdom of saudi arabia study found that while healthcare personnel were at high risk for infection, most illness was relatively mild and could be unrecognized, highlighting potential undetected transmission of the virus to others [ ] . in our investigation, case patients tended to be younger ( - years) , and most reported working in a healthcare setting days prior to their diagnosis where they were exposed to a similar to previous studies, case patients with severe disease had higher frequency of comorbid conditions and required intensive care, including intubation [ ] [ ] [ ] . in a recent investigation from south korea, patients with a higher host infectivity, which included evaluation of pcr cycle threshold values, along with higher numbers of contacts, were more likely to transmit mers-cov [ ] . it is likely that most of the primary case patients in this investigation had lower host infectivity. while our investigation found that some asymptomatic or mildly symptomatic case patients had detectable antibodies, we did not find any detectable antibodies in asymptomatic and mildly symptomatic case patients. other studies also did not find detectable antibodies in some asymptomatic and mildly ill cases [ , ] . if seroconversion is to occur in case patients, studies have demonstrated that this usually occurs within the first month of illness [ ] [ ] [ ] . for the majority of case patients with detectable antibodies, we found persistence of antibody response for several months after the initial diagnosis, even close to a year. additionally, these case patients had a longer duration of mers-cov pcr positivity than those who did not have detectable antibodies, indicating a potential relationship between longer viral shedding and seroconversion. previous studies have demonstrated that asymptomatic and mildly symptomatic case patients can test pcr positive > weeks from lower respiratory tract specimens [ , ] . our investigation's serology results do not provide additional evidence of transmission to household contacts, though there is evidence from other settings to suggest limited household transmission [ ] . also, very low rates of household transmission have been reported during hospital-based outbreaks [ , ] . more robust transmission studies involving larger numbers of case patients representing a range of clinical and demographic characteristics and their contacts are needed to further investigate risk exposures. there are several limitations to this investigation. first, serum samples were collected at varying intervals after illness onset for each case patient, potentially affecting serology results. the duration of antibody response is unknown. second, recall bias might have led to the misclassification of symptom severity among household contacts; however, for case patients, to minimize this bias, we relied upon retrospective medical chart review, though this also might not be as complete since it depended on the initial healthcare provider's history and physical. third, these case patients were immediately isolated in hospitals after pcrpositive results were discovered. the removal from the household setting might have reduced exposure to household contacts although the case patients were residing with household contacts at the time of the contact investigations. last, because our investigation did not detect household transmission, we cannot comment on any behaviors or exposures that would increase risk among household contacts of case patients. in summary, we did not document additional household transmission in this investigation that included a preponderance of asymptomatic and mildly symptomatic confirmed mers-cov case patients. our investigation findings support the recommendation to consider home isolation for asymptomatic and mildly ill cases that do not require hospitalization while using proper precautions, including face masks, frequent hand washing, and minimizing exposure to the case patient in the household [ , , ] . while no vaccines or antivirals against mers-cov are currently available, reducing transmission through effective infection control management remains a major priority. understanding transmission risk for different mers-cov-infected patients who live in different settings will be important data that must be factored into prevention strategies. further studies on human-to-human transmission in different settings should be conducted to inform mers-cov prevention and control guidelines. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov): current situation years after the virus was first identified world health organization. who mers-cov global summary and risk assessment risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia response to emergence of middle east respiratory syndrome coronavirus antibody response and disease severity in healthcare worker mers survivors mers-cov antibody responses year after symptom onset, south korea persistence of antibodies against middle east respiratory syndrome coronavirus comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity mers-cov outbreak in jeddah-a link to health care facilities transmission of mers-coronavirus in household contacts hospital outbreak of middle east respiratory syndrome coronavirus family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus transmission in extended family, saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-update: disease outbreak news abu dhabi emirate: facts and figures statistical yearbook of abu dhabi hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance implementing home care and isolation or quarantine of people not requiring hospitalization for mers-cov transmission of middle east respiratory syndrome coronavirus infections in healthcare settings risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea serologic responses of mers-coronavirusinfected patients according to the disease severity viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker health care worker contact with mers patient, saudi arabia interim guidance for healthcare professionals acknowledgments. the authors gratefully thank the participants; the teams from the disease prevention and screening center-al ain, the disease prevention and screening center-abu dhabi, ghayathi hospital, and silla hospital that supported this investigation; and suvang trivedi and seyhan boyoglu-barnum at the centers for disease control and prevention (cdc) for technical support.disclaimer. the conclusions, findings, and opinions expressed herein are those of the authors and do not necessarily reflect the official position of the us department of health and human services, the us public health service, the cdc, or the authors' affiliated institutions.financial support. this work was supported by the cdc. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -eumuid r authors: widagdo, w.; okba, nisreen m. a.; richard, mathilde; de meulder, dennis; bestebroer, theo m.; lexmond, pascal; farag, elmoubasher a. b. a.; al-hajri, mohammed; stittelaar, koert j.; de waal, leon; van amerongen, geert; van den brand, judith m. a.; haagmans, bart l.; herfst, sander title: lack of middle east respiratory syndrome coronavirus transmission in rabbits date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eumuid r middle east respiratory syndrome coronavirus (mers-cov) transmission from dromedaries to humans has resulted in major outbreaks in the middle east. although some other livestock animal species have been shown to be susceptible to mers-cov, it is not fully understood why the spread of the virus in these animal species has not been observed in the field. in this study, we used rabbits to further characterize the transmission potential of mers-cov. in line with the presence of mers-cov receptor in the rabbit nasal epithelium, high levels of viral rna were shed from the nose following virus inoculation. however, unlike mers-cov-infected dromedaries, these rabbits did not develop clinical manifestations including nasal discharge and did shed only limited amounts of infectious virus from the nose. consistently, no transmission by contact or airborne routes was observed in rabbits. our data indicate that despite relatively high viral rna levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that is known to infect dromedary camels and humans [ , ] . seroepidemiological studies indicate that this virus has been circulating in dromedary camels in the arabian peninsula and africa for decades [ ] [ ] [ ] . mers-cov sequences obtained from these camels are largely similar to those obtained from human mers cases in corresponding regions, thus providing evidence that dromedary camels act as the zoonotic source of this virus [ , ] . however, many primary human mers cases do not have a history of direct contact with these animals [ ] . this suggests the presence of unidentified routes of human-to-human transmission or the involvement of other animal species in spreading the virus to humans. besides dromedary camels, other animal species, i.e. llamas, alpacas, and pigs have been shown to be susceptible and develop upper respiratory tract infection upon experimental intranasal mers-cov inoculation [ ] [ ] [ ] . this is in line with the expression of the mers-cov receptor, dipeptidyl peptidase- (dpp ), in their nasal epithelium [ ] . mers-cov-seropositive llamas and alpacas have been reported in the field, and mers-cov-experimentally-inoculated alpacas have also been shown to transmit the virus via contact [ ] [ ] [ ] . to further understand the zoonotic potential of mers-cov, it is crucial to delineate the factors involved in the spread of the virus among dromedaries as well as other animal species. in order to gain insight into these factors, we performed mers-cov transmission experiments in rabbits. we have previously shown that rabbits are susceptible to mers-cov and develop both upper and lower respiratory tract infection upon virus inoculation [ ] . naïve recipient rabbits were housed with mers-cov-inoculated donor rabbits either in the same or in adjacent cages to determine whether mers-cov can be transmitted via contact or airborne routes, respectively [ ] . donor rabbits were found to shed high levels of viral rna but a limited amount of infectious virus thus potentially restricting mers-cov transmission in these animals. in vivo experiments in this study were performed using passage human isolate mers-cov emc strain (hcov-emc/ ) and passage isolate mers-cov (qatar / ; genbank acc. no. mk ) that were propagated in vero cells as described earlier [ ] . qatar was isolated from a years old qatari man that developed severe pneumonia and was pcr confirmed to have a mers-cov infection [ ] . animal experiments were approved and performed according to the guidelines from the institutional animal welfare committee (no. approved on july , - - approved on september , and avd -wp approved on november ). the studies were performed under biosafety level (bsl ) conditions. to compare whether different routes of mers-cov inoculation generate similar clinical outcomes, twelve -month-old new zealand rabbits (oryctolagus cuniculus), specific pathogen free, and seronegative for mers-cov were divided into four groups. animals were inoculated under ketamine-medetomidine anesthesia either (a) intranasally with µl of × tcid /ml mers-cov; (b) intranasally with ml × tcid /ml mers-cov; (c) intranasally with µl of × tcid /ml mers-cov and intratracheally with ml × tcid /ml mers-cov; or (d) intranasally with ml pbs. the intranasal inoculums were divided equally over both nostrils. nasal and throat swabs were obtained daily from day up to day post inoculation. these animals were then sacrificed on day and respiratory tract tissues were collected. to compare the clinical outcomes of the mers-cov emc strain and the qatar strain, ten new zealand rabbits were divided into two groups and inoculated with ml of × tcid /ml of each mers-cov strain intranasally. nasal and throat swabs were obtained from day up to day post inoculation and these animals were sacrificed on day . to study mers-cov transmission, a modified version of the previously described influenza a virus ferret transmission set-up was used. this set-up consists of two clear polymethyl methacrylate cages of different sizes. donor rabbits and direct contact recipients were housed in a cage of cm × cm × cm (w × h × l), whereas airborne recipients were housed in a cage of cm × cm × cm (w × h × l). these cages are separated by two stainless steel grids cm apart to prevent direct contact but still allow airflow from the donor rabbit to the airborne recipient rabbit. these transmission cages allow the experiment to be conducted in negatively pressured isolators in the bsl facility, with hepa-filtered airflow < . m/s [ ] . since these cages were too small for new zealand rabbits, we chose a smaller-sized breed, the netherland dwarf rabbits (oryctolagus cuniculus), for the mers-cov transmission experiment. we used both male and female rabbits with an age viruses , , of range of months- years in these experiments. first, three mers-cov seronegative netherland dwarf rabbits were inoculated intranasally with ml of × tcid /ml mers-cov qatar strain ( µl per nostril) to show that they are equally susceptible to mers-cov as the new zealand rabbits. nasal and throat swabs were obtained daily up to days post inoculation. these animals were then sacrificed on day and their respiratory tract tissues were collected. for the virus transmission experiment, twelve netherland dwarf rabbits were randomly distributed into four individually housed groups. one naïve rabbit from each group was inoculated intranasally with ml of × tcid /ml mers-cov ( µl per nostril), thus acting as donor rabbits. the other two rabbits were used as direct contact and airborne recipients, respectively. donor and direct contact animals were of the same sex. all animals were sacrificed at days post exposure and blood was collected to assess seroconversion. nasal swabs, throat swabs, and respiratory tract tissue samples were evaluated for the presence of infectious virus by virus titration, and for viral rna by rt-qpcr against the upe gene as previously described [ ] . samples with a cycle threshold less than forty were considered as positive for mers-cov rna. viral rna was quantified as genome equivalents (ge) using mers-cov strain emc (containing tcid /ml) as a calibrator. virus titration was performed in serial -fold dilutions on vero cells. cells were monitored under a light microscope at day for cytopathic effect. the amount of infectious virus in swab samples was calculated by determining the tcid . statistical analysis was performed using the graphpad prism program (la jolla, ca, usa). kruskal-wallis test was applied due to the non-normal distribution of our data as priorly determined by shapiro-wilk test. the significant difference between groups was determined at a p-value < . . respiratory tract tissue samples were collected in formalin and embedded in paraffin for pathological analysis. hematoxylin-eosin staining was performed for histopathological analysis. the presence of mers-cov nucleoprotein and mers-cov rna was detected by immunohistochemistry and in-situ hybridization, respectively, using previously published protocols [ ] . the localization of dpp in the respiratory tract of non-infected new zealand rabbits was analyzed using an optimized immunohistochemical assay [ , ] . collected serum samples were tested for mers-cov neutralizing antibodies using a virus neutralization assay and for mers-cov s -specific antibodies using mers-cov s elisa according to the previously published protocols [ ] . goat anti-rabbit igg conjugated with hrp ( : , dako, glostrup, denmark) was used as a secondary antibody in the elisa. rabbits are the smallest animal species that can be naturally infected by mers-cov. we previously reported that they develop both upper and lower respiratory tract infection upon mers-cov inoculation [ ] , suggesting the expression of the viral receptor at these locations. using immunohistochemistry, we analyzed the dpp expression in rabbit respiratory tract tissues. in the upper respiratory tract, dpp is strongly expressed at the apical surface of both nasal respiratory and olfactory epithelium ( figure ). in the lower respiratory tract, dpp is present in both bronchiolar and alveolar epithelial cells, although some variation in dpp expression was observed throughout the lungs. dpp is either absent, limitedly expressed on alveolar type ii cells, or expressed on both alveolar type i and ii cells ( figure ). thus, these data highlight a broad dpp expression in the respiratory tract different human mers-cov strains have been isolated since the emc/ strain was first characterized [ , ] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar / ) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs ( figure ). following this result, the mers-cov transmission experiment was performed using the qatar strain, the more recent strain of these two. type ii cells are indicated with arrows, and type i cells with an arrowhead. nasal epithelium and bronchiolar epithelium pictures were taken at a × magnification, and alveolar epithelium at ×. the isotype control showed no background signal in our assay. in our previous study, we inoculated rabbits both intranasally and intratracheally [ ] . intratracheal inoculation is quite invasive, and thus requires a skillful operator to minimize procedure-related damage in the respiratory tract. in contrast, intranasal inoculation is less invasive and had been used in other studies to infect rabbits with mers-cov [ , ] . here we investigated whether intranasal mers-cov inoculation is sufficient to induce both upper and lower respiratory tract infection in rabbits, in comparison to combined intranasal and intratracheal inoculation. three new zealand rabbits (oryctolagus cuniculus) were inoculated with mers-cov emc strain either intranasally with µl of × tcid /ml (group a); intranasally with ml of × tcid /ml (group b); intranasally with µl of × tcid /ml and intratracheally with ml of × tcid /ml (group c); or intranasally with ml of pbs as a negative control (group d). all three groups of mers-cov inoculated rabbits developed minimal clinical manifestations and histopathological lesions. the amount of viral rna shed in the nasal and throat swabs did not vary among the inoculated groups (figure a,b) . however, in the lungs of the rabbits, the amount of viral rna was significantly lower in group a than in groups b and c ( figure c ). in line with these observations, fewer mers-cov-infected cells were observed in the lungs of group a animals compared to groups b and c ( figure d ). based on these results, we decided to use intranasal inoculation with ml of × tcid /ml mers-cov for our subsequent experiments. pictures were taken at a × magnification, and alveolar epithelium at ×. the isotype control showed no background signal in our assay. different human mers-cov strains have been isolated since the emc/ strain was first characterized [ , ] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar / ) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs (figure ). following this result, to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, cm apart [ ] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar strain to determine their susceptibility to the virus. similar to the new zealand rabbits [ ] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure a,b) . identical to the new zealand rabbits [ ] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day or post inoculation/exposure for the donor and direct contact rabbits, respectively, and day post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~ - tcid ge/ml) and the throat swabs (~ - tcid ge/ml). the nasal and throat swabs were obtained from day (before inoculation) until day post inoculation. the amount of viral rna is displayed in genome equivalents per ml (ge/ml). dashed lines depict the detection limit of the assay. all error bars represent standard deviations. to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, cm apart [ ] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar strain to determine their susceptibility to the virus. similar to the new zealand rabbits [ ] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure a,b) . identical to the new zealand rabbits [ ] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. donor rabbits at day post inoculation, and in one of the donors up to day . in the throat swabs, infectious virus was only detected in two donors on day , up to day in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration (figure c,d) . serological analysis of samples collected days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [ , ] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day or post inoculation/exposure for the donor and direct contact rabbits, respectively, and day post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~ - tcid ge/ml) and the throat swabs (~ - tcid ge/ml). the amount of viral rna shed by the inoculated rabbits remained high until day post inoculation. on the other hand, recipient rabbits housed in the same cage (direct contact recipients), or adjacent cage (airborne recipients), shed limited amounts of viral rna (~ tcid ge/ml) in both nasal and throat swabs. among four animals in each group, only two direct contact recipient and two airborne recipient rabbits had detectable viral rna up to day post inoculation in the nasal swabs, while in the throat swabs, viral rna was only detected in one direct contact recipient and one airborne recipient rabbit at day post inoculation ( figure a,b) . infectious virus was detected at low level (~ tcid /ml) both in the nasal and throat swabs of the donor rabbits; in the nasal swabs of all donor rabbits at day post inoculation, and in one of the donors up to day . in the throat swabs, infectious virus was only detected in two donors on day , up to day in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration ( figure c,d) . serological analysis of samples collected days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [ , ] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [ ] [ ] [ ] ] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [ ] [ ] [ ] ] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of view, it is important to know whether other animal species in the region may also spread the virus to humans or other animal species. in vitro, mers-cov has been found to infect cells from a broad range of animal species including old and new world camelids as well as primates, bats, cows, sheep, goats, pigs, horses, and rabbits [ , , ] . the dpp viral receptor of these species, especially rabbits, has high similarity to that of humans and dromedary camels, especially in the region that interacts with the spike protein, and thus can facilitate mers-cov infection [ ] [ ] [ ] . the new world camelids, i.e. llamas and alpacas, have been shown to seroconvert to mers-cov when present in regions where mers-cov is circulating and may transmit the virus [ ] [ ] [ ] . it is currently unclear why, besides camelids, other livestock animals do not seem to transmit the virus to humans [ , [ ] [ ] [ ] . to further understand the transmission potential of mers-cov, we performed virus transmission experiments using rabbits as animal model. to perform the virus transmission experiments, we housed mers-cov-inoculated rabbits together with naïve contact rabbits either in the same or adjacent cages. rabbits developed both upper and lower respiratory tract infection upon mers-cov inoculation [ ] , either via intranasal or combined intranasal and intratracheal routes, in line with the localization of dpp in their respiratory tract epithelium. the amount of viral rna being shed by the inoculated rabbits during the first three days post inoculation is almost as high as that of the mers-cov-inoculated dromedary camels [ , ] . however, none of the direct contact and airborne-exposed rabbits developed any clinical signs, shed significant levels of viral rna, shed infectious virus, nor did they seroconvert. one possible reason for this lack of transmission is the limited amount of infectious virus being shed by the inoculated rabbits [ ] . previous studies have shown that in the nasal and lung tissues of experimentally infected rabbits, infectious virus was generally detected in a limited amount despite the abundant presence of viral rna and virus nucleoprotein [ , ] . alternatively, low levels of infectious virus transmitted to recipient animals may be unable to initiate a productive infection due to the presence of host proteins that restrict replication. comparable to rabbits, mers-cov-infected pigs and goats develop minimal clinical signs, hardly shed infectious virus, and barely spread the virus to naïve animals [ , ] . in contrast, mers-cov-infected dromedary camels develop nasal discharge and shed a high amount infectious virus ( - tcid /ml), almost equal to the amount of viral rna being shed ( - ge/ml), during the first days post inoculation [ , ] . these interspecies differences may indicate presence of nasal discharge and infectious virus shedding as critical factors in mers-cov transmission. in humans, levels of infectious virus shed by mers-cov patients have rarely been reported. however, mers-cov patients that transmit the virus have been shown to shed a higher amount of viral rna in their swabs compared to those that do not, supporting the quantity of virus shed as an important factor in the transmission of mers-cov between humans [ ] . for influenza a viruses, infectious virus shedding has been documented as one of the main determinants of airborne virus transmission. using ferrets as an animal model, it has been reported that a reduction in infectious virus shedding in the nasal swabs can subsequently limit virus transmission [ ] [ ] [ ] . our results show that despite the presence of dpp in the upper respiratory tract, accompanied by mers-cov replication at this site, a limited amount of infectious virus was shed. similarly, titration of rabbit lung homogenates that show high levels of viral rna and presence of nucleoprotein ( figure c ,d) resulted in only low levels of infectious virus, in line with earlier observations [ , ] . at this stage, it is not clear which host mediated mechanisms limit the production of infectious virus while allowing viral rna to still be shed at high levels. since restriction of infectious virus shedding in the rabbits already occurred one day post inoculation, activation of host innate immune responses, including type i interferon induction, may be relevant. in vitro studies have shown that mers-cov is relatively sensitive to type i interferon-mediated antiviral activities [ , ] . in human plasmacytoid dendritic cells, mers-cov inoculation leads to secretion of large amount of type i and iii interferons and production of viral rna, but hardly any infectious virus is being produced [ ] . it is also possible that most infectious virus shed by these rabbits is defective, lacking the capacity to efficiently infect target cells. further studies are needed to elucidate the mechanisms that restrict mers-cov replication in rabbits compared to dromedary camels. potentially, some of the mers-cov accessory proteins shown to antagonize immune responses including production of interferon, may not work efficiently in some mers-cov susceptible species, including rabbits. it is intriguing to investigate whether a similar phenomenon occurs in some human mers-cov infections and whether this is linked to the development of asymptomatic to mild clinical manifestations [ ] . this might partly explain why mers-cov transmission in humans is rather inefficient in comparison to dromedary camels [ , ] , and why camelids that secrete high levels of infectious virus are the only known zoonotic source of mers-cov [ , , , ] . deciphering these mechanisms could potentially offer insight into understanding mers-cov transmission as well as developing novel treatments to tackle the ongoing outbreaks. isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels zoonotic origin and transmission of middle east respiratory 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mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment inhibition of novel beta coronavirus replication by a combination of interferon-alpha b and ribavirin high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus world health organization mers situation update transmission of mers-coronavirus in household contacts mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -tsxniu j authors: sha, jianping; li, yuan; chen, xiaowen; hu, yan; ren, yajin; geng, xingyi; zhang, zhiruo; liu, shelan title: fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: tsxniu j middle east respiratory syndrome coronavirus (mers-cov) was first isolated in . the largest known outbreak outside the middle east occurred in south korea in . as of june , laboratory-confirmed cases ( deaths; . % case fatality rate [cfr]) had been reported from countries, particularly in the middle east. however, the cfr for hospital outbreaks was higher than that of family clusters in the middle east and korea. here, we compared the mortality rates for nosocomial outbreaks in the middle east and one outbreak of mers-cov in south korea. our findings showed the cfr in the middle east was much higher than that in south korea ( . % [ / ] vs. . % [ / ], p = . ). infected individuals who died were, on average, older than those who survived in both the middle east ( years [ – ] vs. years [ – ], p = . ) and south korea ( years [ – ] vs. . years [ – ], p = . ). similarly, the co-morbidity rates for the fatal cases were statistically higher than for the nonfatal cases in both the middle east ( . % [ / ] vs. . % [ / ], p = . ) and south korea ( . % [ / ] vs. . % [ / ], p = . ). the median number of days from onset to confirmation of infection in the fatal cases was longer than that for survivors from the middle east ( days [ – ] vs. days [ – ], p = . ). thus, older age, pre-existing concurrent diseases, and delayed confirmation increase the odds of a fatal outcome in nosocomial mers-cov outbreaks in the middle east and south korea. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. the first report of middle east respiratory syndrome (mers) was identified in saudi arabia in june . the middle east respiratory syndrome coronavirus (mers-cov) isolated from this patient was similar to severe acute respiratory syndrome coronavirus (sars-cov), which caused an epidemic in - [ ] . both novel viruses are single-stranded rna viruses belonging to the genus betacoronavirus [ , ] , and the diseases they cause share common clinical characteristics, including fever, cough, diarrhea, and shortness of breath [ ] . jianping sha, yuan li, and xiaowen chen equally contributed to this work. as of june , the world health organization (who) had been notified of laboratory-confirmed cases with mers-cov (globally), including at least deaths; the case fatality rate (cfr) was . % ( / ) [ ] . a total of countries in the world have been affected, including countries in the middle east (egypt, iran, jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates, yemen), africa (algeria, tunisia), europe (austria, france, germany, greece, italy, the netherlands, turkey, the united kingdom), asia (china, the republic of korea, malaysia, philippines, thailand) and north america (united states) (http://www.who.int/ emergencies/mers-cov/en/). so far, all cases of mers have been linked through travel to or residence in countries in or near the middle east. generally, the middle east is the primary region where mers-cov originates, circulates and is exported. in contrast, since the first report of sars-cov in china in , a total of sars cases, including deaths, have been reported to who. these have involved countries, predominantly in south east asia, with only one case identified in kuwait in , and no cases were found in the middle east since then (http:// www.who.int/csr/sars/country/table _ _ /en/). the fatality risk for mers-cov is much higher than that for sars-cov, which has a cfr of . % [ , ] . furthermore, the cfr for patients with co-morbidities is greater ( % in mers vs. % in sars) than those without preexisting diseases [ ] . generally, the cfr is attributed to both host factors and virus factors (e.g. virus replication and mutation) and local medical expertise [ , ] . one unique common epidemiological characteristic of these two diseases is that the spread of both mers-cov and sars-cov infection has been largely driven by human-to-human transmission in healthcare settings [ ] . failures in infection prevention and control in healthcare settings have occasionally resulted in large numbers of secondary cases in nosocomial outbreaks. the earliest identified nosocomial mers outbreak was traced back to march from clusters of severe respiratory illness among healthcare personnel (hcp) in jordan [ ] . since then, a series of nosocomial mers outbreaks in small numbers have been identified in the middle east (jordan in , saudi arabia in - ) [ , , , , ] in this study, we conducted a preliminary mortality risk factor analysis for nosocomial mers-cov outbreaks in south korea and the middle east. the findings from this study might help to reduce the severity and number of deaths from hospital-clustered cases by leading to the adoption of appropriate control measures. in , scientists in the republic of korea and china completed full-genome sequencing of coronaviruses from the mers outbreak in korea. the findings were analysed by a group of virologists convened by who, and preliminary results suggested that the mers cov viruses isolated in the republic of korea were similar to those isolated in the middle east (http://www.who.int/mediacentre/news/ mers/briefing-notes/update- - - /en/). mers-covs associated with the korean and middle east outbreak belong to lineage of mers-cov, which has been the predominant infectious agent in saudi arabian camels since november [ ] . the mers-cov variants associated with the recent outbreak of human infections in south korea (e.g., chinagd -v / and kor/knih/ - / ) show the highest similarity ( . - . %, full genome) to a camel virus (camel/riyadh/ry / ) sampled in march , followed by the latest strain (kt ) prevalent in saudi arabia ( . % identified) [ ] . however, the mers-covs in korea have the ability to cause large outbreaks in environments that are different from that of the middle east (http://www.who.int/emer gencies/mers-cov/en/). the national health and family planning commission of china determined that the collection of data from one human mers-cov infection imported from korea was part of the public health investigation of an outbreak and was exempt from institutional review board assessment. all other mers cases were obtained from publicly available data sources. all data were supplied and analysed without access to personal identifying information. information on all laboratory-confirmed mers cases was obtained from various publicly available sources, including who global alert and response updates, documents officially released by the local health bureau, news releases from middle eastern and south korean authorities, the weekly epidemiological record, promed posts, and literature published from april to june (http:// www.who.int/csr/don/archive/disease/coronavirus_infections/ en/). the latest cases that had not been officially announced by who were identified by searching promed posts, which confirmed announcements by individual countries' ministries of health. based on the available data, we initially established a database of a line list of human nosocomial mers outbreaks (supplementary tables s , s and s ). according to the who's july interim reporting definition (http://www.who.int/csr/disease/coronavirus_ infections/case_definition/en/), a person with mers has a laboratory-confirmed mers-cov infection, irrespective of clinical signs or symptoms. a case may be laboratoryconfirmed by detection of viral nucleic acid or serology. the presence of viral nucleic acid can be confirmed by either a positive reverse transcription polymerase chain reaction (rt-pcr) result on at least two specific genomic targets or a single positive target with sequencing of a second target. a case confirmed by serology requires demonstration of seroconversion in two samples, ideally taken at least days apart, by enzyme-linked immunosorbent assay (elisa), by indirect fluorescent antibody (ifa) screening, or by a neutralization assay [ , ] . a direct epidemiological link with a confirmed mers-cov patient may include ( ) healthcare-associated exposure, including providing direct care for mers-cov patients, working with healthcare workers infected with mers-cov, visiting patients or staying in the same close environment of individuals infected with mers-cov; ( ) working together in close proximity or sharing the same classroom environment with individuals infected with mers-cov; or ( ) travelling together with individuals infected with mers-cov in any kind of conveyance or living in the same household as individuals infected with mers-cov. in addition, the epidemiological link may have occurred within a -day period before or after the onset of illness in the case under consideration [ ] . we used a comparative epidemical analysis of the dates of onset of illness and the characteristics of the fatal and surviving cases. all statistical analysis was conducted using the statistical analysis system, version . (sas institute, cary, nc, usa). quantitative measurements are presented as the median and range of the observed values, and qualitative measurements are presented as relative and absolute frequencies. an analysis of variance (f test) was applied to the measurement data. v tests were used to compare the distribution of the different variables of qualitative measurements between fatalities and survivors. fisher's exact test was used in the analysis of contingency tables when the sample sizes were small (the expected values in any of the cells of a contingency table were below ; the number of total samples was no more than ; the data were very unequally distributed among the cells of the table). any p-values given were two-sided and considered statistically significant at . . as of march , we had identified human laboratory-confirmed clusters with mers-cov, involving cases, of which were fatal. all clusters had been reported to who or published by the local authority or in pubmed. these mers-clustered cases were distributed in nine countries: clusters from the kingdom of saudi arabia (ksa), six from the united arab emirates (uae), four from jordan, three from qatar, and one each from france, iran, italy, tunisia, and the united kingdom (uk). the numbers of clusters per year were as follows: three clusters including cases in , clusters including cases in , and clusters including cases in . for the control groups, we chose a total of sporadic cases of mers-cov, composed of fatal and nonfatal cases from the following countries: cases from the ksa, cases from the uae, cases from jordan, from qatar and from tunisia. the numbers of sporadic cases per year were as follows: cases in , cases in and cases in . fatality risks for nosocomial mers outbreaks the results showed that the percentages of hcp in mers clusters were much higher than those in sporadic cases ( . % [ / ] vs. . % [ / ], p = . ) ( table and table s ). however, the hcp-specific cfr was much lower than the overall cfr from mers clusters ( , p = . ). the median age in fatal cases in hcp was much lower than in fatal cases overall ( . years vs. years , p = . ) ( table ) . we stratified the age groups between the fatal and nonfatal cases groups. the results showed a statistical difference in the distribution of the - , - , - , - , and ? year-olds between the two groups (p = . ). males dominated both the fatal and nonfatal groups of the clustered and sporadic cases (p [ . ) ( table ) . a history of exposure to camels prior to onset of disease was not significantly correlated with survival ( . five time periods useful for public health surveillance were evaluated. the median time from onset to confirmation of infection in the fatal groups was much longer than that for survivors in mers clusters ( . days [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] vs. days [ - ], p = . ) and in sporadic mers cases ( days vs. days [ - ], p = . ). however, there were no statistical differences in the median time from onset to hospital admission, onset to hospital discharge, and subsequent death or the number of hospitalized days between the fatal and nonfatal cases for the two groups (table ) . by march , we had obtained data on nosocomial outbreaks involved in confirmed cases (all nosocomial outbreaks were from the middle east; the above clusters were not included in these outbreaks), including iran (one cluster), ksa ( clusters), jordan (three clusters), france (one cluster) and uae (five clusters). we also had one nosocomial outbreak with confirmed cases with mers-cov in south korea (table and table s ). the observed average cluster size ( ) for mers from south korea was much greater than that for the middle east ( , range - ). human nosocomial outbreaks with mers-cov in the middle east occur throughout the year and peak in the spring, especially february to april. mers outbreaks in south korea were reported from march to june , concomitant with peaks in the reporting of mers nosocomial outbreaks in the middle east ( table ) . the average age in the fatal groups was much higher than that in the survival groups ( years old vs. table ) . we found no difference between the fatal and nonfatal cases with respect to exposure to camels and other animals (horses, sheep and goats). in contrast, the level of humanhuman transmission was much higher in the nonfatal cases in the middle east than in the fatal cases ( . table ) . the middle east group showed a statistical difference between fatal and nonfatal cases for the median days from ], p = . ). however, there were no differences in the percentage of total deaths between the index and secondary cases ( table ) . the mean age of the deaths was significantly higher than that of the survival cases for the index ( years vs. years [ - , p = . ) and secondary cases ( years vs. years , p = . ). patients in the age groups c and - years were the most common in the fatal and survival cases, respectively, for the index group, while the - and - -year age groups were the common groups in the fatal and nonfatal cases, respectively, for the secondary cases. there was no significant difference in gender distribution between the fatal and nonfatal cases in the index and secondary groups ( table ) . the ratio of co-morbidity was much higher in the fatal groups than in the non-fatal groups from the secondary cases ( . % [ / ] vs. . % [ / ], p = . ); however, there was no difference between the fatal and nonfatal groups from the index cases. similarly, a history of exposure prior to onset was common for the fatal and nonfatal groups from the index and secondary cases ( table ) . there were no differences between fatal and nonfatal cases in the median time from onset to hospitalization, onset to confirmation, onset to discharge or death or hospitalized days (table ) . however, the median time from onset to hospitalization was shorter in the secondary cases compared to the index cases ( days the secondary cases was slightly shorter than in the index cases ( days vs. days , p = . ); however, the median time from onset to hospital discharge for secondary survivors was days ( - ), which was significantly shorter than the days ( - ) for index survivors (p = . ). acute respiratory tract infections with mers-cov cause considerable morbidity and mortality and pose a threat of repeated outbreaks in healthcare facilities [ , , , [ ] [ ] [ ] ] . the resulting transmission among patients, visitors, and hcp has been a defining feature of mers-cov epidemiology since its emergence in [ ] . in this study, we compared the mortality risk factors in two different nosocomial outbreaks, based on nosocomial outbreaks of mers-cov infection in the middle east and one large outbreak identified in south korea. our findings showed the final cfr for the middle east ( . %) was significantly higher than that for south korea ( . %). both estimated cfrs were significantly lower than that for one hospital outbreak of mers (cfr % [ / ] ) in saudi arabia in and another nosocomial outbreak (cfr . % [ / ]) in saudi arabia [ , ] . the cfr of this latter outbreak was also much higher than that of one extended family cluster ( . % [ / ]) in saudi arabia in [ ] . these results demonstrate that the survival rate of clustered patients with mers-cov in korea was higher than in the middle east. there are several possible explanations for the observed differences between the cfrs in south korea and the middle east. first, there may be disparities in national surveillance and available expertise [ ] . second, the cfr for the middle east might have been overestimated because a large number of mild and asymptomatic cases are likely to go undetected there [ ] . third, it is possible that primary cases accounted for a higher percentage of the cluster patients in the middle east than in south korea [ ] . the findings on age were consistent in hospital outbreaks in the middle east and from south korea. our results showed that the median age in fatal cases was much higher than that in nonfatal cases. this is in agreement with a saudi arabian case series report that showed individuals older than years had a greater association with mortality. a multivariate logistic regression model estimated that for every -year increase in age, the odds of dying increased by % [ ] . in all, this indicates that older age is associated with death in cases of mers-cov infection [ , , ] . in particular, the median age in fatal hcp cases was also much higher than that in nonfatal hcp cases, but lower than the overall average. this is in agreement with the findings of liu et al. [ ] . the reasons for the higher fatality rates in older individuals are not understood but have been attributed to cultural practices that result in an increase in the exposure risk that older people are willing to take [ ] . in addition, older people may be more likely to smoke and to have underlying diseases and impaired immune functions, which may increase susceptibility and progression of infections and even increase the chance of death [ ] . the sex characteristics of mers outbreaks in the middle east are similar to those in south korea. the patients in mers outbreaks in both areas were predominantly male, and the proportion of males in the study populations did not differ [ ] . furthermore, there was no difference in the male-specific cfr between the mers clusters of the two groups, a finding that is similar to other reports [ , , , ] . our findings suggest that the gender distribution is not linked to a fatal risk factor in mers outbreaks. hcp are at high risk of acquiring emerging mers infections due to occupational exposure and are affected mostly by nosocomial outbreaks [ , , , , ] [ ] . in total, the fatality risk for hcp was significantly lower than the overall fatality risk in the middle east and south korea. these findings can be attributed to three facts: first, the majority of hcp developed asymptomatic or mild symptoms and moderate symptoms [ ] ; second, hcp were confirmed as secondary cases under medical investigation, which led to earlier confirmation and good outcomes [ ] ; third, epidemiological analysis showed that hcp were much younger and had fewer co-morbidities compared to total mers cases [ ] . in contrast with sars, about % of patients with mers had at least one additional illness, and patients who died were more likely to have an underlying condition ( % of patients who died vs. % of recovered or asymptomatic patients) [ , ] . similar to the middle east, this study showed that the odds of dying were four times higher for individuals with concurrent health conditions than for those without these conditions in south korea. the odds of fatality were much lower than those estimated by the logistic regression model (seven times) [ ] . this is in part due to higher viral loads in the respiratory tract and longer shedding in patients with underlying diseases compared to cases without co-mortalities [ , ] . human-to-human transmission of mers-cov has been confirmed by epidemiological and genomic studies of cases associated with hospital and household mers outbreaks [ ] . spread was assumed to occur largely via large droplets and contact [ ] . our study indicated that the percentage of human-to-human transmission in nonfatal cases was slightly higher ( . % vs. . %) than in fatal cases in mers clusters, and two reasons could explain this: first, the survivors in secondary cases were younger and had fewer co-morbidities [ , , , , ] ; second, most of the secondary cases were under medical investigation, and therefore, the infection could be confirmed early once symptoms were observed, making timely treatment possible [ , , , , , , ] . overall, human-to-human transmission seems to have had a positive effect on the outcome of the secondary cases from the mers nosocomial outbreaks in the middle east. rapid diagnosis and providing supportive care may be of marginal consequence in the mers clusters [ , ] . the progression of illness in fatal and nonfatal infections in nosocomial outbreaks with mers-cov in the middle east does not follow the typical pattern of south korea infections [ ] . in the middle east, the median time from onset to confirmation in fatal cases ( days) was clearly longer than in nonfatal cases ( days). in south korea, however, there was no difference in the median time between fatal and nonfatal cases. this is consistent with other retrospective studies of mers virus infections [ , , ] . furthermore, the time between suspected symptom onset and laboratory confirmation ( . days) in the fatal clusters was also slightly longer than the overall average [ ] . in particular, this finding indicated that delayed confirmation is a high-risk factor for human nosocomial outbreaks with mers-cov in the middle east. in conclusion, the overall cfr for nosocomial outbreaks in the middle east was much higher than in south korea. however, the mortality risk factors for mers infections in the middle east were similar to those identified for nosocomial outbreaks in south korea. older age, underlying diseases and delayed confirmation were the major risk factors for fatal outcome in human nosocomial outbreaks. in contrast, person-to-person transmission was associated with a good outcome for secondary cases during nosocomial outbreaks. interestingly, gender, exposure history and median days were not indicators of death with mers nosocomial outbreaks. the severity of nosocomial outbreaks and the risk of fatal infection in hcp were significantly lower than the overall rate in the middle east and south korea. nosocomial outbreaks of mers-cov infection are associated with knowledge deficits, unrecognized disease, insufficient infection control measures, poor compliance, and an overwhelming number of patient cases [ , , , , ] . therefore, early and rapid detection of suspected cases, especially in older people and hcp, along with appropriate infection control practices, education and timely preparedness, are important strategies to reduce nosocomial transmission and to improve the clinical outcome in health settings in the future [ , , , ] . hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome coronavirus transmission in extended family epidemiological, demographic, and clinical 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corticosteroids critical role of nosocomial transmission in the toronto sars outbreak fatality risks for nosocomial mers outbreaks middle east respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mers-related betacoronavirus in vespertilio superans bats middle east respiratory syndrome acknowledgements we thank the reporting countries with the confirmed mers cases. we appreciate their assistance with field investigations, administration and data collection and sending the data to who. conflict of interest none declared. key: cord- -tm gds h authors: gardner, lauren m.; chughtai, abrar a.; macintyre, c. raina title: risk of global spread of middle east respiratory syndrome coronavirus (mers-cov) via the air transport network date: - - journal: j travel med doi: . /jtm/taw sha: doc_id: cord_uid: tm gds h background: middle east respiratory syndrome coronavirus (mers-cov) emerged from the kingdom of saudi arabia (ksa) in and has since spread to countries. all cases reported so far have either been in the middle east or linked to the region through passenger air travel, with the largest outbreak outside ksa occurring in south korea. further international spread is likely due to the high travel volumes of global travel, as well as the occurrence of large annual mass gathering such as the haj and umrah pilgrimages that take place in the region. methods: in this study, a transport network modelling framework was used to quantify the risk of mers-cov spreading internationally via air travellers. all regions connected to mers-cov affected countries via air travel are considered, and the countries at highest risk of travel-related importations of mers-cov were identified, ranked and compared with actual spread of mers cases. results: the model identifies all countries that have previously reported a travel acquired case to be in the top at-risk countries. india, pakistan and bangladesh are the highest risk countries which have yet to report a case, and should be prepared for the possibility of (pilgrims and general) travellers returning infected with mers-cov. in addition, the uk, egypt, turkey and the usa are at risk of more cases. conclusions: we have demonstrated a risk-analysis approach, using travel patterns, to prioritize countries at highest risk for mers-cov importations. in order to prevent global outbreaks such as the one seen in south korea, it is critical for high-risk countries to be prepared and have appropriate screening and triage protocols in place to identify travel-related cases of mers-cov. the results from the model can be used by countries to prioritize their airport and hospital screening and triage protocols. a novel coronavirus, middle east respiratory syndrome coronavirus (mers-cov) emerged from the kingdom of saudi arabia (ksa) in and has since spread to countries. as of march, around laboratory confirmed cases of mers-cov have been reported to the world health organization (who), with a case fatality rate of cfr %. the vast majority of cases in middle east have been reported from ksa, followed by united arab emirate (uae), jordan and qatar. all cases of mers-cov reported so far have either been reported in the middle east or can be linked to the region through passenger travel, with the largest outbreak outside of the middle east occurring in south korea, following an imported case. in that instance a failure in identification, hospital triage and infection control resulted in cases ( in the republic of korea and in china). the large number of highly travelled airports in mers-cov affected countries poses a risk of further international spread through infected air passenger travellers. in addition, ksa hosts annual mass gathering events such as the haj and umrah pilgrimages, during which millions of pilgrims from around the world travel to and congregate in regions where mers-cov is actively being reported. to further complicate the risk of spread, the origin of mers-cov is unknown, and there are still many uncertainties surrounding the sporadic epidemic patterns and transmission mechanisms. [ ] [ ] [ ] mers-cov is phylogenetically related to bat coronaviruses, and known to have been circulating for decades in dromedary camels in northeast africa. serological surveys in camels during the recent outbreaks also report presence of anti-mers-cov antibodies in camels in egypt, kenya, oman, saudi arabia and uae. [ ] [ ] [ ] [ ] previous studies have also identified dromedary camel exposure to be a risk factor for mers-cov and transmission from a dromedary camel to human has been confirmed. it is generally believed that the mers-cov entered human populations from direct and indirect contact with camels or camel-related products. however, details of camel-to-person transmission are unclear, many cases have not reported camel contact or epidemiologic links, and large studies of handlers of infected camels fail to show transmission to humans. with the exception of south korea, large epidemics have not seeded outside of the middle east. travel-related cases have been reported from the uk, germany, austria, france, turkey, greece, netherlands, malaysia, philippines, china, tunisia, algeria and the usa, with the number of secondary cases in all locations very small. south korea represents the largest outbreak outside of the middle east, resulting in cases and deaths (cfr: %). all secondary cases in south korea were linked to a single chain of transmission and associated with health care facilities. as was the case in south korea, human-to-human transmission has so far been mainly nosocomial, and modelling studies have revealed a -fold higher risk of transmission in healthcare setting compared with community settings. yet, there is still no evidence of sustained human-to-human transmission. without a clear understanding of local transmission combined with ongoing cases in the middle east, the risk of global spread will continue. as such, risk analysis to identify countries at high risk of imported cases is critical for disease control. network models have been used previously to quantify the risk of disease transmission posed by the global air traffic system, [ ] [ ] [ ] [ ] [ ] and airline travel data have been previously used to model the potential spread of mers-cov through air traffic network. , khan et al. utilized the flight itinerary and historic haj pilgrim data to predict the spread of mers-cov to other countries during haj season. the study-based risk on travel volumes departing all airports in four countries, saudi arabia, jordan, qatar and the uae. in addition, poletto et al. assessed the risk of mers-cov, where the focus was estimating local level transmission parameters, and the global level risk analysis was simply based on the capacity of traffic volumes between the middle east and other countries. this study, similarly, proposes a global transport network modelling framework that accounts for international air travel originating in mers-cov-affected regions to quantify the importation risk of mers-cov posed to other countries via air travel passengers, but defines the risk more precisely, and at a more spatially disaggregate scale than the previous studies. the countries at highest risk of travel-related importations of mers-cov are identified, and the relative risk posed to each is quantified. the results provide a country level ranking and corresponding expected relative risk, which can be used by public health authorities in each country to ensure the appropriate screening and triage protocols are in place to identify travel-related cases of mers-coronavirus. the proposed model quantifies the relative risk of disease spread by mers-cov-infected travellers departing from the middle east and arriving at any given world airport. in the modelled network structure, airports are represented as nodes, and the links in the network represent directed air travel routes between airports (with and without stopovers). the risk of mers-cov spread posed by air travel between an origin airport i and destination airport j is r ij , and defined in equation ( ): equation ( ) is specific to the origin-destination (od) pair (i,j), and is dependent on the origin being in an active region (i.e. the virus is assumed to be in circulation and/or in the environment, and therefore this region poses a risk of local infection), the outbreak intensity at the origin (x i ), and the total passenger volume (v ij ) travelling between (i,j). the variable, a i , is a binary variable which indicates whether mers-cov is active in a given region. if a region is assumed to be active then the status of a i is set to one for all airports in that region, otherwise it is set to . active airports therefore pose the risk of exporting infected travellers. the outbreak intensity, x i , is equal to the relative outbreak size at the origin, and is normalized to the largest outbreak size across all active regions. this variable is assumed to be correlated with the outgoing travel risk posed by a region, and thus inflates the risk per outgoing traveller at a constant relative rate. the passenger flow variable, v ij , or the total passenger volume originating at airport i and travelling to airport j, captures the potential dispersal for the disease, and includes travel on both direct routes and indirect routes with stopovers between airport i and airport j. the od level travel risk can be aggregated across all origin airports, i, which are connected via travel routes to destination airport j, to quantify the risk posed to a destination airport j. the aggregated risk posed to destination j is defined by equation ( ): the destination level risks are then normalized (as shown in equation ) by dividing by the highest value computed over all destinations, j; thus what is being estimated is the expected relative risk posed to a destination airport j from all incoming travel: finally, the country-level risk is computed by aggregating the risk posed to all airports in a given country. similar to the airport-level destination risk, the country-level risk is normalized by dividing by the highest country-level risk across all countries. the final outcome is the expected relative risk of mers-cov-infected passengers arriving in each country. similar measures for importation risk have been used previously in the context of vector borne diseases. the model requires passenger air travel data and case report data for mers-cov. passenger air travel data were purchased from the international air transport association (iata), and includes the calibrated passenger travel volumes for all international air travel routes, where a route is defined by the origin, destination and stopover airports. the route-specific passenger travel volumes supplied by iata were calibrated based on data from airlines comprising % of global air traffic, and includes over airports (iata). the passenger volumes were available at a monthly temporal resolution, which thus determined the temporal resolution of the model. the analysis was conducted using travel volumes from january to august, . this data are used for the passenger flow variable, v ij . the mers-cov case data used in the model were collected from flutrackers. detail of each case was sought from various sources including world health organization (who) and european centre for disease prevention and control (ecdc). , . the location and number of cases reported in each city in ksa between january and august of was provided by ecdc. these data define the outbreak intensity variable for each region, x i . the complete set of cities where mers-cov has been reported since was collected from who, which is used to determine the status of region, a i . two case studies are evaluated to quantify the global risk posed by travellers departing from two different specified regions (denoted as scenarios and ). thus, the scenarios differ by the set of active regions specified in the model. scenario quantifies the global risk posed by travellers departing mers-cov affected cities in ksa. in scenario all cities in ksa which have reported cases in are assumed to be an active transmission region (a i ¼ ), and the outbreak intensity variable, x i , is set equal to the number of reported cases for each city between january and august of . while this estimate is likely an underestimation of the actual number of people infected with mers in a given city, the value acts as a proxy for the number of infected individuals in a given city and inflates the outgoing (per person) travel risk proportionally. in scenario , all regions outside ksa are designated as inactive, and considered potentially at risk of imported cases. the input data for scenario , including the list of cities in ksa with confirmed cases, corresponding number of cases and assigned airport for each city are listed in supplementary table s . the airports were selected by identifying the closest (in terms of vehicle travel distance) major airport to each active city. this analysis is of particular relevance for events such as the haj and umrah, which attract millions of pilgrims to ksa, and more importantly, to cities where mers-cov is known to be in circulation. after congregating in masses at these events, the pilgrims return to their countries of origin. the results from scenario can be used to help the home countries be better prepared for the return of potentially infected pilgrims post such mass gatherings in ksa. the relative risk posed to each country is computed, allowing those countries at highest risk to be identified and targeted for increased surveillance. scenario increases the set of active regions identified in scenario to include all regions that have reported non-travelrelated cases since mers-cov was first diagnosed in humans in . scenario is based on the assumption that the cases in these regions could have been contracted from either an infected human or alternatively, an animal or environmental source. thus our active regions are assumed to still pose a risk because the virus may still be in circulation in the environment, even if there has not been a recently reported case. this assumption is further supported by the evidence of mers circulating in animal populations in these active regions, which was noted previously. in efforts to conduct a conservative risk analysis, we consider all such regions potential sources of infection for travellers. the set of active transmission regions in scenario includes a more comprehensive list of cities in ksa (listed in supplementary table s ) in addition to cities in jordan, kuwait, yemen, qatar, oman, uae, lebanon and iran. this scenario excludes south korea as an active transmission region because all cases in the south korea outbreak were traceable to the original human source, thus knowingly not contracted from the local environment. our list of active transmission regions is restricted to those in which cases were locally acquired and the source of infection is unknown (i.e. not from an infected traveller). supplementary table s lists the set of countries defined as active (in addition to ksa) in scenario and the corresponding airports assumed to pose an outgoing risk. because it is impossible to accurately estimate the number of infected individuals in each active region at any given time, the outbreak intensity variable is set to a constant for each active region in scenario . therefore, in scenario , each active region is considered equally likely to have an infected traveller departing the city. while this is a major simplifying assumption of the model, it is still able to capture the risk as a direct function of the relative connectivity to regions with confirmed local cases, and the method is illustrated to predict the likelihood of imported infection cases accurately. for each scenario evaluated, the results are presented in both tables and figures. tables and list the top countries atrisk of importing a mers-cov infected traveller from an active region, and corresponding relative expected risk for scenarios and , respectively. figures and present the same information on a global map. to validate the model the results are compared with the set of confirmed travel imported cases that have been reported. the set of travel-related cases reported since are listed in supplementary table s . this list excludes cases that were reported in jordan, kuwait, yemen, qatar, oman, uae, lebanon and iran because the existence of locally acquired cases in these regions makes it impossible to confirm whether the reported cases in patients with travel histories acquired the virus locally or abroad. the airport-level risks which were aggregated to generate the country level risk are provided in supplementary tables s and s for scenarios and , respectively. the tables include the top airports at-risk of importing a mers-cov-infected traveller from an active region, and their corresponding relative expected risk. the country-level results are presented in tables and and figures and for scenarios and , respectively. each table includes the top at-risk countries, their respective ranking in terms of the relative risk posed, and the last column specifies if the country has previously reported a travel related mers-cov case (from the specified set of active regions which is scenariospecific), and if so how many. in scenario , where ksa is the only source of infected travellers considered in the model, india is identified to be at the highest risk, which has surprisingly not yet reported a case. the uae and egypt rank nd and rd, respectively, and both have reported travel-acquired cases from ksa. in total there have been travel-imported cases confirmed from ksa in different countries; of the countries were included in the top atrisk countries, and all were captured in the top . of the top at-risk countries in scenario , ( %) have previously reported cases from ksa. additional countries in the top for scenario that have not yet reported imported cases from ksa included pakistan, sudan and bangladesh. in scenario , where the set of mers-cov source regions is expanded to include all middle east countries that have previously reported locally acquired cases, of the travelimported cases reported outside the middle east ( %) were reported in one of the countries falling in the top of our list, and of the top ranked countries ( %) have previously reported travel acquired cases. india is again identified as the most at-risk country, and along with pakistan which is ranked third, have yet to report mers-cov cases. countries such as uae and jordon, which were identified as high-risk in scenario , are no longer included in table because they are treated as high-risk travel origins (rather than considered potentially atrisk destinations). in addition, because scenario considers a more comprehensive set of travel sources, the risk is increased to highly connected regions such as the uk and germany, which both appear in the top at-risk countries. the proposed model identifies the set of countries at greatest risk of importing mers-cov-infected travellers. two scenarios are evaluated, which differ by the set regions from which infected travellers are assumed to depart from. scenario limits the outgoing travel risk to be from ksa only, while scenario considers all regions which have reported locally acquired cases as potential sources. scenario results are more likely to represent the expected risk posed globally by mers-cov. scenario may be more appropriate for evaluating the risk during and immediately after major mass gatherings in ksa. for both scenarios, the quantified relative risk and ranking can be useful for informing public health authorities on the optimal locations for airport screening and travel protocols. for both scenarios, india is identified as the highest at-risk country, while pakistan and bangladesh are also included in the top in both scenarios. critically, none of these countries have reported mers-cov cases as of june , and may therefore be unprepared to diagnose and treat a case were one to arise. furthermore, each of these countries has substantial muslim populations who may travel to ksa for religious pilgrimages, and should be prepared for the possibility of (pilgrims and general) travellers returning infected with mers-cov. in addition to the pilgrimage, a large number of people from these countries work in the middle east, and travel back and forth regularly. although these countries are already preparing for mers outbreaks and have issued policy documents, actual capacity to diagnose and treat cases as they arrive is questionable. it is likely that mers-cov cases may have already been imported to these countries, but the cases were not picked up by local surveillance due to mild disease, lack of diagnostic capacity and reporting. lack of proper diagnosis can pose significant harm to a country, as was illustrated by the episode in south korea, where the index case visited four hospitals before properly diagnosed, by which time he had spread infection to many people. surveillance systems should be improved to identify cases, and travellers from high risk areas should be screened and monitored for associated symptoms. a rapid response system should be developed accordingly, and healthcare workers should be trained to identify symptoms and manage the cases safely. in contrast to india, pakistan and bangladesh, the remaining seven of the top at-risk countries across the two scenarios, have previously reported travel-imported cases, and these countries are likely to remain at-risk of importing infected travellers, and should continue surveillance and public travel health awareness campaigns, especially for religious pilgrims. for both scenarios all countries that have previously reported travel acquired cases were identified in the top , with the majority of countries reporting travel imported cases identified in the top . these results suggest that the model is able to capture the risk posed to most countries for importing mersinfected travellers. as further validation, the model results are consistent with previous travel-related studies. poletto et al. reported the highest air traffic from middle east was to india ( . %), bahrain ( . %), pakistan ( . %), uk ( . %), oman ( . %) and egypt ( . %). in addition, khan et al. found india, egypt, pakistan, uk, kuwait, bangladesh, iran and bahrain to be the highest risk countries of importing mers-cov cases. each of these countries were identified as high-risk in scenario ranking as well, but in scenario iran and kuwait were included as potential sources of infection in our model (which was not the case in ). while our country-level rankings are similar to khan et al., the variation is due to three main factors, (i) our model incorporates a weighting for each travel origin based on the outbreak intensity variable (included only in scenario ) which serves to differentiate the outgoing traveller infection risk posed by different regions in the middle east where mers-cov has been reported, (ii) outgoing infected travellers departures are restricted to airports nearest the set of regions with reported locally acquired cases, rather than the entire middle east and (iii) more current air travel data are used. since , when khan et al. conducted their study, the regions where mers-cov has been locally acquired have grown, and the travel patterns within the middle east have also changed. thus, the results in this study should be more accurate estimates of importation risk to countries connected to the middle east via the air traffic network. it should be noted, however, that several high-risk countries identified by the model have not had an imported case as of yet, whilst lower risk countries have. this outcome has substantial implications: (i) countries with lower risk should not be complacent, as south korea was ranked below the top countries, yet experienced a large epidemic, and (ii) countries such as india and pakistan, identified at highest risk but yet to report any cases, should be prepared for potential imported cases. this model is subject to various limitations. the first limitation results from the uncertainty surrounding mers-cov transmission. the analysis quantifies the relative expected risk of mers-cov-infected (air travel) passengers arriving at airports based on a set of active transmission regions, the outbreak size at each and travel patterns; the model does not include the potential importation of infected intermediary hosts or intermediary host by-products since the influence of that possibility is yet to be established. second, an estimate of the true number of cases in each region based on the confirmed reported case count is not included, and the reported number of cases is instead used as a proxy. third, the potential harm posed to a region by local transmission (if successfully introduced into the population by an infected traveller) is not accounted for in the risk assessment. fourth, the model is limited by the available travel data. air travel reliably captures human mobility patterns at large spatial scales such as travel between countries and across large bodies of water; however, it does not fully capture travel patterns within countries due to the availability of alternative modes of travel such as road and rail. because the proposed model is solely based on air travel data, the intra-country mobility patterns are not fully captured. for this reason the risk is modelled and validated at the country level rather than a more disaggregate spatial scale, such as the city level, which cannot be reliably quantified without a more comprehensive multi-modal data set. the same issue regarding travel via alternative modes can also arise at the intercountry level, especially for smaller neighbouring countries. these limitations may all be factors in why several high-risk countries identified by the model have not had an imported case as yet, whilst lower risk countries have. other modelling approaches which focus on intra-country transmission are needed to inform internal disease control policy for a particular country. in summary, mers-cov has persisted in human populations for more than years. , despite uncertainty about transmission, a mixed pattern of both sporadic and epidemic spread continue to be observed, suggesting the virus is still present in the environment in various regions of the middle east. the risk of travel-related cases will therefore remain as long as mers-cov transmission persists in the region. furthermore, the haj pilgrimage to mecca in ksa and other regional mass gatherings pose an ongoing risk of spread by international travellers. although travel cases were not reported after haj in - despite increase likelihood of secondary infection during such mass gathering, several travel acquired cases of mers-cov were reported in travellers who had returned from umrah, a minor pilgrimage, in . luckily, the numbers of secondary cases resulting from these umrah travellers was not high, and the number of secondary cases resulting from infected travellers is typically very low. a recent modelling study analysed the data of travel-related cases and estimated . % risk of secondary transmission and . % risk of tertiary transmission in event of importation of a mers case from middle east. none the less, the potential harm posed by a single mers-cov-infected traveller to an unprepared country can be significant, as was exemplified by the south korean epidemic, in which cases resulted from a single index case. in this instance, failure to recognize mers coronavirus infection at the hospital and lack of appropriate, timely triage and infection control procedures resulted in a large epidemic. for this reason, it is critical for all countries to be prepared and have appropriate screening and triage protocols in place to identify travel-related cases of mers-cov, and for risk stratification to be utilized to prioritize awareness and preparedness for mers-cov. countries higher on the risk scale, such as india and pakistan could invest more in preparedness and review existing protocols and policies. middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus outbreaks and emergencies. mers-cov in republic of korea at a glance as of air passenger market analysis a scenario-based evaluation of the middle east respiratory syndrome coronavirus and the hajj the discrepant epidemiology of middle east respiratory syndrome coronavirus (mers-cov) unanswered questions about the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease mers coronavirus neutralizing antibodies in camels middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study mers coronavirus in dromedary camel herd, saudi arabia mers coronaviruses in dromedary camels antibodies against mers coronavirus in dromedary camels risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia evidence for camel-to-human transmission of mers coronavirus lack of middle east respiratory syndrome coronavirus transmission from infected camels severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) synthesizing data and models for the spread of mers-cov, : key role of index cases and hospital transmission a global airport-based risk model for the spread of dengue infection via the air transport network a predictive spatial model to quantify the risk of air-travel-associated dengue importation into the united states and europe the risk of dengue spread from the philippines after typhoon haiyan inferring infection-spreading links in an air traffic network assessment of the potential for international dissemination of ebola virus via commercial air travel during the west african outbreak potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia assessment of the middle east respiratory syndrome coronavirus (mers-cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach passenger intelligence services (paxis) - case list of moh/who novel coronavirus mers ncov announced cases world health organisation (who) mers may not be sars; but india is still vulnerable interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response assessing the risk of observing multiple generations of middle east respiratory syndrome (mers) cases given an imported case conflicts of interest: none declared. key: cord- - wkbjtji authors: da'ar, omar b.; ahmed, anwar e. title: underlying trend, seasonality, prediction, forecasting and the contribution of risk factors: an analysis of globally reported cases of middle east respiratory syndrome coronavirus date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: wkbjtji this study set out to identify and analyse trends and seasonal variations of monthly global reported cases of the middle east respiratory syndrome coronavirus (mers-cov). it also made a prediction based on the reported and extrapolated into the future by forecasting the trend. finally, the study assessed contributions of various risk factors in the reported cases. the motivation for this study is that mers-cov remains among the list of blueprint priority and potential pandemic diseases globally. yet, there is a paucity of empirical literature examining trends and seasonality as the available evidence is generally descriptive and anecdotal. the study is a time series analysis using monthly global reported cases of mers-cov by the world health organisation between january and january . we decomposed the series into seasonal, irregular and trend components and identified patterns, smoothened series, generated predictions and employed forecasting techniques based on linear regression. we assessed contributions of various risk factors in mers-cov cases over time. successive months of the mers-cov cases suggest a significant decreasing trend (p = . for monthly series and p = . for quarterly series). the mers-cov cases are forecast to wane by end . seasonality component of the cases oscillated below or above the baseline (the centred moving average), but no association with the series over time was noted. the results revealed contributions of risk factors such as camel contact, male, old age and being from saudi arabia and middle east regions to the overall reported cases of mers-cov. the trend component and several risk factors for global mers-cov cases, including camel contact, male, age and geography/region significantly affected the series. our statistical models appear to suggest significant predictive capacity and the findings may well inform healthcare practitioners and policymakers about the underlying dynamics that produced the globally reported mers-cov cases. this study set out to identify trends and seasonal variations; made a prediction based on the globally reported cases of the middle east respiratory syndrome coronavirus (mers-cov), extrapolated into the future by forecasting the trend and assessed contributions of various risk factors for the mers-cov cases. specifically, we consider the questions: ( ) what is the underlying growth or trend of the globally reported mers-cov cases? ( ) are there seasonal variations present in globally reported mers-cov cases over time and how do they affect the series? ( ) what are the contributions of various risk factors for mers-cov cases? the motivation for this study is that to date, the world health organisation (who) places the mers-cov among the list of blueprint priority diseases. although a survey of the literature shows a rapid increase in research activities related to mers-cov [ ] , yet there is still a general paucity of empirical literature examining trends and seasonality. to the best of our knowledge, there is a single study, which anecdotally examined seasonality and time series patterns of mers-cov to date [ ] . given mers-cov remains a potential pandemic disease globally, it is important to understand the dynamics of the underlying growth or trend of the globally reported cases. the motivation for this study also comes from the aetiology of mers-cov, especially its causes, spread, the complexity of its diagnosis and mortality. mers-cov is a virus that causes severe viral pneumonia in humans, known to have a high mortality rate [ ] [ ] [ ] [ ] [ ] and has clinical symptoms similar to severe acute respiratory syndrome coronavirus [ , ] . it was first reported in saudi arabia [ ] and after that, the virus exhibited outbreaks in several regions of the world, particularly saudi arabia and the republic of korea [ , ] . additionally, the complexity of mers-cov and its diagnosis of infection have been acknowledged in the literature [ , ] . according to who, laboratory-confirmed cases of mers-cov were reported at the end of january , including associated deaths (case-fatality rate: . %) that were reported globally [ ] . the majority of these cases were reported in saudi arabia. this is a time series analysis using publicly reported mers-cov monthly global cases. the who receives confirmed mers-cov cases from countries across the world. to date, new cases continue to be identified and reported to the who, specifically from the middle east region. these data are available at http:// www.who.int/csr/don/archive/disease/coronavirus_infections/en/. the latest report included one case from malaysia on january . we used time series of mers-cov cases reported between january and january , where who began using standard case report. a research assistant retrieved data from who webpage and reviewed for quality by the second study author. the data retrieved include patient and clinical data such as age, gender, healthcare worker, comorbidity, the source of infection and geographical regions. the main outcome was the number of cases reported on a monthly basis from january to january . the analysis was performed using stata (stata corp., texas, usa) and microsoft excel . using the classical multiplicative time series model, we decomposed the original series into seasonal, irregular and trend components and examined their effects. additionally, we identified patterns, smoothened series, generated predictions and employed forecasting techniques based on linear regression. finally, we assessed contributions of various risk factors in mers-cov cases over time. p-values < . were considered statistically significant. figure shows that although cases of mers-cov are decreasing in the range period selected, the series exhibits peaks and spikes. figure also reveals a negative trend of mers-cov series from january to january . we investigated direction and significance of this trend, as well as stationarity of the series. while a unit root test for nonstationarity confirmed the mers-cov cases had a negative and statistically significant trend, the series was found to be stationary. the negative and statistically significant trend was also confirmed by subsequent regressions. we collapsed the monthly series into quarters and then smoothened out the series using a centred moving average in order to understand underlying growth component. we assumed the classical multiplicative time series model by decomposing original series into seasonal, irregular, and trend components ( table ) our decomposition of the mers-cov cases shows that in q , the seasonality and irregularity components of the series were % below the baseline (the centred moving average). decomposing further, seasonality component was % above the baseline in q , while it was % below the baseline in q . our analysis also shows the de-seasonalised series of the original mers-cov by removing seasonality and irregularity components. using a linear regression, we then estimated the effect of time on the deseasonalised series to capture the underlying growth or trend component using a linear regression in order to make predictions. since the last available data was in january , we also made a forecast of three more quarters ( q , q , q ), an additional months into the future. the forecast of the series revealed that mers-cov cases would approach zero by end of or beginning of , making further extrapolation into the horizon infeasible. figure shows decomposition of mers-cov series of the original series, centred moving average (smoothed series), forecast omar b. da'ar and anwar e. ahmed and linear forecast. together, after accounting for irregular and trend components of the series, seasonality was found to range between % below the baseline i.e. the centred moving average (of order ) in some quarters and % above the baseline in other quarters. the average seasonality component was found to be % below the baseline. however, regression estimation revealed that, unlike the trend component, seasonality was not statistically significant, a fact also backed by our statistical test, which showed the monthly global mers-cov cases series were stationary. regressions table shows results of the effect of trend and seasonal on mers-cov cases. we compared the seasonal dummy variables, interpreted by comparing them with quarter (q ) (base season) while holding time constant. time, in this analysis, was represented by successive months and was interpreted as the effect of the linear trend on mers-cov cases over time, holding the effect of the seasons constant. the regression results revealed that after accounting for the trend, mers-cov cases quarter (q ) each year averaged about cases more than q cases, although the effect was not found to be statistically significant. similarly, after adjusting for the trend, cases in quarter (q ) averaged around cases more than q cases. quarter (q ) cases averaged cases more than q cases after accounting for the trend component time. it is important to note that the effects of seasonality were not statistically significant. however, what was revealed statistically significant is the negative trend. consistent with the negative trend shown in figure , the regression results revealed that each additional quarter registered approximately an average decrease of one case, after adjusting for the season. in other words, the mers-cov cases decreased, on average, by four ( × − . ) per quarter year to year. the model as a whole appears to suggest statistically significant predictive capacity. we analysed fluctuations of reported mers-cov cases from period to another by graphing the residuals (generated from the regression of trend and seasonality on mers-cov cases) against time, as is the convention with time series analysis. the results indicated no clear patterns, suggesting that correlated errors are not a problem with this model. other diagnostic tests also revealed neither violation of the classical linear assumptions nor correlation between the reported mers-cov cases in each month with cases reported in earlier months. we further examined the effects of various risk factors of mers-cov cases such as camel contact, healthcare worker contact, exposure, gender and region. table shows regression that adjusts for these factors. the results reveal camel contact, saudi table ). the model in this estimation also appears to suggest statistically significant predictive capacity. using linear time series models and their application to the modelling and prediction of the globally reported mers-cov data, the present study identified trends, analysed seasonality, predicted and forecast evolution of mers-cov cases and assessed the contribution of various risk factors. the decomposition of the time series of mers-cov cases into trend and seasonality components and making predictions have not hitherto been studied in the context of mers-cov pandemic. in this study, we set out to understand the dynamics of its growth over time. the results of our time series analysis of globally reported mers-cov cases suggest a significant negative trend that is forecast to be eradicated in the near future unless something unexpected happens. our study showed that although seasonality oscillated below or above the baseline i.e. the centred moving average (of order ) over time, the average seasonality component was found to be % below the baseline. even then, our analysis showed that, unlike the trend component, seasonality did not affect the series over time. many risk factors are associated with mers-cov cases, mortalities, or complications. our results indicate those aged under years (reference category) are less likely to be a mers-cov case than those aged over , consistent with several studies that associated mers-cov with elderly patients [ , ] . surprisingly, comorbidity did not show a statistically significant contribution to mers-cov cases. however, there are studies that showed mers-cov cases were associated with patients with comorbidities [ ] [ ] [ ] . a recent systematic study, for example, suggests the prevalence of comorbidities of mers-cov cases, diabetes, hypertension and cardiac diseases [ ] . while our analysis suggests males contribute to the global reported mers-cov cases, gender was reported to have a mixed effect on mers-cov mortalities in the literature. some studies showed men as high risk [ , ] and mers-cov infects more males than females [ , [ ] [ ] [ ] . other literature indicated that the frequency of deaths was less in men [ ] . the literature showed that mers-cov can be spread via human-human [ ] [ ] [ ] [ ] [ ] , or healthcare facilities [ , [ ] [ ] [ ] . other studies revealed animal to human [ , ] as the primary culprit of mers-cov virus transmission. specifically, the literature showed camels act as a direct source of human mers-cov infection [ , ] , while healthcare workers were reported to be at higher risk [ , , , ] . the results of our study in this regard were mixed. while our study indicated that the effect of camel cases on overall mers-cov reported cases are positive and significant, the contribution of healthcare workers was not. our analysis also showed evidence of geographical contributions to mers-cov cases such as saudi arabia and greater middle east compared with south korea. this can be seen as somewhat consistent with earlier studies that demonstrated a link between mortality associated with mers-cov and geography [ ] . this finding is also intuitive in that saudi arabia and middle east, in general, remain the global epicentre of mers-cov, hence the name. the contribution of our study is that it adduces empirical evidence by making inferences and predictions based on the globally reported cases of mers-cov and extrapolated into the future by forecasting the trend. unlike previous studies that descriptively analysed seasonality patterns of mers-cov and influenza in the middle east [ ] , our study presents statistically significant results of trends of global mers-cov cases, consistent with regularities underlying the empirical dynamics and classical time series analysis. however, there are limitations of this study. first, the data used for this study comprised months (january to january ). while this was just enough for several years' worth of monthly observations to appropriately model seasonality, time series analysis can be sensitive to the number of observations. hence, sufficiently large number of observations might have provided a better fit and results. additionally, the analysis utilised who open source globally reported data, which may lack harmonisation from the various country sources. this study contributes to the time series analysis of mers-cov literature. in particular, our analysis of trends and seasonality components the series, the prediction based on the globally reported cases of mers-cov and extrapolation into the future by forecasting the trend is envisaged to help in understanding the dynamics of the reported cases over time. the study findings suggest a significant negative trend of the monthly and quarterly data from to . however, a further extrapolation into the future reveals that the mers-cov cases are forecast to be zero by end or beginning of unless something unexpected happens. seasonality component of the series oscillated below or above the baseline, i.e. the centred moving average but did not affect the series over time. the results demonstrated that camel contact, exposure, gender, age and geography/region significantly contributed to the overall global reported mers-cov cases. the findings may well inform healthcare practitioners and policymakers about the underlying dynamics that produced the globally reported mers-cov cases. data. these dataset used and/or analysed are available at http://www.who.int/ csr/don/archive/disease/coronavirus_infections/en/. global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis differences in the seasonality of mers-cov and influenza in the middle east the predictors of -and -day mortality in mers-cov patients development of a risk-prediction model for middle east respiratory syndrome coronavirus infection in dialysis patients epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study mers-cov infection in humans is associated with a pro-inflammatory th and 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coronavirus infections the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections mers-cov outbreak in jeddah-a link to health care facilities human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia acknowledgements. the authors are grateful for the collegiality and research support at the college of public health and health informatics, king saud bin abdulaziz university for health sciences. the information and opinions contained in this work do not necessarily reflect the views or policy of these institutions. this research is supported by king abdullah international medical research centre (kaimrc), king saud bin abdulaziz university for health sciences, national guard health affairs, riyadh, saudi arabia.author contributions. obd analysed the data and wrote manuscript. aea retrieved the data from the who /registry website. aea reviewed analysis and manuscript. all authors approved final manuscript for submission. ethical standards. not applicable. key: cord- -jd k z authors: ababneh, mustafa; alrwashdeh, mu’men; khalifeh, mohammad title: recombinant adenoviral vaccine encoding the spike subunit of the middle east respiratory syndrome coronavirus elicits strong humoral and cellular immune responses in mice date: - - journal: vet world doi: . /vetworld. . - sha: doc_id: cord_uid: jd k z background and aim: middle east respiratory syndrome coronavirus (mers-cov) has rapidly spread throughout the middle east since its discovery in . the virus poses a significant global public health threat with potentially devastating effects. in this study, a recombinant adenoviral-based vaccine encoding the spike (s ) subunit of the mers-cov genome was constructed, and its humoral, and cellular immune responses were evaluated in mice. materials and methods: mice were immunized initially by intramuscular injection and boosted weeks later by intranasal application. expression of the s protein in the lungs and kidneys was detected using conventional polymerase chain reaction (pcr) and immunohistochemistry (ihc) targeting specific regions within the s subunit at weeks , , , and after the first vaccination. antigen-specific humoral and cellular immune responses were evaluated in serum and in cell culture following in vitro stimulation with a specific -mer epitope within the s protein (cysslildy). results: s protein expression was only detected by ihc in the kidneys of the ad-mers-s group at week from first immunization, and in both lungs and kidneys of ad-mers-s group by conventional pcr at weeks and post-prime. the vaccine elicited a specific s -immunoglobulin g antibody response, which was detected in the sera of the vaccinated mice at weeks and from the onset of the first immunization. there was a significant increase in the amount of th -related cytokines (interferon-γ and interleukin [il] ), and a significant decrease in the th -related cytokine il- in splenocyte cell culture of the vaccinated group compared with the control groups. conclusion: the results of this study suggest that this recombinant adenovirus vaccine encoding the s subunit of mers-cov elicits potentially protective antigen-specific humoral and cellular immune responses in mice. this study demonstrates a promising vaccine for the control and/or prevention of mers-cov infection in humans. middle east respiratory syndrome coronavirus (mers-cov) is a newly emerging human coronavirus that was discovered in in a -year-old saudi arabian man [ ] . following its discovery, many cases were identified in different regions of the arabian peninsula and worldwide thereafter [ , ] . the most recent outbreak occurred in june in south korea and was linked to a south korean man who had recently traveled to the middle east [ ] . the infection then rapidly spread to persons through close contact in a hospital. within a few months, many cases (n= ) were reported in hospitalized and non-hospitalized persons in south korea [ ] . the disease showed a high mortality rate that reached up to %, which was higher than that of the severe acute respiratory syndrome coronavirus (sars-cov) outbreak in - ( %) [ ] . coronaviruses belong to the subfamily coronavirinae within the family coronaviridae of the order nidovirales [ ] . five human coronaviruses were identified ( e, oc , nl , hku , and sars-cov) before mers-cov. lineage c of betacoronaviruses includes bat coronaviruses, which give a primitive impression regarding the origin of the virus [ ] . the detection of mers-cov and its neutralizing antibodies in the sera of dromedary camels has shed light on the role of the camel as a possible animal reservoir, which may transmit the virus to humans [ ] [ ] [ ] [ ] . indeed, researchers isolated the same mers-cov strain from both a camel "in a barn" and its infected owner in saudi arabia, thus providing further evidence of the potential airborne and direct contact transmission of the virus between camels and humans [ ] . there have been several attempts to develop a protective vaccine against mers-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . researchers around the world are focused on the spike protein as the main target for vaccine development against mers-cov. the spike protein of mers-cov attaches to the host dipeptidyl peptidase- (dpp ) receptor, which is expressed on several types of human cells [ ] . many studies published since suggesting vaccine models were constructed based on the spike protein and its receptor-binding domain (rbd) region to elicit a strong and protective immune response in vitro and in vivo [ ] . recombinant adenoviral vector vaccines are one of the most effective vaccines and showed interesting results during sars-cov outbreaks [ , , ] . since , several studies were published, in which different viral vectors (e.g., adenoviruses and vaccinia virus) were used to develop recombinant vaccine candidates based on full spike gene or part of it and tested their ability to produce protective immunity against mers-cov infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, further investigations are needed on these suggested vaccines including testing their ability to elicit neutralizing antibodies in different animal models, stimulation of both innate and adaptive immune responses and their corresponding cytokine and chemokine profiles, distribution within the host, and their safety and duration profiles. in this study, a recombinant adenoviral-based vaccine encoding the spike (s ) subunit of the mers-cov genome was constructed, and its humoral, and cellular immune responses were evaluated in mice. all procedures performed in this study were approved by the jordan university of science and technology animal use and care committee. the recombinant human adenovirus type (de /e ) vaccine was designed to express the s domain of the spike protein of mers-cov isolate hu/ jordan- / (genbank accession number kt ) under the control of the cytomegalovirus (cmv) promoter. this vaccine was constructed in the laboratories of vector biolabs (the gene delivery company, pa, usa) [ ] . six to eight week-old male balb/c mice (n= ) were purchased from animal house at jordan university of science and technology (irbid-jordan) and distributed into three groups in a replicate manner ( each). animals in the first group were immunized with the ad-cmv-mers-cov-spike "ad-mers-s ." animals in the second group received the vector-only "ad-cmv," and animals in the last group served as a control and received × phosphate-buffered saline (pbs). for the first immunization, the ad-mers-s vaccine was injected intramuscularly at a dose of . ml containing × pfu/ dose/mouse. the same volume of ad-cmv or pbs was used for the other two groups. the booster dose was carried out weeks later by intranasal route at a dose of . ml containing × pfu/dose/mouse. all experimental procedures used in this study were approved by the animal care and use committee at the faculty of veterinary medicine (irbid-jordan). lung, kidney, and spleen tissue samples were harvested from each mouse in all groups after , , , and weeks from the first immunization. parts of lung and kidneys were preserved in % formalin solution for -h, then embedded in paraffin blocks for immunohistochemical analysis, while the other parts were stored at − °c for polymerase chain reaction (pcr) analysis. spleen tissues were harvested and stored at − °c for cell culture analysis. blood samples were drawn through facial vein route, left min at room temperature, and centrifuged at rpm for min. collected sera were stored at − °c for subsequent analysis. to study the distribution of the recombinant vaccine, total of - mg of lung and kidney tissues at weeks and post-prime immunization was cut and homogenized in ml of diethyl pyrocarbonate-treated water. homogenized samples were then centrifuged at rpm for min, and µl of the supernatant was used for the extraction. the extraction was performed using the viral gene-spin rna extraction kit (intron biotechnology, korea) according to the manufacturer's instructions. the extracted rna was used for cdna synthesis through a reverse transcription system (promega, madison, wi, usa) according to the manufacturer's instructions. the cdna was kept at − °c until pcr analysis. the dna template of the constructed vaccine was also purified and used as a positive control in this experiment. heminested pcr was carried out using specific primers targeting specific sequences within the s region, as shown in table- [ ] . the first pcr targeted a bp sequence. pcr mixture contained . µm of each primer and using µl of the cdna template. touchdown pcr assay was programmed on the mycycler thermal cycler (bio-rad laboratories, hercules, ca, usa) as the following conditions: °c for min, followed by cycles of °c for s, - °c for s, and °c for min and s, then cycles of °c for s, °c for s, °c for min and s, and a final extension step at °c for min. two microliters from the first pcr product were used as a template for the second (heminested) pcr to amplify the bp product following the first pcr conditions. the bands were visualized in . % agarose gels using an ultra-violet transilluminator. localization of s protein expression was investigated in both kidney and lung tissues from all groups at week after the first vaccination. ihc using rabbit-specific hrp/dab (abc) detection ihc kit (abcam, cambridge, uk) was adapted, with using antinovel coronavirus spike protein s polyclonal antibodies ( : ; sino-biological, pa, usa). a -µm thick section of formalin was fixed, and paraffin-embedded tissues were cut and loaded on specially coated slides. the prepared slides were put in an oven at °c for min followed by min in a xylene solution for deparaffinization. the sections were dried and surrounded by liquid blocker (dako pen). the antigen retrieval (pretreatment) step was executed by immersing the sections in × reveal decloaker reagent (biocare medical, pacheco, ca, usa) and heating in a microwave several times for min then cool at room temperature for - min. after that, staining procedure was carried out by following the manufacturer's instructions of the abc kit. to eliminate the effect of internal renal biotin, . % avidin was applied (sigma-aldrich, st. louis, mo, usa) only on kidney sections for min at room temperature [ , ] . the immunoreactivity of the expressed s protein was visualized using a light microscope at different magnifications. spleen tissues from each group were collected, pooled, and homogenized to obtain single-cell suspensions. the cells were centrifuged for min at rpm, and the pellet was re-suspended in roswell park memorial institute medium (rpmi) (gibco, grand island, ny, usa), supplemented with % fetal calf serum, mm hepes, µg/ml penicillin/streptomycin, mm l-glutamine, µm -mercaptoethanol, and ng/ml amphiostat b (complete rpmi) [ ] . the cell suspension was washed again by centrifugation as described earlier. spleen cells were re-suspended in red blood cell lysis buffer (trisbuffered nh cl) to remove erythrocytes. cells from each group ( × ) were re-suspended in ml of complete rpmi and plated in -well plates in triplicate in parallel, adjacent wells. the first set of samples was stimulated with µg of specific peptide "epitope" cysslildy per well. this antigen was defined as the highest potential t-cell epitope from a total of eight potential epitopes within the rbd region of the s subunit of the spike protein of mers-cov genome and was expected to display interactions with the maximum number of major histocompatibility complex (mhc) class i molecules, and to show the highest peak in the b-cell antigenicity plot [ ] . the second set of samples was stimulated with µg of non-specific stimulant phytohemagglutinin to confirm the viability and productivity of the cultured cells. the third set of samples was not stimulated as a control set. cells were incubated in round-bottom -well microtiter plates at °c in % co . the cultured media were collected after h, and the samples were centrifuged for min at rpm. the supernatants were stored at − °c before cytokine analysis, while the pellets were stored at − °c until real-time (rt)-pcr analysis. detection and measurement of specific mers-s igg were achieved using a semi-quantitative anti-mers-cov elisa (igg) (euroimmun ag, luebeck, germany) with a slight modification using universal enzyme conjugate, anti-mouse iggconjugated hrp, to be able to detect mouse igg antibodies instead of human igg antibodies. microtiter plates were pre-coated with purified s antigen of mers-cov. mouse diluted serum samples ( : ) were incubated in each well from the three experimental groups (ad-mers-s , ad-cmv, and pbs) for min at room temperature by following the manufacturer's instructions. the color intensity was measured using an elisa plate reader (biotek, winooski, vt, usa). cell culture supernatants from stimulated and corresponding non-stimulated samples of the three groups were tested for the level of mouse cytokines at weeks and post-prime using a quantitative sandwich enzyme immunoassay technique (quantikine elisa mouse cytokines immunoassay, minneapolis, mn, usa) according to the manufacturer's instructions. relative quantification of mouse ifn-γ and il- was normalized to the housekeeping gene β-actin in the cell culture yield of stimulated groups. the collected pellets from cell culture were used to extract total rna using the sv total rna isolation system (promega) according to the manufacturer's instructions; this extraction kit has a dnase incubation step to eliminate any dna in the sample. isolated rna ( µl) was stored at − °c before rt-pcr analysis. rna concentration was adjusted to ng/µl using te buffer. purified rna ( µl) was used as a template to produce µl of cdna using the reverse transcription system (promega) kit. relative rt-pcr assay was performed to determine the mrna expression levels of mouse ifn-γ and il- in the cell culture. the fold changes were calculated using the −ΔΔct method normalized to β-actin [ ] . specific primer sets were used, as shown in table- [ ] . extracted rna ( µl) was used as a template in the pcr reaction mixture ( µl), which was composed of . µm each of the forward and reverse primers, µl of powerup sypr green master mix (applied biosystems, foster city, ca, usa), and µl of nuclease-free water. rt-pcr conditions were programmed in rotor gene-q (qiagen, hilden, germany) as follows: °c for min, °c for min, followed by cycles of °c s, °c for min, and a melting step at - °c for s. data for elisa and fold changes were expressed at mean ± standard deviation. one-way anova and t-test were used to compare different values in all groups using openepi software (emory university, usa). parameter differences were considered statistically significant at p< . . parameter differences were indicated by small letters labeled within each group; different letters indicated significant differences at p< . . distribution and expression of the s subunit of the mers-cov spike protein in mice tissues were detected at weeks and post first immunization in the kidneys and lungs of the vaccinated group but not in control groups using conventional pcr (figure- ). using ihc, s protein expression was detected in the cytoplasm of proximal tubule epithelium in the vaccinated mice at week post first immunization but not in the control groups (figure- ) . s expression was not detected in lung tissues in either the vaccinated or the control groups (figure- ) . there was a significant level (p< . ) of mers-s specific igg antibodies detected in the mice sera of the vaccinated group at weeks and post-prime. the combined results of weeks and revealed that the igg antibody response was significantly higher (p< . ) in the vaccinated group than in the control groups (figure- ) . available at www.veterinaryworld.org/vol. /october- / .pdf the level of mouse cytokines at weeks and after the first vaccination in the cell culture supernatants of stimulated and corresponding non-stimulated samples of the three groups was tested by sandwich elisa assay. mice cytokines (ifn-γ, il- , and il- ) production in the ad-cmv and pbs groups were combined and expressed collectively as one group (control groups) due to the non-significant differences shown across these time points between these two groups (figure- ) . therefore, the levels of each cytokine at the two tested time points (weeks and after the first vaccination) were also combined and expressed as one unit. regardless of in vitro stimulation, production of ifn-γ was significantly (p< . ) higher in the vaccinated group compared with the control groups (ad-cmv and pbs) at all tested time points (figure- ) . in terms of il- production, in vitro stimulation with the mers-specific peptide "cysslildy" resulted in a significant (p< . ) upregulation in production when compared with the antigen-stimulated and non-stimulated cell culture in the vaccinated group only (figure- ) . although there was no significant difference in il- production between the control groups and the vaccinated group in non-stimulated cell culture, antigen-specific stimulation clearly revealed a higher production of this cytokine in the control groups above the vaccinated group level of production (p< . ). in addition, in vitro stimulation with the mers specific peptide "cysslildy" resulted in a significant upregulation in this production of this cytokine in the control groups while the same antigen stimulation resulted in a significant decrease in il- production in the vaccinated group (p< . ) (figure- ) . at week post first immunization, the fold change in ifn-γ gene expression was significantly higher than that in the control groups (ad-cmv and pbs) (p< . ). however, there was a prominent increase in ifn-γ gene expression at week postprime in both the vaccinated and ad-cmv groups compared to that in week (p< . ). the expression of ifn-γ was slightly higher but not statistically significant in the vaccinated group compared to that in the ad-cmv group at week (p> . ) (figure- ) . the only significant change (p< . ) in il- gene expression was observed in the ad-cmv group at week (figure- ) while no significant changes were observed in the vaccinated group at any time point. in this study, potentially protective humoral and cellular immune responses were elicited in mice by immunization using a recombinant adenoviral-based vaccine encoding the s subunit of the mers-cov spike gene. several studies have been published that constructed similar vaccines against mers-cov and tested their ability to induce production of neutralizing antibodies and other immune system components [ , , , [ ] [ ] [ ] [ ] [ ] . the results of these studies were encouraging and showed that ] , and il- ) in vitro. regardless of in vitro stimulation, production of ifn-γ was significantly (p< . ) higher in the vaccinated group with ad-middle east respiratory syndrome (mers)-s compared with the control groups (ad-cytomegalovirus and phosphate-buffered saline) at all tested time points. in vitro, antigen-specific stimulation resulted in a significant upregulation of il- production when compared with the antigen-stimulated and non-stimulated cell culture in the vaccinated group only. in vitro stimulation using the mers-specific peptide "cysslildy" resulted in a significant upregulation of il- production in the control groups while the same antigen stimulation resulted in a significant decrease in il- production in the vaccinated group. ns: non-antigen stimulated, ag: antigen-stimulated. different letters mean significantly different at p< . . available at www.veterinaryworld.org/vol. /october- / .pdf such vaccines may be promising to prevent the global threat of mers-cov infection [ , , , [ ] [ ] [ ] [ ] [ ] . however, the safety and efficacy of these vaccines still to be confirmed. it was confirmed that the s protein is responsible for the production of neutralizing antibodies [ , ] . the s subunit contains the rbd, which binds to its host receptor dpp (also known as cd ) and induces production of specific neutralizing antibodies. several studies have supported the role of this domain to elicit protective immunity against mers-cov challenge [ , , ] . in this study, we were able to demonstrate the distribution and expression of the s protein in the vaccinated group but not in the control groups (ad-cmv and pbs) by detection of the truncated protein-encoding segment in kidney and lung tissues following vaccination using conventional pcr and ihc. these findings support the ability of this vaccine candidate to distribute effectively within host tissues and express the target protein. it has been established that dpp is expressed on multiple cell types in the human body including cells in the kidneys and lungs [ ] . however, the n glycoprotein, but not the s glycoprotein, of mers was the target of investigation of mers-cov localization using ihc [ ] . the n glycoprotein was clearly present in lung tissues following mers exposure. in fact, the current study can be considered pioneering given the use of the s glycoprotein to express viral distribution in tissues. therefore, the expression of s protein in kidney tissues and not in lung might be used to explain previous in vitro experiments, which found a cytopathic effect of the mers-cov infection inside primary human kidney cells but not in primary human bronchial epithelial cells [ ] . in a recent study, the adenoviral vector with enhanced green fluorescent protein but not the adenoviral vector with mers in a hdpp mice experiment after intramuscular or intranasal injection [ ] . this supporting our finding that this construct was able to reach the lung tissues, but the s protein was not expressed in lung tissues. however, s gene detection in lung tissues by conventional pcr and not by ihc indicates further investigation is necessary regarding the level and intensity of s expression in this tissue. additional studies are also needed to explore s protein expression in other tissues such as the liver and intestine given the expression of dpp in these tissues [ ] . in this study, there was a significant serum level of the s -specific igg antibody in the vaccinated group but not in the control groups (ad-cmv and pbs). similar results have been shown previously: the sars-cov vaccine had a strong ability to elicit the s -specific igg antibody response [ ] . the results presented in this study also supported the findings of recent publications applying a similar vector vaccine construction [ , , , [ ] [ ] [ ] [ ] [ ] . the t helper immune response is known as the dominant immune response in the case of intracellular pathogens, such as viruses and bacteria, while the t helper immune response is the dominant immune response in cases of extracellular pathogens and allergic reactions [ ] . many pathogens, especially viruses, shift the host immune response toward th dominance over the th response to evade cellular immunity. in this study, ad-mers-s was able to provoke pro-inflammatory but not anti-inflammatory cytokine release. both th and th responses were elicited following immunization. cytokines such as ifn-γ and il- , which represent the th- response, were upregulated, while il- , which represents the th- response, was downregulated. the ad-mers-s was able to induce production of a significant amount of ifn-γ regardless of antigen-specific stimulation. a similar increase in il- was apparent in vaccinated animals in response to specific in vitro antigen stimulation. the mrna expression of ifn-γ was detected as early as weeks after the first immunization. in contrast, il- production in cell culture showed a significant increase in control animals after antigen stimulation, while the production of this cytokine was significantly decreased in the at week , the fold change in ifn-γ gene expression was significantly higher in the ad-middle east respiratory syndrome (mers)-s group than that of both control groups (ad-cytomegalovirus [cmv] and phosphate-buffered saline). however, there was a prominent increase in ifn-γ gene expression at week in both ad-mers-s and ad-cmv groups compared to that in week . the only significant change in il- gene expression was in the ad-cmv group at week , while no significant changes were observed in the ad-mers-s group at any time point. different letters indicate statistically significant differences at p< . . vaccinated group. the results demonstrated neither positive nor negative impact on il- expression in mice vaccinated with the ad-mers-s or mice that received only pbs. interestingly, there was a significant increase in il- and ifn-γ gene expression in antigen-stimulated cell culture obtained from mice vaccinated with ad-cmv at week . this might be due to the ability of adenoviruses to elicit strong humoral and cellular immune responses [ , ] . therefore, it can be concluded that the presence of the s protein gene in the adenovirus vector genome disturbs the immune stimulation ability of the adenovirus vector to elicit il- cytokine. two studies have shed light on the ability of the s subunit, and specifically, the ability of the rbd domain, to shift the host immune response toward th [ , ] . it was demonstrated in these two studies that igg , which is produced mainly during the th immune response, was decreased, while igg a, which is produced mainly during the th immune response, was upregulated following vaccination with their vaccine candidate. the truncated rbd fused with the fc portion of human igg was able to elicit both isotypes with a relatively higher amount of the igg a isotype (th response) [ ] . recombinant adenovirus vector carrying the s subunit or the whole s gene was also able to induce both types of the immune response. the igg a level was detected earlier and higher in mers-s than mers-s at week post-vaccination but not after week [ ] . accordingly, these findings lead to the dominance toward the th cellular immunity. several in vivo and in vitro studies have revealed the ability of mers-cov to evade the host innate immune response through downregulating the antigen-presenting pathway and proinflammatory cytokines such as ifn-γ and il- [ , [ ] [ ] [ ] . several mers-cov proteins were previously identified as potent ifn antagonists, including the m structural protein and the orf a, orf b, and orf accessory proteins [ ] . in addition, a comparison of the immune response of two mers-infected patients [ ] , one of whom with a poor outcome (died) that had elevated levels of il- and il- which promote the th immune response, while the other patient who overcame the infection had increased levels of ifn-α, ifn-γ, and il- , which promote the th immune response [ ] . in general, it is crucial to induce early and strong innate immune responses against mers-cov infection to save lives. our vaccine candidate was able to induce production of the key cytokines of activated t lymphocytes toward cd + th cells, which is also an indication of upregulation of the antigen-presenting pathway. the vaccine also did not trigger the production of il- , which is involved in the th response, which may be referred to the counter effect of the produced ifn-γ in il- producing cells [ ] . finally, it is important to clarify that the in vitro antigen stimulation was done using the specific -mer peptide "cysslildy," this peptide is located at position - within the conserved region of the s glycoprotein among all identified mers-cov strains. this was selected in a computerized simulation that showed that this sequence has the highest b cell antigenicity plot and has the ability to form the greatest number of interactions with mhc-class i alleles [ ] . although this epitope was not able to increase ifn-γ production level after in vitro stimulation, it resulted in a significant increase in il- production and a significant decrease in il- production in the vaccinated group. in addition, this epitope was able to increase il- production after 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coronavirus infection in dromedary camels in saudi arabia endogenous biotin expression in renal and testicular tumors and literature review retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry effects of gamma interferon, interleukin- , and transforming growth factor beta on the survival of mycobacterium avium subsp. paratuberculosis in monocyte-derived macrophages from naturally infected cattle computer-aided prediction and identification of potential epitopes in the receptor-binding domain (rbd) of spike (s) glycoprotein of mers-cov analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method comparison of intranasal and transcutaneous immunization for induction of protective immunity against chlamydia muridarum respiratory tract infection structure of mers-cov spike receptor-binding domain complexed with human receptor dpp a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv th /th balance: the hypothesis, its limitations, and implications for health and disease adenoviruses as vaccine vectors replication-defective adenovirus serotype vectors elicit durable cellular and humoral immune responses in nonhuman primates intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus the structural and accessory proteins m, orf a, orf b, and orf of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? this study was supported by the deanship of research at jordan university of science and technology (grant no. / ). ma and mua designed the adenovirus vaccine for the mers-s vaccine. ma and mk designed the mice experiments. mua, ma, and mk took the different tissue samples from the mice. mua performed the molecular assays and the immunohistochemical staining. ma and mua prepared the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord- -cp qr f authors: matsuyama, ryota; nishiura, hiroshi; kutsuna, satoshi; hayakawa, kayoko; ohmagari, norio title: clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis date: - - journal: bmc public health doi: . /s - - - sha: doc_id: cord_uid: cp qr f background: while the risk of severe complications of middle east respiratory syndrome (mers) and its determinants have been explored in previous studies, a systematic analysis of published articles with different designs and populations has yet to be conducted. the present study aimed to systematically review the risk of death associated with mers as well as risk factors for associated complications. methods: pubmed and web of science databases were searched for clinical and epidemiological studies on confirmed cases of mers. eligible articles reported clinical outcomes, especially severe complications or death associated with mers. risks of admission to intensive care unit (icu), mechanical ventilation and death were estimated. subsequently, potential associations between mers-associated death and age, sex, underlying medical conditions and study design were explored. results: a total of eligible articles were identified. the case fatality risk ranged from . to %, with the pooled estimate at . %. the risks of icu admission and mechanical ventilation ranged from . to % and from . to %, with pooled estimates at . and . %, respectively. these risks showed a substantial heterogeneity among the identified studies, and appeared to be the highest in case studies focusing on icu cases. we identified older age, male sex and underlying medical conditions, including diabetes mellitus, renal disease, respiratory disease, heart disease and hypertension, as clinical predictors of death associated with mers. in icu case studies, the expected odds ratios (or) of death among patients with underlying heart disease or renal disease to patients without such comorbidities were . ( % confidence interval (ci): . , . ) and . ( % ci: . , . ), respectively, while the ors were . ( % ci: . , . ) and . ( % ci: . , . ), respectively, in studies with other types of designs. conclusions: the heterogeneity for the risk of death and severe manifestations was substantially high among the studies, and varying study designs was one of the underlying reasons for this heterogeneity. a statistical estimation of the risk of mers death and identification of risk factors must be conducted, particularly considering the study design and potential biases associated with case detection and diagnosis. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. cases of middle east respiratory syndrome (mers), caused by mers-associated coronavirus (mers-cov), have continuously been reported since june . as of june , the total number of laboratory-confirmed cases notified to the world health organization (who) reached , cases, including deaths [ ] . particularly large outbreaks of mers-cov infection have been reported in the kingdom of saudi arabia (ksa) and the republic of korea (rok), while smaller outbreaks and importation events have been reported in other countries [ ] . of these, countries are located in the middle east, countries in europe, countries in africa, countries in southeast and east asia, and in north america (the united states of america) [ ] [ ] [ ] . because of the regular reporting of mers cases in the middle east, countries across the world are now facing a continuous threat of mers outbreak. to understand the clinical burden of mers, it is necessary to quantify the risk of developing severe clinical manifestations. the case fatality risk (cfr) is a measure of the risk of death among those who satisfy the case condition [ ] , while risks of admission to an intensive care unit (icu) and that of requiring mechanical ventilation are also useful to measure the extent of developing severe mers complications. however, it is not only necessary to estimate such risks, but it is also critically important to identify epidemiological determinants of those risks to then predict the risk of severe complications for each patient before the onset of disease exacerbation [ ] . in previous studies, the risk of death among secondary cases was estimated based on statistical modelling and was found to range from to %, approximately [ ] [ ] [ ] [ ] [ ] . meanwhile, among the primary cases, the risk of death was estimated to be greater at approximately %, perhaps because of biases associated with case detection and diagnosis [ ] [ ] [ ] . as for epidemiological determinants of mers death, elderly patients with underlying comorbidities have been identified as the most susceptible population with a high risk of death [ , , ] . despite our further understanding of the risk of developing severe mers, the abovementioned estimates are mostly based on a subset of mers cases; for instance, some of the risk estimates are a result of the analysis of cases diagnosed in in the rok or ksa alone. published articles with different study designs and populations have yielded different estimates and effect sizes associated with mers death. because of this variability, it is valuable to comprehensively and systematically analyze published mers studies that have recorded the clinical prognoses of cases. a systematic review is a highly informative review method that combines published results from different studies, thereby merging and contrasting results across multiple studies and answering study questions using the pooled estimates [ ] . thus, we aimed to perform a systematic review to assess risks of death and other severe complications and determine the risk factors for mers-associated death and contrast these results by study population and study design. the present study was a systematic review conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (prisma) statement [ ] . pico statement: our study question is focused on laboratory confirmed cases of mers regardless of their treatment status, and thus, involves only retrospective observational studies, measuring their risks of admission to intensive care unit (icu) and death and comparing those risks by age, gender and underlying comorbidities. our systematic review protocol is summarized as additional file . published studies that referred to the clinical prognosis of mers cases were retrieved from medline (pubmed) and web of science electronic databases on may . the following search terms were used in "all fields" to identify relevant published articles: . "mers" or "middle east respiratory syndrome" or "novel coronavirus" or "novel coronavirus " . "sever*"or "fatal*"or "death" or "mortalit*" . "hospitalization" or "intensive care" or "icu" . and and we limited the search to articles published between april (i.e., after the first mers case was reported) and june . additional studies reporting associated outcomes that were not identified by the abovementioned search strategy were manually retrieved by tracking the references of included articles (i.e., ancestry and discordancy approach). we restricted ourselves to publications written in english. all titles identified by the abovementioned search strategy were independently screened by two authors (rm and hn). abstracts of potentially relevant articles were subsequently reviewed for eligibility, and if a description of severe or lethal mers was available, articles were selected for closer examination of the full text. to be eligible for inclusion, published studies were required to meet the following characteristics: (i) studies focused on patients infected with mers-cov and (ii) explicitly documenting clinical outcomes (i.e., prognosis) and characteristics of both surviving and deceased patients. studies that allowed us to stratify the risk of severe or fatal mers by demographic or medical condition were preferred, but this was not an essential inclusion criterion. to calculate the risk of severe mers or mers death, we excluded case reports that documented only one or two cases (i.e., case reports with a sample size n ≥ were eligible). included studies were further classified into five groups based on the study design and population studied: (i) case reports comprising published studies that described the clinical course of individual patients including mild cases; (ii) studies including only icu cases (hereafter referred to as icu studies): case reports or retrospective studies that reported outcomes of patients admitted to the icu only; (iii) hospital studies: retrospective or descriptive studies that aimed to document the outbreak in a hospital or healthcare-associated facility; (iv) retrospective studies: published studies that retrospectively analyzed the series of mers cases that were registered in the patient database or tracked medical records; and (v) surveillance studies: published studies that extracted data from a database of cases, systematically gathering epidemiological data, as coordinated by a country or who. the primary data extracted were the proportions of deceased mers patients, patients admitted to the icu and patients undergoing mechanical ventilation. all of these outcomes were dealt with as dichotomous variables, and thus, we calculated the % confidence interval (ci) for each included study using the binomial distribution. whenever possible, we stratified the risk of death by age, sex, underlying medical condition and study design. for the analysis of the effect of each covariate on the outcome, the odds ratio (or) for death among those with underlying conditions was calculated and compared with those without comorbidities. stratified analysis could not have been made for the proportions of icu admission and mechanical ventilation because the dataset of such covariates was not commonly available for these two outcomes. we employed a fixed effects inverse variance weighted model. weighted means (i.e. pooled estimate) of the abovementioned proportions and the or for death by each covariate were calculated using the inverse of variance estimates from each study. the heterogeneity among identified studies was statistically assessed by the i statistic. to explore the possible sources of heterogeneity, we stratified pooled estimates by study design. a forest plot was used to illustrate the distribution of the outcome and effect size obtained from each published study. the flow diagram of the search and study selection process is shown in fig. . among a total of potentially relevant articles, and articles were excluded by screening of the titles and abstracts, respectively. one article was excluded by full-text screening. following the same process for additional manually identified articles, a total of articles were selected as eligible articles [ , and all were subject to meta-analysis. of these, four studies were classified as case reports, four as reports of icu cases, four as hospital outbreak studies, eight as retrospective studies and five as surveillance study. the majority of included articles were reported either from the ksa or the rok, except for one study conducted in jordan [ ] and the who the estimated cfr was reported in articles, ranging from . to % (fig. ) . the pooled cfr was . % ( % ci: . , . ), but the i was as large as . %. the sample size of case reports ranged from to , while studies with other designs tended to have larger samples, with or more cases, except for one icu study, one retrospective study and one hospital outbreak study. the proportions of icu admission and mechanical ventilation among all cases were available in and articles, respectively. the proportion of icu admission ranged from . to % with the pooled estimate at . % ( % ci: . , . ) and an i value of . %. the proportion of mechanical ventilation ranged from . to % with the pooled estimate at . % ( % ci: . , . ) and an i value of . %. [ ] age and sex distributions are shown in relation to the risk of death by mers in fig. . in the majority of the studies (except for a study from jordan), survivors were younger than those who died of mers. although not generally, infected men tended to die more often than women, and the pooled or of death among men compared with women was . ( % ci: . , . ). the i value of the sex difference for the risk of death was . %. the risks of death, icu admission and mechanical ventilation were stratified by study design and are shown in fig. , in which the pooled estimate for each study design was compared. the risk of death in the hospital outbreak and surveillance studies was significantly smaller than in icu case and retrospective studies. risks of icu admission and mechanical ventilation were the highest among icu case studies, followed by case report and retrospective studies. hospital outbreak studies yielded the smallest pooled risks of icu admission and mechanical ventilation. when comparing surveillancebased data between ksa and rok (fig. ) , the risk of death in rok (i.e., . - . % [ , , ] ) tended to be lower than that in ksa (i.e., . % by alsahafi and cheng [ ] ), perhaps reflecting the presence of the contact tracing effort in the rok. figure shows the possible association between five selected underlying medical conditions and the risk of death by mers. pooled estimates of the or were greater than the value of for all five comorbidities, including diabetes mellitus (n = studies), renal disease ( studies), respiratory disease ( studies), heart disease ( studies) and hypertension ( studies). among a total of five predictors, heart disease yielded the greatest or value at . ( % ci: . , . ) followed by respiratory disease with an or of . ( % ci: . , . ). figure shows the potential association between the risk of death by mers and potential predictors, including sex, heart disease and renal disease. men from icu studies tended to yield a greater or for death compared with other study designs. conversely, expected values of ors for death among those with heart disease and renal disease compared with those without appeared to be lower than the value of . . the present study systematically reviewed the risk of severe manifestations and death by mers by systematically searching and analyzing published articles from the ksa and the rok and calculating not only the cfr but [ ] . icu represents intensive care unit also the risks of icu admission and requiring mechanical ventilation. several clinical predictors of death were identified including older age, male sex and underlying medical conditions, including diabetes mellitus, renal disease, respiratory disease, heart disease and hypertension. the risk estimate appeared to vary by study design. in particular, studies focusing on patients in the icu yielded the greatest estimates, while the cfrs for surveillance and hospital outbreak studies were smaller. these findings indicate that ascertainment biases in surveillance and hospital outbreak studies, frequently involving case finding effort, were smaller than in other types of studies. the importance of case finding effort is likely reflected in the different cfr estimates based on surveillance data between ksa and rok. although the presently identified clinical predictors are in line with previously published studies [ , , ] , the present study is the first to systematically analyze published studies, including clinical research studies, and extract findings that echo those of published articles. as was observed in this study, systematic search and analysis of the transmission characteristics [ ] and spatial spreading patterns of mers [ ] have been successful. an important contribution of the present study is that we demonstrated that the risk of death or severe manifestations is highly heterogeneous for various reasons, including different study designs. it is recognized that mers involves asymptomatic infection [ ] , and thus, studies must be clear as to how the risk is estimated, including the definition and diagnostic methods used to identify infected individuals. depending on the study design, the clinical predictors of death also differed. for fig. estimated risks associated with middle east respiratory syndrome (mers) by study design. panels show the risk estimates by study outcome: (a) risk of death, (b) risk of admission to intensive care unit (icu) and (c) risk of mechanical ventilation. cfr represents the case fatality risk. the estimate for each study design represents the pooled risk of death calculated using the inverse variance of the risk of death in each published study. the size of the diamonds reflects the sample size, and the whiskers extend to the lower and upper values of the % confidence interval (ci). the diamond without fill represents the pooled estimate using the inverse variance of the risk of death. i measures the extent of the heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity example, renal and heart diseases might not predict the risk of death in an icu setting, but they may be critically important in other settings that involve milder cases. not only studies in icu settings, but also retrospective studies yielded relatively high risk estimates for severe manifestations and death. our finding raises concerns regarding the retrospective analysis of confirmed cases in registered databases without referring to biases associated with case detection and diagnosis, which could yield a biased risk estimate of mers severity. in fact, that could explain why the cfr of confirmed cases among registered cases in patients' database has been as high as %, while the cfr of secondary cases in the presence of contact tracing has been estimated at about % [ ] [ ] [ ] [ ] [ ] . the comorbidities identified in our study are in line with those already identified elsewhere [ , ] . the identification of comorbidities is not only stressed based on previous and present findings [ ] , but it is critically important to understand the underlying pathophysiological mechanisms. high representation of men among deceased cases may reflect the interaction of factors related to sex-specific lifestyle (e.g., smoking habits in the middle east). older age might reflect the greater likelihood of having underlying medical conditions. diabetes, renal and respiratory diseases could predispose patients to be immunologically vulnerable and heart disease could induce water retention (e.g., secondary aldosteronism), both exacerbating the systemic condition. hypertension could have been confounded by some other explanatory factor (s), for example, obesity could have likely led to both hypertension and mers death. nevertheless, identified predictors are accompanied by reasonable biological explanations. the present study is not free from limitations. the biggest concern is, given the absence of identifying information, the included articles most likely referred to the same cases multiple times, potentially overestimating the . the vertical dashed line shows the threshold value of or = . the diamond without fill represents the pooled estimate using the inverse variance of the or. i measures the extent of heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity number of cases. in fact, the total number of diagnosed and reported cases of mers as of june is approximately , cases, but our systematic review included as many as , cases. thus, it is likely that multiple reports from rok (e.g., cowling et al. [ ] , kcdcp [ ] and majumder et al. [ ] ) reported on the same cases multiple times. rather, we did not avoid any overlap of cases in datasets because that adjustment forced us to adjust the overlap among the cases from the ksa in a similar manner. for this reason, the pooled estimate would never represent the actual pooled outcome data because the same case was counted multiple times. if we remove cowling et al. [ ] and majumder et al. [ ] from our analysis and include kcdcp [ ] , which had the largest sample size, the pooled estimate of the cfr would be increased to . % ( % ci: . , . ). this is understandable owing to the diminished impact of the extensive contact tracing effort in the rok. despite these overlaps, we conducted this systematic review to demonstrate that ascertainment biases likely act as a key factor that characterizes differential mortality across countries. to avoid any overlap of cases and better identify risk factors of icu admission and death, it is advised to set up a common case registration system across countries and allocate identity number for each individual case. as the second technical limitation to remember, it should be noted that the access to individual data was not achieved, and thus, for instance the age-related analysis did not rest on individual age data, and similarly, we have had limitations in the precision of the majority of outcome evaluations. third, clinical predictors of death have been classified only at organ level, and moreover, individual behavioral factors or habitat [ ] have not been examined in relation to the risk of mers death. fourth, non-english language manuscripts have been missed, and they include at least a few publications in korea and one from jordan. despite these problems, we cannot help but consider that the present study successfully and systematically . odds ratio (or) represents the odds ratio of death among men with underlying medical condition compared with women without comorbidities, respectively. the size of the diamonds reflects the sample size, and the whiskers extend to the lower and upper values of the % confidence interval (ci). the diamond without fill represents the pooled estimate using the inverse variance of the risk of death. i measures the extent of heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity evaluated the risk of severe manifestations and death by mers by collecting published information on clinical predictors of the risk of death. an important consideration is that the associated risk estimation and identification of risk factors of mers call for particular care in terms of study design, especially in aiming to eliminate biases associated with detection and diagnosis. heterogeneity in risks of death and severe manifestations secondary to mers was substantial. differential study design was one of underlying reasons for the large heterogeneity. statistical estimation of the risk of mers death and identification of risk factors must be conducted with particular careful attention paid to study design, especially accounting for biases associated with case detection and diagnosis. additional file middle east respiratory syndrome coronavirus (mers-cov). geneva: world health organization clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus (mers-cov) into the united states 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transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, : a retrospective epidemiological analysis funding hn received funding support from the japan agency for medical research and development, the japanese society for the promotion of science (jsps) kakenhi grant numbers kt , k and , the japan science and technology agency (jst) crest program and ristex program for science of science, technology and innovation policy. no received funding support from the ministry of health, labor, and welfare, japan (h -shinkogyosei -shitei- ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. collected datasheet is available from the authors (rm) upon request. authors' contributions hn conceived the systematic review. rm and hn implemented systematic search. rm and hn performed statistical analyses. rm and hn drafted the early version of the manuscript and hn substantially rewrote the text. sk, kh and no further revised the manuscript. all other authors gave comments on the revised manuscript and approved the final version of the manuscript. the authors are experts with interest in infectious disease epidemiology and also in clinical infectious diseases, and the team of lead author is led by professor from hokkaido university graduate school of medicine. the authors declare that they have no competing interests. not applicable.ethics approval and consent to participate not applicable.author details graduate school of medicine, hokkaido university, kita jo nishi chome, kita-ku, sapporo - , japan. crest, japan science and technology agency, - - , honcho, kawaguchi-shi, saitama - , japan.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - ybfiz f authors: decaro, nicola; lorusso, alessio title: novel human coronavirus (sars-cov- ): a lesson from animal coronaviruses date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: ybfiz f the recent pandemic caused by the novel human coronavirus, referrred to as severe acute respiratory syndrome coronavirus (sars-cov- ), not only is having a great impact on the health care systems and economies in all continents but it is also causing radical changes of common habits and life styles. the novel coronavirus (cov) recognises, with high probability, a zoonotic origin but the role of animals in the sars-cov- epidemiology is still largely unknown. however, covs have been known in animals since several decades, so that veterinary coronavirologists have a great expertise on how to face cov infections in animals, which could represent a model for sars-cov- infection in humans. in the present paper, we provide an up-to-date review of the literature currently available on animal covs, focusing on the molecular mechanisms that are responsible for the emergence of novel cov strains with different antigenic, biologic and/or pathogenetic features. a full comprehension of the mechanisms driving the evolution of animal covs will help better understand the emergence, spreading, and evolution of sars-cov- . eighteen years after the emergence of severe acute respiratory syndrome (sars) in china and years after the emergence of middle east respiratory syndrome (mers) in saudi arabia, a novel coronavirus (cov) epidemic, recently classified as pandemic by the who, is threatening the human population worldwide (zhou et al., ) . the disease, now referred to as coronavirus disease , is caused by a novel human cov, which was initially denominated novel coronavirus ( -ncov) and later renamed as sars coronavirus (sars-cov- ) the by coronavirus study group of the international committee on taxonomy of viruses (gorbalenya et al., ) . covid- emerged in december in wuhan city, hubei province, china, in humans exposed to wildlife at the huanan seafood wholesale market, which is the largest seafood market in central china, and where different species of farm and wild animals are commonly sold (lorusso et al., ) . the epidemic has then expanded not only to neighbouring asian countries, but also to other continents (https://www.who.int/ docs/default-source/coronaviruse/situation-reports/ -sitrep- -covid- .pdf?sfvrsn=c ea _ ). . a list of human covs is showed in table . historically, only two human covs (hcovs) had been known before the sars emergence, namely hcov- e, an alphacoronavirus originated in bats and transmitted to humans through alpacas, and hcov-oc , a betacoronavirus which had passed from rodents to humans through cattle (corman et al., (corman et al., , . after - sars epidemic, the renovated interest in hcovs allowed the discovery of two additional viruses, the alphacoronavirus hcov-nl and the betacoronavirus hcov-hku , derived from bats and rodents, respectively (tao et al., ) . all these four viruses are usually responsible for mild respiratory symptoms in immunocompetent patients. sars-cov and mers-cov are two unrelated betacoronaviruses originated in bats and transmitted to humans by wild carnivores and dromedary camels, respectively. in contrast to other hcovs, these two viruses displayed an increased virulence, causing severe pneumonia and even the death of affected people, with mortality rates of about % and %, respectively (guarner, ) . the occurrence of three highly pathogenic covs with a zoonotic origin in less than two decades, highlights the role of animals in generating covs with increased virulence that can adapt to humans, causing epidemics (and eventually pandemics) with high impact on human health. indeed, cov infections of veterinary interest have been known since almost a century (cavanagh, ; pedersen, ; decaro et al., ) , so that animal covs are paradigmatic of how this large family of viruses evolves, generating strains with different biological properties. in addition, the efforts done in veterinary medicine https://doi.org/ . /j.vetmic. . received march ; received in revised form april ; accepted april evolve rapidly, changing their antigenic profile, tissue tropism or host range by means of two distinct mechanisms. the viral replicase (an rna dependent-rna polymerase) does not possess a good proof reading activity, therefore the incorporation of wrong nucleotides at each replication cycle and the consequent accumulation of mutations in the viral genome lead to a progressive differentiation of the viral progeny from the parental strain. this mechanism, which is well known for influenza viruses being responsible for the so called antigenic drift, may cause the progressive adaptation of the viral surface proteins to the cell receptors of new animal species, increasing the viral fitness. in addition, the particular replicating machinery of covs facilitates recombination events due to the presence of consensus sequences upstream each gene. therefore, in the case of coinfection by more than one cov strain, the rna polymerase can jump from the rna of a strain to that of the other one, synthetizing a hybrid rna containing sequences from both viruses. recombination can occur not only with genomic sequences of other covs (homologous recombination), but also with rnas of different viruses and other organisms (heterologous recombination) (luytjes et al., ; banner and lai, ; lai, ; zeng et al., ; huang et al., ) . recombination is an alternative mechanism that let covs acquire novel biological properties in terms of virulence, host range and tissue tropism, so that cov strains, which are non-pathogenic or lowpathogenic in the original host, may increase their pathogenicity in the same species or adapt to different species spreading in the new host with exceptional rapidity (banner and lai, ) . the occurrence of three human cov epidemics in less than years, along with the emergence of less pathogenic human covs, arises some questions on how these viruses that have their reservoirs in bats and rodents may overcome the species barriers jumping to humans. the animal-to-human transmission of viruses has been already occurred in the past, but it seems that its frequency has been increased in the last decades, involving in a short time span not only covs, but also a plethora of genetically and biologically different viruses with zoonotic potential, such as ebola virus, influenza viruses, flaviviruses, hendra and nipah viruses (mcmahon et al., ) . climate changes that are intensifying in this first quarter of the st century are favouring the spread of vector-borne diseases through increasing the proliferation of vectors and predisposing to their occupation of new ecological niches. the emergence in temperate climate areas such as europe of vectorborne diseases caused by viruses considered exotic until few years ago (west nile virus, usutu virus, chikungunya virus) accounts for a progressive geographic expansion of tropical diseases thanks to the ongoing phenomenon of tropicalisation (mcmahon et al., ) . deforestation and urbanization are other major factors that facilitate the spill-over of zoonotic agents to humans by reducing the habitat of wildlife and increasing the chances of contacts between wild animals (like bats, rodents and birds) and human beings (beena and saikumar, ; lorusso et al., ) . this could be the case of ebola virus, hendra and nipah viruses, hantavirus and coronavirus infections. in addition, the close contact between human beings and different animal species sold at the wet markets of east asia represents the optimal situation for the host species jump and adaptation to humans of potentially zoonotic agents like covs. it is not a coincidence that two of the most severe zoonoses of the last two decades (highly pathogenic h n avian influenza and sars) have emerged in the same chinese province of guangdong where the contact between humans and animals is closer (lorusso et al., ) . table reports the most important avian cov species recognised so far and their associated diseases. the number of avian species in which covs have been detected in the last years is humongous. since the emergence of sars-cov in , there has been increased interest in table main coronaviruses in domestic and domesticated avian species. schalk and hawn ( ); beach and schalm ( ) ; beaudette and hudson ( ) turkey ( respiratory and kidney disease spackman et al. ( ) n. decaro and a. lorusso veterinary microbiology ( ) covs in other species, including birds. prior to that time, our knowledge of covs in avian species was limited largely to three birds of the order galliformes, i.e., domestic fowl (gallus gallus), turkeys (genus meleagris) and pheasants (phasianidae), with their infectious bronchitis virus (ibv), turkey coronavirus (tcov), and pheasant coronavirus (phcov), respectively. these three viruses were considered for a long-time different species for several reasons such as the diverse pathotype (enterotropic or respirotropic), host range and genetic relatedness of the s protein (cavanagh, ) . this scenario radically changed after the discovery of several novel covs with high genetic diversity from different avian species and the novel rules for species designation of the coronavirus study group (csg https://talk.ictvonline.org/ictv-reports/ ictv_ th_report/positive-sense-rna-viruses- /w/posrna_viruses/ /coronaviridae). all these viruses as well as analogous ibv-like covs detected in other birds including penguins, pigeons, peafowl, parrots, waterfowl, teal, quail, duck and whooper swan (cavanagh et al., ; circella et al., ; domanska-blicharz et al., ; torres et al., ; hughes et al., ; liu et al., ; wille et al., ; jordan et al., ; bande et al., ; suryaman et al., ) have been assigned to the same viral species known as avian coronavirus (acov) within the subgenus igacovirus of genus gammacoronavirus. ibv and ibv-like strains are commonly detected in both gallinaceous and non-gallinaceous birds, also asymptomatically (cavanagh, ) . this might suggest that these species would act as wild reservoirs, spreading ibv strains over the world (de wit et al., ) . as for the huge economic impact of the disease it causes, ibv is one of the most studied covs over the last decades. ibv causes the infectious bronchitis (ib), a term adopted in for describing the main clinical characteristics of a transmissible respiratory disease of poultry detected for the first time in north dakota (usa). ib has been now diagnosed worldwide and is one of the most important viral diseases of poultry characterised by respiratory signs, but it can also affect the kidneys and reproductive tract following viremia with a severity that differs depending on the involved viral strain (cavanagh and gelb, ) . the disease also affects wild and ornamental birds chen et al., ) . ib control has been hampered by the intricate ibv evolution, which has been entailed, over the years, by the emergence of many different antigenic or genotypic types, commonly referred to as variants, with divergent molecular, biological, and antigenic properties. being a cov, ibv has, indeed, a considerable ability to change both by mutation and by homologous recombination events, which may cause, along with replicase stuttering or slippage, also insertions and deletions in the genome (cavanagh and gelb, ) . if these mechanisms involve the hypervariable region s , they frequently result in the emergence of new ibv variants. although many new variants are not successful, a few may emerge, spread, causing devastating disease either worldwide or in limited geographic areas. currently lineages have been recognized, categorized into six genotypes (gi to gvi) (valastro et al., ) . through their s protein, ibv and ibv-like viruses recognise as cellular host receptor the α , -linked sialic acid glycan, widely distributed in the respiratory tract and in several other host tissues, factor which may explain the tropism also for several organs of the infected host (winter et al., (winter et al., , shahwan et al., ; ambepitiya wickramasinghe et al., ) . extensive use of vaccines has greatly contributed to the high variability of ibv strains thorough recombination between vaccine and field viruses and viral selection pressure resulting from vaccination and presence of partially immune birds (gandon and day, ; gandon et al., ; bande et al., ) . important ibv-like strains are tcov, responsible for enteritis in turkey (also known as bluecomb disease), guinea fowl coronavirus (gfcov) and quail coronavirus (qcov) responsible for fulminating enteric disease in guinea fowl and quail, respectively cavanagh, ; liais et al., ) . tcov, gfcov and qcov are evolutionarily distant from acov based on the s protein. while ibv is a primarily respiratory pathogen, tcov causes gastrointestinal disease (liais et al., ; guy, ) . enterotropism has also been observed for some ibv serotypes; however, all ibv strains infect primarily the respiratory tract, resulting in mild to severe inflammation of the nasal and tracheal epithelia (cavanagh, (cavanagh, , . the s domain of the s protein is highly variable, with the amino acid sequences of ibv and tcov from the usa sharing < % sequence identity. phylogenetic analysis of the s gene shows, indeed, grouping of ibv and ibv-like viruses on the one hand and tcov-us, gfcov and qcov on the other hand (ambepitiya wickramasinghe et al., a) . accordingly, the emergence of covs in turkeys in the usa was proposed to have resulted from recombination events involving ibvs and an as-yet-unidentified cov donating a novel s gene. this switch contributed largely to determine the in vivo tissue tropism of tcov and related viruses. intriguingly, the s protein of these covs requires nonsialylated type poly-lacnac structures on n-glycan cores for binding. this is in marked contrast to the α , -linked sialic acid glycan binding of ibv and ibv-like viruses (ambepitiya wickramasinghe et al., b) . the s subdomain of a tcov isolate from france in (tcov-fr) had only % sequence identity to that of the tcov-us strain (maurel et al., ) . this diversity was biologically evident by the prominent tropism for the epithelium of the bursa of fabricius and only mild tropism for the small intestine of turkey. tcov-fr s protein did not show, indeed, affinity for nonsialylated type poly-lacnac (ambepitiya wickramasinghe et al., a) . this genetic diversity between tcovs is in accordance with several recombination events involving ibvs on different continents with several unknown covs. on the one hand, the s genes of gfcov/fr/ (isolated in france in ) and tcov-us share significant genetic relationships, and thus these viruses must have acquired their s gene from a common ancestor. on the other hand, gfcov/fr/ and fr tcov have a very similar genetic background in other genes. two recombination events may be responsible for the genesis of tcov-us and fr tcov. a first event occurred between an ibv eu recipient strain and an unknown acov donor, resulting in a virus with a new s gene, whose evolution would have resulted in fr tcov and gfcov/fr/ . a second recombination event involving a us ibv recipient and gfcov/fr/ would have generated us tcov viruses, which share a stronger s gene similarity with gfcov/fr/ than with fr tcov . additional covs distinct from acovs and mainly circulating in ducks (duck coronavirus, dcov), pigeons (pigeon coronavirus, pcov), or geese (goose coronavirus, gcov) have been identified (cheng et al., ; jonassen et al., ; muradrasoli et al., ; kim and oem, ; zhuang et al., ; papineau et al., ) . although their genome seems to fulfill the official ictv criteria required to distinguish a new species within the gammacoronavirus genus, ictv approval is still pending. historically, covs of birds were all included in the gammacoronavirus genus and, in turn, all covs belonging to this genus were identified only in birds. however, this suggestion was rebutted by the evidence of a cov belonging to the gammacoronavirus genus in a beluga whale first discovered in (viral species beluga whale coronavirus sw species, subgenus cegacovirus, genus gammacoronavirus) (mihindukulasuriya et al., ) , and of three novel covs, bucov hku , thcov hku , and mucov hku in birds of the order passeriformes, namely bulbuls (pycnonotus jocosus), thrushes (turdidae) and munias (lonchura punctulate), respectively, which did not cluster phylogenetically with extant covs identified in birds. these latter three viruses were distinct from known covs forming a unique cluster in the phylogenetic tree, which was the basis for generation of the deltacoronavirus genus (woo et al., ) . importantly, additional novel viruses belonging to this novel genus were detected in wild birds chu et al., ; durães-carvalho et al., ; torres et al., ) . these viruses cluster with previously unclassified covs detected in various asian carnivores, i.e., the asian leopard cat (prionailurus bengalensis) and chines ferret badger (nyctereutes procyonoides) (dong n. decaro and a. lorusso veterinary microbiology ( ) et al., ) . covs belonging to the betacoronavirus genus, which are strictly related to mouse hepatitis virus (mhv), were also described in wild birds, including parrots, in brazil (durães-carvalho et al., ) . interestingly, this was not the first detection of viruses belonging to the betacoronavirus genus in birds. often overlooked is the discovery over years ago of a cov from the manx shearwater (puffinus puffinus), a bird that visits the shores of britain in summer (nuttall and harrap, ; cavanagh et al., ) . this virus was also related to mhv. however, at that time, considering the unusual finding and that the virus was isolated by passage of shearwater material in the brains of mice, it was speculated that the detected virus was an mhv strain already present in the mice before inoculation (cavanagh, ) . bats are an ancient and heterogeneous group of ecologically important mammals, representing nearly a quarter of all mammalian diversity on earth. they belong to the order chiroptera and further classified in two suborders yinpterochiroptera and yangochiroptera. the first includes the non-echolocating pteropodidae family (megabats) and five echolocating rhinolophoidea microbat superfamilies. yangochiroptera contain thirteen echolocating microbat families (tsagkogeorga et al., ) . bats are thought to host a large plethora of viruses. these include, amongst the others, lyssaviruses, filoviruses, henipaviruses, and reoviruses (calisher et al., ) . before sars-cov epidemic, bats were not known to host covs. indeed, the first evidence of a bat cov was published in . after the sars epidemic, there was a boost in interest regarding searching for novel covs in various animals, including bats. to date, over novel covs have been identified in bats and approximately % of the bat virome sequenced to date is composed of covs (chen et al., ) . this data has been made available following the massive surveillance, coupled with the advent of next-generation sequencing (ngs) technology, which has been performed in wild animals banerjee et al., ) . just a small portion of these covs have been officially recognised by the ictv; many others are still pending for official designation. cov species detected in bats and officially recognised by the ictv are listed in table and the following chapter reasonably discusses only officially recognized bat cov species. bats can carry and transmit covs into local bat populations via migration even though little is known about the migratory patterns of these animals. closely related covs can be detected in the same bat species living at locations separated by thousands of miles (drexler et al., ) and different cov species or genera can be found in different bat species living at the same roosting sites. however, some covs have been shown to be species-specific. accordingly, regional patterns of bat cov outbreaks at species level can be deduced from the population distribution of their respective bat hosts. although bats seem to develop clinical diseases induced by several viruses and bacteria (mühldorfer et al., ) , generally covs do not cause apparently overt disease in these mammals, also experimentally. this phenomenon seems to be related with peculiar characteristics of their immune system (ahn et al., ; brook et al., ) . based upon genomic data available so far, it is widely accepted that while birds represent the reservoir for covs belonging to genera gammacoronavirus and deltacoronavirus, bats are the natural reservoir for alpha-and betacoronaviruses. however, only betacoronaviruses of subgenera sarbecovirus, merbecovirus, nobecovirus and hibecovirus have been detected in bats so far. given that several betacoronaviruses from the subgenus embecovirus have been discovered in rodents, it was speculated that rodent covs may be the ancestors of currently circulating viruses belonging to this subgenus . covs have been detected at high frequency in bats in all continents, with alphacoronaviruses being more widespread than betacoronaviruses . subgenus colacovirus (genus alphacoronavirus) officially comprises the viral species bat coronavirus cdphe , so far composed by two bat covs strains named cdphe /usa/ and myotis lucifugus cov (myl-cov), which share a . % nucleotide identity across the whole genome. both strains have been detected in myotis lucifugus bats (vespertilionidae) also known as the northern american little brown bats. the former was detected in in colorado (genbank acc. no. kf ), while the latter was reported in in canada. this virus was identified in the intestines and lungs and associated with minimal pathology or inflammation (subudhi et al., ) . subgenus decacovirus (genus alphacoronavirus) comprises the species rhinolophus ferrumequinum alphacoronavirus hub- composed so far by btms-al-phacov/gs and btrf-alphacov/hub strains discovered in china in myotis spp. and rhinolophus ferrumequinum bats, respectively. these two viruses share very high sequence identities (higher than %), which dramatically decrease in the s genes (only % nucleotide identity) (wu et al., ) . woo et al., ) shares an % sequence identity with severe acute diarrhoea syndrome-coronavirus (sads-cov) of pigs . these two viruses are now included in the same viral species rhinolophus bat coronavirus hku (subgenus rhinacovirus, genus alphacoronavirus). viral strains btkynl - a, btkynl - b, btkynl - and btkynl - a, identified in in triaenops afer bats from kenya, form the viral species nl -related bat coronavirus strain btkynl - b that is part of the subgenus setracovirus (genus alphacoronavirus) along with human coronavirus nl (tao et al., ) . in this regard, a bat origin has been strongly suggested for two of the less-pathogenic hcovs causing mild respiratory symptoms in immunocompetent people, namely hcov- e and hcov-nl , both belonging to the alphacoronavirus genus. whereas hcov- e (subgenus duvinacovirus) recognises as direct ancestor an alphacoronavirus from alpacas, which in turn derives from e-related covs identified in hipposiderid bats (corman et al., ) , hcov-nl is likely a recombinant virus originating from the distantly related e-related covs associated with hipposiderid bats and covs associated with triaenops afer bats (tao et al., ) (table ). the s protein of hcov-nl is more closely related to that of e-related covs, whereas the rest of the genome with covs included in the nl -related bat coronavirus strain btkynl - b species (tao et al., ) . different from the bovine coronavirus (bcov)-like viruses that cause enteric disease, in a novel alpaca cov was associated to respiratory disease in california, usa. full-length genome analysis showed that this respiratory alpaca cov was closely related to the alphacoronavirus hcov- e (subgenus duvinacovirus) (crossley et al., ) . more recently, close relatives of hcov- e were detected in african hipposiderid bats. interestingly, both bat and alpaca viruses displayed an intact accessory gene orf located at the genomic ' end, while hcov- e retained only a conserved trs preceding remnants of this orf, suggesting its loss after acquisition of a e-related cov by humans. therefore, hcov- is likely a descendant of the alpaca alphacoronavirus (corman et al., ) . strains forming the viral species bat hp-betacoronavirus zhejiang (subgenus hibecovirus, genus betacoronavirus) were discovered in hipposideros pratti bats from china in (wu et al., ) . strain ro-batcov gccdc was identified from stools of rousettus leschenaultii, a species of fruit bats (pteropodidae) of southern asia, which were collected in yunnan province, china, in (huang et al., ) . ro-batcov gccdc shows a small intact orf of nucleotides embedded between the n and ns a genes. this orf has no homology to any known coronavirus, and the encoded protein exhibited . % amino acid identity with the p protein encoded by the first orf of segment s of bat fusogenic orthoreoviruses (genus orthoreovirus, species nelson bay orthoreovirus, also known as pteropine orthoreovirus). these viruses are double-stranded segmented rna viruses, belonging to the family reoviridae, and are able to cause severe pneumonia in humans (chua et al., ; lorusso et al., ) . ro-batcov gccdc is included in the viral species rousettus bat coronavirus gccdc within the subgenus nobecovirus, genus betacoronavirus. rousettus bat coronavirus hku , belonging to subgenus nobecovirus, was also identified in rousettus leschenaultii and in other bat species (mendenhall et al., ) . this virus was first detected in in guangdong province in china (woo et al., ) . subsequent studies suggested that the virus was widely distributed and is circulating in different bat species (ge et al., ) . covs from the bthku -like cluster were also detected in hipposidereos commersoni and rousettus aegyptiacus bats in kenya (tong et al., ) . being a fruit bat, rousettus leschenaultii has a wider flying range than most of the insectivorous bats in china, thus it may carry viruses over long distances. a comparison of the reported hku -cov sequences showed a high genetic diversity within this viral species (luo et al., a, b; lau et al., ; ge et al., ) . when mers-cov was first isolated in the middle east in and its genome sequenced, it was found that it was most closely related to ty-batcov hku discovered in tylonycteris pachypus and pi-batcov hku discovered in pipistrellus abramus, which were the only known members of subgenus merbecovirus at that time. these two viruses are now the prototype strains of tylonycteris bat coronavirus hku and pipistrellus bat coronavirus hku viral species, respectively, within subgenus merbecovirus, genus betacoronavirus. although mers related covs (mers-rcovs) were lately discovered, mers-cov was much closer in the s region to hku -cov than to mers-rcov or hku -cov. indeed, dipeptidyl peptidase (dpp ), the receptor for mers-cov, is also the receptor for hku , but neither for hku nor for early discovered mers-rcovs. however, hku prefers bat dpp over human dpp , whereas mers-cov shows the opposite trend . so far, hku -covs are only carried by tylonycteris spp. bats (t. pachypus and t. robustula) and are relatively conserved; hku -covs are found in different pipistrellus spp. bats, including p. abramus, p. pipistrellus and p. minus . due to the current sars-cov- pandemic, attention should be given to the viral species severe acute respiratory syndrome-related coronavirus (sars-rcov,subgenus sarbecovirus, genus betacoronavirus) and middle east respiratory syndrome-related coronavirus (mers-rcov, subgenus merbecovirus, genus betacoronavirus), which enclose sars-cov and mers-cov, the first two highly pathogenic covs that were discovered in humans. in , at the beginning of the sars epidemic, almost all early human index patients had animal exposure in a market place, in guangdong province, before developing disease. after sars-cov was identified, its rna and/or specific antibodies were found in masked palm civets (paguma larvata) and animal handlers in a market place. however, later investigations of farmed and wild-caught civets revealed that sars-cov strains found in market civets were transmitted to them by other wild animals (tu et al., ; kan et al., ) . subsequently, novel covs related to human sars-cov (sars-rcovs) were discovered in horseshoe bats (genus rhinolophus) in china and hong kong lau et al., ) . these sars-rcovs showed genome sequence identity of - % among themselves and - % identity to human or civet sars-cov isolates. sars-rcovs were detected in rhinolophus spp. bats of other regions of china (tang et al., ; woo et al., ; yuan et al., ; ge et al., ) . sars-rcovs with higher genetic diversity with respect to chinese strains were also detected in rhinolophid bats from slovenia, bulgaria and italy in europe (drexler et al., ; rihtaric et al., ; balboni et al., ) . covs related to sars-rcov were also detected in hipposideros spp. and chaerophon spp. bats from ghana, kenya and nigeria (hu et al., ) . these evidences suggested that bats may be the natural hosts for sars-cov and that wild carnivores were only intermediate hosts. although these sars-rcovs showed high sequence identity to sars-cov, they were demonstrated to be unable to bind to the human cell angiotensin converting enzyme ii (ace ) receptor, the receptor of sars-cov, as a consequence of deletions in their s protein (ren et al., ) . besides, the theory of bat origin of sars-cov lacked a powerful support due to the failure of direct isolation of this virus from bats. thus, considering that no direct progenitor of sars-cov was found in bats and that rna recombination is the fuel for cov evolution, it has been proposed that sars-cov emerged through recombination of bat sars-rcovs. this hypothesis was made after the evidence of a single bat cave in yunnan, china, with very high covs diversity and considering that, within the identified covs, all genetic elements needed to form sars-cov have been identified in that single cave (ge et al., ) . recombination analysis also strongly supported the hypothesis that the civet sars-cov strain sz originated following a recombination event of two existing bat strains, wiv and rf (hu et al., ) . moreover, wiv , the closest relative to sars-cov that has been found in bats so far (more than % nucleotide identity, higher than that of any other bat sars-rcovs ( - %)), likely arose through recombination of two other prevalent bat sars-rcov strains. the most frequent recombination breakpoints were within the s gene and upstream of orf , which encodes an accessory protein. these genes were also involved in the crucial adaptation pathways of sars-cov from bats to wild carnivores, from wild carnivores to humans, and from human to human (cui et al., ) . wiv has been shown to have the capacity to bind to the human, civet and bat cell ace receptor (ge et al., ) . the isolation in cellculture of a highly related sars-cov strain, coupled with the evidence of a functional s protein capable of using the same ace receptor, provided robust and conclusive evidence for the bat origin of sars-cov. an additional sars-rcov strain has been shown, by reverse genetics studies, to have the capacity to bind to the human ace receptor (menachery et al., ) . quite the opposite, a direct bat cov highly related to mers-cov of humans was never detected. indeed, the genome sequences of mers-cov in human and dromedaries possess only around - % nucleotide identities to those of the other members of subgenus merbecovirus from different bats. human mers-covs were instead almost identical to mers-covs identified in dromedary camels (camelus dromedaries). lately, genomic sequence analyses indicated that covs now belonging to the mers-rcov species were found in several bat species from two bat families, vespertilionidae and nycteridae (lelli et al., ; de benedictis et al., ; corman et al., a, b; anthony et al., ; moreno et al., ; wong et al., ) . however, none of these mers-rcovs is a direct progenitor of mers-cov, as their s proteins differ substantially from that of the human virus. the closest relative to mers-cov of humans and dromedary camels is mers-rcov strain neoromicia/ isolated from neoromicia capensis bats in south africa (geldenhuys et al., , table ). a short sequence (around nucleotides) of viral rna identical to that of mers-cov was also detected in a taphozous perforates bat in saudi arabia (memish et al., ) . overall, although it is widely accepted that mers-cov ancestor is in bats, further studies are warranted in order to discover the precise mechanisms of its emergence in dromedary camels and humans. it was suggested that mers-cov ancestors had been circulating in bats for very long time. mers-cov has evolved to adapt to use human receptor and the dpp -recognising bat coronaviruses like hku may follow up, thereby posing a serious risk to human health. recent mers-rcovs were shown to have the capacity to bind to the dpp as entry cell receptor as they acquired the s through recombination with hku -like viruses (luo et al., a, b) . as for the recent and threatening covid- outbreak in humans, we certainly know that sars-cov- belongs to the species sars-rcov together with sars-cov from humans and sars-rcovs from wild carnivores and horseshoe bats (genus rhinolophus) (gorbalenya et al., ; zhou et al., ; wu et al., ) . epidemiological investigations revealed that many initial patients were exposed to wildlife at the huanan seafood wholesale market (south china seafood market), which is the largest seafood market in central china (lorusso et al., ) . sars-cov- has been assigned to an existing species of hundreds of known viruses largely isolated from bats. these viruses have names derived from sars-cov, but only the viral isolates originating from the - outbreak have been confirmed to cause sars in humans (gorbalenya et al., ) . importantly, it has also been confirmed that sars-cov- uses the ace receptor through the receptor binding domain (rbd) of the s protein (hoffmann et al., ; zhou et al., ) . likely, also sars-cov- has a bat origin. according to genome sequences available so far, the most closely related virus ( . % of nucleotide sequence identity) to sars-cov- is strain batcovratg identified from a bat, rhinolophus affinis, from yunnan province, china, followed by sars-rcovs identified from pangolins (tang et al., ) . the receptor-binding spike protein of sars-cov- is highly divergent from other covs with less than % nucleotide sequence identity to all previously described sars-rcovs, except for a . % nucleotide identity to batcovratg (zhou et al., ) . although sars-cov- uses the ace receptor, five out six critical amino acid residues in rbd were different between sars-cov- and sars-cov; the same residues were instead identical to those of pangolin sars-rcovs and, in turn, only one of these residues was identical to those of batcovratg (tang et al., ) , although this latter shows the highest nucleotide sequence identity with sars-cov- along the whole genome. thus, it was tempting to speculate that sars-cov- rbd region might have originated from recent recombination event in pangolins or that sars-cov- and sars-rcovs of pangolins represent the result of coincidental evolution (lam et al., ; tang et al., ) . overall, it remains to be solved whether also sars-cov- needed an intermediate (and amplification) host before being able to infect humans as it was the case for sars-cov and other hcovs. since a mammal reservoir has not yet been identified, a prudent use of specific antigens is strongly recommended for serological diagnosis of sars-cov- in animals as cross-reactions with viruses of the alphacoronavirus genus, widespread in animals, might occur (sun and meng, ) . analogously to bats, but with a lesser extent, also rodents have been recently demonstrated to play a significant role in the evolution of cov, in particular of those belonging to subgenus embecovirus of genus betacoronavirus. rodentia (rodents) is the largest order of mammals with more than species worldwide, representing a major source of zoonotic infectious diseases (han et al., ) . for decades, only one species of coronavirus, murine coronavirus (subgenus embecovirus, genus betacoronavirus), has been associated with rodents. the prototype virus, which was named mouse hepatitis virus (mhv), was first isolated in mice in (cheever et al., ) . a mhv variant was lately identified in rats in (parker et al., ) . rat coronavirus (rcov) causes epidemics of respiratory disease in laboratory rat colonies. the two prototype strains of rcov are sialodacryoadenitis virus (sdav) and parker's rcov (rcov-p) (bhatt et al., ; parker et al., ) . both strains infect the respiratory tract, and sdav can also infect the eye, salivary and lacrimal glands. young rats are especially susceptible to rcov with the infection occurring in the lower respiratory tract and developing into interstitial pneumonia (parker et al., ) . together with feline infectious peritonitis virus (fipv) and ibv, mhv has been one of the most strictly animal cov studied ever. mhv is a natural pathogen of mice, normally infecting the liver, gastrointestinal tract, and central nervous system, causing a wide range of disease, including hepatitis, gastroenteritis, and acute and chronic encephalomyelitis. importantly, it served as model for cov replication and pathogenesis, with emphasis for neuro-invasion and neurovirulence (weiss and navas-martin, ) . as for the additional structural protein he, some strains (such as jhm) of mhv contain the he protein, while others (such as a ) do not yokomori et al., ) . the role of rodents in the evolution of covs belonging to embecoviruses has been recently highlighted by means of the discovery of a novel betacoronavirus in norway rats (rattus norvegicus) in china. this virus forms a separate species named china rattus coronavirus hku (chrcov hku ) within the embecovirus subgenus. although designated as a novel species, this virus possessed genome characteristics that resemble to those of both betacoronavirus- and murine coronavirus, suggesting that chrcov hku represents the murine origin of betacoronavirus- , with interspecies transmission from rodents to other mammals having occurred centuries ago (lau et al., ) . genus betacoronavirus consists of five subgenera, with bat covs being including in all but one of subgenus embecovirus, where rodent, human and bovine covs are included (https://talk.ictvonline.org/ taxonomy/). this supports the hypothesis that rodent covs were the ancestors of embecoviruses of other animals, while bats are the natural reservoirs for all other betacoronaviruses. importantly, rodent covs are not restricted to genus betacoronavirus. a deep virological screening was performed in rodents sampled in zhejiang province, china, during - , with nearly % of rodents testing positive for cov . in particular, covs were detected in striped field mice (apodemus agrarius), norway rats, lesser ricefield rats (rattus losea), asian house rat (rattus tanezumi) and chinese white-bellied rat (niviventer confucianus). amplicons of the replicase gene sequences were recovered from ( %) of the cov rna positive rodent samples described above and whole genome or nearly whole genome sequences (> %) were recovered from and cov positive samples, respectively. by means of whole genome sequence analysis, authors were able to identify a divergent alphacoronavirus, which was lately officially designated as species lucheng rn rat coronavirus (lrnv) within the subgenus luchacovirus, and two novel betacoronaviruses termed longquan aa mouse coronavirus (lamv) and longquan rl rat coronavirus (lrlv) and assigned to the two established species betacoronavirus- and murine coronavirus, respectively . moreover, lrnv seems to be a recombinant virus as its n protein gene is more closely related to those of the genus betacoronavirus. overall, the discovery of rodent-associated covs belonging to subgenera that are distinct from those including bat covs warrants further investigations upon the role played by rodents in the evolution and emergence of these viruses. sars-cov replication has been studied in mice, syrian golden and chinese hamsters. the most severe symptoms of sars were observed in aged animals. indeed, aged mouse model of sars-cov has been generated (gretebeck and subbarao, ) . transgenic mice expressing human ace were also developed to closely mimic sars-cov infection in humans. some animal models have been tested and analysed on the genomic and proteomic level to study the pathogenesis of sars-cov. therefore, we have reason to believe that such models would work also for sars-cov- . quite the opposite, studies have demonstrated that mice, guinea pigs and hamsters are not susceptible to experimental mers-cov infection, mainly because their homologous dpp molecules table coronaviruses in domestic swine and associated diseases. do not function as receptors for mers-cov entry (cockrell et al., ) . the first mouse model of mers infection reported in involved transducing animals with recombinant adenovirus encoding human dpp (hdpp ) molecules intranasally, and this resulted in replication of mers-cov in the lungs. this mouse model also showed clinical symptoms of interstitial pneumonia, including inflammatory cell infiltration, and thickened alveolar and mild oedema (song et al., ) . currently, six covs are circulating in swine (table ). these include four alphacoronaviruses, transmissible gastroenteritis virus of swine (tgev) and its derivative porcine respiratory coronavirus (prcov) (subgenus tegacovirus), porcine epidemic diarrhoea virus (pedv) (subgenus pedacovirus) and sads-cov (subgenus rhinacovirus), one betacoronavirus, porcine haemagglutinating encephalomyelitis virus (phev) (subgenus embecovirus), and one deltacoronavirus, porcine deltacoronavirus (pdcov) (subgenus buldecovirus). tgev, pedv, sads-cov and pdcov are responsible for acute gastroenteritis in swine, with fatal infections in piglets born to seronegative sows, prcov causes a mild respiratory disease and phev is the causative agent of neurological and/or digestive disease in pigs (mora-díaz et al., ; wang et al., ). tgev was first described in uk in s, representing the oldest known swine cov. tgev and prcov are closely related to canine coronavirus (ccov) and feline coronavirus (fcov) forming with these carnivore covs a unique species, referred to as alphacoronavirus- . based on the analysis of the accessory protein gene orf , it has been postulated that tgev has originated from ccov type ii (ccov-ii), since while ccov type i (ccov-i) exhibits an intact gene, both ccov-ii and tgev, which are strictly related in the s gene, have only remnants of orf (lorusso et al., ) . prcov, in turn, has derived from tgev through the deletion of ≈ nucleotides at the ' end of the s gene (corresponding to ≈ amino acids at the n-terminus of the spike protein) and consequent change of the major tissue tropism from the enteric to the respiratory epithelium. this large deletion caused the loss of sialic acid binding activity that allows the attachment to mucins and mucin-type glycoproteins, so that tgev but not prcov is able overcome the intestinal mucus barrier, having access to the gut mucosa . prcov shares some epitopes for neutralising antibodies with tgev, so that its extensive circulation in swine herds has resulted in a drastic reduction of tge outbreaks worldwide. pedv was introduced in the pig population in the s, likely as a consequence of a spillover event from bats. the virus was first described in europe and had been primarily maintained as an endemic pathogen in european and asian swine populations until its introduction into north america in . pedv is more strictly related to a scotophilus bat coronavirus than to other known alphacoronaviruses, including tgev and human alphacoronaviruses hcov- e and hcov-nl . therefore, pedv and btcov/ / likely have a common evolutionary precursor and a cov cross-species transmission may have occurred between bats and pigs (banerjee et al., ) . accordingly, pedv contains signature motifs at the ′-untranslated region that are shared by bat covs, thus providing further support of the evolutionary origin of pedv from bats and potential cross-species transmission (huang et al., ) . currently, different pedv genotypes are described based on the s gene: i) g a pedv, including classical european and asian strains with moderate virulence; ii) g pedv, also called "original us pedv", comprising highly virulent strains that originated in asia and are now widespread in the usa; iii) g b pedv, which is represented by the so-called s-indel strains, i.e., strains presenting insertions and deletions in the s gene that are associated with mild clinical outbreaks. these strains are natural recombinant pedvs with a g -like genomic backbone carrying an s region of g a strains; iv) s n-terminal domain-deletion (ntd-del) strains that are g -like strains containing a to -aa deletion within the n-terminal domain of the s subunit, also associated to mild clinical forms (hou and wang, ) . recombinant strains between pedv and tgev have been also reported in europe (akimkin et al., ; belsham et al., ; boniotti et al., ) . sadv-cov, now referred to as swine enteric alphacoronavirus (seacov), is another virulent swine enteric alphacoronavirus that originated from bats, sharing an % sequence identity with a bat alphacoronavirus hku -cov. since viruses displaying a - % sequence identity to sads-cov were detected in rhinolophus spp. bats, sads-cov and hku -cov likely descend from a common ancestor . accordingly, both viruses now belong to the unique species rhinolophus bat coronavirus hku . in contrast, phev, which was first described in in nursery pigs with encephalomyelitis in ontario, canada, has not derived from bat covs, but its evolutionary history is tightly intermingled with other two closely related betacoronavirus, hcov-oc and the oldest known bcov, with which phev may have common ancestors (vijgen et al., ) and is included in the same viral species, betacoronavirus- (corman et al., ) . most probably, hcov-oc and phev descend from a rodent betacoronavirus through preliminary adaptation to bcov, from which they may have emerged in the context of a pandemic recorded historically at the end of the th century (corman et al., ) . pdcov was recently detected in in hong kong during cov molecular surveillance in avian and mammalian species. this swine deltacoronavirus seems to recognise another different ancestor, likely emerging from a host-switching event between avian and mammal covs. the most closely related pdcov relative has been identified in quail deltacoronavrus uae-hku and the virus has been proposed to be a recombinant between other two avian deltacoronaviruses, sparrow cov hku and bulbul cov hku . all these deltacoronavirus are now members of the same species coronavirus hku (lau et al., ) . pigs were found to be susceptible to experimental infection with the betacoronavirus mers-cov (vergara-alert et al., ), while sars-cov rna was detected in pigs and wild boars wang et al., ) . in contrast, a recent experimental infection demonstrated that pigs are not susceptible to sars-cov- . few studies have been carried out to assess the circulation of covs in farmed or free-ranging wild boars (sus scrofa). antibodies against tgev/prcov were detected in some animals in slovenia (vengust et al., ) and croatia (roic et al., ) and pedv rna was demonstrated in south korea (lee et al., ) . a wild boar sold at a live animal market of guangzhou, china, was positive for sars-cov rna . the main covs infecting ruminants are reported in table . the oldest known ruminant cov is bcov, which is also the prototype of the species betacoronavirus- (subgenus embecovirus, genus betacoronavirus). this virus is able to cause a variety of clinical forms, including enteric disease with high mortality rates in neonate calves, winter disease (a severe enteric form) in lactating cows (decaro et al., b) , and a respiratory disease, also known as shipping fever, in cattle of all ages, with a higher prevalence in - month-old calves (decaro et al., a) . it was postulated that the presence of genetic signatures differentiates enteric and respiratory bcovs (hasoksuz et al., ) , but it was ultimately evident that the same virus strain could be responsible for simultaneous appearance of enteric and respiratory disease in the same animals (chouljenko et al., ) . it has been postulated that bcov originated from a rodent cov (corman et al., ) . very recently, a novel cov, representing a new viral species, referred to as china rattus coronavirus hku (chrcov-hku ), was detected in norway rats in china. this virus was phylogenetically distinct from mhv and hcov-hku and displayed genome features that were intermediate between bcov and mhv. therefore, chrcov hku may represent the murine origin of bcov and rodents are likely an important reservoir for ancestors of subgenus embecovirus (lau et al., ) . bcov is paradigmatic of how covs are able to cross the interspecies barriers, establishing its derivatives as separate viral lineages affecting the respiratory and/or enteric tract of humans (hcov-oc ), swine (phev), horses (equine coronavirus, ecov), and dogs (canine respiratory coronavirus, crcov). a number of bcov-related viruses, all currently included in the unique species betacoronavirus- , have been detected in the enteric and/or respiratory tract of domestic and wild ruminants. these bcov-like covs include viruses of domestic and domesticated ruminants that were reported in sheep and goats (reinhardt et al., ; yang et al., ) , water buffalo (bubalus bubalis) (decaro et al., c) , llamas (lama lama) and alpacas (vicugna pacos) (cebra et al., ; jin et al., ) . in the wild, bcov-like covs were demonstrated in six species of the cervidae family, which are caribou/ reindeer (rangifer tarandus caribou), elk/wapiti (cervus elephus), samber deer (cervus unicolor), white-tailed deer (odocoileus virginianus), sika deer (cervus nippon yesoensis) and water deer (hydropotes inermis) (amer, ) . similar viruses were also found to circulate in the giraffe (giraffa camelopardalis) (hasoksuz et al., ) , several species of antelopes (alekseev et al., ; chung et al., ) , wisent (bison bonasus), himalayan tahr (hemitragus jemlahicus) (chung et al., ) , and dromedary camels (camelus dromedarius) (woo et al., ) . the last strain, detected in the united arab emirates and consequently named dromedary camel coronavirus uae-hku- (dccov uae-hku ), was slightly divergent from other bcov-like viruses (woo et al., ) . dromedary camels are susceptible to mers-cov infection, developing asymptomatic infections or mild upper respiratory disease, so that they are considered the natural host of mers-cov, with adult animals in many countries in the middle east as well as in north and east africa showing > % seroprevalence to the virus (hemida et al., b) . although human-to-human transmission has occurred outside middle east due to travel-associated patients with mers and has caused large clusters of human cases within healthcare facilities in saudi arabia, jordan and united arab emirates, it remains inefficient and sustained community transmission has not being documented so far, thus suggesting multiple virus introduction into the human population by infected dromedaries (hemida et al., b) . more recently, a phylogenetic study of mers-cov full-genome sequences revealed recombination signatures that defined five major phylogenetically stable lineages, all of which contained human and camel mers-cov sequences (sabir et al., ) . in the same study, an alphacoronavirus strictly related to hcov- e was found in the respiratory tract of dromedary camels of saudi arabia (sabir et al., ) . although some studies ruled out the susceptibility of other domestic ruminants to mers-cov (reusken et al., ; adney et al., ) , a recent study detected specific antibodies and rna in sera and nasal secretions, respectively, of domestic ruminants raised in africa, including sheep, goats and cattle (kandeil et al., ) . llamas were found to be susceptible to experimental infections with mers-cov (vergara-alert et al., ). the only cov that has been so far known in horses is ecov, which is a bcov-descendant betacoronavirus (subgenus embecovirus). ecov was first isolated from the faeces of a diarrhoeic foal in (ecov-nc ) in north carolina, usa (guy et al., ) , and was initially believed to only affect foals. since , the virus has been recognised in japan, europe and the usa as a new, clinically important, enteric virus of adult horses (pusterla et al., ) . despite mers-cov was successfully adapted to the in-vitro growth in equine cell lines (meyer et al., ) , serological and molecular table coronaviruses in domestic and domesticated ruminants and associated diseases. sabir et al. ( ) n. decaro and a. lorusso veterinary microbiology ( ) investigations have demonstrated that horses are not naturally infected by mers-cov (meyer et al., ; hemida et al., a) , nor they are susceptible to experimental infection (adney et al., ; vergara-alert et al., ) . however, surprisingly, mers-cov rna was detected in respiratory specimens of three donkeys of from egypt (kandeil et al., ) , a finding that requires further confirmation. a molecular survey aimed to assess cov circulation in horses in saudi arabia and oman has detected two dccov uae-hku strains in enteric samples of horses (hemida et al., a) . scarce data are available about cov circulation in donkeys. these equids are susceptible to ecov infection since positive rt-pcr results were obtained from a donkey in ireland (nemoto et al., ) . in addition, three donkeys ( . %) of from egypt tested positive for mers-cov rna in their nasal secretions (kandeil et al., ) . covs of carnivores are listed in table . three covs are known in dogs, i.e., two alphacoronaviruses of the subgenus tegacovirus, namely ccov-i and ccov-ii, and one betacoronavirus of the subgenus embecovirus, namely crcov. ccovs (species alphacoronav-irus- ) are commonly responsible for mild, self-limiting enteritis in pups . although they are neglected viruses and vaccination is not recommended due to the absence of an effective challenge model, two independent studies have demonstrated their significant involvement in the onset of acute canine enteritis (duijvestijn et al., ; dowgier et al., ) . the evolutionary history of ccovs is tightly intermingled with that of tgev and fcovs. ccov-i possesses a divergent spike protein and the intact form of an additional gene, orf , whose remnants are present in ccov-ii and, at a lesser extent, in tgev. therefore, ccov-ii has likely emerged as a consequence of recombination between the original ccov-i and an unknown cov in the s gene and of progressive loss of orf (lorusso et al., ) . a further recombination occurred in the very ' end of the s gene between ccov-ii and tgev, giving rise to back recombinant ccov-ii strains, also known as tgevlike ccovs, having a spike protein n-terminus of tgev in a ccov-ii backbone (decaro et al., , . consequently, the ccov taxonomy was revised, with classical and tgev-like strains being referred to as ccov-iia and ccov-iib, respectively. while ccovs are usually involved in mild forms of diarrhoea, there are some hypervirulent strains that are associated to severe, haemorrhagic, sometimes fatal gastroenteritis. in addition, ccov-iia strains, designated pantropic ccov, that are able to spread systemically and cause severe disease and the death of infected dogs have been reported in italy (buonavoglia et al., ; alfano et al., ) , other european countries (decaro et al., ) and south america (pinto et al., ) . genomic sequences from pantropic ccovs were analysed, but no obvious genetic signatures that may have caused the switch in pathogenicity were found (decaro and buonavoglia, ; decaro et al., ) . different from ccov-i and ccov-ii, the betacoronavirus crcov is associated with mild respiratory signs and has been proposed as an etiological agent of canine infectious respiratory disease (cird) together with other viral and bacterial agents . the virus was first detected firstly in uk in (erles et al., ) and subsequently in other european and extra-european countries (decaro et al., (decaro et al., , mitchell et al., ; maboni et al., ; piewbang et al., ; more et al., ) . being a bcov derivative, crcov possesses the same genomic organisation, with some differences in accessory orfs located between the s and e protein genes. in particular, while some crcovs possess a unique . kda protein gene directly downstream of the s protein gene, other canine bcov-like covs display the canonical set of bcov accessory genes but with truncated forms of the . kda protein gene . in cats, two alphacornavirus- genotypes are known, namely fcov type i (fcov-i) and fcov type ii (fcov-ii), the latter being generated as table coronaviruses in domestic and domesticated carnivores and associated diseases. jakob, jacob ( , pedersen et al. ( ) cat ( erles et al. ( ) n. decaro and a. lorusso veterinary microbiology ( ) a consequence of recombination events between ccov-ii and fcov-i that generated viruses with a ccov-ii genomic region, encompassing orf b, orf (s gene), orf abc, orf (e gene), and partial orf (m gene), in the context of an fcov-i backbone (pedersen, ) . both genotypes are involved in the development of feline infectious peritonitis (fip), a perivascular pyogranulomatosis of cats that may occur in two clinical forms, effusive and non-effusive fip, which are characterised by prevalence of effusions in the body cavities and of pyogranulomatous lesion in organs, respectively. fip occurs as a consequence of a change in tissue tropism of an enteric fcov strain (feline enteric coronavirus, fecv), infecting enterocytes of the intestinal villi, that acquires the ability to infect monocytes/macrophages switching to the more virulent fipv, which is responsible for systemic infections and dysregulation of the proinflammatory cytokines (addie et al., ) . the changes responsible for the pathogenetic shift have been investigated for many decades, being suggested to be variably represented by point mutations located in the s gene (rottier et al., ) , deletion/insertion in the group-specific genes c (vennema et al., ; chang et al., ) , b (vennema et al., ) or a (kennedy et al., a) . however, none of these differences appeared to consistently correlate with disease phenotype. more recent studies have identified specific genetic signatures in the s gene of fcov-i that are implicated in monocyte/macrophage tropism. two amino acid substitutions, m l and/or s a, corresponding to nucleotide mutations a t/c and t g, respectively, in the viral genome, together distinguished fcovs found in the tissues of fip cats from those found in the faeces of healthy cats without fip in > % of cases (chang et al., ) . however, subsequent studies concluded that these mutations are likely to be markers of systemic fcov infection rather than fip per se (porter et al., ; barker et al., ) . two alphacoronaviruses, both belonging to subgenus minacovirus, are currently known in mustelids, namely mink coronavirus (mcov- ) and ferret coronavirus (frcov). mcov- has been recently identified as the etiological agent of mink epizootic catarrhal gastroenteritis (ecg), an infectious disease of farmed american (neovison vison) and european (mustela lutreola) mink first described in (larsen and gorham, ) and later affecting several million mink in different countries (vlasova et al., ) . the disease is observed at greater frequency in mink of ≥ months and is characterised by seasonality, high morbidity (approaching %) and low mortality (< %). recent full-genome analysis demonstrated that mcov- is phylogenetically distant from ccovs and fcovs, being closely related to frcov (vlasova et al., ) . presently, the two viruses are considered separate species within subgenus minacovirus (https://talk.ictvonline.org/taxonomy/). frcov has been recognised as the causative agent of epizootic catarrhal enteritis (ece), first described in in domestic ferrets (mustela putorius furo) in the eastern part of the usa (williams et al., ) and subsequently reported in domestic and laboratory ferrets throughout the world (murray et al., ) . analogous to fcov, frcov exists in two different pathotypes: i) ferret enteric coronavirus (frecv) is associated to ece, a highly contagious diarrhoeal disease also known as green slime disease, which affects mainly young ferrets with morbidity and mortality rates similar to those of ecg; ii) ferret systemic coronavirus (frscv) is responsible for a systemic diseases of ferrets, which is characterised by pyogranulomatous perivasculitis and peritonitis resembling to those of fip (murray et al., ) . similar to fip, wise et al. ( ) have shown that frecv and frscv differ significantly in spike protein and that deletions in frcov c may also correlate with the severe pathotype of frscv. recombination in the s, c and e genes between different frcov has been also reported (lamers et al., ) . different covs were found to circulate in wild carnivores. ccovs were detected in wolves (canis lupus), red foxes (vulpes vulpes), eurasian otters (lutra lutra), common genets (genetta genetta) (alfano et al., ; rosa et al., ) . ccov-like viruses were also found in african wild carnivores, including spotted hyenas (crocuta crocuta) and silver-backed jackals (canis mesomelas) (goller et al., ) . fcovs have a wide circulation in non-domestic felids (kennedy et al., (kennedy et al., , , with fip cases being reported in servals (felis serval) (juan-salles et al., ) , cheetah (acinonyx jubatus) (kennedy et al., b) , mountain lion (puma concolor) (stephenson et al., ) , and european wildcat (felis silvestris) (watt et al., ) . divergent alphacoronavirus- viruses were detected in chinese ferret badger (nyctereutes procyonoides) and raccoon dog (melogale moschata) (dong et al., ) . the same study reported the identification in asian leopard cat (prionailurus bengalensis) and chinese ferret badger of an unclassified cov, which was closely related to gammacoronaviruses in most parts of the genome, whereas the s gene displayed the highest sequence identity to alphacoronaviruses (dong et al., ) . with the discovery of deltacoronaviruses, these viruses were later included in this novel genus along with avian and porcine strains (woo et al., ; wang et al., ) . some domestic and wild carnivores are also susceptible to sars-cov infection. while the potential natural reservoirs are horseshoe bats, sars-like cov strains were found to be widespread in masked palm civets (paguma larvata) and raccoon dogs, which were suspected to be intermediate hosts (guan et al., ) . full-genomic comparative analysis has shown that sars-like covs isolated from palm civets are under strong selective pressure and are genetically most closely related to sars-cov strains infecting humans early in the outbreaks (song et al., ) . sequence analysis of the sars-cov-like virus in masked palm civets indicated that they were highly homologous to human sars-cov with nucleotide identity over . %, indicating the virus has not been circulating in the population of masked palm civets for a very long time (shi and hu, ) . a chinese ferret-badger (melogale moschata) was found to have neutralising antibodies against sars-cov (guan et al., ) , whereas sars-cov rna was detected in naturally infected cats and red foxes (vulpes vulpes), but not in domestic dogs . there was, however, a single dog testing positive for sars-cov (https://apps.who.int/iris/bitstream/handle/ / /who_cds_csr_gar_ . _eng.pdf). among carnivores, sars-cov- is able to infect cats, ferrets and, at a lesser extent, dogs . in , a highly divergent cov, tentatively named sw , was discovered a deceased beluga wale (delphinapterus leucas) with pneumonia and hepatic necrosis (mihindukulasuriya et al., ) . the virus was only distantly related to ibv, so that it now represents the prototype of the single mammalian cov species belonging to the genus gammacoronavirus, namely beluga wale coronavirus sw (bwcov-sw ) (subgenus cegacovirus). few years later, related gammacoronaviruses were retrieved from faecal samples of three indo-pacific bottlenose dolphins (tursiops aduncus), which were named bottlenose dolphin cov (bdcov) hku . comparative genome analysis showed that bdcov-hku and bwcov-sw have similar genome characteristics and structures, displaying a % nucleotide sequence identity each to other (woo et al., ) . a novel betacoronavirus distantly related to mers-cov was detected in the faeces of european hedgehogs (erinaceus europaeus), an insectivorous mammal belonging to a related order of chiroptera, from germany. the virus was tentatively referred to as erinaceus cov (ericov) (corman et al., b) and covs found in hedgehogs in france, england and italy had an identity from % to % with the ericov (monchatre- leroy et al., ; saldanha et al., ; delogu et al., ) . these hedgehog covs are are now included in a unique species, hedgehog coronavirus (subgenus merbecovirus). the virus was not associated to any form of disease, so that western european hedgehog is a reservoir host of ericov in the absence of apparent disease, suggesting that hedgehogs in addition to bats may contribute to the evolution of merbecovirus (saldanha et al., ) . a slightly divergent merbecovirus was later found in amur hedgehogs (erinaceus amurensis) in china and was poposed as a prototype of a separate species, namely erinaceus amurensis hedgehog coronavirus hku (ea-hedcov hku ) . a novel coronavirus, named wénchéng shrew coronavirus (wesv) was detected in shrews (suncus murinus) in china . wesv is highly divergent from other alphacoronaviruses, exhibiting less than . % amino acid similarity to any known members of the genus alphacoronavirus in the coronavirus-wide conserved domains of the replicase polyprotein pp ab and less than . % amino acid similarity to the other three coronavirus genera. however, taking into account the current ictv criteria, wesv is sufficiently divergent to be considered a distinct member of the genus alphacoronavirus, but not a new genus of the subfamily orthocornavirinae . covs have been known in veterinary medicine since many decades; some of these viruses, such as ibv, swine enteric covs, bcov and mustelid covs, can cause diseases that have a great impact on the farm industry. other covs, namely fipv, frscv and mhv, cause severe disease in companion (cats, ferrets) or laboratory (mice) animals. animal covs are paradigmatic on how covs evolve through accumulation of point mutations and homologous (and heterologous) recombination, generating different genotypes and pathotypes. these virus variants may have different antigenic properties, escaping the host immunity induced by vaccines, as is the case of ibv. alternatively, they may have a different tissue tropism in the same host that can increase or decrease the virus pathogenicity, as observed for the virus pairs fecv/fipv or frecv/frscv and tgev/prcov, respectively. in other circumstances, the cov evolution may result in the switch of the host range from one animal species to another one or from animals to humans. the former event is well documented in veterinary medicine, with a plethora of viruses being originated from ibv and bcov that adapted to different animal species. however, the most interesting scenario is the jumping and further adaptation of an animal cov to humans. there is increasing evidence that all hcovs currently known recognise an animal origin, with bat or rodent covs being the most probable ancestors. in most instances, it was suggested that other mammals served as intermediate hosts prior to final adaptation to humans, i.e., alpacas and cattle for the low-pathogenic hcov- e and hcov-oc , respectively, and wild carnivores and dromedary camels for the high-pathogenic sars-cov and mers-cov, respectively. other two hcovs, namely hcov-nl and hcov-hku , were likely derived from bats and rodents, respectively, but whether this transmission required an intermediate mammalian host is presently unknown. the origin of sars-cov- should be zoonotic, since highly related sequences were detected in bats, but a definitive intermediate host has been not identified so far. what should we expect from the current pandemic? when hcov-oc crossed the species barrier to infect humans from domestic livestock around , an epidemic of respiratory infection was recorded. even though, several years later, influenza was suspected to be the cause of it, in that pandemic involvement of central nervous system was more pronounced than in other influenza outbreaks. this evidence is further supported by molecular studies claiming that the most recent common ancestor of bcov and hcov-oc emerged around (vijgen et al., ) and by the fact that hcov-oc can be neuroinvasive (arbour et al., ) . likely, hcov-oc crossed species to infect dogs becoming established in this species as crcov . a similar scenario could be observed with sars-cov- with dogs and, at a greater extent, cats. apparently, cats represent, within the domestic animals which have been experimentally infected, the host, together with ferrets, which is able to sustain more efficiently sars-cov- replication . furthermore, based on structural studies and biochemical experiments, sars-cov- seems to have an rbd that binds with high affinity to ace also from ferrets and cats (andersen et al., ) . reasonably, a full comprehension of the animal cov molecular evolution, host range and pathobiology is beneficial to better understand the mechanism driving the emergence and adaptation to humans of zoonotic covs. the present review has highlighted that in the last years, also thanks to the availability of novel sequencing technologies, we have witnessed a large number of novel covs being discovered in a large number of animals. truth to be told, it was difficult for us to summarise, in this single review, all covs detected in animals and the tight interaction existing between them and human covs. among animals, it is evident that bats are the group of mammals that harbor the largest number of covs and that many other animal covs recognise their ancestors in bat covs. in an excellent review (cui et al., ) written by the group coordinated by dr. zheng-li shi of the wuhan institute of virology, hubei, (china), city infamously known for being the epicenter and origin of the covid- outbreak, authors stated that "...given the prevalence and great genetic diversity of bat sars-rcovs, their close coexistence and the frequent recombination of covs, it is expected that novel variants will emerge in the future". this forecasting statement was not surprising to coronavirologists and it was not, importantly, surprising to those scientists that daily deal with the plethora of viruses existing at the human/animal health interface. although scientists were well aware of this hazard, no substantial actions were taken forward the limitations of strict and repeated contacts between humans and wildlife. indeed, whereas biological mechanisms underlying viral evolution are not under human control, social and cultural habits can be modified accordingly through a deep and pounding informative campaigns. if to the human habits we sum the impact of modern agricultural practices and urbanization and the decrease of vital space for wildlife, it is quite easy to understand that, if countermeasures are not taken, we will face novel serious health emergencies of animal origin in the following years with tremendous social and economic impact on our lives. as clearly demonstrated by the sars-cov- emergence, covs are the main characters of this intricate puzzle characterised by the interactions of viral biological mechanisms and human habits. our review was reasonably prepared also to highlight (once more!) how covs originate, evolve, jump, mutate and infect their host. could have the current covid- outbreak been avoided? answering this question is not relevant now, but actions to avoid the next viral spillover from animals to humans is certainly a priority. this task needs to be coupled with massive genomic surveillance in wild animals not limited to covs. massive sequencing of sars-cov- strains detected in humans and covs of wildlife will help further assess the origin of this novel human pandemic and plan future measures able to reduce the risk of emergence of new cov spillover events. however, additional tasks should be provisionally addressed in order to reduce the risk of future cov pandemic like the current one. these include: i) prevention of animal-to-human infections through a ban of the wet markets and a more 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transmission of mers coronavirus biosynthesis, structure, and biological activities of envelope protein gp of murine coronavirus intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans structure of coronavirus hemagglutinin-esterase offers insight in corona and influenza virus evolution isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic analysis and surveillance of the coronavirus dominant in ducks in china funding were provided by the italian ministry of health, izs am / rc , ricerca corrente "ngs e diagnostica molecolare in sanità animale: fast d ", recipient alessio lorusso. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . key: cord- -pztrwvib authors: choi, won suk; kang, cheol-in; kim, yonjae; choi, jae-phil; joh, joon sung; shin, hyoung-shik; kim, gayeon; peck, kyong ran; chung, doo ryeon; kim, hye ok; song, sook hee; kim, yang ree; sohn, kyung mok; jung, younghee; bang, ji hwan; kim, nam joong; lee, kkot sil; jeong, hye won; rhee, ji-young; kim, eu suk; woo, heungjeong; oh, won sup; huh, kyungmin; lee, young hyun; song, joon young; lee, jacob; lee, chang-seop; kim, baek-nam; choi, young hwa; jeong, su jin; lee, jin-soo; yoon, ji hyun; wi, yu mi; joung, mi kyong; park, seong yeon; lee, sun hee; jung, sook-in; kim, shin-woo; lee, jae hoon; lee, hyuck; ki, hyun kyun; kim, yeon-sook title: clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea date: - - journal: infect chemother doi: . /ic. . . . sha: doc_id: cord_uid: pztrwvib background: from may to july , the republic of korea experienced the largest outbreak of middle east respiratory syndrome (mers) outside the arabian peninsula. a total of patients, including deaths, had been diagnosed with mers-coronavirus (mers-cov) infection as of september th, . materials and methods: we obtained information of patients who were confirmed to have mers-cov infection. mers-cov infection was diagnosed using real-time reverse-transcriptase polymerase chain reaction assay. results: the median age of the patients was years (range, to ). a total of . % of the patients had one or more coexisting medical conditions. the most common symptom was fever ( . %). at admission, leukopenia ( . %), thrombocytopenia ( . %), and elevation of aspartate aminotransferase ( . %) were observed. pneumonia was detected in . % of patients at admission and developed in . % during the disease course. antiviral agents were used for . % of patients. mechanical ventilation, extracorporeal membrane oxygenation, and convalescent serum were employed for . %, . %, and . % of patients, respectively. older age, presence of coexisting medical conditions including diabetes or chronic lung disease, presence of dyspnea, hypotension, and leukocytosis at admission, and the use of mechanical ventilation were revealed to be independent predictors of death. conclusion: the clinical features of mers-cov infection in the republic of korea were similar to those of previous outbreaks in the middle east. however, the overall mortality rate ( . %) was lower than that in previous reports. enhanced surveillance and active management of patients during the outbreak may have resulted in improved outcomes. middle east respiratory syndrome (mers), which is caused by β-coronavirus of the c lineage (mers-cov), ranges in severity from asymptomatic to a severe respiratory illness with rapid progression to respiratory failure [ ] . since the identification of the first case in saudi arabia, the world health organization (who) has been notified of , laboratory-confirmed cases, including fatal cases, in countries as of february th, . these confirmed cases have been directly or indirectly linked to countries in the arabian peninsula [ ] . although the exact mode of transmission remains unknown, primary mers-cov infections are presumably associated with community zoonotic exposure and may be passed to family members via limited secondary transmission [ ] . human-to-human spread of mers-cov is assumed to occur through close contact, such as caring for or living with an infected person [ , ] . as infected people have spread mers-cov to others in hospitals, the importance of vigilant surveillance and appropriate infection control measures must be emphasised to prevent transmission in healthcare settings. from may to july , the republic of korea experienced the largest outbreak of mers outside the arabian peninsula. infection was confirmed in a -year-old businessman returning from the middle east on may th, , when he presented with atypical pneumonia and he failed to response to anti-microbial therapy for community-acquired pneumonia. prior to the confirmation of mers-cov infection, he had visited three hospitals where more than people (including other patients, care givers, and healthcare workers) were exposed, resulting in cases of mers-cov infection [ ] . as of december th, , , individuals had been quarantined and cases of mers-cov had been confirmed. all confirmed cases were associated with a total of healthcare facilities, and approximately % of the confirmed cases were caused by 'super-spreading' events that originated from five patients in four hospitals [ ] . we aimed to determine the clinical features and outcomes of korean patients confirmed to be infected with mers-cov, and the risk factors contributing to mortality. this retrospective observational study focused on the clinical characteristics of confirmed cases of mers-cov infection in the republic of korea. approval for this study and a waiver for requiring informed consent were obtained from institutional review board of chungnam national university hospital. all patients with laboratory-confirmed mers-cov infection were identified during the outbreak in the republic of korea, during which confirmatory tests were performed only for suspected cases. suspected cases were defined as follows: ) fever and pneumonia or acute respiratory distress syndrome (based on clinical or radiologic evidence) and either a history of travel from countries in or near the arabian peninsula within days before symptom onset or close contact with a symptomatic traveller who developed fever and acute respiratory illness (not necessarily pneumonia) within days after traveling from countries in the arabian peninsula. ) fever and symptoms of respiratory illness (e.g., cough, shortness of breath) and presence in a healthcare facility (as a patient, worker, or visitor) within days before symptom onset in a country in or near the arabian peninsula. ) fever or symptoms of respiratory illness (e.g. cough, shortness of breath) and close contact with a confirmed symptomatic mers case [ ] . since june th, , symptomatic people who visited a healthcare facility within days before the onset of symp-toms in two or more confirmed healthcare-associated mers cases were also included as suspected cases. one of the hospitals that treated patients with laboratory-confirmed mers-cov infection also performed active surveillance of asymptomatic health care workers who were involved in the care of confirmed cases. in the republic of korea, real-time reverse-transcriptase polymerase chain reaction (rrt-pcr) assays for mers-cov diagnosis were performed exclusively at the korea centre for disease control and prevention until may th, . medical centres and referral laboratories began to perform rrt-pcr assays for mers-cov on may th and june rd, , respectively. these rrt-pcr tests targeted the upstream e protein (up-e) and open reading frame a (orf a) gene segments of mers-cov, as described in the who guideline [ ] . patient information was collected using a standardized case-report form. gathering of data was carried out by the physicians who cared for the mers-cov-infected patients of each hospital. a major means of obtaining data was review of medical records, including doctors' and nurses' charts, imaging findings, and laboratory results. in those cases where additional information was necessary, re-interview of patients was performed. collected data were categorized with respect to demographics, clinical symptoms, comorbidities, laboratory test results, image findings, treatments performed, and clinical outcomes. we carried out descriptive analysis for the aforementioned categories. continuous variables were reported as means with standard deviations or medians with ranges. for categorical variables, the proportion of patients was calculated for each variable. comparison analysis for subgroups was performed using student's t-test, pearson's chi-square test, or fisher's exact test, and cox-regression analysis was also performed to evaluate risk factors of mortality. for cox-regression analysis, the data was censored at september , for survivors. a p-value < . was considered to indicate statistical significance. all analysis was performed with spss software (spss version . , spss inc., chicago, il, usa) for windows. we described the clinical characteristics of all patients with confirmed mers-cov infection during the outbreak in the republic of korea (table ) . we also compared the clinical characteristics of survivors and deceased at days after symptom onset. the median age of the patients was years (range, to ); . % of the patients were years of age or older (table ) . male patients predominated in number over female patients, with a sex ratio of approximately : . a total of . % of the patients had one or more coexisting medical conditions. hypertension, diabetes, and solid organ malignancy were the most common coexisting medical conditions. thirty-nine patients ( . %) were health care workers (hcws), of which nurses were most common ( . %), and two cases ( . %) were asymptomatic. compared with survivors, the deceased were older and more frequently had coexisting medical conditions. one hundred fifty-two patients ( . %) manifested fever and more than % had developed cough at admission. almost all of the patients ( . %) developed fever during the course of disease. unstable vital signs were observed in approximately a quarter of the patients at admission. compared with survivors, the deceased more frequently presented with dyspnea, hypotension, and tachypnea at admission. at admission, ( . %) of the patients underwent one or more blood tests (table ) including complete blood counts in ( . %), c-reactive protein (crp) analyses in ( . %), and liver function and blood chemistry tests in ( . %). among those, . % had leukopenia (< , cells/mm ), . % had leukocytosis (> , /mm ), . % had anemia (< g/dl), and . % had thrombocytopenia (< , /mm ). crp was elevated (> mg/dl) in . % of patients. elevated level of aspartate aminotransferase (ast) was observed in . %, whereas elevated creatinine was observed in only . % of patients. among the patients who underwent urinalysis, ( . %) had proteinuria and ( . %) had hematuria. forty-four patients underwent arterial blood gas analysis, which revealed a decreased pao :fio ratio (< ) in . % of patients. compared with survivors, the deceased more frequently exhibited leucocytosis, hypoalbuminaemia, elevated serum creatinine and crp levels, and hypoxemia (pao :fio ratio < ). abnormalities in chest radiography were detected in of patients ( . %) who underwent chest radiography at admission and ( . %) of the patients manifested pneumonia during the course of disease. the most common features of the abnormalities were ground glass opacity or consolidation. the abnormal findings appeared in peripheral ( . %), focal ( . %), and unilateral ( . %) patterns. multifocal or bilateral location was relatively rare ( . % and . %, respectively). the abnormalities had disappeared in only onethird of patients by the time of discharge. antiviral therapy was administered to patients ( . %; table ). the median time from the onset of illness to the initiation of antiviral therapy was days (range, - days), although . % of patients received antiviral therapy within hours of symptom onset. the most commonly prescribed antiviral regimen was a combination of interferon (ifn), ribavirin, and lopinavir/ritonavir. one hundred thirty-eight patients ( . %) received antibiotic therapy. in addition, patients ( . %) were treated with mechanical ventilation, ( . %) with haemodialysis, and ( . %) with extracorporeal membrane oxygenation (ecmo). seven ( . %) patients were treated with convalescent serum. acute respiratory distress syndrome, acute kidney injury, and shock occurred in . %, . %, and . % of patients, respectively. among the patients, a total of ( . %) died in hospitals. from the day of symptom onset, seven patients ( . %) died within days and ( . %) died within days. the median interval from symptom onset to death was days (range, - days). two patients ( . %) died before confirmation of mers. among cured patients, the median interval from symptom onset to discharge was days (range, - days), the median duration of fever was days (range, - days), and the median time to a negative conversion of virus as determined via rrt-pcr analysis of sputum was days (range, - days). in univariate analysis, the deceased were older than the survivors (median age, vs. years), and included a smaller portion of hcws than did the survivors ( . vs. . %). the deceased had fever, dyspnea, or decreased consciousness more frequently at admission. they had coexisting medical conditions more frequently, especially diabetes, chronic heart disease, chronic lung disease, and solid organ malignancy. hypotension and tachypnea were observed more frequently in the deceased. laboratory abnormalities, namely leukocytosis, thrombocytopenia, hypoalbuminemia, elevated serum creatinine, and elevated crp, were reported more frequently in the deceased at admission and during the course of disease. however, leukopenia was reported more frequently in survivors. in multivariate cox-regression analysis, age ≥ years, occurrence of dyspnea during the disease course, presence of coexisting medical conditions including diabetes or chronic lung disease, systolic blood pressure < mm hg at admission, leukocytosis at admission, and the use of mechanical ventilation were found to be independent predictors of death (table ). the use of mechanical ventilation was assumed to be an independent indicator of severe disease. among the patients with mechanical ventilation, ( . %) died. the deceased among patients with mechanical ventilation were older than the survivors, and the proportion of hcws was smaller than among the survivors (table ) . they also had underlying diseases more frequently, especially chronic lung disease and solid organ malignancy. elevation of serum creatinine was observed more frequently in the deceased. in contrast, the occurrence of diarrhea and the elevation of alt were observed more frequently in survivors. ecmo or convalescent serum was employed more frequently among survivors. since its initial identification in saudi arabia in , , cases of laboratory-confirmed mers-cov infection have been reported as of february th, . in that time interval, another multi-facility outbreak in an endemic area has occurred since the first healthcare facility-related outbreak was reported by assiri, et al [ ] . here, we describe the clinical findings of mers patients in the republic of korea from may through september . to the best of our knowledge, this is the largest report of an outbreak outside of the middle east. in this study of the mers outbreak in the republic of korea, the reported data suggest that mers-cov is most likely to be transmitted as a healthcare-associated infection. no communi- ty-associated infections lacking any history of contact with mers-cov were found in this outbreak. even though the overall transmissibility of mers-cov was relatively low, there were several so-called 'super-spreading' events posing a serious threat to public health. rapid transmission and high attack rates in dialysis units were noted, in a previous report, in saudi arabia [ ] . however, no additional cases in dialysis units were reported in this korean outbreak, even though many patients undergoing hemodialysis were exposed to mers in dialysis units. this apparent heterogeneity in transmission, with many infected patients not transmitting disease at all and several patients transmitting disease to others, may be characteristic of mers-cov infections, similar to sars [ , , , ] . the clinical features of patients with mers-cov infection in the republic of korea were generally similar to those reported in saudi arabia [ , , ] . asymptomatic patients were rare in this outbreak, presumably because confirmatory mers-cov tests were performed only for symptomatic patients, although active surveillance of asymptomatic hcws involved in patient care was conducted at one hospital. in a previous outbreak in jeddah, . % of laboratory-confirmed cases were reported to have been asymptomatic; however, a telephone survey revealed that about % of those who were reached by telephone had experienced at least one symptom [ ] . the proportions of patients with underlying medical conditions such as diabetes ( . %), chronic lung disease ( . %), or chronic kidney disease ( . %) were smaller in this study, which might explain why cases with complicating respiratory or renal failure were relatively uncommon in the outbreak in the republic of korea, compared to those reported in saudi arabia. the incidence of pneumonia ( . %) in this outbreak is similar to that reported in saudi arabia, although the proportion of patients requiring mechanical ventilation was smaller ( . % in the republic of korea vs. - % in saudi arabia) [ , ] . in addition to the reduced presence of comorbidities, many patients in this study were diagnosed relatively early because rrt-pcr tests for mers-cov were actively performed for those with an epidemiological linkage as soon as fever or respiratory symptoms occurred. this early diagnosis might have led to early treatment initiation and thus prevented disease progression to a severe status. the case fatality rate observed in this study was lower than that reported in other studies of previous mers outbreaks ( . % vs. . - %) [ , , ] . this low case fatality rate could be attributed to the application of aggressive treatment measures, including antiviral agents, mechanical ventilation, or ecmo. antiviral treatments were administered to . % of the patients, although clinical decisions for their use were made by attending physicians. during the mers-cov epidemic in the republic of korea, the korean society of infectious diseases and the korean society for chemotherapy collaborated to generate and distribute recommendations for the use of antiviral treatments based on existing available data from experiences with sars and mers [ , ] . we were not able to assess the clinical impact of antiviral therapy on outcomes, as most patients with severe pneumonia received combination antiviral therapy along with comprehensive supportive care. the therapeutic efficacy of combination antiviral therapy should be evaluated in further studies. our study has several limitations. first, clinical data were retrospectively collected through electronic medical records and chart review, although cases were enrolled prospectively by means of active surveillance of outbreaks. the present study was observational, and thus unknown risk factors and bias might have been unequally distributed between the two groups in the survival analysis. the possibility of limitations that preclude accurate comparisons should be kept in mind given the observational nature of this study. clinical judgments regarding management of patients were made by attending physicians, not by researchers. second, we have presented months of clinical data after control of the outbreak, and thus the data might represent acute complications of mers-cov-infected patients. only . % of patients showed resolution of abnormal findings on chest radiologic evaluation, and there are concerns regarding chronic complications such as pulmonary fibrosis. further follow-up prospective cohort studies are warranted in the future. third, although this study was performed with a nationwide database, a relatively small number of patients with mechanical ventilation were included, with limited statistical power. the impact of ecmo and convalescent serum therapy on survival may not be fully adjusted for in the survival analysis. finally, this study was conducted mainly at large referral centers in korea, and our findings may not be generalizable to other institutions outside of korea. in conclusion, a mers-cov outbreak in the republic of korea was initiated by a patient recently returned from saudi arabia, and transmitted as a healthcare-associated infection. although clinical features did not differ significantly from those of previous outbreaks in the middle east, the overall mortality rate was . %, which was lower than that reported in other countries. the early detection of cases with mers-cov infection and active management of patients during this outbreak may have improved patient outcomes. our data sug-gest that mers-cov infections pose an important public health threat in regions outside the middle east, and that more information on the emergence and dissemination of this virus is necessary in order to prevent its further spread. isolation of a novel coronavirus from a man with pneumonia in saudi arabia spread, circulation, and evolution of the middle east respiratory syndrome coronavirus family cluster of middle east respiratory syndrome coronavirus infections mers-cov outbreak in jeddah--a link to health care facilities hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus outbreak in the republic of korea response guidelines for middle east respiratory syndrome (the rd revision) laboratory testing for middle east respiratory syndrome coronavirus -interim recommendations (revised) multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome rapid response team. antiviral treatment guidelines for middle east respiratory syndrome ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study this work was supported by a grant ( p ) from the research of korea centers for disease control and prevention (kcdc). we express the deepest thanks to physicians, field epidemiologists, and the officials of kcdc, who did not spare the dedicated efforts for the control of the mers outbreak in korea. http://orcid.org/ - - - jae-phil choi http://orcid.org/ - - - key: cord- -p a chn authors: kelly-cirino, cassandra; mazzola, laura t; chua, arlene; oxenford, christopher j; van kerkhove, maria d title: an updated roadmap for mers-cov research and product development: focus on diagnostics date: - - journal: bmj glob health doi: . /bmjgh- - sha: doc_id: cord_uid: p a chn diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. ► the middle east respiratory syndrome-coronavirus is a high-priority pathogen identified by the who r&d blueprint because of its high fatality rate, large geographical range of the dromedary camel reservoir and lack of medical interventions. ► accurate and accessible diagnostic tests are essential to outbreak containment and case management, as well as surveillance in both humans and animals, but available diagnostic tests are limited by inconsistent quality assessment, specimen acquisition issues and infrastructure requirements. ► diagnostic research and development (r&d) needs to include point-of-care testing options, syndromic panels for differential diagnosis, a greater understanding of viral and antibody kinetics, improved access to clinical specimens, and establishment of international reference standards. diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. the middle east respiratory syndrome-coronavirus (mers-cov) is an emerging virus associated with severe respiratory illness, first detected in in saudi arabia. provides an overview to the current status of mers-cov diagnostics, including feedback from subject matter expert and developer interviews on the common challenges with test development and implementation, and identifies gaps for further research and development (r&d). mers-cov is a zoonotic virus, and dromedary camels (camelus dromedarius) are the reservoir host and the source of zoonotic transmission to humans. [ ] [ ] [ ] dromedaries appear to be only mildly symptomatic following infection and present a significant reservoir risk for spillover events. mers-cov rna has been detected in dromedary camels in a number of countries, including egypt, oman, qatar and saudi arabia, with evidence suggesting that mers-cov is also widespread in the middle east, africa and south asia. - infection in camels is notifiable to the oie. individuals with close and frequent contact with dromedaries are at a higher risk for mers-cov infection than the general population. clinical indications and management coronaviruses are a family of viruses that can cause diseases in humans, ranging from the common cold to severe acute respiratory syndrome (sars). the clinical spectrum of mers ranges from no symptoms (or asymptomatic infection), mild symptoms including fever, cough, gastrointestinal illness and shortness of breath, to severe disease including pneumonia, acute respiratory distress syndrome and death. severe cases of mers can result in respiratory failure, requiring mechanical ventilation and support in intensive care. risk factors for severe disease include a weakened immune system, older age (> years), and comorbidities such as diabetes, cancer, renal disease and chronic lung disease. human-tohuman transmission spreads through close and unprotected human contact, and more than half of reported mers cases have occurred through nosocomial transmission. [ ] [ ] [ ] [ ] to prevent nosocomial infections, who and others recommend using standard infection and prevention control measures when caring for patients. [ ] [ ] [ ] who also recommends that contact tracing of all symptomatic and asymptomatic close contacts of the primary patient should be conducted routinely. the molecular epidemiology for mers-cov has not changed significantly since the initial human cases were detected in . the current virus remains % identical to the sequences seen in the first human cases from as well as archived camel sera from , with no increase in pathogenicity observed in the animal host. [ ] [ ] [ ] as genetic mutations could impact detection, bmj global health immunotherapy and vaccine development efforts, sequencing of mers-cov strains from camels and humans (after a zoonotic spillover) is important and is regularly being conducted in affected member states (who, personal communication, ). there are currently no prophylactic or therapeutic interventions of proven efficacy for any coronavirus infections. without a specific therapy for mers, treatment is supportive. effective mers therapeutics are still in the early stages of research and evaluation. several broad-spectrum antiviral agents including nitazoxanide, viral methyltransferase inhibition and nucleotide prodrugs have shown in vitro activity against mers-cov. early results for novel mers-specific therapeutics that inhibit viral replication or have specific neutralising activity are promising. the who r&d blueprint for mers has called for three types of vaccines: ( ) dromedary camel vaccine to prevent zoonotic transmission, ( ) human vaccine for long-term protection of persons at high exposure risk and ( ) human vaccine for reactive use in outbreak settings. mers-cov vaccines are in the early stages of development, with one candidate vaccine in phase i clinical trials (nct ). neutralising monoclonal antibodies have been designed to target the mers-cov spike protein, with chadox and modified vaccinia ankara vectors also strong vaccine candidates, but none have yet advanced to clinical trials. to accelerate the process, the coalition for epidemic preparedness innovation has recently launched a call for proposals for the development of a human mers-cov vaccine in order to engage with developers interested in supporting these efforts. the who laboratory guidelines recommend nucleic acid amplification tests (naat) for diagnosis, using serology for diagnosis only when naat is not available. in suspected patients, a single negative test result does not exclude diagnosis. repeat sequential sampling and testing is strongly recommended. the kinetics of mers-cov infection has been shown to vary widely across cases, - prompting a more detailed investigation of viral and antibody dynamics across the broad range of sample types, disease states and host factors. the best naat test sensitivity is achieved using specimens from the lower respiratory tract (sputum, tracheal aspirates or bronchoalveolar lavage), where mers-cov replication occurs at higher and more prolonged levels of mers-cov rna, typically between and copies/ml. mers-cov viral load is generally higher for severe cases, with more prolonged viral shedding than mild cases. viral load concentrations, which may be undetectable at early-stage infection, generally peak in the second week after symptom onset, and then drop to undetectable in survivors by the fourth week from onset. upper respiratory tract specimens (nasopharyngeal or oropharyngeal swabs) may also be used, but demonstrate ×- × lower viral load and can test negative for mild cases. if possible, both upper and lower respiratory tract sampling are advised. specimens outside the respiratory tract are not recommended for diagnosis, as they can test negative in both severe and mild presentation. viral rna has been detected in stool samples ( copies/ml), serum samples ( copies/ml) and urine ( copies/ml), more likely an indicator of severity as it typically precedes a poor clinical outcome. serological diagnosis can be made using paired samples, more often used for research rather than diagnostic purposes, preferably with the initial sample collected in the first week of illness and the second collected - weeks later. if only a single serum sample can be collected, this should occur at least - weeks after onset of symptoms for determination of a probable case. table presents an overview of the implementation requirements for mers-cov diagnostics (detailed commercial product information is presented in online supplementary tables s and s ). molecular diagnostics such as naat (eg, pcr) typically require sophisticated laboratory infrastructure including biosafety cabinets, while most serological tests (elisa, indirect immunofluorescence test (iift)) can be run on the benchtop in a more modest laboratory environment, depending on sample preparation precautions. point-of-care (poc) tests are designed to be used outside of a traditional laboratory; near-poc tests are defined for rapid use in a laboratory near the patient, but are more automated and easy to use than the traditional laboratory test. poc tests such as low-complexity rapid diagnostic tests (rdts) can be used at the bedside, typically with non-invasive samples after minimal training. inhouse tests are described in sections below; commercial sources are listed in online supplementary tables s and s . naats are currently the standard for mers-cov diagnosis, as these tests (typically reverse transcriptase pcr (rt-pcr)) have the highest sensitivity at the earliest time point during the acute phase of infection. following the who guidelines, two different targets on the mers-cov need to be detected by rt-pcr to confirm a case. mers-cov assays to detect the upstream envelope gene (upe) followed by confirmation of open reading frame a (orf a), b (orf b) genes or nucleocapsid (n) genes for confirmation have been developed. most commercial pcr tests perform parallel screening for the upe gene with confirmation by the orf a, orf b or n genes (most commonly upe + orf a). initial naat tests for mers-cov were developed as inhouse tests, following the first detection of mers-cov in the middle east. [ ] [ ] [ ] [ ] inhouse tests are not necessarily subject to quality control or regulation, and may not be rigorously validated; in some cases, inhouse tests are eventually developed into commercial products through collaboration and licensing efforts. - commercial assays may undergo an international and/or incountry regulatory process; once on the market they can be independently evaluated for sensitivity, specificity and limit of detection. as of , there are several commercial naat tests available for mers-cov, including duplex and multiplex panels (see online supplementary table s ). serology is not widely performed for diagnosing acute mers-cov infection; however, it has been a useful tool bmj global health to determine the extent of infection around clusters and in seroepidemiological studies in animals and humans. seroconversion typically occurs during the second and third week after symptom onset; data suggest that low antibody titre in the second week or delayed seroconversion is more closely associated with mortality than high viral load. mers-cov seroconversion may not be observed for some patients, notably with mild or asymptomatic infection, and can show cross-reactivity with antibodies to other coronaviruses. serological methods for the detection of antibodies against mers-cov include elisa, iift and neutralisation tests. mers-cov serological assays can employ commercial reagents or proprietary monoclonal antibodies as capture agents. many mers-cov elisa tests are labelled for research use only, with little or no clinical validation data available. similar to the elisa, iift is used when it is difficult to evaluate specific antigens individually by enzyme immunoassays or there is a preference for broader analysis of an immobilised specimen. iift microscopy assay can probe the entire antigen spectrum of the specimen, and is often designed for simultaneous detection of antibodies against biochemically distinct antigens. neutralisation is a method for detecting anti-mers-cov antibody activity via inhibition of infection or replication, performed as plaque reduction neutralisation, microneutralisation (mn) and pseudoparticle neutralisation (ppnt). mn is labour-intensive and slow, requiring at least - days for results; neutralisation techniques other than ppnt require biosafety level containment as they involve live virus cultures. rdts can leverage the same antibody/antigen capture agents as elisa but in a lateral flow strip cartridge. this enables a fast - min time to result, but with a -fold lower detection sensitivity than elisa. follow-up confirmatory testing is therefore required. rdts are typically paired with minimally invasive specimen collection (blood, oral fluid, nasal swabs) so that they can be used with minimal training outside of laboratory settings. early prototypes for mers-cov rdts have been developed, with commercial rdts for detection of mers-cov in camels and humans available (online supplementary table s ). the human mers-cov rdt does not appear to be widely used, perhaps due to the more invasive processing required for lower respiratory specimens, as well as sensitivity issues for upper respiratory specimens. the camel mers-cov rdt is used with upper respiratory specimens; however, test sensitivity varies depending on specimen sampling and infection kinetics. multiplex panels at the early stages, the symptoms of mers-cov infection can mimic diseases such as influenza, pneumonia, sars and other respiratory infections. a syndromic approach involves testing for pathogens based on a syndrome such as fever or acute respiratory distress; a shift from individual tests to multiplex panels can quickly identify or eliminate likely pathogens from a single specimen. for analysis of circulating reservoirs, multiplex microbead-based immunoassays have been used to detect igg antibodies for multiple pathogens. multiplex, syndromic panels that include mers-cov have been demonstrated using pcr-based panels including mers-cov, showing similar limits of detection to single assays. commercial respiratory panel tests including mers-cov have also recently been developed (see online supplementary table s ). there is a need for international consensus and adoption of minimum standards for tests used in diagnosis, surveillance and research, following who's recommended algorithm for human cases and oie recommendation for animal health. harmonisation of the testing process can be achieved by building consensus and capacity across international and incountry laboratories. in order to enable and sustain the capacity for a rapid outbreak response, laboratories must have access to high-quality reagents and instrumentation, along with technical support and cold-chain transport when necessary. in addition, international reference panels would achieve a more standardised training for external quality assessment (eqa) and quality control. building on mandatory case reporting, an international mers-cov data sharing platform that includes case exposure history and sequence data would greatly facilitate the knowledge base across the mers-cov community. [ ] [ ] [ ] [ ] clinical validation understanding mers-cov viral dynamics across a broad range of specimen types is critical to establishing the limits of detection and timing of diagnostics in order to make the greatest impact for diagnosis, case management and surveillance. ensuring a test has appropriate sensitivity and specificity is a major challenge in the development of diagnostics for novel and rare pathogens, as there is often a very limited supply of well-characterised clinical material. especially during the early stage of an outbreak, clinical evaluation must often be performed in the affected countries by laboratories working closely with the ministries of health. typically only a small number of patient specimens are shared outside of the affected countries due to strict import and export regulations, particularly for 'dual-use' pathogens. specifically, the provisions of the nagoya protocol have significant impact on the access to genetic materials for both commercial and non-commercial applications. in particular, the development and validation process for new diagnostics could be accelerated if well-characterised specimens and reference standards could be more easily obtained. eqa can be useful for evaluation of test performance, as shown with evaluations of both inhouse and commercial assays for mers-cov, [ ] [ ] [ ] and bmj global health more recently a global proficiency testing programme used to assess laboratory detection of mers-cov. even after validation, a substantial amount of reference material is required for quality control; often manufacturers must develop their own calibration standards to maintain supply and to control lot-to-lot variability. international reference standards and qualified specimen panels can accelerate the development and validation of diagnostic tests. in particular, the who international biological reference preparations (as provided by member states) serve as reference sources for ensuring the reliability of in vitro biological diagnostic procedures used for diagnosis of diseases and treatment monitoring, including mers-cov. several international institutes also provide specimens for validation; these groups typically have a defined pathogen/disease focus with a corresponding archive of biological reference materials; however, the supplies may be limited (see online supplementary material ). currently, mers-cov diagnosis by pcr requires a laboratory with sophisticated facilities and biosafety cabinets. the turnaround time to receive a test result can take days to weeks, depending on laboratory proximity, sample transport options and laboratory processing capacity, and infrastructure requirements place most pcr systems in reference laboratories, which may not be ideal for diseases like mers-cov that recommend immediate isolation for infections detected across a variety of settings. a more nimble approach is needed for mers-cov case detection and triage, and at border crossings for animal surveillance, quarantine and targeted vaccination. the fao-oie-who mers technical working group has given a clear call for the development of an rdt to improve identification and isolation of primary human cases in healthcare facilities. serological rdts are ideal for low infrastructure settings such as a primary health clinic, home or field testing. however, specimen collection remains a key challenge for mers-cov, as the recommended lower respiratory specimens are difficult to obtain outside of a hospital setting. upper respiratory specimens such as nasal swabs are easy to obtain and work well in conjunction with rdts for camels, but these specimens generally have low virus titre in humans, thus limiting current use of rdts to animal testing. improvement of the current rdt detection chemistry, if feasible, may support the future use of these tests in humans, at least for rapid triage in highly infectious cases. poc and near-poc microfluidic platforms enable a more flexible, but still highly sensitive approach for near-patient naat testing in decentralised settings. near-poc naat platforms are compact and self-contained, with automated sample preparation for processing in minimal laboratory settings, which most healthcare workers can be trained to operate within a day. [ ] [ ] [ ] recent publications describe mers-cov assays designed for poc pcr, loop-mediated isothermal amplification assay and paper-based sensor detection ; however, no mers-cov assays are currently available for the existing near-poc platforms. given that pcr is now the standard for mers-cov diagnosis, it would be highly desirable to have an automated, self-contained naat assay that can be readily deployed in a field or clinic setting. syndromic testing can be valuable during the early stages of an outbreak, in order to distinguish mers-cov from other respiratory infections or identify cases of coinfection. a syndromic panel could be effective in expediting pathogen and outbreak identification, especially with technologies that can screen for multiple pathogens simultaneously. using the panel approach, a definitive diagnosis could enable timely decisions about triage, treatment, infection control and contact tracing. while the per-test cost rises with test complexity, including additional reagents and more sophisticated instrumentation, a rapid and efficient diagnosis scheme can impact intervention and infection control and can be cost-saving overall. as respiratory diseases are both regional and seasonal, - region-specific panels may be more cost-effective. multiplex panels offer the alternative for a 'bundled' testing paradigm; however, if not routinely used (if the market is small), then developers may be reluctant to support the test for diagnostic use, which requires additional investment for validation and regulation. surveillance can be an effective method to identify the initial stages of outbreak, but it requires routine and effective sampling. the impact of surveillance testing depends on the test sensitivity and specificity, sampling rates, kinetics of the disease, and whether the target is animal or human populations. most surveillance sampling is performed in the field, either through population-based or 'hot spot' sampling. for mers-cov, it may be difficult and expensive to implement routine surveillance in dromedary camel stock, as they represent a significantly large reservoir but may suffer only mild effects from mers-cov infection, if any. the ideal surveillance tool would be a highly sensitive and field-appropriate screening test. per-test cost is also an important factor along with ease of implementation. this review has identified diagnostics currently available for mers-cov and highlighted ongoing challenges caused by critical gaps in diagnostics to support outbreak management. rdts offer the potential for rapid poc screening for mers-cov; however, there are practical limits to implementing lower respiratory sample acquisition outside of a hospital setting, limiting feasibility. poc or near-poc naat platforms provide an opportunity for implementation of automated, self-contained bmj global health testing in hospitals and clinics with limited training in endemic-prone areas. expansion of test menu options for existing poc or near-poc naat platforms will strengthen incountry response capacity to endemic diseases and simultaneously ensure countries are prepared for future pandemics. syndromic multiplex panels may expedite differential diagnosis of mers-cov from other endemic respiratory diseases, but further analysis is needed to inform implementation and cost-effectiveness in the context of regional and seasonal detection. there is also a need for more sensitive serological assays with lower cost and minimum cross-reactivity that can be used as surveillance tools. a more detailed understanding of mers-cov viral and antibody kinetics is needed across the broad range of sample types in order to optimise the use of existing assays and to address ongoing technical challenges in the detection of mild and asymptomatic infections. surveillance continues to be important for the detection of mers-cov spillover events; however, questions remain on the cost-effectiveness of routine screening of the large reservoir camel population. in addition, support towards sample biobanks with well-characterised specimens and reference standards will facilitate diagnostic development and quality assurance for mers-cov diagnostics worldwide. in order to achieve the goals of the r&d blueprint efforts, who is identifying key target product profiles for diagnostics in order to mobilise funding and resources to support the development and implementation of the most critically needed tests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who | middle east respiratory syndrome coronavirus (mers-cov). who mers-cov r&d blueprint plan of action r&d blueprint for action to prevent epidemics progress on the global response, remaining challenges and the way forward evidence for camel-tohuman transmission of mers coronavirus human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection middle east respiratory syndrome 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should be first-line tests for detection of respiratory and intestinal pathogens cost analysis of multiplex pcr testing for diagnosing respiratory virus infections impact of a rapid respiratory panel test on patient outcomes pcr for detection of respiratory viruses: seasonal variations of virus infections global mortality estimates for the influenza pandemic from the glamor project: a modeling study prevalence and seasonal distribution of respiratory viruses during the - season in istanbul development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay acknowledgements we gratefully acknowledge input to the roadmap from all those who attended the fao-oie-who global technical meeting on mers-cov in september . the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organisations with which they are affiliated. editorial assistance for later drafts was provided by rachel key: cord- -kv n qj authors: rabaan, ali a.; alahmed, shamsah h.; bazzi, ali m.; alhani, hatem m. title: a review of candidate therapies for middle east respiratory syndrome from a molecular perspective date: - - journal: journal of medical microbiology doi: . /jmm. . sha: doc_id: cord_uid: kv n qj there have been laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) in countries, with a mortality rate of . %. there is no specific therapy. the current therapies have mainly been adapted from severe acute respiratory syndrome (sars-cov) treatments, including broad-spectrum antibiotics, corticosteroids, interferons, ribavirin, lopinavir–ritonavir or mycophenolate mofetil, and have not been subject to well-organized clinical trials. the development of specific therapies and vaccines is therefore urgently required. we examine existing and potential therapies and vaccines from a molecular perspective. these include viral s protein targeting; inhibitors of host proteases, including tmprss , cathepsin l and furin protease, and of viral m(pro) and the pl(pro) proteases; convalescent plasma; and vaccine candidates. the medline database was searched using combinations and variations of terms, including ‘middle east respiratory syndrome coronavirus’, ‘mers-cov’, ‘sars’, ‘therapy’, ‘molecular’, ‘vaccine’, ‘prophylactic’, ‘s protein’, ‘dpp ’, ‘heptad repeat’, ‘protease’, ‘inhibitor’, ‘anti-viral’, ‘broad-spectrum’, ‘interferon’, ‘convalescent plasma’, ‘lopinavir ritonavir’, ‘antibodies’, ‘antiviral peptides’ and ‘live attenuated viruses’. there are many options for the development of mers-cov-specific therapies. currently, mers-cov is not considered to have pandemic potential. however, the high mortality rate and potential for mutations that could increase transmissibility give urgency to the search for direct, effective therapies. well-designed and controlled clinical trials are needed, both for existing therapies and for prospective direct therapies. middle east respiratory syndrome coronavirus overview middle east respiratory syndrome coronavirus (mers-cov) was first isolated in jeddah in the kingdom of saudi arabia (ksa) from a -year-old male hospital patient, who died june , days after presenting with acute pneumonia and subsequent renal failure [ ] . since then, the who have been notified of laboratory-confirmed cases, including deaths [ ] . while most cases have arisen in the middle east, cases have also emerged in countries worldwide in travellers from the middle east and/ or in their contacts [ ] . most human mers-cov infections are considered to be the result of multiple zoonotic transfers. bats are the most likely mers-cov natural reservoir, as with other mammalian coronaviruses (covs), while camels are likely to be the major zoonotic source for human infections [ ] [ ] [ ] . secondary human-to-human transmission is considered to be limited, occurring mainly within family and healthcare settings. the first cluster of cases in humans was retrospectively identified to have occurred in a public hospital in jordan in april [ ] . multiple healthcare facility-associated outbreaks have since occurred in the middle east, most notably in ksa, often linked to deficiencies in infection control procedures [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although cases outside the middle east have mainly been isolated, a large outbreak occurred in korea in june , in which human-human transmission resulted in cases and deaths [ ] . increased vulnerability to either cross-species or trans-human transmission could result from viral adaptations [ ] . mers-cov infection is often accompanied by acute viral pneumonia, and sometimes gastrointestinal symptoms. clinical severity varies from asymptomatic to death, and the extent of asymptomatic spread is unclear. the high mortality rate is mainly accounted for by acute respiratory distress syndrome (ards) [ , , ] . higher mortality is observed among vulnerable patients, such as older individuals and those suffering from comorbid illness, and is also associated with high viral load [ , , , ] . in one study in a ksa hospital, intensive care unit (icu) admission among mers-cov patients was associated with a mortality rate of . % [ ] . while mers-cov is not currently considered to have pandemic potential, it is clear that human-human transmission does occur. the exact mechanisms by which mers-cov is transmitted from animals to humans have not been fully elucidated. in the south korean outbreak, the virus emerged in second-and third-generation contacts, resulting in the first human case to be imported into china [ ] . this raised concern that viral mutations were contributing to humanhuman transmission. given its high mortality and poor outcomes for vulnerable patients, and the potential for viral mutations, there is no room for complacency in the search for therapeutic options for mers-cov. there is currently no specific therapy. many of the therapeutic options used have been adapted from approaches used to treat severe acute respiratory syndrome (sars-cov) during the outbreak of , and/or the h n influenza virus during the outbreak of [ ] . however, while mers-cov and sars-cov are phylogenetically related betacoronaviruses, they differ in many important respects. mers-cov utilizes human dipeptidyl peptidase (dpp ; cd ) receptors, with binding mediated by the viral spike (s) protein, not the angiotensin-converting enzyme (ace- ) receptors used by sars [ ] [ ] [ ] [ ] [ ] . mers-cov also has a wider cellular tropism [ ] [ ] [ ] . therapies currently used include broad-spectrum antibiotics, corticosteroids and anti-viral treatments, such as interferons (ifn), ribavirin, lopinavir-ritonavir, or mycophenolate mofetil [ , , [ ] [ ] [ ] [ ] [ ] . however, the efficacy and/or safety of many of these therapies is unclear, and none are specific to mers-cov. ribavirin monotherapy, for example, is associated with multiple side-effects in the treatment of other viral illnesses, including sars-cov, has uncertain efficacy, and has not been tested in animal studies or randomized control trials for mers-cov [ , , ] . corticosteroids have not been successful in the treatment of respiratory distress or lung fibrosis in mers-cov [ , ] . meanwhile, studies in sars-cov and h n patients suggest that corticosteroid use may in fact increase viral replication in airways, and sars patient and animal studies indicate that it contributes to immunosuppression [ ] [ ] [ ] . mycophenolate mofetil has been associated with fatal disease and high viral loads in a marmoset model of mers-cov infection [ ] . ifn therapy, alone or in combination with ribavirin or lopinavir-ritonavir, has shown greater promise in in vitro, animal and human studies [ ] [ ] [ ] [ ] . however, clinical studies on ifns vary with respect to factors such as time of administration and type of patient [ , , ] . overall, there is a lack of randomized control trials (rcts) designed to test the safety and efficacy of any potential therapies specific to mers-cov, and much of the information available for existing therapies is based on in vitro and/or animal studies [ , , ] . a position paper on the evidence base for specific mers-cov therapies, published by public health england (phe) and the world health organization-international severe acute respiratory and emerging infection consortium (isaric-who), suggested that benefit was likely to exceed risk for convalescent plasma, lopinavir-ritonavir, ifns and monoclonal/polyclonal antibodies, while, by contrast, for ribavirin monotherapy and corticosteroids it was considered that the risks would outweigh the benefits [ ] . for interferon/ribavirin combination therapy, nitazoxanide and chloroquine, the available data were considered to be inadequate for assessment [ ] . in this review, we consider potentially effective mers-cov therapies, including ifns, lopinavir-ritonavir and inhibitors of proteases, including tmprss and cathepsin l, as well as mers-cov-specific potential therapies, including convalescent plasma, monoclonal antibodies (mabs), antiviral peptides and candidate vaccines. these therapies will be considered from a molecular perspective, in the context of the infection and replication mechanisms of mers-cov. the therapies are summarized in table . mers-cov lineage and structure mers-cov is a betacoronavirus belonging to clade c (lineage ) of the betacoronaviruses [ ] . its closest known coronavirus relatives are the prototypic clade c betacoronaviruses, tylonycteris bat virus hku , pipistrellus bat hku virus and neoromicia zuluensis bat pml/ (neocov) virus [ , [ ] [ ] [ ] [ ] [ ] . in common with other coronaviruses, the genome of mers-cov is a single, positive-stranded rna of over nucleotides. it encodes predicted open reading frames (orfs) and genes for structural proteins, namely the spike (s), nucleocapsid (n), membrane (m) and envelope (e) proteins (figs and ) [ ] [ ] [ ] . orf a and b encode virus replication-related proteins (pp a, pp ab), which are cleaved to give non-structural proteins (nsps) involved in synthesis of viral rna and recombination ( fig. ) [ ] [ ] [ ] . these include nsp- , which contains a -to- exoribonuclease (exon) domain that is important in viral proofreading and in determining the sensitivity of rna viruses to mutagens. thus small-molecule inhibitors references s /dpp binding antibody (mouse): s rbd in vitro [ ] antibody (human): s rbd m , m , m in vitro/in vivo (mouse, rabbit -m ) [ ] [ ] [ ] antibody (human): s rbd in vitro [ , ] antibody (mouse-humanized): s rbd in vitro/in vivo (mouse) prophylactic and therapeutic [ ] antibody (mouse-humanized): s rbd hms- in vitro/in vivo (mouse) [ ] antibody (human): s rbd in vitro/in vivo (mouse) potential for more mers-specific agents [ ] of exon activity could be candidates for mers-cov and other coronavirus therapies [ ] . as with other coronaviruses, the mers-cov s protein is critical to host cell receptor binding and cell entry, and is considered to have been under strong positive selection pressure when the virus was transmitted to humans [ , ] . hence the s protein is a major target for potential anti-mers-cov therapies [ ] . the s protein of mers-cov is composed of s and s subunits ( fig. ) [ ] . in common with other coronaviruses, entry into host cells depends on the s subunit, which contains a receptor-binding domain (rbd) comprising a core subdomain and a receptor-binding motif (rbm). the mers-cov rbm differs from that of sars-cov and dictates that mers-cov uses the dpp receptor, as opposed to the ace- receptor [ , ] . the infection process is shown in fig. . dpp , which is widely expressed in tissues, including the lung and kidneys, is critical in the species tropism of mers-cov infection; bat, human, camel, non-human primate and swine cells, for example, are permissive for mers-cov infection, whereas mouse, hamster and ferret are not [ , ] . host species restriction has been attributed to differences in five amino acids involved in dpp -rbd binding, with glycosylation of the mouse dpp also identified as being important in the inhibition of mers-cov infection [ ] [ ] [ ] . the human dpp receptor is therefore a potential target for mers-covspecific therapeutics, in particular anti-dpp mabs (fig. , table ) [ ] [ ] [ ] [ ] . adenosine deaminase (ada), which is a dpp -binding protein, competes with mers-cov for dpp binding and hence is a natural mers-cov antagonist; this gives potential insights for the development of therapeutic antagonists [ ] . mers-cov binds to the permissive host cell dpp via the rbd of the s domain, one of the major targets for potential mers-cov therapies [ ] . similarly to other coronaviruses, mers-cov then uses the s subunit for virus-host membrane fusion (fig. ) . fusion results in cleavage of the s protein at the s /s boundary by host proteases [ ] . the s subunit contains the fusion peptide, two heptad repeat domains termed hr and hr , and a transmembrane (tm) domain ( fig. ) [ ] . membrane fusion requires conformational rearrangement of s , the formation of a sixhelix bundle ( hb) fusion core, of which hr and hr are essential elements, and exposure of the fusion peptide, which inserts itself into the host cell membrane [ , , ] . hr -derived peptides have been identified as potentially effective anti-viral agents for treatment of mers-cov [ ] ( fig. ; table ). the availability of host cell proteases is essential for mers-cov entry into cells [ , ] . the host proteases responsible for s protein cleavage at the s /s boundary include the serine protease tmprss , endosomal cathepsins such as cathepsin l, and furin protease [ , , [ ] [ ] [ ] . in vitro studies suggest that uncleaved mers pseudovirus can enter host cells by cathepsin l-dependent endocytosis, but that cleavage of virus during maturation by host proteases such as tmprss results in viral entry at neutral ph and the formation of massive syncytia [ ] . host cell proteases are therefore potential molecular therapeutic targets for mers-cov prophylaxis and/or treatment (fig. , table ). the tmprss inhibitor camostat, for example, has been identified as a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [ ] . following host cell entry, mers-cov pp a and pp ab are synthesized and then cleaved by two viral proteases, the main protease (mpro/ clpro) and the papain-like protease (plpro) (fig. ) [ , ] . thus viral proteases represent further potential molecular targets for therapy ( table ) . the recently described mers-cov mpro crystal structure resembles other coronavirus mpro proteases [ ] . the sars-cov pl(pro) inhibitors, -mercaptopurine ( mp) and -thioguanine ( tg), can inhibit mers cov protease activity in vitro, as can the immunosuppressant drug mycophenolic acid [ ] . however, caution is required, as the results of studies on marmosets have associated use of mycophenolate mofetil with fatal disease and high viral loads [ ] . other mers-cov proteins involved in helping the virus to circumvent the immune system also present potential molecular targets (fig. ) . for example, the accessory protein products of orf a, b and are interferon (ifn) antagonists [ , ] . the orf a protein both inhibits type i ifn production via cytoplasmic and nuclear mechanisms, and interferes with the ifn-mediated interferon-stimulated response element (isre) promoter element signalling pathways [ , ] . this gives a molecular level rationale for the use of ifn as a therapeutic option in mers-cov treatment. mers-cov can also infect dendritic cells and macrophages [ , , ] . endosomal uptake of mers-cov by dendritic cells following binding via dpp prompts these cells to produce abundant amounts of type i and iii ifns [ ] . this gives context to the ifn antagonism exhibited by mers-cov accessory proteins. recently, mers-cov has also been shown to infect t cells, which are rich in dpp , both in vitro and in marmoset spleen [ ] . this results in t cell apoptosis and could contribute significantly to viral pathogenesis and further emphasizes the potential therapeutic utility of molecular targeting of dpp and/or the mers-cov s protein. both convalescent plasma containing virus-specific antibodies and the use of specific mabs provide options for targeting mers-cov infection at a molecular level. the use of convalescent plasma [or hyperimmune iv immunoglobulin (hvig) from the plasma of convalescent donors] for infectious disease treatments has a long history, including in the treatment of respiratory diseases [ ] [ ] [ ] [ ] . for influenza and sars-cov infection, early convalescent plasma treatment within - days of symptoms is associated with decreased viral load and reduction in mortality [ ] [ ] [ ] [ ] . however, for sars-cov the quality of studies has been inconsistent and the results have been inconclusive, with a lack of adequate clinical trials [ , ] . according to the phe and isaric-who position paper, convalescent plasma (or high neutralizing antibody titre products) is indicated for the treatment of serious mers-cov infection [ ] . one rct on critically ill patients with h n infection identified a significant reduction in viral load and mortality in patients who received hvig within days of the onset of symptoms [ ] . to date, no rcts have been completed on the use of convalescent plasma/hvig in mers-cov patients. in the light of results from sars and influenza patients, an ongoing clinical trial on the safety and efficacy of convalescent plasma treatment for critically ill mers-cov patients was initiated in may and is due to report in june [ ; clinicaltrials.gov identifier: nct ]. this trial is being carried out in ksa. however, as is common for convalescent plasma therapies, the trial has been affected by logistical and technical issues, including the availability of sufficient donors [ , ] . issues can also arise in the collection of convalescent plasma that has sufficient levels of mers-cov antibodies, particularly outside the middle east [ , ] . while there are two case reports in which intravenous immunoglobulin (ivig) was used in treatment of mers-cov, it is uncertain as to whether this contributed to patient recovery [ , ] . thus, while convalescent plasma is a promising potential therapy for mers-cov, the available clinical evidence is very limited and the results of the ongoing clinical trial will be vital in guiding any future use [ ] . focused development of neutralizing monoclonal antibodies targeted against specific mers-cov proteins has meanwhile yielded promising in vitro and/or in vivo results. monoclonal antibodies: s -dpp binding a number of mouse and human neutralizing mabs against the s region of mers-cov have been developed and tested in vitro and/or in animal models [ ] . targeting of s protein for therapeutic purposes was recently comprehensively reviewed by du et al. [ ] [ ] . in particular, the s rbd is a popular target, as mabs directed against this region have the most potent neutralizing capacity. however, in terms of vaccine development, neutralizing antibodies raised by immunization with full-length s or s protein expression vectors may produce a more effective immunogenicity through the targeting of multiple epitopes and the reduction of the possibility of escape mutations [ ] . nevertheless, many mouse and human mabs targeting the s rbd have given promising results in vitro and in mouse models [ ] . (table ) . a mouse monoclonal antibody, mersmab , blocks mers pseudovirus cell entry in vitro by binding to the rbd and preventing s binding to dpp [ ] . meanwhile, the human monoclonal antibodies m , m and m , which target overlapping epitopes in the rbd, all potently neutralize pseudovirus and live mers-cov in vitro [ ] . significantly, intraperitoneal injection of m has also been shown to have both prophylactic and therapeutic protective effects against mers-cov infection in a wellestablished human dpp (hdpp )-expressing transgenic mouse model, and in rabbits [ , ] . other anti-rbd human antibodies, mers- and mers- , which recognize distinct rbd regions and block binding to dpp , likewise have potent in vitro neutralizing activity against pseudovirus and live virus infection, and they also act synergistically [ , ] . mers- has anti-syncytia formation activity [ ] . the crystal structure of mers- bound to the dpp receptor revealed two critical rbd residues [ ] . the crystal structure of another anti-rbd antibody c , which was raised in mice, has also been elucidated. this has allowed the identification of an epitope that partially overlaps the rbd receptor binding unit [ ] . c was consequently humanized to give an antibody with prophylactic and therapeutic properties, shown by a reduction of mers-cov lung viral titres in an ad -hcd (hdpp ) transgenic mouse model [ ] . another humanized anti-rbd antibody, hms- , similarly has potent in vivo protective properties against fatal mers-cov infection in a transgenic hdpp mouse model [ ] . the human antibody lca targets both the n-terminal domain (ntd) and the rbd of the s region, and was isolated from b cells of a mers-cov-infected human donor before being used to rapidly establish a stable cho cell line that can be used to reliably produce clinical grade antibody [ ] . this is a promising candidate for clinical development, given the antibody's potent prophylactic and therapeutic activities against mers-cov infection in ad /hdpp transgenic mice and type i interferon receptor (ifnar)-ko mice [ ] . the human anti-rbd antibody b -n is another promising candidate that prophylactically reduces lung pathology in rhesus monkeys infected with mers-cov [ ] . targeting the s -dpp interaction from the host side through the development of anti-dpp (cd ) antibodies is another possible therapeutic option. the anti-cd antibodies f , f and ys target the mers-cov entry into cells in vitro [ ] . the f epitope maps close to the binding site of ada, a natural dpp binding protein and mers-cov antagonist, while the f and ys epitopes lie outside this region [ , ] . thus, targeting of the s -dpp interaction by use of mabs is a promising strategy for the clinical development of molecular therapeutics against mers-cov. another molecular approach involves targeting of the s -mediated mers-cov-host cell fusion element of the mers-cov infection cycle by use of antiviral peptides. the role of hr and hr in viral fusion makes them potentially effective molecular therapeutic targets. this has been borne out by in vitro and in vivo results obtained using hr peptides (table ). hr peptides are ineffective antivirals as they aggregate in physiological solutions [ ] [ ] [ ] . a peptide named hr p, which spans residues - of hr , effectively inhibits viral replication and s proteinmediated cell fusion in vitro [ ] . a hr p analogue named hr p-m is an even more potent fusion blocker in vitro, and inhibits mers cov-expressing pseudovirus infection [ ] . hr p-m interacts with an hr peptide to effectively block hb bundle formation. in vivo hr p-m intranasal administration to ad /hdpp transgenic mice protected them from mers-cov infection, as evidenced by the reduction of the lung viral titres by more than -fold [ ] . the addition of ifn-b along with hr p-m enhanced the protective effect [ ] . thus, s hr peptides have potential as mers-cov intranasal antiviral treatments. the s protein is also the focus of a number of candidate vaccines (table ) [ , [ ] [ ] [ ] . a fusion product combining truncated rbd and the fc portion of human igg can bind human dpp and inhibit mers-cov infection in an in vitro cell culture model [ ] . importantly, this rbd-igg fusion product can induce a humoral response in mice vaccinated by subcutaneous injection, hence blocking rbd-dpp binding and inhibiting mers-cov infection [ ] . further in vivo studies have indicated that intranasal administration to mice induces similar long-term igg humoral responses to those achieved with subcutaneous injection, but superior cellular immune responses and local mucosal responses in lungs [ , ] . this suggests that this type of construct is both potentially effective and readily deliverable by intranasal means. use of an adjuvant, particularly mf , significantly improves the humoral and t cell immunogenicity of the rbd s - -fc igg fusion construct in subcutaneously immunized mice [ ] . the possibility of using the s rbd as a vaccine molecular target for a range of divergent mers-cov strains and escape mutants has also been explored recently [ ] . the use of five recombinant rbds with mutations observed in different mers-cov outbreaks or in camel strains induced potent neutralizing antibody responses against several mers-cov pseudoviruses [ ] . however, while the rbd of the s subunit is a logical and promising target for mers-cov vaccine development, the epitope scope is relatively limited and full-length s protein may be a preferable option [ ] . technical difficulties in stably expressing abundant quantities of full-length s protein have presented a barrier. however, studies on delivery options, including the use of adjuvants and nanoparticles, may help in overcoming such issues. one study undertaken by novavax (gaithersburg, maryland, usa) showed that the inoculation of mice by intramuscular injection with fulllength s protein proprietary nanoparticles produced a relatively low neutralizing antibody response after days [ ] . however, the addition of the adjuvants alum or matrix m resulted in a robust and sustained anti-mers-cov neutralizing antibody response [ ] . [ ] . these viruses are expected to enter clinical trials as a proposed prophylactic mers-cov vaccine. likewise, intramuscular injection of ad or ad expressing full-length s protein induces both antigen-specific t cell and neutralizing antibody responses in mice [ ] . finally, intraperitoneal injection of measles virus expressing either membraneanchored, full-length s protein or soluble s protein lacking the tm domain induces robust mers-cov antigen-specific neutralizing antibody and cytotoxic t lymphocyte in interferon-a/b receptor (ifnar)-deficient mice [ ] . recently, an mva-based vaccine expressing s protein has been shown to induce mucosal immunity in mers-cov-infected dromedary camels, with a reduction in excreted virus and viral transcripts [ ] . this has potential for veterinary use and the reduction of cross-species infection of humans by camels [ ] . dna plasmids gls- is a dna-plasmid vaccine that encodes mers-cov s protein (table ) [ , ] . it was co-developed by inovio, geneone life science, inc. and the walter reed army institute of research, and has become the first potential mers-cov vaccine to enter human testing [ , ] . a phase i clinical trial in healthy volunteers commenced in for the evaluation of its safety and ability to generate antibody and cellular immune responses over a -year period, using one of three dosages in a three-injection regimen [ ] . the vaccine has already undergone pre-clinical trials in mice, camels and macaques [ ] . it induced robust and antigen-specific cytotoxic t lymphocyte and neutralizing antibody responses, which effectively protected animals against viral infection [ ] . gls- and other potential vaccine candidates provide an opportunity to develop a prophylactic mers-cov vaccine. however, the barriers to development of a prophylactic vaccine include the current relatively low mers-cov incidence in humans, as well as sourcing suitable small animal models [ , , ] . these factors complicate the definition of a target population for mass prophylactic vaccination and pre-clinical demonstration of vaccine efficacy [ ] . in this context, the monoclonal antibodies described above may be invaluable resources in an outbreak situation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in vitro and animal studies while mers-cov-specific therapies are offering promising pre-clinical results, and gls- has entered clinical trials, there is as yet no specific evidence-based therapy or vaccine clinically available for mers-cov. as described in the mers-cov infection and replication section, mers-cov accessory protein products are ifn antagonists [ , ] . attenuation of the ifn response is an important mers-cov immune response circumvention mechanism [ ] . the orf a in particular inhibits ifn-b production via the inhibition of interferon regulatory transcription factor (irf)- and nuclear factor (nf)-kb actions, and thus irf- -activating small molecules, for example, may be potential therapeutic agents for restoring ifn responses [ , ] . toll-like receptor- (tlr- ) is also involved in the immune response of mice to sars-cov and mers-cov, recognizing viral molecular patterns and initiating the innate response that leads to ifn production (fig. ) [ ] . thus, tlr- agonists are another possible candidate for mers-cov-specific anti-viral agents [ ] . therapeutically, ifn itself is particularly useful prophylactically or during the early days of viral exposure, including for coronaviruses [ , ] . in vitro and animal studies have confirmed the potential efficacy of ifns in mers-cov therapy, in particular in combination with other therapeutic agents such as ribavirin and/or lopinavir. in vitro, mers-cov was substantially more susceptible to ifn-a than sars-cov [ ] . while mers-cov in vero or llc-mk cells was sensitive to both ifn-a b and ribavirin separately, relatively high concentrations were required to reduce viral replication [ ] . however, combination therapy allowed the concentrations of each to be substantially reduced [ ] . combination therapy of ifn-a b and ribavirin in macaques administered hours after mers-cov infection reduced systemic and local viral effects, and reduced viral genome copy number and gene expression levels [ ] . bioinformatics data from microarray analysis recently showed that ifn-a b and ribavirin treatment impacts on mers-cov gene expression in different pathways, including genes involved in recognition of pathogens, immune responses and release of cytokines [ ] . both ifn-b b and lopinavir treatment, alone or in combination, also protected marmosets from the adverse clinical, radiological and pathological effects of mers-cov infection [ ] . clinically, the use of ifn monotherapy, or ifn therapy in combination with ribavirin and/or lopinavir/ritonavir, has shown some promise (table ) [ ] [ ] [ ] [ ] . however, the interpretation of clinical studies has been complicated by variability in factors such as the stage of infection at which therapy was administered. the available data are limited to case studies and retrospective cohort studies [ , ] . in one case study on a patient who died in a greek hospital, pegylated ifn along with ribavirin and lopinavir was administered as part of the treatment regime, but not until the thirteenth day of the illness [ ] . by contrast, in another preliminary study on two patients, the first patient was treated with ifn-a b and ribavirin within a day of admission prior to mers-cov diagnosis, but he was also being treated with antibiotics, steroids and non-invasive ventilation [ ] . patient , the wife of patient , was treated prophylactically after developing a low-grade fever and poorly defined lung infiltrates, but a diagnosis of mers-cov was not formally made [ ] . thus, while patient survived and patient had only a mild course of illness, it is difficult to draw any firm conclusions regarding the efficacy of the treatment. in another case study on a patient in korea, administration of pegylated ifn-a a along with ribavirin and lopinavir days after hospital admission was deemed to have been effective in viral clearance and patient survival [ ] . these case studies do not overall provide firm evidence for the efficacy or otherwise of ifn combination therapy for mers-cov. in one case involving a series of five patients who were critically ill with mers-cov infection and on mechanical ventilation and corticosteroids, ifn-a b and ribavirin was administered on average days after admission [ ] . all five patients died, but they may not have benefited, as they were treated late in their illness and were already critically ill [ ] . the benefit of earlier treatment in less vulnerable patients was suggested in another series of six patients in which three who received ifn-a b and ribavirin early in the illness survived, while three other patients who were older and had comorbid conditions received the combination treatment later and all died [ ] . however, in another study in which mechanically ventilated patients with severe mers-cov infection who received pegylated ifn-a a and ribavirin early in treatment were compared to patients who did not receive the combination therapy, the -day survival rate was significantly higher in the treatment group, but the -day survival rate was equivalently low in the two groups [ ] . in another retrospective analysis of results from a series of patients who received either ifn-a a or ifn-b a in combination with ribavirin, no significant difference in outcome between the two types of ifn was shown, and there was no survival benefit due to use of either ifn [ ] . however, most of the patients in this study were aged more than years and some had comorbid conditions, including end-stage renal disease [ ] . thus the retrospective studies that have been carried out are heterogeneous in terms of type of patient, stage of disease and type of ifn used, including whether or not it was pegylated or short-acting. there is an urgent need for well-controlled clinical trials for ifn combination therapy in mers-cov, preferably early in the illness, as ifns are routinely available agents whose safety and efficacy is established for other viral illnesses and whose use has a sound molecular basis for mers-cov treatment. another type of therapy with a logical molecular basis for mers-cov treatment is the targeting of proteases, both host and viral (table ; fig. ) [ , , , , , [ ] [ ] [ ] [ ] . camostat, an inhibitor of tmprss , is a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [ ] . in a pathogenic mouse model of sars-cov infection, viral spread and pathogenesis was effectively blocked by camostat, and it is likely that it would have a similar impact on mers-cov [ ] . as camostat is already in clinical use for the treatment of chronic pancreatitis, it represents a potentially safe and effective therapeutic option. recently, another tmprss inhibitor, nafamostat, was identified in a split protein-based cell-cell fusion assay as a potent inhibitor of mers-cov s protein-mediated hostviral membrane fusion in vitro [ ] . nafamostat is already clinically approved for use by the us food and drug administration (fda) and is used as an anticoagulant [ ] . the cathepsin l inhibitor teicoplanin, a glycopeptide antibiotic, was recently shown, via high throughput screening of fda-approved drugs, to block entry of mers-cov, sars-cov and ebola pseudoviruses into the cytoplasm [ ] . teicoplanin is currently used clinically as an antibiotic in both prophylaxis and the treatment of serious grampositive bacterial infections. it also has derivatives, including dalbavancin, oritavancin and telavancin, all of which also block viral entry. the viral proteases, mpro ( clpro) and pl(pro), also represent potential molecular therapeutic targets [ , ] . as well as its role in viral maturation, the mers-cov pl(pro) causes deubiquitination of ifn regulatory factor (irf- ), and hence suppression of ifn b production, which contributes to viral suppression of the innate immune response (fig. ) [ , ] . the x-ray d crystal structure of mers-cov pl(pro) is similar to that of sars-cov, and includes ubiquitin-like and catalytic core domains [ ] . thus the sars-cov pl(pro) inhibitors, -mercaptopurine ( mp) and -thioguanine ( tg), can inhibit mers cov protease activity in vitro [ ] . however, the mers-cov pl pro crystal structure also has unique aspects, including the oxyanion hole, and s and s subsites, which may be viable molecular targets for antivirals specifically designed against mers-cov [ ] . a commercial compound termed compound (commercial code f - ,life chemicals) has been identified as an inhibitor of mers-cov and sars-cov plpro activity [ , ] . the critical binding interactions and mode of inhibition differ between the two viral proteases, with the compound acting as a competitive inhibitor against mers-cov pl(pro), but an allosteric inhibitor of sars-cov pl[pro) [ ] . however, f - may lose potency in physiological reducing environments [ ] . lopinavir is a protease inhibitor with activity against the sars-cov main protease m pro [ ] . in a screen of a library of fda-approved drugs to identify anti-mers-cov activity in cell culture, lopinavir emerged as one of four compounds that inhibited viral activity in a low micromolar range [ ] . however, the clinical efficacy of lopinavir in mers-cov treatment has not yet been fully established. as mentioned above, it has usually been used clinically in combination with ifn and data are only available from case studies and series. however, notably, lopinavir-ritonavir treatment resulted in better clinical, radiological and pathological outcomes and reduced mortality in marmosets infected with mers-cov [ ] . lopinavir has also been identified in a position paper from phe and isaric-who as a potential mers-cov therapy whose benefits are likely to exceed its risks [ ] . thus far, mers-cov has not been considered to have pandemic potential. most cases have occurred in the middle east, particularly in ksa. outbreaks have been primarily linked to healthcare institutions, and shortcomings in infection control and prevention procedures [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, potential viral mutations could facilitate expanded viral host range and enhance cross-species and human-human transmission [ , , ] . the outbreak in korea resulted in mers-cov emergence in second-and third-generation contacts, highlighting the potential for mutational changes that could increase the likelihood of human-human transmission [ , ] . mers-cov also exacts a high mortality rate, mainly due to the development of ards [ ] [ ] [ ] . these factors emphasize the importance of developing targeted therapies and/or vaccines. the most promising advances in the development of specific molecular mers-cov therapies relate to targeting of the viral s protein by means of anti-s monoclonal antibodies, hr-targeted antiviral peptides and viruses or plasmids bearing s protein as potential vaccine candidates [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the use of ifns, usually in combination with other therapies such as ribavirin or lopinavir, also has a logical molecular basis given that ifn antagonism is an important mechanism by which the virus circumvents the innate immune system [ , , , ] . targeting of host and viral proteases is also a sound molecular approach, as host proteases are important in viral-host membrane fusion, while viral proteases are key to viral maturation and are also involved in targeting ifns [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the therapies currently used for mers-cov have mainly been extrapolated from those used for sars-cov treatment, regardless of the important differences in receptor usage and cellular tropism between the viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] . none of these therapies have been subject to well-controlled trials, and in some cases the risks are likely to outweigh any poorly defined benefits [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in general, the clinical research response to mers-cov may have been too slow [ ] . thus, while there are many promising lines of research in terms of specific molecular targeting of mers-cov, no potential therapies have yet been subject to welldesigned clinical trials, and none have been approved for clinical use, apart from the gls- dna-plasmid vaccine [ , ] . continuing outbreaks of mers-cov, with possible increases in human-human transmission, are likely to galvanize the research community to push ahead with the design and performance of clinical trials for some of the available monoclonal antibodies and/or antiviral peptides for use in outbreak situations. there are various challenges inherent in the development of specific mers-cov therapies. these include the difficulty of identifying a target population for potential prophylactic vaccines, limited small animal model availability and dependence on transgenic mouse models, and the current relatively low incidence of infection, which complicates the performance of adequate clinical trials [ , , , ] . for example, one currently ongoing trial on convalescent plasma therapy has been affected by logistical and technical issues, including insufficient available donors and difficulty in collecting convalescent plasma containing sufficient mers-cov antibody levels [ , ] . thus, while numerous monoclonal antibodies have been raised with anti-mers activity, in particular against the s protein [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and promising antiviral hr peptides have been synthesized [ , ] , the available data are thus far limited to in vitro and animal studies. despite these issues, there is cause for optimism, given the many candidate antibody and peptide therapies. there is also some promising in vitro and animal model evidence suggesting that use of ifns, which are well-established therapies in other viral illnesses, may be of benefit if used sufficiently early in mers-cov treatment, or as a prophylactic, especially in combination with other therapies, including ribavirin or lopinavir-ritonavir [ ] [ ] [ ] [ ] . likewise, other drugs that are currently in clinical use for other conditions have been shown to be potentially useful for mers-cov treatment, including camostat and nafamostat; teicoplanin and its derivatives dalbavancin, oritavancin and telavancin; and the sars-cov pl(pro) inhibitors, -mercaptopurine ( mp) and -thioguanine ( tg) [ , , [ ] [ ] [ ] [ ] . these drugs have already been shown to be safe and well-tolerated by humans. repurposing of existing drugs may therefore prove to be the most viable option in mers-cov therapy. for example, screen of approved drugs uncovered candidates with in vitro activity against both mers-cov and sars-cov, including oestrogen receptor inhibitors and dopamine receptor inhibitors [ ] . thus, there are 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respiratory syndrome coronavirus infection repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection mechanisms of coronavirus cell entry mediated by the viral spike protein the authors received no specific grant from any funding agency. the authors declare that there are no conflicts of interest. this is a review article and no experimental work with humans was performed. five reasons to publish your next article with a microbiology society journal . the microbiology society is a not-for-profit organization. . we offer fast and rigorous peer reviewaverage time to first decision is - weeks. . our journals have a global readership with subscriptions held in research institutions around the world. . % of our authors rate our submission process as 'excellent' or 'very good'. . your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord- -d l cgc authors: nan title: mers: progress on the global response, remaining challenges and the way forward date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: d l cgc this article summarizes progress in research on middle east respiratory syndrome (mers) since a fao-oie-who global technical meeting held at who headquarters in geneva on – september . the meeting reviewed the latest scientific findings and identified and prioritized the global activities necessary to prevent, manage and control the disease. critical needs for research and technical guidance identified during the meeting have been used to update the who r&d mers-cov roadmap for diagnostics, therapeutics and vaccines and a broader public health research agenda. since the meeting, progress has been made on several key actions in animal populations, at the animal/human interface and in human populations. this report also summarizes the latest scientific studies on mers since , including data from more than research studies examining the presence of mers-cov infection in dromedary camels. since its identification in the kingdom of saudi arabia (ksa) (zaki et al., ) and jordan (hijawi et al., ) in , middle east respiratory syndrome (mers) has become a global public health threat. typical of an emerging zoonosis, middle east respiratory syndrome coronavirus (mers-cov) has an animal reservoir, i.e. dromedary camels in which the virus causes little to no disease (mohd et al., ) . many details about the extent of circulation and the mechanisms of transmission within dromedary camel herds, or factors related to zoonotic transmission and differences in circulating mers-cov strains, remain unknown. the virus has repeatedly spilled over from dromedary camels to humans, principally in countries on the arabian peninsula, causing significant morbidity and mortality (world health organization, a; azhar et al., ) . clusters of cases in the community and among family members are rare (world health organization, a; . however, delays in diagnosis in hospitals has sometimes led to secondary cases among health care workers, patients sharing rooms or family members as a result of unprotected direct contact with a patient before isolation. this humanto-human transmission in health care facilities can sometimes be amplified, causing very large outbreaks, as has been seen in the middle east and in the republic of korea, with significant public health and economic impacts (hijawi et al., ; assiri et al., ; drosten et al., ; al hosani et al., ; ki, ; park et al., ) . as of august , more than human cases from countries have been reported to the world health organization (who) (world health organization, a) . the fao, oie and who tripartite have regularly brought together affected member states, public health and animal officials, and academics to discuss what is known and unknown about the zoonotic origin of mers-cov (world health organization, ; fao, fao, , ; who regional office for the eastern mediterranean, a). the purposes of these meetings and workshops have been to advocate for more surveillance and research on mers-cov in animals and humans, to share information about how mers-cov is transmitted between animals, from animals to humans and between humans, to describe the diseases it causes, and to develop policies and guidelines for detection, https://doi.org/ . /j.antiviral. . . received august ; accepted september reporting of animal and human infections, and prevention of human cases and clusters. in the two years since the last international technical consultation on mers-cov in , there have been notable improvements in surveillance and reporting of human cases, multidisciplinary research, cross-sectoral collaboration at country level, public awareness about the disease, and laboratory and surveillance capacity in affected countries. in addition, a number of countries in the arabian peninsula and in africa have engaged in research activities and surveillance of camel populations to shed light on the wider distribution of this virus or investigate transmission patterns and routes for viral shedding. as a follow-up to previous meetings (world health organization, ; fao, fao, , ; who regional office for the eastern mediterranean, ; who regional office for the eastern mediterranean, b, c), fao, oie and who tripartite held a global technical meeting on mers-cov with representatives from ministries of health and ministries of agriculture, subject matter experts, researchers, funders and industrial partners from to september in geneva, switzerland (see supplementary information) (world health organization et al., ) . the objectives were to review the latest scientific evidence on mers-cov, further enhance cross-sectoral collaboration and communication during preparedness and response activities, and identify research priorities given the advancements in our knowledge. with participants, this was the largest mers-cov technical meeting to date and the first meeting attended by representatives from both affected and at risk countries. that is, countries which have reported human infection, countries with evidence of mers-cov in dromedary camels but no reported human cases, and countries at risk for importation (countries without infected camels that have close ties to affected countries through expatriate workers, travel to affected countries for medical procedures and/or frequent international travel). there is strong consensus among all stakeholders that dromedary camels are the main source of transmission to humans. in , oie identified mers-cov as an emerging disease with zoonotic potential in camels and thereby creating expectations of reporting positive camels by countries (oie, a) and recently published a mers-cov case definition (oie, ) for the reporting of confirmed and suspected infection in camels. not all countries face the same risks. for example, countries that have the infected reservoir (dromedary camels) differ from those countries in which dromedary camels show no evidence of current or past infection (fig. ). there may also be differences in spillover potential in countries with documented zoonotic transmission, compared to those without, due to several factors including potential differences in husbandry practices, cultural, social, medicinal, occupational exposures, prevalence of underlying chronic medical conditions, or genetic factors in human populations, and mers-cov viral differences (wong et al., ) . as such, technical and risk mitigation guidance to protect human health and research priorities differ by region. the findings from the global technical meeting are summarized below: i. surveillance needs: surveillance in animals and humans to limit zoonotic transmission routine human surveillance for mers-cov in ksa (abdulaziz et al., ) and throughout the middle east has improved since the identification of the virus in humans in , but there is significant variation in the quality and extent of surveillance between countries. in other parts of the world, surveillance is limited. since it is known that mers-cov is enzootic in areas of africa and asia where dromedary camels are found, heighted awareness and surveillance for zoonotic mers is required. this is currently lacking and remains a knowledge gap. one exception is the notable effort to identify potential mers-cov infection among pilgrims travelling back from the middle east. since , event-based surveillance among pilgrims returning from hajj, umrah and other religious events in ksa has been conducted by ksa and countries sending pilgrims. while many return reporting respiratory symptoms, no mers-cov infections have been identified among returning pilgrims (muraduzzaman et al., ; barasheed et al., ; atabani et al., ; koul et al., ; annan et al., ; ma et al., ; memish et al., a memish et al., , b refaey et al., ; al-abdallat et al., ; matthew et al., ; alqahtani et al., ; win et al., ; yavarian et al., ; kapoor et al., ) . among animals, field surveys conducted to date have included several domestic and wildlife species including dromedary camels (camelus dromedarius) and bactrian camels (camelus bactrianus), goats, bats, cattle, sheep, chickens, swine, ducks, buffalo and equids. field studies in dromedary camels have been conducted in a number of countries (table ) . to date, mers-cov rna or mers-cov-specific antibodies have been identified in dromedary camels a number of countries (table ) except australia (hemida et al., ) , kazakhstan (miguel et al., ) , and the netherlands (reusken et al., a) . other livestock such as alpacas (vicugna pacos), llamas (llama pacos), young goats, rabbits and pigs have been shown to be susceptible to experimental infection (crameri et al., ; adney et al., ; vergara-alert et al., ) . despite improvements, routine surveillance in dromedary populations is limited. the lack of surveillance information about mers-cov circulation in dromedary camels restricts our understanding of the transmission dynamics and epidemiology in dromedary camel populations. meeting participants agreed that surveillance should be integrated into existing surveillance systems, particularly in at-risk countries, similar to one health approaches developed for avian influenza, and existing human respiratory disease surveillance systems set up for influenza-like illness (ili) or severe acute respiratory infections (sari). currently, a limitation in our ability to mitigate spillover from dromedary camels to humans is a lack of clarity on the mode(s) of transmission between dromedary camels and humans, the extent and epidemiology of mers-cov circulation in dromedary camels in large parts of africa and south asia, and on why zoonotic transmission is limited across africa, large parts of the middle east, and some parts of south asia despite high seroprevalence in dromedary camels (chu et al., ) (table ) . fao has outlined the meeting participants conclusions on priorities for mers-cov surveillance and management of pcr positive dromedary camels, coordinated outbreak investigation of community acquired cases with dromedary exposure, testing of animals at quarantine and entry points, food safety and environmental contamination, risk communication and awareness raising for mers-cov among animal owners and intersectoral collaboration and coordination in an updated doha declaration first published in (fao, ) (ref/hyperlink). in dromedary camels, longitudinal studies to evaluate the natural history, shedding profile and immunity were highlighted as key research priorities. meeting participants agreed that further understanding of differences in viral strains and transmission dynamics, including the role of immunity in acquiring infection and shedding virus, the geographic range of spillover events, and environmental, behavioral or host-related risk factors for zoonotic transmission should be prioritized. countries face significant challenges in the early identification and diagnosis of mers in humans due to the non-specificity of clinical symptoms ( the spectrum of illness ranges from no symptoms (or asymptomatic infection) to severe disease including pneumonia, acute respiratory disease syndrome, organ failure and death, with a case fatality ratio . % among reported cases (world health organization, ). the delay in identification and recognition of signs and symptoms compatible with mers and delay in early isolation of patients has reduced the ability to prevent transmission between people in health care settings, notably in emergency departments, cardiac care centers and renal dialysis units (hijawi et al., ; assiri et al., ; drosten et al., ; al hosani et al., ; ki, ; park et al., ; ahmed et al., ; amer et al., ) . our understanding of human-to-human transmission in health care settings has improved through experimental and observational studies conducted in countries during such outbreaks. for example, studies of respiratory pathogens (yu et al., ; tran et al., ; thompson et al., ) and mers-cov conducted in the middle east (assiri et al., ; oboho et al., ; hunter et al., ; balkhy et al., ) and the republic of korea (bin et al., ; kim et al., a kim et al., , b nam et al., ) illustrate that aerosol-generating procedures and non-invasive ventilation, combined with inappropriate infection prevention and control practices and lack of adherence to standard practices had an important role in facilitating human-to-human transmission in health care settings. the role of environmental contamination has been evaluated in a number of hospitals following the outbreak in the republic of korea and collaborative, experimental studies are being conducted to evaluate the viability and persistence of mers-cov on surfaces and in the air (bin et al., ; kim et al., a; van doremalen et al., ) . the role of mild or asymptomatic cases in transmission chains, however, remains unclear (omrani et al., ; memish et al., c; al-gethamy et al., ; moon and son, ; al-abdely et al., ) , warranting targeted epidemiological and clinical studies to be conducted among contacts during outbreaks, especially in health care facilities. countries which have reported health care-associated outbreaks have implemented a variety of strategies to improve infection prevention and control and reduce human-to-human transmission in hospitals, including the introduction of a visual triage system prior to entrance to the emergency departments, the restructure of emergency department layouts for better triage of patients with respiratory symptoms, the standardization and training and re-training of infection prevention and control practices at facilities with high hospital staff turnover, and the auditing of health care facilities for adherence to infection prevention and control measures. iii. product research and development needs: clinical management, diagnostics and medical interventions the who r&d blueprint, a global strategy and preparedness plan that allows the rapid activation of r&d of epidemic pathogens, aims to fast-track the development and use of effective point-of-care diagnostic tests, vaccines and medicines that can be used to save lives and avert large scale crises. since , mers-cov has been included in the annual who r&d blueprint list of prioritized pathogens for accelerated research and development on diagnostics, vaccines and therapeutics (world health organization). in addition, mers-cov has a specific roadmap for product research and development, outlined by who in ( the nonspecific, and sometimes unusual, clinical presentation of mers in humans, makes early diagnosis difficult in health care facilities. while several highly specific and sensitive molecular and serologic assays exist for diagnosis in animals and humans corman et al., a corman et al., , b lu et al., ; perera et al., a; reusken et al., b; müller et al., ; song et al., ) , there was a clear call from representatives from affected countries for the development of a rapid diagnostic test to improve identification and isolation of primary human cases in health care facilities. a full landscape analysis of mers-cov diagnostics will be published separately (van kerkhove, personal communication). at the global technical meeting, several therapeutics (including convalescent plasma, lopinavir/ritonavir, ribavirin, interferon and novel therapies including polyclonal antibodies and broad-spectrum antivirals) in development were presented. however, small case numbers make the evaluation of their impact on morbidity and mortality from mers-cov infection difficult (arabi et al., a; arabi et al., b; al-dorzi et al., ; sheahan et al., ; ko et al., ; arabi et al., c) . several pre-clinical and phase i-iii studies are under way or in the design phase (outlined by the who r&d blueprint: http://who-blueprint-mapping-tool.surge.sh/). who is currently evaluating all available evidence on therapeutics to update guidance on clinical management of patients and in the process to develop standardized clinical trial protocols that could be used in affected countries to evaluate promising therapeutic candidates. who has identified target product profiles for mers-cov vaccines which include a dromedary camel vaccine for the reduction of zoonotic transmission, a human vaccine for long term protection of high risk individuals, such as those working with infected dromedary camels or health care workers, and a human vaccine for reactive use in outbreak settings (world health organization, b) . currently, no mers-cov-specific or licensed human vaccines are available (modjarrad et al., ; world health organization, c) . several human vaccine candidates for coronaviruses, including mers-cov, are at various stages of development and five general vaccine technology platforms have been developed and target the mers-cov spike protein (modjarrad et al., ; okba et al., ) . who, the ministry of health in ksa and the international vaccine institute (ivi) have continued to further align efforts to develop coronavirus vaccines (excler et al., ) and the coalition for epidemic preparedness and innovation (cepi) has included mers-cov as one of three priority pathogens for financing of a human vaccine. understanding correlates for protection and having a reliable animal model remain essential for evaluating coronavirus vaccine candidates, including mers-cov. there was a clear call from global technical meeting participants to accelerate the development of a dromedary camel vaccine in order to evaluate the potential to reduce spillover transmission to humans. the acceptability, cost-effectiveness and feasibility of a dromedary camel vaccine will also need to be evaluated and compared to other intervention strategies, such as human vaccination of high risk groups (e.g., those with occupational exposure). because mers-cov is endemic in dromedary camel populations in the middle east and elsewhere, multiple intervention strategies, including personal protective measures and the strategic implementation of a camel vaccine, are likely needed to reduce transmission from dromedary camels to humans. (farag et al., ) antibodies to mers-cov s were found in of ( . %) dromedary camels tested by micro-array technology (farag et table list of prioritized research and progress on mers-cov research, as discussed at the september meeting. *based on an enhanced understanding of the virus, the doha declaration (fao, ) is undergoing revision with a focus on guiding surveillance techniques, management of dromedary camels shedding the virus, research, regional and inter-sectoral coordination, risk communication, food and environmental safety practices, and biosecurity measures. the update includes explicit guidance on import testing, quarantine procedures, and management of shedding animals. these recommendations and priority actions in the doha declaration will be delivered as a separate document after validation by stakeholders in affected and at risk countries. at least two promising camel vaccine candidates are currently in development and being evaluated in field trials (haagmans et al., ; alharbi et al., ) . stakeholders agreed that the current funding mechanisms need to include risk-mitigating options that target the animal-human interface to prevent zoonotic transmission. these funding pathways would enable the development of camel vaccine candidates. stakeholders also agreed that prior to camel vaccine implementation, consultation with camel owners and government agencies, feasibility studies including exploring opportunities for commercial manufacturing and incentives for camel vaccination and an assessment of potential trade implications, would all be critical. in june , who and the international vaccine institut (ivi) held a joint workshop update the status of human and animal mers-cov vaccine development, and identify and prioritize activities to accelerate vaccine research and development. the meeting was held in seoul, korea on - june and included experts and professionals from industry, academia, international organizations and government agencies around the world, including the coalition for epidemic preparedness innovations (cepi), the korean ministry of food and drug safety (kmfds) and the korea centers for disease control and prevention (kcdc). the global technical meeting served as an opportunity to review the available evidence and best practices for control of this epidemic threat six years after the virus was first detected in humans. this covered our understanding of the virus, our ability to detect and respond to cases in animal and human populations, how we communicate our findings and how our work impacts policy decisions to protect animals and prevent human infections. during the global technical meeting, the latest findings from scientific studies and knowledge gained from collaborative research and surveillance were shared across animal, environmental and human sectors. fao, oie and who strongly believe that to effectively address zoonoses, including mers-cov, a one health approach to prevent, detect, contain, eliminate and respond to animal and public health risks from zoonotic high threat respiratory pathogens such as mers-cov and should involve all relevant sectors, the public and animal health and academic research community, industry and affected communities. we acknowledged the progress that has been made, and importantly, discussed the challenges that need to be addressed so that we can minimize the future public health and economic impacts of this epidemic prone virus. our aim was to articulate a clear action plan to address these remaining unknowns and to foster better collaboration between sectors and with subject matter experts willing to support member states. given the marked expansion in research related to mers-cov conducted in the past two years, fao, oie and who agree that global surveillance and research activities must now be focused on achieving the following major public health goals: reducing zoonotic transmission, detecting and identifying suspected cases early, providing safe and effective treatment to reduce human morbidity and mortality, and significantly reducing human-to-human transmission in health care settings. the critical needs for research and technical guidance identified during the global technical meeting have been used to inform the who r&d mers-cov roadmap (modjarrad et al., ) and a broader research agenda for mers-cov and other high threat coronaviruses. this research agenda serves as a catalyst to focus, align and mobilize partners to address outstanding knowledge gaps in relation to mers-cov across five technical areas: i) virus origin and characteristics, ii) epidemiology and transmission, iii) clinical management and infection prevention and control, iv) product development and implementation (modjarrad et al., ) and v) impact of interventions and operational research. the meeting identified a large number of remaining priorities and the organizing committee has summarized the main research needs in table . now is the time to devote more 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role of super-spreaders in infectious disease world health organization. who r&d blueprint list of priority diseases middle east respiratory syndrom coronavirus (mers-cov summary report on the intrenational scientific meeting on middle east respiratory syndrome coronavirus (mers-cov). who regional office for the eastern mediterranean middle east respiratory syndrome coronavirus (mers-cov) who target product profiles for mers-cov vaccines landscape analysis of mers-cov research and project development for diagnostics, theraputics and preventives press release: countries agree next steps to combat global health threat by mers-cov influenza virus but not mers coronavirus circulation in iran, - : comparison between pilgrims and general population why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? isolation of a novel coronavirus from a man with pneumonia in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia we gratefully acknowledge the input from all those who attended the fao-oie-who global technical meeting on mers-cov in september . the authors also thank malik peiris for his review of the final manuscript. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated. the authors have no competing financial interests. supplementary data to this article can be found online at https:// doi.org/mmcdoino. key: cord- -eb a ln authors: aghazadeh-attari, javad; mohebbi, iraj; mansorian, behnam; ahmadzadeh, jamal; mirza-aghazadeh-attari, mohammad; mobaraki, kazhal; oshnouei, sima title: epidemiological factors and worldwide pattern of middle east respiratory syndrome coronavirus from to date: - - journal: int j gen med doi: . /ijgm.s sha: doc_id: cord_uid: eb a ln background: middle east respiratory syndrome coronavirus (mers-cov) is an emerging threat to global health security with high intensity and lethality. this study was conducted to investigate epidemiological factors and patterns related to this disease. methods: full details of mers-cov cases available on the disease outbreak news section of the world health organization official website from january to november were retrieved; demographic and clinical information, global distribution status, potential contacts, and probable risk factors for the mortality of laboratory-confirmed mers-cov cases were extracted and analyzed by following standard statistical methods. results: details of , laboratory-confirmed cases were recorded, including related deaths. significant differences were observed in the presentation of the disease from year to year, and all studied parameters differed during the years under study (all p-values < . ). evaluation of the effects of various potential risk factors of the final outcome (dead/survived) revealed that two factors, namely, the morbid case being native and travel history, are significant based on a unifactorial analysis (p < . ). from to , these factors remained important. however, factors that were significant in predicting mortality varied in different years. conclusion: these findings point to interesting potential dimensions in the dynamic of this disease. furthermore, effective national and international preparedness plans and actions are essential to prevent, control, and predict such viral outbreaks; improve patient management; and ensure global health security. middle east respiratory syndrome coronavirus (mers-cov) is an emerging threat to global health security. this infection is caused by a zoonotic single-stranded, positivesense rna virus of the β-coronavirus family that causes epidemics or even pandemics with substantial morbidity and mortality. , this emerging disease was first detected in september in a patient with fatal pneumonia in jeddah, saudi arabia, a country where a large number of muslims from all over the world gather to attend the hajj pilgrimage (one of the largest islamic rituals ) each year. , following the initial case, some cases in europe, africa, south east asia, the united states, and several arabian countries were reported to be sporadic. the majority of infected patients ( %) are from saudi arabia. world health organization (who) travel advisory has not issued travel and trade restrictions or screening in saudi arabia. however, the health ministry of saudi arabia recommends aghazadeh-attari et al pilgrims over years; those with chronic diseases, such as heart, kidney, and respiratory diseases, diabetes, and immune deficiency; pregnant women; and children under years, who are planning to attend the hajj, to postpone their visit. , the case fatality rate (cfr) of mers-cov differed but remained high : %, , %, and %. incubation period varied from to days. , - as a result, who and the us centers for disease control and prevention have recommended that individuals who are returning from the arabian peninsula and other affected countries should be monitored for mers-cov infection for at least days. several studies indicate that health care staff, including physicians, nurses, and laboratory personnel, has a higher risk of contracting, developing, and transmitting this infection. for this reason, the health ministry of saudi arabia developed and implemented standard guidelines for the prevention, infection control, and rapid response to mers-cov outbreaks according to the who recommendations. , , the transmission mechanisms of mers-cov are still unknown. however, previous studies suggest that humanto-human transmission through sneezing, coughing, contact with contaminated items, and consumption of raw camel milk may play a major role in the transmission of infection. the risk factors of mers-cov are not fully understood, and a definite risk factor in the initial human-to-human or animal-to-human transmission has not been confirmed by epidemiological studies yet. globally, awareness of mers-cov is low. the disease has high intensity and lethality with unknown epidemiological factor and pattern. every year, millions of muslims travel to the epicenter (saudi arabia) of this infection to attend hajj. upon returning home, pilgrims hold ceremonies, which are attended by family and friends. long-standing traditions of sharing and hospitality on such occasions increase the likelihood of mers-cov transmission to others. therefore, unknown epidemiological patterns and the probable risk factors of mers-cov should be investigated to prevent its spread and devise effective interventions. authorization from who and the ethical committee of urmia university of medical sciences authorities was obtained. by census method, data related to laboratory-confirmed mers-cov cases between september , , and november , , were extracted from the disease outbreak news on mers-cov section of the who website. from september , , to november , , the occurrence of , laboratory-confirmed cases of mers-cov infection, including deaths, was reported to who by the national ihr focal points of countries in europe, north africa, the middle east, the united states of america, and asia. due to the small number of cases in (n= ), to avoid random bias, we merged occurred cases with . details for , laboratory-confirmed cases of infection with mers, including related deaths, were recorded in the disease outbreak news section of the who website for this time. extracted data comprised demographic information, such as age, gender, nationality; and country of origin; clinical data on the year of morbidity, day/month of the onset of symptom; day/month of admission to the hospital and icu or negative pressure isolation room; potentially hazardous contacts, including background of contact with morbid cases at home or hospital and health care facilities - days prior to the onset of symptoms and background of direct or indirect contact with a camel; and consumption of raw camel products. other probable risk factors included travel history, initial or secondary case, and comorbidity. the authors would like to confirm that all data of mers-cov cases retrieved on the who website were anonymous or de-identified. statistical analysis was performed using spss version . quantitative and qualitative measures were expressed as absolute frequencies and percentages. logistic regression was used to assess the potential relationship between probable risk factors and the final outcome (dead/survived) of the laboratory-confirmed mers-cov cases. p-values < . were considered statistically significant, and for the model, a value of . was considered significant. table shows the characteristics of laboratory-confirmed cases of infection with mers. in this table, we see that the cases of the disease are increasing year by year, and all studied parameters related to the cases differ for the years under study (all p-values < . ). table displays and compare the effects of various potential risk factors on the final outcome (dead/survived) of laboratory-confirmed mers-cov cases worldwide. this table shows that among the possible variables under consideration in this study, some factors such as nativity and travel history (p= . and p= . , respectively) that are important in predicting the disease mortality have been identified. there was no significant difference between the two groups (dead/ survived) of laboratory-confirmed mers-cov in aspects of other probable factors investigated in this study. table presents that the effects of probable risk factors on the mortality of laboratory-confirmed mers-cov cases this report stated that the chance of death due to mers-cov infection in native morbid case was . times higher than non-native cases. there were statistically significant differences between an infected individual who had travel history and in those who had not, for dying from mers-cov infection. the chance of death due to mers-cov infection in an individual who had travel history was . ( % ci: . - . ) times higher than those who had not. the occurrence of a large number of mers-cov cases and its death related in the world indicates that this disease must be considered as a threat to public health. results of the present study show the outbreaks of mers-cov infection in recent years. because of the occurrence of a large number of mers-cov cases in the world and deaths related to it, this disease was considered as a threat to public health. our results support the high fatality of this disease, and the cfr ( . %) of mers-cov infection should be a major health concern at the global scale; thus, the characteristics of this disease and potential factors contributing to its fatality should be comprehensively studied to effectively know its health risk. evidence of person-to-person transmission (particularly during close contact) has already been emphasized in the study by perlman and mccray. in our study, of , cases, . % involved close contact with laboratory-confirmed cases of infection with mers-cov days prior to the onset of symptoms. this finding highlights the notion that early cases have enormous potential for disseminating mers-cov among health care workers and other community members. furthermore, who recommendations on surveillance and control should be practiced with great vigilance, and hospitals must utilize negative pressure isolation rooms for morbid cases of mers-cov. the analysis of the global distribution status of mers-cov in the world suggests that outbreaks of this disease emanate primarily from saudi arabia and coincides with the largest annual mass gathering of muslims from around the world in this country to perform hajj and umrah rituals. , [ ] [ ] [ ] [ ] in addition, the tradition of consuming raw camel milk is observed in arab countries with high occurrence rates of mers-cov infection; several studies have also addressed this point. , as demonstrated in this study (table ) , the presentation of the disease from year to year has a significant difference, and all studied parameters related to the cases differ for the years under study (all p-values are < . ). these parameters include demographic factors, such as gender, mean age, nationality of the patients, and salient characteristics, and features related to the disease, i.e., travel history, background of contact with camel or camel milk days prior to the onset of symptoms and contact with morbid case at home or hospital days prior to the onset of symptoms, admission to a hospital, need for admission to a negative pressure isolation room or icu, and the final outcome during or after occurrence (dead/survived). in table we also see that in all years, the proportion of males was higher than in females, more of them had the saudi nationality, and more number of mers-cov cases occurred approximately at age near and up to years. one of the reason for this is the people near and up to years of age are at a greater risk of complications resulting from mers-cov or viral infections than other people in low and middle ages. on the other hand, men in comparison to women are likely to spend more time outdoors and therefore have a higher risk of exposure to a source of infection. the comparison of characteristics of the cases and the effect of various potential risk factors on the final outcome (dead/survived) of laboratory-confirmed mers-cov cases in the world (table ) reveal that two factors, namely, morbid case being native and travel history, are considered significant in a unifactorial analysis (p-values are < . ) and with the potential of bearing on the dynamics of the disease. as we follow the dynamics of the disease from to (table ) , these factors remain important. however, some interesting factors such as nativity and travel history that are significant in predicting mortality varied in different years. with attention to this fact, the majority of mers-cov cases that occurred outside of the middle east had travel history to countries in or near the arabian peninsula within days before symptom onset. therefore, this can be an evidence for the human-tohuman transmission of mers-cov infection. for nativity, there is a need for further studies on genetic features of the culprit virus. some limitations were recognized in our study. first, the design of the study was cross-sectional so that no causal inferences could be made. further prospective studies are necessary to explain the effects of all potential risk factors investigated in this study for the outcome (dead/survived) in individuals with laboratory-confirmed mers-cov the international journal of general medicine is an international, peer-reviewed open-access journal that focuses on general and internal medicine, pathogenesis, epidemiology, diagnosis, monitoring and treatment protocols. the journal is characterized by the rapid reporting of reviews, original research and clinical studies across all disease areas. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. epidemiological factors of mers-cov infection. second, possible misclassification in the categorization of cases may be due to the respondents, declaration such as background of contact with morbid case at home or hospital days prior to the onset of symptoms household, comorbidity, and background of contact with camel or camel milk days prior to the onset of symptoms, which potentially may occur as a result of the measurement bias. third, it should be mentioned that from mers-cov cases occurred in republic of south korea, only details related to cases were published in the disease outbreak news on the who website. this might introduce a negligible selection bias in the results. in summary, these findings point to interesting and potential dimensions of the dynamic evolution of this disease, and the need for further studies on genetic features of the culprit virus and the epidemiological parameters and risk factors of mers-cov mortality is also emphasized. furthermore, effective national and international preparedness plans and actions are essential to prevent, control, and predict such viral outbreaks; improve patient management; and ensure global health security. ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus hadj ritual and risk of a pandemic transmission of mers-coronavirus in household contacts fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea the knowledge and attitude about hiv/aids among jordanian dental students: (clinical versus pre clinical students) at the university of jordan risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia middle east respiratory syndrome coronavirus: a comprehensive review middle east respiratory syndrome and severe acute respiratory syndrome transmission of middle east respiratory syndrome coronavirus infections in healthcare settings epidemiologic parameters of the middle east respiratory syndrome outbreak in korea abbas zadeh bazzi m. evaluation the effect of health education on knowledge, attitude and practices of zabol's women barbers about aids in middle east respiratory syndrome: a new global threat middle east respiratory syndrome coronavirus during pregnancy mers-cov scenario and modeling working group. estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia person-to-person spread of the mers coronavirus-an evolving picture evidence for camel-to-human transmission of mers coronavirus evidence for camel-to-human transmission of mers coronavirus this work was funded by the urmia university of medical sciences (grant number ). this research is dedicated to dr. m. m. dilar for her sincerity and honesty and for her endeavor in motivating us to do the research. the authors would like to thank rana sidani, senior communication officer in the who regional office for the eastern mediterranean (cairo, egypt) for guidance and help. all authors contributed toward data analysis, drafting and critically revising the paper, and agreed to be accountable for all aspects of the work. the authors report no conflicts of interest in this work. key: cord- -g vcchrh authors: agrawal, anurodh shankar; ying, tianlei; tao, xinrong; garron, tania; algaissi, abdullah; wang, yanping; wang, lili; peng, bi-hung; jiang, shibo; dimitrov, dimiter s.; tseng, chien-te k. title: passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: g vcchrh middle east respiratory syndrome coronavirus (mers-cov) has repeatedly caused outbreaks in the arabian peninsula. to date, no approved medical countermeasures (mcm) are available to combat mers-cov infections. several neutralizing human monoclonal antibodies (mabs), including m , a germline-like human mab, have been chosen as promising mcm for mers-cov. however, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. we assessed the prophylactic and therapeutic efficacy of m by using well-characterized transgenic mice shown to be highly sensitive to mers-cov infection and disease. we found that mice treated with m prior to or post lethal mers-cov challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. taken together, these results support further development of m and other human monoclonal antibodies as potential therapeutics for mers-cov infection. scientific reports | : | doi: . /srep and long-term threat to people, particularly those who interact closely with camels in the arabian peninsula. even though mers-cov presently has limited human-to-human transmission , , the high mortality rate of this virus and limited information on the mechanism able to confer increased human-to-human transmission have raised concerns of a potential mers pandemic. indeed, the recent outbreaks in korea and the appearance of super-spreading events indicate that mers-cov has the ability to cause large outbreaks outside of the arabian peninsula [ ] [ ] [ ] . currently, no approved vaccines or drugs are available to treat this viral infection. these facts highlight an urgent need to develop potent prophylactic and therapeutic agents to fight this lethal virus. similar to other coronaviruses, mers-cov uses the envelope spike (s) glycoprotein, a class i transmembrane protein, for interaction with its cellular receptor for binding, fusion and entry into the target cell . the receptor binding domain (rbd) located in the s domain of the mers-cov spike is responsible for binding to the well-characterized cellular receptor identified as dpp (cd ) and is, therefore, critical for binding and entry of the virus [ ] [ ] [ ] . therefore, neutralizing antibodies capable of blocking such interaction could be promising preventive and/or therapeutic candidates. recently, human monoclonal antibodies (mabs) capable of neutralizing mers-cov have been identified and characterized by several research groups [ ] [ ] [ ] [ ] [ ] [ ] . these antibodies have been isolated from naive human antibody libraries, from transgenic "humanized" mice, or from b cells of an infected individual, and they recognize different epitopes on mers-cov rbd. one of the most potent mabs, m , is a germline-like antibody identified from a very large (~ size) phage-displayed antibody library derived from b cells of healthy donors. this mab exhibits exceptionally potent neutralizing activity (ic = . μ g/ml) in vitro . moreover, because its epitope almost completely (~ %) overlaps with the receptor-binding site of dpp on mers-cov rbd, as is evident by its recently solved crystal structure , the probability of generation of resistant mutants may be absent or very low. notably, although the functions of these mabs have been extensively characterized in vitro, their further clinical development has been hindered by the lack of an effective animal model of mers-cov infection. mers-cov cannot infect small laboratory animals (e.g., mice, hamsters and ferrets) as a consequence of species-specific differences in dpp , while only causing mild-to-moderate symptoms in rhesus macaques. marmosets, which are more susceptible to mers-cov, developed a moderate-to-severe disease, but limited availability and high cost have hampered their use . rabbits can be infected, but the infectious virus is challenging to detect , . it was found that the expression of human dpp could overcome the lack of susceptibility in normal mice. with prior transduction of adenoviral human dpp -expressing vectors, mice became susceptible to mers-cov infection without revealing any measurable clinical manifestations . in contrast, transgenic (tg) mice with the human dpp gene integrated into the genome readily developed acute morbidity (weight loss), and uniform death occurred within a week , , making it an ideal preclinical model for the development of vaccines and treatments against mers. some of the aforementioned human neutralizing monoclonal antibodies have been shown to protect engineered human dpp -expressing mice and the naturally permissive rabbits, entirely based on their ability to inhibit mers-cov infection and/or alleviate histopathology of the lungs , , , . to further verify the protective efficacy of these human monoclonal antibodies, particularly m , against mers-cov infection, it is highly desirable to use the well-characterized human dpp tg mice known to result in acute disease (weight loss) and death , . by using this highly permissive tg mouse model, we evaluated the prophylactic and therapeutic efficacy of m mab in vivo. we report in this study for the first time that treatment of tg mice with a single-dose of m antibody prior to or after challenging with , ld of mers-cov protected mice from the lethality in a dose-dependent manner, thereby representing the first antibody tested for its protective efficacy against lethal mers-cov infection. prophylactic efficacy of mers-cov rbd-specific human monoclonal antibody, m . we established a tg mouse model which is profoundly sensitive and susceptible to mers-cov infection, as determined by high viral titers in the lungs, as well as a high rate of morbidity and mortality , . equipped with this small animal model of human mers-cov, we investigated the protective efficacy of mab m . to accomplish this, each group (n = ) of mice was treated via the intraperitoneal (i.p.) route with two different doses: . mg and mg per mouse diluted in μ l pbs, and challenged intranasally (i.n.) at h post treatment with tcid (i.e., , ld ) of mers-cov in a volume of μ l . challenged mice were monitored daily for clinical manifestations (weight loss) and mortality. as shown in fig. , the group treated with mg mab survived viral infection without showing any clinical symptoms. these mice initially showed either no weight loss or recovered from mild weight loss within three days (fig. a) . on the other hand, the group treated with . mg mab showed a gradual weight loss ( - %) until day just before starting to recover (fig. a ). all surviving mice (one died on day in mice treated with . mg of m ) continued to recover and appeared well up to dpi when the experiment was terminated (fig. b) . all mers-cov-challenged mice pretreated with a high dose ( mg) of irrelevant mab m . exhibited profound weight loss (> %) and succumbed to infection with % mortality by day p.i. (fig. a,b ). therapeutic efficacy of mers-cov rbd-specific human monoclonal antibody, m . to determine the therapeutic potential of this human monoclonal m antibody, groups of mice (n = per group) were challenged (i.n.) with tcid of mers-cov (i.e., , ld ) in a volume of μ l and then treated (i.p.) hours later with a single dose of either mg or . mg of m or mg of m . antibody (control) in μ l per mouse, followed by monitoring daily for wellbeing (weight loss and other clinical manifestations) and mortality of mice. we noted that whereas treatment with mg of m antibodies was effective in the protection against the lethality caused by mers-cov infection, it failed to protect mice fully from the onset of clinical illness (weight loss). specifically, all of the challenged mice treated with mg of m antibody suffered an attenuated (< %), and transient weight loss until day , and gradually recovered to day when the experiment was terminated (fig. ) . similarly, challenged mice treated with a low dose of . mg of m antibodies suffered from attenuated and transient weight loss until day p.i. and gradually recovered. however, we noted a single death at day in this low dose treatment group (fig. ) . as expected, all mice treated with a single dose of mg of control m . antibody exhibited profound weight loss (> %) and succumbed to mers-cov infection with % mortality by day p.i. (fig. ) . taken together, these results indicate that this mers-cov rbd-specific human m antibody can be highly effective as prophylactic or therapeutic modalities in protecting highly permissive transgenic mice against mers-cov infection and disease. we also investigated the protective mechanism of m against mers-cov by determining the lung virus titers in challenged mice at day after treatment. specifically, we sacrificed two mice (out of ) in each group, as described above for figs and and their lung specimens were harvested for determining viral titers by using via vero e cell-based infectivity assay and quantitative pcr (q-pcr)-based assay targeting the upstream e gene of mers-cov. as shown in fig. a we were unable to recover infectious virus from any mouse treated with mg of m antibody either before or after challenge with mers-cov. however, we were able to detect a barely detectable infectious virus, with the limit of detection (lod) of . log ticd /g, from a single mouse receiving . mg of m prior to viral challenge. these results indicated that mab m most likely confers protection from lethal challenge by restricting viral replication within the lungs, thereby preventing viral infection in the brains and other organs. titers of viral rna copy number, as shown by qrt-pcr assays, were also compared among groups having different doses of mabs. lungs of infected mice were harvested on day post-and pre-virus challenge group. all groups exhibited detectable viral rna. titers were significantly lower than those in the control group in all m -treated groups. in the pretreatment group, mice treated with mg of m showed a -log reduction in viral rna detection, while a ~ log reduction in viral numbers was seen in mice treated within . mg m when compared to mice receiving control mab m . . in the post-treatment group, a smaller (~ log) difference in viral rna copy number (compared to that in the pretreatment group) was observed between mice treated with mg antibody compared with those receiving control antibody, while a more than log reduction in viral rna number was seen in mice treated with . mg m when compared to mice receiving control mab (fig. b) . these data indicate that m confers significant protection to mice when administered pre-or post-viral challenge. taken together, these results suggest to us that the epitope targeted by this exceptionally potent rbd-specific m antibody has a great potential for further development as a potent preventive and therapeutic agent in the future. treatment with m attenuates lung pathology associated with mers-cov infection. the effect of m antibody treatment on the pulmonary pathology associated with mers-cov infection was evaluated by using formalin-fixed, paraffin embedded, and hematoxylin/eosin (h&e)-stained lung specimens harvested at day p.i. pulmonary pathology was noted in all mice that were treated with different doses of m or control m . antibodies either before or after viral infection. on a severity scale of to (none, mild, moderate, severe), h&e-stained samples from mice pretreated with mg and . mg of m antibody were graded and , respectively, for perivascular and intra-alveolar infiltration of mononuclear cells, including lymphocytes, macrophages/monocytes (fig. , middle panel) , whereas those obtained from mice that received post-infection lung specimens collected at day after viral challenge were processed for assessing the viral titers by using both vero e -based infectivity assay and qrt-pcr targeting upstream e gene of mers-cov, and expressed as log tcid /gram and log tcid equivalent (eq.)/gram, respectively. (a) prophylactic and therapeutic efficacy of human m antibody treatment in reducing the lung titers of infectious virus. (b) prophylactic and therapeutic efficacy of human m antibody in reducing the titers of viral rna. the data shown are representative of at least two independently conducted assays using the same samples. data is presented as mean ± standard error (se). * * * p < . as determined by using student's t test. scientific reports | : | doi: . /srep treatment with either dose of m were graded (fig. , right panel) , compared to the grade assigned to mice received control antibody treatment prior to infection (fig. , left panel) . mers-cov has attracted significant basic research and clinical studies since it was first discovered in early . even though the transmissibility of mers-cov among humans remains low at present, as a mutation-prone rna virus, it could eventually evolve into a highly communicable and more virulent human pathogens. this emphasizes the urgent need for the development of an effective antiviral therapy which could restrict the spread of this deadly disease. in other viral infections, neutralizing antibodies have been shown to protect the host from disease progression and/or reduce the severity of clinical symptoms. passive immunotherapy for prophylaxis and treatment of infectious viral diseases has been widely used for many decades [ ] [ ] [ ] [ ] [ ] . passive transfer of neutralizing antibodies is also a promising strategy for both prophylaxis and treatment against mers-cov infection. to this end, we and others have successfully demonstrated the protective efficacy of specific human neutralizing monoclonal antibodies in animal models of mers-cov infection , , , . among a panel of mers-cov-specific mabs generated by using a vast phage display library , we identified three mabs which specifically bind to the mers-cov rbd with very high affinity. among these three identified, we noted that mab m exhibited the highest potency in neutralizing live mers-cov. here, we further characterized this novel human mab in our tg mouse model of mers-cov infection and showed prophylactic and therapeutic protection of mice treated with m before and after a lethal challenge with the virus, respectively. thus, mab m is highly promising as a potent inhibitor for urgent prophylaxis in adjunctive treatment for patients infected with mers-cov. in our studies, we noted that passively transferred with mg and . mg of m monoclonal antibodies to individual mice h prior to challenge with , ld of mers-cov resulted in % and % protection against lethality, respectively (fig. ) , suggesting that using . mg m /mouse as a prophylaxis is suboptimal to completely neutralize viral infection, thereby allowing residual viruses to replicate within lungs during the course of infection. these data demonstrate that m confers a dose-dependent reduction of mers-cov infection, corroborating lower viral rna levels and live virus isolation determined for these mice when compared to control mice. our study also confirmed the therapeutic efficacy of m in a dose-dependent manner. similar to the prophylactic studies, administration of a single-dose of m antibody at a concentration of either or . mg per mouse at h after mers-cov challenge provided % and % protection, respectively, against infection-induced lethality, accompanied by reduced viral loads (both infectious virus and viral rna) within the lungs. however, we also noted the recovery of bodyweight loss and the reduction of viral loads in mice treated with mg of m at hrs after infection were slower than those treated with . mg of m , as shown in figs a and b, respectively. while there is no clear evidence showing an adverse impact on the overall wellbeing of mice imposed upon treatment with mg of m antibody before mers-cov challenge (fig. ) , it is difficult to completely rule out the existence of subtle "yet-to-be investigated" high-dose drug toxicity. we speculate that such a subtle high-dose drug toxicity in the phase of acute and dynamic mers-cov infection initiated at hrs before treatment with mg of m could exacerbate drug toxicity, resulting in reduction of appetite and antiviral capacity. however, such a negative impact imposed upon high-dose treatment of virally infected mice appeared to be transient and did not irreversibly alter the final outcome of infection, as judged by the mortality (fig. b) . additional studies, especially the pharmacokinetics and the dosing frequency of m are warranted in the future to optimize preventive and therapeutic strategies with this promising antibody. the transgenic mice that we used for evaluating the prophylactic and, especially, the therapeutic efficacy of this m antibody are extremely sensitive to mers-cov infection and disease, with ld and id of . and . tcid of mers-cov, respectively (data not shown), titers which are lower than our original estimations . such a striking ability of this m antibody, as a prophylactic or therapeutic agent, to significantly protect these transgenic mice against challenge with ld of mers-cov is highly impressive. the rbd of the mers-cov, targeted by this m antibody, is highly conserved among various clinical isolates and the mutation rate of this rbd appears to be extremely low, compared to that of other rna viruses , , thereby making the development of escape mutants to m unlikely. however, a combination treatment with multiple neutralizing mabs targeted at different epitopes or the mers-cov-specific hr p fusion inhibitor targeting the hr domain of the s subunit of the mers-cov s protein , could be desirable. by immunizing mice with rbd of mers-cov s protein, li, y. et al. recently developed a humanized mab, named c h, that exhibited strong neutralizing activity with nd of ~ . and ~ . μ g/ml against the pseudotyped and live mers-cov, respectively , which are about -fold less potent than m (nd = . and . μ g/ml against the pseudotyped and live mers-cov, respectively) . using ad -hcd -transduced mouse model , they demonstrated that intravenous administration of a single dose of c h one day before or after the mers-cov challenge resulted in reduction of viral titer by log at dpi. however, intraperitoneal administration of m to our hdpp tg mice lead to the reduction of viral titer as high as log at dpi. since mers-cov challenged ad -hcd -transduced mice showed no severe disease, the effect of c h on the weight loss and mortality in these mice is unavailable. additionally, unlike hdpp transgenic mice that we used in this study with well-defined hdpp expression as well as % lethal dose (ld ) and infectious dose (id ), the intensities of hcd expression among the ad -hcd -transduecd mice are variable, ranging from undetectable to a high level . although both c h and m bind to the rbd of mers-cov s protein, some of the critical amino acid residues recognized by these two mabs are different , . the epitope of m overlaps extensively with the dpp -binding site, which is composed of mers-cov rbd residues n -k , s , f , d , e , w -r , w , v , s and s . the epitope of mab c , the parental mouse mab of c h, only overlaps with partial of dpp -binding site, which is composed of five rbd residues, w -e and d -r . most of other rbd amino acids recognized by c , including y -n , k , l -k , p and v -s , are not located on the dpp -binding site, indicating that the neutralization efficacy of c h is largely attributed to the steric hindrance created by its binding with mers-cov rbd .these results suggest that combinational use of c h and m may exhibit synergistic antiviral effect against both wild-type strains and escape mutants (if any) of mers-cov. taken together, these results suggest to us that the mers-cov rbd protein-specific m mab is an excellent candidate for passive immunotherapy to provide immediate and effective protection to individuals who may be exposed to mers-cov and to treat patients who have been exposed. testing in humans is needed for its potential use as a therapeutic for the treatment of mers-cov-infected patients. monoclonal antibody production. for expression of m igg , the previously described m igg vector was used to infect cho-k cells (atcc, manassas, va) with polyfect transfection reagent (qiagen, valencia, ca). after screening of clones for antibody productivity by elisa and subsequent characterization, a stable cell line was generated and inoculated into a bioflo bioreactor (new brunswick scientific, nj) for large-scale production of m igg . purification was carried out by using a protein g column (ge healthcare), and endotoxin was removed by detoxi-gel endotoxin removing columns (thermo scientific) according to the manufacturer's instructions. transgenic mice expressing human dpp established by us were used throughout the study. animals were housed in on-site animal facilities at galveston national laboratory under a : light/dark cycle with room temperature and humidity kept between - °c and - %, respectively, and with ad libitum access to food and water. all experiments were performed in accordance with the guide of nih and aaalac and were approved by the institutional animal care and use committee at the university of texas medical branch, as described previously . briefly, groups of - -weeks tg mice were challenged intranasally (in) with tcid /ml (~ , ld ) of mers-cov-emc/ , originally provided by heinz feldmann (nih, niaid rocky mountain laboratories, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, netherlands). the titers of individual virus stocks, stored at − °c, were determined by using vero e -based infectivity assays and expressed as % tissue culture infectious doses (tcid )/ml. scientific reports | : | doi: . /srep viral infections and isolation. all of the animal studies involving infectious mers-cov were conducted within approved animal biosafety level (absl- ) at the galveston national laboratory. experimental designs and strategies in different tg mouse groups involving intranasal challenge with live mers-cov were described in individual experiments in the results section. for live virus isolation, lung tissues were collected at day post mers-cov challenge, weighed, and homogenized in phosphate-buffered saline (pbs) containing % fetal calf serum (fcs) by using tissuelyser (qiagen, retsch, haan, germany), as previously described . the resulting suspensions of infected tissues were tittered in the standard vero e cell-based infectivity assays to quantify yields of infectious virus expressed as log tcid per gram (g) of tissue. rna extraction and viral titers determination by real-time q-pcr. lung tissue samples from each group of mice were transferred to individual vials having rna later solution (qiagen) and subsequently homogenized and subjected to total rna isolation, by using trizol reagent (life technologies), to assess mers-cov-specific genome targeting of virus-specific upstream e gene (upe) and endogenous control gene (mouse β -actin) by using a one-step rt-pcr kit (invitrogen), as previously described . ct values for each sample were analyzed against ct values generated in our lab from the standard curve of mers-cov mrna copy number. relative mers-cov upe mrna expression value was calculated for each replicate and expressed as the equivalent of log tcid per gram (g) of the tissue by the standard threshold cycle (∆∆ct) method. ct value analysis was done by using bio-rad cfx manager . software. is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sarslike pandemic? hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory 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protein human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the growth and potential of human antiviral monoclonal antibody therapeutics the spike protein of sars-cov-a target for vaccine and therapeutic development passive immunity in prevention and treatment of infectious diseases prophylaxis and therapy for chikungunya virus infection post-exposure treatment of ebola virus using passive immunotherapy: proposal for a new strategy structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme virus receptor we thank dr. heinz feldmann, national institute of health at hamilton, montana, and dr. ron a. fouchier, erasmus medical center at rotterdam, the netherlands for the mers-cov. this research was supported in part by a national institutes of health grant, r ai - (to c-t.k.t), and pilot grants from the center for biodefense and emerging infectious diseases and from the galveston national laboratory (grant number: uc ai - . project title: national biocontainment laboratories (nbls) operations support), university of texas medical branch, galveston, tx (to c-t.k.t.), intramural funding of nci (to dsd), and the national natural science foundation of china (# to sj, # to ty). t.g was supported in part by a t biodefense training program ( t ai - ) awarded to utmb by nih. histopathology. mice were necropsied, lung tissues were inflated and fixed in % neutral buffered formalin for days before paraffin-embedded and processed for routine hematoxylin and eosin stain (h&e) for assessing the histopathology . key: cord- -wbd s fo authors: shehata, mahmoud m.; gomaa, mokhtar r.; ali, mohamed a.; kayali, ghazi title: middle east respiratory syndrome coronavirus: a comprehensive review date: - - journal: front med doi: . /s - - - sha: doc_id: cord_uid: wbd s fo the middle east respiratory syndrome coronavirus was first identified in and has since then remained uncontrolled. cases have been mostly reported in the middle east, however travel-associated cases and outbreaks have also occurred. nosocomial and zoonotic transmission of the virus appear to be the most important routes. the infection is severe and highly fatal thus necessitating rapid and efficacious interventions. here, we performed a comprehensive review of published literature and summarized the epidemiology of the virus. in addition, we summarized the virological aspects of the infection and reviewed the animal models used as well as vaccination and antiviral tested against it. coronaviruses (cov) became known to cause human disease in the twentieth century. hcov- e and hcov-oc were discovered in the s and shown to cause respiratory infections in humans [ , ] . with the emergence of sars-cov in [ ] , two other human coronaviruses were discovered, hcov nl , hcov hku [ ] . in , a new type of coronavirus was detected as the cause of severe respiratory illness in humans. the first case was a -year-old male from saudi arabia admitted to hospital with acute respiratory illness leading to pneumonia and acute renal failure. the virus initially named as human corona virus-emc [ ] , is currently known as the middle east respiratory syndrome coronavirus (mers-cov) [ ] . classification and nomenclature of mers-cov phylogenetically, mers-cov is a lineage c β coronavirus (β-cov) and is closely related to bat coronaviruses hku and hku . the rooted phylogenetic analysis showed that mers-cov had an amino acid sequence identity less than % to all other known covs [ ] . the virus initially named by many different working groups as novel coronavirus, human coronavirus emc, human b coronavirus c emc, human b coronavirus c england-qatar, human b coronavirus c jordan-n , and b coronavirus england , which represented the places where the first complete viral genome was sequenced (erasmus medical center, rotterdam, the netherlands) or where the first laboratory-confirmed cases were identified or managed (jordan, qatar, and england) was later named as mers-cov by the coronaviruses study groups of ictv [ , , ] . mers-cov is an enveloped virus with a positive sense rna genome. coronavirus genomes range between to kb in size. the complete sequence of hcov-emc- resulted in nucleotides sequence [ ] . coronavirus genomes are polycistronic with large replicase open reading frames orf a and orf b which are subsequently cleaved into or nonstructural proteins (nsps). the region downstream of orf b encode smaller genes including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural protein [ ] [ ] [ ] . the functional receptor for mers-cov is the dipeptidyl peptidase (dpp ) which is present on human nonciliated bronchial epithelial cells surfaces [ ] . the dpp protein displays high amino acid sequence conservation across different species, including the sequence that was obtained from bat cells. cell lines susceptibility studies showed that mers-cov infected several human cell lines, including histiocytes as well as respiratory, kidney, intestinal, and liver cells [ ] . the range of tissue tropism in vitro was broader than that for any other known human coronavirus [ ] . mers-cov can also infect nonhuman primate, porcine, bat, civet, rabbit, and horse cell lines all possessing the dpp receptor [ ] . the replication cycle of mers-cov consists of numerous steps as illustrated by lu et al. [ ] . the mers-cov s protein is a class i fusion protein composed of two subunits: the amino n-terminal receptor binding s and carboxyl c-terminal membrane fusion s subunits. the s /s junction is a protease cleavage site which is responsible for membrane fusion activation, virus entry, and syncytium formation. the s c domain contains the receptor binding domain (rbd), and an n domain [ ] . neutralizing monoclonal antibodies against the rbd may inhibit virus entry into cells and receptor-dependent syncytium formation in cell culture, hence vaccines containing the rbd induced high levels of neutralizing antibodies in mice and rabbits [ ] [ ] [ ] . dpp is the cell key functional receptor for the mers-cov s protein [ ] . mers-cov is the first cov that has been identified to use dpp as a receptor [ , ] . dpp has important roles in glucose metabolism, t cell activation, chemotaxis modulation, cell adhesion, and apoptosis [ , ] . the s subunit contains five domains: a fusion peptide, the heptad repeat (hr ) and hr domains, a transmembrane domain, and a cytoplasmic domain, which form the stalk region of s protein that facilitates fusion of the viral and cell membranes [ , ] . the binding of the s subunit to the cellular receptor triggers conformational changes in the s subunit, which inserts its fusion peptide into the target cell membrane to form a six-helix bundle fusion core between the hr and hr domains that approximates the viral and cell membranes for fusion. mers-cov utilizes many pathways for membrane fusion depending on available host proteases, such as transmembrane protease serine protease (tmprss ), trypsin, chymotrypsin, elastase, thermolysin, endoproteinase lys-c, and human airway trypsin-like protease. proteases cleave the s protein into the s and s subunits to activate the mers-cov s protein for endosome-independent host cell entry at the plasma membrane [ ] [ ] [ ] . in addition to the pervious fusion proteases furin has been identified recently to play an essential role in the mers-cov s protein cleavage activation into their biologically active forms [ , ] . after cell entry, the virion particle disassembles to release the nucleocapsid and viral rna into the cytoplasm for expression of viral polyproteins pp a and pp ab. doublemembrane vesicles and convoluted membranes are formed by the attachment of the hydrophobic domains of the mers-cov replication machinery to the limiting membrane of auto-phagosomes [ ] . the viral polyproteins pp a and pp ab are cleaved by papain-like protease and c-like protease into nsp to nsp [ , , ] . these nonstructural proteins form the replication-transcription complex, which regulates transcription and viral protein expression [ ] . after the production of abundant viral rna and structural and accessory proteins, the n protein binds to the genomic rna in the cytoplasm to form the helical nucleocapsid (viral core). the viral core is enveloped by budding through intracellular membranes between the endoplasmic reticulum and golgi apparatus [ ] . the s, e, and m proteins are transported to the budding virion, where the nucleocapsid probably interacts with m protein to generate the basic structure and complexes with the s and e proteins to induce viral budding and release from the golgi apparatus [ ] . mers-cov replication cycle is completed by releasing the progeny virions through the cell membrane via exocytosis pathway. mice mers-cov strain hcov-emc/ was inoculated to three different mouse strains (immunocompetent balb/c mice, s /svev and innate immune-deficient / stat -/mice) intranasally. no significant weight loss was observed and infectious virus could not be detected in the lungs. only moderate pathological lesions were observed in the lungs. hence no viral replication was observed in these strains of mice [ ] . zhao et al. developed a mouse model transduced with a recombinant adenovirus vector expressing hdpp (ad -hdpp ) in lung tissue. inoculation of mers-cov in these mice resulted in mers-cov replication but without mortality. young mice cleared from mers-cov in - days and old mice in - days. perivascular and peribronchial lymphoid infiltration was observed, with progression to an interstitial pneumonia postinfection [ ] . in another study, transgenic mice expressing hdpp were susceptible to mers-cov infection. infectious virus was isolated from lung and brain tissue and weight loss was observed [ ] . pascal et al. developed humanized transgenic mouse. no mortality or clinical signs was observed but interstitial pneumonia and significant lung disease were observed histopathologically, suggesting that humanized dpp mouse is a model for mers-cov infection in which pathological changes resembles mers-cov infection in humans [ , ] . the rhesus macaque was the first animal model used for mers-cov infection as it possessed dpp receptor [ , ] . in infected animals, an increase in respiratory rates, body temperature, cough and reduced appetite was observed with mild to moderate severity. infectious virus isolated only from the lower respiratory tract. viral rna was detected in the conjunctiva, nasal mucosa, tonsils, pharynx, trachea, bronchus and lungs. mild to marked interstitial pneumonia with dark red lesions appeared in lungs. seroconversion of neutralizing antibodies began at dpi and increased in titer with time. the development of a transient pneumonia, rapid replication, and tropism of mers-cov for the lower respiratory tract resembled the severity of the disease observed in humans [ , , ] . similarly, the common marmoset was shown to possess the dpp receptor [ ] . radiographic imaging showed mild to severe bilateral interstitial infiltration and extensive bronchointertitial pneumonia in infected animals. infectious virus was detected in lower and upper respiratory tract tissue and viral rna was detected in nasal mucosa, oropharyngeal swabs, blood, conjunctiva, lymph nodes, gastrointestinal tract, kidney, heart, adrenal gland, liver, spleen, brain and lungs [ ] . inoculation of syrian hamsters and ferrets with mers-cov did not result in infection [ , ] . rabbits may be used as a model to study pathogenesis, transmission, and disease control strategies of mers-cov in vivo as they seroconvert and shed virus after inoculation [ ] . in september , a novel coronavirus infection was noted in promed mail [ ] . the virus was isolated from the sputum of a -year-old saudi male, who was admitted to a hospital with pneumonia and acute kidney injury in june . a few days later, another report appeared describing an almost identical virus detected in a patient in qatar with acute respiratory syndrome and acute kidney injury. the patient had a recent travel history to saudi arabia and then traveled to uk for further medical care [ , , ] . two cases from jordan (april ) were retrospectively diagnosed as mers patients. since that time, more than cases of mers-cov infection have been reported including deaths [ ] . the actual number of cases could be higher than those reported [ ] . an outbreak of more than confirmed cases including deaths occurred in south korea in may and june . the median age of korean cases were years (range: to years), % were men, and % were health care professionals. the index case was a -year-old male who had recently traveled to several countries in the arabian peninsula [ ] . disease control and prevention (cdc), and the ministry of health of saudi arabia (mohsa) as asymptomatic, mild, severely symptomatic, or mortal. cases may be classified into suspected, probable, and confirmed [ , ] . any person with laboratory confirmation of infection with mers-cov irrespective of clinical signs and symptoms is considered as a confirmed case. who criteria for laboratory confirmation require detection of viral rna or acute and convalescent serology. the presence of nucleic acid can be confirmed by positive results from at least two sequence-specific rrt-pcrs or a single sequence-specific rrt-pcr test and direct sequencing from a separate genomic target [ ] . a case confirmation by serological methods requires demonstration of seroconversion in two samples collected at least days apart using at least one screening assay (enzyme-linked immunoassay, immunofluorescence assay) and a neutralization assay. a probable case is defined by the following criteria, a febrile acute respiratory illness as pneumonia or acute respiratory distress syndrome, direct contact with a confirmed mers-cov case and unavailability of mers-cov testing or results being inconclusive for a single inadequate specimen. any person who developed a fever and pneumonia or acute respiratory distress syndrome with a history of travel to countries in or near the arabian peninsula within days before symptom onset or was in contact with a traveler from this region who developed a febrile respiratory illness is considered as a mers-cov suspected case. the who, cdc, and mohsa recommended laboratory diagnostics for mers-cov infection [ , , , , ] . mers-cov cases must be confirmed by at least two positive qrt-pcr tests on two different specific genomic regions or single positive qrt-pcr with a sequence of another positive genome fragment [ ] . the who algorithm for testing mers-cov relies on qrt-pcr and sequencing [ ] . available real-time tests include an assay targeting the rna upstream of the e gene (upe) as a highly sensitive screening assay and three confirmatory assays targeting open reading frames (orf a and b) and/ or n gene. the orf a assay is of equal sensitivity to the upe assay. the orf b assay is less sensitive but is useful for confirmation. these assays are specific for mers-cov and have not shown cross-reactivity with other respiratory human coronaviruses. for sequencing, two target genes, the rna-dependent rna polymerase (rdrp, present in orf b) and n genes are enough to confirm the existence of mers-cov rna in the samples of a patient [ ] . several serologic assays including immunofluorescence assays, protein microarray assay, enzyme-linked immunosorbent assay (elisa) have been developed for the detection of mers-cov antibodies [ , [ ] [ ] [ ] . any positive test by one of these assays should be confirmed with a neutralization assay. single serological result may be valuable for definition of probable case and should be followed by further testing for confirmation of mers-cov infection [ ] [ ] [ ] . incubation period of mers-cov infections was studied by assiri et al. in . the median incubation period was . days ( % ci . - . days) [ ] . in another report from france of a secondary case, a patient who shared a room with an infected patient, the incubation period was estimated at to days [ ] . in the recent outbreak in south korea during may/june , the median incubation period was . days [ ] . who and cdc recommended that individuals that returned from the arabian peninsula and other affected countries must be evaluated for mers-cov infections up to at least days [ ] . clinical features of mers-cov infections range from asymptomatic cases to mildly ill, severe pneumonia, acute respiratory distress syndrome, septic shock and mortal with multi-organ failure (table ) [ , ] . many other clinical features such as gastrointestinal symptoms (anorexia, nausea, vomiting, abdominal pain, diarrhea), pericarditis, and disseminated intravascular coagulation were reported [ , , ] . specific clinical conditions (comorbidities) were apparently proportionate with high severity of mers-cov infections. a study by assiri et al. in saudi arabia showed that of a total of patients with mers-cov infection in , ( %) had underlying clinical conditions, including diabetes mellitus ( %), hypertension ( %), chronic cardiac disease ( %), and chronic kidney disease ( %) [ ] . this high rate of comorbidities must be interpreted with some caution, since diabetes mellitus is common in saudi arabia, and because approximately half of those were part of an outbreak in a hemodialysis unit, where rates of comorbidities might be high due to chronic kidney disease [ , ] . in another study, being on dialysis, diabetes mellitus, and age > years was associated with mortality [ ] . in this study, testing positive for mers-cov in a plasma sample was a predictor of severe outcome [ ] . younger adults and children appeared to be less susceptible to mers-cov infection. only one study described mers-cov infection in children [ ] . all of those children were discovered during contact investigations of older patients. only of children developed symptoms of mers-cov infection. these two children had underlying conditions (cystic fibrosis and down syndrome). the other children were asymptomatic. there are few reports of mers-cov infections in pregnant women. a five-month pregnant female developed vaginal bleeding and abdominal pain after one week, then delivered a stillborn infant [ ] . another case in the united arab emirates was near term phase, she gave birth to an apparently healthy baby, and died after delivery [ ] . mild and asymptomatic mers-cov infections have been reported, a majority of whom were identified among the contacts of patients [ , , ] . in a report from mohsa, more than contacts of patients were screened using qrt-pcr and seven healthcare workers with mers-cov infection were identified, two of whom were asymptomatic and five of whom had mild upper respiratory tract symptoms [ ] . epidemiological and virological studies were conducted in attempts to determine person to person transmission of mers-cov. they studied case clustering in household and hospital outbreaks in the uk, tunisia, italy, and in healthcare facilities in saudi arabia, france, iran, and lately in south korea. those studies provided strong evidence that human-to-human transmission occurs [ , [ ] [ ] [ ] [ ] . the number of contacts infected by individuals with confirmed infections, however, appears to be limited [ ] , except the outbreak of south korea in may/ june , where most cases were secondary and some cases were tertiary infections [ , ] . secondary cases often were milder or symptomless [ ] . possible modes of transmission include droplet and close contact transmission, air borne transmission, and fomite transmission [ ] . the majority of all laboratory-confirmed secondary cases have been associated with healthcare settings [ ] . the majority of cases of jeddah, saudi arabia hospital outbreak during the spring of were acquired through human-to-human transmission due to systematic weaknesses in infection control [ ] . secondary transmission rates were assessed within households and the transmission rate was around %, suggesting that the actual number of infection is greater than reported [ ] . during the outbreak in south korea during may/june , secondary infections were associated with the index case, who was hospitalized from may to may and were tertiary [ ] . the median incubation period was six days for secondary cases and six days for tertiary cases. this outbreak also clearly demonstrated roles of "superspreaders," who may be responsible for a high proportion of cases [ ] . for instance, a single patient infected more than other people while being treated in the emergency room of a hospital in south korea for three days, - may . transmissibility and epidemic potential studies of mers-cov revealed that the reproduction number (r ) of patients infected with mers-cov ranged between . to . [ , ] . the finding of an r < suggests that mers-cov does not yet have pandemic potential. other study suggested that r values might reach to . to . in the absence of infection control [ ] . shedding periods of mers-cov in humans was reported to be long as viruses were detected in lower respiratory samples of symptomatic patients for more than two weeks [ ] . at instances, prolonged shedding for weeks was detected in an asymptomatic healthcare worker. these findings raise concerns that asymptomatic persons could transmit infection to others in a silent manner [ ] . the majority of cases have occurred in saudi arabia and united arab emirates [ ] [ ] [ ] . many cases have also been reported outside the arabian peninsula in north africa, europe, asia, and north america as shown in table . almost all cases reported outside the arabian peninsula had a travel history to it. the first cluster was in october/november in four men of the same family in riyadh, saudi arabia, two of whom died [ ] . the second cluster was reported in jordan in april involving healthcare workers exposed to fatal patients. in addition, seven surviving hospital contacts seroconverted suggesting that they had mers-cov infection [ ] . the third cluster was reported in uk during january/ february . an english resident had a travel history to saudi arabia and pakistan in january, developed a severe respiratory illness, and tested positive for both mers-cov and h n influenza a, and died in march after infecting several contacts [ ] . a cluster of cases of mers-cov was reported in al-hasa in saudi arabia during april . all those cases were directly linked to human to human contact in the same hospital. there were only two confirmed cases of healthcare workers, and three family members were detected by a survey of over household contacts that visited this hospital [ ] . in france, may , an infection of mers-cov was reported in a patient who recently traveled to the united arab emirates. a second case who shared the hospital room with the first case tested positive. the first patient died and the second patient was critically ill. a survey of healthcare workers found no other infections with mers-cov, despite the lack of use of personal protective equipment [ ] . a surge in mers-cov cases was reported in saudi arabia and the united arab emirates during march and april [ , ] . the majority of cases were associated with hospital-based outbreaks jeddah, riyadh, tabuk, and madinah in saudi arabia as well as in al ain, and abu dhabi in united arab emirates. cases included several healthcare workers, visitors, patients, and ambulance staff. person to person transmission was confirmed in > % of cases. the majority of infected health care workers developed mild symptomatic or asymptomatic infection, but about % had severe illness or died [ ] . the recent outbreak of south korea occurred in may . the index case was a man who had recently traveled to bahrain, the united arab emirates, saudi arabia, and qatar [ ] . as of late july , > secondary cases were reported including death and many cases had been reported among household and hospital contacts [ , ] . in china, one case occurred in a man who traveled to china from korea following exposure to two relatives with mers-cov infection [ ] . in spite of reporting of mers-cov infections throughout the year, some evidence on disease seasonality occurred. the first identified cases of mers-cov infection were reported in april and june [ , , ] . a high increase in cases was reported in april and may followed by a surge in case reporting in april and may . increase in case reporting in march to may were attributed to infection from infected young camels [ , ] sources and modes of transmission of mers-cov are still unclear. initially, a bat origin of mers-cov was suggested based on the relation of genome sequences between mers-cov and bat coronaviruses [ ] . cell tropism studies showed that both bat coronavirus hku and mers-cov shared the same cell type receptors, dpp [ , , ] . mers-cov grows readily in several bat-derived cell lines [ ] . there is no evidence for direct or indirect transmission of mers-cov from bats to humans. virological studies performed in europe, africa, and asia, including the middle east, have shown that coronavirus rna sequences are found frequently in bat feces. some of the sequences were closely related to mers-cov sequences [ ] [ ] [ ] . in a survey from saudi arabia, fecal and rectal samples were tested by pcr for mers-cov, many coronaviruses sequences were detected [ ] . most of the detected sequences were unrelated to mers-cov, but one sequence of nucleotide in the rna-dependent rna polymerase (rdrp) gene had a % identity with a mers-cov. this sequence was detected from feces of a taphozous perforatus bat captured near the home of the index saudi patient. uncommon contact of humans with bats indicates that bats are not the intermediate host of mers-cov transmission but may be the reservoir of the virus [ ] . dromedary camels (camelus dromedarus) appear to be the source of mers-cov. other animals like sheep, goats, and cows tested negative to anti-mers-cov antibodies. camel sera from oman, canary islands, and egypt were positive for anti-mers-cov antibodies in about %, %, and > % of the samples respectively [ , , ] . retrospective studies on archived human sera showed no evidence of infection with mers-cov before [ ] , but anti-mers-cov antibodies were detected in archived camel sera in saudi arabia in [ ] , and united arab emirates in [ ] , indicating circulation of mers-cov in camels for many years. bactrian camels in mongolia tested negative for mers-cov antibodies [ ] . serologic studies from around the middle east suggested that camels are one of the sources of mers-cov as > % of adult camels tested positive and had high titers of antibodies. seropositivity was different in juvenile camels and was usually lower than in adults. these results suggested that mers-cov infections in camels occurred in young ages followed by frequent boosting [ , , , ] . camels in other parts of the world, far from the middle east like in europe, australia, and the americas do not have mers-cov antibodies and have no evidence of infection [ ] . table summarizes camel serologic studies. in a study aimed to evaluate virus infectivity and shedding in camels, three adult dromedary camels were inoculated with mers-cov intratracheally, intranasally, and conjunctivally. those camels shed large quantities of virus from the upper respiratory tract and infectious virus was detected in nasal secretions for days post-inoculation and viral rna for up to days post-inoculation [ ] . human infections with mers-cov were linked to camels. the first evidence was a study in saudi arabia in which the mers-cov full genome sequences of isolates from a man with fatal infection and from one of his camels were identical. this patient had a direct contact with his deceased camels some days before the onset of symptoms. these results suggested that mers-cov can infect dromedary camels and can be transmitted from them to humans by direct close contact [ ] . in other studies, phylogenetic analyses of camel and human isolates of the mers-cov genome demonstrated that the viruses were highly identical or in some cases were similar to each other [ , , ] . seroepidemiological studies shown low prevalence of mers-cov antibodies in humans in saudi arabia [ , ] . a survey of individuals representative of the general population of saudi arabia resulted in seropositive subjects ( . %), however, seropositivity increased - folds in camel-exposed individuals [ ] . in a separate report, of camel shepherds and slaughterhouse workers ( . %) tested positive for mers-cov antibodies [ ] . an overview of mers-cov transmission routes is illustrated in fig. . the development of an effective vaccine is critical for prevention of a mers-cov pandemic. some investigators have indicated that the rbd protein of mers-cov s protein is a good candidate antigen as a subunit vaccine. various rbd fragments showed the highest dpp binding affinity and induced the highest-titer of igg ab and neutralizing ab in mice and rabbits [ , , [ ] [ ] [ ] [ ] [ ] . a robust neutralizing antibody response was elicited in balb/c mice against mers-cov after immunization with purified full s protein nanoparticles produced in sf cells infected with specific recombinant baculovirus containing the s gene [ ] or a recombinant human adenoviral vectors (rad or rad ) containing the s or s genes [ , ] . vaccinia ankara was encoded with full s protein and inoculated to balb/c mice that developed high levels of neutralizing antibodies and had induction of humoral and cell-mediated immunity [ , ] . another study using ad -hdpp -transduced balb/c mice immunized with venezuelan equine encephalitis virus replicon particles containing s protein elucidated a reduction of viral titers to nearly undetectable levels and increased neutralizing antibodies [ ] . recently, wang et al. developed two candidate vaccines, a subunit (full s and s protein fraction) and a dna vaccine (full s and s gene in a mammalian vrc vector). the vaccine containing the full s dna and s protein was the most efficacious in mice and rhesus macaques [ ] . using antibodies to deter mers-cov infection appears to have some promise. transfer of sera containing anti-mers-cov-s protein to or seropositive camel sera to ad -hdpp -transduced mice accelerated virus clearance, inhibited virus attachment, and reduced weight loss [ , , ] . recently, corti et al. successfully isolated monoclonal antibodies from serum obtained from a mers-cov survivor after days of infection [ ] . transduced ad -hdpp balb/c mice were immunized with mg/kg of the mab and showed decreased lung [ ] viral titers, no weight loss, and decreased peribronchial lymphoid infiltration [ ] . no approved antivirals for use against mers-cov infection are yet available. the first approach performed when a new unknown virus like mers-cov emerges is testing drugs used as antiviral for similar viruses [ , , ] . type i interferons and ribavirine combination exhibited acceptable results in cell culture and rhesus macaques by decreasing the host inflammatory response, replication of virus, and improved clinical outcome [ , , ] . a human cohort study in saudi arabia showed that treatment with combination of ribavirin and interferon-α b to did not improve clinical outcomes but this may have been due to late treatment or due to the immunocompromised state of the patients [ ] . in a retrospective study of mers-cov infected patients treated with ribavirin and interferon α- a, results showed -day and -day survival was improved by % and % in the treated group as compared to an untreated group [ ] . the second approach is screening of approved drugs with known safety profiles and transcriptional signatures in different cell lines. several drugs, including antiparasitics, neurotransmitters, antibacterials, inhibitors of clathrinmediated endocytosis estrogen receptor, lipid or sterol metabolism, protein processing, and dna synthesis or repair were tested on culture cells [ , [ ] [ ] [ ] [ ] [ ] . lopinavir-ritonavir combined with pegylated interferon and ribavirin therapy showed improved outcomes in infected marmosets [ ] . the third approach involves in vitro inhibition of s protein to block virus entry into host cells using designed antiviral peptides targeting the hr domain of the s subunit of the mers-cov and preventing the interaction between the hr and hr domains required for the formation of the heterologous six-helix bundle in viral fusion core formation [ , ] . other drugs that act as inhibitors for viral proteases and helicase to suppress mers-cov infection were tested [ ] [ ] [ ] [ ] [ ] . other investigators studied inhibition of mers-cov infection by competitive inhibition of dpp cell receptor using compounds such as sitagliptin, vildagliptin, and saxagliptin [ , ] . more than three years have passed since the first detection of mers-cov human infection and the virus, uncontrolled, continues to cause major outbreaks in the middle east. the recent outbreak in korea demonstrated that a single index case can lead to more infections in a short period of time, hence raising questions about the accuracy of the number of cases being reported in the middle east. furthermore, the korean outbreak confirmed the high fatality rate of mers-cov infection as being true rather than overestimated in case only the more severe cases are detected. in all, public health, veterinary health, and research efforts need to be consolidated in order to answer the following high priority questions: -what is the true extent of human infection with mers-cov? -what antivirals and vaccines are to be used in humans? -what infection control measures are needed in healthcare settings to prevent nosocomial outbreaks? -what measures 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inhibitors for the ability to block viral entry screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset prediction and biochemical analysis of putative cleavage sites of the c-like protease of middle east respiratory syndrome coronavirus assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and c-like proteases using luciferase-based biosensors evaluation of ssya - as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus the newly emerged sars-like coronavirus hcov-emc also has an "achilles' heel": current effective inhibitor targeting a c-like protease inhibition of middle east respiratory syndrome coronavirus infection by anti-cd monoclonal antibody mahmoud m. shehata, mokhtar r. gomaa, mohamed a. ali, and ghazi kayali declare that they have no conflict of interest. this manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. key: cord- -zbqfs n authors: sikkema, r. s.; farag, e. a. b. a.; islam, mazharul; atta, muzzamil; reusken, c. b. e. m.; al-hajri, mohd m.; koopmans, m. p. g. title: global status of middle east respiratory syndrome coronavirus in dromedary camels: a systematic review date: - - journal: epidemiol infect doi: . /s x sha: doc_id: cord_uid: zbqfs n dromedary camels have been shown to be the main reservoir for human middle east respiratory syndrome (mers) infections. this systematic review aims to compile and analyse all published data on mers-coronavirus (cov) in the global camel population to provide an overview of current knowledge on the distribution, spread and risk factors of infections in dromedary camels. we included original research articles containing laboratory evidence of mers-cov infections in dromedary camels in the field from to april . in general, camels only show minor clinical signs of disease after being infected with mers-cov. serological evidence of mers-cov in camels has been found in countries, with molecular evidence for virus circulation in countries. the seroprevalence of mers-cov antibodies increases with age in camels, while the prevalence of viral shedding as determined by mers-cov rna detection in nasal swabs decreases. in several studies, camels that were sampled at animal markets or quarantine facilities were seropositive more often than camels at farms as well as imported camels vs. locally bred camels. some studies show a relatively higher seroprevalence and viral detection during the cooler winter months. knowledge of the animal reservoir of mers-cov is essential to develop intervention and control measures to prevent human infections. middle east respiratory syndrome (mers) is a highly fatal respiratory tract disease in humans that was first detected in in the kingdom of saudi arabia (ksa) [ ] . after its first detection, mers-coronavirus (mers-cov) was being reported in human patients across the arabian peninsula, with occasional travel-related cases in other continents. as of the end of march , a total of human laboratory-confirmed cases from countries have been reported to the world health organisation (who), including associated deaths [ ] . dromedary camels (camelus dromedaries) have been shown to be the natural reservoir from where spill-over to humans can occur [ , ] . human-to-human infection is also reported frequently, especially in healthcare settings [ ] . sustained human-to-human transmission outside of hospital settings has not been shown yet [ ] . direct or indirect human contact with camels has resulted in repeated introductions of mers-cov into the human population [ ] . it has been suggested that camels may have acquired mers-cov from a spill-over event from a bat reservoir, but evidence for that remains inconclusive [ ] . infections with mers-cov generally are thought to be mild or inapparent in camels [ ] , and are therefore of low economical or animal welfare significance. this systematic review was done to compile and analyse all published data on mers-cov in the global camel population to provide an overview of current knowledge on the distribution, spread and risk factors of mers-cov infections in dromedary camels as a basis for the design of intervention and control measures to prevent human infections. on may , a literature search on pubmed was performed, using the terms 'middle east respiratory syndrome coronavirus' and 'mers-cov'. using the term 'mers' did not result in any additional articles that fit the scope of this review. only articles published in english were included. two reviewers individually selected all original research articles containing laboratory evidence of mers-cov infections in dromedary camels in the field. articles that were mentioned in food and agriculture organization (fao) updates [ ] or in the references of included publications, but did not appear in the pubmed search were added. subsequently, abstracts, follow-up studies of mers-cov-positive camels and genome studies without prevalence data were excluded from the analysis. data on variables such as year of sampling, country, region, age, sex and animal origin were extracted and analysed. for each variable, the number of positive camels, total number of camels tested and the median percentage positivity was calculated. data from experimental infection studies were not included in this analysis, but they were included in the review to provide additional information and context to the field studies. additional information on the distribution and trade of dromedary camels was collected from references in the publications on mers-cov in camels and extracted from official fao and world organisation for animal health (oie) databases [ , ] . the additional literature on camel trade was collected in a less systematic way from pubmed. the literature search resulted in a total of papers (fig. ). forty-three research papers described the results of crosssectional studies in dromedary camel populations, six papers described outbreak investigations, including an analysis of camel samples, and four papers described longitudinal studies. in total, papers describe camel studies in the middle east, studies investigated camels from africa and the remaining seven surveys were from spain, australia, japan, bangladesh and pakistan (table ) . most recent fao statistics estimate the world population of camel to be around million [ ] , of which approximately % are dromedary camels [ ] . however, it is believed that the true population size is even larger due to inaccurate statistics and feral camels, such as the feral dromedary camel population in australia that is estimated to be around million [ ] . over % of the camel population lives in africa. the main camel countries are chad ( ), ethiopia ( ), kenya ( ), mali ( ), mauritania ( ), niger ( ), sudan ( ), somalia ( ) and pakistan ( ) [ ] (table ) . a large number of camels are being transported from the horn of africa to the middle east each year. these are mainly meat camels coming from the east of africa going to egypt, libya and the gulf states, and sudanese camels that are being imported into the middle east to participate in camel racing competitions [ ] . for example, the fao reported that somalia exported camels in [ ] . the largest camel market in africa is the birqash market near cairo (egypt), where camels from sudan and ethiopia are most common, but trade routes include animals from chad, somalia, eritrea and kenya [ ] . imported camels are usually quarantined for - days at the border before they are allowed to enter egypt [ ] . most somali and sudanese camels that are exported to the ksa are shipped from the ports of berbera and bosaso in north somalia to the ksa ports of jizan and jeddah [ ] . in general, only minor clinical signs of disease have been observed in animals infected with mers-cov and most mers-cov infections do not appear to cause any symptoms [ ] . disease symptoms that have been described after experimental and field infections are coughing and sneezing, respiratory discharge, fever and loss of appetite [ ] [ ] [ ] . although mers-cov rna can be detected in several organs after experimental infection, in studies of natural infectious virus it has only been detected in the tissues of the upper and lower respiratory tract and regional lymph nodes of the respiratory system in part of the infected camels. histologically, a mild-to-moderate inflammation and necrosis could also be seen on the upper and lower respiratory tract. no viral antigen or lesions were detected in the alveoli. histopathological examination showed that the nasal respiratory epithelium is the principal site of mers-cov replication in camels [ , ] . in one study investigating experimental infection of camels, mers-cov shedding started - days post-infection (dpi). in that study, infectious virus could be detected until dpi, and viral rna until dpi in nasal swab samples and, in lower amounts, in oral swab samples [ ] . no infectious virus or viral rna was detected in faecal or urine samples [ ] . viral rna detection in nasal, but also rectal swabs of camels after experimental infection until day , has been confirmed in a recent vaccine study [ ] . in the field surveys included in this review, mers-cov rna has been described in rectal swab samples, although other field studies report negative results [ , [ ] [ ] [ ] and when viral rna can be detected, the positivity rate of rectal swabs is lower compared with nasal swab samples [ , [ ] [ ] [ ] . oral swabs are usually negative or show a lower positivity rate even when nasal swabs test positive for mers-cov rna [ , , ] . some studies have reported mers-cov rna in milk samples [ , ] . longitudinal studies of camel herds show that pcr results of nasal swabs can remain positive after weeks [ , ] . when an interval of sampling of or months was maintained, nasal swabs become negative for viral rna in the next sampling round [ , ] . mers-cov infections have also been detected in camels with mers-cov antibodies, both in calves with maternal antibodies as well as older camels that had already acquired antibodies from a previous infection. however, virus replication and thus the virus load is generally lower in infected seropositive animals compared with seronegative camels [ , , , , , ] . little is known about the longevity of antibody titres after infection from longitudinal studies. a study following camels on a closed farm found that neutralizing antibodies remained consistent during a year [ ] , while other studies found that antibody titres rapidly drop by - -fold within a period often as short as weeks [ , ] . the first evidence of mers-cov in camels described so far is the detection of antibodies to mers-cov in camel sera from somalia and sudan from of which % tested positive [ ] . additional serological evidence of the widespread presence of mers-cov infection in camels, included in this review, has been found in additional countries: bangladesh, burkina faso, egypt, ethiopia, iraq, israel, jordan, kenya, ksa, mali, morocco, nigeria, oman, pakistan, qatar, spain, tunisia and the uae (fig. ). in addition, promed mail reported that virus-positive camels had been found in kuwait and iran, the latter reportedly in imported animals (archive number . and . ). in countries, serological findings were complemented with the finding of viral rna in dromedary camels: burkina faso, egypt, ethiopia, iraq, jordan, ksa, morocco, nigeria, oman, qatar and the uae. investigations of mers-cov circulation amongst dromedary camels in australia, japan, kazakhstan, usa and canada did not find any proof of mers-cov circulation. all countries where mers-cov circulates in the camel population, with the exception of spain (canary islands), pakistan and bangladesh, are located in the middle east or africa [ , ] . one out of camels that had mers-cov antibodies in bangladesh was born in bangladesh, others were imported from india [ ] . however, there have not been any additional reports of mers-cov in camels in india. there is no record of foreign origin of the seropositive camels from pakistan [ ] . moreover, in previous studies there had already been evidence of seropositive camels that originate from pakistan [ , ] . when combining serology data from all papers included in this review, the overall median seroprevalence of camels in africa is % ( / ; range - %), compared with a median seroprevalence of % ( / ; range - %) in camels from the middle east. based on viral shedding studies from african countries, the median rate of viral shedding was % ( / ; range - %), compared with % in camels from the middle east ( / ; range - %). the seroprevalence of mers-cov antibodies increases with age in camels, while the fraction of camels that test positive for mers-cov rna in their nasal swabs decreases with age [ , , , , ] . when all serological results of papers that included sufficient age information is combined, the median seroprevalence of camels aged under years is % ( / ; range - %), while the age groups - years ( / ; range - %) and over years old ( / ; range - %) had a combined median seroprevalence of %. in the virological studies reporting age breakdown, the median rate of nasal shedding in - years old camels was % ( / ; range - %) of cases, compared with % ( / ; range - %) in camels older than years. some individual studies show a significantly higher seroprevalence in female camels compared with males [ , ] , while others show the opposite [ ] or do not find any significant difference [ , ] . similar disagreeing results are published for the presence of mers-cov rna in male vs. female camels [ , , , ] . in the studies in this review where sex of camels was recorded, a total of serum samples from female camels and samples from male camels were collected and analysed for mers-cov antibodies, compared with vs. nasal swabs for viral rna testing. approximately three times more female camels were sampled at farms, while male camels were in the majority in studies that looked at mers-cov prevalence of camels at slaughterhouses, live animal markets and quarantine areas. the overall median seroprevalence of male and female camels in our review is % and %, respectively (range - %; excluding results from israel and kazakhstan). the median percentage of presence of viral rna is % in nasal swabs of male camels (range - %) compared with % in female camels (range - %), in our review. in several studies, camels that were sampled at animal markets or quarantine facilities were seropositive more often than camels at farms [ , , , ] . combining serological laboratory results of camels in our review with sufficient background information with regard to the sampling location does not result in the same pattern, with a median seroprevalence of % ( / ; range - %; excluding australia and spain) in camels from farms and % ( / ; range - %) in the camel population sampled at markets and quarantine facilities. studies in egypt found a significantly higher pcr positivity rate in camels sampled in abattoirs or quarantine facilities, but these results could not be confirmed by other papers in this review [ , ] . when comparing differences in seroprevalence or virus rnapositive rate in nomadic vs. sedentary camel herds, some authors did not find a statistical difference between the two herd management types [ , ] , while others found some evidence of higher seroprevalences in nomadic herds [ , ] . one study in kenya looked at the differences between herds with different levels of isolation, and did not find significant differences in mers-cov antibody levels [ ] . most studies that compared local camels with imported camels suggested that imported camels are seropositive for mers-cov more often [ , , , , ] , although not all differences were significant. two studies in egypt found a significantly higher rna positivity rate in imported camels from east africa compared with domestically bred camels [ , ] , while another study executed in the ksa found a significantly higher number of mers-cov rna-positive results amongst local camels vs. camels from sudan and somalia [ ] . although mers-cov was detected almost year-round in camels, some studies show a relatively higher seroprevalence and viral detection during the cooler winter months [ , , , ] . mers-cov antibodies have been detected in llamas and alpacas in israel and in alpacas in qatar [ , ] . to date, no mers-cov antibodies or viral rna have been detected in bactrian camels [ , , [ ] [ ] [ ] [ ] (table and table ). swine, goats and horses that were included in the field surveys in our review all tested negative for mers-cov rna and antibodies [ , , , [ ] [ ] [ ] [ ] [ ] . mers-cov antibodies were detected in two studies in sheep in egypt and qatar, although in very low numbers [ , ] . however, most surveys that investigated sheep did not find evidence of mers-cov infection or exposure [ , , , , , [ ] [ ] [ ] [ ] ] . the publications in this review show that the mers-cov mainly circulates in dromedary camel populations in the middle east and part of africa, and has been infecting dromedary camels in africa for more than three decades. antibodies have also been found in arabic camel sera from the early s [ , ] . however, mers-cov was discovered until , after the first human cases appeared [ ] , which is probably due to the minor clinical symptoms of mers-cov infections in camels [ ] . most camel surveys were conducted in the middle east and some northern and eastern african countries, but significant data gaps currently still exist in the north and west of africa, in countries that have camel populations of to more than a million animals, such as algeria, libya, mauritania and niger. even less is known about the central asian region. some evidence of mers-cov circulation in camels of pakistan and bangladesh was recently published, but data is lacking from afghanistan and india. knowledge on the presence of mers-cov in the animal reservoir is a crucial first step to assess whether mers-cov could be a relevant public health threat in these regions. mers-cov infections are mainly detected in calves and young camels [ , ] . the research included in this review shows that the igg positivity rate increases gradually in dromedary camels of increasing age while the mers-cov rna detection rate decreases. maternal igg antibodies in camels are acquired through the intake of colostrum during the first h post-parturition. after h, antibody levels in the dam's milk decrease rapidly [ ] . one study showed that maternal antibodies in calves peak at days postparturition and decline in the following months. after - months, over half of the calves did not have maternal neutralizing antibodies in their serum any longer [ ] . however, in other field studies, the titre of mers-cov-specific antibodies is still low at month of age and increases with age in dromedary calves [ , ] . a lower or undetectable antibody levels in young camels is likely to explain the higher mers-cov rna detection rate. in adult camels, a much higher mers-cov seroprevalence can be found, which is probably due to a long-lasting immune response against a mers-cov infection or multiple re-infections with mers-cov. immunity is not sterilizing, as mers-cov infection and shedding have also been shown in adult camels that have mers-cov antibodies [ , , , , , ] . several articles have analysed seroprevalence and virus shedding data in relation to factors, other than age, that may explain differences in seroprevalence and mers-cov rna-positive rate in camels, such as sex, sampling location, herd characteristics and animal origin. our review shows that there is considerable heterogeneity in results. in addition, comparison between studies is difficult given the lack of standardisation of study designs. a key factor to consider when comparing studies is the difference in distribution of male and female camels amongst different disciplines of camel husbandry. females are mainly used for milking and reproduction. as a result, they often stay at farms. male camels, especially of young age (< year old), are the predominant sex in slaughterhouses and amongst camels used for transport [ , ] . this also influences the risk profile of acquiring a mers-cov infection. female camels are in closer contact with calves, who are more susceptible to infection and shed virus in higher quantities compared with older camels [ ] . on the other hand, meat and transport camels (predominantly male) travel more, leading to increased contact with other camels and camel herds, and therefore a higher chance of exposure to mers-cov. some papers in this review suggest that there is a generally lower infection rate of domestically bred camels and camels on farms compared with imported camels and camels on animal markets or in quarantine facilities. this may be explained by the same increased contact rate and mixing of camel herds, leading to an increased chance of mers-cov exposure and spread. the increase in mers-cov circulation in winter and spring can have multiple explanations. firstly, the winter is the calving season [ ] , which leads to a larger proportion of young animals that usually have a higher number of mers-cov infections and virus excretion. moreover, in winter season, there is a major increase of camel and human movements due to camel racing competitions, camel breeding, trading and movements to grazing grounds, which increases the chance of virus spread. additionally, cooler temperatures may facilitate coronavirus survival in the environment [ ] . in experimental studies, llama's and alpaca's are shown to be susceptible to infection with mers-cov [ , ] , which was confirmed by two papers in our review, describing serologically positive llamas and alpacas in israel and alpacas with mers-cov neutralizing antibodies in qatar [ , ] . in experimental settings, animal-to-animal transmission has been shown for alpacas, making them a possible risk population for human infections [ ] . two studies in our review also found anti-mers-cov antibodies in sheep [ , ] but experimental inoculation of sheep did not result in mers-cov replication or antibody development [ , ] . however, the dpp receptor, the entry receptor for mers-cov, is present in sheep tissues, making it possible for the virus to bind to the sheep respiratory tract which may explain the finding of mers-cov antibodies [ ] . pigs also express the dpp receptor in their respiratory tract, and viral replication in experimental settings has been shown for pigs, but no antibodies or mers-cov rna have been found in pigs during field surveys [ , ] . this may be explained by the limited viral shedding in pigs and the absence of animal-to-animal transmission [ , ] . we show that dromedary camels are present in large parts of the african and asian continent, and that mers infections in dromedary camels are widespread. however, human infections due to spill-over from the dromedary camel reservoir have not been reported in africa [ ] . several explanations for the difference in human cases between the arabian peninsula and africa have been suggested, such as differences in cultural habits, camel husbandry, prevalence of comorbidities, under detection or genetic factors in the local population [ ] . moreover, west african viruses were found to be phylogenetically and phenotypically distinct from the mers-cov viruses that caused human disease in the middle east [ ] . increased knowledge on the animal reservoir of mers-cov needs to be combined with research on mers prevalence and risk factors in humans to assess the true public health risk. moreover, the absence of human disease, combined with the mild symptoms in camels, caused by mers, will likely have a negative effect on the willingness to implement interventions and the cost-effectiveness of possible interventions in some areas. since the discovery of mers-cov in , the dromedary camel has been identified as the animal reservoir of human infections with the mers-cov. however, the exact route of human primary infections is still unknown. moreover, the scale of the spread and prevalence of mers-cov in the camel reservoir is not fully known yet since there is still a lack of mers-cov prevalence data in some countries that harbour a very significant proportion of the world camel population. however, knowledge of the animal reservoir of mers-cov is essential to develop intervention and control measures to prevent human infections. prospective studies that include representative sampling of camels of different age groups and sex, within the different husbandry practices, are needed to fully understand the patterns of mers-cov circulation. such studies are important as they may give more information on critical control points for interventions to reduce the circulation of mers-cov and/or exposure of humans. author orcids. r. s. sikkema, - - - financial support. this study was financially supported by the european commission's h programme under contract number (http:// www.compare-europe.eu/). none. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers situation update march middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation 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linked to infected dromedaries imported from oman to united arab emirates middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate phylogenetic analysis of merscov in human and camels in iraq no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection in livestock and temporal relation to locations and seasons key: cord- -str r a authors: al ghamdi, mohammed; alghamdi, khalid m.; ghandoora, yasmeen; alzahrani, ameera; salah, fatmah; alsulami, abdulmoatani; bawayan, mayada f.; vaidya, dhananjay; perl, trish m.; sood, geeta title: treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: str r a background: middle eastern respiratory syndrome coronavirus (mers-cov) is a poorly understood disease with no known treatments. we describe the clinical features and treatment outcomes of patients with laboratory confirmed mers-cov at a regional referral center in the kingdom of saudi arabia. methods: in , a retrospective chart review was performed on patients with a laboratory confirmed diagnosis of mers-cov to determine clinical and treatment characteristics associated with death. confounding was evaluated and a multivariate logistic regression was performed to assess the independent effect of treatments administered. results: fifty-one patients had an overall mortality of %. most patients were male ( %) with a mean age of years. almost a quarter of the patients were healthcare workers ( . %) and % had a known exposure to another person with mers-cov. survival was associated with male gender, working as a healthcare worker, history of hypertension, vomiting on admission, elevated respiratory rate, abnormal lung exam, elevated alanine transaminase (alt), clearance of mers-cov on repeat pcr polymerase chain reaction (pcr) testing, and mycophenolate mofetil treatment. survival was reduced in the presence of coronary artery disease, hypotension, hypoxemia, cxr (chest x-ray) abnormalities, leukocytosis, creatinine > · mg/dl, thrombocytopenia, anemia, and renal failure. in a multivariate analysis of treatments administered, severity of illness was the greatest predictor of reduced survival. conclusions: care for patients with mers-cov remains a challenge. in this retrospective cohort, interferon beta and mycophenolate mofetil treatment were predictors of increased survival in the univariate analysis. severity of illness was the greatest predictor of reduced survival in the multivariate analysis. larger randomized trials are needed to better evaluate the efficacy of these treatment regimens for mers-cov. coronaviruses cause a spectrum of illness from asymptomatic disease to respiratory failure. early reports of coronavirus infections suggested that most infections were mild until the sars epidemic that was associated with significant morbidity and mortality [ ] . in september , a novel coronavirus was identified in a -year old man in saudi arabia [ ] . a second case was identified in a qatari patient hospitalized in the united kingdom [ ] . the two coronaviruses were genetically identical and similar to isolates obtained from bats [ ] . in july , the coronavirus study group named this new virus middle east respiratory syndrome coronavirus (mers-cov) [ ] . as of december , , there have been cases worldwide with deaths [ ] . the epidemiology and clinical manifestations of this disease have described a spectrum of illness from asymptomatic infection to severe respiratory failure and death. the overall mortality rate remains at % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . importantly, there are no known effective treatments. in there was an increase in mers-cov cases reported from the jeddah region of saudi arabia. to describe the changing epidemiology and outcomes, we report the clinical features and treatment outcomes of patients admitted to a regional referral hospital in jeddah, saudi arabia. king fahd general hospital is an -bed hospital in jeddah, kingdom of saudi arabia and is a regional coronavirus referral center. there are icu beds and one infectious disease physician that serves the hospital. between january through december , all patients admitted or transferred to king fahd hospital with a positive mers coronavirus pcr from clinical nasal swabs or nasopharyngeal aspirates were included. all pcr testing was performed at the ministry of health regional lab in jeddah. the magna pure compact/ magna pure (roche) automated system was used to extract rna from samples. primers and probes for upe and orf a targets of mers-cov were used from tib molbiol (germany) along with master mix from roche for the light cycler ii (roche) were used to amplify upe and orf a gene targets. samples that tested positive for both upe and orf a gene targets with a cycle threshold time of less than were considered confirmed cases. positive and negative controls were used to monitor the amplification process & to check for any inhibition of amplification. medical charts for all patients were reviewed and data abstracted on standardized data collection forms by an infectious disease trained physician. demographic, clinical and laboratory data were entered into a database. to understand the epidemiology, age was categorized as < , - and > . hypotension was defined as blood pressure < / mm hg, tachypnea as a respiratory rate greater than , hypoxia as an oxygen saturation < %, thrombocytopenia as platelets < , / cubic millimeter, leukopenia was defined as a white blood cell count < cells/cubic millimeter and leukocytosis as a white blood cell count > , cells/ cubic millimeter. renal insufficiency was defined as a creatinine > . mg/dl. liver function abnormalities were defined as a lactate dehydrogenase (ldh) > u/liter, alanine transaminase (alt) > u/liter and aspartate aminotransferase (ast) > u/liter. immunosuppression was defined as aids, history of organ transplant, neutropenia, known malignancy, taking immunosuppressive medication and congenital immunodeficiency. pregnancy was considered an immunosuppressed state. a modified acute physiologic and chronic health evaluation (apache ) score was calculated using age, temperature, mean arterial blood pressure, respiratory rate, potassium, creatinine, acute renal failure, and comorbid conditions to estimate severity of illness [ ] . pao was estimated using pulse oximetry oxygen saturation results and hematocrit was calculated by multiplying the hemoglobin times three. all statistical analyses were performed using stata software (version . , college station, tx). the percent distribution of clinical variables among patients who survived and those who died were compared using the fisher exact test. a multivariate logistic regression was done on treatments administered and severity of illness to determine which treatments were associated with survival. mycophenolate mofetil was not included in this logistic regression analysis because % of patients receiving mycophenolate mofetil survived. the association between severity of illness and treatments administered was assessed by performing a linear regression of treatments administered onto the modified apache score. there were a total of cases, thirty patients ( . %) of whom were saudi nationals, and ( . %) were foreign nationals. the median age was years old (iqr . - ). most were male (n = , . %). twenty-one patients ( . %) had exposure to a known patient with mers coronavirus and ( . %) were healthcare workers. none of the patients had animal exposure. two patients ( . %) were on pilgrimage to mecca. overall, % of patient had at least one co-morbid condition. seventeen patients had diabetes ( . %), had hypertension ( %), ( . %) had end stage renal disease, eight ( . %) had coronary artery disease and six ( . %) patients were immunosuppressed, two of whom were pregnant. patients received a variety of novel treatments including immunosuppressants and antivirals. forty-two ( . %) patients received broad-spectrum antibiotics and five ( . %) received hydrocortisone. thirty one patients received antiviral treatment. twenty-three patients ( . %) were treated with interferon beta, eight ( . %) were treated with interferon alpha. a variety of anti-viral combinations were used. eight patients ( . %) received mycophenolate mofetil, seven of these patients received it in combination with interferon beta. nineteen ( . %) patients required intensive care unit (icu) care, and patients received extracorporeal membrane oxygenation (ecmo). all patients treated in the icu and all patients receiving ecmo died. in this recent cohort, when comparing survivors to nonsurvivors, survival was associated with male gender, vomiting on admission, elevated respiratory rate, abnormal lung exam on physical exam, working as a healthcare worker, history of hypertension, elevated alt, clearance of mers cov on repeat pcr testing, and receiving mycophenolate mofetil or beta interferon (table ). in contrast, markers of severe disease like hypotension, hypoxemia, chest radiographic abnormalities, leukocytosis, elevated creatinine, thrombocytopenia, anemia, renal failure were associated with death. treatments given were based as indicated based on the clinical assessment of the infectious disease consult team. thirty-one patients received antivirals, ribavirin or alpha or beta interferon, and patients received immunosuppressive medication. most patients received a combination of alpha interferon and ribavirin ( , . %), beta interferon and ribavirin ( , . %) or beta interferon alone ( , . %). two patients received alpha interferon alone ( . %). eight patients received mycophenolate mofetil ( . %) and seven of them received this in combination with beta-interferon. five patients received hydrocortisone; two in combination with beta interferon and ribavirin and in combination with alpha interferon and ribavirin. all eight patients given mycophenolate mofetil survived therefore mycophenolate mofetil could not be evaluated in this model. while the results of the univariable analysis demonstrated improved survival in patients treated with betainterferon and mycophenolate mofetil, the multivariable analysis which included a marker of severity of illness, demonstrated a strong association between severity of illness and reduced survival, and no association between treatment with beta interferon and survival. mycophenolate mofetil was not evaluable in this model ( table ). in analyzing the relationship between severity of illness and treatments administered, beta interferon and mycophenolate mofetil were given to less severely ill patients (table ) discussion mers-cov is an emerging disease for which the initial epidemiology has been described, but in-depth clinical studies and the role of therapy in incompletely understood. while the clinical features for mers-cov have been described in several large case series [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , there is a paucity of literature on therapy. our results from a relatively large number of patients demonstrate similar clinical features and mortality to previous studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in our cohort, treatment with beta interferon and mycophenolate mofetil may be predictive of survival, but the greatest predictor of survival is the severity of illness on presentation. improved diagnostics have demonstrated an expanded spectrum of disease that includes less severe cases than previously reported. we now understand that mers-cov causes an acute respiratory disease syndrome and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this may be partially related to the epidemiology of increased disease transmission in healthcare settings rather than a true host risk factor. laboratory findings have been nonspecific and consistent with other viral infections. thrombocytopenia ( %) and lymphopenia ( %) have been commonly described in these patients [ , [ ] [ ] [ ] [ ] [ ] ] . forty three percent had acute kidney injury [ , [ ] [ ] [ ] ] and - % had cxr abnormalities with bibasilar infiltrates as the most common finding [ - , , ] . the outcomes in these more severely ill patients remain poor. between - % required icu care [ , , , ] and - % in the icu setting required invasive ventilation for a median of - days [ , , ] . in addition to mechanical ventilation, several patients have received extracorpeal membrane oxygenation (ecmo) to support ventilation. from non-randomized data from the world health organization, five out of six patients receiving ecmo died [ ] . fifty-eight to % required renal replacement therapy [ , , ] and - % of hospitalized patients died [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the severity of illness can be partially explained by the widespread lung disease caused by mers-cov and it appears that mortality in those patients requiring intensive care is extremely high. although no autopsy data is available, in explanted lung, infection with mers-cov causes widespread infection and alveolar disease [ , ] . the clinical features in our cohort similarly also show a high proportion of patients with fever ( %) and cough ( . %) shortness of breath ( %), and almost one third of patients ( . %) with gastrointestinal symptoms. our cohort consisted of ill patients with hypotension ( . %), tachypnea ( . %) and hypoxia ( %). thirty seven percent required icu care and patients received ecmo. similar to previous results, all of the patients who received ecmo died [ ] . there is no known effective treatment for mers cov. many compounds have been screened in vitro for possible activity against this coronavirus [ ] [ ] [ ] [ ] , however, the in vivo efficacy has not been subjected to clinical investigation. in vitro data suggests that mers-cov inhibits host interferon production through various molecular pathways [ ] [ ] [ ] [ ] [ ] [ ] mycophenic acid, the active agent of prodrug mycophenolate mofetil, and cyclosporine strongly inhibit mers coronavirus in human and monkey cell lines even more so than they inhibit sars coronavirus [ , [ ] [ ] [ ] . interferon alpha and interferon beta reduce mers coronavirus replication in explanted lung tissue [ ] . in vivo, comparing host response in two patients with mers coronavirus and differing outcomes, the patient who was able to clear mers cov infection was able to mount an interferon response and the patient who died had low levels of interferon alpha suggesting a therapeutic role for interferon [ ] . the combination of interferon alpha and ribavirin has been used successfully in rhesus monkeys infected with mers coronavirus [ ] , and in a few small case series [ ] [ ] [ ] . beta-interferon seems to be an even more potent inhibitor of mers coronavirus in vitro [ ] [ , [ ] [ ] [ ] . one small study with exceptionally high mortality rates using interferon beta for treatment found no difference in mortality between interferon beta use and interferon alpha use [ ] . our data, albeit from a retrospective cohort support the findings that interferon beta is associated with a decrease in mortality. there are limited data on the efficacy of treatment regiments for this virulent disease. we present data from a retrospective cohort of ill patients with mers-cov and the results of the evaluation of the clinical efficacy of beta interferon beta, alpha interferon, ribavirin and mycophenolate mofetil in addition to routine supportive care. forty five percent of patents ( patients) received interferon beta and in this cohort, sixteen percent of patients received interferon alpha ( patients) and % of patients ( patients) received ribavirin, either in conjunction with interferon alpha or interferon beta, and patient received mycophenolate mofetil. patients receiving beta interferon and mofetil had improved survival, however this was confounded by the severity of illness on presentation for beta interferon. all of the patients who received mycophenolate mofetil survived however because of the small number, we could not analyze the independent efficacy of mycophenolate mofetil. while this is a relatively large series of mers-cov cases, the primary limitation of our study is that it is a retrospective review of cases and not a randomized trial and thus subject to confounding as seen in our cohort. we used a modified apache score without all of the clinical variables, which may have underestimated the association of severity of illness with reduced survival. importantly, the mortality in patients receiving additional therapies that modulate the immune response was low. all of the eight patients who received mycophenolate mofetil in our study survived. hence, it may be reasonable to further study this agent in controlled trials. this observational study investigates novel treatment options like beta interferon and mycophenolate mofetil for mers-cov in humans which have in vitro activity. our cohort demonstrated severity of illness is an important effect modifier and needs to be considered in evaluating novel agents. to better assess the efficacy of these therapies, international prospective randomized trials with adequate numbers of patients are needed to further evaluate the impact of these treatments in addition to routine supportive care when compared to other treatment options. this study was reviewed and approved by johns hopkins university institutional review board and the directorate of health affairs. data supporting the findings are in the manuscript, additional data available upon request. abbreviations aids: acquired immune deficiency syndrome; alt: alanine transaminase; apache : acute physiologic and chronic health evaluation; ast: aspartate aminotransferase; cxr: chest x ray; ecmo: extracorporeal membrane oxygenation; icu: intensive care unit; ldh: lactate dehydrogenase; mers co-v: middle eastern respiratory syndrome coronavirus; pcr: polymerase chain reaction. the authors declare that they have no competing interests. authors' contributions ma conceived of the study, participated in its design and helped draft the manuscript. ka participated in data collection and analysis, and reviewed the manuscript. yg participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. fs participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. mb participated in data collection and analysis, and reviewed the manuscript. tmp participated in the design and analysis as well as the writing of the manuscript. dv participated in the statistical analysis of the study. gs helped analyze the data and write the manuscript. all authors read and approved the final manuscript. the severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia patient with new strain of coronavirus is treated in intensive care at london hospital latest outbreak news from promed-mail: novel coronavirus -middle east middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group who summary family cluster of middle east respiratory syndrome coronavirus infections ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection jordan mers-cov investigation team. hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients apache ii: a severity of disease classification system in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection evaluation of ssya - as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses screening of an fda-approved compound library identifies four smallmolecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus molecular pathology of emerging coronavirus infections middle east respiratory syndrome coronavirus accessory protein a is a type i interferon antagonist pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the structural and accessory proteins m, orf a, orf b, and orf of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists the orf b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? treatment with interferon-α b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon (ifn)-alpha- b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α a or ifn-β a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study there are no acknowledgements. there was no external funds provided for this project. key: cord- - oykgp h authors: omrani, ali s; saad, mustafa m; baig, kamran; bahloul, abdelkarim; abdul-matin, mohammed; alaidaroos, amal y; almakhlafi, ghaleb a; albarrak, mohammed m; memish, ziad a; albarrak, ali m title: ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study date: - - journal: lancet infect dis doi: . /s - ( ) -x sha: doc_id: cord_uid: oykgp h background: middle east respiratory syndrome coronavirus (mers-cov) infection is associated with high mortality and has no approved antiviral therapy. we aimed to compare ribavirin and interferon alfa- a treatment for patients with severe mers-cov infection with a supportive therapy only. methods: in this retrospective cohort study, we included adults (aged ≥ years) with laboratory-confirmed mers-cov infection and pneumonia needing ventilation support, diagnosed between oct , , and may , , at the prince sultan military medical city (riyadh, saudi arabia). all patients received appropriate supportive care and regular clinical and laboratory monitoring, but patients diagnosed after sept , , were also given oral ribavirin (dose based on calculated creatinine clearance, for – days) and subcutaneous pegylated interferon alfa- a ( μg per week for weeks). the primary endpoint was -day and -day survival from the date of mers-cov infection diagnosis. we used χ( ) and fischer's exact test to analyse categorical variables and the t test to analyse continuous variables. findings: we analysed patients who received ribavirin and interferon (treatment group; initiated a median of days [range – ] after diagnosis) and who did not (comparator group). baseline clinical and laboratory characteristics were similar between groups, apart from baseline absolute neutrophil count, which was significantly lower in the comparator group ( · × ( )/l [sd · ] vs · × ( )/l [ · ]; p= · ). ( %) of patients in the treatment group had survived after days, compared with seven ( %) of in the comparator group (p= · ). after days, six ( %) of and four ( %) of , respectively, had survived (p= · ). adverse effects were similar between groups, apart from reduction in haemoglobin, which was significantly greater in the treatment group than in the comparator group ( · g/l [sd · ] vs · g/l [ · ]; p= · ). interpretation: in patients with severe mers-cov infection, ribavirin and interferon alfa- a therapy is associated with significantly improved survival at days, but not at days. further assessment in appropriately designed randomised trials is recommended. funding: none. since it was fi rst described in september, , cases of middle east respiratory syndrome coronavirus (mers-cov) infection have been confi rmed, of which were fatal. , cases occur sporadically, as community clusters or as hospital outbreaks, and range in severity from asymptomatic or mild illness to rapidly progressive and fatal disease. [ ] [ ] [ ] [ ] [ ] the management of patients with mers-cov infection consists of a combination of supportive measures, antimicrobial therapy for any associated bacterial or viral infections, and strict implementation of appropriate infection control precautions. so far, no antiviral therapy has been approved for the treatment of patients with mers-cov infection. several therapeutic interventions for coronavirus were investigated during the large multinational outbreak of severe acute respiratory syndrome (sars) in . , reviews of the available scientifi c literature suggest that a combination of ribavirin and interferon might be of benefi t in patients with severe mers-cov infection. , , furthermore, this combination was shown to inhibit mers-cov in cell culture and seemed to improve outcomes in an animal study. , both agents are associated with substantial potential adverse eff ects and hence their clinical use should be carefully balanced against any potential harm. we aimed to assess outcomes of a treatment programme for patients with severe mers-cov infection that consisted of oral ribavirin and subcutaneous pegylated interferon alfa- a. we report the results and outcomes in patients given treatment in accordance with this protocol by comparison with a historical group who received supportive therapy only. this single-centre, retrospective cohort study included individuals who were diagnosed with laboratoryconfi rmed mers-cov infection between oct , , and may , , at the prince sultan military medical city (riyadh, saudi arabia). eligible patients were those aged years or older with severe pneumonia needing invasive or non-invasive ventilation. no exclusion criteria were applied at this stage. mers-cov infection was diagnosed by rt-pcr testing of respiratory tract samples for mers-cov upe, orf b, and n genes. all rt-pcr tests for mers-cov were done at the saudi ministry of health regional laboratory in jeddah and riyadh, saudi arabia. pneumonia was defi ned as new, otherwise unexplained, lower respiratory tract symptoms such as cough or shortness of breath with at least one systemic feature such as fever or chills, and new focal chest signs on examination, in addition to new or progressive pulmonary infi ltrates on chest radiograph. from sept , , all eligible patients were off ered treatment with oral ribavirin and subcutaneous pegylated interferon alfa- a after informed written consent had been obtained from the patients themselves or their next of kin. the treatment protocol was approved by pharmacy and therapeutics committee. the study was approved by the research ethics committee at the prince sultan military medical city to allow retrospective access to patients' records and fi les. pegylated interferon alfa- a (pegasys; roche pharmaceuticals, basel, switzerland) was given by subcutaneous injection at a dose of μg per week for weeks. the dose of oral ribavirin (copegus; roche pharmaceuticals) was adjusted according to calculated creatinine clearance and continued for - days. patients with a creatinine clearance of greater than · ml/sec/m received a mg loading dose, followed by mg every h for days then mg every h for - days; those with a creatinine clearance of · - · ml/sec/m² received a mg loading dose, followed by mg every h for days then mg every h for - days; and those with a creatinine clearance of < · ml/sec/m² or on dialysis received a mg loading dose, followed by mg every h for days then mg every h for - days. patients did not receive ribavirin and interferon alfa- a therapy if they were diagnosed before sept , , or if they declined consent. all patients received appropriate supportive care such as supplementary oxygen, vasopressor therapy, and renal replacement as needed. hydrocortisone mg daily was given to patients with refractory septic shock and continued until vasopressor therapy was no longer needed. in addition to regular clinical monitoring, renal function, liver enzymes, and blood count were assessed at baseline and daily throughout the treatment course. conscious patients were monitored for any clinical signs of depression or acute confusion. patients who received ribavirin and interferon alfa- a therapy were classifi ed as being in the treatment group and those who did not made up the comparator group. two investigators, aso and kb, both of whom were masked to group allocation and the patients' clinical outcomes, compared baseline characteristics of the two groups. immunosuppressive therapy ( %) ‡ ( %) · data are number (%) or mean (sd). apache ii=acute physiology and chronic health evaluation ii. sofa=sequential organ failure assessment. *only patients were assessed. †only patients were assessed. ‡only patients were assessed. the primary endpoints for the study were -day and -day survival from the date of mers-cov infection was diagnosis. we used χ² and fischer's exact tests for categorical variables, whereas we used the student's t test for continuous variables to assess the diff erences in means of the two groups. the log-rank test was used for assessing survival diff erences between the two groups. our cutoff for statistical signifi cance was · . the graphical and statistical tests suggested that the pro portionalhazard assumption was not violated. we did statistical analyses using microsoft excel and stata statistical software, release . no external funding was received for this study. zm had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. individuals were diagnosed with mers-cov infection between oct , , and may , . baseline characteristics were generally similar between patients who received ribavirin and interferon alfa- a therapy and those who did not (table ) , with the exception that end-stage renal failure was present in three patients who did not receive study treatment and in none who did. after excluding ineligible patients, patients were included in the study: in the treatment group and in the comparator group (fi gure ). the mean age of all patients was · years (sd · ), and ( %) were men (table ) mean absolute neutrophil count was signifi cantly lower in the treatment group than in the comparator group (table ) ; however, no other statistically signifi cant differences in baseline characteristics or support measures were noted between the two groups (tables , ). of all patients, ( %) were still alive days after diagnosis of mers-cov infection, whereas at days only ten ( %) had survived. ( %) of patients in the treatment group were alive days after diagnosis, compared with seven ( %) of in the comparator group (p= · ). however, six ( %) of patients in the treatment group survived up to days from diagnosis of mers-cov infection, whereas four ( %) of did in the comparator group (p= · ; fi gure ). ribavirin and pegylated interferon therapy was well tolerated by the treatment group with no premature discontinuation secondary to adverse eff ects. however, the mean drop in haemoglobin over the treatment course was signifi cantly greater in the treatment group ( · g/l [sd · ]) than in the comparator group eff ective treatment interventions for patients with severe mers-cov infection are still urgently needed. in critically ill patients with severe mers-cov infection, our study shows that ribavirin and pegylated interferon alfa- a therapy is associated with a signifi cant -day survival benefi t compared with standard treatment. -day survival also seemed to improve with ribavirin and pegylated interferon alfa- a therapy, but the diff erence between groups was not signifi cant (panel). the loss of a signifi cant survival diff erence over time might be partly explained by most patients in our cohort having several comorbidities with high apache ii and sofa scores. mortality is known to be very high in patients with severe mers-cov infection who need critical-care support. therefore, long-term survival benefi t, if present, might be diffi cult to show in smaller studies. treatment with ribavirin and interferon was well tolerated in our study. the only adverse event that was signifi cantly worse in the treatment group was mean decrease in haemoglobin ( . g/l in the treatment group compared with · g/l in the comparator group). anaemia is a well recognised complication of ribavirin therapy and was noted previously in studies investigating the role of ribavirin in the treatment of sars coronavirus infection. , of note, receipt of packed red blood cells was not signifi cantly diff erent between the treatment and comparator groups in our study. furthermore, no treatment discontinuations occurred as a result of anaemia. therefore, the risk of ribavirin-associated anaemia-although substantial and in need of careful monitoring-might not hinder the use of ribavirin for patients with severe mers-cov infection, especially if a survival benefi t can be confi rmed. baseline absolute neutrophil count was signifi cantly lower in the treatment group and therefore a signifi cantly lower minimal absolute neutrophil count during the course of the illness is not surprising. several investigators showed that interferon α has useful in-vitro activity against mers-cov. , , however, when compared with interferon α and interferon γ, interferon β seems to have the most potent inhibitory in-vitro activity against mers-cov. ribavirin has slight anti-mers-cov activity in vitro when used alone or in combination with interferon α. , mycophenolic acid is another compound that exhibits signifi cant in-vitro activity against mers-cov. of compounds screened, were active in cell culture against mers-cov. although only the combination of interferon α plus ribavirin has so far undergone in-vivo assess ment against mers-cov, many others are potential candidates for further clinical assessment. one of the limitations of our study is its small size. however, only two previous reports of clinical use of ribavirin and interferon for mers-cov infection have been published. , in a retrospective report, fi ve patients with severe mers-cov infection, all of whom had signifi cant comorbidities and needed mechanical ventilation, received a com bination of ribavirin and pegylated interferon alfa- b a median of days after admission. none of the patients survived and the investigators concluded that late commencement of therapy might not be benefi cial. in another report, a patient with severe mers-cov infection received ribavirin and interferon therapy with good clinical response and no signifi cant adverse eff ects. our study, albeit small, is the largest clinical investigation so far to assess the use of this combination in the treatment of patients with severe mers-cov infection. although baseline characteristics of our treatment and comparator groups seem to be reasonably balanced, substantial diff erences might not be apparent because of the small number of patients in the study. our study is also limited by its retrospective, nonrandomised nature. inevitably, selection and unmeasured confounding bias cannot be completely excluded. undoubtedly, new interventions should ideally be assessed in randomised, controlled clinical trials. however, such an approach is generally accepted to not always be practically feasible in the context of an emerging and relatively uncommon disease such as mers-cov infection. we carefully selected our comparator group, ensuring that the two cohorts were matched as closely as possible in their clinical characteristics and treatment interventions other than the receipt of ribavirin and interferon. we removed three individuals who had outlying baseline serum creatinine from the comparator group to minimise the risk of any spurious conclusions driven by clinical characteristics that might be potentially detrimental to clinical outcome. clinical outcomes for each individual were masked from investigators who selected patients and did matching assessments. the absence of serial viral load measurement in lower respiratory tract samples in our study makes it impossible to show any association between temporal viral load changes and antiviral therapy. such measurements should be included in any future clinical studies exploring the therapeutic benefi t of any antiviral intervention for patients with mers-cov infection. severe mers-cov is associated with poor overall survival. treatment with oral ribavirin and subcutaneous pegylated interferon alfa- a is associated with signifi cantly improved survival at days, but not at days. the combination is associated with signifi cant falls in haemoglobin, but no other signifi cant adverse eff ects were noted. treatment with ribavirin and pegylated interferon might be considered in patients with severe mers-cov infection, provided that adequate monitoring and assessment can be ensured. further assessment, including in patients with less severe mers-cov infection, in appropriately designed randomised trials, is recommended. this study was initiated and designed by aso, mms, zam, and ama. aso, mms, ab, ma-m, aya, gaa, mma, zam, and ama obtained and collated patient data. kb undertook all statistical analyses for the study. aso and kb prepared all tables and fi gures. aso, mms, zam, and ama wrote the fi rst draft of the manuscript and all authors reviewed and contributed to subsequent drafts and the fi nal report. aso has received consultancy fees from gilead, pfi zer, msd, and viiv; payment for lectures from pfi zer, msd, glaxosmithkline, and sanofi -aventis; and sponsorship to attend international meetings and conferences from msd, pfi zer, biopharma, bristol-myers squibb, and janssen-cilag. ab has received travel funding to attend an international meeting from pfi zer. gaa has received travel funding to attend an international meeting from edwards lifesciences. all other authors declare no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov)- th update epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case hospital outbreak of middle east respiratory syndrome coronavirus 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international guidelines for management of severe sepsis and septic shock ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon (ifn)-alpha- b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada severe acute respiratory syndrome: report of treatment and outcome after a major outbreak we thank staff of prince sultan military medical city, riyadh, saudi arabia, for the clinical care given to the patients and for facilitating access to the relevant medical records. we searched pubmed for reports published in english any time before june , , with the search term "[(mers-cov or hcov-emc or novel coronavirus) and (therapy or interferon or ribavirin]". we found one animal study, two small case series in human beings, , and several in-vitro studies. , [ ] [ ] [ ] [ ] the data suggested that combination therapy with ribavirin and interferon alfa could have potential benefi ts for patients with severe mers-cov infection. this is, to our knowledge, the largest clinical study done so far assessing the potential benefi t and safety of combination therapy with pegylated interferon alfa- a plus ribavirin in patients with severe mers-cov infection. because we noted a signifi cant -day survival benefi t in patients who received the combination compared with those who received supportive therapy only, but no survival benefi t at days, we recommend further assessment in appropriately designed randomised clinical trials to provide further information about the role of this combination in the treatment of patients with severe mers-cov infection. key: cord- - u ki dv authors: xu, ping; sun, guo-dong; li, zhi-zhong title: clinical characteristics of two human to human transmitted coronaviruses: corona virus disease versus middle east respiratory syndrome coronavirus. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: u ki dv after the outbreak of the middle east respiratory syndrome (mers) worldwide in . currently, a novel human coronavirus has caused a major disease outbreak, and named corona virus disease (covid- ). the emergency of mres-cov and covid- has caused global panic and threatened health security. unfortunately, the similarities and differences between the two coronavirus diseases remain to be unknown. the aim of this study, therefore, is to perform a systematic review to compare epidemiological, clinical and laboratory features of covid- and mers-cov population. we searched pubmed, embase and cochrane register of controlled trials database to identify potential studies reported covid- or mers-cov. epidemiological, clinical and laboratory outcomes, the admission rate of intensive cure unit (icu), discharge rate and fatality rate were evaluated using graphpad prism software. thirty-two studies involving patients (covid- = , mers-cov = ) were included in this study. the present study revealed that compared with covid- population, mers-cov population had a higher rate of icu admission, discharge and fatality and longer incubation time. it pointed out that fever, cough and generalised weakness and myalgia were main clinical manifestations of both covid- and mers-cov, whereas ards was main complication. the most effective drug for mers-cov is ribavirin and interferon. coronaviruses are rna viruses with envelope and non-segmented positive-sense, causing respiratory and intestinal tract infections in humans and other mammals [ ] . despite . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint although many previous studies have reported clinical characteristics of covid- or mers-cov diseases [ ] [ ] [ ] [ ] , systematic comparison of clinical features between covid- and mers-cov diseases has yet been published. thus, the purpose of this study is to perform a systematic review of epidemiological, clinical and laboratory characteristics of patients infected by covid- or mers-cov disease, and to compare covid- and mers-cov in the context of their incubation, laboratory features, admission rate of intensive cure unit (icu) and rate of discharge and fatality, which will provide a comprehensive reference for clinical physicians in treatment of coronavirus diseases. a comprehensive and systematic search was performed using pubmed, embase and or ( novel coronavirus)). if necessary, we also contacted corresponding author to obtain accurate data. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint the study that met following criteria were included: ( ) reporting clinical characteristics of covid- or mers-cov disease, ( ) minimum sample size of five, ( ) confirmed covid- or mers-cov disease, ( ) english literature. studies were excluded as following criteria: duplicate publications, case report, meta-analysis, letter, review, technology report, commentary, animal trial, correspondence, predictive study, guidence, radiograph study and meeting report. at the beginning, potential publications were identified. we removed duplicates and reviewed the titles and abstracts of remaining publications. publications were excluded for following reasons: not involved research point (n = ), review (n = ), no english (n = ), case report (n = ), meta-analysis (n = ), letter (n = ), technology report (n = ), commentaries (n = ), animal study (n = ), correspondence (n = ), predictive study (n = ), guidence (n = ), meeting report ( n = ) and radiograph (n = ). then, a comprehensive review of full-text was conducted for remaining publications. two reviewers independently screened eligible literature, and any argument was solved by discussion with a third reviewer. finally, thirty-two studies were included in this study . the process was shown in figure . two reviewers extracted independently common, clinical and laboratory characteristics of included studies, with disagreements were solved by discussion with a third reviewer. the extracted data included incubation time, white blood cell (wbc) count, lymphocyte count, creactive protein (crp), alamine aminotransferase (alt), aspartate aminotransferase (ast), . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint creatinine, creatine kinase (ck), the admission rate of intensive cure unit (icu), rate of discharge and fatality, symptom, comorbidity, complications and cure rate of drug. for normality distribution data, outcomes were extracted directly. for skewness distribution data, the outcomes was extracted after being converted as specific formula [ ] . the quality assessment of included studies was performed through the newcastle-ottawa quality assessment scale (nos), as recommended by the cochrane non-randomized studies [ ] . the nos includes three parts for risk of bias, with nine points in total: ( ) selection of research groups (four points); ( ) inter-group comparability (two points); and ( ) ascertainment of exposure and outcomes (three points) for case-control and cohort studies, respectively. study that scored or more was qualified for systematic review [ ] . the assessment process was completed by two reviewers independently. all debates were solved by discussion with a third reviewer. all statistical analyses and graphs were generated and plotted using graphpad prism version . software (graphpad software inc). the p value < . suggests significant difference. all included studies were retrospective study . among ten studies reported covid- , one trial was performed in netherlands [ ] and remaining nine trials were conducted in china [ - , - , - ] . the sample size ranged from to , and had a total . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint [ - , , ] , one trial was performed in iran [ ] and one trial was conducted in japan [ ] . the sample size ranged from to , and had a total of patients. the year of publications ranged from to . clinical and laboratory characteristics of included study were shown in table and table respectively. among thirty-two included studies, four studies obtained points of nos [ , , , ] , and remaining twenty-eight studies obtained points of nos or more [ - , , - , - , - ] . the result of quality assessment was presented in table . for covid- population, the number of patients with fever was ( . %), cough was ( . %), generalised weakness and myalgia was ( . %), stuffy or rhinorrhoea was ( . %), pharyngalgia was ( %), chest pain was ( . %), diarrhoea or anorexia was ( . %), dyspnoea was ( . %) and dizziness or headache was ( . %). for mers-cov population, the amount of patients with fever was ( . %), cough was ( . %), generalised weakness and myalgia was ( . %), stuffy or rhinorrhoea was ( . %), pharyngalgia was ( . %), chest pain was ( %), diarrhoea or anorexia was ( . %), dyspnoea was ( %) and dizziness or headache was ( . %). the above results were shown in table . for covid- population, the main complications included shock, arrhythmia, acute respiratory distress syndrome (ards), acute cardiac injury, acute kidney injury and acute . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint liver injury, and the amount of which was ( . %), ( . %), ( . %), ( %), ( . %) and ( . %) respectively. for mers-cov population, the number of individuals presented shock was ( . %), arrhythmia was ( . %), ards was ( . %), acute cardiac injury ( . %), acute kidney injury was ( . %), acute liver injury was ( . %) and neurological symptoms was ( . %). the results were shown in table . table . systematic review was performed for clinical and laboratory outcomes of coronavirus disease. there was no significant difference in age ( . ± vs. . ± . , p = . ), wbc table . . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint the higher admission rate of icu was found in mers-cov population than in covid- population ( . % vs. . %, figure ). there was a higher discharge rate in mers-cov populations compared with covid- population ( . % vs. . %, figure ). the lower fatality rate was found in covid- population compared with mers-cov population ( . % vs. . %, figure ). coronavirus is an important pathogen causing respiratory and intestinal infection. of seven identified coronaviruses, the two very pathogenic viruses, sars-cov and mers-cov, cause severe ards and even acute respiratory failure. with a mortality rate of more than % and more than % respectively [ ] [ ] . the four other human coronaviruses in addition, we found that the number of males is more than that of females in either covid- or mers-cov population. the possible reason of reduced susceptibility of females to viral infection is that females have a lot of x chromosome and estrogen that are vital components in development of innate and adaptive immunity [ ] . meanwhile, numbers of patients with covid- infection had chronic comorbidities, mainly hypertension, diabetes and cardiovascular disease, which is similar to mers-cov population. those results indicate that older adult males with chronic underlying disease might be more susceptibility to covid- or mers-cov. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint in terms of laboratory testing, reduced lymphocytes and increased crp were found in both covid- and mers-cov patients. this result indicates that covid- might be associated with cellular immune response, mainly act on lymphocytes like mers-cov does [ ] . the cells infected by viruses induce the release of numbers of pro-inflammatory cytokines and inflammation storm in the body. moreover, increased cytokines might make damage to related organs such as liver [ ] . our results demonstrated that abnormal value of ast was found in mers-cov population, but not in covid- population. the possible reason is that the follow-up time of covid- population was too short, and the liver might remain to be in compensatory stage. for this result, a long follow-up time study is urgently needed. on the other hand, our results suggested that mers-cov population had a higher rate of icu admission and fatality than covid- population, indicating that compared with mers-cov, covid- has less toxic and more easily cured. however, lower discharge rate was found in covid population than in mers-cov population. the possible explanation is that most of covid- patients remained to be hospitalized at the time of manuscript submission, and data on those patients could not be obtained in time. thus, careful interpretation is urged for this result. up to now, no effective strategy has been found for treatment of covid- infection [ ] . currently, the measure to covid- is to control the source of the infection; taking personal protective works to reduce the risk of transmission; and early diagnosing, isolating and supportive treating for confirmed patients. in the present study, our systematic results of cure rate of drug for mers-cov infection indicated that ribavirin and interferon, oseltamivir, antivirals and intravenous immunoglobulin all had been effective for mers-cov infection, with the cure rate of those drugs was . %, . %, . % and . % respectively. thus, we . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint assume that those drugs might also be effective at covid- infection. however, further studies are needed to confirm this idea. this study has several limitations. first, many patients infected by covid- remained to be hospitalized at the time of manuscript submission, leading to the unavailable of some data. second, the follow-up time of covid- population is too short to get related data from long-term observations of this disease. third, finding of statistical tests and p values between covid- and mers-cov populations should be interpreted with caution. fourth, the number of mers-cov patients treated by drugs is small, careful understanding is needed for the cure rate of drug for this disease. finally, as covid- is still developing around the world and remains to have many unknowns, the results of this study are staged and need to be carefully understood. more large-sample, multicentre, high-quality research should be performed to update this study. our systematic review reveals that main clinical manifestations of both covid- and mers-cov populations are fever, cough and generalised weakness and myalgia. ards is main complication of both two populations. covid- population has a shorter incubation time and lower rate of icu admission, discharge and fatality compared with mres-cov population. this study was supported by grants from the national natural science 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risk factor for colon cancer and rectal cancer ? sars and other coronaviruses as causes of pneumonia author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint from sars to mers, thrusting coronaviruses into the spotlight origin and evolution of pathogenic coronaviruses epidemiology, genetic recombination, and pathogenesis of coronaviruses sexual dimorphism in innate immunity prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis liver manipulation during liver surgery in humans is associated with hepatocellular damage and hepatic inflammation sars and mers: recent insights into emerging coronaviruses author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint key: cord- -gpb fga authors: hashem, anwar m; algaissi, abdullah; agrawal, anurodh shankar; al-amri, sawsan s; alhabbab, rowa y; sohrab, sayed s; s. almasoud, abdulrahman; alharbi, naif khalaf; peng, bi-hung; russell, marsha; li, xuguang; tseng, chien-te k title: a highly immunogenic, protective, and safe adenovirus-based vaccine expressing middle east respiratory syndrome coronavirus s -cd l fusion protein in a transgenic human dipeptidyl peptidase mouse model date: - - journal: j infect dis doi: . /infdis/jiz sha: doc_id: cord_uid: gpb fga background: infection control measures have played a major role in limiting human/camel-to-human transmission of middle east respiratory syndrome coronavirus (mers-cov); however, development of effective and safe human or camel vaccines is warranted. methods: we extended and optimized our previous recombinant adenovirus (rad )–based vaccine platform characterized by in vivo amplified and cd -mediated specific responses to generate mers-cov s subunit-based vaccine. we generated rad constructs expressing cd -targeted s fusion protein (rad -s /f/cd l), untargeted s (rad -s ), and green fluorescent protein (rad -gfp), and evaluated their efficacy and safety in human dipeptidyl peptidase transgenic (hdpp tg(+)) mice. results: immunization of hdpp tg(+) mice with a single dose of rad -s /f/cd l elicited as robust and significant specific immunoglobulin g and neutralizing antibodies as those induced with doses of rad -s . after mers-cov challenge, both vaccines conferred complete protection against morbidity and mortality, as evidenced by significantly undetectable/reduced pulmonary viral loads compared to the control group. however, rad -s – but not rad -s /f/cd l–immunized mice exhibited marked pulmonary perivascular hemorrhage post–mers-cov challenge despite the observed protection. conclusions: incorporation of cd l into rad -based mers-cov s vaccine targeting molecule and molecular adjuvants not only enhances immunogenicity and efficacy but also prevents inadvertent pulmonary pathology after viral challenge, thereby offering a promising strategy to enhance safety and potency of vaccines. the middle east respiratory syndrome coronavirus (mers-cov) is a novel zoonotic virus that was first isolated from a fatal human case in saudi arabia in [ ] . the virus can cause severe acute and fatal respiratory symptoms associated with systemic infection and occasional multiorgan failure [ ] . as of october , mers-cov has caused > laboratoryconfirmed infections with an approximately % mortality rate in countries, with saudi arabia being the most affected country [ ] . most global cases are linked to the arabian peninsula, where mers-cov continues to be endemic for > years with the potential to spread globally, as seen in the outbreak in south korea [ ] . the spike (s) glycoprotein of mers-cov ( aa) is a type i trimeric membrane protein expressed on the virus surface, and binds to dipeptidyl peptidase (dpp ) on target cells [ ] . it is comprised of an n-terminal globular s subunit (aa - ) containing the receptor-binding domain (rbd) and a membraneproximal s subunit (aa - ) consisting of a transmembrane domain and an intracellular cytoplasmic domain (cd) involved in viral fusion with target cells [ , ] . given its critical role in viral replication, the s protein has been the focus for mers-cov vaccine development similar to severe acute respiratory syndrome coronavirus (sars-cov), where it has been the main target for vaccines that led to robust induction of neutralizing antibody (nab)-mediated protection in immunized animals [ ] [ ] [ ] . several vaccine candidates based on full-length or truncated s protein including vectored, dna, and nanoparticle vaccines, as well as s or rbd protein-based subunit vaccines, have been developed and investigated in animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although some of these candidates can induce high levels of nabs in vaccinated animals and have entered phase clinical trials [ ] [ ] [ ] , most of these candidates are associated with low immunogenicity requiring multiple doses [ ] . importantly, vaccine research on other members of the coronaviruses including sars-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] revealed several safety concerns associated with the use of s-based vaccines, including inflammatory and immunopathological effects such as pulmonary eosinophilic infiltration and antibody (ab)-mediated disease enhancement following subsequent viral challenge of vaccinated animals. recently, we also showed that whole inactivated mers-cov vaccine (wiv) is associated with significantly increased pulmonary eosinophilic infiltration accompanied by elevated levels of th cytokines in vaccinated human dpp transgenic (hdpp tg + ) mice in response to virus challenge [ ] . given these previous observations, a mers-cov vaccine based on the full-length s protein could pose potential safety concerns similar to those associated with sars-cov vaccines. while the neutralizing epitope-rich s region could be considered as an alternative target for an effective and safe mers vaccine, s -based protection could clearly be enhanced through appropriate immunological modulation. cd ligand (cd l), a type ii membrane protein, is a key co-stimulatory molecule and an essential regulator of the immune system. it is expressed transiently on activated cd + t cells mainly [ , ] . cd l and its receptor cd , which is constitutively expressed on all antigen-presenting cells (apcs) [ ] [ ] [ ] , represent a crucial link between innate and adaptive immunity [ , ] . the potential of cd l as a molecular adjuvant has been investigated by several groups using multiple strategies [ ] [ ] [ ] [ ] . we have previously developed a nonreplicating recombinant adenovirus (rad ) vectored prototype vaccine in which secreted viral proteins are targeted to cd -expressing apcs using cd l [ , ] . such studies provided a proof of concept for a vaccine platform characterized by enhanced durability, breadth, potency, and universal protection against influenza viruses in different mouse models. here, we further extended and optimized our previous platform using rad expressing a secreted and cd -targeted fusion protein comprised of head globular ectodomains of both mers-cov s protein and murine cd l (rad -s /f/cd l) using a trimerization motif to express the fusion protein as a trimer and a nonpolar linker to provide flexibility. the vaccine was then subjected to systematic analyses of protection and safety in the highly mers-cov permissive hdpp tg + mouse model. the fusion gene was designed and codon-optimized to express a secreted and cd -targeted consensus mers-cov s subunit (amino acids - ). in brief, all available mers-cov s sequences were downloaded from the genbank database, and the dataset was filtered by removing sequences containing ambiguous amino acid codes (bjouxz). the final dataset was multiply aligned using clustalw, the shannon entropy for each position was determined, and the consensus s subunit sequence was obtained. the signal peptide from the mers-cov s protein was maintained at the n-terminus to secrete the fusion protein from rad -infected cells. the synthetic fusion gene was synthesized by linking the coding region of mers-cov s to a histidine tag coding sequence, followed by coding region for the fibritin trimerization motif [ ] connected via a nonpolar amino acid linker to the ectodomain of cd l (amino acids - ) coding region to express the fusion protein s /f/cd l. the fusion gene was then used to generate the proposed rad construct (rad -s /f/cd l) in addition to rad vaccines expressing secreted and consensus s protein alone (rad -s ) and a vector control expressing green fluorescent protein (rad -gfp) as shown in figure . vero e and huh . cells were cultured and maintained in complete dulbecco's modified eagle's medium (dmem) supplemented with % heat-inactivated fetal bovine serum. mers-cov-emc/ was provided by heinz feldmann (national institutes poly a p s f tag cd l l poly a p s poly a p gfp rad -s /f/cd l rad -s rad -gfp figure . schematic representation of middle east respiratory syndrome coronavirus vaccine candidates. the proposed recombinant adenovirus (rad ) construct (rad -s /f/cd l) was engineered to express a fusion protein with the s subunit containing the s protein signal peptide at the n-terminal, followed by a trimerization motif derived from t bacteriophage fibritin (f) fused with the ectodomain of murine cd ligand at the c-terminus (cd l). thus, the s and cd l are expressed as a trimerized fusion protein via f (s /f/cd l). the construct was placed under the control of the cytomegalovirus promoter (cmv) and in front of the bovine growth hormone-poly a site (poly a). other control constructs included rad -s and a control rad vector expressing green fluorescent protein (rad -gfp). ) assay as previously described [ ] . all work involving infectious mers-cov was conducted within approved biosafety level (bsl- ) at the galveston national laboratory (gnl) at the university of texas medical branch (utmb) in galveston. the immunogenicity and the protective efficacy of the rad based vaccine candidates were evaluated against mers-cov infection in the hdpp tg + mouse model. a total of hdpp tg + mice aged - months were used ( mice/group). mice were intramuscularly immunized twice, days apart, with of rad vaccine candidates ( figure ). blood samples were collected weeks after each immunization via retro-orbital bleeding. four weeks after the second immunization, mice were intranasally challenged with tcid (~ median lethal dose [ld ]) of mers-cov. on days and postinfection, mice from each group were killed to assess the virus titer and lung pathology. the remaining mice were monitored for morbidity and mortality on a daily basis for up to days. all animal studies involving infectious mers-cov were conducted within approved animal bsl- laboratories at gnl in accordance with the guide for the care and use of laboratory animals of the nih and association for assessment and accreditation of laboratory animal care, and with institutional animal protocol approval from utmb. animals were housed in on-site animal facilities under a : light:dark cycle with room temperature and humidity kept between °c and °c and %- %, respectively, with ad libitum access to food and water. at day and after challenge, mice in each group were euthanized and their lungs were collected and examined for pathological changes after immunization and viral challenge. in brief, lung tissues were fixed in % buffered formalin for hours, transferred to % ethanol, and later paraffin embedded. histopathological evaluation was performed on deparaffinized sections stained by routine hematoxylin and eosin staining. evaluations for histopathology were done by pathologists who were blinded to each specimen source. numeric scores were assigned to assess the extent of pathological damage as follows: , no observed pathology (undetectable infiltration); , mild pathology (up to % infiltration); , moderate pathology (up to % infiltration); , severe pathology (> % infiltration). lungs collected from challenged mice on day postchallenge were used to determine viral titer by tcid . in brief, pieces of collected lung tissue were weighed and homogenized in phosphate-buffered saline containing % fetal calf serum with a tissuelyser (qiagen). after clarification of the cellular and tissue debris by centrifugation, the titers of infectious virus in the suspensions of infected tissues were determined using tcid assay. the virus titers of individual samples were expressed as log tcid per gram of tissue and the minimal detectable level of virus was . log tcid as determined by lung size. lung samples from each group of mice (n = per group) were transferred to individual vials containing rnalater solution and subsequently homogenized and subjected to total rna isolation using trizol reagent. to determine the viral titer in the lung, mers-cov-specific upstream e gene (upe) and endogenous control gene (mouse β-actin) were quantified using -step reverse-transcription quantitative polymerase chain reaction (rt-qpcr monitoring for morbidity and mortality upe mrna expression value was calculated for each replicate and expressed as the equivalent of log equivalents per gram (tcid eq/g) of the tissue by the standard ct (∆∆ct) method using bio-rad cfx manager . software. the end-point titers of anti-s total immunoglobulin g (igg) as well as its isotypes including igg , igg a, and igg b from immunized mice were determined by enzyme-linked immunosorbent assay as described previously [ ] . serum samples were tested in a -fold serial dilution starting from : . end-point titers were determined and expressed as the reciprocals of the highest dilution that gave an optical density signal higher than the cutoff value, defined as the mean of prebleed samples plus standard deviations (sd). mers-pseudotyped viral particles (merspp) were produced and titrated using the huh . cell line as described previously [ ] . plasmids for generating merspp were kindly provided by dr nigel temperton at the university of kent, united kingdom. heat-inactivated serum samples were prepared in a -fold serial dilution starting from : and tested in duplicates in -well nunc white plates. a standard amount of merspp (~ relative light units) was added to each well and plates were incubated for hour at °c. then, approximately huh . cells were added to each well and incubated for hours. cells only and cells with merspp only were included in quadruplicate as controls in all plates. following incubation, cells were lysed and luciferase activity was developed and measured using the bright-glo luciferase assay system (promega) according to the manufacturer's instructions. log median inhibitory concentration (ic ) neutralization titers were calculated for each serum sample using graphpad prism software. the standard live virus microneutralization (mn) assay was used as previously described [ ] . in brief, starting at a dilution of : , μl volumes of serial -fold dilutions of heat-inactivated sera were mixed with equal volume of media containing tcid of mers-cov. each dilution was tested in duplicate in -well plates. the antibody-virus mixtures were incubated for hour at °c before transfer of μl into confluent vero e cell monolayers in -well plates. vero e cells cultured with dmem medium with or without virus were included as positive and negative controls, respectively. after hours of incubation, the nab titer of each serum sample was determined as the reciprocal of the highest dilution capable of completely preventing virus-induced cytopathic effect in % of the wells (mn ). statistical analysis was conducted using -way or -way analysis of variance with bonferroni posttest to adjust for multiple comparisons between groups using graphpad prism software. to evaluate the immunogenicity of the generated rad vectorbased mers vaccines, we immunized mice intramuscularly with doses of individual vaccines and measured the levels of circulating s -specific igg and its isotypes before and at weeks after each immunization. here, we observed that both rad -s /f/cd l and rad -s induced s -specific abs from all isotypes in hdpp tg + mice after primary or secondary immunization ( figure ). as expected, control vector (rad -gfp) failed to elicit any s -specific ab response. notably, a single dose or doses of rad -s /f/cd l consistently elicited significantly higher levels of total igg as well as igg , igg a, and igg b isotypes compared with the control group immunized with rad -gfp (figure ), whereas immunization with a single dose of rad -s induced significant titers of total igg, igg , and igg a but not igg b compared to the control group, with a second dose of rad -s eventually inducing significantly higher levels of igg b compared to the control group. interestingly, immunizing mice with a second dose of rad -s /f/cd l enhanced the levels of circulatory igg and all tested isotypes compared with the rad -s -vaccinated group. furthermore, doses of rad -s only enhanced igg b significantly compared to dose, with mean igg :igg a ratios being . (sd, . ) and . (sd, . ) after priming and boosting, respectively. in contrast, boosting with a second dose of rad -s /f/cd l resulted in a significant increase in total igg, particularly in igg a and igg b isotypes but not igg , compared with a single dose of rad -s /f/cd l, suggesting a bias toward th response as demonstrated by mean igg :igg a ratios of . (sd, . ) and . (sd, . ) after priming and boosting, respectively. together, these data showed that targeting the secreted s to cd -expressing apcs via cd l (rad -s /f/ cd l) not only enhanced s -specific igg abs levels but also boosted th responses, as demonstrated by markedly elevated titers of s -specific igg a and igg b antibodies, especially after boosting. to extend our analysis and to evaluate the effector function of induced abs, we measured their neutralizing activities before and after each immunization. as expected, all immunized mice from all groups showed no detectable levels of nabs in samples collected before immunization. as shown in figure , a single dose of either rad -s /f/cd l or rad -s elicited high levels of nabs compared to the control group (rad -gfp), with rad -s /f/cd l, but not rad -s , being consistently capable of inducing significantly higher levels of nabs against both live and pseudotyped mers-cov virus, suggesting that rad -s /f/cd l is better than rad-s vaccine in promoting strong humoral response and neutralizing activity against mers-cov. however, after boosting once, all rad -s /f/ cd l-and rad -s -immunized animals elicited robust and significant levels of nabs, indicating that at least doses of rad-s are required to induce nab levels similar to those obtained by a single dose of rad -s /f/cd l. these findings clearly confirm that incorporation of cd l as molecular adjuvant could effectively enhance the immunogenicity of s -based vaccines and may represent a very promising vaccine platform to induce protective immunity with a single dose. having demonstrated that cd -targeted vaccine was superior in eliciting immune responses in hdpp tg + mice, we next investigated if these s -based vaccines could effectively protect these highly mers-cov permissive mice from viral challenge. to this end, the vaccinated mice were challenged with ld of mers-cov and subsequently monitored for weeks. it was clear that mice immunized twice with either rad -s or rad -s /f/cd l were completely protected based on clinical signs of disease (weight loss) and mortality ( figure ), whereas rad -gfp-immunized animals expectedly succumbed to lethal infection within days, likely due to encephalitis [ , ] . these findings suggest that both rad -s and rad -s /f/cd l vaccines are protective in this mouse model. to further confirm that immunized hdpp tg + mice were protected from mers-cov infection after challenge, we measured lung viral titer, both infectious progeny virus and viral rna, on day postchallenge by vero e -based infectivity assay and rt-qpcr, respectively. analysis of infectious viral titer revealed that immunization with both vaccines significantly protected against viral replication to undetectable levels compared with rad -gfp-immunized and -challenged mice ( figure ). these results were further confirmed by an average of -to -log reduction in pulmonary viral rna, compared to that of the rad -gfp-immunized group ( figure ). it should be mentioned that, albeit not statistically significant, rad -s /f/cd l-immunized animals had a viral rna load lower by log compared with the rad -s group, consistent with the overall stronger immune response we observed in this group. finally, investigations were carried out to assess safety of these vaccine candidates to rule out any vaccine-associated pulmonary injuries as observed with some vaccines against other coronaviruses. evaluation of lung pathology in immunized and challenged mice showed up to % multiple monocytic and lymphocytic infiltrations (moderate pathology) in the lungs of the rad -gfp group and days postchallenge compared to other groups, which showed minimal to no inflammatory infiltration ( figure ) . surprisingly, although rad -s immunization protected animals from death and weight loss and prevented the lung infiltration similar to rad -s /f/cd l, we observed perivascular hemorrhage in the whole lung of all examined mice immunized with rad -s but not those vaccinated with rad -s /f/cd l (figure ) . together, these results suggest that although s alone could protect the animals from viral infection, it may inadvertently induce lung pathology, and that using cd l as targeting molecule and molecular adjuvant does not only enhance immunogenicity and protective efficacy but also prevents vaccine-associated pulmonary pathology. the rapid spread and persistence of mers-cov in the arabian peninsula, in addition to the associated high mortality rates, represent a serious global public health concern, particularly that the elimination of the zoonotic reservoir is extremely unlikely at the current setting. this threat is further complicated by the absence of prophylactic or therapeutic measures. therefore, development of an effective and safe vaccine is urgently needed to prevent or contain mers-cov. several groups have investigated various mers-cov vaccine platforms in several animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . however, serious safety concerns are associated with vaccines for several coronaviruses including mers-cov and need to be elucidated and better understood [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we showed in this study that although rad expressing s or cd -targeted s were both capable of inducing significant levels of igg and nabs specific to mers-cov in immunized mice, incorporation of cd l substantially enhances the immunogenicity of s , as demonstrated by the effectiveness of a single immunization dose, which was sufficient to elicit stronger and robust immune responses compared to control groups, consistent with our previous reports [ , ] . importantly, both s and cd -targeted s vaccines provided complete protection, prevented virus replication significantly, and prevented monocytic and lymphocytic lung infiltration as compared to control group. however, the non-cd -targeted vaccine (rad -s ) uniformly induced varying degrees of severe perivascular hemorrhage within the lungs of all examined mice following viral challenge, regardless of the exhibited full protection against lethal mers-cov challenge. while these data suggest that there might be yet to be explained lung pathology associated with rad vector-and/or mers-cov s -based vaccines, our results clearly show that including cd l as a fusion protein of s -based mers-cov vaccine could prevent or at least mitigate the safety concern of undesirable immunopathology while affording complete protection in hdpp tg + mice. at least groups have developed different rad-vectored mers-cov vaccines [ , , ] . however, these studies have mainly focused on the immunogenicity and/or the protective efficacy, and may have overlooked any possible vaccine-associated pathology, especially after viral challenge. our finding that rad -s immunized animals were completely protected despite the observed pathology is reminiscent of the immunopathology documented using wiv for mers-cov, sars-cov, and respiratory syncytial virus after viral challenge [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is of note that lung pathology was also observed in some previous studies involving rhesus macaques immunized with high doses of dna vaccines expressing mers-cov s protein [ ] , as well as in mouse models vaccinated with rbd or vectored vaccines encoding s protein [ ] [ ] [ ] . nevertheless, this is the first report of vaccine-associated severe pulmonary perivascular hemorrhage after viral challenge. although the exact molecular mechanisms underlying such vaccine-induced pulmonary injury associated with rad -s vaccination remain to be determined, several factors could have been involved. specifically, the likely higher levels of residual infectious viruses in rad -s -immunized mice as detected by rt-qpcr, and the quantitative and perhaps qualitative difference of specific antibody and t-cell responses, as evidenced by the lower levels of ig a and igg b found in this group, could have played a role. . pulmonary viral load. viral load was determined in the lungs of immunized and challenged mice with middle east respiratory syndrome coronavirus (mers-cov). viral load was determined as median tissue culture infectious dose per gram (tcid /g) and tcid equivalent per gram (tcid eq/g) by tcid and reverse-transcription quantitative polymerase chain reaction (rt-qpcr) assays, respectively. limit of detection in the tcid assay was . log (dashed line) . tcid eq/g was determined using a standard curve generated from dilutions of rna from a mers-cov with known virus titer. titers are shown as mean with standard deviation from mice per group from experiment. ***p < . ( -way analysis of variance with bonferroni posttest); ns, not significant. see the figure legend for explanation of the recombinant adenovirus (rad ) constructs. rad -gfp rad -s rad -s /f/cd l μm therefore, future studies should include systematic analyses of t-cell responses as we have previously conducted [ , ] , to fully explore the mechanisms of immunization-induced hemorrhage of the lungs. collectively, these previous data, along with our report here, indicate that viral components (ie, within the s protein) could be involved in inadvertent adverse reactions similar to those associated with the different s-based sars vaccines [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our study suggests that although rad -s could be a potential vaccine candidate, it might be associated with severe lung pathology upon viral challenge or infection. these findings are of great importance in order to develop safe and effective human mers-cov vaccine. moreover, there is a huge gap in our understanding of the correlates of protection in camels, and the waning nature of nabs in these animals [ , ] might hinder the "one health" approach in tackling the mers epidemic. therefore, it is critical to better understand and elucidate any safety concerns that might be associated with mers-cov vaccine to develop a safe and effective human vaccine. our previous studies have mainly focused on the development of candidate universal influenza vaccines using vectored vaccines by targeting antigens to cd -expressing cells via cd l. this platform was modified, optimized, and utilized to generate an immunogenic, protective, and safe mers-cov vaccine based on the s subunit of the mers-cov s protein, thus representing a promising platform for vaccine development against a broad range of pathogens. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia disease outbreak news outbreaks of middle east respiratory 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vaccine a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase (hdpp ) transgenic mice from mers-cov infection protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study key: cord- -kptkmgdm authors: crameri, gary; durr, peter a.; klein, reuben; foord, adam; yu, meng; riddell, sarah; haining, jessica; johnson, dayna; hemida, maged g.; barr, jennifer; peiris, malik; middleton, deborah; wang, lin-fa title: experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: kptkmgdm we conducted a challenge/rechallenge trial in which alpacas were infected with middle east respiratory syndrome coronavirus. the alpacas shed virus at challenge but were refractory to further shedding at rechallenge on day . the trial indicates that alpacas may be suitable models for infection and shedding dynamics of this virus. (mers-cov) was first reported in september ( ); since then, > , confirmed cases have been reported to the world health organization (http://www.who.int/csr/ don/ -february- -mers-saudi-arabia/en). the role of domestic animals as an intermediate host for humans was initially suggested by case histories of infected patients who had visited farms or tended sick animals shortly before onset of infection ( ) . this suggestion was given credence by a study of camel serum samples that showed high levels of neutralizing antibodies in disparate camel populations ( ) ; the findings were subsequently confirmed by virus detection and sequencing ( ) . infection trials in camels have been limited ( , ) , mainly because of difficulties in housing and handling the animals in a high-containment facility, which is necessary because the virus has a biosafety level classification ( ) . however, the alpaca, a close relative within the camelidae family, may provide a temperamentally suitable and valuable animal model for mers-cov infection, particularly for developing and testing vaccine candidates for camels. we sought to assess whether alpacas could be infected by means of a natural (oronasal) route, to determine whether viral shedding occurred after reinfection, and to evaluate the development of serologic markers of protection. we obtained adult female alpacas (vicugna pacos) from a commercial supplier in victoria, australia, and housed them in the biosafety level containment facility at the csiro australian animal health laboratory. before experiments, the alpacas were allowed to acclimatize for days; during this time, intrauterine temperature data loggers were implanted according to a previously published procedure ( ) . we found no previous mers-cov challenge trial reported in alpacas, so we chose a preliminary dose and rechallenge time on the basis of our experience with other virus infection trials for other emerging infectious diseases ( ) . we used a camel isolate of mers-cov (dromedary_ mers-cov_al-hasa_kfu-hku / ; genbank accession nos. kj -kj ) for infection; the isolate was prepared in vero cells as described ( ) . the alpacas were exposed oronasally to a % tissue culture infective dose of mers-cov in ml of phosphate-buffered saline. the animals were monitored for days, reexposed to a replicate challenge of mers-cov, and observed for more days. clinical samples of blood (in edta for obtaining serum) and swabs (deep and superficial nasal, oral, rectal, and urogenital) were collected immediately before inoculation and thereafter on days , , , , , , , , , , , and . alpacas were electively euthanized, on day and the others on day . the animals remained clinically healthy except for a reduced condition score that occurred by day in animal (alpaca ); no signs of upper or lower respiratory tract disease appeared in any animal. increased temperature was noted in alpaca during days - , but fever (rectal temperature > °c) was not recorded. gross abnormalities at postmortem examination were found only in alpaca and comprised extensive adhesions of the caudal sac of compartment of the stomach to the umbilicus; clinical findings in this animal were attributed to this lesion. rna extraction and real-time pcr were performed by following specimen-handling procedures established for hendra virus ( ) and were used to identify shedding patterns after each challenge. after initial challenge, (table ) . virus isolation was undertaken with vero cells by using published protocols ( ) and was successful for all animals from all types of samples. virus recovery was successful from oral and superficial nasal swab samples through day ; deep nasal swab samples were positive only through day . all urogenital and rectal swab samples were negative by both real-time pcr and virus isolation. after rechallenge, viral rna was not detected with confidence from any sample (figure) . serum samples were assessed for immunologic responses by using a virus neutralization test (vnt) and a luminex bead assay to the nucleocapsid protein. we used in-house assays modeled after those previously developed to assess the serologic status of feral camels in central australia ( ) . all animals were seronegative by both luminex and vnt before challenge. antibody was first detected by luminex on day or day in each animal ( table ) ; neutralizing antibody titers were : to : in alpaca from day . neutralizing antibody titers of : to : were detected in alpaca from day on but not in alpaca at any time during the study. for controls, we used mers-cov positive and negative serum samples from egypt and australia (online technical appendix table, http://wwwnc.cdc.gov/eid/article/ / / - -techapp .pdf). our study confirms that alpacas are susceptible to mers-cov infection; this finding is consistent with a previous report showing that alpaca kidney cell lines possessing the dipeptidyl peptidase- receptor could be infected in vitro ( ) . our challenge/rechallenge trial was planned as a first stage in the assessment of the alpaca as a potential surrogate for camels for mers-cov vaccine testing. consequently, the trial was not designed for direct comparison with previous mers-cov challenge trials reported in camels ( , ). our trial used a lower challenge dose and a different timeframe for observation; nevertheless, some preliminary comparative observations may be useful. in the previous studies, as in ours, the animals were inoculated by the oronasal route, and live virus was detected through day postinfection. similarly, neutralizing antibodies were detected beginning - days postinfection. however, findings in the trials with camels differed considerably from findings in our trial. the trials with camels detected live virus from nasal washes at days - , a nasal discharge, and transient temperature rises; viral rna was detected by real-time pcr for an extended period. furthermore, the vnt titers for camels were much higher than those for the alpacas in our study. these differences possibly represent underlying dissimilarities in immune responses to mers-cov for the species but may also result from the higher infecting dose ( % tissue culture infective dose) used in the camel studies. our study showed that alpacas secreted live virus after oronasal infection and that the immune response to the initial infection prevented further excretion following reinfection. an underlying assumption in our trial is that the initial infection equates to natural vaccination and that the lack of viral excretion thus follows an induced immune memory response. however, our results indicate that this immunologic response is complex; although a strong serologic response developed in only alpaca, all alpacas were refractory to reinfection. this study has several limitations. first, it was a preliminary study with only animals and functioned more as proof of concept than a definitive study of the use of alpacas as a model for studying infection dynamics of mers-cov in camelids. second, our observation period of days before rechallenge is informative but does not provide complete information on duration of protective immunity. future studies should have a larger sample and a longer period of study postinoculation. third, our study did not seek to understand the pathogenesis of infection; we did not conduct histopathology or immunohistochemistry to understand the site of initial viral replication and the role of mucosal immunity in mounting an effective immune response upon infection. notwithstanding these limitations, we believe that the alpaca might be a useful model that could greatly facilitate the development and testing of vaccine candidates. we recommend further research and trials to substantiate this potential. experimental infection of alpacas with mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels inactivation and safety testing of middle east respiratory syndrome coronavirus hendra virus vaccine, a one health approach to protecting horse, human, and environmental health mers coronavirus in dromedary camel herd, saudi arabia absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread replicative capacity of mers coronavirus in livestock cell lines infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas we thank kaylene selleck, leah frazer, jean payne, rachel arkinstall, and mahen perera for providing technical assistance and support for this study. mr. crameri is a virologist with the csiro australian animal health laboratory. he has pioneered work in the high biocontainment facility at csiro and worked on hendra, nipah, sars, mers, ebola, and many other viruses with high impact to human and animal health.note added in proof: adney et al. also report infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas in this issue of emerging infectious diseases ( ) . key: cord- -gnqbyc t authors: hemida, maged gomaa; ali, mohammed; alhammadi, mohammed; alnaeem, abdelmohsen title: the middle east respiratory syndrome coronavirus in the breath of some infected dromedary camels (camelus dromedarius) date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: gnqbyc t dromedary camels remain the currently identified reservoir for the middle east respiratory syndrome coronavirus (mers-cov). the virus is released in the secretions of the infected camels, especially the nasal tract. the virus shedding curve through the nasal secretions was studied. although human transmission of the virus through the respiratory tract of close contact people with dromedary reported previously, the exact mechanism of transmission is still largely unknown. the main goal of this study was to check the possibility of mers-cov shedding in the exhaled air of the infected camels. to achieve this goal, we conducted a follow-up study in one of the dromedary camel herds, december –april . we tested nasal swabs, breath samples from animals within this herd by the real-time pcr. our results showed that some of the tested nasal swabs and breath were positive from march until april . the phylogenetic analysis of the obtained s and n gene sequences revealed the detected viruses are clustering together with some human and camel samples from the eastern region, especially from al-hufuf city, as well as some samples from qatar and jordon. these results are clearly showing the possibility of shedding of the virus in the breath of the infected camels. this could explain, at least in part, the mechanism of transmission of mers-cov from animals to humans. this study is confirming the shedding of mers-cov in the exhaled air of the infected camels. further studies are needed for a better understanding of the mers-cov. it has been almost years since the emergence of the middle east respiratory syndrome coronavirus (mers-cov) in in the arabian peninsula. the majority of human cases were reported in the arabian peninsula [ ] [ ] [ ] . more recently, there are many reports about the potential risk of infection with mers-cov in africa [ ] . the only known reservoir so far is the dromedary camels [ ] . there are many reports about the animal-human transmission in the context of mers-cov [ , ] . it is now well documented that mers-cov is released in the respiratory secretions detected in the nasal swabs by several research groups [ , [ ] [ ] [ ] [ ] [ ] . although no report on the secretion of the virus in the milk, it is highly recommended to boil it before consumption to avoid any potential contamination of the raw milk during the process of milking and handling [ ] [ ] [ ] . similar concept for eating the meats and raw organs, particularly the liver, it should be cooked well before consumption to avoid any potential hazards of infection as suggested by the world health organization (who) [ , ] . the virus was also detected in the air around some positive camel herd. detection of the mers-cov-rnas of identical sequences from the owner and the herders of this particular herd was also reported [ , ] . however, some other studies failed to detect the virus in the urine of some infected animals [ ] . earlier studies succeeded in the detection of the mers-cov nucleic acids in the air circulated by an infected camel herd as well as in the people of close contact of this particular herd [ , ] . meanwhile, several reports showed the possibility of detection of the mers-cov nucleic acids in the circulating air in some health care settings during the korean outbreak of mers-cov in [ , ] . the main goal of the current study was to check the possibility of mers-cov shedding in the infected animal breath. we conducted this study according to the guidelines of the animal ethics protocols and the national committee of bio-ethics, king abdul-aziz city of science and technology, royal decree no. m/ (http://www.kfsh.med.sa/kfsh_website/users uploadedfiles% cncbe% regulations% english.pdf). the animal experiments were approved by the animal care committee of the deanship of scientific research, king faisal university, saudi arabia. we conducted molecular surveillance for the mers-cov in a dromedary camel herd during . this herd consists of animals held together in wire fenced yards. the male animals were kept in a separate individual partition while the female was kept together in multiple partitions. there is a shared open yard where all females may be allowed to be mixed together. the males only approach females during the mating season from november to march per each year. during the time from march until april , we randomly selected nine animals out of the ( %) from the camel herd to conduct our molecular surveillance. all animals were of variable ages ranging from to years old. the nine animals selected from different colour coat-based breeds, including magaheem, wodoah and sofr, were selected. these nine animals were mixed with the rest of the animals in the herd all the time. our molecular surveillance lasts from march to april , with a biweekly sampling interval. we collected nasal swabs, breath samples from these nine animals, as previously described. a paired nasal and breath samples were collected per each animal at each time point, as described in detail below. nasal swabs were collected from at least % of the dromedary camel herd from november until march . swabs were transferred into the tubes containing viral transport media including the dmem media containing % foetal bovine sera and antibiotic cocktail (penicillin and streptomycin). the processing of the collected swabs was done as previously described. briefly, the swabs were vortexed, then centrifuged at rpm for min in a cooling centrifuge at °c. the supernatants were collected and stored at − °c for further testing. the breath samples were collected in sterile tubes containing viral transport media as used for the collection of the nasal swabs. the breath collection technique was done in a simple way. briefly, the animals were secured in a specially designed camel crush. the animals were allowed to settle down for min. with the help of an assistant, the nasal orifice is dilated, and the tube containing the media is slightly inserted inside ( - cm) the orifice without touching the skin or the mucous membranes of these animals during sampling. the collection tubes were held in a slope position facing the air stream coming from the animal during the exhalation. the tubes were kept in such position until the animal completes at least five cycles of breathing, including five exhalation streams. any container that touched the skin or the mucous membrane of the animals was discarded and not included in our analysis. the collection tubes were shaken immediately for min and inverted upside down several times, then placed into ice tanks until transferred to the laboratory. the viral rnas were extracted from paired swabs and breath samples collected from dromedary camels using the qiagen viral rna (rneasy mini kit, qiagen, hilden, germany) extraction kit, according to the manufacturer's protocol. the concentration of the extracted viral rnas was measured by the nanodrop machine (thermo scientific nanodrop , applied biosystems, lincoln centre drive, foster city, ca , usa). the eluted rnas were stored at − °c for further testing. the rt-pcr targeting the upstream gene (up-e) of mers-cov was used for screening [ ] . confirmation was done using the open reading frame (orf) a. five μl of the extracted rna was subjected to the real-time pcr testing using upe primers using lightmix molecular dx mers-cov-upe kits (roche, roche molecular systems, inc, pleasanton, ca , usa) according to the manufacturer's protocol. all positive samples by the upe assay were confirmed by orf a, as previously described [ ] . positive samples were considered when there is an overlapping between the results from two targets. samples (nasal swabs and breath) that had ct values < were considered positive per each target. we used several sets of primers to amplify the partial mers-cov-s, mers-cov-n genes. the sequences of these primers are as follow: nf- ′ -cct tcg gta cag tgg agc ca- ′ and nr- ′ -gat ggg gtt gcc aaa cac aaa c- ′ , primers for the mers-cov-s gene sf- ′ -ccaattta-cgccaggatgat- ′ and sr- ′ -aatagaggcgg aaatagcac- . we used the primers listed above to amplify the partial mers-cov-s and n genes using the qiagen one-step rt-pcr kit (cat no./id: ) to synthesis the cdna then doing the pcr in one reaction. the procedure of this one-step reaction was carried out as per the kit's instructions as well as previously described. we ran μl per each amplified mers-n and s amplicons on a % agarose gel containing sybr® safe dna gel stain (invitrogen, thermo fisher scientific, waltham, ma, usa). the amplicons were visualised under ultraviolet light, then the gel pictures were photographed with the gel documentation system (bio-rad laboratories, inc., hercules, ca, usa). we purified the desired pcr products from the target bands by using the gel-based pcr extraction kits (qiagen, cat no/id: ), as per the instructions of the kits. we eluted the purified pcr products in μl elution buffer as suggested by the kits, then stored at − °c. we sequenced some positive samples from dromedary camels having low ct values (≤ ) on the real-time pcr. the sequencing was done by the sanger method using the applied biosystems® we used the obtained sequences from dromedary camels to develop the phylogenetic tree based on the reported partial mser-cov-s and n genes sequences. the phylogenetic trees were built using the neighbour-joining method by the mega- software, as previously described. the scale bar represents the tree distance corresponding to . nucleotide substitution/nucleotides. a series of two by two tables, utilizing the fisher's exact test, were used to test the association between the results of the molecular surveillance, the sampling intervals and the sampling technique [ ] . all the statistical data analyses were performed by spss version . (ibm corp, ). the values (< . ) were considered significant (fig. ) . our molecular testing for the selected animals at three time points starting march , until april revealed that / ( %) of the tested nasal swabs were positive; all the nine animals were negative from the breath samples (table ) . two weeks later, on march, / ( %) nasal swabs were positive, and / ( %) breath samples were positive (table ) . finally, on april, / nasal swabs ( %) were positive, while / ( %) of the breath samples were positive ( table ). the prevalence of positive nasal and breath samples was higher on the second sampling batch ( march) compared to first ( march) and third ( april) batches. however, the difference was statistically significant (p < . ) only between the first vs. second sampling batches. similarly, consolidated results obtained from both nasal swaps and breath were significantly higher in the second batch vs. the first batch (p < . ). no significant statistical differences were observed between the prevalence of positive results obtained from nasal swabs and breath samples. the detection of the mers-cov-rnas in the breath of all three time intervals was statistically significant (p > . ) ( table ) . the sequencing analysis of the partial mer-cov-s and n genes (figs and , respectively) from this positive mers-cov herd showing the detected viruses were closely related to the sequence from dromedary camels and humans from the arabian peninsula. the reported partial mers-cov-s gene clustered together with the other human sequences reported from al-hufuf city in the same regions in as well as sequences from qatar and jordon- (fig. ). the mers-cov continues to pose a significant risk for humans, especially some of those who may come in close contact with dromedary camels [ , ] . despite the emergence of sars-cov- late [ ] , mers-cov still has the high case fatality rates among the affected populations compared to sars-cov and sars-cov- . the mers-cov case fatality rate started at % in then dropped down to % in [ ] . the presence of mers-cov in the air of the proximity of patients and infected dromedary camels was previously documented independently by many research groups [ , , ] . this result confirms that mers-cov may be transmitted through droplet infection. however, little is still known about the mechanism of transmission of mers-cov from the dromedary camels to humans. the roles of some camel secretions and excretions in the context of the mers-cov infection have been studied [ , , ] . our results show that about % of the tested nasal swabs were positive during the first batch of the collection, while none of the breath samples was positive ( table ). the reasons behind the inability to detect the viral rnas in the breath during the first round of sampling may be attributed to the dilution of the viral rna in the breath, and the rna may be present at the nondetectable limit by the real-time pcr. a high consistency was observed between the detection of the viral nucleic acids in both the nasal and breath samples in all three tested batches (p > . ), indicating similar opportunities to detect the virus nucleic acid in both methods (table ). this may be attributed to several factors. first, during the early stage of the virus infection, the rna concentration in the breath may be at an undetectable level by the real-time pcr. second, the virus replication reached the peak of each animal; therefore, high virus concentration was achieved as previously reported [ , ] . these results showed that mers-cov could be shed in the breath of infected animals for a while. however, the nasal swabs are still the sample of choice in the diagnosis of mers-cov among the infected dromedary camel population. spreading of the virus from animal to another in the same herd could potentially happen through the respiratory routes taking into consideration the very close contact between animals within the same herd. it may also occur through sharing the contaminated food and water from infected animals; however, all these aspects still need further investigations. detection of the virus in the air of positive camel's herd [ , ] may suggest the virus is excreted in the breath of the infected animals in high concentration. however, this hypothesis was never tested before. detection of the foot and mouth diseases virus (fmdv) in the breath of some infected cattle was previously documented [ ] . this study confirmed the potential spread of the fmdv between animals and among various herds in close proximity or within a distance. the aim of our study was to test the possibility of mers-cov shedding in the breath of the infected dromedary camels. our results are clearly showing the potential secretion of mers-cov in the breath of infected animals ( table ) . the phylogenetic analysis based on the partial mers-cov-s and n genes in these infected animals revealed high identity to other mers-cov previously detected in the arabian peninsula (figs and ) [ , , ] . a recent study showed that the sars-cov- could be transmitted through the droplet infection in the air [ ] . although detection of the mers-cov-rnas in the breath does not conclude the viability of the detected virus particles. this may require virus isolation and the plaque assay to ensure the virus infectivity in the collected samples. these techniques should be done at a biosafety contaminant- laboratory. however, the overlapping between the viral detection in the nasal swabs and breath, as one of the gold standard techniques for the detection of the virus as well as the high degree of identity of the reported sequences in this study, may provide a piece of strong evidence about the potential shedding of the mers-cov in the breath of infected animals at various levels. further large-scale studies are highly recommended to document the curve of the mers-cov shedding in various body secretions and execrations as well as in the breath. this will lead to a better understanding of the dynamics of the virus spread among certain dromedary camel populations as well as in the environment. taken together all these evidence, we may conclude that mers-cov could be transmitted through the breath of infected animals. the virus spread from animal to animal and from animal to human come in their close contact (fig. ) . however, large-scale controlled studies are still required to further enrich our understandings about the mechanism of mers-cov transmission between animals as well as from animals to humans. middle east respiratory syndrome coronavirus (mers-cov) neutralising antibodies in a high-risk human population comparative serological study for the prevalence of anti-mers coronavirus antibodies in high-and low-risk groups in qatar middle east respiratory syndrome coronavirus during pregnancy middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia evidence for camel-to-human transmission of mers coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in africa and middle east genetic evidence of middle east respiratory syndrome coronavirus (mers-cov) and widespread seroprevalence among camels in kenya prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate mers-cov at the animal-human interface: inputs on exposure pathways from an expert-opinion elicitation middle east respiratory syndrome coronavirus and the one health concept dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) failure to detect mers-cov rna in urine of naturally infected dromedary camels risk of global spread of middle east respiratory syndrome coronavirus (mers-cov) via the air transport network airflow as a possible transmission route of middle east respiratory syndrome at an initial outbreak hospital in korea handbook of biological statistics a novel coronavirus from patients with pneumonia in china some one health based control strategies for the middle east respiratory syndrome coronavirus detection of foot-and-mouth disease virus in the breath of infected cattle using a hand-held device to collect aerosols airborne transmission of sars-cov- : the world should face the reality acknowledgements. we wish to thank the king abdul-aziz city for science and technology (kacst), saudi arabia, for their generous funding through the mers-cov research grant programme (number - / - ), which is a part of the targeted research program (trp). data availability statement. data are available upon request. key: cord- -ycmr prw authors: lee, jae hoon; lee, chang-seop; lee, heung-bum title: an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) date: - - journal: j korean med sci doi: . /jkms. . . . sha: doc_id: cord_uid: ycmr prw nan the editorial, "better understanding on mers coronavirus (mers-cov) outbreak in korea, " was previously published by lee ( ) . he briefly summarized the ongoing status of the middle east respiratory syndrome (mers) outbreak and emphasized close monitoring of medical staffs, patients, and visitors, and timely well-designed briefings to mass media. he pointed out critical aspects to control the mers-cov outbreak in korea. the present opinion proposes a topic on mers by focusing on early diagnosis of patients via appropriate specimen collection. as on july , , there have been confirmed cases of mers and fatalities reported in the republic of korea ( ). this national outbreak of mers has not been controlled yet but there is a rapid decrease in the number of current infections and fatalities. all patients in korea acquired their disease in hospital settings where they came in direct or indirect contact with mers patients. the ministry of health and welfare and the korea centers for disease control and prevention have carried out strong enforcement measures including quarantine care for up to days (the maximum incubation period of the disease) in cases of symptomatic travelers from the arabian peninsula who arrived in korea within the past weeks and individuals who came in close contact with confirmed cases of mers ( ) . suspicious cases are transferred to an assigned hospital if symptoms occur during the observation period. real-time reversetranscription polymerase chain reaction (rt-pcr) assay conducted in -hour-intervals using respiratory samples is the standard diagnostic tool for mers. a -year-old man developed fever, chills, and myalgia on june , . on may , he visited the emergency department of a hospital that had previously reported several cases of mers. during this time, he stayed in close contact with a laboratoryconfirmed mers patient. he was quarantined on may , at his home and he developed fever and chills, nausea, anorexia, and myalgia after the quarantine isolation. he was then moved to an institutional isolation care unit where real-time rt-pcr was conducted twice on sputum samples in -hour-intervals and resulted in negative findings. he was afebrile but his gastrointestinal discomfort and myalgia continued, and the isola-tion location was changed to his home on june , because he was improving. on june , he became febrile with symptoms of cough, dyspnea, and myalgia, which gradually deteriorated. he was tested again and the results returned positive for mers-cov on june . radiographic findings revealed far-progressed bilateral peripheral radio-opacities. some reasons that could be considered for the delayed diagnosis of mers include: ) low initial viral load and shedding, and ) poor sample collection in patients with "no cough" or dry cough. rapid and correct diagnosis of infection and control measures are critical in preventing the possible spread of mers-cov. however, the early symptoms of mers are non-specific and are the same as those of common pneumonia. in this context, it is very important to conduct thorough and careful patient interviews with a high interest of mers in mind and to obtain optimal samples for diagnosis. in the clinical findings of patients infected with mers-cov, patients with dry cough are more common than patients with productive sputum ( , ) . therefore, optimal sample collection is frequently limited, particularly in patients with no cough or dry cough. dipeptidyl peptidase (dpp ) has been identified as the receptor for mers-cov ( ). dpp is expressed in type i and ii alveolar cells, ciliated or non-ciliated bronchial epithelium, and bronchial submucosal glands ( , ) . this corresponds with viral tropism in ex vivo human lung cultures ( ) ( ) ( ) . in studies with rhesus macaques, intra-tracheally inoculated virus was present in the lungs but neither in the upper respiratory tracts, trachea, nor other organs ( , ) . all of marmosets infected with a high dose of mers-cov via various routes showed multifocal to coalescing, moderate to marked acute broncho-interstitial pneumonia centered on small calibre and terminal bronchioles which further extended into the adjacent pulmonary parenchyma. in summary, in vitro and in vivo studies concluded that mers-cov had adherence to the lower respiratory tract and high viral loads were mainly detected in distal respiratory tissues. therefore, lower respiratory tract specimens such as bronchoalveolar lavage fluid, deep tracheal aspirates, and induced sputum contain the highest viral loads which are optimal for increasing di- agnostic accuracy ( ) ( ) ( ) ( ) . in re-evaluating the patient's diagnostic history, his viral load could have been low due to the early phase of disease and/or could have been falsely negative due to inadequate dry coughlinked respiratory samples. in any occasion, his diagnosis was quite delayed. delayed diagnosis of patients is inevitably linked to delayed quarantine care of persons with contact history. to obtain an accurate diagnosis, the circumstances in the early phase of mers-cov infection should be considered and at least two repeated diagnostic tests during the late incubation period should be considered for patients with less severity but persistent symptoms. the nationwide pneumonia census has been conducted to identify hidden mers-cov infections. however, inadequate sputum specimens must have resulted in false negatives. for accurate surveying, appropriate specimens should have been obtained by collecting sputum from the lungs or bronchi and not saliva. in suspicious patients who are unable to produce sputum for examination, aerosol administration of a hypertonic saline solution may be used to increase the flow of secretions and stimulate coughing. however, during the procedure of induced specimen collection, clinicians must consider the high risk of contamination of the surroundings because the procedure would create a large amount of aerosols and increase the risk of transmitting the virus to other individuals. the optimum time for collection of a sputum specimen is in the early morning before eating or drinking. at this time secretions accumulated in the bronchi through the night are more readily available. in conclusion, to control the mers-cov infection completely, delicate history taking related with mers is very important, and appropriate specimen collection is essential as well. moreover, even two real-time rt-pcr tests in negative results during the early stage of the disease process cannot rule out silent mers-cov infections. clinicians should consider the possibility of false negative real-time rt-pcr findings resulting from inappropriate sample collection. finally, a sufficient period and strict quarantine of suspected cases are critical for control of the mers outbreak in korea. better understanding on mers corona virus outbreak in korea middle east respiratory syndome information middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs pathogenesis of middle east respiratory syndrome coronavirus an animal model of mers produced by infection of rhesus macaques with mers coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome kinetics and pattern of viral excretion in biological specimens of two mers-cov cases first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities key: cord- -lh czr a authors: ng, dianna l.; al hosani, farida; keating, m. kelly; gerber, susan i.; jones, tara l.; metcalfe, maureen g.; tong, suxiang; tao, ying; alami, negar n.; haynes, lia m.; mutei, mowafaq ali; abdel-wareth, laila; uyeki, timothy m.; swerdlow, david l.; barakat, maha; zaki, sherif r. title: clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates, april date: - - journal: the american journal of pathology doi: . /j.ajpath. . . sha: doc_id: cord_uid: lh czr a middle east respiratory syndrome coronavirus (mers-cov) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal mers-cov infection is unknown. we describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of mers-cov in the world, which was related to a hospital outbreak in the united arab emirates in april . the main histopathologic finding in the lungs was diffuse alveolar damage. evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. double staining immunoassays that used anti–mers-cov antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of mers-cov antigen; double immunostaining with dipeptidyl peptidase showed colocalization in scattered pneumocytes and syncytial cells. no evidence of extrapulmonary mers-cov antigens were detected, including the kidney. these results provide critical insights into the pathogenesis of mers-cov in humans. middle east respiratory syndrome coronavirus (mers-cov) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal mers-cov infection is unknown. we describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of mers-cov in the world, which was related to a hospital outbreak in the united arab emirates in april . the main histopathologic finding in the lungs was diffuse alveolar damage. evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. double staining immunoassays that used antiemers-cov antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of mers-cov antigen; double immunostaining with dipeptidyl peptidase showed colocalization in scattered pneumocytes and syncytial cells. middle east respiratory syndrome coronavirus (mers-cov) was initially isolated from a sputum specimen of a patient who died of respiratory and renal failure in saudi arabia in . recent data have indicated that dromedary camels are likely to transmit mers-cov to humans. , human-to-human mers-cov transmission is well documented, particularly in the setting of nosocomial outbreaks. , as of july , , the world health organization (who) was notified of laboratory-confirmed cases of mers-cov infection with reported deaths ( . %) from countries, primarily in men with a median age of years. most cases (> %) were reported from the kingdom of saudi arabia. mers-cov infection can result in a wide clinical spectrum from asymptomatic infection, upper respiratory tract illness, to severe pneumonia and multiorgan failure. e mers-cov binds to dipeptidyl supported by cdc operational funds. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. disclosures: none reported. current address of m.g.m., national cancer instituteeshady grove, rockville, md. peptidase (dpp ) receptors that are primarily in the lower respiratory tract but also distributed in other tissues. although there have been numerous cases and fatalities, the pathologic changes and viral distribution in humans associated with severe mers-cov illness are unknown, and knowledge of pathogenesis remains limited. this report provides the first autopsy, clinicopathologic, immunohistochemical (ihc), and ultrastructural description of a fatal case of mers-cov. this patient, who had multiple close contacts, was identified as part of an epidemiologic investigation of a large cluster of mers-cov infections at hospital in the united arab emirates (uae) in april . this outbreak occurred in the context of a surge of cases in the arabian peninsula in which mers-cov cases were identified in saudi arabia from april to june , (world health organization, http://www.who.int/csr/don/ _ _ _ mers/en, last accessed october , ). tissues obtained at autopsy were fixed in % buffered formalin, paraffin-embedded, sectioned at mm, and stained by routine hematoxylin and eosin. an immunostaining protocol was applied with use of several mouse antibodies and one human antibody to mers-cov as described previously. ihc assays that used mouse anti-mers antibodies ( table ) lung tissue samples were excised from a paraffin block with the use of a -mm punch and processed for electron microscopy examination as previously described. full genome sequencing by the sanger method with the use of direct genome walking pcr and next-generation sequencing (illumina miseq; illumina, san diego, ca) were performed as previously described on confirmed positive lung tissue, named as abu dhabi_uae_ _ and deposited into genbank (http://www.ncbi.nlm.nih.gov; genbank accession number kp ). clustalw was used to align the full genome with the respective available complete or near complete mers-cov genomes in the american journal of pathologyajp.amjpathol.org the patient was a -year-old, nonsmoking, obese (body mass index, . kg/m ) filipino man with no relevant past medical history, recent travel, or exposure to sick contacts or farm animals. he worked in a storage room at a paramedic station with no patient care duties. he shared housing with five roommates from the paramedic department. he presented to an emergency department in abu dhabi, uae, on april , , with a -day history of fever, rhinorrhea, and productive cough. at evaluation, he was afebrile with an oxygen saturation of % on room air, and a chest x-ray showed a left-sided opacity ( figure a) . the patient was diagnosed as acute bronchitis, prescribed mg prednisolone daily for days and paracetamol, and discharged. he returned to the emergency department on april , , with persistent cough and shortness of breath; a chest x-ray showed a left-sided opacity and air bronchograms, and the patient was diagnosed with pneumonia, given a prescription for levofloxacin, and discharged ( figure b) . he returned to the emergency department the same day, with worsening shortness of breath and was admitted. he had a temperature of . c, pulse of beats per minute, a respiratory rate of breaths per minute, blood pressure of / , an oxygen saturation of % on room air, and left basal crackles and rales. an arterial blood gas on room air showed ph of . , partial pressure of carbon dioxide of . mm hg, partial pressure of oxygen of . mm hg, and bicarbonate of . meq/l, consistent with respiratory alkalosis. laboratory evaluation showed a normal white blood cell count with lymphopenia ( . k/ml) and creatinine of . mg/dl (supplemental table s ). blood, sputum, and urine cultures were obtained, and a nasopharyngeal swab specimen was sent for mers-cov testing. on april , day of admission, oseltamivir, ceftriaxone, and azithromycin were started empirically. the day after admission he was transferred to the intensive care unit for tachypnea and respiratory distress and was intubated for mechanical ventilation; a portable chest x-ray showed multiple patchy airspace opacities ( figure c ). the patient became hypotensive, requiring inotropic support, and developed acute kidney injury and renal failure, requiring dialysis. on april , rt-pcr assay of the nasopharyngeal swab specimen collected on april was reported to be positive for mers-cov for upe and orf a gene targets. bacterial cultures of blood, sputum, and urine specimens obtained at admission after antibiotic administration were negative. on april , he was given mg hydrocortisone intravenously every hours and started on nitric oxide at ppm. the patient's condition continued to deteriorate, and he died april . the body was kept refrigerated at c, and an autopsy was performed days after death. notable findings included massive pleural effusion ( l), substantial pericardial effusion ( ml), and abdominal effusion; edematous and consolidated lungs; and generalized congestion. the predominant pulmonary histologic pattern was exudative-phase diffuse alveolar damage with denuding of bronchiolar epithelium, prominent hyaline membranes, alveolar fibrin deposits, type pneumocyte hyperplasia, rare multinucleated syncytial cells, and alveolar septa involved by edema and lymphocytes with fewer plasma cells, neutrophils, and macrophages (figure , aec) . dispersed foci of necrotic debris were seen both subpleurally and within alveoli. no viral inclusions were seen, and moderate anthracosis was present. all four antibodies ( / , sections of trachea and bronchi showed mild-to-moderate lymphocytic mucosal and submucosal inflammation with a few neutrophils and plasma cells ( figure g ). occasional clusters of noninvasive and extracellular candida yeast and hyphae were seen in septa, alveolar spaces, and overlying respiratory epithelium, suggesting postmortem overgrowth. bronchial submucosal glands were focally necrotic ( figure h ). immunostaining for mers-cov antigens was identified in both unremarkable and necrotic bronchial submucosal glands ( figure i ). multiple double immunostaining immunoassays were performed to assess for colocalization of mers-cov and cells labeling for cytokeratin, cd , surfactant, and dpp . double staining with cytokeratin confirmed the presence of viral antigens within pneumocytes and epithelial syncytial cells, whereas double staining with cd showed two distinct populations with no colocalization of mers-cov and macrophages (figure , a and b) . surfactant double staining revealed type pneumocytes and syncytial cells with intracytoplasmic viral antigens ( figure c ). dpp antigens were detected in pneumocytes, syncytial cells, mononuclear leukocytes, and vascular endothelium, although colocalization of mers-cov and dpp was observed in scattered pneumocytes and syncytial cells ( figure d ). electron microscopy showed infected and degenerated pneumocytes encased between hyaline membranes, composed of fibrin and basement membranes ( figure e ). clusters or individualized, predominately spherical, to nm in diameter, viral particles were observed in membrane-bound vesicles ( figure f) . the kidney showed increased globally sclerotic glomeruli ( % to % of glomeruli), thickened bowman capsules, severe atherosclerosis and hyaline arteriolosclerosis, patchy interstitial inflammation, and intratubular proteinaceous and granular casts. diminished lymphoid follicles and a robust interfollicular proliferation of pleomorphic immunoblasts intermixed with a polymorphous population of reactive lymphocytes were seen in multiple lymph nodes; the spleen also contained numerous immunoblasts and reactive lymphocytes. the bone marrow was normocellular with maturing trilineage hematopoiesis and left-shifted the american journal of pathologyajp.amjpathol.org granulopoiesis. the heart revealed diffuse myocyte hypertrophy, moderate coronary atherosclerosis, and patchy fibrosis. moderate steatosis, scattered calcifications, and mild portal tract and lobular lymphocytic inflammation were identified in the liver. the sections of the cerebrum and cerebellum were unremarkable. mers-cov ihc was negative in multiple specimens from different organs, including kidney, liver, spleen, several lymph nodes, bone marrow, small intestine, and colon. overall the histology was well-preserved; although the sections of brain showed minimal-to-mild autolysis and kidney showed moderate autolysis. the genome sequence is similar (> %) to other known mers-covs and clustered closely with camel-derived mers-cov strains (kj to kj ) obtained in al-hasa, saudi arabia, in , suggesting recent origin in camels, although the patient had no known camel exposures. as indicated from the phylogenetic tree (supplemental figure s ), the sequences from this case and close contacts cluster most closely in the same clade, further supporting their transmission link between these cases. this report provides invaluable insights as the first description in the published literature of the clinicopathologic, ihc, and ultrastructural findings of a fatal case of mers-cov infection. the histopathologic pattern observed in the lungs was diffuse alveolar damage. mers-cov ihc and double staining techniques showed viral antigens were predominantly localized to type pneumocytes and epithelial syncytial cells. although the pathogenesis of severe and fatal mers-cov infection is unknown, these postmortem findings provide critical insights, including evidence that pneumocytes are important targets, suggesting that direct cytopathic effects contribute to mers-cov respiratory symptoms. an ex vivo study that examined mers-coveinfected human lung tissue found evidence of pneumocyte damage by electron microscopy, including detachment of type pneumocytes, and membrane blebbing, suggestive of apoptosis. however, ihc staining for mers-cov was patchy, implying that other causes such as immune dysfunction may be relevant. acute renal failure is commonly observed in critically ill mers-cov patients e , and mers-cov rna was detected in urine , ; however, no evidence of extrapulmonary mers-cov dissemination was observed, suggesting that acute renal failure in this patient was not caused by direct renal infection but likely by other factors, such as hypoperfusion or cytokine dysregulation. the presence of mers-cov antigens in submucosal glands provides a mechanism by which the virus enters respiratory secretions and becomes transmissible. the pathologic features of mers-cov are shared by other similar respiratory illnesses, such as severe acute respiratory syndrome (sars)-cov. several reports that evaluated fatal cases of sars-cov describe diffuse alveolar damage in various stages as the most characteristic feature, with ihc staining of sars-cov antigen primarily in alveolar epithelial cells. syncytial cells may also be seen with other coronavirus infections, including sars-cov and paramyxovirus infections. mers-cov entry is mediated by dpp , which is expressed throughout the lower respiratory tract but also numerous other organs, including the kidney. double staining with mers-cov and dpp ihc was seen in pneumocytes and syncytial cells, consistent with the identification of these cells as targets of mers-cov infection. in addition, ex vivo models have detected mers-cov antigen in bronchial epithelial cells, pneumocytes, and endothelium and dpp in bronchiolar epithelium, endothelium, pneumocytes, and alveolar macrophages, , correlating with our findings. in contrast, the functional receptor for sars-cov is angiotensin-converting enzyme . similar to ddp , angiotensin-converting enzyme has a broad distribution, with heavy expression in alveolar epithelial cells, enterocytes, and endothelial cells. although evidence shows that sars-cov also infects type pneumocytes, , sars-cov was detected in several extrapulmonary sites by in situ hybridization and electron microscopy, including circulating lymphocytes, lymphoid tissues, renal distal tubules, and small intestinal mucosa. because mers-cov rna was detected in blood, and dpp is widely distributed in different tissues, extrapulmonary dissemination could be possible, but we did not detect any evidence of mers-cov spread outside the respiratory tract. pulmonary findings of consolidation, diffuse alveolar damage, and pleural effusions with no evidence of bacterial pneumonia are consistent with the clinical features reported in other critically ill adults with mers-cov infection. e pathologic evidence of several chronic diseases in this patient, including myocardial fibrosis, atherosclerosis, and hepatic steatosis, correlates with reports of individuals with underlying comorbidities, such as end-stage renal disease, diabetes mellitus, hypertension, or cardiac disease, having an increased risk of developing severe mers-cov infection. , the impact of low-dose oral prednisolone before admission and intravenous hydrocortisone on the clinical course and fatal outcome of this mers-cov patient is unknown. follicular depletion in lymph nodes, consistent with corticosteroid use, was noted, and no bacterial coinfections were identified. the hydrocortisone dosing of mg every hours in the intensive care unit before death was higher than recommended for refractory septic shock by the surviving sepsis campaign guidance, but the patient only received a few doses. a systematic review of high-dose corticosteroids for treatment of sars-cov reported evidence of harm without survival benefit. in patients with influenza a(h n )pdm virus infection, corticosteroid treatment was associated with increased mortality, although further studies are needed. therefore, until further evidence is available, high-dose corticosteroid treatment for mers-cov pulmonary disease should be avoided. current clinical management of mers-cov patients is based on supportive care of complications and adherence to recommended infection prevention and control measures (cdc, http://www.cdc.gov/coronavirus/mers/infection-preventioncontrol.html, last accessed october , ). our findings provide invaluable and unprecedented insights into the histologic changes, pathogenesis, and viral dissemination of mers-cov in humans. further studies, including additional postmortem examinations, evaluation of the host immune response, and identification of sites of viral replication, are necessary to strengthen knowledge on pathogenesis, transmission patterns, and effective treatment strategies for optimal clinical management and infection control practices. isolation of a novel coronavirus from a man 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management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance we thank cynthia goldsmith for reviewing the electron microscopy and azaibi tamin for providing the mers-coveinfected vero cell cultures. supplemental material for this article can be found at http://dx.doi.org/ . /j.ajpath. . . . key: cord- - vw a authors: darling, n. d.; poss, d. e.; schoelen, m. p.; metcalf-kelly, m.; hill, s. e.; harris, s. title: retrospective, epidemiological cluster analysis of the middle east respiratory syndrome coronavirus (mers-cov) epidemic using open source data date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: vw a the middle east respiratory syndrome coronavirus (mers-cov) is caused by a novel coronavirus discovered in . since then, cases, including deaths, have been reported by the kingdom of saudi arabia (ksa) and affected countries as of june . previous literature attributed increases in mers-cov transmission to camel breeding season as camels are likely the reservoir for the virus. however, this literature review and subsequent analysis indicate a lack of seasonality. a retrospective, epidemiological cluster analysis was conducted to investigate increases in mers-cov transmission and reports of household and nosocomial clusters. cases were verified and associations between cases were substantiated through an extensive literature review and the armed forces health surveillance branch's tiered source classification system. a total of clusters were identified, primarily nosocomial ( · %) and most occurred in ksa ( · %). clusters corresponded temporally with the majority of periods of greatest incidence, suggesting a strong correlation between nosocomial transmission and notable increases in cases. middle east respiratory syndrome coronavirus (mers-cov) is a respiratory illness caused by a novel coronavirus originally discovered in . mers-cov can cause severe acute respiratory symptoms, including fever, cough, and shortness of breath, and is fatal in approximately one-third of reported cases. presently, there is no vaccine to prevent infection and no specific antiviral treatment for those infected with the virus [ ] . mers-cov is the sixth strain of human coronavirus identified. although mers-cov cases were first reported from the kingdom of saudi arabia (ksa) in september , the first two known cases were retrospectively discovered in jordan from april [ ] . since its discovery in , mers-cov cases have predominately been reported from ksa; however, cases have also been reported from algeria, austria, bahrain, china, egypt, france, germany, greece, iran, italy, jordan, kuwait, lebanon, malaysia, the netherlands, oman, philippines, qatar, republic of korea (rok), thailand, tunisia, turkey, united arab emirates (uae), united kingdom (uk), united states (usa), and yemen [ ] . according to the armed forces health surveillance branch (afhsb), which is an organization under the defense health agency that utilizes biosurveillance to protect and promote the health of the us armed forces, as of june , cases of mers-cov have been reported, including at least deaths [ ] . afhsb's death count (case fatality proportion - %) includes only those deaths which have been publicly reported and verified. the dynamics of the transmission of mers-cov is essential to understanding the risk posed by the virus as well as instituting effective infection control and prevention practices in areas where humans are at a greater risk of exposure. despite beliefs that camels are the most likely reservoir of the virus, the limited camel-to-human transmission has been reported by cdc and who [ , ] . many published findings suggest that camel calves play a potential role in mers-cov transmission [ ] [ ] [ ] . they found the 'rate of virus isolation [was] significantly higher in calves', and calves are often more acutely infected with mers-cov than adult camels, suggesting increased infectivity among calves [ ] [ ] [ ] . although camel breeding season has been a proposed contributor to mers-cov transmission among camels, there is insufficient literature to support that human infections are more common during this time of year. human-to-human transmission of mers-cov has been investigated as a potentially significant route of spread. researchers have found that close contact with infected individuals is required to transmit the virus from one human to another, supporting the role of limited human-to-human transmission in the mers-cov epidemic and, more specifically, its role in nosocomial and household clusters [ ] . while it is known that the virus can be transmitted through respiratory secretions, the exact routes through which the virus spreads are not well understood [ ] . in an effort to better understand the patterns of transmission, a retrospective analysis of epidemiological clusters identified throughout the ongoing mers-cov epidemic was conducted using open-source data. epidemiological literature, classified by the tiered-source classification system (tscs), addressing mers-cov cluster analyses was collected and reviewed. several key search terms were utilized to capture all cluster-related literature, including 'mers-cov', 'nosocomial', 'cluster', 'transmission', 'superspreader', 'contact tracing', and 'healthcare worker'. see supplemental for a comprehensive list of key search terms. publications selected for inclusion in the literature review were classified using tiers. this system was developed by afhsb to categorize the sources used in the literature review by their credibility. literature published by official sources, including the who and cdc, was considered a tier source. literature published by reputable sources other than the who and cdc (e.g. all peer-reviewed journals regardless of perceived impact factor, including the lancet or nature) was considered a tier source. literature from a foreign source, such as the ksa ministry of health (moh) or a media source, was classified as a tier source. inclusion of a source required two or more of the key terms in supplemental . the literature included in the analysis encompassed translatable studies, situation reports from public health agencies, and publications updated to include more recent cluster information. studies related to mers-cov that were identified as having one or more of the following characteristics were excluded from the analysis: an investigational period prior to ; published in a non-translatable language; molecular-based; a focus on viral reservoirs, genealogy, or genetics, preventive measures, or other coronaviruses. in total, studies were selected for inclusion. of these, were classified as tier sources, as tier , and as tier (table ). these sources were used to identify clusters as well as verify and characterize associations between cases. a mers-cov cluster was defined as two or more persons with onset of symptoms within a -day incubation period who are associated with a specific setting [ ] . clusters were further categorized as exported, nosocomial, and/or household clusters. an exported cluster was defined as any cluster that resulted from verified travel of an index case (from an area of known mers-cov transmission) within one incubation period ( days) of symptom onset. if the index case was asymptomatic, verified travel from an area of known mers-cov transmission within days prior to the date of the index case was laboratory confirmed for mers-cov. a nosocomial cluster was defined as a cluster associated with a healthcare or hospital setting. a household cluster was defined as a cluster associated with the same family and/or physical household. case identification and data collection were performed on an ongoing basis by epidemiologists at afhsb beginning with the emergence of the mers-cov outbreak in . case demographics, including city and country of origin, age, gender, date of symptom onset (if any), asymptomatic status, mortality, comorbidities, healthcare worker (hcw) status, and date reported, were collected on a daily basis. each case was verified using tiers and sources. if a tier or source failed to verify a case reported by a tier source, it was not included in the afhsb case line list. if an epidemiological link between cases was identified through a tier source, tiers and sources were used to verify the link. if a tier or source was not available, supporting data from at least three separate tier sources were used as verification. due to the lack of data available at the local level in the arabian peninsula, in the event that multiple ongoing nosocomial outbreaks were known to have been occurring in one area, all cases reported to have been associated with that area and possibly epidemiologically linked to one of the ongoing clusters were categorized under one cluster (e.g. riyadh, jeddah). for all exported mers-cov clusters, the city and country of origin were determined by the reported travel history of the index case. if travel history was only available in the country level, the capital city of the country of travel was used as the point of origin for the index case. for two identified clusters, the index case had to travel to multiple countries with a history of confirmed autochthonous mers-cov transmission. as a result, the country of most probable exposure, determined by the duration of stay in the country as well as active transmission reported in that country at the time of travel, was used as the point of origin. in order to visually display the overlap of clusters on the epidemiological curve, the start and end dates of each identified cluster were defined. the start date of each cluster was the date of symptom onset of the index case. in the absence of symptom-onset data, the 'report date', or the date a case was publically reported, was used instead. the end date of each cluster was determined by adding days to the date of symptom onset or date of death of the last case identified in the cluster. the -day period is representative of the maximum incubation period of a mers-cov case, ensuring no additional cases could have been associated with a given cluster [ ] . for asymptomatic cases, date of diagnosis was used in place of date of symptom onset. if the date of diagnosis was not publically available, date reported was used. april and june , ( · %) cases were determined to have been associated with at least one of the clusters identified in this analysis [ , ] . a small portion of cases associated with one or more clusters were hcws (n = ), and clusterassociated cases were asymptomatic ( from another mers-cov case, including nosocomial infections and household infections. as the ksa moh did not specify which mers-cov case these were acquired from, it is unclear if any of these batched cases were associated with the identified mers-cov clusters or were part of separate clusters. of the identified clusters, ( · %) were classified as nosocomial clusters; ( · %) were household clusters; and eight ( · %) were exported (table ). ten clusters were classified as more than one type of cluster, including four exported nosocomial clusters, three exported household clusters, and three clusters with both nosocomial and household characteristics. three clusters displayed both nosocomial and household transmission characteristics, four clusters were classified as both exported and nosocomial, and three clusters were classified as both exported and household. the two countries reporting the greatest number of nosocomial clusters were ksa and rok, with and nosocomial clusters, respectively. of the eight exported clusters, two (rok , tunisia ) had index cases with travel to multiple countries with a history of confirmed autochthonous mers-cov transmission (see table , technical appendix). the average duration of each cluster was · days, with a range of - days. cluster size ranged from two cases to cases, with an average of individuals affected. over % of the cluster-associated cases were acquired in ksa (n = , · %) and rok (n = , · %), supporting the notion that nosocomial transmission, which accounted for % of the clusters identified in rok and % of the clusters identified in ksa, was a prominent driver of clusters in this epidemic. figure depicts the epidemiological curve of the mers-cov outbreak using the estimated epidemiological week of illness onset for each case. symptom-onset date was available for cases ( · %), including ( · %) of the clusterassociated cases. for asymptomatic cluster-associated cases, date reported was used for of the cases, and date of diagnosis was used for eight cases. the cluster bands seen above the epidemiological curve in figure illustrate the duration of transmission within each cluster and the corresponding overlap of identified clusters with peak mers-cov incidence over the epidemic period of this study. most temporal peaks correspond with at least two ongoing clusters (see fig. ). the time periods of greatest incidence (> incident cases at the period's peak) in and correspond with at least four ongoing mers-cov clusters, of which at least two were classified nosocomial in each of these periods. see table (technical appendix) for individual cluster data, such as cluster duration and a number of cases affected. the clusters identified in this analysis spanned the duration of the epidemiological curve, demonstrating the consistency of cluster-driven transmission throughout the epidemic. clusters also corresponded with each period of increased mers-cov incidence and accounted for over % of the total confirmed cases reported, supporting the notion that human-tohuman transmission is a prominent driver of the mers-cov epidemic. the alignment of nosocomial clusters with the time periods of greatest incidence (> incident cases at the period's peak) suggests nosocomial transmission is a key driver of transmission in the mers-cov epidemic as opposed to seasonal events, such as the hajj or camel-breeding season that occur in the fall and spring, respectively. the absence of apparent seasonality in the identified clusters further supports the significant role humanto-human transmission in healthcare settings plays in the propagation of mers-cov. classification of each cluster into one or more of the three disease transmission categories (nosocomial, household, or exported) elucidated common characteristics among the clusters, including the overall concentration of case clusters in cities and healthcare institutions. with nosocomial transmission accounting for of the identified clusters ( · %), the role of healthcare facilities, transportation protocols during inter-and intra-hospital transfers, and the contribution of cultural norms such as 'doctor shopping' became apparent themes observed throughout this analysis. in rok, practices such as seeking healthcare at multiple facilities, or 'doctor shopping', and being cared for by relatives while hospitalized are believed to have contributed to the emergence of the largest outbreak outside of ksa during the mers-cov epidemic [ , ] . additionally, four superspreaders, defined as a confirmed mers-cov case responsible for infecting or more secondary cases, played a critical role in the perpetuation of transmission in rok. the mers-cov outbreak in rok included clusters at separate healthcare institutions, each with an index case related to an ongoing mers-cov cluster at another facility. between may and july , laboratory-confirmed mers-cov cases were reported. the geographic distribution and rapid pace at which mers-cov spread in rok demonstrates the susceptibility of healthcare environments to human-to-human transmission of the virus and their catalytic role in mers-cov transmission. in ksa, clusters were primarily concentrated in cities, specifically active transmission at healthcare facilities within those cities, as illustrated by the largest identified clusters in the country (ksa , ksa , ksa , ksa , and ksa ; see table , technical appendix). the most common theme in the perpetuation of transmission among nosocomial clusters was the role of more transient departments in the healthcare setting, such as dialysis units and emergency departments, which were implicated in a majority of the nosocomial clusters identified in this analysis. at least seven of the clusters identified in ksa occurred in designated mers-cov treatment centers, which were presumably best equipped to handle these infectious cases. however, the nosocomial transmission that persisted in these clusters suggests inconsistent infection control practices across different departments of those designated hospitals [ ] . hcws likely contributed to the continued transmission among patients and between wards within a hospital, as hcws represent ( · %) of the clusterassociated cases. of the clusters classified as nosocomial, clusters ( · %) involved hcws. the use of tier data collected as the mers-cov outbreak progressed is one of the greatest strengths of this comprehensive cluster analysis. collection of the data and emerging literature in real time allowed for an inclusive literature review from which cluster identification and evaluation of key components of the outbreak could be analyzed. the availability of opensource data and information provided the opportunity to precisely map each confirmed case temporally and geographically. utilization of the date of symptom onset for the epidemiological curve created an accurate representation of the progression of the epidemic and corresponding cluster durations (fig. ) . the availability of open-source data from certain regions also served as a limitation in this analysis. due to limited demographic information, it was occasionally necessary to broaden the case criteria for a particular cluster, specifically clusters ksa , ksa , and ksa (see table , technical appendix). if a case was reported from the city during the estimated time in which there was ongoing nosocomial transmission, had no travel or camel exposure in the days prior to illness onset, and had no known household contact with a confirmed mers-cov case, the case was included in the case count for that particular nosocomial cluster. although the particular epidemiological details regarding the exact location of exposure were generally not provided for cases during the three aforementioned clusters, tier sources often denoted if a case was under investigation for a possible link to a hospital with the known ongoing transmission of mers-cov, which reinforced the inclusion of these cases in their respective clusters. in addition to limitations on precise case inclusion in the larger clusters denoted above, availability of date of symptom onset may have created an artifact in the cluster date and duration, potentially altering its appearance on temporal scales. of the known symptomatic cases, illness onset data were missing for cases, including cluster-associated cases, and date reported was used as an approximation. on average, afhsb observed an - -day lag from the date of symptom onset to the date a case was reported. considering our analysis aggregated these cases by estimated epidemiological week of illness onset, the impact of this possible artifact is likely to be relatively insignificant to the overall trends observed in this analysis. additionally, ksa retrospectively released information on confirmed mers-cov cases on june and on cases on september with minimal geographic and demographic data. all cases released on june occurred between may and may , with a majority of the cases (n = ) occurring after march . the rest of the cases (n = ) occurred between may and february . as no other date was available, the date reported was used to represent the cases released in these two batches on the epidemiological curve. asymptomatic cases accounted for · % (n = ) of the total cases in this study population. over % (n = ) of these asymptomatic cases were related to a cluster. this finding may support who's assessment that contact tracing efforts intensified as the epidemic progressed and are responsible for the detection of asymptomatic cases [ , ] . a study performed by cdc analyzing case data between september and january found that there was likely an underrepresentation of asymptomatic cases reported from the countries of the arabian peninsula. estimations suggest that the total number of mers-cov cases from the region may be · times greater than the total number of cases recorded to date [ ] . inconsistencies in reporting of asymptomatic cases from entities such as the ksa moh may have contributed to an underrepresentation of not only the total number of mers-cov cases in the outbreak but also the number of clusters, as well as the breadth and duration of the identified mers-cov clusters. the supplementary material for this article can be found at https://doi.org/ . /s . middle east respiratory syndrome (mers): prevention & treatment centers for disease control and prevention military-health-topics/ health-readiness/armed-forces-health-surveillance-branch/integrated-biosurveillance/surveillance-summaries) world health organization. a roadmap for research and product development against middle east respiratory syndrome-coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir dromedary camels and middle east respiratory syndrome: mers coronavirus in the 'ship of the desert' co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia acute middle east respiratory syndrome coronavirus infection in livestock dromedaries guidance for monitoring and movement of persons with potential middle east respiratory syndrome coronavirus (mers-cov) exposure surveillance for human infection with middle east respiratory syndrome coronavirus (mers-cov): interim guidance novel coronavirus infection update middle east respiratory syndrome (mers) in the republic of korea middle east respiratory syndrome coronavirus outbreak in the republic of korea infection prevention and control guidelines for the middle east respiratory syndrome coronavirus (mers-cov) infection moh's command and control center forms rapid response teams including health specialists middle east respiratory syndrome coronavirus (mers-cov) global summary and risk assessment accessed estimation of severe middle east respiratory syndrome cases in the middle east the authors acknowledge the support provided by afhsb's us government partners, including the centers for disease control and prevention and the united states forces korea. this study received no specific grant from any funding agency, commercial, or not-for-profit sectors. none. key: cord- -n s dzgp authors: al-tawfiq, jaffar a.; memish, ziad a. title: update on therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) date: - - journal: expert rev anti infect ther doi: . / . . sha: doc_id: cord_uid: n s dzgp introduction: the middle east respiratory syndrome coronavirus (mers-cov) is an important emerging respiratory pathogen. mers-cov resulted in multiple hospital outbreaks within and outside the arabian peninsula. the disease has a high case fatality rate, with the need for a therapeutic option. areas covered: in this review, we provide an overview of the progress in the development of therapeutic strategies for mers. we searched pubmed, embase, cochrane, scopus, and google scholar, using the following terms: ‘mers’, ‘mers-cov’, ‘middle east respiratory syndrome’ in combination with ‘treatment’ or ‘therapy’. expert commentary: there are multiple agents tried in vitro and in vivo. none of these agents were used in large clinical studies. available clinical studies are limited to the use of the combination of interferon and other agents. these clinical studies are based solely on case reports and case series. there are no prospective or randomized trials. there is a need to have prospective and randomized clinical trials for the therapy of mers-cov. however, this strategy might be hampered by the sporadic cases outside the large hospital outbreaks. the middle east respiratory syndrome coronavirus (mers-cov) emerged as an important virus in and since then has caused multiple outbreaks in hospitals especially in the kingdom of saudi arabia and outside the arabian peninsula [ ] [ ] [ ] . since the emergence of mers-cov, a total of cases including deaths were reported by the world health organization (who) [ ] . due to the increased morbidity and mortality of mers-cov infection, the attention was directed toward the development of prevention strategies and the establishment of therapeutic modalities. an earlier review was based on the severe acute respiratory syndrome (sars) experience and had suggested few possible options for the treatment of mers-cov infection [ ] . in this review, we provide an overview of the progress in the development of therapeutic strategies for mers. we searched pubmed, embase, cochrane, scopus, and google scholar using the following terms: 'mers,' 'mers-cov,' 'middle east respiratory syndrome' in combination with 'treatment' or 'therapy. ' we also reviewed the references of each article to further include other studies or reports not identified by the search. we classified the studies into the following categories: in vivo and in vitro studies, animal studies, and human case reports or case series. for clinical studies, we graded the level of the evidence based on the 'oxford centre for evidence-based medicine' [ ] . in vitro studies showed variable activity of various agents against mers-cov (table ) . these agents include: interferon, ribavirin, hiv protease inhibitors (nelfinavir, ritonavir, and lopinavir). interferon is antiviral type i ifn system, a major part of the innate immune response [ , ] . in vitro studies showed that ifn-β has an ic of . u/ml and that ifn-β has anti-mers-cov activity of -, -, -, and -fold higher than ifn-α b, ifn-γ, ifn-universal type and ifn-α a, respectively [ ] . in vitro studies showed that ifn-β has a lower ic for mers-cov compared to ifn-a b [ ] . ribavirin is a nucleoside analog that is activated by host kinases to a nucleotide [ , , ] . it was shown that in vitro doses of ribavirin required to inhibit mers-cov replications are too high to be achieved in vivo [ , ] . nelfinavir and lopinavir inhibit mers-cov in vitro [ , ] . the mean % effective concentration (ec ) of lopinavir using vero e and huh cells was . μm [ ] . camostat and the heptad repeat peptide (hr p) are two mers-cov fusion inhibitors that were tested in vitro [ , ] . the fusion inhibitor, camostat, inhibited viral entry into human bronchial submucosal gland-derived calu- cells but not the immature lung tissue [ ] . the second fusion inhibitor, hr p, inhibits mers-cov replication and the spike protein-mediated cell-cell fusion [ ] . cyclosporin affects the function of many cyclophilins that act as chaperones and facilitate protein folding [ , ] . in vitro, cyclosporine inhibited mers-cov replication [ , ] . nitazoxanide, a broad-spectrum antiviral agent, and teicoplanin, an inhibitor of cathepsin l in the late endosome/lysosome and blocker of the entry of mers-cov, also showed inhibitory effect of mers-cov in vitro [ , ] . there are few studies evaluating various agents as therapy for mers-cov in animal models (table ) [ , [ ] [ ] [ ] [ ] . in the rhesus macaques model, interferon-α b-ribavirin combination decreased viral replication within h of mers-cov infection [ ] . in a primate model, the mortality rate at h post-inoculation was reduced from % in untreated to - % in animals treated with a combination of interferon-β b and either lopinavir or ritonavir [ ] . intranasal use of an hr p analog with improved pharmaceutical property, hr p-m , was protective in mice model [ ] . in an animal model using mers-cov infected mice, the use of high titer mers immune camel serum was effective in reducing lung injury and acceleration of virus clearance [ ] . mycophenolate has a direct and indirect antiviral activity by modulation of ifn response [ ] . the use of mycophenolate in the common marmoset animal model resulted in higher mortality than untreated animals [ ] . a monoclonal antibody designated as m is an antibody derived from a large phage-displayed antibody library from b cells of healthy donors [ ] . the use of this m in mice showed promising results as a therapeutic and a prophylactic agent [ ] . currently, there is no animal model that completely reflects the course of mers-cov disease in humans and thus the data obtained from these animal models are to be interpreted cautiously. and animal models utilize therapy shortly after infection. based on analysis of sars data, interferon-ribavirin combination was suggested as a possible therapeutic option for the treatment of mers-cov infections [ ] . limited data are available regarding the clinical efficacy of antiviral agents [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( table ). the first use of the combination of ribavirin-interferon therapy was in five patients with mers infection [ ] . the therapy was started late in the course of the disease with a median time from admission to therapy of days [ ] . of the included patients, none responded to therapy [ ] . in a subsequent retrospective cohort study, mers patients received ribavirin-interferon compared to patients who did not [ ] . the -day survival rate was better in those who received the combination therapy ( % vs. %, p = . ); however, the -day survival rate was not statistically different ( % vs. %) ( table ) [ ] . in another case series, mers patients had ribavirin and peg-interferon α- a [ ] . ribavirin-ifn-α a was compared to ribavirin-ifn-β a in a study of and patients, respectively [ ] . the mortality rate was not statistically different between the two groups ( % vs. %) [ ] . in a large cohort study of patients, various combinations of interferon and ribavirin were used with different outcomes (table ) [ ] . in a case series of patients, patients received ribavirin and interferon-alfa b within - days of admission and they survived compared to the other patients who died as they received the therapy - days after admission [ ] . another study evaluated the use of interferon beta, interferon alpha, or ribavirin and showed survival rates of / ( . %), / ( %), and / ( . %), respectively (table ) [ ] . the combination therapy was also used in other case reports (table ) [ , ] . the role of the combination of ribavirin and ifn was also tried as a treatment and a prophylaxis [ ] . the current studies of the use of ribavirin and ifn combination therapy for mers-cov infection rely on small number of patients but there is a trend for improvement. thus, it was suggested that the combination of type interferon and ribavirin could be used [ ] . due to the inhomogeneous nature of available studies and the limited data that are available, a precise recommendation on therapy of mers could not be established. the combination of lopinavir/ritonavir, ribavirin and interferonalpha was used in one case [ ] . one patient received pegylated interferon, ribavirin, and lopinavir/ritonavir from day of illness and the patient had continued mers-cov in the respiratory tract secretions until the fourth week of illness [ ] . however, viremia was detected for only days after initiation of triple therapy [ ] . in a case series, eight patients received mycophenolate mofetil and all survived [ ] . in the sars epidemic, passive immunotherapy with neutralizing antibodies was considered as a therapeutic approach. there are multiple antibodies against mers-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table ). in the mers-cov infection, the production of large quantities of mers-cov neutralizing human polyclonal antibodies was possible using gamma-irradiated whole-killed virion vaccine or a spike protein nanoparticle vaccine in a bovine model [ ] . utilizing one dose of these antibodies prevented infection in mice [ ] . these antibodies were effective when given h before or and h after mers-cov infection [ ] . corti et al. isolated a potent mers-cov-neutralizing antibody (lca ) from memory b cells of an infected individual. the lca antibodies bind to a site on the spike protein and neutralize mers-cov infection [ ] . these lca antibodies were used successfully in mice model [ ] . similarly, utilizing a humanized mouse model of mers-cov infection, antibodies against the spike protein were efficacious as prophylaxis [ ] . antibodies obtained from the sera of mers immune camels were supportive of the clearance of the virus, and reduction of the severity of the disease in mers-covinfected mice [ ] . however, the purification and safety of these antibodies in humans has not been established yet. the cellular dipeptidyl peptidase iv (ddp iv; known as cd or adenosine deaminase (ada)-complexing protein- ) [ ] is an important receptor that mediates mers-cov infection through the viral spike (s) protein [ , ] . the mers-cov receptor binding domain (rbd), present on the surface spike protein (s), binds to the host cells receptor dpp iv [ , , ] . in humans, dpp iv is present mainly on the lower respiratory tract area such as the bronchial epithelial and alveolar cells [ , ] . although ddp iv is important for the viral entry into host cells, the use of dpp iv inhibitors, sitagliptin, vildagliptin, and saxagliptin, does not block the infection of mers-cov [ ] . in vitro use of monoclonal antibodies (mers- ) exhibited ic of . µg/ml [ ] . other possible human mab (m , m , and m ) neutralize pseudovirus and live virus [ ] . in rhesus model of mers-cov infection, a human monoclonal antibody, b -n, against mers-cov was effective in reducing the pathology of mers-cov [ ] . the use of polyclonal antibody (pab) against cd inhibits mers-cov infection in vitro [ ] . humanized anti-cd monoclonal antibodies (mab) such as mab ys and f significantly inhibit mers-cov infection in vitro [ ] . polyclonal antibodies against the mers-cov s domain neutralize the virus infection [ ] . many other mers-cov antibodies are being developed and tested [ ] . in the sars epidemic, convalescent plasma was thought to improve the outcome of sars patients [ ] . previous studies suggest that convalescent plasma may be used for patients with sars and severe influenza and may result in decreased viral load and a lower mortality rate [ ] [ ] [ ] [ ] . however, most of the studies were of low or very low quality, lacked control groups, and had risk of bias [ ] . two patients with mers-cov infection received intravenous immunoglobulin in an attempt to treat the infection, one patient was in saudi arabia [ ] and the other was in the usa [ ] . a protocol for the use of convalescent plasma as a therapeutic option for mers was suggested [ ] . plasma donors were identified as those with anti-mers-cov indirect immunofluorescence assay (ifa) antibodies (titer of ≥ : ) with no evidence of active mers-cov infection [ ] . in nine confirmed survivors of mers-cov infection, %, %, and % of them had positive mers antibodies by ifa at , , and months, respectively [ ] . the two patients who had long lasting antibodies had severe disease; however, the titer of the ifa antibodies was not measured in the study [ ] . in a larger study, mers-cov neutralizing antibodies were produced at low levels and were short-lived [ ] . further studies of the kinetics of the mers-cov antibodies showed that all surviving patients and % of fatal cases produced igg and neutralizing antibodies [ ] . the presence of antibodies did not lead to the elimination of virus from the lower respiratory tract [ ] . in a study of patients from south korea, nine patients had prnt titers > : by day and two had titers > : by day [ ] . in a study of samples, ( . %) had reactive elisa results, and of those had reactive indirect fluorescent antibody and microneutralization assay titers [ ] . thus, the use of convalescent plasma for the treatment of mers-cov in a clinical trial may be challenging due to a small pool of potential donors with sufficient antibody titers [ ] . based on sars experience, some authors suggested that steroid therapy might be beneficial for severe mers-cov infection [ ] . corticosteroid use for patients with sars showed that early use of corticosteroids significantly increased viral load, and . -fold increase in risk of icu admission and mortality [ ] . in another study of non-icu patients, the median time for sars-cov to become undetectable in plasma was days compared to days in patients who did and did not receive early corticosteroid therapy [ ] . corticosteroids were used as adjunct therapy for many patients with mers-cov [ , ] . in a study of patients with mers-cov infection, one patient received steroid and intravenous immunoglobulin for thrombocytopenia [ ] . initial study of five patients, three patients received a combination of interferon and ribavirin in addition to adjunct steroid on day - and all died during hospitalization [ ] . however, steroids were not evaluated systematically as therapy for mers patients. the host protease, furin, is an important factor to break down the s -s region of the mers-cov [ ] . in vivo studies showed activity of multiple agents against mers-cov and include: chloroquine, chlorpromazine, cyclosporine, and mycophenolic acid [ , ] . in addition, the us fda approved repurposed agents with broad antiviral activity including in vitro activity against mers-cov [ ] . these agents include the polymerase inhibitor bcx and the helicase inhibitor ssya - , spike binding (immunoadhesin (dpp -fc)) [ ] . there are many other agents currently in preclinical investigation such as: fluspirilene, thiothixene, fluphenazine hydrochloride, promethazine hydrochloride, astemizole and chlorphenoxamine hydrochloride [ ] . the emergence and continued cases of mers-cov infection require the availability of mers therapy. there is an urgent need for the development of standardized animal models and the establishment of standardized clinical therapeutic protocols. the current clinical studies are limited to case reports and case series with no control arm. the quality of evidence these studies offer is too low to make a conclusion. it is difficult to draw conclusion in the face of these limited studies. the most used combination of therapy was interferon and ribavirin as developed initially in . the development of novel therapeutic agents or the repurposing old therapeutic agents against mers-cov are needed as alternative pathways for testing and clinical trials. it was thought that convalescent sera may provide an exciting alternative as a therapeutic agent; the present data does not support the wide adaptation of this therapy. the current mers therapy relies on supportive care and providing circulatory and ventilation support. it is expected that the development and the use of repurposed drugs would allow the development of therapeutic agents for mers-cov. the best location to provide a randomized controlled trail was thought to be the intensive care units for the use of convalescent sera; the presence of milder disease may necessitate the development of therapeutic protocols for patients with severe and those with milder disease. monoclonal and polyclonal antibodies may offer further therapeutic options for the disease in humans. since the prospect for a randomized clinical trial is low due to the sporadic nature of the disease outside hospital outbreaks, it is prudent to have well-conducted prospective clinical studies. the proteins involved in mers-cov entry and replication are attractive targets for the development of antiviral therapeutics. clinical studies utilizing anti-mers-cov antibodies as therapeutic options would add to the prospect to develop therapeutic agents for this syndrome. although many drugs appear to be effective in vitro, a consideration of their availability, pharmacokinetic/pharmacodynamic properties, and side effects should be taken into consideration. of medications with an attractive use, lopinavir, interferon, and mycophenolate are among these agents. accelerated and preferably randomized controlled trails should be conducted. neutralizing antibodies are also promising and the use of these agents in humans. targeting the ddp receptor may be of particular importance; however, it is important to keep in mind that the development of any mutation in the binding sites may limit the use of these agents [ ] . few monoclonal antibodies showed protective efficacy as a prophylaxis in animal models [ , ] . the development and testing of monoclonal antibodies are associated with high costs and lack of an undefined population for their use [ ] . these monoclonal bodies had not been used in phase clinical trials and that further development of these agents require time and cost. • mers-cov emerged in and has caused multiple hospital outbreaks. this paper was not funded. managing mers-cov in the healthcare setting • a review of mers-cov in healthcare setting 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arabia clinical recommendations from an observational study on mers: glucocorticoids was benefit in treating sars patients the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mechanisms of coronavirus cell entry mediated by the viral spike protein repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection development of medical countermeasures to middle east respiratory syndrome coronavirus towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for middle east respiratory syndrome coronavirus (mers-cov) • a study of use of human neutralizing monoclonal antibodies for mers-cov research and development activities for middle east respiratory syndrome: the current landscape. n d the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. key: cord- -n tkf authors: altamimi, asmaa; abu-saris, raghib; el-metwally, ashraf; alaifan, taghreed; alamri, aref title: demographic variations of mers-cov infection among suspected and confirmed cases: an epidemiological analysis of laboratory-based data from riyadh regional laboratory date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: n tkf introduction. middle east respiratory syndrome coronavirus was first recognized in september in saudi arabia. the clinical presentations of mers and non-mers sari are often similar. therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of mers-cov is essential. however, the real challenge is to flag these patients through some demographic markers. the nature of these markers has not previously been investigated in saudi arabia, and hence, this study aims to identify them. methods: it was a surveillance system-based study, for which data from a total of , suspected patients in riyadh and al qassim regions were analyzed from january until december to estimate the prevalence of mers-cov among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. results: of , suspected cases, ( . %) were confirmed by laboratory results. these confirmed cases ( . % of which were males) had a mean age of . years (sd ± . ). around . % of the confirmed cases were aged between and years and about % of confirmed cases had their suspected specimen tested in the summer. the study identified three significant and independent predictors for confirmation of the disease: an age between and years, male gender, and summer season admission. conclusion: the study provides evidence that the mers-cov epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. future studies should aim to confirm such findings in other regions of saudi arabia as well and explore potential preventable risk factors. a respiratory viral disease caused by the middle east respiratory syndrome coronavirus (mers-cov) was first isolated in , in a -year-old man who died in jeddah, ksa due to severe acute pneumonia and multiple organ failure [ ] . since then, countries have reported the presence of this virus, including the countries of the eastern mediterranean region. several outbreaks have occurred in multiple countries including saudi arabia, the united arab emirates and the republic of korea [ ] . recent fatality rate (cfr) of % [ , ] . very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [ ] . e literature shows that mers-cov infects males more than females [ , ] . e casefatality rate of men ( %) is higher than that of women ( %) [ ] . males with a history of serious medical conditions are highly susceptible to this infection. moreover, the mean age of infection in adults is years [ ] . e mode of transmission is not entirely understood yet [ ] ; however, human-to-human [ ] and zoonotic sources of transmission [ ] have been documented in many studies. dromedary camels are the major animal source of mers-cov transmission to humans. interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [ ] . clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [ ] . severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in icus across different healthcare settings [ ] . studies have suggested an incubation period of days with a mean of - days [ , ] , while the median time until death is - days (range - days) among severely ill patients [ ] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rrt-pcr) assays [ ] . ere is no specific treatment for mers-cov. like most viral infections, the treatment options are supportive and symptomatic [ ] . at present, no vaccine exists for preventing the infections of mers-cov. e cdc indicated that preventative actions should be taken for any type of respiratory illness [ ] . such actions include washing hands with water and soap for around seconds or using hand sanitizers with alcohol if no water is available. one must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [ ] . many studies have been conducted in recent years in saudi arabia to combat this deadly disease. a large multicentre study showed that it is nearly impossible to differentiate between patients of mers-cov and non-mers-cov just on the basis of clinical presentation [ ] . another cohort study, which was hospital-based ( cases vs. controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [ ] . similarly, two more single-centre case control studies reported that the presenting symptoms of mers-cov infection were not specific [ , ] . physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between and hours). identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [ ] . mild symptomatic cases, which result in a positive pcr, may be isolated at home. severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. both mild and severe cases are retested after days, and the test is subsequently repeated after every days until a negative result is obtained [ ] . identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of mers-cov infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of mers-cov. e nature of these markers has not been investigated in saudi arabia, and hence this study aims to identify them. a cross-sectional study was conducted at the regional laboratory and blood bank, located at shumaisi hospital in riyadh, ksa. e laboratory has received the central blood banks and reference laboratories accreditation program saudi central board for accreditation of healthcare institution (cbahi) [ ] . technique. data were collected during the period of january to december . all patients in riyadh and al-qassim regions who had their samples tested at riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. for the descriptive aim, we estimated the prevalence of mers-cov. for the analytical aim, a binary logistic regression model was developed. in this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. data were cross-checked with a labcomputerized database. further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. we collected data from , cases, of which , suspected cases of mers-cov were included in the final analysis. data were cleaned, entered, stored, and managed with an excel database and ibm spss version . e statistical analyses consisted of descriptive counts and percentages. for those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. a logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. at first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. to determine significant factors, a p value below . and a % confidence interval were considered. a confirmed case is defined as a suspected case with laboratory confirmation of mers-cov infection [ ] . a total of , of mers-cov suspected cases were included in this study, of which . % were males (n � ) and . % were females (n � ). e age of individuals with suspected cases ranged between to years with a mean age of . e adjusted odds of mers-cov remained significant among different age groups; the odds of patients aged between - years increased threefold (a.or: . , % ci: . - . , p value � . ), whereas in the age group of - years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of mers-cov infection laboratory-based data was conducted in riyadh over a one-year period ( ). a total of , suspected cases were included in the results. of the total suspected cases, cases had been confirmed via laboratory results. all the confirmed cases are reported to moh through hesn (health electronic surveillance networks) and to the world health organization (who) through the international health regulations (ihr), national focal point of saudi arabia. we found that mers-cov infection was found significantly in people aged between and years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. during the study period, i.e., the year , only confirmed cases were reported, which means that the number of mers-cov infection cases has decreased in riyadh and al-qassim regions in comparison to that of the last three years. from to , there was a . % decrease, whereas from to , it decreased by . %, which translates into a % decrease between the two periods. is also complements the findings reported by of da'ar and ahmed in their paper [ ] . e predominance of infection in males was also observed in another study pwefromed in ksa ( ), which reported the percentage of confirmed cases among males to be %, compared with % among females [ ] . it is worth mentioning that saudi arabia defines age categories differently from the who (children: - , adult: otherwise) [ ] . however, unlike the classification used in saudi arabia, we have followed the who categorization of age to differentiate between children/adolescents ( to years) and adults ( years and older) as indicated in who reports for age-standardized population and in infectious diseases [ ] . is categorization was also followed by aly and his collaborators in their recent paper published in [ ] . adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of and age of ) [ ] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of mers-cov were reported among people aged and above [ ] . in , only of cases ( . %) of mers-cov infection were found among pediatric patients. moreover, the study which was conducted in king fahad medical city in riyadh (kfmc) between january and december did not report any mers-cov cases among children [ ] . e study which was conducted across the gulf countries for four years by mahmoud aly et al. between and suggests that the prevalence and distribution of mers-cov were the highest-risk in elderly aged years or above [ ] . similar to our results, this study also reported the highest number of confirmed cases during the summer season [ ] . among confirmed cases, only . % were healthcare workers, whereas around % were non-healthcare workers. is is in agreement with the study done by ahmad to estimate the survival rate in mers-cov globally prior to january ; . % were not health-care workers compared with . % confirmed cases of healthcare workers [ ] . similarly, other studies also reported a lower prevalence in healthcare workers [ ] [ ] [ ] . our data reported a higher prevalence of infection among saudi nationals as compared with non-saudi. another study also showed similar results but with a much higher percentage among saudis, which may be due to the fact that it included saudis from all regions [ ] . ere is no finding basis for comparison as such, because our study was focused on the riyadh and al qassim regions only. in our study, we detected a low prevalence ( . %). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the real star kit from altona was reported to be % with no cross reactivity with other respiratory pathogens [ ] . moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. in fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. to the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. however, it is worth mentioning that ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (ast) [ ] . however, further investigations are needed to confirm our findings. one of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of mers-cov in the riyadh and al-qassim regions. secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in saudi arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. in addition to this, quality assurance policies are implemented at hesn in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. according to our knowledge, this is one of the few studies that have specifically investigated mers-cov risk factors in the riyadh and al-qassim areas (two major regions in ksa). given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. it must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed mers-cov with those with suspected mers-cov who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. in conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged , presenting in winter will be less likely to be diagnosed than a male, aged , presenting in the summer, or, to give another example, a -year-old male who is presenting mers-cov signs with a negative lab result may need retesting. our study covered two main regions in saudi arabia and provides evidence that the mers-cov epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged to years, and presented to healthcare facilities in the summer. future studies should aim to confirm such findings in other regions in saudi arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged - more susceptible than others. e laboratory data used to support the findings of this study were provided by riyadh regional laboratory under license and are not freely available. however, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who, middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome (mers), who cov outbreak largest outside kingdom of saudi arabia middle east respiratory syndrome coronavirus in children hospital outbreak of middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection e pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive 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coronavirus or other causes development of a risk-prediction model for middle east respiratory syndrome coronavirus infection in dialysis patients mers-cov diagnosis: an update underlying trend, seasonality, prediction, forecasting and the contribution of risk factors: an analysis of globally reported cases of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): impact on saudi arabia - ) standard-standard populations-seer datasets [internet], acute myeloid leukemia-cancer stat facts acute viral respiratory infections among children in mers-endemic riyadh estimating survival rates in mers-cov patients and days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study diagnostic delays in symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia e predictors of -and -day mortality in mers-cov patients risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel clinical validation of commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of middle east respiratory syndrome coronavirus from upper respiratory tract specimens acknowledgments e authors would like to thank dr. waleed alsalem, dr. ahmed hakawi, and dr. mutaz mohammed from ministry of health, saudi arabia, dr. kamel al-dossari from riyadh regional lab for their help in this research, hatim al-mutairi for data cleaning, dima zailaey for structuring, and dr. munazza jawed from dow university of health sciences, karachi, for proofreading. e authors would also like to thank miss laila mohamed ghoneim from the american university cairo for english-language editing. all authors contributed to the writing of the manuscript and had access to the data. all authors read and approved the final manuscript. key: cord- - s i f authors: al-sehaibany, fares s. title: middle east respiratory syndrome in children: dental considerations date: - - journal: saudi med j doi: . /smj. . . sha: doc_id: cord_uid: s i f as of january , , laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection and mers-related deaths have been reported by the world health organization globally. middle east respiratory syndrome coronavirus may occur sporadically in communities or may be transmitted within families or hospitals. the number of confirmed mers-cov cases among healthcare workers has been increasing. middle east respiratory syndrome coronavirus may also spread through aerosols generated during various dental treatments, resulting in transmission between patients and dentists. as mers-cov cases have also been reported among children, pediatric dentists are at risk of mers-cov infection. this review discusses mers-cov infection in children and healthcare workers, especially pediatric dentists, and considerations pertaining to pediatric dentistry. although no cases of mers-cov transmission between a patient and a dentist have yet been reported, the risk of mers-cov transmission from an infected patient may be high due to the unique work environment of dentists (aerosol generation). middle east respiratory syndrome coronavirus is a novel beta coronavirus of the coronaviridae family that causes a severe respiratory disease with a high fatality rate. [ ] [ ] [ ] [ ] as of january , laboratoryconfirmed cases of mers-cov infection and related deaths have been reported by the world health organization (who) globally. the male-to-female ratio of the affected patients was : , with a median age of years. the incubation period for human-tohuman transmission ranges from - days. the symptoms of mers-cov infection range from being asymptomatic to severe pneumonia, acute respiratory distress syndrome, septic shock, and multi-organ failure, leading to death. the symptomatic patients may have fever, cough, chills, throat soreness, myalgia, arthralgia, vomiting, or diarrhea. furthermore, men over years of age, patients with comorbidities, and healthcare workers have been reported to be high-risk groups for mers-cov infection. the virus has been detected in respiratory, gastrointestinal and other bodily secretions, , as well as in air samples, which indicates the possibility for airborne transmission. several studies have reported mers-cov infections among healthcare workers , , , and children. , viral infections, such as severe acute respiratory syndrome saudi med j ; vol. ( ) www.smj.org.sa (sars-cov), may be transmitted to healthcare workers from infected patients through aerosols. considering that several types of dental equipment, such as handpieces, air-water syringes and ultrasonic scalers, produce considerable amounts of aerosols, the potential for the spread of infections from patients to dentists or dental assistants is high. this review is an attempt to discuss mers-cov infection among children and those providing dental treatment to them, including precautions and considerations pertaining to the practice of pediatric dentistry. mers-cov infection in healthcare workers. the patterns of mers-cov transmission suggested in the literature include the following: a) sporadic cases occurring in communities; b) transmission within families; and c) nosocomial transmission. healthcarerelated mers-cov transmission is associated with high morbidity, extended use of mechanical ventilation and fatality rates of up to %; this type of transmission can be attributed to shortcomings in observing stringent infection control protocols. , , , furthermore, mers-cov has been reported to be viable in hospitallike environments for up to hours with a stability that is unaltered during aerosolization. among healthcare workers' contacts identified in al-hassa, saudi arabia, mers-cov infection was confirmed in cases. a saudi study compared healthcare worker and family contact with laboratory-confirmed mers-cov patients and reported a lower rate ( . %) of infection among the healthcare workers than among the families ( . %). healthcare workers could be infected with mers-cov through exposure in the community or at their workplace, , , they could be diagnosed late, , and they could remain asymptomatic or mildly symptomatic. , , furthermore, unsuspected cases entering healthcare facilities have been considered the main source of mers-cov. considering these aspects and that healthcare workers may continue to work regardless of being symptomatic, the possibility of transmitting the infection to their patients is also high. memish et al reported that the age of saudi pediatric patients who presented clinically with symptoms of mers-cov infection ranged from - years with a female-to-male ratio of . to . of the cases, were symptomatic, and were asymptomatic with no underlying comorbidities. another report by thabet et al concluded that although mers-cov infection presents with a wide range of clinical manifestations, the mortality rate in children is lower than that in adults. a review on the effects of coronavirus infection in children concluded that human coronavirus infections may be associated with respiratory symptoms and may also involve central nervous system. the authors suggested that the clinical and genetic traits of human coronavirus infection among children should be monitored closely for early prevention. dental considerations. bioaerosols in dental practice. bioaerosols are defined as a suspension of biological particles in gaseous media. apart from radiation exposures, dermatitis, eye injuries, and musculoskeletal and respiratory disorders, other occupational health risks exist in dentistry. these risks include percutaneous exposure incidents and exposure to infectious diseases, such as those that may be present in bioaerosols. subgingival scaling of periodontally involved teeth with ultrasonic scalers may produce aerosols containing blood. one study reported that ultrasonic scalers and tips produced significantly more aerosol and splatter than a handheld curette, regardless of the scaler type used. miller et al studied the characteristics of blood-containing aerosols generated by common powered dental instruments. the author concluded that all the recovered particles could contain the hepatitis b virus and be inhaled and retained ( - %) in the human respiratory system. the repeated and chronic exposure to bioaerosols generated during certain dental procedures and the relatively small particle size contribute to an increased risk of infection among dental professionals. furthermore, the protection provided by surgical masks worn by dental professionals may be low for small particles; in addition, these masks may not fit perfectly in clinical practice. a study reported that - % of plasma aerosol particles ranging from . - . microns in size passed through the filters of makes of surgical masks used by dentists. these factors may play a role in the airborne route of viral infections among dentists and clinical dental auxiliaries. the likelihood of detecting, reporting, documenting and publishing dentistry-associated infections is relatively less. although no definitive evidence of an extensive public health hazard from exposure to dental unit waterlines has been reported. few case reports have suggested a definitive link between exposure to contaminated dental unit waterlines and legionella infection. , disclosure. authors have no conflict of interest, and the work was not supported or funded by any drug company. infection control in dental practice. a review by scully and samaranayake on the emerging and fluctuating viral diseases in the new millennium concluded that infection control plays an equally important role in the practice of dentistry as do the understanding of oral manifestations and the diagnosis and management of viral infections. while differences exist in the virology, epidemiology, and clinical outcomes of mers-cov and sars-cov infections, the clinical symptoms of mers-cov resemble those of sars-cov, apart from acute renal failure. although these results cannot be directly interpreted for a definitive conclusion, the management protocols for different mers-cov infection-associated with clinical scenarios for dental professionals may be similar to those of sars-cov. the implications of sars-cov for general dental practitioners, the significance of droplets and aerosols in disease transmission, the recommended management protocols for sars-cov infection and specific infection control measures have been well described by li et al. a comprehensive review on universal and standard precautions has been published elsewhere and is beyond the scope of this article, infection control measures pertaining only to pediatric dental practice in the context of mers-cov infection are discussed here. in pediatric dental practice, effective infection control measures for the prevention or minimization of viral infection transmission can be implemented by a) controlling the gag or cough reflex; b) reducing aerosol/ splatter generation; c) managing contaminated air and; d) improving personal protection. the gag or cough reflex may be stimulated by certain procedures, such as posterior intraoral and bite-wing radiographs and taking impressions. orthopantomographs or oblique lateral views may be considered instead of intraoral radiographs for screening, whereas oral mucosa in very sensitive patients may be anesthetized before taking impressions. sedation may also be considered to control gag reflex. varying levels of physical, intellectual, emotional and social development of children and adolescents are major challenges for dentists especially attending to their psychological needs within adequate infection control regimen. toys provided for pediatric patients may be a potential source of cross-infection. soft toys are more likely to be contaminated, difficult to disinfect and may re-contaminate quickly compared to hardsurfaced toys. furthermore, restraining devices used to control movements of pediatric patients such as velcro fasteners may also be contaminated and should be disinfected accordingly. extra-oral evacuation devices and special aerosol reduction devices may be used in combination with ultrasonic scalers to reduce the amount of aerosol produced. , in high-risk cases, chemomechanical caries removal or the atraumatic restorative technique , may be utilized to prevent or reduce aerosol and splatter generation. in addition, high-volume evacuation removes infectious droplets at the source as they are emitted; thereby, minimizing, or preventing their dispersion in the air. to maintain their efficacy, the filters in the suction apparatus should be cleaned every day, and the exhaust air should be vented outside to prevent the recirculation of contaminated air. contaminated air can be managed by improving dental clinic ventilation and/or by disinfecting the air. an ideal airflow pattern combined with a minimum of air changes per hour has been recommended for dental clinics. [ ] [ ] [ ] moreover, although its use in dental clinics is unconfirmed, ultraviolet germicidal irradiation may be installed and is effective against fungi, viruses, and bacteria, namely, tubercle bacilli and anthrax. , on the other hand, measures of improving personal protection include washing hands frequently before and after treatment, using disposable barriers, dispensing instruments and materials just before treatment (thereby preventing particles from settling on the surfaces), and sterilizing soiled instruments. after each patient visit, surfaces may be disinfected using hospital-grade disinfectants, which are effective against coronavirus. personal protective equipment, such as gowns, hair covers, masks, gloves, shielded face masks and shoe covers, should be used as appropriate. a higher level of respiratory protection should be considered, especially with aerosol-generating procedures. the use of n respirators offers a certain level of protection against the airborne transmission of sars-cov, although the exact level of protection offered by these respirators for individuals may vary. examples of personal protective equipment such as face mask with plastic shield (figure ) and n respirator (figure ) . these respirators may also provide some level of protection against mers-cov and may be utilized instead of surgical masks in dental clinics. apart from these measures, vaccination of pediatric dentists and staffs working in pediatric dental clinics against measles, mumps, varicella and rubella may be necessary for their protection from these viral infections. in conclusions, considering the unique work environment of dentists, which involves close patient contact and aerosol production, the risk of mers-cov transmission from an infected patient is high. children are also prone to mers-cov infection. as the number of mers-cov cases may increase in future, pediatric dentists should be well informed and educated about not only the signs and symptoms of 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legionellosis among participants in the national nosocomial infections surveillance system legionella contamination of dental-unit water emerging and changing viral diseases in the new millennium middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection severe acute respiratory syndrome (sars) and the gdp. part ii: implications for gdps guidelines for infection control in dental health-care settings essentials of dental radiography and radiography oral bioscience. london (uk): churchill-livingstone sedation in dentistry. part : management of the gagging patient infection control issues related to pediatric dentistry reduction of bacteria-containing spray produced during ultrasonic scaling the usefulness of the modified extra-oral vacuum aspirator (eova) from household vacuum cleaner in reducing bacteria in dental aerosols chemochemical caries removal: a review of the techniques and latest developments the atraumatic restorative treatment (art) technique: does it have a place in everyday practice? atraumatic restorative treatment (art): rationale, technique, and development the application of ultraviolet germicidal irradiation to control transmission of airborne disease: bioterrorism countermeasure infection control for the dental team. copenhagen: munksgaard additional precautions for tuberculosis and a self-assessment checklist managing sars amidst uncertainty possible sars coronavirus transmission during cardiopulmonary resuscitation safety management and infection control in pediatric dentistry key: cord- -hmj qu v authors: wiwanitkit, somsri; wiwanitkit, viroj title: korean mers: a new cross continent emerging infectious disease date: - - journal: asian pacific journal of tropical disease doi: . /s - ( ) - sha: doc_id: cord_uid: hmj qu v abstract middle east respiratory syndrome is a new emerging infectious disease that was firstly detected in the middle east. this disease can cause severe respiratory illness and was proved to be a coronavirus infection. the infection was firstly confined within the middle east. however, the new emergence of this infection is in korea and korean middle east respiratory syndrome becomes the new concern in public health. in this brief article, the authors discuss on this new cross continent emerging infectious disease. middle east respiratory syndrome (mers) is a new emerging infectious disease that was firstly detected in the middle east [ ] [ ] [ ] . the infection was firstly confined within the middle east. the first case was from saudi arabia in . as noted by banik et al. [ ] , since its discovery in , mers coronavirus has reached countries affecting about people, including a dozen children, and claiming over lives. this disease can cause severe respiratory illness and was proved to be a coronavirus infection [ , ] . acute respiratory distress can be difficult to clinically differential diagnose from other acute respiratory viral disease [ ] . in , the new emergence of this infection is in korea and korean mers becomes the new concern in public health [ ] . in this brief article, the authors discuss on this new cross continent emerging infectious disease. as noted, mers, by its name, is primary existed in the middle east. the identification of the virus is reported from many countries in this area and can also be seen in the nearby area. the report of virus in the camels from egypt is an interesting finding indicating that the virus has already moved out of the middle east for many years [ ] . in , there are case reports of infected patients in france [ ] . the first patient visited dubai and carried disease back to france [ ] . the second patient got nosocomial infection without travel history [ ] . in , the first human infection in usa was reported and this brought several attentions from medical scientist due to the migration of disease to the western hemisphere [ , ] . the case was a physician who imported disease from the middle east. kapoor et al. [ ] in , there is a new problem existed in korea. a patient was identified to have mers and the local centers for disease control could not early identify this risk patient. the disease firstly occurred in may , , then it spread to others and this resulted in local policy for school closing [ ] . quarantine of thousand people was also done. the widespread of the disease in this outbreak is believed to be due to the poor disease control system at the airport and the lack of concern on mers and poor quarantine technique on the first group of patients. of interest, the disease is still continuous ( june, ) and there is a note to avoid travel to korea due to mers. of interest, the outbreak generated from the first korea case caused nationwide spreading and panic. but the disease already attacked many ones who contacted with the first patient including his friend from china [ ] . the middle east respiratory syndrome novel middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps the emergence of a new corona virus--mers-cov: hind sight is always / are animals a bane for the spread of the deadly malady, the corona virus (mers)? chest ct findings in mers south korea scrambles to contain mers virus mers coronaviruses in dromedary camels clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission first patient with mers in us is improving, but number of cases worldwide doubles in six weeks to may first us mers-cov cases underscore need for preparedness clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states spread of mers to south korea and china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china genomic sequencing and analysis of the first imported middle east respiratory syndrome coronavirus (mers cov) in china imported case of mers-cov infection identified in china mers in south korea and china: a potential outbreak threat? we declare that we have no conflict of interest. key: cord- -ujq mxk authors: chen, bin; tian, er-kang; he, bin; tian, lejin; han, ruiying; wang, shuangwen; xiang, qianrong; zhang, shu; el arnaout, toufic; cheng, wei title: overview of lethal human coronaviruses date: - - journal: signal transduct target ther doi: . /s - - - sha: doc_id: cord_uid: ujq mxk coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. seven coronaviruses that infect humans have been identified: hcov- e, hcov-oc , hcov-nl , hcov-hku , sars-cov, mers-cov, and sars-cov- . among them, sars-cov and mers-cov caused outbreaks in and , respectively. sars-cov- (covid- ) is the most recently discovered. it has created a severe worldwide outbreak beginning in late , leading to date to over million cases globally. viruses are genetically simple, yet highly diverse. however, the recent outbreaks of sars-cov and mers-cov, and the ongoing outbreak of sars-cov- , indicate that there remains a long way to go to identify and develop specific therapeutic treatments. only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. this paper mainly focuses on sars-cov, mers-cov, and sars-cov- . here, recent studies are summarized and reviewed, with a focus on virus–host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment. coronaviruses are crown-like particles with spikes protruding from their surface. they are enveloped viruses with single-stranded, positive-sense rna (+ssrna) genomes of approximately - kb, which is currently the largest known genome size for an rna virus. rna viruses are divided into five branches. , certain groups in a particular branch might be related to and/or share features with viruses classified in another branch. in branch are the leviviruses and eukaryotic relatives (e.g., mitoviruses, narnaviruses, ourmiaviruses); branch represents several +rna virus families (of eukaryotes) (e.g., orders picornavirales and nidovirales, and the families caliciviridae, potyviridae, astroviridae, and solemoviridae) and several dsrna viruses (e.g., partitiviruses and picobirnaviruses); in branch are certain +rna viruses such as the supergroups of alphaviruses and offlaviviruses, the nodaviruses, tombusviruses, and many small and novel groups; in branch are dsrna viruses such as the cystoviruses, reoviruses, and totiviruses; branch consists of −rna viruses. coronaviruses were classified as most likely related to branch , and belong to the order nidovirales, and the family coronaviridae. in , the international committee on taxonomy of viruses divided the coronaviridae into the orthocoronavirinae and letovirinae subfamilies. according to their host targets, coronaviruses can also be divided into animal and human coronaviruses. many diseases in domestic animals are related to animal coronaviruses, such as canine respiratory coronavirus, which causes respiratory disease in dogs. the highly pathogenic human coronaviruses belong to the subfamily coronavirinae from the family coronaviridae. the viruses in this subfamily group into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the classic subgroup clusters are labeled a- b for the alphacoronaviruses and a- d for the betacoronaviruses. to date, including the recently discovered sars-cov- , there are seven coronaviruses that infect humans. human coronavirus (hcov)- e, hcov-nl , hcov-oc , or hcov-hku cause only the common cold, whereas the severe acute respiratory syndrome coronavirus (sars-cov) or the middle east respiratory syndrome coronavirus (mers-cov) cause relatively high mortality and emerged in and , respectively. notably, sars-cov- is currently causing a worldwide epidemic. sars-cov and mers-cov belong to subgroups b and c of the betacoronaviruses, respectively, , whereas sars-cov- is a new member of the betacoronaviruses distinct from sars-cov and mers-cov. figure shows the phylogenetic tree of rna viruses. the estimated mutation rates of coronaviruses (covs) are moderate to high compared to those of other ssrna viruses. there are two gene loci that are sites of variation in sars-cov. one of these sites is located in the spike (s) protein gene. the second major site of variation is the accessory gene open reading frame orf . in mers-cov, the major sites of variation are located in the s, orf b, and orf genes. the major differences in the sequence of the s gene of sars-cov- are three short insertions in the n-terminal domain and changes in four out of five of the key residues in the receptor-binding motif. the organizations of the genome and gene expression are similar for all coronaviruses. orf a/b, located at the ′ end, encodes nonstructural proteins (nsps) (named nsp -nsp ); other orfs at the ′ end encode structural proteins, including the s, envelope (e), membrane (m), and nucleocapsid (n) proteins. the mutations in sars-cov- have become a hot research topic. the surface proteins of sars-cov and batcov-ratg , the nearest potential bat precursors of sars-cov- , have and % sequence identity, respectively, to that of sars-cov- . compared to that of sars-cov, the antigenic surface of sars-cov- is highly divergent compared to other covs. since its outbreak began at the end of , sars-cov- has acquired mutations throughout its genome, and there are already hundreds of virus strains distributed worldwide. according to gisaid.org, as of may , there were , full genome sequences available, and the s clade of the full genome tree contains the largest group of viruses, indicating that mutations in orf -l s are most frequent. the temporal resolution assumes an average nucleotide substitution rate of × − substitutions per site per year. based on these mutations, researchers found that sars-cov- can be divided into two subtypes. they reported that the snps at locations and , show strong linkage; the "ct" haplotype was defined as the "l" type because t , encodes leucine, while the "tc" haplotype was defined as the "s" type because c , encodes serine, which is in accordance with another study that divided sars-cov- into two types. the "l" type seems to be more prevalent and aggressive, but the "s" type may be ancestral, as sites and , were identical to the orthologous sites in the most closely related viruses. patients may be infected by either or both types. from the gisaid update, compared with ratg , there are differences in the receptor-binding interface, which may have favored host switching. data related to the sequence and mutations of sars-cov- are also available and continuously updated online through the china national center for bioinformation (cncb) (https://bigd.big.ac.cn/ncov). the history of sars-cov, mers-cov, and sars-cov- the first case of sars was discovered in foshan, china, in november . infections occurred through either direct or indirect contact with patients. by july , sars-cov had spread to over countries, causing reported cases and deaths. after that, no additional infections were detected, and the sars pandemic was declared over. sars-cov was first isolated from himalayan palm civets (hpcs) from a live-animal market in guangdong, china. , other animals, such as raccoon dogs (nyctereutes procyonoides), along with human workers from the same market, also showed evidence of viral infection. multiple studies have demonstrated that the reservoirs of several coronaviruses, including sars-cov-like and mers-cov-like viruses, were bats. , although palm civets might have been intermediary hosts of sars-cov, researchers often concluded there was no evidence that these animals were the ultimate sources of sars-cov, and the viruses cannot circulate directly in palm civets in the wild. in , guan and zheng's team investigated a live-animal retail market in guangdong, focusing on recently captured wild animals and human consumption. the animals sampled included seven wild and one domestic animal species. they collected nasal fig. phylogenetic tree of coronaviruses based on full-length genome sequences. all complete genome sequences of coronavirus were downloaded from the ncbi reference sequence database, refseq. the tree was constructed using maximum likelihood estimation (mle) by mega x, with clustal omega as the multiple sequence alignment method, and bootstrap replicates. only bootstraps ≥ % values are shown. the seven known human-infecting coronaviruses are indicated with a red star and fecal samples with swabs and then used reverse transcriptionpolymerase chain reaction (rt-pcr) to test for viral nucleic acid from the n gene of the human sars-cov. the rt-pcr assay results showed that samples from four of six hpcs were positive; the other seven species (beaver, chinese ferret-badger, chinese hare, chinese muntjac, domestic cat, hog-badger, and raccoon dog) sampled were negative. mers-cov was first found in in a lung sample from a year-old patient who died of respiratory failure in jeddah, saudi arabia. on september , a similar type of virus named human coronavirus was isolated from a patient with severe respiratory infection. cases have also been reported in other countries. mers has a % mortality rate, and since it emerged in the human population in june , it has caused substantial morbidity and mortality. , from until january , , the total number of laboratory-confirmed mers-cov infection cases reported globally to the world health organization (who) was , with associated deaths, covering countries (www. who.int/csr/don/ -january- -mers-united-arab-emirates/en). cases of mers from other countries were linked to travel to the middle east. , mers-cov was identified from the saliva of a patient with acute pneumonia and renal failure in jeddah (ksa). mers-cov can be detected in respiratory tract secretions, as well as in feces, serum, and urine. [ ] [ ] [ ] in addition, it has been isolated from environmental objects such as bedsheets, bedrails, intravenous fluid hangers, and x-ray devices. in the case of known camel infection, mers-cov was transmitted from a camel to a human, which was confirmed by rna sequencing. at the end of december , the first clusters of patients with pneumonia caused by sars-cov- were reported in wuhan, china. the basic reproduction number of sars-cov- is approximately . , indicating that each patient would on average spread the infection to . people. human-to-human transmission of sars-cov- occurred rapidly, and the atypical symptoms during the early stage may be a further disadvantage. moreover, human infection might have begun months prior to the official announcement of the outbreak. to prevent further spread, stricter screening and surveillance are needed at travel hubs. early detection, diagnosis, and treatment are also effective measures to contain the epidemic. nonetheless, the fatality rate of sars-cov- is lower than that of sars, with an estimated mortality risk of %. sars-cov- was first isolated from clinical specimens using human airway epithelial cells and the vero e and huh- cell lines. approaches to assess virions include isolation from lower respiratory tract specimens and artificially infected specific pathogen-free human airway epithelial cells. fluorescence quantitative pcr with primers designed according to specific sequences and serological testing aimed at igg and igm can be used to detect the virus. sars-cov, mers-cov, and sars-cov- pose major challenges to global health, causing infections in large parts of the world. sars-cov was found in countries and mers-cov in countries, while sars-cov- was found in countries by th may, . details on the pandemics caused by sars-cov- are shown in box , highlighting the status of the coronavirus-caused diseases worldwide. epidemiological analysis and symptoms of sars, mers, and coronavirus disease (covid- ) the clinical manifestations of sars include hypoxia, cyanosis, high fever (above °c), accelerated breathing or respiratory distress syndrome, and shortness of breath. x-rays show changes to the lungs to varying degrees. the who case definition ( ) includes the following: ( ) fever higher than °c or history of such in the past days, ( ) radiological evidence of new infiltrates consistent with pneumonia, ( ) chills, cough, malaise, myalgia, or known history of exposure, and ( ) positive test for sars-cov by one or more assays. similar to sars-cov infection, mers-cov infection manifests as a severe lower respiratory tract infection with extrapulmonary involvement and high fatality rates. symptomatic patients may present fever, chills, stiffness, myalgia, discomfort, cough, shortness of breath, and gastrointestinal symptoms of diarrhea, vomiting, and abdominal pain. pneumonia is common, and severe infection with acute respiratory failure, renal failure, and shock is particularly frequent among older patients. previous studies have estimated that approximately . - % of mers-cov infections may be asymptomatic. , it must be noted that immunocompromised people are at high risk of mers-cov infection. for covid- , fever, cough, myalgia, and fatigue are most commonly observed at illness onset; less commonly observed are sputum production, hemoptysis, and headache, among others. , similar to sars and mers, some patients develop acute respiratory distress syndrome (ards). around % of infected patients only develop mild symptoms, with mild pneumonia or no pneumonia. and % are severe and critical status, respectively. patients requiring icu care tend to be much older and are more likely to have underlying comorbidities, such as hypertension and diabetes. additionally, symptoms such as pharyngeal pain and anorexia are reported at higher rates among those admitted to the icu than among non-icu patients. histopathologically, symptoms such as diffuse alveolar damage (dad), loss of cilia, squamous metaplasia, denudation of bronchial epithelia, and giant-cell infiltrate often occur in the lungs of patients with sars. the number of macrophages increases prominently in the alveoli and the interstitium. according to the morphological changes, observed, most authors have subclassified lung lesions in sars into two or three consecutive phases: an acute exudative inflammatory phase, a fibrous proliferative phase, and a final fibrotic stage, although there is considerable overlap in histological findings among these phases. the main features of the first phase include extensive hyaline membrane formation, edema, alveolar hemorrhage, and fibrin exudation in alveolar spaces. the second phase includes widening of septae, pneumocyte hyperplasia, and organizing fibromyxoid and cellular exudates. , the third phase includes septal and alveolar fibrosis. several autopsies of sars patients have demonstrated secondary bronchopneumonia, and the pathogens identified mainly consist of staphylococcus aureus, mucor sp., aspergillus sp., pseudomonas aeruginosa, and cytomegalovirus. sars-cov also greatly affects the immune system. for example, hemorrhagic necrosis is usually obvious in the lymph nodes and spleen. as described above, sars-cov attacks immune cells, especially t cells and macrophages, with approximately % of lymphocytes and % of monocytes in the circulation becoming infected. , under the influence of the virus, the patient's germinal centers disappear, and both t and b lymphocytes are depleted. in the spleen, the white pulp shrinks, hemorrhage and necrosis occur in the red pulp, and the periarterial lymphatic sheaths decrease. , , with regard to the immune system, sars-cov can infect the lymphoid component of the intestine. for this reason, the patient's submucosal lymphoid tissue atrophies. sars-cov can also infect epithelial cells in the mucosa of the small and large intestines, and the digestive tract may undergo mild diffuse inflammation and atrophy of the submucosal lymphoid tissues. according to previous reports, symptoms in the liver include extensive hepatocyte mitosis, balloon degeneration of hepatocytes, mild to moderate lymphocytic infiltrates, fatty degeneration, and central lobular necrosis. additionally, apoptosis of liver cells has been confirmed in some cases. on autopsy, the kidneys of some sars patients showed focal bleeding and acute tubular necrosis of different degrees. one of the major complications of ards is acute kidney injury. , , in many other cases, the features are nonspecific, including benign hypertensive nephrosclerosis and autolysis. moreover, researchers have detected viral particles and genome sequences in the cytoplasm of neurons of the hypothalamus and cortex in the brain, which suggests that the virus can cross the blood-brain barrier and cause degeneration and necrosis of neurons, edema, extensive glial cell hyperplasia, and cellular infiltration. pneumocytes and epithelial syncytial cells are important targets of mers-cov. the lung tissue presents dad. severe peripheral vascular diseases, patchy cardiac fibrosis, and hepatic steatosis have also been found in other organs. another symptom is bronchial submucosal gland necrosis. renal biopsy may show acute tubulointerstitial nephritis and acute tubulosclerosis with protein casts. in covid- patients, a bilateral (sometimes unilateral) distribution of patchy shadows and ground glass opacity in the lungs is typical, based on ct scans. plasma concentrations of interleukins (ils) , , and , granulocyte-colony stimulating factor, interferon (ifn)-γ-inducible protein , monocyte chemoattractant protein , macrophage inflammatory protein α, and tumor necrosis factor α are increased in most dying patients. , in addition, neutrophilia related to cytokine storms induced by virus invasion, coagulation activation related to a sustained inflammatory response, and acute kidney injury related to the effects of the virus, hypoxia, and shock, may be involved in mortality. those who survive intensive care may experience long-term lung damage and fibrosis due to aberrant and excessive immune responses. moreover, researchers found that as sars-cov- spread and changed across generations, clinical symptoms started to differentiate those infected previously from those infected more recently. the initial symptoms are becoming more insidious, which indicates that the virus may gradually evolve into an influenza-like virus and lie dormant for longer in asymptomatic patients. in conclusion, in terms of outbreak times, sars-cov was the earliest, followed by mers-cov, then sars-cov- . to date, mers-cov has the longest duration of infection, and sars-cov- appears to be the most infectious. sars-cov, mers-cov, and sars-cov- infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. the common transmission methods and symptoms of the three coronavirus infection are shown in fig. . structural studies of sars-cov, mers-cov, and sars-cov- sars-cov is a coronavirus with a diameter of approximately nm. coronaviruses are the largest +ssrna viruses and contain at least orfs, protein combines with viral rna to form a nucleocapsid, which is involved in the replication of sars-cov and is the most abundant protein in virus-infected cells. , the m protein is a membrane glycoprotein that is involved in budding and envelope formation. the s protein of sars-cov is a type i transmembrane (tm) glycoprotein that is responsible for viral binding, fusion and entry into cells, as well as antibody induction. , the s protein contains a signal peptide (sp: amino acids - ) and three domains called the extracellular domain (amino acids - ), the tm domain (amino acids - ) and the intracellular region (amino acids - ). the extracellular domain consists of two subunits: s and s . the fragment located in the middle region of the s subunit (amino acids - ) is the angiotensin-converting enzyme (ace ) receptor-binding region. the s subunit comprises a fusion peptide (fp) and two x -valent element repeats (hr and hr ) that are responsible for fusion between the virus and the target cell membrane. thus, the s protein may be key to develop a vaccine to generate antibodies and block virus binding and fusion in the coronaviruses (fig. ) . the ′ end of the sars virus genome encodes structural proteins and helper proteins, of which orf a, orf , orf a, and orf b have been proven to be viral structural proteins involved in the formation of viral particles. the ′ end of the genome encodes nonstructural proteins (nsps), which are important for virus assembly, and may enable the design of small molecule drugs and/or vaccines. mers-cov belongs to lineage c of the genus betacoronavirus (βcov) in the family coronaviridae, order nidovirales, and it is the first lineage c cov and the sixth cov known to cause human infection. mers-cov is an enveloped +ssrna virus similar to other covs, but the amino acid sequence homology between mers-cov and other known covs is less than %. orf a and orf b located in the first two-thirds of the mers-cov genome are involved in virulence and encode nsps (nsp - ). the remaining third of the genome encodes four structural proteins, the s, e, m, and n proteins, as well as five accessory proteins (orf , orf a, orf b, orf , and orf b). [ ] [ ] [ ] the flanking regions of the genome contain the ′ and ′ untranslated regions (utrs). most mers-cov proteins are found in the cytoplasm. to date, the ns b protein and the orf a-encoded protein are the only known coronavirus proteins to be detected in the nucleus. nsp suppresses host gene expression in infected cells, and its rna cleavage-inducing function promotes virus assembly/budding. nsp is a viral ′-o-methyltransferase ( ′-o-mtase) that encodes critical functions in immune modulation and infection, suggesting that nsp is a conserved universal candidate for rapid design of a live attenuated vaccine. orf a's protein can mask viral dsrna to play a role in immune evasion, including type ifn and protein kinase r-mediated antiviral stress responses. mers-cov nsp is a full-length coronavirus helicase that contains multiple domains, including an n-terminal cys/his rich domain with three zinc atoms, a beta-barrel domain and a c-terminal superfamily (sf) helicase (sf ) core with two reca-like subdomains. this protein might interfere with the nonsensemediated mrna decay pathway to avoid degradation of viral rna. the mers-cov s protein can recognize dipeptidyl peptidase (dpp ) on the cell surface, promoting the fusion of the viral envelope and the cell membrane. the mers-cov s protein, a -amino acid type i tm glycoprotein located on the viral envelope surface, is composed of s and s subunits. the structure of the s protein consists of four parts: an sp located at the n terminus (amino acids - ), an extracellular domain (amino acids - ), a tm domain (amino acids - ), and an intracellular domain (amino acids - ). the s subunit contains two independent domains, an n-terminal domain (ntd, amino acids - ) and a c-terminal domain (amino acids - ), both of which may function as receptor-binding domains (rbds); these domains serve as critical targets for the development of vaccines and therapeutics , and facilitate the involvement of the s subunit in cell receptor (dpp ) binding. the core structure of the mers-cov s protein rbd is a five-stranded antiparallel sheet with several short helices, in which three disulfide bonds stabilize the core by connecting c to c , c to c , and c to c . , the accessory subdomain of the c-domain is a hypervariable region for receptor recognition fig. clustering of the spike protein of each coronavirus. fifty-four spike protein sequences filtered from each coronavirus coding sequences were clustered using the clans (cluster analysis of sequences) program on the website of mpi bioinformatics toolkit. each colored dot represents the spike protein sequence of each coronavirus. dots in the same color mean they are of the same genus, and each line shows the similarity of two sequences, with darker lines indicating higher similarity (lower e values). the coronaviridae family includes the following genera: alphacoronavirus (colored in green); betacoronavirus (red); gammacoronavirus (orange), and deltacoronavirus (blue). the indicated sars-cov, mers-cov, and sars-cov- belong to the genus of betacoronavirus and is called the receptor-binding motif (rbm). the accessory subdomains in sars-cov and mers-cov differ, resulting from divergent evolution and leading to the use of different receptors. dpp consists of an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets and the rbd. the s subunit of mers-cov contains an fp (residues - ), an hr domain (residues - ), an hr domain (residues - ), a tm domain (residues - ), and an intracellular domain (residues - ). the s subunit is responsible for fusion between the virus and the target cell membrane. after the s subunit binds to dpp , the s subunit inserts its fp into the target cell membrane to change its conformation. its hr helix then forms a homotrimer with an exposed surface that has three highly conserved hydrophobic grooves, and binds with hr to form a six-helix bundle ( -hb) core structure, which facilitates fusion by bringing the viral and cell membranes into close proximity. the genetic material of mers-cov then enters the host cell via the fusion pore. sars-cov- , which causes covid- , is a spherical betacoronavirus with a diameter of - nm and unique spikes ranging from to nm. its rna has the typical betacoronavirus organization, containing a ′ utr, a gene encoding the replicase complex (orf a/b), the s gene, e gene, m gene, and n gene, a ′ utr, and several unidentified nonstructural orfs. , the length of the sequences is nt for the ′ terminal region, nt for the ′ terminal region, nt for the s gene, nt for the e gene, nt for the m gene, and nt for the n gene. the , nt-long orf a/b gene contains predicted nsps. similar to sars-cov, there is a predicted orf gene that is nt in length and located between the m and n orf genes. nucleotides , , and may be the recombination breakpoints because based on the fragments from nt to nt and nt to the end, sars-cov- seems to be related to bat-sl-covzc and bat-sl-covzxc ; however, when considering the region from nt to nt , sars-cov- is mostly likely to be grouped with sars-cov and bat sars-like covs. more research is required to analyze the recombination of the genome as a whole. with over % of its conserved replicase complex being different from those of other betacoronaviruses, sars-cov- solely occupies a newly created subclade within the sarbecovirus subgenus. , bats may be its natural host, but owing to several arguments including the complicated environment in the wet markets in wuhan, this remains unclear, and further research is needed. , [ ] [ ] [ ] the free energy of the spike protein of sars-cov- is much lower than that of sars-cov, indicating that sars-cov- is more stable than sars-cov. the high similarity of the rbd sequences and of the spike protein structures between sars-cov- and sars-cov, , , also the simulation of the spike protein of sars-cov- binding to ace , the receptor of sars-cov, and results shown that ace plays a vital role in sars-cov- entry into hela cells, indicating that sars-cov- also uses ace for cell entry. structural modeling of the ace -b at complex (b at is used to obtain stable ace ) suggests that the complex can bind two s proteins simultaneously, providing important clues to the molecular basis of coronavirus recognition and infection. the rbd-ace binding free energy for sars-cov- is significantly lower than that for sars-cov, in accordance with the fact that sars-cov- is more infectious than sars-cov. a recent study found that cd , a kind of tm glycoprotein, facilitates cell entry of sars-cov- functionally, and its affinity constant with the s protein is . × − m. table shows a comparison of the structures of sars-cov, mers-cov, and sars-cov- . the specific function of the sars-cov- proteins, including s, e, m, and n proteins, need further study. in conclusion, sars-cov, mers-cov, and sars-cov- are similar in structure. their major proteins are structural proteins, including the s, e, m, and n proteins, accessory protein, and nsps (nsp - ). all these viruses rely on the s protein to identify cell targets and fuse with membranes. their s proteins are composed of s and s subunits. the s subunit contains an ntd and a c-domain, in which there is an rbd. the accessory subdomain of the c-domain is a hypervariable region for receptor recognition and is called the rbm. the difference is that sars-cov recognizes ace , sars-cov- recognizes ace and cd , but mers recognizes dpp , which results from the difference between the accessory subdomains of sars-cov, sars-cov- , and mers-cov. the function of the accessory and nonstructural proteins of these three viruses is related to viral replication and assembly, hence linked to virulence. pathogenesis of sars-cov, mers-cov, and sars-cov- sars-cov and sars-cov- are nearly identical in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov- is ace , plus cd for sars-cov- , while that of mers-cov is dpp . the human ace protein is a typical zinc metallopeptidase that comprises amino acids and contains a single catalytic domain. it is a type i integral membrane glycoprotein that is oriented with an extracellular n terminus and a catalytic site that functions in metabolizing circulating peptides. it is believed that there is a virus-binding hot spot on ace that is not pre-existent or preorganized but is induced to form upon virus binding. the hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. the general structural features of the hot spot favor virus binding: it is located in a region on ace that is furthest from the membrane, relatively flat, and free of glycosylation and is thereby easily accessible to viruses. the hot spot is mainly an intrinsic property of ace , but it is also a dynamic structure and receives structural contributions from both ace and the viruses, with the contributions of ace being more pronounced. dpp is a type ii transmembrane glycoprotein that consists of approximately amino acids. dpp contains an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets. the s protein rbd binds to the side surface of the dpp propeller and the amino acid residues in lancets and , including k , q , t , r , r , q , a , l , and i . the pathogenic mechanism of sars-cov is believed to include two parts: the virus injures target cells directly, and subsequent immune system dysfunction leads to indirect injury. sars-cov spreads via droplet and contact transmission and via the fecal-oral route. through droplet inhalation, the virus enters the respiratory tract and invades epithelial cells. infection and replication of the virus and local inflammatory changes contribute to lung tissue damage. subsequently, sars-cov infects circulating and resident immune cells. the key cells in this step consist mainly of t cells and macrophages. the viruses are then carried to other organs, including secondary lymphoid organs, by these circulating immune cells. sars-cov resembles hiv because both viruses attack immune cells and cause immunodeficiency. sars-cov causes lung injury mainly by inhibiting the function of ace . the virus binds to ace via the spike protein, downregulating its function and contributing to lung injury. ace is an important component of the renin-angiotensin system (ras). in this system, angiotensinogen is first converted to angiotensini (angi) by renin, and then angiis converted to ang ii under the influence of ace . ace downregulates the levels of angi and ang ii, which can bind to the ang ii type i (at ) receptor and cause certain types of lung injury, mainly pulmonary hypertension, pulmonary fibrosis, and acute lung injury. ang ii can directly induce pulmonary artery smooth muscle cells to grow or proliferate rapidly through at , thus causing pulmonary hypertension. ang ii contributes to pulmonary fibrosis by promoting the expression of the profibrotic cytokine transforming growth factor-b , causing fibroblasts to convert to myofibroblasts and accumulate collagen. several strategies can be used by the virus to escape the innate immune response. normally, viral pathogen-associated molecular patterns (pamps) are detected by host pattern recognition receptors (prrs). pamps include viral dsrna and mrna, and prrs include retinoic acid-inducible gene i protein (rig-i) and melanoma differentiation-associated protein (mda ). production of type i ifn is induced via the nuclear factor-κb (nf-κb) pathway. when ifn binds to the ifnα/β receptor, signal transducer and activator of transcription (stat) proteins are activated, which increases the production of other antiviral proteins and thus blocks sars-cov replication for further infection. additionally, sars-cov could encode several proteins that hinder the signaling cascades downstream of prrs. the cell receptor of mers-cov is dpp , which is widely expressed on epithelial cells in the prostate, alveoli, kidneys, liver, and small intestine and on activated leukocytes; thus, the range of mers-cov tissue tropism is broader than that of any other similar coronavirus. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of proinflammatory cytokines and chemokines (such as tnf-α, il- , cxcl- , ccl- , ccl- , ccl- , and il- ). compared to sars-cov infection, mers-cov infection can cause higher expression levels of il- , ifn-γ, ip- /cxcl- , mcp- /ccl- , mip- α/ccl- , rantes/ccl- , and il- . induction of immune cell-recruiting cytokines/chemokines might lead to the infiltration of a large number of immune cells into the lower respiratory tract, causing severe inflammation and tissue damage. mers-cov can also infect t cells and lead to apoptosis, avoiding the host immune response and facilitating faster spread. mers-cov has also evolved a mechanism to escape the host cell immune system. when host sensors initiate signaling pathways, transcription of type i ifn genes begins, and the type i ifn response, which is an essential component of antiviral innate immunity, is initiated. [ ] [ ] [ ] ifn activates the transcription of numerous ifn-stimulated genes (isgs) and synthesizes ′, ′oligoadenylate ( - a), which causes rnase l activation. [ ] [ ] [ ] activated rnase l can cleave both viral and host ssrna, leading to translation stagnation and apoptosis and limiting virus replication and transmission in vitro and in vivo. , however, the mers-cov protein ns b has phosphodiesterase activity and antagonizes rnase l via enzymatic degradation, leading to inference with ifn signaling. although ns b is mainly expressed in the nucleus, the expression level of the ns b protein in the cytoplasm is sufficient to prevent activation of rnase l. in addition, the mers-cov m, orf a, orf b, and orf proteins are reported to be strong ifn antagonists, among which the orf a, orf b, and orf proteins inhibit both type i ifn induction , , and nf-κb signaling pathways, which are similar to the type i ifn signaling pathway, providing a possible mechanism for mers-cov evasion of innate immunity. , for sars-cov- , the first cluster of patients was believed to be associated with a seafood market. however, due to the identification of patients without direct exposure to the market, transmission between humans was proven. hospital-related transmission has also been detected via infected medical workers and hospitalized patients. main routes of transmission include respiratory droplets, close contact, and extended exposure to aerosol environments loaded with high concentrations of virions. in addition to transmission through the respiratory tract, exposure of unprotected eyes to sars-cov- might cause acute respiratory infection. notably, the virus can be transmitted during the incubation period. the tm protease serine type (tmprss ) primes the s protein of sars-cov- for cell entry. based on analysis of the ace rna expression profile in normal human lungs, sars-cov- mainly infects type ii alveolar (at ) cells (only . % of human lung cells can express ace , % of which are at cells), and they express many other genes that facilitate sars-cov- infection; moreover, cd was also reported that it probably functionally facilitates cell entry of sars-cov- . high expression of ace has also been detected in cells of the digestive system, such as upper esophageal cells; accordingly, this system is a potential route of infection. in addition to the lungs and intestines, other organs, such as the heart, esophagus, kidney, bladder, testis, ileum, and adipose tissue, also express ace , and the expression level is higher than that in the lungs. , certain tumor tissues have higher expression of ace , making cancer patients more vulnerable than other people. in conclusion, sars-cov and sars-cov- bind cells expressing ace . they affect the ras system due to the affinity for ace (much stronger with sars-cov- than with sars-cov), leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. the specific pathogenic mechanisms of these three viruses are illustrated in fig. . diagnostic methods in addition to clinical manifestations, definitive diagnosis and confirmation of a coronavirus infection requires specific testing. the criteria for the diagnosis of sars from the who include ( ) fever higher than °c or history of such in the past days, ( ) radiological evidence of new infiltrates consistent with pneumonia, ( ) chills, cough, malaise, myalgia, or known history of exposure, and ( ) positive tests for by at least one assay. as for mers, the criteria for the diagnosis according to the who include ( ) fever, cough, and hospitalization with suspicion of lower respiratory tract lesion, ( ) history of contact with probable or confirmed cases, ( ) history of travel or residence within the arabian peninsula, and ( ) positive tests by a pcr-based detection method using respiratory samples, such as nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal) and induced sputum, or by serological tests such as the rapid immunochromatographic assay using highly selective monoclonal antibodies at room temperature. the initial criteria for the diagnosis of sars included ( ) a history of travel or residence in wuhan within weeks before the onset of illness, ( ) exposure to patients from wuhan with fever and respiratory symptoms within weeks before the onset of illness or the existence of clusters of diseases, ( ) fever accompanied by radiographic features of pneumonia, a normal or decreased total number of white blood cells, or a decreased lymphocyte count, and ( ) fluorescence pcr testing positive for nucleic acids of sars-cov- . , , [ ] [ ] [ ] early diagnosis of mers is difficult, but several highly specific and sensitive molecular and serologic assays exist for diagnosis in both animals and humans. among them, a rapid immunochromatographic assay can detect mers-cov antigens based on the detection of the mers-cov nucleocapsid protein in a short time frame; the relative sensitivity and specificity of this test are . and %, respectively. nucleic acid tests, such as real-time pcr tests, were mostly used to detect virus in the early stage of the outbreak of sars-cov- , although this approach requires a relatively long time and is not conducive to accelerating diagnosis or large-scale application. therefore, alternative, rapid diagnostic kits for sars-cov- may be used. for example, the colloidal gold detection kit uses serum, plasma, or whole blood samples to detect igm/igg antibodies for diagnosis on the th day of infection or the rd day after onset and requires only min. the nucleic acid detection kit for six respiratory viruses, including sars-cov- and influenza a virus, uses thermostatic amplifier chips for diagnosis. by detecting different infections, it may help distinguish people with influenza from those with other viral infections. a newly developed fig. pathogenesis of sars-cov, mers-cov, and sars-cov- . sars-cov and sars-cov- play a pathogenic role by inhibiting ace . under the influence of renin and ace, angiotensinogen is converted into ang ii. through the at receptor, ang ii acts as a lung injury-promoting factor, and in some cases, may cause vascular constriction, an inflammatory response, cell proliferation, fibrosis, and apoptosis of alveolar epithelial cells, resulting in diseases such as pulmonary hypertension, pulmonary fibrosis, and acute lung injury. ace converts ang ii into ang ( - ), and through the mas receptor, ang ( - ) play roles in vasodilation, antiproliferative activity, and antioxidant activity. sars-cov downregulates the activity of ace and causes an increase in the amount of ang ii and lung injury. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of pro-inflammatory cytokines and chemokines, leading to a large number of immune cells infiltrating a patient's lower respiratory tract, causing severe inflammation and tissue damage. mers-cov can infect t-cells from human lymphoid organs and causes the peripheral blood inducing apoptosis by intrinsic and extrinsic pathways, thus avoiding host immune response detection method, nanopore targeted sequencing, also has the potential for efficiently detecting viruses in a reasonable time. moreover, it could identify other respiratory viruses and monitor changes in virulence and transmissibility caused by viral mutations. therapeutic agents all patients should be treated in isolation. based on previous studies, drugs may be developed to inhibit cell entry. several strategies have been investigated to identify specific antivirals targeting the s protein, including mers-rbd-targeted nabs that block viral attachment, peptide inhibitors targeting s to prevent the formation of the fusion core, and small molecules without defined mechanisms. sdpp has been found to bind mers-cov rbd with high affinity, suggesting that sdpp could serve as a blocker for mers-cov attachment to and entry into cells. , one study reported a monoclonal antibody, d , that binds to the ntd to inhibit cellular entry of mers-cov. experiments have shown that d not only inhibits the binding of mers-cov to cells but also has effects after viral-cell attachment. d inhibits virus invasion by blocking the binding of the s subunit to the receptor and interfering with the conformational transformation of the s subunit when the virus fuses with the cell membrane. a peptide fragment spanning residues - of the s protein exerts neutralization activity by inhibiting membrane fusion and the entry of mers-cov, suggesting that it is possible to develop vaccines targeting this neutralizing epitope. griffithsin, a lectin derived from red algae, binds to oligosaccharides on the surface of sars-cov s protein and may also prove effective against sars-cov- . , researchers found that antibodies raised against sars-cov, such as css- , - , - , and - , could cross-neutralize sars-cov- by reducing s-driven cell entry, although the efficiency was lower than that observed for sars-cov. meplazumab, an anti-cd humanized antibody, binds with the cd in competition with the s protein, thus significantly inhibiting virus from invading cells and reducing the time of negative conversion. , chloroquine could effectively inhibit sars-cov- in vitro, and hydroxychloroquine was even more potent than chloroquine. , hydroxychloroquine could significantly help reduce the virus load, and azithromycin could reinforce its effect. arbidol could also contribute to condition improvement. note that these studies were generally limited and further trials and development are necessary. protease inhibitors also have the potential for coronavirus therapeutics. protease inhibitors, including disulfiram, lopinavir, and ritonavir, have been reported to be effective against sars and mers, and disulfiram, a drug for alcohol dependence, was reported to inhibit the papain-like protease (plpro) of mers-cov and sars-cov in cell cultures. , in vitro, sars-cov can be inhibited by both -mercaptopurine and -thioguanine targeting plpro. when used together with ribavirin, the protease inhibitors lopinavir and ritonavir have improved the outcomes of patients with sars compared with those achieved with the use of ribavirin alone. such inhibitors are also being tested in patients infected with sars-cov- , but whether these inhibitors effectively suppress clpro and plpro of sars-cov- remains controversial. drugs such as colistin, valrubicin, icatibant, bepotastine, epirubicin, epoprostenol, vapreotide, aprepitant, caspofungin, perphenazine, prulifloxacin, bictegravir, nelfinavir, and tegobuvir bind to the main protease of sars-cov- , and thus, are potential candidates. lianhua-qingwen formula, a kind of traditional chinese medicine, has been also recently described theoretically as a potential treatment candidate against the main virus protease. the serine protease inhibitor camostat mesylate could partly block sars-cov- infection of lung cells by inhibiting tmprss . viral rna synthesis blockers also have potential. remdesivir has broad-spectrum activity against mers-cov and sars-cov in cell cultures and animal models and is currently in clinical development for the treatment of ebola virus disease. , it was also reported positively based on a patient with sars-cov- infection in the us, and another study showed that it was able to inhibit the virus. ribavirin is often used to treat sars and mers, but it is uncertain whether it is potent enough against covid- . , regardless, when used alone, ribavirin has minimal activity against sars-cov and may cause strong side effects. it is often used together with corticosteroids. , a preclinical study of galidesivir (bcx ), an adenosine analog, revealed antiviral activity against sars-cov and mers-covcov. a study of favipiravir (t- ), a guanine analog, proved its activity against sars-cov- , and randomized trials of favipiravir plus interferon-α (chictr ), and favipiravir plus baloxavir marboxil (chictr ) are in progress. corticosteroids may be used to treat sars patients to suppress lung inflammation; although this therapy is not associated with mortality in patients with mers, it is associated with delayed clearance of mers-cov rna. , moreover, clinical evidence does not support the use of corticosteroids for covid- treatment. regarding antibodies, plasma therapy using convalescent plasma from fully recovered patients is effective against these coronaviruses, including sars-cov- . , tocilizumab (antihuman il- receptor) may curb immunopathology and trials are ongoing now. a study of novel monoclonal antibodies against the mers-cov spike protein suggests that mabs can be utilized for the identification of specific mutations of mers-cov. wang et al. provided an all-hydrocarbon-stapled peptide that likely mimics the native conformation of the c-terminal short α-helical region of the mers-cov s protein, which can block the formation of the hexameric coiled-coil fusion complex to inhibit viral-cell membrane fusion. , current treatments for sars mainly include ribavirin, ifn α, plasma therapy, host-directed therapies, and systemic corticosteroids. , due to the lack of clinical trial data, adequate supportive care supplemented with different combinations of drugs remains the main treatment for patients. , for mers, despite many studies in humans, there is no consensus on the best treatment; thus, randomized clinical trials are needed to assess potential treatment options. several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn and novel therapies, including polyclonal antibodies and broad-spectrum antivirals. antimicrobial peptides (amps) may be used as alternative therapeutic agents, and many have been effective even against bacterial proteases agents. antiviral drugs with effective activity in vitro include neutralizing monoclonal antibodies, antiviral peptides, mycophenolic acid, lopinavir, ifn, ribavirin, nitazoxamide, mycophenolate mofetil (mmf), alisporivir, silvestro, and corticosteroids. , , although there is no agreement on the most ideal treatment option to cure covid- , much research activities and pursued routes are underway. potential host-targeted agents the host-directed strategy is another approach for limiting viral replication. host proteases such as cathepsin b and cathepsin l can cleave the spike protein of sars-cov. drugs such as camostat inhibit host serine proteases and then interfere with the entry of sars-cov, but they may cause many significant side effects. , pegylated ifn α- a and - b may be used to stimulate innate antiviral responses in patients, and chloroquine, an approved immune modulator, was reported to have inhibitory effects against sars-cov- under certain conditions. researchers have also proposed that some commercially available drugs with suitable safety profiles, such as metformin, glitazones, fibrates, sartans, and atorvastin, as well as nutrient supplements and biologics, might reduce immunopathology, boost immune responses, and prevent or curb ards. in addition, ongoing cellular therapies using mesenchymal stromal cells from allogeneic donors have been shown to reduce nonproductive inflammation and affect tissue regeneration and are being evaluated in phase / trials in patients with ards; these therapies may be assayed in sars-cov- -infected patients. , , expansion of antiviral t cells as cellular drugs could aid in preparing t cell products for adjunct treatment of patients with severe infection. overall, there are similarities and differences among the treatments for these three coronaviruses. notably, we summarize the potential drugs and vaccines in table . vaccination could be used to prevent infection or to reduce disease severity. different kinds of vaccines, such as dna vaccines, recombinant proteins, subunit vaccines, and inactivated viruses, were described. however, the highly sophisticated immune evasion mechanisms of viral pathogens and their high mutation rates make human vaccine development a major challenge. the four nsps ( clpro, plpro, helicase, and rdrp), which are key enzymes in the viral life cycle, and the s protein, which is responsible for receptor binding during cell entry, are attractive targets for developing vaccines against coronaviruses. among them, the s protein is most commonly targeted. based on previous studies, it is believed that the s protein receptor-binding domain (s-rbd) located in the s subunit is an important target for the development of a sars vaccine, especially the key neutralizing region, cnd. indeed, cnd can induce a strong neutralizing antibody response and crossprotection against sars-cov mutants. one study showed that a recombinant fusion protein (designated rbd-fc) containing the -amino acid rbd and a human igg fc fragment can induce highly potent antibody responses in immunized rabbits. the antibodies inhibited sars-cov infection (serum dilution of : , ), and they are believed to be safer than other types of vaccines. additionally, with chloroplast transgenic technology, it is possible to combine the fusion gene s-rbd and the carrier molecule cholera toxin b subunit into the tobacco chloroplast genome to obtain a chloroplast transgenic tobacco plant that stably expresses the oral sars-cov subunit vaccine. for mers, vaccines based on the viral s protein include full-length s or strimer protein-based vaccines such as a full-length s-based simian adenovirus vector vaccine (chadox ) and dna vaccine (gls- ), s -based vaccines such as an ad vector encoding mers-cov s extracellular domain (ad .mers-s ) and an rabv vector encoding an s -elicited antibody, , rbd-based vaccines, and vaccines based on other regions. it has been confirmed that the s proteins of sars-cov and sars-cov- are quite similar, but researchers recently found that the three monoclonal antibodies developed to bind to the s protein of sars-cov, s , m , and r do not cross-react with the s protein rbd of sars-cov- , simian adenovirus vector vaccine (chadox );an ad vector encoding mers-cov s extracellular domain (ad .mers-s ); an rabv vector encoding s elicit antibody; rbd-based vaccines , , , overview of lethal human coronaviruses chen et al. suggesting that tailored vaccines and antibodies against sars-cov- must be designed. dna vaccines are also quite promising. specific igg antibodies against sars-cov can be promoted by the s gene dna vaccine. as mentioned above, the s protein of sars-cov plays an important role during the pathogenic process, and synthetic peptides induced by dna vaccine in escherichia coli elicit specific antibodies against the sars-cov s protein which might provide another approach for further developing sars-cov vaccines. , to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls- ) that expresses the s protein of mers-cov, a phase i clinical trial on healthy volunteers was conducted in , but the results were not reported. another phase i trial utilizing the viral vector, chimpanzee adenovirus, oxford university # (chadox ), containing the mers-cov s protein expression gene was started by oxford university in january . in addition, camel vaccines against mers-cov are a consideration. at present, at least two promising candidate camel vaccines are undergoing development, and field trial evaluation is in progress. , one study found that the rbd fragment covering spike residues - is a key neutralizing receptor-binding fragment and an ideal candidate for mers vaccines. another potential neutralizing epitope is a peptide fragment covering - residues of the s protein which blocks the membrane fusion and cellular entry of mers-cov (fig. ) . other approaches recombinant viruses may be employed to generate an immune response against the viruses. there are two kinds of recombinant viruses which are promising for designing a protective vaccine. the first is a defective or nonpathogenic vector capable of expressing viral proteins, and the second is a vector that can be assembled in a test tube to stimulate the assembly of virus-like particles. adenosine deaminase (ada), a kind of human enzyme, could interact with dpp , therefore, ada could compete with mers-cov as a natural antagonist when the virus attempts to bind to cells. in addition, anti-dpp can play a similar role, however, it is not practical to use this method in vivo because dpp has important functions in the regulation of several different signaling pathways. questions concerning the coronavirus-host interactions this topic has intrigued the community. understanding certain mechanisms about virus interactions will support drug research activities. for instance, it was found that sars-cov- has a stronger, more rapid spreading ability than many known viruses. is it possible that it invades host cells via additional novel routes, not limited to ace ? for example, cd is probably involved in virus invasion. is there an s-like protein involved in invasion? the affinity of the human ace protein for the rbd of the new coronavirus is - times higher than it is for that of the sars virus, which likely explains why sars-cov- spreads more swiftly. there is a distinct insert present in the peptide fragment spanning of s /s loop of the sars-cov- s protein, which is not shared the similarity with sars-cov or any sars-related viruses. does this peptide loop impact on the entry pathway type of sars-cov- , compared to other known betacoronavirus lineage b? furthermore, the s protein is likely cleaved during virus assembly and delivery to the cell surface by golgi-resident proprotein convertases such as furin, which is different from the behavior of its close relatives. furin is found in a variety of human tissues, including the lungs, liver, and small intestine. therefore, which are exactly the cell types that sars-cov- may attack? yet, in relation to the last two questions, it should be noted that studies are also necessary to investigate any possibility of receptor-independent virus entry. the sars-cov- genome encodes nonstructural proteins, structural proteins, and accessory proteins. clpro, plpro, helicase, and rdrp, which belong to the first type, and the s protein, which belongs to the second, have been recognized as promising targets for developing antiviral agents against sars-cov and mers-cov, which therefore may be applied to sars-cov- . nonetheless, the rapid identification of effective interventions against sars-cov- is a major challenge. however, the subject of using currently known antiviral drugs has been frequently brought up by the community as a potential short-term strategy to combat sars-cov- . drugs affecting such targets and their status for sars-cov- are listed below. the main candidates as inhibitors of clpro include other agents for sars-cov- fig. the targets of the different drug candidates against the three coronaviruses. common targets against the three coronaviruses are mainly the s protein and the s /s subunits, pl protein, rdrp, cl protein, and helicase. the figure shows drug candidates (in black) and vaccines (in red). among them, remdesivir has been trending in the news recently. it inhibits the rdrp, is in phase iii for sars-cov- , and may have an effect on the three viruses. ribavirin in combination with a pegylated interferon may also have an effect against the three viruses. ritonavir and lopinavir, which inhibit the clpro and are in phase iii for sars-cov- , have an effect on both sars-cov- and mers-cov. dna vaccines and vaccines based on the s protein or subunits of the s protein are in development lopinavir, ritonavir, darunavir, cobicistat, and asc f (phase iii, in combination with oseltamivir, for sars-cov- ). candidates as inhibitors of plpro include thiopurine analogs, compound (preclinical), and remdesivir (phase iii for mars-cov). , favipiravir, ribavirin (randomized trial for sars-cov- ), and remdesivir are among the candidate inhibitors of rdrp. previously, compounds that may interfere with atpase and helicase activities were reported (before covid- ), such as bananins, -hydroxychromone derivatives, and triazole derivatives (preclinical). , candidates that may suppress viral entry by targeting the s protein include peptide p and α-helical lipopeptides (preclinical), and those targeting the s subunit of the s protein mainly include hr p, hr m, hr l, hr l, hr p, and hr l (preclinical). due to the strong affinity between ace receptor and the rbd of sars-cov- , another opportunity might be through the development of antibodies to block ace . the second method is to use a large amount of soluble ace to directly block the spike protein. the third method is to find a drug that directly inhibits the spike membrane fusion process, such as the aids drug enfuvirtide. the fourth approach is to identify an agent that inhibits the activity of disintegrin and metalloproteinase (adam ), which may also prevent viral release and proliferation in cells and protect ace and the lungs. mrna vaccine technology sars-cov- vaccines are already under development. the mrna and dna vaccines are promising approaches to prevent cellular invasion by similar coronaviruses in the future. in january , the coalition for epidemic preparedness innovations (cepi) announced three partnerships to develop a new coronavirus vaccine. among them, moderna and inovio presented mrna and dna vaccine technologies, respectively. the goal of the three teams is to have at least one candidate vaccine capable of preventing coronavirus infection in weeks, to be tested in a phase i clinical trial. moderna's vaccine model is designed to use mrnas to safely pre-expose the immune system to a small amount of encoded proteins usually generated by the pathogen, so that the immune system becomes prepared to fight future pathogens and prevent infection. the candidate genes developed by moderna contain mrnas, and combining multiple mrnas into a single vaccine might be useful to rapidly induce responses, which is an approach that may be applied for future, emerging pandemic threats too. at the same time, because the mrna in the cell will be degraded over time, the mrna vaccine will not continue to produce antigen components for a long time, which can avoid the risk of continued stimulation of the immune system. generally, mrna vaccines are also described as simple to prepare and to yield remarkable results. additional advantages, as well as disadvantages, of nucleic acid-based vaccines were described in detail. furthermore, the basis of traditional vaccines, proteins or polysaccharides, cannot be straight forward applied at present against many pathogens. therefore, mrna vaccines would be suitable for achieving rapid, mass production of emergency vaccines in the event of a major epidemic. however, because rna is easily degraded, inducing cells to absorb mrna quickly is a challenge. in recent years, the method for delivery of mrna into cells has been greatly improved, and stemirna and moderna have adopted nanolipid (lpp or lnp (lipid nanoparticle)) drug-loading technology. however, this is still to be developed for mrnas. furthermore, ensuring safety and effectiveness is another challenge for viral mrna vaccines, and clinical validation will have to be carried out carefully. moderna is a worldwide leader in mrna therapy, with currently nine mrna-type vaccine candidates (e.g., mrna- against sars-cov- ). the national institutes of allergy and infectious diseases (niaid) of the national institutes of health (nih) and the mrna vaccine giant moderna have teamed up to bring the new coronavirus vaccine possibly to phase ii within a few months, and phase iii late this year. simultaneously, in january , the translational medicine platform of dongfang hospital affiliated with tongji university cooperated with stemirna (shanghai) biotechnology co., ltd. to rapidly promote the development of a new coronavirus mrna vaccine. in february , the chinese scientific research team announced that animal testing of the newly developed coronavirus vaccine has begun. if animal testing goes well, this new vaccine will enter human clinical trials within a few months. also in february , zhuhai livanda biotechnology co., ltd. announced that the first batch of new coronavirus mrna vaccine standard samples completed during the spring festival had been delivered to relevant national authorities on for animal testing and efficacy verification. the researchers detected the production of target antibodies in mouse serum at day following immunization. the company claimed that this was also the first time worldwide that new coronavirus vaccines developed based on mrna technology have resulted in antibodies in animals. livanda is currently pushing ahead with the project through extensive cooperation with the academy of military medical sciences, the guangdong provincial institute of supervision, and the macau university of science and technology. the antigen used in its development is the same as that used by moderna. dna vaccine technology dna vaccine technology may have many advantages (e.g., safe, fast, less technical barriers), along with potential limitations too. a phase i human clinical trial of the mers-cov vaccine has proven its safety and effectiveness. the dna vaccine based on the sars-cov s protein developed by the team of barney graham and gary nabel of the us-nih vaccine research center (vrc) has achieved positive results in animal experiments and a clinical phase i trial. , inovio pharmaceuticals has accumulated extensive safety and immunogenicity data for mers-cov vaccine studies. because of the high degree of similarity between mers-cov and sars-cov- , lessons from such a vaccine development process may be beneficial for the development of a dna vaccine against sars-cov- . since january , inovio has been collaborating with several experts and companies (some in china), and has currently already begun phase i clinical trials of the dna vaccine ino- . china's cansino biologics might be the leader as it was announced it has moved to phase ii testing of a vaccine called ad -ncov. the latter is currently often being reported as a dna vaccine, but to be precise, it uses viral vectors (adenovirus) to deliver dna related to sars-cov- . part of the project is carried out currently with the chinese military medical research institute. the vaccine candidate is constructed using genetic engineering methods and defective human type adenovirus as a vector that can express the sars-cov- s antigen, to stimulate the body to produce strong humoral or cellular immunity. sars-cov and mers-cov belong to subgroups b and c of the betacoronaviruses, respectively, and sars-cov- is a new member of betacoronaviruses, distinct from sars-cov and mers-cov. sars-cov and sars-cov- are similar in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov- is ace , plus cd for sars-cov- , while that of mers-cov is dpp (cd ). all of them evolved a mechanism to escape the host cell immune system. sars-cov and sars-cov- affect the ras system by suppressing ace , leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. sars-cov, mers-cov, and sars-cov- infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. current treatments for sars mainly include ifn-α, antiviral treatments (e.g., ribavirin), plasma therapy, host-directed therapies, and systemic corticosteroids. for mers, several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn, and novel therapies, including polyclonal antibodies, broad-spectrum antivirals, and amps. for covid- , candidates mainly include drugs targeting the s protein, nonstructural proteins ( clpro, plpro, helicase, and rdrp), and viral rna synthesis blockers such as remdesivir and ribavirin. vaccines such as dna vaccine, mrna vaccine, and recombinant vaccines are being rapidly developed. so far this century, human beings have experienced several epidemic outbreaks, and each outbreak had a negative impact at different levels, including health, economy, and even psychology and human behavior. in the future, more precautious measures should be available to guide individuals and groups to take effective emergency measures and to support social stability, and physical and mental health. furthermore, additional studies of coronaviruses and disease epidemics should continue, to support the preparation for future responses, medical therapies, vaccines, and methods of relieving personal anxiety. this review has highlighted several approaches, and the names and progress related to several compounds and biologics currently under research and development, as well as the companies and researchers involved in these efforts. for future directions, it has also described the differences and similarities, as well as the potential routes 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international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -zwh xj u authors: al-dorzi, hasan m.; aldawood, abdulaziz s.; khan, raymond; baharoon, salim; alchin, john d.; matroud, amal a.; al johany, sameera m.; balkhy, hanan h.; arabi, yaseen m. title: the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study date: - - journal: ann intensive care doi: . /s - - -z sha: doc_id: cord_uid: zwh xj u background: middle east respiratory syndrome coronavirus (mers-cov) has caused several hospital outbreaks, including a major outbreak at king abdulaziz medical city, a -bed tertiary-care hospital in riyadh, saudi arabia (august–september ). to learn from our experience, we described the critical care response to the outbreak. methods: this observational study was conducted at the intensive care department which covered icus with single-bedded rooms. we described qualitatively and, as applicable, quantitatively the response of intensive care services to the outbreak. the clinical course and outcomes of healthcare workers (hcws) who had mers were noted. results: sixty-three mers patients were admitted to mers-designated icus during the outbreak (peak census = patients on august , , and the last new case on september , ). most patients had multiorgan failure. eight hcws had mers requiring icu admission (median stay = days): seven developed acute respiratory distress syndrome, four were treated with prone positioning, four needed continuous renal replacement therapy and one had extracorporeal membrane oxygenation. the hospital mortality of icu mers patients was . % ( % for the hcws). in response to the outbreak, the number of negative-pressure rooms was increased from to rooms in mers-designated icus. patients were managed with a nurse-to-patient ratio of : . . infection prevention practices were intensified. as a surrogate, surface disinfectant and hand hygiene gel consumption increased by ~ % and n masks were used per patient/day on average. family visits were restricted to h/day. although most icu staff expressed concerns about acquiring mers, all reported to work normally. during the outbreak, . % of nurses and . % of physicians working in the mers-designated icus reported upper respiratory symptoms, and were tested for mers-cov. only / ( . %) icu nurses and / ( . %) physician tested positive, had mild disease and recovered fully. the total sick leave duration was days for nurses and days for physicians. conclusions: our hospital outbreak of mers resulted in patients requiring organ support and prolonged icu stay with a high mortality rate. the icu response required careful facility and staff management and proper infection control and prevention practices. the middle east respiratory syndrome (mers) coronavirus is a recently identified virus that is closely related to the severe acute respiratory syndrome coronavirus (sars-cov) [ ] , causes severe hypoxemic respiratory failure with multiorgan failure and frequently requires admission to the intensive care unit (icu) [ , ] . as of september , , the world health organization (who) reported laboratory-confirmed cases, including related deaths [ ] . the majority (~ %) of mers cases occurred in saudi arabia [ ] , where several hospital outbreaks happened [ , ] with % of all cases taking place within healthcare facilities [ ] . the outbreak in the republic of korea illustrated the global threat of this disease. it started in may and resulted from a case with travel history to the arabian peninsula [ ] . human-to-human transmission occurred to close contacts [family members, other patients and healthcare workers (hcws)] and led to cases of mers-cov infections with % fatality rate. in our hospital, king abdulaziz medical city-riyadh, an outbreak of mers disease occurred in august to september [ ] , led to significant disruption of hospital functions and resulted in mers cases [ ] with patients requiring icu admission. the outbreak was attributed to crowding, movement of infected but undiagnosed patients and breaches in infection prevention and control practices [ ] . details of the hospital outbreak have been published elsewhere [ ] . most of the medical literature on mers has focused on describing the characteristics and outcomes of affected patients. preparedness and the response of the healthcare system at its different levels are crucial to contain this disease and manage its associated outbreaks. nevertheless, little is published about how the healthcare system responded to the disease and hospital outbreaks. the objective of this study was to describe the response of the icu to a hospital mers outbreak, the associated changes in its workflow and the impact on its hcws. this study was an observational study conducted at the intensive care department of king abdulaziz medical city, a -bed tertiary-care referral hospital in riyadh, saudi arabia, that was accredited by the joint commission international. the hospital had an infection prevention and control department. the intensive care department covered units: an -bed trauma icu (unit a), a -bed medical-surgical intensive care unit (unit b), a -bed surgical icu (unit c), an -bed neurologic icu (unit d) and a -bed stepdown unit (unit e). the department also provided coverage to boarding patients in the -bed resuscitation area in the emergency department (ed). the hospital had also an -bed burn icu. the icus were operated as closed units with -h, -day onsite coverage by boardcertified critical care intensivists [ ] . normally, medical teams covered the units during the day with each team consisting of one intensivist consultant, one registrar/ fellow and - residents. the nurse-to-patient ratio in all the icus was mostly : . one certified respiratory therapist covered a maximum of six ventilated patients. additionally, the department had a rapid response team, which covered the hospital wards and was activated according to predefined criteria and is covered by a sixth separate team [ ] . the department has had several ongoing quality improvement projects and indicators. infection prevention and control practices, such as hand hygiene and the ventilator care bundle, were monitored by multidisciplinary icu teams and the infection prevention and control department [ ] . for intubated patients, ventilator care was provided by specialized respiratory therapists and included using closed endotracheal suctioning systems which were changed every h or as clinically indicated [ ] , changing the ventilator circuits in between patients or if they became soiled or damaged [ ] , and using heat and moisture exchangers which were changed every days or when visibly soiled [ ] . due to the high prevalence of multidrug-resistant organisms and prior cases of influenza and mers infection in the hospital, droplet precautions were applied to all icu patients since february [ ] . sporadic cases of mers cases have been managed in our icu since february . the characteristics, management and outcomes of the initial mers cases managed in our unit were described previously [ ] . all hcws were required to undergo fit testing for the n respirators (table ). an infectious disease epidemic plan (idep) was initially released in may and was revised in march . table describes selected plan elements. according to the idep, one unit (unit a) was designated as the primary receiving unit for mers patients, because of its geographic location being away from main hospital traffic and because of its rooms were negative-pressure airborne infection isolation rooms (aiir). the plan was not explicit about which units would be used if the number of cases exceeded the capacity of this unit. however, unit b had negative-pressure rooms. in our hospital, mers was suspected based on clinical presentation and confirmed by laboratory testing as recommended by the who and the saudi ministry of health [ , ] . in our icu, the workup of patients having lower respiratory tract infections was standardized to include bacterial gram stain and culture, mers-cov polymerase chain reaction (pcr), h n pcr, bacterial and viral multiplex pcr on respiratory samples, mycoplasma, chlamydia and legionella serology and, if suspected, tuberculosis workup. the respiratory samples were routinely obtained by blind deep tracheal aspirate in intubated patients. in the hospital biosafety level laboratory, mers-cov screening was performed by real-time reverse-transcription (rrt) pcr on respiratory samples by checking for the upstream e protein genome (roche modular dx coronavirus) and infection confirmation by detecting the open reading frame a genome (mers-cov kit from tib molbiol) [ ] . laboratory workers were fit-tested and applied n respirators while handing respiratory samples. positive samples were sent to the saudi ministry of health reference laboratory for confirmation. viral culture was not performed as biosafety level is needed. we noted mers cases admitted to the icu from july to october , , and collected data on the clinical characteristics, management and outcomes of the affected hcws. we also obtained data about the rrt-pcr performed for icu patients. we identified the physicians and nurses who reported sick leave for respiratory illnesses. as surrogate for infection control practices, we obtained data on the related consumables for units a and b before and during the outbreak (april- september , ) . we also collected qualitatively our own observations and those of other hcws on the icu response during the outbreak using interviews and open discussions. descriptive data were presented as means and standard deviations or frequencies and percentages, as appropriate. the infection prevention and control consumables were compared in the months before (april to july) and the months during (august and september) the outbreak. the plan will be activated by the chair of the outbreak response committee based on the phase definition phase i - cases of suspected or confirmed in the hospital phase ii - cases of suspected or confirmed in the hospital phase iii > cases of suspected or confirmed in the hospital phase i confirmed mers-cov cases requiring intubation will be assigned a negative-pressure room and cohorted in one icu confirmed cases that have been diagnosed with mers-cov in any icu other than the trauma icu (unit a), shall be transferred to the trauma icu (unit a) as soon as possible. phase ii all mers-cov patients will be cohorted in one unit. if the number of patients exceeds its capacity, then other units are identified to receive the additional cases closure of all nonessential hospital functions phase i all services run without interruptions except for certain precautions for mers patients phase ii all elective surgery shall be canceled to free more icu beds phase iii all elective cardiac surgery shall be canceled outpatient clinic visits shall be limited to urgent visits only healthcare worker (hcw) management all hcws shall be aware of . relevant infection prevention and control policies and procedures . their annual influenza immunization status. if not vaccinated, please contact the employee health clinic to arrange for an appointment . their n fit check/test status. if have not been fit-tested, please contact the employee health clinic to arrange for an appointment hcws exposed to a confirmed mers-cov case shall be assessed according to a predetermined protocol hcws requiring isolation at home and happen to share a room with another hcw will be provided a room in the designated accommodation for isolation till cleared by the infection prevention and control department an infection prevention and control officer is available h per day, days per week in july , five cases of acute respiratory failure were referred to the icu team from the ed and wards and were diagnosed to have mers pneumonia. as the number of mers patients increased, the idep was activated on august , and included strict implementation of infection control measures, including airborne and contact isolation for confirmed and probable mers cases, and droplet and contact isolation for suspected cases [ ] . on august , phase iii of the idep was activated (table ) , which included ed closure, elective surgical procedure cancelation and outpatient clinic suspension [ ] . meanwhile, the icu maintained full operations. figure suspected mers cases had rrt-pcr on nasopharyngeal swabs in nonintubated patients and on deep tracheal aspirates in intubated patients. fiberoptic bronchoscopy was not performed for the diagnostic workup of mers or ventilator-associated pneumonia. repeated testing was frequently needed to make the diagnosis. among our critically ill mers patients, the initial mers-cov testing was performed on nasopharyngeal swabs in and deep tracheal aspirates in the rest (n = ). in the first sample, mers-cov was detected in only / ( . %) nasopharyngeal samples and / ( . %) deep tracheal aspirates. after initial negative or equivocal nasopharyngeal swabs (n = ), a second nasopharyngeal swab was performed in patients (positive in . %) and deep tracheal aspirate in (positive in %). our microbiology laboratory extended its working hours and prioritized testing samples coming from the icu and the rest of the hospital. the number of mers-cov tests performed on icu patients went up from an average of . before to . per day during the outbreak with a maximum of tests on september , . in patients with suspected mers and negative rrt-pcr, the test was repeated after - days. there was a general consensus among our intensivists that three negative lower respiratory samples and low clinical pretest probability were needed to exclude mers-cov infection. for patients with confirmed mers, the test was repeated twice weekly until consecutive tests were negative. the mean age of the patients was . ± . years with the majority ( . %) being males. eight hospital workers required icu admission after acquiring mers. table summarizes their characteristics. half of them did not have direct contact with patients. one of them was a pregnant nurse that worked in the ed. all but one required intubation. the medical management of mers patients was largely supportive. most ( . %) mers patients required endotracheal intubation, which was performed by the most experienced available physician with airborne precautions. lung-protective ventilation was implemented for acute respiratory distress syndrome with tidal volumes ( - ml/kg of ideal body weight). to reduce the airborne generating procedures, we discouraged the use of noninvasive ventilation for suspected mers cases. nevertheless, it was used in the initial management of ( . %) patients. these patients were either suspected to have concomitant cardiogenic pulmonary edema or had milder disease. intubation was needed for patients. highflow oxygen therapy was not used as it was unavailable. when needed, bronchodilators were used via metered dose inhalers rather than nebulizers. most ( . %) mers patients required vasopressors. renal replacement therapy was provided for ( . %) patients. for the hospital workers acquiring mers (n = ), cisatracurium infusion was used in ( . %), early prone positioning in ( %), continuous renal replacement therapy in ( %) and extracorporeal membrane oxygenation in ( table ) . none of the patients received ribavirin, interferon therapy or high-dose steroids. the hospital mortality of mers patients was . % with all deaths occurring in the icu. all hospital workers who had mers survived and were discharged to home. the icu and hospital length of stay were prolonged ( . ± . and . ± . days, respectively). during the outbreak, our hospital established a command center, which met twice daily, and oversaw all interventions in accordance to the idep. the intensive care department chairman was a member of the command center and presented daily the number of suspected and confirmed cases in the icu, bed and staff management issues and any challenges. the department chairman attended all morning handover meetings in the icu where he received input from the icu teams and provided feedback from the command center. the hospital provided an intranet page that had educational material on mers, mers management guidelines and proper infection control practices. the page was regularly updated. additionally, the hospital frequently informed staff about the mers outbreak status through emails. staff could communicate with the command center regarding any outbreak-related concern or question. the intensive care department communicated with the medical staff about the saudi ministry of health and who practice guidelines. before and during the outbreak, the who interim guidance for the management of suspected and confirmed mers-cov infection [ ] was circulated to our icu staff. moreover, a letter expressing gratitude and encouragement was sent from the department chairman to all hcws. family visiting to mers patients was restricted from an open visiting policy to h per day during the evening with visitors not allowed to enter patient rooms. visiting outside these hours was allowed in selected cases if the clinical condition required. to update the patients' families, the icu consultant contacted and updated the next of kin by phone every day and addressed the family concerns. a nurse was assigned to screen all staff and visitors entering each unit by asking for symptoms of acute respiratory infection and measuring temperature. staff and family members with symptoms of acute respiratory illness or fever were not allowed to enter. the initial mers cases were admitted to the designated mers unit (unit a). as the number of patients increased, the icu leadership identified other icus as potential placement units. the hospital clinical engineers converted a total of standard rooms in unit b and unit c to negative-pressure rooms by increasing air exhaust more than supply by cubic feet per minute. as the number of suspected and confirmed mers patients increased, unit b and then unit c were used. as the number of our mers patients increased beyond unit a capacity, patients without mers were transferred to other units or hospitals to increase bed capacity. the old pediatric icu, which was recently vacated in june after the opening of a new pediatric hospital, was used to care for stable and long-term patients. during the outbreak, and patients from units a and b, respectively, were transferred to the other icus (units c-e and the old pediatric icu), and patient to another hospital. the care for mers patients was demanding. for example, of the affected hcws required prone positioning for the management of acute respiratory distress syndrome ( table ). also of them required continuous table ) . the care was also associated with significant exposure risk. this can be reflected in the duration of mechanical ventilation for the hcws who required intubation ( - days) and length of icu stay ( - days) ( table ) . during the outbreak, the nurse-to-patient ratio was mostly : except for one patient on ecmo ( : ). additionally, - additional nurses were deployed in each unit to assist in procedures such as prone positioning and to monitor and correct infection control practices. in unit a, for instance, the nurse-to-patient ratio was : . before the outbreak and became : . during the outbreak. we restricted medical management to the attending physicians, senior registrars and critical care fellows. rotating residents were not allowed to work in the icu during the outbreak. entry of nonclinical staff, such as research coordinators, was restricted and the ongoing clinical trials were put on hold, except for mers studies, to reduce staff exposure. during the outbreak, concerns among icu staff were raised about acquiring mers and transmitting the virus to their families; a concern that was substantiated by seeing hospital workers infected and developing critical illness. however, many felt privileged to be part icu team managing the outbreak and taking care of mers patients; none refused to report to work as per schedule. two pregnant icu nurses were redeployed to low-risk units. staff members who developed fever, respiratory symptoms or gastrointestinal illness were asked not to present to work, but rather to report to the ed or the employee health clinic depending on their illness severity. of the bedside nurses covering units a and b, ( . %) nurses had symptoms of acute respiratory infection during the outbreak and consequently had nasopharyngeal swabs obtained for mers-cov; all tested negative. their total sick leave duration was days (range: - days per nurse). in comparison, in the months before the outbreak, ( . %) nurses had sick leaves for a total of days (range - days per nurse). of the nonresident icu physicians, ( . %) physicians received sick leave for a total of days (range - days per physician). in unit c, two nurses ( . %) of nurses who worked in mers units (units a, b and c) and one rotating resident ( . %) out of physicians covering the icus tested positive for mers-cov. the resident and one of the nurses were symptomatic and required hospitalization in a mers ward for approximately week and both recovered fully. in february , long before the mers outbreak, droplet precautions had been added to the standard precautions for all icu patients, mainly to prevent the transmission of influenza. during the outbreak, airborne precautions were added for all confirmed and suspected mers cases. although all staff were required to be fit-tested for the n respirators before the outbreak (table ) , we discovered that many icu staff were not tested. during the outbreak, a clinic was emergently opened to fit test hcws and the results were documented. specific policies and procedures were developed or updated for donning and doffing personal protective equipment (ppe). related visual instructions were provided inside each icu room. outside patient rooms, carts containing ppe were organized to facilitate donning in the correct sequence. during the outbreak, additional training on hand hygiene techniques and ppe application was provided. housekeepers were also retrained on proper cleaning techniques and ppe use. the intensive care department worked closely with the infection prevention and control department on all aspects of infection control. the implementation of such infection control measures required having adequate ppe supplies, such as respirators, goggles, face shields and gowns. table describes the consumption of surface disinfectants, antiseptic alcohol for hand hygiene, n masks and other ppe before and during the mers-cov outbreak. during the outbreak, the consumption of detergent surface disinfectant and ethyl alcohol for alcohol-based hand rub increased by almost % and the use of n masks increased by > times compared to the preceding months. the number of examination gowns per patient per day decreased during the outbreak probably due to staff avoiding unnecessary exposure. twenty-four powered air-purifying respirators were made available to staff who failed the n respirator fit test. they were used by physicians, nurses and respiratory therapists. training sessions on their application were conducted. in this report, we described how the icu responded to a mers-cov outbreak at a tertiary-care hospital. the outbreak led to mers patients requiring prolonged icu care and most received invasive mechanical ventilation, vasopressors and renal replacement therapy. the overall mortality was %, but all affected hospital workers survived. the outbreak management included almost tripling the icu capacity of negative-pressure rooms and intensifying infection prevention and control practices. even though icu staff had significant exposure risk, very small number acquired mers-cov. response to incidents such as an infectious hospital outbreak requires a robust hospital-wide command and control structure that is able to make rapid informed decisions across an institution. as it was the case in our hospital, the control of outbreak may require major interventions such as closing the ed, suspending elective surgeries, preventing inter-facility patient transfers, canceling ambulatory clinics and outpatient diagnostic procedures, preventing hospital staff from working at other institutions and restricting hospital visitors [ ] . our hospital had a preexisting idep, which facilitated managing and containing the mers-cov outbreak. such a plan is mandatory for every hospital. mers infection is associated with several challenges. its presenting symptoms overlap with those of other severe acute respiratory illnesses and include fever ( %), cough ( %), dyspnea ( %) and diarrhea ( %) [ ] , often in older adults with preexisting chronic comorbidities [ , ] . common laboratory abnormalities include leukopenia, lymphocytopenia, thrombocytopenia and elevated serum creatinine, lactate dehydrogenase, and liver enzymes [ ] . the initial chest radiographs show minimal abnormality to extensive bilateral infiltrates [ ] . unfortunately, many frontline physicians are unfamiliar with the mers case definition, probably because cases are sporadic, leading to delayed or even missed diagnosis. delayed recognition may lead to exposing many other patients, visitors and hcws to the infection as it was the case in our hospital. mers-cov nosocomial transmission is thought to be via respiratory droplets, and contact spread is suspected [ ] . in korea, the delayed diagnosis of an infected traveler to the arabian peninsula led to mers cases and resulted in intraand inter-hospital transmission [ ] . in saudi arabia, % of mers cases were acquired in the healthcare setting with % of all cases being hcws [ ] . therefore, taking a detailed history, knowing mers case definitions, standardizing pneumonia workup, obtaining lower respiratory tract specimens [ ] and implementing droplet isolation for suspected cases are crucial interventions to break the transmission chain in the healthcare setting. admitting mers patients in single-bedded negativepressure rooms and cohorting them in selected units are recommended to facilitate providing care and monitoring [ ] . during an outbreak, clinical engineering should have expedient plans to convert standard rooms. retrofitting the rooms with externally exhausted hepa filters may be a solution [ ] . outbreaks can lead to significant increase in the need for icu beds, but may simultaneously reduce the available beds. the sars outbreak in toronto led to -day closures of icu beds, which represented % of the tertiary-care university medical-surgical icu beds in toronto [ ] . hence, hospitals should always have plans to augment icu bed capacity, such as by transforming general wards. the ability of any hospital to deal with an infectious outbreak is decided by the availability of icu beds [ ] . caring for mers patients represents a substantial exposure risk for icu staff because of three reasons: high exposure dose, long daily contact hours and prolonged icu stay with viral shedding. mers-cov patients requiring icu admission have higher viral load than other mers patients [ ] . aerosol-generating procedures, such as noninvasive ventilation, suctioning and bronchoscopy, add to the exposure and transmission risk [ ] . extended bedside care is needed for mers patients due to the requirement of organ support such as mechanical ventilation, vasopressor therapy, continuous renal replacement therapy, prone positioning and extracorporeal membrane oxygenation [ , ] . in this study, we observed an increase in the nurse-to-patient ratio from : . to approximately : . during the outbreak. the sars epidemic was also associated with increases in the nurse-topatient ratio [ ] . stay in the icu can last for weeks [ ] , which we observed. additionally, mers-cov shedding can be prolonged and may last for > days [ ] . the exposure risk to mers-cov can exert significant psychosocial stress on hcws. death has occurred in young hcws who acquired mers-cov infection [ ] , which adds to this fear. during the sars outbreak in toronto, a survey of hcws found that > % of respondents reported sars-related concern for their own or their family's health [ ] . moreover, % of respondents had probable emotional distress [ ] . during our outbreak, many icu staff expressed concerns about acquiring mers. staff safety should be a primary goal in a hospital infectious outbreak. pregnant and immunocompromised staff should be redeployed to lower-risk areas [ ] , which we did. proper exposure management should be pre-planned, which was determined in our idep. n respirator fit testing should be performed at hiring of new staff and done for all other staff before any outbreak. although fit testing was required for all staff, many did not have fit testing done. however, in response to the outbreak, fit testing was performed to all staff and powered air-purifying respirators were provided to those who failed fit testing. strict infection prevention and control practices should be implemented and audited. this was performed in our units. repetitive training is recommended [ ] . despite intensive infection prevention practices, of our icu staff had mers-cov infection during the outbreak likely due to suboptimal ppe use while intubating yet undiagnosed patients. mers management was supportive and largely adhered to the who recommendations [ ] . it included intubation, early prone positioning and neuromuscular blockade for moderate-to-severe acute respiratory distress syndrome as these interventions have been shown to improve the outcomes of ards patients [ , ] . although noninvasive ventilation use was discouraged, it was used in patients. the who considers noninvasive ventilation an option in selected mers cases [ ] . it should be used as a short trial without delaying intubation if unsuccessful [ ] . moreover, high-flow oxygen by nasal cannula may be another option [ , ] ; however, the associated transmission risk as a result of aerosol generation is unknown. systematic corticosteroids, ribavirin and interferon were avoided as they have no proven benefit [ , ] . our mers patients had high mortality ( %). the previously reported mortality of mers patients who had critical illness ranged from to % [ , , ] . none of our hcws who developed mers died, which was gratifying to our staff. our mers-cov hospital outbreak stressed our system to unprecedented limits. we learned many lessons from it ( table ). the successful management of outbreak required integrating icu functions with the hospitalwide plans, having preparedness plans, implementing proper infection control practices and managing staffing and staff exposure. every hospital should have an infectious disease epidemic plan that should govern the response to an infectious disease outbreak. the response should cover organizing patient services, implementing infection control, managing employee exposure and communicating with national health services and with hospital staff hospital leaders should be prepared to increase the capacity of negative-pressure airborne infection isolation rooms in the case of an infectious disease outbreak all healthcare workers should receive training on proper hand hygiene and personal protective equipment application. hand hygiene and personal protective equipment practices should be monitored. education should be repeated periodically all healthcare workers should be fit-tested for n respirators on hire with the result documented in their files. periodic audit of this requirement should be done hospitals should make plans to acutely increase personal protective equipment supplies as consumption increases tremendously during an infectious disease outbreak hospital and icu leaders should have plans to cover healthcare workers who are exposed or become symptomatic to avoid potential staff shortage severe acute respiratory syndrome vs. the middle east respiratory syndrome clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities the national command control center; ministry of health-kingdom of saudi arabia. mers-cov in ksa notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia weekend and weeknight admissions have the same outcome of weekday admissions to an intensive care unit with onsite intensivist coverage impact of an intensivist-led multidisciplinary extended rapid response team on hospital-wide cardiopulmonary arrests and mortality a multifaceted approach to improve hand hygiene practices in the adult intensive care unit of a tertiary-care center comprehensive evidence-based clinical practice guidelines for ventilator-associated pneumonia: diagnosis and treatment ministry of health kingdom of saudi arabia. infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection assays for laboratory confirmation of novel human coronavirus (hcovemc) infections clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected-interim guidance identification and containment of an outbreak of sars in a community hospital middle east respiratory syndrome: knowledge to date middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions mers outbreak in korea: hospital-to-hospital transmission an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus hospital preparedness and sars association of higher mers-cov virus load with severe disease and death, saudi arabia aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review clinical review: sars-lessons in disaster management middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications middle east respiratory syndrome coronavirus infections in health care workers psychosocial effects of sars on hospital staff: survey of a large tertiary care institution sars and hospital priority setting: a qualitative case study and evaluation infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease prone positioning in severe acute respiratory distress syndrome neuromuscular blockers in early acute respiratory distress syndrome high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study none. the authors declare that they have no competing interests. written informed consent was not obtained for publication of these data. the institutional review board of the ministry of national guard health affairs approved the retrospective clinical data collection on mers patients, and no consents were required. abbreviations ed: emergency department; hcw: healthcare worker; icu: intensive care unit; idep: infectious disease epidemic plan; mers: middle east respiratory syndrome; ppe: personal protective equipment; rrt-pcr: real-time reversetranscription polymerase chain reaction; who: world health organization. hmd was involved in conception and design, data collection, statistical analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content and approval of the final version to be published. asa contributed to the analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. rk helped in data collection, analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. sb contributed to the analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. jda helped in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. aam was involved in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. smj helped in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. hhb contributed to data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. yma helped in conception and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content and approval of the final version to be published. all authors read and approved the final manuscript.author details icu and ticu, intensive care department, king abdulaziz medical city, king key: cord- -eeyqh nc authors: zhou, yusen; yang, yang; huang, jingwei; jiang, shibo; du, lanying title: advances in mers-cov vaccines and therapeutics based on the receptor-binding domain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eeyqh nc middle east respiratory syndrome (mers) coronavirus (mers-cov) is an infectious virus that was first reported in . the mers-cov genome encodes four major structural proteins, among which the spike (s) protein has a key role in viral infection and pathogenesis. the receptor-binding domain (rbd) of the s protein contains a critical neutralizing domain and is an important target for development of mers vaccines and therapeutics. in this review, we describe the relevant features of the mers-cov s-protein rbd, summarize recent advances in the development of mers-cov rbd-based vaccines and therapeutic antibodies, and illustrate potential challenges and strategies to further improve their efficacy. middle east respiratory syndrome (mers) coronavirus (cov) is an infectious virus that was first reported in june [ ] . mers-cov may infect people of any age, but older age, underlying comorbidity (such as diabetes mellitus, renal disease, respiratory disease, heart disease, and hypertension), and delayed confirmation or late diagnosis are all factors that affect mers disease outcomes and mortality [ ] [ ] [ ] [ ] [ ] [ ] . sex could be a factor in mers epidemiology, as more males seem to be affected than females [ ] [ ] [ ] . mers-cov infection of women during pregnancy has adverse outcomes, with fetal mortality of~ %; however, only a limited number of pediatric mers-cov infections occur [ ] [ ] [ ] [ ] . at the end of december , , laboratory-confirmed mers infections were reported globally (in countries), leading to deaths, and a mortality of . %. among these infections, , ( . %) were reported in saudi arabia, with mortality in individuals ( . %) (http://www.emro.who.int/health-topics/mers-cov/mers-outbreaks.html). the largest mers outbreak outside saudi arabia occurred in south korea in , with cases and deaths [ , , ] . the most recent mers cases were reported in in south korea, the united kingdom, and malaysia, in addition to saudi arabia, the united arab emirates, and oman (http://www.who.int/emergencies/mers-cov/en/). mers-cov is thought to have originated in bats [ ] [ ] [ ] [ ] . mers-like viruses have been isolated from bats that use (at lower efficiency) the same receptor for cell entry as the mers-cov isolated from humans [ ] [ ] [ ] . dromedary camels are potential intermediates for long-term evolution of mers-cov and seasonal zoonotic transfer of virus to humans [ ] [ ] [ ] [ ] . antibodies specific to host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [ , ] . the dpp receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to speciesspecific variations [ ] [ ] [ ] . in addition to dpp , mers-cov can bind to sialic acid via the s subunit of s protein, or utilize the membrane-associated kda glucose-regulated protein (grp ) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [ , ] . the structures of mers-cov rbd alone and complexed with dpp have been determined ( figure ) [ , , ] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [ ] . a number of rbd residues are located at the dpp -binding interface, and they have a critical role in rbd-dpp binding [ , , ] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp . notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp [ ] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd events [ ] . in configurations with one, two, or three rbds rotated out, rbd determinants are exposed at the apex of the rbd-dpp complex, and they are accessible for interaction with dpp ( figure ) [ ] . mers-cov s protein has an important role in viral pathogenesis, determining host tropism and entry into host cells [ , , ] . the s protein contains an s subunit at the n terminus and an s subunit at the c terminus. the s subunit is composed of the n-terminal domain (ntd) and rbd [ , , ] . the rbd has a key role in the mediation of binding of mers-cov to cells expressing dipeptidyl peptidase (dpp ) receptor, enabling the virus to enter into target cells by fusing with cell membranes through the formation of a fusion core ( figure c ) [ ] [ ] [ ] [ ] . the s protein requires host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [ , ] . the dpp receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to species-specific variations [ ] [ ] [ ] . in addition to dpp , mers-cov can bind to sialic acid via the s subunit of s protein, or utilize the membrane-associated kda glucose-regulated protein (grp ) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [ , ] . the structures of mers-cov rbd alone and complexed with dpp have been determined ( figure ) [ , , ] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [ ] . a number of rbd residues are located at the dpp -binding interface, and they have a critical role in rbd-dpp binding [ , , ] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp . notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp [ ] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd the mers-cov rbd core is colored in blue, the rbm is colored in red, and dpp is colored in green. the rbm residues directly involved in dpp binding are shown as sticks. dpp , dipeptidyl peptidase ; rbd, receptor-binding domain; rbm, receptor-binding motif; s, spike protein. the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus (ad )-directed expression of human dpp (ad /hdpp ), hdpp -transgenic (hdpp -tg) mice, and non-human primates (nhps) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table . the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus (ad )-directed expression of human dpp (ad /hdpp ), hdpp -transgenic (hdpp -tg) mice, and non-human primates (nhps) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table . a soluble nanoparticle vaccine formed in escherichia coli by the rna-mediated folding of a rbd-ferritin (fr) hybrid elicits robust rbd-specific antibody and cellular immune responses in mice, producing antisera that effectively block the binding of rbd to hdpp in vitro [ ] . the adjuvants alum and the squalene-based mf significantly augment the antibody titers and t-cell responses induced by rbd-fr nanoparticle vaccines engineered with or without a ssg linker [ ] . similarly, a chimeric, spherical vlp (svlp) vaccine expressing mers-cov rbd induces specific antibody and cellular immune responses in mice, preventing pseudotyped mers-cov entry into susceptible cells [ ] . the protective efficacy of these two types of mers vaccine does not yet seem to have been investigated in a viral-challenge animal model. recombinant vaccines involving rbd subunits have been extensively studied for protection against mers-cov infection in mers-cov-susceptible animal models [ , [ ] [ ] [ ] , ] . a recombinant rbd (rrbd) fragment (residues - ) expressed in insect cells elicits an antibody response and the production of neutralizing antibodies in mice and nhps [ , ] . it gives incomplete protection in mers-cov-challenged nhps, with the alleviation of pneumonia and clinical manifestations, as well as the reduction of viral load in lung, trachea, and oropharyngeal swabs [ ] . a mers-cov s-protein rbd fragment containing residues - has been identified as a critical neutralizing domain [ ] . a treatment regimen involving two doses of a fusion of this fragment and the fc region of human igg (s - -fc) four weeks apart is able to induce strong, long-term antibody responses (including production of neutralizing antibodies) in mice [ ] . these responses are significantly greater than those with a single dose or two doses at intervals of one, two, or three weeks [ ] . rrbds with single or multiple mutations corresponding to s-protein sequences of mers-cov strains isolated from humans or camels from to have also been studied [ ] . all these rrbds bind rbd-specific neutralizing monoclonal antibodies (mabs) and dpp , and are highly immunogenic, eliciting the production of s -specific antibodies in mice, which cross-neutralizes multiple mers pseudoviruses and live mers-cov [ ] . a trimeric rbd-fd protein formed by fusing a mers-cov rbd fragment (residues - ) to the foldon trimerization motif, binds strongly to dpp , and elicits robust and long-term responses with the production of mers-cov s -specific antibodies and neutralizing antibodies in mice, and protects hdpp -tg mice against mers-cov infection [ ] . the protection provided by existing subunit vaccines based on wild-type mers-cov rbd is not complete, with survival rates in hdpp -tg mice after a mers-cov challenge of~ % for s - -fc and % for rbd-fd [ , ] . however, a variant rbd (t n) vaccine produced by masking a non-neutralizing epitope at residue with a glycan probe has both functionality in binding dpp , and antigenicity in binding four potent mers-cov rbd-specific neutralizing mabs (hhs- , m , m , and m ) [ ] . the t n vaccine has significantly greater efficacy than the wild-type rbd vaccine, and it fully protects against a lethal mers-cov challenge in immunized hdpp -tg mice [ ] , demonstrating the possibility of developing rbd-based mers-cov vaccines with high efficacy. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these antibodies generally have greater neutralizing activity against mers-cov infection than non-rbd s -based or s -based antibodies [ , , , ] . the prophylactic and therapeutic efficacies of rbd-targeting antibodies have been tested in ad /hdpp mice, hdpp -tg mice, and nhps [ , , [ ] [ ] [ ] . in an earlier review, we described the antiviral mechanisms, in vivo protection, and crystal structures of previously reported mers-cov rbd-specific mabs, including mouse mabs mersmab , e , c , f , and d , and human mabs lca , mers- , mers- , regn , regn , e , f , a , b , b , b -n, c , m d , m , m , m , hms- , and c h [ ] . in this review, we focus on newly reported antibodies targeting mers-cov s-protein rbd, or on newly identified features of existing mabs that were not described previously (table ) [ , [ ] [ ] [ ] [ ] . rbd-targeting human mabs have been extensively reported. most of these mabs can neutralize pseudotyped or live mers-cov in vitro, and some have shown protection against mers-cov infection in animal models in vivo [ , [ ] [ ] [ ] [ ] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure ) [ , [ ] [ ] [ ] [ ] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp (as is the case for gd- , mca , and cdc -c ), or with binding to the rbd residues outside of the dpp -binding interface (as seen with mers- ) ( table ) . infection in animal models in vivo [ , [ ] [ ] [ ] [ ] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure ) [ , [ ] [ ] [ ] [ ] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp (as is the case for gd- , mca , and cdc -c ), or with binding to the rbd residues outside of the dpp -binding interface (as seen with mers- ) ( table ) . the human mabs mers-gd and mers-gd each recognize distinct regions of the rbd [ ] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic ) for their use in combination than separately [ ] . an analysis of crystal structures has indicated that mers-gd binds rbd at the dpp -binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m [ , ] . the mers-gd mab protects hdpp -tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day postchallenge, and higher survival rates ( % for pre-challenge vaccination and % for post-challenge vaccination) relative to control mice treated with an irrelevant mab [ ] . the human mab mca was isolated from a mers survivor via the construction of a phagedisplay antibody library from peripheral b cells [ ] . crystal structure analysis indicates that mca binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp ( figure a ) [ ] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced the human mabs mers-gd and mers-gd each recognize distinct regions of the rbd [ ] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic ) for their use in combination than separately [ ] . an analysis of crystal structures has indicated that mers-gd binds rbd at the dpp -binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m [ , ] . the mers-gd mab protects hdpp -tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day post-challenge, and higher survival rates ( % for pre-challenge vaccination and % for post-challenge vaccination) relative to control mice treated with an irrelevant mab [ ] . the human mab mca was isolated from a mers survivor via the construction of a phage-display antibody library from peripheral b cells [ ] . crystal structure analysis indicates that mca binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp ( figure a ) [ ] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced lung disease, compared with unvaccinated controls, and undetectable virus titers days post-challenge [ ] . a probe-based single-b-cell cloning strategy has been used for the isolation of cdc -c and cdc -c mabs from a patient convalescing from mers, as well as for the isolation of jc - and jc - mabs from nhps immunized with mers-cov full-length s dna and protein [ ] . all these antibodies have neutralizing activities against both pseudotyped and live mers-cov. among them, cdc -c is the most potent against pseudotyped mers-cov strains, with neutralization ic values ranging from . µg/ml to . µg/ml [ ] . crystal-structure analysis of the cdc -c and jc - fab-rbd complexes indicates that both mabs bind rbd in the "out" (exposed) position, with the cdc -c rbd binding overlapping with the dpp -contacting residues ( figure b ,c) [ ] . in addition, cdc -c prophylactically protects hdpp -tg mice from mers-cov infection, resulting in no detectable viral replication in the lungs three days post-challenge, and no fatalities over days of observation [ ] . the human mab mers- also neutralizes pseudotyped mers-cov and, notably, displays synergistic neutralization in combination with the mers-cov s-protein rbd-targeting mers- and m mabs [ , ] , as well as the s-protein ntd-targeting f mab, in each case with dramatic reduction of the ic compared with individual mabs [ ] . structural analysis of a mers- -fab-rbd complex revealed that mers- binds the rbd from outside the dpp -binding interface, rather than competing with dpp ( figure d ). unlike mers- , which binds rbd regardless of its conformational state within the s trimer, mers- binds rbd in the "standing" position where its epitopes are readily exposed and accessible [ ] . thus, mers- displays unique epitope specificity, and an unusual mechanism of action involving indirect interference with dpp binding through conformational changes, which may explain the observation of synergistic neutralization in combination with other mabs [ ] . single-domain antibody fragments (vhhs), or nanobodies, are the antigen-recognition regions of camelid heavy-chain-only antibodies (hcabs), which do not contain light chains. vhhs are easily expressed with high yield, and they have intrinsic stability, strong binding affinity, and specificity to target antigens, and they have therefore been developed as important therapeutic tools against viral infection, including that of mers-cov [ , , [ ] [ ] [ ] [ ] [ ] . four vhhs (vhh- , vhh- , vhh- , and vhh- ) have been identified from bone marrow cells of dromedary camels immunized with modified vaccinia virus (mva) expressing mers-cov s protein, and challenged with mers-cov [ ] . these vhhs bind mers-cov s protein with low k d values ( . - nm), recognize an epitope at residue d of rbd, and neutralize mers-cov (prnt , . - . µg/ml) [ ] . these four monomeric vhhs have each been fused with a c-terminal human igg tag to generate four hcabs (hcab- , hcab- , hcab- , and hcab- ), with a higher binding affinity and a longer half-life than the free vhhs [ ] . studies of protective efficacy show that hdpp -tg mice (k ) injected with monomeric vhh- ( or µg per mouse) lose weight, and die within seven days post-infection, possibly because of the short half-life of the vhh. however, when the mice are injected with hcab- ( µg per mouse), which has an extended half-life (~ . days), protection against mers-cov is complete, with no viral titers or pathological changes in the lungs of virally challenged mice [ ] . by immunizing llamas with a recombinant rbd fragment (residues - ) fused to a c-terminal human igg fc tag (s - -fc), we constructed a vhh library, and we used it to generate a monomeric vhh, nbms , and a human fc-fused vhh, nbms -fc [ ] . both vhhs can be expressed in a yeast expression system to high purity, and bind rbd with high affinity, recognizing a conformational epitope (residue ) at the rbd-dpp interface, and blocking the binding of rbd to dpp . these vhhs, particularly nbms -fc, potently cross-neutralize pseudotyped mers-cov strains isolated from different countries, hosts, and time periods [ ] . importantly, the fc-fused nbms -fc significantly improves the serum half-life of nbms , and a single-dose treatment of hdpp -tg mice with this agent completely protects them against lethal mers-cov challenge [ ] . these single-domain vhhs demonstrate the feasibility of developing cost-effective, potent, and broad-spectrum therapeutic antibodies against mers-cov infection. compared with vaccines based on mers-cov full-length s protein, which have the potential to attenuate neutralizing activity or enhance immune pathology, vaccines developed from mers-cov s-protein rbd are safer, and they do not cause immunological toxicity or eosinophilic immune enhancement [ , , , ] . moreover, rbd-based therapeutic antibodies are generally more potent than non-rbd s -based or s -based antibodies [ , , ] . hence, rbd-based vaccines and therapeutic antibodies have the potential for further development as effective tools to prevent and treat mers-cov infection. despite their acknowledged advantages, there are some issues associated with rbd-based interventions that need to be addressed. for example, rbd is under a high level of pressure of positive selection, and mutations occur in the rbd-dpp binding interface that might reduce the efficacy of these treatments [ , [ ] [ ] [ ] . one possible way to avoid this effect, and to delay the emergence of escape mutants is to combine rbd-targeting therapeutics with those targeting other regions of the s protein, or to combine antibodies recognizing distinct epitopes within the rbd [ , ] . such combinatorial strategies could also dramatically reduce antibody neutralization doses, providing feasible means to combat the continual threat of mers-cov. some recent advances have been made in the structure-guided design of anti-mers-cov interventions. structurally designed inhibitors of the cl protease have demonstrated potency against mers-cov [ ] . also, a structurally designed s-protein trimer in the optimal prefusion conformation is shown to elicit production of high titers of anti-mers-cov neutralizing antibodies [ ] . indeed, based on the previous studies on the structural design of mers-cov rbd, non-neutralizing epitopes in the rbd can be masked, to refocus the immunogenicity of the rbd on the neutralizing epitopes, and thus to enhance its ability to confer immune protection [ ] . results from these structure-based studies will help to inform the design of innovative rbd-based anti-mers-cov vaccines and therapeutics with improved efficacy. isolation of a novel coronavirus from a man with pneumonia in saudi arabia fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea risks of death and severe disease in patients with middle east respiratory syndrome coronavirus impact of comorbidity on fatality rate of patients with middle east respiratory syndrome clinical determinants of the severity of middle east respiratory 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dromedary camels (camelus dromedaries) in laikipia county reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission unusual presentation of middle east respiratory syndrome coronavirus leading to a large outbreak in riyadh during outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study outbreak of middle east respiratory syndrome at tertiary care hospital transmission of middle east respiratory syndrome coronavirus infections in healthcare settings hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections family cluster of middle east respiratory syndrome coronavirus infections healthcare-associated infections: the hallmark of middle east respiratory syndrome coronavirus with review of the literature clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus persistence of antibodies against middle east respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase randomised, double-blind, single-dose-escalation study prospects for a mers-cov spike vaccine current advancements and potential strategies in the development of mers-cov vaccines is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sars-like pandemic? cov spike protein: a key target for antivirals genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus the endonucleolytic rna cleavage function of nsp of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines mers coronavirus nsp participates in an efficient propagation through a specific interaction with viral rna middle east respiratory syndrome coronavirus nonstructural protein is necessary for interferon resistance and viral pathogenesis structural and biochemical characterization of endoribonuclease nsp encoded by middle east respiratory syndrome coronavirus structural insights into the middle east respiratory syndrome coronavirus a protein and its dsrna binding mechanism middle east respiratory coronavirus accessory protein a inhibits pkr-mediated antiviral stress responses inhibition of stress granule formation by middle east respiratory syndrome coronavirus a accessory protein facilitates viral translation, leading to efficient virus replication sola, i. mers-cov b protein interferes with the nf-kappab-dependent innate immune response during infection proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein structure, function, and evolution of coronavirus spike proteins mers-cov spike protein: targets for vaccines and therapeutics dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus spike protein is not activated directly by cellular furin during viral entry into target cells receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase adaptive evolution of mers-cov to species variation in dpp middle east respiratory syndrome coronavirus and bat coronavirus hku both can utilize grp for attachment onto host cells identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein structure of mers-cov spike receptor-binding domain complexed with human receptor dpp molecular basis of binding between novel human coronavirus mers-cov and its receptor cd cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles novel chimeric virus-like particles vaccine displaying mers-cov receptor-binding domain induce specific humoral and cellular immune response in mice recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase (hdpp ) transgenic mice from mers-cov infection searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design receptor-binding domain-based subunit vaccines against mers-cov optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection engineering a stable cho cell line for the expression of a mers-coronavirus vaccine antigen recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies middle east respiratory syndrome: current status and future prospects for vaccine development evaluation of candidate vaccine approaches for mers-cov a novel human mab (mers-gd ) provides prophylactic and postexposure efficacy in mers-cov susceptible mice ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein chimeric camel/human heavy-chain antibodies protect against mers-cov infection a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov structural basis for the neutralization of mers-cov by a human monoclonal antibody mers- application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies(r) as inhaled biotherapeutics for lung diseases generation and characterization of alx- , a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection nanobodies as therapeutics: big opportunities for small antibodies nanobodies: natural single-domain antibodies vaccines for the prevention against the threat of mers-cov evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd during the south korean outbreak mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization combining a fusion inhibitory peptide targeting the mers-cov s protein hr domain and a neutralizing antibody specific for the s protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd structure-guided design of potent and permeable inhibitors of mers coronavirus cl protease that utilize a piperidine moiety as a novel design element this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this study was supported by the nsfc grant , and the nih grants r ai , r ai , and r ai . the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the authors declare no competing interests. key: cord- -jtx ni b authors: uyeki, timothy m.; erlandson, karl j.; korch, george; o’hara, michael; wathen, michael; hu-primmer, jean; hojvat, sally; stemmy, erik j.; donabedian, armen title: development of medical countermeasures to middle east respiratory syndrome coronavirus date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: jtx ni b preclinical development of and research on potential middle east respiratory syndrome coronavirus (mers-cov) medical countermeasures remain preliminary; advancements are needed before most countermeasures are ready to be tested in human clinical trials. research priorities include standardization of animal models and virus stocks for studying disease pathogenesis and efficacy of medical countermeasures; development of mers-cov diagnostics; improved access to nonhuman primates to support preclinical research; studies to better understand and control mers-cov disease, including vaccination studies in camels; and development of a standardized clinical trial protocol. partnering with clinical trial networks in affected countries to evaluate safety and efficacy of investigational therapeutics will strengthen efforts to identify successful medical countermeasures. f rom september through april , , a total of , laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) infections, leading to deaths ( % case-fatality proportion), had been reported to the world health organization (who) ( ) . most infections ( %) have been identified in saudi arabia ( ) . zoonotic transmission from exposure to mers-cov-infected arabian camels, known as dromedaries, or their raw milk and limited, nonsustained human-to-human transmission have been reported, including large outbreaks in healthcare facilities ( ) ( ) ( ) . the recovery of infectious mers-cov in virus cultures of specimens from bed sheets, bedrails, intravenous fluid hangers, and radiograph equipment indicates the potential for fomite transmission of the virus in hospitals providing care for mers-cov patients ( ) . however, sustained human-to-human transmission has not been documented, and some case-patients have no identified source of exposure to mers-cov. as of april , a total of countries had reported locally acquired or exported cases from the arabian peninsula, including cases in the united states identified during may in healthcare personnel who became ill after working in saudi arabia ( , ) . a traveler who visited saudi arabia, qatar, the united arab emirates, and bahrain and then returned to south korea infected with mers-cov in mid- triggered mers-cov cases, resulting in deaths in multiple health facilities and additional case in a person who traveled to china ( , ) . human infections with mers-cov are expected to continue to occur on the arabian peninsula because of the prevalence of mers-cov in dromedaries and the cultural importance of these camels (i.e., for food, milk, and racing purposes) in the region. during the outbreak of severe acute respiratory syndrome (sars) in china, civet cats, the suspected reservoir of sars coronavirus (sars-cov), were culled aggressively; no outbreaks were identified after . in contrast, culling of camels is culturally impractical in the middle east, and mers-cov zoonotic infections of humans have continued since . the potential for emergence of mers-cov mutations that could facilitate sustained community transmission and global dissemination cannot be predicted. no vaccines against or specific treatments for human infection with sars-cov, mers-cov, or other coronaviruses have been approved. since , efforts have focused on furthering development of animal models, vaccines, and therapies against mers-cov ( , ). in this report, we update the current state of development for mers-cov medical countermeasures, including regulatory challenges in the united states, and draw attention to areas in immediate need of increased infrastructure support for development of these countermeasures. persons at higher risk for more severe disease, including persons > years of age and those with chronic medical conditions. therapeutic drugs with specific activity against mers-cov (e.g., antiviral drugs, immunotherapeutic treatments) or that target the host immune response could be used for treatment of human illness caused by mers-cov infection or for pre-or postexposure prophylaxis. before human clinical trials of potential mers-cov medical countermeasures are started, proof-of-concept data must be obtained from in vivo studies of experimentally infected animals. such data may indicate a product's potential efficacy and provide a mechanism for selection of available medical countermeasure candidates. in addition, mers-cov vaccines could be developed for animals and used for vaccination of dromedaries on the arabian peninsula and in source countries for camel imports to the horn of africa to reduce mers-cov transmission among camels and possibly from camels to humans. preclinical development of mers-cov medical countermeasures has been hindered by several factors, including limited data on the natural history of mers-cov infection in humans; the lack of a small animal model that is naturally susceptible to mers-cov; and the inability to consistently replicate severe human disease in mers-cov-infected nonhuman primates (nhps). another factor is limited access to clinical samples and recent virus isolates; for example, a mers-cov strain isolated from a patient in , rather than a more recently isolated strain, is currently used by most investigators worldwide. small animal and nhp models are useful for testing potential medical countermeasures for efficacy (table ) . studies in mice, both dipeptidyl peptidase- (dpp or cluster differentiation ) transduced and transgenic, and in rabbits, hamsters, and ferrets have been reviewed elsewhere ( , , ) . these small animal models have been used for screening potential mers-cov medical countermeasures ( , , ) . the major nhp models under development include rhesus macaques and common marmosets ( , , ) . overall, common marmosets appear to be better suited than rhesus macaques for therapeutic studies designed to target severe disease because marmosets show slightly slower onset of illness and longer duration and severity of disease and their small size requires lower doses of therapeutic drugs. however, the marmoset model has not been standardized and is not consistent between laboratories ( , , ) . furthermore, the size of marmosets substantially limits sequential blood sampling for virologic or pharmacokinetic testing. challenges to the development of nhp models include determination and standardization of the optimal mers-cov challenge dose and of the volume and route of exposure, as well as the limited availability of nhps, especially marmosets. large animal models in development include camels and camelids such as alpacas ( , , ) . these models may be vital in understanding the virology and immunology of mers-cov infection in dromedaries, a natural host. in addition, serologic evidence of mers-cov infection in alpacas has been reported in qatar ( ) . major gaps for all animal models include a lack of consensus and availability of the optimal animal model to replicate severe human illness from mers-cov infection; limited availability of currently or recently circulating mers-cov strains; the lack of understanding of clinically relevant symptoms that can be incorporated into clinical scores or used as a signal to begin treatment in animal models; and competition for funding, laboratory space, availability of animals, and expertise with other emerging or reemerging infectious diseases, such as ebola virus disease and zika virus disease. ( , ) . the secretary of the us department of health and human services declared a potential public health emergency on may , , regarding mers-cov infection that could have a high potential to affect national security or the health and security of us citizens living abroad. the us food and drug administration (fda) subsequently issued an emergency use authorization to the centers for diseases control and prevention (cdc) for an in vitro molecular diagnostic test to diagnose mers-cov infection in multiple types of clinical specimens from symptomatic patients. the use of this test was later expanded to include the ability to test asymptomatic contacts of a person infected with mers-cov who traveled from saudi arabia to the united states. the cdc made this test available to multiple us public health laboratories, the us department of defense, and who laboratories worldwide. although the test has been distributed extensively, it is limited in terms of the cdc's ability to scale up the supply of reagents to support a surge in mers-cov cases in the united states and in other countries where the test has been made available. therefore, an emergency use authorization was issued on july , , for the commercially developed realstar mers-cov rt-pcr kit u.s. (altona diagnostics gmbh, hamburg, germany) for use in the in vitro qualitative detection of mers-cov rna in tracheal aspirate or tracheal secretion samples ( ) . although this commercial assay is a first step in bridging the diagnostic test availability gap in case of a surge scenario, the current coverage, at least in the united states, is insufficient until alternative, fda-cleared commercial tests are available (table ) . a worldwide gap exists in the lack of readily available, simple, rapid, and accurate diagnostic tests for use in outpatient and inpatient clinical settings where the ability of the facility to use currently available, higher complexity molecular tests is limited. the lack of commercial development of mers-cov assays may be partially related to the limited availability of clinical specimens and mers-cov isolates from infected patients. availability of serum specimens from rt-pcrconfirmed mers-cov patients who survived can help facilitate development of serologic tests. if paired acute and convalescent serum samples are available, serologic tests can be used to confirm mers-cov infection when viral shedding is not detectable, and for surveillance purposes such as measuring population exposures and immunity to mers-cov infection. no investigational therapeutic drugs have been evaluated for treatment of mers-cov patients in prospective randomized controlled clinical trials. potential therapeutic drugs for mers-cov patients include available approved drugs with nonspecific properties, such as immunomodulators, small-molecule drugs with broad antiviral activity, repurposed fda-approved small-molecule drugs that show activity against mers-cov in vitro (table ) ( , ) , and newly developed monoclonal or polyclonal antibody therapies with specific activity against mers-cov (table ) ( ). one promising approach has been to investigate libraries of drugs approved by the fda and the european medicines agency. considering development times and manufacturing requirements for new products, repurposing of existing drugs might potentially facilitate a rapid response to outbreaks of emerging viruses (see regulatory section for a discussion on repurposing). other early-stage work on mers-cov therapeutics includes studies focusing on the essential viral replication steps of fusion, proteolysis, and rna polymerization (table ) ( ) . immunotherapeutics under evaluation consist of convalescent plasma and monoclonal and polyclonal antibodies. most of the monoclonal antibodies in development have specific neutralizing activity against the mers-cov spike protein ( , ) . platforms are being developed to rapidly discover monoclonal antibodies, either from fully human convalescent blood or from transgenic animals, which can be manufactured on a large scale and are likely to have a good safety profile. the most advanced immunotherapeutic for mers-cov uses a transchromosomal bovine production system to produce fully human polyclonal mers-cov antibodies; a phase i study of this product was recently implemented ( ; https://clinicaltrials.gov/ct / show/nct ). preliminary results from immunoprophylaxis or treatment studies have shown efficacy of fully human monoclonal or polyclonal antibodies in mers-cov-infected mice and nhps ( profile and a defined set of preclinical toxicology studies, challenges to development of immunotherapeutics include ensuring the absence of antibody-dependent enhancement of disease and reducing the risk for generation of escape mutant viruses that would be resistant to treatment. development of mers-cov candidate vaccines was initiated by the national institute for allergy and infectious diseases at the national institutes of health, academic investigators, and several companies (table ). most candidate vaccines are still being evaluated in animal models. they have generally targeted the spike protein of mers-cov and are recombinant virus, subunit, dna, or virus-like vector vaccines ( , - ). one live-attenuated mers-cov candidate vaccine is in early development ( ) . preliminary studies for several other mers-cov vaccine candidates have been initiated, and early results demonstrate immunogenicity; have progressed to nhp challenge, and a phase clinical study in adults of different doses of a dna plasmid vaccine that expresses the mers-cov spike protein was started in january ( ) . ongoing assessment of antigenic evolution of circulating mers-cov strains is essential for informing vaccine development ( ) . a concern that must be addressed in the development of mers-cov vaccines is the potential for causing antibody-dependent enhancement of disease upon virus challenge, such as what was observed with a sars-cov candidate vaccine upon sars-cov challenge ( ) . the lack of a precedent of coronavirus vaccines for humans poses another challenge for the evaluation of mers-cov vaccines for humans, although vaccines against other animal coronaviruses are safe and in use in animals. considering the cultural importance of dromedaries on the arabian peninsula for meat, milk, and racing, prevention of camel-to-camel mers-cov transmission and reduction of spread from dromedaries to humans by camel vaccination is being investigated by government, academic, and commercial investigators (table ). young camels appear to be at high risk for mers-cov infection and could be a priority group for vaccination ( , ) ; the loss of maternal mers-cov antibodies ≈ - months after birth suggests a short time window for vaccination ( ) . a major challenge to this approach is that dromedaries can be reinfected with mers-cov; a study by farag et al. found no correlation between mers-cov rna levels and neutralizing antibodies in camels ( ) , suggesting that antibodies may not be protective against infection. because older camels can be reinfected, a camel vaccination strategy may require multiple dosing and booster vaccination to increase effective- in the united states and overseas ( ) . in addition, doses of a dna vaccine containing the mers-cov spike protein induced humoral immunity in dromedaries ( ) . in a recent study, a modified vaccinia virus ankara vaccine that expresses the mers-cov spike protein was administered intranasally and intramuscularly to dromedaries; when challenged intranasally with mers-cov, vaccinated dromedaries had fewer signs of respiratory infection and lower mers-cov titers in the upper respiratory tract compared with unvaccinated dromedaries ( ) . alpacas (new world camelids) are being investigated as a suitable proxy for camels because of the lack of available dromedaries in the united states, the high cost of acquiring dromedaries, and the relatively smaller size of alpacas ( , ) . regulatory considerations for mers-cov medical countermeasures in the united states are focused on a pathway to human clinical trials for drugs and vaccines through submission of investigational new drug applications. investigational new drug submissions must adhere to requirements set forth in the code of federal regulations, title , part ( cfr ; http://www.ecfr.gov/cgibin/text-idx?tpl=/ecfrbrowse/title / cfr _main_ . tpl). several guidance documents exist on the fda website related to virology, microbiology, pharmacology and toxicology, and clinical and medical considerations ( ). the most appropriate approval pathway is likely to be product-specific and will require consideration of existing product data, proposed intended use and population for use, and validated endpoints for efficacy predictive of clinical benefit, if any. likewise, data needed for consideration of an emergency use authorization, including dose finding and dose ranging, duration, and safety, can be obtained through sources such as investigational new drug clinical trials. repurposing of drugs approved by the fda for other illnesses for a mers-cov indication can potentially be expedited or accelerated if ) the mechanism of action for antiviral activity is defined, ) there is no change to the approved final drug form and route of administration, ) dosing does not exceed the currently approved dose and duration for the currently indicated population and adequate pharmacokinetics data support this dosing, and ) the risk-benefit profile is acceptable for the intended population and indication. for example, the risk-benefit profile for an approved drug with an oncology indication may be unacceptable if the drug is repurposed for administration to a healthy population for mers-cov postexposure prophylaxis. however, data requirements to initiate human trials will depend on the characteristics of the drug product and its intended use against mers-cov. as such, sponsors should consider prioritizing drug development on the basis of the totality of scientific evidence and merit of the drug alone, not on whether the drug has been previously approved. in the absence of a standardized and accepted animal model that simulates human disease from mers-cov infection, it is unclear how the fda may be able to expedite licensure or approval when data are lacking. the best approach may be collection of preclinical safety data and implementation of adaptive human clinical trials. this approach was taken for medical countermeasures in response to the - ebola virus disease outbreak. for diagnostic devices, the current emergency use authorization pathway serves as a fast approach to make products available for emergency public health purposes. after an emergency has been terminated, premarket notifications for these products should be submitted to fda for a more thorough evaluation as (k)s (http://www.fda.gov/ e emerging infectious diseases • www.cdc.gov/eid • vol. , no. , july medicaldevices/productsandmedicalprocedures/deviceap provalsandclearances/ kclearances/default.htm). the overarching goal for clinical research of mers-cov patients is to optimize clinical management and to identify effective therapies to improve survival. although clinical data on some mers-cov patients have been published in case series ( , , ) , there is a need for much more epidemiologic, clinical, virologic, and immunologic data to improve the limited understanding of the pathogenesis of mers-cov infection in humans. gaps include information on viral load and duration of viral shedding in blood, urine, respiratory, and other clinical specimens from infected persons; understanding of the innate and adaptive immune response to mers-cov infection; pathology data on the distribution of mers-cov in respiratory and extrapulmonary tissues in fatal cases; information from autopsies of persons who died of mers-cov; and an overall improved understanding of the pathogenesis of mers-cov in humans. only study has investigated mers-cov infection in autopsy tissues of a patient who died from the disease ( ) . collaborations are especially needed to pool and systematically collect serial clinical specimens from mers-cov patients for virologic, immunologic, and biomarker analyses to correlate with clinical illness, and to conduct long-term follow-up of survivors of severe disease ( ) ( ) ( ) . detailed understanding of host factors and cofactors associated with disease severity from asymptomatic infection to fatal illness is needed. efforts to promote international sharing of clinical specimens and mers-cov isolates are needed to foster development of diagnostics, therapeutics, and vaccines. use of standardized clinical data collection instruments and common biologic sampling protocols for serial prospective data collection will facilitate data pooling from mers-cov cases and comparisons across clinical sites and countries. global collaborations among clinical networks are also needed to implement clinical trials, preferably randomized controlled clinical trials, of mers-cov investigational therapeutics ( ) ( ) ( ) ( ) . without an international agreement on protocols and systematic standardization of case reporting and data collection methods, haphazard or anecdotal reporting and analysis of disease course and outcome may continue. who and the international severe acute respiratory and emerging infection consortium are collaborating in adapting standardized protocols for controlled clinical trials for mers-cov ( ) . prospective controlled clinical trials (ideally randomized clinical trials) of potential mers-cov therapies and vaccines in humans are needed urgently; however, there is uncertainty in estimating timelines for the development of potential mers-cov medical countermeasures because of the need to further characterize existing and new animal models, the unpredictability of demonstrating a favorable risk-benefit outcome during preclinical testing, and competition for resources with other emerging infectious diseases. in addition, the risk for antibody-dependent enhancement of disease may interrupt the timeline for conducting human clinical trials of mers cov vaccines and immunotherapeutics. researchers of all potential mers-cov medical countermeasures should have preclinical toxicology data available before initiating human clinical trials. although animal efficacy data are not technically required before implementing human clinical trials of potential countermeasures, such data are considered important for identifying the most promising medical countermeasure candidates, justifying risk in human volunteers, and informing the design of future clinical studies. timeframes for the production of specimen panels and repositories to aid commercial diagnostic development are also contingent on obtaining adequate funding and clinical samples. although preclinical development and research on potential mers-cov medical countermeasures has achieved appreciable progress to date, such development is preliminary, and substantive challenges must be overcome before most potential countermeasures are ready for human emerging infectious diseases • www.cdc.gov/eid • vol. , no. , july e results of substantial progress in 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management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance we thank john beigel, wendy carter, david cho, su-young choi, eric donaldson, heinz feldmann, barney graham, lisa hensley, cynthia kelley, peter miele, vincent munster, david spiro, kanta subbarao, and melissa willis for participating in foundational discussions and for help assessing potential therapeutic drugs and methods; vaccines under development or investigation; timelines for animal studies; scale-up of medical countermeasures; when human trials are to be started; regulatory issues for investigational new drug (ind) and emergency use authorization (eua) applications, and clinical studies; and safety issues and prioritization for clinical trials. we also thank dean erdman for insightful discussions on mers-cov diagnostic assay development, rick bright, tom dreier, and byron rippke for helpful commentary, and thuy phan for organizational assistance.dr. uyeki is the chief medical officer for the influenza division, national center for immunization and respiratory diseases, cdc. his research interests are in the epidemiology and clinical management of influenza and emerging infectious diseases. key: cord- -l kywzro authors: adney, danielle r.; van doremalen, neeltje; brown, vienna r.; bushmaker, trenton; scott, dana; de wit, emmie; bowen, richard a.; munster, vincent j. title: replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: l kywzro in , a novel coronavirus associated with severe respiratory disease in humans emerged in the middle east. epidemiologic investigations identified dromedary camels as the likely source of zoonotic transmission of middle east respiratory syndrome coronavirus (mers-cov). here we provide experimental support for camels as a reservoir for mers-cov. we inoculated adult camels with a human isolate of mers-cov and a transient, primarily upper respiratory tract infection developed in each of the animals. clinical signs of the mers-cov infection were benign, but each of the camels shed large quantities of virus from the upper respiratory tract. we detected infectious virus in nasal secretions through days postinoculation, and viral rna up to days postinoculation. the pattern of shedding and propensity for the upper respiratory tract infection in dromedary camels may help explain the lack of systemic illness among naturally infected camels and the means of efficient camel-to-camel and camel-to-human transmission. in , a novel coronavirus associated with severe respiratory disease in humans emerged in the middle east. epidemiologic investigations identified dromedary camels as the likely source of zoonotic transmission of middle east respiratory syndrome coronavirus (mers-cov). here we provide experimental support for camels as a reservoir for mers-cov. we inoculated adult camels with a human isolate of mers-cov and a transient, primarily upper respiratory tract infection developed in each of the animals. clinical signs of the mers-cov infection were benign, but each of the camels shed large quantities of virus from the upper respiratory tract. we detected infectious virus in nasal secretions through days postinoculation, and viral rna up to days postinoculation. the pattern of shedding and propensity for the upper respiratory tract infection in dromedary camels may help explain the lack of systemic illness among naturally infected camels and the means of efficient camel-to-camel and camel-tohuman transmission. t he middle east respiratory syndrome coronavirus (mers-cov) was first recognized in related to a fatal human case of pneumonia in saudi arabia ( ) . currently, > cases of mers have been identified, and the estimated case-fatality rate is ≈ % ( ) . most cases have been identified on the arabian peninsula, but several travel-associated cases have been reported ( ) ( ) ( ) . human-to-human transmission has been reported, predominantly among persons in health care facilities and households; the rate of human infection by zoonotic transmission from a reservoir source is currently not known ( ) ( ) ( ) . the close phylogenetic relationship of human mers-cov isolates with those obtained from bats initially suggested a direct link between the emergence of mers-cov and a putative natural reservoir ( ) ( ) ( ) . anecdotal reports mentioned contact of mers-cov-infected patients with camels and goats, suggesting that livestock might be the intermediate reservoir host for mers-cov ( , - ). serologic studies revealed widespread prevalence of mers-cov-specific antibodies in dromedary camels from several countries that reported mers cases ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . further, mers-cov rna was detected in nasal swab samples obtained from camels on a farm linked to human mers-cov cases, and the virus was isolated from nasal swab samples from dromedary camels in qatar ( ) . mers-cov isolation and subsequent full genome sequencing directly linked a dromedary camel and a fatal mers-cov case in a person in saudi arabia ( , ) . despite these associations, the role of camels as a primary reservoir for mers-cov is still debated ( , ) . here we report on the experimental inoculation of camels with a human isolate of mers-cov. mers-cov (strain hcov-emc/ ) was provided by the department of viroscience, erasmus medical center, rotterdam, the netherlands. the virus was propagated in vero e cells cultured in dulbecco modified eagle medium (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum, mmol/l glutamine, u/ml penicillin, and µg/ml streptomycin. three native-born adult male dromedary camels (camelus dromedarius) were obtained through private sale; the animals tested negative by neutralization assay for mers-cov and for bovine coronavirus by elisa. camels , , and were , , and years old, respectively. camels and were intact males, and camel had been castrated. animals were housed in an animal biosafety level facility for the duration of the experiment and fed ad libitum. camels were acclimated to the facility for weeks before virus inoculation. we sedated the camels with xylazine, then inoculated them with a total dose of % tissue culture infective dose (tcid ) of mers-cov (strain hcov-emc/ ) in a total volume of ml, by way of intratracheal ( ml using transcutaneous catheter), intranasal ( . ml in each nostril by expulsion from a syringe), and conjunctival ( . ml in each conjunctival sac) routes. the routes of inoculation and infectious dose were chosen to reflect a combination of most likely routes of exposure and to increase the potential of infection. the animals were observed at least × daily for the duration of the experiment for behavior, food consumption, activity level, and nasal discharge. rectal temperature was taken daily from to days postinoculation, then × weekly until the animals were euthanized. nasal and oral swab samples and fecal samples were collected into virus transport medium or virus lysis buffer daily from to days postinoculation (dpi), then × weekly until the animal was euthanized. blood was collected into evacuated edta and serum-separating tubes daily at - dpi and × weekly thereafter. urine was collected by convenience and at necropsy. to evaluate whether virus is exhaled from infected camels, a funnel was placed over the muzzle of each camel and connected to a vacuum pump to capture exhaled air in tissue culture media ( ml dulbecco modified eagle medium, % fetal bovine serum, . % se- (anti-foam) with an all glass impinger (ace glass inc., vineland, nj, usa). exhaled breath was collected for ≈ minutes and analyzed by quantitative real-time pcr (qpcr) and virus titration. on days , , and , camels , , and , respectively, were euthanized, and samples were collected from nasal turbinates, lungs, trachea, larynx, pharynx, liver, spleen, kidney, bladder, urine, duodenum, jejunum, colon, rectum, abomasum, forestomachs, prescapular lymph node, retropharyngeal lymph node, tracheobronchial lymph node, mediastinal lymph node, mesenteric lymph node, medulla, and olfactory cortex. we extracted rna from swab samples, fecal samples, and serum samples using the qiaamp viral rna kit (qia-gen, valencia, ca, usa) according to the manufacturer's instructions. for detection of viral rna, we used ml of rna in a one-step real-time reverse transcription pcr upe assay ( ) using the rotor-genetm probe kit (qiagen) according to manufacturer's instructions. standard dilutions of a titered virus stock were run in parallel, to calculate tcid equivalents in the samples ( ) . we titrated swab samples in viral transport medium, whole blood, and homogenized tissues (≈ % wt/vol) for mers-cov virus by plaque assay. briefly, -fold serial dilutions of samples were prepared in ba- medium (mem, % bovine serum albumin, mg/l sodium bicarbonate, mm tris, ph . , mg/l phenol red) containing mg gentamicin, , u penicillin g, mg streptomycin, and mg amphotericin/l; plaque assay was conducted as horizontal lines indicate the normal temperature range observed among these dromedary camels as calculated by mean ± ×, the sd before inoculation. described for west nile virus ( ) . plaques were counted on days and after the second overlay and virus titers were expressed as pfus per ml or gram. we determined neutralizing antibody titers by plaque reduction neutralization test as described ( ), using a % neutralization cutoff. tissues were fixed for > days in % neutral-buffered formalin and embedded in paraffin. tissue sections were stained with hematoxylin and eosin. to detect mers-cov antigen, we completed immunohistochemical testing using a rabbit polyclonal antiserum against hcov-emc/ ( : , ) as a primary antibody. each camel showed minor clinical signs of disease, consisting of rhinorrhea (figure , panel a) and a mild elevation in body temperature at dpi and - dpi ( figure , panel b) ; no other clinical signs were observed. rhinorrhea developed in all camels beginning at (camels and ) and (camel ) dpi, and persisted < weeks. the nasal discharge drained from both nares and varied in character from serous to purulent; minor hemorrhage was observed on some occasions, but may have been caused by trauma that occurred during collection of samples. mers-cov shedding started during - dpi, as detected by the presence of infectious virus and viral rna by qpcr in nasal swab samples. infectious virus shedding was detected < dpi, and shedding of viral rna was detected < dpi in nasal swab samples (figure ). low concentrations of infectious virus and viral rna were detected in oral samples, likely originating in drainage from the nasal cavity ( figure ) . no viral rna was detected in fecal samples or in urine samples collected by convenience or at necropsy at , , , , , , and dpi from the camels. no infectious virus or viral rna was detected in any of the serum or whole blood samples. small quantities of mers-cov rna were detected in exhaled breath by qpcr ( . and . tcid equivalent/ml) at and dpi, but infectious virus was not detected. infectious virus was detected in tissues from camel , which was euthanized on dpi, but not in tissues obtained from camels and , which were euthanized at and dpi, respectively. infectious virus was detected in tissues of the upper respiratory tract (urt), including nasal turbinates, olfactory epithelium, pharynx, and larynx. in the lower respiratory tract, infectious virus was detected in the trachea and in of lung lobes tested. infectious virus was also detected in the retropharyngeal, mediastinal, mesenteric, and tracheobronchial lymph nodes (figure ). on necropsy of camel at dpi, histologic lesions were found in the pseudostratified epithelial cells in the urt and the lower respiratory tract (trachea, bronchi, and bronchioles) but not in the alveoli ( figure ) . the lesions were characterized as mild to moderate acute intraepithelial and submucosal inflammation with multifocal necrosis and loss of pseudostratified epithelial cells, comparable to the common cold among humans. multifocal loss of epithelial polarity and cilia with squamous metaplasia were observed. the epithelium was infiltrated by small-to-moderate numbers of neutrophils with fewer macrophages; similar inflammatory cells also permeated the submucosa. the submucosal glands of the trachea were multifocally necrotic and infiltrated by small numbers of neutrophils. viral antigen was detected within the epithelial cells of the nasal turbinates, larynx, trachea, bronchi, and bronchioles, but not the alveoli. in addition, viral antigen was present at the follicular mantle zone of the tonsils and mediastinal and retropharyncheal lymph nodes ( figure ). the nasal turbinates, larynx, and trachea of camel (necropsied at dpi) had similar but milder lesions when compared with those of camel . the nasal turbinate, larynx, and bronchus showed small numbers of infiltrating neutrophils; however, in contrast with the condition of camel , the cilia and goblet cells were intact. the remainder of the respiratory tract of camel was unaffected. immunohistochemical testing revealed the presence of limited viral antigen in the nasal turbinate but not in any of the other tissues at that time. no lesions or viral antigens were detected in camel at dpi. serum samples were collected weekly from the camels to monitor the generation of neutralizing antibodies specific to mers cov. each of the camels was seronegative before inoculation. robust mers-cov specific antibody responses developed in camels and (euthanized on and dpi, respectively), detected first on dpi with a plaque-reduction neutralization test titer from to that increased to at dpi (table) . camel was euthanized at dpi and was not tested for development of antibodies against the virus. tissues were collected at days postinoculation (dpi) for camel , dpi for vamel and dpi for camel . detectable infectious virus in the collected tissues was found only in camel . nasal turbinates were sampled in different sections: anterior, medial, and posterior. infectious titers were determined by plaque assay. ln, lymph node. epidemiologic and surveillance data on the emergence of mers-cov strongly point toward a role for dromedary camels as a reservoir for zoonotic transmission ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , ) . to understand the ecology of mers-cov in the most likely reservoir host, we experimentally inoculated young adult dromedary camels with mers cov. the disease observed was clinically benign, in agreement with the absence of overt illness reported from field surveillance studies ( , , , ) . a large quantity of mers-cov and viral rna was detected in nasal swab specimens from each of the camels. infectious virus was detected through dpi, and rna was detected through dpi in camel , which was euthanized on day . this route of shedding is consistent with data on naturally infected camels ( , , , , ) , and the pattern of shedding suggests that the infectious period of camels may be short. mers-cov was not detected in either urine or feces, again consistent with field observations ( , ) . the large quantities of mers-cov shed in nasal secretions by each of the camels suggest that camel-tocamel and camel-to-human transmission may occur readily through direct contact and large droplet, or possibly fomite transmission. histopathologic examination revealed that the urt, specifically the respiratory epithelium in the nasal turbinates, is the predominant site of mers-cov replication in camels. neutralizing antibodies were detected from dpi onward, reaching a maximum neutralizing titer of after days. serologic studies in camels in the field have reported mers-cov neutralizing titers as high as , ( , ) , potentially indicative of repeated exposure and re-infection. the study reported here was done on the basis of inoculation of male animals with a human isolate of mers-cov, and the study design we used imposed several limitations on how these data inform what occurs in natural infections. the camels we inoculated were exposed to a high dose of virus by simultaneous routes of inoculation. in retrospect, the inoculation dose does not seem excessive, based on the large quantity of virus shed nasally in all animals ( figure ). the total dose inoculated was relatively equivalent to the amount of virus present in a single nasal swab sample taken during the first days postinoculation, and it seems probable that a camel shedding this quantity of virus would readily infect other camels or humans with which it had direct contact. the fact that we inoculated the camels with the virus by routes precludes drawing conclusions regarding efficiency of transmission by a particular route, which is a topic that should be addressed in future studies. the influence of camel age on susceptibility and dynamics of virus shedding is another notable parameter that requires further study. it seems likely that productive infection and shedding of virus in natural settings occurs predominantly in juvenile camels ( ) . this could be the result of an intrinsic difference in age-related susceptibility, but is more likely related to the immunologically naïve status of the animals in the context of a high force of infection after decay of passively acquired antibodies. the animals we infected were young adults, but were seronegative and therefore probably as susceptible as juveniles from mers-cov-endemic regions. another aspect of pathogenesis not addressed here is whether virus is present in milk or meat from infected camels and thereby poses another potential route of exposure to humans who consume such products. despite these limitations, the magnitude and pattern of virus shedding was essentially identical in all animals and supports the available epidemiologic data indicating that camels are likely a major reservoir host for mers-cov. additional experimental and field studies are clearly required to address the duration of shedding of infectious mers-cov from infected camels, to determine whether infection results in protective immunity, and to clarify the burden of illness among humans resulting from transmission from camels. emerging infectious diseases • www.cdc.gov/eid • vol. , no. , december isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. global alert and response. middle east respiratory syndrome coronavirus (mers-cov)-update investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in florence the emergence of the middle east respiratory syndrome coronavirus hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections genomic characterization of a newly 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coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through wholegenome analysis and virus cultured from dromedary camels in saudi arabia we thank bart haagmans and ron fouchier, for providing mers-cov (isolate hcov-emc/ ); tina thomas, dan long, and rebecca rosenke for histopathologic examination; and anita mora and ryan kissinger for figure preparation. all animal work in this study was approved by the institutional animal care and use committee of colorado state university and was performed in compliance with recommendations in the guide for the care and use of laboratory animals of the national institute of health.ms adney is a graduate student at colorado state university in fort collins, colorado. her research focus is on the pathogenesis of emerging infectious diseases. key: cord- -mwj qby authors: mackay, ian m.; arden, katherine e. title: mers coronavirus: diagnostics, epidemiology and transmission date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: mwj qby the first known cases of middle east respiratory syndrome (mers), associated with infection by a novel coronavirus (cov), occurred in in jordan but were reported retrospectively. the case first to be publicly reported was from jeddah, in the kingdom of saudi arabia (ksa). since then, mers-cov sequences have been found in a bat and in many dromedary camels (dc). mers-cov is enzootic in dc across the arabian peninsula and in parts of africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. the ksa is the focal point of mers, with the majority of human cases. in humans, mers is mostly known as a lower respiratory tract (lrt) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in % to % of those infected. however, mers-cov has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. older males most obviously suffer severe disease and mers patients often have comorbidities. compared to severe acute respiratory syndrome (sars), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, mers progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. most human cases of mers have been linked to lapses in infection prevention and control (ipc) in healthcare settings, with approximately % of all virus detections reported among healthcare workers (hcws) and higher exposures in those with occupations that bring them into close contact with camels. sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics have been available almost from the start of the emergence of mers. while the basic virology of mers-cov has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. an email from dr ali mohamed zaki, an egyptian virologist working at the dr soliman fakeeh hospital in jeddah in the kingdom of saudi arabia (ksa) announced the first culture of a new coronavirus to the world. the email was published on the website of the professional emerging diseases (promed) network on th september [ ] (fig. ) and described the first reported case, a year old man from bisha in the ksa. this information led to the rapid discovery of a second case of the virus, this time in an ill patient in the united kingdom, who had been transferred from qatar for care [ ] . the new virus was initially called novel coronavirus (ncov) and subsequentlty entitled the middle east respiratoy syndrome coronavirus (mers-cov). as of nd of september , there have been , detections of viral rna or virus-specific antibodies across countries (additional file : figure s ) confirmed by the world health organization (who), with over a third of the positive people dying (at least , %) [ ] . since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over % of adult dromedary camels (dc; camelus dromedarius) in the ksa [ ] , also dcs across the arabian peninsula and parts of africa that are a source of dc imports for the ksa [ ] . to date, mers-cov has not been detected in dcs tested in zoos or herds from other parts of the world [ ] [ ] [ ] [ ] . occasionally, virus is transmitted from infected dcs to exposed humans. subsequent transmission to other humans requires relatively close and prolonged exposure [ ] . the first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [ , ] . however, sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [ ] . subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase (dpp ; also called cd ); that mers-cov has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than sars-cov; is widespread in dcs; has the potential to infect other animals and that mers kills its human host more often than sars did ( - % versus % for sars [ ] ) [ ] [ ] [ ] [ ] [ ] . in humans, overt disease was given the name middle east respiratory syndrome, with the acronym mers. from intermittent animal-to-human spill-over events, the mers-cov spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. the spread of mers-cov among humans has often been associated with outbreaks in hospitals, with around % of all cases to date involving healthcare workers (hcws). although dcs appear to suffer the equivalent of a 'common cold' from mers-cov infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to % of reported cases. it has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [ ] . studies have established that the mean incubation period for mers is five to six days, ranging from two to days, with to days between when illness begins in one person and subsequently spreads to another [ ] [ ] [ ] [ ] . among those with progressive illness, the median time to death is to days, ranging from five to days [ , ] . fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [ , ] . the first who case definition [ ] defined probable cases of mers based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (lrt) involvement. it also included roles for contact with a probable or confirmed case or for travel or residence within the arabian peninsula. if strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [ ] . from july , the revised who case definition included the importance of seeking out and understanding the role of asymptomatic cases and from june , the who definition more clearly stated that a confirmed case included any person whose sample was rt-pcr positive for mers-cov, or who produced a seroconversion, irrespective of clinical signs and symptoms. [ ] [ ] [ ] apart from the who and the ksa ministry of health reports, asymptomatic or subclinical cases of mers-cov infection were documented in the scientific literature although not always as often as occurred early on [ , ] . the ksa definition of a case became more strict on th may , relying on the presence of both clinical features and laboratory confirmation [ ] . testing of asymptomatic people was recommended against from december [ ] , reinforced by a case definition released by the ksa ministry of health in june [ ] . the ksa has been the source of % of human cases. severe mers is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [ ] [ ] [ ] . interestingly in june , an outbreak in south korea followed a similar distribution [ , ] . among laboratory confirmed cases, fever, cough and upper respiratory tract (urt) signs and symptoms usually occur first, followed within a week by progressive lrt distress and lymphopaenia [ ] . patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [ , ] . disease can progress to acute respiratory distress syndrome and multiorgan system failure [ ] . mers has reportedly killed approximately % of all reported cases, % of cases in the ksa, yet only % of cases in south korea, where mortality ranged from % among younger age groups to % among those aged years and above [ ] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [ ] . general supportive care is key to managing severe cases [ ] . children under the age of years are rarely reported to be positive for mers-cov, comprising only . % (n = ) of total reported cases. between st september and nd december , a study described the then tally of paediatric cases in the ksa, which stood at (two to years of age; median years); nine were asymptomatic ( %) and one infant died [ ] . in amman, jordan, , samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for mers-cov rna, despite being collected at a similar time to the first known outbreak of mers-cov in the neighbouring town of al-zarqa [ ] . a second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not rt-rtpcr positive, the mother did subsequently develop antibodies to mers-cov, suggestive of recent infection [ ] . her exposure history to a mers-cov rt-rtpcr positive relative and an antibody-reactive husband, her incubation period and her symptom history met the who criteria for being a probable mers-cov case [ ] . diagnostic methods were published within days of the promed email announcing the first mers case [ ] , including several now gold standard in-house rt-rtpcr assays (fig. ) as well as virus culture in vero and llc-mk cells [ , , ] . a colorectal adenocarcinoma (caco- ) epithelial cell line has since been recommended for isolation of infections mers-cov [ ] . we previously [ ] .). open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (utr; grey rectangles). fs-frame-shift. predicted regions encompassing recombination break-points are indicated by orange pills. created using geneious v . [ ] and annotated using adobe illustrator. beneath this is a schematic depicting the location of rt-pcr primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest rt-rtpcr screening assays and conventional, semi-nested (three primers) rt-pcr confirmatory sequencing assays [ , ] . publication order is noted by first [ th september ; red] and second [ th december ; orange] coloured rectangles; both from corman et al. [ , ] those assays recommended by the who are highlighted underneath by yellow dots [ ] . the nseq reverse primer has consistently contained one sequence mismatch with some mers-cov variants. an altered version of that from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res vol : - with permission from elsevier [ ] reviewed the broad tropism of mers-cov [ ] . however, as is well described, cell culture is a slow, specialised and insensitive method [ ] while pcr-based techniques are the preferred method for mers-cov detection. the first open reading frames (orf a and b; fig. ) have become a key diagnostic and taxonomic target for cov species identification. with less than % identity between the amino acid sequence of mers orf ab and betacoronavirus relatives, tylonycteris bat hku and pipistrellus bat hku , it can be concluded that it is a novel and distinct virus. mers-cov is predicted to encode ten open reading frames with ' and ' untranslated regions [ ] . the structural proteins include the spike (s), envelope (e), membrane (m) and nucleocapsid (n) [ ] . the products of orf a and orf b are predicted to encode nonstructural proteins. the majority of specimen testing to date has employed validated rt-rtpcr assays shown to be sensitive and specific [ , , ] . the realstar® kit uses these whorecommended assays [ ] . the target sequences of these screening assays have not changed among genomes examined until at least mid- (imm observation). other rt-rtpcr assays have been developed and validated for use as laboratory-based diagnostic tools [ ] [ ] [ ] . additionally, loop-mediated [ , ] or recombinase polymerase [ ] isothermal assays have been designed for field deployment. the detection of mers-cov antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. detection of viral proteins rather than viral rna indicates the likely presence of infectious virus. the first rapid immunochromatographic tool described could detect recombinant mers-cov nucleocapsid protein from dc nasal swabs with % sensitivity and % specificity compared to rt-rtpcr [ ] . a different approach used a monoclonal antibody-based capture elisa targeting the mers-cov nucleocapsid protein with a sensitivity of tcid and % specificity [ ] . demonstration of a seroconversion to a mers-cov infection meets the current who definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior mers-cov infection and help support transmission studies. because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral rna [ ] . strategic, widespread sero-surveys of humans using samples collected after are infrequent. much of the arabian peninsula and all of the horn of africa lack baseline data describing the proportion of the community who may have been infected by a mers-cov. however, sero-surveys have had widespread use in elucidating the role of dcs as a transmission source for mers-cov. because of the identity shared between dc and human mers-cov (see molecular epidemiology: using genomes to understand outbreaks), serological assays for dc sero-surveys should be transferrable to human screening with minimal re-configuration. also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested mers-cov isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single mers-cov serotype [ ] . the development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to mers-cov, as well as to likely sources of cross-reaction [ ] . obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [ ] . a number of commercial elisa kits, immunofluorescent assays (ifa) kits, recombinant proteins and monoclonal antibodies have been released [ , [ ] [ ] [ ] [ ] . initially, conventional ifas were used for human sero-surveys. these relied on mers-cov-infected cell culture as an antigen source, detecting the presence of human anti-mers-cov igg, igm or neutralizing antibodies in human samples [ , , ] . no sign of mers-cov antibodies was found among , sera from patients visiting hospital in jeddah, from through , prior to the description of mers-cov [ ] . nor did ifa methods detect any sign of prior mers-cov infection among a small sample of healthy blood donors from another hospital in jeddah (collected between jan and dec ) [ ] . of slaughterhouse workers, only eight ( . %) were positive by ifa, and those sera could not be confirmed by virus neutralization (nt) test. the study indicated that hcov-hku was a likely source of crossreactive antigen in the whole virus ifa [ ] . whole virus mers-cov ifa also suffered from some cross-reactivity with convalescent sars patient sera and this could not be resolved by an nt test which was also cross-reactive [ ] . ifa using recombinant proteins instead of whole-virus ifa, has been shown to be a more specific tool [ ] . since asymptomatic zoonoses have been posited [ ] , an absence of antibodies to mers-cov among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering dcs [ ] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. ifa using recombinant proteins instead. some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the mers-cov nucleocapsid and spike proteins [ , ] , or using a recombinant lentivirus expressing mers-cov spike protein and luciferase [ , ] . a pseudo particle neutralization (ppnt) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (mnt) test. [ , , [ ] [ ] [ ] ] studies using small sample numbers and ppnt found no evidence of mers-cov neutralizing antibody in sera from children with lrt infections between may and may , sera from to year old male blood donors and selfidentified animal workers from the jazan region of the ksa during [ , ] . similarly, a study of four herdsmen in contact with an infected dc herd in al-ahsa, eight people who had intermittent contact with the herd, veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in al-ahsa and controls who were not exposed to dcs in any professional role, found none with serological evidence of past mers-cov infection using the ppnt assay [ ] . a delay in the neutralizing antibody response to mers-cov infection was associated with increased disease severity in south korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [ ] . the implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. a jordanian outbreak of acute lrt disease in a hospital in was retrospectively found to be associated with mers-cov infection, initially using rt-rtpcr, but subsequently, and on a larger scale, through positivity by elisa and ifa or mnt test. [ , , ] this outbreak predated the first case of mers in the ksa. the elisa used a recombinant nucleocapsid protein from the group betacoronavirus bat-cov hku to identify antibodies against the equivalent crossreactive mers-cov protein [ ] . it was validated using sera collected from people with prior hcov-oc , hcov- e, sars-cov, hcov-nl , hrv, hmpv or influenza a(h n ) infections but was reportedly less specific than the recombinant ifa discussed above. it was still considered an applicable tool for screening large sample numbers [ ] . a protein microarray expressing the s protein subunit has also been validated and widely used for dc testing [ , ] . detection of mers-cov infection using elisa or s subunit protein microarray [ ] is usually followed by confirmatory ifa and/ or a plaque-reduction neutralization (prnt) [ , , ] or mnt test. [ , , ] this confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in dcs (bovine cov, bcov) or humans (hcov-oc , hcov- e, hcov-nl , hcov-hku , sars-cov). in the largest study of human sera, a tiered diagnostic process assigned both recombinant ifa and recombinant elisa positive sera to 'stage ' seropositivity. a stage seropositive result additionally required a suitably titred prnt result [ ] . the study found sera collected in to from , ( . %) people in ksa provinces contained mers-cov antibodies, but significantly higher proportions in occurred in camel shepherds (two of ; . %) and slaughterhouse workers (five of ; . %) [ ] . contemporary surveys are needed. mers-cov does not appear to be easily transmitted from dcs to humans, or perhaps it is [ ] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. this was reinforced when a false positive us case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, nt assay and was subsequently retracted [ , ] . the who recommends sampling from the lrt for mers-cov rt-rtpcr testing, especially when sample collection is delayed by a week or more after onset of symptoms. [ ] lrt samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [ ] . recommended sample types include bronchoalveolar lavage (bal), tracheal/tracheobronchial aspirate, pleural fluid and sputum [ , ] . fresh samples yield better diagnostic results than refrigerated material [ ] and if delays in testing of ≥ h are likely, samples (except for blood) should be frozen at − °c [ ] . if available, lung biopsy or autopsy tissues can also be tested [ ] . the urt is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when urt sampling is to be conducted [ ] . paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first - days if conducting rt-rtpcr [ , ] . human urine and stool have been found to contain mers-cov rna to days after symptom onset [ , , ] and are listed as samples that should be considered [ , ] . in two cases that arrived in the netherlands, urine was rt-rtpcr negative but faeces was weakly positive and sera were rt-rtpcr positive for five days or more [ ] . the finding of mers-cov viral rna in serum provides an avenue for retrospective pcr-based studies if respiratory samples are unavailable [ ] . rnaaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [ ] . clinically suspected mers cases may return negative results by rt-rtpcr. data have shown one or more negative urt samples may be contradicted by further urt sampling or the use of lrt samples, which is preferred [ , , ] . higher viral loads occur in the lrt compared to the urt. [ , , , ] this fits with the observation that the majority of disease symptoms are reported to manifest as systemic and lrt disease [ ] . however, on occasion, even lrt specimens from mers cases may initially be negative, only to later become positive by rt-pcr [ ] . this may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [ ] . despite this both the largest human mers-cov studies [ , [ ] [ ] [ ] and smaller ones [ , , ] , use samples from the urt. it is then noteworthy that one study reported an association between higher loads in the urt and worse clinical outcome including intensive care and death [ ] . at writing, no human data exist to define whether the virus replicates solely or preferentially in the lrt or urt, or replicates in other human tissues in vivo although mers-cov rna has been detected from both the urt and lrt in a macaque monkey model [ ] .the distribution of dpp in the human upper airways is also not well described. individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [ , , , ] in one instance, a hcw shed viral rna for days in the absence of disease [ ] . it is an area of high priority to better understand whether such cases are able to infect others. over three quarters of mers cases shed viral rna in their lrt specimens (tracheal aspirates and sputum) for at least days, while only % of contacts were still shedding rna in their urt specimens [ , ] . in the only study to examine the effect of sample type on molecular analysis, nasopharyngeal aspirates (npa; an urt sample), tracheal aspirates, sputa and three bal were examined. the tracheal aspirates and bal returned the highest viral load values followed by npa and sputum. unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in npa testing, were significantly correlated with severe disease and death [ , , ] . this study demonstrated the importance of lrt sampling for whole genome sequencing. when tested, samples positive for mers-cov are often negative for other pathogens [ , , , ] . however, many studies make no mention of additional testing for endemic human respiratory viruses [ , , , ] . when viruses are sought, they have included human herpesvirus (hhv), rhinoviruses (hrv), enteroviruses (ev), respiratory syncytial virus (rsv), parainfluenzavirus types , and (pivs),influenzaviruses (ifvs), endemic hcovs, adenoviruses (advs) metapneumovirus (mpv) and influenza a\h n virus; co-detections with mers-cov have been found on occasion [ , , , , ] . bacterial testing is sometimes included (for example, for legionella and pneumococcus) but the impact of bacterial co-presence is also unclear [ , [ ] [ ] [ ] . further testing of the lrt sample from the first mers case used ifa to screen for some viruses (negative for ifv, pivs, rsv and advs) and rt-pcr for others (negative for adv, evs, mpv and hhvs) [ ] . rt-pcr also detected mers-cov. the who strongly recommends testing for other respiratory pathogens [ ] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both mers cases and their contacts. little is known of other causes of mers-like pneumonia in the ksa or of the general burden of disease due to the known classical respiratory viruses. testing of adult pilgrims performing the hajj in to has not detected any mers-cov. in , nasal swabs from pilgrims collected prior to leaving for or departing from the ksa were tested [ ] . in , testing was significantly scaled up with , nasopharyngeal swabs from , incoming pilgrims and , swabs from outgoing pilgrims tested [ ] . it should be noted that most pilgrims arrived from mers-free countries. a further swabs were taken from pilgrims with influenza-like illness [ , ] . in earlier hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [ ] [ ] [ ] over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among hajj pilgrims. [ ] a lrt sample is often not collected for these studies [ , , ] , so false negative findings are a possibility although little is known about the initial site of mers-cov infection and replication; it may have been assumed it was the lrt because disease was first noticed there but the urt may be the site of the earliest replication. in jeddah between march and july (hereafter called the jeddah- outbreak; fig. ), there was a rapid increase in mers cases, accompanied by intense screening; approximately , samples from in and around the region were tested in a month yielding around mers-cov detections (~ % prevalence) [ ] . among , individuals sampled and tested across the ksa between october and september , ( . %) detections were made in a hospital-centric population which included hospitalized cases (n = , ; . %), their families (n = ; . %) and associated hcws (n = , ; . %) [ ] . among the detections, ( . %) were hcws and ( . %) were family contacts [ ] . the - % prevalence of active mers-cov infections is not dissimilar to the hospital-based prevalence of other human covs. [ ] however, the proportion of deaths among those infected with mers-cov is much higher than that known for the hcovs nl , hku , e or oc in other countries, and even above that for sars-cov; it is not a virus that could reasonably be described as a "storm in a teacup". it is the low transmission rate that has prevented worldwide spread, despite many "opportunities". very early in the mers outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of mers-cov with three of the first five cases having contact with dcs [ , , ] . today, animal mers-cov infections must be reported to the world organization for animal health as an emerging disease [ ] . a summary of the first mers cases reported by the who defined animal contact with humans as being direct and within days prior to symptom onset [ ] . this definition made no specific allowance for acquisition from dcs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [ ] . camels are known to produce high levels of mers-cov rna in their urt and lungs [ ] . providing support for a droplet transmission route and perhaps indicating the presence of rna in smaller, drier droplet nuclei, mers-cov rna was identified in a high volume air sample collected from a barn housing an infected dc [ ] . the precise source from which humans acquire mers-cov remains poorly studied but it seems likely that animal and human behavioural factors may play roles (fig. ) [ ] . these factors may prove important for human cases who do not describe any dc contact [ ] nor any contact with a confirmed case. whether the who definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. wording focuses on consumption of dc products but does not specifically ascribe risk to a droplet route for acquisition of mers-cov from dc [ ] . some mers patients are listed in who disease notices as being in proximity to dcs or farms, but the individuals have not described coming into contact with the animals. no alternative path for acquiring infection is reported in many of these instances. what constitutes a definition of "contact" during these interviews has been defined for one study [ ] . despite this lack of clarity, the who consider that evidence linking mers-cov transmission between dcs to humans is irrefutable (fig. ) [ ] . the possibility that bats were an animal host of mers-cov was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [ ] [ ] [ ] [ ] . conclusive evidence supporting bats as a source for human infections by mers-cov has yet to be found, but bats do appear to host ancestral representatives [ , ] . however, these are not variants of the same virus nor always within the same phylogenetic lineage as mers-cov; they are each a genetically distinct virus. bat-to-human infection by mers-cov is a purely speculative event. the only piece of mers-cov-specific evidence pointing to bats originates from amplification of a nt fragment of the rnadependent rna polymerase gene of the mers-cov genome, identified in a faecal pellet from an insectivorous emballonuridae bat, taphozous perforatus found in bisha, the ksa [ ] . while very short, the sequence of the fragment defined it as a diagnostic discovery. subsequently a link to dcs was reported [ ] and that link has matured into a verified association [ , ] (fig. ) . (see figure on previous page.) fig. monthly detections of mers-cov (blue bars) and of cases who died (red bars) with some dates of interest marked for to th september . an approximation of when dc calving season [ ] and when recently born dcs are weaned is indicated. spring (green) and summer (orange) in the arabian peninsula are also shaded. note the left-hand y-axis scale for and which is greater than for / . sources of these public data include the who, ministries of health and flutrackers [ ] [ ] [ ] . earlier and subsequent versions of this chart are maintained on a personal blog [ ] . modified and reprinted from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res vol : - with permission from elsevier [ ] dcs, which make up % of all camels, have a central presence in the arabian peninsula where human-dc contact ranges from little to close [ ] . contact may be commonplace and could occur in variety of ways (fig. a) . there are several large well-attended festivals, races, sales and parades which feature dcs and dcs are also kept and bred close to populated areas in the ksa [ , ] . dc milk and meat are widely consumed and the older dc is an animal of ritual significance after the hajj pilgrimage [ ] . however, mers-cov infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing dc products. daily ingestion of fresh unpasteurized dc milk is common among the desert bedouin and many others in the ksa. dc urine is also consumed or used for supposed health benefits. despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among mers cases; this may simply be a reporting issue rather than an unexplainable absence of mers. a small case-control study published in identified direct dc contact, and not ingestion of products, to be associated with onset of mers [ ] . the first sero-survey of livestock living in the middle east region was conducted during - [ ] . dcs were sampled from a mostly canary island-born herd and from omani dcs (originally imported from the horn of africa) [ ] . a neutralising antibody assay found only % of strongly seropositive canary island [ ] . b camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor ipc in healthcare settings where transmission is amplified, accounting for the bulk of cases. there are human mers cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) mers-cov infection may be implicated in transmission dc sera could neutralise mers-cov while all omani dc sera had high levels of specific mers-cov neutralizing antibody [ ] . this indicated that dcs had in the past been infected by mers-cov, or a very similar virus. since this study, a host of peer-reviewed reports have looked at both dcs and other animals, and the possibility that they may host mers-cov infection. seropositive dcs have been found throughout the arabian peninsula including oman, the ksa, qatar, jordan, the united arab emirates (uae), kuwait as well as sudan, somalia, egypt, tunisia, nigeria, kenya and ethiopia in africa and the canary islands [ , [ ] [ ] [ ] [ ] [ ] . other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, bactrian camels, llamas and guanaco (south american camelids) but none had detectable neutralising antibody against mers-cov [ , , , , , , ] . no virology or serology studies of human samples from areas in africa where there are camels with a history of mers-cov have been reported to date. however,an absence of unexplained pneumonia that may be attributable to mers-cov infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. it is thus unclear whether mers-cov, or an antigenically related cov, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the arabian peninsula [ ] . mers-cov rna has also been detected in dc samples, and recovery of infectious virus has also been achieved from dc samples [ , , , , [ ] [ ] [ ] [ ] [ ] . from some of these, full or majority length genomes of mers-cov have been sequenced [ , , ] . dc versions of mers-cov were found to be as similar to each other, as were variants detected from different humans over time and across distance. antibody screening assays have also detected crossreactive antibodies in sera. these were identified as such by screening sera against similar viruses, for example bcov or hcov-oc (as an antigenic facsimile for bcov). it is possible that other mers-cov-like viruses also reside within dcs, but this does not detract from the definitive finding of mers-cov genetic sequences in both dcs and humans [ , , ] . screening studies have shown that juvenile dcs are more often positive for virus or viral rna while older dcs are more likely to be seropositive and rna or virus negative [ , , ] . in adult dcs, mers-cov rna has been detected among animals with pre-existing antibody, suggesting re-infection is possible [ , ] . viral loads among positive dcs can be very high [ , , , , ] and dcs have been found positive both when ill with urt respiratory signs [ , , , ] or when apparently healthy [ ] . these findings indicate dcs host natural mers-cov infections. furthermore, stored dc sera have revealed signs of mers-cov in dcs which date back over three decades (the earliest collected in ) [ , , ] . older sera have not been tested and so precisely how long dcs have been afflicted by mers-cov, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in africa or the arabian peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. researchers sought to determine a direction for infection; were dcs transmitting virus to humans or were humans infecting dcs? at a qatari site, a farm owner and his employee became ill in mid-october and tested positive for mers-cov rna in a sputum and throat swab sample, respectively. rt-rtpcrs found mers-cov rna in of positive dc nasal swabs at the farm; six ( %) positive by two or more assays [ ] . the results indicated a recent outbreak had occurred in this herd; the first indication of mers-cov rna found within dcs with a temporal association to human infections. three positive dc samples were confirmed by sequencing a nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and dc mers-cov sequences [ ] . the dcs and human contacts yielded orf a and orf b sequences differing by only a single nucleotide each, clustering closely with the hafr-al-batin_ _ variant [ ] . subsequent case studies found evidence of a concurrent human and dc infection and the direction of that infection was inferred to be from the ill dcs and to their human owners [ , , ] . partial genome sequences indicated that a human and a mers-cov rt-rtpcr positive dc had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [ ] all nine dc in the owner's herd, serially sampled, reacted in a recombinant s antigen elisa, with the two animals that had been rt-rtpcr positive showing a small, verifiable rise in antibody titre [ ] . a rise in titre theoretically begins to days after dc infection [ ] . the authors suggested that the rise in titre in dc sera which occurred alongside a declining rna load, while the patient was actively ill and hospitalized, indicated that the dcs were infected first followed by the owner [ , ] . bcov antibodies were also present, and rising in one of the two rt-rtpcr positive animals but no animal's antibodies could neutralise bcov infection [ ] . camel calving season occurs in the winter months (between late october and late february; fig. ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve dc populations [ ] . what role maternal camel antibody might play in delaying infection of calves remains unknown [ , ] . juvenile dcs appear to host active infection more often than adult dcs and thus the sacrificial slaughter of dcs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. in contrast to earlier results, slaughterhouse workers who kill both younger and older dcs, may be an occupational group with significantly higher incidence of seropositivity to mers-cov when animals have active mers-cov infections [ , , [ ] [ ] [ ] . expanded virological investigations of african dcs may lead to more seropositive animals and geographic areas in which humans may be at risk. it is possible that there are areas where humans already harbour mers-cov infections that have not been identified because of an absence of laboratory surveillance. virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [ , ] . infectious mers-cov added to dc, goat or cow milk and stored at °c could be recovered at least h later and, if stored at °c, recovery was possible for up to h [ ] . mers-cov titre decreased somewhat when recovered from milk at °c but pasteurization completely ablated mers-cov infectivity [ ] . in a subsequent study, mers-cov rna was identified in the milk, nasal secretion and faeces of dcs from qatar [ ] . a single study has examined the ability of mers-cov to survive in the environment [ ] . plastic or steel surfaces were inoculated with tcid of mers-cov at different temperature and relative humidity (rh) and virus recovery was attempted in cell culture. at high ambient temperature ( °c) and low rh ( %) mers-cov remained viable for h [ ] . by comparison, a well known and efficently transmitted respiratory virus, influenza a virus, could not be recovered in culture beyond four hours under any conditions [ ] . aerosol experiments found mers-cov viability only decreased % at low rh at °c. in comparison, influenza a virus decreased by % [ ] . mers-cov survival is inferior to that previously demonstrated for sars-cov [ ] . for context, pathogenic bacteria can remain viable and airborne for min in a coughed aerosol and can spread m. mers-cov's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [ ] . whether mers-cov can remain adrift and infectious for extended periods (truly airborne) remains unknown. such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. the nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the al-ahsa hospital, the ksa and the south korean outbreaks [ , , , ] . by extrapolation, aerosol-generating events involving dcs (urination, defecation, and preparation and consumption of dc products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. the provision of evidence supporting the best formulation of personal protective equipment to be worn by hcws who receive, manage or conduct procedures on infectious cases remains a priority. mers-cov was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to mers-cov transmission chains. however, transmission of mers-cov is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [ , , , , , , ] in a household study, of ( %) contacts of mers-cov positive index patients were rna or antibody positive; the rate of general transmission, even in outbreaks is around % [ ] . it seems that the majority of human cases of mers-cov, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of mers-cov has not been self-sustaining [ ] [ ] [ ] [ ] [ ] . that is to say, the basic reproduction number (r ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. if r was greater than , a sustained increase in case numbers would be expected. some r o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. that mers has had a constant presence in the arabian peninsula since is due to ongoing, sporadic spill-over events from dcs amplified by poorly controlled hospital outbreaks. the first known mers human-to-human transmission event was one characterized by acute lrt disease in a healthcare setting in jordan. in stark contrast, a sero-survey of hcw who were sometimes in close and prolonged contact with the first, fatal mers-cov case in [ ] , found none of the hcw had seroconverted four months later, despite an absence of eye protection and variable compliance with required ppe standards [ ] . early on in the mers story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. contacts of confirmed mers cases were often observed for clinical illness, but not tested. these omissions may have confounded our understanding of mers-cov transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. case-control studies were not a focus. as testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [ ] . a rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [ ] ) resulted in a drop in the proportion of fatal cases during the jeddah- outbreak. historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. upon follow-up, over three-quarters of such mers-cov rna positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [ ] raising some question over the reliability of other reported data. the proportion of fatal mers cases within the ksa compared to outside the ksa, as well as the age, and sex distribution change in different ways when comparing mers outbreaks. approximately % of mers cases ( of ) in the ksa were fatal betwen and december while % ( of ) died among those occurring outside of the ksa. the total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. however the proportion of male deaths from total males with mers is a similar figure to that for females. in the ksa, there is a greater proportion of younger males among cases and deaths than were observed from the south korean or the jeddah- outbreaks (additional file : figure s ). why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [ ] . as a group, hcws comprised % of mers cases in the ksa and south korea. it is apparent that the weekly proportion of infected hcws increases alongside each steep rise in overall detections (fig. ) . in may , the who published guidelines for ipc during care of probable or confirmed cases of mers-cov infection in a healthcare setting [ ] . this is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [ ] . these rises in mers-cov detections can decrease the average age during each event because hcws are usually younger than inpatients with mers. healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (ipc) procedures [ , ] . most of the analysis of mers-cov genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [ ] [ ] [ ] . mers-cov was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. the technique can produce genomic [ ] [ ] [ ] . earlier and subsequent versions of this chart are maintained on a personal blog [ ] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [ , ] . despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during mers-cov characterization [ ] . as more genomes from both humans and dcs have been characterized, two clades have become apparent; a and b (fig. ) . clade a contains only human-derived mers-cov genomes from jordan, while clade b comprises the majority of human and camel genomes deduced thus far [ ] . two studies during , one looking at jeddah- mers-cov variants and another looking at a variant exported from south korea to china, have now identified signs of genetic recombination among mers-cov variants. while human and camel whole genome sequences have retained > % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [ ] [ ] [ ] . shared identity implies that the major source for human acquisition is the dc, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. over a month, a dc virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that dcs are the natural, rather than intermediate, host for the mers-cov we know today [ ] . to date, recombination has been localised to breakpoints near the boundary between orf a and orf b regions, within the spike gene [ ] and in the orf b region (fig. ) [ ] . it is not unexpected that recombination should occur since it is well known among other covs [ ] and because the majority of mers-cov whole genomes collected from samples spanning three years ( - ) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [ , , , , , [ ] [ ] [ ] . changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. if we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely fig. the genetic relationship between mers-cov nucleotide sequences (downloaded from genbank using the listed accession numbers and from virological.org [ ] ). this neighbour joining tree was created in mega v using an alignment of human and dcderived mers-cov sequences (geneious v . [ ] ). clades are indicated next to dark (clade a) or pale (clade b) blue vertical bars. camel icons denote genomes from dcs. healthcare or community outbreaks are boxed and labelled using previously described schemes [ , ] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. genetic mutations noted during the largest of human outbreaks, jeddah- , did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [ , ] . however, we understand very little of the phenotypic outcomes that result from subtle genetic change in mers-cov genomes. to date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [ ] . but vigilance and larger, more contemporary and in vivo studies are needed. genome sequence located to a distinct clade were identified from an egyptian dc that was probably imported from sudan. this does not fit into either of the current clades [ , , ] . a virus sequenced from a neoromicia capensis bat was more closely related to mers-cov than other large bat-derived sequences had been to that point, but the genome of a variant of a mers-cov has yet to be discovered and deduced from any bat [ ] . analyses of mers-cov genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (fig. ) , which encodes the spike protein and accessory proteins [ ] . at least nine mers-cov genomes contained amino acid substitutions in the receptor binding domain (rbd) of the spike protein and codons (n-terminal region), (rbd), (in heptad repeat ), and bear investigation as markers of adaptive change [ , ] . the spike protein had not changed in the recombinant mers-cov genome identified in china in but was reported to have varied at a higher rate than that for complete mers-cov genomes, among south korean variants [ , ] . this highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. despite this, one assay amplifying a nucleotide fragment of the spike s domain gene for sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [ ] . genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (fig. ) [ ] . this approach was employed when defining the geographically constrained mers hospital outbreak in al-ahsa, which occurred between st april and rd may , as well as clusters in buraidah and a community outbreak in hafr al-batin, the ksa. genomic sequencing identified that approximately mers-cov detections from a community outbreak in hafr al-batin between june and august may have been triggered by an index case becoming infected through dc contact [ ] . sequencing mers-cov genomes from the al-ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [ ] . however, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [ ] . in riyadh- , genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [ , , ] . riyadh is a nexus for camel and human travel and has had more mers cases than any other region of the ksa to date but also harbours a wide range of mers-cov variants [ , , ] . however the south korean outbreak originated from a single infected person, resulting in three to four generations of cases [ , ] . studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [ ] . for many mers cases detected outside the arabian peninsula, extensive contact tracing has been performed and the results described in detail. contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. results of contact tracing to date have found that onward transmission among humans is an infrequent event. for example, there were contacts, both symptomatic and asymptomatic, of a case treated in germany who travelled from the uae but no sign of virus or antibody were found in any of them [ ] . the very first mers case had made contact with hcws and others, but none developed any indication of infection [ ] . in a study of contacts of a case treated in france, only seven matched the definition for a possible case and were tested; one who had shared a m hospital room while in a bed . m away from the index case for a prolonged period was positive [ ] . none of the contacts of the first two mers cases imported into the usa in contained any mers-cov footprint [ ] and none of the contacts of two travellers returning to the netherlands developed mers-cov antibodies or tested rna positive [ , ] . analyses of public data reveal many likely instances of nosocomial acquisition of infection in the arabian peninsula and these data may be accompanied by some details noting contact with a known case or facility. one example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [ ] . contact tracing was a significant factor in the termination of a outbreak involving multiple south korean hospitals [ ] . such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (fig. c) . the hospital-associated outbreak in jeddah in was the largest and most rapid accumulation of mers-cov detections to date. the greatest number of mers-cov detections of any month on record occurred in jeddah in april. the outbreak was mostly (> % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [ , , ] . a rise in fatalities followed the rapid increase in case numbers. in two large outbreaks occurred. south korea was the site of the first large scale outbreak outside the arabian peninsula and produced the first cases in both south korea and china, occurring between may and july . this was closely followed by a distinct outbreak in ar riyad province in the ksa which appeared to come under control in early november. after staying in bahrain for two weeks, a year old male ( m) travelled home to south korea via qatar, arriving free of symptoms on the th may [ ] . he developed fever, myalgia and a cough nearly a week later ( th ). he visited a clinic as an outpatient between the th and th of may and was admitted to hospital a on the th [ ] . he was discharged from hospital a on the th then visited and was admitted to the emergency department of hospital b on the th . during this second stay, a sputum sample was taken and tested positive for mers-cov on the th [ , ] , triggering transfer to the designated isolation treatment facility. over a period of days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the south korean outbreak. approximately people were infected during this time [ ] . in total cases were generated in this outbreak, all linked through a single transmission chain to m; cases died [ ] . in south korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [ ] . other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with mers, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of korean society, crowded emergency rooms, poor ipc measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [ , [ ] [ ] [ ] . all of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [ , , , [ ] [ ] [ ] . few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. the hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [ , [ ] [ ] [ ] . early in the outbreak, a infected traveller, the son of an identified case in south korea, passed through hong kong on his way to china where he was located, isolated and cared for in china [ , , ] . no contacts became ill. the outbreak was brought under control in late july/ early august [ ] after improved ipc measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [ , ] . a review of public data showed that, as for mers in the ksa, older age and the presence of underlying disease were significantly associated with a fatal outcome in south korea. [ ] even though r is < , super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over , such as this one, are not unexpected from mers-cov infection [ ] . the dynamic of an outbreak depends on the r and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [ ] . in the region of ar riyad, including the capital city of riyadh, a hospital based cluster began, within a single hospital, from late june [ ] . by mid-september there had been approximately cases reported but the outbreak appeared to been brought under control in november. it became apparent early on that mers-cov spread relatively ineffectively from human-to-human. despite ongoing and possibly seasonal introduction of virus to the human population via infected dcs and perhaps other animals yet to be identified, the vast majority of mers-cov transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. this opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. it remains unclear if this group are uniquely affected by mers-cov or if other respiratory virus infections, including those from hcovs, produce a similarly serious impact. in south korea, a single imported case created an outbreak of cases and deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. household human-to human transmission occurs but is also limited. educational programs will be essential tools for combatting the spread of mers-cov both within urban and regional communities and for the health care setting. vigilance remains important for containment since mers-cov is a virus with a genetic makeup that has been observed for only three years and is not stable. among all humans reported to be infected, nearly % have died. continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. whole genome sequencing has been used extensively to study mers-cov travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. mers and sars have some clinical similarities but they also diverge significantly [ ] . defining characteristics include the higher pfc among mers cases (above % in and currently at - %; well above the % of sars) and the higher association between fatal mers and older males with underlying comorbidities. for the viruses, mers-cov has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to sars-cov. there appears to be a - % prevalence of mers-cov in the ksa with a % chance of secondary transmission within the household. there is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any r would predict on face value. nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of mers or mers-cov during or immediately after these events. there is no evidence that mers-cov is a virus of pandemic concern. nonetheless, hospital settings continue to describe mers cases and outbreaks in the arabian peninsula. as long as we facilitate the spread of mers-cov among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. the mers-cov appears to be an enzootic virus infecting the dc urt with evidence of recent genetic recombination. it may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in . rt-pcr testing of lrt samples remains the gold standard for mers-cov confirmation. serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. similarly, the important question of whether those who do shed mers-cov rna for extended periods are infectious while appearing well, continues to go unanswered. it is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. while the basic virology of mers-cov has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. additional file : figure s . the severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome: an emerging coronavirus infection 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emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands first cases of middle east respiratory syndrome coronavirus 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middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case association of higher mers-cov virus load with severe disease and death, saudi arabia an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj , despite a high rate of respiratory symptoms health protection agency ukncit. evidence of person-to-person transmission within a family cluster of novel coronavirus infections prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the hajj a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications respiratory tract samples, viral load and genome fraction yield in patients with middle east respiratory syndrome ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response a case of imported middle east respiratory syndrome coronavirus infection and public health response viral respiratory infections among hajj pilgrims in influenza a and b viruses but not mers-cov in hajj pilgrims acute respiratory infections in travelers returning from mers-cov-affected areas changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a -year retrospective analysis of data soaring mers cases cause pandemic jitters, but causes are unclear co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in recovery from severe novel coronavirus infection human isolate . who statement on the fifth meeting of the ihr emergency committee concerning mers-cov | who statement mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia evidence for camel-to-human transmission of mers coronavirus taking stock of the first mers coronavirus cases globally-is the epidemic changing? human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection. zoonoses public health summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus in bats, saudi arabia emerging infectious diseases associated with bat viruses bats and their virome: an important source of emerging viruses capable of infecting humans coronavirus diversity, phylogeny and interspecies jumping rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease link between mers virus and camels worries breeders dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) fiqh us-sunnah antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries mers coronavirus neutralizing antibodies in camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus antibody reactors among camels in dubai serologic assessment of possibility for mers-cov infection in equids mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate human infection with mers coronavirus after exposure to infected camels, saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels evidence for camel-to-human transmission of mers coronavirus the holy qur'an sacrificing an animal at mina occupational exposure to dromedaries and risk for mers-cov infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels the effects of temperature and relative humidity on the viability of the sars coronavirus viability of pseudomonas aeruginosa in cough aerosols generated by persons with cystic fibrosis middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? an observational, laboratory-based study of outbreaks of mers-coronavirus in jeddah and riyadh, kingdom of saudi arabia assessment of the mers-cov epidemic situation in the middle east region assessing the pandemic potential of mers-cov interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility transmission dynamics and control of ebola virus disease (evd): a review health care worker contact with mers patient, saudi arabia middle east respiratory syndrome coronavirus: epidemiology and disease control measures age-specific and sex-specific morbidity and mortality from avian influenza a(h n ) mers-cov outbreak in jeddah-a link to health care facilities infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers-cov recombination: implications about the reservoir and potential for adaptation middle east respiratory syndrome coronavirus recombination and the evolution of science and public health in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of reliable typing of mers-cov variants with a small genome fragment variations in spike glycoprotein gene of mers-cov molecular epidemiology of hospital outbreak of middle east. respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) in the republic of korea microevolution of outbreak-associated middle east respiratory syndrome coronavirus, south korea first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities followup of contacts of middle east respiratory syndrome coronavirus-infected returning travelers, the netherlands press release -who, korea-who joint mission on mers cov call for infection control to stem mers infection control and mers-cov in health workers an outbreak of middle east respiratory syndrome coronavirus infection in south korea middle east respiratory syndrome coronavirus (mers-cov) -republic of korea samsung hospital to invest w b in post-mers improvements why the panic? south korea's mers response questioned intensified public health measures help control mers-cov outbreak in the republic of korea south korean mers outbreak spotlights lack of research complete genome sequence of middle east respiratory syndrome coronavirus isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china urgent call for research on middle east respiratory syndrome (mers) in korea middle east respiratory syndrome (mers) in asia: lessons gleaned from the south korean outbreak no respite for korean economy even as mers patients recover middle east respiratory syndrome coronavirus (mers-cov) -china mers in south korea and china: a potential outbreak threat? objective determination of end of mers outbreak, south korea updates on mers outbreak (as of : on surveillance operation for the st confirmed case of middle east respiratory syndrome coronavirus in response to the patient's prior travel to jeju island the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission moh holds the first press conference to update community and media on mers severe acute respiratory syndrome vs. the middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) - case list of moh/who novel coronavirus mers ncov announced cases geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data recent evolution patterns of the middle east respiratory syndrome coronavirus (mers-cov) preliminary analysis of middle east respiratory syndrome coronavirus (mers-cov) sequences from korea and china any unreferenced opinions expressed herein are those of the authors and do not necessarily represent the views of any employer or institution. our thanks go to andrew rambaut, maia majumder, marion koopmans and hale abdali for helpful discussions on social media and cmam and riam for patience. the authors declare that they have no competing interests.author's contributions imm and kea contributed equally to this work and read an approved the final manuscript. key: cord- - qj rrgd authors: lvov, dimitry konstantinovich; shchelkanov, mikhail yurievich; alkhovsky, sergey vladimirovich; deryabin, petr grigorievich title: single-stranded rna viruses date: - - journal: zoonotic viruses in northern eurasia doi: . /b - - - - . - sha: doc_id: cord_uid: qj rrgd in this chapter, we describe zoonotic viruses that were isolated in northern eurasia and that belong to the different families of viruses with a single-stranded rna (ssrna) genome. the family includes viruses with a segmented negative-sense ssrna genome (families bunyaviridae and orthomyxoviridae) and viruses with a positive-sense ssrna genome (families togaviridae and flaviviridae). among them are viruses associated with sporadic cases or outbreaks of human disease, such as hemorrhagic fever with renal syndrome (viruses of the genus hantavirus), crimean–congo hemorrhagic fever (cchfv, nairovirus), california encephalitis (inkv, tahv, and khatv; orthobunyavirus), sandfly fever (sfcv and sfnv, phlebovirus), tick-borne encephalitis (tbev, flavivirus), omsk hemorrhagic fever (ohfv, flavivirus), west nile fever (wnv, flavivirus), sindbis fever (sinv, alphavirus) chikungunya fever (chikv, alphavirus) and others. other viruses described in the chapter can cause epizootics in wild or domestic animals: geta virus (getv, alphavirus), influenza a virus (influenzavirus a), bhanja virus (bhav, phlebovirus) and more. the chapter also discusses both ecological peculiarities that promote the circulation of these viruses in natural foci and factors influencing the occurrence of epidemic and epizootic outbreaks single-stranded rna viruses the bunyaviridae family was named after the prototypical bunyamwera virus (bunv) isolated in from mosquitoes (aedes spp.) in bunyamwera, uganda. currently, the bunyaviridae family includes four genera of animal viruses (orthobunyavirus, phlebovirus, nairovirus, and hantavirus) and one genus (tospovirus) of plant viruses. bunyavirus virions are spherical in shape (size, about À nm) and have an outer lipid bilayer with the viral envelope glycoproteins gn and gc exposed on the surface. the genome consists of three segments of single-stranded, negative-sense rna with a total length from , to , nt. depending on the size, the segments are designated l (large), m (medium), and s (small). the viral proteins are synthesized on the mrna that is produced during replication and that is complementary to the genomic rna. the length of segments varies for different genera, but in general, they have a common structure. the l-segment, whose length is from , nt (phlebovirus) to , nt (nairovirus) , has a single open reading frame (orf) encoding rna-dependent rna polymerase (rdrp). the m-segment of all of the genera also has a single orf, which encodes a polyprotein precursor of envelope glycoproteins gn and gc. the length of the m-segment ranges from , nt for some of the phleboviruses to , À , nt for the nairoviruses. the mature glycoproteins gn and gc of the bunyaviruses are derived during complex endoproteolytic events leading to cleavage of the polyprotein precursor by cellular proteases. the s-segment of the bunyaviruses encodes a nucleocapsid protein. additional nonstructural (nss) protein is encoded by the s-segment of viruses of the phlebovirus, tospovirus, and orthobunyavirus genera. , the bunyaviruses are widely distributed in the world and are one of the most numerous known zoonotic viruses. most of the zoonotic bunyaviruses are transmitted to animal or humans by bloodsucking arthropod vectors, usually mosquitoes or ticks. viruses of the hantavirus genus are the exception, being transmitted mainly by aerosol formed from virus-laden urine, feces, or saliva of infected rodents or insectivores that are their natural hosts. À the genus hantavirus consists of those bunyaviruses of vertebrates which do not have the ability to replicate in an arthropod's cell and which are transmitted by respiratory route through the formation of aerosols from urine or feces containing the virus. the morphology of the virion and the genome structure of the hantaviruses are common to all bunyaviruses. the size of the negative-sense ssrna genome of the prototypical hantaan virus (htnv) is , nt for the l-segment, , nt for the m-segment, and , nt for the s-segment (figure . ). in nature, hantaviruses persist asymptomatically in rodents and insectivores, with each type of hantavirus associated predominantly with one host species. the phylogenetic relationships of hantaviruses enable virologists to divide them into three lineages, which correspond in general to their main hosts. in the s-segment of some hantaviruses carried by arvicolinae and sigmodontinae rodents, there is an additional orf-encoded nonstructural protein nss. but nss is absent in the hantaviruses of the murinae rodents. À history. hemorrhagic fever with renal syndrome (hfrs) was originally described as a separate nosological category (called "endemic (epidemic) hemorrhagic nephroso-nephritis" at that time) by anatoly smorodintsev (figure . ) during À in the far east. later, japanese scientists described hfrs in northeastern china as "songo fever" and swedish scientists as "epidemic nephropathy"; a similar disease was described in in china. the abbreviation "hfrs" was suggested by mikhail chumakov (figure korea. hantaviruses. the hantaviruses are members of the hantavirus genus of the bunyaviridae family. the first serotype, -htnv, included strains isolated from mouselike rodents (muridae) in south korea, china, and the southern part of the russian far east (primorsky krai). À the second serotype, puumala virus (puuv), was isolated from hamsterlike rodents (cricetidae), mainly the bank vole (myodes glareolus) in finland and then in other european countries and the western part of russia, as well we from maximowicz's vole (microtus maximoviczii) in the far east). À the third serotype, seoul virus (seov), was isolated from brown rats (rattus norvegicus), black rats (rattus rattus), and laboratory albino rats (rattus norvegicus f. domestica) in south korea and elsewhere, including the united states. , the fourth serotype, dobravaÀbelgrade virus (dobv), was isolated from the striped field mouse (apodemus agrarius) in slovenia and yugoslavia. the fifth serotype, sin nombre virus (snv), literally "nameless virus" in spanish, was isolated from the meadow vole (microtus pennsylvanicus). in addition to the main serotypes, other serotypes are known today, including in eurasia: amur virus (amrv), isolated from asiatic forest mice (apodemus peninsulae) in the far east of russia and in china ; tula virus (tulv), from common voles (microtus arvalis) in central russia , ; khabarovsk virus (khav), from from reed voles (microtus fortis) and siberian brown lemmings (lemmus sibiricus) in the far east ; thottapalayam virus (tpmv), from asian musk shrews (suncus murinus) in india ; thailand virus (thaiv), from bandicoots (bandicota indica) in thailand ; and a newfound hantavirus, from chinese mole shrews (anourosorex squamipes) in vietnam. virion and genome. the size of the negative-sense ssrna genome of the prototypical htnv is , nt for the l-segment, , nt for the m-segment, and , nt for the s-segment (figures . and . ). epizootiology. rodents (order rodentia) are the main natural reservoir of hantaviruses. nevertheless, strains have been isolated from birds in the far east and from bats in china. infection in rodents is asymptomatic, but the virus is expelled with saliva, urine, and excrement, most intensively during the first month after inoculation. (during this period, virus antigen can be detected in the lungs.) the evolution of hantaviruses is closely related to that of its rodent host (figure . ). , , at least species of rodents (rodentia), species of lagomorphs (order lagomorpha), species of insectivores (order insectivora), species of predators (order carnivora), and species of artiodactyls (order artiodactyla) are known to take part in hantavirus circulation on the territory of northern eurasia. , , the main species of rodents, which are the hosts of hantaviruses in russia, are presented in table . . the infection rate of mouselike rodents and insectivores lies within the limits . . %. hantavirus antigens have been detected in birds as well: the oriental turtle dove (streptopelia orientalis), coal tit (parus ater), marsh tit (parus palustris), daurian redstart (phoenicurus auroreus), nuthatch (sitta europaea), black-faced bunting (emberiza spodocephala elegans), eurasian jay (garrulus glandarius), hazel grouse (tetrastes bonasia), pheasant (phasianus colchicus), ural owl (strix uralensis), green-backed heron (butorides striatus), and grey heron (ardea cinerea). hantavirus (magboi virus, or mgbv) was isolated in from the hairy slit-faced bat (nycteris hispida) in africa (sierra leone), but the role of bats in the circulation of hemorrhagic fever with renal syndrome virus (hfrsv) is yet to be investigated in detail. in western siberia, the main natural reservoir of hfrsv is rodents of the hamsterlike (cricetidae) family-in particular, bank voles (myodes glareolus), with a susceptibility up to %; red-backed voles (myodes rutilus), susceptibility %; and, in the north, siberian brown lemmings (lemmus sibiricus), %. the infection rate of other rodents and insectivores is about . À . %. , in eastern siberia, the maximum susceptibility is demonstrated in grey red-backed voles (myodes rufocanus), %; house mice (mus musculus), %; water voles (arvicola terrestris), %; and tundra voles (microtus oeconomus), %. in the far east, hfrsv was revealed to circulate among field mice (apodemus agrarius) with a susceptibility of about %; asiatic forest mice (a. peninsulae), susceptibility %; reed voles (microtus fortis), À %; grey redbacked voles (myodes rufocanus), %; and other rodents (rodentia), . À . %. , , epidemiology. hfrsv infection starts by aerogenic penetration of the virus during the inhalation of waste products (saliva, urine, excrement) of latently infected animals. an alimentary pathway (with contaminated food and water) of the infection is also possible. , , , , hfrs is distributed over eurasia (russia, belarus, ukraine, moldova, the baltic countries, the czech republic, slovakia, bulgaria, romania, serbia, slovenia, england, france, germany, belgium, hungary, denmark, fennoscandia, kazakhstan, georgia, azerbaijan, china, north and south korea, japan), as well as american and african countries. , , during À , in of regions in russia, , cases of hfrs were registered (table . ). annual morbidity of hfrs in russia is in the range from , to , cases ( . À . %) and is decreasing. about % of cases take place in european forest landscapes. puuv associated with the bank vole (myodes glareolus) provokes about % of hfrs cases in russia (especially in bashkortostan, udmurtia, mari el, tatarstan, the chuvash republic, orenburg, ulyanovsk, and the penza region). , morbidity in the urban population is higher ( %) than in the rural one. the peak of the disease occurs during julyÀoctober in forests and in gardens and kitchens closely situated to the forests. , À dobv associated mainly with field mice (apodemus agrarius) and small forest mice (a. uralensis) is of leading epidemiological significance in the central and southwestern sectors of the european part of russia (the voronezh, lipetsk, orel, and belgorod regions), as well as in georgia. , , , puuv and tulv are associated with the common vole (microtus arvalis) and the bank vole (myodes glareolus) and are also distributed over this territory. , , a similar situation is observed in other regions of the central federal district: in the moscow, yaroslavl, ryazan, tver, kaluga, vladimir, ivanov, kostroma, smolensk regions. hfrs morbidity in the moscow region is associated with puuv, the infection rate of which is À % among bank voles (myodes glareolus), À % in the common vole (microtus arvalis), % in major's pine vole (microtus majori), and in À % other rodent species. in krasnodar krai, the black sea field mouse (apodemus ponticus) and major's pine vole (microtus majori) play the main role in human morbidity. , human morbidity in the european part of russia is registered beginning at a relatively low level in marchÀapril, decreasing to yet a lower level in mayÀaugust, increasing in septemberÀnovember, and then increasing again during decemberÀjanuary. the hyperendemic territory is the southwestern ural region (especially the bashkortostan republic and the chelyabinsk and orenburg regions), the volga-vyatka economic region (especially the udmurt republic), the chuvash republic, and the tatarstan, mari el, samara, penza, saratov, and ulyanovsk regions. , the main human morbidity occurs among those À years old (chiefly men). in russia, hfrs represents a significant part of all naturalfoci zoonotic diseases. the immune layer to hfrsv in the european part of russia is a mean . %; in the bashkortostan republic, it reaches up to % (mean, %). the immune layer among the populations of western and eastern siberia is about % for the entire region, . % in krasnoyarsk krai, . % in the irkutsk region, . % in the omsk region, and . % in the tyumen region. , the far east provides about % of all hfrs cases in russia. the highest morbidity was revealed in khabarovsk krai, primorsky krai, and the amur region. in khabarovsk krai and primorsky krai, las in china and japan, -htnv is associated with grey red-backed voles (myodes rufocanus). , , , the morbidity of seov (the third serotype) associated with the synanthropic brown rat (rattus norvegicus) and black rat (r. rattus) was examed in both the far east and the european part of russia. the researchers found that seov provoked hfrs more often among the urban population, whereas htnv did so more often among the rural population, of primorsky krai. morbidity in the far east has a small uptick in mayÀjuly and reaches its main peak in novemberÀdecember. the immune stratum in the far east is about % (ranging from . % in the amur region to . % in primorsky krai). , pathogenesis. capillary damage is the basis of hfrs pathogenesis. in the first part of the disease, toxicoallergic phenomena predominate, caused by viral infection of the walls of vegetative centers, venules, and arterioles. lesions on the sympathetic nodes of the neck are followed by hyperemia of the face and neck. irritation of the vagus nerve leads to bradycardia and a fall in arterial pressure. damage to the vascular permeability is accompanied by hemorrhages in mucous membranes and the skin. the cause of death is cardiovascular insufficiency, massive hemorrhages into the vital organs, plasmorrhea into the tissues, collapse, shock, swelled lungs, spontaneous rupture of the kidneys, a hypertrophied brain, and paralysis of the vegetative centers. , clinical features. the incubation period is À days. hfrs starts with fever, headache, muscular pain, dizziness, nausea, vomiting, hyperemia of the face and neck, bradycardia, and a fall in arterial pressure. abnormalities of the central nervous system (cns) in the form of block, excitement, hallucinations, meningeal signs, and visual impairments often occur. hemorrhagic syndrome becomes apparent as plasmorrhea into the tissues, together with microthrombosis; exanthema; petechial skin rash; nasal, pulmonary, and uterine bleeding: vomiting blood, hematuria, and visceral bleeding. in some cases, pasternatsky syndrome, pain in the kidneys, oliguria, and albuminuria become morphologically apparent as interstitial and tubular nephritis. the duration of fever is À days. two-wave temperature dynamics is possible. , analyses of , cases of hfrs etiologically linked with puuv in sweden during À found . % mortality in the first three months of the disease. , defense immunity remains for at least years. , diagnostics. laboratory diagnostics are based on the fluorescent antibody method (fam), enzyme-linked immunosorbent assay (elisa), and reverse transcription polymerase chain reaction (rt-pcr) testing. the virus can be isolated with the use of vero e (green monkey kidney cell line), bs (diploid human embryo lung cell line), a- (human lung carcinoma cell line), or rlc (rat lung tissue primary cell culture). , control and prophylaxis. treatment of hfrs can be symptomatic, pathogenetic, or etiotropic (or any combination thereof). during the fever period, early hospitalization, disintoxical therapy, and strengthening of the walls of vessels are necessary. during the oliguria period, transfusion with desalinated human albumin, hemodes, a % glucose solution, and an isotonic nacl solution (under the control of the emitted volume of urine) are given. in case of shock, antishock therapy is applied, and hemodialysis is prescribed for kidney insufficiency. , vaccination is the most effective approach to the prophylaxis of hfrs. the efficacy of vaccination was demonstrated in china and in north and south korea. nevertheless, it must be mentioned that vaccines in these countries are produced from htnv and seov stains and do not defend against puuv infection, which is the main etiological agent of hfrs in the european part of russia (where % of all russian morbidity occurs) . for a long time, anti-hfrs vaccine was difficult to produce because there were no sensitive cell lines to accumulate hantavirus. however, the recent adaptation of puuv and dobv to the certified vero e cell line affords an opportunity to produce candidate vaccines against hfrs. experimental series of "combi-hfrs-vac" vaccine have passed compliance tests for medical immunoglobulin preparations for use in humans. , , the genus nairovirus includes the ticktransmitted bunyaviruses, whose genome is the largest in the family bunyaviridae. the size of l-segments of the dugbe virus (dugv), a prototypical species of the nairoviruses, is , nt. the m-and s-segments are , and , nt, respectively (figure . ) . as with other bunyaviruses, the l-segment of the nairoviruses encodes an rdrp, the m-segment encodes a polyprotein precursor of the envelope glycoproteins gn and gc, and the s-segment encodes the nucleocapsid (n) protein. , the genus nairovirus was established on the basis of antigenic relationships among viruses of the six antigenic groups of arthropod-borne viruses: the crimeanÀcongo hemorrhagic fever (cchf), nairobi sheep disease (nsd), qalyub (qyb), sakhalin (sak), dera ghazi khan (dgk), and hughes (hugv) groups. À subsequently, a seventh, thiafora (tfa), group was assigned to the genus. , currently, about viruses are assigned to the genus nairovirus, now united in the aforementioned seven groups. sequence analysis of previously unclassified bunyaviruses revealed that the nairoviruses actually number much more than . three additional groups of nairoviruses-issyk-kul (isk), artashat (artsv), and tamdy (tam)-were established on the basis of phylogenetic analysis (table . ). cchfv belongs to the nairovirus genus of the bunyaviridae family and is the etiological agent of crimeanÀcongo hemorrhagic fever (cchf). history. cchf was first mentioned as "hunibini" and "hongirifta" by tajik physician abu-ibrahim djurdjani in the twelfth century. the viral nature of cchf was originally established in during an expedition to crimea headed by mikhail chumakov at the time of an outbreak. À the modern history of cchfv investigation starts in june with an epidemic of the disease in the northwestern steppe part of the crimean peninsula. more than severe cases of the disease broke out, all exhibiting hemorrhagic syndrome, known in that time as "severe infectious capillary toxicosis." mikhail chumakov headed an expedition to the region, and much research revealed that the disease is transmitted by hyalomma plumbeum (marginatum) ticks of the ixodidae family. the disease in , the historical hodzha strain was isolated from a patient with hemorrhagic fever in uzbekistan, as was a set of strains from h. marginatum larvae and nymphs in the astrakhan region, near the caspian sea. , in , the similarity between the etiological agent of crimean hemorrhagic fever and that of congo virus, isolated from a patient in in zaire (congo), was demonstrated, so the virus was renamed cchfv. , genome and taxonomy. like the genomes of all nairoviruses, that of cchfv consists of three segments of negative ssrna: a signed small (s) ( , nt) segment, a medium (m) ( , nt), and a large (l) ( , nt) segment. each segment has a single orf that encodes the nucleocapsid protein (n, aa, s-segment), a polyprotein precursor of envelope glycoproteins gn and gc ( , aa, m-segment), and rdrp ( , aa, l-segment). genetic diversity among cchfv strains may reach % nt and % aa differences for m-segment sequences, a reflection of pressure on the immune system and adaptation to various ecologic zones with different prevalences of hyalomma tick species. the s-and l-segments are more conservative: the level of divergence of s-segment sequences is % nt and % aa, and that for l-segment sequences is % nt and % aa. phylogenetic analysis based on sequence data comparisons of s-segments shows that cchfv isolates from different regions can be clustered into seven phylogeographic groups: west african isolates (group i), as well as isolates from central africa (uganda and the democratic republic of the congo) (group ii); south africa and west africa (group iii); the middle east and asia (group iv) (the asian strain can be divided to two distinct subgroups: asia (iva) and asia (ivb)); europe and turkey (group v); and greece (group vi), a separate group detached from the rest of europe (figure . ) . À in general, the genotypic structure defined on basis of the s-segment analysis is correlated strictly with geography. cases of isolation of strains not typical for a given territory were attributed to possible transmission of the virus by infected ticks carried by migratory birds. the tree topology based on the l-segment comparison is, on the whole, similar to that generated on the basis of the s-segment. exceptions are the viruses from senegal, which represent a separate lineage in the s-segment analysis, and those clustered within group iii in the l-segment analysis. similarly, the division of group iv into group iva (asia ) and ivb (asia ) is not clear (figures . and . ) . in russia, most of the strains of cchfv that were isolated were isolated in the country's southern regions (astrakhan, volgograd, and stavropol districts). phylogenetic analysis showed that all of them are closely related to european and turkish strains (group v). À epizootiology. up to today, cchfv has been found to circulate in countries in europe, africa, and asia. , À cchfv was isolated from at least species of mainly ixodidae ticks, but their roles in maintaining virus circulation are different (tables . and . ). the main significance for cchfv reservation and transmission belongs to ticks of the hyalomma genus: h. marginatum in the south of he european part of russia, h. anatolicum and h. detritum in the middle east and asia, and h. asiaticumin kazakhstan. according to our data, the viral load among imagoes of h. marginatum in the astrakhan region in À was . %; among nymphs, the load was . %. the presence of transphase and transovarial transmission of cchfv provides a reservation for viruses during the interepidemic period. three hostsfor larvae (ground birds, mainly corvidae; table . isolation of cchfv from ixodidae ticks mouselike rodents; and hares), nymphs (also ground birds, mouselike rodents, and hares), and imagoes (large mammals-mainly cattle, sheep, and camels)-provide a variety of ecological links of cchfv to vertebrates. , À in nigeria, cchfv was isolated from midges (culicoides sp.) the distribution of h. marginatum is limited by the isotherm of effective temperatures such that sum (Σ t $ c ) , c, or days with mean temperature $ c per year. so, the northern boundary of the distribution of cchfv in the south of the european part of russia lies in the dry steppe subzone. in russia and south africa, cchfv is often isolated from hares. , cchfv was isolated from hedgehogs (atelerix spiculus) in nigeria. hares and mouselike rodents play the main role in cchfv circulation. , , viremia in birds is not sufficient for vector transmission (although specific antibodies appear); nevertheless, ground birds are an important element of cchfv transmission because they are the hosts for the preimaginal phases of h. marginatum development. , , during field investigations of chatkalsky ridge in kirgizia, nymphs and larvae of h. marginatum dominated among field-collected materials from birds. the highest number of ticks was found on rollers (coracias garrulus), crested larks (galerida cristata), tree sparrows (passer montanus), and blackbilled magpies (pica pica). in the astrakhan region, rooks (corvus frugilegus) are the main hosts for h. marginatum preimaginal phases. during migrations, birds can take part in dispersing preimago ticks that carry the virus. for example, in spain in , cchfv of african origin (probably introduced by migrating birds) was isolated from h. lusitanicum. european birds overwintering in africa were also found to harbor ticks that carried the virus. cchfv infection rates found as the result of an investigation of , domestic animal sera are presented in table . . domestic animals are one of the main reservoirs of cchfv among vertebrates. viremia ( . À . (log ld )/ mcl) sufficient for the infection of ticks was detected À days after experimental inoculation of sheep. viremia after up to days post inoculation was detected in small gophers (citellus pygmaeus), long-eared hedgehogs (hemiechinus auritus), and wood mice (apodemus sylvaticus). experimental infection was revealed only in nymphs, and that is why hares and corvidae birds-the main hosts for nymphs-play the chief role in cchfv circulation. astrakhan volgograd dagestan rostov stavropol krasnodar total , of ixodidae (at first, h. marginatum) ticks in this region as the result of climatic changes. during À , , cases of cchf were recorded in russia, including in stavropol krai, in the rostov region, in the kalmyk republic, in the astrakhan region, in the volgograd region, in the dagestan republic, in the ingush republic, and in the karachaevoÀcherkesskaya republic (table . ). in , cases of cchf were recorded on the territory of the southern federal district and the north caucasian federal district (table . , figure . ). the absence of any recorded cases of cchf in krasnodar krai could be explained by a lack of attention to cchf diagnostics. a decrease in the proportion of severe clinical forms with hemorrhagic syndrome occurred after . the drop could have been due to the introduction of high-grade express diagnostics methods into clinical practice and an intensification in seeking out and diagnosing those suspected of having cchf. at the same time, the disease extended its incidence into the new territories of the volgograd region, with nosocomial cchf cases recorded there once again. pathogenesis. pathogenesis is defined by lesions of the vascular and nervous systems. , , clinical features. the incubation period after transmissive cchfv inoculation (as the result of a tick bite) is À days, whereas that after contact inoculation is À days. the difference is due to a much higher quantity of virus entering the system during contacts inoculation. , , cchf starts rapidly, with the temperature increasing to À c and the appearance of fever, skin hyperemia in the top half of the trunk, headache, lumbar pain, abdominal and epigastric pains, generalized arthralgia, conjunctivitis, pharingitis, and diarrhea. about % of cases have two obvious waves of increasing temperature, with the temperature decreasing in À days after the end of the incubation period. petechial rash appears in the majority of all cchf patients in À days after the incubation period and is a marker of the second increasing-temperature wave. hemorrhagic diathesis with nasal bleeding (in two-thirds of cases), bloody vomiting, blood in the sputum, and hematuria, all starting À days after the end of incubation period, are characteristic in % of cases. the duration of the hemorrhagic period is À days. meningitis symptoms and signs of psychosis (depression, sleepiness, lassitude, photophobia) could develop as well. lethality is À % for transmissive inoculation and up to % for contact inoculation. nevertheless, lethality is decreasing as the result of the introduction of modern testing systems and treatment with ribavirin. the convalescent period is about a month. , , , e.v. leshchinskaya has suggested the following clinical classification of cchf: (i) severe form with hemorrhagic syndrome ( .a. without band bleeding; .b. with band bleeding); (ii) without hemorrhagic syndrome ( .a. medium-severe form; .b. light form). , diagnostics. diagnosis is based on the detection of both specific antibodies via elisa (igm after days post disease progression and igg) and virus rna via rt-pcr testing (earlier than days post disease progression). , both tests must be conducted for a definitive diagnosis of cchf to be made. during the first week of infection with cchf, positive results via rt-pcr are obtained in % of cases; during the second week, the percentage is %. during the second week of the disease, positive results in igm via elisa are obtained in % of cases; during the third week, the percentage of positive reults in igg via elisa is %. À control and prophylaxis. ribavirin is the most effective drug prescribed today. , À ribavirin is used for days after symptoms first appear: , mg ( capsules) or mg/kg for the first time, then mg times a day if the weight of the patient is more than kg or mg times a day if the weight of the patient is is less than kg). the duration of treatment is À days. ribavirin must not be used by pregnant women, except when the disease is considerd life threatening. vaccine development is currently just in the experimental stages, À so prophylaxis involves early detection of sick humans and the prevention of further contact infections. nonspecific prophylaxis includes the eradication of ixodidae ticks on livestock and acaricide treatment of locations inhabited by domestic animals. in pastures with large numbers of ixodidae ticks, animals have to be led into box stalls and the humans leading them there must use special suits. history. artashat virus (artsv, strain leiv- ar) was originally isolated from ornithodoros alactagalis ticks (family argasidae) collected in the burrows of a small five-toed jerboa (allactaga elater) near arevashat village ( absence of antigenic relationships with any known viruses, it was referred to as an "unclassified bunyavirus." À taxonomy. three strains of artsv were sequenced. a full-length genome comparison revealed that artsv has À % nt similarity to other nairoviruses. phylogenetic analysis revealed that the virus is a new species in the nairovirus genus and forms a distinct genetic lineage on the nairovirus tree, which was constructed for all three segments of the genome (figures . À . ) . the phylogeny of the nairoviruses is based mainly on analyses of the partial sequence of the conservative catalytic core domain of rdrp. , the similarity of this domain of artsv to other nairoviruses is À % nt and À % aa. the phylogenetic tree constructed by the maximum-likelihood method on the basis of the amino acid alignment of the rdrp catalytic core domain of nairoviruses confirms the topology of artsv on a newly formed genetic lineage (figures . À . ). the nairoviruses on the tree can be divided into two main phylogenetic groups. the first group includes the nairoviruses, which are transmitted predominantly by ixodids: the crimeanÀcongo hemorrhagic fever group (hyalomma and haemaphysalis, as well as dermacentor, rhipicephalus, and ixodes), the dugbe group (mainly amblyomma, but also hyalomma, rhipicephalus, and haemaphysalis), the sakhalin group (ixodes), and the tamdy group (hyalomma). the first group also includes erve virus (ervev), whose vectors are unknown. , the second phylogenetic lineage includes the nairoviruses from the hughes, issyk-kul, dera ghazi khan, and qalyub groups, whose vectors are argasids: argas and ornithodoros. the tree topology of artsv shows that the virus is in the lineage of the nairoviruses transmitted predominantly by ixodidae ticks, although all isolations of artsv were obtained from the argasidae ticks o. alactagalis and o. verrucosus (table . ). it can be assumed that the adaptation of artsv to argasids is the result of the the narrow ecologic niche occupied by those ticks, which are ticks of the subgenera theriodoros and pavlovskyella. note that, although ervev, a european nairovirus, is phylogenetically close to the nairovirus transmitted by ixodids, the association of ervev with ixodes spp. ticks has not been established in endemic areas (southern europe). ervev has been isolated from shrews (crocidura russula). arthropod vectors. the adaptation of viruses to argasidae ticks facilitates the possibility of survival of viral populations in winter at low temperatures and in dry periods. the ability of argasids to fast (up to years and more), the long life cycle of these ticks (up to À years), and their polyphagia and ecological plasticity determine the stability of the natural foci of arboviruses transmitted by argasids. these foci are confined mainly to the arid regions of the southern part of the temperate and subtropical zones. , , the northern border of the range of argasids coincides with isolines denoting a frost-free period of À days per year and an average daily temperature above c for no less than À days per year. tick species from the subgenera theriodoros (ornithodoros alactagalis, o. nereensis) and pavlovskyella (o. papillipes, o. verrucosus, o. cholodkovskiy, o. tartakovskiy) are associated mainly with burrows of rodents. this ecological peculiarity narrows the possibility of the spread of viruses that are adapted to ticks from the theriodoros and pavlovskyella subgenera. it also applies to artsv associated with burrowÀ shelter biomes and found only in transcaucasia. history. caspiy virus (casv, prototypical strain leiv- az) was originally isolated from the blood of a sick herring gull (larus argentatus) caught on gil island in the baku archipelago, off the western coast of azerbaijan in the caspian sea ( n, e; figure . ) in . À on the basis of electron microscopy, casv was classified as a member of the bunyaviridae family, but antigenic relationships with known bunyaviruses have not been found. thus, casv was categorized into the unclassified bunyaviruses. , À at the same time, and in the same place, three strains of casv were isolated from ornithodoros capensis (family argasidae) ticks taxonomy. the genome of the prototypical strain leiv- az of casv was sequenced, and it has been shown that casv is a member of the hugv group of the nairovirus genus. the s-segment of casv is about , nt in length and has a single orf that encodes the nucleocapsid protein (n, aa). the second start codon, in position , is located in the n-protein orf of casv. the identity of the amino acid sequence of the n-protein of casv with those of other nairoviruses is only %, on average. the cleavage site for caspase- (d evd ) that has been found in the n-protein of cchfv is absent in casv. cleavage of n by caspase- is required for effective replication of cchfv. note that caspase cleavage sites in the nucleocapsid protein are also necessary for replication of human influenza a viruses. the m-segment of casv, like that of the other nairoviruses, has a single orf-encoded polyprotein precursor of the envelope glycoproteins gn and gc. the length of the gn/gc precursor of casv is , aa. according to the results of an analysis of polyprotein in the program signalp server . , the first aa constitute the signal peptide that is cleaved on the ssa/sy site. the cleavage site between pre-gn and pre-gc is in position (vsg/ik). these data are confirmed by the location of transmembrane domains in mature proteins gn and gc that was defined with the use of the program tmhmm server . . six potential sites of n-glycosylation are predicted in the mature gn protein of casv, only one in the gc protein. in general, the level of identity of polyprotein in casv is À % aa with that of other members of the nairovirus genus (table . ). the l-segment of casv has an orf ( , aa) that encodes the viral enzyme rdrp, which is the most conservative viral protein. the similarity of the rdrp of casv to that of other nairoviruses for which complete genome sequences were available is . À . % aa. phylogenetic analysis based on the predicted full-length amino acid sequences revealed that casv is equidistant from other nairoviruses, and forms a distinct branch, on the trees (figures . À . ). for many nairoviruses, only short sequences of the catalytic core domain of rdrp are available in genbank. this domain of rdrp is very conservative and relevant to phylogenetic analysis. , , the highest level of similarity ( % aa) that the rdrp core domain of casv has is with the same sequences in viruses of hug. on the dendrogram, constructed on the basis of a comparison of rdrp core domains, casv is located on the branch of the hug group (figures . À . ). note that viruses of this group (as well as casv) have been isolated from ornithodoros (carios) ticks that are associated with seabirds on the coasts and islands of the world's oceans. , thus, the phylogenetic relationship of casv with hug group viruses reflects the ecological features of those coasts and islands. arthropod vectors. ornithodoros capensis ticks inhabit the coasts and islands of the atlantic, indian, and pacific oceans from the southern part of the temperate zone to the equator, as well as some large inland ponds. , , o. capensis ticks feed on many bird species, mainly those of the order charadriiformes: gulls (family laridae) and terns (sturnidae), but also cormorants (phalacrocoracidae) and pelicans (pelecanidae). , these argasid ticks have a life cycle made up of six to eight stages: egg, larva, three to five stages of nymphs, and imago. according to laboratory study, the cycle is from to days and so can be completed during a single breeding season. these ecological peculiarities provide stability to the natural foci of the viruses, which are adapted to the o. capensis tick viruses and their transcontinental transfer by migrating birds. vertebrate host. in , during the collection of field material on islands in the baku archipelago, an epizootic among herring gulls was observed. the first strain of casv was isolated from sick birds. migrations in search of food, including migration between the western and eastern coasts of the caspian sea, result in a sharing of the argasids and viruses ranging over the area. history. the prototypical strain leiv- uz of the chim virus (chimv) was isolated from ornithodoros tartakovskyi ticks collected in july in the burrows of great gerbils (rhombomys opimus) in the vicinity of the town of chim in the kashkadarinsky region of uzbekistan) ( n, e; figure . ). À isolation of chimv was carried out during monitoring of these arboviruses' foci on the territory of central asia and kazakhstan. chimv was investigated through serological testing with viruses from different families and with unclassified viruses isolated earlier in the ussr. because no antigenic relationships of chimv were (and still have not been) found, chimv was assigned to the category of unclassified viruses. , later, four strains of chimv were isolated from the ticks o. tartakovskyi, o. papillipes, and rhipicephalus turanicus (rhipicephalinae) respectively collected in the burrows of great gerbils in the kashkadarya, bukhara, and syrdarya districts of uzbekistan in À . , three strains of chimv also were isolated from hyalomma asiaticum (hyalomminae) ticks and from the livers of great gerbils, which were collected in the floodplains of the or river and karatal river (dzheskazgan district, kazakhstan) in april (figure . ). , taxonomy. the genome of the prototypical strain leiv- uz of chimv was sequenced, and, on the basis of sequence analysis, the virus was classified as a novel member of the nairovirus genus. phylogenetic analysis based on a partial sequence of a catalytic center of rdrp placed chimv on the genetic branch of the qybv group. , the amino acid sequence of this domain of chimv has an % identity with qybv, geran virus (gerv), and bandia virus (bdav), the other members of the qybv group. À all these data are consistent with the fact that viruses of the qybv group, as well as chimv, have an environmental connection to ticks of the ornithodoros genus and to the burrows of rodents. qybv has repeatedly been isolated from o. erraticus ticks, collected in burrows of the african grass rat (arvicanthis niloticus) in the nile valley and the nile delta in egypt. to date, only short sequences of the rdrp of qybv are available in genbank, but recently we gave a genetic characterization of gerv, isolated in transcaucasia and, apparently, closely related to qybv. the full-length amino acid comparison of chimv with gerv showed that their nucleocapsid proteins n (s-segment) have only a . % identity. the similarity of complete amino acid sequences of rdrp (l-segment) is . %. the similarity of the polyprotein precursor of gn/gc is . %. the proteins of chimv have . À . % aa (n-protein), . À . % aa (gn/gc precursor), and . À . % aa (rdrp) identities with their counterpart proteins in other nairoviruses. among these nairoviruses, chimv has the highest level of similarity with iskv, which is associated with bats in central asia (figures . À . ) . arthropod vectors. most isolations of chimv were obtained from ornithodoros tartakovskyi ticks. these ticks are common in the irano-turanian and mountain provinces of asia (kazakhstan, the central asian republics, northeastern iran, and china (xinjiang)). the western border of the area in question is the eastern shore of the caspian sea ( À e), the eastern border is in xinjiang ( e) , and the northern border is À n. the typical biotopes that o. tartakovskyi ticks inhabit are the foothills of dry steppes with loess soils. the ticks also inhabit meadow steppes and deserts (floodplain terraces and canals). o. tartakovskyi ticks prefer burrows of small diameter (inhabited by rodents, including jerboas, ground squirrels, small predators, and hedgehogs, as well as by turtles and birds). synanthropic biotopes are rarely inhabited. vertebrate hosts. the great gerbil (family muridae, subfamily gerbillinae, genus rhombomys) is distributed from the shores of the caspian sea on the plains of central asia and southern kazakhstan, to the deserts of central asia, iran, and afghanistan, and on eastward to northern china and inner mongolia. great gerbils are typical inhabitants of sandy deserts and form a colony with complex multistory burrows that have a large number of entranceways and egresses (up to À ). these burrows are a specific biotope that exists for many decades, and they maintain natural foci (in particular, of plague) in arid areas. , animal infection. the significance of chimv in the pathology of humans is unknown. antibodies to chimv have been found in camels ( . %) in the kashkadarya region in uzbekistan. this finding shows the ability of chimv to infect camels, as does qybv, but additional studies are necessary to clarify the pathogenicity of chimv in humans and cattle. history. grnv (strain leiv- az) was isolated from ornithodoros verrucosus (family argasidae, subfamily ornithodorinae) ticks collected in a burrow of red-tailed gerbils (meriones (cricetidae) erythrurus) near geran station, goranboy district, azerbaijan; figure . ). serological methods have failed to identify grnv, but the virus has been sequenced and classified into the nairovirus genus (family bunyaviridae). taxonomy. the genome of grnv was sequenced by a next-generation sequencing approach. full-length genome analysis revealed that the genetic similarity of grnv to other known nairoviruses is, on average, À % aa for the nucleocapsid protein (n, s-segment), À % aa for the polyprotein precursor of the proteins gn and gc (m-segment), and . À . % aa for rdrp (l-segment). the highest level of similarity all three proteins of grnv have is to that of chimv ( . À . % aa identity) and that of iskv ( . À . % aa identity). , further analysis based on a comparison of partial sequences of the conservative core domain of rdrp of the nairoviruses showed that grnv and chimv were most closely related to qybv, which is the prototypical virus of the group of the same name. the nucleotide sequence of the rdrp core domain of grnv has . % nt and . % aa identities with the counterpart sequence of qybv. the data obtained allow grnv to be classified as a virus of the qybv group (figures . À . ). the phylogenetic relationship between grnv and qybv corresponds to their similar ecological characteristics. qybv was first isolated in by r. taylor and h. dressler from argasid ornithodoros erraticus ticks collected in a rodent burrow in the nile river delta near qalyub village, egypt ( n, e). À complementbinding antibodies to qybv were found in humans ( . %), camels, donkeys, pigs, buffalos, dogs, and rodents. , the antigenic group of qalyub, a group that includes qybv and antigenic-related bdav, is one of the prototypical groups of the nairovirus genus. , previously, qybv had been repeatedly isolated from o. erraticum collected in the burrows of rodents (arvicanthis) in africa. the second member of the qybv group, bdav, was isolated from o. sonari (a member of the o. erraticus group) collected in the burrows of rodents (mainly mastomys) in senegal. , the isolation of gerv, which is closely related to qybv, is the first confirmation of the circulation of qybv group viruses in transcaucasia. arthropod vectors. the area of distribution of o. verrucosus ticks covers the southern part of moldova as well as ukraine and the caucasus region, and is limited by n latitude. the area includes the southern part of russia (the krasnodar and stavropol regions), the northern and eastern foothills of dagestan, the foothills and lowland hills of georgia, the valleys of the hrazdan river in armenia, the foothills of the lesser caucasus mountains in azerbaijan, and the gobustan plateau and the absheron peninsula, also in azerbaijan. o. verrucosus ticks inhabit shelter biotopesin particular, the burrows of red-tailed gerbils (meriones (cricetidae) erythrurus), animals that are common in central asia, southern kazakhstan, and eastern transcaucasia. redtailed gerbils tends to inhabit desert and semidesert landscapes. their burrows are deep and may have À entranceways and egresses. history. iskv (prototypic strain, leiv- k) was originally isolated from a pool of internal organs (liver, spleen, brain) of nyctalus noctula bats, and their ticks (argas (carios) vespertilionis) were collected near issyk-kul lake in kyrgyzstan in (figure . ). , subsequently, iskv was isolated from other bat species of the vespertionidae family (vespertilio serotinus, vespertilio pipistrellus, myotis blythii, rhinolophus ferrumequinum), and from birds, in different regions of kyrgyzstan and tajikistan. À two strains were isolated from anopheles hyrcanus mosquitoes and culicoides schultzei biting midges, respectively (figure . , table . ). , , complement-fixation testing showed that iskv is closely related or identical to the keterah virus, which was isolated from scotophilus temminckii bats and a. pusillus ticks in malaysia in . , a strain that has a close, one-sided antigenic relationship to iskv, leiv- taj (named garm virus), was isolated from a common redstart (phoenicurus phoenicurus) caught in the village of garm, tajikistan, morphological studies by electron microscopy characterized iskv as a member of the bunyaviridae family, and because no antigenic relation to any known viruses was found, it was assigned to the unclassified bunyaviruses. taxonomy. the genome of the prototypical strain of iskv, leiv- k, was sequenced, and, on the basis of sequence analysis, the virus was classified into the nairovirus genus. like the genomes of other nairoviruses, that of iskv consists of three segments of rna (in negative polarity), each of which has a single orf-encoded nucleocapsid protein (n, aa, s-segment), a polyprotein precursor of the envelope glycoproteins gn and gc ( , aa, m-segment), and a rdrp ( , aa, l-segment). a pairwise comparison of the full-length nucleotide and deduced amino acid sequences of the iskv orfs with those of other nairoviruses revealed . À . % nt ( . À . % aa), . À . % nt ( . À . % aa), and . À . % nt ( . À . % aa) identity for rdrp, the precursor of gn and gc, and the n protein, respectively (table . ) . phylogenetic analysis carried out for the fulllength amino acid sequences by the maximumlikelihood nearest-neighbor method showed that iskv occupies a new and distinct branch on the phylogenetic trees relevant to all three nairovirus proteins (rdrp, gn/gc, and n) (figures . À . ) . for the many known nairoviruses (i.e., qybv, dgkv, and hugv, as well as for a new nairovirus that was found in european bats by a metagenomics approach), there are only partial sequences of the conservative catalytic core domain of rdrp. , , the level of identity for this domain of iskv with other nairoviruses ranged from . À . % for the nucleotide sequence and . À . % for the amino acid sequence (table . ). the iskv rdrp core domain has the highest level of identity with qybv ( . % nt and . % aa). the phylogenetic tree constructed on the basis of the amino acid alignment of the rdrp core domain of nairoviruses confirms the topology of iskv on a new genetic branch of the nairoviruses (figures . À . ) . arthropod vectors. most isolates of iskv were obtained from argas vespertilionis ticks, and we can assume that these ticks are the main natural reservoir of the virus. the range of ticks of the a. vespertilionis group covers territory in central asia, africa, oceania, and australia ( figure . ) . vertebrate hosts. the natural vertebrate hosts of iskv are apparently bats-specifically, the genera nyctalus, vespertilio, rhinolophus, and myotis (family vespertilionidae). these bats are common in the temperate and subtropical zones of europe, asia, and north africa, and widespread iskv transmission and the appearance of an emergency are possible in all of their territories. human pathology. the first case of issyk-kul fever was registered in tajikistan in august when a staff member became ill after catching bats during surveillance for arbovirus. iskv was isolated from his blood on the second the disease occurs with fever ( À c), headache ( %), dizziness ( %), hyperemia of the throat ( %), cough ( %), and nausea ( %). the outcome is generally favorable, and no deaths have been registered. most of the cases were associated with the presence of bats in the attic of the residence. the primary route of human infection was apparently by argasid ticks, but respiratory or alimentary routes (via the feces and urine of bats) could not be excluded. furthermore, a laboratory experiment showed that iskv can be transmitted by aedes caspius mosquitoes. the percentage of the population immune to iskv in the southern part of tajikistan is . %. in kyrgyzstan, antibodies to iskv have been found in . À . % of the human population. the highest percentage ( %) with antibodies to iskv was found in the southeastern part of turkmenistan. history. uzun-agach virus (uzav), strain leiv-kaz , was isolated from the liver of a myotis blythii oxygnathus (order chiroptera, family vespertilionidae) bat caught in the vicinity of the village of uzun-agach, alma-ata district, kazakhstan, during the virological sounding of territory in central asia and kazakhstan in (figure . ). À on the basis of virion morphology, uzav was classified into the bunyaviridae family. no serological study of uzav was ever conducted, but the place of uzav isolation, uzun-agach, is close to where iskv was originally isolated, namely, near issyk-kul lake, and the source of both viruses is the same: bats. , taxonomy. the full-length genome of uzav was sequenced, and, on the basis of phylogenetic analysis, the virus was classified into the nairovirus genus. the genome of uzav, like those of other nairoviruses, consists of three segments of ssrna with negative polarity. the l-segment encodes rdrp ( , aa), the m-segment encodes a polyprotein precursor of the envelope glycoprotein gn and gc ( , aa), and the s-segment encodes the nucleocapsid protein n ( aa). a pairwise comparison of the sequence of the uzav genome with those of other nairoviruses showed that the virus is related most closely to iskv. full-length sequences of the l-and m-segments of uzav have, respectively, . % nt and . % nt identities with those of iskv. amino acid sequences of rdrp (s-segment) of uzav and iskv have . % aa similarity. the similarity of the amino acid sequences of the precursor of gn and gc for uzav and iskv is . % aa. a comparison of the s-segments of uzav and iskv revealed that they are almost identical ( . %). thus, we can conclude that uzav is a reassortant virus that got an s-segment from iskv. phylogenetic analysis based on l-and m-segments placed uzav in the lineage of iskv (figures . À . ). , vertebrate hosts. the vertebrate host of uzav is apparently bats, but because only a single isolation was obtained, this assertion is speculative. the finding that uzav is a reassortant virus closely related to iskv suggests that uzav occupies the same ecological niche as iskv and therefore is associated with bats and their argasid ticks. myotis blythii oxygnathus, the bat from which uzav was isolated, is common in the southern parts of the russian plain and in western siberia, caucasia, kazakhstan, southern europe, northern africa, middle and central asia, iran, and iraq. bats are important natural reservoir of emerging viruses. À iskv and uzav are the first nairoviruses that appear to be associated with bats. sakhalin virus (sakv) has been isolated from ixodes (ceratixodes) uriae (family ixodidae, subfamily ixodinae) ticks, which are obligate parasites of auks (family alcidae). the prototypical strain of sakv (leiv- c) was isolated in from i. uriae ticks collected in a colony of the common murre (uria aalge) on tyuleniy island near the southeastern coast of sakhalin island in the sea of okhotsk ( n, e; figure . ). À subsequently, strains of sakv were isolated from i. uriae ticks on tyuleniy island and iona island in the sea of okhotsk, the commander islands in the barents sea, and the southeastern coast of the chukotka peninsula in the bering strait (table . ). À on the basis of virion morphology, sakv has been classified into the bunyaviridae family. sakv was the first of the eponymous viruses, which together have formed a basis for the nairovirus genus. paramushir virus (pmrv), prototypical strain, leiv- , a virus of the sakv group, was originally isolated from ixodes signatus ticks collected in in a colony of cormorants (phalacrocorax pelagicus) on paramushir island (in the kuril islands) ( n, e; figure . ). , later (in À ), strains of pmrv were isolated from i. uriae ticks, collected in the nests of auks (family alcidae) on tyuleniy island in the sea of okhotsk and on the commander islands in the bering sea (table . ). À at least five nairoviruses are included in the sakv group. , , À avalon virus (avav), which was isolated from engorged imagoes and nymphs of i. uriae collected in l. argentatus nests on great island, newfoundland, , in , is apparently identical to pmrv. , several strains of avav were isolated in in cap sizun, brittany, france. clo mor virus (cmv) was isolated in from nymphal i. uriae ticks collected in a uria aalge colony of clo mor, cape wrath, scotland. cmv was found to be closely related to sakv in a complementfixation test. two strains of cmv were isolated from i. uriae collected in seabird colonies on lundy island (england) and the shiant isles (scotland) ( table . ) . , rukutama virus (rukv) (strain leiv- s), which previously had been included in the sakv group, is now classified into the uukuniemi virus (uukv) group in the phlebovirus genus. , taxonomy. complete genomes of sakv (strain leiv- c) and pmrv (leiv- k) were sequenced. also, partial sequences of rdrp of tillamook virus (tillv, identical to sakv), isolated from i. uriae ticks on the pacific coast (oregon) of the united states, are available (table . ). a full-length genome comparison showed that sakh and pmrv respectively share . % nt and . % aa identities in rdrp (l-segment), . % nt and . % aa in the precursor of gn and gc (m-segment), and . % nt and . % aa in the nucleocapsid protein (s-segment). sakv n-protein ranges from % (casv, hugv) to % (cchfv) similarity to other nairoviruses. the similarity of rdrp and the precursor of gn and gc proteins of sakv to other nairoviruses ranges from . % (casv, hugv) to . % (cchfv), respectively, and from . % (ervev, tfav) to . % (nsdv, dugv), respectively. arthropod vectors. it has been shown that the infection rate of infected ixodes uriae imagoes is times higher than of the species' nymphs and times higher than that of the larval stage. transovarial transfer of sakv has been found to be %. the infection rates of male and female ticks are approximately the same. the hypostome of male i. uriae ticks is vestigial; therefore, they cannot be infected by breeding on infected birds. the infection rate of i. uriae imagoes is at least times higher than that of i. signatus imagoes. À , , some other species of ixodes ticks are parasites of seabirds and may be an additional reservoir of sakv. i. auritulus and i. zealandicus ticks are distributed from alaska to cape horn in south and north america. laboratory experiments have demonstrated that aedes aegypti and culex pipiens molestus mosquitoes can be infected by sakv as they suck blood. the virus was found in mosquitoes on , , and days after infection in titers . , . , and . log (ld )/ μl, respectively. however, it was shown that infected mosquitoes could not transmit the virus to mice through a bite. , vertebrate hosts. ixodes uriae ticks and their host, the common murre (uria aalge), are a natural reservoir of sakv. pelagic cormorants (phalacrocorax pelagicus) and their obligate parasites (i. signatus) likely have only an additional influence. antibodies to sakv have been found in the common murre (u. aalge), pelagic cormorants (p. pelagicus), fulmars (fulmarus glacialis), tufted puffins (lunda cirrhata), and black-legged kittiwakes (rissa tridactyla) in the far east. À , a serological examination of birds via an indirect complement-fixation test revealed that the northern boundary of the range of sakv is the commander islands, where antibodies have been found in . % of birds. the southernmost place where antibodies have been detected ( . % birds) is kunashir island in the kuril islands. antibodies were found most often (in . À . % of birds) in the central part of the basin of the sea of okhotsk (on sakhalin island, tyuleniy island, and iona island). antibodies were also found in the red-necked phalarope (phalaropus lobatus), sanderling (calidris alba), the long-toed stint (c. subminuta) (up to . % of the population), fulmars (f. glacialis) ( . %), leach's petrels (oceanodroma leucorhoa), tufted puffins (l. cirrhata) ( . %), the common murre (u. aalge) ( . %), japanese , , neutralizing antibodies to avav, a virus closely related to pmrv, have been found in . % of puffins (fratercula arctica), petrels (calonectris leucomelas), and herring gulls (larus argentatus) in canada. , findings of antibodies to sakv in seabirds carrying out their annual seasonal migration to the southern hemisphere suggest the possibility of transcontinental transfer of the virus to the southern hemisphere. the closely related taggert virus (tagv) was isolated from ixodes uriae ticks in penguin colonies on macquarie island, a phenomenon that may indicate a transfer of viruses by birds and their ticks between the northern and southern hemispheres. human infection. three human cases of cervical adenopathy associated with avav were described in france. serological examination of farmers in cap sizun, brittany, france, found only % of the population positive. history. tamv (prototypal strain, leiv- uz) was originally isolated from hyalomma asiaticum asiaticum (family ixodidae, subfamily hyalomminae) ticks collected from sheep in the arid landscape near the town of tamdybulak subsequently strains of tamv were isolated in uzbekistan, À turkmenistan, À kyrgyzstan, , kazakhstan, , , armenia, , and azerbaijan , À in À (table . ) . most of the strains were obtained from h. asiaticum ticks, but several were isolated from birds, mammalians (including bats), and sick humans. on the basis of virion morphology, tamv has been classified into the bunyaviridae family. serological studies by complement-fixation and neutralization tests revealed no antigenic relationships of tamv with any known viruses. taxonomy. three strains of tamv isolated in uzbekistan (leiv- uz), armenia (leiv- ar), and azerbaijan (leiv- az) were completely sequenced. phylogenetic analysis of the full-length sequences showed that tamv is a novel member of the nairovirus genus, forming a distinct phylogenetic lineage (figures . À . ). the similarity of the amino acid sequence of tamv rdrp (l-segment) with those of other nairoviruses is % aa, on average. the similarity of the rdrp of tamv with that of the nairoviruses associated predominantly with ixodid ticks (cchfv, hazara virus (hazv), and dugv) is higher ( % aa) than that with viruses associated with argasid ticks (iskv and casv) ( % aa). the similarity of the tamv polyprotein precursor of cn and gc with that of other nairoviruses is less than % aa. the similarity of the amino acid sequence of the nucleocapsid protein (s-segment) of tamv is % aa with ixodid nairoviruses and % aa with argasid nairoviruses. phylogenetic analysis of the catalytic core domain of the rdrp of the nairoviruses confirms that tamv forms a novel group in the nairovirus genus (figures . À . ). genetic diversity among the three sequenced strains of tamv is low. the prototypic strain leiv- uz, isolated in central asia, has % nt identity in the l-segment with leiv- az from transcaucasia. the l-segment of the strain leiv- ar has . % nt and . % aa identity with the l-segment of leiv- uz. the similarity of the m-segment of leiv- uz with those of leiv- az and leiv- ar is % nt and % aa, respectively. the similarity of the s-segment among the three strains is À % nt. arthropod vectors. h. asiaticum ticks are apparently a main reservoir of tamv. more than half ( %) of tamv isolations were obtained from h. asiaticum asiaticum ticks, % from h. asiaticum, % from h. anatolicum, % from h. marginatum, % from rhipicephalus turanicus, and % from haemaphysalis concinna. the infection rates of male and female ticks in endemic territory were : and : , respectively. the infection rate of h. asiaticum nymphs was times lower. , , , furthermore, tamv was isolated from larvae of h. asiaticum, which were hatched from eggs in the laboratory, indicating transovarial transmission of the virus. h. asiaticum asiaticum ticks are the most xerophilous subspecies of the hyalomma genus (ixodinae subfamily), a characteristic that allows tamv to be distributed over the karakum desert in turkmenistan, the moinkum desert in kazakhstan, and the central part of the kyzyl kum desert in kazakhstan and uzbekistan. . animal hosts. the larvae of h. asiaticum feed on ruminants, hoofed animals, small predators, hedgehogs, birds, and reptilians. one of the major hosts of h. asiaticum preimagoes is the great gerbil (rhombomys opimus). wild animals, as well as sheep and camels, are the hosts for h. asiaticum imagoes and may be involved in the circulation of tamv (table . ). human pathology. sporadic cases of the disease associated with tamv was registered in kyrgyzstan in october , when tamv was isolated from the blood of a patient with fever ( c), headache, arthralgia, and weakness. h. asiaticum asiaticum ticks rarely attack humans, and no outbreaks of tamv fever have been registered; however, human infection by h. asiaticum ticks is still possible concinna (ixodidae, haemaphysalinae) during À . , according to preliminary information, burv is not able to agglutinate erythrocytes of birds and mammals and has no antigenic relationships with arboviruses from different groups of the togaviridae, taxonomy. the genome of burv was sequenced, and the virus was classified into the nairovirus genus, family bunyaviridae. the genome consists of three segments: an l-segment (orf, , nt; encodes rdrp); an m-segment (orf, , nt; encodes a polyprotein precursor of the envelope proteins gn and gc); and an s-segment (orf, , nt; encodes the nucleocapsid protein n). , a comparison of rdrp sequences of burv with those of other nairoviruses demonstrated that the virus is distantly related to tamv ( % aa similarity). the similarity of the rdrp catalytic core domain of burv to that of tamv is % aa, compared with about % aa for viruses in other phylogenetic groups. the level of similarity for the nucleotides sequences of this part of the rdrp of burv is % nt with those of tamv and À % nt with those of other viruses (figure . ). the m-segment of burv has a long orf and encodes a polyprotein precursor of the envelope glycoproteins gn and gc. the size of the polyprotein precursor is , aa. the mature gn and gc proteins of nairoviruses are formed by complex processes involving cellular peptidases. by the netnglyc . server, potential glycosylation sites were predicted, with only within mature gn or gc proteins. , the level of similarity of the amino acid precursor of gn and gc in burv is % with that of tamv and no more than % with viruses of other phylogenetic groups. phylogenetic analyses based on a comparison of the full-length polyprotein precursor demonstrated the position of burv on the tamv branch and was consistent with the rdrp data ( figure . ). the s-segment of nairoviruses encodes a nucleocapsid protein (n). , the size of the burv nucleocapsid protein is aa, corresponding to the average size of the n protein of other nairoviruses ( À aa). the level of similarity of the amino acid sequence of burv n protein with that of tamv is %, and that with the amino acid sequences of other nairoviruses is À %. phylogenetic analyses of burv n protein are represented in figure . . the phylogenetic position of burv is on the tamv branch, despite the virus's having the lowest level of similarity of the n protein compared with that of other virus proteins. arthropod vectors. as mentioned earlier, six strains of burv were isolated from the ticks haemaphysalis punctata (five strains) and haem. concinna (one strain) in À . the rate of infected ticks was . À . %. burv is associated with haem. punctata and haem. concinna ticks in pasture biocenoses. the virus is phylogenetically close to tamv, which is also associated with ixodes ticks in pasture and desert biocenoses. the orthobunyavirus genome consist of three segments of single-stranded negative-sense rna designated as large (l), medium (m), and small (s) (figure . ). the l-segment of the prototypical bunv ( , nt in length) encodes the viral rdrp. the m-segment ( , nt) encodes two surface glycoproteins (gn and gc) and a nonstructural protein (nsm). , the s-segment ( nt) encodes the nucleocapsid protein (n) and a nonstructural protein (nss). the nss protein is considered a pathogenic factor for vertebrates, because it may act as an antagonist of interferon, which is involved in blocking the host's innate immune responses. À , to olyka virus, isolated in from an. maculipennis mosquitoes collected in western ukraine; À and to chittoor virus, isolated in from an. barbirostris mosquitoes collected in brahmanpally, chittoor district, andhra pradesh state, india. the african ngari virus (nriv) is reassortant between batv and bunv. , in russia, batv was repeatedly isolated in different regions (figure . ). anadyr virus (anadv), strain leiv- , was isolated by s.d. lvov from a pool of aedes mosquitoes collected in september in a swamp tundra landscape near the village of ukraine, and russia are members of the european group. two strains of batv-leiv-ast - - and leiv-ast - - -isolated in russia were completely sequenced and placed into the cluster of the european strains. within this group, they are phylogenetically close to strain , isolated in the volgograd region in from anopheles messeae (maculipennis) mosquitoes, for which the partial nucleotide sequences of the l-and m-segments are known. between the strains leiv-ast - - and leiv-ast - - , there is very high level of nucleotide and amino acid identity of three segments of the genome: . / . % (l-segment/rdrp), . / . % (m-segment/ polyprotein predecessor), and . / . % (s-segment/nucleocapsid). the levels of nucleotide identity of strain with these strains on partial sequences of l-and m-segments are . / . % and / %, respectively; that is, for the m-segment, all available nucleotide polymorphisms are synonymous. the lowest observed genetic differences and the temporal and geographical proximities of the various strains of these viruses suggest a common origin as different isolates of the same strain of batv circulating in the southern part of russia. phylogenetic analysis of anadv (strain leiv- ) revealed its similarity to batv. the l-segment of anadv is from . % to . % identical with those of the different batv strains (figure . , table . ). the identity of the l-segment of anadv with the l-segments of other viruses of the bunyamwera group is . % (bunv), . % (cvv), and . % (tenv). the amino acid and is about . % with tenv and cvv. the amino acid similarity of the nucleocapside protein is . % with that of batv from uganda. phylogenetic analysis of the nucleotide sequences of the s-, m-, and l-segments conducted with the use of a maximum-likelihood algorithm placed anadv (leiv- ) on a distinct branch of the dendrogram that considers it a new representative of the bunyamwera group. arthropod vectors. batv has been reported in sudan, africa. the distribution of batv in southeastern asia includes malaysia, india, sri lanka, thailand, cambodia, and japan, , while in europe batv is distributed over austria, germany, yugoslavia, moldova, ukraine, belarus, and other countries. , À in central europe, batv was isolated from anopheles claviger, an. maculipennis (an. messeae), coquillettidia richiardii, aedes (ochlerotatus) punctor, and ae. communis. , , a wide distribution of batv in different landscape belts of the european part of russia, as well as in siberia and the far east, was demonstrated: in the temperate belt the main source of batv isolation was the zoophilic anopheles genus, whereas in high latitudes (tundra, northern taiga) it was the aedes genus. , À in the european part of russia, batv has been isolated in the northern (komi republic), middle (vologda region), and southern (leningrad, yaroslavl, and vladimir regions; , , , , in the southern hyperendemic regions of russia, the main vector of batv is an. messeae. according to our data, the infection rate of an. messeae in the middle belt of the volga delta (astrakhan region) reaches . % (approximately infected mosquito out of ). because this species of mosquito attacks mainly domestic animals, it serves as a biological barrier, reducing risk of infection to humas. in the northern areas (the subarctic, the northern taiga), batv circulation is due mainly to aedes mosquitoes: ae. communis complex and ae. punctor. under experimental conditions, batv was isolated from hibernating females of an. messeae. hibernation is one of the mechanisms by which batv survives during the winter. , vertebrate hosts. in anthropogenic biocenoses of the southern regions of russia, domestic animals are the main vertebrate reservoir, because they (especially cattle) are the main hosts for an. messeae. batv-neutralizing antibodies were found in india among rodents (mus cervicolor ( . %), rattus exulans ( . %), rattus rattus ( . %), bandicota indica ( . %)) and bats (cynopterus sphinx) ( . %). , this indicator is significantly higher in india among domestic animals: goats ( . %), camels ( %), cows ( . %), and buffalos ( . %). in finland, anti-batv antibodies occasionally were found among cows ( . %), but not among reindeers. the chittoor strain is associated with mild illness, but is pathogenic to sheep and goats. batv was isolated from birds: crows (corvus corone), coots (fulica atra), and grey partridges (perdix perdix). persistent avian infection was established experimentally with reactivation of viremia by cortisone six months after the acute infection period. an investigation of , sera of domestic animals in russia during À revealed anti-batv antibodies among these animals significantly more often than among people (table . ). the largest immune layer was found in populations of horses (up to %), cattle ( À %), sheep (up to %), and camels in forestÀsteppe, semidesert, and desert landscape belts. in contrast to the situation in finland, antibodies were found in reindeer sera in a tundra landscape belt of the chukotka peninsula. no examinations of vertebrates in natural biocenoses were conducted. epidemiology. epidemic outbreaks and sporadic cases caused by batv, as well as outbreaks of hemorrhagic infection caused by ngari virus, have been reported. , , , , to date, no cases of laboratory infection are known. according to a serological examination of , people in the endemic regions of russia, about À % withstand batv infection in an asymptomatic form. the highest infection rate was established in forestÀsteppe and steppe belts. (however, as a rule, the rate is higher for domestic animals than humans.) some northern areas in russia became hyperendemic for no apparent reason. , , pathogenesis. no pathogenetic mechanism during batv infection in humans has yet been described in detail. there are experimental data, however, on batv infection in primates: green monkeys (chlorocebus sabaeus) were found to be carriers of the virus days after inoculation (the observation period); the virus was pantropic, destroying small vessels and producing vasculitis and perivascular focal lymphohistiocytic infiltrates. clinical features. the disease etiologically linked with batv proceeds mainly as influenzalike disease complicated by meningitis, malaise, myalgia, and anorexia. , , , , at the same time, ngari virus (reassortant between batv and bunv) infection in east africa appears as outbreaks of hemorrhagic fever. diseases associated withtheclosely related ilev in africa and madagascar also proceed with hemorrhagic phenomena and with lethal outcomes. , diagnostics. a highly specific test based on rt-pcr has been developed, as have elisa tests for the detection of specific anti-batv igm and igg. genome and taxonomy. the genome of the ce group of viruses consists of three segments of ssrna with negative polarity. the l-segment of lacv, a prototypical virus of the group, is , nt in length, the m-and s-segments , and nt, respectively. as in other bunyaviruses, the l-segment encodes rdrp, the m-segment a polyprotein precursor of the envelope glycoproteins gn and gc, and the s-segment nucleocapsid protein (n). two nonstructural proteins are found in infected cells: nss, which encodes by adding an orf in the s-segment; and nsm, which forms during the maturation of the gn and gc proteins from the precursor. , but viruses of the ce serocomplex were isolated from ae. albopictus (a known vector for at least arboviruses), which was imported from southeastern asia and spread into states of the united states. , transovarial transmission was established in ae. vexans and cs. annulata. overwintering of tahv was documented in cx. modestus and cs. annulata females. mosquito species have been defined and classified only partially in connection with the huge volume of this laborious work. the majority of strains were isolated from pools of mosquitoes belonging to different species. of strains that were isolated ( strain was isolated from a wild population of the common house mouse, mus musculus), only were isolated from strictly defined species (table . ). the other strains were isolated from aedes mosquitoes of unidentified species: % of strains were from ae. communis, % from the mixed pools, in which ae. communis prevailed. strains were isolated from other species significantly less often. only one strain was isolated from anopheles maculipennis (an. messeae) and culiseta alaskaensis. the dynamics of the seasonal infection rate of mosquitoes was investigated for two years on the model of the northern part of the russian plain and the eastern part of fennoscandia. in tundra, the epizootic period begins with the second decade of july and proceeds to the beginning of august, when the activity of mosquitoes comes to an end. in forest tundra, the epizootic period begins with the first decade of july and proceeds for . months; in the northern taiga, this period lasts at least months (julyÀaugust); in the middle and southern taiga, the first strains began to be isolated in the second decade of june. the mosquito infection rate increases significantly in the third decade of july and reaches a maximum in the middle to end of august, when the total number of mosquitoes decreases. , the data collected testify to an almost universal distribution of ce serocomplex viruses in all landscape belts, except the arctic, in all six physicogeographical lands examined in the north of russia, located on a territory of more than million km . the infection rate of mosquitoes increases (р , . ) in moving from the subarctic (tundra) ( . . %) to the landscape belt of the middle taiga ( . . %). this indicator in tundra and in the forest tundra is close to that in the southern taiga of the russian plain ( . %), in north america ( . %), and in the forest steppes of the russian plain ( . À . %). in the steppe belt of the russian plain, the infection rate of mosquitoes appeared to be the smallest ( . %). in the leaf forests of the russian plain ( . %) and of the former czechoslovakia ( . %), the infection rate of mosquitoes is comparable to that for landscape belts of the northern and middle taiga. to date, at least s ce serocomplex virus strains were isolated from mosquitoes in the central and southern parts of the russian plain. among them, strains were isolated from the blood and spinal fluid of patients, and strains from the internal parts of rodents ( from the bank vole, myodes glareolus; and from the wood mouse, apodemus sylvaticus). the infection rate of mosquitoes depends on the landscape belt and the particular season in which field material was collected. the rate decreases, as a rule, from the north to the south. data indicating an absence of viruses in semideserts can be explained by an insufficient quantity of mosquitoes collected, but in wet subtropical zones in azerbaijan ce serocomplex viruses were isolated from anopheles hyrcanus. in the southern taiga belt and mixed forests, the infection rate of mosquitoes was defined to be from the third week of may to the second week of august and two peaks were noted: at the end of june (the emergence of the first generation of aedes mosquitoes) and at the end of july to the beginning of august (the emergence of the second generation of aedes mosquitoes). in the majority of the southern belts, the infection rate was registered from the second week of june until the end of august with a small peak in the first week of august caused by the emergence of the second generation of aedes mosquitoes and by the peak of activity of culex, coquillettidia, and anopheles mosquitoes. in steppe and forestÀsteppe belts, ce serocomplex viruses were isolated from mosquitoes collected in the rostov and orenburg regions, as well as in the foothills of the caucasus mountains (krasnodar krai). most of the strains were obtained from aedes mosquitoes, which play the leading role in virus circulation. in these regions, anopheles mosquitoes join the virus population maintenance (three strains were isolated), being ecologically connected with agricultural animals and, because of that connection, playing an important role as an indicator species in anthropogenic biocenoses. in the center and south of the russian plain, there is a mix of populations of inkv, tahv, khtv. , vertebrate hosts. the principal vertebrate hosts of tahv in europe are lagomorpha (hares (lepus europaeus), rabbits (oryctolagus cuniculus), hedgehogs (erinaceus roumanicus), and rodents (rodentia)). experimental viremia has been established in lagomorphs, hedgehogs, ground squirrels (citellus citellus), muskrats (ondatra zibethicus), squirrels (sciurus vulgaris), martens (martes foina), polecats (putorius eversmanni), foxes (vulpes vulpes), badgers (meles meles), bats (vespertilio murinus), piglets, and puppies. , , , in total, strains of ce serocomplex viruses were isolated within all landscape belts of all physicogeographical lands (figure . , table . ). according to our data, the susceptibility of mosquitoes increased from the tundra to the northern and middle taiga; however, the highest indicators were noted to be in the forestÀsteppe and the steppe of western siberia (in altai krai). identification of these strains revealed at least three viruses of the ce complex: strains of tahv, of inkv, and strains of khtv. in all landscape belts east of the yenisei river (central and northeast siberia and the physicogeographical lands bordering the north pacific ocean), only khtv strains have been isolated. west of the yenisei river, inkv strains predominated in the tundra and the forestÀtundra of western siberia, whereas khtv prevailed in other landscapes located to the south. in the eastern part of fennoscandia and in the north of the russian plain, inkv and khtv strains were isolated in about equal proportions. the pattern of distribution of tahv, inkv, and khtv over northern eurasia suggests that the emergence of the ancestor of ce serocomplex viruses probably is connected to oligocene chineseÀmanchurian fauna of the deciduous forests of eastern siberia evolving into okhotsk fauna during the upper tertiary period. the okhotsk fauna, in its turn, extended in early glacial times to the north, the west, and partially to the east in tundra through ancient beringia and on into north america. the ancestral virus could then penetrate into north america together with this fauna and gradually extend in the southern direction, in the process laying the foundation for the appearance of some other viruses of the ce serocomplex now circulating mainly in north america. mercurator, nigripes, excrucians . maculipennis b . total a one strain was isolated from the genus culiseta. b one strain was isolated from the genus anopheles. the introduction of the virus population to the western hemisphere probably occurred through two pathways around the central siberian plateau: (i) through the tundra lying to the north of the plateau and (ii) through southern taiga and forestÀsteppe territories. these pathways can explain the modern predominance of khtv in the forestÀsteppe belt of siberia and in a taiga belt west of the yenisei river. in moving to other ecological systems further to the west, khtv could have been transformed partially to inkv and tahv. the inkv population penetrated into the western part of the eurasian subarctic through the taiga belt and occupied that part of eurasia, whereas tahv proceeded into the deciduous forests of europe, where it now prevails. epidemiology. cev is endemic in the united states in california, new mexico, texas, the southwestern part of virginia, tennessee, and kentucky. , sporadic morbidity with cns lesions occurs in those states, but the main morbidity is linked to lacv, which is endemic in states, predominantly the u.s. census bureauÀdefined east north central states (ohio, wisconsin, minnesota, iowa, and indiana), where morbidity reaches . À . %. cases of lacv-associated encephalitis are within the distribution of the main vector-aedes triseriatus-eastward from the rocky mountains. during the last few decades, natural foci in west virginia, north carolina, and tennessee, with sporadic cases occurring in louisiana, alabama, georgia, and florida, joined with previously known ones in wisconsin, illinois, minnesota, indiana, and ohio. thus, having traversed the distance from southeastern asia to north america, ae. triseriatus is now part of the north american virus circulation. the clinical picture varies from an acute fever syndrome (in some cases with pharyngitis and other symptoms of acute respiratory disease) to encephalitis. lethality is about . %. from to cases occur annually. generally, the virus attacks children age and under ( %), a phenomenon that may be explained by the existence of a layer of immunity in up to % of adults. jcv (in the united states and canada) and sshv (in the northern part of the united states and in canada) are associated with sporadic cases of fever and encephalitis. domestic dogs are susceptible to lacv, which provokes encephalitis. , , , the role of deer in virus circulation has been established as well. horizontal and vertical transmission of viruses provides an active circulation of the virus, a high rate of infection in mosquitoes, and stability of natural foci under the relatively rough conditions of the central and northern parts of the temperate climatic belt. all three viruses (inkv, khtv, and tahv) of the ce serocomplex distributed in eurasia have significance in human pathology. , these viruses were found in czechoslovakia in , , austria in , finland in , , romania in , norway in , the former ussr(in transcaucasia) in , and elsewhere in the european and asian parts of russia. , , , , , , À in europe, human disease associated with tahv presents as an influenzalike illness mainly in children with sudden-onset fever, headache, malaise, conjunctivitis, pharingitis, myalgia, nausea, gastrointestinal symptoms, anorexia, and (seldom) meningitis and other signs of cns lesions. , , À the circulation of ce serocomplex viruses was established in china, where they provoke human diseases with encephalitis as well as acute respiratory disease, pneumonia, and acute arthritis. in north america (the united states and canada), lacv is the most important of these viruses, but sshv also is associated with human disease. between and in the united states, , cases of ce were reported. , so, ce serocomplex viruses have circumpolar distribution. in russia, these viruses are found from subarctic to desert climes ( figure . , table . ). , according to our summary data for , sera, the number of people with specific antibodies to ce serocomplex viruses in the tundra and forestÀtundra belts ( . %) is significantly lower than the number in the north and middle taiga belts ( % and %, respectively). these data correlate with the infection rate of mosquitoes in those landscape belts. , results obtained from serological investigation of the human population correlate with those obtained from virological investigation of the mosquitoes (figure . ). the maximum immune layer of the healthy population is registered in the southern taiga. in the landscape and geographical zones located south of that landscape, a gradual decrease in this indicator takes place. specific antibodies to inkv are seen everywhere that this virus circulates. in forest-Àsteppes, specific antibodies to tahv and inkv are marked out with an identical frequency. in semideserts, anti-tahv antibodies are found twice as often as anti-inkv ones. the small number of strains isolated in these natural zones precludes establishing a relationship between the circulation of viruses and an immune layer of the population. active circulation of ce serocomplex viruses on the territory of russia results in regular registration of the diseases caused by these viruses. more than % of all seasonal fevers are etiologically linked to such viruses, and in some natural zones (the southern taiga and the mixed forests), this indicator increases to À %. in mixed forests, the main etiological role most often belongs to inkv ( . %), and in semideserts (astrakhan region) to tahv ( . %). the diseases caused by ce serocomplex viruses in the center and south of the russian plain start appearing during the middle of may and reach a maximum in almost equal titers of specific antibodies to more than one virus were revealed in patients ( . %) in a neutralization test. , , diseases were registered from may to september: in may, cases ( . %); in june, ( . %); in july, ( . %); in august, ( . %); and in september, ( . %). the seasonal dynamics in all landscape zones were identical: the maximum number of diseases is noted in julyÀaugust. diseases were registered everywhere in the form of sporadic cases and small outbreaks, but more often in the taiga and the deciduous forests of the european part of russia and western siberia. most patients were À years old, with those up to years making up . % of all people infected. pathogenesis. a systematic destruction of small vessels, together with the development of vasculitis and perivascular focal lymphohistiocytic infiltrates, underlies the pathogenesis of the diseases caused by ce serocomplex viruses. lesions in the lungs, brain, liver, and kidneys are the most frequent complications. , clinical features. the incubation period lasts from to days, but in some cases is only days. three main forms of disease linked with ce serocomplex viruses have been proposed: (i) influenzalike; (ii) with primary compromise of the bronchiopulmonary system; (iii) neuroinfectious, which proceeds with a syndrome of serous meningitis and encephalomeningitis. analysis of the clinical picture of cases examined showed that . % of cases proceeded without signs of cns lesion, . % with a syndrome of acute neuroinfection, and . % with radiologically uncovered signs of changes in the bronchiÀlung system. a comparison of clinical forms and etiologic agents showed that inkv and tahv often cause disease without cns lesions ( . % and . %, respectively) and that inkv plays the leading role in acute neuroinfection ( . %). the etiological role of khtv was established in cases without cns symptoms of lesions. eighty-three patients had an influenzalike form of the disease etiologically linked to ce serocomplex viruses. the incubation period was À days. the disease began abruptly, with a high temperature that reached a maximum of À c in . % of patients on the first day. the duration of the fever was . . days. one of the main symptoms was an intensive headache ( . . days in duration) that developed in the first few hours and was often accompanied by dizziness, nausea ( . %), and vomiting ( . %). , À a survey of patients revealed infection of the sclera ( . . %), hyperemia of the face and the neck ( . . %), and, in some cases ( . %), a spotty and papular rash on the skin of the trunk and the extremities. violations of the upper respiratory airways were characterized by hyperemia of the mucous membranes of the fauces ( . . %)and congestion of the nose and a dry, short cough ( . . %). with regard to the lungs, . . % of patients exhibited rigid breathing a dry, rattling cough during auscultation, and a strengthening of the bronchovascular picture on roentgenograms. among cns symptoms, the most common were a decrease in appetite, a stomachache without accurate localization and with liquid stool, and a small increase in the size of the liver with a short-term increase in aminotransferase activity in the blood. inflammatory changes in the bronchiÀlung system (bronchitis and pneumonia) occurred as well. in all cases in which it appeared, pneumonia had a focal character with full the etiological role of different ce serocomplex viruses has been established in % of cases with acute diseases of the nervous system (serous meningitis, encephalomeningitis, arachnoiditis, acute encephalomyelitis, and seronegative tick-borne encephalitis (tbe)): inkv ( . . %), tahv ( . . %), and unidentified ( . . %). the age of patients with cns lesions was from to years, with the majority ( . %) from age to . serous meningitis was observed in patients who arrived at the hospital a mean . days after symptoms appeared. the disease began abruptly. the majority ( . %) of patients complained of a high temperature that reached a maximum the first day, the duration of the fever was . . days, with a critical ( . %) or steplike ( . %) decrease. headache was noted in % of patients and was accompanied by dizziness in %. vomiting developed on the first ( . %) or the second ( . %) day and continued in . % of patients. meningeal signs appeared in . % of patients but were weak and dissociated in most cases, with only . % of patients exhibiting rigidity of the occipital muscles. the duration of the meningeal signs was . . days. the cells of the spinal fluid (investigated on the . th . day of the disease) was lymphocytic, mostly reaching three digits and up to cells ( . %); the protein concentration was reduced ( . . g/l) in . % of cases but was within the normal range ( . . g/l) in other cases. in . % of patients exhibiting acute neuroinfection symptoms of bronchitis and focal pneumonia, their condition was confirmed radiologically. encephalomeningitis caused by inkv was characterized by an abrupt beginning and fast development of focal symptomatology (ataxy, horizontal nystagmus, and discoordination) against a background of common infectious and meningeal syndromes, including inflammatory changes to the spinal fluid. , À the variability of the clinical picture of the diseases caused by ce serocomplex viruses and its similarity-especially at early stagesto that of other infections suggest the necessity of carrying out differential clinical diagnostics with a number of diseases. the influenzalike form needs to be differentiated, first of all, from influenza, especially in the presence of symptoms of neurotoxicity, as well as from other acute respiratory diseases (parainfluenza, adenoviral and respiratoryÀsyncytial diseases), pneumonia (including a mycoplasma and chlamydia etiology), and enteroviral diseases. the main epidemiological features and clinical symptoms that lend themselves to carrying out differential clinical diagnostics for the influenzalike diseases described here are presented in table . . note that considerable difficulties arise in implementing differential clinical diagnostics of the diseases that proceed with acute neuroinfection syndrome (serous meningitis, encephalomeningitis), especially when those diseases occur in the same season (tables . and . ). , , the main criteria in differential clinical diagnostics of the disease etiologically linked with ce serocomplex viruses are as follows (see tables . and . ): acute onset; high short-term fever ( À days, on average) reaching a maximum on the first day and decreasing critically at the end of the feverish period; and intensive headache, nausea, vomiting, and weakness. also observed are insignificant catarrhal phenomena (nose congestion, rare dry cough) or their complete absence. a radiograph of the chest reveals signs of bronchitis and focal pneumonia with poor clinical symptomatology. an examination of the liver shows that its size, as well as its aminotransferase activity, has increased. changes in urine, such as albuminuria and, in some cases, cylindruria, are frequently reported. finally, symptoms relating to the vegetative nervous system (hyperemia of the face and the neck, subconjunctival hemorrhage, bradycardia, and persistent tachycardia) can be observed, as can both cns lesions in the form of serous meningitis and encephalomeningitis in combination with compromise of the bronciopulmonary system, liver, and kidneys. diagnostics. specific diagnostics of the diseases etiologically linked with ce serocomplex viruses could be based on virological testing (using sensitive biological models of newborn mice or cell lines to isolate the strains) or on serological testing. in the presence of the sera taken from patients during the acute period of the disease (the first À days) and in À weeks, the best method of retrospective inspection is a neutralization test. a hemagglutination inhibition test is considerably less sensitive. both complement-binding reactions and diffuse precipitation in agar have no diagnostic value today. for serological reactions, it is necessary to utilize hktv, tahv, and inkv antigens simultaneously. (in reference labs, sshv antigen should be used as well.) a quadruple (or greater) increase in the titers of specific antibodies or the detection of specific antibodies in the second serological test in their absence in the first test are diagnostic criteria. elisa for igg indication and monoclonal antibody capture elisa (mac-elisa) for igm indication provide good diagnostic opportunities. control and prophylaxis. supervision of morbidity and of the activity of natural foci linked with ce serocomplex viruses offers the following instructions: (i) monitor the patient clinically and the disease epidemiologically. (ii) provide well-timed diagnostics and seroepidemiological investigations. (iii) track the number and specific structure of mosquito vectors and possible vertebrate hosts. history. khurdun virus (khurv), strain leiv-ast - (deposition certificate n , . . , in the russian state collection of viruses), was isolated from a pool of internal parts of the coot (fulica atra; order gruiformes, family rallidae), collected august , , in natural biomes in the western part of the volga river delta, in khurdun tract, ikryaninsky district, astrakhan region. later, nine more strains of khurv were isolated from f. atra and the cormorant phalacrocorax pygmaeus; order pelecaniformes: family phalacrocoracidae) in À (figure . ). at least six viruses associated with birds have been shown to circulate in the volga river estuary. , khurv has not been identified by any serological method, including sera against viruses of the flaviviridae, togaviridae, bunyaviridae, and orthomyxoviridae families. taxonomy. the genome of khurv was sequenced, and phylogenetic analysis revealed that it is a new representative of the orthobunyavirus genus (figures . À . ). the genome consists of three segments of ssrna with negative polarity-an l-segment ( , nt), an m-segment ( , nt), and an s-segment ( nt)-and has only À % identity with those of other orthobunyaviruses. the terminal -and -sequences of khurv genome segments, determined by rapid amplification of cdna ends, are canonical for the orthobunyavirus ( -ucaucacaug and cgtgtgatga- ). the l-segment of khurv has a single orf ( , nt) that encodes rdrp ( , aa). the similarity of khurv rdrp with those of the orthobunyaviruses is %, on average. the similarity of the conservative polymerase domain iii (a, В, c, d, and e motifs) in rdrp reaches % (in bunv). the М-segment of khurv is shorter than those of the orthobunyaviruses ( , nt vs. , nt for bunv). the М-segment of khurv has a single orf ( , nt), which encodes a polyprotein precursor ( aa) of the envelope glycoproteins gn and gc. apparently, the m-segment of khurv does not contain a nonstructural protein nsm, which is common in most of the orthobunyaviruses. , the putative cleavage site between gn and gc of khurv was found in position / aa (asa/en). this site corresponds to the cleavage site between nsm/gc of the orthobunyaviruses and the conservative amino acid a/Е (vaa/ee in bunv). the size of the gn protein of khurv is the same as that of the other orthobunyaviruses, aa. the similarity of khurv gn is À % aa, on average, to that of the other orthobunyaviruses ( . % aa to bunv). the size of the gc protein of khurv, aa, is shorter than that of the other orthobunyaviruses (cf. aa for the gc protein of bunv). the c-part (approx. aa) of the gc protein, which includes the conservative domain g (pfam ), has about % aa similarity to the c-part in the other orthobunyaviruses, whereas the n-part (approximately aa) has no similarity to that of any proteins in the genbank database. the s-segment of khurv is nt in length and encodes a nucleocapsid protein ( aa). the similarity of the n protein to that of the orthobunyaviruses is À aa%. most orthobunyaviruses have an additional orf that encodes arthropod vectors. there are no known arthropod vectors of khurv; the virus has been isolated only from birds. more than , aedes, culex, and anopheles mosquitoes were examined during the survival period for arboviruses in this region, and no khurv isolations were obtained. the family ceratopogonidae of biting midges is a potential vector of khurv, but these insects have not been surveyed. vertebrate hosts. all isolations of khurv were obtained from birds. nine strains of the virus were isolated from coots (fulica atra). (one hundred seventeen birds were examined and were found to have an infection rate of . %.) one strain was isolated from the pygmy cormorant (phalacrocorax pygmaeus). (two hundred eighty-nine cormorants, mostly ph. carbo, were examined and were found to have an infection rate of . %.) the phlebovirus genus comprises about viruses that are divided into two main groups based on their ecological, antigenic, and genomic properties: mosquito-borne viruses and tick-borne viruses. , the genome of the phleboviruses consists of three segments of ssrna with negative polarity: l (about , nt), m (about , À , nt), and s (about , nt) (figure . ). in general, the structure of the genome is the same for mosquito-borne and tick-borne phleboviruses, but the m-segment is shorter in tick-borne viruses and it does not encode the nonstructural protein nsm. phylogenetically, the phleboviruses can be divided into two branches in accordance with their ecological features. the tick-borne phleboviruses comprise viruses of the uukuniemi group, the bhanja group, and the two novel related viruses severe fever with thrombocytopenia syndrome virus (sftsv) and heartland virus (hrtv), which form separate clusters and are unassigned to any group (figures . À . ). the uukv serogroup currently comprises viruses, but the status of some of them may be revised with the accumulation of more genomic and serological data. history. bhanja virus (bhav) was originally isolated from haemaphysalis intermedia ticks that were collected from a paralyzed goat in the town of bhanjanagar in the ganjam district in the state of odisha, india, in and was assigned to the unclassified bunyaviruses. in europe, the first isolation of bhav was obtained from adult haem. punctata ticks collected in italy ( ) and then in croatia and bulgaria. , , palma virus (palv), a virus closely related to bhav, was isolated from haem. punctata ticks in portugal. two viruses-kismayo virus (kisv) and forécariah virus (forv)-antigenically related to bhav were isolated in africa. , these viruses have been merged into the bhanja group on the basis of their serological cross-reactions. , in transcaucasia, bhav (strain leiv- az) was isolated from ixodidae ticks rhipicephalus bursa collected from cows in ismailli district, azerbaijan, in ( figure . ). closely related to bhav, razv (strain leiv- arm) was isolated from ixodid ticks dermacentor marginatus collected from sheep near the village of solak in the razdan district of armenia ( figure . ). , serological methods (detection of antibodies in animals and humans) have shown that bhav circulates in many mediterranean countries, the middle east, asia, and africa. , taxonomy. viruses of the bhav group are not antigenically related to any of the other bunyaviruses, but they were assigned to the phlebovirus genus on the basis of a genetic analysis of their full-length genome sequences. , , weak antigenic relationships were found between bhav and sftsv, a novel phlebovirus isolated in china. , , sftsv, in its turn, is antigenically related to viruses of the uukuniemi group. the genomes of certain viruses of the the m-segment of bhav ( , nt) encodes a polyprotein precursor ( , aa) of the envelope glycoproteins gn and gc. like the m-segments of other tick-borne phleboviruses, that of bhav has no nsm proteins that are common to mosquitoes-borne phleboviruses. the predicted cleavage site between gn and gc proteins has been found by signal ip software (http://www.cbs.dtu.dk/services) to be in position / of the polyprotein precursor (motif mhmalc/cdesrl). a dipeptide cd in the cleavage site is also typical for sftsv and hrtv, which were associated with human disease in china and the united states, respectively. , , other phleboviruses, including uukv and rvfv, contain a dipeptide cs in this position. the s-segment ( , nt) of bhav has two orfs (n and nss proteins) disposed in opposite orientations (an ambisense expression strategy) and separated by an intergenic spacer ( nt) . the similarity of the nucleocapsid vertebrate hosts. the ungulates, including domestic cows, sheep, and goats, are apparently involved in the circulation of bhav. usually, bhav infection in adult animals is asymptomatic, but it is pathogenic to young ones (lamb, calf, suckling mouse), causing fever and meningoencephalitis. , À experimental infection of rhesus monkeys by bhav induced encephalitis. several strains of bhav were isolated from the four-toed hedgehog (atelerix albiventris) and the striped ground squirrel (xerus erythropus) in africa. antibodies have been detected in dogs, roe deer (capreolus capreolus), and wild boars (sus scrofa). human pathology. bhav infection in human is mainly asymptomatic, but several cases of fever and meningoencephalitis caused by bhav have been described. À history. gissar virus (gsrv) was isolated from argas reflexus ticks collected in a dovecote in the town of of gissar in tajikistan ( n, taxonomy. the genome of gsrv (strain leiv- taj) has been sequenced. phylogenetic analysis shows that gsrv is a member of the phlebovirus genus of the uukuniemi group (figures . À . ). gsrv is closely related to grand arbaud virus (gav), which was isolated from a pool of argas reflexus ticks collected in a dovecote near gageron in arles in the rhô ne river delta in the camargue region of france in . gav is classified as virus belonging to the uukuniemi group. the identity of the nucleotide and amino acid sequences of gsrv and gav is % nt for the s-segment ( % aa for the nucleocapsid protein), % nt for the m-segment ( % aa for the polyprotein precursor of gn/gc), and % nt for the l-segment ( . % aa for rdrp). arthropod vectors. regardless of their geographical distribution, gsrv and gav occupy a narrow ecological niche associated with ticks (argas reflexus) and birds (most likely, pigeons (columbidae)). in laboratory experiments, gsrv reproduced in a. reflexus ticks in days with titers up to . log (ld )/ mcl. the distribution of argas reflexus ticks is limited between n on the north and n on the south. the a. reflexus metamorphosis cycle is about three years. the ticks inhabit pigeons' habitats, which are also used by other birds, such as swallows and swifts. a. reflexus larvae were found in europe on a rock swallow (ptyonoprogne rupestris), in egypt on a little owl (athene noctua), in israel on a rock dove (columba livia) and a fan-tailed raven (corvus rhipidurus), and in crimea on the western jackdaw (corvus monedula). the mass reproduction of mites in a dovecote has a negative impact on pigeons' bereeding behavior. worse, at night the ticks can go down to the living space and bite people if the dovecote is built into a house. vertebrate hosts. the main vertebrates involved in the circulation of gsrv are apparently birds, particularly the columbidae. in laboratory experiments, gsrv was isolated from the blood of small doves (streptopelia senegalensis) , , , and days after infection. the virus titer in the blood was . À . log (ld )/ mcl, on average. serological examination of birds in tajikistan found antibodies to gsrv % of doves (columba livia). history. khasan virus (khav) was isolated from haemaphysalis longicornis ticks collected from spotted deer (cervus nippon) in in the forest in khasan district in the south of primorsky krai, russia (figure . ). morphologic studies showed that khav belongs to the bunyaviridae family. the virion of khav has structural elements (filaments up to nm) that are typical for uukv, but no antigenic relationships between khav and uukv (as well as zaliv terpeniya virus, ztv) have been found. , in a complement-fixation test, khav did not react with serum used in the identification of certain bunyaviruses, so it was categorized in with the unclassified bunyaviruses. taxonomy. the genome of khav (strain leiv-prm ) was sequenced, and the virus was classified into the phlebovirus genus of the bunyaviridae family. the genome of khav consists of three segments of ssrna whose size and orf structure correspond to the size and orf structure of the other tick-borne phleboviruses. a full-length pairwise comparison of l-segments revealed a . % nt identity between khav and uukv and . % between khav and rvfv. the predicted amino acid sequence of rdrp of khav has . % and . % aa identities with uukv and rvfv, respectively. as in other tick-borne phleboviruses, the m-segment of khav does not contain any nsm protein. the similarity between the m-segments of khav and uukv is . % nt, and that between the polyprotein precursors of khav and uukv is . % aa. the s-segment of khav has % nt ( . % aa for the n-protein) identity, on average, with that of the uukuniemi group viruses and % nt ( . % aa), on average, with the mosquitoborne phleboviruses. on phylogenetic trees constructed on the basis of the alignment of full-length genome segments, khav forms a distinct branch external to the uukuniemi group viruses (figures . À . ). at least viruses with unsettled taxonomy are included in the uukuniemi group. some of them can be considered variants of the species uukv, manawa virus (mwav), precarious point virus (ppv), and gav. two tick-borne phleboviruses, sftsv and hrtv, are more closely related to the bhanja group than the uukuniemi group. , arthropod vectors. only a single isolation of khav was ever obtained, and the ecology of the virus has not been studied. haemaphysalis longicornis ticks, from which khav was isolated, are distributed in the far east of russia, the northeastern part of china, the northern islands of japan, korea, fiji, new zealand, and australia. haem. longicornis ticks also are the main vector of sftsv (oterwise called huaiyangshan virus, hysv), which caused a large outbreak of febrile illness with a high mortality rate ( %) in in china. vertebrate hosts. the principal vertebrate host of khav is unknown. khav was isolated from ticks collected on deer. haemaphysalis longicornis ticks are repeatedly found on cows, goats, horses, sheep, badgers, and dogs. history. the sandfly fever virus group includes naples and sicilian subtypes. epidemics of the comparatively mild acute febrile disease of short duration brought on by these viruses in countries bordering the mediterranean have been known since the napoleonic wars. the same disease was common among newly arrived austrian soldiers on the dalmatian coast each summer. experiments conducted by an austrian military commission proved that the disease was caused by a filterable agent in the blood of patients and that the sandfly phlebotomus papatasi can serve as a vector to transmit the disease. during world war ii, epidemics occurred among troops in the mediterranean and two antigenically distinct strains were isolated from the blood of patients in in sicily and naples. these strains have been designated the sandfly fever sicilian virus (sfsv) and sandfly fever naples virus (sfnv), with prototype virus tosv. , dr. a. sabin gave a clinical description of the disease and demonstrated that immunity developed to one type of virus does not protect from infection caused by the other type. later, several viruses related to sfnv (anhanga (anhv), bujaru (bujv), candiru (cduv), chagres (chgv), icoaraci (icov), itaporanga (itpv), and punta toro (ptv)) were isolated from humans and rodents in south america. , , to date, viruses related to tosv have been found in all regions of the world, including the palearctic, neotropical, ethiopian, and oriental zoogeographical regions. the prototype strain of tosv was isolated from phlebotomus papatasi sandflies in in monte argentario in central italy. two viruses antigenically related to tosv-karimabad virus (karv) and salehabad virus (salv)-were isolated from phlebotomus flies collected in near karimabad village and salehabad village, respectively, in iran. , several related viruses were isolated in the mediterranean: sandfly fever cyprus virus (sfcv; adria virus (adrv, salehabad-like), isolated in saloniki (alternatively, thessaloniki), greece; and massilia virus, isolated near marseilles, france. epidemic outbreaks of sandfly fever whose agents could not be typified occurred in some central asian countries and in crimea during and after world war ii and in turkmenistan after the devastating earthquake of . antibodies to sfsv, sfnv, and karv were found in the blood of humans in tajikistan, azerbaijan, and moldova. antibodies were also found in wild animals in turkmenistan: the great gerbil (rhombomys opimus), the long-clawed ground squirrel (spermophilopsis leptodactylus), and the hedgehog (erinaceus auritus). three strains of sfnv and two strains of sfsv were isolated in À from the blood of patients in afghanistan. , taxonomy. the genome of tosv consists of three segments of negative-polarity ssrna: l-segment ( , nt in length), m-segment ( , nt) and s-segment ( , nt). phylogenetic analysis revealed that viruses of the sfnv complex are divided into five genetic clades that differ in their geographical distribution: (i) from africa (saint floris virus and gordil virus (gorv)); (ii) from the western mediterranean (punique virus (punv), granada virus (grv), and massilia virus); (iii) tosv; (iv) viruses from italy, cyprus, egypt, and india; (v) strains from serbia and tehran virus. distribution. sfnv and sfsv are distributed over those areas of the southern parts of europe and asia, and over those areas of africa, which are within the range of the vector. , À tosv is distributed over italy; spain; portugal; the south of france; slovenia; greece, including the ionian islands: cyprus; sicily; and turkey. , , À both the naples and sicilian strains were isolated from the blood of patients with febrile illness in the vicinity of aurangabad, maharashtra state, in northern india. sandfly virus fever also circulates in western india, as well as in pakistan. the cocirculation of two tosv genotypes was uncovered in the southeast of france. , , a case of disease associated with tosv befell a tourist returning from elba to switzerland in , and another struck an american tourist returning from sicily the same year. tosv from france is genetically different from that in spain. , , , periodic outbreaks of sandfly fever occurred in the first half of the twentieth century in some central asian republics, transcaucasia, moldova, and ukraine. arthropod vectors. the primary vector of sfnv and sfsv is phlebotomus papatasi; for tosv, the primary vectors are ph. perniciosus and ph. perfiliewi. the viruses can be transmitted by the transovarial route and therefore may not require amplification in wild vertebrate hosts. the infection rate of sandflies can reach : . the active period of phlebotomus in the southern part of europe lasts from may to september. sandflies are peridomestic; the immature stages feed on organic matter in soil and do not require water, but are sensitive to desiccation and therefore are often found in association with humid rodent burrows. vertebrate hosts. the main vertebrates involved in the circulation of sfnv are rodents, particularly the great gerbil (rhombomys opimus) and the long-clawed ground squirrel (spermophilopsis leptodactylus), as well as a hedgehog (erinaceus auritus). the great gerbil is distributed over areas ranging from near the caspian sea to the arid plains and deserts of central asia. the northern border of the animal's distribution is from the . family bunyaviridae mouth of the ural river on northward to the aral karakum and betpak-dala deserts, to the southern coast of lake balkhash, and thence to northern china and inner mongolia. the habitats of rh. opimus are sandy and clayey deserts. tosv was isolated from the brain of the bat pipistrellus kuhlii. animal and human pathology. sandfly virus fever does not cause disease in domestic or wild animals. the hosts of phlebotomus sandflies are usually rodents, which may develop antibodies. over human experimental volunteers were infected at the time of world war ii. , the incubation period is between and days, and the onset of fever and headache in those patients was sudden. nausea, anorexia, vomiting, photophobia, pain in the eyes, and backache were common and were followed by a period of convalescence with weakness, sometimes diarrhea, and usually leucopenia. viremia was present h before and h after the onset of fever. tosv was established as the cause of one-third of previously undiagnosed human aseptic meningitis and encephalitis cases examined in central italy. sfcv was associated with a large outbreak in the ionian islands of greece. adrv is associated with serious illness with tonic muscle spasms, convulsions, difficulty urinating, and temporary loss of sight. human disease frequently goes unrecognized by local health-care workers. studies of antibodies in people indicate that the most infections occur in children. when large numbers of unimmunized adults are introduced into an endemic area, the incidence of disease can be high. human exposure to sandflies can be reduced by repellents, air-conditioning, and screens on windows. because sandflies have a flight range of not more than m, human habitats can be constructed at a distance from potential domestic sandflies' breeding places, such as chicken houses and quarters for other farm animals. history. uukv was originally isolated from ixodes ricinus ticks collected in from cows in southeastern finland. , antigenically similar isolates (strains leiv- az and leiv- az) have been obtained from blackbirds (turdus merula) and i. ricinus ticks collected in the foothills of the talysh mountains in the southeast of azerbaijan in and , respectively. À uukv is distributed in the mid-and southern boreal zones of fennoscandia and adjacent areas of the russian plain. twelve strains of uukv were isolated from i. ricinus ticks (the infection rate was . %), and one strain from aedes communis mosquitoes, in landscapes in the mideastern region of fennoscandia. , three strains were isolated from i. persulcatus ticks collected in belozersky district, vologda region, russia, in . , uukv was also isolated from the mosquitoes ae. flavescens and ae. punctor in the west of ukraine, as well as at the border between poland and belarus. , twenty-eight strains of uukv were isolated from i. ricinus ticks collected in lithuania and estonia in À . , , À uukv was isolated as well from birds and i. ricinus ticks in western ukraine and belarus. , , in central europe, uukv was found in the czech republic, slovakia, and poland. À the prototypical strain leiv- c of ztv was isolated from ixodes uriae ticks collected in in a colony of common murres (uria aalge) in tyuleniy island in zaliv terpeniya bay in the sea of okhotsk). , in accordance with the results of electron microscopy, ztv was assigned to the bunyaviridae family. complement-fixation testing revealed that ztv is most closely related to uukv, but the two viruses are easily distinguishable in a neutralization test. , more than strains of ztv were isolated from i. uriae ticks collected in colonies of seabirds on the shelf and islands of the sea of okhotsk, the bering sea, and the barents sea (table . , figure . ). , , , two strains of ztv were isolated from i. signatus ticks collected on ariy kamen island in the commander islands, but their infection rate was less than : , (, . %). a similar virus was found in norway. one strain of ztv (leiv- az) was isolated from the mosquito culex modestus collected in in a colony of herons (genus ardea) in the district of kyzylagach in the southeastern part of azerbaijan (figure . ). natural foci of ztv and uukv associated with bloodsucking mosquitoes (subfamily culicinae) are found in continental areas in the european part of russia, particularly murmansk region. taxonomy. the viruses of the phlebovirus genus can be divided into two main ecological groups: those transmitted by bloodsucking mosquitoes (subfamily culicinae) and midges (subfamily phlebotominae), together called mosquito borne, and those transmitted by ticks (tick borne). uukv is a prototypical virus of the uukuniemi antigenic group, which includes at least related tick-borne phleboviruses (figures . À . ). the genome of uukv consists of three segments of ssrna: an l-segment , nt long, an m-segment , nt long, and an s-segment , nt long. the m-segment of uukv, and indeed, that of all tick-borne phleboviruses, is shorter than the m-segment of mosquito-borne phleboviruses, owing to the absence of the nonstructural protein nsm, which is common in the mosquitoborne phleboviruses. originally, ztv was described as a virus closely related to uukv. a full-length sequence comparison showed that the similarity of ztv to uukv is . % nt identity of the l-segment ( . % aa of rdrp) and . % nt identity of the m-segment ( . % aa). arthropod vectors. most isolations of uukv and ztv were obtained from ixodes ricinus and i. uriae ticks, respectively. the infection rates of nymphs and larvae of i. uriae are and times lower, respectively, than that of the imago. these rates indicate a high frequency ( À %) of transovarial transmission of ztv. , probably, ztv has a more pronounced ability to replicate in mosquitoes that are active in the subarctic climate zone (tundra landscapes) in july through the first half of august at temperatures sufficient for the accumulation of virus in the salivary glands. islands. in the murmansk region, which lies to the north of the european part of russia, antibodies were found in % of common murres (u. aalge), % of black-legged kittiwakes (rissa tridactyla), and % of voles (microtus oeconomus). , apparently, ruminants could be infected by mosquitoes or by eating fallen birds. on the north coast of the kola peninsula, antibodies were found in % of thick-billed murres (u. lomvia), in % of blacklegged kittiwakes, and in % of voles. , in central and eastern europe, a number of vertebrate hosts are involved in the circulation of uukv: forest rodents (myodes glareolus, apodemus flavicollis) and terrestrial passerine birds-the blackbird (turdus merula), pale trush (t. pallidus), ring ouzel (t. torquatus), european robin (erithacus rubecula), hedge sparrow (prunella modularis), wheatear (oenanthe oenanthe), european starling (sturnus vulgaris), carrion crow (corvus corone), magpie (pica pica), brambling (fringilla montifringilla), hawfinch (coccothraustes coccothraustes), yellow bunting (emberiza sulphurata), turtle dove (streptopelia turtur), and ringnecked pheasant (phasianus colchicus). , À viremia and long-term persistence of the virus were demonstrated in experimentally infected birds of many species. specific antibodies were detected in cows and reptiles. human pathology. an association was revealed between uukv and different forms of disease, including neuropathy. , a serological survey of , people in lithuania concluded that antibodies existed in . À . % of the population. human antibodies to uukv were detected in less than % of the human population in central europe À and À % in belarus. the people living in the tundra landscape had antibodies to ztv in . % of cases, while in the forest no such antibodies were detected (via a neutralization reaction). (table . ). in previous studies, rukv was mistakenly included in the sakhalin serogroup in the nairovirus genus. taxonomy. the genome of komv (strain leiv- ) and rukv (strain leiv- ) were completely sequenced, and the two viruses were classified into the phlebovirus genus. , a full-length comparison showed that the genetic similarity between komv and rukv is . À . % nt. among other tick-borne phleboviruses, komv and rukv are most closely related to mwav, which was isolated from argas abdussalami ticks in in pakistan. the similarities of the genomes of komv and rukv to that of mwav are . % nt for the l-segment ( . % aa for rdrp), . % nt of the m-segment ( % aa for the polyprotein precursor), and . % nt for the s-segment ( . % aa for the n-protein). in phylogenetic trees, komv and rukv were placed into the uukuniemi group (figures . À . ). the ecology and area of distribution of komv and rukv are the same as those of ztv, which is closely related to uukv. several strains of ztv isolated on the commander islands were sequenced, and no reassortants of ztv with komv were found. , arthropod vectors. all isolations of komv and rukv were obtained from ixodes uriae ticks, the obligate parasite of alcidae birds. the commander islands are located on the border of the temperate and subarctic climatic zones, and many different viruses belonging to the bunyaviridae (ztv, sakv, pmrv), flaviviridae (tyuleniy virus, tyuv), and reoviridae (okhv) families have been isolated from i. uriae ticks collected from birds living in colonies there. À note that the komv infection rate of the i. uriae ticks in the commander islands is times less than the ztv ( : ) and tyuv (family flaviviridae, genus flavivirus) infection rates of the same ticks. vertebrate hosts. the main vertebrate host of komv and rukv is apparently alcidae birds, especially the common murre (uria aalge), but their involvement in the circulation of komv and rukv has not been studied sufficiently. human pathology. uukv group viruses, in general, do not play a role in human infectious pathology, although serological studies have detected antibodies to various viruses of this group in people. the flaviviridae family (from the latin flavus, "yellow," as well as from yellow fever virus (yfv)) includes three genera: flavivirus, pestivirus, and hepacivirus. the flaviviridae are small ( À nm) enveloped viruses. the genome is represented by ssrna the flavivirus genus includes more than viruses classified into antigenic groups. , the flavivirus virion is spherical ( nm) and consists of a nucleocapsid ( nm) and a lipid bilayer envelope covering it. the lipid envelope contains two transmembrane glycoproteins: m (matrix protein, kd) and e (envelope protein, kd). the genome of the flaviviruses is a single molecule of rna about , nt in length and capped on the terminus. the genomic rna encodes a long orf of a polyprotein precursor flanked by and untranslated regions. mature viral proteins are produced during a complex process of proteolytic cleavage of the polyprotein precursor by cellular and viral proteases. structural proteins (core, m, and e) occupy one-third of the rna (the n part of the polyprotein) on the part of the genome, followed by nonstructural proteins (ns -ns b) (figure . ). , most of the flaviviruses are arboviruses; that is, they can be transmitted to vertebrate hosts by bloodsucking arthropod vectors (figure . ). approximately % of known flaviviruses are transmitted by mosquitoes, about % by ticks. the arthropod vectors of some flaviviruses are unknown. there is also a group of flaviviruses that infect only insects and not vertebrates. some flaviviruses (e.g., west nile virus, wnv) have ecological plasticity and can be transmitted either by mosquitoes or by ticks. flaviviruses are distributed over all continents, with mosquito-borne viruses found mainly in regions with an equatorial and tropical climate and tick-borne viruses found mostly in regions with a temperate climate zone. many flaviviruses are associated with birds, which can transfer them during the birds' seasonal migration. flaviviruses belongs to natural foci zoonoses. certain flaviviruses, such as yfv, dengue virus (denv), and west nile virus (wnv), pose a serious threat to humans. À history. the first hint that omsk hemorrhagic fever (ohf) was etiologically linked figure . ) an area with a wide network of lakes. about cases with two lethal outcomes ("atypical tularemia" and "atypical leptospirosis") were investigated (without the expedition produced prodigious results: the prototype strain ohfv/kubrin was isolated from the blood of one patient; the mechanism of transmission of the virus by the ixodidae tick dermacentor reticulatus was established; the epidemiological and clinical features of ohf, as well as its pathogenesis and pathomorphology, were described; and inactivated vaccine from mouse brain was developed and prepared for epidemiological trials. later, the role of another species of ixodidae ticks (d. marginatus) as an ohfv vector was revealed. , taxonomy. ohfv belongs to the phylogenetic branch of the mammalian tick-borne virus group (figure . ). the ohfv genome has a length of , nt, and its organization is common to the flaviviruses. two genotypes of ohfv are known today: prototypical strains for the first one are ohfv/kubrin and ohfv/bogolubovska, which have an extremely small genetic distance between them; the prototypical strain for the second genotype is ohfv/uve. À only six nucleotide substitutions, which encode four amino acids, have been found in the entire genome. three of four amino acid changes were located in the envelope glycoprotein e. phylogenetic analysis based on a comparison of partial sequences of the e gene available in genbank showed that ohfv isolates can be divided to three genetic lineages (figure . ). the genetic diversity among strains of different lineage is up to . %. arthropod vectors. the natural foci of ohfv are found in the forestÀsteppe landscape zone of western siberia, an area with numerous bogs and a wide network of lakes within the omsk, novosibirsk, kurgan, and tyumen regions (figure . ). the natural foci border the area of distribution of tbev, and the two virus's natural foci are intermingled. À the principal ixodidae tick vectors for ohfv are dermacentor pictus (in the northern forestÀsteppe subzone) and d. marginatus (in the southern forestÀsteppe subzone). , , the infection rate of d. pictus in epidemic years reaches %, in interepidemic years . À . %. the main host for preimago phases of d. pictus is the narrow-headed vole (microtus gregalis). this species of rodent is host to À % of d. pictus nymphs and larvaein the northern forestÀsteppe subzone. in À , when the number of microtus gregalis voles fell significantly, there was a concomitant decrease in the number of d. pictus ticks in the center of an epidemic zone that was accompanied by a sharp decrease in the infection rate of ticks and an attenuation of the meadow natural foci of ohfv. in some of those years, however, a high number of ixodes apronophorus, all phases of which feed on the water vole (arvicola terrestris), become involved in the virus's circulation on a par with d. pictus ticks. ar. terrestris makes fodder migrations in juneÀaugust from damp locales (where their infection takes place) to coastal meadows (where peak activity of the larvae and nymphs of d. pictus is observed during those months). small animals living in those meadows become infected as they feed on the d. pictus larvae and nymphs. in damp locales, i. apronophorus could infect muskrats. also, d. marginatus, whose optimum zone lies in a steppe landscape belt, plays some (though largely insignificant) role in the lake areas of the southern forestÀsteppe subzone. during epizootic and epidemic activity of ohf natural foci, gamasidae ticks, as well as aquatic organisms belonging to the hydracarinae, take part in ohfv circulation. their involvement is confirmed by the identity of isolated strains with those isolated from muskrats and sick humans. experiments with experimentally and spontaneously ohfvinfected gamasidae ticks testify to the ability of longitudinal (more than six months) virus preservation. vertebrate hosts. the principal vertebrate host of ohfv, which is able to directly infect humans, is the muskrat (ondatra zibethicus). this species was introduced into western siberia from canada in . their population density reached a modern-day high in the s. close interactions among o. zibethicus and local populations of arvicola terrestris emerged. ar. terrestris has periods of rapid population growth followed by epizootics of tularemia, leptospirosis, and ohfv. muskrats suffered these epizootics together with other local species of rodents: microtus oeconomus, m. gregalis, myodes rutilus, apodemus agrarius, and ar. terrestris. the ofv infection rate among muskrats is about % in both the autumnÀwinter and the springÀsummer periods. latent infection was established in all rodents except the muskrat. ohfv was detected in birds and in mosquitoes, but the role of these two animals in virus circulation is not clear. À epidemiology. ohfv is transmitted both by ixodidae tick bites and as the result of direct contact with infected muskrats, their flesh, and fresh fells. , ohf morbidity during À reached . À . %. then there was a gradual decrease down to single cases. most ohf cases ( . %) were detected in the lake forestÀsteppe, in the south of the forestÀsteppe landscape zone, which occupies . % of the territory where . % of country people in the omsk region live. the northern forestÀsteppe landscape zone is the youngest landscape of western siberia, having evolved in place of the former southern taiga landscape zone. , in the south of western siberia, the following territorial zones can be marked out: (i) the . family flaviviridae preferred territory of tick-borne encephalitis virus (tbev) (the southern taiga); (ii) intermediate territory (the boundary of the southern taiga with the northern forestÀsteppe); (iii) the preferred territory of ohfv (the northern and southern forestÀ steppe); and (iv) the territory of sporadic cases of ohf (part of the southern forestÀsteppe and steppe). , in the first zone, more than % of all cases of tbe in western siberia are registered and only single ohf cases are found; in the second zone, % each of cases of tbe and ohf; in the third zone, % of tbe and % of ohf; and in the fourth zone, % of tbe and single cases of ohf. the seasonal incidence of ohf distinctly correlates with the activity of the principal ixodidae tick vectors. cases (a few) of ohf acquired by direct contact with muskrats occur mainly during the season in which the animals are hunted, in octoberÀjanuary. in the springÀ summer season, ohf cases occur chiefly in rural areas. the age of patients ranges from to years, but cases occur mainly among middle-aged persons ( À years old). in the autumnÀwinter period, ohf occurs mainly among muskrats trappers ( %), adult members of their families ( %), and children ( %). it appears that all patients infected directly from muskrats develop symptomatic illness. seroprevalence ranges from to % in populations of endemic regions. , , in the last decade of the twentieth century, an increase in ohf natural foci activity took place in the tyumen ( ), omsk ( , À ), novosibirsk ( À ; regular epidemic activity took place on the territory of only four administrative districts), and kurgan ( ) regions. in the absolute majority of laboratoryconfirmed cases, the nontransmissive pathway (direct contact with muskrats) of the infection dominated. pathogenesis is determined first of all by the destruction of capillaries, the vegetative nervous system, and the adrenal glands. , clinical features. the incubation period of ohfv is À days long. the disease begins abruptly, with fever, head and muscular pain, hyperemia, and injection in the sclera. the body temperature increases up to À c and stays that way for À days, then decreases a little and critically falls on the th to th day after symptoms appear. from the first days of the illness, there are diapedetic bleedings, especially in the nose. recovery is usually complete, without any residual phenomena; lethal outcomes are possible, but are rare. , À control and prophylaxis. ohfv survives up to days in lake water. water can be contaminated by urine and feces of the infected muskrats or some other rodents. the water pathway in human infection has been discussed in the literature. , prevention of the infection depends on the use of protective respirators and rubber gloves in processing muskrat pelts and on personal protective measures against tick bites. tbe vaccine offers a high degree of protection against ohf. , cases of laboratory-acquired ohf have been reported in unvaccinated persons, and tbe vaccine is recommended for laboratory personnel working with either virus. interferon and its inductors have shown a high efficiency in preventing ohf in experiments using animal models. the genome of powv is a about , nt in length. the virus comprises two genetic lineages, formed by powv (lineage i) and the closely related deer tick virus (dtv, lineage ii) (figure . ). phylogenetic analysis based on partial sequences of the e gene showed that the population of powv in russia has a low genetic diversity. the strains of powv isolated in russia have a high genetic similarity to the strains of lineage i isolated in north america. a full-length genome comparison revealed that far eastern isolates (leiv- prm, spassk- , and nadezdinsk- ) have a . % identity with strain powv/lb from canada (figure . ) . arthropod vectors. powv was isolated from ixodidae ticks collected in the russian far east and in the u.s. states of california, colorado, connecticut, massachusetts, south dakota, and west virginia. serological investigations of wild mammals indicate that powv also circulates in the canadian provinces of alberta, british columbia, and nova scotia. , À in north american natural foci, powv was isolated from ixodes cookei, i. spinipalpus, i. marxi, and dermacentor andersoni ticks. , , in the far east, known vectors of powv are haemaphysalis longicornis, haem. concinna, haem. japonica, d. silvarum, and i. persulcatus ticks. , , , transphase and transovarial transmission of powv in ixodidae ticks has been established. vertebrate hosts. in north america, powv was isolated from wild mammals: the woodchuck (mormota monax, the main reservoir), american red squirrel (tamiasciurus hudsonicus), deer mouse (peromiscus maniculatus), red fox (vulpes fulva), eastern gray squirrel (sciurus carolinensis), north american porcupine (erethizon dorsatum), striped skunk (mephitis mephitis), raccoon (procyon lotor), long-tailed weasel (mustela frenata), and gray fox (urocyon cinereoargenteus). , , infection of wild vertebrates most often is inapparent. , in the south of the russian far east (in primorsky krai), powv was isolated from aquatic birds: the common teal (anas crecca) and the mallard (anas platyrhynchos). , , , epidemiology. human infections of powv were reported in canada (ontario and quebec), the united states (new york and pennsylvania), and russia (primorsky krai). , , nevertheless, human infection rarely develops. clinical features. the clinical picture of developing meningitis and encephalomeningitis includes high temperature, dryness in the gullet, drowsiness, headache, disorientation, convulsions, vomiting, difficulty breathing, coma, and paralysis, with % lethality in the severe phase of the disease. autopsy has revealed widespread perivascular and focal parenchymatous infiltration. in % of recoveries, consequent damage to the cns develops, which could lead to death in À years. , control and prophylaxis. the vaccine against tbev is not effective against powv. history. in À , the russian military medical doctorÀneuropathologist a.g. panov, together with his colleagues a.n. shapoval and d.a. krasnov, described a neuroinfection with a high level of mortality in the far east. this neuroinfection later was called "springÀ summer encephalitis." , during field expeditions in À , the historical strain tbev/ sofjin was isolated from the brain of a patient with encephalitis who died in khabarovsk krai (figure . ) . in that period, the main vector of tbev-ixodes persulcatus tickswas established, epidemiological peculiarities of tbe were studied, and the first anti-tbev vaccine was developed on the basis of intracerebrally infected mouse brain and was successfully used in medical practice. complex expeditions were undertaken by a number of prominent virologists (l.a. zilber (figure . strain tbev/leiv- kaz (the former aav) was isolated from ixodes persulcatus in the low-mountain part of southeastern kazakhstan (alma-ata region) in . preliminary investigation revealed a one-sided antigenic relation between aav and powv. aav was associated with human cases of meningitis. specific antibodies to aav were found among ground squirrels (citellus fulvus), agricultural animals, and humans. later, the aav genome was sequenced (genbank id: kj ). a full-length genome comparison showed that aav has the highest similarity ( . % nt and . % aa identities) to the tbev/ chita- , tbev/irkutsk- , tbev/aino, and tbev/vasilchenko strains belonging to the siberian genotype (figure . ) . recent genetic studies of tbev revealed two additional genotypes of this virus on the territory of eastern siberia (irkutsk region): for the first one, only a single strain is known today; for the latter, there are five strains in mongolia. thus, tbev has a high level of genetic diversity in northern eurasia. tbev-sib genotype dominates in europe, western siberia, and eastern siberia, tbev-fe in the far east. , the tbev-fe genotype, which was widely distributed in siberia and northeastern europe, is now being displaced by tbev-sib. tbev-fe strains are often pathogenic to laboratory mice, whereas tbev-sib frequently provokes severe and lethal disease. local populations of all genotypes of tbev could be stable for a long time. distribution. tbev is distributed within the areas of distribution of its main vectors: ixodes persulcatus and i. ricinus ticks (figure . -see details in the detailed work of e.i. korenberg norway; À in the rest of europe, the czech republic, , slovakia, , , bulgaria, hungary, , poland, , croatia, latvia, lithuania, estonia, , denmark, germany, À austria, slovenia, france, italy, , and spain (table . ); and in asia, the russian far east and siberia, , , japan (hokkaido), north and south korea, , china, mongolia, kazakhstan, and kyrgyzstan. arthropod vectors. natural tbev infection has been observed in species of ixodidae ticks. the principal arthropod vectors for tbev in russia are the ixodidae ticks ixodes persulcatus (in the far east, siberia, and the north of the european part of the country) and i. ricinus (in the south of the european part) (figure . ). the northern boundary of i. persulcatus and i. ricinus lies within the limits of an effective temperature sum isoline of about , À , c (the middle taiga landscape belt). the most suitable climatic conditions for these ticks are within the south taiga. imago tick activity begins in the third d decade of april and reaches a maximum in the second and third decades of may or in the first and second decades of june, with activity beginning to decrease in the third decade of june. this time frame correlates with morbidity dynamics having an -to -day lag (figure . ). the ecological links of tbev during its circulation in natural foci are extremely diverse as the result of wide distribution of this virus (figures . and . ). ixodidae ticks, mainly i. persulcatus, are the natural reservoir of tbev and the core of natural foci. , , , from the very beginning of the tick's larval stage, a suctional, tarlike liquid appears around the hypostome and becomes rosin. , the quantity of virus in this rosin plug is comparable to that in the tick's body ( À pfu/mcl). the place of suction on the body of the host is significant for the development of infection; for example, suction in the axillary hollow results in the highest lethality ( . %, . times more in comparison to suction in the neck and in the head. ticks become infected as they suck blood from a vertebrate host with a level of viremia that is equal to or higher than the threshold required for infection. ticks can also become infected from an uninfected vertebrate host as they suck blood together with infected ticks. , transovarial and transphase transmission of tbev has been described in the literature; nevertheless, the effectiveness of vertical transmission of tbev is low. (about % of progeny turn out to be infected). , the sexual pathway of tbev transmission from male to female is quite effective (about %). À the aggressiveness and activity of tbev-infected ixodidae ticks increases with the tbev titer in their bodies. , infected ticks have been found on the clothing of figure . trends in the incidence of tbe in russia, by month (as a percentage of the amount of disease for the year, according to long-term data). humans at a fequency À times higher than uninfected ticks have been found. , , tbev has been isolated from the mosquitoes anopheles hyrcanus in kyrgyzstan and aedes sp. in western siberia. the strain tbev/malyshevo was isolated from aedes vexans nipponii mosquitoes collected in on the coast of petropavlovskoe lake in khabarovsk krai in the russian far east ( ʹn, ʹe ). À a preliminary investigation concluded that this strain belonged to negishi (negv) virus, and later the possibility was discussed that the strain belonged to a separate, malyshevo virus. then, phylogenetic analysis using a next-generation sequencing approach revealed that malyshevo is a strain of tbev and is closely related to tbev strains isolated in the far east: tbev/ , tbev/ spassk- , tbev/primorye- . tbev has been isolated many times fromticks and fleas of the superfamily gamasoidea living in nests of rodents and birds (table . ), even during the winter period. , , À vertebrate hosts. hosts for the preimago stage of ixodidae ticks-asian chipmunks (tamias sibiricus), shrews (members of the soricidae family), bank voles (myodes glareolus), field voles (microtus agrestis), mountain hares (lepus timidus), and species of birds (table . )-have great significance in tbev circulation. , , , , , , , persistent tbev infection in bank voles and field voles has been found during the winter period. infection among vertebrates occurs mainly by tick bites. in rare instances, alimentary transmission of tbev through milk containing viruses is possible. , epidemiology. there are two basic modes of human infection by tbev: (i) as the result of being bitten by infected ixodidae ticks (the main mode); and (ii) as the result of consuming infected raw goat, sheep, and cow meat, milk, or dairy products (mainly in natural foci linked to ixodes ricinus). , , the latter pathway of tbev distribution often involves whole families. as much as % of cases in belarus have been alimentary. tbev can persist in milk at c for more than min, and some of the viruses can remain viable even after pasteurization at c for min. nor is tbev inactivated after h at c and ph . . many laboratory infection cases (usually by aerosol) have been described. several hundred cases are recorded in europe (table . ) and in russia (table . ) each year, with considerable interannual variation. , , À the highest level of tbe morbidity is registered in the baltic states (latvia, . À . per , population); lithuania, . À . ; and estonia, . À . ) and in slovenia ( . À . ) and the czech republic ( . À . ). in neighboring austria, where the vaccination rate is higher, the index is lower ( . À . ). seasonal tbe morbidity in russia is connected with seasonal activity of the ixodidae tick vectors (figure . ) . the risk of infection depends upon the frequency of tick bites, which is different for populations living in the different landscape belts. results of an investigation of almost , people demonstrate that the highest risk is for the population living in the southern taiga belt, where about % of adults were found to have tick bites during one epidemic season (table . in rural localities of the southern taiga belt, about half of schoolchildren and about % of adults have antibodies to tbev. for comparison, only À % of adult citizens of kemerovo, a city of about half a million in western siberia, and À % of citizens of moscow have antibodies specific to tbev (table . ) . a mathematical model for evaluating the infection rate and the probability of developing the disease as a function of the density of the tick population, its infection rate and biting activity, and the level of the immune human layer was developed by d.k. lvov and coauthors. À the same approach, which is also suitable for other arboviral infections, was used for landscape-epidemiological zoning of tbev natural foci in altai krai in the southern part of western siberia: more than , residents living in the different landscape belts on a territory about , km were tested by serological methods (figure . ) . the tests produced a good fit between calculated and registered morbidity data (table . ) . pathogenesis. tbe can be realized in several pathogenetic variants. an inapparent clinical form is characterized by short-term localization of tbev in lymph nodes and immune cells, as well as by extranervous reproduction without viremia. infection is terminated by the development of stable immunity. about % of cases of infection are inapparent. clinical fever is expressed as a common infectious process, but both the central and the peripheral nervous system are involved in the pathology. neuroinfection is characterized by lesion of the envelope and substance of the spinal cord and cns. clinical features. the incubation period ranges from to days, but usually is À days. the onset of illness in typical cases is abrupt and with a headache. the temperature clinical symptoms of tbe, as well as the severity of the disease, are at least partially determined by biological properties of the virus. there are two main clinical forms of tbe: the far eastern variety, associated with far eastern and siberian strains of the virus, and the european variety (also known as western biphasic meningoencephalitis or biphasic milk fever), associated chiefly with european strains. human disease of the first type is usually clinically more severe in the acute phase, but is associated with a lower rate of chronic cns sequelae. the first phase starts with sudden fever, flulike symptoms (pronounced headache, weakness, nausea, myalgia, arthralgia), and conjunctivitis. the second phase appears after À days of apparent recovery, but then the cns is affected (meningoencephalitis appears), accompanied with fever, retrobulbar pain, photophobia, stiff neck, sleeping disorders, excessive sweating, drowsiness, tremors, nystagmus, meningeal signs, ataxia, pareses of the extremities, dizziness, confusion, psychic instability, excitability, anxiety, disorientation, and/or memory loss. tbev produces diffuse degenerative changes in neurons, perivascular lymphocytic infiltration, and damage to purkinje cells in the cns. mortality ranges from % (tbev-eur), to % (tbev-sib), to À % (tbev-fe). convalescence is prolonged, and neurological and psychotic sequelae often include paresis and atrophic paralysis of the neck and shoulders. , , a chronic form of the disease occasionally combines with a progressive course (called kozhevnikov's epilepsy), in which progressive neuritis of the shoulder plexus, multiple sclerosis, and progressive muscle atrophy often develop. , the chronic form is registered in À % of all tbe cases and is said to be the result of virusÀimmunity interactions. many authors have noted a decreasing number of severe tbe cases. diagnostics. laboratory diagnosis of tbe involves both serological (elisa, hemagglutination inhibition test (hit), neutralization testing) and virological methods (virus isolation using a biological model of intracerebrally inoculated newborn mice, À g mice, cell culture), as well as highly sensitive rt-pcr and real-time rt-pcr. control and prophylaxis. specific and nonspecific prophylaxis tools are highly efficient if they are utilized correctly. personal safety includes protection against ticks. vaccination against tbev has a long history of success. mass vaccination of populations in the endemic territory is necessary. a full course of vaccination provides % safety. all vaccines produced in russia are effective in the entire area of distribution of tbev, independently of the strain used to prepare the vaccine. vaccination has reduced tbe morbidity down to single cases in austria, the czech republic, and slovakia. single cases of tbev among vaccinated persons need to be investigated because possible causes are personal peculiarities of the immune system and errors in the control of vaccine production. the presence of brain tissue in vaccines produced on the basis of intracerebrally inoculated newborn mice was a source of danger for a long time: demyelinating encephalitis could develop. this danger was eliminated after vaccines were developed which used tbev strains that reproduced in cell cultures. in the s, cell culture vaccines against tbev were developed by e.n. levkovich history. japanese encephalitis virus (jev) was originally isolated by h. hayashi in from a patient who died with encephalitis and then, again, in from a patient who died with a fever in tokyo. , before that, however, japanese encephalitis (je) epidemics was documented in japan in and onward as "ioshiwara cold." in the south of the russian far east, strains of jev were known since the end of the s (figure . ). je played a role in the historical events of world war ii. american military personnel massed on okinawa and preparing to invade japan were demoralized by an outbreak of encephalitis among the indigenous people. a fictionalized account of the risk from je for american soldiers during world war ii underscores the military risk. taxonomy. phylogenetic studies indicated that jev isolates be divided into five genotypes, the distributions of which overlapped (figure . ). genotypes i, ii, and iii are most prevalent and are spread throughout asia (japan, china, india, korea, malaysia, and vietnam), the far east of russia, and northern australia. genotypes iv and v are rarer and were isolated in indonesia and india, respectively. genotypes i and iii are found mostly in temperate zones, whereas genotypes ii and iv predominate in tropical zones. À genetic diversity between strains of the different genotypes ranges from . % to . %. arthropod vectors. jev circulation in the equatorial and subequatorial climatic zones is year-round and is seasonal in the tropical, subtropical, and temperate belts, with a peak at the end of summer and the beginning of fall. jev is brought from the equatorial and tropical climatic belts to the subtropical and temperate belt during the spring migration of birds. about species of mosquitoes are able to transmit jev; nevertheless, only some of them are effective vectors. the main vector in japan, the philippines, the korean peninsula, china, the indochinese peninsula (except malaysia), indonesia, sri lanka, india, and nepal is epidemics usually develop after plentiful precipitation and a long rise in environmental temperatures until they are no less than c (but within the range À c). for a long time, the main vector for jev in the south of primorsky krai in russia was culex tritaeniorhynchus. in the s, as a result of both improvements in agriculture and meteorological changes, this species of mosquitoes consisted about % of all field collections. in subsequent years, however, their numbers abruptly declined, and by the s the species represented only . À . % of all mosquitoes collected. cx. pipiens is an accessory vector, and aedes togoi transmits jev in seashore areas. jev was also isolated in from ae. vexans. , vertebrate hosts. aquatic and semiaquatic birds (especially herons) have the main significance in the natural cycle of jev circulation. regular transfer of jev in migratory birds from endemic territories with year-round circulation of the virus to regions of the southern part of the temperate climatic belt (in particular, the southern part of primorsky krai, to the south from lake khanka ) is likely. jev transfer over hundreds of kilometers by infected mosquitoes is possible as well, especially in areas with a monsoonal climate (e.g., in australia through the torres strait À ). birds transfer jev from natural to synantropic biocenoses, where, thanks to culex tritaeniorhynchus mosquitoes willingly attacking wild birds, pigs, persons, synantropic birds, and domestic animals (chiefly pigs), these all join into jev circulation. infection in pigs could be inapparent, or it could be clinically expressed with encephalitis and a lethal outcome. the level of viremia in infected pigs is enough to infect mosquitoes. such epizootics among pigs are, in effect, amplifiers for jev, serving as prerequisites for the development of epidemics, first of all among people living in the countryside, but then among city dwellers as well. antibodies to jev specifically were revealed among wild boars ( %), raccoons ( %), and dogs ( %). in the south of china, jev was isolated from both leschenault's rousette (rousettus leschnaulti), a species of fruit bat, and the little tube-nosed bat (murina aurata), and anti-jev antibodies were identified in the blood of those animals. jev preservation in bats could be one of the mechanisms of the year-round circulation of the virus in its natural foci, with activation in the spring and subsequent replication and spreading in the summer and autumn. in natural foci, birds are the principal vertebrate hosts contributing to transmission of the virus; in synantropic foci, pigs are the most important vertebrate hosts. , jev has been isolated from the grey-headed bunting (emberiza fucata), great cormorant (phalacrocorax carbo), japanese thrush (turdus cardis), azure-winged magpie (cyanopica cyana), japanese wagtail (motacilla grandis), barn swallow (hirundo rustica), and night heron (nicticorax nicticorax). natural foci are situated in meadows. of late, culex tritaeniorhynchus has become more abundant in connection with intensive rice cultivation, portending the possibility of increased jev circulation and epidemics. , epidemiology. all the territory of japan, except for northern part of hokkaido, is endemic, but most diseases are registered near islands in a closed sea, as well as in tokyo and adjacent prefectures. before , outbreaks of je emerged in japan practically every year, with , À , patients seen. later, morbidity began to decrease to tens of cases per year. in the a and s, morbidity fell to the level of single cases per year. the main cause of the decrease was a significant drop in the population of the main jev vector-culex tritaeniorhynchus mosquitoes-as the result of a reduction in the acreage of rice fields as well as water pollution in places of mosquito habitation. in addition, the program of mass vaccination carried out annually among children of school age and a change in the structure of pork farms lessening the availability of pigs played a significant role in the falloff in the mosquito population. je is a serious problem in countries of southeast asia and oceania. during the last few years, more than , cases per year were registered, with about % lethality. morbidity increases annually in bangladesh, indonesia, laos, myanmar, north korea, and pakistan. , in addition, , the occurrence of an epidemic in southeastern asian countries is becoming more and more likely because those countries are now seeking to increase their production of rice. the greatest risk of je is said to be in china, nepal, sri lanka, thailand, laos, and vietnam. je is of the highest importance among all kinds of endemic encephalitis, potentially threatening nearly % of the population of our planet. the disease especially affects military contingents, as it did the american army during the concentration of armies in okinawa and the soviet army during the battle of lake khasan (also called the changkufeng incident) in the south of primorsky krai. precursors of jev circulated in indonesia and then evolved into six genotypes. genotype iii is widespread in a moderate climatic belt and often provokes epidemic outbreaks in eastern and southeastern asia. genotype i originated in indonesia, circulated in thailand and cambodia in the s and in south korea and japan in the s, and has now completely replaced genotype iii. genotype i got into japan in two ways: from southeastern asia and from mainland china. , two island territoriesthe philippines and taiwan, in both of which genotype iii circulates-were free of genotype i-and the philippines remains free-but the genotype appeared in taiwan in . the evolution of jev led to the emergence of two new subclusters in À ; the two together have replaced genotype iii. until recently, the qinghai-tibet plateau, in china, was free of jev, but in august the virus was isolated from culex tritaeniorhynchus mosquitoes there. during an epidemic in septemberÀnovember , genotype i circulated in japan. in nepal, on the northern border of india, je has been known since , after which outbreaks were observed annually. jev circulates in the north of australia as well. , je claimed morbidity in the south of the russian far east (in primorsky krai) in during an expedition headed by p.g. sergiev and i.i. rogosin. epidemics of jev broke out in the region in , , and . more than cases were recognized between and , with % reported in the extreme south of primorsky krai. the northern extent of this area is limited by the southern part of the ussuri lowland (about À n, À e). enzootic jev circulation without human morbidity has been documented, with the seroprevalence of residents estimated at about À %. , , , je cases occur mainly in augustÀseptember (but also when heavy rains are combined with high temperatures from april to september: $ c in april, $ c in june, $ c in august, and $ c in september). clinical features. the clinical picture of je varies from asymptomatic and easy feverish forms to an encephalitis syndrome. the ratio of clinical to asymptomatic forms is from : to : , , although the ratio in india in the s and s was from : to : . À the start of the disease is sudden, with fever ( %), headache, vomiting ( %), and symptoms of cns destruction (most often, hemiplegia and articulation lesions)-in % of cases, and at the height of the illness in % of cases. about one-third of patients with cns lesions recover completely. lethal outcomes are preceded by unconsciousness and then coma ( À % of the total number of patients). death comes in two-thirds of cases during the first week, in one-fourth during the second week, and in the rest of the cases in one month, from the onset of symptoms. after the disease, residual phenomena in the form of paralysis and mental issues are quite often observed. , control and prophylaxis. inactivated vaccines are used to immunize people, , , , À live vaccines to immunize pigs and horses. vaccination and protection of pigs from mosquito attack and protection of humans from mosquitoes (through the use of repellents, mosquito nets, bed curtains, etc.) are recommended during epidemics among people. mass vaccination has been carried out successfully in japan, south korea, china, and india. , , , À live vaccine manufactured on the basis of the chinese strain sa À is is given in china, south korea, and other countries in government programs aimed at expanding immunization of children. the complete genomes of tyuv and kamv (genbank id: kf and kf , respectively) were presented in a article in the journal of medical entomology, and it was established that kamv was a new virus within the tyuv group of the flavivirus genus. virion and genome. tyuv is a prototypical virus of the tyuleniy antigenic complex. the viruses of that complex belong to the ecological group of seabird tick-borne flaviviruses, which forms a distinct branch on the phylogenetic tree. four species are known in the tyuleniy antigenic complex: tyuv (in russia and the united states), meav (in europe), srev (in oceania) and kamv (in russia). the genetic similarity between the seabird tick-borne flaviviruses and the mammalian tick-borne flaviviruses is about % nt. a full-length genome comparison showed that the similarity among the four viruses in the tyuleniy antigenic complex is % nt and % aa, on average. tyuv leiv- c, isolated in the russian far east, has % nt and % aa identities with tyuv isolated on the pacific coast of the united states. kama virus (strain leiv-tat ) has % nt identity with the other viruses of the tyuleniy antigenic complex (meav, srev, tyuv). the similarity of the polyprotein precursor of kamv is % aa with each of tyuv and srev, % aa with meav. arthropod vectors. tyuv is distributed over the basins of the sea of okhotsk and the bering and barents seas. the infection rate of ixodes uriae in the pacific part of the virus's distribution is . times greater than in the atlantic part (table . ). , À outside of northern eurasia, tyuv is distributed over the west coasts of the united states (chiefly in oregon) and canada. , the infection rate of nymphs and larvae of i. uriae is one-twentieth to one-half the infection rate of the imago. the infection rates of i. uriae females and males (the males have only a rudimentary hypostome and do not feed) are practically the same. these data testify to the transphase and transovarial transmission of tyuv. (the efficiency of this type of transmission is about %.) attempts to isolate tyuv from i. signatus ticks were unsuccessful. the presence of antibodies to tyuv among local cows and indigenous people of the commander islands , indicates the possible role of sanguivorous mosquitoes (e.g., aedes communis, ae. punctor, and ae. excrucians) in infection. mosquitoes could also take part in virus circulation: their infection rate from the end of july to the beginning of august reaches . % in nesting colonies of seabirds and . % on the seacoast. experimental infection of tyuv on the model of aedes aegypti demonstrated the presence of the virus À days after inoculation, with . À . lg ld / mcl on days À ; . À . lg ld / mcl on days À ; and . lg ld / mcl on day . the transmission of tyuv during the feeding of infected mosquitoes on mice was established À days after infection of the mosquitoes. in culex pipiens molestus, tyuv was detected À days (the period of observation) after infection, with . À . lg ld / mcl. vertebrate hosts. migratory seabirds play a role in the exchange of tyuv group flaviviruses between the northern and southern hemispheres. , investigation with the help of indirect complement-binding reactions of sera samples from , birds collected in the far east revealed that the maximum tyuv infection rate takes place in brü nnich's guillemots (uria lomvia), common murres (u. aalge), and tufted puffins (fratercula cirrhata). lower rates were seen in pelagic cormorants (phalacrocorax pelagicus), redfaced cormorants (ph. urile), glaucous-winged gulls (larus glaucescens), kittiwakes (rissa tridactyla), northern fulmars (fulmarus glacialis), and sandpipers (scolopacidae). , , , , , the presence of specific anti-tyuv antibodies among sandpipers-red-necked phalaropes , , considering the annual migrations of these birds, tyuv can be found within the i. uriae area of distribution in nesting colonies of puffins. about % of adult and % of juvenile northern fur seals (callorhinus ursinus) on the commander islands have specific anti-tyuv antibodies, implying that these animals are involved in the circulation of that virus. a tyuv strain was isolated from the arctic ground squirrel (citellus (urocitellus) parryii) on the southeastern coast of the chukotka peninsula ( n, e). this event is one more argument for virus splash into the continent, with rodents included in virus circulation. in the tundra of the kola peninsula seacoast, antibodies specific to tyuv were detected among cattle ( . %) as well as red-necked phalaropes (phalaropus lobatus), snow buntings (plectrophenax nivalis), ruffs (philomachus pugnax), and rodents: tundra voles (microtus oeconomus). thus, in the atlantic part of its distribution, tyuv also tends to penetrate into the continent. experimental infection of kittiwakes (rissa tridactyla), herring gulls (larus argentatus), and brü nnich's guillemots (uria lomvia) was followed by the development of clinical features with cns lesions and lethal outcomes. epidemiology. the indigenous population in the far eastern part of tyuv distribution has specific anti-tyuv antibodies: . % in tundra on the coast of the chukotka peninsular, . % in forestÀtundra on the coasts of the sea of okhotsk and the bering sea, . % -in taiga on sakhalin island, and . % in tundra on the coast of the kola peninsula. the development of fever in humans visiting nesting colonies of seabirds on the coast of the barents sea has been described in the literature. ecological peculiarities of tyuv and kamv distribution. penetration of tyuv from the northern to the southern hemisphere is carried out by about species of birds, mostly turnstones (arenaria interpres), that nest in the north of asia and overwinter in australia and new zealand. wedge-tailed shearwaters (puffinus pacificus) nest in the southern hemisphere and carry out an annual migration along the coasts of the pacific ocean up to northern eurasia and north america. , close genetic relations found between tyuv and kamv have not been explained yet because information is lacking about ecological links between alcidae birds in the north and bank swallows in the central part of the russian plain. nontheless, the closeness demonstrates an ancient link between the flaviviruses and ixodidae ticks-obligatory parasites of colonial and burrow-shelter birds not only on the ocean coast, but also on the continental part of the distribution of those viruses. , , , , meav and srev, which are genetically close to tyuv, , could be intermediate evolutionary branches between tick-borne viruses of seabirds and later mammalian viruses transmitted by ticks. , the main vector of tyuv in subarctic regions-ixodes uriae, adapted to seabirds-is replaced by the ornithodoros capensis complex or argas spp. in the subtropics and tropics. , the northern boundary of the argas genus distribution is limited by a july isotherm of À c and of the ornithodoros genus by À c in europe and À c in asia. the vector of kamv-the i. lividus tick-has transpaleoarctic distribution, from the british isles in the west to japan in the east and from n down to s. this species of tick has an extrazonal distribution in the diggings of bank swallows (riparia riparia) made in the soft ground of steeps along the banks of rivers and lakes in taiga, leaf forest, forestÀsteppe and . family flaviviridae steppe climatic belts. i. lividus ticks are typical parasites of-burrow-shelter birds and relate strictly to the life cycle of the host: after the appearance of birds in the nesting areas in may, larvae begin to feed. in june, nymphs feed on the nestlings; female imagoes also feed on the nestlings, but male imagoes do not. given the presence of kamv-a virus closely related to tyuv-in the central part of the russian plain, it is worthwhile, and even necessary, to carry out a wider search for tyuv analogues on the continental part of northern eurasia. history. dengue fever (denf), etiologically linked to dengue virus (denv) (family flaviviridae, genus flavivirus), has been known in asia, africa, and america since the end of the eighteenth century. , wide epidemics of denf appeared in southeastern asia after world war ii. according to who data, denf morbidity, including imported cases, has been detected in more than countries of asia, africa, and europe. more than . billion people on earth are under the threat of denf. about million people fall victim to denf annually. american armies sustained heavy losses as the result of denf during world war ii, as well as during À in vietnam, the philippines, somalia, and haiti. simultaneous outbreaks of denf and chikungunya fever often occur. the virus etiology of denf and its transmission by mosquitoes was established by p.m. ashburn and c.f. craig in experiments using volunteers at the beginning of the twentieth century. denv- was isolated in from the blood of patients with fever on the hawaiian islands, denv- in from the blood of patients with fever on new guinea, denv- in from the blood of patients with fever in the philippines, and denv- in from the blood of patient with fever during epidemics in manila. taxonomy. four different serotypes of denv form a distinct phylogenetic lineage on the mosquito-borne flavivirus lineage (figure . ). genetic variation among different strains suggested that denv be divided into distinct genetic clusters considered as genotypes. the genetic diversity of denv is best exemplified in denv- , the different strains of which are divided into four genotypes: asian , asian , american/asian and so-called cosmopolitan. denv- strains are divided into five genotypes (iÀv), and denv- strains form three genotypes. in general, a particular genotype is linked to specific geographical regions and that genotype may be used in describing imported cases of denv infection. arthropod vectors. denf belongs to natural-foci diseases. its vectors are anthropophilic species of mosquitoes: aedes aegypti and ae. albopictus in synantropic natural foci. humans are the only vertebrate hosts in synantropic natural foci, whereas wild mammals are involved in virus circulation in sylvatic natural foci. vectors in equatorial africa are ae. furcifer, ae. vittatus, ae. tailori, and ae. luteocephalus. vertebrate hosts. in southastern asia, the vertebrate hosts of denv are macaques (genus macaca) and surilis (genus presbytis) living in the rain forests of equatorial climatic belts; the main vector is aedes niveus; a circulation of denv-{ , , } has been identified. natural foci of denv were also found in the eastern part of equatorial africa, in senegal and nigeria. the vertebrate hosts are patas monkeys (erythrocebus patas); wild strains are considered possible precursors of epidemic ones. among humans, wild strains provoke slight clinical forms of dengue fever. À epidemiology. denf has an epidemic character involving tens of thousands of people in southeastern asia, oceania, the caribbean basin, central and south america, and africa. the transmission pathway is a mosquito bite, mainly by members of the aedes genus. these mosquitoes are able to transmit denv in À days after feeding on a sick person. about À % of the human population falls victim to denf during epidemics. denv continues to circulate actively and to provoke wide epidemics. for example, all four types of denv exist in sri lanka, with new clades replacing old ones, accompanied by a severe clinical picture. , in the s, a new wave of denf epidemics began to develop in sri lanka, india, pakistan, and central and south america. , these epidemics were linked mainly to the relatively new denv- , but to denv- and denv- as well. in some cases-for instance, in myanmar and china -all four types of denv circulated simultaneously. clinical features. the incubation period is À days. the start of the disease is quick, with fever and with frontal and retroorbital headache. lymphadenopathia, rash in macule and papule forms (not always), leukopenia, skin hyperesthesia, changes in taste, loss of appetite, and muscle and joint pains gradually develop. then, after À days of normal body temperature, the second wave of fever develops, accompanied by a measleslike rash. the palms and soles are rash free. severe cns complications have been described to arise in endemic regions (e.g., brazil). the hemorrhagic clinical form of denf, with shock and a high level of lethality (especially among children), was originally seen in the philippines in . later, this clinical form was registered in india, malaysia, singapore, indonesia, vietnam, cambodia, and sri lanka, as well as on islands in the pacific. according to who data, more than . million patients had hemorrhagic denf from to , with , lethal outcomes. starting from , hemorrhagic denf has become the main cause of hospitalization and deaths among children in the countries of southeastern asia. the hemorrhagic form of denf usually develops after a secondary infection by a type of denv different from the primary one. the primary type of denv is not neutralized, but fragments antigen binding (fab)associated enhancement of the infection occurs. for example, in french polynesia in , two years after epidemics of denv- , an outbreak etiologically linked to denv- emerged and hemorrhagic denf was detected among children À months and À years old. five symptoms are characteristic of the hemorrhagic clinical form of dengue: high temperature, rash, hemorrhagia, hepatomegalia, and insults to the circulatory system. thrombocytopenia with blood condensation also occurs. hemorrhagic denf can be without shock or can precede it. shock develops in À days of the disease, wheninsults to the circulatory system appear: the skin becomes cold, sticky, and cyanochroic; the pulse rate increases; and drowsiness appears. in the absence of antishock actions, patients die within À h. the severity of the disease depends on a number of factors: the infection titer in the blood, the type of denv, its biological properties, and more. À imported cases of dengue. there is a high risk of denv infection for visitors to endemic regions, with consequent penetration of the virus into nonendemic regions. , denf has occurred in spain in the past (e.g., in cádiz in ). several tens of human cases are introduced into the country each year from equatorial and subequatorial regions. denv- and denv- caused a huge outbreak in greece in augustÀseptember of both and : in those periods, about , of , inhabitants of athens and piraeus contracted denf, including hemorrhagic forms and about , lethal outcomes. penetrations of denv also took place in the netherlands in À and in japan, france, northern italy, and germany in . during À in russia, among patients with fever from the risk group that visited tropicalÀequatorial countries, cases of denf were identified with the help of serological investigation ( cases arrived from indonesia; from thailand; each from vietnam and india; each from venezuela and the dominican republic; and each from sri lanka, malaysia, singapore, sierra leone, and costa rica). À in in russia, cases of denf were identified in moscow, in st. petersburg, and imported strains of denv were isolated. the risk of denf for europe has appeared again with the introduction of aedes albopictus and ae. aegypti mosquitoes in the countries of the mediterranean and black sea basins. stable populations of both these species were found on the southeastern coast of the black sea (in krasnodar krai, russia, as well as in abkhazia). À control and prophylaxis. the main approach to prophylaxis is to struggle against mosquito vectors. during the s and s, a program against ae. aegypti mosquitoes that was unprecedented in terms of scale and expense was conducted in america, but it was stopped in ; as a result, in the number of ae. aegypti mosquitoes was estimated to be same as that before the program began. the struggle against mosquito vectors in singapore turned out to be more successful, but still did not prevent denf morbidity. investigations into four-component vaccines are far from completion today. , express methods of denf diagnostics are used in airports. who issues a reference guide for the diagnosis, treatment, prophylaxis, and control of denf. (table . , figure . ). further serological investigations with the help of hit revealed that sokv belongs to the flaviviridae family, and with the help of complement-fixation testing (but not neutralization testing), to the entebbe bat serogroup. À a prototypical strain of this serogroup was isolated from a kenyan big-eared free-tailed bat (tadarida lobata) collected near entebbe, uganda, in july . taxonomy. the genome of sokv was sequenced, and genome analysis showed that the virus is related most closely ( % nt and % aa identities) to entebbe bat virus (entv). sokv has about % nt and % aa identities with other flaviviruses, except viruses of the rio bravo (rbv) and modoc (modv) groups (, % similarity). no arthropod vector of entv and sokv has been established; however, phylogenetic analysis based on a full-length genome comparison placed sokv and entv together on a distinct branch of mosquito-borne flaviviruses related to yfv and sepik virus (sepv) (figure . ) . arthropod vectors. according to serological data, domestic animals do not take part significantly in sokv circulation, although antibodies to sokv were detected among cows and sheep. isolation of sokv from birds that were known not to have made contact with obligatory parasites of bats, as well as the presence of positive sera from humans and domestic animals, suggest the participation of mosquitoes in sokv circulation. transmission of the virus by bats could be carried out by argas vespertilionis and ixodes vespertilionis. À vertebrate hosts. more than flaviviruses were isolated from bats (order chiroptera); about half are unique to these mammals. ; and yokose virus (yokv). the insectivorous bats vespertilio pipistrellus, from which sokv was isolated, belong to the evening bats family (vespertilionidae), which is active during the evening and at night. their daylight shelters are situated mostly in house garrets. v. pipistrellus is distributed over europe, the mediterranean, the caucasus region, and central asia. a part of the population overwinters in africa, where infection by local viruses (e.g., bbv, dbv, entv) could occur. experimental infection of sparrows (passer montanus) resulted in sokv being detected in internal parts of infected birds on the th and th days after inoculation. epidemiology. there are no laboratoryconfirmed human cases of sokv infection. nevertheless, the proximity of sokv hosts (bats) to human habitats, as well as the presence of encephalitis and hemorrhagic fever agents among the flaviviruses, suggest that sokv may be dangerous to humans. complement-binding specific anti-sokv antibodies were detected among humans in kyrgyzstan and turkmenistan ( . % and . %, respectively), testifying to recent infection events. À , , , , , À history. wnv (family flaviviridae, genus flavivirus), theetiological agent of west nile fever (wnf), was first isolated during research on yfv in from the blood of a native of uganda who was suffering a mild fever. the strain isolated, b , belongs to genetic lineage ii. (see "taxonomy" next.) strain eg , isolated from the sera of a child without clinical signs in egypt, is the prototype for african genetic lineage i, widely used for investigations. wnv belongs to the jev group, has the broadest antigenic properties, and, on theoretical grounds, appears to be the most ancient member of the flavivirus genus. lowpassaged wnv strains are known by many investigators to be common causes of laboratory infection, apparent or inapparent. taxonomy. phylogenetic analysis revealed that different geographic isolates of wnv could be grouped into two major genetic lineages (figure . ). lineage i includes strains from africa, southern and eastern europe, india, and the middle east. lineage ii includes isolates from west, central, and east africa, as well as madagascar. lineage can be subdivided into three clades: clade a consists of strains from europe, africa, the united states, and israel. the topotypic isolates of wnv in australia-kunjin virus (kunv)belong to clade b, and clade c is formed by isolates from india. subsequently, two genetically divergent rabensburg strains- À (isolated in the czech republic) and leiv-krnd - (isolated in russia)-were proposed to form novel lineages iii and iv, respectively. À a fifth lineage was formed by strains from india. phylogenetic analyses based on complete genomic sequences revealed that the various lineages differed from each other by À %. a putative novel sixth lineage has been detected in spain in , but only a partial sequence of the ns gene of this isolate is available in genbank. world distribution. the distribution of wnv in northern eurasia, and indeed, in the whole world, covers vast territories within the equatorial, tropical, and temperate (the southern part) climatic belts in africa, europe, asia, australia, and north america (the last starting from ). in africa, it is very difficult to find a country or landscape in which wnv has not been detected by either a virological or serological approach. the isolation of this virus from a wide array of species of birds, mosquitoes, ixodidae and argasidae ticks, and domestic animals as well as humans testifies to the ecological plasticity of the virus and therefore to its ability to adapt to different ecological conditions. two genetic lineages circulate in africa: the first, which dominates, and the second. sporadic morbidity and epidemic outbreaks permanently take place in a number of african countries, especially the republic of south africa, where a wide outbreak with at least , human cases occurred in after an active period of rain. according to a report from the pasteur institute, during the last À years alone, epidemic outbreaks were registered in algeria (in , with more than cases and deaths, and in , with cases), in tunisia (during À , with cases), morocco (in and ; the epidemic reached both humans and horses), in senegal (in ), and in kenya (in ). new centers of infection continue to be arise in africa-for example, in in morocco, where morbidity among people and horses was observed and . % of birds had specific anti-wnv antibodies, and in in the republic of south africa, where there were a number of lethal outcomes. the wide distribution of wnv in africa and its circulation among populations of the majority of the continent's species of local and migrating birds indicates that the virus is able to penetrate to southern europe and western siberia through the birds' migration pathways. most of the birds nesting in or migrating through the volga delta overwinter in africa. thus, africa is the main source of penetration of wnv genotypes i and ii into southern europe and western siberia. in asia, a peculiar third genotype of wnv appears to be circulating in the indian subcontinent. a prototypical strain of wnv genotype was isolated from xculex vishnui mosquitoes in southeastern india, and human morbidity was identified in india, pakistan, and israel. taking into account the fact that most of the birds from western siberia and many from eastern siberia overwinter in india and other countries of southern asia, there is a high probability that wnv genotype has penetrated into siberia. also in asia, both epidemics and sporadic cases etiologically linked with the first genotype of wnv have arisen regularly in israel since at least . one such outbreak was observed in À . surveillance in south korea does not indicate any wnv circulation in that country. in australia and oceania, the kunjin variant of the first genotype of wnv appears to be circulating. À kunv could be introduced into northern eurasia (in eastern siberia and the far east) by migrating birds overwintering in southeastern asia and australia. , in , an outbreak among horses in new south wales, australia, was identified. in central europe, for a long time only two strains of wnv were known: one isolated in from aedes cantans in in western slovakia and the other isolated from ae. vexans, ae. cinereus, and culex pipiens in in the czech republic, near the austrian border. anti-wnv antibodies were identified in the czech republic among . À . % of birds, including crows, daws, turtle doves, common kestrels, ducks, coots, and thrushes. later, two strains of the so-called rabensburg genotype of wnv were isolated from cx. pipiens in and in the czech republic. À the strain belonging to the second lineage of wnv was isolated from a goshawk in hungary. in in tuscany, italy, usutu virus (usuv), which is closely related to wnv, was isolated during an epizootic episode among birds, especially thrushes (turdus merula), and then, again, in in austria. later, this virus was found in hungary, switzerland, and germany. practically all of the southern european countries are endemic for wnv. , especially tragic events unfolded in romania, where there was an epidemic in julyÀoctober with a peak at the end of august to the beginning of september in the southeastern part of the country, downstream of the danube river. six administrative units and bucharest were affected, among other jurisdictions. human morbidity reached . %, and patients with cns insult were hospitalized. the number of patients with fever was at least times more, and the number of infected individuals À times more. the outbreak, which dragged on until , testifies to the development of a city epidemic form of wnf. the virus belonged to the first genotype of wnv and probably was brought to romania by birds from africa. wnv distribution in europe indicates an especially high risk of a wvf outbreak in deltas of the large rivers-the rhô ne in france and the danube in romania-through which the main migratory paths of birds overwintering in africa lie. in the recent past, wnv has been active in europe in italy, , greece, , spain, poland, the czech republic, , and france. infected mosquitoes were imported into great britain from the united states by airplane travel. as for north america, before that continent was free of wnv. penetration of wnv into america most likely happened by infected mosquitoes in the holds of ships from ports in the mediterranean sea or black sea. fifty-six cases of human wnf were revealed in new york city and its surroundings at the end of julyÀseptember , with a peak in the second half of august. seven cases ( . %) had a lethal outcome. the virus was found in culex sp. and aedes vexans mosquitoes caught in septemberÀoctober in new york city and in the states of new jersey and connecticut. positive results were obtained by rt-pcr during an investigation of brain tissues of dead birds: crows, seagulls, storks, herons, ducks, cuckoos, pigeons, jays, robins, hawks, and eagles. the genomes of the strains that were isolated were found to belong to the first genotype and were close to the strains isolated in in romania and in in israel. in , wnv was registered in the united states, probably translocated there by migrating birds or by infected mosquitoes inhabiting the holds of visiting ships. wnv was found in by À , practically all the territory of the united states, southern canada, and latin america became endemic with high morbidity and mortality. the greatest morbidity in the united states was found in the states of northdakota, south dakota, and nebraska. , the number of diseased individuals reached , À , cases in separate years. during À in the united states, more than , wnf cases were identified, with more than ( %) succumbing to the disease. the economic damage was estimated in billions of dollars. , today, wnv continues to circulate in the united states. , morbidity grew in the states of louisiana and mississippi after hurricane katrina. in montana, the infection rate of people living in close proximity to a colony of pelicans (pelecanus erythrorhynchos) is five times higher than in other regions of the state. in a sea park in texas, grampuses (orcinus orca) contracted encephalitis and died, and previous episodes of polyencephalomyelitis were revealed among seals (phoca vitulina). also in texas, three new genetic clades of wnv were found, testifying to rapid evolution of the virus on the american continent. in , an epidemic arose again, accompanied by a large number of lethal outcomes. in texas, a state of emergency was declared. northern eurasia. in northern eurasia, on the basis of the results of multiple investigations, the distribution of wnv includes moldova, ukraine, belarus, armenia, azerbaijan, georgia, kazakhstan, tajikistan, kyrgyzstan, uzbekistan, turkmenistan, the south of the european part of russia (the desert, semidesert, steppe, and forestÀsteppe landscape belts), and western siberia. , , the first data on wnv isolation were obtained from hyalomma marginatum ticks collected in the astrakhan region in . data were also obtained in azerbaijan from a blackbird (turdus merula) and a european nuthatch (sitta europaea) and, later, from a herring gull (larus argentatus) and argasidae ticks (ornithodoros coniceps) parasitizing it. wnf morbidity is now a permanent feature in the astrakhan region, kazakhstan, central asian countries (republics of the former ussr), ukraine, and azerbaijan. virological, entomological, zoologicoornithological, and epidemiological investigations of wnv in the astrakhan region and the kalmyk republic were conducted especially actively. , , À virus activity in the volga river delta was found at least as far as years ago. , , but interactions between wnv, on the one hand, and animal and vector populations, on the other, were not investigated in detail as well as genetic characteristics of the virus; indeed, the latter began to be studied well only during the first decade of twenty-first century, when suckling mice and vero-e cell culture were used to isolate the virus and serological investigations were employed to detect viral rna (neutralization testing, elisa, hit) and to sequence genes (rt-pcr). wnv endemic territories in southern russia were known from the moment the virus was isolated in the astrakhan region in . (the number of cases confirmed by elisa in the southof the european part of russia is presented in table . .) sporadic cases with a moderate clinical picture and minor outbreaks were observed in the area practically annually, as well as in other southern regions of the former soviet union. the immune structure to wnv among humans in the ussr was also known, with the most immunity occurring in the south of russia, mainly the astrakhan region (figure . , table . ). all this familiarity with wnf is why an outbreak in in volgograd was not exactly unexpected, even though it originally was identified by regional experts as an enterovirus infection. still, laboratory-confirmed wnf cases reached more than that year, and according to our estimations, the number of infected patients exceeded , (table . ) . mortality (about %) was also unusually high. large deltas of european rivers such as the rhô ne, danube, and volga rivers are known to be transit hubs for migrating birds and places of introduction of viruses linked with birds. the main natural focus in russia is the volga delta. the volga delta and contiguous territories around the northern caspian basin have been endemic for wnf for many years (tables . À . ) , and other arboviruses have been ecologically linked with aquatic and semiaquatic birds frequenting the region. ninety percent of these species of birds overwinter on the african continent. up to , birds pass over the region daily during their seasonal migrations via the volga delta main line of the eastern europe migratory route. (see figure . .) the problem is that the volga delta is the place from which viruses are introduced into anthropogenic biocenoses in close vicinity to human habitation. one consequence of this scenario was epidemic outbreaks in the astrakhan and volgograd regions in À . the volga delta consists of three basic belts, each with its own unique ecosystem features (figure . ) . the lower volga delta borders the caspian sea and is characterized by extensive exposed spaces with water. the water depth usually does not exceed . À . m, a situation that is highly conducive to the mass propagation of mosquitoes and one that also provides nesting opportunities for aquatic and semiaquatic birds. near where it empties into the caspian sea, the volga bed turns significantly to the west, so the western part of the delta, including both the reed bed of the northwestern caspian coast (up to lagan in the kalmyk republic) and some flooded islands, is more extensive than the central and eastern parts. the extreme eastern part of the delta lies in kazakhstan. a number of hunters and fishermen could be infected in the lower delta of the volga. the middle volga delta is more distant from the sea, has powerful currents, and consists of shallow lake ecosystems with reeds and shrubs. water ecosystems adjoin semidesert ones. within the limits of this zone, wild biocenoses combine with anthropogenic areas around a number of settlements, whose inhabitants keep cattle, sheep, and camels. wnf is widely registered among the native population. the upper volga delta adjoins the volgaÀ akhtuba lowlands and semideserts. large cities, including astrakhan, are located within the limits of this zone. some species of wild birds that are common in the middle delta also occur in this zone, coming into close contact with domestic animals and synanthropic birds. analysis of retrospective data collected before revealed that the main locus of native-population morbidity by wnf is in the volga delta (table . ). viruses could be introduced into the northern part of the volgaÀakhtuba lowland up to volgograd and maybe even higher. thus, in the future it will be necessary to control the introduction of the virus into the volgaÀakhtuba lowland from astrakhan to volgograd. arid landscapes occupying contiguous terrian to the west of the volgaÀakhtuba system and the volga delta are situated within the boundaries of the caspian seaÀturanian basin physicogeographical area (figure . ) . every year at the end of july, a group of specialists from the d.i. ivanovsky institute of virology in moscow has traveled to the astrakhan region and the kalmyk republic to organize and conduct a joint scientific expedition with local centers of sanitaryÀepidemiological inspection for ecologo-virological monitoring of the northwestern caspian region (figures . À . ) . the main goal of the expedition is to contain the ecological and epidemiological situation after suppression of wnv circulation in the previous epidemiological season as the result of a combination of natural factors. the plan for the collection of field material took into account the results of previous expeditions, when key milestones and marker species of mosquitoes and wild and domestic animals were identified. in particular, the researchers planned to investigate the role of the ixodidae tick hyalomma marginatum (figure . ) in wnv and other arbovirus circulation in anthropogenic and wild biotopes. both federal and local heads of various services, as well as virologists, epidemiologists, veterinarians, hunters, and frontier guards, were supplied with materials containing evaluations of ecologo-virological monitoring of their respective territories in the previous epidemiological season. practical recommendations were given for prophylaxis of wnf, cchf, and other arboviral diseases. field materials-bloodsucking mosquitoes, ixodidae ticks, internals (blood, serum, liver, spleen, and brain) of wild birds and mammals, and sera from donors and domestic animalswere collected on the territory of the astrakhan region and the kalmyk republic from the end of july to the beginning of august À within the boundaries of the volga delta, the volgaÀakhtuba valley, and adjacent arid landscapes. field materials were collected in the biotopes of the west volga coast and the east akhtuba coast, including internal waterÀmeadows of the upper and lower volgaÀakhtuba zones, hydromorphic and adjacent meadowÀsteppe biotopes of the upper and meddle belts of the volga, the volga avandelta, the territory of the sarpa lakes, and the east side of ergeny (see figures . À . ). during À , the expedition collected , bloodsucking mosquitoes (of the order diptera and family culicidae: genera culex, aedes, coquillettidia, and anopheles); , ixodidae ticks (of the taxon acari and family ixodidae: genera hyalomma, rhipicephalus, and dermacentor), mainly h. marginatum; internal parts of , birds and hares (lepus europaeus); sera from , human donors ( , in the astrakhan region and , in the kalmyk republic); and sera from , domestic animals ( , in the astrakhan region and , in the kalmyk republic) (figure . ). the field materials that were collected were stored and transported to the d.i. ivanovsky institute of virology in liquid nitrogen in dewars, in accordance with all requirements for the handling and transport of infectious samples. internal parts of , wild birds were investigated by virological methods (table . ). twelve wnv strains (tables . and . ) were isolated. according to the bioprobe method used, the total wnv infection rate among wild birds is about . %, with the highest level ( . %) reached in the middle and rt-pcr testing for any indication of wnv rna was carried out on samples of internal parts collected from wild mammals on the territory of the northwestern caspian region. positive results are presented in tables . detected in anopheles messeae ( . %), a common visitor to houses with domestic animals in anthropogenic biocenoses, as well as in an. hyrcanus ( . %) in rushes in natural biocenoses. as is illustrated in figure . , the highest intensity of wnv circulation takes place among sanguivorous mosquito populations in anthropogenic biocenoses on the territory of the volga delta (figure . ). rt-pcr testing was carried out for the detection of wnv rna in , samples of hyalomma marginatum ticks (taxon acari, wnf cases began to be registered starting in june , with the maximum reached in august (figure . ) . durint the first three . % of patients in the latter group had intracranial hypertension syndrome. there were two cases of severe disease: a -year-old patient with seromeningitis and an -year-old child with neurotoxic syndrome during the acute period. all of the cases had a favorable result: no lethal cases were registered. sera from , farm animals collected in the astrakhan region during À were tested by hit and neutralization testing in order to detect specific anti-wnv antibodies. in addition, hit-positive sera underwent neutralization testing. anti-wnv antibodies were found by hit in all species investigated: horses (mean positive result for the entire observation period, . %; coincidence with neutralization testing, . %), cattle ( . %; . %), camels ( . %; . %), pigs ( . %; cattle are the main host of anopheles messeae, and cowsheds offer favorable conditions for the mosquitoes to reproduce. cattle-specific antigens could often be found in the intestines of culex pipiens females (but not an. messeae females), which inhabitat damp basements. town utilities adjoin with farm utilities in all settlements of the astrakhan region, so cattle are the hosts both for an. messeae and for cx. pipiens. both species of mosquitoes are active vectors of wnv in anthropogenic biocenoses. horses were the only species of farm animals with clinically expressed wnf. in contrast to cattle, whose pastures are situated close to human settlements, horses browse far from settlements, often grazing in natural biocenoses. a significant portion of horse livestock in the astrakhan region are of the kushum breed, bred for meat and racing, and browse freely all year. pedigree horses (don, akhaltekinsky and arabian race horses) are kept in bloodstock farms in a stall, or they browse locally. draft horses are kept in settlements. horse-specific antigens have been found in the intestines of replete females of all mosquitoes species (except for culiseta annulata, which are relatively fewer). the total ( À ) distribution of hitpositive horses increases from the upper volgaÀakhtuba to the lower, with the highest number found in the middle belt of the volga delta (where the epicenter of the natural foci is located). pigs are the animals closest to human settlements, so pig-specific antigens are often found in the intestines of replete females of the anthropogenic mosquito species anopheles messeae and culex pipiens. pigs are kept in individual yards or on pig farms. the latter are situated far from human settlements. as they are in cattle housing, an. messeae are the main mosquito species on the pig farm; nevertheless, all mosquitoes collected here by probe were negative for wnv. in , we collected sera on the pig farms, and all probes were hit negative. in , we collected sera both on pig farms and in individual yards. sheep are the most numerous species of farm animal in the astrakhan region. sheep pastures are in the dry steppe, where conditions are favorable for the ixodidae tick hyalomma marginatum. only a couple of species of mosquito could live in the saltish, dry steppe il'mens: aedes caspius and cx. modestus. the latter is an active vector for wnv. a stable and low level of infection rate among sheep (about %) reflects the low level of intensity of wnv circulation in arid landscapes of the astrakhan region. kalmyk racing camels inhabit more arid landscapes than sheep inhabit; consequently, one might expect a lower level of seropositive camels. however, hit often demonstrates a high percentage of positive results: . % in and . % in . so, we instead collected sera from camels during À in semiwild pastures, and the percentage of seropositive results decreased. the coincidence between the results of hit testing and neutralization testing is presented in table . . horses are the best marker of wnv circulation, because they have the largest percentage of hit-positive results and the greatest coincidence between hit and nt results. kushum race horses are the most significant marker. monitoring the infection rates among farm animals will be continued, taking into account the relationships and phenomena described. it has been found that wnv can remain viable during interepidemiological periods in overwintering imagoes of sanguivorous mosquitoes (e.g., anopheles messeae, culex pipiens and culiseta annulata) as well as overwintering imagoes of the ixodidae tick hyalomma marginatum. the scheme of wnv circulation on the territory of the northwestern caspian region is presented in figure . . after the À outbreak of wnf in four administrative units in southern russia, a significant outbreak with more than cases arose in the summer and autumn of . the disease spread up to km to the north and northeast from an earlier known endemic area and now includes an additional two administrative units (tables . the orthomyxoviridae includes six genera of enveloped viruses with a segmented, negative-polarity ssrna genome. the genome of the orthomyxoviruses consists of six (thogotovirus and quaranjavirus), seven (influenza c virus), or eight (influenza a virus, influenza b virus and isavirus) segments. , all orthomyxoviruses encode three enzymes formed of viral rdrp: pb (figure . ) , pb , and pa. these proteins are about % similar among viruses of different genera. common structural proteins are np, associated with genomic rna; matrix protein; and two envelope proteins: hemagglutinin, or ha (possesses hemagglutinating activity) and neuraminidase, or na (also called sialidase) in the influenza viruses. viruses of the thogotovirus and quaranjavirus genera are transmitted by arthropod vectors, predominantly ixodidae and argasidae ticks, respectively. viruses of the influenza a virus, influenza b virus and influenza c virus genera are important human pathogens transmitted by a respiratory route. genus isavirus has only one species: infectious salmon anemia virus, which strikes fish in the salmonidae family. genus influenza a virus has just one named species: influenza a virus, represented by numerous antigenic and genetic subtypes. the genome of influenza a virus consists of segments of ssrna that encode or more proteins. À influenza a viruses are divided into distinct subtypes based on the antigenic and genetic properties of their ha and na proteins. sixteen subtypes of ha (ha À ) and subtypes of na (na À ) have been found worldwide in aquatic birds. two additional subtypes of ha (ha and ha ) and na (na and na ) are seen in new world bats. , , h and ha form a clade distinctly history. influenza as a human disease was originally described in b.c. by hippocrates (figure . ) in his book epidemics, but the "father of medicine" did not consider influenza to be an infectious disease. instead, the famous english physician thomas sydenham (figure . ) was the first who suggested the infectious nature of the disease. , the term "influenza" has been around since the first half of eighteenth century and derives from the italian "influenza di freddo" ("influence of the cold") or from spanish "influencia de las estrellas" ("influence of the stars"), the latter reflecting the contemporaneous belief in astrological reasons for the emergence of disease. up to the nineteenth century, the archaic terms "catarrhus epidemicus," "cephalgia contagiosa," "febris catarrhalis" and "febris comatose" had wide currency. the english word "grippe" (related to the russian "грипп") is related to the german "greifen" ("to catch hold") and derived from the french "gripper" ("to catch hold," "paralyze"); the word gained currency at the beginning of nineteenth century. (cf., e.g., the passage from volume , chapter of tolstoy's famous novel war and peace: "she was, as she said, suffering from la grippe; grippe being then a new word in st. petersburg, used only by the elite."). before the nineteenth century, influenza a epidemics were described only qualitatively. subtypes of the etiological agent were retrospectively revealed for the À epidemic (h n ), the À epidemic (h n ), and the À pandemic (h n , the so-called spanish flu) À -retrospectively only because influenza a virus wasn't found until by richard shope (figure . ) on the model of swine (sus scrofa) flu. , human flu was found two years later , by a group of english scientists: wilson smith (figure . ), christopher andrewes (figure . ) and patrick laidlaw (figure . ) . during the pandemic of À , it was suggested that the etiological agent of influenza a was the socalled afanasievÀpfeiffer bacillus," À named after the russian bacteriologist mikhail afanasiev (figure . ) and the german bacteriologist richard pfeiffer (figure . )-the modern haemophilus influenzae bacillus. , three influenza a pandemics were described after the discovery of the etiological agent: the avian flu has been known under the name "lombardian disease" since the beginning of the nineteenth century. À in , the italian veterinarian edoardo perroncito (figure . ) described a highly contagious disease (previously named "exsudative typhus of chickens") among chickens, with % lethality in the vicinity of turin. the terms "classic fowl plague" and "bird pest" came into wide use in , when a large epizootic outbreak in tyrol province, italy, did away with the population of farm birds there. the term "braunschweig disease" was used to identify an analogous disease among guinea fowls in europe. in , the italian scientists eugenio centanni and ezio savonuzzi demonstrated that the etiological agent of classic fowl plague is a filtrated substance. nevertheless, classic fowl plague wasn't identified as influenza a virus until , by werner shäfer (figure . ) on the example of the historical strain a/chicken/brescia/ / (h n ). , w.b. becker was the first who identified influenza a virus among wild birds when he subtypes of influenza a virus in northern eurasia. at present, we know that numerous avian influenza viruses are abundant in the bird populations of northern eurasia. however, until the end of the s, these data were absent. at that time in the former ussr, avian influenza a virus was being isolated only from poultry. one of the first avian viruses isolated in the ussr-a/duck/ukraine/ / -was destined to play an important role in the development of the theory of influenza virus evolution. in À , a group of researchers in the ukrainian soviet republic isolated several influenza virus strains from ducklings affected with sinusitis. the first three strains were isolated in in crimea and in the kharkov astrakhan region volgograd region institute of virology in moscow. as early as , the duck strains ya- , b- , z- , and c- were analyzed with respect to their antigenic specificity by hit and were found to be antigenically distinct from the human h and h viruses. after the appearance of the h pandemic virus in , some of the ukrainian duck strains were shown to be antigenically , moreover, hit testing also showed that the b- and bv strains of the virus reacted with human sera, including those collected in À and in À . on the basis of this phenomenon, the authors suggested that an avian virus similar to the strains b- and bv was the precursor of the human pandemic strain and that this antigenic variant had appeared in humans several times in the past. formerly known as ya- , strain a/ duck/ukraine/ / was shown to belong to the h n subtype, whereas a/duck/ ukraine/ / was identified as h n and a/duck/ukraine/ / as h n . the highly pathogenic h n and h n strains were isolated from chickens in the moscow region. , several virus strains producing enteritis in chickens were isolated in and in in chicken farms and identified as h n strains, , , an unusual antigenic formula for a pathogenic virus affecting poultry. six h n isolates were obtained in a chicken farm in kamchatka from chickens affected with rhinitis, conjunctivitis, and laryngotracheitis. , in , isolates identified as h n viruses were isolated from sick chickens and ducks in the russian federation and uzbekistan in the former ussr. in , h n strains were isolated in the western part of the ukrainian soviet republic from the lungs of ducklings affected with pneumonia. the isolation was the only one of an h influenza virus in the ussr (lvov dk, unpublished data). in , a large-scale series of virus isolations from wild birds, combined with some serological studies, was initiated as a part of the coordinated program of the national committee on the studies of viruses ecologically linked to birds together with the virus ecology center of the d.i. ivanovsky institute of virology. by the end of the s, the pattern of circulation of avian viruses in the territory of the ussr was identified. , , , in the ensuing years, the pattern of the influenza a virus subtypes (including h and h ) circulating in northern eurasia was amplified (figure . ). blood sera collected in the spring and autumn of near lake khanka and peter the great bay (both in primorsky krai) from birds-mainly mallards (anas platyrhynchos), common teals (an. crecca), baikal teals (an. formosa), garganeys (an. querquedula), falcated ducks (an. falcata), pintails (an. acuta), grey herons (ardea cinerea), coots (fulica atra), black guillemots (cepphus grylle) and blacktailed gulls (larus crassirostris)-were hittested against h , h , h , h , h , and h avian influenza viruses. no antibodies were found in the sera of grey herons and coots, nor were any found against h in any species. antibodies against all the other subtypes tested were encountered occasionally in the sera of gulls, black guillemots, and ducks. in some species, such as teals, falcated ducks, and black guillemots, antibodies against several subtypes were detected. in , sera were collected from gulls, cormorants, murres, and tufted puffins in the commander islands. antibodies against h , h , h , and h viruses were detected. in À , sera from gulls, cormorants, and murres were collected in the kamchatka, sakhalin, and magadan regions and antibodies to h , h , h , h , h , and h viruses were detected. antibodies against h , h , h , h , and h were identified in sera taken from arctic terns (sterna paradisaea), black-throated loons (gavia arctica), mallards (anas platyrhynchos), common teals (anas crecca), tufted ducks (aythya fuligula), greylag geese (anser anser), skuas (stercorarius sp.), and a blue whistling thrush (myophonus caeruleus) collected in the white sea basin in the estuary of the pechora river in the arkhangelsk region of russia in À . the serologic studies suggested a wide range of avian influenza viruses circulating in wild birds in northern eurasia. this suggestion was confirmed and extended by the isolation of virus strains from other wild birds. many avian species proved to be hosts of h viruses. a virus belonging to the h n subtype was isolated in from a tern in the southern part of the caspian sea basin. in , an h n strain was isolated from a common teal (anas crecca) in the russian republic of buryatia in eastern siberia. several h n viruses were isolated in kazakhstan in from waterfowl, including the common teal (an. crecca), garganey (an. querquedula), shoveler (spatula clypeata), and coot (fulica atra), as well as in from tree sparrows (passer montanus) and hooded crows (corvus cornix). in , an h n virus was isolated from a hawfinch (c. coccothraustes) in mongolia. in the same year, an h n strain was isolated from a black-headed gull (larus ridibundus) on an island in the northern part of the caspian sea. the avian viruses belonging to the h subtype seem not to be abundant in russia. in fact, for a long time the only virological evidence of the presence of this subtype in russia was the isolation of an h n virus in from a pintail (anas acuta) in primorsky krai. however, serological data suggested that h viruses circulated in wild birds not only in primorsky krai, but also in other regions of the far east, including the commander islands as well as the kamchatka, sakhalin, and magadan regions. , avian influenza a viruses belonging to the h subtype are widespread in northern eurasia. an h n virus was isolated from a common murre (uria aalge) in on sakhalin island, and another h n strain was isolated in from a pintail (anas acuta) in primorsky krai. two h n strains were isolated in in the ukrainian soviet republic from unusual hosts for avian viruses: the white wagtail (motacilla alba) and the european turtle dove (streptopelia turtur). h n strains were also isolated from grey crows (corvus cornix) in in the volga basin and from a shelducks (tadorna ferruginea) in in kazakhstan. an h n virus was isolated from a tree sparrow (p. montanus) in in the ukrainian soviet republic. in À , h n and h n viruses were isolated from ducks and herons in khabarovsk krai. one of the viruses closely resembled a strain isolated a year later in central asia. this resemblance demonstrated that h n viruses circulated in regions fairly distant from one another. in À , h n viruses were isolated in khabarovsk krai from wild ducks (anas sp.), tufted puffins (fratercula cirrhata), and horned puffins (f. corniculata) and in the arkhangelsk region in the pechora river estuary (white sea basin) from arctic terns (sterna paradisaea) and black-throated loons (gavia arctica). in , h n strains were isolated in the republic of buryatia from a mallard (an. platyrhynchos) and a pintail (an. acuta), as well as in khabarovsk krai from the common murre (u. aalge) and from black-headed gulls (larus ridibundus). avian viruses of the h subtype were isolated in À mostly in a narrow belt stretching from the lower volga, through kazakhstan, and on to the south of eastern siberia. several h n strains were isolated in from slender-billed gulls (chroicocephalus genei) in the volga delta and from great black-headed gulls (ichthyaetus ichthyaetus) on the islands in the northern part of caspian sea. in , h n virus was isolated from the black tern (chlidonias niger) in central kazakhstan. in the republic of buryatia, h n strains were isolated in from the common goldeneye (bucephala clangula). isolations of h influenza viruses from wild birds were scarce. in , several h n strains were isolated from terns (common terns and little terns) and a slender-billed gull in the volga river delta. a detailed description of the penetration of the h n strain of of highly pathogenic avian influenza (hpai) a into northern eurasia and its further dissemination is presented shortly. the strains belonging to the h subtype seem not to be abundant, but their geographic distribution is wide. an h n strain was isolated in from the arctic tern (sterna paradisaea) in the arkhangelsk region (white sea basin). one h n strain was isolated in from the pintail (anas acuta) in primorsky krai, and an h n strain was isolated from the common tern (s. hirundo) in in the caspian sea basin. in , two h n strains were isolated on kunashir island (the southernmost of the kuril islands) and four were isolated on sakhalin island. an h n strain was isolated in from a sandpiper (a member of the scolopacidae family) in the arkhangelsk region of russia. one strain of h n was isolated in in the republic of buryatia, and one strain in in mongolia. an h n strain was isolated from a mallard (anas platyrhynchos) in primorsky krai in and in khabarovsk krai in . over h n strains were isolated from a wide array of bird species near alakol lake in east central kazakhstan in . the strains were isolated from several species of ducks (anas sp.), from shorebirds (members of the order charadriiformes), to passerine birds (members of the order passeriformes), to coots (fulica atra), plovers (members of the family charadriidae, subfamily charadriinae), and chukars (alectoris chukar). this situation is a rare case of an isolation of closely related viruses from an extremely wide array of avian species. the viruses identified as h n strains were isolated in from the arctic tern (sterna paradisaea) and the red-throated diver (gavia stellata) in the estuary of the pechora river in the northern part of european russia. several h n strains were isolated from the common teal (anas crecca), the european widgeon (an. penelope), and the european golden plover (pluvialis apricaria) in in eastern siberia. in , h n strains were isolated from mallards, a pintail, and european widgeons south of issyk-kul lake in kyrgyzstan. two strains of h n were isolated from wild ducks (subfamily anatinae) in kyrgyzstan. the results of virus isolation and serological studies in the territory of the ussr in À suggested a wide circulation of avian influenza viruses in wild birds and enabled researchers to construct a map of avian influenza viruses encountered in different regions of northern eurasia. the general pattern of distribution of influenza virus subtypes in wild birds was fairly evident by the end of the decade. virus isolation was continued in the ensuing years, and it brought . single-stranded rna viruses several major results. isolations were performed mostly in the central and southern parts of european russia, in western and eastern siberia, and in the russian far east. overall, , strains were isolated from wild birds in russia in À (table . ). about samples were taken yearly from to birds in each geographic region. the mean percentage of successful isolations ranged from . % to . %. over % of the isolates were h viruses (h n , h n , h n , and h n ) isolated mostly from gulls and shorebirds in the northern part of the caspian sea. the viruses of the h subtype (over % of the total number of isolates) were isolated in several regions. many strains isolated in À from great black-headed gulls (ichthyaetus ichthyaetus), herring gulls (larus argentatus) and caspian terns (hydroprogne caspia) on the island of maly zhemchuzhny in the northern part of the caspian sea were not identified at the time of isolation with respect to the subtype of their ha. as it turned out, the strains belonged to the subtype h , was first described in , and in the mysterious caspian isolates were identified as h n , h n , and h n . to characterize the h subtype molecularly and antigenically, the complete nucleotide sequence of the ha of the strain a/great black-headed gull/astrakhan/ / was used for comparison with the has of two american strains isolated from a gull and a pilot whale. virus isolation studies in the northern caspian basin were continued in the s and s. materials were collected from wild birds in the area of the northern coast of the caspian sea (including maly zhemchuzhny island) from the delta of the terek river in the north caucasus region to the emba river in western kazakhstan. most of the strains that were isolated belonged to the h subtype, including h n , h n , h n , and h n isolates; besides these strains, only single isolates belonging to the h n , h n , h n , and h n subtypes were isolated. , in , a new, previously unrecognized, subtype of influenza virus h ha was described on the basis of the characterization of two strains isolated in from mallards (anas platyrhynchos) in the ural river delta. the h n and h n subtypes were isolated from mallards and gulls in astrakhan. a partial sequencing revealed that ns gene of the h strains isolated from the gulls was closely related to the ns gene of h and h strains isolated previously from gulls and terns in the caspian sea basin and to the h n strain isolated in the russian far east. the ns gene of an h n strain isolated from a mallard was much more distantly related to the ns gene of the viruses isolated from gulls. the results suggest that reassortment events play a significant role in the evolution of h viruses, with the ns gene being an important determinant of the range of the host. a large-scale isolation of avian influenza viruses from fecal samples was performed in À in eastern siberia and the far east by a group that included both russian and japanese researchers. scientific contacts between russian and japanese researchers of avian influenza a virus were ongoing during the eighth russianÀjapanese consultations at a conference titled "protection of migratory wild birds in the asiaÀpacific region" held at the russian ministry of natural resources in moscow april À , . at the conference, the d.i. ivanovsky institute of virology took the initiative to renew the international meetings on medical ornithology at the level of experts of asiaÀpacific countries that had been taking place regularly during the and s. as a result, the first international meeting for medical ornithology in the asiaÀpacific region was held in tokyo, japan, on june , . the meeting was devoted to the topic of hpai h n distribution in asia. a second meeting was conducted in moscow at the d.i. ivanovsky institute of virology march À , (figure . ) . in the summer of in a valley in the sayan mountains in southeastern siberia, the strains h n , h n , h n , h n , and h n were isolated. the h n and h n strains were isolated from ruddy shelducks (tadorna ferruginea) and common redshanks (tringa totanus), the h n strains from common pochards (aythya ferina), and the h n strains from northern shovelers (anas clypeata) and great crested grebes (podiceps cristatus). the h n strains were isolated from all of the aforementioned species, as well as from teals, ducks, and terns. in À , the subtypes h n , h n , h n , h n , h n , h n , h n , h n , and h n were isolated in the same region; , samples were taken from bird species. a strain isolated from the muskrat (ondatra zibethicus) in in the republic of buryatia was identified as an h n virus closely resembling the h n strains isolated from ducks in the same year and the same region. the has of the h strains (including the muskrat strain) isolated in buryatia formed a separate group of the eurasianÀaustralian branch in the phylogenetic tree of h ha (figure . ). they had a c-terminal proline residue in their ha subunit, in contrast to the serine residue of most eurasian strains. the ha genes of the h n isolates turned out to have cleavage peptides lrnvpqretr/gl identical to the ones of the low-pathogenic strains isolated from ducks in hong kong and malaysia. in contrast, the has of h and h strains isolated from teals in and from mallards in near lake chany in novosibirsk region western siberia, were related to the has of the european h and h strains. , interestingly, the has of the h strains were closely related to the ha of a/duck/ukraine/ / (h n ). however, unlike the has of h and h , the has of h strains isolated in the same area in from mallards resembled the has of h strains isolated in in japan from mallards (anas platyrhynchos). in , influenza a virus strains belonging to a rare subtype h n were isolated in mongolia from the great cormorant (phalacrocorax carbo), white wagtail (motacilla alba), and magpie (pica pica). penetration of hpai h n into northern eurasia: reasons and consequences. during longitudinal wide-scale monitoring of influenza a viruses among wild bird populations in northern eurasia, several h n and h n strains were isolated in and in the caspian sea basin. , more recently, in À , strains belonging to the same subtypes were isolated in siberia, and their features proved to be relevant to h virus circulation. onn the one hand, the has of the strains isolated from teals in in primorsky krai, as well as the has of strains isolated from a mallard in lake chany in western siberia in , were shown to be closely related to has of h strains isolated in in italy from poultry. , on the other hand, the ha of the h n strain isolated from a wild duck as early as in altai krai in southwest siberia was closely related to the ha of a/duck/malaysia/f - / (figure . ). the ha of the altai ( ) and lake chany ( ) viruses had a monobasic ha Àha cleavage site, and, accordingly, it had a low-pathogenic avian influenza (lpai) phenotype. , , , besides the amino acid sequence of the ha, the sequences of other genes of the h viruses isolated in russia proved to be relevant. the np genes of the h n and h n strains isolated in primorsky krai in formed a separate cluster in the phylogenetic tree, together with the np genes of the h n strains isolated from common shelducks (tadorna ferruginea) and common pochards (aythya ferina) in the republic of buryatia in , the h n strain isolated from the northern pintail (anas acuta) in primorsky krai in , and the , however, they were very distantly related to the np genes of h n , h n , and h n strains isolated from poultry and humans in southeast asia in À and to the np genes of h n viruses isolated from wild ducks in the caspian sea basin in the european russia in . by contrast, unlike the np genes, ns genes of the strains from primorsky krai were closely related to the ns genes of the h n and h n viruses isolated in southeastern asia in À , as well as to the ns genes of an h n virus isolated in the caspian sea basin in (figure . ) . , an abundance of influenza a subtypes in the avian populations of northern eurasia provides excellent conditions for gene exchange. the extent of the exchange is demonstrated by the relatedness of different genes of the russian isolates to the genes of european strains, on the one hand, and south asia isolates, on the other. , , , the exchange is to a certain extent restricted by host specificity, but this restriction is not rigid, and the virus genes frequently traverse interspecies barriers. avian migration routes crossing russian territory are an important factor in the gene flow. the extensive intra-and interspecies contacts in the natural habitats of wild birds in russia stimulate rapid virus evolution and the appearance of new variants through reassortment events and, presumably, through the postreassortment adjustment of genes, thereby restoring the functional intergenic match. , another factor may be the occurrence of avian influenza viruses in lake water, first registered in in eastern siberia. this phenomenon might provide a means for the temporal as well as territorial transfer of genes, as suggested by the recent detection of influenza thus, the sequencing data suggest that there exists an extensive exchange of genes of the avian influenza viruses circulating in europe, siberia, and southeast asia along the avian migration routes connecting europe, through the russian territory, with southeastern asia, the cradle of potentially pandemic reassortant viruses. after the highly pathogenic h n viruses began disseminating from southeastern our second prediction was that overwintering migrating birds could transmit the hpai virus into northern eurasia during their spring migration. we discussed two possible routes by which the birds might introduce the virus: the dzungarian (indianÀasian) migration route and the asianÀpacific route. preparing for these two possibilities, we increased our surveillance in the southern part of western siberia (through the russian foundation for basic research project -а - ) and in primorski krai (through the international scienceÀtechnical center project ) in the spring of . in april of , a wide epizootic outbreak emerged at kukunor lake (also called qinghai lake) in qinghai province, china, and from this location the virus could spread through the dzungarian gate, which links the northwestern mountain ranges of tibet with the western siberian lowland. our second prediction was confirmed as well, when hpai h n first appeared in northern eurasia, in western siberia (novosibirsk region, russia) in the summer of (figure . ). although the official start of the epizootic among poultry was dated july , (table . ), that one occurred among wild birds about weeks before was retrospectively established. the outbreak spread quickly and caused over % lethality among poultry. the virus isolations in the area were performed independently by two groups of researchers. a number of strains were isolated in zdvinsky district, novosibirsk region, by a group of researchers from the d.i. ivanovsky institute of virology in moscow. the materials for isolation (cloacal and tracheal swabs, pools of internal organs, and blood) were taken from dead, sick, and healthy birds at the farm where the epizootic occurred and from wild birds in the vicinity. , three strains were isolated from dead chickens (gallus gallus domesticus), two strains from sick or dead ducks (anas platyrhynchos domesticus), and one strain from a healthy great crested grebe (podiceps cristatus). all of the strains were deposited into the russian state collection of viruses functioning under the auspices of the d.i. ivanovsky institute of virology ( several features of the primary structure of virus proteins, such as lys residue in pb and glu residue in ns , characteristic of highly virulent variants of h n viruses, correlated with the high pathogenicity of the novosibirsk isolates. a deletion in the na gene in amino acid positions À indicated that the strains belonged to the genotype z, which dominated in in southeastern asia. the other group of strains was isolated by a team of researchers from the state research center of virology and biotechnology vector (also known as the vector institute) in koltsovo, novosibirsk region. two strains were isolated from chickens and one strain from a turkey in the village of suzdalka, dovolnoe district, in july . the viruses were isolated from homogenates guangdong province, china. the viruses were highly pathogenic to chickens in a laboratory test. our third prediction was that the virus would move with the migrating birds to their overwintering locations. as it turned out, coincident with this prediction, epizootic outbreaks occurred along the main migration routse in the urals, the russian plain, europe, africa, central asia, and india figure . ), indicating the distribution of the virus through the eastern european flyway of birds (figure . ), connecting western siberia, the russian plain, eastern europe, the middle east, and africa. our fourth prediction was that the virus would return in birds migrating from their overwintering places to northern eurasia in the spring of , with a widening of the epizootic. dramatic events occurred june À , , at uvs-nuur lake, which is situated on the boundary between the great lakes depression of mongolia and the tyva republic of russia (figure . ). an estimated , -plus birds died in the russian part of this lake, which is only about % of the total area of the lake. the species most affected was the great crested grebe (podiceps cristatus); as also affected were coots (fulica atra) and cormorants (phalacrocorax carbo). terns and gulls were involved in the epizootic to a significantly less extent. the absence of poultry farms in the vicinity of uvs-nuur lake precluded outbreaks among poultry. the tyva strains appeared to be the beginning of a new genetic lineage in the qinghaiÀsiberian genotype . . the lineage was designated as a tyvaÀsiberian subgroup (figure . ) that was isolated not only in siberia, but also in europe. it is believed (table . ) from dead and sick poultry, and all the isolates were identified as hpai h n (table . ) with a high level of sequence similarity to the qinghaiÀsuberian genotype . (figure . ) . this outcome implied a common source of infection for all the local outbreaks ( figure . ) , and subsequent epidemiologic investigation demonstrated a link to live-bird markets in moscow, where the affected farmers had purchased poultry several days before. a complete genome analysis of the prototype a/ chicken/moscow/ / revealed group of strains is shown with the use of braces: designations common to all strains in the given group are shown outside the braces; the variable part of the designations is cited inside the braces; the asterisk "*" means "any designation." only mutations that are found in all the strains of the given group are listed in the table. b bold font indicates substitutions with respect to hpai/h n / . consensus; the frame -substitutions unique to northern eurasian strains (tables À )-that is, they did not occur among northern eurasian strains previously; the frame with grey background -substitutions unique to all hpai/h n / . genotypes (strains isolated in both northern eurasia and other places); {kc-substitution that takes place in the strains of the given epizootic outbreak only; {£c-substitution that takes place in the strains of both the given and later or previous epizootic outbreaks. valley ecosystem in the north or south caucasus in the winter of and was introduced into the live-bird market through contaminated poultry cages or contaminated grain. in september , an outbreak was detected in the northeastern part of the basin of the sea of azov on a chicken farm called "lebyazhje-chepiginskaya" in the krasnodar region of russia (figure . ) . the virus isolates-a/ chicken/krasnodar/ / from poultry and a/cygnus cygnus/krasnodar/ / from a sick whooper swan (cygnus cygnus) found in a "liman" (shallow gulf) near the farm-were closely related to each other (they had two synonymous nucleotide substitutions in pb , two synonymous in pb , one nonsynonymous in m , two nonsynonymous in na, and one nonsynonymous in ns ) and belonged to the iranÀnorth caucasian subgroup of qinghaiÀsiberian genotype . (figure . ). the isolated strains contained unique amino acid substitutions with respect to a qinghaiÀsiberian consensus in pb , pa, ha, na, and ns , suggesting that regional variants were continuing to emerge. in december , a poultry farm called "gulyai-borisovskaya" in the rostov region became infected (figure . ) . unfortunately, the infection was not reported in time, and infected poultry manure was spread on adjacent fields, where wild terrestrial birds could be infected. this exposure is thought to have contributed to the infection of a number of species. including rooks (corvus frugilegus), jackdaws (corvus monedula), rock doves (columba livia), common starlings (sturnus vulgaris), tree sparrows (passer montanus), house sparrows (passer domesticus), and more. surveillance of these species by rt-pcr detected h virus in % of pigeons and crows, in around % of starlings, and in % of tree sparrows, all without clinical features. these results were confirmed by viruses isolated from wild birds and poultry (table . ). birds whose infection was confirmed by rt-pcr and virus isolation seemed reluctant to move and had ruffled feathers. on necropsy, the birds were observed to have had conjunctivitis; hemorrhages on the lower extremities and in muscle, adipose, intestine, mesentery, and brain tissue; and changes in the structure of the pancreas and liver. wide involvement of wild terrestrial birds in virus circulation, presumably from the exposure to infected chicken manure, distinguished this outbreak from others. genome analysis ( the qinghaiÀsiberian clade includes viruses that have infected and caused severe disease and mortality in humans, but currently they do not appear to be transmitted efficiently in humans. upon analyzing representative viruses in our collection for their potential to replicate in mammals, we found that isolated strains replicated effectively in mammalian cell culture lines bhk- , lech, vero e , mdck, and spev. , , pb has consensus k that promotes virulence in mammalian cells. on the basis of the amino acid sequence of ha receptor-binding sites of qinghaiÀsiberian isolates containing e , q , and g , its affinity of qinghai siberian isolates for α - À sialic acids was predicted. however, a double mutation q- l and g- s or just a single mutation e- d could switch ha receptor-binding affinity from avian to human receptors. all the qinghaiÀsiberian isolates are sensitive to amantadine, rimantadine, and oseltamivir, as has been confirmed by both direct biological experiments in vitro the first overwintering area could be the source for the iranÀnorth caucasian subgroup, the second for the tyvaÀsiberian subgroup. returning to their nesting areas in northern eurasia in the spring of , wild birds afforded a mixed virus population the opportunity to spread (figure . ) . , , , , , a decrease in the potential of isolated strains to reproduce in vitro (figure . ) is more evident in poultry (tcid . À . t) than in to wild birds (tcid although hpai h n has penetrated into northern eurasia through the dzungarian flyway of wild birds, this fact did not exclude the possibility of the virus transferring through other flyways -(e.g., through the far eastÀpacific flyway). indeed, in april with wild waterfowl. one initial theory of the introduction of the virus to poultry was from the birds' exposure to hunted ducks, but the direct interaction of wild birds with poultry seems more likely. the isolates (see table . ) from dead chickens and the common teal (anas crecca) collected in the vicinity of epizootic farms were identical and indicated a direct role of migrating birds in the introduction of the virus. the teal, which appeared to be the most likely source of infection of poultry, had no obvious behavior changes but did have hemorrhagic lesions in the intestines on necropsy. it is interesting to underline the fact that common teals were the source of isolation of h (figure . ) . , fortunately, both clades ( . and . . . ) of hpai h n that had penetrated into northern eurasia had low epidemic potential because their receptor specificity did not switch from α À -to α À -sialoside affinity, a fact that was revealed by the primary structure of the ha receptor-binding region and direct testing in sialoside-based experiments in vitro. , thus, we discuss the epizootic event provoked by hpai h n in northern eurasia during À as a model of an emer-gingÀreemerging situation in need of permanent ecologo-virological monitoring. influenza a viruses among mammals. the circulation of influenza a viruses among swine (order artiodactyla: family suidae, genus sus) was originally established in by richard shope (figure . ): his investigations not only established the viral etiology of swine flu and isolated the first historical strain a/swine/iowa/ / (h n ), but also serologically demonstrated the close relation between human infection agents and those of swine. shope's findings gave rise to a number of isolations of swine respiratory disease agents. many of these agents later turned out not to be influenza a virus; for example, "kö be porcine influenza virus," isolated in germany; "infectious pneumonia of pigs;" , "beveridgeÀbetts virus" (more often, these pathogens belonged to chlamydia sp.); and "hemagglutinating virus of japan," , which initially was named "influenza d virus" and was later identified as sendai virus (sev) (family paramyxoviridae, genus respirovirus). nevertheless, a number of strains isolated at the end of s in korea (strain oti), and in the s and s in lithuania (prototype a/swine/kaunas/ / ), estonia, poland, and russia were identified as influenza a (h n ) virus. also, in the middle of twentieth century, influenza a strains closely related to a/ swine/iowa/ / (h n ) were isolated in czechoslovakia , and hungary. finally, after the beginning of the "asian flu" pandemic in , swine influenza a (h n ) virus strains were isolated initially in china and later in czechoslovakia the principal peculiarity of pigs is the presence of both α À -sialosides (typical of human cells) and α À -sialosides (typical of avian cells) on the surface of respiratory tract cells. this feature permits both human (or adapted swine) and bird influenza a virus strains to circulate simultaneously, giving rise to conditions favorable to the reassortment and emergence of virus variants with suddenly appearing new properties. , À avian influenza a virus strains have been demonstrated to initiate productive infection in swine under experimental conditions. , À the great number of reassortment forms of influenza a viruses isolated from swine constitute evidence of the extremely high reassortment potential of the swine viral population. thus, a/swine/england/ / , isolated from nasal swabs of sick pigs in great britain in , belongs to the unique h n subtype, which was formed by the reassortment of a/ussr/ / (h n ) (the source of pb , pb , pa, ha, np, and ns segments) and a/equine/prague/ / (h n ) (the source of na and m segments). the most evident illustration of the reassortment potential of swine populations is the emergence of the pandemic "swine flu" h n pdm in as the result of the reassortment of two swine genotypes of the h n subtype: the "american swine genotype" (the source of pb , pb , pa, ha, np, and ns segments) and the "european swine genotype" (the source of na and m segments) (figure . ). À using different receptor-mimicking sialosides (table . ), we investigated the evolution of receptor specificity in influenza a (h n ) pdm virus during pandemic and postpandemic epidemiological seasons. different types of sialoside specificity spectra are presented in figure . . to compare α À -and α À -sialoside specificities, we introduced the special parameter w / , which is the ratio of the optical density for flat α À -sialosides ( sl and sln) to the optical density for flat α À -sialosides ( sl and sln): if w / is , (w / , . ), then α À -specificity dominates. in contrast, if w / . . , then α À -specificity dominates. (strains with w / % . have approximately equal α À -and α À specificities.) the sialoside specificity of the first pandemic strains isolated in our study, a/california/ / (h n ) pdm , demonstrates dual affinity to both α À -and α - -sialosides (figure . ) . therefore, such strains might be able to effect swineÀhuman and humanÀhuman transmission, and their pathogenicity is higher than that of seasonal influenza viruses (w / % pigs could be the source of influenza a virus not only in humans, but also in synantropic animals. s. agapov published an article on the pathogenic properties of influenza a virus specimens isolated from brown rats (rattus norvegicus) in pigsties. experimental infection of swine influenza a virus strains in rodentsmice (subfamily murinae) and hamsters (subfamily cricetinae)-has been described in a number of publications. , , , À rodents have become a widely used laboratory model for influenza a virus. productive infection in laboratory mice (order rodentia: family muridae, genus mus) was revealed in a pioneer publication of w. smith (figure . ), c. andrewes (figure . ) and p. laidlaw (figure . ). adapted to mice, influenza a virus strains are widely used to investigate infectious process, pathology, and the efficiency of antivirals. , À in , the strain influenza a/muskrat/ buryatia/ / (h n ) was isolated from muskrat (ondatra zibethicus) hunted in the selenga river delta, near where it empties into lake baikal. despite mountain relief along the lake coast, the delta represents a sandbank wedge overgrown with low reeds where the conditions are conducive to a mass nesting of ducks and a high density of population of muskrats. as a result, there is a high level of interaction between the populations of aquatic birds and muskrats. in particular, a/muskrat/buryatia/ / (h n ) has the highest homology with a/pochard/buryatia/ / (h n ). the strain from muskrat turned out to be virulent to mice without any preliminary adaptation, like the majority of h strains from siberian ducks. it was suggested that virulence was promoted by an r g mutation in ha. , the russian state collection of viruses contains the influenza a/sciurus vulgaris/ su- sln -su- -sialyllactose: -su-neu acα - galβ - glcβ primorje/ / strain with an undetermined subtype isolated from a red squirrel (sciurus vulgaris). weasels (order carnivora: family mustelidae) are another sensitive group of hosts for influenza a viruses. the sensitivity of the domestic ferret (mustela putorius furo), an albino form of the forest polecat (mustela putorius), to the virus was explored even in the earliest scientific publications devoted to influenza a virus. , today, ferrets are the best animal model of influenza a virus infection. in particular, sera of infected ferrets (as well as infected rats) are widely utilized for influenza a virus subtype identification. in , japanese scientists demonstrated that the epidemic strain a/kumamoto/ / (h n ) was able to provoke disease in the european mink (mustela lutreola), and perhaps it was this virus that caused a respiratory disease epizootic on japanese fur farms during À . in À , during an epizootic among minks in sweden, six strains of influenza a (h n ) virus (prototype a/ mink/sweden/e / ) were isolated and turned out to have an avian origin. in , an influenza a/stone marten/germany/r / (h n ) strain was isolated from the internals of a stone marten (martes foina) that was found dead in a place where there was mass mortality of birds in germany. , the circulation of influenza a virus among cats (order carnivora: family felidae) was originally established in by the japanese virologists j. nakamura and t. iwasa: strain a/cat/fusan/ / (known as "chiba virus") turned out to be an avian strain of the h n subtype. in , c.k. paniker and c.m. nair described the successful experimental infection of adult cats and eight-monthold kittens by a/hong kong/ / (h n ), of the "hong kong flu" pandemic strain. a number of h n strains from felidae members-tigers (panthera tigris), À leopards (p. pardus), and domestic cats (felis catus) À -were described after . the first experiment involving the infection of dogs (order carnivora: family this strain had an avian origin, but provoked lethal pneumonia in dogs. it is noteworthy that influenza a virus can be isolated from nasal swabs of dogs during inapparent infection, so this virus might be more widely distributed among dogs than is usually considered. influenza a virus is often the cause of pericarditis in dogs. the circulation of influenza a viruses among horses (order perissodactyla: family equidae, genus equus) was originally explored in by a group of czechoslovakian scientists headed by bella tumova (figure . ). in that year, a widespread epizootic emerged among horses (equus ferus caballus) and the historical strain a/equine/prague/ / was isolated. a subtype of this strain was given an initial designation h eq n eq and later was identified as h n (but, for a long time, veterinarians designated this subtype as equine influenza type ). later, influenza a (h n ) strains were isolated in other european countries and the united states. during the "asiatic flu" pandemic of À , a number of strains of influenza a (h n ) were isolated from sick horses in the moscow region of the former ussr hungary. , it was shown that these strains were significantly different from a/equine/ prague/ / (h n ), belonged to the h n subtype, and had a human origin. equine influenza a type was originally found in in miami, florida, in the united states, when the prototypical strain a/equine/ miami/ was isolated and designated as subtype h eq n eq . later, this subtype was identified as h n and was multiply isolated À in both north and south america. in the former ussr, influenza a (h n ) virus strains were isolated from horses in the ukrainian soviet republic during a widespread epizootic in in the vicinity of kiev. the russian state collection of viruses contains the influenza a/equine/mongolia/ / (h n ) strain, which originates from birds and over came the interspecies barrier to penetrate into the equine population. the circulation of influenza a virus among camels (suborder tylopoda: family camelidae, genus camelus) was originally established by d. k. lvov (figure . ) in . in december , an epizootic of "contagious cough" among bactrian camels (camelus bactrianus) emerged in mongolia. thirteen strains were isolated from nasal swabs; , tajikistan, and the ukrainian soviet republic in the former ussr. the circulation of influenza a viruses among cattle has been confirmed by multiple serological data. , À the first isolation of influenza a strain from sick sheep (ovis aries) (order artiodactyla: family bovidae, subfamily caprinae) was carried out in by a group of hungarian scientists under the direction of g. takatsy during an epizootic among farm animals. , the strain a/sheep/hungary/b / (h n ) isolated by takatsy was later utilized by j.l. mcqueen and f.m. davenport for experimental infection in lambs, but they observed no clinical symptoms. the circulation of influenza a viruses among deer (order artiodactyla: family cervidae) was originally established by t.v. pysina and d.k. lvov when they isolated the a/rangifer tarandus/chukotka/ / (h n ) strain from slowed reindeer (rangifer tarandus) in the chukotka peninsula. the russian state collection of viruses in the d.i. ivanovsky institute of virology contains the strains a/ deer/primorje/ / (h n ), isolated from red deer (cervus elaphus) in primorsky krai, and a/rangifer tarandus/yamal/ / (h n ), isolated from reindeer (r. tarandus) on the coast of the barents sea. specific antibodies towards influenza a (h n ) and a (h n ) were detected in the sera of red deer (c. elaphus) and elks (alces alces) in the north of germany. , s.q. li established the presence of about a % immune layer toward influenza a (h n ) and a (h n ) among cervidae in the northeastern provinces of china. the strain influenza a/whale/pacific ocean/ / (h n ) (or, alternatively, a/ whale/po/ / ) from a whale belonging to the balaenopteridae family (order cetacea, suborder mysticeti) and bagged in the south pacific ocean was isolated by a group of soviet virologists under the direction of d.k. lvov (figure . ) in . this strain turned out to be reassortant between human and avian virus variants. two strains of influenza a virus were isolated by a group of american virologists under the direction of r. webster (figure . ) from slowed long-finned pilot whales (globicephala melaena) near portland, maine, in the united states in : a/whale/maine/ / (h n ) (from periapical lymph nodes in the lungs) and a/whale/maine/ b/ (h n ) (from the lungs). further molecular genetic investigation, carried out by a russianÀamerican group of scientists, revealed that influenza a variants in gulls (family laridae) were the source of these strains. a number of influenza a virus strains were isolated on the coast of north america: h n , h n , and h n . , thus, one could expect to find influenza a viruses among seals in northern eurasia as well. pathogenesis. epithelial cells of mucous membranes are the main targets of influenza a viruses. degeneration, necrosis, and further apoptosis, followed by tearing away of the epithelial cell layer take place as a result of the infection. nevertheless, the main element of influenza a virusÀinduced pathogenesis is lesions on the system of vessels; the lesions emerge as the result of the toxic effect of the virus, an effect that includes the multiple formation of active oxygen forms. the latter provoke the generation of hydroperoxides, which interact with lipids and phospholipids of the cell wall to oxidize their peroxide, thereby hindering transport across the cell membrane. À a subsequent increase in the permeability of vessels, the fragility of their walls, and a violation of the body's microcirculation result in hemorrhagic manifestations, from nasal bleeding to hemorrhagic hypostasis of the lungs and hemorrhages in the substance of the brain. , frustration of the circulation, in turn, defeats the nervous system. the pathomorphological picture is characterized by the existence of lymphomonocytic infiltrates around small and average-size veins, hyperplasia of glial elements, and a focal demyelinization that testifies to the toxic and allergic nature of the pathological process in the cns during influenza. , , the most significant factors involved in cell tropism of the influenza a virus are the receptor assembly on the surface of the potential target cell and the ability of cell proteases to cleave ha into two subunits (ha and ha ) followed by fusion peptide rescue. À for example, for avian influenza a virus variants, there is an obvious threshold in the virulence level: so-called lpai and hpai. hpai strains strike vascular endothelial and perivascular parenchymal cells as well as the cardiovascular system, quickly reproduce high titers in practically all internal organs, and cause systemic disease leading to death of a bird À days after infection. lpai strains, to the contrary, reproduce in low titers, have a narrow tropism toward mucous in the digestive and respiratory tracts (figure . ), and cause enteritis or rhinitis with low mortality. (however, bird diseases connected with lpai also cause significant damage to agriculture and can break the interspecies barrier, resulting in diseases that are dangerous to people). wild aquatic and semiaquatic birds, which are natural reservoirs of influenza a viruses, can have inapparent disease during either lpai or hpai infection. , , , , , À , , , À the ability of ha to be cleaved by proteases depends on the amino acid composition of the proteolytic cleavage site: lpai strains contain only one or two positively charged basic amino acids (k or r), whereas hpai strains have an enriched amount of basic amino acids. , , , , , , À nevertheless, pandemic strains with extremely high virulence in humans have only single basic amino acids within the limits of the proteolytic cleavage site (table . ). still, it is noteworthy that lpai could provoke human disease as well. except for the amino acid composition of the proteolytic cleavage site of ha, the efficiency of the cleavage process depends on glycosylation of ha in the vicinity of this site. , amino acid substitutions that switch virus tropisms from avian to mammalian cells in different influenza a virus proteins have been described: e k, , , , the classic diagnostic approach is to isolate the virus with the use of sensitive biological models (ferrets, developing chicken embryoa, and cell lines). influenza a virus infection could be retrospectively detected by hit or neutralization testing, but the most effective diagnostic methods are rt-pcr and biological microchips. control and prophylaxis. vaccination, together with the forced slaughter of livestock. is the most effective and accessible approach to influenza a prophylaxis among domestic animals. each country chooses its own strategy for combining these methods. for example, in russia only livestock in small and individual farms is to be vaccinated whereas birds in poultry farms are not vaccinated, but are killed if either hpai or lpai is detected. the genome of the quaranjaviruses consists of six segments of negative ssrna. segments À encode the proteins of a replicative polymerase complex (polymerase basic protein , or pb ; polymerase acidic protein, or pa; and polymerase basic protein , or pb , respectively). the pb protein (polymerase basic protein, rdrp) is one of the most conservative proteins of all viruses with a segmented rna genome. the amino acid sequence similarity of the pb protein among the viruses of different genera in the orthomyxoviridae family is À %, on average, but the similarity of the functional domains of rdrp (pre-a, a, b, c, d, and e motifs) is À % (figure . ) . the envelope glycoprotein gp (ha, segment ) of the quaranjaviruses has a very low similarity to the homologous protein (ha, segment ) of influenza viruses. however, it has some similarities tgo the surface glycoprotein of the baculoviruses. the amino acid sequences of thogotovirus genus members have about % identity with qrfv and tlkv. two other segments of the genome (segments and ) of the quaranjaviruses encode two proteins whose function is unknown. these proteins are probably structural proteins, which act as nucleocapsid (n) and matrix protein (m), respectively, but currently their function is not well known. other viruses of the quaranjavirus genus have been found in south africa, nigeria, egypt, iran, afghanistan, and oceania. the quaranjaviruses are associated with argasidae ticks (argas arboreus, a. vulgaris, ornithodoros capensis), which are obligate parasites of birds. tlkv has been classified into the quaranfil group of the orthomyxoviridae family on the basis of its antigenic reactions. À taxonomy. like the other members of the quaranjavirus genus, tlkv has a genome that consists of six ssrna segments. the pb protein amino acid sequence of tlkv has % and % identities with qrfv and jav, respectively (table . ). the similarity of the pb and pa proteins of tlkv to those of orf virus (orfv) is %, on average. the envelope glycoprotein (gp, segment ) of the quaranjaviruses has very low similarity to the homologous protein (ha) of influenza viruses. however, it has some similarities to the surface glycoprotein of the baculoviruses. the similarity of the gp of tlkv to that of qrfv is % nt and % aa (table . ). segment of tlkv has one orf and encodes a protein with unknown function ( aa). its similarity to the same protein of qrfv is % aa. segment encodes a protein aa long, which has no homology with any of the virus's proteins that are deposited in the database genbank. the similarity of this protein in tlkv and the same protein in qrfv is %. figures . and . show the results of phylogenetic analysis based on a comparison of pb and the envelope protein (gp and ha, respectively). on the phylogenetic trees, tlkv is grouped with qrfv and jav within the quaranjavirus genus. arthropod vectors. natural foci of tlkv associated with argas vulgaris ticks in kyrgyzstan are located below the northern border of the area of distribution of argasidae ticks ( % on). this boundary coincides with the line of a frost-free period of À days a year and an average daily temperature above for no less than À days per year. the ability of these ticks to withstand prolonged starvation (up to years), as well as their long life cycle ( À years), polyphagia, and ability to transfer viruses transovarially, provides stability of the virus's natural foci. À , À animal hosts. tlkv was isolated from argasidae ticks collected in the nesting burrows of birds. complement-fixation testing of the birds from these colonies revealed that qrfv have been found in . % of the human population. the genus thogotovirus currently includes four viruses: thogoto virus (thov), dhori virus (dhov), araguari virus (argv), and jos virus (josv). , the viruses of thogotovirus are arboviruses, transmitted mainly by ixodidae ticks; therefore, the genus had previously been called orthoacarivirus, to emphasize these viruses' association with ixodids (taxon acari: order parasitiformes, family ixodidae). thov was originally isolated from the ticks rhipicephalus (boophilus) decoloratus and rh. evertsii collected from cattle in thogoto forest, nairobi, kenya, in . subsequently, it was isolated from human, cows, camels, and ticks in many countries in africa. , the genome of the thogotoviruses consists of six segments of negative-polarity ssrna that encode seven proteins. (segment encodes two forms of matrix protein.) , the most conservative proteins of the replicative complex (pb , pb , pa) of thogotoviruses have À % identity with those of the influenza a virus genus. history. dhori virus (dhov) was originally isolated from hyalomma dromedarii ticks collected from camels in india. dhov has also been isolated in egypt, portugal, russia, and transcaucasia. À in russia, several strains of dhov were isolated from h. plumbeum ticks, anopheles hyrcanus mosquitoes, and lepus europaeus hares, all in the volga river estuary. , one strain of dhov was isolated from the cormorant phalacrocorax carbo in maly zhemchuzhnyi island in the caspian sea ( n, e; figures . and . ). the prototypical strain of batken virus (bknv), leiv-k , was isolated from hyalomma marginatum ticks collected from sheep near the town of batken in kyrgyzstan in april . other strains of bknv were isolated from a mixed pool of aedes caspius and culex hortensis mosquitoes in kyrgyzstan and from ornithodoros lahorensis and dermacentor marginatus ticks in transcaucasia. antigenic studies showed that bknv is closely related to dhov, but differs from it. taxonomy. the similarity of the structural homologous proteins of the thogotoviruses (thov, dhov, argv, and josv) ranges from % (m-protein, segment ) to % (np, segment ). the envelope protein ha (segment ) has À % identity, on average. the similarity of the nonstructural proteins (pb , pb , and pa) ranges from % to %. bknv has a high similarity to dhov. the proteins are % (pb , pa, np, m) and % (pb ) identical. the similarity of the envelope protein ha of bknv to that of dhov is %, a percentage that explains the antigenic differences between these two closely related viruses. because the homology of the other structural and nonstructural proteins of bknv and dhov is À %, it can be concluded that bknv is a variant of dhov, typical to central asia and transcaucasia. a phylogenetic analysis based on a comparison of the pb and ha proteins is presented in figures . and . . arthropod vectors. apparently, the main arthropod vector of dhov and bknv is hyalomma sp. ticks-in particular, h. marginatum. dhov has also been isolated from h. dromedarii, dermacentor marginatus, and ornithodoros lahorensis ticks. rare isolations of dhov and bknv from mosquitoes (anopheles hyrcanus, aedes caspius, and culex hortensis) suggest that they play some role in the circulation of these viruses. vertebrate hosts. antibodies to dhov were found in % of camels, % of horses, and % of goats in the indian state of gujarat, where the virus was first isolated. antibodies to bknv were found in . % of sheep and . % of cattle in kyrgyzstan. two strains of dhov were isolated from a hare (lepus europaeus) and a cormorant (phalacrocorax carbo) in natural foci of the virus. , the bird, from which dhov was isolated on maly zhemchuzhnyi island, was ill with respiratory failure, inability to fly, and loss of coordination (figure . c) . human disease. several cases of disease caused by dhov have been registered. the disease occurred with fever, encephalitis ( %), headache, and weakness. antibodies to dhov were found in À % of the population in the volga river delta (in the south of russia) and in . % in the south of portugal. antibodies to bknv were found in the sera of . % of the human population of kyrgyzstan. five cases of laboratory infection were identified. the togaviridae family consists of two genera (alphavirus and rubivirus) of enveloped rna viruses. the virion of the togaviruses ( nm) contains a core particle ( nm) formed by a capsid protein and comprising a single-stranded, positive-sense genomic rna , À , nt long. the lipid bilayer contains the heterodimers of two surface glycoproteins e and e , which form an icosahedral surface of the virion. the genomic rna has a cap structure at the -and poly-a tail at the -end, as well as two orfs encoding nonstructural and structural proteins. the nonstructural proteins are encoded by the -orf (which occupies two-thirds of the genome), whereas the structural proteins are encoded by the subgenomic -orf. most viruses of the alphavirus genus are arboviruses and can replicate in either a vertebrate host and or an invertebrate vector. , the rubivirus genus consists of one species-rubella virus-that is transmitted by aerosol and is the causative agent of disease known as rubella. , the genome of the alphaviruses is a singlestranded rna with positive polarity about , nt in length. the viral rna has a cap at the -end and a poly-a tail at the -end. a large part of the genome of the alphaviruses (about two-thirds, beginning from one-third into the genome and extending to the -end) encodes nonstructural proteins that form the viral replicative complex nsp , nsp , nsp , and nsp ). structural proteins (core, e , e , k, and e ) are translated from subgenomic rna ( s rna), which is formed in the process of replicating the virus and corresponds to the other one-third of the genome (figure . ). the alphaviruses can infect a wide range of vertebrates. most of the alphaviruses are arboviruses and are associated with mosquitoes (genera culex, culiseta, aedes, coquillettidia, and haemogogus) and birds, the latter of which can transfer viruses during migration. À other vertebrate hosts of the alphaviruses are ruminants, reptiles, amphibians, and fish. , the alphaviruses are divided into antigenic complexes. among the alphaviruses are dangerous pathogens of humans or animals, such as eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), sindbis virus (sinv), chikungunya virus (chikv), and others. history. chikv (family togaviridae, genus alphavirus, semliki forest group) is the etiological agent of a fever that is mortally dangerous to humans. this disease is accompanied by joint and muscle pains (right up to complete immobilization of the patient) and a two-wave course of the fever, together with a macu-larÀpapular rash emergency (usually during the second wave). the etymology of the name "chikungunya" is {chee-kungunyalac, which, in the makonde local language, means "doubled up," owing to the severe joint pains. chikv was originally isolated by r.w. ross from the serum of a patient with fever during the decoding of an epidemic outbreak in tanzania in februaryÀmarch . À the close relation of chikv to mayaro virus (mayv), from the semliki forest group, was demonstrated in by serological methods. , distribution. chikv was also isolated in cambodia in southeastern asia in , in hindustan in , , and in the eastern part of new guinea in . the basic area over which chikv is distributed (table . taxonomy. chikv belongs to the togaviridae family, alphavirus genus, semliki forest group. on the basis of comparative analysis of the e gene, chikv was classified into three genotypes: a (asian), cesa (centre, east, and south african), and wa (west african) , À (table . vertebrate hosts. rodents, bats, and monkeys are the natural reservoir of chikv. , À , there is substantial evidence, that, in africa, wild primates play an important role in the natural transmission cycle, but it is not clear whether infection in primates is incidental to or necessary for the maintenance of the virus. in uganda, chikv was frequently isolated from aedes africanus mosquitoes, which preferto feed on monkeys in the forest canopy. specific anti-chikv antibodies were found among chimpanzees (pan troglodytes) in equatorial and savanna forests in the democratic republic of the congo (kinshasa) and in savannas in southern africa. antibodies were found over a wide area in vervet monkeys (cercopithecus aethiops) and baboons (pipio ursinus), and in both species the virus could circulate in the blood for two to three days at high concentrations without evidence of illness. so, wild animals could play an important role as amplifying hosts. chikv was isolated in dakar , senegal, from bats, which developed viremia after experimental infection. but in india, inoculation of the virus into two species of fruiteating bats was followed by low virulence. , antibodies were found among donkeys, bats, and wild rodents in africa and among domestic animals in asia. , inoculation of african strains into cattle, sheep, goats, and horses failed to produce viremia. apart from chickens, adult fowl and several species of wild birds did not develop viremia after experimental infection. but experimental infection of vervet monkeys and baboons led to high viremia (up to log pfu/ml) during six days, which is sufficient for the infection of mosquitoes. arthropod vectors. chikv is transmitted by bloodsucking mosquitoes. the main vectors for this virus during epidemics are aedes aegypti and ae. albopictus in urban regions and mosquitoes from the aedes, culex, and coquillettidia genera in rural landscapes. , À , chikv has been multiply isolated from ae. africanus, ae. luteocephalus, ae. furciferÀtaylori, cx. fatigans, and coq. fuscopenatta, all of which could preserve the virus and realize virus circulation in natural foci. , , epidemiology. a high level of viremia in humans (up to log pfu/ml) makes it possible for mosquitoes to transmit chikv from human to human -a plausible reason that large epidemic outbreaks have been known in big cities of southern and southeastern asia since the s. , , À beginning in the middle of the s, epidemiological processes linked to chikv have intensified (table . ), although this fact could be explained by improvements in laboratory diagnostics: previously, chikungunya fever was often confused with dengue. in any event, chikvprovoked lethality has increased, in some cases up to . %). , increases in the frequency of imported chikungunya fever cases seen at the beginning of the twenty-first century (table . ) are most dangerous, especially when the possibility of chikv penetration into local mosquito populations is taken into account. since , imported cases of chikungunya fever have become more frequent in europe (italy, , , , spain, france, , , belgium, switzerland, germany, the czech republic, norway ); the americas (canada, the united states, , brazil ); eastern asia (hong kong, south korea, japan , ); and australia. outbreaks in brazilian cities emerged with infections from aedes aegypti, whereas in rural regions aedes albopictus was the vector, introduced from southeastern asia, including japan. imported cases of chikungunya fever in russia. a -year-old patient arrived in russia september , , and suddenly fell ill, with a body temperature of . c. antipyretic drugs were not effective. early in the morning on september , , the patient was delivered to a moscow infection hospital with a diagnosis of "fever with unknown etiology." the fever had mid-level severity, and the patient complained of shivering, headache, and asthenia. hyperemia of the conjunctivae, papularÀhemorrhagic rash on the abdomen, and cruses were found. a medical radiolograph (figure . ) of the lungs of the patient revealed decreased clarity at the back of the lung field and diffuse reticular pneumosclerosis in the right lower lobe pyramid, as well as local changes with expressed peribronchial and perivascular alterations. a round shadow was detected near (i.e., peribronchially to) the intermediate bronchus. the roots were intensified. the heart was enlarged at the left. thus, the medical radiography portrait was consistent with rightside pneumonia with lymphadenopathy. several peculiarities of the case were the bareness of clinical symptoms (pneumonia was diagnosed only via medical radiography), a rapid progression of changes in the lungs, and the absence of inflammation markers in the peripheral blood. three days later, positive dynamics were detected: the basal parts of the right lung were restored to their previous level of clarity, although the shadow indicating a hypertrophic lymph node and right root broadening remained. bioprobes (blood swabs and nasopharyngeal swabs) were delivered to the d.i. ivanovsky institute of virology. the absence of influenza a and b viruses was established by rt-pcr. the strain chikv/leiv-moscow/ / was isolated with the use of intracerebrally inoculated newborn mice and was identified with the help of a completegenome (genbank id: kf ) nextgeneration sequence approach. phylogenetic analysis (figure . , table . ) revealed that the chikv/leiv-moscow/ / strain belonged to an asian genotype. this strain was deposited into the russian state collection of viruses (deposition certificate n with a priority of november , ). serological methods revealed eight cases of imported chikungunya fever that had previously been described in russia: from indonesia, singapore, india, the island of réunion, and the maldives islands. the chikv/leiv-moscow/ / strain was found to belong to the a-genotype, whereas most of the cases imported into europe belong to the cesa genotype, reflecting the "bridge" role of russia between europe and asia. the modern-day intensification of both international links and transport flows among countries increases the probability of imported cases of infection emerging. the penetration of aedes aegypti and aedes albopictus to the russian black sea coast , , suggests the emergence of seasonal outbreaks in the dynamically developing greater sochi region as well. history. getah virus (getv) was originally isolated in western malaysia from culex gelidus and cx. tritaeniorhynchus mosquitoes. À this virus is widespread in southeastern asia and in australia. À the first isolation of getv in northern eurasia was carried out by m.p. the genome of getv is , nt long. the strains of getv, circulating in different geographical regions of northeastern and southeastern asia, have a high level of similarity. À a pairwise comparison of complete genome sequences revealed that isolates from malaysia, south korea, china, mongolia, japan, and russia have À % nt identities, suggesting that the rate of getv evolution is low. phylogenetic analysis of the e gene ( figure . ) is not conducive to dividing the getv strains into distinct clusters. analyses of numerous strains isolated in japan showed that genetic differences were determined by the time of isolation more than the place of isolation. an analysis of strains of getv isolated in different regions of russia revealed their high degree of similarity, but still, they could be divided into three groups on the basis of minimal differences. the first group comprises strains from tundra and for-estÀtundra in the magadan region and the sakhaÀyakutia republic in the north of asia. the second group encompasses strains from leaf-bearing forests of khabarovsk krai. the third group consists of isolates from forestÀsteppe and steppe landscape belts of khabarovsk krai, the republic of buryatia, and mongolia. , distribution. according to our data, , , À getv is distributed over eastern siberia and north pacific physicogeographical lands (figure . ) . the most intensive virus circulation was revealed in the steppe landscape belt of mongolia, as well as in the mixed forests of khabarovsk krai and in the northern taiga of the magadan region and the sakhaÀyakutia republic. getv circulation intensity is significantly lower in tundra and forestÀtundra landscapes, a phenomenon that could be explained by the temperature there. getv is the only member of the alphavirus genus whose distribution extends to the rough climatic conditions of the high latitudes of northern eurasia. , getv has penetrated to the north of asia from the overwintering places of birds, which regularly migrate by the east asian flyway , (figure . ) . the distribution of the virus in the north coincides with that of aedes mosquitoes, which are the effective vector of getv. getv and closely related viruses are known outside of northern eurasia in japan, various countries in southeastern asia, and australia. À , , À human infection. the pathogenicity of getv to humans has not yet been described. nevertheless, the antigenically close rrv has been associated with large epidemic outbreaks of polyarthritis in australia and sarawak. , vertebrate animal infection. symptomatic and subclinical infections of animals were reported in in japan, where there was a large outbreak involving racehorses. , among the clinical features seen were fever, rash on various parts of the body, and edema on the hind legs. virus isolates were more similar to the prototypical malaysian strain than to the japanese sagiyama strain. getv has been implicated in illness and abortion or stillbirths in pigs. , disease among horses was described in india. infection in cattle is usually subclinical. arthropod vectors. getv has been isolated from culex gelidus, cx. tritaeniorhynchus (malaysia, cambodia, china), cx. bitaeniorhynchus, anopheles amictus (australia), cx. vishnui (philippines); the sagiyama subtype of getv was isolated from cx. tritaeniorhynchus and aedes vexans, as well as from pigs with fever, in japan. , although their natural transmission cycle is not known in details, mosquitoes acquire getv mainly while feeding on domestic mammals and fowl. there may also be a jungle cycle involving wild vertebrates. the bebaru subtype was isolated from culex lophoceratomyia and aedes spp. mosquitoes collected in mangrove swamp forests of western malaysia. the main vectors in russia (i.e., in northern eurasia) are aedes nigripes, ae. communis, ae. impiger, ae. punctor, and ae. excrucians. , kfv was first noted in the summer of in the central and southwestern parts of fennoscandia, including russia, finland, sweden, and southern norway (figure . ). the prototypical strain leiv- a of kyzv was first isolated from culex modestus mosquitoes collected in a colony of ardeidae birds (herons) in kyzylagach reservation, located on the coast of kyzylagach bay in the caspian sea ( n, e; figure . ). taxonomy. on the basis of a comparison of a partial sequence of the e gene, isolates of sinv can be divided into five genotypes (figure . ). genotype i includes viruses from europe and africa, genotype ii isolates from australia and oceania, and genotype iii viruses from india and the philippines. together with the chinese strain sinv xj- , kyzv was assigned to genotype iv. genotype v consists of only the strain m from new zealand. the strains of genotype i form two subclusters, one of which comprises sinvs from northern europe and sub-saharan africa and the second of which consists of strains from the mediterranean region (southern europe, northern africa, and the middle east). the genetic distance between the viruses of the different genotypes of sinv (e.g., between the european and australian isolates) is not more than % nt (table . ). at the same time, sinvs isolated in the same geographic region are characterized by a high degree of similarity (figure . ) . thus, sinv strains isolated in russia, germany, sweden(ockv), and finland have about % similarity (table . ). , , , babanki virus, which is from cameroon, has % similarity to the european strains of sinv. despite the high degree of similarity among the different genotypes of sinv, known cases of human disease are caused only by strains of the europeanÀafrican subcluster of genotype i (karelian fever, a disease of ockelbo, a disease of babanki). kyzv has a high similarity ( %) to the chinese isolate sinv xj- , isolated from anopheles sp. mosquitoes in the xinjiang uighur autonomous region in the northwest of china. the divergence of kyzv and xj- from the european isolates of sinv is % nt and % aa of the entire genome sequence (table . ). the geographic isolation of kyzv and xj- and their genetic divergence from the european and australian isolates suggest that kyzv is a variant of sinv that is typical to central asia. distribution. sinv has been isolated in many regions of southern europe, the middle east, africa, southeastern asia, the philippines, and australia. , , the african continent is almost all endemic for sinv: strains are known from egypt, the republic of south africa, uganda, the central african republic, sudan, nigeria, and zimbabwe. as for asia, there are strains from turkey, india, malaysia, and the philippines. in australia, sinv strains were multiply isolated in the north of the continent. in europe, sinv has been isolated in sicily (italy) and slovenia. on the territory of the former ussr, sinv strains were multiply isolated in belarus, ukraine, azerbaijan, tajikistan, and western siberia (in the areas around the central region of the ob river valley). À . vertebrate hosts. the main vertebrate hosts of sinv are different species of birds, predominantly of the orders passeriformes, pelecaniformes, ciconiiformes, and anseriformes. sinv infection in birds can chronic, allowing them to transfer the virus during their seasonal migration. À migratory birds play an important role in the wide distribution of this virus. sinv has been known to persist for as much as two months after experimental infection. sinv strains have been multiply isolated from aquatic and semiaquatic birds in the delta of the nile river in egypt, from the white wagtail (motacilla alba) and the common hill myna (gracula religiosa) in india, and from the reed warbler (acrocephalus scirpuceus) in the western part of slovakia. in zimbabwe, sinv has been isolated from insectivorous bats of the rhinolophidae and hipposideridae families. occasionally, sinv has been isolated from rodents and amphibians. on the territory of the former soviet union, sinv was originally isolated from a yellow herons (ardeola ralloides) caught out of a bird colony in the southeastern part of azerbaijan in . serological methods have revealed sinv circulation in the astrakhan region among aquatic and semiaquatic birds, especially those of the orders pelecaniformes ( %), ciconiiformes ( %), and anseriformes ( %). neutralizing antibodies to sinv were found in coots (fulica atra) ( . %) from natural foci of the middle belt of the volga river delta. in the kuban river delta in krasnodar krai, specific anti-sinv antibodies were found among eight species of aquatic and semiaquatic birds, most frequently mallards (anas platyrhynchos) and purple herons (ardea purpurea). in belarus, anti-sinv antibodies were detected in % of birds in the summer and in . % in the fall. antibodies to sinv have been detected among farm animals (table . cattle ( . %) and horses ( . %) in the middle belt of the volga river delta. arthropod vectors. sinv is closely associated with ornithophilic mosquitoes. in egypt, this virus was isolated from culex univittatus, cx. antennatus, and anopheles pharoensis; in uganda, from coquillettidia spp.; in sarawak, (malaysia), from cx. bitaeniorhynchus; in australia, from cx. annulirostris, aedes normanensis, and ae. vigilax; in india, from coq. fuscopennata; in sudan, from cx. quinquefasciatus; and in europe, from cx. pipiens, cx. torrentium, culiseta morsitans, coq. richiardii, ochlerotatus communis, oc. excrucians, ae. cinereus, and an. hyrcanus. , according to our data, in the volga river delta sinv is transferred by culex pipiens in anthropogenic biocenoses and by anopheles hyrcanus and coquillettidia richiardii in natural ones. in the natural foci of the middle belt of the volga delta, strain can be isolated from approximately , an. hyrcanus or , coq. richiardii mosquitoes; in the low belt of the delta the ratio is in about in a power less, and in anthropogenic biocenoses it is strain per , cx. pipiens mosquitoes. sinv strains from gamasidae ticks (ornithonyssus bacoti) in india and from ixodidae ticks (hyalomma marginatum) in sicily (italy) are known. productive experimental infections were described in the argasidae ticks ornithodoros savignyi and argas persicus (although infected ticks did not transmit the virus during feeding). most likely, ticks do not play an important role in sinv circulation or as a reservoir for this virus. human pathology. sinv causes acute fever in humans but has a favorable outcome. antibodies to sinv are widely detected in human sera (table . ), although in eastern siberia and the far east cross-reactions with getv (another member of the semliki forest serogroup) can take place. the start of the disease is sudden. clinical symptoms include fever, muscle and joint pain, and rash. severe progressive arthritis of large joints could develop several years after the disease and could lead to disability. this pathology appears in À % of citizens of endemic territories. outbreaks of sindbis fever in egypt and israel emerged at the same time as west nile fever; hence, it is necessary to distinguish these infections in the laboratory. a neurotropic virus isolated from aedes mosquitoes caught in the semliki forest virus taxonomy: classification and nomenclature of viruses: 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investigation of virus evolution in the limits of the problems of biosafety and socially significant infections. moscow: d.i. ivanovsky institute of virology of rams characterization of dobrava virus: a hantavirus from slovenia, yugoslavia epidemic hemorrhagic fever with renal syndrome in yugoslavia genetic characterization of hantaviruses transmitted by the korean field mouse (apodemus peninsulae), far east russia complete genome sequence of an amur virus isolated from apodemus peninsulae in northeastern china genetic variation of wild puumala viruses within the serotype local rodent populations and individual animals phylogenetic relations of western siberian hantaviruses belonging to tula and puumala genotypes khabarovsk virus: a phylogenetically and serologically distinct hantavirus isolated from microtus fortis trapped in far-eastern russia development of serological assays for thottapalayam virus, an insectivore-borne hantavirus genetic analysis of thailand hantavirus in 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diagnostics algorithm of crimean-congo hemorrhagic fever and west nile fever. methodical handbook nosocomial infection of crimean hemorrhagic fever crimean-congo hemorrhagic fever (clinical aspect, diagnostics, treatment, organization of medical help). handbook. stavropol handbook of virology: viruses and viral infection of human and animals development and introduction of pcr-test-systems for the detection of west nile virus and crimean-congo hemorrhagic fever virus molecular diagnostics of viral hemorrhagic fevers novel one-step real-time rt-pcr assay for rapid and specific diagnosis of crimean-congo hemorrhagic fever encountered in the balkans detection of crimean-congo hemorrhagic fever, hanta, and sandfly fever viruses by real-time rt-pcr diagnostic assays for crimean-congo hemorrhagic fever efficacy of oral ribavirin treatment in crimean-congo haemorrhagic fever: a quasi-experimental study from turkey evaluation of the efficacy of ribavirin therapy on survival of crimean-congo hemorrhagic fever patients: a caseÀcontrol study the efficacy of oral ribavirin in the treatment of crimean-congo hemorrhagic fever in iran a novel vaccine against crimean-congo haemorrhagic fever protects % of animals against lethal challenge in a mouse model healthy individuals' immune response to the bulgarian crimean-congo hemorrhagic fever virus vaccine proceedings of the institute of poliomyelitis and viral encephalitis ecology of crimean-congo hemorrhagic fever virus and clinical picture of the disease in russia and its neighboring countries natural foci of arboviruses in the ussr arboviral infections in subtropics and on south of temperate zone in ussr uspekhi nauki i tekhniki. ch. {virology arboviruses and arboviral infectionc taxonomic status of artashat virus (artsv) (bunyaviridae, nairovirus), isolated from ticks ornithodoros alactagalis issaakjan, и o. verrucosus olenev, sassuchin et fenuk, (argasidae koch, ), collected in transcaucasia the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts virus taxonomy: ninth report of the international committee of taxonomy of viruses genetic characterization of erve virus, a european nairovirus distantly related to crimean-congo hemorrhagic fever virus the erve virus: possible mode of transmission and reservoir erve virus, a probable member of bunyaviridae family isolated from shrews (crocidura russula) in france the role of ecovirological zoning in prediction of the influence of climatic changes on arbovirus habitats moscow-leningrad: ussr academy of sciences; vol. ( ). arachnoidea. argas ticks (argasidae) crimean-congo hemorrhagic fever virus-encoded ovarian tumor protease activity is dispensable for virus rna polymerase function tick-borne viruses in sea-birds birds parasitizing ticks ornithodoros koch. digest of parasitology of the zoological institute ussr academy of sciences moscow-leningrad: ussr academy of sciences; vol. ( ). arachnoidea. argas ticks (argasidae) migration of the birds and transduction of infection agents. moscow: nauka; p preliminary dates about isolation of three of new arboviruses in caucuses and central asia natural foci of arboviruses in the ussr the new arboviruses, isolated in ussr in À ecology of tick-borne viruses in colonies of birds in the ussr some features of the new arboviruses, isolated in uzbekistan and turkmenia genetic characterization of caspiy virus (casv) (bunyaviridae, nairovirus), isolated from seagull larus argentatus (laridae vigors, ) and ticks ornithodoros capensis neumann in eastern and western cost of caspian sea structure of crimean-congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase- cleavage influenza virus pathogenicity is determined by caspase cleavage motifs located in the viral proteins ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts immunofluorescence studies on the antigenic interrelationships of the hughes virus group (genus nairovirus) and identification of a new strain ticks (ixodoidea) on birds migrating from africa to preliminary data about isolation of three novel arboviruses in caucasus and central asia chim virus, a new arbovirus isolated from ixodid and argasid ticks collected in the burrows of great gerbils on the territory of the uzbek ssr isolation viruses from natural foci in the ussr international catalogue of arboviruses and some others viruses of vertebrates results of searching of the arboviruses in uzbek ssr all-union conference for natural foci infection some features of the ecology of novel arboviruses, isolated in uzbekistan and turkmenia isolation of chim virus and karshi virus from rodent in kazakhstan arboviruses in the mediterranean countries taxonomic status of chim virus (chimv) (bunyaviridae, nairovirus, qalyub group) isolated from ixodidae and argasidae ticks collected in the great gerbil (rhombomys opimus lichtenstein, ) (muridae, gerbillinae) burrows in uzbekistan and kazakhstan the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts genetic characterization of geran virus (gerav) (bunyaviridae, nairovirus, qalyub group) isolated from ticks ornithodoros verrucosus (olenev, zasukhin et fenyuk, ) (argasidae), collected in the burrow of meriones libycus (lichtenstein, ) (muridae, murinae) in azerbaijan international catalogue of arboviruses and some others viruses of vertebrates qalyub virus, a member of the newly proposed nairovirus genus (bunyavividae) international catalogue of arboviruses and some others viruses of vertebrates taxonomy of issyk-kul virus (iskv, bunyaviridae, nairovirus), the etiologic agent of issyk-kul fever, isolated from bats (vespertilionidae) and ticks argas (carios) vespertilionis (latreille, ) fauna of ussr. moscow/leningrad: ( ) arachnoidea. argas ticks (argasidae) serological studies of qalyub virus in certain animal sera in egypt genetic characterization of the geran virus (gerv, bunyaviridae, nairovirus, qalyub group), isolated from ticks ornithodoros verrucosus olenev, zasukhin and fenyuk, (argasidae), collected in burrow of meriones erythrourus grey, in azerbaijan taxonomy of issyk-kul virus (iskv, bunyaviridae, nairovirus), the etiologic agent of issyk-kul fever, isolated from bats (vespertilionidae) and ticks argas (carios) vespertilionis (latreille, ) taxonomy of previously unclassified chim virus (chimv-chim virus) (bunyaviridae, nairovirus, qalyub group), isolated in uzbekistan and kazakhstan from ixodes (acari: ixodidae) and argas (acari: argasidae) ticks, collected in a burrows of great gerbil rhombomys opimus lichtenstein, (muridae, gerbillinae) the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts qalyub virus, a member of the newly proposed nairovirus genus (bunyavividae) experimental studies on the replication and dissemination of qalyub virus (bunyaviridae: nairovirus) in the putative tick vector, ornithodoros (pavlovskyella) erraticus international catalogue of arboviruses including certain other viruses of vertebrates international catalogue of arboviruses including certain other viruses of vertebrates the bandia forest virus (ipd-a ), a new arbovirus prototype isolated in senegal issyk-kul" virus, a new arbovirus isolated from bats and argas (carios) vespertilionis (latr., ) in the kirghiz s international catalogue of arboviruses incuding certain other viruses of vertebrates issyk-kul virus disease arbovirus infections in the subtropics and on the south of temperate zone of ussr migration of birds and the transfer of the infectious agents. moscow: nauka viruses, ecologically associated with birds and chiroptera in transcaucasian moscow: d.i. ivanovsky institute of virology of ussr academy of medical sciences results and prospects of study of arboviral infection in kazakhstan the problems of arbovirus conservation in the interepidemic period the results of the virus scan in tajikistan study of issyk-kul virus ecology ecology of viruses. moscow: d.i. ivanovsky institute of virology of ussr academy of medical sciences isolation of the issyk-kul virus from bloodsucking biting flies culicoides schultzei enderlein in southern tadzhikistan international catalogue of arboviruses incuding certain other viruses of vertebrates keterah virus infections in four species of argas ticks (ixodoidea: argasidae) virus taxonomy: ninth report of the international committee of taxonomy of viruses taxonomy of issyk-kul virus (iskv, bunyaviridae, nairovirus), the etiologic agent of issyk-kul fever, isolated from bats (vespertilionidae) and ticks argas (carios) vespertilionis (latreille, ) a preliminary study of viral metagenomics of french bat species in contact with humans: identification of new mammalian viruses the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts isolation of an arbovirus antigenically related to issyk-kul virus from the blood of a human patient outbreak of arbovirus infection in the tadzhik ssr due to the issyk-kul virus (issyk-kul fever) experimental infection of aedes caspius caspius pall. mosquitoes on dwarf bats, vespertilio pipistrellus, infected with the issyk-kul virus and its subsequent transmission to susceptible animals arboviral zoonoses of northern eurasia (eastern europe and the commonwealth of independent states ecological soundings of the former ussr territory for natural foci of arboviruses isolation of the viruses from natural sources in the ussr arboviruses in kazakhstan region. alma-ata/moscow: d.i. ivanovsky institute of virology rams natural foci of arboviruses in the ussr genetic characterization of the uzun-agach virus 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