key: cord-0802957-1huoe4dp authors: Yan, Ying; Chang, Le; Wang, Lunan title: Laboratory testing of SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2 (2019‐nCoV): Current status, challenges, and countermeasures date: 2020-04-17 journal: Rev Med Virol DOI: 10.1002/rmv.2106 sha: 4e9162c60a8f6d42ae89e4c56a55e8252ce44fb3 doc_id: 802957 cord_uid: 1huoe4dp Emerging and reemerging infectious diseases are global public concerns. With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus, SARS‐CoV‐2 has been attracting tremendous attention. Rapid and accurate laboratory testing of SARS‐CoV‐2 is essential for early discovery, early reporting, early quarantine, early treatment, and cutting off epidemic transmission. The genome structure, transmission, and pathogenesis of SARS‐CoV‐2 are basically similar to SARS‐CoV and MERS‐CoV, the other two beta‐CoVs of medical importance. During the SARS‐CoV and MERS‐CoV epidemics, a variety of molecular and serological diagnostic assays were established and should be referred to for SARS‐CoV‐2. In this review, by summarizing the articles and guidelines about specimen collection, nucleic acid tests (NAT) and serological tests for SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2, several suggestions are put forward to improve the laboratory testing of SARS‐CoV‐2. In summary, for NAT: collecting stool and blood samples at later periods of illness to improve the positive rate if lower respiratory tract specimens are unavailable; increasing template volume to raise the sensitivity of detection; putting samples in reagents containing guanidine salt to inactivate virus as well as protect RNA; setting proper positive, negative and inhibition controls to ensure high‐quality results; simultaneously amplifying human RNase P gene to avoid false‐negative results. For antibody test, diverse assays targeting different antigens, and collecting paired samples are needed. European region. 4 Rapid and accurate diagnosis of the causative viral pathogen is essential for early discovery, early reporting, early quarantine, early treatment, and cutting off epidemic transmission. CoVs are enveloped single-stranded positive-sense RNA (+ssRNA) viruses that can infect respiratory, gastrointestinal, hepatic, central nervous systems of humans, other mammals and birds. 5 CoVs belong to the subfamily Coronavirinae in the family Coronaviridae. 6 This subfamily consists of four genera-alpha, beta, gamma, and delta-CoVs. 7 Seven CoVs are known to cause respiratory diseases in humans. Among them, Alpha-CoVs HCoV-NL63, HCoV-229E, and beta-CoVs HCoV-OC43, HKU1 can induce mild upper respiratory disease in immunocompetent individuals, while the other three CoVs SARS-CoV, MERS-CoV, and SARS-CoV-2 belonging to beta-CoVs are more pathogenic. 6, 8 The genome of a typical CoV contains a 5 0 untranslated region (UTR), a conserved replicase domain (ORF 1ab), four genes S, E, M, and N to encode structural proteins spike, envelope, membrane, and nucleocapsid proteins, a 3 0 UTR, and several unidentified nonstructural ORFs (Figure 1 ). 6, 9 Person-to-person spread of SARS-CoV, MERS-CoV, and SARS-CoV-2 mainly occurs via respiratory droplets produced when an infect person coughs or sneezes. 10 Fever was the most prevalent symptom that occurred in 86% to 97% SARS-CoV-2 infected patients, followed by dry cough (59%-76%), fatigue (34-68%) , and dyspnea (20%-40%). 11 The median time from first symptom to dyspnea was only 5 days. 12 Apart from acute respiratory syndrome, some other organ dysfunctions, including gastrointestinal symptoms, hepatic dysfunction, splenic atrophy, seizures, and lymphadenopathy have been found in SARS and MERS. [13] [14] [15] [16] Since SARS-CoV-2 shares the same receptor angiotensin converting enzyme 2 (ACE2) with SARS-CoV, 17, 18 it is reasonable to speculate that these organ dysfunctions may also be found in SARS-CoV-2 infected patients. Actually, a retrospective, observational study showed that in 52 critically ill adult patients who were admitted to the intensive care unit (ICU), 15 (29%) had acute kidney injury, 12 (23%) cardiac injury, and 15 (29%) liver dysfunction. 19 The most severe sequelae after rehabilitation from SARS were femoral head necrosis and pulmonary fibrosis. High-dose steroid pulse treatment was used to suppress inflammation, which caused subchondral osteonecrosis in about 5% of SARS patients. 20 The risk of osteonecrosis was 0.6% for patients receiving less than 3 g and 13% for patients receiving more than 3 g prednisolone-equivalent dose. 20 In addition, the use of high dose of hydrocortisone or methylprednisolone for an extended duration was shown to be a significant risk factor for osteonecrosis. 21 RefSeq GCF_000864885.1 It has been reported that viral loads of SARS-CoV and MERS-CoV in respiratory specimens always peak in the second week after symptom onset, and viral loads in LRT specimens were higher than in URT specimens. [43] [44] [45] As seen in Table 3 23 As seen in Tables 1 and 3 , differently to SARS-CoV and MERS-CoV, SARS-CoV-2 viral loads in respiratory specimens often peak in the first week of illness and decrease thereafter. Evidence showed that viral RNA could be detected in blood and feces, 33, 46 raising the possibilities of blood transmission and oral-fecal transmission. A study of 2134 SARS-CoV infected specimens showed that the rate of viral shedding in feces was low in the first 5 days of illness (up to 28%), but rose gradually to peak at around 70% at 9 to 14 days with very high titers, even higher than in nasopharyngeal aspirates. 44 Other studies also showed that the positive rate for stool specimens peaked at Weeks 2 and 3, with a higher diagnostic yield than pooled throat and nasal swabs, and nasopharyngeal aspirate specimens. 47 or 291 copies/mL of sample, which was considered highly sensitive and recommended for screening. 63 The ORF 1a assay was considered of equal sensitivity with the upE assay, while the ORF 1b assay was considered less sensitive than the ORF 1a assay. 63, 64 An alternative approach involved in two rRT-PCR assays targeting the MERS-CoV N gene with a sensitivity of ≤10 copies/reaction, which can complement upE and ORF 1a assays for screening and confirmation has also been published and authorized for emergency use as an in vitro diagnostic test for MERS-CoV by the US Food and Drug Administration (FDA). 65 Several commercial and in-house assays that detect SARS-CoV-2 RNA have been developed. Chu, et al reported two one-step quantitative rRT-PCR assays targeting ORF1b and N gene that could detect SARS-CoV-2 < 10 copies/reaction, while the N gene assay was about 10 times more sensitive than the ORF1b gene assay in detecting positive clinical specimens. 11 It might be possible that clinical samples contain infected cells expressing subgenomic mRNA, resulting in more N gene copies. 66 71 The US CDC has developed assays including three pairs of primers targeting the N gene of SARS-CoV-2, and authorized emergency use by the US FDA. 72 Details of these assays are summarized in Table 4 In 2002, Jansen et al. firstly discovered clustered regularly interspaced short palindromic repeats (CRISPR) system. 94 In 2003, it was confirmed for the first time that CRISPR-Cas9 can be used to edit human genes. 95 In 2017, combining RPA and CRISPR-Cas13a, SHER-LOCK system was developed for NAT. 57 SHERLOCK detected viral particles down to 2 aM, and could discriminate between similar viral strains. 57 Recently, a protocol for detection of SARS-CoV-2 using SHERLOCK system has been published. 96 (Table 5) . Thus, we recommend RNase P as internal control when amplifying SARS-CoV-2 RNA. detection of viral genome by molecular testing, making it not suitable for early diagnosis. In SARS, higher neutralizing antibody response was associated with a longer illness. 13 In most MERS patients, the levels of IgG and neutralizing antibodies were weekly and inversely correlated with LRT viral loads. 51 In addition, an early MERS-CoV antibody response was associated with reduced disease severity. 98 Thus, testing antibody is helpful for surveillance, prediction of disease outcome and epidemiological investigation, but not for early diagnosis. week 30, while only anti-S3 showed significant neutralizing activity. 101 Anti-N appeared earlier than anti-S, indicating that S proteinbased assays may be preferable for use with convalescent sera. 102, 103 Sera tend to react against both antigens with higher sensitivity. 102 Cross reaction also happened in assays using recombinant antigens. Moderate cross-reactivity between SARS-CoV and porcine CoVs was mediated through amino acids 120 to 208 of the N protein. 104 Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of S protein of SARS-CoV and HCoV-EMC, 105 which is known to harbor an epitope for broadly neutralizing antibody in the case of SARS-CoV. 106 As a result, convalescent SARS sera cross reacted with HCoV-EMC. 105 Thus, for SARS- In SARS, IgG seroconversion was documented in 93% patients at mean of 20 days. 43 Positive antibody, neutralizing antibody titer of most patients was shown to peak between Weeks 5 and 8 after onset and to decline with a half-life of 6.4 weeks. 13 In most MERS patients, robust antibody responses developed by the third week of illness, and were delayed further in severely ill patients requiring mechanical ventilation. 51 From SARS to MERS, thrusting coronaviruses into the spotlight A novel coronavirus from patients with pneumonia in China Severe acute respiratory syndrome-related coronavirus: The species and its viruses -a statement of the Coronavirus Study Group Coronavirus disease (COVID-2019) situation reports Coronavirus pathogenesis Origin and evolution of pathogenic coronaviruses SARS and other coronaviruses as causes of pneumonia Epidemiology, genetic recombination, and pathogenesis of coronaviruses Coronavirus: epidemiology, genome replication and the interactions with their hosts A novel coronavirus outbreak of global health concern Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China Multiple organ infection and the pathogenesis of SARS Severe acute respiratory syndrome associated coronavirus is detected in intestinal tissues of fatal cases Histopathology of Middle East respiratory syndrome coronovirus (MERS-CoV) infectionclinicopathological and ultrastructural study Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a singlecentered, retrospective, observational study Osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids Steroid-induced osteonecrosis in severe acute respiratory syndrome: a retrospective analysis of biochemical markers of bone metabolism and corticosteroid therapy Chinese National Health Commission. 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