key: cord-0929739-sr7oa93x
authors: Peng, Minhua
title: Outbreak of COVID-19: An emerging global pandemic threat
date: 2020-07-04
journal: Biomed Pharmacother
DOI: 10.1016/j.biopha.2020.110499
sha: dc99fc5726bdc4275a595eeae69f2d46b6323134
doc_id: 929739
cord_uid: sr7oa93x

Since the first outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Wuhan, Hubei, China in December 2019, it is now recognized as a pandemic by the World Health Organization (WHO) as more than 200 countries and territories worldwide are affected with an increasing incidence. The SARS-CoV-2 infection results in a spectrum of non-specific signs and symptoms, ranging from asymptomatic infection, to flu-like illness such as fever, cough, dry cough and fatigue, to pneumonia, acute respiratory distress syndrome, and even multi-organ failures with high morbidity and mortality. SARS-CoV-2 is mainly transmitted through respiratory droplets that infected people exhale during incubation and onset period. By 12 June 2020, over 7.5 million confirmed cases of Coronavirus disease 2019 (COVID-19) with more than 421,000 deaths in the world have been reported to the WHO. No specific medication is approved to treat COVID-19, raising the urgent need for antiviral drug development. By 12 June 2020, there are over 1000 clinical trials registered in clinicaltrials.gov for treatment of COVID-19. This review summarizes the epidemiology, virology, clinical presentation, pathophysiology, diagnosis, and particularly the antiviral drugs currently under clinical trials for treatment of SARS-CoV-2 infection, together with the challenges and perspectives of this disease are also discussed.

cases were cured and more than 421,000 infected people were died with global death-to-cases ratio 5.61%. Updated cases throughout the world can be found in the following websites: https://coronavirus.jhu.edu/map.html and https://www.who.int/emergencies/diseases/novelcoronavirus-2019/situation-reports.

Estimation of the incubation period and viral shedding duration is helpful to assess the risk of transmission, make measures of isolation and quarantine, and provide rational antiviral interventions for treating patients. Based on the current epidemiological investigations, the incubation period of COVID-19 is about 1-14 days, with a median time of 3-7 days, and can be up to 24 days in a tiny minority of cases from exposure to symptoms onset [1] [2] [3] . Viral shedding may begin 2-3 days before the presence of the first symptom [4] . Median duration of viral shedding has found to be 20.0 days and the longest duration could be up to 37 days in survivors, while the viral RNA in non-survivors could be detected until death [5] . In the cases of severe COVID-19 patients, the median duration of viral shedding has shown to be 31.0 days from illness onset.

Novel coronavirus is mainly transmitted through respiratory droplet, aerosol, direct or indirect contact. Several studies have provided epidemiological evidences that human-to-human transmission of novel coronavirus in family, hospitals, and even across cities were occurred [6] [7] [8] .

Guan et al found that patterns of human-to-human transmission included familial aggregation [6] , asymptomatic virus carrier [9] , and the three-phase outbreak [10] , which were in accordance with other findings. The "super-spreaders" may occur in this pandemic [1] . Prominently, infected persons without symptoms during the incubation period could be sources of infection, suggesting the asymptomatic transmission of SARS-CoV-2 [9, [11] [12] [13] [14] [15] . The rapid and uncontrollable spread of this virus may ascribe to the "silent transmission" by some patients with atypical symptoms.

More and more evidences have proved that this virus can spread through multiple routes as well. To et al detected the self-collected saliva, a non-invasive specimen, by one-step real-time RT-qPCR assay targeting the S protein [6] . They obtained the positive results that were consistent with those detected through nasopharyngeal or sputum specimens, which may be due to the infection of epithelial cells in saliva gland ducts or the secretions from nasopharynx and lung in the saliva. Therefore, virus in saliva may allow transmission via speaking or coughing [16] .

Zhang et al firstly reported the presence of viral nucleotide in anal swabs and blood. During the later stage of infection, the level of viral RNA was higher in anal swab than in blood, suggesting the possibility of multiple routes transmission of virus including oral-fecal or body fluid route [17] . Studies of Xiao et al have shown that the viral RNA tested in feces was positive even in gastrointestinal tracts was negative [18] . Another two findings emphasized that the diarrhea as onset symptom should not be ignored and the fecal-oral transmission route should be taken seriously [19, 20] . Chen et al analyzed the viral RNA levels in pharyngeal, swab, blood, and the anal swab by real-time PCR. The concentrations of viral RNA in blood and anal swabs were positively correlated with the severity of COVID-19. Since the spike protein of novel coronavirus interacts with the host ACE2 receptor for viral attachment and entry to establish infection, the presence of viral RNA in digestive tract in which ACE2 is abundantly expressed confirmed the viral replication in extrapulmonary sites. Early detection of viral RNA in the blood and digestive tract may be helpful in predicting the progression of COVID-19 [21] .

A clinician who wore an N95 mask but not safety goggles was infected with SARS-CoV-2 while working with COVID-19 patients in Wuhan, suggesting the potential transmission through mucous membranes in the eyes [22] . Despite no detectable viral RNA was in the conjunctival swabs samples from infected patients reported in the further studies [23] , Deng et al have confirmed that SARS-CoV-2 could be transmitted through ocular conjunctival route in a rhesus macaques model, cautioning us the necessity of eye protection to prevent the spread of SARS-CoV-2 [24] .

Chen et al reported potential vertical transmission in nine pregnant women with laboratory confirmed COVID-19 infection. When the newborns were delivered by caesarean section, amniotic fluid, cord blood, throat swabs from newborns, and breastmilk were collected for testing and all of them were negative for the novel coronavirus. Currently there is no conclusive evidence supporting that women with COVID-19 in the late trimester of pregnancy could cause intrauterine infection due to vertical transmission. However, this investigation is restricted to the small number of cases and the pregnant women in the third trimester [25, 26] . It should be noted that infants and pregnant women remain susceptible to COVID-19 infection. Special care should be taken to prevent neonatal infections from mothers with COVID-19.

Overall, the above studies warn the virus transmission through multiple routes including respiratory tract, alimentary tract, nasolacrimal system, oral-fecal, and body fluid route, and that the potential maternal-fetal vertical transmission should not be ignored. The properties of viral shedding before symptoms appear and via multiple routes indicate the strong infectiousness of SARS-CoV-2 and pose a huge challenge in controlling the spread of disease.

SARS-CoV-2 belongs to the family coronaviruses and subgenus sarbecovirus (beta-CoV lineage B) and is a new strain virus distinct from Severe Acute Respiratory Syndrome (SARS-CoV) and

Middle East Respiratory Syndrome (MERS-CoV). The genome sequence of SARS-CoV-2 shows more than 85% identity to a bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1) [27] . Its The ORF1ab gene accounts for two-thirds of the genome and encodes 16 nonstructural proteins (NSP1-NSP16) while the remaining genome encodes 4 structural proteins (S,   M , E, N) and 6 accessory proteins (ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF10 ) [28] [29] [30] [31] .

Currently more than 40,000 SARS-CoV-2 genomes publicly available (https://www.gisaid.org/ https://bigd.big.ac.cn/ncov/release_genome# ) represent the genomic diversity of the virus in the world. Though all sequenced SARS-CoV-2 genomes share more than 99% similarity, it has been found that there are at least two hypervariable genomic hotspots. One is a silent mutation in the ORF1ab, the other one is an amino acid polymorphism (Serine/Leucine) in ORF8 which is predicted to induce structural disorder of the protein in the C-terminal portion [30] . 149 mutations were identified in 103 SARS-CoV-2 genome sequences and through population genetic analyses, investigators uncovered two major types of SARS-CoV-2 in circulation (L and S type) based on two tightly linked SNPs at position 8,782 and 28,144. The S type (~30%) is the ancestral version while L type (~70%) is derived from S type, although L type is more prevalent and more aggressive in the outbreak. Based upon the evolution of novel coronavirus, there may be great distinctions in transmissibility, pathogenicity, and virulence between S and L type [32] . Forster et al analyzed 160 complete SARS-CoV-2 genomes by phylogenetic network and found three central variants named A, B, and C. The genome of type A is the most closely related to the bat coronavirus, which is supposed to be the "root of the outbreak". Type A is mainly found in United States and Australia. Type B is distinguished from type A by two mutations T8782C and C28144T, and is prevalent in Wuhan and East Asia. It seems that type B is resistant outside Ease Asia populations, since type B is not intended to spread outside East Asia without further mutated. Type C is derived from type B by the mutation G26144T and mainly found in Europeans, and also found in Singapore, Hong Kong, Taiwan, and South Korea but absent in mainland China [33] .

In addition to the viral mutations mentioned above, the human genetic variation may partly contribute to the geographical variations in the prevalence and mortality of COVID-19 pandemic. Prevalence and mortality data of the COVID-19 infections from European, African, Mediterranean, Middle East and Asian countries were included in the study. They found that the frequency of ACE1 D-allele was negatively correlated with prevalence of COVID-19, suggesting the confounded role of ACE1 D/I polymorphism in the circulation of SARS-CoV-2 and the outcome of the infection [34] [35] [36] [37] . While complement component 3 (C3) polymorphism, a central component of the innate immune system, has been found to be a principal component of gene frequencies among European populations and a crucial determinant for COVID-19 prevalence and mortality [37] . Dai's paper suggested infected patients carrying A allele of ABO blood group type especially those with cardiovascular diseases in particular hypertension, tend to develop severe COVID-19 [38] . SARS-CoV-2 virion particles are enveloped, roughly spherical or moderately polymorphic with diameter ranging from 80-160 nm [39] . SARS-CoV-2 has four structural proteins including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins necessary for virion particle formation, and four highly conserved nonstructural proteins including papain-like protease (PLpro, nsp3), 3CL-protease (3CLpro, nsp5), RNA-dependent RNA polymerase (RdRp, nsp12), and helicase (nsp 13) [40] that are needed for viral RNA replication [41, 42] . The N protein forms a ribonucleoprotein complex with viral RNA. The S protein is responsible for virus entry to the host cell by binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2). The width of the S protein is about 7 nm, and the length is about 23 nm [39] . S protein has unique insertion of four amino acids (PRRA), which is a furin-like or TMPRSS2 cleavage site [43, 44] . S protein can be cleaved into S1 and S2 subunits.

When the S1 subunit is dissociated, S2 undergoes a conformational change, extending itself from a compressed form to a nail shape. S1 is the receptor binding domain that helps the virus attach to the surface of the host cell, then the cellular proteases prime the S protein and cleave it at specific site, thereby promoting the S2 mediated fusion process of virus with host cell membrane. The Nsp5 may affect the transport of endoplasmic reticulum and mitochondria. Some viral proteins also showed interaction with natural immune signaling proteins involved in IFN pathway, NF-κB pathway, etc, which may take part in cytokine storm. 69 compounds were found to target these protein interactions including FDA approved drugs, compounds in clinical trials, and preclinical compounds. Based on screening assays, two broad sets of drugs and compounds: mRNA translation inhibitors and Sigma1 and Sigma2 receptor modulators, were showed to exert antiviral activity [47] .

The clinical manifestations of COVID-19 range from asymptomatic infection, to flu-like illness such as fever, cough, dry cough and fatigue, to pneumonia, acute respiratory distress syndrome, and even multi-organ failures with high morbidity and mortality. The clinical diversity of non-J o u r n a l P r e -p r o o f specific signs and symptoms is poorly understood and seems to be correlated to the age, underlying comorbidities, and immunity status of the individual.

Wang et al reported 138 hospitalized cases of 2019-nCoV infected pneumonia in which most patients had fever, fatigue, dry cough, lymphopenia, prolonged prothrombin time, and higher level of lactate dehydrogenase [14] . Specifically, abnormal coagulation features including higher D-dimer and fibrin degradation product levels, longer prothrombin time and activated partial thromboplastin time, are significantly associated with poor prognosis [48] . Chest CT scans in the lungs of all patients showed ground glass opacity or bilateral patchy shadows [14] . But a larger scale of research on 1,099 laboratory-confirmed COVID-19 patients from 552 hospitals in 31 provinces throughout China showed that a certain amount of patients did not have fever or abnormalities on chest CT, making the diagnosis more complicating [1] . Severe patients who received intensive care unit (ICU) care were more likely to have two or more underlying comorbidities that usually associated with poor clinical outcomes. Hypertension, cardiovascular disease, and diabetes were the most common comorbidities, while chronic obstructive pulmonary disease (COPD) was less common. Other comorbidities include pharyngeal pain, dizziness, abdominal pain, dyspnea, anorexia, cerebrovascular diseases, hepatitis B infections, chronic kidney diseases, and malignancy, and complications include acute respiratory distress syndrome [47] , arrhythmia, and shock [14, 49] .

The fact that SARS-CoV could attack the nervous system was confirmed by the viral genome sequence detected in the infected patients' cerebrospinal fluid. In the findings of Mao et al, more than 30% patients showed the neurological manifestations including central nervous system symptoms (headache, dizziness, disturbance of consciousness, epilepsy, etc), peripheral nervous system symptoms (decreased taste, decreased smell, decreased appetite, neuralgi, etc), and skeletal muscle injury. The neurological symptoms were more frequent in severe patients than in non-severe patients. It is worth noting that the first symptoms of some patients occurred in the nervous system, such as facial paralysis, hallucinations, etc. The virus circulation in the blood and J o u r n a l P r e -p r o o f crossing the blood-brain barrier, as well as the highly expression levels of ACE2 in vascular endothelial cells of nervous system and in smooth muscle cells of skeletal muscle, may contribute to the viral infection in nervous system [50] . On March 4, a 56-year-old COVID-19 patient in China was firstly reported to present with viral encephalitis (https://www.caixinglobal.com/2020-03-06/new-research-sparks-debate-over-whether-coronavirus-can-infect-the-nervous-system-101524986.html). Though this is a rare case, in terms of clinical treatment, it provides some important indications. When the central nervous system is infected, cerebral edema or even hernia may occur, which is life-threatening. It is necessary to monitor the neurological indicators to avoid the death. Since the primary damage of SARS-CoV-2 infection is mainly in the lungs, whether viral infection leading to pathological damage in other tissues and organs is primary or secondary remains to be further investigated.

Laboratory abnormalities provide some vital implications that may serve as predictors for severity of disease. Older age, higher sequential organ failure assessment score, and levels of D-dimer greater than 1 µg/mL were three potential risk factors related to poor prognosis, as reported by Zhou et al [5] . White blood cells count, neutrophil count, and levels of D-dimer, creatine kinase, and creatine were higher in ICU patients compared to non-ICU patients [14] . Liu et al found that the viral load of SARS-CoV-2 from patients' respiratory tracts was positively correlated to the lung disease severity. Albumin, percentage of lymphocytes and neutrophils, lactate dehydrogenase, and C-reactive protein were highly associated to the acute lung injury and may be useful to predict the disease severity. Notably, the angiotensin II levels in the plasma of the infected patients were markedly higher than that in healthy persons and strongly related to viral load and pulmonary injury, suggesting the potential treatment with angiotensin receptor blockers for COVID-19 [51] . , levels of IL2, IL7, IL10, GCSF,   IP10 , MCP1, MIP1A, and TNFα in plasma were higher in ICU patients as compared to non-ICU patients [54] . However, the questions that remains unanswered are: How does the body perceive the virus and release cytokines? Why and how higher levels of cytokines cause the deterioration of clinical conditions and even mortality? Which cytokines and their pathways can be potential therapeutic targets? In response to viral infections, the host needs to effectively mobilize the immune system to clear virus and avoid the cytokine storm triggered by over activation. Our understanding of virus-induced cytokine storms and the balance between immune system and virus is still limited. To address these problems, we still have a long way to go.

Currently, nucleic acid testing kits are commercial available and still the gold standard to confirm the presence of the SARS-CoV-2. Chu's group has established two one-step quantitative reverse transcription-PCR assays. Primers binding to the ORF1b and N genes of subgenus sarbecoviruses respectively were used to amplify the viral genome. But these developed assays couldn't distinguish SARS-CoV, SARS-CoV-2, and other sarbecoviruses, further sequence analyses of amplicons that are positive in RT-PCR results are required to confirm the species [55] . Shirato et al have developed two nested RT-PCR assays with primers bounding to the ORF1 and S protein, and two real-time RT-PCR assays with primers binding to the sequence encoding N protein, successfully detecting 25 SARS-CoV-2 positive infected patients in Japan [56] . Corman et al have designed a workflow for diagnosis of novel coronavirus without the virus isolates available.

Making use of the genetically similarity to 2003 SARS-CoV and synthetic nucleic acid technology, they have developed three RT-PCR assays as follows, primers binding to E gene can detect bat-associated SARS-related coronaviruses; specific probe hybridizing to RNA-dependent RNA polymerase (RdRp) was used to make a distinction between SARS-CoV-2 and SARS-CoV; the RT-PCR assay designed to amplify the region of N gene was less sensitive so that it was not further validated [57] .

Normally the throat and nasal swab in the upper respiratory tract of the patients are sampled for nucleic acid testing, which is fast and convenient for physicians to operate and causes no trauma to the patients. Nonetheless, novel coronavirus mainly infects the lower respiratory tract and the viral load in the upper respiratory tract may not be high enough for detection, resulting in false negatives [58] . In addition, given the viral replicative activity is influenced by the host immune system, it is possible that one has clinical symptoms but the quantity of virus is not sufficient for detection, leading to the occurrence of false negative results as well. What's more, the progression and severity of pneumonia cannot be concluded based on the real-time RT PCR results. Infected patients with false negative RNA results bring enormous challenges for medical workers to provide appropriate treatment and take isolation measures in time. Diagnosing the confirmed COVID-19 cases merely by nucleic acid testing is obviously not accurate enough.

The most common manifestations of chest CT in patients with novel coronavirus pneumonia are ground glass opactities and patchy consolidation in bilateral lungs. According to the distributions and types of lesions, chest CT manifestations can be divided into three stages: at the early stage, only a single ground-glass opacity or irregular solid nodules of varying sizes can be seen. As the infection progresses, ground-glass opactities with a rounded morphology, reticulation, or crazy paving pattern and patchy, parenchyma or mixed consolidation distributed in the middle and peripheral area of bilateral lungs may present. Nodules involving multiple lobes are increased and enlarged. A few patients may have small pleural effusions. When the disease progresses to a critical stage, more extensive pulmonary consolidation and ground-glass opactities, diffuse pulmonary lesions, increased density of bilateral lungs and even "white lung" may appear. Pleural J o u r n a l P r e -p r o o f effusions, lymphadenopathy and an air bronchogram sign may present in a small number of patients [59] [60] [61] [62] [63] [64] [65] . A clinical trial involving in using CT scores to predict the mortality of patients with SARS-CoV-2 pneumonia have been completed (NCT04284046).

It is not surprising that patients have false negative results in nucleic acid testing but the CT images show some features of novel coronavirus infection [65] . Therefore, in the latest edition of China's diagnosis and treatment guidelines for new coronavirus pneumonia, chest CT scan is one of the clinical diagnosis standards of suspected cases and the nucleic acid testing kit is then used to make a clinical diagnosis of confirmed cases of novel coronavirus infection.

Not as nucleic acid testing or Chest CT images that can detect acute infection, antibody tests detects the person ever infected, especially those with asymptomatic infection. IgG and IgM antibodies in serum of SARS-CoV-2 infected patients arise simultaneously or sequentially within around 1 to 3 weeks after illness onset [66] . N and different forms of S protein are the major antigens for detecting the antibodies induced by SARS-CoV-2 infection. Different types of assays for antibody testing can be applied to assess different aspects of immunological response and function of antibodies induced by virus infection.

Zhao et al have developed a serology ELISA kit using CHO cell expressing fully glycosylated, full length SARS-CoV-2 S1 protein for surveillance of the infection at the early stage.

Glycosylation of S1 protein is important to facilitate the protein folding properly and increase the protein's affinity to the receptor, ensuring the great sensitivity and high specificity of the ELISA kit [67] . Padoan Without PCR amplification, the results can be read out in less than an hour (https://www.broadinstitute.org/files/publications/special/COVID19%20detection%20(updated). iLACO is rather sensitive that can detect as low as 10 copies of the targeted ORF1ab gene in 15-40 minutes. Compared to RT-PCR, these two approaches are more rapid, accurate, and can be conducted with limited facilities [73] .

Currently no specific antiviral medication or vaccine is approved for treating patients with 

Lopinavir/ritonavir is a fixed dose combination medication approved by FDA acting as a potent protease inhibitor for HIV-1 infection [83] . Lopinavir/ritonavir are potential candidates for SARS-CoV and MERS-CoV infection [84, 85] . Molecular dynamics simulations studies have showed that lopinavir/ritonavir could bind to the active site of SARS-CoV 3CLpro [86] . An open clinical trial conducted in Hong Kong during the SARS-CoV outbreak in 2003 revealed that patients treated with lopinavir/ritonavir had a lower viral load and were associated with better clinical outcomes and fewer adverse effect [84] . Lopinavir/ritonavir combined with recombinant interferon beta-1b is also being investigated for treatment of MERS-CoV infection (NCT02845843) [87] . Since the protein sequence of main protease of SARS-CoV and SARS-CoV-2 are closely related with 96% identity, it is possible that lopinavir/ritonavir can be applied J o u r n a l P r e -p r o o f to novel coronavirus infection. However, a clinical trial conducted in hospitalized adult patients with severe COVID-19 in China showed no benefit with lopinavir/ritonavir treatment over standard care (ChiCTR2000029308) [88] . Whether combination of lopinavir/ritonavir with other antivirals would have improvement of clinical outcomes remains to be further determined.

Chloroquine, a FDA-approved drug for anti-malarial and autoimmune disease [89] , is also known as a potent antiviral of SARS-CoV infection in vitro for interrupting the glycosylation of cell surface receptor, ACE2 [90] . Chloroquine also showed inhibitory effect on the replication of MERS-CoV in vitro [91] . Recently, chloroquine were tested against SARS-CoV-2 with EC 90 at low micromolar concentration and may restrict the viral entry and post entry processes in vitro [75] . Chloroquine is an immunomodulation agent that may enhance its antiviral activity as well.

In light of the safety and potent efficacy, clinical study testing chloroquine against pneumonia caused by SARS-CoV-2 is being conducting (NCT04261517). Preliminary results from the multicenter clinical trials conducted in China have demonstrated that chloroquine phosphate effectively inhibited the progression of pneumonia, eliminated the virus, and shortened the duration of diseases without any severe adverse effects [92, 93] . However, data of an open label, randomized controlled trial conducted in China (ChiCTR2000029868) enrolling patients with mild to moderate COVID-19 and two observational studies involving a large number of consecutive hospitalized patients with COVID-19 in New York City did not support the use of hydroxychloroquine because it was not associated with either a significantly higher or lower risk of intubation or death, or lower in-hospital mortality [94] [95] [96] . In the absence of evidence of therapeutic effects, we particularly need to understand whether the drug's safety risks are worth taking.

ACE2, an important regulatory component in the renin-angiotensin-aldosterone system (RAAS), not only mediates virus infection, but also directly participates in the progression of acute lung injury that highly correlated with the high mortality [97] . SARS-CoV-2 infection has been shown to significantly reduce the expression of ACE2 in lung, resulting in aggravated acute lung injury. 

Except for ACE2 as a receptor of host cells for SARS-CoV-2 attachment and entry, it is reported that CD147, which expresses in many human cell types including epithelial cells, endothelial cells and leukocytes, also facilities the invasion of SARS-CoV-2 via binding to spike protein [99] .

Blocking the interaction between spike protein of SARS-CoV-2 and CD147 via anti-CD147 humanized antibody was proved to be able to inhibit the replication of SARS-CoV-2 in vitro, offering a novel target for antiviral therapy (NCT04275245) [100] .

Methylprednisolone is a corticosteroids medication for inhibiting the immunity and reducing inflammation [101] . New coronavirus infected patients are complicated by acute lung injury and acute respiratory distress syndrome, which are partly caused by host overactive immune responses [102] . Corticosteroids can decrease inflammation of the lungs but also suppress the immune response and increase viral replication. Treatments of SARS and MERS-CoV infected patients with corticosteroids showed that inflammation persisted after clearance of virus. No sufficient evidence supported that SARS-CoV-2 infected patients would benefit from corticosteroids, and contrarily they may suffer from this therapy [103, 104] . Two trials are recruiting critically ill patients with severe acute respiratory failure caused by novel coronavirus However, corticosteroids should be used with caution for treating novel coronavirus. While considering the possible benefits, the risk of complications including psychosis, diabetes, avascular necrosis must be balanced against.

Immunoglobulin therapy is to use a mixture of normal human immunoglobulins for improving the immunity, which is especially effective for acute infections. This passive immunity through immunoglobulin injection can be acquired immediately but the duration is short [105] .

Intravenous immunoglobulin suggested for treating severe and critically ill COVID-19 patients was proved to improve the clinical outcomes and prognosis [82] . Whereas SARS-CoV-2 is a novel virus and almost no antibodies are present in the healthy populations, intravenous immunoglobulin acting as a non-specific immune enhancer on mild or common patients may only have limited efficacy.

After virus infects the body, many different antibodies against viral proteins can be produced in the body, but most antibodies do not actually have an antiviral effect except for those that recognize proteins on the surface of the virus particles [106] . During the outbreak of SARS-CoV, infected patients treated with convalescent plasma carrying SARS-CoV-specific antibodies from cured patients have been suggested to yield clinical benefits with reduced viral load and lower mortality rate [107] . The underlying mechanism for the protective effect of convalescent plasma seems to be that the neutralizing antibodies produced in previously infected patients can directly bind to the spike protein on the virus particles to prevent them from entering the cells [108] . In 

Virus attacking the respiratory system and immune system causes the damage of immunological function with lymphocytopenia, decreased CD8 and CD4 T cell count that associated with severe SARS-CoV-2 pneumonia [14, 51, 54, 103] . The host fights against the viral infection through virus-specific cytotoxic T lymphocytes. Reversion of T cell exhaustion is helpful to promote the antiviral immunity. PD-1 blocking antibody is one of the anticancer drugs that prevents the interaction between PD-L1 on the tumor cells and PD-1 on the T cells. Blocking the interaction of these cell surface proteins can modulate the immunological tolerance of tumors, thus preventing the cancer cells from evading the surveillance of immune system [110] . Diao et al reported that the expression levels of T cell exhaustion marker PD-1 from COVID-19 patients were much higher as compared to healthy controls [111] . Therefore, blocking PD-1 to prevent T cell exhaustion resulting in activation of antiviral immune responses may be a novel therapeutic strategy for COVID-19. However, before applying the PD-1 blocking antibody for treatment of lymphocytopenia induced by SARS-CoV-2 infection (NCT04268537), the correlation between PD-1 and T cell function should be measured to predict the response rate and the adverse effects such as cytokine-release syndromes and pneumonitis. -2 admitted to the intensive care unit had cytokine storms with   increased secretion of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα, associated with severe illness including acute respiratory distress syndrome and organ failure [102] .

Mesenchymal stem cells (MSCs) can be an immunomodulatory agent inhibiting the secretion of cytokines, suppressing the overactive immune responses, and decreasing the inflammation. MSCs are safe and seldom induce rejection reactions to human body due to the inherently poor immunogenicity properties [112] . Regenerative potential of MSCs is helpful to repair the alveolar epithelial cells and vascular endothelial cells, leading to reduction of lung damage [113] . Owing to the immunoregulation, anti-inflammation, damaged tissue repair and regeneration properties, MSCs-based treatments have been applied to a variety of refractory diseases and are expected to have a critical role in novel coronavirus-induced severe pneumonia (NCT04293692, NCT04288102, NCT04252118, NCT04273646, NCT04269525).

MSCs-derived exosomes contain abundant biologically active substances such as signaling lipids, growth factors, cytokines, and RNA. Recent studies have revealed that exosomes are also important mediators for cell-cell communication via paracrine or endocrine manner [114] . MSCsderived exosomes have similar biological functions as mesenchymal stem cells, for example, repairing and regeneration of tissues, suppression of inflammatory response and regulation of immunity [115] . MSCs-derived exosomes have been applied to the treatment of various lung diseases including pulmonary fibrosis, pulmonary arterial hypertension, lung cancer, bronchopulmonary dysplasia, and acute respiratory distress syndrome [116] . Studies have demonstrated that MSCs-derived exosomes could significantly reduce lung pathologic damage and increase the survival rate of mice with pulmonary fibrosis [117] . Compared to MSCs-based therapy, its derived exosomes as cell-free therapy is much safer and easier to pass through the physical barriers due to their nano size [118] , suggesting a broad application prospect in a variety of diseases especially the pulmonary diseases (NCT04276987).

TCMs have a long history of medical use and are frequently used for the prevention and treatment of infection diseases in China. Based on TCM therapy concept of "multiple compounds-multiple targets-multiple effects", a TCM prescription often contains multiple herbs working together to battle a disease. Within these mixture components, some work directly on the viral targets involving the life cycle including attachment, entry, transcription of viral mRNA, replication of viral genome, translation of viral protein, and the assembly, maturation and release of progeny virus particles [119] . To infect the host efficiently, viruses have evolved a series of strategies to escape the host's innate and adaptive immune responses against viral infection. On the other hand, to fight against the virus, the host may release excessive or uncontrolled proinflammatory cytokines that correlated to tissue damage or organ failure [102] . Except working as direct-acting antivirals, most of the molecules in TCM are immunomodulators that could block the viral immune evasion or enhance the host antiviral immune responses, while the others may contribute to reducing the toxicity or enhancing the bioavailability [119] . Despite not often being recognized as a real medicine, TCMs actually have exhibited excellent anti-SARS-CoV-2 efficacy during the outbreak of COVID-19 as evidenced by the high recovery rate and low mortality rate of confirmed cases taking TCM in China [82, 120] . Xiyanping is a licensed TCM injection in China used in the treatment of bronchitis, tonsillitis, bacterial dysentery. It mainly contains active compounds extracted from Andrographis paniculata (Chuan Xin Lian) [121] . In the 7 th edition of the diagnostic and treatment guidelines for the COVID-19 issued by Chinese health authorities, Xiyanping injection is recommended for treating severe and critically ill patients with bacterial co-infection attributed to its antiviral, anti-inflammatory, antibacterial, and immune regulation activities. In addition to the significant antiviral efficacy, what calls for special attention is that the indication, dosage, and solvents of TCM injection should be used rationally to reduce the occurrence of side effects [122] . Specific antibodies of anti-SARS-CoV-2 were found to mainly distribute in the lungs after immunization. SARS-CoV-2 S protein-specific T cell responses were also stimulated in immunized mice [125] . 

ACE2 not only expresses in the lung tissue, but also distributes in the intestinal epithelium [126] .

Apart from invading the host's lung tissues, SARS-CoV-2 also interferes with the absorption of nutrients in the human body by binding to ACE2 of the intestinal epithelium, causing gastrointestinal discomfort [14] . The intestinal flora can regulate various lung diseases through the intestinal-lung axis with immunomodulatory effect, including viral pneumonia, asthma, tuberculosis, and chronic obstructive pulmonary disease. Maintaining the normal communication of intestinal-pulmonary axis is helpful to alleviate lung disease [127, 128] . Intestinal malnutrition often occurs in patients with COVID-19 and sometimes develops to secondary bacterial infections and antibiotic-related diarrhea, suggesting the crucial connection between intestinal tract and respiratory tract. Washed microbiota transplantation as a rescue therapy for critically ill patients with diarrhea may have important clinical benefits (NCT04251767).

Vitamin C, a kind of antioxidant, is essential for tissue repair and immune system function [129] .

Vitamin C can prevent oxidative damage induced by virus infection and improve the immunity of patients with pneumonia. It has been reported that enterovirus/rhinovirus infection can cause J o u r n a l P r e -p r o o f devastating acute lung injury. High dose of intravenous vitamin C could attenuate the acute respiratory distress syndrome and relieve lung damage produced by enterovirus/rhinovirus infection. Down-regulation of reactive oxygen, nitrogen species, and pro-inflammatory genes by vitamin C may contribute to these curative effect [130, 131] . Nevertheless, the role of vitamin C as monotherapy in the recovered patients is still uncertain. Before vitamin C can be applied as a therapy, the side effects of high dosage, such as nausea, indigestion, abdominal cramps, and diarrhea owing to the unabsorbed vitamin C in the intestine should be considered. Since the leading causes of death of SARS-CoV-2 infected patients are associated with acute respiratory distress and multiple organ failure, further investigations and larger trials should be performed to uncover the underlying mechanism and evaluate the exact efficacy of vitamin C.

The strong infectivity of SARS-CoV-2 highlights the need of early intervention with effective treatment. Although abundant clinical trials are registered and underway since the outbreak of COVID-19, the exact pathogenesis of SARS-CoV-2 has not been elucidated clearly yet, along with no approved drug or vaccine for either SARS-CoV or MERS-CoV as a reference, raising a significant challenge for specific and effective anti-SARS-CoV-2 drug development. 

Clinical Characteristics of Coronavirus Disease 2019 in China

Incubation Period and Other Epidemiological Characteristics of

Novel Coronavirus Infections with Right Truncation: A Statistical Analysis of Publicly Available Case Data

The Incubation Period of Coronavirus Disease 2019 (COVID-19) From Publicly Reported Confirmed Cases: Estimation and Application

Temporal dynamics in viral shedding and transmissibility of COVID-19

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Pattern of early human-to-human transmission of Wuhan 2019 novel coronavirus (2019-nCoV)

Potential of large "first generation" human-to-human transmission of 2019-nCoV

Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia

A familial cluster of infection associated with the 2019 novel coronavirus indicating potential person-to-person transmission during the incubation period

The Extent of Transmission of Novel Coronavirus in Wuhan, China, 2020

Presumed Asymptomatic Carrier Transmission of COVID-19

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in

Evidence Supporting Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 While Presymptomatic or Asymptomatic

Consistent detection of 2019 novel coronavirus in saliva

Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes

Evidence for gastrointestinal infection of SARS-CoV-2

SARS-CoV-2 induced diarrhoea as onset symptom in patient with COVID-19

Diarrhoea may be underestimated: a missing link in 2019 novel coronavirus. Gut

Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity

2019-nCoV transmission through the ocular surface must not be ignored

Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection

Rhesus macaques can be effectively infected with SARS-CoV-2 via ocular conjunctival route

Clinical characteristics and intrauterine vertical transmission potential of COVID-19 infection in nine pregnant women: a retrospective review of medical records

What are the risks of COVID-19 infection in pregnant women?

A Novel Coronavirus from Patients with Pneumonia in China

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Genomic variance of the 2019-nCoV coronavirus

The COVID-19 Pandemic: A Comprehensive Review of Taxonomy, Genetics, Epidemiology, Diagnosis, Treatment, and Control

On the origin and continuing evolution of SARS-CoV-2

Phylogenetic network analysis of SARS-CoV-2 genomes

The host's angiotensinconverting enzyme polymorphism may explain epidemiological findings in COVID-19 infections

ACE Ins/Del genetic polymorphism and epidemiological findings in COVID-19

COVID-19 infections are also affected by human ACE1 D/I polymorphism

C3 and ACE1 polymorphisms are more important confounders in the spread and outcome of COVID-19 in comparison with ABO polymorphism

ABO blood group predisposes to COVID-19 severity and cardiovascular diseases

Viral Architecture of SARS-CoV-2 with Post-Fusion Spike Revealed by

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys

Therapeutic options for the 2019 novel coronavirus

COVID-2019: The role of the nsp2 and nsp3 in its pathogenesis

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

Mutations, Recombination and Insertion in the Evolution of 2019-nCoV

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia

Comorbidity and its impact on 1,590 patients with COVID-19 in China: A Nationwide Analysis

Neurological Manifestations of Hospitalized Patients with COVID-19 in Wuhan, China: a retrospective case series study

Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Clinical Features and Treatment of 2019-nCov Pneumonia Patients in Wuhan: Report of A Couple Cases

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia

Development of Genetic Diagnostic Methods for Novel Coronavirus 2019 (nCoV-2019) in Japan

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

False-Negative Results of Real-Time Reverse-Transcriptase Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2: Role of Deep-Learning-Based CT Diagnosis and Insights from Two Cases

CT Imaging Features of 2019 Novel Coronavirus (2019-nCoV)

CT Manifestations of Two Cases of 2019 Novel Coronavirus (2019-nCoV) Pneumonia

Chest CT Findings in 2019 Novel Coronavirus (2019-nCoV) Infections from Wuhan, China: Key Points for the Radiologist

CT Imaging of the 2019 Novel Coronavirus (2019-nCoV) Pneumonia. Radiology

Initial CT findings and temporal changes in patients with the novel coronavirus pneumonia (2019-nCoV): a study of 63 patients in Wuhan, China

Chest CT for Typical 2019-nCoV Pneumonia: Relationship to Negative RT-PCR Testing

Antibody responses to SARS-CoV-2 in patients with COVID-19

Early detection of SARS-CoV-2 antibodies in COVID-19 patients as a serologic marker of infection

Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics

Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect

Development and Evaluation of A CRISPR-based Diagnostic For 2019-novel Coronavirus

Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification

Rapid colorimetric detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic plat-form: iLACO

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

First Case of 2019 Novel Coronavirus in the United States

Remdesivir for the Treatment of Covid-19 -Preliminary Report

Compassionate Use of Remdesivir for Patients with Severe Covid-19

Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial

Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol

Arbidol as a broad-spectrum antiviral: an update

Clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined Chinese and Western medicine treatment

Evaluation of pharmacokinetic interactions between long-acting HIV-1 fusion inhibitor albuvirtide and lopinavir/ritonavir, in HIV-infected subjects, combined with clinical study and simulation results

Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings

Treatment With Lopinavir/Ritonavir or Interferon-beta1b Improves Outcome of MERS-CoV Infection in a Nonhuman Primate Model of Common Marmoset

Molecular dynamic simulations analysis of ritonavir and lopinavir as SARS-CoV 3CL(pro) inhibitors

Treatment of Middle East respiratory syndrome with a combination of lopinavir/ritonavir and interferon-beta1b (MIRACLE trial): statistical analysis plan for a recursive two-stage group sequential randomized controlled trial

A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19

Antimalarial drug resistance in Africa: strategies for monitoring and deterrence

Chloroquine is a potent inhibitor of SARS coronavirus infection and spread

Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies

A systematic review on the efficacy and safety of chloroquine for the treatment of COVID-19

Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial

Association of Treatment With Hydroxychloroquine or Azithromycin With In-Hospital Mortality in Patients With COVID-19 in

Observational Study of Hydroxychloroquine in Hospitalized Patients with Covid-19

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Angiotensin-converting enzyme 2 protects from severe acute lung failure

Dealing with the family: CD147 interactions with cyclophilins

SARS-CoV-2 invades host cells via a novel route: CD147-spike protein

Methylprednisolone pulse therapy as an adjuvant treatment of Streptococcus pneumoniae meningitis complicated by cerebral infarction-a case report and review of the literature

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet

Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province

Clinical evidence does not support corticosteroid treatment for 2019-nCoV lung injury

Use of intravenous immunoglobulin in human disease: a review of evidence by members of the Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and Immunology

Role of antibodies in controlling viral disease: lessons from experiments of nature and gene knockouts

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

Convalescent plasma as a potential therapy for COVID-19

The convalescent sera option for containing COVID-19

De-novo and acquired resistance to immune checkpoint targeting

Reduction and Functional Exhaustion of T Cells in Patients with Coronavirus Disease

Mesenchymal Stem Cell-Based Immunomodulation: Properties and Clinical Application

Concise Review: MSC-Derived Exosomes for Cell-Free Therapy

Exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm

Mesenchymal stem cell-derived exosomes for clinical use

Human mesenchymal stem cell microvesicles for treatment of Escherichia coli endotoxin-induced acute lung injury in mice

Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats

Traditional Chinese herbal medicine as a source of molecules with antiviral activity

Traditional Chinese medicine for COVID-19 treatment

Potential anti-angiogenic sulfates of andrographolide

Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, firstin-human trial

Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine induces equivalent preclinical antibody titers and viral neutralization to recovered COVID-19 patients. bioRxiv

Immunogenicity of a DNA vaccine candidate for COVID-19

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

The Lung Microbiome, Immunity, and the Pathogenesis of Chronic Lung Disease

The Gut-Lung Axis in Respiratory Disease

Vitamin C as an antioxidant: evaluation of its role in disease prevention

Intravenous vitamin C as adjunctive therapy for enterovirus/rhinovirus induced acute respiratory distress syndrome

Oudemans-van Straaten, Vitamin C supplementation in the critically ill patient

Data were collected from https://clinicaltrials.gov by June 9, 2020