Focus on a 2019-novel coronavirus (SARS-CoV-2)

A new coronavirus, severe acute respiratory syndrome coronavirus 2, was first discovered in Wuhan, China, in December 2019. As of April 7, 2020, the new coronavirus has spread quickly to 184 countries and aroused the attention of the entire world. No targeted drugs have yet been available for intervention and treatment of this virus. The sharing of academic information is crucial to risk assessment and control activities in outbreak countries. In this review, we summarize the epidemiological, genetic and clinical characteristics of the virus as well as laboratory testing and treatments to understand the nature of the virus. We hope this review will be helpful to prevent viral infections in outbreak countries and regions.

The virus can be transmitted not only from animals to humans but also from humans to humans [23]. A Lancet report demonstrated that the virus could have recently acquired the ability to transmit between humans [6]. A report of five patients in a family cluster who traveled to Wuhan and were infected with SARS-CoV-2 was the first report directly illustrating that the virus is capable of person-to-person transmission in hospital and family settings [23].
SARS-CoV-2 can spread via direct contact and respiratory droplets. Respiratory particles are spread while breathing, speaking, coughing or sneezing [24]. In addition, aerosol and fomite transfer may promote transmission of the virus according to a study in the New England Journal of Medicine [25]. Aerosolized virus may be generated by respiratory and surgical procedures. The study showed that the half-life of the virus is about 1.1-1.2 h, and it remained viable for 3 h in aerosols. Meanwhile, the virus on plastic, stainless steel, copper and cardboard remained stable for 4-72 h [25]. These results indicate that aerosol or fomites may be able to spread SARS-CoV-2 [25]. A fluid-resistant (Type-R) surgical face mask is used to protect against droplets.
Fecal-oral transmission may also play an important role in SARS-CoV-2 spread [26]. Xiao and colleagues showed that 53.42% of 73 hospitalized COVID-19 patients had SARS-CoV-2 RNA in stool specimens, and the duration time of positive stool results ranged from 1 to 12 days [27]. In addition, viral nucleic acid in 64.29% patients remained positive in the feces after SARS-CoV-2 RNA in pharyngeal swabs turned negative. Furthermore, positive SARS-CoV-2 RNA in stool specimens was not associated with gastrointestinal symptoms [28]. Together, these findings indicate the possibility of SARS-CoV-2 via the fecal-oral transmission.
The spike glycoprotein plays an important role in binding to receptors on host cells and, therefore, is involved in host tropism [40]. SARS-CoV-2, SARS-CoV and MERS-CoV have S proteins containing 1,273, 1,255 and 1,270 aa, respectively ( Figure 1A) [36]. The S protein mediates entrance into human respiratory epithelial cells by interacting with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) [35,41]. It is comprised of S1 and S2 subunits ( Figure 1C), and the S1 subunit shares approximately 70% identity with that of human SARS-CoV and bat SARS-like CoVs (SL-COVZXC21 and ZC45). The S1 subunit has an N-terminal domain and a receptorbinding domain (RBD) that are both responsible for the binding of virions to host cells [16]. Both SARS-CoV-2 and SARS-CoV bind to ACE2 through the C-terminal domains (CTD) of their S1 subunits, and MERS-CoV utilizes the CTD to bind proteinaceous dipeptidyl peptidase 4 (DPP4) [42]. The RBD of SARS-CoV-2 has 73% identity to that of SARS-CoV [43]. The transmissibility of the SARS-CoV-2 virus is greater than that of SARS-CoV, which may be because the RBD of SARS-CoV-2 is slightly different from that of SARS-CoV. The S2 subunit shares 99% identity with two bat SARS-like CoVs and human SARS-CoVs [38]. The S2 subunit contains a fusion peptide (FP) and heptad repeats (HRs) 1 and 2 ( Figure 1C). After the S1 RBD binds to the ACE receptor on the host cell, the FP of S2 is inserted into the host cell membrane, and then HR1 and HR2 form a six-helix bundle (6-HB), which helps the virus fuse with host cell membranes [16,44]. SARS-CoV-2 envelope (E) protein, matrix protein, accessory proteins p6 and p8, nonstructural protein 7 (nsp7), and nsp13 are homologous with those of SARS virus [39]. Therefore, the SARS-CoV-2 virus has a high level of identity with SARS-CoV. This suggests that an anti-SARS-CoV antibody, which could cross-react with the SARS-CoV-2 S protein, may be useful to treat patients with the virus.
Another clinical study containing 138 patients showed that common symptoms were fever, fatigue, dry cough, lymphopenia, prolonged prothrombin time and an increased lactate dehydrogenase level (Table 1 & Figure 2) [5]. Common complications included shock, acute respiratory distress syndrome, acute renal injury, acute liver failure, arrhythmia, RNAaemia and acute cardiac injury [45]. In addition, it is now understood that SARS-CoV-2 can infect children as well as adults [46].
One study showed that during the early infection period, most patients had normal white blood cell counts; however, 56.8% of patients had leukopenia in cases of serious infection [46]. Patients with dyspnea were more frequently admitted to the intensive care unit (ICU) [46]. Most chest computed tomography (CT) showed bilateral patchy shadows or ground-glass opacity (GGO) in the lungs [5,6]. In another study, 86% of patients showed GGO by chest CT, and 29% of patients had consolidation [47]. Following the appearance of GGO, 75% of patient lung CTs showed reticular or interlobular septal thickening [48]. However, no direct cavitation, pleural effusion, lymphadenopathy or nodules were observed in the lungs of COVID-19 patients [49] (Table 1).
The mortality rate of SARS-CoV-2-infected patients varies in different studies. A study of 41 patients showed a mortality rate of 15% [6], and the fatality of 138 patients in a clinical study was 4.3% [5]. However, Chen showed that the mortality was 3%, a number closer to the official national statistics of China (2.01%) [51]. These differences in mortality rates are possibly due to the difference in sample size. The reported mortality rate of SARS-CoV was 10%, and that of MERS-CoV was about 36% [11]. The currently estimated mortality rate of SARS-CoV-2 is therefore lower than that of SARS-CoV and MERS-CoV [51,52] (Table 1). In summary, SARS-CoV-2 spreads more rapidly but has a relatively lower fatality rate as compared with two other related coronaviruses.

Hematology testing
The number of total leukocytes, lymphocytes and monocytes has been detected from hospitalized patients with COVID-19 [53]. Approximately 25% of cases formed leucopenia [6]. Moreover, lymphopenia was observed in 63-70.3% of patients [5,6]. CD4 + or CD8 + T-cell numbers decreased as the disease severity increased [6,54]. Patients with severe cases had more prominent abnormalities than those with non-severe cases.  Patients infected with COVID-19 had some unique clinical features including rhinorrhoea, sneezing, sore throat. Most patients had haemoptysis dyspnea, fever, headache, fatigue, sputum production, pneumonia and ground-glass opacities. However, only a low percentage of patients developed intestinal symptoms such as diarrhea and vomiting. Leucopenia, lymphopenia, pro-inflammation cytokines increasing, acute respiratory distress syndrome and acute organs damages (such as cardiac, liver or kidney) were common features in some intensive care unit patients.

Nucleic acid testing
Virus from throat swabs, blood, urine, stool or respiratory tracts have been assessed by fluorescent reverse transcription-polymerase chain reaction (RT-PCR) methods [55]. Primers and probes targeting RdRp/helicase (Hel), E, S, N and replicase ORF 1a/b genes were designed and tested for SARS-CoV-2 [55]. RT-PCR showed that primers of the E and RdRp genes demonstrated better sensitivity than that of the N gene, and the limit of detection (LOD) of RdRp/Hel and N gene was lower than that of S gene and RdRp-P2 [18]. Chan et al. showed that the COVID-19-RdRp/Hel assay (RdRp/Hel probe, FAM-TTAAGATGTGGTGCTTGCATACGTAGAC-lABkFQ) was significantly more sensitive than the RdRp-P2 assay for the detection of SARS-CoV-2 RNA, and detection targeted ORF1a/b, ORF1b-nsp14, RdRp, S, E or N genes was with less specificity for SARS-CoV-2 [18].
Moreover, a previous study showed that the specific probe RdRP SARSr-P2 (FAM-CAGGTGGAACCTCATCAGGAGATGC-BBQ) detected only the SARS-CoV-2 RNA transcript but not the SARS-CoV RNA [55]. However, a novel RT-PCR assay showed that targeting RdRp2 was a non-specific assay for SARS-CoV-2, because this detected other betacoronaviruses such as SARS-CoV [18]. The COVID-19-RdRp/Hel assay had the lowest LOD in vitro and higher sensitivity and specificity, which helped to reduce the false-negative rate and improve the laboratory diagnosis of COVID-19 [18].
SARS-CoV-2 viral load is detectable from throat-and lung-derived samples; however, blood and urine have not yet yielded virus [56]. Patients with COVID-19 produced the highest viral load near symptom presentation, which may be the reason for the fast spread of the virus [57]. An observational cohort showed that viral load in saliva was highest following symptom onset in the first week, then gradually declined with time [56]. Endotracheal aspirate viral load was available from day 8 after symptom onset and did not significantly decline thereafter [57]. Following symptom onset, virus load in 33% patients could be detected for 20 days or longer in that study. Viral load in respiratory tract specimens was about sixfold higher than that in the nonrespiratory tract specimens [57]. In addition, elder patients reportedly had a greater virus load than that of younger patients. Studies showed that higher initial viral load was related to the severity of COVID-19 symptoms [57].
Although the positive test ratio was only 47.4% in previous study, it was improved by novel assay methods [58]. The sample quality, collection time, detection kits and technical abilities of clinical doctors may affect the accuracy of detection. Therefore, precise diagnosis of COVID-19 should be combined with CT scans and nucleic acid testing.

Viral genome sequencing
Bronchoalveolar lavage fluid or throat swabs from patients have been sequenced, and viral genomes were searched via BLAST with the SARS-CoV-2 sequence [38,39]. Genome sequencing has also been used to identify patients with suspected infection.

Serology testing
In addition to hematological detection and nucleic acid testing, serological diagnosis is important for patients who present late with a very low viral load, below the detection limit of RT-PCR assays [59]. IgM and IgG titers were relatively increased on day 5 and rapidly raised 10 days after symptom onset in most patients [59,60]. The IgM-positive rate increased from 50 to 94%, whereas the IgG-positive rate increased from 81 to 100% [60]. In addition, the IgM-and IgG-positive rates were not significantly different before and after patients were found to be virally negative [61]. Therefore, virus-specific IgM and IgG serological testing may be used to confirm current or previous infection with SARS-CoV-2.

Animal models for COVID-19
An ideal animal model for COVID-19 would reflect the clinical signs, viral replication and pathology displayed in humans. Non-human primate models (rhesus, cynomolgus macaques, African green monkeys, common marmoset, squirrel monkeys and mustached tamarins) have all been evaluated as models of SARS-CoV infection [62]. All non-human primates had pneumonia, cough and respiratory distress after virus infection [15]. Mice (BALB/c, C57BL6 and 129S strains) supported SARS-CoV replication and showed clinical signs of SARS [15]. Rag1 −/− mice, CD1 −/− mice, STAT1 −/− mice and Beige mice have been used to determine the role of immune effectors of the virus [63]. AC70 and AC63 transgene-positive mice also showed clinical manifestation after SARS-CoV infection, demonstrating their usefulness to study the pathogenesis and evaluation of vaccines and other therapeutics [63]. Hamsters also have been used to study immuno-prophylaxis and drug research as they harbored high viral titers and pulmonary histopathology upon virus infection [63]. Ferrets are another appropriate animal model to study this respiratory virus because the clinical symptoms, viral titers and histologic changes were similar to those of patients with virus infection [63]. ACE2, the receptor of SARS-CoV, was also identified as the functional receptor for SARS-CoV-2, therefore, mice, hamsters and ferrets may be animal models for studying the SARS-CoV-2 [63]. An article reported in Science shows that SARS-CoV-2 can replicate in the upper respiratory tract of ferrets, indicating that ferrets represent an ideal animal model for evaluating antiviral drugs or vaccine candidates against COVID-19 [64]. In addition, the domestic cat has shown multifocal pulmonary consolidation with infection of SARS-CoV, and SARS-CoV-2 can replicate efficiently in cats and transmit between cats via the airborne route [64]. By contrast, dogs, pigs, chickens and ducks are poorly susceptible to SARS-CoV-2 [64].

Treatment for COVID-19
No specific therapeutic medicine has been approved for the treatment of SARS-CoV-2 infection. All patients are given empirical antibiotic or antiviral drugs. Moxifloxacin or levofloxacin are empirically used to treat early coinfections with bacteria [65]. Linezolid is effective against Streptococcus pneumoniae and Staphylococcus aureus and is combined with nemonoxacin in cases of severe infection [65].
A combination of lopinavir and ritonavir has been used to treat COVID-19 because the lopinavir/ritonavir (LPV/r) combination has been confirmed to be effective against SARS-CoV and MERS-CoV [6,66,67]. Another antiviral drug, remdesivir (RDV), was predicted to be efficacious against COVID-19 by target-based virtual ligand screening [68]. RDV is a novel nucleotide analog prodrug that is in development and has demonstrated effective pan-CoV therapy. The first case of SARS-CoV-2 infection in the USA was successfully treated with RDA. Moreover, a randomized, double-blind, parallel-controlled Phase III clinical trial was conducted to recruit SARS-CoV-2-infected patients [4,69].
Favipiravir is a nucleoside analog that can lead to lethal viral mutagenesis, chain termination or the inhibition of nucleotide biosynthesis [70]. Favipiravir in combination with oseltamivir, which was given to patients infected with SARS-CoV-2, has been used to treat severe influenza [45]. Oseltamivir, a neuraminidase inhibitor, is recommended as an antiviral treatment for influenza and has been widely used to inhibit COVID-19 in China [45,70]. Other neuraminidase inhibitors, zanamivir and peramivir, are also effective treatments for MERS-CoV [71]. At this time, these are all empirical therapies for COVID-19. However, whether oseltamivir or zanamivir are effective treatments for COVID-19 also needs further study.
Arbidol (ARB) was licensed for the treatment of influenza and other respiratory viral infections in Russia and China [72,73]. Blaising and coauthors consider ARB to be a broad spectrum antiviral drug [72]. In addition, ARB has been reported to inhibit SARS-CoV in vitro. Therefore, a clinical trial of ARB-treated COVID-19-positive patients has been registered [65].
Glucocorticoids have been commonly used in patients with SARS-CoV or MERS-CoV infection [74,75]. Glucocorticoids prolonged the survival time of SARS cases [75]. Patients with COVID-19 were given glucocorticoids in some hospitals in China. However, the mortality rate did not decrease with corticosteroid treatment in patients infected with SARS-CoV-2, and viral clearance was not delayed [6,74]. Therefore, it is still controversial whether corticosteroids should be used to treat SARS-CoV-2 infections [69].
Chloroquine/hydroxychloroquine was used as an antimalarial, broad spectrum antiviral drug, and has been broadly used in autoimmune diseases including lupus and rheumatoid arthritis [76]. A recent study indicates that chloroquine and the antiviral drug RDV-inhibited SARS-CoV-2 in vitro [77]. Clinical symptoms of patients treated with chloroquine were obviously relieved: for example, more rapid decline in fever and improvement of lung CT [77]. Chloroquine was suggested to treat COVID-19 in the SARS-CoV-2 treatment guidelines by the Chinese medical advisory. Chloroquine has been highly effective in reducing SARS-CoV-2 viral replication by increasing endosomal pH and interfering with the glycosylation of cellular receptors [76]. Moreover, chloroquine was probably the first molecule to be used to treat COVID-19.
Another antiviral drug, nelfinavir, an HIV protease inhibitor, was predicted to be a potential inhibitor of COVID-19 [26]. In addition, cytokine immunotherapy with IFN-α, a broad spectrum antiviral drug, has been used to inhibit HBV. IFN-α was used to treat patients infected with SARS-CoV-2 according to established guidelines [70]. IFN-α combined with LPV/r was shown to be beneficial for treatment of COVID-19 [78].
Tocilizumab is a humanized anti-IL-6-receptor (IL-6R) monoclonal antibody that inhibits IL-6 signaling and is used as a treatment in rheumatoid arthritis [79]. IL-6 is one of the most important cytokines involved in COVID-19-induced cytokine storms. Tocilizumab (TCZ) has been used to treat COVID-19 in China and Italy. TCZ was recommended for COVID-19 patients to prevent or treat cytokine storms and could reduce the mortality of COVID-19 [80].
Other drug types such as RNA synthesis inhibitors (TDF and 3TC) and an FP (EK1), have also effectively inhibited SARS-CoV-2 in vitro. In addition, traditional Chinese medicines (Lian Hua Qing Wen Capsules, Shu Feng Jie Du Capsules) have also been used to treat COVID-19 in the latest version of the Diagnosis and Treatment of pneumonia induced by COVID-19 [81]. In addition, human seroalbumin and γ-immunoglobulin were given to some patients with severe infections [82]. In conclusion, there are specific vaccines or antiviral drugs for COVID-19. All of the drugs described above have shown some usefulness in treating SARS-CoV-2 infections, and their efficacy merits further study.

Conclusion & prospects
COVID-19 is a serious human infectious disease of global concern. As of 7 April 2020, SARS-CoV-2 has infected a total of 134,784 patients globally at least ( Figure 3D & G). The COVID-19 outbreak poses a serious challenge to China and the whole world; it has profoundly affected public health. Although the overall mortality rate of SARS-CoV-2 appears to be lower than that of SARS-CoV and MERS-CoV (Table 1), the transmissibility of COVID-19 is more rapid. Moreover, the fatality rate of elderly patients with reduced immunity or chronic diseases is as high as 15% [6]. In addition, asymptomatic carriers may be a potential source of infection, sustaining a local 10  epidemic and global spread [83]. Therefore, COVID-19 may cause disruptions to the global public health system for an extended period of time.
The outbreak of COVID-19 has shown that there are still shortcomings in the prevention of public health diseases such as the lack of awareness of the frontline doctors and the early vigilance and attention from the government. Meanwhile, the awareness of the general public to health concerns also must be improved. Individuals should develop good living habits: for example, keeping away from wild animals, not consuming wild animals, maintaining hand hygiene -among others. In addition, the development of targeted antiviral drugs should be anticipated and accelerated in the future.
Certainly, vigorous measures to prevent contagion have been taken in China and other countries. To prevent the spread of infection, the Chinese government imposed a full lockdown and canceled public events such as the new year festival; contact with wild animals was also restricted, and travel was reduced with screening at airports, railway stations and subway stations [84]. Moreover, two emergency hospitals in Wuhan were constructed, and army medical units and medical staff from other areas were deployed to help prevent infections in Wuhan [84]. The WHO and various governments advised people to reduce public activity, maintain social distance, wear masks, wash their hands frequently, and practice respiratory hygiene. As of 7 April 2020, the number of fully recovered patients was 277,420 worldwide ( Figure 3E & H), and the number of recovered patients was 77,468 in China ( Figure 3B). Therefore, we are confident that the outbreak of COVID-19 will be effectively curbed.
Most countries have isolated any suspected cases as rapidly as possible to contain infection and prevent local outbreaks. The ability to rapidly test patients suspected of having a SARS-CoV-2 infection is the cornerstone of case isolation. The experience gained from SARS-CoV and MERS-CoV by the health community over the last 20 years could also help in dealing with SARS-CoV-2 infections throughout the world.

Future perspective
To date, no SARS-CoV-2-specific antiviral drugs or vaccines have been described for COVID-19. Therefore, a safe and stable vaccine for COVID-19 is urgently needed, and it is expected to be ready within 18 months [81]. Vaccines specific for SARS-CoV-2 will immunize people worldwide in the future. It is hoped that drugs specifically targeted for COVID-19 also will be widely developed. Meanwhile, the origin, intermediate host, structure and pathogenesis of SARS-CoV-2 will be the focus of future research. Moreover, prediction of whether the similar coronaviruses can infect humans will be import for all the world. We hope that vaccines specific for the similar coronaviruses will be developed in advance to prevent epidemics in the world.

Executive summary
• Coronavirus disease 2019 (COVID-19) was first reported in China and currently poses a serious challenge worldwide. The pneumonia was caused by a novel coronavirus named SARS-CoV-2. • More than 1,347,804 cases of COVID-19 and 74,596 deaths have been reported as of April 7, 2020 according to data from Johns Hopkins Resource Center. • SARS-CoV-2 belongs to Betacoronavirus, a large genus of viruses prevalent in nature. SARS-CoV-2 has 82% nucleotide identity with human SARS-CoV and 96% nucleotide identity with bat SARS coronavirus (SARSr-CoV-RaTG13). • The fatality rate of COVID-19 has been approximately 3.4% and the R0 has ranged from 1.4 to 6.4. Compared with previous coronavirus outbreaks,  has been reported to have a lower mortality rate and more rapid transmissibility and caused severe acute respiratory syndrome similarly to SARS. • The main symptoms are fever, cough, shortness of breath, leukopenia and pneumonia.
• Diagnostic laboratory testing of COVID-19 includes hematology testing, nucleic acid testing, viral genome sequencing and serology testing. To date, no specific antiviral drugs or vaccines for COVID-19 have been developed. Therefore, empirical antiviral drugs (lopinavir/ritonavir, favipiravir, oseltamivir, zanamivir and peramivir, arbidol), antibiotic drugs (moxifloxacin, levofloxacin, linezolid), chloroquine/hydroxychloroquine, glucocorticoids, monoclonal anti-inflammatory antibody (tocilizumab), have been used to treat SARS-CoV-2 infection. Traditional Chinese medicines are also used for therapy during infection with SARS-CoV-2. • This review is presented in the hope of helping the public effectively recognize and combat COVID-19 and to provide a reference for future studies or outbreaks.

Acknowledgment
The authors gratefully acknowledge all doctors who participate in the fight against COVID-19 on the frontline.
Financial & competing interests disclosure No writing assistance was utilized in the production of this manuscript.