Scientific research progress of COVID‐19/SARS‐CoV‐2 in the first five months

Abstract A cluster of pneumonia (COVID‐19) cases have been found in Wuhan China in late December, 2019, and subsequently, a novel coronavirus with a positive stranded RNA was identified to be the aetiological virus (severe acute respiratory syndrome coronavirus 2, SARS‐CoV‐2), which has a phylogenetic similarity to severe acute respiratory syndrome coronavirus (SARS‐CoV). SARS‐CoV‐2 transmits mainly through droplets and close contact and the elder or people with chronic diseases are high‐risk population. People affected by SARS‐CoV‐2 can be asymptomatic, which brings about more difficulties to control the transmission. COVID‐19 has become pandemic rapidly after onset, and so far the infected people have been above 2 000 000 and more than 130 000 died worldwide according to COVID‐19 situation dashboard of World Health Organization (https://covid19.who.int). Here, we summarized the current known knowledge regarding epidemiological, pathogenesis, pathology, clinical features, comorbidities and treatment of COVID‐19/ SARS‐CoV‐2 as reference for the prevention and control COVID‐19.

age groups (6.4% [5.7-7.2], ≥60 years) and up to 13.4% (11.2-15.9) in those aged 80 years or older. Estimates of case fatality ratio from international cases stratified by age were consistent with those from China (4.5% [1.8-11.1] in those aged ≥60 years [n = 151]). 10 SARS-CoV-2 has strong transmission ability, and it has been occurred human-to-human transmission. The basic reproductive number (R 0 ) of SARS-CoV-2 was estimated ~2.2 based on early patients and a subsequent study based on 75 815 individuals (from 31 December 2019 to 28 January 2020) estimated that R 0 was 2.68. 5,8 Recent study from the Los Alamos National Laboratory has collected extensive individual case reports and designed mathematical modelling, which calculated the median R 0 value as 5.7 (95% CI 3.8-8.9). 11 Therefore, the R 0 of SARS-CoV-2 is rising with the increased number of confirmed cases and so far it has exceeded the R 0 of MERS (R 0 = 0.6) and SARS (R 0 = 1). 12 Scientists have predicted the trend of COVID-19 development by studying its epidemic dynamics. It was indicated that Wuhan epidemic would peak around April 2020 and local epidemic across cities in mainland China would lag by 1-2 weeks in a study. 5 In another study, researchers estimated the epidemic peak would be on 17 February 2020 in China and an obvious rise could occur from 25 to 29 February 2020 overseas. 13 Asymptomatic cases with COVID-19 accounted for ~1% of the total number of patients and the viral load of asymptomatic patients was similar to that of symptomatic patients, which indicated that asymptomatic patients also had the potential to transmit virus. 7,14 SARS-CoV-2 is mainly transmitted by droplets, which invade the human body through contaminating the human conjunctival epithelium such as nose and eyes and cause infection. 15 In addition to droplet transmission, faecal-oral spread is also a potential mode of transmission. 16 Although no studies have found COVID-19 in newborns, specific antibodies have been detected in some newborns, 17,18 which indicated the possibility of transmission from the mother to her baby. Due to the particularity of droplet transmission, close contact activities, such as family clustering, usually increase the possibility of infection. 19,20 To block the viral transmission by isolation, wearing masks and other ways of reducing close contact are recommended. There are also at least 6 accessory ORFs. 21,22 SARS-CoV-2 belongs to the β coronavirus and its sequence significantly differs from that of SARS-CoV and MERS-CoV, which have caused epidemics previously. 1 Sequence alignment displays that SARS-CoV-2 has 79% and 50% identity with SARS-CoV and MERS-CoV, respectively, 23 whereas 96.3% identity with a bat's coronavirus. 24 Phylogenetic analysis suggests that the closest relatives of SARS-CoV-2 are several viruses originated from bats, such as Bat-CoV RaTG3, Bat SARSr CoV-ZC45 and Bat SARSr CoV-ZXC21. Therefore, bats are assumed to be the original host of SARS-CoV-2. However, there should be an intermediate host, who transfers the novel coronavirus to human because the sequence of SARS-CoV-2 and bat virus Bat-CoV RaTG3 has minor difference. 23,25 The suspicious intermediate host has been suggested to be the pangolin by Chinese scientists on the basis of genetic analyses, which has not been confirmed until now with the release of new pangolin coronavirus genome studies. 26 A recent study has found that the SARS-COV-2 was easy to infect cats and ferrets, but weakly infectious to dogs, pigs, chickens and ducks, which could be important for the traceability of the SARS-COV-2. 27 Multiple sequence analysis studies have shown that the genome sequences of SARS-CoV-2 exhibit more than 99% sequence identity. 22,23,25 As a typical RNA virus, the average substitution rate for coronavirus is 10 −4 substitutions per year per site. 28 The low heterogeneity among sequences suggests that all these SARS-CoV-2 originated from the same ancestor within a very short period. 23 Despite the low heterogeneity, the number of viral genome variants will consistently increase with the continuous spread of COVID-19. So far, 4007 variants have been identified and include single nucleotide polymorphism (SNP), deletion, indel and insertion with the mutant numbers of 3951, 82, 10 and 20, respectively (https://bigd.big.ac.cn/ncov). A deletion of eight amino acid has been reported in viral polyprotein 1ab (pp1ab) protein, encoded by ORF1ab gene of SARS-CoV-2.

| Viral genome information and viral origin
The pp1ab protein is composed of 16 mature non-structural proteins (NSPs) and seldom reported to have mutations previously.
This mutant was isolated from a Japanese COVID-19 patient, who did not show critical symptoms. 29 Domenico group described the presence of two mutations of SARS-CoV-2, affecting the NSP6 (amino acid position 3691) and ORF10 adjacent regions (amino acid position 9659), respectively, and both mutations could confer lower stability of the protein structures. 30 Another two mutations of nt28144 in ORF8 and nt8782 in ORF1a of SARS-CoV-2 have been noticed to have higher mutation rate of 30.53% and 29.47%, respectively. 31 A study analysed 103 SARS-CoV-2 genomes and also found nt28144 mutant in ORF8. They classified the SARS-CoV-2 virus as type L (28144T) and S (28144C) based on the different amino acid of leucine and serine in nt28144. S type was indicated to be the ancestral version although the L type (mutation rate ~70%) was more prevalent than the S type (mutation rate ~30%). 32 L type was speculated to be more aggressive and contagious. By analysis of 160 complete human SARS-CoV-2 genomes, researchers from the University of Cambridge found three central variants distinguished by amino acid alteration, named A (8782T, 28144C), B (derived from A, 8782C, 28144T) and C (derived from B, 26144T), with A being the ancestral type according to the bat out-group coronavirus. 33 The A and C types were found mainly in Europeans and Americans, whereas the B type was the most common type in East Asia.
Currently, the reported mutation rate of SARS-CoV-2 is generally low and there is no evidence that viral recombination has occurred.

| The receptor of SARS-CoV-2: Angiotensin converting enzyme II (ACE2)
The envelope spike (S) protein is crucial for determining host tropism and transmission capacity. 23 Although the sequence identity of SARS-CoV-2 and SARS-CoV is only 79%, their spike proteins have a highly similar three-dimensional structure in the receptor-binding domain (RBD), 35,36 which makes it possible that SARS-CoV-2, like SARS-CoV, invades cells through the receptor ACE2. Viral infectivity studies using HeLa cells with and without ACE2 expression from humans, Chinese horseshoe bats, civets, pigs and mice reveal that SARS-CoV-2 is able to use all ACE2 except that of mouse as an entry receptor. 25 To further understand the interaction of SARS-CoV-2 and ACE2 receptor, scientists from the United States and China separately analysed the cryo-electronic microscope (cryo-EM) structures of SARS-CoV-2 S protein 37 and ACE2 receptor, as well as the three-dimensional structure of S protein RBD and the full-length protein complex of ACE2 receptor. 38 Molecular modelling reveals that SARS-CoV-2 RBD interacts more strongly with ACE2 receptor than that of SARS-CoV. 35 Surface plasmon resonance analysis reveals that the affinity of human ACE2 protein binding to the novel coronavirus S protein is 10-to 20-fold higher than that of ACE2 binding to SARS-CoV S protein, which results in a stronger SARS-CoV-2 transmission capacity. 37 These evidences strongly support that SARS-CoV-2 infects host cells by binding ACE2 receptor.
ACE2 belongs to a type I membrane protein expressed highly in lungs, oesophagus upper and stratified epithelial cells, intestine, absorptive enterocytes from ileum and colon, cholangiocytes, myocardial cells, kidney proximal tubule cells and testicle, which will be preferentially attacked. [39][40][41] COVID-19 transmission through faecal-oral has been proved by evidences that diarrhoea and other alimentary system symptoms are the main manifestations of some patients 6 and the stool samples of patients with diarrhoea are positive for SARS-CoV-2 nucleic acid test. 42 The renal and testicle, highly expressing ACE2, may be potential to be invaded by SARS-CoV-2. 43 The interaction between viral S protein and cell surface receptor ACE2 could activate the downstream renin-angiotensin-aldosterone system (RAAS) since ACE2 is one of the crucial components of RAAS ( Figure 2). RAAS could be involved in the COVID-19 pathogenesis and could balance the RAAS by increasing ACE2 or blocking the interaction between AngII, and AT1/AT2 could be a potential therapeutic target for COVID-19. 44

| Cytokine storm
It was found that before death, the level of neutrophil count, D-dimer, blood urea and creatinine levels of COVID-19 patients continued to increase and the lymphocyte counts continued to decrease. These

F I G U R E 2
The mechanism of SARS-CoV-2 and ACE2 interaction based on renin-angiotensin-aldosterone system (RAAS) and potential therapeutic strategies in COVID-19. SARS-CoV-2 invades cells via ACE2 receptor, which may lead to the down-regulation of ACE2 expression. The down-regulation of ACE2 expression could destroy the balance between ACE/ACE2 and lead to the tissue injury. Potential therapeutic approaches include a SARS-CoV-2 spike protein-based vaccine and small-molecule inhibitors to block the interaction between S protein and ACE2 changes were related to cytokine storm, suggesting the activation of coagulation system, continuous inflammatory response and the occurrence of acute kidney injury, which could be involved into the pathogenesis of COVID-19 exacerbation and explain the death of COVID-19 patients. 45

| PATHOLOGY
Although greatly resembled pathological features of COVID-19 with SARS and MERS coronavirus infection, COVID-19 patients were found to have the unique pathological feature presenting as bilateral diffuse alveolar damage with cellular fibromyxoid exudates. Interstitial mononuclear inflammatory infiltrates dominated by lymphocytes were seen in both lungs, and multinucleated syncytial cells with atypical enlarged pneumocytes characterized by large nuclei, amphophilic granular cytoplasm and prominent nucleoli were identified in the intra-alveolar spaces, showing viral cytopathic-like changes. 46

| CLINIC AL FE ATURE S
COVID-19 is characterized by fever, weakness, dry cough, mus- There are many COVID-19 risk factors, and age, gender and blood type seem to be more important among them. In a retrospective, multicentre cohort study, older age is significantly correlated with COVID-19, which is consistent with the higher incidence of older people. High Sequential Organ Failure Assessment (SOFA) score and D-dimer >1 μg/L are also risk factors. 47 Gender is another risk factor, and a lot of research have disclosed that higher prevalence of COVID-19 in men than women. 48 It may be related to sex-based immunological or behavioural difference, such as smoking and so on. 49 According to a research based on large population, A and O blood group displayed different association risks for COVID-19. COVID-19 patients with blood group A account for 37.75%, whereas the proportion is 32.16% in normal people. Respectively, the proportion of O group in COVID-19 patients and normal people is 25.80% and 33.84%. 50 This result is similar to the study focused on the relationship between ABO blood group and SARS-CoV, which showed O blood group with a lower infection chance and anti-A antibodies specifically inhibit the adhesion of SARS-CoV S protein-expressing cells to ACE2-expressing cell lines. 51,52 These studies may contribute to the therapeutic strategy design of COVID-19, but further studies need to be done to clarify the detailed mechanism.
In the early stage of COVID-19, the total number of peripheral blood leucocytes was normal or decreased and the lymphocyte count was decreased. In some patients, liver enzyme, lactate dehydrogenase (LDH), muscle enzyme, aspartate transaminase level and myoglobin were increased. 53 The numbers of helper T cells, suppressor T cells and regulatory T cells were significantly reduced in patients of severe COVID-19, whereas the percentage of naïve T cells increased. 4 Pneumonia is the typical symptom of COVID-19, and the character of pulmonary image from high-resolution computed tomography (HRCT) includes multiple small patchy shadows, interstitial changes, multiple ground glass opacities and infiltration in both lungs and so on. 9,54 In severe cases, pulmonary consolidation may occur whereas pleural effusion is rare. 54,55

| COVID-19 and cardiovascular system
A study of 41 patients diagnosed with COVID-19 showed that hypersensitive troponin I (hs-cTnI) was increased substantially in five patients with the diagnosis of virus-related cardiac injury. Four out of the five patients were admitted to the ICU, accounting for 31% of the total ICU patients. 2 In a study of 138 COVID-19 patients admitted to Zhongnan Hospital of Wuhan University, from 1 January to 28 January 2020, compared with non-ICU patients, 36 severe patients receiving care in the ICU were more likely to have one of the complications such as cardiac injury and arrhythmia. 45 Recently, a systematic review and meta-analysis for 1813 COVID-19 patients indicated that cardiovascular disease and hypertension were strongly predictive for both severe disease and ICU admission. However, they found that COPD was the most strongly predictive comorbidity for both severe disease (pOR 6.42, 95% CI 2.44-16.9) and ICU admission (pOR 17.8, 95% CI 6.56-48.2).
Studies have found that patients with the underlying cardiovascular diseases account for a large proportion of COVID-19 patients.
A study of the epidemiological and clinical characteristics of 99 COVID-19 cases indicated that around 40 per cent had cardiovascular and cerebrovascular diseases. 6 In one study of 138 patients, 58.3% patients with severe symptoms had hypertension. 45 Another study analysed the clinical characteristics of 140 COVID-19 patients and found that hypertension, diabetes and cardiovascular and cerebrovascular diseases were the most common underlying diseases in all patients. 56 Therefore, it is important to improve the vigilance of patients with cardiovascular diseases.
Multiple evidences suggest that SARS-CoV-2 infects host cells by binding ACE2 receptor widely expressed in the cardiovascular system. 36 Therefore, ACE2-related signalling pathway may play a crucial role in cardiac injury. In addition, some scholars consider that there may be an imbalance between Th1 and Th2 responses in COVID-19 patients and the resulting cytokine storm could also be another mechanism of cardiac injury. 2 On the other hand, pneumonia caused by SARS-CoV-2 can lead to respiratory dysfunction and hypoxaemia, which can also bring about cardiomyocyte injury. 57

| COVID-19 and tumour
A study of 1590 COVID-19 patients indicated that patients with cancer had a higher risk of COVID-19 (1% vs 0.29%) and a higher risk of severe events (a composite endpoint defined as the percentage of patients being admitted to the intensive care unit and requiring invasive ventilation or death) (39% vs 8%) than those without cancer. 58 Current evidence remains insufficient to explain a conclusive association between cancer and COVID-19. However, a recent communication in the Lancet refutes this viewpoint, arguing that current evidence is insufficient to explain the conclusive association between cancer and COVID-19. 59

| COVID-19 and kidney
Data from a clinical study involving 59 COVID-19 patients showed that 19% (11/59) and 27% (16/59) of patients had an elevated level of plasma creatinine and urea nitrogen, respectively. All patients who underwent renal parenchymal CT had abnormal imaging of the kidney, mainly manifesting as low CT values of the renal parenchyma. 60 A study of 1099 COVID-19 patients found that the incidence of creatinine >133 micromol/L and acute kidney injury (AKI) were 4.3% and 2.9% in severe patients, respectively, whereas they were only 1% and 0.1%, respectively in non-severe patients. 9 Data from another study of 710 patients with COVID-19 demonstrated that 44% of patients have proteinuria haematuria, 26.9% have haematuria and the prevalence of elevated serum creatinine and blood urea nitrogen were 15.5% and 14.1%, respectively on admission. During the study period, AKI occurred in 3.2% patients. Cox proportional hazard regression confirmed that elevated serum creatinine, elevated urea nitrogen, AKI, proteinuria and haematuria were the independent risk factors for in-hospital death. 61 Single-cell RNA sequencing data revealed that ACE2 was highly expressed in kidney tubule cells, suggesting that the kidney was at high risk of coronavirus invasion. 62 Moreover, compared with non-ICU patients, ICU patients had higher plasma levels of IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1A and TNF-α, suggesting that the cytokine storm may be associated with disease severity. 2
As above-mentioned, SARS-CoV-2 has 79% and 50% identity with SARS-CoV and MERS-CoV, respectively 23 whereas 96.3% identity with a bat's coronavirus. 24 Compared the genomic sequences of SARS-CoV-2 with SARS-CoV, regions coding ORF1ab gene, S gene, ORF7b, ORF8 genes and N genes are different. 63 In S gene, SARS-CoV-2 shows lower GC% and higher AT% than SARS-CoV and MERS.
As for N gene, SARS-CoV-2 shows little difference in GC% and AT%  (Table 1).
Although the viral nucleic acid test is currently the standard method for diagnosis of COVID-19, the obvious limitations restrict

| Antibody detection
Rapid and accurate detection of SARS-CoV-2-specific antibodies in the blood of patients is one of the options for supplemental nucleic acid testing. IgM provides the first line of defence during viral infection, followed by the production of IgG that is important for long-term immunity and immunological memory. 68 (Table 1).
According to a recent study, an increase of viral antibodies can be seen in almost all patients on day 5 after COVID-19 onset and the positive rates of IgM and IgG were 81% and 100%, respectively. 69 Therefore, dynamic monitoring of viral IgM or IgG can be considered as a complementary way for nucleic acid detection and could also help to diagnose the COVID-19 patient with negative result of nucleic acid test but positive antibody. 70 In addition, antibody test could be crucial to inform public policymakers how many asymptomatic cases have occurred in a population 71 since it is the most convenient and fast approach to identify the asymptomatic cases so far.

| Antiviral drugs
Antiviral drugs can be classified as viral protease inhibitors, viral nucleoside analogs and agents that disrupt the interaction mechanism between the virus and the host ( Table 2).

| Viral protease inhibitors
Lopinavir/ritonavir and disulfiram are the well-known viral protease inhibitors and have been shown to be effective against SARS and MERS. 76 Lopinavir/ritonavir were reported to reduce mortality, intubation rate and the use of methylprednisolone when introduced as a treatment of SARS patients in early stage. 76

| Virus-host fusion inhibitors
In a study involving more than 100 patients, chloroquine was superior to the control group in suppression of COVID-19 pneumonia exacerbation, reduction of symptom duration and delay of viral clearance without severe side effects, 85  to guide policymakers and clinicians. These studies should report medium-and long-term follow-up results and safety data. 90 Arbidol is an anti-influenza drug and also used in anti-HCV treatment. An in vitro study has revealed that Arbidol could effectively inhibit SARS-CoV-2 infection at a concentration of 10-30 µmol/L, which brought arbidol the potential to treat COVID-19 clinically. 82 Nitazoxanide, as an anti-diarrhoea medicine, has been shown inhibition activity of the SARS-CoV-2 in in vitro studies. 81 In the future, combining the molecular mechanisms of these two agents could develop more effective compounds to prevent emerging infections. 91 The mechanism of Chinese herbal medicines against COVID-19 may include the following process, such as antiviral, immune/inflammatory, hypoxic reactions and so on, and it may also directly inhibit the SARS-CoV-2. 94

| Convalescent plasma therapy
Convalescent plasma or immunoglobulins have been shown to improve the survival rate of SARS patients with progressive deterioration. 95 In the pandemic of 2009 influenza A H1N1 (H1N1pdm09) infection, convalescent plasma therapy was found to help to reduce the relative risk of mortality significantly. 96 101 Two of these antibodies were tested and showed strong anti-SARS-CoV-2 capabilities by the reduction of the binding of SARS-CoV-2 S protein RBD to human ACE2 with reduction rate of 99.2% and 98.5%, respectively. 102 However, more clinical research should be conducted to prove the safety and effectiveness of convalescent plasma therapy among COVID-19 patients. 103

| Vaccine
Genomic sequence studies displayed that the spike protein of SARS-CoV-2 has high identity with that of SARS and MERS, which might indicate the similarity of immune evasion mechanism among SARS-CoV-2, SARS and MERS. 104,105 On the basis of knowledges obtained from SARS and MERS vaccine development, several research groups have announced to start SARS-CoV-2 vaccine R&D immediately after the global outbreak. 106,107 As was reported, receptor-binding domain in full-length spike (S) or S1 was crucial for SARS-CoV-2 entry into the host cell. 25 Table). To develop SARS-CoV-2 vaccine successfully, important information related to vaccine development and evaluation should be well defined, including targeted antigen(s), immunization route, related immune protection, animal models, outbreak forecasting and targeted population. 113,114 However, the production process and preclinical information of vaccines should be assessed to ensure volunteers' safety prior to clinical testing. 114 In a recent interview of the Harvard Office of Technology Development (OTD), researchers suggested that further research and development on a class of molecules called bisphosphonates might turbocharge a vaccine against SARS-CoV-2 and help bring immunity to huge populations more quickly. 115

| CON CLUS ION
The pandemic of COVID-19 has lasted for around 5 months, and so far, the main affected areas have from the original outbreak China to outside regions, especially American and Europe. Fortunately, we already accumulated some knowledge about the pathogen virus, its epidemiological and clinical features, pathogenesis and some effective therapeutic approaches, summarized above, which have contributed to the success against COVID-19 and will help human being completely control the epidemic finally.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
HL and ZL collected, analysed and interpreted the data. HL and J. G.
conceived and supervised the study. HL wrote the manuscript. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data, models and code generated or used during the study appear in the submitted article.