SARS coronavirus 2: from genome to infectome

Coronaviruses (CoVs) were first isolated from humans in 1962 [1]. The CoVs were thought to cause only mild respiratory and gastrointestinal infections in human and animals [2]. The outbreaks of Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) in 2002–2003 in Guangdong province, China [3] and the Middle East respiratory syndrome coronavirus (MERS-CoV) in the Middle Eastern countries, particularly Saudi Arabia in 2012 [4], changed the prevailing concept on coronavirus infections. Both the viruses originated in bats and their chain of transmission established between animal to human and human to human [5, 6]. A similar outbreak of pneumonia like respiratory infections, reported from the Wuhan city, Hubei province, China in late December 2019 made a new addition to the list of human CoVs. The International Committee on Taxonomy of Viruses (ICTV) has named the novel Coronavirus as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the disease caused by this virus has been officially named as COVID-19 by WHO [7]. The COVID-19 has been regarded as Public Health Emergency of International Concern (PHEIC) on January 30, 2020 by WHO [8]. SARS-CoV-2 shares 96% genome similarity with a bat Coronavirus [9‐11]. The primary targets are the type-II alveolar epithelial cells and airway-epithelial cells, which highly express the Angiotensin converting enzyme-2 (ACE2) receptor on their surface. The ACE2 receptor is used for internalization, similar to SARS-CoV-1 and Human Coronavirus-229E (HCoV-229E) [12]. The SARS-CoV-2 quickly replicates inside the cells and kick-start the plethora of signaling cascade, from activating the pro-inflammatory response to antiviral response leading to cytokine storm. The virus rapidly disseminates through peripheral blood to other organs like, heart, kidney, liver, spleen, etc. [12, 13]. However, the pathogenicity of SARS-CoV-2 is notably less than SARS-CoV-1 and MERS-CoV, but its high transmissibility led to the pandemic, which resulted in the global lock-down and affected the global health scenario adversely [14]. The rapid development of the diagnostic tools and therapeutics in the form of antivirals and vaccines are the need of an hour to overcome the present situation.

SARS-CoV-2 has been identified as a third zoonotic-human coronavirus [15]. The bats are the natural host for SARS-CoV-2 while the intermediate reservoir is still under debate [16‐18]. As per the global scenario, about 53.9 million people have been reported to be positive for COVID-19 with 1.31 million confirmed deaths and 34.7 million recovered till November 14, 2020 [19]. The top five worst COVID-19 affected countries include, United States, India, Brazil, France and Russia with > 1.5 million cases till November 14, 2020 [19].

The early reports from USA, China and Italy indicated the SARS-CoV-2 infection among people > 60 years of age [20‐22]. However, the recent reports (June–August, 2020) indicated the increased rate of infection (4.5% to 15%) among age groups of 15–29 years [23]. This dramatic shift in the infection cases in terms of age groups may be due to the reversion of younger population to their work place, Universities, colleges and schools etc. [23]. Therefore, the COVID-19-related mortality has a varying age distribution starting from 10 to 80 years of age with a greater number of cases reported among patients suffering from other co-morbid conditions [24]. The case fatality rate (CFR) is defined as total number of deaths to the total number of cases reported. In case of COVID-19 the CFR differs from country to country due to the differences in the medical and health infrastructure, co-morbidities and population age.

The major co-morbid conditions leading to the severity of COVID-19 include 10.5% for cardiovascular disease (CVD), 37.3% for diabetes, 8.3% for chronic obstructive pulmonary diseases (COPD), and 55.4% for hypertension, and 8.1% for cancer patients [25‐27].

The sex-disaggregated COVID-19 data, collected from 26 countries indicate that males and females are almost equally susceptible to SARS-CoV-2 infection, however the mortality rate is 2.4 times higher in males compared to females [14, 28, 29]. The high mortality rates among males may be correlated to the co-morbidities like, diabetes, hypertension, cardiovascular diseases, and chronic kidney diseases etc. [30]. The higher levels of circulating ACE2 have been reported in the plasma of males suffering from COVID-than females. This condition indicates the higher levels of ACE2 receptor expression on tissues, which help in virus internalization [31]. Overall, the COVID19 related mortalities have been reported to be higher in males than females due to the differences in immunological, genetic, endocrinological, social and behavioral factors [32].

The SARS-COV-2 transmission was most probably due to cross-species jump from animal to humans, which first started from wet animal market in Wuhan province, China [33, 34]. The person-to-person transmission established from the visitors who visited Huanan animal market [35]. Therefore, the mode of transmission is either through direct human contact, or through the droplets generated during sneezing and coughing of an infected individual (Fig. 1) [36]. The presence of viral RNA in stool samples suggest another route of transmission but so far it has not established very well due to the absence of the live virus in stool samples [37]. The vertical transmission of virus was reported during SARS-CoV-1 and MERS-CoV outbreaks, while the same could not be established so far in the case of SARS-COV-2 [36, 38, 39]. The testing of symptomatic and asymptomatic patients, containment procedures and other precautionary measures like, wearing masks in public places, maintain social distancing, regular use of handwash and hand sanitizers should be adopted to break the chain of transmission of SARS-CoV2 [40].

Coronaviruses (CoVs) belong to Coronaviridae family (subfamily Coronavirinae), order Nidovirales [10]. The subfamily Coronavirinae has been divided into four genera: -Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus [10, 41]. The Alphacoronavirus and Betacoronavirus are known to infect humans [2]. Bats serve as the evolutionary hosts for the Alphacoronavirus and Betacoronavirus [42]. The whole genome sequencing and phylogenetic analysis classified SARS-CoV-2 as Betacoronavirus from the sub-genus Sarbecovirus, which also includes SARS-CoV-1 [9, 43]. The mutations, recombination and re-assortments routinely occur in the RNA viruses as a part of the evolutionary process for increasing the genetic diversity. The Betacoronavirus have been reported to undergo recombination within bats. The SARS-CoV-2 belongs to Sarbecovirus and shares similarity with two bat derived Coronavirus strains bat-SL-CoVZC45 and bat-SL-CoVZXC21 [43]. The SARS-CoV-2 genome shows 96% similarity to horse-shoe bat virus RaTG13 Rhinolophus affinis [9, 44]. The ecological separation of bats from human population makes an obvious note on the presence of an intermediate host, where SARS-CoV-2 develops adaptive changes, before transmitting to humans. This is supported by the difference in the key genomic features of SARS-CoV-2 from RaTG13 and SARS-CoV-1 [45]. Although RaTG13 is 96% similar to SARS-CoV-2, but the receptor binding domain (RBD) of SARS-CoV2 spike protein shares only 85% similarity with the RaTG13 and only one out of six critical amino acid residues of RBD is similar in RaTG13 and SARS-CoV-2 [46‐48]. The five of the six amino acid residues differ between the SARS-CoV-1 and SARS-CoV-2 [48]. The SARS-CoV-2 spike proteins contain a polybasic furin cleavage site insertion (residues PRRA) at the junction of S1 and S2, which is probably enhancing the infectivity of the SARS-CoV-2 and is not present in any other Coronavirus [46‐48]. The coronaviruses reported in Pangolin exhibit a strong similarity to SARS-CoV-2. The Malayan pangolins Manis javanica illegally imported into southern China (Guangdong and Guangxi provinces) were reported to be infected with SARS-CoV-2 related virus [44, 49]. Several SARS-CoV-2 related viruses have been reported in Malayan Pangolins. The sequencing data from these strains show them to be very closely related to SARS-CoV-2 and share 92.4–99.8% sequence identity. The Receptor Binding Motif (RBM) of Spike protein of these strains is also identical to SARS-CoV-2 and differs only in one out of five critical amino acid residues [49‐51]. Therefore, SARS-CoV-2 might be a recombinant form of bat and pangolin coronaviruses, and the homologous recombination events might have occurred in spike glycoprotein genes between bat and pangolin CoVs [11, 45]. It has been reported that the cats and ferrets can also get infected with SARS-COV-2 and are susceptible to air-borne transmission.

However, the virus replicates poorly in dogs, pigs and chicken [52‐54]. Though, to ascertain the exact pattern and genomic ancestors of the recombination events, a wider sampling of the viral diversity is required to resolve the evolutionary events.

The SARS-CoV-2 is a single stranded positive RNA virus of ~ 29.9 kB in size. The SARS-CoV-2 has 14 open reading frames (ORFs), which encodes for 27 different proteins [55]. It has 5′ untranslated region (UTR), replication complex (ORF1a and ORF1b), Spike (S) gene, Envelope (E) gene, Membrane (M) gene, Nucleocapsid (N) gene, 3′ UTR, several unidentified non-structural ORFs and a poly (A) tail [56, 57]. The ORF1a gene is located at the 5′UTR, encodes for polyprotein pp1a, which contains 10 nsps. The ORF1b gene, located next to ORF1a, encodes for polyprotein pp1ab which contains 16 nsps [55]. The pp1ab and pp1a protein undergoes autoproteolytic cleavage to form the viral replication complex. The 3′UTR contains the four structural genes and eight accessory genes. The accessory genes are distributed between the structural genes and their function is mostly unknown [55, 57]. The SARS-CoV-2 is a non-segmented enveloped virus with a diameter of 50–200 nm [58]. Structurally, it has a double-layered lipid envelope, including Spike glycoprotein (S), Envelope protein (E), Membrane glycoprotein (M), and Nucleocapsid protein (N) [59, 60]. The viral genome having a RBD for the interaction with host cell receptors is covered by the Spike glycoprotein [46]. The M glycoprotein is responsible for the assembly of viral particles has three domains, the cytoplasmic domain, the transmembrane domain, and the N hydrophilic domain [61]. The Envelope protein is reported to play role in pathogenesis as it interacts with the tight junction related protein PALS1 [62]. The nucleocapsid protein packs the viral genome into a ribonucleoprotein complex [63]. The nucleocapsid, a phosphoprotein plays role in viral genome replication and the cell signaling pathway (Fig. 2).

The positive sense RNA genome serves as a template for both replication and protein synthesis. The virus enters through membrane fusion and releases its positive sense RNA into the cytoplasm. The CoVs control the relative expression of their proteins through a conserved molecular mechanism, known as -1 programmed ribosomal frameshifting (-1 PRF) [64]. The SARS-CoV-2 -1PRF and SARS-CoV1-1PRF is nearly similar with a single nucleotide difference which does not impact the rate of -1 PRF in SARS-CoV-2 [65].

The two ORFs of the viral genome, ORF1a and ORF1b translate to non-structural proteins (nsps) in the cytoplasm. The ORF1a produces a polypeptide pp1a, which proteolytically cleaved to produce 10nsps while the -1PRF of SARS-CoV-2 allows continued translation till ORF1b [2, 65, 66] and yields a larger polypeptide (pp1ab) which gets cleaved into 16 nsps. The proteolytic cleavage of the polypeptides is carried out by the viral proteases 3CL^pro and M^pro [2, 67]. The functions of different nsps are listed in Table 1.

The replication and transcription of the viral genome is mediated by the activity of RNA dependent RNA polymerase (RdRP/nsp12). The RdRP catalyzes the synthesis of viral RNA, with the assistance of nsp7 and nsp8 as cofactors [68]. The RNA viruses lack the proofreading capacity. Although, Smith et al. reported that, an exoribonuclease domain (ExoN) in non-structural protein 14 provides proofreading activity that protects the SARS-CoV1 from mutagenesis [69]. The ExoN deletion leads to reduced replicative fidelity [69]. The replication complex generates a full-length negative sense RNA intermediates from the viral genome, which serve as the template for the synthesis of positive sense genomic RNAs (gRNA) and the sub-genomic RNA (sgRNA). The nucleocapsid protein encapsulates the gRNA, the S, M and E proteins in the endoplasmic reticulum-Golgi intermediate compartment. The assembly of mature virion occurs inside the Golgi and the virion containing vesicles fuse with plasma membrane and release the virus by exocytosis (Fig. 3b) [2, 66]. The SARS-CoV-2 expresses nine sgRNAs (S, 3a, E, M, 6, 7a, 7b, 8, and N) which form the structural and accessory proteins. These sgRNAs are produced by the canonical Transcription Regulatory Sequence (TRS) mediated mechanism for discontinuous transcription [57, 70].

The COVID-19 clinical presentations are similar to that of SARS-CoV1 and MERS, which are mild and self-limiting in 80% of the cases. Only 20% of the cases aggravate to secondary complications of ARDS or multiple organ failure [97, 98]. The virus affects people differently depending upon the genetic pre-disposition, immune status and diseases associated with respiratory system [99, 100]. The people > 60 years of age are at higher risk of exacerbating the disease [58, 83, 101].

The incubation period for SARS-COV-2 has been estimated up to 14 days, which is longer than SARS-CoV-1 and MERS [102, 103]. The longer incubation period supports the asymptomatic and subclinical infection rate [14]. The common symptoms include fever, dry cough, fatigue, myalgia, dyspnea, runny nose, nausea, joint pain and gastrointestinal symptom [104]. In addition, patients with co-morbidities like, diabetes, hypertension [28, 83, 84], acute kidney disease, cardiac problem, cerebrovascular disease or liver dysfunction may be more susceptible to infection [97, 105].

The severity of COVID19 directly co-relates to lymphopenia, eosinopenia and hypercytokinemia, similar to SARS-CoV-1 and MERS [83, 84, 97, 106]. The serological reports of COVID-19 patients show a sharp increase in their C reactive proteins, lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR), creatinine kinase, alanine aminotransferase (ALT), aspartate transaminase (AST), D-dimer and low serum albumin [13, 105, 107] indicating sepsis which may lead to multiorgan failure during the later stages of infection. The higher levels of pro-inflammatory cytokines like, IL2, IL7, IL10, granulocyte-colony stimulating factor (GCSF), interferon-gamma protein-10 (IP10), monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein-1α (MIP1A), and TNFα may contribute to “cytokine storm” similar to SARS-CoV1 and MERS [83, 107‐110]. The CT-scans and X-Ray reports of COVID-19 patients revealed the opacities and bilateral diffuse alveolar damage followed by cellular exudates, pleurisy, pericarditis, lung consolidation and pulmonary edema [13, 109]. The autopsy reports revealed the atypical enlarged pneumocytes, interstitial mononuclear infiltration with significant cytopathic effects and the presence of lymphocytes in the affected area and thick alveolar wall [83]. The other deformities include degeneration of neurons, atrophy of spleen, infiltration of immune cells in liver, hyaline thrombus in blood vessels leads to cardiac arrest and hemorrhage in kidney are observed in severely affected COVID-19 patients [13, 109‐111]. The nasopharyngeal swab/oropharyngeal swab (upper respiratory tract), sputum, lavage or aspirate (lower respiratory tract) is used for diagnosis. In addition, blood, stool and urine sample are also used for need based diagnosis [112]. The diagnosis is carried by RT-qPCR or by high throughput sequencing of viral genome. The primers and probes against ORF1b and N genes are used for the detection of SARS-COV-2 from respiratory fluid [113]. The asymptomatic patients are identified by the presence of viral nucleic acid, and are responsible for human-to human transmission of SARS-COV-2 infection [101, 114‐116]. Many molecular based diagnostics and immunoassays are used for the detection of SARS-COV-2 (Table 2).

Scientists across the world are aiming to instigate therapeutics and vaccine against SARS CoV-2. The vaccine should be useful for all age groups and people with various co-morbidities. According to WHO report; there are 48 vaccine candidates under the advance stages of the clinical trials (Table 3). Among them 11 are currently in clinical trial Phase-III. The vaccine produced by BioNTech/Pfizer BNT162b2, is in the late stage of clinical trial. The BNT162b2is an mRNA-based vaccine; which exploits for RBD of SARS CoV-2 spike protein for eliciting immune response. This mRNA vaccine is lipid encapsulated for its effective delivery into the cells [117]. Another RNA based vaccine mRNA1273 is in clinical phase-III trial. This vaccine has been developed by Moderna Incorporation. The mRNA1273 vaccines lipid-nanoparticle encapsulated mRNA encoding for the SARS-CoV-2 spike glycoprotein [118]. The other potential vaccine is of University of Oxford with AstraZeneca (AZD1222 vaccine) is also currently at the final stages of the clinical trials. The AZD1222 vaccine has been developed by using the Chimpanzee adenovirus viral vector, ChAdOxo-1s, which encodes for SARS-CoV-2 spike protein [119]. Another viral vector-based vaccine Sputnik V has been developed by Gamaleya Research Institute in Russia. The vaccine contains 2 viral vectors recombinant adenovirus type 26 (rAd26) and recombinant adenovirus type 5 (rAd5) encoding the full length S glycoprotein of SARS-CoV-2 [120].

The different classes of therapeutics that can be used for the treatment of COVID-19 patients include, protease inhibitors, nucleoside analogue, neutralizing-monoclonal antibody, immune modulator, RNA polymerase inhibitor, interferon alpha, endonuclease Inhibitor, fusion inhibitor, convalescence plasma therapy and Immunosuppressant [121].

The antimalarial drug, Ivermectin has been tried in combination with Remedesivir for the COVID-19 treatment. Ivermectin has been reported to inhibit the nuclear translocation of viral protein and prevents the inhibition of antiviral response [122, 123].

Remedesivir (GS-5734) is an adenosine analog, which suppresses the viral replication by interfering in the activities of RdRp. Remedesivir is also used against MERS, SARS-CoV1 [124‐126]. Bamlanivimab (LY-CoV555), the monoclonal antibody produced by Eli Lilly & Company has been granted an emergency approval by FDA for COVID-19 treatment. It is a recombinant human monoclonal neutralizing antibody IgG1 produced against SARS-CoV-2 spike protein [127]. The Hydroxychloroquine (HCQ) a well-known antimalarial drug has been under trial for COVID 19 treatment but it was not found to have any beneficial effect for the COVID19 patients. HCQ was reported to inhibit the viral infection by glycosylating the ACE2 receptor of SARS-CoV-1 and increases the endosomal pH, rendering the membrane fusion [124, 128]. Favipiravir has also been under trial for the treatment of COVID 19 patients as it was previously tested against Ebola and Influenza virus. Favipiravir inhibits the RNA polymerases and halts the viral replication [129]. The convalescent plasma therapy has been used for treating the critical COVID-19 patients during the early phases of this outbreak. Previously it was used against SARS-CoV-1, Ebola and H1N1 but the use of convalescent plasma therapy against SARS-CoV2 has been quite debatable issue [130]. Another type of therapeutic, tocilizumab has also been used against SARS-CoV2. Tocilizumab has been used for the treatment of severe rheumatoid arthritis, systemic juvenile idiopathic arthritis and giant cell arteritis. Tocilizumab, an immunosuppressant binds to the IL-6 receptor and hinders the inflammatory responses [131]. In addition, there are many other therapeutic trials for COVID-19 ongoing and some of them with positive responses have been listed in Table 4.