SARS冠状病毒的病原生物学分析及其启示

Severe acute respiratory syndrome (SARS) is the first new epidemic of the twenty - first century. A novel coronavirus (SARS - CoV) has been identified as the causative agent of SAP, S. The genome of SARS - CoV has 29,727 nucleatides in length. The genome organization, with 11 open reading flames, is similar to that of conronaviruses.Phylogenetic analyses and sequence comparisons showed that SARS- CoV is not closely related to any of the known coronaviruses, indicating neither a mutant nor recombinant of well -characterized coronaviruses. It is a complete new coronavirus from nonhuman hostPathological studies show that severe immune response, associated to cytokine dysregulation, may be related to the lung damage of fatal SRAS. Recombination of genomes of wild - type strains with vaccine coronavirus is a potential risk associated with the application of living attenuated coronavirus vaccines. The proteinases, controlling the activities of the SARS- CoV replication, and spike protein, involved in viral entry and pathogenesis, represent attractive targets of anti- SARS drug development. Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates suggests a remarkable genetic conservation of the virus. Anti - SARS vaccine and drug development will benefit from this genetic conservation. SARS-CoV is not likely to change rapidly and thus may not readily mutate to a benign infection. The progress in anti - SARS research has been impressive. However, one of the most effective tools in the control of the SARS is quickly tracing and isolating the contacts of stricken patients before they spread the virus further.     

Complete genome sequence of bat coronavirus HKU2 from Chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome

Apart from bat-SARS-CoV, we have identified a novel group 1 coronavirus, bat-CoV HKU2, in Rhinolophus sinicus (Chinese horseshoe bats). Since it has been suggested that the receptor-binding motif (RBM) of SARS-CoV may have been acquired from a group 1 coronavirus, we conducted a surveillance study and identified bat-SARS-CoV and bat-CoV HKU2 in 8.7% and 7.5% respectively of R. sinicus in Hong Kong and Guangdong. Complete genome sequencing of four strains of bat-CoV HKU2 revealed the smallest coronavirus genome (27164 nucleotides) and a unique spike protein evolutionarily distinct from the rest of the genome. This spike protein, sharing similar deletions with other group 2 coronaviruses in its C-terminus, also contained a 15-amino acid peptide homologous to a corresponding peptide within the RBM of spike protein of SARS-CoV, which was absent in other coronaviruses except bat-SARS-CoV. These suggest a common evolutionary origin in the spike protein of bat-CoV HKU2, bat-SARS-CoV, and SARS-CoV.     

Phylogenetic analysis of the full-length SARS-CoV sequences: evidence for phylogenetic discordance in three genomic regions

The origin of the severe acute respiratory syndrome-coronavirus (SARS-CoV) remains unclear. Evidence based on Bayesian scanning plots and phylogenetic analysis using maximum likelihood (ML) and Bayesian methods indicates that SARS-CoV, for the largest part of the genome ( approximately 80%), is more closely related to Group II coronaviruses sequences, whereas in three regions in the ORF1ab gene it shows no apparent similarity to any of the previously characterized groups of coronaviruses. There is discordant phylogenetic clustering of SARS-CoV and coronaviruses sequences, throughout the genome, compatible with either ancient recombination events or altered evolutionary rates in different lineages, or a combination of both.     

A review of studies on animal reservoirs of the SARS coronavirus

In this review, we summarize the researches on animal reservoirs of the SARS coronavirus (SARS-CoV). Masked palm civets were suspected as the origin of the SARS outbreak in 2003 and was confirmed as the direct origin of SARS cases with mild symptom in 2004. Sequence analysis of the SARS-CoV-like virus in masked palm civets indicated that they were highly homologous to human SARS-CoV with nt identity over 99.6%, indicating the virus has not been circulating in the population of masked palm civets for a very long time. Alignment of 10 complete viral genome sequences from masked palm civets with those of human SARS-CoVs revealed 26 conserved single-nucleotide variations (SNVs) in the viruses from masked palm civets. These conserved SNVs were gradually lost from the genomes of viruses isolated from the early phase to late phase human patients of the 2003 SARS epidemic. In 2005, horseshoe bats were identified as the natural reservoir of a group of coronaviruses that are distantly related to SARS-CoV. The genome sequences of bat SARS-like coronavirus had about 88-92% nt identity with that of the SARS-CoV. The prevalence of antibodies and viral RNA in different bat species and the characteristics of the bat SARS-like coronavirus were elucidated. Apart from masked palm civets and bats, 29 other animal species had been tested for the SARS-CoV, and the results are summarized in this paper.     

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.     

The phylogeny of SARS coronavirus

Summary. Different tree-building methods consistently place the SARS corona-virus (SARS-CoV) as a basal Group 2 coronavirus rather than as an ungrouped species as concluded by others. Detailed comparisons of the SARS-CoV genomic sequence with those of six other coronaviruses failed to find evidence of recombination or genomic rearrangement using computational methods designed for that purpose.     

Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus

The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein-protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168-208) in the N protein may be critical for such protein-protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.     

Overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses SARS-CoV,MERS-CoV,and 2019-nCoV

First reported from Wuhan, The People's Republic of China, on 31 December 2019, the ongoing outbreak of a novel coronavirus (2019-nCoV) causes great global concerns. Based on the advice of the International Health Regulations Emergency Committee and the fact that to date 24 other countries also reported cases, the WHO Director-General declared that the outbreak of 2019-nCoV constitutes a Public Health Emergency of International Concern on 30 January 2020. Together with the other two highly pathogenic coronaviruses, the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), 2019-nCov and other yet to be identified coronaviruses pose a global threat to public health. In this mini-review, we provide a brief introduction to the pathology and pathogenesis of SARS-CoV and MERS-CoV and extrapolate this knowledge to the newly identified 2019-nCoV.     

The neuroinvasive potential of SARS-CoV2 may play a role in the respiratory failure of COVID-19 patients

Following the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), another highly pathogenic coronavirus named SARS-CoV-2 (previously known as 2019-nCoV) emerged in December 2019 in Wuhan, China, and rapidly spreads around the world. This virus shares highly homological sequence with SARS-CoV, and causes acute, highly lethal pneumonia coronavirus disease 2019 (COVID-19) with clinical symptoms similar to those reported for SARS-CoV and MERS-CoV. The most characteristic symptom of patients with COVID-19 is respiratory distress, and most of the patients admitted to the intensive care could not breathe spontaneously. Additionally, some patients with COVID-19 also showed neurologic signs, such as headache, nausea, and vomiting. Increasing evidence shows that coronaviruses are not always confined to the respiratory tract and that they may also invade the central nervous system inducing neurological diseases. The infection of SARS-CoV has been reported in the brains from both patients and experimental animals, where the brainstem was heavily infected. Furthermore, some coronaviruses have been demonstrated able to spread via a synapse-connected route to the medullary cardiorespiratory center from the mechanoreceptors and chemoreceptors in the lung and lower respiratory airways. Considering the high similarity between SARS-CoV and SARS-CoV2, it remains to make clear whether the potential invasion of SARS-CoV2 is partially responsible for the acute respiratory failure of patients with COVID-19. Awareness of this may have a guiding significance for the prevention and treatment of the SARS-CoV-2-induced respiratory failure.     

Severe acute respiratory syndrome coronavirus spike protein counteracts BST2-mediated restriction of virus-like particle release

BST2/tetherin, an interferon-inducible antiviral factor, can block the cellular release of various enveloped viruses. We previously reported that human coronavirus 229E (HCoV-229E) infection can alleviate the BST2 tethering of HIV-1 virions by downregulating cell surface BST2, suggesting that coronaviruses are capable of encoding anti-BST2 factors. Here we report our new finding that severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) glycoprotein, similar to Vpu, is capable of antagonizing the BST2 tethering of SARS-CoV, HCoV-229E, and HIV-1 virus-like particles via BST2 downregulation. However, unlike Vpu (which downmodulates BST2 by means of proteasomal and lysosomal degradation pathways), BST2 downregulation is apparently mediated by SARS-CoV S through the lysosomal degradation pathway only. We found that SARS-CoV S colocalized with both BST2 and reduced cell surface BST2, suggesting an association between SARS-CoV S and BST2 that targets the lysosomal degradation pathway. According to one recent report, SARS-CoV ORF7a antagonizes BST2 by interfering with BST2 glycosylation¹. Our data provide support for the proposal that SARS-CoV and other enveloped viruses are capable of evolving supplementary anti-BST2 factors in a manner that requires virus replication. Further experiments are required to determine whether the BST2-mediated restriction of authentic SARS-CoV virions is alleviated by the SARS-CoV spike protein.     

Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases

Severe acute respiratory syndrome (SARS) is a new human infectious disease with significant morbidity and mortality. The disease has been shown to be associated with a new coronavirus (SARS-CoV). The clinical and epidemiological aspects of SARS have been described. Moreover, the viral genome of SARS-CoV has been fully sequenced. However, much of the biological behaviour of the virus is not known and data on the tissue and cellular tropism of SARS-CoV are limited. In this study, six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS-CoV using an in-situ hybridization (ISH) technique. Among all the tissues studied, positive signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel. Infected pneumocytes were further confirmed by immunofluorescence-fluorescence in-situ hybridization (FISH) analysis. These results provide important information concerning the tissue tropism of SARS-CoV, which is distinct from previously identified human coronaviruses, and suggest the possible involvement of novel receptors in this infection. Whereas the lung pathology was dominated by diffuse alveolar damage, the gut was relatively intact. These findings indicated that tissue responses to SARS-CoV infection are distinct in different organs.     

Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication

All coronaviruses encode a small hydrophobic envelope (E) protein, which mediates viral assembly and morphogenesis by an unknown mechanism. We have previously shown that the E protein from Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) forms cation-selective ion channels in planar lipid bilayers (Wilson, L., McKinlay, C., Gage, P., Ewart, G., 2004. SARS coronavirus E protein forms cation-selective ion channels. Virology 330(1), 322-331). We now report that three other E proteins also form cation-selective ion channels. These E proteins were from coronaviruses representative of taxonomic groups 1-3: human coronavirus 229E (HCoV-229E), mouse hepatitis virus (MHV), and infectious bronchitis virus (IBV), respectively. It appears, therefore, that coronavirus E proteins in general, belong to the virus ion channels family. Hexamethylene amiloride (HMA)--an inhibitor of the HIV-1 Vpu virus ion channel--inhibited the HCoV-229E and MHV E protein ion channel conductance in bilayers and also inhibited replication of the parent coronaviruses in cultured cells, as determined by plaque assay. Conversely, HMA had no antiviral effect on a recombinant MHV with the entire coding region of E protein deleted (MHVDeltaE). Taken together, the data provide evidence of a link between inhibition of E protein ion channel activity and the antiviral activity of HMA.     

Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.     

Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection

Before the emergence of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in 2003, only 12 other animal or human coronaviruses were known. The discovery of this virus was soon followed by the discovery of the civet and bat SARS-CoV and the human coronaviruses NL63 and HKU1. Surveillance of coronaviruses in many animal species has increased the number on the list of coronaviruses to at least 36. The explosive nature of the first SARS epidemic, the high mortality, its transient reemergence a year later, and economic disruptions led to a rush on research of the epidemiological, clinical, pathological, immunological, virological, and other basic scientific aspects of the virus and the disease. This research resulted in over 4,000 publications, only some of the most representative works of which could be reviewed in this article. The marked increase in the understanding of the virus and the disease within such a short time has allowed the development of diagnostic tests, animal models, antivirals, vaccines, and epidemiological and infection control measures, which could prove to be useful in randomized control trials if SARS should return. The findings that horseshoe bats are the natural reservoir for SARS-CoV-like virus and that civets are the amplification host highlight the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections.     

Phylogenetic and recombination analysis of coronavirus HKU1, a novel coronavirus from patients with pneumonia

Phylogenetic trees constructed using predicted amino acid sequences of putative proteins of coronavirus HKU1 (CoV-HKU1) revealed that CoV-HKU1 formed a distinct branch among group 2 coronaviruses. Of the 14 trees from p65 to nsp10, nine showed that CoV-HKU1 was clustered with murine hepatitis virus. From nsp11, the topologies of the trees changed dramatically. For the eight trees from nsp11 to N, seven showed that the CoV-HKU1 branch was the first branch. The codon usage patterns of CoV-HKU1 differed significantly from those in other group 2 coronaviruses. Split decomposition analysis revealed that recombination events had occurred between CoV-HKU1 and other coronaviruses.     

Recombinational histories of avian infectious bronchitis virus and turkey coronavirus

Phylogenetic analysis of complete genomes of the avian coronaviruses avian infectious bronchitis (AIBV) and turkey coronavirus (TCoV) supported the hypothesis that numerous recombination events have occurred between these viruses. Although the two groups of viruses differed markedly in the sequence of the spike protein, the gene (S) encoding this protein showed no evidence of positive selection or of an elevated mutation rate. Rather, the data suggested that recombination events have homogenized the portions of the genome other than the S gene between the two groups of viruses, while continuing to maintain the two distinct, anciently diverged versions of the S gene. The latter hypothesis was supported by a phylogeny of S proteins from representative coronaviruses, in which S proteins of AIBV and TCoV fell in the same clade.     

Emergence of a group 3 coronavirus through recombination

Analyses of turkey coronavirus (TCoV), an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were conducted to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Strains of TCoV that were pathogenic in poults and nonpathogenic in chickens did not adapt to cause disease in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses.     

Full genome analysis of a novel type II feline coronavirus NTU156

Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated with fatal feline infectious peritonitis (FIP). Despite nearly six decades having passed since its first emergence, different studies have shown that type II FCoV represents only a small portion of the total FCoV seropositivity in cats; hence, there is very limited knowledge of the evolution of type II FCoV. To elucidate the correlation between viral emergence and FIP, a local isolate (NTU156) that was derived from a FIP cat was analyzed along with other worldwide strains. Containing an in-frame deletion of 442 nucleotides in open reading frame 3c, the complete genome size of NTU156 (28,897 nucleotides) appears to be the smallest among the known type II feline coronaviruses. Bootscan analysis revealed that NTU156 evolved from two crossover events between type I FCoV and canine coronavirus, with recombination sites located in the RNA-dependent RNA polymerase and M genes. With an exchange of nearly one-third of the genome with other members of alphacoronaviruses, the new emerging virus could gain new antigenicity, posing a threat to cats that either have been infected with a type I virus before or never have been infected with FCoV.     

Composition of human-specific slow codons and slow di-codons in SARS-CoV and 2019-nCoV are lower than other coronaviruses suggesting a faster protein synthesis rate of SARS-CoV and 2019-nCoV

Translation of a genetic codon without a cognate tRNA gene is affected by both the cognate tRNA availability and the interaction with non-cognate isoacceptor tRNAs. Moreover, two consecutive slow codons (slow di-codons) lead to a much slower translation rate. Calculating the composition of host specific slow codons and slow di-codons in the viral protein coding sequences can predict the order of viral protein synthesis rates between different virus strains.Comparison of human-specific slow codon and slow di-codon compositions in the genomes of 590 coronaviruses infect humans revealed that the protein synthetic rates of 2019 novel coronavirus (2019-nCoV) and severe acute respiratory syndrome-related coronavirus (SARS-CoV) may be much faster than other coronaviruses infect humans. Analysis of host-specific slow codon and di-codon compositions provides links between viral genomic sequences and capability of virus replication in host cells that may be useful for surveillance of the transmission potential of novel viruses.