key: cord-0807882-apc0lm5e
authors: Shi, Z.; Wang, L.-F.
title: Evolution of SARS Coronavirus and the Relevance of Modern Molecular Epidemiology
date: 2017-01-20
journal: Genetics and Evolution of Infectious Diseases
DOI: 10.1016/b978-0-12-799942-5.00026-3
sha: d078065e748bdfbd159a9acb9fcddcbab3b5f084
doc_id: 807882
cord_uid: apc0lm5e

The severe acute respiratory syndrome (SARS) is the first major zoonotic infectious disease of the 21st Century. The disease, originally termed “atypical pneumonia,” was first recognized in November 2002 in Guangdong Province, China, which spread rapidly to 26 countries within 5 months and eventually led to 8096 human infection cases and 774 deaths. A previously unrecognized coronavirus, named SARS coronavirus (SARS-CoV), was later identified as the causative agent of SARS. Subsequent investigation indicated that the source of the human infections originated most likely from infected wildlife animals traded in the live animal markets, with masked palm civets considered the most prominent and important carrier. Large-scale culling of civets appeared to be effective in preventing further outbreaks in the region. Subsequent epidemiological studies showed that civets are intermediate host of SARS-CoV and bats are the likely natural reservoir of SARS-CoV and a large number of SARS-like coronaviruses (SL-CoVs). Detailed analysis of the massive SARS-CoV genomic sequence data indicated that SARS-CoV experienced a strong selection pressure during different outbreak phases in humans as well as interspecies transmission from animals to humans. The spike glycoprotein (S) of SARS-CoV plays a key role in virus–host interaction and hence is a key determinant of interspecies transmission. It has been shown that minor changes of amino acid (aa) residues in the S protein could lead to dramatic changes in virus susceptibility in animal and human hosts. This chapter focuses on the genetics and evolution of SARS-CoVs and SL-CoVs in humans, civets, and bats. The events of SARS outbreaks and the accompanying response activities highlight the importance of modern molecular epidemiology in disease investigation and the urgent need to broaden the screening and investigation of unknown viruses in wildlife animals.

outbreaks were effectively under control by July 5, 2003 . This was the first powerful demonstration of the kind of devastation a new infectious disease can cause worldwide and the effectiveness of an international organization when it is running at its peak.

Following the major SARS outbreaks of 2003e2004, there were several minor outbreaks with much smaller impacts. Between December 2003 and January 2004, four independent SARS cases were reported in Guangdong, and none of them led to fetal infection or widespread transmission. Subsequent epidemiological tracing revealed that all cases could be linked to civet trading activities. 3 In addition, there were three laboratory outbreaks in September 2003, December 2003, and April 2004 in Singapore, Taiwan, and Beijing, respectively. The most severe outbreak was associated with the incident in Beijing that resulted in a total of nine infection cases with one death. None of the other two laboratory infections resulted in further spread of the virus. 4 

Rapid identification of causative agent in an outbreak caused by unknown pathogen is the key for an effective response. However, in the case of SARS outbreak, this was not the case. Due to the association of nonspecific clinical symptoms associated with SARS patients, several pathogens were initially "identified" as the potential causes of SARS, which included Chlamydia, influenza virus, and paramyxovirus. 5 The confusion continued until March, 2003 when three laboratories independently confirmed that a previously unknown coronavirus was the most likely etiological agent of SARS. 6e8 Coronaviruses are enveloped viruses with the largest single-stranded, positivesense RNA genomes currently known, ranging in size from 27 to nearly 32 kb in length. Coronaviruses can infect and cause disease in a broad array of avian and mammal species, including humans. The name "coronavirus" is derived from the Greek word, meaning crown, as the virus envelope appears under electron microscopy to be crowned by a characteristic ring of small bulbous structures. Within the virion, the ssRNA genome is encased in a helical nucleocapsid composed of many copies of the nucleocapsid (N) protein. The lipid bilayer envelope contains three proteins: envelope (E) and membrane (M) protein, which coordinate virion assembly and release, and the large spike (S) protein, which confers the virus's characteristic corona shape as well as serves as the principal mediator of host cell attachment and entry via virusand host-specific cell receptors. The size of the SARS-CoV viral particle is approximately 80e90 nm and its genomic size is around 29.7 kb. 9,10 The SARS-CoV genome contains 14 open reading frames (ORFs) flanked by 5 0 -and 3 0 -untranslated regions of 265 and 342 nucleotides, respectively. While all CoVs carry strain-specific accessory proteins encoded by their downstream ORFs, the order of essential genesdthe replicase/transcriptase gene, S gene, E gene, M gene, and N genedis highly conserved. 11 Similar to other known coronaviruses, the SARS-CoV genome expression starts with two long open reading frames (ORFs), ORF1a and ORF1b, which account for twothirds of the genomic capacity, followed by ORFs encoding S, E, M, and N proteins ( Fig. 26.1 ). In addition to these conserved core genes in coronaviruses, the SARS-CoV genome contains several accessory genes that are specific to SARS-CoV and their encoded products have no homologue to known proteins. Phylogenetic analysis based on the most conserved gene ORF1b indicated that SARS-CoV is distantly related to the group 2 coronaviruses (now the genus Betacoronavirus) in the family The phylogenetic tree is generated based on full-length genome sequences of selected SARS-CoVs and bat SL-CoVs using the Neighbor-Joining algorithm in the MEGA4 program 78 with a bootstrap of 1000 replicates. A bat coronavirus BtCoV HKU9 is used as an outgroup. 79 outbreaks, and it was therefore impossible to trace the origin of the virus. However, through retrospective investigation, it emerged that the majority of the early index cases were limited in several cities of the Guangdong province and most of them have history of contact directly or indirectly with wildlife animals, including handling, killing, and selling wildlife animals as well as preparing and serving wildlife animal meat in restaurants. 14e16 As these epidemic regions have a unique dietary tradition favoring freshly slaughtered game meat, there is a huge trafficking and trading industry dedicated to live animal trading in specialized market, the "wet market." Immediately after SARS-CoV was identified as the etiological agent of SARS, studies were conducted in those markets for evidence of SARS-CoV in market animals. One of the earliest and most important studies was conducted by a joint team from Hong Kong and Shenzhen in mainland China. 14 In this investigation, out of 25 samples collected from market animals, SARS-CoV-like viruses were isolated from four out of six masked palm civets (Paguma larvata) and one raccoon dog (Nyctereutes procyonoides). Antibodies against SARS-CoV were detected in masked palm civets, raccoon dog, and Chinese ferretbadgers (Melogale moschata). Genome sequencing indicated that the viruses isolated from civets were almost identical to those from human, suggesting a highly possible zoonotic transmission of SARS-CoV from animal(s) to human. 14 These data indicated that at least three different animal species were infected by a coronavirus that is closely related to SARS-CoV. This important study provided the first direct evidence that SARS-CoV existed in animals, pointing to an animal link of the SARS outbreaks.

Although three animals were identified as susceptible to SARS-CoV infection, the larger sale volume of civets in comparison to other animals in the market made them the target animals of subsequent surveillance studies. The role of civets as a major carrier of SARS-CoV in the markets was further confirmed by serological studies involving much large samples. 17, 18 The most detailed epidemiological data proving a direct civet to human transmission of SARS-CoV was obtained during the investigation of the second wave of SARS outbreaks during December 2003 to January 2004. There were two lines of evidences suggesting a direct transmission. First, all four independent cases had the history of direct or indirect contact with civets. Second, sequencing analysis indicated that sequences derived from human samples were more closely related to those in the civets during that period than those from human samples obtained in the major 2002e2003 outbreaks. 3 In summary, based on the previously mentioned study findings, it was concluded that the civet to human transmission is a major, if not the only, source of SARS-CoV introduction into the human population. 19e21

Natural reservoir refers to the long-term host of the pathogen of an infectious disease. It is often the case that hosts do not get the disease carried by the pathogen, or the infection in the reservoir host is subclinical, asymptomatic, and nonlethal. Once discovered, natural reservoirs elucidate the complete life cycle of infectious diseases, which in turn will help to provide effective prevention and control strategies.

As stated earlier, it is clear that civets played a pivotal role in the 2002e2004 outbreaks of SARS in southern China. Culling of civets seemed to be effective in controlling further outbreaks in the region. However, the role of civets as a potential natural reservoir host was less evident and eventually ruled out by several studies. Serological and molecular studies indicated that only civets in the markets were infected with SARS-CoV whereas the populations of civets in the wild or on farms were free of major infections. 18, 22, 23 Civets produced overt clinical syndromes when experimentally infected with SARS-CoV. 24 Comparative genome sequence analysis indicated that SARS-CoVs in civets experienced rapid mutation, suggesting that the viruses were still adapting to the host rather than persisting in equilibrium expected for viruses in their natural reservoir species. 17, 25 Continuing search for the potential reservoir host of SARS-CoV resulted in the simultaneous discovery of SARS-like coronaviruses (SL-CoVs) in bats by two independent teams in 2005. Using serological and PCR surveillance, both groups discovered that SL-CoVs were present in different horseshoe bats in the genus Rhinolophus. 22, 26 Complete genome sequence analysis revealed that bat SL-CoVs have an identical genome organization and a nucleotide sequence identity of 88e92% to SARS-CoV ( Fig. 26.1 ; Table 26 .2). Except for the S, ORF3, and ORF8 gene products, all deduced aa sequences of the other gene products have a sequence identity above 93% with those of SARS-CoV. The variable regions between SARS-CoV and bat SL-CoV are mainly located in the coding regions for the nonstructural protein 3 (Nsp 3), S protein, ORF3, and ORF8, the products of these genes have aa sequence identity of 87e95%, 76e78%, 82e90%, and 34e80%, respectively. Among the different bat SL-CoVs, the coding regions for these proteins also represent the most variable regions. 27e29 The phylogenetic analysis indicated that bat SL-CoVs were grouped in the same cluster of SARS-CoV and were only distantly related to other previously known coronaviruses ( Fig. 26.2 ). To date, these bat SL-CoVs represent naturally occurring CoVs that are most closely related to the SARS-CoVs isolated from humans and civets.

Analysis of nonsynonymous and synonymous substitution rates in bat SL-CoVs suggests that these viruses are not experiencing a positive selection pressure that would be expected if horseshoe bats are new host to these viruses. Instead, these data would argue that these viruses have been associated with the bat hosts for a long time. 27, 29, 30 These observations would support the notion that bats in the genus Rhinolophus are the likely natural reservoir hosts of bat SL-CoVs. It can be further postulated that similar bat species may serve as natural reservoirs of viruses with closer evolutionary relationship to the viruses that were responsible for the 2002e2004 SARS outbreaks.

In this context, we and other groups continued the search for the direct progenitor of SARS-CoV and made great progress in the last 10 years following the initial discovery of SL-CoVs in horseshoe bats. First, highly diverse SL-CoVs have been found not only in Chinese but also in European and African bats, indicating a much wider geographic distribution and long evolutionary history of SL-CoVs in different bat populations The region of nsp3 is highly variable and was calculated alone.

Evolution of SARS Coronavirus (Table 26. 2). 31e37 Second, great genetic diversity of SARS-CoVs were discovered in one particular population of R. sinicus in China by a longitudinal surveillance. 31, 38 Third and most importantly, two SL-CoV strains were isolated in Vero cells. These two isolates are closely related to the progenitor of the SARS-CoV not only in genomic sequences but also in receptor usage 31 

Analysis of the large number of SARS-CoV and SL-CoV sequence datasets accumulated since 2004 has clearly demonstrated the importance of virus evolution in cross-species transmission and in pathogenesis. The following is a summary of the major evolutionary findings in host switching, recombination, and virusereceptor interactions.

On the basis of the epidemiological data, the Chinese SARS molecular epidemiology consortium divided the course of the 2002e2004 outbreaks into three stages, the early, middle, and late phases, respectively. 1 The early phase is defined as the period from the first emergence of SARS to the first documented SSE. The middle phase refers to the ensuing events up to the first cluster of SARS cases in a hotel (Hotel M) in Hong Kong, while cases following this cluster fall into the late phase. Analysis of all the viral sequences available from human patients and animals revealed two major hallmarks of rapid virus evolution during the initial stages of the 2002e2003 outbreaks: (1) All isolates from early patients and market animals contained a 29-nucleotide (nt) sequence in ORF8 that is absent in most of the publicly available human SARS-CoV sequences derived from later phases of the outbreaks;

(2) characteristic motif of single-nucleotide variations (SNVs) were identified in SARS-CoVs of different phases and all these SNVs were located in the S gene that codes for the spike protein responsible for attachment to the host cellular receptor. 25 All SARS-CoV isolates from epidemic countries and regions outside mainland China could be traced to Guangdong or Hong Kong based on the S-gene SNV motif. 23, 39 During the second sporadic outbreaks of 2003e2004, it was shown that the SARS-CoV sequences from index patients were almost identical to that from civets collected in the same period and all retained the 29-nt sequence in the ORF8 gene. The mild disease symptoms associated with these viruses and the lack of rapid human-to-human transmission provided further evidence that the rapid adaptation of the SARS-CoV in the first major outbreak of 2002e2003 was essential for its establishment and pathogenesis in humans.

With the available genomic variation data and the sampling time, it is now possible to calculate the neutral mutation rate and to estimate the date for the most recent common ancestors (MRCAs) of SARS-CoV. The estimate obtained is around 8.00 Â 10 À6 nt À1 day À1 , suggesting that SARS-CoV evolves at a relatively constant 

At the present time, at least 33 full-length genome sequences of bat SL-CoVs were determined. 22,26e29,32,34e37,40 Shown in Fig. 26.1 is a comparison of the genome structures for seven selected bat SL-CoVs and one each of civet and human SARS-CoV isolates. All bat SL-CoVs, with the exception of HKU3-8 29 and BM48-31, 32 contain the 29-nt sequence in ORF8, which is present in SARS-CoV from early-phase patients and civets, indicating the common ancestor between civet SARS-CoV and bat SL-CoV. The SL-CoV HKU3-8 contained a 26-nt deletion that is located 14 nt downstream from the commonly observed 29-nt deletion, and the BM48-31 completely lost the ORF8, indicating that the ORF8 coding region is a "hotspot" for deletions.

SL-CoVs from different bat species share 88e97% nt identity among themselves, indicating that the genetic diversity of SL-CoVs in bats is much greater than that observed among civet or human isolates. The most dramatic sequence difference between human SARS-CoV and bat SL-CoV is in the S protein that has 76e97% aa identity for the whole S protein and 64e95% aa identity for the N-terminal region (or the S1 region; Table 26 .2). This great genetic diversity observed among bat SL-CoVs and the major difference between the S1 regions of SL-CoV and SARS-CoV S proteins clearly demonstrated that bats are natural reservoirs of human SARS-CoV.

It is well documented that the positive-sense ssRNA genomes of coronaviruses are prone to homologous recombination during coinfection of different coronaviruses and that recombination plays an important role in generating new coronavirus species, in facilitating cross-species transmission and in modulating virus virulence.

Several studies provided evidence for coinfection and recombination came from analysis of SL-CoVs in bats. 29,41e44 It was further revealed that recombination can occur at multiple sites along the SL-CoV genome. 11, 28, 29, 31, 34, 41 For example, detailed sequence analysis of two genotypes of bat SL-CoV, Rp3 and Rs672 (both were identified from R. sinicus), suggested that they may represent a recombinant of two bat SL-CoVs and one of them is more closely related to the human SARS-CoVs. 28, 41 During 2015 and 2016, two teams reported a full-length ORF8 that shares higher sequence similarities to the SARS-CoV GZ02 and civet SARS-CoV SZ3 than previously detected SL-CoVs. 37, 40 These results suggest that SARS-CoV most likely originated from different bat SL-CoVs via a complicated evolutionary path that involved recombination events.

The S protein of coronavirus is responsible for attachment to cellular receptor to initiate the first step of virus infection. The angiotensin-converting enzyme 2 (ACE2) was identified as a main functional receptor for SARS-CoV. 45 Further analysis demonstrated that the region covering aa 318e520 of S protein is the key receptor-binding domain (RBD), which is both essential and sufficient to bind the human ACE2 molecule in vitro. 46 Detailed analysis of the crystal structure of the RBDeACE2 complex revealed that 19 key residues have close contact with the receptor molecule, which are located from aa 424 to 474. This region is termed the receptor-binding motif (RBM). 47 When the existing epidemiological data was analyzed in combination with the data on infectivity of SARS-CoV isolated in humans at the different phases of the outbreaks and SARS-CoV isolates in civets, a clear correlation could be established between the evolution of the S proteins and virus infectivity. It was observed that the S protein is the fastest evolving protein of SARS-CoV during interspecies transmission from animal to human and in the following phases of human to human transmission. The majority of the mutations are located in the S1 domain (31 of a total of 48 SNVs), particularly in the RBD. 1, 46 The interaction analysis between the S proteins of different isolates and the ACE2 molecules demonstrated that two aa residues in the S protein, aa 479 and aa 487, played a key role in virus infectivity. 48 (Fig. 26.3) . When examined using an HIV-based pseudovirus infection assay, S proteins with all combinations of residues 487/479 could efficiently use the civet ACE2 as an entry receptor, but showed different infectivity in human ACE2mediated infection. 48, 49 The combination of N479/T487 had the highest infectivity, N479/S487 medium infectivity, and K479/S487 the lowest, which almost abolished the infection. These results demonstrated elegantly at the molecular interface that the rapid evolution of the S protein, especially at the aa positions important for host receptor engagement, was essential for the adaptation to and establishment of an effective and productive human infection.

When the genome sequences of SL-CoVs were analyzed, it became evident that the N-terminal regions of their S proteins are the most divergent among themselves, as well as with the SARS-CoV. As shown in Fig. 26.3 , bat SL-CoVs can be grouped into three groups based on the RBM sequences. The strains discovered early are close to each other and have a major sequence difference involving deletions of 17e18 aa right in the middle of RBM. We have since demonstrated experimentally that SL-CoV S proteins are unable to use ACE2 molecule, regardless of its origin, as a functional receptor. The second group, identified from European bats, has deletions of 4 aa. 32 The third group, discovered recently, has no deletion and contains an identical size as the SARS-CoV in the S protein (Fig. 26.3) . 31, 34, 35 As predicted from their S sequences, three isolates from the third group, SL-CoVdWIV1, WIV16, and SHC014, have been shown to be able to use ACE2 for cellular entry, even though these S proteins still have slight difference at the key aa involved in direct interaction with ACE2. 31, 34, 50 Most importantly, the SHC014 can replicate well in transgenic mice containing human ACE2, and it caused tissue damage in tested animals. 50 

Zoonosis contributes to the majority of emerging disease in the last 30 years, many of them originated from wildlife animals. 51e55 The story of SARS is just one of such examples that spectacularly demonstrated the seamless evolution of a bat virus into a human pathogen responsible for one of the most severe global pandemic outbreaks in modern history of mankind. In general, pathogens carried by wildlife reservoir animals usually do not cause clinical symptoms and they lie dormant until they spill over into and cause diseases in domestic animals or humans. Classical outbreak response measures, such as those deployed during the SARS outbreaks, are still useful, but no longer sufficient for early detection and prevention of major infectious disease outbreaks in the 21st century.

With the demonstration of an increasing number of spillover events that led to severe disease outbreaks in human and domestic animals, we believe it is paramount that from now on we include active surveillance of wildlife animals as part of an integrated infectious disease prevention and control strategy. Surveillance of wildlife animals has also been made more feasible and productive, thanks to the advance in modern molecular techniques including PCR with virus group-specific primers, virus discovery using next generation high-throughput sequencing technologies, and high density virus microarrays. 56e63 Since the SARS outbreaks, especially after the discovery of SL-CoVs in bats, there is a significant surge in international effort for surveillance of coronaviruses in wildlife animals. Before the SARS outbreak, there were only 10 coronaviruses with complete genomes sequenced. This number has increased more than sixfold as a result of the active surveillance works conducted around the world. 27e29, 31,32,34,40,55,64e73 Although this only marks the beginning of our understanding of coronaviruses in wildlife animals, it is fair to say that we have learnt a lot more about coronaviruses in general than the past 50 years or so; during that period studying of viruses was only possible and called for in response to disease outbreaks. Based on phylogenetic analysis of the large number of bat coronavirus sequences available presently, it is postulated that all known disease-causing coronaviruses previously identified in humans or animals originated from bat strains. 31, 34, 43, 55 This hypothesis was unfortunately proved by the outbreak of another SARS-like disease, Middle East Respiratory Syndrome (MERS), which was caused by a novel coronavirus (previously named HCoV-EMC, now MERS-CoV) and supposed to originate from bats. 74 Even though the MERS-CoV-like viruses found in bats are not the direct progenitor of the MERS-CoV, the highly genetic diversity of these bat viruses is likely the gene sources for the deadly pathogen in humans, just like that for SARS-CoV. 71,72,75e77 

The emergence of SARS-CoV has had a huge impact on the global health and economy. It served as a warning to what may come out of a seemingly harmless virusereservoir equilibrium in bats or any other wildlife species. At the same time, the experience gained from the SARS outbreaks and the following in-depth studies on SARS-like coronaviruses has provided and will continue to provide invaluable knowledge and guideline to our future fight against new and emerging infectious diseases. One of the major lessons is that we need to pay much more attention to the reservoir species in understanding the genetic diversity of different viruses, the intricate interplay at the virusehost interface, and the major factors responsible for the disturbance of viruse host equilibrium, which in turn trigger spillover events leading to disease outbreaks.

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