Zhengli Shi

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

The 2002–3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

Metagenomic Analysis of Viruses from Bat Fecal Samples Reveals Many Novel Viruses in Insectivorous Bats in China

ABSTRACT Increasing data indicate that bats harbor diverse viruses, some of which cause severe human diseases. In this study, sequence-independent amplification and high-throughput sequencing (Solexa) were applied to the metagenomic analysis of viruses in bat fecal samples collected from 6 locations in China. A total of 8,746,417 reads with a length of 306,124,595 bp were obtained. Among these reads, 13,541 (0.15%) had similarity to phage sequences and 9,170 (0.1%) had similarity to eukaryotic virus sequences. A total of 129 assembled contigs (>100 nucleotides) were constructed and compared with GenBank: 32 contigs were related to phages, and 97 were related to eukaryotic viruses. The most frequent reads and contigs related to eukaryotic viruses were homologous to densoviruses, dicistroviruses, coronaviruses, parvoviruses, and tobamoviruses, a range that includes viruses from invertebrates, vertebrates, and plants. Most of the contigs had low identities to known viral genomic or protein sequences, suggesting that a large number of novel and genetically diverse insect viruses as well as putative mammalian viruses are transmitted by bats in China. This study provides the first preliminary understanding of the virome of some bat populations in China, which may guide the discovery and isolation of novel viruses in the future.

Review of Bats and SARS

TOC Summary: The discovery of SARS-like coronaviruses in horseshoe bats highlights the possibility of future outbreaks caused by different coronaviruses of bat origin.

A review of studies on animal reservoirs of the SARS coronavirus.

Abstract 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.

Antibodies to Nipah or Nipah-like Viruses in Bats, China

To the Editor: Hendra virus (HeV) and Nipah virus (NiV), the only known members of the genus Henipavirus, are 2 emerging paramyxoviruses that are highly pathogenic in a variety of vertebrate animals, including humans (1). Since the initial discovery of the viruses in Australia and Malaysia (2,3), sporadic HeV outbreaks have been reported from 1995 to 2007 in Australia (4), and regular NiV outbreaks have occurred in Bangladesh (5) and India (6). Numerous frugivorous bat species (genus Pteropus), and some insectivous bat species have been found to be reservoir hosts of henipaviruses in Australia and Asian countries (7–9). In this study conducted during 2004–2007, bats were trapped within their natural habitat from 10 provinces in mainland People’s Republic of China. Serum, pharyngeal, and fecal swab samples were collected and stored as described previously (10). An ELISA was developed to detect antibodies to the NiV nucleocapsid (N) and attachment glycoprotein (G) proteins. For confirmation, ELISA-positive samples were tested by using Western blot against a recombinant NiV G fragment (aa 71–193) fused with the maltose-binding protein. Virus neutralization tests were conducted with live NiV and HeV under Biosaftey Level 4 containment in Australia. In addition, a surrogate neutralization test was developed by using recombinant env– HIV-1, pseudotyped with NiV G and F. RNA was extracted by using the QIA amp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription–PCR (RT-PCR) was performed by using primers against the NiV N gene as described previously (3). In total, 692 bat serum specimens were screened for antibody to NiV N or G protein (or both) by ELISA, and 33 were positive (Appendix Table). These specimens were from 9 of the 23 bat species examined in this study. Of the 33 serum samples reactive in ELISA, 25 with sufficient quantity left were further tested by Western blot, and 17 of 25 serum samples were reactive with MBP-NiV G fusion fragment, but not with the control MBP. None of the samples inhibited entry of NiV F/G-pseudotyped virus or neutralized either HeV or NiV. No NiV-specific RNA was detected by RT-PCR among 479 fecal swab samples and 67 throat swab samples tested; therefore, virus isolation was not attempted. This study systematically investigated NiV presence among bats in China. The detection of henipavirus antibody suggests that several bat species have been exposed to NiV or a closely related virus. The prevalence of antibody was especially prominent among Myotis species from Yunnan Province. Antibody was detected in samples from 3 of 4 Myotis species captured in the same location in 2006 and 2007. A relatively high prevalence of henipavirus antibody was also found among Rousettus leschenaultia samples from Hainan Province in 2007. Notably, Yunnan and Hainan are both located in southern China. Although pteropid bats are not found in China, these data suggest henipaviruses could be introduced into China by other susceptible bat species that overlap their habitat with pteropid bats in neighboring countries. Several possibilities may explain the failure to detect neutralizing antibodies. One might be the unique immune response among those nonpteropid bats, which results in a low level of neutralizing antibodies that are difficult to detect in the current assay systems. Alternatively, and perhaps more likely, >1 Nipah-like viruses could be circulating among the bat populations sampled in this study, producing antibodies that cross-react with, but do not neutralize, the prototype Malaysian NiV virus isolate. This phenomenon has been observed previously by our group for severe acute respiratory syndrome (SARS)–like viruses in horseshoe bats, whose sera cross-reacted with, but did not neutralize, the SARS virus in humans (10). Obtaining serologic evidence of viruses in bat populations is typically more successful as a screening tool than either nucleic acid based assays or virus isolation; this is likely attributable to the often low-level of virus replication, the transient nature of the infection in bats, or both. The inability to amplify NiV sequences may have been attributable to the viral RNA present among these samples being below the threshold of detection in our assay or to the absence of infection in the population at the time of sampling. In addition, the primers used in the PCR may target regions of the NiV N protein that exhibit substantial sequence divergence in a Nipah-like virus. Bat species in the genera Rousettus, Myotis, Miniopterus, and Hipposideros naturally reside in trees, buildings, and caves that can be in close proximity to human residential areas, which increases the potential of transmission of zoonotic pathogens from bats to humans. The increased risk for these zoonotic infections to spread from bats to humans in areas of cohabitation is best illustrated by the repeated spillover events involving NiV in Bangladesh (5). Given the present initial evidence of exposure among bats in mainland China shown here, there is an urgent need to continue and expand surveillance studies for henipaviruses in China and elsewhere on the Asian continent.

Host range, prevalence, and genetic diversity of adenoviruses in bats.

ABSTRACT Bats are the second largest group of mammals on earth and act as reservoirs of many emerging viruses. In this study, a novel bat adenovirus (AdV) (BtAdV-TJM) was isolated from bat fecal samples by using a bat primary kidney cell line. Infection studies indicated that most animal and human cell lines are susceptible to BtAdV-TJM, suggesting a possible wide host range. Genome analysis revealed 30 putative genes encoding proteins homologous to their counterparts in most known AdVs. Phylogenetic analysis placed BtAdV-TJM within the genus Mastadenovirus, most closely related to tree shrew and canine AdVs. PCR analysis of 350 bat fecal samples, collected from 19 species in five Chinese provinces during 2007 and 2008, indicated that 28 (or 8%) samples were positive for AdVs. The samples were from five bat species, Hipposideros armiger, Myotis horsfieldii, M. ricketti, Myotis spp., and Scotophilus kuhlii. The prevalence ranged from 6.25% (H. armiger in 2007) to 40% (M. ricketti in 2007). Comparison studies based on available partial sequences of the pol gene demonstrated a great genetic diversity among bat AdVs infecting different bat species as well as those infecting the same bat species. This is the first report of a genetically diverse group of DNA viruses in bats. Our results support the notion, derived from previous studies based on RNA viruses (especially coronaviruses and astroviruses), that bats seem to have the unusual ability to harbor a large number of genetically diverse viruses within a geographic location and/or within a taxonomic group.

Genetic diversity of novel circular ssDNA viruses in bats in China

Novel circular ssDNA genomes have recently been detected in animals and in the environment using metagenomic and high-throughput sequencing approaches. In this study, five full-length circular ssDNA genomes were recovered from bat faecal samples using inverse PCR with sequences designed based on circovirus-related sequences obtained from Solexa sequencing data derived from a random amplification method. These five sequences shared a similar genomic organization to circovirus or the recently proposed cyclovirus of the family Circoviridae. The newly obtained circovirus/cyclovirus-like genomes ranged from 1741 to 2177 bp, and each consisted of two major ORFs, ORF1 and ORF2, encoding putative replicase (Rep) and capsid (Cap) proteins, respectively. The potential stem-loop region was predicted in all five genomes, and three of them had the typical conserved nonanucleotide motif of cycloviruses. A set of primers targeting the conserved Rep region was designed and used to detect the prevalence of circovirus/cyclovirus sequences in individual bats. Among 199 samples tested, 47 were positive (23.6%) for the circovirus genome and two (1.0%) were positive for the cyclovirus genome. In total, 48 partial Rep sequences plus the five full-length genomes were obtained in this study. Detailed analysis indicated that these sequences are distantly related to known circovirus/cyclovirus genomes and may represent 22 novel species that belong to the family Circoviridae.

Evidence of the Recombinant Origin of a Bat Severe Acute Respiratory Syndrome (SARS)-Like Coronavirus and Its Implications on the Direct Ancestor of SARS Coronavirus

ABSTRACT Bats have been identified as the natural reservoir of severe acute respiratory syndrome (SARS)-like and SARS coronaviruses (SLCoV and SCoV). However, previous studies suggested that none of the currently sampled bat SLCoVs is the descendant of the direct ancestor of SCoV, based on their relatively distant phylogenetic relationship. In this study, evidence of the recombinant origin of the genome of a bat SLCoV is demonstrated. We identified a potential recombination breakpoint immediately after the consensus intergenic sequence between open reading frame 1 and the S coding region, suggesting the replication intermediates may participate in the recombination event, as previously speculated for other CoVs. Phylogenetic analysis of its parental regions suggests the presence of an uncharacterized SLCoV lineage that is phylogenetically closer to SCoVs than any of the currently sampled bat SLCoVs. Using various Bayesian molecular-clock models, interspecies transfer of this SLCoV lineage from bats to the amplifying host (e.g., civets) was estimated to have happened a median of 4.08 years before the SARS outbreak. Based on this relatively short window period, we speculate that this uncharacterized SLCoV lineage may contain the direct ancestor of SCoV. This study sheds light on the possible host bat species of the direct ancestor of SCoV, providing valuable information on the scope and focus of surveillance for the origin of SCoV.

Evolutionary Relationships between Bat Coronaviruses and Their Hosts

Host shifting has occurred among Rhinolophus spp., with potential implications for emergence of SARS.

Difference in Receptor Usage between Severe Acute Respiratory Syndrome (SARS) Coronavirus and SARS-Like Coronavirus of Bat Origin

ABSTRACT Severe acute respiratory syndrome (SARS) is caused by the SARS-associated coronavirus (SARS-CoV), which uses angiotensin-converting enzyme 2 (ACE2) as its receptor for cell entry. A group of SARS-like CoVs (SL-CoVs) has been identified in horseshoe bats. SL-CoVs and SARS-CoVs share identical genome organizations and high sequence identities, with the main exception of the N terminus of the spike protein (S), known to be responsible for receptor binding in CoVs. In this study, we investigated the receptor usage of the SL-CoV S by combining a human immunodeficiency virus-based pseudovirus system with cell lines expressing the ACE2 molecules of human, civet, or horseshoe bat. In addition to full-length S of SL-CoV and SARS-CoV, a series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone. Several important observations were made from this study. First, the SL-CoV S was unable to use any of the three ACE2 molecules as its receptor. Second, the SARS-CoV S failed to enter cells expressing the bat ACE2. Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to enter cells via human ACE2, albeit with different efficiencies for different constructs. Fourth, a minimal insert region (amino acids 310 to 518) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to human ACE2 binding, indicating that the SL-CoV S is largely compatible with SARS-CoV S protein both in structure and in function. The significance of these findings in relation to virus origin, virus recombination, and host switching is discussed.