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Short Communication |
1 State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan, Hubei 430071, China
2 Graduate School of CAS, Beijing 100039, China
3 Institute of Zoology, CAS, Beijing 100080, China
4 CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia
5 Shanghai Center for Bioinformation Technology, Shanghai 200235, China
6 School of Life Sciences, Fudan University, Shanghai 200433, China
Correspondence
Zhengli Shi
zlshi{at}wh.iov.cn
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; Ksiazek et al., 2003; Marra et al., 2003; Peiris et al., 2003; Rota et al., 2003; Zhong et al., 2003). The rapid identification of highly similar viruses in masked palm civet and racoon dog in the live-animal markets provided strong evidence of an animal origin of SARS-CoV and played an important role in the prevention of further outbreaks (Guan et al., 2003). However, subsequent epidemiological studies on civets from market, farm and wild populations demonstrated that there was no widespread infection among wild or farmed civets, implying that wild animal(s) other than civets may serve as the natural reservoir(s) of SARS-CoV (Tu et al., 2004; Kan et al., 2005; Poon et al., 2005).
Recently, we and another independent group have simultaneously reported the detection of SARS-like coronavirus (SL-CoV) or SARS coronavirus-like virus in different horseshoe bat species in the genus Rhinolophus, providing evidence that suggests bats as a natural reservoir of this group of viruses (Lau et al., 2005; Li et al., 2005b). Due to the close genetic and antigenic relationship of SARS-CoVs and SL-CoVs, this group of viruses has been named the SARS cluster coronaviruses or group 2b coronavirus (G2b-CoV) in differentiation from other group 2 coronaviruses in the genus Coronavirus (Gorbalenya et al., 2004; Lau et al., 2005; Li et al., 2005b; Woo et al., 2006). Molecular and serological studies indicated that at least five different horseshoe bat species in mainland China and Hong Kong harbour G2b-CoVs. They include Rhinolophus sinicus, Rhinolophus pearsonii, Rhinolophus ferrumequinum, Rhinolophus macrotis and Rhinolophus pusillus. Full-length genome sequences were published for three isolates, one from R. pearsonii (Rp3) and two from R. sinicus (HKU3-1 and HKU3-2). The sequences of the HKU3-1 and HKU3-2 genomes were almost identical and they probably represented different isolates of the same genotype. The Rp3 and HKU3 isolates share an overall nucleotide sequence identity of 92 and 88 % to the outbreak SARS-CoVs isolated from civets and humans, respectively.
In this paper, we describe the characterization of full-length genome sequences for two additional G2b-CoV isolates, Rf1 from R. ferrumequinum and Rm1 from R. macrotis, and present genome-comparison data of all known G2b-CoV genome types to demonstrate further the great genetic diversity among this group of novel coronaviruses and to identify potential genetic features that might be associated with host specificity, transmission in non-bat species and virus virulence. It should be noted that there seems to be a large number of different coronaviruses present in different bat species. At least seven other novel bat coronaviruses have been discovered among bat populations in Hong Kong (Poon et al., 2005; Woo et al., 2006). As these coronaviruses are not related to the G2b-CoVs, the focus of this study, and there were no full-length genome sequences available for them, they are not included in the current comparative study.
The collection, processing and storage of bat samples, as well as the determination of the full-length genome sequence, were conducted as described previously (Li et al., 2005b). Sequence alignment was performed by using CLUSTAL_X version 1.83 (Thompson et al., 1997) and corrected manually. Phylogenetic trees based on nucleotide sequence were constructed by using the neighbour-joining (NJ) method with a bootstrap of 1000 replicates implemented in MEGA version 3.1 (Kumar et al., 2004). The mean non-synonymous substitution rate (Ka), synonymous substitution rate (Ks) and the ratio of Ka/Ks for four protein-coding sequences (ORF1a, ORF1b, ORF3a and S) were calculated by K-Estimator 6 (Comeron, 1999). The Kimura two-parameter substitution model was used and other parameters were as default settings in MEGA 3.1. Fisher's exact test of positive-selection analysis implemented in MEGA 3.1 and the CODEML program implemented in the PAML package (Yang & Swanson, 2002) were also used to detect potential positive selection for genes P1a, P1b, ORF3a and S of bat and human/civet G2b-CoV.
The full-length genomes of Rf1 and Rm1 are 29 690 and 29 733 nt [excluding the poly(A) tail], respectively. The genome organization and the predicted gene products of both viruses are similar to those of other characterized G2b-CoVs (Fig. 1; Table 1). However, Rf1 seems to have a unique feature that may represent an evolutionary intermediate between bat G2b-CoVs and human/civet G2b-CoVs. As shown in Fig. 1, there is an ORF3b of 154 aa (overlapping ORF3a) in the human/civet isolates that is absent from most bat G2b-CoVs. In the corresponding region in the Rf1 genome, there were two ORFs, of 113 and 32 aa. The four bat G2b-CoV genomes share a sequence identity of 88–90 % among themselves. Similar sequence identity, 88–92 %, exists between bat and human/civet isolates. Nucleotide variations are scattered along the whole genome, but the most variable regions were located in the genes encoding non-structural protein 3 (nsp3), S (the N-terminal S1 domain), ORF3a and ORF8. This is also true for deletion/insertion mutations in nsp3, S and ORF8. For nsp3 genes, the deletion/insertion mutations seem to be concentrated in the region encoding a unique domain originally identified by Snijder et al. (2003) that is present in SARS-CoV, but absent in other coronaviruses (Fig. 1). The sequence identity of the S genes among four bat G2b-CoVs is 89–95 %. The sequence identity drops to 76–78 % between S genes of bat G2b-CoVs and human/civet G2b-CoVs, and even lower (63–64 %) for the putative S1 domain. There are one 6 aa insertion and three deletions of various lengths in the S1 domains of bat isolates in comparison to those of the human/civet isolates (Lau et al., 2005; Li et al., 2005b). Two deletion sites (5 and 12 aa, respectively) are located in the receptor-binding domain (RBD) region, and overlap with the so-called receptor-binding motif (RBM; aa 424–494 of the Tor2 S protein), which is identified as being critical for receptor binding (Li et al., 2005a). Human G2b-CoV isolates are known to use angiotensin-converting enzyme-2 (ACE2) as the main receptor for cell entry (Li et al., 2003). It is not known whether the bat G2b-CoVs are able to use the bat ACE2 homologue as receptor or whether they use an alternative receptor molecule for cell entry, as speculated by Li et al. (2006).
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. Depending on the sequences used, several different phylogenetic patterns were observed. When the full-length genome sequences were used, bat isolate Rp3 grouped closer to the human/civet isolates than to other bat isolates, with a high bootstrap support (Fig. 2a). Similar observations were also made for trees based on P1a and P1b gene sequences (data not shown). When the full-length S genes were analysed, all bat G2b-CoVs clustered together and were separated from human/civet isolates (Fig. 2b). A third pattern was observed for trees based on ORF3a, M, ORF6 and ORF8 sequences (the representative tree of ORF3a is shown in Fig. 2c). In these trees, the Rf1 sequence does not group with other bat isolates; instead, it sits between the bat isolates and human/civet isolates, and for ORF8, the Rf1 sequence is related much more closely to human/civet isolates than to other bat isolates. Poorly resolved trees were observed for genes E, ORF7a, ORF7b and N among four different bat isolates (a representative tree of ORF7a is shown in Fig. 2d). These incongruent phylogenetic trees seem to suggest potential recombination events among these G2b-CoVs. However, when these sequences were analysed by using a recombination-detection program (RDP2; Martin et al., 2005), we were unable to obtain conclusive evidence for any definitive recombination event (data not shown). We aim to collect more G2b-CoVs and related coronaviruses of bat to continue the search for recombination points in the G2b-CoV genomes.
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), suggesting a recent species-crossing event.
Among the five complete bat isolates sequenced so far, HKU3-1 and HKU-2 were almost identical in genome sequence, which was not unexpected considering that they were isolated from the same species (R. sinicus) within a small geographical location in Hong Kong (Lau et al., 2005). For that reason, we considered them to be of the same genome type. We noted that the genome sequence of Rf1 displayed a more distant evolutionary relationship to other bat isolates. Whether these different G2b-CoV genotypes from different bat species are linked to their host evolution needs further investigation when more G2b-CoVs from different bat species become available. Based on the current data, it can be hypothesized that there is a wide spectrum of genetically diverse G2b-CoVs present in their natural reservoir hosts, and viruses with a much closer evolutionary relationship to the SARS outbreak strains from civets and human may be present in different Rhinolophus species or other bat species in China or neighbouring countries.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Comeron, J. M. (1999). K-Estimator: calculation of the number of nucleotide substitutions per site and the confidence intervals. Bioinformatics 15, 763–764.
Fouchier, R. A. M., Kuiken, T., Schutten, M. & 7 other authors (2003). Aetiology: Koch's postulates fulfilled for SARS virus. Nature 423, 240.[CrossRef][Medline]
Gorbalenya, A. E., Snijder, E. J. & Spaan, W. J. M. (2004). Severe acute respiratory syndrome coronavirus phylogeny: toward consensus. J Virol 78, 7863–7866.
Guan, Y., Zheng, B. J., He, Y. Q. & 15 other authors (2003). Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276–278.
Kan, B., Wang, M., Jing, H. & 27 other authors (2005). Molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms. J Virol 79, 11892–11900.
Ksiazek, T. G., Erdman, D., Goldsmith, C. S. & 23 other authors (2003). A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348, 1953–1966.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Lau, S. K. P., Woo, P. C. Y., Li, K. S. M. & 7 other authors (2005). Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 102, 14040–14045.
Li, W., Moore, M. J., Vasilieva, N. & 9 other authors (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454.[CrossRef][Medline]
Li, F., Li, W., Farzan, M. & Harrison, S. C. (2005a). Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864–1868.
Li, W., Shi, Z., Yu, M. & 14 other authors (2005b). Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676–679.
Li, W., Wong, S.-K., Li, F., Kuhn, J. H., Huang, I.-C., Choe, H. & Farzan, M. (2006). Animal origins of the severe acute respiratory syndrome coronavirus: insight from ACE2-S-protein interactions. J Virol 80, 4211–4219.
Marra, M. A., Jones, S. J. M., Astell, C. R. & 56 other authors (2003). The genome sequence of the SARS-associated coronavirus. Science 300, 1399–1404.
Martin, D. P., Williamson, C. & Posada, D. (2005). RDP2: recombination detection and analysis from sequence alignments. Bioinformatics 21, 260–262.
Peiris, J. S. M., Lai, S. T., Poon, L. L. M. & 13 other authors (2003). Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 1319–1325.[CrossRef][Medline]
Poon, L. L. M., Chu, D. K. W., Chan, K. H. & 9 other authors (2005). Identification of a novel coronavirus in bats. J Virol 79, 2001–2009.
Rota, P. A., Oberste, M. S., Monroe, S. S. & 32 other authors (2003). Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394–1399.
Snijder, E. J., Bredenbeek, P. J., Dobbe, J. C. & 7 other authors (2003). Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331, 991–1004.[CrossRef][Medline]
Song, H.-D., Tu, C.-C., Zhang, G.-W. & 56 other authors (2005). Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human. Proc Natl Acad Sci U S A 102, 2430–2435.
Spaan, W. J. M., Brian, D., Cavanagh, D. & 8 other authors (2005). Family Coronaviridae. In Virus Taxonomy: Eighth Report of the Committee on Taxonomy of Viruses, pp. 947–964. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & C. A. Ball. London: Elsevier Academic Press.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Tu, C., Crameri, G., Kong, X. & 11 other authors (2004). Antibodies to SARS coronavirus in civets. Emerg Infect Dis 10, 2244–2248.[Medline]
Woo, P. C. Y., Lau, S. K. P., Li, K. S. M. & 7 other authors (2006). Molecular diversity of coronaviruses in bats. Virology 351, 180–187.[CrossRef][Medline]
Yang, Z. & Swanson, W. J. (2002). Codon-substitution models to detect adaptive evolution that account for heterogeneous selective pressures among site classes. Mol Biol Evol 19, 49–57.
Zhong, N. S., Zheng, B. J., Li, Y. M. & 13 other authors (2003). Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003. Lancet 362, 1353–1358.[CrossRef][Medline]
Received 20 May 2006;
accepted 17 July 2006.
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