MERS–Related Betacoronavirus in Vespertilio superans Bats, China

To the Editor: Middle East respiratory syndrome coronavirus (MERS-CoV), a novel lineage C betacoronavirus, was first described in September 2012, and by April 16, 2014, the virus had caused 238 infections and 92 deaths in humans worldwide (1). Antibodies against MERS-CoV in dromedary camels were recently reported (2), as was the full genome of MERS-CoV from dromedary camels (3). Finding the natural reservoir of MERS-CoV is fundamental to our ability to control transmission of this virus to humans (4). 
 
We report a novel lineage C betacoronavirus identified from Vespertilio superans bats in China. The full-length genome of this betacoronavirus showed close genetic relationship with MERS-CoV. Together with other evidence of MERS-CoV–related viruses in bats (5–8), our findings suggest that bats might be the natural reservoirs of MERS-related CoVs. 
 
In June 2013, we collected anal swab samples from 32 V. superans bats from southwestern China. A small proportion of each sample was pooled (without barcoding) and processed by using virus particle–protected nucleic acid purification and sequence-independent PCR for next-generation sequencing analysis with the Illumina (Solexa) Genome Analyzer II (Illumina, San Diego, CA, USA). Redundant reads were filtered, as described (9), from the raw sequencing reads generated by the genome analyzer and then aligned with the nonredundant protein database of the National Center for Biotechnology Information (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) by using BLAST (http://blast.ncbi.nlm.nih.gov). The taxonomy of these aligned reads was parsed by using MEGAN 4 (http://ab.inf.uni-tuebingen.de/software/megan/). 
 
On the basis of the BLAST results, 8,751,354 sequence reads 81 nt in length were aligned with the protein sequences of the nonredundant protein database: 72,084 of the reads were uniquely matched with virus proteins. Of these 72,084 reads, 32,365 were assigned to the family Coronaviridae, primarily to lineage C of the genus Betacoronavirus, and found to share 60%–97% aa identity with MERS-CoV. 
 
The MERS-CoV–related reads were extracted and assembled by using SeqMan software from the Lasergene 7.1.0 program (DNASTAR, Madison, WI, USA), resulting in a draft CoV genome. Reverse transcription PCR selective for the partial RNA-dependent RNA polymerase (RdRp) gene of this novel lineage C betacoronavirus suggested that 5 of the 32 samples (≈16%) were positive for the novel betacoronavirus, and the PCR amplicons shared >98% nt identity with each other. Using a set of overlapped nested PCRs and the rapid amplification of cDNA ends method, we determined the full-length genome of 1 strain of this V. superans bat–derived betacoronavirus (referred to as BtVs-BetaCoV/SC2013, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KJ473821","term_id":"627792518","term_text":"KJ473821"}}KJ473821). 
 
The betacoronavirus strain had a genome length of 30,413 nt, excluding the 3′ poly (A) tails, and a G+C content of 43.1%. Pairwise genome sequence alignment, conducted by the EMBOSS Needle software (http://www.ebi.ac.uk/Tools/psa/emboss_needle/) with default parameters, suggested that the genome sequence of BtVs-BetaCoV/SC2013 showed 75.7% nt identity with that of human MERS-CoV (hCoV-MERS); this shared identity is higher than that for other lineage C betacoronaviruses (from bats and hedgehogs) with full genomes available. hCoV-MERS showed 69.9% nt identity with bat CoV (BtCoV) HKU4-1, 70.1% nt identity with BtCoV-HKU5-1, and 69.6% nt identity with hedgehog CoV EriCoV-2012–174. 
 
Compared with those lineage C betacoronaviruses, which had an 816-bp partial RdRp sequence fragment available, BtVs-BetaCoV/SC2013 shared 96.7 % aa identity with hCoV-MERS. Pipistrellus BtCoVs found in Europe (BtCoV-8-724, BtCoV-8-691, BtCoV-UKR-G17) shared 98.2 % aa identity with hCoV-MERS, and Eptesicus BtCoV found in Italy (BtCoV-ITA26/384/2012) and other lineage C betacoronaviruses shared 96.3 % aa and <95% aa identity, respectively, with hCoV-MERS. 
 
To clarify the evolutionary relationship between BtVs-BetaCoV/SC2013 and other lineage C betacoronaviruses, we performed phylogenetic analyses based on the deduced RdRp and the spike, envelope, membrane, and nucleocapsid proteins by using MEGA5 (http://www.megasoftware.net/) (Figure; Technical Appendix). For RdRp and the envelope, membrane, and nucleocapsid proteins, BtVs-BetaCoV/SC2013 always clustered with hCoV-MERS with short branch lengths, reflecting their high sequence similarities. 
 
 
 
Figure 
 
Phylogenetic trees based on the deduced amino acid sequences of the partial RNA-dependent RNA polymerase (RdRp; an 816-nt sequence fragment corresponding to positions 14817–15632 in human Middle East respiratory syndrome coronavirus [hCoV-MERS; ... 
 
 
 
In the spike protein phylogenetic tree, BtVs-BetaCoV/SC2013 clustered with a clade defined by BtCoV-HKU5, with which it shares 74.8% aa identity. The spike proteins of hCoV-MERS form a sister clade of the clade defined by HKU5 BtCoVs and BtVs-betaCoV/SC2013, and the spike proteins share 69.0% aa identity with BtVs-betaCoV/SC2013. Spike proteins of BtVs-BetaCoV/SC2013, HKU5 BtCoVs, HKU4 BtCoVs, and hCoV-MERS, rather than EriCoV-2012-174, EriCoV-2012-216, and BtCoV-KW2E-F93, form a super clade. Spike protein is the critical factor for receptor recognition, binding, and cellular entry of CoVs in different host species (10), which may explain why the spike proteins in our study were relatively conserved within the same host species. 
 
We identified a novel lineage C betacoronavirus from a V. superans bat and determined its full-length genome sequence. This novel betacoronavirus represents one of the most MERS-like CoVs that have been identified in bats as of the end of March 2014. The full-length genome sequence of the novel virus showed a closer genetic relationship with hCoV-MERS and camel MERS-CoV than with any other fully sequenced lineage C betacoronaviruses previously identified in bats or hedgehogs. Further studies of CoVs from more bat species worldwide may, therefore, help provide additional clues to the origins of pathogenic hCoV-MERS. 
 
Technical Appendix: 
Phylogenetic trees of a novel Middle East respiratory syndrome–related coronavirus, human Middle East respiratory syndrome coronaviruses, severe acute respiratory syndrome virus, and various other coronaviruses. 
 
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MERS-Related Betacoronavirus in Vespertilio superans Bats, China
To the Editor: Middle East respiratory syndrome coronavirus (MERS-CoV), a novel lineage C betacoronavirus, was first described in September 2012, and by April 16, 2014, the virus had caused 238 infections and 92 deaths in humans worldwide (1). Antibodies against MERS-CoV in dromedary camels were recently reported (2), as was the full genome of MERS-CoV from dromedary camels (3). Finding the natural reservoir of MERS-CoV is fundamental to our ability to control transmission of this virus to humans (4).
We report a novel lineage C betacoronavirus identified from Vespertilio superans bats in China. The fulllength genome of this betacoronavirus showed close genetic relationship with MERS-CoV. Together with other evidence of MERS-CoV-related viruses in bats (5)(6)(7)(8), our findings suggest that bats might be the natural reservoirs of MERS-related CoVs.
In June 2013, we collected anal swab samples from 32 V. superans bats from southwestern China. A small proportion of each sample was pooled (without barcoding) and processed by using virus particleprotected nucleic acid purification and sequence-independent PCR for next-generation sequencing analysis with the Illumina (Solexa) Genome Analyzer II (Illumina, San Diego, CA, USA). Redundant reads were filtered, as described (9), from the raw sequencing reads generated by the genome analyzer and then aligned with the nonredundant protein database of the National Center for Biotechnology Information (ftp://ftp. ncbi.nlm.nih.gov/blast/db/) by using BLAST (http://blast.ncbi.nlm.nih. gov). The taxonomy of these aligned reads was parsed by using MEGAN 4 (http://ab.inf.uni-tuebingen.de/software/ megan/).
On the basis of the BLAST results, 8,751,354 sequence reads 81 nt in length were aligned with the protein sequences of the nonredundant protein database: 72,084 of the reads were uniquely matched with virus proteins. Of these 72,084 reads, 32,365 were assigned to the family Coronaviridae, primarily to lineage C of the genus Betacoronavirus, and found to share 60%-97% aa identity with MERS-CoV.
The MERS-CoV-related reads were extracted and assembled by using SeqMan software from the Lasergene 7.1.0 program (DNASTAR, Madison, WI, USA), resulting in a draft CoV genome. Reverse transcription PCR selective for the partial RNA-dependent RNA polymerase (RdRp) gene of this novel lineage C betacoronavirus suggested that 5 of the 32 samples (≈16%) were positive for the novel betacoronavirus, and the PCR amplicons shared >98% nt identity with each other. Using a set of overlapped nested PCRs and the rapid amplification of cDNA ends method, we determined the fulllength genome of 1 strain of this V. superans bat-derived betacoronavirus (referred to as BtVs-BetaCoV/ SC2013, GenBank accession no. KJ473821).
In the spike protein phylogenetic tree, BtVs-BetaCoV/SC2013 clustered with a clade defined by BtCoV-HKU5, with which it shares 74.8% aa identity. The spike proteins of hCoV-MERS form a sister clade of the clade defined by HKU5 BtCoVs and BtVs-betaCoV/SC2013, and the spike proteins share 69.0% aa identity with BtVs-betaCoV/SC2013. Spike proteins of BtVs-BetaCoV/ SC2013, HKU5 BtCoVs, HKU4 Bt-CoVs, and hCoV-MERS, rather than EriCoV-2012-174, EriCoV-2012-216, and BtCoV-KW2E-F93, form a super clade. Spike protein is the critical factor for receptor recognition, binding, and cellular entry of CoVs in different host species (10), which may explain why the spike proteins in our study were relatively conserved within the same host species.
We identified a novel lineage C betacoronavirus from a V. superans bat and determined its full-length genome sequence. This novel betacoronavirus represents one of the most MERS-like CoVs that have been identified in bats as of the end of March 2014. The fulllength genome sequence of the novel virus showed a closer genetic relationship with hCoV-MERS and camel MERS-CoV than with any other fully sequenced lineage C betacoronaviruses previously identified in bats or hedgehogs. Further studies of CoVs from more bat species worldwide may, therefore, help provide additional clues to the origins of pathogenic hCoV-MERS.