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. 2018 Jun 13;92(13):e00116-18.
doi: 10.1128/JVI.00116-18. Print 2018 Jul 1.

Discovery of Novel Bat Coronaviruses in South China That Use the Same Receptor as Middle East Respiratory Syndrome Coronavirus

Chu-Ming Luo  1   2   3 , Ning Wang  1   2 , Xing-Lou Yang  1 , Hai-Zhou Liu  1 , Wei Zhang  1 , Bei Li  1 , Ben Hu  1 , Cheng Peng  1 , Qi-Bin Geng  3 , Guang-Jian Zhu  4 , Fang Li  5 , Zheng-Li Shi  6
Affiliations

Discovery of Novel Bat Coronaviruses in South China That Use the Same Receptor as Middle East Respiratory Syndrome Coronavirus

Chu-Ming Luo et al. J Virol. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) has represented a human health threat since 2012. Although several MERS-related CoVs that belong to the same species as MERS-CoV have been identified from bats, they do not use the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4). Here, we screened 1,059 bat samples from at least 30 bat species collected in different regions in south China and identified 89 strains of lineage C betacoronaviruses, including Tylonycteris pachypus coronavirus HKU4, Pipistrellus pipistrelluscoronavirus HKU5, and MERS-related CoVs. We sequenced the full-length genomes of two positive samples collected from the great evening bat, Ia io, from Guangdong Province. The two genomes were highly similar and exhibited genomic structures identical to those of other lineage C betacoronaviruses. While they exhibited genome-wide nucleotide identities of only 75.3 to 81.2% with other MERS-related CoVs, their gene-coding regions were highly similar to their counterparts, except in the case of the spike proteins. Further protein-protein interaction assays demonstrated that the spike proteins of these MERS-related CoVs bind to the receptor DPP4. Recombination analysis suggested that the newly discovered MERS-related CoVs have acquired their spike genes from a DPP4-recognizing bat coronavirus HKU4. Our study provides further evidence that bats represent the evolutionary origins of MERS-CoV.IMPORTANCE Previous studies suggested that MERS-CoV originated in bats. However, its evolutionary path from bats to humans remains unclear. In this study, we discovered 89 novel lineage C betacoronaviruses in eight bat species. We provide evidence of a MERS-related CoV derived from the great evening bat that uses the same host receptor as human MERS-CoV. This virus also provides evidence for a natural recombination event between the bat MERS-related CoV and another bat coronavirus, HKU4. Our study expands the host ranges of MERS-related CoV and represents an important step toward establishing bats as the natural reservoir of MERS-CoV. These findings may lead to improved epidemiological surveillance of MERS-CoV and the prevention and control of the spread of MERS-CoV to humans.

Keywords: MERS-related coronavirus; bat; dipeptidyl peptidase 4; virus discovery.

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Figures

FIG 1
FIG 1
Geographic locations of collection sites, phylogeny of lineage C betacoronaviruses, and genomic organization of novel bat MERS-related CoVs. (A) Map of sampling locations and lineage C betacoronaviruses detected. Names of these counties, districts, and cities are noted. (B) Phylogenetic analysis of the 228-bp RdRp genes of the newly detected lineage C betacoronaviruses. The tree was constructed using the maximum likelihood method, with the Kimura 2-parameter model with bootstrap values calculated from 100 trees, and was midpoint rooted. Scale bar indicates the estimated number of substitutions per 10 nucleotides. Viruses characterized in this study are shown in boldface and marked with black diamonds. MERS-CoVs are shown in boldface. (C) Genomic organization of BtCoV/Ii/GD/2013-845 (845) and BtCoV/Ii/GD/2014-422 (422). Genomes are represented by black lines, and ORFs are indicated as gray blocks. Transcription regulatory core sequence (TRS) locations are marked with labeled dots. The nucleotide position of the ribosomal frameshift site (RFS) is marked with a black arrow.
FIG 2
FIG 2
Phylogenetic trees derived from the amino acid sequences of lineage C betacoronaviruses for PP1, S, RBD, and N. Trees were constructed using the maximum-likelihood method, with the JTT model with bootstrap values calculated from 100 trees, and were midpoint rooted. For PP1, S/N, and RBD, scale bars indicate the estimated number of substitutions per 10, 5, or 2 amino acids, respectively. Viruses characterized in this study are shown in boldface.
FIG 3
FIG 3
Genomic sequence identities between novel MERS-related CoVs and other lineage C betacoronaviruses (A) and evidence for recombination in BtCoV/Ii/GD/2013-845 and BtCoV/Ii/GD/2014-422 (B). Similarity (window of 400 nt, step size of 40 nt) and recombination (window of 1,500 nt, step size of 300 nt) plots were generated using Simplot (v3.5.7) with default settings (25).
FIG 4
FIG 4
BtCoV/Ii/GD/2014-422 RBD analysis and DPP4-binding assay. (A) Sequence alignment of the partial S1 domains (including all positions with direct interactions with human DPP4) of selected lineage C betacoronaviruses. Asterisks indicate positions with fully conserved residues. Colons indicate positions with strongly conserved residues. Periods indicate positions with weakly conserved residues. Positions that have direct interactions with human DPP4 according to data from MERS-CoV are in gray bars. Residues identical to corresponding MERS-CoV residues are in red. (B) AlphaScreen assay showing the direct binding interactions between the coronavirus spike RBD and hDPP4 or bDPP4. Binding affinity was characterized as AlphaScreen counts. Error bars indicate standard errors of the means (SEM) (*, P < 0.05 by two-tailed t test; n = 3). (C) Dot blot hybridization assay showing the direct binding interactions between the coronavirus spike RBD and hDPP4 or bDPP4. His8-tagged hDPP4, bDPP4, or hACE2 was dotted and then incubated with each of the Fc-tagged coronavirus RBDs, followed by anti-IgG4 monoclonal antibody detection.
FIG 5
FIG 5
BtCoV/Ii/GD/2014-422 spike-mediated pseudovirus entry and inhibition assay. Anti-hDPP4 antibodies competitively block interactions between BtCoV/Ii/GD/2014-422 spike and hDPP4 but not bat DPP4. Error bars indicate SEM (*, P < 0.05 by two-tailed t test; n = 4). (A) Anti-hDPP4 antibodies did not neutralize BtCoV/Ii/GD/2014-422 spike-mediated and MERS-CoV spike-mediated pseudovirus entry into bDPP4-expressing cells. (B) Anti-hDPP4 antibodies strongly neutralized BtCoV/Ii/GD/2014-422 spike-mediated and MERS-CoV spike-mediated pseudovirus entry into hDPP4-expressing Tb1-Lu cells.
FIG 6
FIG 6
Cross-neutralization assay of MERS-CoV antibodies to BtCoV/Ii/GD/2014-422 spike-mediated pseudovirus. Anti-MERS-spike antibodies could not inhibit the entry of BtCoV/Ii/GD/2014-422 spiked pseudovirus. Error bars indicate SEM (N.S indicates a P value of >0.05 by two-tailed t test; n = 4). (A) Anti-MERS-spike antibodies neutralize MERS-CoV spike-mediated pseudovirus entry into bDPP4-expressing cells but not BtCoV/Ii/GD/2014-422 spike-mediated pseudovirus entry. (B) Anti-MERS-spike antibodies neutralize MERS-CoV spike-mediated pseudovirus entry into hDPP4-expressing cells but not BtCoV/Ii/GD/2014-422 spike-mediated pseudovirus entry.
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