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. 2015 Oct;89(20):10532-47.
doi: 10.1128/JVI.01048-15. Epub 2015 Aug 12.

Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination

Susanna K P Lau  1 , Yun Feng  2 , Honglin Chen  1 , Hayes K H Luk  3 , Wei-Hong Yang  2 , Kenneth S M Li  3 , Yu-Zhen Zhang  2 , Yi Huang  3 , Zhi-Zhong Song  2 , Wang-Ngai Chow  3 , Rachel Y Y Fan  3 , Syed Shakeel Ahmed  3 , Hazel C Yeung  3 , Carol S F Lam  3 , Jian-Piao Cai  3 , Samson S Y Wong  1 , Jasper F W Chan  1 , Kwok-Yung Yuen  1 , Hai-Lin Zhang  4 , Patrick C Y Woo  5
Affiliations

Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination

Susanna K P Lau et al. J Virol. 2015 Oct.

Abstract

Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission.

Importance: Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats (Rhinolophus sinicus) exhibited <40% amino acid identities to human/civet SARS-CoV in the ORF8 protein. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, China, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed ORF8 proteins with exceptionally high amino acid identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 protein, which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination.

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Figures

FIG 1
FIG 1
Map showing five locations of bat sampling in four autonomous prefectures in Yunnan Province, China. Sampling locations in Yunnan are in red. The location of SARSr-Rs-BatCoV strains Rs3367 and RsSHC014, detected in a previous study (42), is in blue.
FIG 2
FIG 2
Phylogenetic analysis of the nucleotide sequences of the 267-nt fragment of RdRp gene regions of the 46 positive samples identified in bats in Yunnan in this study. The tree was constructed by the maximum likelihood method with the model GTR+G. Bootstrap values were calculated from 1,000 trees, and only values of >700 are shown and given at the nodes. The scale bar represents 5 nucleotide substitutions per site. The two SARSr-Rf-BatCoV strains, YNLF_31C and YNLF_34C, are in red. The potentially novel bat CoVs are in purple. AntelopeCoV, sable antelope coronavirus (EF424621); BatCoV CDPHE15/USA/2006, bat coronavirus CDPHE15/USA/2006 (NC_022103.1); BatCoV/SC2013, betacoronavirus/SC2013 (KJ473821.1); Erinaceus CoV/VMC/DEU/2012, betacoronavirus Erinaceus/VMC/DEU/2012 (NC_022643); BCoV, bovine coronavirus (NC_003045); BdHKU22, bottlenose dolphin coronavirus HKU22 (KF793826); BuCoV HKU11, bulbul coronavirus HKU11 (FJ376619); BWCoV SW1, beluga whale coronavirus SW1 (NC_010646); CCoV, canine coronavirus strain CCoV/NTU336/F/2008 (GQ477367.1); CRCoV, canine respiratory coronavirus strain K37 (JX860640.1); CmCoV HKU21, common moorhen coronavirus HKU21 (NC_016996); CoV Neoromicia/PML-PHE1/RSA/2011, coronavirus Neoromicia/PML-PHE1/RSA/2011 (KC869678); DcCoV HKU23, dromedary camel coronavirus HKU23 (KF906251); ECoV, equine coronavirus (NC_010327); FIPV, feline infectious peritonitis virus (AY994055); GiCoV, giraffe coronavirus US/OH3-TC/2006 (EF424622.1); HCoV-229E, human coronavirus 229E (NC_002645); HCoV-HKU1, human coronavirus HKU1 (NC_006577); HCoV-NL63, human coronavirus NL63 (NC_005831); HCoV-OC43, human coronavirus OC43(NC_005147); Hi-BatCoV HKU10, Hipposideros bat coronavirus HKU10 (JQ989269); IBV-beaudette, Beaudette coronavirus (AY692454); Human MERS-CoV, Middle East respiratory syndrome coronavirus (NC_019843.3); Human MERS-CoV EMC/2012, human betacoronavirus 2c_EMC/2012 (JX869059.2); Camel MERS-CoV KSA-CAMEL-363, Middle East respiratory syndrome coronavirus isolate KSA-CAMEL-363 (KJ713298); MRCoV HKU18, magpie robin coronavirus HKU18(NC_016993); BatCoV 1A, Miniopterus bat coronavirus 1A (NC_010437); BatCoV 1B, Miniopterus bat coronavirus 1B(NC_010436); Mi-BatCoV HKU7, Miniopterus bat coronavirus HKU7 (DQ249226); Mi-BatCoV HKU8, Miniopterus bat coronavirus HKU8 (NC_010438); Mink CoV strain WD1127, mink coronavirus strain WD1127 (NC_023760.1); MunCoV HKU13, munia coronavirus HKU13 (FJ376622); MHV-A59, murine hepatitis virus (NC_001846); My-BatCoV HKU6, Myotis bat coronavirus HKU6 (DQ249224); NH CoV HKU19, night heron coronavirus HKU19 (NC_016994); PEDV, porcine epidemic diarrhea virus (NC_003436); PHEV, porcine hemagglutinating encephalomyelitis virus (NC_007732); Pi-BatCoV-HKU5-1, Pipistrellus bat coronavirus HKU5 (NC_009020); PorCoV HKU15, porcine coronavirus HKU15 (NC_016990); PRCV, porcine respiratory coronavirus (DQ811787); RbCoV HKU14, rabbit coronavirus HKU14 (NC_017083); RatCoV parker, rat coronavirus Parker (NC_012936); Rs-BatCoV HKU2, Rhinolophus bat coronavirus HKU2 (EF203064); Ro-BatCoV-HKU9, Rousettus bat coronavirus HKU9 (NC_009021); Ro-BatCoV HKU10, Rousettus bat coronavirus HKU10 (JQ989270); Human SARS-CoV TOR2, SARS-related human coronavirus (NC_004718); Civet SARS-CoV SZ16, SARS-related palm civet coronavirus (AY304488); Badger SARS-CoV, SARS-related badger coronavirus CFB/SZ/94/03 (AY545919.1); SARSr-Rs-BatCoV HKU3, SARS-related Rhinolophus bat coronavirus HKU3 (DQ022305); Scotophilus BatCoV 512, Scotophilus bat coronavirus 512 (NC_009657); SpCoV HKU17, sparrow coronavirus HKU17 (NC_016992); TCoV, turkey coronavirus (NC_010800); TGEV, transmissible gastroenteritis virus (DQ443743); ThCoV HKU12, thrush coronavirus HKU12 (FJ376621); Ty-BatCoV-HKU4-1, Tylonycteris bat coronavirus HKU4 (NC_009019); WECoV HKU16, white-eye coronavirus HKU16 (NC_016991); WiCoV HKU20, widgeon coronavirus HKU20 (NC_016995).
FIG 3
FIG 3
Multiple alignment of the amino acid sequences of the receptor-binding motifs of the spike proteins of human and civet SARSr-CoV and the corresponding sequences of SARSr-BatCoVs in different Rhinolophus species. Asterisks indicate positions that have fully conserved residues. Amino acid deletions among some SARSr-BatCoVs are highlighted in yellow. The five critical residues for receptor binding in human SARS-CoV, at positions 442,472,479,487,491, are highlighted in pink.
FIG 4
FIG 4
Phylogenetic analyses of nsp2, nsp3, nsp5, RdRp, S, ORF3, ORF8, and N gene nucleotide sequences of SARSr-BatCoVs from different bat species. The trees were constructed by the maximum likelihood method using GTR+G (A), GTR+G (B), GTR+G+I (C), TN93+G (D), GTR+G (E), TN93+G (F), T92 +G (G), and GTR+G (H) substitution models, and bootstrap values were calculated from 1,000 trees. Except for ORF3 and ORF8, all trees were rooted using the corresponding sequences of HCoV HKU1 (GenBank accession number NC_006577). Only bootstrap values of >70% are shown. Nucleotide positions 1736 (A), 5019 (B), 908 (C), 2777 (D), 3638 (E), 804 (F), 345 (G), and 1222 (H) were included in the analyses. The scale bars represent 50 (A), 10 (B), 20 (C), 20 (D), 10 (E), 20 (F), 10 (G), and 200 (H) substitutions per site. Human and civet SARSr-CoVs are in green, SARSr-Rs-BatCoVs from R. sinicus are in blue, and SARSr-Rs-BatCoVs from R. ferrumequinum are in red. The two SARSr-Rf-BatCoV strains YNLF_31C and YNLF_34C detected in this study are in bold.
FIG 5
FIG 5
(A) Bootscan (upper panel) and Simplot (lower panel) analysis using the genome sequence of civet SARSr-CoV strain SZ03 as the query sequence. Bootscanning was conducted with Simplot version 3.5.1 (F84 model; window size, 1,000 bp; step, 200 bp) on a gapless nucleotide alignment, generated with ClustalX. The red line denotes SARSr-Rf-BatCoV strain YNLF_31C, the blue line denotes SARSr-Rs-BatCoV strain Rs3367, and the black line denotes SARSr-Rs-BatCoV strain HKU3-1. The ORF8 region with potential recombination is highlighted in yellow. (B) Multiple alignment of nucleotide sequences from genome positions 27000 to 28700. Bases conserved between civet SARSr-CoV SZ03 and SARSr-Rf-BatCoVs (strains YNLF_31C and Rf1) are marked in yellow boxes. Bases conserved between civet SARSr-CoV SZ03 and SARSr-Rs-BatCoVs (strains Rs3367 and HKU3-1) are marked in green boxes. The 29-nt deletion in human SARS coronavirus TOR2 is highlighted in orange. The start codon and stop codon of ORF8 are labeled with black boxes.
FIG 5
FIG 5
(A) Bootscan (upper panel) and Simplot (lower panel) analysis using the genome sequence of civet SARSr-CoV strain SZ03 as the query sequence. Bootscanning was conducted with Simplot version 3.5.1 (F84 model; window size, 1,000 bp; step, 200 bp) on a gapless nucleotide alignment, generated with ClustalX. The red line denotes SARSr-Rf-BatCoV strain YNLF_31C, the blue line denotes SARSr-Rs-BatCoV strain Rs3367, and the black line denotes SARSr-Rs-BatCoV strain HKU3-1. The ORF8 region with potential recombination is highlighted in yellow. (B) Multiple alignment of nucleotide sequences from genome positions 27000 to 28700. Bases conserved between civet SARSr-CoV SZ03 and SARSr-Rf-BatCoVs (strains YNLF_31C and Rf1) are marked in yellow boxes. Bases conserved between civet SARSr-CoV SZ03 and SARSr-Rs-BatCoVs (strains Rs3367 and HKU3-1) are marked in green boxes. The 29-nt deletion in human SARS coronavirus TOR2 is highlighted in orange. The start codon and stop codon of ORF8 are labeled with black boxes.
FIG 6
FIG 6
Estimation of tMRCA of SARSr-CoVs based on ORF1ab (A) and nsp5 (B). The mean estimated dates are indicated. The taxa are labeled with their sampling dates.
FIG 7
FIG 7
SARSr-Rf-BatCoV YNLF_31C mRNA leader-body junction and flanking sequences. The subgenomic ORF8 mRNA sequences are shown in alignment with the leader and genomic sequences. The start codon AUG in subgenomic RNA is depicted in red. The putative TRS is depicted in boldface type and underlined. Identical bases between leader sequence and subgenomic mRNA sequence are in blue. Identical bases between genome and subgenomic mRNA sequences are in green.
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