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


OBJECTIVE
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.
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Susanna K P
SK
Lau
Yunnan Institute of Endemic Diseases Control and Prevention, Dali, Yunnan, China Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention, Dali, Yunnan, China.
Yun
Y
Feng
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Honglin
H
Chen
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Hayes K H
HK
Luk
Yunnan Institute of Endemic Diseases Control and Prevention, Dali, Yunnan, China Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention, Dali, Yunnan, China.
Wei-Hong
WH
Yang
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Kenneth S M
KS
Li
Yunnan Institute of Endemic Diseases Control and Prevention, Dali, Yunnan, China Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention, Dali, Yunnan, China.
Yu-Zhen
YZ
Zhang
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Yi
Y
Huang
Yunnan Institute of Endemic Diseases Control and Prevention, Dali, Yunnan, China Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention, Dali, Yunnan, China.
Zhi-Zhong
ZZ
Song
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Wang-Ngai
http://orcid.org/0000-0003-1275-2464
WN
Chow
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Rachel Y Y
RY
Fan
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Syed Shakeel
SS
Ahmed
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Hazel C
HC
Yeung
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Carol S F
CS
Lam
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Jian-Piao
JP
Cai
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Samson S Y
SS
Wong
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Jasper F W
JF
Chan
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Kwok-Yung
KY
Yuen
Yunnan Institute of Endemic Diseases Control and Prevention, Dali, Yunnan, China Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention, Dali, Yunnan, China pcywoo@hku.hk zhangHL715@163.com.
Hai-Lin
HL
Zhang
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China Department of Microbiology, The University of Hong Kong, Hong Kong, China pcywoo@hku.hk zhangHL715@163.com.
Patrick C Y
PC
Woo
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002681:China; D002685:Chiroptera / Q000821:virology; D018352:Coronavirus Infections / Q000235:genetics / Q000635:transmission / Q000662:veterinary* / Q000821:virology; D019143:Evolution, Molecular; D015870:Gene Expression; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D058974:Phylogeography; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D045473:Severe acute respiratory syndrome-related coronavirus / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D045169:Severe Acute Respiratory Syndrome / Q000235:genetics / Q000378:metabolism / Q000635:transmission / Q000821:virology; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D045949:Viverridae / Q000821:virology
D012333:RNA, Messenger; D012367:RNA, Viral; D014763:Viral Matrix Proteins; C488151:sars7a protein, SARS virus