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

Abstract

Despite the identification of horseshoe bats as the reservoir of SARS-related-coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nt signature deletion among human strains, remains obscure. Although two SARSr-Rs-BatCoVs, RsSHC014 and Rs3367, previously detected from Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, possessed 95% genome identities to human/civet SARSr-CoVs, their ORF8 exhibited only 32.2-33% aa 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% nt identities to human/civet SARSr-CoV genomes. Although they displayed lower similarities to civet SARSr-CoVs than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein, their ORF8 demonstrated exceptionally high (80.4-81.3%) aa identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2-37.3%). 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 this protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSs-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs at approximately 1990. SARS-CoV ORF8 is originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission.