Kenneth S. M. Li

Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and analysis of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were observed between human and civet SARS-CoV genomes. In addition, the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals.

S eventeen years after the severe acute respiratory syndrome (SARS) epidemic, an outbreak of pneumonia, now called coronavirus disease , was reported in Wuhan, China. Some of the early casepatients had a history of visiting the Huanan Seafood Wholesale Market, where wildlife mammals are sold, suggesting a zoonotic origin. The causative agent was rapidly isolated from patients and identified to be a coronavirus, now designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (1). SARS-CoV-2 has spread rapidly to other places; 113,702 cases and 4,012 deaths had been reported in 110 countries/areas as of March 10, 2020 (2). In Hong Kong, 130 cases and 3 deaths had been reported. SARS-CoV-2 is a member of subgenus Sarbecovirus (previously lineage b) in the family Coronaviridae, genus Betacoronavirus, and is closely related to SARS-CoV, which caused the SARS epidemic during 2003, and to SARS-related-CoVs (SARSr-CoVs) in horseshoe bats discovered in Hong Kong and mainland China (3-5). Whereas SARS-CoV and Middle East respiratory syndrome coronavirus were rapidly traced to their immediate animal sources (civet and dromedaries, respectively), the origin of SARS-CoV-2 remains obscure. SARS-CoV-2 showed high genome sequence identities (87.6%-87.8%) to SARSr-Rp-BatCoV-ZXC21/ ZC45, detected in Rhinolophus pusillus bats from Zhoushan, China, during 2015 (6). A closer-related strain, SARSr-Ra-BatCoV-RaTG13 (96.1% genome identity with SARS-CoV-2), was recently reported in Rhinolophus affinis bats captured in Pu'er, China, during 2013 (7). Subsequently, Pangolin-SARSr-CoV/ P4L/Guangxi/2017 (85.3% genome identity to SARS-CoV-2) and related viruses were also detected in smuggled pangolins captured in Nanning, China, during 2017 (8) and Guangzhou, China, during 2019 (9). To elucidate the evolutionary origin and pathway of SARS-CoV-2, we performed an in-depth genomic, phylogenetic, and recombination analysis in relation to SARSr-CoVs from humans, civets, bats, and pangolins (10).

Despite the identification of severe acute respiratory syndrome-related coronavirus (SARSr-CoV) in Rhinolophus Chinese horseshoe bats (SARSr-Rh-BatCoV) in China, the evolutionary and possible recombination origin of SARSr-CoV remains undetermined. We carried out the first study to investigate the migration pattern and SARSr-Rh-BatCoV genome epidemiology in Chinese horseshoe bats during a 4-year period. Of 1,401 Chinese horseshoe bats from Hong Kong and Guangdong, China, that were sampled, SARSr-Rh-BatCoV was detected in alimentary specimens from 130 (9.3%) bats, with peak activity during spring. A tagging exercise of 511 bats showed migration distances from 1.86 to 17 km. Bats carrying SARSr-Rh-BatCoV appeared healthy, with viral clearance occurring between 2 weeks and 4 months. However, lower body weights were observed in bats positive for SARSr-Rh-BatCoV, but not Rh-BatCoV HKU2. Complete genome sequencing of 10 SARSr-Rh-BatCoV strains showed frequent recombination between different strains. Moreover, recombination was detected between SARSr-Rh-BatCoV Rp3 from Guangxi, China, and Rf1 from Hubei, China, in the possible generation of civet SARSr-CoV SZ3, with a breakpoint at the nsp16/spike region. Molecular clock analysis showed that SARSr-CoVs were newly emerged viruses with the time of the most recent common ancestor (tMRCA) at 1972, which diverged between civet and bat strains in 1995. The present data suggest that SARSr-Rh-BatCoV causes acute, self-limiting infection in horseshoe bats, which serve as a reservoir for recombination between strains from different geographical locations within reachable foraging range. Civet SARSr-CoV is likely a recombinant virus arising from SARSr-CoV strains closely related to SARSr-Rh-BatCoV Rp3 and Rf1. Such frequent recombination, coupled with rapid evolution especially in ORF7b/ORF8 region, in these animals may have accounted for the cross-species transmission and emergence of SARS.

Twelve complete genomes of three novel coronaviruses-bat coronavirus HKU4 (bat-CoV HKU4), bat-CoV HKU5 (putative group 2c), and bat-CoV HKU9 (putative group 2d)-were sequenced. Comparative genome analysis showed that the various open reading frames (ORFs) of the genomes of the three coronaviruses had significantly higher amino acid identities to those of other group 2 coronaviruses than group 1 and 3 coronaviruses. Phylogenetic trees constructed using chymotrypsin-like protease, RNA-dependent RNA polymerase, helicase, spike, and nucleocapsid all showed that the group 2a and 2b and putative group 2c and 2d coronaviruses are more closely related to each other than to group 1 and 3 coronaviruses. Unique genomic features distinguishing between these four subgroups, including the number of papain-like proteases, the presence or absence of hemagglutinin esterase, small ORFs between the membrane and nucleocapsid genes and ORFs (NS7a and NS7b), bulged stem-loop and pseudoknot structures downstream of the nucleocapsid gene, transcription regulatory sequence, and ribosomal recognition signal for the envelope gene, were also observed. This is the first time that NS7a and NS7b downstream of the nucleocapsid gene has been found in a group 2 coronavirus. The high Ka/Ks ratio of NS7a and NS7b in bat-CoV HKU9 implies that these two group 2d-specific genes are under high selective pressure and hence are rapidly evolving. The four subgroups of group 2 coronaviruses probably originated from a common ancestor. Further molecular epidemiological studies on coronaviruses in the bats of other countries, as well as in other animals, and complete genome sequencing will shed more light on coronavirus diversity and their evolutionary histories.