Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats

Abstract

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.

Fig. 1.

Surveillance for bat-SARS-CoV in the wild areas of the HKSAR. (a) Map of HKSAR showing locations of wild animal surveillance indicated by stars. Areas belonging to Shenzhen of mainland China are shaded. Red stars represent the two locations with bats positive for bat-SARS-CoV, both being water tunnels. Other sampling sites include abandoned mines, sea caves and forested areas. SZDM, Shenzhen Dongmen market, the nearest wildlife market with SARS-CoV-infected civets. (b) A group of Chinese horseshoe bats roosting in a water tunnel in the HKSAR. The common name describes the horseshoe-shaped area of thick skin surrounding the nostrils that covers the upper lip (close-up view)

Fig. 2.

Phylogenetic analysis of chymotrypsin-like protease (3CL^pro), RNA-dependent RNA polymerase (Pol), spike (S), and nucleocapsid (N) of bat-SARS-CoV. Bootstrap values were calculated from 1,000 trees. Included for analysis were 306, 932, 1242, and 421 amino acid positions in 3CL^pro, Pol, S, and N, respectively. The scale bar indicates the estimated number of substitutions per 10 amino acids. PEDV, porcine epidemic diarrhea virus; TGEV, porcine transmissible gastroenteritis virus; MHV, murine hepatitis virus; BCoV, bovine CoV; IBV, infectious bronchitis virus

Fig. 3.

Western blot analysis with animal sera against the purified (His)₆-tagged recombinant bat-SARS-CoV N protein antigen. Prominent immunoreactive protein bands of about 50 kDa, consistent with the expected size of 49.5 kDa of the recombinant protein, were detected with a convalescent serum sample from a patient with SARS as positive control and four of the eight bat serum samples shown, indicating antigen-antibody interactions between the recombinant bat-SARS-CoV N protein and serum antibodies. Results of neutralization tests for SARS-CoV and RT-PCR of anal swabs for bat-SARS-CoV also are shown