Prevalence and genetic diversity of coronaviruses in bats from China

Abstract

Coronaviruses can infect a variety of animals including poultry, livestock, and humans and are currently classified into three groups. The interspecies transmissions of coronaviruses between different hosts form a complex ecosystem of which little is known. The outbreak of severe acute respiratory syndrome (SARS) and the recent identification of new coronaviruses have highlighted the necessity for further investigation of coronavirus ecology, in particular the role of bats and other wild animals. In this study, we sampled bat populations in 15 provinces of China and reveal that approximately 6.5% of the bats, from diverse species distributed throughout the region, harbor coronaviruses. Full genomes of four coronavirues from bats were sequenced and analyzed. Phylogenetic analyses of the spike, envelope, membrane, and nucleoprotein structural proteins and the two conserved replicase domains, putative RNA-dependent RNA polymerase and RNA helicase, revealed that bat coronaviruses cluster in three different groups: group 1, another group that includes all SARS and SARS-like coronaviruses (putative group 4), and an independent bat coronavirus group (putative group 5). Further genetic analyses showed that different species of bats maintain coronaviruses from different groups and that a single bat species from different geographic locations supports similar coronaviruses. Thus, the findings of this study suggest that bats may play an integral role in the ecology and evolution of coronaviruses.

FIG. 1.

Map of China showing 15 provinces where coronavirus surveillance in bats was conducted. Numbers indicate number of sites positive over the total number of sites sampled in each province.

FIG. 2.

Phylogenetic relationships of 64 coronaviruses isolated from bats in China. The tree was generated based on 440 nucleotides of the RNA-dependent RNA polymerase region by the neighbor-joining method in the MEGA program. Numbers above branches indicate neighbor-joining bootstrap values (percent) calculated from 1,000 bootstrap replicates. Terminal nodes containing bat coronaviruses isolated in this study are collapsed and represented by a blue triangle with the number of viruses indicated within. The tree was rooted to Breda virus (AY427798). Scale bar, 0.05 substitution per site. Red text indicates provinces from where viruses were isolated. Abbreviations: AH, Anhui; FJ, Fujian; GD, Guangdong; GX, Guangxi; HA, Hainan; HB, Hubei; HE, Henan; JX, Jiangxi; SC, Sichuan; SD, Shandong; YN, Yunnan.

FIG. 3.

Linear representation of the ORFs of the bat coronaviruses and representative known coronaviruses from each group. Conserved functional domains in ORF1a and ORF1b are indicated by yellow boxes. The following predicted domains are shown: pepain-like proteases 1 and 2 (PL1 and PL2), 3C-like protease (3CL), RdRp, metal ion-binding domain (MB), and helicase (Hel). Putative ORFs are indicated by blue boxes and numbered according to their order in the genome: BtCoV/R. ferrumequinum/Hubei/273/04 (BtCoV/273/04), BtCoV/R. macrotis/Hubei/279/04 (BtCoV/279/04), BtCoV/T. pachypus/Guangdong/133/05 (BtCoV/133/05), BtCoV/S. kuhlii/Hainan/512/05 (BtCoV/512/05), SARS-CoV, PEDV, avian IBV, and human coronavirus OC43 (HCoV-OC43).

FIG. 4.

Similarity histogram of RdRp (A) and spike (B) genes based on alignments from the program TransAlign.

FIG. 5.

Phylogenetic relationships of the helicase (A) and spike (B) genes of representative coronaviruses isolated from bats in China. Trees were generated by the neighbor-joining method in the PAUP program. Numbers above branches indicate neighbor-joining bootstrap values (percent) calculated from 1,000 bootstrap replicates. Analyses were based on 1,833 nucleotides for the helicase gene and 3,510 nucleotides for the spike gene. The trees were rooted to Breda virus (AY427798). Scale bar, 0.1 substitution per site.