Genetic characterization of Betacoronavirus lineage C viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus HKU5 in Japanese pipistrelle: implications for the origin of the novel Middle East respiratory syndrome coronavirus

Abstract

While the novel Middle East respiratory syndrome coronavirus (MERS-CoV) is closely related to Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat CoV HKU5 (Pi-BatCoV HKU5) in bats from Hong Kong, and other potential lineage C betacoronaviruses in bats from Africa, Europe, and America, its animal origin remains obscure. To better understand the role of bats in its origin, we examined the molecular epidemiology and evolution of lineage C betacoronaviruses among bats. Ty-BatCoV HKU4 and Pi-BatCoV HKU5 were detected in 29% and 25% of alimentary samples from lesser bamboo bat (Tylonycteris pachypus) and Japanese pipistrelle (Pipistrellus abramus), respectively. Sequencing of their RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes revealed that MERS-CoV is more closely related to Pi-BatCoV HKU5 in RdRp (92.1% to 92.3% amino acid [aa] identity) but is more closely related to Ty-BatCoV HKU4 in S (66.8% to 67.4% aa identity) and N (71.9% to 72.3% aa identity). Although both viruses were under purifying selection, the S of Pi-BatCoV HKU5 displayed marked sequence polymorphisms and more positively selected sites than that of Ty-BatCoV HKU4, suggesting that Pi-BatCoV HKU5 may generate variants to occupy new ecological niches along with its host in diverse habitats. Molecular clock analysis showed that they diverged from a common ancestor with MERS-CoV at least several centuries ago. Although MERS-CoV may have diverged from potential lineage C betacoronaviruses in European bats more recently, these bat viruses were unlikely to be the direct ancestor of MERS-CoV. Intensive surveillance for lineage C betaCoVs in Pipistrellus and related bats with diverse habitats and other animals in the Middle East may fill the evolutionary gap.

Fig 1

Phylogenetic analysis of RdRp, S, and N genes of Ty-BatCoV HKU4 and Pi-BatCoV HKU5 strains and those of other betaCoVs with available complete genome sequences. The trees were constructed by the maximum-likelihood method with bootstrap values calculated from 100 trees. The analysis included 937, 1,535, and 546 aa positions in the RdRp, S, and N genes, respectively. The scale bars indicate the estimated number of substitutions per 5 or 20 aa. HCoV-HKU1, human coronavirus HKU1; HCoV-OC43, human coronavirus OC43; MHV, murine hepatitis virus; BCoV, bovine coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; GiCoV, giraffe coronavirus; RCoV, rat coronavirus; ECoV, equine coronavirus; RbCoV HKU14, rabbit coronavirus HKU14; AntelopeCoV, sable antelope coronavirus; SARS-CoV, SARS coronavirus; SARSr-Rh-BatCoV HKU3, SARS-related Rhinolophus bat coronavirus HKU3; SARSr-CiCoV, SAR-related civet coronavirus; SARSr CoV CFB, SARS-related Chinese ferret badger coronavirus; Ty-BatCoV HKU4, Tylonycteris bat coronavirus HKU4; Pi-BatCoV HKU5, Pipistrellus bat coronavirus HKU5; MERS-CoV EMC, Middle East Respiratory Syndrome Coronavirus EMC; MERS-CoV England1, Middle East Respiratory Syndrome Coronavirus England1; Ro-BatCoV HKU9, Rousettus bat coronavirus HKU9.

Fig 2

Distribution of amino acid changes in the spike protein of Ty-BatCoV HKU4 (upper panel) and Pi-BatCoV HKU5 (lower panel). The positions of the amino acid changes are depicted by vertical lines. SS, predicted signal peptide; RBD, receptor binding domain; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain.

Fig 3

Graphical representation of multiple sequence alignment showing the amino acid changes in the spike protein of Pi-BatCoV HKU5. The height of each symbol indicates the relative frequency of each amino acid at the position. Polar amino acids are indicated in green; neutral amino acids are indicated in purple; basic amino acids are indicated in blue; acidic amino acids are indicated in red; hydrophobic amino acids are indicated in black. The figure was generated using WebLogo (91).

Fig 4

Distribution of positively selected sites in S proteins identified using REL in Ty-BatCoV HKU4 (upper panel) and Pi-BatCoV HKU5 (lower panel). Positively selected sites with posterior probability greater than 0.5 are shown.

Fig 5

Estimation of the tMRCA of Ty-BatCoV HKU4 and Pi-BatCoV HKU5. The time-scaled phylogeny was summarized from all MCMC phylogenies of the complete RdRp (A), complete N (B), and 904-bp RdRp (C) sequence data sets analyzed using the relaxed clock model with an exponential distribution (Uced) in BEAST v 1.7.4. Viruses characterized in this study are bolded.