Zoonotic origin and transmission of emerging betacoronaviruses

Abstract

The animal reservoir of SARS coronavirus (SARS-CoV) was traced to civets as the amplification host and horseshoe bats as primary reservoir. A potentially similar situation is observed in Middle East Respiratory Syndrome Coronavirus (MERS-CoV) with dromedary camels as the immediate source and bats possibly be the natural reservoir, given its close genetic relationship with Tylonycteris pachypus coronavirus HKU4 (Ty-BatCoV-HKU4) as well as the ability of Ty-BatCoV-HKU4 to utilize the host receptor of MERS-CoV, dipeptidyl peptidase 4 (DPP4). However, the evolutionary origin of these emerging Betacoronaviruses were still not fully elucidated. In this study, two novel strains of Rhinolophus ferrumequinum bat SARS-related CoVs (SARSr-Rf-BatCoVs) from Yunnan province closely related to human/civet SARS-CoV were identified. These two SARSr-Rf-BatCoVs were phylogenetically closely clustered to human/civet SARS-CoV in the replicase region, which was absent in other SARSr-Rf-BatCoVs. Remarkably, these two viruses could possess the origin of the Open Reading Frame 8 (ORF8) of human/civet SARS-CoV with its exceptionally high 81.3% amino acid identity. This phenomenon was subsequently observed among SARSr-Rf-BatCoVs detected in different regions and filled the evolutionary gap between bat SARS-related CoVs and human/civet SARS-CoV. As for the evolutionary origin of MERS-CoV, two novel strains of Hypsugo pulveratus bat coronavirus HKU25 (Hp-BatCoV-HKU25) from Guangdong province were also discovered. The virus possessed high genome identity to MERS-CoV of 73% with their spike genes phylogenetically close to that of MERS-CoV, only second to that of Ty-BatCoV-HKU4. Hp-BatCoV-HKU25 could utilize DPP4 for cell entry in pseudovirus assays despite a lower efficiency compared to Ty-BatCoV-HKU4. Our data filled the evolutionary gap and supported the hypothesis of MERS-CoV being originated from vesper bats. The results also suggested that bat CoVs previously detected in Africa were unlikely the immediate bat origins of MERS-CoV, given that their spike protein were distantly related to that of MERS-CoV and were in fact more closely related to that of European Hedgehog CoV. We also discovered two novel strains of Erinaceus amurensis Hedgehog CoVs (Ea-HedCoV-HKU31) in China. The spike gene of Ea-HedCoV-HKU31 also clustered with the African bat MERS-related CoV, suggesting that the spike genes of African bat MERS-related viruses were acquired from Hedgehog CoVs during the evolution of their ancestors. To further understand the possible bat origin of MERS-CoV, primary bat cell lines were developed. MERS-CoV could infect bat cells from Pipistrellus, Rhinolophus, Myotis and Rousettus bats but not the cells from T.pachypus. Cell line susceptibility correlated with the bat DPP4 expression. The receptor binding regions of the bat DPP4 genes were generally conserved except the dispensable polymorphism at residue 336 and the possibly detrimental mutation I295K in T.pachypus cells. Binding affinity assays between DPP4 and CoV Receptor binding domains (RBDs) confirmed that Hp-BatCoV-HKU25 could interact with human DPP4. Both MERS-CoV and Ty-BatCoV-HKU4 could bind to camel DPP4. Interestingly, RBD of MERS-CoV did not bind to T.pachypus DPP4, whereas RBD of Ty-BatCoV-HKU4 could weakly bind to T.pachypus DPP4. The results suggested that DPP4-utilizing ability in Betacoronaviruses from vesper bats could be involved during evolution and interspecies transmission of the ancestors of MERS-CoV.