Studying the emergence potential of betacoronaviruses using serological and reverse genetics approaches

Abstract

Middle East Respiratory Syndrome coronavirus (MERS-CoV) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), two human betacoronaviruses that were emerged in the last decade, are both believed to be of animal origins. However, the definite source of human infections as well as the pathway and mechanisms of interspecies transmission are yet to be fully understood. For MERS-CoV, dromedary camel was found to be the animal reservoir soon after its first emergence, while a more recent study showed that Bactrian camels could be infected by MERS-CoV experimentally. Therefore, I hypothesize that MERS-CoV could also naturally infect and circulate in Bactrian camels. Indirect enzyme-linked immunosorbent assays (ELISA) based on receptor binding domain (RBD) and nucleocapsid (N) proteins were established to detect MERS-CoV specific antibodies in Bactrian and hybrid camels from Dubai, UAE and Bactrian camels from Xinjiang, China. Neutralization antibody test was used to confirm the seropositivity. The results showed that 41.4% (12/29) of the serum samples from Bactrian camels and 54.5% (6/11) of the serum samples from hybrid camels in Dubai were seropositive to MERS-CoV, suggesting that Bactrian and hybrid camels are also potential source of MERS-CoV infections. In 2019, SARS-CoV-2 emerged and caused the worst known coronavirus pandemic in human history. It is hypothesized that ancestors of SARS-CoV-2 may have been circulated in animals. Hence, a competitive ELISA (cELISA) based on N protein was developed for identification of the potential reservoirs of SARS-CoV-2. Ninety-four serum samples of different animals from an amusement park were tested and none of these animals were seropositive to SARS-CoV-2. This in-house developed cELISA can be applied on future field settings for large scale animal serosurveillance, without the need of generating secondary antibodies against different animal species. To prepare for and prevent future coronavirus disease pandemics, it is also necessary to understand the mechanism of interspecies transmission of coronaviruses with zoonotic potential. Tylonycteris bat coronavirus (Ty-BatCoV) HKU4 is phylogenetically closely related to MERS-CoV and utilizes the same cellular receptor, human dipeptidyl peptidase 4 (hDPP4), for cell entry. I hypothesize that recombination between zoonotic coronaviruses may expand host/tissue tropism which enhances its emergence potential in humans. Reverse genetics system was employed to construct a recombinant virus, rMERS-HKU4S, with Ty-BatCoV HKU4 Spike (S) gene substituted in the context of MERS-CoV backbone. The cell susceptibility, replication efficiency, virulence, and pathogenicity of the recombinant virus were compared with those of MERS-CoV and Ty-BatCoV HKU4. The in vitro replication efficiency of rMERS-HKU4S was lower than that of MERS-CoV but higher than that of Ty-BatCoV HKU4. rMERS-HKU4S replicated efficiently in the human lower respiratory tract cell line which is non-susceptible to Ty-BatCoV HKU4, implying the expansion of the cellular tropism after switching Ty-BatCoV HKU4 S gene into MERS-CoV backbone. Moreover, rMERS-HKU4S exhibited a lower in vivo virulence and pathogenicity than MERS-CoV. The inoculation of rMERS-HKU4S could cross-protect the mice from MERS-CoV re-challenge. The study illustrated that recombination of coronaviruses in wild animals could enhance the possible emergence of zoonotic coronaviruses of bat origin in humans.