Evolution of Avian coronavirus (AvCoV) in BHK-21 and VERO cells

Abstract

Avian coronavirus (AvCoV) infects a range of tissues in chickens and several other avian species. Although the virus can be isolated in chicken embryos, only a few strains of the 6 genotypes/33 lineages can grow in cell lines, with the Beaudette strain (GI-1 lineage) being the most used for in vitro studies. Considering the differences between cell lines and chicken embryos as habitats for AvCoV, this study aimed to assess the diversity of the genes coding for the nonstructural protein 3 (nsp3) and spike envelope protein (S) after serial passages in BHK-21 and Vero cells. After 14 passages of an embryo-adapted Beaudette strain, the virus loads fluctuated in both cell lines, with the highest loads being 8.72 log genome copies/µL for Vero and 6.36 log genome copies/µL for BHK-21 cells. No polymorphisms were found for nsp3; regarding S, not only aa substitutions (Vero: 8th passage A150S, and 14th S150A; BHK-21: 4th S53F, 8th F53Y, and 8th S95R), but also minor variants could be detected on chromatograms with fluctuating intensities. As the regions of these aa substitutions are within the receptor-binding domain of S, it can be speculated that differences in cell receptors between Vero and BHK-21 cells and the speed of cell death led to the selection of different dominant strains, while the stability of nsp3 supports its function as a protease involved in AvCoV replication. In conclusion, AvCoV quasispecies evolution is influenced by the biological model under consideration, and a gradual transition is seen for minor and major variants.