Emergence of Avian coronavirus Escape Mutants Under Suboptimal Antibody Titers

To perform a quasispecies assessment of the effect of vaccine combinations and antibody titers on the emergence of Avian coronavirus (AvCoV) escape mutants, 5-week-old males from a commercial chicken breeder lineage were vaccinated intramuscularly with one dose of a monovalent (genotype GI-1) or a bivalent (genotypes GI-1 and GI-11 (n = 40 birds/group) AvCoV vaccine. Seven birds were kept as controls. Six weeks later, pools of sera of each group were prepared and incubated at virus neutralization doses of 10 and 10^-1 with the Beaudette strain (GI-1) of AvCoV in VERO cells. Rescued viruses were then submitted to genome-wide deep sequencing for subconsensus variant detection. After treatment with serum from birds vaccinated with the bivalent vaccine at a titer of 10^-1, an F307I variant was detected in the spike glycoprotein that mapped to an important neutralizing region, which indicated an escape mutant derived from natural selection. Further variants were detected in nonstructural proteins and non-coding regions that are not targets of neutralizing antibodies and might be indicators of genetic drift. These results indicate that the evolution of AvCoV escape mutants after vaccination depends on the type of vaccine strain and the antibody titer and must be assessed based on quasispecies rather than consensus dominant sequences only because quasispecies may be otherwise undetected.