Evolution of avian infectious bronchitis vaccine viruses reisolated from vaccinated broilers

Genetic and phenotypic diversity among infectious bronchitis virus (IBVs) is a key element of the epidemiology of avian infectious bronchitis. Virtually, all studies have focused on the evolution of wild viruses. Live-attenuated vaccine strains deserve a special attention. Two strains of IBV, 2T/17 and 16T/17, detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in samples from H120 vaccinated chickens showing respiratory signs, were genetically analysed targeting the S1 gene. The total RNA was extracted by the classical Trizol method. The whole S1 gene was amplified in an end-point RT-PCR using a specific primer pair, before being sequenced. The obtained sequences were compared to those of vaccine strains and reference viruses and finally subjected to a phylogenetic analysis. The two isolates turned out to be vaccine strains as evidenced by the monophyletic cluster they formed with the H120 vaccine strain (100% bootstrap). Also, they shared with this vaccine strain 99.60% and 99.47% of nucleotides (nt) for strain 2T/17 and 16T/17 respectively. The sequence alignment revealed, for each of the two strains, one non-synonymous nt substitution outside the hypervariable regions: C56T and T39G leading to two amino acid (aa) substitutions: Alv19Val and Cys13Trp, respectively for the strain 2T/17 and 16T/17. The second hypervariable region of strain 2T/17 displayed 2 nt substitutions at positions 345 and 347, resulting in an aa substitution Lys116Thr. As for 16T/17 strain, it harboured two non-synonymous nt substitutions (T353G and T380G), leading to two aa substitutions: Val118Gly and Met127Arg. The chromatogram analysis revealed minor and major peaks at all positions where nt changes were observed. Nucleotides associated with minor peaks matched with those observed in the sequence of the H120 vaccine strain at the same positions, indicating subpopulation selection. Only the substitution T353G results in a mutation as evidenced by the absence of minor peak at this position. Viral subpopulations contained in IBV live-attenuated vaccines are susceptible to be in vivo selected and emerge as persistent pathotype with reversion to virulence. This draws attention to the need of monitoring the evolution of IBV vaccine strains.