Isolation and molecular characterization of nephropathic infectious bronchitis virus isolates of Gujarat state, India

Infectious bronchitis (IB) is a common, highly contagious, acute, and economically important viral disease of chickens caused by Infectious bronchitis virus (IBV, sp. Avian coronavirus). Five pooled tissue suspensions of 50 layer birds and one reference Massachusetts vaccine strain were inoculated into specific pathogen free (SPF) chicken egg for isolation of IBV. Reverse-transcription polymerase chain reaction (RT-PCR) was carried out using post inoculated allontoic fluid to amplify the spike (S) glycoprotein of S1 subunit of IBV. All the eggs inoculated with five pooled tissue samples and vaccine sample showed dwarfing and curling of SPF embryos indicative of IBV. All the five samples and the vaccine sample produced the expected amplicons of 466 bp by RT-PCR. The sequencing of five isolates revealed that all the five sequences were 99.09-100 % similar among themselves and showed 99.10-100 % nucleotide identity with the vaccine strain. On multiple sequence alignment it was found that our isolates were more similar at S1 subunit nucleotide level with the reference Ma5 and H120 vaccine strains than the reference Mass41 strain. The sequences of Anand isolates revealed further genetic changes in the circulating IBV in comparison to previous isolate of Gujarat as well as higher differences with the strains isolated in other states showing substantial changes at genetic level in Indian IBV isolates, which may partially explain the increasing incidences of IB in the country in spite of the vaccination.