Investigation of Infectious Bronchitis Virus Strains by Real Time RT-PCR and S1 Gene Sequencing in Broilers

Engin Alp Onen and Yakut Oz

Abstract

In this study, detection of infectious bronchitis virus (IBV) from chicken tracheal swab samples by real time RT-PCR, which was reported as a rapid and sensitive virus detection method, was aimed. Phylogenetic relations in different field and variant strains of IBV by sequencing of hyper variable regions (HVRs) of S1 gene of IBV was determined. Tracheal swabs were taken from 120 broiler chickens with respiratory signs, randomly. Samples were inoculated into the allantoic fluids (AF) of SPF embryonated chicken eggs. RNAs were extracted by AF. RNAs were translated to ss cDNAs. Real time RT-PCR was performed with double labelled Taqman probe, IBV 5 GU391 and IBV5 GL533 primer pairs. Positive results were obtained in 33 samples of 120 (27.5 %) by real time RT-PCR. cDNAs from positive samples were used for amplification HVRs (Hyper Variable Regions) in S1 gene region with C2U and Ag0723 primers. Samples were amplified by S1 5’mod and CK2 primers, too. RT-PCR products were sequenced directly. Sequencing results have been compared with NCBI’s nucleotide database. Sequencing results were matched to some variant IBV strains in gen banks database at the rate of 72-86 %. These results support that we are faced with variant IBV strains. Also, we observed a lot of SNP mutations in alignment. This study shows that birds can be infected by IBV variant strains at rate of 27.5 %, although they were vaccinated by classic type vaccine. These results demonstrate that vaccination by classical vaccine strains of IBV cannot provide enough cross protection against variant strains. Therefore, outbreaks of the disease still occur in vaccinated flocks. Also, we found these primers are not enough for all IBV field strains to be sequenced and classified. Thus, different primers should be developed and used primer pairs more than two. In this experiment, we found virus load is more in AF than swab samples because of IBV replication in SPF egg AF. We observed dwarfism and hemorrhages at 14 days old chick embryos