MOLECULAR DETECTION OF INFECTIOUS BRONCHITIS VIRUS IN CHICKEN
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Date
2019
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College of Veterinary and animal Science,Mannuthy
Abstract
Avian infectious bronchitis, an acute highly contagious disease of chickens,
affects birds of all age groups. The present study envisaged the isolation,
identification and molecular characterisation of IBV. For the study, samples were
collected from chickens of different ages with signs of respiratory tract infection. Out
of the 95 samples collected, 42 were positive for IBV using specific UP and DOWN
primers targeting the highly conserved 5’UTR region. The samples were inoculated
into nine to eleven day-old embryonated chicken eggs via., allantoic cavity. Virus
inoculated embryos after six passages exhibited the characteristic curling and
dwarfing. The harvested allantoic fluid revealed the presence of IBV by RT-PCR in
42 samples. Reverse transcriptase polymerase chain reaction identified IBV in three
more allantoic fluid. A Taqman probe based real time PCR was developed targeting
the highly conserved 5’UTR and N genes which detected 45 positives in suspected
tissue samples and allantoic fluid. The hypervariable regions 1, 2 and 3 of spike gene
of all the positive samples were amplified by RT-PCR using three different primers
and the respective amplicons were sequenced to study the genetic diversity of the
isolates. The sequences of the isolates were compared with those of commercial
vaccine strain (H120) and other Indian isolates available in NCBI GenBank database.
All the sequences showed more than 95 per cent similarity with the commercial
vaccine strain as well as with the Indian isolates from various regions. Phylogenetic
tree was constructed based on the obtained sequences. On phylogenetic analysis of
hypervariable regions 1 and 2, all the four Thrissur isolates clustered with each other
and also with the IBV isolates from different regions. Whereas, on phylogenetic
analysis of hypervariable region 3, all the four Thrissur isolates were branched
separately among themselves and also with the IBV isolates from different regions of
the country. Thus, a comprehensive analysis of these variations could aid in the
selection of a suitable candidate vaccine strain.