title: Rapid detection and characterisation of infectious bronchitis virus (IBV) from New Zealand using RT-PCR and sequence analysis.
abstract: OBJECTIVE
  To develop a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect infectious bronchitis virus (IBV) from commercially-raised poultry in New Zealand and compare results with those from virus isolation. To characterise the IBV isolates using sequence analysis.
  
  
  METHODS
  Pooled tissue samples (trachea, kidney, caecal tonsils and cloacal swabs) from 164 broiler and 53 layer flocks located throughout New Zealand were collected in transport medium containing antibiotics. Tissues were homogenised and the resultant supernatant used directly in a RT-PCR assay, and also inoculated into the allantoic cavity of 10-day-old embryonated eggs for virus isolation. Primers for the RT-PCR were selected from an area close to the N-terminus of the S1 (spike) gene and bracketed the hypervariable region 1 (HVR 1). The RT-PCR amplimers were sequenced from both termini, and alignment was constructed and analysed.
  
  
  RESULTS
  From the 217 field samples that were subjected to RT-PCR, 42 (19%) were positive. Twenty-nine (69%) of these RT-PCR-positive, and none of the RT-PCR-negative, samples yielded virus by isolation in chicken embryos. A phylogenetic tree constructed from these amplimers, that spanned the HVR of the S1 gene, revealed the IBV isolates clustered into two demarcated groups which had <60% homology. It is likely that the isolates of one group were derived from the live attenuated vaccine commonly used in New Zealand.
  
  
  CONCLUSIONS
  The RT-PCR assay exhibited higher sensitivity than virus isolation and could be used for rapid diagnosis of IBV in the field. The prevalence of IBV appears to be surprisingly high in New Zealand although the use of pooled samples in the study did not allow accurate calculation of the prevalence in birds. Sequence analysis of a hypervariable region from the S1 gene was informative for the differentiation of closely-related strains.
authors.affiliation: Agriculture and Life Sciences Division, Lincoln University, PO Box 84, Lincoln, New Zealand.
authors.lastname: Ramneek
authors.forename: N L
authors.initials: NL
authors.lastname: Mitchell
authors.forename: R G
authors.initials: RG
authors.lastname: McFarlane
mesh_terms: D000818:Animals; D001483:Base Sequence; D002645:Chickens; D018352:Coronavirus Infections / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary*; D005260:Female; D001351:Infectious bronchitis virus / Q000145:classification / Q000302:isolation & purification*; D008297:Male; D009520:New Zealand / Q000453:epidemiology; D010802:Phylogeny; D011201:Poultry Diseases / Q000175:diagnosis* / Q000453:epidemiology; D015995:Prevalence; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000662:veterinary
chemical_list: D012367:RNA, Viral