New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses

In Egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (IBV) are circulating with detrimental effects for poultry industry. A sensitive real-time RT-PCR assay targeting the IBV nucleocapsid gene (N) was developed to screen clinical samples for presence of IBV. Conventional RT-PCRs amplifying hypervariable regions (HVRs 1–2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples. In total, fifty samples from poultry comprising swabs, tissues and allantoic fluid were examined. Twenty eight samples from chickens showed IBV-positive results. Genetic analysis of the HVRs 1–2 of seven samples revealed closest amino acid homology of 83.3-89.7% in four viruses and 96.1–97.7% in the others to the previously described Egyptian variant II (EG/12197B/2012), while all seven samples shared >98.2% amino acid homology at the HVR3 locus with that genotype. In addition, in most of samples a high degree of co-infections with highly pathogenic AIV H5N1, low pathogenic H9N2 and Newcastle disease was found. Mixed infections in this study were detected in 19 out of 28 IBV positive samples. This indicates an intricate situation in Egyptian poultry populations with unknown putative synergistic effects on pathogenicity and spread of these pathogens. Effective control measures including vaccination may be severely compromised.