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Abstract
ABSTRACT
Infectious bronchitis (IB) remains a major problem in the Egyptian poultry industry despite the usage of many different vaccines. IB caused by Infectious Bronchitis Virus (IBV) which has positive sense single stranded RNA genome with a high genetic diversity, mainly in the S glycoprotein gene. The S gene has specific RNA sequences in the different serotypes-genotypes. In Egypt, IBV strain typing is a difficult process owing to the widespread distribution of four genotype lineages (GI-1, GI-13, GI-16, and GI-23) which may contribute to IBV vaccination failure. This study aimed to develop a duplex real-time quantitative reverse-transcription polymerase chain reaction (Duplex RT-qPCR) that targets the highly conserved areas of the S1 gene in order to detect the Classic (G1) and the Egyptian Variant II (G23) strains in allantoic fluids and clinical samples. The viral genotyping technique was assessed using three commercially available vaccines, four reference strains for other avian pathogens, nine negative samples whereas nine clinical samples and sixteen field isolates were investigated for clinical applicability. The assay was found to be specific for the detection of Classic, and VAR II strains and did not detect VAR I strain nor other avian pathogens like Newcastle Disease virus (NDV), Avian Influenza virus (H9N2 and H5N8) as well as Infectious Bursal Disease virus (IBDV). The results also showed that 28/41 samples tested positive for IBV utilizing rt-qRT-PCR targeting the N gene, and 26 out of the 28 positive samples were genotyped by duplex RT-qPCR targeting the S1 gene, whereas the remaining two non-genotyped samples were VAR 1 (4/91) and VAR I (793/B). Interestingly, the testing could identify combined infections in one sample, indicating a mixed infection with both genotypes. The detection limit of the developed assay was as low as 102 EID50 /1ml for both Classic and Variant II sets. This assay appears to be a valuable tool in regular disease monitoring in order to detect and differentiate IBV. This study also discussed the genetic changes that may occur in spike protein as a result of some field practices such as co-inoculation of two live attenuated vaccines in the same chicken (H120 and IB Var 2 vaccines). After ten passages of both vaccines in SPF chicks, viruses isolated from the 6th and 10th passage were identical to each other and shared (96%) and (83 %) amino acid identity to IB Var 2 vaccine and H120 respectively. However, amino acids substitutions were observed at twenty-six positions in the N terminal domain (S1) compared to IB Var 2 vaccine. Majority of amino acid changes occurred in the receptor binding domain of S1 gene. Hyper variable region II (HVR2) has seven amino acid changes compared to IB Var II vaccine. Isolates of 6th and 10th passages were lacked IBV glycosylation site at position 139 which was observed in IBV/EG/1212B/2012 and IB Variant II vaccine.
Keywords
Infectious Bronchitis Virus, Duplex RT-qPCR, Specificity, Sensitivity, Live attenuated vaccine, Receptor binding domain