Pathogenicity And Molecular Characterization Of A New Infectious Bronchitis Virus Isolate In China

Infectious bronchitis virus(IBV)is a protectotype,enveloped and positive-strand RNA virus of family coronaviridae.It is a highly contagious virus of chicken,and produces respiratory,urinary and reproductive tract infection called infectious bronchitis disease(IB).It enters in the body through the oral,nasal and ocular routes,replicates in the cytoplasm of the respiratory epithelial cells and spreads to all body tissues such as the ovaries,kidneys,testes,muscles,gastro-intestinal tract and proventriculus.It is excreted through the respiratory secretions and feces.Infected chicken show depression,decreased feed intake,nasal discharge,sneezing,tracheal rale and caceous plug formation in the trachea,leading to asphyxia and death.IBV causes 100%morbidity and 25-80%mortality in young chicken.IBV has a high mutation rate and new strains always emerge by insertion,deletion and recombination.The spike gene has major antigenic determinants which makes it attractive as a vaccine candidate.Presently,vaccines available in the market do not provide complete protection.There is dire need to prepare a new vaccine,which can protect circulating IBV strains.In the present study,a new IBV XDC-2 was isolated from the vaccinated flock,pathogenicity and molecular characterization was analyzed.The contents of the study are as follows:1.Pathogenesis of an infectious bronchitis virus isolate in ChinaIn China,IBV is one of the most important viral pathogen of poultry since 1980.A new IBV XDC-2 strain was isolated and inoculated in 10-day-old chicken embryonated eggs.The pathological lesions in the developing embryos were detected.IBV was confirmed using RT-PCR from the allantoic fluid.The virus titer of 50%embryo infectious dose(EID50)was calculated 5×10-5.33/ml.In another experiment,one-day-old specific pathogen free chickens were inoculated with a lethal dose of the same strain of IBV by different routes(oral,nasal and ocular),to observe the pathogenicity,morbidity and mortality rates. Eighty one-day-old SPF chicks were divided into four groups with 20 chicks per group.The three groups were inoculated with 200?l egg albumin through the oral,nasal and ocular routes while 20 chicks were inoculated with orally sterile PBS as a control group.In infected chicken,clinical signs observed were similar to the nephropathogenic IBV.The dead or diseased chicken's lungs did not show any prominent macroscopic lesions and the control group also did not show morbidity and mortality.However,kidneys were highly inflamed,visibly pale and distended with massive urate deposits.The histopathological examination revealed nephritis,hyaline degeneration,tubular dilatation,necrosis of the epithelial cells and severe infiltration of the interstitial inflammatory monocyte cells.The virus was reisolated and detected from the chicken kidneys by RT-PCR.It confirmed that IBV XDC-2 strain had much tropism to kidneys and predominantly nephropathogenic strain isolates from China.2.Full length genome sequencing of an infectious bronchitis virus isolate XDC-2The full length genome sequence of IBV XDC-2 was obtained by RT-PCR.The genome sequence was 27714 nt in length,and had a similar genome organization to other IBV strains.The S glycoprotein gene(S1 and S2)had the highest nucleotide identity with the nephropathogenic BJ strain.However,S1 gene amino acids compared with available IBV vaccine strains H120,H52 and Ma5 showed eight amino acid insertions(YSNGNSDV)at 73-80 site and three amino acid deletions at(126-128).Moreover,the full length genome sequence compared pairwise,minimum divergence,phylogenetic analysis and distance matrix results showed that it had the maximum relationship to the indigenous BJ strain.Further,S gene recombination analysis confirmed that this isolate evolved by the recombination with previously circulating BJ strain and KM91 vaccine strain.All results indicated that XDC-2 was a new nephropathogenic IBV strain.3.Construction of recombinant plasmids containing IBV S1 and Ii-key segment of chicken major histocompatibility complex?geneNephropathogenic IBV XDC-2 strain S1 gene and Ii-key segment of chicken major histocompatibility complex(MHC-?)gene tagged with Flag were amplified(S1-F and Ii-F)with the expected band size(1684bp,705bp)and confirmed through agarose gel electrophoresis and purified.S1,Ii and DNA cloning vector pcDNA3.1 were digested by restriction endonuclease enzymes and the DNA fragment Sl-F and Ii-F were cloned into pcDNA3.1 vector.Recombinant plasmids were confirmed by colony-PCR,enzyme digestion and sequencing.Further,recombinant plasmids were transfected into BHK-21 cells for protein expression.Cells lysate were used for immune-blot analysis,S1 gene and Ii gene were expressed with expected protein bands of 61KDa and 26KDa respectively.However,in this study,the primary animal experiment results showed that the humoral and cellular responses of pcDNA-S1-F induced more significant responses than pcDNA-Ii-F or co-administration of both recombinant plasmids.The animal immune experiments should be done in the future.