JHALA, M. K.PATEL, BHARATKUMAR HIRALAL2018-06-112018-06-112014http://krishikosh.egranth.ac.in/handle/1/5810051036Infectious bronchitis (IB) is a highly contagious acute viral disease of chicken characterized by respiratory signs in growing chickens. IB is a worldwide respiratory disease of major economic importance associated with losses from production inefficiencies and mortality. Some strains of the virus are nephropathogenic and produce interstitial nephritis and mortality. IB is caused by the virus belonging to the Coronaviridae family, Coronavirus genus with more than 26 serotypes. Present work was aimed to carry out isolation, identification, molecular detection and characterization of SI geneof ncpliropathogenicIBV. Fifty tissues samples of kidneys, lungs, trachea and cloaca were collected from dead birds at the Poultry Complex, AAU, Anand suspected for IBV infection during postmortem examination. Tissues from 10 birds were pooled and processed as single sample, and 5 such pooled samples were processed for isolation of IBV in SPF egg embryo. Five tissue homogenates, vaccine sample and PBS (negative control) were passaged thrice in Specific Pathogenic Free (SPF) embo^onated eggs of 9-11 days incubation. Isolates and vaccine sample produced typical lesions of dwarfing and curling in embryos suggestive of presence of IBV in the samples. RNA isolation was done from the allantoic fluids collected after third passage by using QIAamp viral RNA mini kit. RT-PCR was carried out from the infected allantoic fluids of 5 pooled samples, vaccine sample and negative control by using primers XCE2(F) and XCE2(R), targeting SI gene of IBV. All the five samples and the vaccine sample produced the expected amplicons of approximately 466 bp by RT-PCR, where as no amplification was observed in negative control. Direct sequencing of the products generated by RT-PCR was carried out by Sanger sequence method. The obtained sequences were aUgned with each other by using SeqScapc v 5.2.0 sequence analysis software. Sequences were compiled and analyzed using BioEdit software. Deduced amino acid sequences of IBV SI gene were obtained using ExPASy proteomics tools. Length of the nucleotide sequences for ANDGUJIBVl, ANDGUnBV2, ANDGUJIBV3, ANDGUnBV4, ANDGUHBVS, and ANDGUJmVvac were 444 bp, 440 bp, 447 bp, 439 bp, 441 bp, 442 bp and length of the amino acid sequences were 148, 146, 148, 146, 146 and 147 amino acids respectively. The GenBank accession numbers of the nucleotide sequences of Anand isolates obtained were KJ577258 (ANDGUJIBVl); KJ577259 (ANDGUJIBV2); KJ577260 (ANDGUJIBV3); KJ577261 (ANDGU.TIBV4); K.T577262 (ANDGUJIBV5) andKJ577263 (ANDGUnBVvac) obtained. The sequencing of five field samples (Anand isolates) revealed that all the five sequences were 99.09-100% similar among themselves, and 99.10-100% similar with the vaccine sample. The consensus sequence (length 437 bp) was obtained. The multiple sequence alignment of Anand isolates and vaccine sample was done with the three reference vaccine strains (Ma5, HI20 and Mass 41). The nucleotide sequences of Anand isolates were found to have >97% identity with M41,11120, Mass 41 and Ma5 strains of Massachusetts serotype of IB virus. The three reference vaccine strains i.e. Ma5, H120 and Mass41 showed SNVs at positions 708, 1143 and 1146 with ' C ,'T' and 'A' respectively, while the Anand isolates including the vaccine showed 'T', ' C and G at the same positions. Mass41 showed additional 7 SNVs at positions 737, 846, 861, 1017, 1104, 1119 and 1136 with T, T, T, C, T, T, and C nt respectively, while all other strains showed C, A, C, T, C, C and T at the respective positions. Multiple amino acid sequence aligimient revealed that all the strains under comparison had similar amino acid sequences except the reference vaccine virus strain Mass41, which showed I (isoleucine) and S (serine) at positions 246 and 379, while all the other strains including Anand isolates showed T (threonine) and L (leucine) at the same positions. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 6. Nucleotide sequences of 16 IBV isolates/strains with different pathogenicity were retrieved from the Genbank database and were aligned for sequence analysis and phylogenetic study using the Clustal W programme. The Phylogenetic analysis of nucleotide and aminoacid sequences of selected 16 IBV isolates/strains revealed that the Anand IBV isolates were less similar to Arkansas and Cormecticut strains of USA. The Anand isolates were found to have similarity with nephropathogenic strains 4-91 pathogenic (UK), Australia T (Australia) and CK/CH/Chongqing/0909 (China) with average identity of >78%, >85% and >78% respectively. In addition, the Anand isolates also showed maximum similarity (>99%) with the mass type strain M41 reported from Andra Pradesh, India. This indicates that the Anand isolate are of nephropathogenic Massachusetts serotype. The Anand isolates showed similarities in the range of 77.59% to 78.08% with earlier reported isolate of Gujarat and also lower similarities with isolates from Maharashtra, Orissa, Punjab and Assam. The sequence of Gujarat IBV isolate was reported in the year 2009 and the lower similarity as observed in the current study indicates further genetic changes in the circulating IBV in Gujarat. Phylogenetic analysis revealed that all the Anand IBV isolates were very similar and probably had a common origin. The results of present study revealed that different strain variants of IBV may be circulating among chicken populations in India and recent outbreaks of nephropathogenic IB were due to this reason. Continuous genotyping and phylogenetic analysis of IBV from field outbreaks would be required so as to update the IB vaccines for better protection of chicken populationsenVERTINARY MICROBIOLOGYA STUDYThesis