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Genomic Sequence of IBV ck/CH/LNM/091017 Isolated in China and Protection Conferred by Attenuated Vaccine

Author: SunChuYang
Tutor: LiuShengWang
School: Northeast Agricultural University
Course: Zoology
Keywords: Infectious Bronchitis Virus Whole-genome sequence analysis Immune Efficacy Evaluation
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 121
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Abstract

Our group incidence broiler flocks separation from Inner Mongolia in 2009 to get a virus, virus isolates morphology observation, the chick embryo pathogenicity tests, hemagglutination characteristics such as testing the biological characterization and RT-PCR method to detect, identified as chicken infectious bronchitis virus. In this study, using RT-PCR method ck/CH/LNM/091017 isolates genome sequence determined, with 27 reference strains genome sequence analysis and comparison and phylogenetic trees to illustrate ck / CH / LNM / 091 017 isolates with reference strains between phylogenetic relationships and molecular genetic variation between, the immune efficacy evaluation and animal protection test isolates in this study. Diseased tissue samples collected from the onset of chickens, and 9 to 11-day-old SPF chicken chorioallantoic fluid collected within 72 hours (discard 24h dead embryos) treated after inoculation. Passaged chick embryo dwarf embryo, embryo diffuse bleeding, amniotic membrane thickening lesion characteristics. Virus allantoic fluid with 1% chicken peripheral blood agglutination of red blood cells do not occur, electron microscopy virus particle was typical coronavirus, initially identified as the avian infectious bronchitis virus. This study by RT-PCR method the ck/CH/LNM/091017 isolates identification and determination of the complete genome sequence, and 27 reference strains genome sequence comparison analysis results show that the ck/CH/LNM/091017 isolates have typical avian coronavirus gene sequence characterized 5'-Replicase-S-3a-3b-M-5a-5b-of N-3 '. ck/CH/LNM/091017 isolates genome of 27646bp AT content of 61.88%, with 27 reference strains gene homology between 85.7% to 99.9%. Phylogenetic analysis of system evolutionary tree genome sequences of genes in the genome, isolated in this experiment IBV strains (ck/CH/LNM/091017) the Mass-type reference strain distribution in the same gene cluster, is a Mass-type IBV. isolates non-structural proteins of ck/CH/LNM/091017 of ORF1a and ORF1b by 11802 and 7959 nucleotides that encode 3933 and 2652 amino acid polypeptide. The emergence of the IBV genetic variant is a change of a few amino acids of the S protein, wherein the S1 gene diversity resulting from point mutations, insertions, deletions, or recombinant, the main reason for a new serotype, subtype or variant strain is IBV. ck/CH/LNM/091017 S gene open reading frame consisting of 3489 nucleotides, encodes 1162 amino acids, the cleavage site is Arg-Arg-Phe-Arg-Arg, wherein the S1 gene is composed of 1611 nucleotides, encodes a 537 amino acid polypeptide consisting of residues. M protein and E protein, respectively, encode 225 and 109 amino acids. N protein is composed of 1230 nucleotides encoding 409 amino acids. nucleotide sequence homology of nucleotide sequence Mass-type vaccine strains H120 of ck/CH/LNM/091017 isolates are more than 99.0%, kinship very close, so ck/CH/LNM/091017 the strain may be a new strain or vaccine virus isolated occurrence of point mutations generated by the vaccine strain. IB live vaccines exist virulence atavism certain pathogenic and ability to spread, and the vaccine strains, attenuated vaccines have the potential to become the main body and the donor of the recombinant variation of the mutation. The attenuated vaccine H120 isolates of animal protection test of ck/CH/LNM/091017 immune flocks IB onset of symptoms, the incidence rate of 100% of non-immune chickens, the mortality rate was 10%. The the necropsy incidence chicken, death chicken pathological changes in the performance of the trachea flushing, bleeding, catarrhal mucus, cecal tonsil bleeding, no kidney enlargement and urate deposition typical lesions of the kidney-type \IBV antibody detection flocks collected serum samples by indirect ELISA method, to the antibody is detected in the serum samples of the immune chickens from the ninth day after immunization, 90% serum was positive. 20 days after immunization isolates ck/CH/LNM/091017 challenge test. Three days after the challenge, the immune flocks 100% detected antibodies. Non-immune chickens after challenging nine days, serum IBV antibody detected all positive. Five days after the challenge, each the random culling chickens, collecting tissues and organs, virus detection by RT-PCR method, H120 immune ck/CH/LNM/091017 challenged groups flock trachea, cecal tonsil and Proventriculus virus detection rates were 60%, 60%, 20%, and the virus was not detected in the kidneys and lungs; the ck/CH/LNM/091017 attack flocks of venom group the trachea and Proventriculus 100% detection of viruses, kidneys, lungs and cecal tonsil virus detection rate of 20%, 20%, 60%, respectively. These data indicate that the the H120 attenuated vaccine chicks kidney can provide better immune protection, but can not provide complete protection the ck/CH/LNM/091017 isolate.

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