Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology

Molecular Charaterization of Infectious Bronchitis Coronavirus CK/CH/LDL/97Ⅰ Isolated from China

Author LiuZuo
Tutor LiuShengWang;RanDuoLiang
School Xinjiang Agricultural University
Course Preventive Veterinary Medicine
Keywords Infectious bronchitis coronavirus Provenventriculus Molecular characterization Passage in embryonated chicken eggs Pathogenicity
CLC S852.65
Type Master's thesis
Year 2006
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Since 1995, a new disease characterized by stunted growth, the provenventriculus swelling, the proventricular papilla hemorrhage and ulcer had been found in some regions of China. This resulted in huge loss for the poultry Industry. In order to study the molecular epidemiology and the genetic background of infectious bronchitis virus in China, and to further to understand the molecular mechanism of variant IBV strain CK/CH/LDL/97 I, structural protein (S, M, N, E) genes and nonstructural protein genes (3a, 3b, 5a, 5b) in 3’ end of genome of CK/CH/LDL/97 I, isolated in China, were amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned, sequenced and analyzed. In addition, CK/CH/LDL/97 I strain was attenuated by serial 115 passages in embryonated chicken eggs (note: the 115th passage were completed in our laboratory by prof. Rong Jun-gong) . This research aims at exploring the variation mechanism of the pathogencity through comparing with the 5th passage and the 115th passage. All studies were listed below:1. Molecular Charaterization of genome: There were 6868 nucleotides (nt) from 5’ end of S gene to 3’ end of N gene. Partial genome sequence analysis showed that CK/CH/LDL/97 I strain had the S-3-M-5-N gene order that is typical of avian coronavirus. S protein gene of CK/CH/LDL/97 I was composed of 3501nt encoding a polypeptide of 1167 amino acids. The precursor of the S protein was cleavaged into S1 and S2 fragments, which comprised 541 and 626 amino acid residues, respectively. The cleavage site sequence was Arg-Arg-Thr-Gly-Arg (RRTGR). The deduced amino acid sequence of S1 gene of CK/CH/LDL/97 I was most similar to that of three proventriculus IBV isolates, Q1(99.1%), J2(98.9%), T3 (98.9%), which were isolated from China. Phylogenetic analysis of S1 proteins indicated that CK/CH/LDL/97 I, Q1, J2, and T3 formed a separated cluster; The S2 gene was more conservation than the S1 gene, and the variation in S2 gene was mainly point mutation. The ORF (Open reading frame) of N gene were composed of 1230nt for encoding a polyprotein of 409 amino acids. There were no desertion and deletion, but they had some point mutations. Phylogenetic analysis of N gene revealed that CK/CH/LDL/97I belongs to a unique cluster; The ORF of M gene includes 681nt for encoding 226 amino acids. Phylogenetic analysis of M gene indicated that CK/CH/LDL/97 I shared closer relation with SD/97/02 and CK/CH/LSHHI/03, isolated in China; Gene 3 comprised three ORFs (3a, 3b, 3c), they included 174nt, 195nt, and 309nt, respectively, encoding 57, 64 and 102 amino acids, respectively. Gene5 had two ORFs (5a, 5b), they included 198 and 249nt, respectively, encoding 65 and 82 amino acids. Evolution relations of Gene5 were the same as that of M gene. Taken together, these results suggested that CK/CH/LDL/97 I might belong to a novel genotype of IBV.2. Relationship between Variation of the pathogencity and passage in embryonated chicken eggs: Median embryo infectious doses of 5th passage and 115th passage indicated that the virulence of 115th passage was stronger than 5th passage After inoculating 15-old-day specific pathogen free (SPF) chickens with the 5th passage and the 115th passage, respectively, the results showed that the pathogenicity of the

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