| Infectious bronchitis(IB)is characterized by coughing, tracheal rales, sneezing, and lesions in kidney and reproductive tract. This disease can cause mortality in young chickens and result in economic losses due to poor weight gain and reduction in egg quality and quantity. Infectious bronchitis virus(IBV), the causative agent of IB, is the prototype of the Coronaviridae family. The virus has a worldwide distribution, and many different variant have been isolated. IBV is an important pathogen of poultry industry and despite routine use of vaccines, variant stains continue to cause outbreaks in field situations. The continuing emergence of new strains may be explained in part by the apparent common occurrence in nature of recombination between IBV strains.IBV is enveloped virus with a single stranded RNA of positive polarity, approximately 27.6kb in length. IBV RNA encodes four major structural proteins: the spike protein(S), the nucleocapsid protein(N), the membrane protein(M)and the envelope protein(E). Molecular studies with IBV have shown that new IBV serotypes and genotypes could emerge as a result of a few changes or mutation in amino acid sequence of the S gene. Recently, study on the M and N protein showed that, point mutations, deletions and recombination events all contribute to the evolution of IBV. Virus-like particle(VLP)formation by the IBV E and M proteins suggests that interactions between these proteins play a critical role in virus assembly.According to the published IBV gene sequence, a pair of specific primers were designed and synthesized. A fragment of 1.7kb including 5'-unique region of mRNA3, mRNA4 and lower part of S2 gene of IBV HLJ1 or SC strains were amplified, cloned and sequenced. Then the nucleotide(nt)and deduced amino acid(aa)sequence of this fragment were analyzed. The result showed that there were no deletions and insertions in 3a, 3b, 3c and M gene of HLJ1 or SC. Gene3 of HLJ1 and SC contained three ORFs, 3a, 3b and 3c, which consisted of 174, 189 and 327 nt(potentially encoded 57, 62 and 108 aa), respectively. Meanwhile, gene4 just has one ORF, M, which consists of 678 nt(potentially encoded 225 aa). The homologies of 3a, 3b and 3c nt sequence between HLJ1 and SC were 96.0%, 91.5% and 96.0%, the aa sequences shared homology of 91.2%, 88.7% and 95.4%, respectively. The maximum nt and aa differences of 3a, 3b, and 3c between the isolated SC and the others were 19%(26.3%), 27.5%(41.9%)and 19.9%(21.6%), respectively. The IBV reference strains had 71.9%~98.2%, 64.5%~98.4% and 75.5%~95.4% nt sequence similarity when compared whit IBV HLJ1 3a, 3b and 3c genes, respectively. Additional charaterization of one of the isolates, HLJ1, revealed that its M gene bears approximately 95.9% identity at the nt level and 98.2% identity at the aa level with SC. The maximum nt differences of M gene between the isolates and the others were 16.8% and 16.4%, respectively. The result of phylogenetic tree analysis showed that, HLJ1 and SC were in the same group, which shared greater phylogenetic relationship with home strains. The foreign IBV strains lied in different group.Prediction of secondary structure of E protein of SC and HLJ1 showed, that there were 15aa continuous Coil conformation in the secondary structure of HLJ1 E protein, at the same sites, SC behavedα-Helix and Coil alternation. At the region of 21-23aa and 136-146aa, HLJ1 M protein is mostly characterized Coil, but SC are dominated with α-Helix. The missense mutations of 113aa and 148aa were responsible for the change of secondary structural conformation, which chages from Coil to strand.The amino terminus of the M protein lies outside of the virion particle and is glycosylated, whereas the 20-100aa of M spans the membrane envelope at least three times. HLJ1 appeared stronger antigenic exhibition than SC in 78aa, so we thought there was an epitope at 77-83aa site of HLJ1.Candidate: Zhang GuoqingMajor: Basic Veterinary MedicineSupervisor: Prof. Li Guangxing...
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