| Avian Infectious Bronchitis(IB) is an acute.highly contagious respiratory disease characterized by sneeze,fever,depression. AH ages,sexes,types of chickens are susceptible to the disease. Layers is often found with decline in egg production and egg quality. The disease is caused by Infectious Bronchitis Virus (IBV) and happened in most areas of China.Although vaccine strains were used, vaccinated chicken flocks were still susceptible to the disease and caused heavy economic loss.IBV is the typical virus of Coronavirus in the family Coronaviridae which is a single-strand positive-sense RNA genome with poly A tail at the 5' end and cap-structure at the 3' end.There are six types of mRNAs(mRNA1 - mRNA6) in infected cells. The three main structure proteins of IBV are the spike glycoprotein(S protein),the membrane glycoprotein(M protein) and the nucleocapsid protein(N protein)which are encoded by mRNA2, mRNA4, mRNA6 respectively. mRNAl conains two ORFs, namely, ORFla and ORFlb.The two ORFs usually encode polyproteins that relative molecular weight are 441kD and 300kD. The downstream ORFlb with upstream ORFla express a fusion protein of 750kDa ,called pplab, then the pplab is proteolytically processed into functional products.In the study, LH2/01/10(Nephropathogenic type from HeiLongJiang province), D971 (Glandular Stomach type from DaLian) and LX4 (Nephropathogenic strain from Xinjiang province) were used for sequence analysis.The isolates were propagated in 10-day specific-pathogen-free(SPF)embyonated chickens eggs and collected allatoic fluid at the 1:256 haemagglutination titer ,then were purified.The predicted fragments were amplified by reverse-transcription polymerase chain reaction(RT-PCR).The structure and non-structural protein genes of the three isolates were cloned and sequenced according to the predicted primers. Phylogenetic tree were mapped with N,M,S,3c and 5a,5b,3a,3b genes.The sequence analysis showed the N gene of the three-isolates were all composed of 1230bp for encoding a polyprotein of 409 amino acids. There were no desertion and deletion, but they had some point mutations. N genes of the three isolates had highly homology in comparison with that of SD/97/02. but in some gene regions,they share 100% homology compared to HI20 and H52. The phylogenetic tree of N gene indicated that LH2/01/10 and LX4 had closely relation with the strains in China.but D971 was close to the foreign reference strains.From the data we deduced that the three isolates perhaps experienced the evolution in different areas to lead to the difference in genetic characterization. M genes of the three isolates were different in the number of nucleotides, LH2/01/10 had 687bp(228AA), D971 681bp(227AA) and LX4678bp(225AA) respectively,but in the main region they showed a high degree of conservativity. The phylogenetic tree of M gene indicated that the three strains were closer relations with strains in China, but different from foreign strains. S protein is the most important iminunogen of IBV. especially S1 subunit,it could induce immuno system to produce protective antibody,however S1 genes were highly variable.Sequence data demonstrated that the S genes of LH2/01/10 and LX4 were composed of 3495bp, encoding 1164 aa, the cleavage site sequence of S protein among them contained five consecutive basic amino acids, namely, His-Arg-Arg-Arg-Arg (HRRRR),which was typical for the isolates in China. Slgenes of the two isolates contained 1614bp for encoding a polypeptide of 539 aa. S gene of D971 was composed of 3498 bp, encoding 1165 aa, the cleavage site sequence was NSRRR, S1 gene contained 1614bp(538 aa), S2 was 1884bp(627 aa). The phylogenetic tree of S1 gene indicated that the strains of different countries have obvious difference, LH2/01/10 and LX4 were classified as the same group, there were closer relations with HI20 and H52 than D1466 and DE072 which had only 36% homology. D971 was in another group and it deduced different evolution.The number of nucleotides in E genes among the three isolates were 336bp,309bp and... |
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