一株鸭源传染性支气管炎病毒的分离鉴定及结构蛋白基因和血清型分析

Identification and analysis of structural protein genes and serotype of an infectious bronchitis virus in ducks

摘要:

[目的]对从广西某鸭场发生呼吸道感染的11天龄樱桃谷肉鸭分离到的病毒株进行鉴定，并探索此鸭源病毒分离株的遗传变异情况。[方法]通过血凝试验、鸡胚接种实验、3'端非编码区（3'UTR）基因扩增与序列测定对分离株进行鉴定，并对该分离株的结构基因S1、E、M和N分别进行序列测定以及相似性、系统进化树分析和血清型鉴定。[结果]血凝试验为阴性，接种鸡胚盲传5代后出现侏儒胚，3'UTR基因测序结果表明为传染性支气管炎病毒（IBV）序列。该分离株S蛋白的裂解位点为RRSRR，S1、E、M和N基因与IBV毒株H120、4/91、LTD3核苷酸相似性分别为：78.6%–99.7%、85.4%–100.0%、91.6%–93.2%、86.7%–91.7%。除N基因存在点突变外，S1、E和M基因均存在氨基酸的突变、插入和（或）缺失。系统进化树分析显示，其S1基因属于4/91型，E、M和N基因均为LDT3型。血清型分析表明，该分离株的血清型不同于疫苗株H120和4/91。[结论]此鸭源病毒分离株为IBV，且该分离株的基因型与血清型均发生了变异。本研究结果暗示禽类传染性支气管炎的防控面临着更严峻的挑战。

Abstract:

[Objective] It is to identify and explore the genetic variation of an infectious bronchitis virus isolated from 11-day-old cherry valley ducks that developed suspected respiratory infection in Guangxi province. [Methods] The virus was identified by hemagglutinin test, chicken embryo inoculation and gene sequence analysis of the 3ʹ untranslated region (UTR). The S1, E, M, and N genes of the virus were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), cloned, sequenced, similarity comparison, phylogenetic tree and serotype analyais was conducted. [Results] The hemagglutination test was negative and dwarf embryo appeared after 5 passages of inoculation of chicken embryos. The sequencing result of 3ʹUTR gene indicated the virus was IBV. The sequence of cleavage site within S protein of the virus was RRSRR. The nucleotide similarities of S1, E, M, and N genes with those of H120, 4/91 and LTD3 reference strains were 78.6%-99.7%, 85.4%-100.0%, 91.6%-93.2% and 86.7%-91.7%, respectively. Compared with the reference strains, there is no insertion or deletion in N gene of the virus except mutations. However, substitutions, insertions and (or) deletions were found in S1, E, M genes of the isolated strain. According to the phylogenetic tree analysis, it was clustered into 4/91-type based on S1 gene and LDT3-type based on E, M and N genes. The serotype analysis showed that the serotype of the isolated strain was different from vaccine strains H120 and 4/91. [Conclusion] The strain isolated from ducks was IBV and the isolated strain existed variation both in structural genes and serotype. The present study suggests that the prevention and control of IB in poultry is facing more severe challenges.