鸡传染性支气管炎病毒变异株全基因组序列分析

Complete Genomic Analysis of A Novel Infectious Bronchitis Virus Isolate

为了明确传染性性支气管炎病毒（Infectious bronchitis virus,IBV）分离株CK/CH/SD09/005的分子特征,以进一步丰富国内IBV的分子流行病学信息。本研究设计了25对引物对其全基因组进行了序列测定,并与参考株进行了同源性比较和S1基因遗传进化分析。结果显示CK/CH/SD09/005基因组为27 691bp（不包括5′端Cap和3′端Poly A）。全基因组同源性比对发现,CK/CH/SD09/005仅与GenBank中广西2009年分离株GX-NN09032各基因高度同源（97%~99%）。除GX-NN09032外,CK/CH/SD09/005基因组5′端复制酶基因（Gene 1）和3′端非转录区（Untranslated region,UTR）与2个QX基因型参考株ck/CH/LDL/091022和SDIB821/2012同源性最高,分别为97%和98%,但是3′端结构蛋白和非结构蛋白基因（S-3a-3b-3c/E-M-5a-5b-N）与这两个毒株同源性较低,仅为72%~90%。其ORF3c/E、5a、5b和N分别与韩国分离株1011、国内分离株CK/CH/LXJ/02I、DK/CH/HN/ZZ2004和YX10同源性最高,分别为97%、96%、99%和96%,而其ORF3a、3b和M与参考株同源性均低于90%。S1基因遗传进化分析发现,CK/CH/SD09/005和国内外39个参考株形成7个进化分支（基因型）,CK/CH/SD09/005和2007以来几个分离株属于基因Ⅳ型,与其它6个基因型参考株S1和S2基因同源性为66%~69%和72%~81%,S1基因不仅表现广泛性点突变,而且有多处碱基插入和缺失,S2仅表现点突变。本研究结果表明CK/CH/SD09/005是一个变异株,可能是QX基因型IBV流行株与其它毒株重组进化而来,此外还涉及基因突变、插入和缺失等多种变异机制。

The genome of CK/CH/SD09/005,an isolate of infectious bronchitis virus（IBV）,was characterized to enable the further understanding of the epidemiology and evolution of IBV in China.Twenty-five pairs of primers were designed to amplify the full-length genome of CK/CH/SD09/005.The nucleotide sequence of CK/CH/SD09/005was compared with reference IBV strains retrieved from GenBank.The phylogenic relationship between CK/CH/SD09/005and the reference strains was analyzed based on S1gene sequences.The complete genome of CK/CH/SD09/005consisted of 27691nucleotides（nt）,excluding the5′cap and 3′poly A tail.The whole-genome of CK/CH/SD09/005shared 97–99% nucleotide sequence homology with the GX-NN09032strain,which was the only complete genome that was closely related to CK/CH/SD09/005.When compared with all reference strains except GX-NN09032,CK/CH/SD09/005showed the highest similarity to ck/CH/LDL/091022and SDIB821/2012（QX-like）in the replicase gene（Gene 1）and 3′UTR,with a sequence identity rate of 97%and 98%,respectively.However,CK/CH/SD09/005exhibited lower levels of similarity with ck/CH/LDL/091022and SDIB821/2012in S-3a-3b-3c/E-M-5a-5b-N with a sequence identity of 72%–90%.CK/CH/SD09/005showed the highest level of nucleotide identity with Korean strain 1011,and Chinese strains CK/CH/LXJ/02I,DK/CH/HN/ZZ2004and YX10,in ORF 3c/E（97%）,5a（96%）,5b（99%）and N（96%）,respectively.ORFs 3a,3band M of CK/CH/SD09/005exhibited no more than 90% homology with the reference strains,excluding GXNN09032.The phylogenic analysis based on the S1gene revealed that CK/CH/SD09/005and 39published strains were classified into seven clades（genotypes）.CK/CH/SD09/005was distributed in clade IV with several isolates collected between 2007and 2012.CK/CH/SD09/005showed 66%–69%and 72%–81%nucleotide identities with the IBV strains of other six clades in the S1and S2subunits,respectively.Moreover,multiple substitutions were found throughout the entire S gene of CK/CH/SD09/005,while insertions and deletions were located within the S1gene.These results indicated that CK/CH/SD09/005is a novel variant that may be derived from the QX-like strains that are prevalent in China.Multiple genetic mechanisms,including recombinations,mutations,insertions and deletions,are likely to have contributed to the emergence of this IBV strain.