我国一株新型黄头病毒的分子流行病学

Molecular Epidemiology of a New Yellow Head Virus Strain in China

为查明黄头病毒(Yellow Head Virus,YHV)在我国的存在和变异情况,本研究采用世界动物卫生组织(OIE)《水生动物诊断手册》中YHV套式RT-PCR检测方法对2012–2014年采集的299份样品进行了YHV监测,并对部分YHV阳性样品基因进行了克隆测序及系统发育分析。流行病学调查显示,299份样品中YHV的阳性率为11%,我国养殖的中国明对虾、凡纳滨对虾、日本囊对虾以及罗氏沼虾的部分样品中均检出了YHV,中国明对虾和罗氏沼虾是本次调查中新发现的YHV自然宿主,而且YHV在中国明对虾中的检出率最高。对6份较强阳性样品YHV基因组ORF1b内1002 bp的分型片段进行克隆测序和序列分析,序列比对结果显示,阳性样品的YHV与国外报道的YHV的6个基因型相似度为81.0%–90.5%;系统发育分析显示,6份阳性样品归于同一分支,但与已知YHV的6个基因型均不在同一分支内,其与YHV基因1型(YHV-1)亲缘关系较近。对阳性样品YHV基因组ORF3内编码gp116蛋白的一段序列进行克隆测序,得出其序列长度为509 bp,与YHV-1a的545 bp、YHV-1b的383 bp和YHV-2(即鳃联病毒GAV)的476 bp均不同;依据该片段构建的系统发育树显示,6份阳性样品归于同一分支,与YHV已知的6个基因型不同,与YHV-1亲缘关系较近,且与YHV-1a相似度大于YHV-1b。对其中两份样品的ORF2序列进行比对显示,两份样品序列相似性为99.8%,蛋白序列完全相同,与YHV-1的序列相似度为85.9%,与YHV-2相似性为80.9%。样品调查结果对增补YHV的宿主范围有重要意义;YHV核酸检测和序列比对结果表明,感染我国养殖对虾的YHV为一种新的致病株型。

Yellow head virus （YHV） is a highly lethal pathogen that causes majority of shrimp diseases. In this study we conducted the epidemiology study and revealed the existence of YHV and the current infectious situation in China. We collected 299 samples in 2012-2014 and detected YHV using the nested RT-PCR method that was recommended in the World Organization for Animal Health （OIE） manual. Genes of some YHV-positive samples were sequenced and underwent the phylogenetic tree analysis. The epidemiology survey showed that the YHV infection rate was 11% and this virus was found in cultured Litopenaeus vannamei, Fenneropenaeus chinensis, Marsupenaeus japonicus and Macrobrachium rosenbergii in China. F. chinensis showed the highest infection rate. E chinensis and M rosenbergii were newly reported as natural hosts located in the ORFlb region. The alignment results of YHV. We then amplified a 1002 bp fragment suggested that YHVs from the six infected samples shared 81.0%-90.5% sequence identity with the six YHV genotypes archived in NCBI. The phylogenetic tree analysis showed that YHVs from the six samples clustered in one branch, but they did not share the same branch with the six already known YHV strains. YHVs in the 6 samples were relatively closely related to YHV Type 1 （YHV-1） and had higher similarity with YHV-lb than with YHV-1a. We also amplified a 509 bp sequence located in the ORF3 region. It encoded gpll6 and was different from the 545 bp of YHV Type la （YHV-1a）, the 383 bp of YHV-1b and the 476 bp of YHV-2 （GAV）. We next compared the ORF2 sequence of two samples and found that they were 99.8% identical in gene sequence and had the same protein sequence. Their sequence similarities with YHV-1 and YHV-2 （GAV） were 85.9% and 80.9% respectively. Results of gene sequence alignment suggested that the YHV found in China was a new pathogenic strain.