不同动物源脑心肌炎病毒的分离、鉴定和全基因组序列分析

为研究脑心肌炎病毒（encephalomyocarditis virus,EMCV）对不同动物宿主的感染情况。本试验应用改进的＂细胞接种与RT-PCR方法相结合＂技术,成功分离到国内首株地方土猪源EMCV、首株家养野猪源EMCV、1株鼠源EMCV和3株良种猪源EMCV,并进行了全基因组序列测定和分析。结果显示,6株EMCV分离毒的基因组全长从7 724~7 735bp不等,ORF长度均为6 879bp,彼此间核苷酸同源性为99.3%~99.8%,与其他不同动物源EMCV参考毒株的同源性为79.9%~99.9%,与国内猪源、鼠源分离毒的同源性均在99.4%以上;各基因片段中,以VP1和2A变异幅度最大,VP2和3D最为保守;基于EMCV全基因组、ORF和VP1基因序列绘制的系统发育进化树显示,EMCV可分为G1、G2和G3 3个群,猪源EMCV主要分布在G1、G2群,鼠源EMCV在G1、G3群中均有分布;6株EMCV分离毒与其他国内参考毒株同属于G1群,但分布并不完全集中。结果表明,地方土猪和家养野猪可感染EMCV并引起发病,提醒在进行地方品种养殖和野生动物家养时要充分考虑人兽共患疫病传播的生态学;鼠在良种猪、家养野猪和地方土猪EMCV之间的交叉感染、传播与流行中起到重要媒介作用;不同EMCV地方流行株间存在较大的地域差异,其传播具有一定的区域限制性;EMCV在感染不同动物时可能会发生某些突变,以适应新的宿主。

Isolation,identification and full length genome sequence analysis of EMCV from different animal source

In order to study the infection status of EMCV in the different animal hosts.Modified＂cell inoculation and RT-PCR＂technology was used in this study,and the 6EMCV isolated strains from local aardvark,domesticated boar,mice and improved breed pig.We performed the full length genome sequencing and molecular characteristic analysis of the EMCV strains.The results showed that the full-genome sequence of 6EMCV isolates generated a sequence of 7 724-7 735 bp in length,ORF length of 6 879 bp,and had 99.3%-99.8%nucleotide identity with each other,but 79.9%-99.4% with reference EMCV strains from different animal sources,99.4% with Chinese strains from pig and mice.Among all gene fragments,VP1 and 2Aare easy to mutation,the most conserved is VP2 and 3D.The phylogenetic tree based on the full length genome,ORF and VP1 gene sequences,EMCV was divided into G1,G2 and G3groups.The results of this study showed the isolates from pig belong to G1 and G2groups,the ones from mice distribute in G1 and G3group,6isolates strains in this study belong to G1 group with other Chinese reference strains,but the distribution is not complete.The results identified that the EMCV infection could cause severe clinical symptoms in local aardvark and domesticated boar,which remind us of taking fully consideration of the zoonosis ecology in the course of wild animal breeding activities.Mice plays an important medium role in cross infection,transmission and prevalence of EMCV infection in improved pigs,domesticated boar and local aardvark.A big regional differences exist in EMCV and the transmission is limited in a range of area,moreover,some mutation may occurred in EMCV infection to adapt new hosts.