2013-2014年黑龙江地区猪流行性腹泻病毒检测及M基因的遗传进化分析

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Heilongjiang,China,2013—2014

猪流行性腹泻是由猪流行性腹泻病毒(PEDV)引起的一种严重的病毒性传染病。本试验于2013 2014年在黑龙江省6个地级市的10个规模化猪场采集新生哺乳仔猪临床表现为腹泻症状的粪便与小肠,进行RT-PCR检测,表明PEDV检出率为56%,PEDV+TGEV检出率为6%,PRV检出率为10%,TGEV检出率为10%,结果表明,引起黑龙江省规模化猪场新生仔猪腹泻的主要病原是PEDV,同时也存在与其他腹泻病毒的混合感染。针对阳性病料扩增出的6株PEDV的M基因序列进行遗传进化分析,6株PEDV-M基因序列之间的核苷酸同源性为98.2%~99%,与疫苗株CV777的核苷酸同源性为97.6%~98.2%。利用MEGA 6.0构建遗传进化树,结果显示,扩增出的6条PEDV-M基因的遗传进化树分为两大系,大部分遗传距离较近,处于同一分支。其中LJJHD-M-13和LJJQ-M-14与泰国KU06RB08、泰国KU07RB08的亲缘关系较近,LJJC-M-14、LJJJ-M-14、LJJH-M-13、LJMM-M-14这4株毒株与韩国M1595、韩国CPF259的亲缘关系较近。同时扩增出的6条PEDV-M基因与CV777毒株非一系,与其亲缘关系较远,说明这些地区的PEDV流行毒株已经发生变异,形成了独特的流行进化分支。因此,本试验通过对变异毒株基因序列进行分析,以期为研制新的疫苗和预防控制该病提供实验数据和理论基础。

Porcine epidemic diarrhea virus（PEDV） is the etiological agent of Pig epidemic diarrhea（PED）. In this study, fecesand small intestine of the neonatal piglets that were noted with signs of diarrhea were collected form 10 large-scale pig farms in sixcities of Heilongjiang province from 2013 to 2014, and the RT-PCR analysis was used to detected the pathogens. The resultsshowed that 56% of the samples were positive for PEDV, 6% of the samples were positive for PEDV and TGEV, 10% of the sampleswere positive for PRV and 10% of the samples were positive for TGEV. Our findings indicated that PEDV was the main pathogen thatcaused diarrhea of neonatal piglets in Heilongjiang province, and there was also a mixed infection with other diarrhea viruses. Therewere six PEDV-M gene sequences were detected by RT-PCR; The results showed that the nucleotide homology was 98. 2% ~99% among these genes, and the nucleotide homology between these stains and vaccine strain CV777 was 97. 6% ~ 98. 2%. Ge-netic evolutionary tree was built by MEGA 6. 0, and the result showed that the 6 PEDV-M genes were divided into two series,and most of the genetic distance was velatively close at the same branch. Compared with other Chinese isolates and foreign isolates,the 6 PEDV isolates had a closer genetic relationship with the Korea isolate M1595, CPF259 and Thailand isolates KU06RB08,KU07RB08, but had more distant genetic relationship with the vaccine strain CV777. Our results indicated that the PEDV epidem-ic strains in these areas had been mutated to form a unique branch of the popular evolution.