广西“猪高热综合征”的病原学调查以及PRRSV部分Nsp2和ORF5的基因序列分析
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摘要
“猪高热综合征”是2006年5月以来,首先在我国江西、湖南等地爆发的一种以高热、呼吸困难和急性发病死亡为主要临床特征的猪“高热病”。该病来势凶猛,无特异防制方法,病原复杂,多呈混合感染,为查明广西“猪高热综合征”的病原和混合感染情况,对广西13个市各个地区的病料分别进行猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪流感病毒(SIV),副猪嗜血杆菌(HPS)等几种常见的猪病毒细菌病进行病原学诊断,结果显示检出率最高的是PRRSV变异株,高达54.23%,其次PCV占检测的38.85%;CSFV占28.1%;SIV占10.38%;PRV和HPS各占2.69%。说明广西目前流行的“猪高热综合征”主要病原为PRRSV变异株,PCV、CSFV扮演着十分重要的角色。且病原PRRSV变异株与其它病原菌混合感染十分严重。其中,二重感染(PRRSV+CSFV、PRRSV+PCV、PRRSV+SIV、PRRSV+PRV、PRRSV+HPS)高达43.08%,三重感染(PRRSV+CSFV+PCV、PRRSV+CSFV+SIV、PRRSV+PCV+SIV)占20.97%。混合感染病毒种类最多达到四重感染(PRRSV+CSFV+PCV+SIV)。在混合感染的病原中,以PRRSV与PCV、CSFV混合感染率最高,分别达21.2%和18.43%。
对广西13个市具有代表性的16个PRRSV毒株的部分Nsp2基因进行基因克隆测序,发现广西PRRSV变异株与国内其他省份PRRSV变异株有完全一致的缺失特征,即Nsp2基因的第482位和534～562位氨基酸发生缺失。广西PRRSV变异株之间的核苷酸同源性性为93.3%～99.2%,广西变异株与其他省份的Nsp2缺失株之间的核苷酸同源性为90.3%～99.7%。而广西PRRSV变异株与2005年以前的PRRSV Nsp2的同源性只有68.5%～94.4%推导广西PRRSV变异株之间的氨基酸同源性性为90.2～99.7,广西变异株与其他省份的Nsp2变异株之间的氨基酸同源性为78.7～99.4%。
对6株PRRSV变异株ORF5片段进行克隆测序。结果表明,所有毒株ORF5基因大小均为603bp。与国内外发表的美洲型PRRSV相应片段有较高的同源性,核苷酸同源性达94.7%～98.7%,推导的氨基酸同源性为88.0%～99.5%,而与LV株之间的同源性只有53.2%～59.8%。ORF5蛋白抗原性分析表明本次研究的毒株与国内分离毒株JXA1、LN、GD、HUB2、HEB1等PRRSV变异株抗原性非常相似,仅在192～200位氨基酸稍有差异。在第35～41、53～60、131～145、151～159、163～191、193～200位和其他PRRSV毒株一样均存在6个抗原表位位点,且抗原表位优势无明显差异。
"swine high fever syndrome" first broke out in Jiangxi,Hunan and other places in China in May 2006,with the symptom of high fever,breathing difficulties and acute death,The disease propagate swift,powerful,mostly mixed infection,and hadn't specific control methods to identify the pathogen of the "swine high fever syndrome" of Guangxi,we detected the pathogen of porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),swine pseudorabies virus(PRV), porcine circovirus type 2(PCV-2),swine influenza virus(SIV),Haemophilus parasuis (HPS),such as several common bacterial and virus of pig disease form 13 cities of Guangxi.The result showed that the highest detection rate of PRRSV mutant strain was that up to 54.23 percent,followed by PCV 38.85%,CSFV 28.1%,SIV 10.38%,PRV and HPS were 2.69 percent respectively.We could know that the main pathogenic of "swine high fever syndrome" in Guangxi was PRRSV mutant strain,PCV and CSFV played an important role.The mixed infection of PRRSV and other pathogens was very serious,dual infection(PRRSV+ CSFV,PRRSV+ PCV,PRRSV+ SIV,PRRSV + PRV, PRRSV+ HPS) as high as 43.08 percent,triplinfections(PRRSV + CSFV + PCV,PRRSV + CSFV + SIV,PRRSV + PCV + SIV) accounted for 20.97 percent.The most mixed infected up to four types of re-infection(PRRSV+ CSFV + PCV + SIV).In the mixed infection of the pathogen,the PRRSV+PCV,PRRSV+ CSFV were the highest infection rate,respectively 21.2 percent and 18.43 percent.
We cloned the partial Nsp2 of 16 strains of PRRSV mutant strain form 13 City of Guangxi,gene analysis show that the PRRSV mutant strains of Guangxi were exactly the same characteristics as other provinces,that was,482 and 534 to 562 amino of Nsp2 were absent.the sequence homology of the Guangxi PRRSV mutation strains was 90.2%to 99.2%,with a similarity of 90.0%to 99.7%in deduced amino acid sequence.The sequence homology of the Guangxi PRRSV mutation strains and other provinces was 90.2%to 99.2%,and with a similarity of 78.0%to 100%in deduced amino acid sequence.
The complete ORF5 gene of 6 strains of PRRSV were cloned and gene analysised. The results showed that ORF5 was the size of 603 bp.And they ware high homology with the North American genotype,the sequence homology was 94.7%to 98.7%,with a similarity of 88.0%to 99.5%in deduced amino acid sequence,but only 53.2%to 59.8% with the LV strains.
Antigen analysis of ORF5 gene showed that the 6 strains of Guangxi were very similar with JXA1,LN,GD,HUB2,HEB1,and other PRRSV mutant strains,only 192 to 200 amino acids were slight differences.The 6 strains had the same amino acid epitope sites with other PRRSV strain in 35 to 41,53 to 60,131 from 145,151 to 159,163 to 191,193 to 200.And there was no significant difference in Epitope advantage.
对广西13个市具有代表性的16个PRRSV毒株的部分Nsp2基因进行基因克隆测序,发现广西PRRSV变异株与国内其他省份PRRSV变异株有完全一致的缺失特征,即Nsp2基因的第482位和534～562位氨基酸发生缺失。广西PRRSV变异株之间的核苷酸同源性性为93.3%～99.2%,广西变异株与其他省份的Nsp2缺失株之间的核苷酸同源性为90.3%～99.7%。而广西PRRSV变异株与2005年以前的PRRSV Nsp2的同源性只有68.5%～94.4%推导广西PRRSV变异株之间的氨基酸同源性性为90.2～99.7,广西变异株与其他省份的Nsp2变异株之间的氨基酸同源性为78.7～99.4%。
对6株PRRSV变异株ORF5片段进行克隆测序。结果表明,所有毒株ORF5基因大小均为603bp。与国内外发表的美洲型PRRSV相应片段有较高的同源性,核苷酸同源性达94.7%～98.7%,推导的氨基酸同源性为88.0%～99.5%,而与LV株之间的同源性只有53.2%～59.8%。ORF5蛋白抗原性分析表明本次研究的毒株与国内分离毒株JXA1、LN、GD、HUB2、HEB1等PRRSV变异株抗原性非常相似,仅在192～200位氨基酸稍有差异。在第35～41、53～60、131～145、151～159、163～191、193～200位和其他PRRSV毒株一样均存在6个抗原表位位点,且抗原表位优势无明显差异。
"swine high fever syndrome" first broke out in Jiangxi,Hunan and other places in China in May 2006,with the symptom of high fever,breathing difficulties and acute death,The disease propagate swift,powerful,mostly mixed infection,and hadn't specific control methods to identify the pathogen of the "swine high fever syndrome" of Guangxi,we detected the pathogen of porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),swine pseudorabies virus(PRV), porcine circovirus type 2(PCV-2),swine influenza virus(SIV),Haemophilus parasuis (HPS),such as several common bacterial and virus of pig disease form 13 cities of Guangxi.The result showed that the highest detection rate of PRRSV mutant strain was that up to 54.23 percent,followed by PCV 38.85%,CSFV 28.1%,SIV 10.38%,PRV and HPS were 2.69 percent respectively.We could know that the main pathogenic of "swine high fever syndrome" in Guangxi was PRRSV mutant strain,PCV and CSFV played an important role.The mixed infection of PRRSV and other pathogens was very serious,dual infection(PRRSV+ CSFV,PRRSV+ PCV,PRRSV+ SIV,PRRSV + PRV, PRRSV+ HPS) as high as 43.08 percent,triplinfections(PRRSV + CSFV + PCV,PRRSV + CSFV + SIV,PRRSV + PCV + SIV) accounted for 20.97 percent.The most mixed infected up to four types of re-infection(PRRSV+ CSFV + PCV + SIV).In the mixed infection of the pathogen,the PRRSV+PCV,PRRSV+ CSFV were the highest infection rate,respectively 21.2 percent and 18.43 percent.
We cloned the partial Nsp2 of 16 strains of PRRSV mutant strain form 13 City of Guangxi,gene analysis show that the PRRSV mutant strains of Guangxi were exactly the same characteristics as other provinces,that was,482 and 534 to 562 amino of Nsp2 were absent.the sequence homology of the Guangxi PRRSV mutation strains was 90.2%to 99.2%,with a similarity of 90.0%to 99.7%in deduced amino acid sequence.The sequence homology of the Guangxi PRRSV mutation strains and other provinces was 90.2%to 99.2%,and with a similarity of 78.0%to 100%in deduced amino acid sequence.
The complete ORF5 gene of 6 strains of PRRSV were cloned and gene analysised. The results showed that ORF5 was the size of 603 bp.And they ware high homology with the North American genotype,the sequence homology was 94.7%to 98.7%,with a similarity of 88.0%to 99.5%in deduced amino acid sequence,but only 53.2%to 59.8% with the LV strains.
Antigen analysis of ORF5 gene showed that the 6 strains of Guangxi were very similar with JXA1,LN,GD,HUB2,HEB1,and other PRRSV mutant strains,only 192 to 200 amino acids were slight differences.The 6 strains had the same amino acid epitope sites with other PRRSV strain in 35 to 41,53 to 60,131 from 145,151 to 159,163 to 191,193 to 200.And there was no significant difference in Epitope advantage.
引文
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