摘要
猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种以母猪繁殖障碍和仔猪呼吸道疾病为特征的传染病。高致病性猪蓝耳病是由PRRSV变异毒株引起的一种急性高致死性传染病,病毒致病力强,传播速度快,猪群发病率和死亡率高。本试验对猪繁殖与呼吸综合征病毒进行分离鉴定及建立起的一套检测方法,并从空间和地域对我国部分省区HP-PRRSV结构蛋白基因进行遗传演化分析。
本试验参考Genbank中收录的猪繁殖与呼吸综合征JXA1株ORF2-7基因的序列,设计了一对引物用于检测猪繁殖与呼吸综合征病毒,目的片段大小为690bp,通过特异性、敏感性、重复性试验和符合性试验,建立适合大规模的检测的RT-PCR方法。通过建立的检测方法,对山东、福建、广西、云南、河南、湖北、湖南、辽宁、陕西、浙江等10个省送检的200份样品进行检测,检测PRRSV阳性89份。
本试验利用第二章建立的RT-PCR方法对送检病料检测为强阳性的6个省区20株样品,经处理后接种Marc-145细胞,结果接种细胞在盲传第二代时出现了PRRSV所致的特征性病变。将分离得到的病毒08SDCXA和09HUB5在Marc-145细胞上传代得到的上扩大培养后测定TCID50为106.50和107.714。将获得的病毒经非线性蔗糖密度梯度离心,分别收集各梯度区带上的蛋白提取物,并进行负染,在电子显微镜下观察,结果在35%与45%蔗糖之间收集的条带观察到大小约为50～60nm的病毒粒子,有囊膜,呈球形,其大小及形态与PRRSV一致,这进一步也证实了所分离的病毒为猪繁殖与呼吸综合征病毒。
本试验选取2007～2009年这三年来源于第三章病原学鉴定为阳性的共6个省区20株细胞毒进行测序分析,证实均为PRRSV序列。经测序获得ORF2-7基因片段序列,利用DNAStar软件对其进行核苷酸和氨基酸序列分析。分析表明20个分离株ORF2-7各段基因序列之间核苷酸同源性如下:ORF2为96.2%～99.7%、ORF3为97.0%～99.7%、ORF4为96.3%～99.6%、ORF5为91.9%～99.5%、ORF6为96.4%～100%、ORF7为99.6%～100%;而ORF2-7各段基因序列之间氨基酸同源性如下:ORF2为94.9%～99.6%、ORF3为95.7%～99.2%、ORF4为94.4%～99.4%、ORF5为89.6%～99.0%、ORF6为96.0%～99.4%、ORF7为97.6%～99.2%。分析表明20个分离株ORF2-7各段基因序列与国内10株参考毒株CH-1a、BJ-4、JXA1、HB-1、HB-2、HUN4、CC-1、GD2007、YN2008、SX2009和国外10株参考毒株VR-2332、LV、MLV、NVSL97-7895、PA8、LMY、EDRD-1、SP、07BJ、01CB1核苷酸同源性分别如下:ORF2为56.4%～99.9%;ORF3为55.3%～99.9%;ORF4为56.1%～100%;ORF5为52.7%～99.8%;ORF6为62.5%～100%;ORF7为57.3%～100%。而氨基酸序列同源性如下:ORF2为57.2%～99.6%;ORF3为52.9%～99.6%;ORF4为64.2%～99.4%;ORF5为54.2%～99.5%;ORF6为75.9%～99.4%;ORF7为56.5%～99.2%。通过对这20株分离结构蛋白进行遗传变异分析,无论是从2007年4月～2009年12月的时间跨度上,还是从山东、福建、湖南、湖北、陕西、辽宁地域跨度上,均表现出分离株之间的同源性比较高,与JXA1亲缘关系近,与代表毒株VR-2332有了较大变异,表明HP-PRRSV为目前我国流行毒株。
Porcine Reproductive and Respiratory Syndrome (PRRS) is a infectious swine disease caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),it was characterized by breeding failure of sows and respiratory disease of piglets.Highly Pathogenic Porcine Reproductive and Respiratory Syndrome(HP-PRRS)is a high deadly infectious swine disease which was triggered by mutative PRRSV. HPRRSV has a strong pathogenic ability, it can spread quickly in pig farms and lead to high mortality and mortality of pigs.This study isolated some PRRSVs and established a detective method for PRRSV detection.We also analyzed the homology and evolutionary characterizations of PRRSV structural protein genes.
A pair of primers was designed to detect PRRSV based on the ORF2-7 genes of JXA1 strian which is available on GenBank.The length of the expected fragment was 690bp. An accurate RT-PCR method proper for large scale PRRSV detection was established after many times improvement according to the specificity assay, sensibility assay, repeat assay and criterion assay. 200 samples from 10 provinces of Shandong, Fujian, Guangxi, Yunnan, Hennan, Hubei, Hunan, Liaoning, Shanxi and Zhejiang were detected by the established RT-PCR method.And 89 samples were positive.
20 PRRSV-suspected samples detected positive by the established RT-PCR method in Chapter 2 from Shandong, Fujian, Guangxi, Yunnan, Hennan, Hubei, Hunan, Liaoning, Shanxi and Zhejiang were incubated on Marc-145 cells after proper treatment.The PRRSV specific CPE appeared in the 2nd passage on Marc-145 cells. TCID50 of PRRSV strains of 08SDCXA and 09HUB5 were 106.50 and 107.714 respectively.The viron particles were observed by TSE (transmission electron microscope) after being separated according nonlinear sucrose density gradient centrifugation and negative staining. 50-60nm PRRSV-like round viron particles were seen with envelope between the sucrose range in the density of 35% to 45% , Which proved it were PRRSV particles.
20 PRRSV strains were isolated from positive samples of 6 provinces obtained during 2007-2009 and proved by etiology identification in chapter 3. The ORF2-7 genes sequences were determined with new designed primers. The nucleotide and amino acid sequences were analyzed by DNAStar software. The analysis result showed the nucleotide identity of each ORF among the 20 PRRSV strains followed as below:ORF2(96.2%-99.7%),ORF3(97.0%-99.7%), ORF4(96.3%- 99.6%), ORF5(91.9%-99.5%), ORF6(96.4%-100%), ORF7(99.6%-100%). The aa identity of each ORF among 20 strains followed as: ORF2(94.9%-99.6%), ORF3(95.7%-99.2%), ORF4(94.4%-99.4%), ORF5(89.6%-99.0%), ORF6(96.0%-99.4%), ORF7(97.6%-99.2%). The 20 obtained ORF2-7 genes were compared with 10 domestic PRRSV strains of CH-1a, BJ-4, JXA1, HB-1, HB-2, HUN4, CC-1, GD2007, YN2008, SX2009 and 10 foreign PRRSV strains of VR-2332,LV, MLV, NVSL97-7895,PA8,LMY,EDRD-1,SP,07BJ,01CB1.The nucleotide identity followed as: ORF2(56.4%-99.9%), ORF3(55.3% -99.9%), ORF4(56.1%-100%), ORF5(52.7%-99.8%), ORF6(62.5%-100%),ORF7(57.3%-100%). The aa identity followed as:ORF2(57.2%-99.6%),ORF3(52.9%-99.6%), ORF4(64.2%-99.4%),ORF5(54.2%-99.5%), ORF6(75.9%-99.4%), ORF7 (56.5%-99.2%). The analysis of 20 structural protein genes suggested the PRRSV strains isolated from April 2007 to December 2009 had high homology with no relationship with time and space.The 20 PRRSV strains also had extreme high homology with JXA1 strain and remarkable mutations extent compared with VR-2332 strain.This suggested HP-PRRSVs were the dominant PRRSV strains in China recently.
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