【作者】 赵鹏；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2010， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的猪的一种高度接触性传染病。自1987年首次在美国报道以来,目前已遍布全球各主要养猪国家。2006年夏季,我国南方部分地区在高温高湿季节发生了以猪繁殖与呼吸综合征病毒为主要病原同时伴发多种病原感染及继发感染引起的猪“高热病”,随后该病在山东亦发生流行,给养猪业造成前所未有的损失。本研究对2006年下半年山东某万头猪场发生的“高热病”进行了诊断并分离鉴定了一株高致病性猪繁殖与呼吸综合征病毒,命名为SD0612,对其在抗体免疫选择压下的基因变异和生物学特性的改变及其准种多样性的演变进行了系统研究。1、高致病性猪繁殖与呼吸综合征病毒山东株SD0612的分离鉴定及其致病性试验利用核酸探针杂交技术结合PCR及RT-PCR技术采用对不同病毒的排除法对2006年山东某万头猪场“高热病”进行了病原性鉴别诊断,确定其主要病原为猪繁殖与呼吸综合征病毒(PRRSV)。利用Marc-145细胞对病毒进行了分离,得到了一株猪繁殖与呼吸综合征病毒,命名为SD0612。对其主要基因NSP2、ORF3-ORF7进行了克隆测序,结果显示该病毒为NSP2缺失90个碱基的变异株,序列分析表明该毒株与同时期其它代表株高度同源,位于进化树上的同一分支。该毒株针对1月龄仔猪的致病性试验显示死亡率高达100%,为高致病性变异株。2、SD0612结构基因ORF3、ORF4、ORF5在抗体选择压下的变异为了确定抗体选择压对PRRSV不同基因变异的影响,将PRRSV野毒株SD0612分别在Marc-145细胞上做两个系列的连续传代,系列Ⅰ培养液中不添加抗SD0612血清,包括A、B、C三个平行的传代系;系列Ⅱ培养液中添加一定比例的抗SD0612高免康复猪血清,包括D、E、F三个平行的传代系。上述传代系连续传代40代,其中有抗体组40代突变株F40再接种无PRRSV感染猪获得抗血清后按同样方法连续传40代进行第二轮抗体选择压试验。各传代系列每隔10代收获病毒并对其ORF3、ORF4和ORF5进行RT-PCR扩增,克隆和序列分析。将SD0612及其两次传代中有抗体组40代毒突变株F40和e40接种猪后收获血清,进行三个毒株之间的交叉血清中和试验测定其抗原性相关系数。序列分析结果显示:两次传代中有抗体组在ORF3、ORF4、ORF5的有义突变与无义突变比值(NS/S)分别为11.0和3.5, 0.5和0.67, 13.0和4.0;无抗体组NS/S比值则仅为0.33-1.40。有抗体组ORF5和ORF3分别有14个和4个稳定的氨基酸突变位点,而这些位点在无抗体组并未发现。交叉血清中和试验结果显示SD0612与有抗体组突变株F40和e40的抗原性相关系数分别为0.625和0.5。上述结果表明,抗体的选择压能够显著影响PRRSV的ORF5和ORF3的基因变异并能使其抗原性发生改变。3、SD0612准种的多样性及其在抗体选择压下的演变为了研究PRRSV野毒株SD0612准种的多样性并比较其在有抗体和无抗体的Marc-145连续传代时准种多样性的演变,将SD0612在Marc-145上分别进行有抗SD0612抗体和无抗SD0612抗体两个系列的连续传代,每个传代系又包括3个平行的传代系列。连传40代后分别得到传代毒A40-F40。将有抗体传代系列40代毒F40按上述方法进行第二轮传代,连传40代后分别得到传代毒a40-f40。将SD0612原始毒及两轮传代40代病毒的细胞培养物做RT-PCR扩增和克隆ORF5基因,每个毒株选取所有阳性克隆子测序,以DNAStar软件对序列进行分析。结果显示:SD0612是由核酸同源性介于97.7%-100%的不同准种组成的种群,该种群中17/60为占主体的优势准种(ORF5基因序列完全一致);SD0612在有抗体的连续传代中准种多样性在减少,其ORF5基因完全一致的优势准种比例在上升;而在无抗体组这种优势准种的比例在下降。有抗体组的优势准种随着传代与原始毒株优势准种的核酸同源性越来越低。该研究显示野毒SD0612是由多个准种组成的种群,抗体的选择压能够显著减少PRRSV的准种多样性,并使某些准种通过基因突变演变为新的优势准种。4. SD0612原始株与不同传代突变株在回归猪体实验中病毒血症和抗体动态的比较为了比较SD0612原始株及其抗体选择压下突变株在回归猪体实验中病毒血症和抗体动态,并初步探讨其作为疫苗候选毒株免疫猪后对强毒的抵抗能力。我们先后进行了两次动物实验。在第一次试验中,将8头健康猪分为四组,分别为SD0612原始株感染组、第一次传代有抗体组突变株F40感染组及第二次传代有抗体组突变株e40感染组及阴性对照组,分别于攻毒后1w、2w、3w、4w、5w采血,其中抗凝血用于测定血常规,另收集血清以ELISA试剂盒测定其抗体水平,并以荧光定量PCR测定全血中病毒含量。在第40天时接种强毒,每天测量体温及观察发病表现。结果显示:SD0612原始株感染组能够诱发较高的抗体水平且耐过猪能够经受强毒的攻击。第一次传代突变株F40感染组及第二次传代突变株e40感染组抗体出现较原始株晚且持续时间较短,第一次传代突变株F40感染组能够经受强毒的攻击,而第二次传代突变株e40感染组全部死亡,显示e40抗原性发生了较大的变化,已不足以保护原始毒的攻击。在实验二中我们比较了第一次传代有抗体代表株F40与其在无抗体下传代40代毒a40在回归猪体实验中病毒血症和抗体动态的差异。将15头猪分为三组,分别为第一次传代有抗体组F40感染组、第二次传代无抗体组a40感染组及阴性对照组,于攻毒后1w、2w、3w、4w、5w采血,收集血清以ELISA试剂盒测定其抗体水平,并以单位体积血清中TCID50来显示血液中病毒含量。在第40天时接种强毒并测量体温,观察其发病表现。结果显示:第一次传代有抗体组的F40感染组诱发的病毒血症要显著高于第二次传代无抗体组a40感染组,且a40感染组病毒血症的高峰期相对于F40感染组要延后一周。两组之间抗体水平差异不大,均能够诱发较高的抗体水平并能全部经受强毒的攻击,而阴性对照组死亡率为60%。该研究显示F40在无抗体的继续传代中抗原性变化较小,但在猪体内复制能力显著降低,a40和F40诱发的抗体均能够保护猪体经受强毒的攻击。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a highly contagious disease in pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). Since the first outbreak in the United States 1987,it has spread to the principal pigs cultivation countries around the world currently. In summer of 2006, pigs "high fever" disease characterized by high fever took place in south of China in hot and humid season, the study showes it was caused mainly by porcine reproductive and respiratory syndrome virus as the main pathogen associated with a variety of pathogenic infection and secondary infection of other pathogen at the same time. The "high fever" epidemic in Shandong also occured and caused unprecedented loss since September 2006. In this study, a pig "high fever" of one big pig farm in Shandong was diagnosised and a highly pathogenic porcine reproductive and respiratory syndrome virus was isolated and identified, named for the SD0612. The evolution of it’s genetic variation and quasispecies diversity under the antibody selective pressure was studied.1、The isolation and identification of porcine reproductive and respiratory syndrome virus SD0612 and it’s pathogenicity testUsing DNA hybridization combined with PCR and RT-PCR, a "high fever" of a pig farms in Shandong was diagnosed and the main pathogen is determined as porcine reproductive and respiratory syndrome virus. Marc-145 cells was used to isolate the virus and a highly pathogenic porcine reproductive and respiratory syndrome virus was isolated and named for the SD0612. its main gene NSP2, ORF3-ORF7 was cloned and sequenced, the results showed that the virus was a deletion mutation with NSP2 missing 90 bp, sequence analysis showed it share highly homologous with other representative strains in the same period. pathogenicity test of the strain for the 1 month old piglets showed that mortality rate up to 100%, indicating that SD0612 is highly pathogenic variant.2、Evolution of porcine reproductive and respiratory syndrome virus SD0612 under antibody selective pressuresTo study effects of antibody immune selective pressures on antigenic evolutions of porcine reproductive and respiratory syndrome virus (PRRSV), the field strain SD0612 was continuously passed in Marc-145 cell cultures without or with anti-SD0612 pig sera in 6 separated lineages for 40 passages. The resistant virus of the lineage F with anti-SD0612 sera at 40th passage was collected as strain F40 and used for the second step of 40 passages again in another 6 lineages with or without anti-F40 sera. The resistant virus of the lineage e at 40th passage was collected to prepare anti-e40 pig sera. Infected cells were collected for extraction of viral RNA every 10 passages. ORF3, 4, 5 genes were amplified, cloned and sequenced. Cross viral neutralization indicated that serological homology between SD0612 and its two resistant mutants F40 or e40 were 62.5%, 50%; homology between F40 and e40 was 80%. Sequence analysis indicated that non-synonymous vs synonymous mutation ratios (NS/S) of ORF3 , 4, 5 in two steps were 11.0 and 3.5, 0.5 and 0.67, 13.0 and 4.0 respectively in passages with anti-PRRSV sera, but only in the range of 0.33-1.4 in passages without anti-PRRSV sera. It indicated that anti-PRRSV sera had strong immune selective pressures on viral evolution in its ORF3 and ORF5 genes. Besides, there were total 14 stable NS mutation sites in ORF5 and 4 in ORF3 in 3 lineages during the 80 passages with anti-PRRSV sera, which were not detected in passages without specific antibodies.3、Comparison of PRRSV quasispecies diversity and it’s evolution under the antibody selective pressureTo study the evolution of quasispecies diversity for PRRSV under the antibody selective pressvre, PRRSV wild strain SD0612 was passed in Marc-145 cells for two series, the series ? was cultured with medium without anti-SD0612 serum, including A, B, C three parallel passage lines; while series ?? was cultured with medium added proportional anti-SD0612 serum, including D, E, F three parallel passage. After 40 continuous passages we obtained the derivants A40-F40. the F40 was passed in a second round passage as above, after 40 continuous passages a40-f40 were obtained. 13 strains from three generation(the original of SD0612, the first-generation A40-F40, the second-generation a40-f40) were cultured and it’s ORF5 gene was amplified and cloned, All positive clones of each strain were sequenced and analyzed with DNAStar software. The results show that SD0612 is a population composed of different quasispecies which nucleic acid homology ranging from 97.7% -100%, among the 60 total sequence 17 sequences were the same and account for the main advantages of the populations; in the two passage trial, the quasispecies diversity are declining while the rate for advantage quasispecies is on the rise for group ?? with antibody, the result for group ? without antibody is on the contrast; there are more points mutation for group ? without antibody than group ?? with antibody. The advantage quasispecies of group ?? with antibody shared lower and lower identity with the original strain with the continuous passage. The study indicates that antibodies selective pressure can significantly affect the quasispecies diversity of PRRSV,particularly causing the changes of advantage quasispecies .4、The comparision of viremia and the antibody response in pigs between SD0612 original strain and different mutants in the passage experimentsTo compare the dynamic of viremia and antibody response in pigs between SD0612 original strain and it’s mutants in the selective pressure passage experiment, two animal experiments were conducted in this study. In the first experiment, 8 healthy pigs were divided into four groups as the SD0612 original strain infection group, the first passage mutant F40 infection group and the second passage mutant e40 infection group and negative control group, antibody was determined by ELISA kit in 1w, 2w, 3w, 4w, 5w after challenge, and virus concentration in blood was tested by fluorescence quantitative PCR. In the 40th days all group was inoculated with virulent PRRSV and temperature was measured and incidence of performance was observed daily. The results showed that: SD0612 original strain infection can induce high antibody response and the antybody can resistance to virulent virus attack. Antibodies of the F40 infection group and e40 infection group is later than the original strain group. The antibody caused by F40 can protect the pig withstand the virulent virus attack, while the e40 infection group all died. Showing e40 antigencity have taken place great changes.The dynamic of viremia and antibody response between F40 and it’s derivants a40 in the passage without antibody was compared in the second experiment. 15 pigs were divided into three groups as F40 infection group, a40 infection group and negative control group. Antibody was determined by ELISA kit in 1w, 2w, 3w, 4w, 5w after challenge, and virus concentration in blood was tested with TCID50 in 100ul blood serum. In the 40th days all group was inoculated with virulent PRRSV and temperature was measured and incidence of performance was observed daily. The results showed that the infection of F40 and a40 can induce high antibody titer and protect the pig withstand the virulent virus attack, while the viremia level of the F40 infection group is higher and earlier than the a40 infection group. 3 pigs of the negative control group died at last.更多还原