摘要
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,C3个平行的传代系;系列Ⅱ培养液中添加一定比例的抗SD0612血清,包括D,E,F3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获得抗血清后按同样方法连续传代40代进行第二轮抗体选择压实验.各传代系每隔10代收获病毒并对其ORF3,ORF4和ORF5进行扩增、克隆和序列分析.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3个毒株之间的交叉血清中和实验以测定其抗原相关性系数.序列分析显示,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和4个稳定的氨基酸突变位点,而这些位点在无抗体组未被发现.交叉血清中和实验结果显示,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.
To study effects of antibody immune selective pressures on antigenic evolutions of porcine reproductive and respiratory syndrome virus （PRRSV）, a 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 I 1.0 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.
出处
《中国科学：生命科学》
CSCD
北大核心
2010年第10期952-962,共11页
Scientia Sinica(Vitae)
关键词
猪繁殖与呼吸综合征病毒
抗体选择压
基因变异
ORF3
ORF5
porcine reproductive and respiratory syndrome virus（PRRSV）, immune selective pressure, viral evolution, ORF3, ORF5
