中文摘要：

为研究高致病性猪繁殖和呼吸综合征病毒（HP-PRRSV）强弱毒之间毒力差异的分子基础,本实验分别以HP-PRRSV强毒HuN-F5株及其传代致弱的疫苗病毒株HuN4-F112为亲本病毒,利用反向遗传操作技术分别将ORF1a、ORF1b或ORF2-7编码序列在强弱毒之间互换。将6种含有不同嵌合基因的全长病毒基因组的重组质粒体外转录后转染BHK-21细胞,然后在Marc-145细胞中传代,拯救的重组病毒经RT-PCR、测序和免疫荧光鉴定,并分别命名为rHuN4-F5-ORF1a、rHuN4-F5-ORF1b、rHuN4-F5-ORF2-7（以强毒为骨架）和rHuN4-F112-ORF1a、rHuN4-F112-ORF1b、rHuN4-F112-ORF2-7（以弱毒为骨架）。进一步测定这些病毒在Marc-145上的生长曲线,结果显示：以强毒为骨架的嵌合病毒rHuN4-F5-ORF1a生长滴度显著高于亲本强毒rHuN4-F5,而以弱毒为骨架的嵌合病毒rHuN4-F112-ORF1a在细胞上的生长滴度低于其亲本弱毒rHuN4-F112,其他片段替换对病毒在细胞上的生长没有明显影响。本实验结果提示ORF1a对于PRRSV在体外细胞培养上的生长调节起重要作用。

英文摘要：

To investigate the molecular mechanisms of the different virulence between highly pathogenic porcine reproductive and respiratory virus（HP-PRRSV） HuN4-F5 strain and its attenuated vaccine strain HuN4-F112,we generated six full length infectious cDNA clones with interchange of ORF1a region,ORF1b region,and ORF2-7 region,respectively,between the twoviruses.The six full length infectious cDNA clones were transcribed in vitro,the transcripts were primarily transfected into BHK-21 cells and then passaged on Marc-145 cells.The rescued viruses were identified and confirmed by genomic sequencing and indirect immunofluorescence assay with antibody against PRRSV and designated rHuN4-F5-ORF1a,rHuN4-F5-ORF1b,rHuN4-F5-ORF2-7（genetic backbone of HuN4-F5 with ORF1a,ORF1b or ORF2-7 from HuN4-F112） and rHun4-F112-ORF1a,rHun4-F112-ORF1b,rHun4-F112-ORF2-7（genetic backbone of HuN4-F112 with ORF1a,ORF1b or ORF2-7 from HuN4-F5）,respectively.The growth kinetics showed that rHuN4-F5-ORF1a had significantly higher titer than its parental virus rHuN4-F5,and rHuN4-F112-ORF1a had significantly lower titer than its parental virus rHuN4-F112,while other chimeric viruses had similar growth curve with their parental viruses on Marc-145 cell.Based on our research,we conclude that ORF1a region plays a very important role in virus adaptation on Marc-145 cell.