高致病性猪繁殖与呼吸综合征病毒HBR株不同代次水平的毒力比较试验

为了阐明高致病性猪繁殖与呼吸综合征病毒(PRRSV)HBR毒株在体外传代过程中发生的毒力及遗传变异情况,采用第5、10和125代PRRSV-HBR株病毒进行了人工感染试验。结果显示,低代次(第5和第10代)病毒接种组可复制出以"高热综合征"为特点的症候群,第5代毒株感染组死亡率达40%(2/5),第10代感染组症状明显减轻且无死亡,第125代毒株感染组基本无临床症状。这3个代次的毒株感染组于感染后第3天检测病毒血症呈阳性,第7～14天到达高峰,第125代于第21天消失,较第5和10代毒株提前1周。病毒感染后抗原主要分布于脾、扁桃体、淋巴结等组织,第5和10代毒株感染组脏器出现严重的病理损伤,第125代毒株感染组仅见轻微病理损伤。对3个代次毒株的ORF5、ORF6和ORF7测序显示,仅在ORF5出现了3个氨基酸位点突变,即第29位:第5、第10代的缬氨酸到了第125代变为丙氨酸;第32位:第5、第125代的丝氨酸到了第10代变为甘氨酸;第196位:第5、第10代的谷氨酰胺到了第125代变为精氨酸。研究表明,低代次毒株能够完全复制出典型的"猪高热综合征",而高代次毒株无明显临床症状,证明该毒株经细胞传代后毒力下降。高代次毒株感染动物仍能够引起毒血症和持续感染的结果表明,高代次毒株仍具一定毒力,作为疫苗种毒需要进一步毒力弱化。

Virulence comparative test of a highly pathogenic isolate HBR of PRRSV at different passage levels

In order to examine the virulence change and genetic variation of a highly pathogenic isolate HBR of porcine reproductive and respiratory syndrome virus(PRRSV) during the process of cell culture passage,the virulence comparative test was performed with two low passage generations(5th and 10th) and a high passage generation(125th) propagated in Marc-145 cells.The results showed that the low passage generation can replicate typical symptom as characterized with high fever syndrome.The group infected with the 5th generation had the highest mortality of 40%(2/5) on pigs,while the 10th had no death impact on pigs with clinical symptom conspicuously relived with the comparison with the former.And the high passage generation(125th) groups showed no clinical symptoms as well.The viremia which wasdetermined by RT-PCR after 3 days post-inoculation(dpi) reached a peak on 7 to 14 dpi.The viremia was disappeared on 21 dpi for the 125th generation,ahead of one week than that of the 5th and 10th generations. Examination of virus distribution in the pig organs showed that the spleen,tonsil and lymph node contained the highest virus load.Pathological analysis identified that the most important microscopic lesion in pigs inoculated with low passage generation was multi-organism pan-tropic,which resulted in severe lesion in most of organs all over the body,however,only slight microscopic lesion appeared in the high passage generation group.Sequencing performed on the nucleotide sequences of ORF5,ORF6 and ORF7 of the passage generations revealed, three point mutations in deduced amino acid sequences of ORF5, but no deletion was found. For ORF6 and ORF7,they were conservative and no mutations were found. High passage HBR strain was obtained through cell passages,with virulence attenuated but it still can lead to viremia and persistent infection similar with those of low generation strains.Therefore,it needs further attenuation as a candidate of vaccine.