为了从细胞水平探讨PRRSV GD强毒株和PRRSV弱毒疫苗接种仔猪后对其外周血淋巴细胞亚群的影响,运用血常规技术和流式细胞技术分析了猪体外周血淋巴细胞亚群的动态变化.将30日龄20头SPF猪分成3组,分别感染HP-PRRSV GD第5代强毒株、GDr180弱毒疫苗株及留作对照.于感染后第0、3、7、10、14、21、28及35天采血进行外周血淋巴细胞亚群测定,分析淋巴细胞亚群的变化.结果表明:HP-PRRSV GD强毒株感染可导致白细胞、淋巴细胞数量、单核细胞、粒细胞、B细胞、Tc细胞、Th细胞、Tm细胞、和γ6T细胞数量的下降;而弱毒疫苗GDr180株免疫则表现为白细胞、淋巴细胞、单核细胞、引起粒细胞、B细胞、Tc细胞、Th细胞和Tm细胞的上升,对γ6T细胞的影响不大;阴性对照组在整个检测期间各种细胞基本上保持稳定状态.从实验结果可以看出:与HP-PRRSV GD强毒株破坏免疫细胞不同,PRRSV弱毒疫苗GDr180株免疫接种能刺激猪免疫细胞增殖,给试验猪提供良好的免疫反应.

The potency detection method of C-strain vaccine based on classical swine fever virus rabbit infection dose( RID) is widely used in China, however, this method is greatly influenced by bread,individual difference and housing environment. In order to establish an alternative potency test strategy avoid using experimental animals, the permissive cell was inoculated with serial diluted vaccine, and then the antigen-capture ELISA, RT-nestPCR and traditional RT-PCR were utilized to detect the progeny virus. The highest positive dilution was termed as the indicator of the vaccine’ s potency. The method of cell infection dose( CID) was established. The results showed that the higher live virus particles in vaccine, the higher dose of cell infection virus. Results from the potency tests of different types of C-strain vaccines indicated that the alternative strategy could be applied to potency test of ST cell-line produced C-strain vaccine and bovine testicular cell produced C-strain vaccine. Potency tests of 12 batches of ST cell-line produced C-strain vaccines and 3 batches of bovine testicular cell produced C-strain vaccines were conducted with both the alternative strategy CID and the RID test parallel. The results showed, the positive dilution of all 12 batches of ST cell-line produced C-strain vaccine were 105 CID/dose, and 2.6×104~3.0×104RID/dose. And the positive dilution of 3 batches of bovine testicular cell produced C-strain vaccine were 104 CID/dose and 7.0×103~8.0×103RID/dose. The potency test results of the alternative strategy CID were at certain extent corresponding to that of RID test, and it could reflect the concentration of infectious virus particles in vaccine. The alternative strategy CID is of potentiality to replace the RID test.

Primers and probe were designed with PrimerExpress 3.0 and synthesized according to the conserved gene sequences of 24 highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRSV) strains and 5 classical PRRSV strains available in GenBank.A real-time fluorescent quantitative PCR assay was established for detection,differentiation and quantitation of HP-PRRSV and classical PRRSV.A known concentration of 10-fold series of dilutions of pGET-258 plasmid DNA were detected by using the established real-time FQ-PCR assay,then compared to the detection result of routine PCR.The developed real-time FQ-PCR assay could detect 1.5 copies with plasmid DNA and its sensitivity was 100 times higher than that of the routine PCR.Three plasmid standards were examined using the real-time FQ-PCR repeatedly at three different time,the coefficient of variations were below 2%,and the results indicated that the real-time FQ-PCR assay was reproducible and could be used for the diagnosis and differentiation of HP-PRRSV infection.

为建立一种能够快速鉴别检测猪繁殖与呼吸综合征病毒（PRRSV）GDr180疫苗株与GD野毒株的双重荧光定量RT-PCR方法,本研究在PPRSV的Nsp2基因和Orf7基因区域分别设计不同荧光标记的MGB探针及特异性引物。这两种MGB探针能够分别特异结合GD野毒株和GDr180疫苗株。通过对PRRSV培养液、感染猪血清和组织样品检测证明了该方法的可行性;采用双重实时荧光定量RT-PCR方法检测PRRSV GDr180疫苗株和GD强毒株时敏感性分别达到19.4拷贝/μL和34.2拷贝/μL;该方法与PRRSV其他8个病毒株和猪瘟病毒、猪伪狂犬病毒、猪圆环病毒2型和猪细小病毒不发生交叉反应,表明该方法具有良好的特异性;因此可以用于PRRSV GDr180疫苗株与野毒株的鉴别检测。