Proliferation Characteristics of Type 2 Porcine Circovirus Strains and Antivirus Activity of Iprs and Aps on PCV2 in Vitro
|School||Henan Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||porcine circovirus type 2 proliferation characteristic fluorescent quantitative PCR Astragalus Polysaccharide isatis root polysaccharide antiviral activity|
PCV2 has the poor multiplication capacity in vitro. There is no cytopathic effect and the viral titer is low in PK15 cell line infected by PCV2. The clinical symptoms vary and the viral load in tissue is significantly different in PCV2 infected pigs. In order to establish a sensitive and specific quantitative method to detect PCV2, A SYBR GreenⅠ–based real time quantitative PCR(Q-PCR) assay was developed. A 276 bp conserved region of the ORF2 gene of PCV2 was amplified by conventional PCR and cloned into pMD-18 vector. The resulted pMD-18T-276 plasmid DNA was used as standard DNA template to optimize assay condition. The standard curve produced a good linear relationship between Ct value and initial amounts of total DNA at a range of 5.60×10~1～2.74×10~9 copies per microlitre, and the correlation coefficient and slope were -1.00 and -3.345, respectively. No cross-reactions were found in specimens containing PCV1, PRV, PRRSV, PPV and CSFV.The Q-PCR was used to detect the Proliferative Kinetics of PCV2 in infected cell. The result showed that the peak of PCV2 replication in PK15 was at 96 hour postinfection . Four high titer PCV2 strains were screened from 11 isolates, which enriches the resources for PCV2 vaccine strains research. 8 isolates which have the approximately equivalent virus quality were chose to challenge six-week-old SPF Kunming mices, the result showed that Henan 5 strain, Ruifeng strain and Taiping strain had more effective invasive and proliferative capacity than the others in target tissues challenged at 14 days postinfection.In order to study the antiviral activity of Astragalus Polysaccharide (APS) and Isatis root polysaccharide (IRPS) on PCV2 in PK15, two groups were designed based on different route of administration.PCV2 was inoculated firstly in the first group, which was to observe whether the Chinese herbal drugs had the ability to inhibit infected PCV2 replication. The Chinese herbal drugs were inoculated firstly in the second group respectively, which was to observe whether the Chinese herbal drugs had the ability to block PCV2 adsorption and entry in the other group. In the safe concentration range, the result showed that IRPS has the blocking and inhibitory character to PCV2 in vitro, the minimum inhibitory concentration is 9.375μg/mL and the minimum blocking concentration of is 2.344μg/mL, APS also has the blocking and inhibitory character to PCV2 in vitro, the minimum inhibitory concentration is 18.750μg/mL and the minimum blocking concentration is 2.344μg/mL.