Porcine Parvovirus Inducing Autophagy to Benefit Its Replication

Porcine parvovirus(PPV) is one of the major causes of reproductive failure in pigs, which poses a great threat to the pig breeding industry and results in tremendous economic losses worldwide. Autophagy is the biological process of cell self-defense and self-protection. Despite many viruses can cause cell autophagy, when they enter cell or copied, the relationship between autophagy and PPV infection has not been reported. In this study, impact of autophagy after swine testicular(ST) cells infected by PPV was studied. Autophagy was demonstrated by the effective replication of PPV through transmission electron microscopy, immunofluorescence and western blot analysis. Moreover, autophagy was confirmed to benefit PPV replication by real-time fluorescence quantitative PCR and determination of median tissue culture infective dose(TCID). For the first time, the complex interaction between PPV infection and autophagy was explored in this study. It indicated that PPV could induce autophagy in ST cells, which in turn facilitated its own replication, which might be one of the mechanisms of the virus infection. These findings could facilitate the study of the pathogenesis of PPV infection and provide new insight into the development of effective therapeutic strategies.

Molecular Cloning and Prokaryotic Expression of Non-Structural Protein NS1 Gene of Porcine Parvovirus

Porcine parvovirus (PPV) is one of the major agents causing swine reproductive failure. NS1 protein is a non-structural protein of PPV and can be used as a reagent for differentiation of vaccinated animals and infected ones. In present study, a recombinant plasmid pET28a/NS1 was constructed by cloning the coding sequence for NS1 of PPV into pET28a, a bacterial expression vector. The NS1 protein was expressed in E. coli BL21(DE3) after induced by IPTG and the recombinant fusion protein was purified with affinity chromatography. Expression amount of NS1 protein was improved by optimizing the inducing parameters. The recombinant NS1 protein is reactive to PPV positive sera in Western blot and ELISA test and therefore can be applicable in differential diagnosis of PPV infections.

Epidemiology Survey of Porcine Parvovirus and Porcine Circovirus Type 2 in Sichuan Province

[ Objective] The study aimed to analyze the prevalence of Porcine Parvovirus （PPV） and Porcine Circovirus Type 2 （ PCV2 ） and the mixed infection in Sichuan Province to lay the foundation for further predicting the tendency of the plague and formulating appropriate prevention and control strategies. [ Method] Two hundred and seventy -three samples were collected from 21 large pig farms in Sichuan province, and epidemiology of PPV and PCV2 were investigated by PCR detecting. [ Result] The positive rate of PPV was 17.22% （47/273）, and positive rate of pig farms was 38.1% （8/21）, meanwhile it also displayed the feature that infection rate of boar was higher than that of piglets; The positive rate of PCV2 was 52.38% （143/273）, and positive rate of pig farms was 85.7% （18/21）, and it showed the trend that the infection rate of PCV2 was rising with the growth of the age. The co-infection rate of PPV and PCV2 was 10.62% （29/273）, and co-infection rate of pig farms was 28.7% （6/21）, which mainly concentrated in the sow and nursery piglets. Only 14.3% （3/21） pig farms was epidemiologically negative of PPV and PCV2. [ Conclusion] PPV and PCV2 and co-infection was widely popular in Sichuan province, and it did more serious harm to the pig-raising industry.

Development and Preliminary Application of SYBR Green I Real-Time Fluorescence Quantitative PCR Method for Detecting Porcine Parvovirus Virus

According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.