Isolation And Identification Of PPV In Swine Serum And Optimization Of Viral Culture Conditions

Porcine Parvovirus (PPV), one of the pathogens cause reproductive failure of sows, which is responsible for stillborn, mummification, embryonic death and infertility. Apart from causing reproductive failure of sows above mentioned, it can also lead to non-suppurative myocarditis of piglets. Furthermore, PPV can co-infection with porcine circovirus type 2 (PCV-2) exacerbating Postweaning multi-systemic wasting syndrome (PMWS).Our lab collected 214 sera of a range of ages from a farm with poor administration of early-middle stage and further extracted the DNA from each serum and amplified by PCR with a pair of primers designed to target PPV. According to the results we selected 7 sera containing PPV isolating the viruses with ST cells. Freezing and thawing 3 times after 3 times of blind serial passages, the cells inoculated serum were detected by PCR with the primers mentioned above and the results showed one of them was positive. We designed the isolate as 16WS and further characterized it by sequencing partial genome, observing CPE and measuring HI titer. The sequence obtained was retrieved in GenBank, which showed high identity with PPV, especially strain YL isolated by Shi in 2012, as high as 99.1%. In the process of viral culture, we found that the cells manifest relatively stable CPE. And the HI titer is 28.Several countries in the world control the disease caused by PPV through immunizing vaccines especially inactivated vaccine. One of the most important issues of developing inactivated viral vaccines were obtaining high titer viruses which can be possibly acquired by optimizing the viral culture conditions. In the research, we investigated the viral culture conditions including the condition and density of ST cells used to inoculation, sensitisation time of inoculating viruses, viral copies of inoculation, time of viral incubation in ST cells, volume of cell culture medium, and the concentration of CO2 in cell lines inoculated PPV etc.. The results showed that ST cells cultured 48 h and 12 h after the cells serial passage, diluting the cells to the density～60%(5.0-6.0×109 cells/ml) before inoculating with 107 copies of PPV by asynchronous inoculation with 1 h incubation in 2 ml medium in 5% CO2 for 72 h will stabilize a relatively higher viruses titer more than 3.59x 109 copies/ml.