Establishment Of A Balb/C Mouse Model For Infection With Porcine Circovirus-2-Like Agent P1

Porcine circovirus (PCV), a non-enveloped, covalently closed, single-stranded circular DNA virus, is classified in the genus Circovirus of the family Circoviridae. Two types of PCV have been identified:PCV-1and PCV-2. PCV-1is non-pathogenic for pigs, while PCV-2is related with many PCV-associated diseases. PCV-2is the most important pathogenes for post-weaning multisystemic wasting syndrome (PMWS), but only PCV-2is not sufficient to cause the typical symptoms.We isolated a novel virus P1in the diagnosing process of PMWS. P1is a circular DNA of648nucleotides, part of them are highly homology with ORF2of PCV-2. Althought there is lots of progress on the research of the biological characteristics and differential diagnosis of the agent P1, the establishment of the P1infection model is of great significance.Firstly, fluorescence quantitative real-time PCR for P1was established. The primer and probe were designed and synthesized according to the P1gene sequence, then a TaqMan fluorescence quantitative real-time PCR was developed by optimizing reaction conditions and parameters.The method had the advantages of specificity, sensitivity and rapidity. In addition, it had a dynamic range of detection between101-108copies/μL. The correlation coefficient of the real-time PCR is greater than0.99, and the amplification efficiency is between90%-105%.Secondly, Balb/c mice were studied as the animal model of P1infection. The health Balb/c mice were intraperitoneally injected P1. Comparing the weight growth rates to detect the direct impact of P1in mice. Using the real-time fluorescent quantitative PCR method, together with the ELISA, and immunohistochemistry (IHC), we studied the dynamic changes of the viral nucleic acid in the tissue of Balb/c mice, the changes of antibody agaist P1agent and the localization of antigenes in the tissue. The results showed that the weight growth rates on the three times challenged group was significantly lower than the once challenged group and control group on day14and21PI. The antibody to P1could be detected throughout the whole challenged cycle by ELISA. P1nucleic acid could be detected in heart, liver, spleen, lung, bladder, testis, brain, thymus and pancreas of infected mice by real-time fluorescent quantitative PCR. Positive cells could be detected in heart, liver, spleen, lung, testis and thymus of infected mice by IHC.All of these results proved that the animal model of P1infection was successfully constructed. This animal model laid a foundation for studying the pathogenicity and the invasion pathogenic mechanism of P1in Balb/c mice.