| Porcine circovirus virus(PCV)and Swine flu virus(SIV)are common viruses in pigs,two viruses were widespread throughout worldwide at present.A variety of pig diseases associated with the two viruses and swine industry is suffering the serious economic loss.PCV is a single circular DNA virus,since PCV1 was found in 1974 for the first time,three kinds of genotypes are currently prevalent in the pig populations.Among the three kinds of genotypes,PCV2 plays a vital role in the pathogenicity of the pig populations,while PCV3 was first discovered in 2016 from the aborted fetuses in sows in the United States,the identity of homology of Cap gene between PCV2 and PCV3 was low,and currently the data of epidemiological survey and the test method about PCV3 are not very perfect merely because PCV3 is a kind of new PCV genotype.SIV is a negative chain RNA virus which has eight segments and currently the predominant genotypes in swine populations are H1N1,H1N2 and H3N2.The influenza viruses from different sources can be recombined in pigs and influenza viruses can be transmitted from pigs to humans at the same time,therefore,the epidemiological investigation and pathogenicity study of SIV have important veterinary public health significance.In this study,we collected and processed tissue samples which were suspected of porcine circovirus-related diseases in pig farms of Tianjin,Shanghai and Shandong province from 2016 to 2018.We detect the collected tissue samples by the establishment of PCV2 and PCV3 double PCR method,and it was found that the positive rate of PCV3 was 32.7% and the situation of co-infection with PCV2 and PCV3 is widespread in swine populations(the positive rate of co-infection with PCV2 and PCV3 was 15.2%),and PCV3 has been shown to be widespread in pig populations.The positive samples were inoculated into pk-15 cells for virus isolation experiment,and only 2 strains of PCV2 were successfully isolated.The genetic evolution analysis of representative strains showed that the two PCV2 isolated strains belonged to two subtypes of PCV2b-1a and PCV2b-1c.The identity of full length and Cap gene homology of PCV3 sequences recorded in GenBank of the seven PCV3 strains in different regions were 97.6-99.4% and 97.1% respectively,both belonging to the PCV3 a subtype.In order to explore the antigenicity of PCV3 Cap protein,this study obtained the soluble expression of the deletion of nuciear localization signal peptide recombinant protein pCOLD TF-dPCV3-Cap by prokaryotic expression.The recombinant protein showed good reactivity and specificity by Western blot.In order to prepare monoclonal antibodies against PCV3 Cap,this study used constructed eukaryotic expression plasmid pCAGGS-PCV3-Cap to immunize mice,after cell fusion,positive hybridoma cell lines were screened by IFA.The identification showed that supernatant of 3 hybridoma cell lines could react specifically with PCV3 Cap protein,and 3G10 had the best response effect.To understand the prevalence and pathogenicity of swine influenza virus,in this study,it has been shown that the tissue samples collected from a pig farm in shandong province in 2018 were separated and identified,and three strains of recombinant H1N1 SIV were obtained.The results of genetic evolution analysis show that two surface gene HA and NA belong to the European avian subtype,and the other six internal gene sources belong to 2009 influenza A virus.In order to explore the pathogenicity of the isolated strains on mammals,we carried out the challenge experiment through nasal drip infection using the representative strain A/Swine/Shandong/L1/2018(H1N1)in the mice model in this study.The results showed that the representative strain presented higher pathogenicity in mice,which could lead to significant weight loss and the mortality is 100%.The representative strain was able to replicate well in the lungs of mice and virus infection caused obvious histopathological damage to the lungs of mice. |
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