The Isolation Of PCV2 Henan Strains,and The Effect Of PCV2 Infection On The PD-1/PD-L1 Pathway

Pengli Xu ABSTRACT: PCV2 which belongs tobelongs to the family Circoviridae,genus Circovirus is a small,non-enveloped,single-stranded,closed circular DNA virus.Its whole genome is about 1.8 Kb and contains two important open reading frames(ORFs),ORF 1 and ORF 2.ORF l encodes a protein involved in PCV2 replication(Rep and Rep’)and ORF2 encodes a viral structural protein(Cap)with good immunogenicity.PCV2 is prevalent all over the world,and causes a variety of porcine circovirus associate diseases(PCVAD),such as post weaning multi-systemic wasting syndrome(PMWS),porcine dermatitis nephrotic syndrome(PDNS),sow reproductive failure and so on.Since discovered in the 1970 s,PCV2 which has severely impaired herds of pigs at various growth stages has rapidly rapidly spread around the world and had caused a variety of disease.At the same time,PCV2 infection can lead to autoimmune inhibition in pigs and could causes a variety of other pathogenic infections.Therefore,it has become one of the major threats to the economy of the pig industry in the world now.In order to understand the prevalence of PCV2 in Henan province and obtain a predominant strain of PCV2,62 samples(lymph node,spleen and lung tissues)suspected PCV2-infected pigs in Henan province from 2015 to 2017 was detected for PCV2 by PCR.Then the PCV2 was isolated with the PCV1-negative PK-15 cell line from the PCV2-positive samples,and identified by indirect immunofluorescence assay(IFA).35 PCV2 isolates were successfully obtained,and the complete gene of these PCV2 isolates was cloned and sequenced.The sequencing results showed that the complete genome of 34 PCV2 isolates was 1767 bp,and that of the remaining 3 strains was 1768 bp.The complete genome homology was 94.6%-99.9%,and the ORF2 gene homology was 88%-100% between the 37 PCV2 strains.These PCV2 isolates were highly homologous to the PCV2 classical representative strains at home and abroad and were relatively conservative.3 belong to PCV2 a,15 belong to PCV2 b and 20 belong to PCV2 a among 37 PCV2 strains.Interestingly,there was a PCV2 b and PCV2 d co-infection phenomenon in the DF-2 strain.Additionally,the results of genetic recombination analysis showed that some novel variations of PCV2 from gene recombination under natural conditions were found in these strains.It shows that the prevalence of PCV2 is relatively complex,and PCV2 d is gradually replacing PCV2 b as a predominant epidemic strain in Henan.Other subtypes also exist in individual regions.According to the whole gene sequences of pig PD-1 and its ligand in Gen Bank,the primers were designed to amplify the whole gene of them.After the whole gene was obtained,its extracellular domain were respectively inserted into p ET-28 a vectors and expressed in Escherichia coli,and were preliminary identified by Western Blot.Then the purified recombinant PD-1 and recombinant PD-L1 proteins were emulsified with Freund’s adjuvant and Injected New Zealand white rabbits with it.Three times after immunization,the polyclonal rabbit serum anti-PD-1 or anti-PD-L1 were obtained.Subsequently,the immunogenicity and reactogenicity of the two polyclonal sera were detected by Western Blot.The serum titer was 1:12 800.The complete sequence of PD-1 and its ligand was successfully cloned,and the extracellular domain of PD-1 and its ligand was successfully expressed.The acquisition of the polyclonal rabbit serum anti-PD-1 or anti-PD-L1 lays the foundation for the next test.KF-2 collected from 35 PCV2 isolates were inoculated into PK-15 cell and proliferated.Virus titers of it were detected by indirect immunofluorescence method(IFA)and KF-2 was obtained as candidate strain with the virus titer of 105.78 TCID50/0.1m L.To investigate the regulation mechanism of PD-1/PD-L1 signaling pathway after PCV2-infected PK-15 cells in vitro.Therefore,PCV2 was inoculated with normal negative PK-15 cells and PCV2-infected cells were harvested at 7 time points(1h,6h,12 h,24h,36 h,48h,72h),and negative cell controls were set at each time point.Subsequently,fluorescence quantitative PCR was used to detect the transcription levels of PD-1,PD-L1,IFN-γ,IL-18,and IL-2 at different time points.Then,PK-15 cells were treated with the polyclonal rabbit serum anti-PD-1 or anti-PD-L1 obtained from the last study at 37°C for 2 hours before PCV2 infection,the negative serum control and the control groups were performed subsequently.These PK-15 cells were harvested at 12 h and the transcriptional changes of PD-1,PD-L1,IFN-γ,IL-18,and IL-2 in each group were detected by fluorescent quantitative PCR.At the same time,PCV2 virus load was detected in PCV2 infected group.The results of this study showed that the transcription levels of PD-1,PD-L1,IFN-γ,and IL-2 in PK-15 cells were down-regulated during PCV2 infection,which were most down-regulated at 36 h,whereas IL-18 transcription was up-regulated at 12 h.However,when the PD-1/PD-L1 pathway is blocked,PCV2 replication was significantly inhibited.At the same time,PD-1,PD-L1,IFN-γ,IL-18,and IL-2 were significantly upregulated at 12 h.Taken together,it was shown that PCV2 infection may evade escape through the regulation of PD-1/PD-L1 signaling pathway,and this study also provides experimental basis for further investigation of the mechanism of PCV2 immunosuppression.