Prevalence Of Porcine Circovirus Type 3 In Zhejiang And Mechanism Of Cell Autophagy Induced By Its Capsid Protein

Since porcine circovirus type 3(PCV3)was first reported in US in 2016,the novel virus has been identified in major pig-producing countries in the world.Pigs infected with PCV3 showed clinical symptoms such as reproductive failure,porcine dermatitis and nephrotic syndrome,myocarditis and multi-system inflammation.PCV3 DNA can also be detected in pigs with no apparent clinical symptoms.PCV3 has a genome size of about 2000 bp,containing three open reading frames(ORFs).Orf1 encodes the Replicase protein,and orf2,the Cap protein.The putative orf3,its coding regions and function,remains unknown.PCV3 can be detected in animals such as wild boars,dogs,cattle,mice,and ticks.At present,most of the studies focus on epidemiological investigations and detection methods.Isolation of PCV3 epidemic strains and rescue of recombinant viruses have rarely been reported,and the pathogenic mechanism is not clear.Autophagy is a highly conserved recovery process of nutrients in eukaryotic cells and plays an important role in maintaining cell homeostasis and preventing stress caused by nutritional metabolism or infections.There has been no report so far on the involvement of autophagy in PCV3 infection.This study is aimed to:(1)establish the Taqman qPCR assay and indirect ELISA method for epidemiological investigation of PCV3 in Zhejiang province,and clarify the sequence characteristics of the orf2 gene of PCV3 field strains;(2)attempt to rescue the PCV3 recombinant virus in pig-derived cells,such as PK-15 and IPEC-J2,and BALB/c mice for studying the pathogenetic mechanisms of PCV3 infection;and(3)investigate whether PCV3 Cap protein induces autophagy in cells,and which specific molecule in the autophagy pathway is involved.1.Epidemiological investigation of porcine circovirus type 3 in Zhejiang provinceTaqman qPCR method was established to detect 283 clinical samples collected from 2014 to 2017 in Zhejiang province.The total positive rate of PCV3 is 67.1%,of which one third of the samples were from single infection with PCV3,and most of them were mixed infections.Co-infection of PCV3 and PEDV(41.6%)is predominant,and the co-infection with other viruses was low.Indirect ELISA method was established using truncated PCV3 Cap protein as the coating antigen.A collection of 2345 pig serum samples from 2011 to 2017 was tested and 52.6%were positive.A total number of 203 pig serum samples were randomly selected for detection with both methods.More positive findings were seen with qPCR than with ELISA(81.3%vs 56.2%).Based on 200 PCV3 orf2 sequences,the virus can be divided into three main genotypes,namely,PCV3a,PCV3b,and PCV3c.Most of the 38 PCV3 orf2 gene sequences obtained in this study clustered into PCV3c genotype.Key amino acid positions(24,27,77,and 150)of the Cap protein of three PCV3 genotypes were analyzed,and only PCV3a genotype showed obvious‘VKSI'signature pattern(about 95%),while the other genotypes did not have apparent signature pattern.This study further proved that the PCV3 infection is severe in Zhejiang province and the infection in this province should have begun before 2014,presumably in 2012,when the PCV3 seropositivity was very high.2.Exploration on rescue of porcine circovirus type 3 recombinant virusesCircular PCV3 DNA,obtained by PCR and in vitro circularization,was transfected into pig-derived cells PK-15 and IPEC-J2,and cells are continuously passaged to the 10th generation.qPCR detection of PCV3 DNA in cells and supernatants of each generation showed decreasing trends over continuous passaging.In the 10th passage,PCV3 nuclei acid was close to the lower limit of qPCR detection(102 copies).There were no PCV3-positive fluorescent cells as detected by IFA.However,the recombinant PCV2 virus,which served as positive control,was successfully rescued at the first passage.BALB/c mice were inoculated with circular PCV3 DNA through tail vein injection.After 14 and 21 d,no PCV3 antibodies were detected in the serum samples,and PCV3 DNA levels in serum,spleen,and liver remained low even with slight decrease.Further attempt was made to rescue PCV3 virus using different vector and viral sequence.The whole genome sequences of the reference strains PCV3/MO2015 and PCV3/Hebei-LY-2015 were cloned into the pBlueScript SK(+)vector for construction of recombinant plasmids pSK-PCV3.PK-15 cells were transfected with pSK-PCV3 and continuously passaged to the fifth generation.PCV3 DNA levels of cells and supernatants decreased over increasing passages.By the fifth generation,the nucleic acid level approached the lower limit of qPCR detection.PCV3 orf2 gene was not transcribed as shown by qPCR.To this end,rescue of PCV3 recombinant virus in pig-derived cells PK-15 and IPEC-J2 and mice was unsuccessful.3.Cap protein of porcine circovirus type 3 induces cell autophagyBecause of unavailability of live viruses for pathogenicity studies,we constructed a recombinant plasmid pCMV-Cap and a recombinant lentivirus LV-Cap that carrying the PCV3 orf2 gene.Whether plasmid transfection or lentivirus-mediated gene transduction,PCV3 Cap expression was low or even not expressed in pig-derived cells PK-15 and IPEC-J2.Considering the high transfection efficiency of HEK293T cells,we explored the effect of PCV3 Cap protein on autophagy in this cell line.By immunoblotting,laser scanning confocal microscopy,and transmission electron microscopy,we have demonstrated for the first time that the PCV3 Cap protein induced autophagy and complete autophagy flux in HEK293T cells.The ubiquitin-proteasome pathway was also involved in the autophagy process.Further research shows that PCV3 Cap protein might induce autophagy in HEK293T cells by inhibiting the phosphorylation of mammalian Rapamycin target protein(mTOR).In summary,Taqman qPCR and indirect ELISA methods were established for epidemiological investigation of PCV3 in Zhejiang province.PCV3 infection is sever on many pig farms in Zhejiang.PCV3 Cap protein induced cell autophagy by inhibiting mTOR phosphorylation,and the ubiquitin-proteasome pathway was also involved.This study provides useful reference for control of PCV3 infection and further research on its pathogenetic mechanisms.