Epidemiological Investigation And Pathogenicity Of Isolate Of Porcine Circovirus 3

Porcine circovirus 3(PCV3)is a new pathogen.The prevalence of PCV3 reported was different in many studies,and the predominant genotype of PCV3 is not clear.Although several PCV3 studies have been conducted,there is little information regarding the pathogenicity of PCV3,in large part because of the small number of PCV3 isolates available.Furthermore,the clinical outcome of PCV3 isolates infection is still controversial.In this study,we conducted research and discussion on the prevalence,predominant genotype,isolation and identification in vitro,and pathogenicity of PCV3.A total of 1201 samples from domestic pigs collected in China from 2020 to 2021 were tested by conventional PCR,the prevalence of PCV3 and PCV2/3 co-infection was 26.3%and 18.6%,respectively,and there is no predominant PCV3 genotype at present.Molecular epidemiological investigation showed that the prevalence of PCV2 was 80.5%,94.6%,86.2%,30.9%and 51.9%,respectively;PCV3 was 21.4%,28.6%,37.9%,21.6%and 32.7%,respectively,in Heilongjiang,Henan,Hebei,Xinjiang and Hubei Provinces in China.The prevalence of PCV2,PCV3 and PCV2/3co-infection was 72.1%,26.3%and 18.6%,respectively,in all samples.Based on the phylogenetic analysis of Cap gene of PCV2 and PCV3,PCV2d has become the predominant genotype in China;PCV3a genotype was the predominant genotype in Heilongjiang and Hebei Province;PCV3b genotype was the predominant genotype in Xinjiang Province;PCV3c genotype was the predominant genotype in Henan and Hubei Province.This study provides information about the molecular epidemiology and genetic diversity,and theoretical basis for the diagnosis and prevention of PCV2 and PCV3 in China.A noval PCV3 strain,named PCV3-DB-1,from positive samples detected by specific PCR,was isolated and identified by limited passage of PK-15 cells.In this study,the genome of PCV3-DB-1isolation was confirmed to be 2000 bp by PCR sequencing.Based on phylogenetic analysis,the PCV3-DB-1 isolation was determined to belong to clade PCV3b.PCV3-DB-1 could not be passed continuously in primary cells and cell lines by specific qPCR,and was isolated to sixth generation in PK-15 cells in vitro.PCV-like particles were observed by electron microscopy,and PCV3 replication in PK-15 cell culture was confirmed by situ hybridization RNAscope.The PCV3-DB-1 genome copies were determined by qPCR assay and plotted against time to generate the one-step growth curves.The buoyant density of PCV3-DB-1 was determined to be 1.37 g/cm~3 by cesium chloride density centrifugation.The PCV3-DB-1 isolation of 24A and 27K of the Cap protein was first reported and identified.To investigate the pathogenicity of PCV3,the PCV3-DB-1 cell isolation and PCV3-infected lung lysate were used to inoculate three-week-old CDCD piglets.The clinical outcome of infected piglets was not obvious.In this study,the PCV3-DB-1 inoculated CDCD piglets exhibited no obvious clinical symptoms or macroscopic lesions.PCV3 displayed a broad histotropism,and the lungs and lymph nodes contained a higher quantity of viral genomes compared to that of the other organs.From 7 days after PCV3 DB-1 inoculation,the piglets showed weak Ig G antibody responses against PCV3rCap-VLPs.PCV3-DB-1 infection could not induce the dynamic change of T lymphocytes in peripheral blood by flow cytometry.Histological pathological observation of PCV3-inoculated piglets showed lymphopenia and inflammatory cell infiltration in the lymph nodes and widening of alveolar septum in the lungs.This study is the first to report the pathogenicity of PCV3 DB-1 isolate with the molecular characterization of 24A and 27K in the Cap protein in vivo,and provided a theoretical basis and insight for the clinical relevance of PCV3.