Study On Molecular Epidemiology In Huadong Region And Cases Of Piglets Naturally Infected With Porcine Circovirus-like Virus P1

Porcine circovirus,belonging to a member of circovirus,is divided into two genotypes including PCVI and PCV2.And only PCV2 was pathogenic for pigs.It's isolated from infected pigs with post-weaning multisystemic wasting syndrome(PMWS)for the first time in Canada.Initial studies show that the disease is mainly caused by PCV2.However,subsequent researches have indicated that pigs only infected with PCV2 donot performance typical symptoms of PMWS,but on the condition that PPV.PRV.PRRS and the like exsisting,it will show the symptome.It leads to mixed infection,and results in severe clinical symptoms and high mortality.Porcine circovirus-like virus P1 was a novel virus separated from clinical detection of suspected PMWS pig samples.The genome of P1 is a closed circular DNA molecule containing 648 nucleotides only,which has a similar structure to that of PCV2.Moreover,the full-length sequence of PI has high homology with the gene fragment of opening read frame 2(ORF2)of PCV2.With continuously thorough of the etiology of P1,it has been pointed out that PI double molecular cloning virus can induce apoptosis of PK-15 cells and cause the symptoms of PMWS in infected pigs.However,the epidemic situation and pathological study related to piglets naturally infected with P1 virus remain to be researched.And the pathogenic mechanism is still not clear.Therefore,the study of cases in pigs naturally infected with P1 helps to clarify the possible responsibilities of P1 for the clinical pathogenicity development.This study inducted a research of molecular epidemiology investigation of P1 in Jiangsu Province and other regions by PCR,a molecular cloning method.After sequencing of separated virus strains,we have obtained 15 sequences of P1 complete genome.Then these sequences are conducted with phylogenetic analysis.At the same time,the cases of piglets naturally infection with PI accompanied with serious nervous tremor are studied by dection of virus load of nucleotides of PI and series of pathological experiments.The main work targets of this paper are shown below:1.Molecular epidemiological investigation of P1 in Huadong region and sequence analysis of cloned virus strainsIn order to master the epidemic characterristics of P1 virus in Huadong Region,254 samples collected from hundreds of medium-sized and small pigfarms between 2015 to 2016 in Jiangsu Province,Hebei Province,Anhui Province and Zhejiang Province were detected by PCR method.The results show that 45 samples detected as P1 positive and the infection rate accounts for 17.70%.It demonstrates P1 exists widespread in pigfarms of Huadong Region.The homogeneous analysis of 15 sequences seperated by molecular cloning and sequencing method and P1 reference strains(KJ612072.1)suggests strains from different areas share a ratio of over 95%on homology.It turns out that there is no significant regionalism and variability of P1 isolate strains in Jiangsu province and surrounding areas.The result of ORF1 sequence analysis shows a ratio of 98%above on homology demonstrating ORF1 of P1 conservative.To understand the evolution history of P1 virus,the phylogenetic tree of P1 and three PCV2 genetic subtypes including four references strains PCV2a(AF055392)?PCV2b(AF055394)?PCV2c(EU148503)and China-BF(AF381175)is conducted.It suggests strains P1(KU356940)and strain China-BF are in the same branch with other three PCV2 subgenotype presenting a separate branch,indicating P1 and PCV2 have a closer genetic relationship.Therefore,the study of pathogenicity of P1 plays an important clinical significance.2.Pathological study on cases of piglets naturally infected with P1 virusThe study on cases of piglets infected with P1 virus accomponed with congenital tremor(CT)is carried out to discuss the role of P1 virus in the pathology of CT clinically.The nuclecide distribution of P1 in tissues such as heart,brain,liver and lymph nodes is detected by real-time PCR method established in the papers before.At the same time observation of pathologic changes and imnunohistochemistry experiments for location detection of viral antigen are conducted.After exclusion of main pathogen resulting in CT,the result of quantitative detection of P1 nucleic acid shows lymph node tissue has the highest copies of 105 copies/g,and brain tissue reached about 103 copies/g,which indicates lymph nodes and brain are likely to be the main organs where PI virus replicates.Histopathological observation shows that different tissues have different degrees of inflammatory cell infiltration.Lymphoid lesions is the most obvious with partial lymphocyte in follicle dissolving and disappearing.Cerebrum tissue in parts showsnerve cells are sieged and swallowed by gitter cells,a small quantity of hyperplasianervegliocyte,and cerebral vascular congestion around.Cerebellar tissue performances as partial Purkinje cells karyolysis and loss.Kidney tissue shows renaltubulesepithelial cell lysis,necrosis and exfoliation with inflammatory exudate in lumen.But changes of other tissues are not obvious.The immunohistochemical experiments of brains of two piglets with serious tremor(NO.1/2)show part of the cytoplasm stained yellow indicating positive signal in brain tissue.The above results state the clinical symptoms and tissue lesions of infected piglets consistent with the result of viral load and viral antigen location detection to some extent.As a result,it can be inferred piglets naturally infected with P1 and congital tremorappear to have a close relationship.P1 is likely to be a potential pathogenic of CT,but the specific pathogenic mechanism remains to be further studied.