The Study On The Role Of Cellular Autophagy Induced By MicroRNAs In PCV2 Replication And Related Mechanism

Porcine circovirus type 2(PCV2)is the primary causative agent of porcine circovirus diseases(PCVDs),has a worldwide distribution and has emerged as one of the most important emerging viral pathogens of economic importance affecting global swine-producing.PCV2 is a single stranded,circular,non-enveloped DNA virus that belongs to the genus Circovirus in the family of Circoviridae.Porcine alveolar macrophages(PAM)is not only the target cells of PCV2 but also the important immune cells.The porcine monocytic cell line 3D4/31 and 3D4/21 supports PCV2 replication in vitro and the PCV2 infection model was established in these cells.Previous studies reported that PCV2 infection can induce autophagy in PK-15 cells.However,PK-15 cell line is not PCV2 target cells.Therefore,here we investigated the role of cellular autophagy in PCV2 infection 3D4/21 cells that are the PAM cell line.In the past several years,considerable advances have been made in understanding the complex interplay between autophagy and viruses.However,whether positive or negative regulation by autophagy of virus remains to be elucidated.Autophagy is a "double-edged sword" in virus,autophagy can play defense effect of itself and regulate the host antiviral immune response though autophagy related genes to remove the virus,as well as the repository for proliferation of virus and the assistant for escaping apoptosis and death of PCV2 infected cells to promote the proliferation of virus.In addition,even the same virus,the relation between autophagy and virus is different as the the specificity of target cells.MicroRNAs(miRNAs)are highly conserved,small noncoding RNA molecules that function to regulate a wide variety of cellular processes by interfering with protein expression or mRNA degradation.miRNAs can regulate autophagy and virus directly or indirectly,so it can be a new perspective to study autophagy and virus.miRNAs are also a"double-edged sword" in the process of autophagy or virus infection.miRNAs can pomote or inhibit the replication of virus and positive or negative regulate autophagy.Therefore,we use the porcine alveolar macrophage cell lines as a model to explore the relation between PCV2,autophagy and PAM,miRNA microarray is used to detect differential expression of miRNAs and mRNAs in the autophage process of PCV2-infected PAM.The way of biological information is used to predict the target gene.And the target gene is tested and verified,then construct the regulatory networks between the specific expression of miRNAs and the target gene to get the critical miRNAs which regulated replication of PCV2 on PAM.The interaction between the miRNA and target gene is validated to analyze the impact of miRNAs on the biological function of the target gene.Then the relationship between PCV2,autophagy and miRNAs is explored by using series of experiments.1.Molecular epidemiological analysis of porcine circovirus type 2 in Jiangsu and surrounding areas during 2010 to 2015384 samples that collected from Jiangsu,Anhui and Zhejiang provinces between 2010 and 2015,were screened by PCR for detection PCV2.The results indicated that the PCV2 infection was widespread and the infection rate was 41.15%.The phylogenetic trees were similar based on the complete genome or ORF2 sequences of 42 isolates and 11 reference strains.Forty-two isolates obtained in this study shared 87.0-100%and 89.6-100%based on comparing the ORF2 sequences and complete genome.Forty-two isolates were further divided into three genotypes as PCV2a,PCV2b and PCV2d.However,there were no obvious relations between the distribution of PCV2 and the areas.Genotype PCV2d were found in three provinces and it is the dominant genotype.The amino acid sequence analysis showed that the Cap protein of 42 isolates have a certain variation in specific epitopes,especially the mutation of aa77 and aa206 which were the key points of recognition of antidody.Whether these mutations affect the pathogenicity of PCV2 also needs to be further studied in the further.Further analysis the Cap protein suggested that the isolates had different seuences in different genotypes and exhibited especially representive mutations in the same genotype.In this study,2010AHCY,a strain of PCV2 was successfully isolated,belonging to the current dominant genotype PCV2d,which also provided the object in the following research.All these results might be useful to study the genetic variation and genetic relatedness among PCV2 strains in China.2.Differential expression of microRNAs in the autophage process of PCV2-infected PAM and functional regulation network of target genePAM is not only the target cells of PCV2 but also an important immune cell.PCV2 could infect PAM,not result in apoptosis,escape the cellular immune identify and cause immunosuppression.Autophagy can inhibit apoptosis and virus can escape cell-mediated immunity by miRNA interfere with the normal immune process of host cells.We assumed that PCV2 infection may lead to changes of miRNA regulation and autophagy of PCV2 and causing immunosuppression of host.We demonstrated that there was a positive correlation between PCV2 infection and autophagy in 3D4/21 cells and autophagy induced by PCV2 infection triggered PCV2 replication.miRNAs and mRNAs gene expression profiling were got by high-throughput sequencing and evaluated by RT-PCR,and miRNA mimics/inhibitors.These results demonstrated that several miRNAs can modulate autophagy and virus infecton.This pape focuses on the function of miR-30a-5p in the regulation of autophagy and virus infecton.Overexpression of miR-30a-5p significantly enhanced PCV2 infection and autophagy in a dose-dependent manner.Blockage of miR-30a-5p significantly decreased PCV2 replication.We provided further evidence that miR-30a-5p regulate the link between PCV2 infection and host immune system.Furthermore,miR-30a-5p may have partly regulatory impact on the cell cycle to regulate PCV2 replication and autophagy by interacting directly with 14-3-3 not PCV2 genome.These data not only provide new insights into virus-host interactions during PCV2 infection but also suggests potential new antiviral therapeutic strategy against PCV2 infection.