Construction Of Porcine Circovirus Type 2 ORF5 Gene Mutant Strain And Research Of Its Infection Characteristics

Porcine circovirus type 2(PCV2)is a single-stranded negative-strand circular DNA virus with a genome length of 1 767?1 768 bp.The virus particle has an icosahedral symmetric structure and has no envelope.The size of the virus particle is about 17 nm.PCV2 causes damage and suppression of the immune system of pigs,severely damages the health of pigs,and often secondary or mixed infections with other pathogens,causing more serious damage.The discovery of the pathogenic mechanism of PCV2 is helpful for better prevention and control.The identification and functional research of the virus-encoded product is the basis for revealing the pathogenic mechanism of PCV2.Bioinformatics analysis found that the genome of PCV2 contains at least 11 potential open reading frames(ORF),of which 6 are currently reported(ORF1-6),of which ORF5 is a new PCV2 encoded protein discovered and identified by the research team.Preliminary research results show that ORF5 protein can cause cell cycle arrest in the host,induce endoplasmic reticulum stress response and autophagy,and promote virus replication.The above results are mainly obtained with ORF5 protein as the research object,and ORF5 is at the viral level The role of this still needs to be studied in depth.In this study,we cloned and obtained the full genome sequence of PCV2,and mutated the start codon(ATG)of ORF5 gene by point mutation technology,and rescued PCV2 cloned strain(rPCV2)and ORF5 gene mutant strain(PCV2^?ORF5).We further studied the role of ORF5 on virus growth characteristics,cell infection,and experimental animal infection.The role of ORF5 was clarified at the virus level and provided new information for the discovery of the pathogenic mechanism of PCV2.The research obtained the following results:(1)Obtained PCV2 clonal strain and ORF5 gene mutant strain.The whole PCV2 gene was cloned,and the recombinant plasmid PUC57-rPCV2 inserted into the whole PCV2 gene sequence and the recombinant plasmid PUC57-PCV2^?ORF5 of the mutant ORF5 gene were constructed respectively.After single enzyme digestion,in vitro self-circularization ligation,the ligation product was transfected into cells and blinded.Through experimental operations such as transmission,rPCV2 and PCV2^?ORF5 strains were obtained respectively.(2)The ORF5 gene mutation reduces virus replication and proliferation and induces endoplasmic reticulum stress and autophagy.Porcine alveolar macrophages 3D4/21 were infected with rPCV2 and PCV2^?ORF5 strains.Transmission electron microscopy,RT-PCR,western blot,flow cytometry and other methods were used to influence virus replication and proliferation and cell function.analysis.The results showed that the replication and proliferation of rPCV2 and PCV2^?ORF5 strains in cells were consistent,but the ORF5 gene mutation significantly reduced the replication and proliferation ability of the virus at 36h(P<0.05);rPCV2 and PCV2^?ORF5 strains all induced endoplasmic reticulum stress,autophagy and apoptosis,but the mutation of ORF5 gene caused endoplasmic reticulum stress,autophagy,late cell apoptosis and cell death of infected cells.The cycle retardation effect was significantly reduced(P<0.05 orP<0.01).The results of the study confirmed from the virus level that ORF5 protein can induce endoplasmic reticulum stress,autophagy and cell cycle arrest in host cells during viral infection.(3)ORF5 gene mutation reduces the replication and proliferation of the virus in experimental animals,induces endoplasmic reticulum stress and autophagy.Inoculate Kunming mice with PCV2,rPCV2,PCV2^?ORF5 venom via intraperitoneal injection,once a day,and collect blood from the tail vein every 7 days after the 4 inoculations,and detect the viral load in the blood by fluorescence quantitative PCR;When high viral load appears in the blood of the mouse(28 days after infection),each group of mice is anesthetized and killed.The mouse heart,liver,spleen,lung,kidney,brain,lymph node and other tissues and organs are taken aseptically.RT-PCR and western blot detection of viral load,endoplasmic reticulum stress and autophagy marker expression in each tissue;tissues and organs were made into electron microscope and common pathological sections for observation.The results showed that the viral loads in the liver,spleen,kidney,and lung of the three virus-infected mice were higher,but the viral load in the same tissues of the PCV2^?ORF5group was lower than that of the PCV2 and rPCV2 infection groups(P<0.01 orP<0.05),indicating that ORF5 gene mutation reduces the ability of the virus to replicate and proliferate in animals;viral load is positively correlated with the expression of endoplasmic reticulum stress and autophagy marker factors in tissues,liver,spleen,The expressions of GRP78,Beclin1 and LC3?/?in kidney and lung were higher than other tissues(P<0.01orP<0.05),but the expression of marker factors in the same tissues of mice infected with PCV2^?ORF5 was significantly lower than that of PCV2 and rPCV2 infections Group,indicating that the ORF5 gene mutation reduces the virus-induced endoplasmic reticulum stress and autophagy.The histopathological observation results showed that there were pathological changes such as inflammatory cell infiltration,lymphopenia,cell apoptosis,and cell degeneration in the mouse tissues of the three virus-infected groups,but there were no significant differences between the groups.The research results show that ORF5 gene mutation has no significant effect on virus infectivity and pathological changes,but it reduces virus replication and proliferation,induces endoplasmic reticulum stress and autophagy.Based on the above results,this study constructed a PCV2 strain that mutated the ORF5gene.The mutation of ORF5 reduced the level of virus replication and proliferation in vivo and in vitro,and reduced the virus inducing endoplasmic reticulum stress and autophagy in vivo and in vitro.But it has no significant effect on the infectivity and pathological damage of the virus.This study further clarified the role of ORF5 in PCV2 infection from viruses,in vivo and in vitro,and provided new data for the elucidation of PCV2 infection and pathogenic mechanisms,which has theoretical and potential application value.