The Influence Of PCV2 Infected Pig Tissues By The CRISPR/Cas9 Mediated Deletion Of The GC1qR Gene

Porcine circovirus type 2(PCV2)is a capsule-free single-stranded circular DNA virus,belonging to the genus Circovirus within the family Circoviridae,is also the main pathogen caused by Postweaning Multisystemic Wasting Syndrome.The PCV2 specific replication and pathogenesis is unclear,but it has been reported that gC1 qR interacts with PCV2 Capsid protein and down-regulates the expression level of gC1 qR in PCV2 infection;PCV2 activates PI3 K / Akt and p38 MAPK pathway through gC1 qR and Cap interaction can promote interleukin-10 production in macrophages.It is not clear what the role of gC1 qR in PCV2 plays in vivo replication and its pathologic changes.In this research,we constructed the CRISPR-Cas9 recombinant lentiviral system targeting piglets gC1 qR gene.That recombinant lentivirus was infected with the piglets in order to obtain the gC1 qR gene partial knockout in lung tissue of piglets.Using PCV2 infected the gC1 qR gene partial knockout in lung tissue of piglets and the PCV2 infection model was copied to explore whether the deletion of gC1 qR would affect the replication of PCV2.Research results are listed as follows:In this study,three different gRNAs(g113,g229 and g246)were designed using the CRISPR-Cas9 system,and three recombinant lentiviral systems rLenti-113,rLenti-229 and rLenti-229 were successfully constructed.Using recombinant lentiviral systems transmit Cas9 and gRNA to infected PK-15 cells,filtrating positive clone cells with to obtain the PK-15 cells which gC1 qR gene was knocked out.A successful PK-15 cell with gC1 qR knockout was identified by western blotting and cell genome sequencing.The results showed that rLenti-113,which was targeted at the 113-point packaging of porcine gC1 qR gene,could be used for subsequent animal experiments.With a large number of 293 T packaging rLenti-113 and rLenti-Blank,then recombination lentivirus infected the 7-day-old piglets.By western blotting and immunohistochemistry analysis to identify the partial deletion gC1 qR gene in porcine lung and hilar lymph nodes.Using PCV2 to continue to infect normal control piglets,gC1 qR gene partial deletion piglets and blank control piglets,the results showed that the PCV2 infection model was successfully replicated in normal piglets and vacant lentivirus-infected piglets.In the piglets with the gC1 qR gene partial deletion of the tissue,the pathological changes induced by PCV2 infection in lung and hilar lymph nodes were reduced.Normal piglets,blank control piglets and gC1qR-partial-deficient in tissue of piglets were infected with the same PCV2 copies.We found that virus copy number in the serum was reduced after 1 week of infection in gC1qR-partial-deficient in tissue of piglets,compared with the blank control piglets;and then viral copies in hilar lymph nodes after 4 weeks of infection decreased,while the lung and liver virus copy number changes are not obvious.The expression of Cap and Rep was lower in gC1qR-partial-deficient piglets in tissue compared to normal piglets infected with PCV2.Based on the above results,rLenti-113 targeting the porcine gC1 qR gene was successfully constructed and the gC1 qR gene was partial knocked out in the hilar lymph nodes of the piglet through the CRISPR-Cas9 lentivirus system.PCV2 infection with gC1 qR partial deletion of piglets in tissue to copy PCV2 infection model,in which clearing the gC1 qR gene in PCV2 replication and pathogenesis of the role,providing a pathogenesis basis of PCV2 for the further.