Study On The Pathogenesis And Inhibition Of Porcine Circovirus Type 2 Replication With Recombinant Adenovirus Expressing ShRNA In Vitro And Vivo

Porcine circovirus type 2 (PCV2) has recently prevailed in the world and induced several clinical disease,such as postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). However, the pathogenesis of the virus was not clear. And there is no effective prevention and control method for the PCV2-associated diseases in China. In this study, the pathogenesis of PCV2 was studied by analyzing the genetic characteristic of PCV2 isolates in China and the immunology of porcine alveolar macrophage (PAM) coinfected by PCV2 and PRRSV. Moreover, the inhibition of PCV2 replication were determinded by recombinant adenovirus expressing shRNA targeting to ORF1 and ORF2 regions of PCV2 in PK-15 cells and mouse model.1. Genetic characterization of PCV2 isolates in China.To fully understand the genetic diversity of PCV2 isolates in China, a retrospective study was performed on natural cases of postweaning multisystemic wasting syndrome described from 2002 to 2004 in 9 provinces or municipalities. The data about characterization of prevalence was collected as well as the clinical and histopathological symptoms were observed. To understand the genetic diversity of PCV2, the causative agent of PMWS, complete genomes of 11 PCV2 isolates were sequenced and aligned against the other 49 sequences of PCV2 isolates from Europe, North America, Asia and China. The results indicated that these isolates from 9 provinces or municipalities shared 93.5-99.7% in complete sequence and 89.9-99.9% in ORF2 homology with other 49 PCV2 sequences published in GenBank. In addition, the 11 PCV2 isolates from PMWS cases in this study showed 94.3-99.7% complete sequence identity and 89.3-99.7% homology in ORF2 compared with other strains from healthy cases, and share 94.5-99.6% nucleotide identity in complete sequence with strains from other PCV2-associated diseases (PDNS and abortion). Phylogenetic analysis of all 60 PCV2 isolates from North America, Europe, Oceania and Asia revealed that these isolates were grouped together in one large tree containing two minor clusters. The Chinese PCV2 isolates were spread in both clusters. It suggested that PCV2 infections and PMWS were common in pig herds in China. The PCV2 isolates in China have many genotypes and closely related to those in other countries in the world. No obvious diversity of geographical origin and time-interval exists. The relation between pathogenesis and genetic diversity of PCV2 was unclear.2. Effect of PCV2 and PRRSV coinfection on immunology of PAM in vitro.At first, we developed real-time PCR assays for PCV2, PRRSV and porcine cytokines (INFα, INFγ, TNFα, IL-8 and IL-10) detection. Then PCV2 and PRRSV were investigated in regard to their effects on monolayer cultures of porcine alveolar macrophages (PAMs). Two viruses infected PAMs alone or together with the different orders. Cytopathic effect (CPE) and livability of PAMs were observed and calculated. And virus titers and cytokines' mRNA (INF-α, INF-γ, TNF-α, IL-8 and IL-10) in PAMs were detected by real-time PCR and indirect immunofluorescence assay (IFA). The results showed as following: (1) PRRSV could replicate in PAMs and induce CPE, but PCV2 could not. (2) PRRSV could not help PCV2 to replicate in PAMs. PCV2 could inhibit the replication of PRRSV in PAMs when infected before or together with PRRSV. But when infected after PRRSV, PCV2 could promote PRRSV replication. (3) Coinfection of PCV2 and PRRSV could produce high level of TNF-α, IL-8 and IL-10 and less INF-γin PAMs than infection of PCV2 or PRRSV alone. It suggested that PRRSV had no effect on replication of PCV2 in vitro. However, the infection of PCV2 after challenge of PRRSV in PAMs could boost up the replication of PRRSV, and suppress the reaction of cellar immunity and humour immunity. The suppression of INF-γand over-expression of TNF-α, IL-8 and IL-10 caused by dual infection of PCV2 and PRRSV might enhance the pathological reaction and induced severer clinical symptom in vivo.3. Adenovirus-mediated RNA interference against PCV2 replication both in vitro and vivo.Firstly, shRNAs (S1 & S2) targeting PCV2 ORF1 and ORF2 were synthesized chemically and cloned into the pSUPER vector following H1 promoter. Then the shRNA expressing cassette was inserted into pAdTrack-CMV vector. The recombinant adenoviral plasmids (pAdS1 and pAdS2) were generated by homologous recombination of recombinant pAdTrack vector and pAdEasy-1 in E. coil BJ5183. Then recombinant adenoviral plasmids were linearized with Pac I and transfected into AD-293 packaging cells to produce recombinant adenovirus, rAdS1 and rAdS2. As the same, rAdH1, the control virus, was constructed by only cloning H1 promoter from pSUPER vector into AdEasy systerm.Secondly, we infected PK-15 cells with recombinant adenovirus rAdS1, rAdS2 and rAdH1. After 24h, the cells were challenged with PCV2. PCV2 antigen was detected by IFA assay at 48h post-infection of PCV2. The result showed that both rAdS1 and rAdS2 could inhibit the replication of PCV2, especially rAdS2 showed stronger inhibition (96%). Delivery of these shRNAs into cultured PK15 cells caused a significant reduction in PCV2 viral RNA production, viral DNA replication and protein synthesis in infected cells by semi-quantitative RT-PCR, real-time PCR and western blot assay, respectively. The antiviral effect was sequence-specific and dose-dependent and could sustain at least for 120h. Furthermore, by combination of treatment with rAdSl and rAdS2, the viral inhibition cloud be significantly increased. In addition, rAdS1 and rAdS2 displayed partial therapy for PCV2 infection beforehand.Lastly, the BALB/c mice were inoculated intranasally and interperitoneally with rAdSl or rAdS2 (10⁸ efu per mouse). After 24h, all the mice were challenged with 10⁴ TCID₅₀ of the PCV2 via the same routes. Then the clinical symptom of mice was observed every day. The necropsies of the mice were examined at 7,14, 21 and 28 days post-infection, and the level of PCV2 DNA in Spleens and blood was detected by real-time PCR. The results showed that no obvious clinical signs and macroscopic lesions were detected in each group. In positive group (PCV2 alone-infection), PCV2 nucleic acid in spleen of mice was increasing and arrived to the tip at 21 days post-infection. However, mice injected with rAdS1 and rAdS2 before PCV2 infection showed substantial and increasing decrease in the level of viral DNA replication in the spleen of the treated mice compared to the controls. This suppression was slight at the first two weeks, while it was significant (91.4%-96.8%) at the 3^rd and 4^th weeks. Meanwhile, no PCV2 was deteced in the blood of nearly all the mice.Taken together, these results indicated that shRNAs mediated by adenovirus could sufficiently and continuously inhibit PCV2 infection both in vitro and in vivo, and might be a potential alternative strategy for controlling PCV2 infection.