Suppression Of Replication Of Porcine Reproductive And Respiratory Syndrome Virus By Recombinant Adenovirus Expressing ShRNA Targeting The Viral Genes Or Viral Receptor Genes On PAM

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single-stranded positive-sense RNA virus of the family Arteriviridae in the order Nidovirales. PRRSV is the causative agent of porcine reproductive and respiratory syndrome (PRRS) which characterized by reproductive problems in sows such as premature farrowings, poor farrowing rates, and increased stillbirths, as well as respiratory problems in piglets such as pneumonia and atrophic rhinitis. PRRSV can persist in pigs for a long period of time after initial infection and it may result in immunosuppression. Thus, PRRSV is one of the most economically significant viral diseases in the swine industry. Recently, a highly pathogenic PRRSV spread wildly in many swine herds in China and caused serious economic losses (Li et al.,2007b; Tian et al.,2007). The current commercial PRRSV vaccines are not sufficiently to protect pigs from PRRSV infection. Therefore, it is imperative to develop new antiviral strategies to prevent and control this viral infection.RNA interference (RNAi) is a sequence-specific mRNA degradation phenomenon that induced by small interference RNA (siRNA). RNAi is involved in the regulation of genetic functions and provides defense against virus at the posttranscriptional level in mammalian cells. siRNA is a 19 to 27 nucleotide (nt) RNA molecules homologous to the target genes. siRNA could be achieved by in vitro transcription or from short-hairpin RNA (shRNA) expressed by DNA vector. RNAi has been used to suppress a variety of viruses originated from human or animals, which implicated the potential of RNAi as an antiviral strategy.shRNAs expressd by pSUPER have been demonstrated could specifically inhibit he replication of PRRSV in MARC-145 cells. However, plasmid shRNA-expressing system is not suitable for in vivo application. In this study, recombinant adenoviruses and plasmids expressing shRNA target to PRRSV genes and viral receptor genes were constructed and the suppression effect on target genes induced by shRNA was examined both in vitro and in vivo The content of this thesis consists of seven parts as the following:1. Development of SYBR Green real-time PCR method for detection of PRRSV genesPrimers specific to the ORFlb of PRRSV S1 and SY0608 isolates were designed, respectively. And a rapid and sensitive method of real-time PCR detection of ORF1b gene was developed after a serial modification of reaction system. The results showed that the real-time PCR detection methods developed in this study were sensitive, specific and repetitive, and the lowest range of the sensitivity of the primers both were 10～100TCID502. Inhibition of PRRSV replication induced by shRNA plasmids targeting ORF1bTwo shRNA expression vectors, pSUPER-P2 and pSUPER-P3, targeting ORF1b gene of PRRSV were transfected into MARC-145 cells, and infected with identical amount of PRRSV 24h later. At 48h or 72h post-infection, the inhibition effect on PRRSV replication induced by shRNA plasmids and the mechanism were investigated TCID50, real-time PCR and IFA test. The results showed that PRRSV induced cytopathic effect (CPE) could be inhibited in the cells transfected with pSUPER-P2 and pSUPER-P3, and the virus titers were reduced by approximately 100-fold compared to those control cells. The expression of PRRSV ORFlb gene was significantly decreased both at RNA and protein levels in the cells comparing to the controls. It indicated that vector based shRNA targeting ORF1 region could effectively inhibit PRRSV replication in MARC-145 cells.3. Construction of recombinant adenoviruses expressing shRNA targeting different PRRSV genesRNA-expression cassettes in recombinant pSUPER (pSUPER-N3, pSUPER-G1, pSUPER-P2 and pSUPER-P3) were amplified by PCR and coloned into adenovirus shuttle vector pAdTrack-CMV, under the control of Pcmv promotor. The recombinant shuttle vectors were identified by sequencing and cotransformed with the pAdEasy-1 into E.coli BJ5183 by electroporation and the recombinant adenoviral vectors were generated by homologous recombination. The resultant adenoviral plasmids were linearized by Pac I, and were transfected into HEK-293A cells. The production of recombinant adenoviruses rAd-N3 (ORF7), rAd-G1 (ORF5), rAd-P2 (ORFlb) and rAd-P3 (ORF1b) were obtained. The mutant shRNA expressing adenoviruses rAd-mN3 and rAd-mP2 were constructed as controls. All the recombinant adenoviruses could express GFP and induce CPE 72h after infecton in HEK-293A cells, and all the adenoviruses could reach a titer of 1010～1011efu/mL. Detection of PH1-shRNA in the genome of the recombinant adenoviruses was all positive.4. Comparison of inhibition effect on PRRSV replication induced by rAd-shRNA targeting different viral genes in vitroTo examine whether the shRNA expressing adenovirus could suppress PRRSV replication effectively, MARC-145 cells were first inoculated with 500MOI rAd-shRNAs individually; 24h later, the cells were challenged withlOMOI PRRSV; At 48h after infection with PRRSV, the cells were examined for GFP expression and CPE, and the culture supernatants were collected and virus yields were detected by TCID50. PRRSV N, ORFlb and GP5 protein were investigated by indirect immunofluorescence (IFA) using specific antibody accordingly, and the shRNA targeting genes were detected by real-time PCR using specific primers. It showed that (1) PRRSV induced CPE and viral titers were decreased in the four rAd-shRNA incoculated cells as compared to rAd-mN3 control (p<0.05); (2) PRRSV ORF7 and ORF1b genes were suppressed by 100-fold relative to control; (3) The level of N, GP5 and ORF1b protein were significantly reduced compared to rAd-mN3 and PRRSV challenge control groups. It indicated that adenoviruses expressing shRNA could inhibit PRRSV replication in MARC-145 cells.To determine whether rAd-shRNAs could inhibit PRRSV replication in primary PAM cells, PAM cells were submitted to the same treatment as in MARC-145 cells; At 48h after PRR.SV infection, the cells were examined for CPE, and PRRSV ORF7 gene expression in the cells was investigated by IFA using N protein monoclonal antibody and real-time PCR. The results demonstrated that (1) the four shRNAs targeting different PRRSV genes could all suppress viral replication in PAM cells by 100～1000 fold;(2)The suppression effect induced by shRNA was reduced over time; (3) The suppression effect was dose-dependent on shRNA quantity as inoculation of more rAd-shRNA could induce stronger antiviral effect; (4)rAd-N3,rAd-G1,rAd-P2 and rAd-P3 could inhibit PRRSV replication in PAM previously or simultaneously infected with PRRSV5. The inhibition of high pathogenic PRRSV replication induced by rAd-shRNA targeting ORF lb gene both in vitro and in vivoThe inhibition effect induced by rAd-P2 and rAd-P3 on the replication of PRRSV traditional S1 strain and high pathogenic SY0608 strain was investigated first in MARC-145 cells. It showed that PRRSV S1 and SY0608 could be both suppressed by rAd-P2, whereas rAd-P3 could not inhibit SY0608 strain as effective as S1 strain.PAM cells were isolated from PRRSV negative pig, and the antiviral effect induced by rAd-P2 was further examined on PAM cells. The results showed that rAd-P2 was competent to inhibit PRRSV CPE in PAM and the level of N protein and ORF1b mRNA were significantly reduced as compared to control groups. And the inhibition effect was dose-dependent on shRNA and reduced over time.Twenty 6-week old PRRSV negative pigs were devided into four groups,5 for each. Group 1 and 2 were intramuscularly inoculated with 4×109TCID50 of rAd-P2 each pig,24h later, pigs in group 1～3 were challenged with 2×1040 TCID50 of PRRSV SY0608, and group 4 was treated as a control. Pigs were observed separately, and body temperature data and blood were collected everyday. At 9d post-challenge, all the pigs were euthanized and necropsied, and submitted to pathological examination and real-time PCR detection. The results showed that (1) clinical signs: The rAd-mP2 inoculated group and virus control group developed high fever (40.0～42℃) from 3 dpc, and displayed a range of clinical signs, including inappetence, lethargy, dyspnoea, red skin, eyelid oedema, lightly diarrhea and rough hair coats. However, piglets inoculated with rAd-P2 showed fever which only started from 6 dpc and clinical sighs were milder (40.0～41℃). At 8 dpc, three pigs in rAd-P2 group also showed light inappetence and lethargy. (2) pathological examination:the extent of lung lesions in rAd-P2 group was significant low than controls, three pigs developed milder interstitial pneumonia and the other two have no clear pathological lesions. Lung tissue chips observation showed that pigs in rAd-mP2 and virus control group developed clear interstitial pneumonia as thickened alveolar walls, infiltration with intensive macrophage lymphomononuclear cells, and increased amounts of bronchiole exudates. But only three piglets in rAd-P2 group showed minor microscopic lesions of lung. (3) viremia:PRRSV in serum samples collected on 1d and 5d post-chanllenge were detected and it demonstrated that the level of viremia in rAd-P2 pigs was much lower than control groups on 5d post-chanllenge (p<0.05); (4) PRRSV quantity in lung:the average amount of PRRSV in lung from rAd-P2 inoculated pigs was significantly lower than control group (p<0.05). The results proved that shRNA expressed by rAd-P2 could suppress PRRSV replication in vivo, and delay the onset of disease for at least 3 days. It demonstrated that shRNA directed by adenovirus could inhibit PRRSV replication both in vitra and in vivo and provide a novel potential anti-PRRSV strategy.6. Construction and identification of plasmids and recombinant adenoviruses expressing shRNA targeting PRRSV receptor genesPrimers were designed according to the sequences of CD151,CD 163 and CD 169 (sialoadhesin) genes. The genes were cloned into pEGFP-N1. Three EGFP-fusion expression plasmids were constructed. siRNAs were designed according to the genes of CD151,CD 163 and CD 169, respectively. Short-hairpin oligos were synthesized and coloned into pSUPER after annealing. PH1-shRNA cassettes were amplified from positive recombinant plasmids by PCR and coloned into recombinant adenovirus vector. The recombinant adenoviruses expressing shRNA (rAd-151-1,rAd-151-2,rAd-163-1,rAd-163-2,rAd-169-1 and rAd-169-2) were obtainedEGFP fusion-expression plasmids were cotransfected with shRNA-expressing plasmid accordingly into HEK-293A cells. The EGFP expression was observed at 72h post-transfection. The results showed that EGFP in cells transfected with shRNA-expressing plasmids was decreased as compared to control cells. CD 163 and CD 169 on PAM inoculated with rAd-shRNA were detected by monoclonal antibody accordingly, and it showed that the amount and intensity of fluorescences were relatively lower than control cells. Furtherly, PAM cells were inoculated with rAd-shRNAs individually before PRRSV infection. At 24h after PRRSV challenge, viral titers showed that there was no significant difference between rAd-shRNA inoculated cells and control cells. The cooperation of shRNA targeting to multiple PRRSV receptor genes is needed to be investigated.