Construction Of Short Hairpin Rna Targeting To1D And3AB Genes Of Encephalomyocarditis Virus And Suppression Of Emcv Replication In Vitro And In Vivo

Encephalomyocarditis virus (EMCV) is a new kind of zoonoses virus. It can infect many host species including pigs, rodents, cattle, elephants, raccoons, marsupials, baboons, monkeys and human, and cause severe economic losses on pig production due to high mortality in piglets as a result of respiratory failure and in sows as a result of myocarditis and reproductive failure. EMCV has been isolated in the South and North of China. But the pathogenicity and molecular epidemiology of the virus have not been deeply understood. And there is no effective prevention and control method for the EMCV-associated diseases. In this study, EMCV strain NJ08were isolated and identified from clinical tissue samples originated from the suspected cases with encephalomyocarditis on a pig farm in Jiangsu Province. The viral complete genome was sequenced and compared with that of other isolates. Meanwhile, plasmids and recombinant adenoviruses expressing shRNA target to EMCV different genes were constructed and the suppression effect on target genes induced by shRNA was examined both in vitro and in vivo. The main content was as following:1. Isolation and identification of an EMCV strain from pigletsIn order to isolate EMCV from piglet, the clinical tissue samples were selected from the pig with clinical sings of encephalomyocarditis in Jiangsu Province. After homogenization, freeze-thawing and then filtrated with filter membrane, the supernatants were collected and inoculated into BHK-21cells for virus isolation. CPE in BHK-21cells could be observed after three passages and the virus titers were10-10.0～10-10.25TCID50/mL after passaged by15～25times. The specific fluorescence was observed in the cytoplasm of BHK-21cells infected with the strain by IF A with the molecular antibody to VP1of EMCV. And EMCV VP1gene was amplified by RT-PCR. The sequencing results of PCR product showed that the VP1gene shared85.7%to99.8%nucleotide identity with those reported in GenBank. It was further demonstrated by electron micrography after discontinuous sucrose gradient centrifugation. The results showed that this virus isolate represented EMCV and named as EMCV NJ08. An animal infection experiment showed that the isolate could cause severe clinical symptoms and pathological changes in the infected BALB/c mice. And EMCV antigen could be examined in the brain tissues of the infected mice. But no obvious clinical symptoms and pathological changes were observed in the piglets infected with the strain, even thought the antibodies to EMCV could be detected in the pig serum. It was confirmed that EMCV really exist in China and provided new epidemiologic data of this virus in our country.2. Complete genomic sequencing of an EMCV isolate NJ08from Mid-eastern ChinaBased on the sequence of EMCV in GenBank,7primers were designed for to amplify the7fragments with some overlapped between the upstream and downstream fragments, which covering the complete genomic sequence. And then five large overlapped fragments (fragments2-6) were amplified from EMCV NJ08isolate by RT-PCR method and cloned into the pMD18-T vector for sequencing. Authentic5’and3’terminal fragments of S1isolate were obtained by5’-Full RACE Core Set and3’-Full RACE Core Set and cloned into the pMD18-T vector and sequenced. Five overlapping genomic fragments of EMCV NJ08strain were amplified by RT-PCR and authentic5’ and31terminal fragments of NJ08strain were obtained by5’-and3’-full race core set. The PCR products were also cloned and sequenced. Then the complete nucleotide sequence of the viral genome was obtained by splicing of each fragment with overlapped gene sequence in order. The results of the sequence analysis showed that the complete genome of NJ08strain was7,724bp in length. It belonged to the sub-genotype la, with more than99%identity with the reference strains in GenBank. It was found that there were some variation of deduced amino acids in the non-structure protein2A and the structure protein VP1. These results could be helpful for understanding the molecular epidemiology information of EMCV in China.3. Establishment of SYBR Green I realtime PCR for detection of1D and3AB Genes of EMCVBased on the sequence of EMCV in GenBank, the primers specific to the1D and3AB of EMCV were designed, respectively. And two SYBR Green I real-time PCR methods were developed for the detection of the1D and3AB genes of EMCV, after a serial modification of the reaction condition and the reagents. The specificity of the primers was identified by melting curve analysis and there was no reaction in the presence of PCV2, PRRSV, CSFV, PRV and FMDV gene. Both pairs of the primers have a threshold range between10-100TCID50of EMCV as well as good repetitive. The result of detecting of the tissues or sera of mice and pigs showed that these methods had highly sensitivity and specificity for the viral detection. It should be useful for diagnosis and investigation of EMCV1D and3AB genes expression both in vitro and in vivo.4. Inhibition of EMCV replication by shRNA targeting to1D and3AB genes in vitro and in vivoBased on the genome sequence of encephalomyocarditis virus (EMCV) NJ08strain, four siRNA sequences targeting to1D and3AB genes of EMCV were selected with aid of web-based tool. And the oligonucleotides with overhang ends with64-nt long were synthesized, annealed and cloned into pSUPER plasmid. And four short hairpin RNA (shRNA) expression plasmids were constructed and identified with Kpn I and EcoR I restriction enzymes digestion and sequencing, and named as pSUPER-1D-1, pSUPER-1D-2, pSUPER-3AB-1and pSUPER-3AB-2. After transfection of these plasmids into BHK-21cells, EMCV replication were examined by virus titers, IFA, and real-time PCR, respectively. The results showed that EMCV induced cytopathic effect (CPE) could be inhibited in the cells transfected with pSUPER-1D-1, pSUPER-3AB-1and pSUPER-3AB-2, and the virus titers were reduced by approximately100-1000fold compared to those control cells. The expression of EMCV3AB gene was significantly decreased both at RNA and protein levels in the cells comparing to the controls. Ninety BALB/c mice were randomly assigned into six groups. And pSUPER-1D-1, pSUPER-3AB-1, pSUPER-3AB-2and pSUPER-mN3were interperitoneally inoculated to mice in group1to4, respectively. Meanwhile, mice in group5were injected with ddF2O as negative control, and mice in group6served as positive control. At6,12and24h after infection, five mice were randomly selected from each group were challenged with EMCV NJ08interperitoneally. At14dpc, all mice were killed and submitted to pathological and real-time PCR examinations for EMCV. The results showed that the clinical signs and pathological lesions of the mice in the groups inoculated with the shRNA constructs were milder obviously, compared with those in pSUPER-mN3and challenge control groups. The loads of EMCV in the brain tissue of the mice pretreated with the constructs were significantly lower than those in other control groups. It indicated that the vector-based shRN A targeting to3AB and1D gene might be a potential anti-EMCV strategy.5. Constrcution and identification of recombinant adenoviruses expressing shRN A tarteting to EMCV1D and3AB genesThe PH1-shRNA expression frames in the recombinant pSUPRE vectors pSUPER-1D-1, pSUPER-1D-2, pSUPER-3AB-land pSUPER-3AB-2were amplified by PCR and cloned into shuttle vector pAdTrack-CMV, respectively. After the homologous recombination of the recombinant shuttle vectors with pAdEasy-1in BJ5183, the recombinant adenoviruses vectors containing the shRNA were obtained, and identified by Pac I digestion. After transfection of the linearized plasmids with Pac I into HEK-293A cells, and4recombinant adenoviruses expressing shRNA (rAd-shRNA) specific to1D and3AB were produced and named rAd-1D-1、rAd-1D-2、rAd-3AB-1and rAd-3AB-2, respectively. Forty-eight hours after inoculation with these rAd-shRNAs,293A cells developed cytopathogenic effect (CPE) as rounding and detached as well as expressed green fluorescence (GFP) observed under fluorescence microscopy. The adenoviruses genome DNA was extracted from the cell culture and the PH1-shRNA expression frames were detected by PCR. The results showed that all the rAd-shRNAs were positive, which confirmed the existence of PH1-shRN A expression frames in adenovirus genome. This study successfully constructed adenovirus expressing shRNA targeting to EMCV different genes that will be useful for evaluating the antiviral effect induced by shRNA both in vitro and in vivo.6. Adenovirus-mediated shRNA inhibit EMCV replication both in vitro and in vivoIn order to evaluate the inhibition efficiency of EMCV replication induced by the recombinant adenovirus-medated shRNA targeting to the ID and3AB genes of EMCV, the four recombinant adenoviruses expressing shRN As were used to inoculate MARC-145cells and their inhibition efficiency on the replication of EMCV was evaluated firstly. The results showed that delivery of these shRNAs rAd-1D-2, rAd-3AB-1, rAd-3AB-2could induce a significant inhibition of EMCV replication in viral RNA and protein levels in MARC-145cells. And the antiviral effect was dose-dependent and sustained for at least72h. Moreover, 96BALB/c mice were randomly assigned into six groups. And rAd-1D-2, rAd-3AB-1, rAd-3AB-2and control rAd-G1were inoculated to mice in group1to4with dose of107efu or108efu per mouse, respectively. Meanwhile, mice in group5were injected with ddH2O as negative control, and mice in group6served as mock control. At12h after infection, the mice in group1-5were challenged with200TCID50EMCV NJ08. At21dpc, all mice were killed and submitted to pathological and real-time PCR examinations for EMCV. The results showed that mice injected with those shRNAs before EMCV infection had low viral load in the brain during the period of21days post-EMCV infection. The clinical signs and pathological lesions in the mice inoculated with the shRNA were milder than those in rAd-G1negative and EMCV control groups. The survival rates of mice inoculated with108efu rAds were up to87.5%, while, in the control groups all mice died. These results indicated that shRNAs mediated by the adenovirus could provide protective efficacy against EMCV challenge in mice. It might be a potential new tool for controlling EMCV infection.