Genetic Variation Analysis Of Rabbit Hemorrhagic Disease Virus Based On Vp60Gene And Oral Vaccine Design

RHD virus (RHDV) is a single positive-stranded RNA virus that belongs to theCaliciviridae family with a genome about7.5kb. Since the first report of RHDV in China, thevirus has become a big threaten to both the domestic rabbits and wild rabbits all over of theword. Because the lack of passage cells line for the virus culture, until now, tissue-sourcevaccine is still used to control the disease. However, this vaccine should be avoided in nextgeneration vaccine for bio-security problems and animal welfare.From fall of2009till spring of2010, several rabbit keeping farms suffered RHD kinds ofdisease in Shaaxi province. Since these farms have performed a good immunize program forRHDV, some RHDV varieties may involve. To reveal the reason of the stituation and findnew more efficient ways to control RHDV, in current study, a sequence analysis of RHDVbased on VP60were conduced and live recombinant oral vaccines were construced. Thiswork contains5different parts and the details are presented in following paragraphs.1. Phylogenetic analysis of RHDV in ChinaThe study was to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60sequences of five RHDVs collected by our team, as well as those of16other publishedChinese RHDV strains, were analyzed. Polygenic analysis using MEGA4software showedthat20of the21Chinese strains could be clustered in an RHDVa subgroup, andWX/China/1984was different from them. The Chinese RHDV strains were further classifiedinto4subgroups, CH1to CH4. Subgroup CH1, represented by the WX/China/1984strain,was not prevalent in China after the first RHDV epidemic strain had been reported. The CH2,CH3, and CH4subgroups far different from the CH1subgroup, formed three separate clustersand were distributed according to the time the strains were collected. Recently collectedstrains formed a new subgroup (CH4), represented by new RHDV varieties. The present workis the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation thatshould be carefully monitored.2. New RHDV variationsRHDV subgroup4(CH4) represent a new variation of RHDV. To analyze the antigen variation of the isolates in this subgroup,3RHDV strains and24rabbits were selected toperform an experiment. YL-2006, XA/China/2010and the vaccine strain NJ/China/1985wereselected. Twelve out of24of the rabbits were immunized by inactived vaccine(NJ/China/1985derived) at30day-old. The other12rabbits were not immunized. At60day-old, these rabbits were challenged using YL-2006and XA/China/2010strain. EachRHDV strain challenged6immunized rabbits and6unimmunized rabbits. The resultsrevealed that all the unimmunized rabbits were dead in36h post challenge. And both virusstrains shared some clinical signs and gross lesions on unimmunized rabbits. But theimmunization provided different level of protection to YL-2006and XA/China/2010. Therabbit challenged with YL-2006were fully protected, but only about50percent of rabbitchallenged with XA/China/2010were protected. This different level of protection may relatedwith amino variation of the challenge strain, as XA/China/2010has16different aminos withvaccine strain and YL-2006has only5amino different in VP60.3. S. typhimurium based DNA Vaccine for RHDV controlThe study investigated the utilities of attenuated Salmonella typhimurium as abactofection vehicle for the oral delivery of a DNA vaccine against rabbit hemorrhagicdisease virus (RHDV). The DNA vaccine plasmid pcDNA3-VP60, encoding viral capsidprotein VP60, was transformed into an attenuated S. typhimurium strain SL7207. Theresulting recombinant bacteria, named SL/pcDNA3-VP60, were used to immunize rabbitsorally. Successful delivery of the DNA vaccine plasmid was shown by the detected VP60transcription in intestines through reverse transcriptase-polymerase chain reaction. In addition,RHDV-specific humoral and cell immune response induced by SL/pcDNA3-VP60weredetected by using enzyme-linked immunosorbent assay, T lymphocyte proliferation assay,and cytokine secretion. Experiments with XA/China/2010strains at42dayspost-immunization demonstrated significant protection in immunized rabbits with a relativesurvival rate of66.7%(4/6). The current study demonstrates, for the first time, the ability ofattenuated Salmonella as a live vector to deliver a DNA vaccine orally against RHDV.4. Adenovirus-based oral vaccine designTo futher improve the immune affect of the vaccine, adenovirus was chosen as a vaccinevector and the recombinant adenovirus expressing the RHDV capsid protein (VP60) wasconstructed. The expression of the recombinant protein was identified by western blotanalysis using RHDV-positive rabbit sera. Eighteen rabbits were immunized by injection,direct oral instillation, or using bait. They were challenged with RHDV isolate three weeksafter boost immunization. In all cases, the rabbits immunized with the recombinantadenovirus developed RHDV-specific antibodies and cell immune response. The rabbits injected with the recombinant adenovirus were completely protected against RHDV challenge.The adenovirus expression system may provide a strategy for the immunization of rabbits,particularly for the control of RHDV in wild rabbits.5. ELISA for RHDV antibodies detectionAn indirect ELISA method for detecting of antibody against RHDV was established.E.coli expressed VP60was analyzed using SDS-PAGE and Western blot. The identifiedrecombinant protein was used for coating after purification. All the conditions for the ELISAmethod were optimized with standard protocol. The results showed that target fusion proteinwas about42.34ku with a good reactogenicity confirmed by Western blot; the best ELISAreaction condition were found out as: coating with2.9μg/mL of antigen at4℃overnightafter37℃2h; blocking with1%BSA at37℃2h, serum sample reacting (1:100) at37℃for1h, second antibody reacting with HRP labeled goat anti-rabbit (1:10000) at37℃for1hand the threshold of positive and negative sera was0.356. The ELISA method show goodrepeatability, sensitivity and specificity. The coincidence rate was74.1%when comparedwith HI or94.8%when compared with commercial ELIAS kt in detecting180clinical sera.The method can be used for large quantities clinical samples detection.In summary, we first comprehensivly ananlyzed the Chinese RHDVs and group theminto four different Chinese subgroups. And based on the rabbit challenge and sequencealignment, a new group of antigenic variation of RHDV was identified. To find out new moreefficient method to replace the tissue-souce RHDV vaccine and control the prevalence ofRHDV, S. typhimurium based DNA Vaccine and adenovirus based live vector vaccine forRHDV were construct and test for RHDV special immune response. We found S.typhimurium based DNA vaccine could induce RHDV special immune response and providepartical protection to RHDV challenge, while adenovirus based vaccine could provide moreeffect protection which may be the useful candidate for RHDV vaccine develop.