| Rabies, induced by rabies virus belonging to the genus lyssavirus of the familyRahbdoviridae, is a kind of zoonosis with a typical symptom of encephalomyelitis.Once the rabies symptoms appeared, almost100%mortality would be caused due tothe lack of therapeutic schedule. Rabies annually causes55000deaths worldwide，most of which occurred in developing countries or area with nearly2000in China。There has been no report on rabies circulation in humans by far. The majorreservoir hosts or rabies in developed areas are wild animals such as raccoon, foxand skunk, while in developing areas, rabies mainly spreads in dogs. Once the virusspilled over from dogs into humans and domestic animals, rabies outbreak wouldoccur. Therefore, animal rabies prevention becomes of crucial significance for thecontrol of intra species rabies spread.Prophylaxis through vaccine inoculation is the principal method for rabies prevention. Vaccines used for inoculation mainly included inactive and attenuated rabiesvaccine, both of which could be used for animal by injection route only. However,inoculation by injection could not be administered to stray dogs and cats in rural areain the developing counties in Asia and Africa produced mainly for the lack of strictbreeding limitation. Similar difficulty existed for wild animals. Thus, vaccination byoral route on these animals was put forward and performed. Oral vaccines derivedfrom attenuated rabies such as ERA and SAG1strain and vaccinia virus vectored andhuman adenovirus type5vectored recombinant viruses were compreensively appliedin the wild in North America and West Europe and evident effect was presented.However, sporadic occurrence of rabies and vector derived infection were reportedrevealing their safety deficiency, besides, these vaccines showed low immunizationefficacy in dogs. As a consequence, to develop an oral rabies vaccine with highefficiency suitable for dogs still kept a problem for rabies control.Canine adenovirus belonging to Adenoviridae, contains two types: canineadenovirus type1(CAV-1) and canine adenovirus type2(CAV-2) respectivelycausing canine infectious hepatitis and canine infectious laryngotracheitis. Both viruses show full crossing-protection. Attenuated CAV-2is clinically used forprophylaxis of the two virus infections, and furthermore, CAV-2can infect the hostby oral route. Partial or complete deletion of the non-essential region (E3) of CAV-2presents no effect on virus propagation, providing conditions for the exogenous geneinsertion and expression. This thesis mainly focused on the feasibility of development of CAV-2vectored rabies vaccine, and the following experiences were performed:1. Development of recombinant CAV-2bait.The oral baits were prepared by mixing the CAV-2-EGFP virus culture with a titerof106.875TCID50/100μL, flour and Peruvian fishmeal with a mass proportion of2:2:1and sub-packaging into mesh bag involving a sphere shape with a cross-sectiondiameter of4cm. The stability of the prepared baits was measured via virus titrationpre-,3d,7d and28d post treatments (freezing, indoor and outdoor). Also theimmunization efficiency of the bait was determined through neutralizing antibodytest of the serum samples collected from the oral immunized dogs.2. Survey on neutralizing antibody against adenovirus of dogs.The neutralizing antibody levels of serum samples collected from Hebei, Beijinand Jilin were determined by neutralizing antibody test and subsequently analized.3. Recombinant CAV-2genome bearing glycoprotein of rabies virus BD06.The BD06glycoprotein expression cassette was transfer into the shuttle vectorpVAX-ΔE3, forming pVAX-ΔE3-CGS which was then inserted into the pPolyII-CAV-2after a range of difficult steps. The recombinant genome pPolyII-CAV-2-CGSwas finally identified by restriction analysis and sequencing.Results1. The titer of the recombinant virus in the fresh bait was determined to be105.875TCID50/100μL. The recombinant virus titers of the baits conserved in differentconditions after3/7/28days were measured as follows: freezing(105.875TCID50/100μL,105.875TCID50/100μL,105.625TCID50/100μL), indoor(105.625TCID50/100μL,105.375TCID50/100μL,103.5TCID50/100μL), outdoor(105TCID50/100μL,104.125TCID50/100μL,102.125TCID50/100μL). Dog immunization showed that the NA titerincreased to over1:243when the pre-immunization NA titer was lower than1:27and decreased when the primary NAtiter was higher than1:2187. 2. NA titers of387dog serum samples was measured and analyzed.27of147samples from rural area in Hebei,135of195samples from Beijin and3of45samplesfrom rural areas in Jilin were CAV antibody positive. Additional statistical analysispresented a higher CAV antibody positive rate in rural area than that in urban area.3. Restriction digestion map of the recombinant CAV genome pPolyII-CAV-2-CGS using Asc I, Cla I, Nru I, Sac I was completely consistent with the expectation.Using two pair of primers respectively distributed in the two sides of the twojunctions between the G protein and the CAV-2vector, two fragments (1433bp and2227bp) were amplified and sequenced, revealing the successful insertion of theexpression cassette into the E3region which beard a1041bp deletion.Discussions1. The bait prepared in this study was suitable for dog and present a good stability,providing key support for the recombinant CAV-2derived vaccine application.2. The CAV-2antibody positive rates in rural areas were significantly lower thanthat in urban area, showing that the natural infection rate or the vaccination rate keptin a low level in rural area. Therefore, the recombinant CAV-2derived vaccines weresuitable to be used in dogs and in addition in stray dogs in rural area.3. A recombinant CAV-2genome bearing BD06G protein with high immunogenicity was successfully constructed.Innovations:1. It was the first time in China that the vaccine bait suitable for dog was explored,and that the stability of the recombinant CAV-2vector in bait was determined.2. Our study firstly illuminated the CAV-2antibody positive rate of dogs in Chinaand in addition determined the feasibility for the application of recombinant CAV-2oral bait in rural areas. |
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