Development Of Swine Pseudorabies Virus Gene Deletion Vaccine And Its Vector Vaccine

Pseudorabies(PR),caused by Pseudorabies virus(PRV),is characterized by abortions in sows and high mortality in newborn pigs.At present,controlling and eradicating PRV from domestic pigs mainly rely on inoculation of gene-deleted PRV vaccine combined with the corresponding serological testing.In late 70's,a live attenuated PRV vaccine strain Bartha-K61 was introduced into China from Hungary,and PR was well controlled with widespread use of the vaccine.Since late 2011,however,a PR associated disease re-emerged in many swine herds which actually immunized with Bartha-K61 vaccine and caused huge economic losses.It was later confirmed that a PRV variant was the causative agent of the emerging outbreaks,the PRV variant was shown more virulent to pigs in comparison with the classical PRV,and the Bartha-K61 vaccination could not provide full protection against the PRV variant.Therefore,it is urgent to develop more effective PRV vaccines to control the emerging PRV variant.1.Development of g E/g I gene deletion vaccine for pseudorabies virus(1)Construction of g E/g I gene deletion of pseudorabies virus(JS-2012): In the study,a fragment covering partial g E and g I genes was deleted by homologous recombination based on a variant PRV isolate JS-2012,generating g E and g I deleted recombinant PRV was named as JS-2012-?g E/g I.(2)Analysis of biological characteristics of JS-2012-?g E/g I :The PRV vaccine candidate,JS-2012-?g E/g I is geneticly stable.After 20 consecutive passages in Vero cells,no further mutations were found in genome of the virus and the absence of g E was consistently confirmed by IFA and Western blotting.The results of growth curves and plaque morphology showed that the growth kinetics of JS-2012-?g E/g I was similar to that of JS-2012,but the plaque formed by JS-2012-?g E/g I was significantly smaller than that of JS-2012.(3)Safety analysis of JS-2012-?g E/g I :The PRV vaccine candidate,JS-2012-?g E/g I is safe to piglets and pregnant sows.The results of animal experiment showed that the JS-2012-?g E/g I could not transmit horizontally or vertically among pigs.A t the same time,the JS-2012-?g E/g I was confirmed being unable to get virulence reversed after passing the virus in sucking piglets for five times.(4)The effectiveness of JS-2012-?g E/g I : The results of animal immunizationogenicitys and protective efficacy showed that the vaccine candidate strain JS-2012-?g E/g I was able to provide completely protection for actively immunized piglets and passively immunized sucking piglets against challenge of virulent PRV variant or classical PRV.2.Development of recombinant PRV live vector vaccine containing E2 gene of CSFV(1)Construction of recombinant PRV live vector vaccine expressing E2 gene of CSFV: To develop a bivalent marked vaccine against PRV and classical swine fever virus(CSFV),the attenuated vaccine strain JS-2012-?g E/g I was further used as a vector to express the E2 protein of CSFV.The E2 expression cassette was inserted into the intergenic region between the g G and g D genes of JS-2012-?g E/g I,generating recombinant PRV expressing E2 protein of CSFV was named as JS-2012-?g E/g I-E2.(2)Analysis of biological characteristics of JS-2012-?g E/g I-E2 :The recombinant vaccine candidate,JS-2012-?g E/g I-E2,was further confirmed no mutations or missing of E2 expression cassette after 20 consecutive passages in Vero cells,which indicated the good stability of JS-2012-?g E/g I-E2.The E2 protein was consistantly detected by IFA and Western blotting during passaging the virus in Vero cells and PK-15 cells.The results of growth curves and plaque morphology showed that the growth properties of JS-2012-?g E/g I-E2 was similar to that of JS-2012-?g E/g I.(3)Safety analysis of JS-2012-?g E/g I-E2 :The results of animal experiment showed that the vaccine candidate JS-2012-?g E/g I-E2 was safe for sucking piglets and pregnant sows and could not transmit horizontally or vertically among pigs.(4)The effectiveness of JS-2012-?g E/g I-E2 :The results of animal immunization and challenge test showed that the recombinant vaccine JS-2012-? g E/g I-E2 was able to provide fully protection(100%)against the challenge of the PRV variant and CSFV.Using the recombinant vaccine to immunize piglets with PRV maternal derived antibodies(MDA)and then challenge with virulent CSFV,80% of piglets could be protected.The result indicated that the recombinant PRV vectored vaccine developed in the study could be resistant against MDA interference.In conclusion,two vaccine candidates,JS-2012-?g E/g I and JS-2012-?g E/g I-E2,were developed in this study.Both JS-2012-?g E/g I and JS-2012-?g E/g I-E2 were safe and efficient for piglets and pregnant sows,and the vaccine viruses could not transmit horizontally or vertically among pigs.Meanwhile,the two vaccine viruses were constructed with molec ular markers which can be used to differentiating naturally infected animals from vaccinated ones,and better for eradicating PRV and CSFV in swine herds.Therefore,the two vaccine condidates wo uld have a wide prospective future in application of the field.