Investigation Of Gene-deleted Vaccine Of Infectious Bovine Rhinotracheitis Virus And Its Application

Bovine herpesvirus 1(BHV-1)infection in cattle named as infectious bovine rhinotracheitis(IBR)which lead to fever,dyspnea,nasal discharges,upper respiratory tract disorders and conjunctivitis.BHV-1 is one of the most important cattle pathogen involved in the development of Bovine Respiratory Disease Complex(BRDC)by transiently suppressing the immune system of infected cattle,which causes significant economic losses to cattle industry worldwide.In addition,BHV-1 latency and reactivation cycle has more impact on the epidemiology and the control of BHV-1.Vaccination is an effective measure to control IBR.Several European countries have initiated control programs based on the use of marker vaccines aiming to eradicate BHV-1.In these vaccines at least one antigenic protein deleted than the parental viruses,and it can detect an antibody response to the specific deleted protein which could allow differentiation of infected from vaccinated animals(DIVA).In addition,BHV-1-based live vectors have been used for development of multivalent vaccines against economically important viral and bacterial diseases.However,several concerns have been raised to the marker vaccines,including remain immunosuppression and latency,the possibility of recombination between two vaccine strains,and low level of sensitivity of the diagnostic test at detecting the specific antibody against serological marker.Furthermore,there is no effective vaccine for IBR control in China.A double mutant BHV-1 gG-/tk-was constructed previously,and it was proved to be a candidate for a marker vaccine against IBR.The aim of the study was to further investigate and optimize the immunogenicity of the double mutant vaccine,and develop a superior vaccine candidate for IBR control.Bovine viral diarrhea virus(BVDV)mainly infects cattle,resulting in a broad range of clinical signs including fever,diarrhea,leucopenia,reduction in milk yield and reproductive problems.In addition,BVDV is an etiological agent of BRDC.The disease is primarily spread and maintained in cattle populations by persistently infected(PI)individuals.Thus,identification and removal of such individuals is critical to the success of eradication campaigns.In addition,vaccination against BVDV is an important component of prevention and control programs.The aim of the study was to study the prevalence and genetic typing of BVDV in China,which facilitate an evidence for developing an effective vaccine on the basis of BHV-1 vectored vaccine.The main contents and results were described as follows:1.Construction and immunogenicity of BHV-1 triple mutants.In order to further reduce the virulence of BHV-1 gG-/tk-and increase the immunogenicity of this mutant,two triple gene mutants BHV-1 gG-/tk-/gE-and BHV-1 gG-/tk-/gN-were constructed.The safety and protective efficacy of these triple mutants were evaluated in rabbits.Following primary infection,no virus shedding from BHV-1 gG-/tk-/gN-inoculated group,while gG-/tk-/gE-inoculated group was only detected in one out of three rabbits at 3 dpi.After challenge with wt BHV-1,BHV-1 gG-/tk-/gN-and BHV-1 gG-/tk-/gE-inoculated group shed virus for 6 and 4 days,respectively,whereas the double mutant BHV-1 gG-/tk-inoculated group shed virus for 2 days.Therefore,both triple mutants were more attenuated and lower immunogenic than double mutant.The safety and protective efficacy of BHV-1 gG-/tk-/gE-were further evaluated in calves.The result also indicated that BHV-1 gG-/tk-/gE-was more attenuated than BHV-1 gG-/tk-.After challenge with wt BHV-1 and BHV-5,the protection rates of the BHV-1 gG-/tk-/gE-immunization against wt BHV-1 and wt BHV-5 were 64.2%and 68.6%,respectively.2.Protection induced by BHV-1 gG-/tk-vaccine used in different immunizing routes in calves.To explore the suitable immunizing routs of BHV-1 gG-/tk-vaccine,the calves were received a primary dose of BHV-1 gG-/tk" vaccine via intranasal(IN)or intramuscular(IM)routes of administration and booster at 21 days intervals.The nasal shedding of virus from IM inoculated groups was significantly lower than that of IN inoculation(p<0.05).Primary IM vaccination stimulates earlier neutralizing antibody and higher level of IL-4 than IN administration(p<0.01).In addition,IM booster induced increased titers of neutralizing antibody that was significantly higher than IN booster(p<0.05),along with significantly higher level of IFN-y in IM booster than that of IN(p<0.01)at 5 days post booster vaccination(26 dpi).However,IN booster vaccination induced a higher level of sIgA in nasal swab than other groups(p<0.05)on 7 days post booster vaccination(28 dpi).After challenge with wt BHV-1,all IN and IM vaccinated animals showed significantly lower clinical scores and lower titer of virus shedding than the unvaccinated control.In addition,fewer viruses without difference and shorter shedding period was showed in IN administration groups than by IM administration groups.In conclusion,both IN and IM administration of BHV-1 gG-/tk-vaccine could provide efficient protection against wt BHV-1 challenge.3.Construction and identification of recombinant virus BHV-1 gG-/tk-/gD+and BHV-1 gG-/tk-/gD5+.To increase the immunogenicity and protection of BHV-1 gG-/tk-vaccine,two recombinant BHV-1 virus expressing extracellular domain of gD genes of BHV-1 and BHV-5 were constructed,respectively.The mixtures of parental virus(BHV-1 gG-/tk-/EGFP+)and transfer vector were co-transfected into MDBK cells using calcium phosphate-mediated transfection.Then the propagated viruses were harvested.The recombinant BHV-1(designated BHV-1 gG-/tk-/gD+and BHV-1 gG-/tk-/gD5+)were obtained by counter selection for GFP.PCR results showed that extracellular domain of gD genes of BHV-1 and BHV-5 were successfully inserted into the genome of BHV-1 gG-/tk-.The expression of extracellular domain of gD genes in infected cells were proved by indirect immunofluorescence assay and western blotting.This research not only provides the method to increase the immunogenicity and protection of BHV-1 gG-/tk-vaccine,but also provided a technical platform and basis for applying BHV-1 as vector for development recombinant vaccine for immunization of cattle against economically important viral and bacterial diseases.4.Prevalence study and genetic typing of BVDVTo determine the nationwide status of persistent BVDV infection in different members from Bos taurus and Bubalus bubalus in China and compare different test methods,a total of 1379 serum samples from clinical healthy dairy cattle(Chinese Holstein),beef cattle,yaks(Bos grunniens),and water buffalo(Bubalus bubalis)were collected in eight provinces of China from 2010 to 2013.The samples were analyzed using commercial,antibody(Ab)and antigen(Ag)detection kits,and RT-PCR based on the 5'-UTR and Npro gene sequencing.Results showed that the overall positive rates for BVDV Ab,Ag and RT-PCR detection were 58.09%(801/1379),1.39%(14/1010),and 22.64%(146/645),respectively,while the individual positive rates varied among regions,animal types,and farms.The average Ab-positive rates for dairy cattle,beef cattle,yaks,and water buffalo were 89.49%(298/333),63.27%(248/392),45.38%(236/520),and 14.18%(19/134),respectively,while the Ag-positive rates were 0.00%(0/116),0.77%(3/392),0.82%(3/368),and 5.97%(8/134),respectively,and the nucleic acid-positive rates detected by RT-PCR were 32.06%(42/131),13.00%(26/200),28.89%(52/180),and 19.40%(26/134),respectively.Phylogenetic analysis of the 5'-UTR sequences indicated tha tall of the 124 BVDV-positive samples were BVDV-1 and subtyped into either BVDV-lb(33.06%),BVDV-lm(49.19%),or a new cluster,designated as BVDV-lu(17.74%).Phylogenetic analysis based on Npro sequences confirmed this novel subtype.In conclusion,this study revealed that the prevalence of BVDV-1 in bovines of China and the dominant subtypes of BVDV,as well as facilitate an evidence-based BVDV control strategy in China.