Immune Efficacy And Biosafety Evaluation Of Recombinant Fowlpox Virus Expressing Infectious Laryngotracheitis Virus GB Gene

Infectious laryngotracheitis(ILT)is an acute and highly contagious disease caused by infectious laryngotracheitis virus(ILTV)with a high mortality rate and a tendency to endemic outbreaks.At present,the common used vaccines on the market for prevention and control of ILT are modified live attenuated ILT vaccine,which generally suffer from problems such as reversing to strong virulence or as a cause of disease in the process of mixed infection.It is urgent to develop a new vaccine for prevention and control of ILT,so construction of a live vector vaccine of Fowlpox virus(FPV)is an ideal alternative.In our previous study,a recombinant fowlpox virus(rFPV-ILTVgB)expressing gB gene of dominant epidemic ILTV was constructed,and showed a 100%protection in SPF chickens against ILTV challenge after immunization,which was higher than that of commercial vaccine(cr-FPV).In this study,we further systematically evaluated the immune efficacy and biosafety of the vaccine candidate strain,which will lay a foundation for its market application.1.The physicochemical properties and genetic stability of rFPV-ILTVgBIn order to evaluate the effect of gB gene insertion in FPV on its physicochemical properties,the rFPV-ILTVgB,and wt-FPV282E4 were treated on the different physical or chemical conditions,and then were inoculated into chicken embryonic fibroblasts(CEF)to detect the TCID50 titers.rFPV was continuously passaged on CEF for 30 generations,and the genetic stability of rFPV was evaluated in vitro by gB gene sequence using sequencing method and gB protein expression using indirect immunofluorescent assay(IFA).rFPV was also serially passaged in SPF chickens to evaluate the pathogenicity of rFPV in vivo.The results showed that both rFPV-ILTVgB and wt-FPV282E4 were sensitive to high temperature,trypsin,and chloroform.The titers of both rFPV-ILTVgB and wt-FPV282E4 were not changeable at pH 3.0?9.0 or ether treatment for 24 hours.After continuous passage of rFPV on CEF,no mutation was found in gB gene sequence,and the gB protein in rFPV was also stably expressed.After continuous passage of rFPV in SPF chickens,the positive detection rate of gB gene was gradually decreased,indicating the the rFPV is difficult to passage in chickens and there is no risk of virulence reversion.2.Immune efficacy evaluation of rFPV-ILTVgB in SPF chickens21-day-old SPF chickens were randomly divided into four groups and immunized with rFPV-ILTVgB,cr-FPV,wt-FPV282E4,or PBS,which was inoculated in the non-vascularized area of the wing.Blood samples were collected on day 7,14,and 21 after immunization to separate serum,and the antibody levels against FPV and ILTV were detected by ELISA.Three chickens were sacrificed on day 7,14,and 21 after immunization,and spleen samples were collected to detect the expression levels of IL-2,IL-6,IL-18,and IFN-y by qRT-PCR.On day 21 after immunization,ILTV 119 or WG strain,FPV 1370 or 102 strain were used for challenge to evaluate the immunoprotection of the vaccine in SPF chickens.On day 3,5,and 7 after ILTV challenge,oropharyngeal were collected to detect viral shedding.The ELISA results showed that the serum antibody against FPV reached a peak on day 21 after immunization,with an average of 0.86±0.11,and the serum antibody against ILTV reached a higher level on day 21 after immunization,with an average of 0.77±0.09.On day 7,14,and 21 after immunization,spleen was collected to detect representative cytokines and the results showed that each group significantly induced the IL-2,IL-18,IFN-?,and IL-6 of transcriptional expression levels compared with PBS group.When the chickens in immunized groups were challenged with FPV 1370 strain or 102 strain,the protection indexes of rFPV-ILTVgB,cr-FPV,wt-FPV282E4,and PBS groups were 90,60,90,0,and 90,80,90,0,respectively.When the chickens in immunized groups were challenged with ILTV 119 strain or WG strain,the protection indexes of rFPV-ILTVgB,cr-FPV,wt-FPV282E4,and PBS groups were 70,60,20,0,and 100,100,16.7,0,respectively.The virus shedding results showed that all chickens in immune groups sheded the ILTVs(P>0.05).3.Biosafety evaluation of rFPV-ILTVgB on the target and non-targetanimals21-day-old SPF chickens were randomly divided into five groups and immunized with rFPV-ILTVgB,10-fold dose of rFPV-ILTVgB,cr-FPV,wt-FPV282E4,and PBS.The biosafety of rFPV in SPF chickens was systematically evaluated by weighing,blood physicochemical index detection,virus distribution in vivo,histopathological observation,environmental transmission,and cohabitation infection ability.The antibodies levels against FPV and ILTV after immunization were detected by ELISA to evaluate the production and duration of antibodies.The results showed that rFPV-ILTVgB had an effect on a few indexs of blood in SPF chickens,but had no effect on weight gain after immunization.The histopathological observation and virus distribution in vivo results showed that no pathological changes and no virus isolation were found in other organs except for mild lesions on the skin at the inoculation site.Cohabitation contact showed no horizontal transmission of rFPV was happened,no virus was isolated from feed,water,and fecal swabs,which was safe for the surrounding environment.Antibody monitoring results showed that the antibodies against FPV and ILTV peaked on 21?28 days after immunization,and still could be detected on 63 days after immunization.Forty-eight ducks were randomly divided into three groups and immunized with rFPV-ILTVgB,10-fold dose of rFPV-ILTVgB and PBS.At different time after immunization,the biosafety of rFPV for ducks was evaluated by weighting,blood physiological and biochemical indexes,and histopathological observation.The results showed that rFPV-ILTVgB had an effect on a few indexes of blood in ducks.There was no difference in body weight gain among the groups,and no pathological changes were found in the tissues and organs of ducks in each group.In summary,rFPV-ILTVgB is genetic stable in gB expression,safe for target animals and non-target animals,and a potential vaccine candicate for prevention of both fowlpox and Infectious laryngotracheitis.