Construction And Protective Efficacy Of A Recombinant Herpesvirus Of Turkey Vaccine Strain Expressing Hemagglutinin-Neuraminidase Of Genotype ? Newcastle Disease Virus

Newcastle disease(ND)is a highly potent and highly contagious disease caused by Newcastle disease virus(NDV),which brings huge economic losses to poultry industry.At present,most of the conventional vaccine strains are NDV genotype I or genotype II,and the current prevalence of NDV was genotype VII,although they belong to the same serotype,but the genetic distance is far worse.Therefore,the prevalence of atypical ND and toxins in immunized flocks is common and immune failure occurs.There is an urgent need to develop ND vaccines that match the genotypes of epidemic strains.The herpesvirus of turkey(HVT)vaccine is a common vaccine for the prevention of Marek's disease in chickens.It has a large number of virus replication non-essential areas for foreign gene insertion,and is widely used in the construction of recombinant virus live vaccine.In this study,a new ND live vaccine was constructed by using HVT FC126 vaccine as vector to construct recombinant turkey herpes virus expressing genotype VII NDV HN glycoprotein.In this study,the HN gene of genotype VII NDV DT-2014 strain was amplified by PCR,and the expression cassette was composed of human cytomegalovirus early promoter(CMV)and TK polyA.The expression cassette was inserted into the US2 non-essential region fragment of the HVT FC126 vaccine strain to construct the transfer vector pHVT-US2-HN.Recombinant HVT FC126 virus(rHVT-EGFP)genomic DNA was transfected into chicken embryo fibroblasts(CEF)by EGV gene,and EGFP gene was replaced by HN gene expression by homologous recombination.And the recombinant virus rHVT-US2-HN expressing the HN protein was obtained.The recombinant virus rHVT-US2-HN was identified by PCR,Southern-blot,Western-blot and indirect immunofluorescence assay(IFA),and its growth characteristics were detected on CEF.The results showed that the recombinant protein rHVT-US2-HN was correctly inserted into the non-essential region of US2 replication and could successfully express the target protein with the size of about 70kD.At the same time,the recombinant virus rHVT-US2-HN showed the same replication level as the HVT FC126 vaccine strain on CEF.In this study,we evaluated the protection efficacy of recombinant rHVT-US2-HN.Eighty 1-day-old SPF chickens were randomly divided into four groups:rHVT-US2-HN immunization group,attenuated vaccine immunization group,attack control group and blank control group.The rHVT-US2-HN immunized group was inoculated with rHVT-US2-HN of 5000 plaque forming units(PFU)subcutaneously at 1 day of age.The attenuated vaccine group was immunized with 1 rhyme/vaccine.Blood samples were collected at 7,14,21 and 28 days of age,and the titer of NDV was measured by hemagglutination inhibition test(HI).In addition to the blank control group,the other three groups inoculated genotype VII NDV DT-2014 strain with dose of 105 EID50 via eye intranasal method at 21 days of age.At the 3rd and 5th day after challenge,the laryngeal and cloacal swabs were collected and the virus was isolated.The detoxification situation of each group was tested.The clinical symptoms,morbidity and mortality were calculated for 7 days.The results showed that the antibody level of rHVT-US2-HN immunized group was increasing after inoculation.Seven days after challenge,the mortality rate in the control group was 100%,the mortality rate in the rHVT-US2-HN immunized group was 50%,and the mortality rate in the attenuated vaccine group was 10%.The results showed that the recombinant virus rHVT-US2-HN had immunoprotective effect on the attack of genotype VII NDV strain,and provided a new candidate vaccine strain for the prevention and control of ND.