Construction Of Heat-resistant Newcastle Disease Virus Vector And Its Application In The Development Of Subtype H9N2 Avian Influenza Vaccine

Avian influenza(AI)and Newcastle Disease(ND)are the most important infectious diseases in Animals,which caused by avian influenza virus(AIV)and newcastle disease virus(NDV)respectively and lead to serious damage in the poultry industry.Vaccination remains the primary means of preventing and controlling these two diseases.H9N2 subtype AIV with a low pathogenic only causes mild respiratory symptoms and decreased egg production rate after infect the chickens.However,when mixed with other pathogens,it will cause synergy pathogenic,resulting in a higher mortality.H9N2 subtype avian influenza has caused great harm to China's poultry industry during its more than 20 years of prevalence.NDV is a single-stranded negative-strand RNA virus,and its research as an exogenous gene expression vector has been rapidly developed under the promotion of reverse genetics.Previous studies have shown that NDV can be used as a live vector vaccine constiucted to express exogenous gene and induce humoral and cellular immunity in animals.It can proliferate in animals and express antigen genes for a long time without integration with the host genome.The research was construct a heat-stable ? genotypes newcastle disease virus expression vector based on the reverse genetics platfonn of rAHR09 which was established in the early stage of the laboratory.Further more,we use the vector expressed the hemagglutinin(HA)gene of H9 subtype avian influenza virus to create a thermostable recombinant vaccine candidate strain and evaluated the immunological efficacy.1 Construction a heat-stable weakened VIII genotypes Newcastle disease vaccine vectorThe unique cleavage site Pmel between the gene P and M as an exogenous gene insert position in the whole genome sequence of the strain was obtained by site-directed mutagenesis obtained rAHR09-P,which based on the established reverse genetic platform rAHR09.Then we construct a recombinant newcastle disease virus p-rHR09-EGFP-1 which can express EGFP gene stably.The growth characteristics,thermostability and virulence of recombinant virus have been determined with no significant changes compared with the attenuated parent strain.2 Construction of recombinant heat-resistant Newcastle disease virus expressing hemagglutinin of subtype H9 avian influenza virusThree forms recombinant virus strain were constructed,named rAHR09-H9HA-O,rAHR09-H9HA-Q and rAHR09-H9HA-T.The recombinant virus strain rAHR09-H9HA-O expressed the HA protein of subtype H9N2 AIV strain YZ1406.The recombinant virus strain rAHR09-H9HA-Q expressed the HA protein which lacked the transmembrane region,and the recombinant virus strain rAHR09-H9HA-T expressed the HA protein whose transmembrane region was replaced by NDV F protein.Three recombinant viruses strains grow steadily on chicken embryos,and the hemagglutination titer can reach more than 28.The heat resistance of three recombinant virus was measured after 15 passages on chicken embryos showed with good thermal stability,and remained hemagglutination activity after treatment at 56? for more than 90 min.The MDT values of the three rescued recombinant viruses were more than 120 hours,and the ICPI index was lower than that of the parent viruses.Compared with the original NDV attenuated strains,it was proved that the insertion of exogenous genes did not affect the virulence of the recombinant viruses.3 Evaluation of immune efficacy of three recombinant virus strains75 seven days old SPF chicks were randomly divided into five groups:recombinant virus strain rAHR09-H9HA-O immunization group,recombinant virus strain rAHR09-H9HA-T immunization group,recombinant virus strain rAHR09-H9HA-Q immunization group,inactivated vaccine immunized group and non-vaccinated group.These groups of recombinant virus immunization were immunized by intranasal and intraocular dropping with an immune dose 106 EID50,and the group of inactivated vaccine was immunized by intramuscular injection with the immune dose of 0.2 mL.21 days after immunization,all groups were challenged with YZ1406 strain by intranasal and intraocular dropping at the dose of 108 EID50.The antibody titer of H9 of experimental chickens were determined on the 7th,14th,21st and 28th day after immunization.The results showed that the antibody level of recombinant virus immunization group increased continuously after inoculation and was always higher than that inactivated vaccine group.The titer of antibody produced by recombinant virus strain rAHR09-H9HA-T reached 24 at 21th day after immunization.Oropharynx and cloaca swabs were collected at third,sixth and ninth days after challenge,and the viral content was determined by fluorescence quantitative PCR.The results showed that recombinant viruses strain rAHR09-H9HA-O,rAHR09-H9HA-T and inactivated vaccine groups could effectively reduce the amount of virus excretion,and the recombinant viruses strain rAHR09-H9HA-T group had the lowest amount of virus shedding.In summary,this study constructed a novel heat-resistant NDV vector rAHR09-P with the capacity of stably expressing exogenous genes.Further,three recombinant viruses strains expressing H9 subtype AIV HA protein were constructed.Among them,the recombinant virus strain rAHR09-H9HA-T has shown good immunoprotective effects in animal experiments and has potential as a recombinant live vaccine against H9 subtype avian influenza.