Development And Evaluation Of H3N2 Subtype Canine Influenza Virus Inactivated Vaccine

In early 2005,a poultry-derived H3N2 influenza virus was isolated from pet dogs in South Korea.This is the first time that H3N2 canine influenza virus has appeared in people's sights.Since 2006,more and more pet dogs in southern China have been infected with the H3N2 CIV virus.Cases had been reported,but the pathogenesis of H3N2 CIV remains unclear.A H3N2CIV strain,abbreviated as C1732 strain,was isolated from a sick dog with respiratory symptoms and used as a vaccine strain.The inactivated vaccine with aluminum adjuvant and 201 adjuvant had been emulsified for vaccination to detect the immune effects.The full-length genome sequencing analysis indicated that the C1732 strain was highly homologous to that of the full-blown canine influenza gene published by BLAST.The experimental results of virus inoculation with MDCK cell culture showed that the optimal dose of C1732 strain was 0.1MOI/T75,and the best time for harvesting virus was 72 hours after inoculation,and the 50%Tissue Culture Infective Dose(TCID50)was 107.3/0.1 mL.The experimental results of virus chicken embryo culture indicated that the optimal dose of C1732 strain was 0.1 mL/piece,and the best time for virus collection was 48 hours after inoculation.The chicken 50%egg infective dose(EID50)was 107.2/0.1 mL.The pathogenicity test results of the virulent strains used in the challenge showed that 50%Tissue Culture Infective Dose(TID50)of the chicken-adapted virulent chicken embryos was 107.5/0.1 mL.Challenge experiments on BALB/c mice showed that The half-infective dose(MID50)for the virulent strains was 105 TCID50,0.05 mL/body,and the clinical symptoms such as coarse hairs and apathy were shown one day after the challenge,and they began to die after 3 days of challenge.The HA titer of chicken embryo virus used to prepare the antigen can reach 7 log2,and the highest titer of cytotoxic HA can reach 10 log2.Therefore,virus cultivated by the cells is selected as the antigen of the inactivated vaccine.The C1732 strain(H3N2 CIV)was inoculated into MDCK cells,and cell fluid were collected separately to prepare an inactivated vaccine.The chicken embryo virus was prepared using sigmen aluminum adjuvant and sepic 201 adjuvant,and the virus cultivated by the cells was also used for the above two methods.The shape,safety and immune effects of the vaccine had been studied after the inactivated vaccine was prepared.The safety experiment showed that the BALB/c mice vaccinated with single and m uliiple doses were safe and unstressed after vaccination.The results of immune antibody level test showed that the antibody could be detected after 7 days of immunization with the recommended dose of antibody.The optimal dose of immunization with 201 adjuvant was 105EID50/0.1 mL,and the optimal immunity with aluminum adjuvant was 106EID50/0.1 mL.There were no significant difference between cell and chicken embryo toxicity;the best immunization was intramuscular;the 201 adjuvant peaked at 9.5 log 2 at 4 weeks and the aluminum salt adjuvant peaked at 9.3 log 2 at 4 weeks,decreasing gradually.The results of the immunoprotective effect test showed that the mice in the challenge group died on the 3rd day,and all the mice in the challenge group had died on the 10th day;the inactivated vaccine(105EID50/0.1 mL)with 201 adjuvant and that of vaccine(106EID50/0.1 mL)with aluminum reduced body weight slightly under the same challenge conditions,but no death occurred,and the protection rate was 100%.However,because the weight loss rate of the aluminum salt adjuvant inactivated vaccine group was significantly greater than that of the 201 oil adjuvant inactivated group,and the price of the aluminum salt adjuvant was very expensive,it was not practical to use the adjuvant as an animal vaccine,so comprehensive analysis believes that seppic201 Adjuvants are more suitable as adjuvants for the H3N2 canine influenza virus inactivated vaccine.