| Avian Influenza(AI)is a rapid and severe course of disease, which is caused by the division are sticky,influenza virus type A.Since the 2013 in China outbreak of h7n9 subtype avian influenza, has the number of infections has more than 600 cases and deaths in more than 200 people, causing serious impact on poultry production and social stability.In order to control the occurrence and epidemic of h7n9 subtype avian influenza from the source, the subject choose G1 strain and L62 strain h7n9 subtype AIV as vaccine strains and studied on biological characteristics,preparation of oil emulsion inactivated vaccine and its immune effect were studied.The full-length genome sequencing analysis showed that the two strains of vaccine virus belongs to the branch of Asia-europe, has highly homology with A/chicken/Zhejiang/C483/2013 H7N9. Alignment of the amino acid sequences showed that there are two basic amino acids in HA protein cleavage site,suggesting the low pathogenic avian influenza virus.Virus in chicken embryo propagation characteristic test results show that, G1 strain and L62 strain of chicken embryos inoculated with optimal dilution of 103, the best inoculation dose of 0.1 ml / pieces, best time virus G1 strain after inoculation 72~84h, L62 line for 60 h after inoculation, 50% embryo infective dose(EID50)of 10-8.5~10-9.0/0.2m L. Virus in chicken embryo continuous reproduction of 20~30 generation, the HA titers among in 8.0 ? 2 ~10.0 ? 2.The virus pathogenic test demonstrated that 5×10-6.0EID50/ m L dose of the virus can lead to chicken 100% infection, detoxification time is attacked the virus after 5~7d, there are not appear clinical symptoms and death.Inoculationed G1 strain and L62 strain h7n9 subtype AIV in embryonated eggs, collect allantoic fluid of chick embryo to preparation of inactivated vaccine, and study on the vaccine of physical properties, stability, safety and immune effect. Results show that, preparation of inactivated vaccine for homogeneous ivory water-in-oil type,then the breeder safe without stress reaction.Immune effect results showed that according to the recommended dose of immune after 7d can detectable of HI antibody,3~4 weeks is reached peak 8.6log2,then decreased gradually, to free after 4 months antibody can still achieve the protection level. For the second time immune chickens in the second free after 2 weeks the antibody level reached the highest 9.5 log2.G1 strain vaccine minimum immune dose of 0.2 m L, L62 strain is 0.3m L. Based on the different HI antibody level in chicken of immune after 3 weeks to wing intravenous of 5 x 10-7.0EID50 / m L virus liquid, and attacked after 5d to acquisition of oral and cloacal swab virus isolation results showed that antibody titer higher rate of protection is better, when the antibody titer is more than or equal to 5log2, on chicken protection rate was 100%.The different immune adjuvant preparation of inactivated vaccine to immune in chickens and detection of the HI antibody level. The results demonstrate that with Montanide isa 71 VG adjuvant of the vaccine within three weeks after the antibody titer was the highest and reached 9.5 log2, but in the 4th week antibody titer decreased faster, with Freund's complete adjuvant and shortening span as adjuvant of inactivated vaccine immunized chickens produced the antibody level is high and stable, thinking of comprehensive analysis, shortening span is more suitable for as h7n9 subtype avian influenza inactivated vaccine adjuvants. |
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