The Correlations Between Ha Gene Sequences And Immune Protections Of H5N1Subtype Avian Influenza Virus And The Application Of Immune Procedures Of Its Inactivated Vaccine

Avian Influenza (AI), caused by avian influenza virus (AIV) that is infectious disease and syndrome with that infected all kinds of avian and birds. Avian Influenza virus, has more and complex serumtypes. The same serumtype has different mutation virus. The pathogenicity of these mutation virus have big difference on different kinds of Avian. According to the pathogenicity of AIV, the AI can be divided as high pathogenic Avian Influenza (HPAI) and low pathogenic avian Influenza (LPAI). HPAI is classified as notifiable disease by the world Organization for Animal Health (OIE). In China, the control of AI is by compulsory vaccination and depopulation method, the vaccine in production ware mainly reassortant bivalent inactivated vaccine(H5N1subtype, Re5+Re4strain), reassortant H5subtype inactivated vaccine(H5N1subtype, Re5strain) and reassortant H5subtype inactivated vaccine(H5N1subtype, Re4strain). As the fast mutation of AIV will produce new subtype and new mutation virus, one attention from the society is that, what is the epidemic virus mutation condition? And if there is new mutation virus strain? As the AIV HA sequencing and homology analysis do not concern live virus and not long time needed, this mature technology aquires much more attention by researchers, but here comes the second pressed need to be resolved concern, whether there is the correlation between challenge test and AIV HA sequence mutation, whether there is more better method to judge the protection of epidemic AIV strain by applied vaccine, and further, whether the applied vaccine will supply efficient protection. And the third concern is to solve the question of how to have good protection result in real product when the good vaccine is available. Thus in this paper the study was done on the correlation between challenge protection and HA sequence difference of H5N1subtype Avian Influenza and inactivated vaccine protection immunity, in order to supply test data for above mentioned three questions and formulation strategy for the AI control. Result and conclusion are as follows: 1The sequencing and analysis of H5N1subtype AIV HA geneAltogether39AI strains from2005till2010are studied in this paper, the strains are from different regions and hosts. The HA were sequenced and homology similarities were analyzed, in the mean time, together with the627strains of AIVs sequencing data isolated by China Animal Health and Epidemiology Center and Harbin Veterinary Research Institute. The study was done on H5N1subtype AIV distribution and the HA gene mutation.From the study result of the39strains of AIVs and analysis result of627strains of AIVs, the host distribution is wide which covers almost all domestic and wild poultry, till now, the isolated AI viruses can be divided into clade2and clade7.2, while clade2can be further divided2.2and2.3clade, and in clade2.3, further clade2.3.2and2.3.4are devided, still further in2.3.2we have2.3.2.a,2.3.2.b and2.3.2.c, and in2.3.4we also have2.3.4.a and2.3.4.b.From the666strains,395strains are from clade2.3.2, about59.3%of the total. When concerning the isolation time, the first one is from duck in2004from Guangdong province, in2005,4strains.2strains are from2006,7from2007,52from2008,126are from2009,202are from2010. All these mean this is the dominant clade.There are160strains from clade2.3.4, possess24%. Concerning the time for isolation, this is the main epidemic clade of2006to2008, while in2009the proportion decrease but from2010, the isolations are found in many provinces and the difference between the strains is bigger than before. There are66strains from clade7, occupies10%of total. This clade isolated in2003from Hunan chicken flocks is not prevalent. Later it is isolated in Changzhi Shangxi (named as north mutation strain), the clade is prevalent in some regions in Henan, Hebei, and Shanxi province. We get this clade isolation in2007-2009, which means this clade still is in prevalence in China.2The study of correlation between HI titer, neutralization titer, vacation protection and H5N1subtype AIV HA gene mutation.Based on H5N1subtype AIV HA gene phylogenetic tree and homology similarity analysis of nucleotide,14strains of AIVs,10strains of epidemic virus(2strains separately from clade2.3.2.b、2.3.2.c、2.3.4.a、2.3.4.b and7.2) and4strains of reassorted virus(separately from2.3.2.b、2.3.2.c.2.3.4.a and7.2) were used to make inactive vaccines and single factor serum separately for cross HI and cross neutralization test. SPF chickens were vaccinated separately by the activated vaccines and21days later, then cross challenge protection test were done by the10viruses separately. From the test result, there is correlation between HI titer, neutralization titer, vacation protection and H5N1subtype AIV HA gene mutation, for most strains, they are positive correlation. The bigger the homology difference, the greater the cross Hemaggluination inhabitation (HI titer)difference, the bigger the neutralization effect(neutralization index) difference and the worse the cross protection. Within the clade, the nucleotide homology similarity is over95%, the cross HI titer difference is within0-3log2, while neutralization titer difference is1-21og2, the protection rate reaches9/10. Yet, between clades, the nucleotide acid homology similarity is between90.1%-96.9%, the cross HI titer difference is within3-61og2, while neutralization titer difference is2-51og2, the protection rate is only40%-60%, for some chickens flocks, there is still typical clinical syndrome or death after challenge when the HI titer reaches26-28. For clade2.3.2, the present Re5and/or Re4reassorted vaccine only have a protection rate of40%-60%. While the inactivated AI vaccine of PCPR-2stain from China Animal Health and Epidemiology center (based on the virus A/Chicken/Shandong/01/2010in clade2.3.2) has a protection rate of over90%for the virus of clade2.3.2The HI test, neutralization test and challenge test are all not so complicated, but for these tests, live virus is needed. When concerning bio-safety, these tests can not be done in ordinary conditions, while the virus HA sequence inspection and analysis has less bio-safety risk and less time needed. Thus, in real research, we can do HA sequencing first, and do HI test, neutralization test and challenge test if needed.The protection result from whole epidemic virus is better than reassortant vaccine, but for the former vaccine, there is certain bio-safety risk, thus only can be used in sudden epidemic and endangered area.3The increase and decrease of antibody titer after being vaccinated bivalent inactivated vaccine (H5N1subtype Re5+Re4strain).The same batch of bivalent inactivated vaccine(H5N1subtype Re5+Re4strain) were vaccinated in SPF chickens, commercial layer, broiler breeder and broiler under different environment and feeding management conditions, and inspect antibody of Re5and Re4continuously after immunization to analyze decease and increase of antibody.. The test result shows that the sustain time of antibody titer, highest antibody titer and summit antibody titer (≥6Log2) has relations with immunizing dose. For0.1mL dose group, the summit antibody titer can reach28.3, and summit antibody titer sustain time can reach over40weeks; for0.2mL dose group, the summit antibody titer can reach290, and summit antibody titer sustain time can reach over50weeks; for0.3mL dose group, the summit antibody titer can reach295, and summit antibody titer sustain time can reach over60weeks. The sustain time of antibody titer and high antibody titer (≥6Log2) have relations with immunizing dose, condition of chicken flocks’ feeding and management, and pollution level of chicken flocks living environment, for chicken flocks under good husbandry conditions, the antibody titer is high and summit antibody titer will sustain longer time, while bad management will cause low titer and shorter sustain time.Based on the research result, we make the vaccination program as follows:for broiler blocks, if marketed on28-day old, no need to vaccinate, but the chickens should be selected from the group that the maternal antibody is over61og2; if marketed after35-day old, the vaccination should be done within1week old; for commercial layer and breeders, if the environment pollution is not so heavy and management is good, the first vaccination should be done within1-2weeks old, second vaccination at6-7weeks old, and third vaccination2weeks before laying,0.2-0.3mL for first vaccination and0.5mL per dose for second and third vaccination, if the environment pollution is heavy, the first vaccination should be done within1-2weeks old, second vaccination at6-7weeks old, and third vaccination2weeks before laying, booster vaccination every10-15weeks,0.2-0.3mL for first vaccination and0.5mL later vaccination; for broiler breeders, the body is bid and time for first laying is later, in order to increase the maternal antibody, the dose should be increased,0.5mL per dose at2weeks old, and0.8mL per dose at about12,20,30,40,50weeks old. This vaccination program have been applied on over600million chickens in Shandong, Henan and other provinces, the program is in line with clinical practice, applicable, and with good result, thus are welcomed by many end-users.