Molecular Evolution And Pathogenicity To Babl/c Rats Of Six Strains Of H9N2 Avian Influenza Viruses Isolated From Broilers In Shandong Province

Avian influenza is caused by influenza virus type A. The clinical manifestations are respiratory infection, decline in egg production and alvi profluvium. H9 subtype of avian influenza is low-pathogenicity avian influenza. It can cause decline in egg production, growth retardation and a small quantity death. The Mutation frequency of the Avian influenza virus is frequently and the speed of propagation is quick, so it is very difficult to prevent the dissemination of the Avian influenza virus. There are important significance to explore the genetic mutations of the hemagglutinin gene and the neuraminidase gene and the regularity of molecular epidemiology of H9 subtype avian influenza for prevention and curing the Avian influenza. In order to explore the genetic mutations of the hemagglutinin gene and the neuraminidase gene and the law of molecular epidemiology of H9 subtype avian influenza in China, Six influenza viruses were isolated from broilers in different parts of Shandong province in 2009,and explored their virulence, immune reference and the genetic mutations. The investigation includes five experiments.Through the determination of hemagglutination properties, infected chicken embryo, the virulence was analyzed and compared. The results show that the virulences of there virus were weak and there were small disparity in antigenicity. The HA potency is between 2⁶ and 2⁹, the HI potency for Standard antigen is between 10log2 and 11log2. The EID₅₀ is between 10^-7.75/0.1mL and 10^-9.25/0.1mL. The EID₅₀ is between 10^-8.00/0.1mL¹0^-8.50/0.1mL. There are not quite different between EID₅₀ and TCID₅₀ of the six H9N2 avian influenza virus. This explains the H9 influenza virus which separate in 2009 are adaptability closer in chicken embryos and mammalian cells.Elected the six strains of virus which representing different regions and different periods and depurated these virues by Microdilution, maked manufacture single-factor serum and maked double cross-hemagglutination inhibition test. The correlation of the double cross-hemagglutination inhibition test is between 0.91 and 1.02. It is little different between the viruses.The whole cDNA fragments of the six influenza viruses isolated from broilers in different parts of Shandong province in 2009 were amplified by reverse transcription polymerase chain reaction, with primers specific to each gens. After cloning and sequencing, gene sequences were analyzed on homology and heredity evolution of molecule. HA gene phylogenetic tree, Six viruses exist in the same evolutionary position, all belong to CK/SH/Y2/2008-like lineage. the NA, NP, M, NS, PB1, PA are same with the HA gene phylogenetic tree with the phylogenetic tree. However, in the PB2 phylogenetic tree, Six viruses can be divided into two distinct evolutionary branches. CK/SD/SG2/2009 and CK/SD/WL/2009 are belong to CK/SH/Y2/2008-like lineage, the other four strains CK/SD/PD/2009, CK/SD/HY/2009, CK / SD/SG1/2009, CK/SD/FX/2009 are belong to CK/SH/F/98-like lineages. Therefore, six genotypes of the virus can be divided into two: CK/SD/SG2/2009, CK/SD/WL/2009 belong to a genotype, similar to CK/SH/Y2/2008-like pedigree; CK/SD/PD/2009, CK/SD/HY/2009, CK / SD/SG1/2009, CK/SD/FX/2009 are belone to the genotype whiche is reassortments of CK/SH/Y2/2008-like and CK/SH/F/98-like.To study the pathogenicity to mammals of six viruses, six-week-old-female BABL/c rats were inoculated with 10⁶EID₅₀ dosage of six viruses (in 50μL), respectively. The results was that all the six viruses can replicate in the lungs of rats, and two of six viruses can cause rats to die at 8 days past inoculation(DPI), which indicates that the virulence of H9N2 to rats have been increased. Therefore this study posed significance in human public health.Experimental chickens were immunized with the vaccine CK/SD/SG2/2009 and new vaccine, with CK/SD/SG2/2009 attack drugs. Two vaccines are protective. In this study, the six strains of H9N2 avian influenza viruses isolated from broilers in Shandong province are no significant difference in antigenicity, but the vaccine can not produce complete protection, and vaccine may not induce protective immunity completely relevant. Therefore, a reasonable choice of antigens targeted for vaccine strains is very important, it is best to choice the current strains in the same strain of the virus antigen as a vaccine strains. At the same time immune to the development of rational procedures, this can increase the antibody levels after immunization, better play the role of vaccine protection.