LG1 strain of avian influenza virus H9N2 was passaged continuously for 40 generations in chicken embryos with anti-LG1 maternal antibodies in 4 parallel experiments, of which 3 experiments had a stable mutation of "G" to "A" at #99 of the neuraminidase gene(NA)from the 20th passage resulting in a change of Met to Ile and 2 had a stable mutation of "A" to "G" at #473 of the NA gene from the 30th passage resulting in a change of Asn to Ser which occurred in the 50th passage of another experiment. Eighty continuous passages in chicken embryos without antibody did not have the same mutation, indicating that the mutations of the 2 positions were associated with selective pressure of antibodies. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S of 4 parallel experiments with antibodies was 4.6 (32/7) compared with 2.0 (16/8) of the 2 experiments without antibodies and this significant difference implied the selective pressure of antibodies.
Animals ; Antibodies, Viral ; immunology ; Chick Embryo ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; Mutation ; Neuraminidase ; genetics

The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.
Animals ; Chickens ; virology ; China ; epidemiology ; Humans ; Influenza A Virus, H7N9 Subtype ; genetics ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; physiology ; Influenza in Birds ; epidemiology ; transmission ; virology ; Influenza, Human ; epidemiology ; transmission ; virology ; Vaccination ; Viral Proteins ; classification ; metabolism

Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high-growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6 + 2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.
Animals ; Chick Embryo ; Chickens ; Genetic Engineering ; Hemagglutinins, Viral/genetics/metabolism ; Humans ; Influenza A Virus, H5N1 Subtype/*genetics/immunology ; Influenza A Virus, H9N2 Subtype/*genetics/immunology ; Influenza Vaccines/*genetics/metabolism ; Influenza in Birds/immunology/virology ; Influenza, Human/immunology/*prevention & control/virology ; Neuraminidase/genetics/metabolism ; Transgenes ; Vaccines, Attenuated/*genetics/metabolism ; Viral Proteins/genetics/metabolism

The H9N2 subtype low pathogenic avian influenza is one of the most prevalent avian diseases worldwide, and was first documented in 1996 in Korea. This disease caused serious economic loss in Korea's poultry industry. In order to develop an oil-based inactivated vaccine, a virus that had been isolated in 2001 (A/chicken/Korea/01310/ 2001) was selected based on its pathogenic, antigenic, and genetic properties. However, in animal experiments, the efficacy of the vaccine was found to be very low without concentration of the antigen (2(7) to 2(10) hemagglutinin unit). In order to overcome the low productivity, we passaged the vaccine candidate virus to chicken eggs. After the 20th passage, the virus was approximately ten times more productive compared with the parent virus. For the most part, the passaged virus maintained the hemagglutinin cleavage site amino acid motif (PATSGR/GLF) and had only three amino acid changes (T133N, V216G, E439D, H3 numbering) in the hemagglutinin molecule, as well as 18 amino acid deletions (55-72) and one amino acid change (E54D) in the NA stalk region. The amino acid changes did not significantly affect the antigenicity of the vaccine virus when tested by hemagglutination inhibition assay. Though not complete, the vaccine produced after the 20th passage of the virus (01310 CE20) showed good protection against a homologous and recent Korean isolate (A/chicken/Korea/Q30/2004) in specific pathogen- free chickens. The vaccine developed in this study would be helpful for controlling the H9N2 LPAI in Korea.
Animals ; Chickens ; Gene Expression Regulation, Viral ; Hemagglutinins/genetics ; Influenza A Virus, H9N2 Subtype/*immunology/pathogenicity ; Influenza Vaccines/*immunology ; Influenza in Birds/epidemiology/*prevention & control/*virology ; Korea/epidemiology ; Neuraminidase/genetics ; Specific Pathogen-Free Organisms ; Time Factors ; Vaccines, Inactivated/*immunology

The purpose of this study is to explore the effects of the HA sequence variation on the pathogenicity and antigenicity of avian influenza virus(AIV). Haemagglutinin (HA) genes from, 6 of 25 avian influenza viruses (AIVs) H9N2 strains with different pathogenicity isolated in central China during last 10 years were amplified by reverse transcriptase PCR (RT-PCR), completely sequenced and phylogenetically analyzed. The purpose of this study was to explore the effects of the HA sequence variation on the pathogenicity and antigenicity of AIV. The results showed that all 6 representative H9N2 isolates belong to low pathogenic AIVs, since none of the amino acid sequences at the cleavage site of the HA of the isolates possessed the basic motif required for highly pathogenic viruses (R-X-R/K-R). There were eight potential glycosylation sites in HA of the isolates, except that 3# and 12# had an extra one. The higher pathogenicity of 3# and 12# was probably due to the extra glycosylation site (145aa-147aa) in HA1, which might alter the conformational structure of HA resulting in the mutation or deletion of the binding sites of anti-HA antibody, and has effects on receptor binding sites thus changed the antigenicity of the virus. Our results suggested that attention should be paid to the transmission and natural evolution of H9N2 AIV in order to control AIV H9N2.
Animals ; Chickens ; China ; Computational Biology ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; immunology ; isolation & purification ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment ; Sequence Analysis, DNA

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.
Animals ; Base Sequence ; Birds ; DNA, Viral/chemistry/genetics ; Hemagglutinin Glycoproteins, Influenza Virus/chemistry/genetics ; Influenza A Virus, H9N2 Subtype/genetics/*immunology ; Influenza Vaccines/genetics/*immunology ; Influenza in Birds/*immunology/prevention & control/virology ; Molecular Sequence Data ; Neuraminidase/chemistry/genetics ; Polymerase Chain Reaction/veterinary ; Republic of Korea ; Sequence Alignment ; Vaccines, Inactivated/genetics/immunology