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

OBJECTIVETo investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 (a reassortant of G1 and G9 viruses isolated from a female patient in 1999) in a mouse model of infection.

METHODSMice were infected with increasing virus titers. Viral load in the lungs and trachea was determined by EID50 assay. Pulmonary histopathology was assessed by hematoxylin-eosin staining. Anti-HI antibody titers and T-cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.

RESULTSMice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 (H9N2) influenza virus. Virus was detected in the trachea and lungs of mice harvested on days 3, 6, and 9 post-infection. A T-cell response to viral HA was detected on day 6 and H9 HA-specific CD(4+) T-cells predominated. Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.

CONCLUSIONOur results suggest that H9N2 (A/Guangzhou/333/99) can replicate in the murine respiratory tract without prior adaptation, and both humoral and cell-mediated immunity play an important role in the immune response.

Animals ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cell Line ; Dogs ; Enzyme-Linked Immunospot Assay ; Female ; Hemagglutination Inhibition Tests ; Hemagglutinins, Viral ; immunology ; Humans ; Infant ; Influenza A Virus, H9N2 Subtype ; immunology ; isolation & purification ; pathogenicity ; Interferon-gamma ; immunology ; Lung ; virology ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections ; immunology ; virology ; Spleen ; immunology ; Trachea ; virology ; Viral Load ; Virulence