The worldwide distribution and continuing genetic mutation of avian influenza virus (AIV) has been posed a great threat to human and animal health. A comparison of 3 isolates of AIV H9N2, A/chicken/Korea/KBNP-0028/00 (H9N2) (KBNP-0028), A/chicken/Korea/SNU8011/08 (H9N2) (SNU 8011) and an inactivated oil vaccine strain A/chicken/Korea/01310/01 (H9N2) (01310), was performed. The former 2 AIVs were isolated from field cases before and after the application of an inactivated H9N2 vaccine in 2007, respectively. The antigenic relationship, viral shedding, tissue tropism and genetic analysis were examined. The comparison of virus shedding from the cloaca and the oropharynx revealed that both isolates were more frequently isolated from the upper respiratory tract (90~100%) 1 day post inoculation (DPI) compared with isolation 5 DPI from gastrointestinal tracts (10~60%). Moreover, the isolate KBNP-0028 were recovered from all organs including bone marrow, brain and kidneys, indicating higher ability for broad tissue dissemination than that of SNU 8011. KBNP-0028 replicated earlier than other strains and with a higher titer than SNU 8011. In full-length nucleotide sequences of the NA gene and a partial sequence of the HA gene of SNU 8011, we found that there might be significant changes in tissue tropism, virus replication and genetic mutation in AIV H9N2 isolates.

The circulation and infection of avian influenza virus (AIV) in zoos and backyard flocks has not been systematically investigated. In the present study, we surveyed the birds including those in live bird markets (LBMs) and evaluated co-circulation of AIVs among them. Overall, 26 H9N2 AIVs and one H6N2 AIV were isolated from backyard flocks and LBMs, but no AIVs were isolated from zoo birds. Genetic analysis of the HA and NA genes indicated that most of the H9N2 AIVs showed higher similarities to AIVs circulating in domestic poultry than to those in wild birds, while the H6N2 AIV isolate from an LBM did to AIVs circulating in migratory wild birds. In serological tests, 15% (391/2619) of the collected sera tested positive for AIVs by competitive-ELISA. Among them, 34% (131/391) of the sera tested positive for AIV H9 antigen by HI test, but only one zoo sample was H9 positive. Although AIVs were not isolated from zoo birds, the serological results indicated that infection of AIVs might occur in zoos. It was also confirmed that H9N2 AIVs continue to circulate and evolve between backyard flocks and LBMs. Therefore, continuous surveillance and monitoring of these flocks should be conducted to control further epidemics.

Newcastle and Influenza diseases are important viral diseases and its occurrence and virulence in Iran has increased in recent years. The purpose of this study was to evaluate the humoral immune responses of chickens vaccinated with inactivated dual oil emulsion Newcastle disease and avian influenza vaccine in two methods of intramuscular and subcutaneous injection as well as to assess the possible changes in serum biochemical factors. In this study, after subcutaneous and intramuscular inoculation of inactivated dual oil emulsion Newcastle-Influenza vaccine, the serum antibody level and hematobiochemical factors of these avian were analyzed on 7, 14, 21, and 28 days after vaccine injection.The results showed that the values of biochemical parameters such as albumin, glucose, total protein, triglyceride, cholesterol, ALT, AST, and ALP enzymes, sodium and potassium minerals, and hematocrit and hemoglobin levels in vaccinated and non-vaccinated chickens were not significantly different. Also, in chickens injected with antigen, Newcastle disease and influenza antibody titers were significantly different with those groups containing adjuvant.The results of this study indicate that the intramuscular and subcutaneous injection of dual combination vaccine is similar in terms of changes in biochemical and hematological factors and Newcastle disease and influenza antibody titers. To ensure safety in inactivated vaccines, the presence of proper adjuvant is essential for immune response.

A primary fibroblast cells from embryos of brown quail Coturnixypsilophora has been established and partially characterized. The cells were maintained in Modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum. The cells were able to grow at temperatures between 35°C and 38°C with optimum temperature of 37°C. The growth rate of primary quail fibroblast cells increased as the FBS proportion increased from 5% to 20% at 37°C with optimum growth at the concentrations of 10% or 15% FBS. The cells showed no microbial contamination throughout the period of experiment and the total chromosome number of a diploid cell was 78, according to karyotyping and chromosome analysis. The susceptibility of quail primary cells for avian viruses was investigated in this study after inoculation with ND and IB viruses. Both viruses showed a satisfactory CPE development and the infectivity were assayed by virus titration (TCID50). This suggests that the quail primary cells can be used for isolation of various avian viruses with further steps of infectivity confirmation in the future.

Test vaccines comprised of inactivated water-in-oil emulsions containing various antigen levels were prepared using a non-pathogenic H5N1 avian influenza (AI) virus, A/duck/Hokkaido/Vac-1/04 (H5N1). The potencies of these test vaccines were evaluated by two experiments. In the first experiment, the triangular relationship among the antigen levels of test vaccines, the hemagglutination inhibition (HI) antibody response, and the protective effect against challenge with a highly pathogenic avian influenza (HPAI) virus, A/chicken/Yamaguchi/7/04 (H5N1), was confirmed. Then lasting immunity of chickens after a single-shot vaccination was confirmed in the second experiment. As a result, complete protection after the challenge was observed in chickens immunized by test vaccines with an antigen level of 160 HA units/dose or higher. Thus, it was ascertained that the minimum antigen level in the AI vaccine was 160 HA units/dose, and the minimum HI antibody titer that could protect chickens from HPAI virus infection-related death was considered to be 1:16. Dose-dependent HI antibody responses were observed in chickens after the vaccination. Thus, 640 HA units/dose were thought to be similar to the optimal antigen level. Alternatively, the HI antibody titers of chickens, injected with the vaccine containing 640 HA units/dose, were maintained at 1:181 or higher for 100 weeks after the single-shot vaccination.

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokhaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 virus as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.