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.

Avian influenza viruses (AIV) have been isolated from a wide range of domestic and wild birds. Wild birds, predominantly ducks, geese and gulls form the reservoir of AIV in nature. The viruses in wild bird populations are a potential source of widespread infections in poultry. Active surveillance for AIV infection provides information regarding AIV distribution, and global AIV surveillance can play a key role in the early recognition of highly pathogenic avian influenza (HPAI). Since 2003 in Korea, there have been four H5N1 HPAI outbreaks caused by clade 2.5, 2.2 and 2.3.2. Therefore, improvement of AIV surveillance strategy is required to detect HPAI viruses effectively. This article deals with the major events establishing the role of wild birds in the natural history of influenza in Korea. We highlighted the need for continuous surveillance in wild birds and characterization of these viruses to understand AIV epidemiology and host ecology in Korea.

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.

Cause of high lethality and dissemination to human being, new development of rapid method for the detection of highly pathogenic Avian Influenza Virus (AIV) is still necessary. For the detection of AIV subtype H5N1, typical pathogenic AIV, new method to confirm sub-typing of this virus is also needed. For the purpose of ultra-rapid detection and sub-typing of hemagglutinin and neuraminidase of AIV, this study was planned. As the results we could demonstrate an ultra-rapid multiplex real-time PCR (URMRT-PCR) for the detection of AIV.

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.