Field Application of the H9M2e Enzyme-Linked Immunosorbent Assay for Differentiation of H9N2 Avian Influenza Virus-Infected Chickens from Vaccinated Chickens

Avian influenza (AI) is an important infectious disease in both animals and humans. Since the late 1990s, H9N2 low-pathogenicity avian influenza (LPAI) virus (LPAIV) has been reported from South Asia, South Africa, Europe, and the Middle East (1, 5, 16, 21, 23, 26), and infection has become increasingly prevalent in domestic poultry (16). H9N2 LPAI virus can be transmitted from poultry to mammalian species, including humans, by direct and indirect contact (4, 25). Therefore, the H9N2 LPAI virus is considered an important candidate source of future human pandemic influenza. In South Korea, the first H9N2 LPAI outbreak in chickens occurred in 1996 and caused slight to moderate mortality (5 to 30%) with apparent clinical signs of depression, edema in the head of the chicken, drop in egg production, and cyanosis in the comb and legs of the chicken (15, 20). H9N2 LPAI reappeared in late 1999 and has become prevalent in South Korea and has caused appreciable economic losses in the poultry industry. Vaccination is being considered more commonly as an alternative control measure to prevent avian influenza, although it can affect serologic surveillance for H9N2 LPAI infection and negatively affect international trade. H9N2 LPAI vaccination in several countries has been a promising control measure (15, 17, 21, 34); inactivated vaccines prevent clinical signs and increase resistance to infection despite the risk of masking circulation due to the shedding of field virus, if vaccinated poultry become infected with a sufficiently high dose of virus (6, 29, 30). In South Korea, an H9N2 inactivated vaccine consisting of inactivated-virus whole-virus vaccine (in oil emulsion) has been used since March 2007 in an effort to combat the widespread distribution of H9N2 LPAI since 2004, with increasing prevalence of clinical signs and infection-related mortality (7). However, an important limitation in the use of homologous inactivated vaccine is that vaccinated chickens cannot be differentiated from naturally infected chickens using serologic tests such as the agar gel immunodiffusion test (AGP), commercial enzyme-linked immunosorbent assay (ELISA), and hemagglutination inhibition (HI) tests (29). Strategies of differentiation of infected from vaccinated animals (DIVA) that have been proposed include the use of unvaccinated sentinel birds, subunit vaccines, vaccination with inactivated virus and a homologous hemagglutinin (HA) to the circulating field virus but a heterologous neuraminidase (NA) and the measurement of the serologic response to nonstructural protein 1 (NS1) (28). In South Korea, unvaccinated sentinel chickens have been placed in vaccinated flocks to determine whether flocks have been exposed to H9N2 LPAI. Although sentinels are thought to provide a sensitive measure of infection of a vaccinated flock, this strategy carries practical difficulties; the sentinel poultry must be marked, and these naive poultry may increase the risk of infection of the flock (28). Therefore, development of a diagnostic method(s) in the DIVA strategy for control of H9N2 LPAI is needed for use of the H9N2 LPAI vaccine. Recently, an alternative to DIVA strategy was reported using the measurement of a differential immune response to the extracellular domain of the third integral membrane protein (M2 protein) of the influenza virus (14). The M2e protein (extracellular domain of M2), like NS1, is also associated with actively replicating virus which is exuberantly synthesized in infected cells but is not incorporated into the infectious viral particle, and M2 protein shares with NS1 protein the potential for elaborated DIVA testing, due to differential epitope density on the surfaces of infected cells and on infectious viral particles (10, 18, 22, 35). This M2 protein consists of 97 amino acids (aa), and 24 N-terminal amino acids (M2e) are exposed outside the membrane surface (9); this M2e region is highly conserved in all human, swine, and avian influenza A viruses, with host restriction specificities (18). The main goal of the present study was to evaluate an ELISA using the extracellular domain of the M2 protein (M2e) of H9N2 LPAI virus (H9M2e ELISA) on the basis of the production of anti-M2e antibodies readily detectable in sera from infected chickens in the differentiation of H9N2 LPAIV-vaccinated chickens from infected chickens with the aim of further field application and, ultimately, replacement of the sentinel strategy used in vaccinated flocks in South Korea.