Passage of LPAIV H5 isolates in chickens results in genotypic changes in the glycoprotein genes and development of a species independent competitive ELISA system

Abstract

The determination of the hemagglutinin (HA) subtype is restricted to inhibition and virus neutralization assays. Both assays are labor intensive and not suitable for automation. The presence of HA5 and HA7 antibodies are of major interest since these subtypes of AIV can develop a highly pathogenic phenotype. To develop a test which can be used in automatic settings the HA5 protein of a wild bird isolate [A/duck/NC/674964/07 (H5N2)] was cloned and subsequently expressed in a baculovirus system. In addition, a monoclonal antibody specific for the HA5 antigen (H5-mAb) was generated. Both reagents were used to establish a competitive ELISA (cELISA) system. The cELISA performed with influenza antibody free sera or sera of animals infected with AIV HA subtypes other than H5 showed no significant inhibition of H5-mAb binding, indicating a high specificity of the test. In contrast, sera of animals (chickens, turkeys, mallards, redheads, wood ducks, and cats) experimentally infected with H5 subtype AIV or vaccinated with inactivated vaccines were able to significantly inhibit the binding of the H5-mAb. The cELISA showed a significant inhibition (> 25%) of mAb binding in hemagglutination inhibition (HI) positive serum samples in chicken, duck, and turkey sera with a reproducibility of >95%. This test provides a platform for further development of other subtype specific HA ELISAs. Additionally, to better understand the mechanisms of LPAIV transmission and adaptation serial passages of LPAIV isolates were performed in chickens. Sequences of the HA and NA genes were determined for the stock viruses and at each subsequent passage. Three groups of 3-week-old SPF chickens were infected with three different LPAIV, one wild bird isolate (H5N1), and two chicken isolates (H5N2, H5N3). At 1 day post inoculation (pi), 5 contact birds were added to each group. In addition, subsequent passages in chickens for each virus were performed. Tracheal and cloacal swabs were taken at 2, 4, 7, and 9 d pi., and used for virus isolation (VI) in embryonated SPF eggs. LPAIV positive allantoic fluid was detected by hemagglutination assay. Existence of HI antibodies was tested on serum samples taken before and 21d after infection. The NA and HA genes of the appropriate virus of VI positive swab and allantoic fluid samples were sequenced and amino acid (aa) sequences were deduced.