Erythrocyte Binding Preference of Human Pandemic Influenza Virus A and Its Effect on Antibody Response Detection

BACKGROUND: Validation of hemagglutination inhibition (HI) assays is important for evaluating antibody responses to influenza virus, and selection of erythrocytes for use in these assays is important. This study aimed to determine the correlation between receptor binding specificity and effectiveness of the HI assay for detecting antibody response to pandemic influenza H1N1 (pH1N1) virus. METHODS: Hemagglutination (HA) tests were performed using erythrocytes from 6 species. Subsequently, 8 hemagglutinating units of pH1N1 from each species were titrated by real-time reverse transcription-PCR. To investigate the effect of erythrocyte binding preference on HI antibody titers, comparisons of HI with microneutralization (MN) assays were performed. RESULTS: Goose erythrocytes showed most specific binding with pH1N1, while HA titers using human erythrocytes were comparable to those using turkey erythrocytes. The erythrocyte binding efficiency was shown to have an impact on antibody detection. Comparing MN titers, HI titers using turkey erythrocytes yielded the most accurate results, while those using goose erythrocytes produced the highest geometric mean titer. Human blood group O erythrocytes lacking a specific antibody yielded results most comparable to those obtained using turkey erythrocytes. Further, pre-existing antibody to pH1N1 and different erythrocyte species can distort HI assay results. CONCLUSIONS: HI assay, using turkey and human erythrocytes, yielded the most comparable and applicable results for pH1N1 than those by MN assay, and using goose erythrocytes may lead to overestimated titers. Selection of appropriate erythrocyte species for HI assay allows construction of a more reliable database, which is essential for further investigations and control of virus epidemics.

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.