The present study showed that horse RBCs were not the choice of RBCs for detection and identification of AI viruses. AI H9N2 and H5N2 viruses did not react with horse RBCs both in HA and HI assays. It has been reported that horse RBCs require more viral particles for agglutination than do turkey RBCs. Similarly, using horse RBCs in HI assays require more virus than HI assay with other avian erythrocytes .Guinea pig RBCs were suitable for both HA and HI assays for all AI virus subtypes, but reading and interpretation of results with guinea pig RBCs was difficult. Goose and other avian RBCs were the next suitable RBCs. Advantage of using avian RBCs was that reading and interpretation of results were easier than mammalian RBCs.
With different types of RBCs, variation in HA and HI titers was observed among isolates of the same subtype. Therefore to conclude overall performance or reactivity of RBCs, calculation of GMT among the same subtype was necessary to predict suitability of RBCs. Overall guinea pig, goose and turkey RBCs were best in HA and HI assays for AI viruses. These results are in agreement with the study by Witthawat et. al.. Suda Louisirirotchanakul et al., have reported that goose erythrocytes confer a greater advantage over other erythrocyte species in both HA and HI assays . Currently, HI-based antigenic analysis is used for influenza virus strains . The present study showed that use of different RBCs results in titer differences in HI assay. Therefore, RBCs giving optimum HA and HI titers would increase sensitivity of detection and would be more appropriate for identification and antigenic analysis of AI viruses.
The direct binding assays based on different gangliosides have been used previously to study the receptor specificity of influenza A and B viruses. Hemagglutination with erythrocytes, which were enzymatically modified to contain sialyloligosaccharides of defined sequences, or hemolysis assays based on ganglioside-coated erythrocytes also have been used. These methods require specific reagents and tend to be technically difficult and time-consuming . In the present study, a simpler assay described by Auewarakul et. al. was employed to determine receptor specificity by using alpha 2, 3 sialidase enzyme. The sialidase enzyme theoretically cleaves the entire alpha 2, 3 sialic acid linked receptors on the RBCs . Goose and turkey RBCs were used for receptor specificity assays and results of both RBCs were in agreement. In receptor specificity assays, eight H5N1 isolates, H4N6 and H7N1 viruses clearly indicated preference to avian receptors. Two H5N1 isolates, H9N2 and H11N1 viruses showed ability to bind both α 2,3 and α 2,6 sialic acid linked receptors, highlighting their potential to cross species barrier.
The amino acid 221 position of HA protein has critical role in deciding receptor specificity of H5N1 virus and change of amino acid S221P is responsible for change in avian receptor specificity . All analyzed amino acids at the receptor-binding domain of hemagglutinin protein were conserved in analyzed AI virus strains except S221P change. Two H5N1 viruses showed S221P amino acid change, out of which only one H5N1 virus showed preference to α 2, 6 sialic acid receptor. One H5N1 isolate showed amino acid S at 221 position, still showed preference to α 2, 6 along with α 2, 3 sialic acid receptor. Possibility of the presence of a minor variant within the viral quasispecies that had altered receptor-binding specificity cannot be ruled out. Amino acid P was found at 221 position of hemagglutinin in AI H9N2, H11N1, H4N6 and H7N1 viruses. AI H9N2 and H11N1 virus showed receptor specificity to α 2, 3 and α 2, 6 sialic acid receptors. Involvement of factor(s) other than S221P mutation in the hemagglutinin protein in determining receptor specificity of H5N1 viruses needs further investigation. In the current scenario of emerging influenza viruses, virological as well as molecular characterization and analysis of receptor preferences of AI viruses would be important to study animal-human interface.