Identification of Non-Haemagglutinating Influenza A/H3 Virus and Characterization of Haemagglutinin (HA) and Neuraminidase (NA) Strain Mutations in Influenza-Like-Illness Cases in Egypt on MDCK Cell Line.

Haemagglutination Inhibition assay “HAI” is one of the routinely used assays in the Influenza virus isolation workflow. Many Influenza A/H3 isolates can be mistakenly considered negatives if HAI is the only tool for culture identification, due to the increase in numbers of non-haemagglutinating influenza strains circulation.
We wanted to explore the presence of non-haemagglutinating influenza A/H3 in Egypt and to discover potential mutations.
481 Oropharyngeal swabs in 2014 were collected from outpatients with Influenza-like-illness from eight hospitals in Egypt.
Samples were tested for influenza viruses by qRT-PCR, then virus isolation. Cultures were tested by HAI, indirect Immunofluorescence assay “IFA” and Sequencing of HA and NA genes.
QRT-PCR identified 84 Influenza A/H3 samples, 12 of which were successfully cultured and confirmed by qRT-PCR, IFA, and sequencing.
Results of HAI showed Six Haemagglutinating and Six non-haemagglutinating cultures. HA and NA sequencing revealed mutations present in non-haemagglutinating and absent in Haemagglutinating isolates in both HA and NA genes.
We could link only one mutation per gene HA (N225D) and NA (D151N) to the agglutination avidity of Influenza A/H3 isolates. We suggest that the loss of ability is due to the N225D mutation in non-haemagglutinating isolates causing steric hindrance nearby the binding site of HA spikes. Whereas the agglutination capacity of haemagglutinating isolates is increased due to D151N found in the haemagglutinating only, suggesting the agglutination through the NA spikes also as referenced in literature.
As we have confirmed the presence of non-haemagglutinating influenza A/H3 in Egypt we recommend the use of IFA as the confirmatory assay in the Influenza virus isolation workflow, recommendations also for further to study virulence and prevalence of the identified virus.