Detection and characterization of human influenza A virus isolates from patients attending Kayunga and Mulago Hospitals in Uganda

Abstract

Human influenza A viruses are known to cause severe illnesses and fatalities. Therefore, their continuous surveillance is crucial for the early detection and appropriate interventions through treatment therapies and vaccination. The aim of this study was to detect and characterize human influenza A virus isolates from Kayunga and Mulago hospitals in Uganda. A total of 450 human respiratory samples were collected. All samples were screened for influenza A virus using a One Step RT-PCR Qiagen kit and M gene specific primers from Applied Biosystems, UK. A volume of 100 µl from each of the samples that tested positive for Influenza A viruses were cultured on MDCK (Madin Darby Canine Kidney) cell line passage 2 in T25 culturing flasks. The isolates that showed cytopathic effect (CPE) were subjected to immune-fluorescence assay (IFA) using a Light Diagnostics ™ Influenza A and B DFA kit. The samples confirmed positive by IFA were then sub-typed by One Step RT-PCR Qiagen kit using H1N1 and H3N2 specific primers from Applied Biosysems. This was followed by whole genome DNA sequencing of PCR products using the Illumina Genome Analyser IIe. There were 51 positive samples for influenza A viruses detected by One Step RT-PCR. The viruses from the samples all showed CPE when cultured on MDCK cell line and were further confirmed positive for influenza A viruses by IFA. The prevalence of the influenza A viruses was 11.3%. The participants who were of the 6 months to 5 years age-group had the highest infection rate, 80%. The infection rates were highest during November 2008 and the Mulago site registered more positive cases. All viruses detected were of the H3N2 subtype. The influenza A viral genes, PB2, PA, and NP, had significant amino acid changes that have been reported to be associated with host transmission and low virulence. All study isolates were of different strains that had nucleotide diversities by each of the eight genes. The human influenza A H3N2 viruses detected were already existent worldwide. Their gene sequences were 99% similar to those of other strain sequences of viruses detected in 2008 and submitted in the genbank database. The study findings indicated the presence of influenza A H3N2 viruses with a prevalence rate of 11.3% in the Ugandan human population. All the study strains were already existent worldwide