Abstract:
Influenza A virus (IAV) is a single-stranded negative-sense RNA virus that is a member of
the Orthomyxoviridae group. IAV has been detected in over 100 bird species from 26
different families, although Anseriformes and Charadriiformes are considered the natural
host of the virus. Surveillance of wild birds for IAV is important as it plays a role in the
early detection system for the introduction of potentially dangerous IAV strains with the
ability to cause damage in the poultry industry, or even affect human health. Fresh wild
bird faecal samples at a wild ducks’ roosts may contain a high concentration of IAV. A
method developed by Zhou et al. in 2009 described the amplification of full IAV genome
in a single tube, and this method has been successfully employed at the University of
Pretoria (UP) in the subtyping of IAV in clinical samples (organs and tracheal swabs).
However, when applied to environmental faecal samples the technique was
unsuccessful, presumably due to PCR inhibitors and a high level of contaminating nucleic
acids from bacteria. Therefore, the first objective of this study was to optimize the IAV
pan-genome RT-PCR for environmental faecal samples. PCR parameters such as MgSO4
concentration, annealing temperatures, and different PCR reagents were optimized on
spiked faecal samples. The second objective was to screen fresh environmental faecal
samples from wild duck at a site in Pretoria to identify positive field samples for testing. A
total of 2,144 faecal swabs were collected from January through-February 2021 and
screened with IAV-specific real-time RT-PCR assay. Two samples with positive results
were submitted to the Central Analytical Facility in Stellenbosch University for Ion Torrent
Next-Generation Sequencing. After assembling the results in the CLC Genomics
Workbench software and verifying the results by BLAST analysis, TP2118 was
conclusively identified as an H9N2 strain, but whereas the presence of IAV-specific
internal genes could be identified for TP2067, the sequence data was insufficient to
identify the subtype. TP2118 represents the first H9N2 virus ever detected in wild ducks
in Gauteng Province.
