In southern Africa, clade 2.3.4.4B H5N1 high pathogenicity avian influenza (HPAI) was first detected in South African (SA) poultry in April 2021, followed by outbreaks in poultry or wild birds in Lesotho and Botswana. In this study, the complete or partial genomes of 117 viruses from the SA outbreaks in 2021–2022 were analyzed to decipher the sub-regional spread of the disease. Our analysis showed that seven H5N1 sub-genotypes were associated with the initial outbreaks, but by late 2022 only two sub-genotypes still circulated. Furthermore, SA poultry was not the source of Lesotho’s outbreaks, and the latter was most likely an introduction from wild birds. Similarly, SA and Botswana’s outbreaks in 2021 were unrelated, but viruses of Botswana’s unique sub-genotype were introduced into SA later in 2022 causing an outbreak in ostriches. At least 83% of SA’s commercial poultry cases in 2021–2022 were point introductions from wild birds. Like H5N8 HPAI in 2017–2018, a coastal seabird-restricted sub-lineage of H5N1 viruses emerged in the Western Cape province in 2021 and spread to Namibia, causing mortalities in Cape Cormorants. In SA ~24,000 of this endangered species died, and the loss of >300 endangered African penguins further threatens biodiversity.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021–2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

A high pathogenicity avian influenza (HPAI) subtype H7N6 virus emerged in South African poultry in 2023 and later spread to Mozambique, the first documented emergence of H7 HPAI in the African continent. A total of 6.82 million birds succumbed to the disease or were culled, representing about 20% of the South African egg‐laying flock and almost 30% of the broiler breeder flock. The complete genomes of 68 outbreak viruses were sequenced and analyzed, tracing the phylogenetic origins of the ancestral H7N6 virus to a reassortment of various subtypes that circulated in southern African wild birds. Molecular clock analysis determined that the virus emerged in the first week of May 2023, probably in a smallholder chicken flock, before spreading to commercial farms, where the disease was first reported in early June. The multibasic hemagglutinin protein cleavage site (HA₀) was derived from a nonhomologous recombination event with chicken 28S ribosomal ribonucleic acid (RNA). Few genetic markers associated with an increased risk to humans were present in the translated viral proteins. The intravenous pathogenicity index (IVPI) value of the index case isolate was 1.67, reflecting that 50% of the specific pathogen‐free chickens died within 4 days of infection. Surviving birds showing mostly mild clinical signs and recovered by day 10 postinfection. Prior to death, chickens shed the virus primarily through the respiratory route, with lower amounts shed from the cloaca, but in the survivors, the virus was still being shed from the cloaca on day 10. Fomites were the likely source of disease spread between farms, and the amount of H7N6 HPAI virus per gram of feces was calculated at ~383,193 (5.58 log₁₀) egg infectious dose 50 (EID₅₀) equivalents, chicken feather follicles contained on average 739,712.43 (5.87 log₁₀) EID₅₀ equivalents, and 20 µg of feather dust contained 14,976.96 (4.175 log₁₀) EID₅₀ equivalents.

Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor. Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (10^6.7 – 10^3.0 vs. 10^6.6 - 10^3.1 EID₅₀ for H5N8 HPAIV and 10^5.5 - 10^1.4 vs. 10^5.1 – 10^1.3 EID₅₀ for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains. Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics.