Molecular and Phylogenetic Analysis of the Highly Pathogenic Avian Influenza Virus H5N1 Detected in Mali During the 2021 and 2022 Outbreaks

Abstract

Highly pathogenic avian influenza (HPAI) H5N1 continues to pose a significant threat to animal health, poultry production, and public health worldwide. Mali experienced two outbreaks of Highly Pathogenic Avian Influenza (HPAI) H5N1 in 2021 and 2022, raising concerns about viral evolution and potential public health risks in West Africa. Suspected samples (Tissue and swab) collected from sick and dead birds during these outbreaks were analyzed at the Central Veterinary Laboratory of Bamako using quantitative real-time PCR. Whole-genome sequencing was performed on thirteen H5N1-positive samples using the Illumina MiSeq platform with a paired-end sequencing approach. Phylogenetic analyses were conducted using the maximum-likelihood method implemented in IQ-TREE v1.6.6. Phylogenetic analysis of the hemagglutinin (HA) gene revealed that all detected viruses belonged to clade 2.3.4.4b and clustered closely with strains previously reported in West Africa. In-depth analysis of the whole genome phylogenetic topology revealed the emergence of an H9N2/H5N1 reassortant strain during the 2022 outbreak. Molecular characterization of all eight genomic segments identified several mutations associated with host specificity, including markers linked to adaptation in gallinaceous poultry and mutations potentially associated with increased zoonotic potential. These findings demonstrate the continued genetic evolution of H5N1 viruses circulating in Mali and emphasize the importance of sustained genomic surveillance. Strengthening hygiene practices and biosecurity measures in poultry farms, live bird markets, and slaughterhouses is essential to reduce the risks posed to both animal and public health.