Phylogenetic Analysis and Assessment of the Pathogenic Potential of Avian Influenza Virus H9N2 in Wild Waterfowl, Wild Birds, and Lagoon Water in Tunisia

Abstract H9N2 avian influenza viruses (AIV) have been isolated from various species of wild birds and domestic poultry worldwide. They have been described to not only infect mammals directly, but also provide gene segments to generate novel but lethal human reassortants. H9N2 AIV has circulated in Tunisian commercial poultry since 2009 and poses a serious threat to the poultry industry. In the present study, we investigate five H9N2 viruses isolated from greater flamingoes (GF) (Phoenicopterus roseus), gray-headed gull (GHG) (Chroicocephalus crirrocephalus), black-winged stilt (BWS) (Himantopus himantopus), and lagoon water (EN) from Maamoura Lagoon, Korba Lagoon, and Kuriat Island in Tunisia from 2014–2018. Full-length genome sequences of AIV H9N2.Viral sequence analyses showed several amino acid changes related to viral adaptation to mammals. Phylogenetic analyses demonstrated that the five isolates belonged to the same sublineage G1and were close to AIV H9N2 poultry strains isolated in North Africa, West Africa, and the Middle East. At the hemagglutinin (HA) cleavage site, viruses (GF/120/15), (BSW/119/18), (GHG/216/18), and (EN/117/18) had the 335RSSR/GLF341 motif. However, (GF/121/14) undergo punctual substitutions at the HA1/HA2genesand acquires the dibasic pattern 335KSSR/GLF341.All Tunisian viruses carried in their hemagglutinins the human-like marker 226L, which is important for avian-to-human viral transmission. The neuraminidase (NA) of the five viruses showed S372A and R403W substitutions previously detected in H9N2 from Asia and the Middle East and in H3N2 and H2N2 strains reported in human pandemics. Many mutations associated with mammalian infections have been detected in internal proteins. Chicken experiments (IPIV) show that the five Tunisian viruses displayed low pathogenicity, causing mild symptoms in chickens. The virus could not kill mice, and merely replicated when administrated to mice. To our knowledge, this is the first study to address the evolution, pathogenicity, and transmission of the H9N2 viruses isolated from wild birds, waterfowl, and lagoon water in North Africa. Our results indicate that we should pay greater attention to H9N2 AIV surveillance in wilds birds. These results provide new insights into the measures that need to be undertaken regarding the role of wild birds in AIV introduction and persistence in Tunisia.