Phylogeographic Dynamics of H9N2 Avian Influenza Viruses in Tunisia.

Avian influenza virus subtype H9N2 is endemic in commercial poultry in Tunisia. This subtype affects poultry and wild birds in Tunisia and poses a potential zoonotic risk. Tunisian H9N2 strains carry, in their hemagglutinins, the human-like marker 226 L that is most influential in avian-to-human viral transmission. For a better understanding of how ecological aspects of the H9N2 virus and its circulation in poultry, migratory birds and environment shapes the spread of the dissemination of H9N2 in Tunisia, herein, we investigate the epidemiological, evolutionary and zoonotic potential of seven H9N2 poultry isolates and sequence their whole genome. Phylogeographic and phylodymanic analysis were used to examine viral spread within and among wild birds, poultry and environment at geographical scales. Genetic evolution results showed that the eight gene sequences of Tunisian H9N2 AIV were characterized by molecular markers involved with virulence and mammalian infections. The geographical distribution of avian influenza virus appears as a network interconnecting countries in Europe, Asia, North Africa and West Africa. The spatiotemporal dynamics analysis showed that the H9N2 virus was transmitted from Tunisia to neighboring countries notably Libya and Algeria. Interestingly, this study also revealed, for the first time, that there was a virus transmission between Tunisia and Morocco. Bayesian analysis showed exchanges between H9N2 strains of Tunisia and those of the Middle Eastern countries, analysis of host traits showed that duck, wild birds and environment were ancestry related to chicken. The subtypes phylodynamic showed that PB1 segment was under multiple inter-subtype reassortment events with H10N7, H12N5, H5N2 and H6N1 and that PB2 was also a subject of inter-subtype reassortment with H10N4.

Phylogenetic analysis and assessment of the pathogenic potential of the first H9N2 avian influenza viruses isolated from wild birds and Lagoon water in Tunisia.

H9N2 avian influenza virus (AIV) has been isolated from various species of wild birds and domestic poultry worldwide. It has been reported since the late 1990s, that H9N2 AIV has infected humans as reported in some Asian and North African countries. This subtype has already been circulating and constituting a serious threat to the poultry industry in Tunisia back in 2009. To investigate zoonotic potential and pathogenicity of H9N2 AIV in chickens and mice in Tunisia, five strains have been isolated during the period from 2014 to 2018. Samples were withdrawn from several wild bird species and environment (Lagoon water) of Maamoura and Korba Lagoons as well as Kuriat Island. Phylogenetic analyzes demonstrated that the isolated H9N2 strains belonged to the G1-like sublineage and were close to AIV H9N2 poultry viruses from North Africa, West Africa and the Middle East. All strains carried in their hemagglutinin the residue 226 L, which is an important marker for avian-to-human viral transmission. The hemagglutinin cleavage site has several motifs: PSKSSR/G, PARSSR/G and HARSSR/G. The neuraminidase showed S372A and R403W substitutions that have been previously detected in H3N2 and H2N2 viruses that were reported in human pandemics. Many mutations associated with mammalian infections have been detected in internal proteins. Pathogenicity evaluation in chickens showed that GF/14 replicates effectively in the lungs, tracheas, spleens, kidneys and brains and that it was transmitted among contact chickens. However, GHG/18 replicates poorly in chickens and has not an efficient transmission in contact chickens. GF/14 and GHG/18 could not kill mice though they replicated in their respiratory tract and caused a significant body weight loss (p < 0.05). This study highlights the importance of H9N2 AIV monitoring in both migratory birds and the environment to prevent virus transmission to humans.