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We report in this paper the first detection of low pathogenic avian influenza (LPAI) subtype H9N2 in houbara bustards and in gamebirds in Morocco. Starting in 2019, an increase in mortality rates related to respiratory distress was recorded in these species. Necropsy of the specimens revealed fibrinous sinusitis and tracheitis with intra-bronchial fibrin casts, which are consistent with H9N2 infection in chickens; therefore, implication of the virus in these outbreaks was strongly suspected. Consequently, between January 2020 and June 2023, birds with respiratory signs were necropsied for pathological lesions, tissue samples were examined by histopathology, and samples of trachea, lungs, and cecal tonsils were analyzed using quantitative real-time PCR for the detection of H9N2 virus. In addition, the sequencing of isolates was performed and lastly differential diagnosis with other respiratory pathogens was carried out. During the study period, 93 samples were collected from suspected H9N2 outbreaks, of which 30 tested positive for H9N2 virus: 23 Houbara bustards, 4 partridges, 2 quails, and 1 pheasant. Moreover, sequencing of the HA gene of the virus showed 97.33% nucleotide identity with strains reported previously in broilers in Morocco in 2017 and in 2022. Phylogenetic analysis grouped the Moroccan partridge isolates in the same cluster as viruses isolated in Morocco between 2016 and 2022, Algeria (2017), Burkina Faso (2017), Nigeria (2019), and Togo (2020). Additionally, 10 house sparrows from the premises of these birds were examined for the presence of H9N2 virus, revealing a 30% positivity rate. In conclusion, LPAIV H9N2 is circulating in houbara bustards and gamebirds in Morocco, and house sparrows might be a possible source of the infection. To our knowledge, this is the first report of LPAI H9N2 in the African species of houbara bustards worldwide and in gamebirds in Morocco.

Avian influenza viruses pose significant threats to both the poultry industry and public health worldwide. Among them, the H9N2 subtype has gained substantial attention due to its high prevalence, especially in Asia, the Middle East, and Africa; its ability to reassort with other influenza viruses; and its potential to infect humans. This study presents a comprehensive phylogenetic and molecular analysis of H9N2 avian influenza viruses circulating in Morocco from 2021 to 2023. Through an active epidemiological survey, a total of 1140 samples (trachea and lungs) and oropharyngeal swabs pooled into 283 pools, collected from 205 farms located in 7 regions of Morocco known for having a high density of poultry farms, were analyzed. Various poultry farms were investigated (159 broiler farms, 24 layer farms, 10 breeder farms, and 12 turkey breeder farms). A total of 21 AI H9N2 strains were isolated, and in order to understand the molecular evolution of the H9N2 avian influenza virus, their genetic sequences were determined using the Sanger sequencing technique. Phylogenetic analysis was performed using a dataset comprising global H9N2 sequences to determine the genetic relatedness and evolutionary dynamics of the Moroccan strains. The results revealed the continued circulation and diversification of H9N2 avian influenza viruses in Morocco during the study period. Real-time RT-PCR showed a positivity rate of 35.6% (73/205), with cycle threshold values ranging from 19.2 to 34.9. The phylogenetic analysis indicated that all Moroccan strains belonged to a G1-like lineage and regrouped into two distinct clusters. Our newly detected isolates aggregated distinctly from the genotypes previously isolated in Morocco, North and West Africa, and the Middle East. This indicats the potential of virus evolution resulting from both national circulation and cross-border transmission. A high genetic diversity at both nucleotide and amino-acid levels was observed among all the strains isolated in this study, as compared to H9N2 strains isolated in Morocco since 2016, which suggests the co-circulation of genetically diverse H9N2 variants. Newly discovered mutations were detected in hemagglutinin positions 226, 227, and 193 (H3 numbering), which highlights the genetic evolution of the H9N2 AIVs. These findings contribute to our understanding of the evolution and epidemiology of H9N2 in the region and provide valuable insights for the development of effective prevention and control strategies against this emerging avian influenza subtype.

The H9N2 virus continues to spread in wild birds and poultry worldwide. At the beginning of 2016, the H9N2 Avian influenza virus (AIV) was detected in Morocco for the first time; despite the implementation of vaccination strategies to control the disease, the virus has become endemic in poultry in the country. The present study was carried out to investigate the origins, zoonotic potential, as well as the impact of vaccination on the molecular evolution of Moroccan H9N2 viruses. Twenty-eight (28) H9N2 viruses collected from 2016 to 2021 in Moroccan poultry flocks were isolated and their whole genomes sequenced. Phylogenetic and evolutionary analyses showed that Moroccan H9N2 viruses belong to the G1-like lineage and are closely related to viruses isolated in Africa and the Middle East. A high similarity among all the 2016-2017 hemagglutinin sequences was observed, while the viruses identified in 2018-2019 and 2020-2021 were separated from their 2016-2017 ancestors by long branches. Mutations in the HA protein associated with antigenic drift and increased zoonotic potential were also found. The Bayesian phylogeographic analyses revealed the Middle East as being the region where the Moroccan H9N2 virus may have originated, before spreading to the other African countries. Our study is the first comprehensive analysis of the evolutionary history of the H9N2 viruses in the country, highlighting their zoonotic potential and pointing out the importance of implementing effective monitoring systems.