Genetic characterization and pathogenesis of the first H9N2 low pathogenic avian influenza viruses isolated from chickens in Kenyan live bird markets

Authors: KARIITHI, HENRY - Orise Fellow; WELCH, CATHERINE - University Of Georgia; FERREIRA, HELENA - Orise Fellow; PUSCH, ELIZABETH - Orise Fellow; ATEYA, LEONARD - Kenya Agricultural And Livestock Research Organization; BINEPAL, YATINDER - Kenya Agricultural And Livestock Research Organization; LICHOTI, JACQUELINE - Ministry Of Agriculture, Livestock And Fisheries, State Department Of Livestock; APOPO, AULERIA - Ministry Of Agriculture, Livestock And Fisheries, State Department Of Livestock; Afonso, Claudio; Suarez, David; DULU, THOMAS - Ministry Of Agriculture, Livestock And Fisheries, State Department Of Livestock

Submitted to: Infection, Genetics and Evolution
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/13/2019
Publication Date: 10/18/2019
Citation: Kariithi, H.M., Welch, C.N., Ferreira, H.L., Pusch, E.A., Ateya, L.O., Binepal, Y.S., Lichoti, J.K., Apopo, A.A., Afonso, C.L., Suarez, D.L., Dulu, T.D. 2019. Genetic characterization and pathogenesis of the first H9N2 low pathogenic avian influenza viruses isolated from chickens in Kenyan live bird markets. Infection, Genetics and Evolution. 78:104074. https://doi.org/10.1016/j.meegid.2019.104074.
DOI: https://doi.org/10.1016/j.meegid.2019.104074

Interpretive Summary: Avian influenza in domestic poultry is a great concern because of the severe clinical disease that it can cause and it can spread rapidly, cause significant economic losses, trade embargos, and even affect humans. In most developing countries, active surveillance and diagnosis of avian influenza is lacking, thus posing a risk for cross-border transmission. As part of a surveillance program funded by the US Defense Threat Reduction Agency (DTRA) and Agricultural Research Service of the USDA, oral and cloacal swabs were collected from chickens sold at live bird markets (LBMs) as well as chickens reared at rural backyard poultry farms in Kenya. The samples were screened for the presence of avian influenza virus (AIV) by real-time PCR. Virus isolation in eggs and Next Generation Sequencing identified H9N2 low pathogenic AIV in five LBM samples. Phylogenetically, the H9N2 belongs to the G1 lineage, and is closely related to a H9N2 virus isolated in Uganda reported in 2017. Further experiments on this virus in chickens showed that the H9N2 reported for the first time here is infectious and transmissible by direct contact. The H9N2 LPAIV from Kenya was also found to contain genetic elements that is associated with virulence and adaptation in mammalian hosts, and thus the virus represents a potential disease threat to poultry and people. This study reiterates the need for continuous monitoring of influenza virus infections in humans and other avian species.

Technical Abstract: Poultry production plays important roles in the economy and livelihoods of rural households in Kenya. As part of a surveillance program, AIV-specific real-time RT-PCR (RRT-PCR) was used to screen 282 oropharyngeal swabs collected from chickens at six live bird markets (LBMs) and 33 backyard poultry farms in Kenya and 8 positive samples were detected. Virus was isolated in eggs from five samples, sequenced and identified as H9N2 LPAIV G1 lineage, with nucleotide sequence identity (98.6-99.9%) to a 2017 Ugandan H9N2 isolate. The H9N2 contained molecular markers for mammalian receptor specificity, implying their zoonotic potential. Virus pathogenesis and transmissibility were assessed by inoculating low and medium virus doses of a representative Kenyan H9N2 LPAIV isolate into experimental chickens and exposing them to naïve uninfected chickens at 2 -days post inoculation (dpi). Virus shedding was determined at 2/4/7 dpi and 2/5 days post placement (dpp), and seroconversion determined at 14 dpi/12 dpp. None of the directly-inoculated or contact birds exhibited any mortality or clinical disease signs. All directly-inoculated birds in the low dose group shed virus during the experiment, while only one contact bird shed virus at 2 dpp. Only two directly-inoculated birds that shed high virus titers seroconverted in that group. All birds in the medium dose group shed virus at 4/7 dpi and at 5 dpp, and they all seroconverted at 12/14 dpp. The Kenyan H9N2 LPAIV reported for the first time is infectious and transmissible in chickens by direct contact and represents a new disease threat to poultry and people.