Antigenic and molecular characterization of low pathogenic avian influenza A(H9N2) viruses in sub-Saharan Africa from 2017 through 2019

Abstract

Sub-Saharan Africa was historically considered an animal influenza cold spot, with only sporadic highly pathogenic H5 outbreaks detected over the last 20 years. However, in 2017, low pathogenic avian influenza A(H9N2) viruses were detected in poultry in Sub-Saharan Africa. Molecular, phylogenetic, and antigenic characterization of isolates from Benin, Togo, and Uganda showed that they belonged to the G1 lineage. Isolates from Benin and Togo clustered with viruses previously described in Western Africa, whereas viruses from Uganda were genetically distant and clustered with viruses from the Middle East. Viruses from Benin exhibited decreased cross-reactivity with those from Togo and Uganda, suggesting antigenic drift associated with reduced replication in Calu-3 cells. The viruses exhibited mammalian adaptation markers similar to those of the human strain A/Senegal/0243/2019 (H9N2). Therefore, viral genetic and antigenic surveillance in Africa is of paramount importance to detect further evolution or emergence of new zoonotic strains.

Keywords: Africa; Influenza virus; antigenic cartography; one health; phylogeny.

Figure 1.

Antigenic and phylogenetic characterization of Sub-Saharan African H9N2 viruses. (A) Antigenic map resulting from HI assays of H9N2 viruses. The viruses are represented by circles and antisera by squares. One grid corresponds to 1 unit of antigenic distance, a 2-fold dilution in HI titres. (B) HA phylogenetic maximum-likelihood tree with synonymous substitutions. Scale bar indicates the number of nucleotide substitutions per site. Underlines depict the AIV H9N2 viruses used to draw the antigenic map. Influenza viruses from Togo are labelled in blue, Benin in red, Uganda in orange, Asia in grey, and Egypt underlined in black. (A/chicken/Uganda/MUWRP-790/2017: Ug/790; A/chicken/Uganda/MUWRP-200162/2017: Ug/200162; A/chicken/Togo/EC-171/2019: Tg/171; A/chicken/Togo/EC-122/2019: Tg/122; A/chicken/Benin/19-A-01-145-E/2019: Bn/145; A/chicken/Benin/19-A-02-303-E/2019: Bn/303; A/chicken/Benin/19-A-04-511-E/2019: Bn/511; A/Bangladesh/0994/2011: Bd/0994; A/chicken/Egypt/A15043/2018: Eg/A15043)

Figure 2.

Time to the most recent common ancestor of Sub-Saharan African H9N2 viruses. Maximum clade credibility phylogenetic tree of the HA gene. The H9N2 viruses from Togo are represented in blue, Benin in red, and Uganda in orange. The mean TMRCA and the 95% highest posterior density intervals of the relevant nodes are indicated in parentheses.

Figure 3.

Replication kinetics of A/H9N2 viruses in Cultured Human Airway Epithelial Cells (Calu-3). Growth kinetics of Sub-Saharan Africa viruses were measured and compared with two A/H9N2 human strains after infection of Calu-3 cells with an MOI of 0.001, for indicated time points. Viruses from Uganda are labelled in orange, Benin in red, Togo in blue and the two Asian human strains in grey. Error bars indicate mean + SD of the combined results of 2 individual experiments performed in triplicate. Statistical significance of replication between virus groups at a given time point was determined by performing a 2-way ANOVA. *** p< 0.0001. (A/chicken/Uganda/MUWRP-790/2017: Ug/790; A/chicken/Uganda/MUWRP-200162/2017: Ug/200162; A/chicken/Togo/EC-171/2019: Tg/171; A/chicken/Benin/19-A-01-145-E/2019: Bn/145; A/Bangladesh/0994/2011: Bd/0994; A/Hong Kong/1073/99: Hk/1073).