Genetic Variations among Different Variants of G1-like Avian Influenza H9N2 Viruses and Their Pathogenicity in Chickens

Abstract

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.

Keywords: antigenicity; avian influenza; genetic characterization; pathogenicity; reassortment.

Figure 1

Different LPAI H9N2 viruses reported in Egypt (2011–2021). (A) The first reported LPAI H9N2 virus variant (A/quail/113413V/2011) in Egypt in 2011. (B) The quail/14864V/2014 variant detected in Egypt in 2014. (C) The recently circulating LPAI H9N2 variant in Egypt that harbors the PB2, PB1, PA, and NS genes from the pigeon variant and the rest of the genes from the Egyptian A/quail/113413V/2011-like variants. The three virus strains (A–C) were used for the chicken experiment. Silhouettes of duck (by Maija Karala) and Pigeon (by Dori (dori@merr.info) and Nevit Dilmento) were retrieved from PhyloPic (http://phylopic.org, accessed on 11 January 2022) under a CC BY-NC-SA-3.0 license (https://creativecommons.org/licenses/by-nc-sa/3.0/). Other silhouettes were downloaded from the same cite under a CC0 1.0 Universal (CC0 1.0) license (https://creativecommons.org/publicdomain/zero/1.0/).

Figure 2

Phylogenetic trees of the nucleotide sequences of the HA (a) and NA (b) gene segments. The whole HA (a) and NA (b) gene segment were used from viruses reported till October 2021 and available at NCBI. Maximum likelihood calculations were done using the IQTree software under the best fit model, according to the Bayesian criterion. Egyptian LPAIV H9N2 virus variants are grey-shaded. Virus strains sequenced in this study are indicated by a black dot.

Figure 2

Phylogenetic trees of the nucleotide sequences of the HA (a) and NA (b) gene segments. The whole HA (a) and NA (b) gene segment were used from viruses reported till October 2021 and available at NCBI. Maximum likelihood calculations were done using the IQTree software under the best fit model, according to the Bayesian criterion. Egyptian LPAIV H9N2 virus variants are grey-shaded. Virus strains sequenced in this study are indicated by a black dot.

Figure 3

Virus shedding of LPAIV H9N2 infected chickens. Oropharyngeal (A) and cloacal (B) swabs were sampled at indicated days post-infection (dpi) and tested by RT-qPCR. Individual results of detected RNA copy numbers are given as EID₅₀ equivalents. Circle indicates primary chicken and triangle indicates secondary/contact chickens.

Figure 3

Virus shedding of LPAIV H9N2 infected chickens. Oropharyngeal (A) and cloacal (B) swabs were sampled at indicated days post-infection (dpi) and tested by RT-qPCR. Individual results of detected RNA copy numbers are given as EID₅₀ equivalents. Circle indicates primary chicken and triangle indicates secondary/contact chickens.

Figure 4

Antigenic cartography based on the hemagglutination inhibition (HI) titers of the sera of the Egyptian H9N2 virus strains used in this study and representative virus isolates of different antigenic clusters. Each point indicates a virus and both axes represent antigenic distance. The spacing between grid lines represents 1 antigenic unit distance, corresponding to a 2-fold dilution in the HI assay.