# Isolation, characterization and phylogenetic analyses of avian influenza A (H9N2) viruses isolated from poultry between 2019 and 2023 in Egypt

## TL;DR

This study analyzed H9N2 avian flu viruses in Egypt from 2019 to 2023, focusing on their genetic traits and potential for cross-species transmission.

## Contribution

The study provides new insights into the genetic evolution and virulence markers of H9N2 viruses in Egypt.

## Key findings

- H9N2 isolates clustered within genotype III and showed glycosylation sites in HA and NA proteins.
- Viruses carried residues associated with avian-to-human transmission and mutations linked to virulence and mammalian infection.
- HA gene was under positive selection pressure, while internal genes were under negative selection.

## Abstract

The current study aimed to investigate the genetic characterization and evolution of low pathogenic avian influenza virus H9N2 in Egypt. Ten H9N2 viruses were recently isolated from samples collected between 2019 and 2023. Phylogenetic analysis of the haemagglutinin (HA) gene segment of the H9N2 isolates showed a relatedness with G1 H9 4.2 lineage and clustered within genotype III of the Egyptian strains identified earlier in 2018. The majority of H9N2 strains had seven and eight glycosylation sites in HA and neuraminidase (NA) respectively. All strains carried H191 and L234 residues in their hemagglutinin which are markers facilitating avian-to-human cross species barrier transmission. No stalk deletions were detected in NA gene. In addition, genetic analysis of the NA and M encoding proteins revealed the absence of substitutions associated with resistance to oseltamivir and amantadine. The NA showed S372A and R403W substitutions which were previously detected in H3N2 and H1N2 viruses that were reported in previous influenza pandemics in 1975 and 2001 respectively. Many mutations associated with virulence and mammalian infection were detected in internal proteins such as PB2(V504), PB1-F2(N66), PA (V127, L672, and L550), M2(S64), and NS1(42S). Analysis showed the presence of full-length PB1-F2 with 227PDZ230 motif which is associated with virus virulence and pathogenesis. Mammalian associated mutations such as PB2 (I 667, T64), PB1-P13, PB1-F2-S82, NP-K214, NP-Q398 and M1-I15 were detected. The HA gene was under positive selection pressure especially at sites 198 and 235 of RBS, while other internal genes were under negative selection pressure. The study highlights the importance of continuous monitoring of H9N2 virus to enable timely implementation of control measures in poultry populations in Egypt.