Genetic and pathogenic potential of highly pathogenic avian influenza H5N8 viruses from live bird markets in Egypt in avian and mammalian models

Abstract

Since its first isolation from migratory birds in Egypt in 2016, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks among domestic poultry in various areas of the country affecting poultry health and production systems. However, the genetic and biological properties of the H5N8 HPAI viruses have not been fully elucidated yet. In this study, we aimed to monitor the evolution of circulating H5N8 viruses and identify the pathogenicity and mammalian adaptation in vitro and in vivo. Three H5N8 HPAI viruses were used in this study and were isolated in 2021-2022 from poultry and wild birds during our routine surveillance. RNA extracts were subjected to full genome sequencing. Genetic, phylogenetic, and antigenic analyses were performed to assess viral characteristics and similarities to previously isolated viruses. Phylogenetic analysis showed that the hemagglutinin genes of the three isolates belonged to clade 2.3.4.4b and grouped with the 2019 viruses from G3 with high similarity to Russian and European lineages. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, several mutations associated with increased virulence and polymerase activity in mammals were observed. Growth kinetics assays showed that the H5N8 isolate is capable of replicating efficiently in mammalian cells lines. In vivo studies were conducted in SPF chickens (White Leghorn), mice, and hamsters to compare the virological characteristics of the 2022 H5N8 isolates with previous H5N8 viruses isolated in 2016 from the first introduction. The H5N8 viruses caused lethal infection in all tested chickens and transmitted by direct contact. However, we showed that the 2016 H5N8 virus causes a higher mortality in chickens compared to 2022 H5N8 virus. Moreover, the 2022 virus can replicate efficiently in hamsters and mice without preadaptation causing systemic infection. These findings underscore the need for continued surveillance of H5 viruses to identify circulating strains, determine the commercial vaccine's effectiveness, and identify zoonotic potential.

Fig 1. Phylogenetic trees of the HA and NA gene segments of the three H5N8 viruses (red dots) compared to previously isolated H5N8 viruses in Egypt and reference strains from different 2.3.4.4. clade.

It was constructed by the neighbor-joining method with 1000 bootstraps using MEGA X software.

Fig 2. Phylogenetic trees of the six internal gene segments of the three H5N8 viruses (red dots) compared to previously isolated H5N8 viruses in Egypt and reference strains.

Different genetic lineages were marked in green for Russian and European-like H5N8 viruses, Red for Eurasian-like H5N8 viruses, and purple for Russian and Asian-like H5N8 viruses.

Fig 3

Growth kinetics of the H5N8/20036 and H5N8/877 viruses in MDCK (A) and A549 cells (B) as titrated by plaque assay. (C) Survival of H5N8 infected and contact chickens after 14 dpi. (D) Viral shedding in oral and cloacal swabs of infected and contact chickens at 1, 3, and 5 dpi.

Fig 4

Photomicrograph of chicken tissue sections from the different experimental groups showing (a) lung sections of the control group displaying normal morphological architecture of the lung with intact lung alveoli and bronchioles. The donor chickens infected with H5N8/877 virus showed remarkable interstitial pneumonia with areas of hemorrhage (H). The contact chickens with H5N8/877 virus showed hemorrhagic pneumonia (H) with areas of collapsed alveoli and others with bronchiectasis (bc) where the alveoli appeared disrupted and irregularly-dilated. The donor chickens infected with H5N8/20036 virus showed inflammatory cellular lymphoid infiltration (I) with disruption and thickening of the bronchial wall (B). The contact chickens with H5N8/20036 showed marked thickening (T) of the inter-alveolar septa and bronchial wall. (b) The cerebral cortex of the control group showed intact neurons, neuropil, and glial cells. The donor and contact groups infected with H5N8/877 or H5N8/20036 virus showed disfigured darkly-degenerated apoptotic neurons (ap). Some capillaries appeared congested (cg) and other blood vessels showed marked dilatation of the peri-vascular space (S) (Virchow Robin’s space). The donor groups infected with H5N8/877 or H5N8/20036 virus showed marked glial cell proliferation (microgliosis) (circles). (c) The intestinal sections of the donor chickens infected with H5N8/877 virus showed inflammatory lymphoid infiltration with shortening of the villi. The contact chickens with H5N8/877 showed invasion of the mucosa and submucosa of the villi with inflammatory cells (I). The donor chickens infected with H5N8/20036 virus showed subepithelial Gruenhagen’s spaces at the tip and the sides of the villi (curved arrow) with marked shedding of the villous epithelium (E). The contact chickens with H5N8/20036 showed severe degeneration necrosis of the intestinal villi (D). (d) The kidney tissue of the control group showed normal glomeruli and tubules. The donor and contact chickens of H5N8/877 and H5N8/20036 virus showed numerous shrunken glomeruli (spiral arrows) with multiple areas of blood extravasation and inflammatory cells. The H5N8/20036 donor and contact chickens showed marked vascular dilatation and congestion (cg) with areas of hemorrhage (H) and tubular degeneration. (e) Grading of the pathological lesions observed in the different study groups. (*: significant versus control, #: significant versus H5N8/877 donor, and @: significant versus H5N8/877 contact at (P<0.05) using ANOVA, Bonferroni post hoc testing).

Fig 5

Pathogenicity and replication of H5N8 viruses in infected BALB/C mice compared to uninfected control, A) change in body weight of infected mice after 14 dpi, B) survival, and C) viral shedding of the two H5N8 viruses in infected mice organs at 3, 5, and 7 dpi.

Fig 6

Pathogenicity and replication of H5N8 viruses in infected and contact hamsters compared to uninfected control, A) change in body weight, and B) survival rates.

Fig 7

Histopathological examination of the lungs of infected mice (I) and hamsters (II). Photomicrographs of lung sections in mice from different experimental groups showed the following: (I.a) control group showing normal morphological architecture of the lung with intact lung alveoli and bronchioles. (I.b) The donor mice infected with H5N8/20036 virus showed peri-bronchial and interstitial inflammatory cellular lymphoid infiltration at days 3 and 5 (I) with vascular congestion (cg). The donor mice infected with H5N8/20036 virus showed hemorrhagic bronchopneumonia at day 7 (H), marked vascular dilatation and congestion (cg), irregularity of the bronchial walls (B), and bridging fibrosis. (I.c) The mice infected with H5N8/877 virus showed moderate interstitial pneumonia with disruption of the inter-alveolar septa and congestion of the bronchial arteries (arrowheads) at days 3, 5, and 7. (I.d) Pathological scoring of lung lesions in the different study groups. (*: Significant versus control group at (P<0.05) using ANOVA, Bonferroni post hoc testing). II) Photomicrograph of hamster lung sections from different experimental groups showed (II.a) control group with normal lung alveoli, alveolar sacs, and bronchioles. (II.b) The donor hamsters infected with H5N8/20036 virus showed inflammatory cellular lymphoid infiltration at days 3 and 7 (I) with irregularity of the bronchial wall (B) and sloughing of the bronchial epithelium. Vascular thrombosis and hyaline degeneration (Hy) were noticed at day 7. (II.c) The H5N8/877 donor hamsters infected with H5N8/877 virus showed severe hemorrhagic bronchopneumonia at days 3 and 7 (H) invading the lung parenchyma with numerous hemosiderin-laden macrophages (M) and vascular congestion. (II. d) Pathological scoring of lung lesions in the different study groups. (*: Significant versus control group at (P<0.05) using ANOVA, Bonferroni post hoc testing).