Comparative Virological and Pathogenic Characteristics of Avian Influenza H5N8 Viruses Detected in Wild Birds and Domestic Poultry in Egypt during the Winter of 2016/2017

Abstract

The surveillance and virological characterization of H5N8 avian influenza viruses are important in order to assess their zoonotic potential. The genetic analyses of the Egyptian H5N8 viruses isolated through active surveillance in wild birds and domestic poultry in the winter of 2016/2017 showed multiple introductions of reassortant viruses. In this study, we investigated and compared the growth kinetics, infectivity, and pathogenicity of the three reassortant forms of H5N8 viruses detected in wild birds and domestic poultry in Egypt during the first introduction wave in the winter of 2016/2017. Three representative H5N8 viruses (abbreviated as 813, 871, and 13666) were selected. The 871/H5N8 virus showed enhanced growth properties in vitro in Madin Darby canine kidney (MDCK) and A549 cells. Interestingly, all viruses replicated well in mice without prior adaptation. Infected C57BL/6 mice showed 20% mortality for 813/H5N8 and 60% mortality for 871/H5N8 and 13666/H5N8, which could be attributed to the genetic differences among the viruses. Studies on the pathogenicity in experimentally infected ducks revealed a range of pathogenic effects, with mortality rate ranging from 0% for 813/H5N8 and 13666/H5N8 to 28% for 871/H5N8. No significant differences were observed among the three compared viruses in infected chickens. Overall, different H5N8 viruses had variable biological characteristics, indicating a continuous need for surveillance and virus characterization efforts.

Keywords: Egypt; H5N8; avian influenza virus; pathogenicity.

Figure 1

Phylogenetic trees of the nucleotide sequences of the PB2, PB1, PA, NP, HA, NA, M, and NS genes of the characterized H5N8 viruses in Egypt from domestic poultry and wild birds.

Figure 1

Phylogenetic trees of the nucleotide sequences of the PB2, PB1, PA, NP, HA, NA, M, and NS genes of the characterized H5N8 viruses in Egypt from domestic poultry and wild birds.

Figure 2

The gnome constellation of the three H5N8 forms detected in poultry and wild birds during our active surveillance study in Egypt. Three forms of H5N8 viruses are represented by triangles containing horizontal bars that represent eight gene segments and the most identical strains.

Figure 3

Growth kinetics of the three forms of H5N8 viruses in A549 (A), and Madin Darby canine kidney (MDCK) (B) cells. The cells were infected with the virus at a MOI of 0.001. At the time points indicated, the supernatant was taken and titrated by 50% tissue culture infectious dose assay (TCID₅₀/_mL) assay on MDCK cells. (C) Plaque phenotypes of the three forms of H5N8 viruses on MDCK cells were shown. (D) Average plaque sizes were determined from 20 plaques for each virus. Meanwhile, the growth kinetics of the three forms of H5N8 viruses were assessed in embryonated eggs using 10³ EID₅₀ at 12 hpi. The viral titers in collected allantoic fluids were titrated for their HA assay (E) and EID₅₀/_mL (F).

Figure 4

Replication of the three forms of H5N8 viruses in infected mice. C57BL/6 mice were infected with 10⁶ EID₅₀ of 871, 813, and 13666 H5N8 viruses. Survival (A), change in body weight (B), and titers of virus in lungs, intestines and livers at 3 and 5 dpi (C) were determined.

Figure 5

Viral replication and pathogenicity of the three forms of Egyptian H5N8 viruses in ducks and chickens.

Figure 6

Histopathological changes in lungs of infected mice (a–d), ducks (f–i) and chickens (k–n), with different forms of H5N8 viruses compared with the control animals. Histological scoring of pulmonary lesions of three H5N8 viruses (e,j,o). (*: statistically significant relative to the control group; #: statistically significant relative to virus 813; scale bar 200 µm).(b) Infected mice with 871 virus show degeneration of bronchioles lining (arrowheads), inflammatory cells (I) and vascular wall degeneration (black arrows). (c) 813 infected mice bronchioles (Br) show disruption of their walls (arrow), and inflammatory cells in the lumen (arrowheads). Some alveoli are collapsed (C), while others appear dilated (D). (d) Virus 13666 shows massive inflammatory infiltration around bronchioles (Br) and blood vessels (bv), degeneration of the blood vessel wall (arrow) and bronchial epithelial cell desquamation (arrowhead) and severely collapsed alveoli (c). (g) 871-infected ducks show inflammatory lymphoid infiltration (arrows) and rupture of arteriolar walls (arrowheads). (h) 813 shows severe vascular congestion (Co), hemorrhages (hg) and blood extravasation from ruptured vascular wall (arrowhead), dense lymphocytic infiltration (I) and peri-arteriolar metaplasia (arrows). (i) Virus 13666 shows thickening of interalveolar septa (arrows), hemorrhage (hg) and irregularly-dilated alveolar ducts (AD). In chickens, 871, 813 and 13666 viruses show inflammatory lymphoid infiltration (asterisk) with degeneration in the vessel wall (arrow).

Figure 7

Histopathological changes in liver of infected mice (a–d), ducks (f–i) and chickens (k–n) with different forms of H5N8 viruses compared with the control animals. Histological scoring of intestinal lesions (e,j,o). (*: statistically significant relative to the control group). (b) 871-infected mice show hepatocellular necrosis (arrows) and congestion of the central vein (CV). (c) 813 shows hemorrhagic infarctions (arrows) with edema in the space of Disse (arrowheads). (d) 13666 shows mononuclear cellular infiltration (arrows). In ducks, Virus 871 shows (g) hepatocellular degeneration (D), hemorrhagic necrosis (arrows) and marked dilatation of the central vein (CV). (h) 813 shows vacuolar degeneration of hapatocytes (V), and (i) 13666 shows mononuclear cellular infiltration (arrows). In chickens, (l) Virus 871 shows thrombus formation (T), while (m) 813 shows bile duct hyperplasia (arrows) with thrombus formation (T), and (n) 13666 shows thrombus formation (T) and focal lymphoid cell reaction (arrows). (Scale bar 200 µm).

Figure 8

Histopathological changes in intestine of infected mice (a–d), ducks (f–i) and chickens (k–n) with different forms of H5N8 viruses compared with the control animals. (e,j,o) Histological scoring of intestinal lesions (*: statistically significant relative to the control group, scale bar 200 µm). In mice, (b) Virus 871 shows Lymphocytic infiltration (arrows). (d) Virus 13666 shows subepithelial Gruenhagen’s spaces at the tip of the villi (arrowhead), epithelial lifting down the side of the villus (arrows) and lymphocytic infiltration (circle). In ducks, (g) Virus 871 shows shedding of the villous epithelium (E) and loss of intestinal brush borer (arrowheads). (h) Virus 813 and (i) 13666 shows marked increase in goblet cells (arrows). In chickens, Virus 871 shows necrotic tissue in the intestinal lumen (astrisk), while 813 shows sloughing of the intestinal epithelial lining (arrow).