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. 2017 Nov 14;91(23):e01300-17.
doi: 10.1128/JVI.01300-17. Print 2017 Dec 1.

Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets

Mahmoud M Naguib  1   2 , Reiner Ulrich  1 , Elisa Kasbohm  1 , Christine L P Eng  3 , Donata Hoffmann  1 , Christian Grund  1 , Martin Beer  1 , Timm C Harder  4
Affiliations

Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets

Mahmoud M Naguib et al. J Virol. .

Abstract

The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation.IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported. Here, the principal compatibility of the two subtypes is shown by forcing the reassortment between copassaged H5N1 und H9N2 viruses in embryonated chicken eggs. The resulting reassortant viruses displayed a wide range of pathogenicity including attenuated phenotypes in chickens, but did not show enhanced zoonotic propensities in the ferret model.

Keywords: Egypt; highly pathogenic avian influenza; reassortment; viral fitness; zoonosis.

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Figures

FIG 1
FIG 1
Host tropism and zoonotic prediction between the Egyptian HPAIV H5N1 and LPAIV H9N2 strains. (A) The host tropism signatures of proteins from both parent strains depict the individual host tropism predictions of 11 viral proteins (HA, M1, M2, NA, NP, NS1, NS2, PA, PB1, PB1-F2, and PB2), determined using deduced protein sequences. The intensity of the color represents the confidence in the avian or human tropism prediction by the individual protein prediction models. Both parent strains carry a typical avian tropism signature with 11 avian protein tropisms. (B) The signatures were next used for a second layer of machine learning prediction for classification of avian (A), human (H), or zoonotic (Z) strains, where both strains were predicted to show low zoonotic risks.
FIG 2
FIG 2
Genotype (A) and plaque morphology (B) of five reassortant clones of HPAIV H5N1 and H9N2 strains from Egypt generated by coinfection of embryonated chicken eggs. (A) The eight viral genome segments are indicated by dashes, which indicate, from top to bottom, PB2, PB1, PA, HA, NP, NA, M, and NS segments. Gene segments of HPAIV H5N1 parent strain A/chicken/Egypt/AR236/2015 (AR236) are colored in green, and those of the H9N2 subtype are shown in purple. The genotypes of five reassortant clones (C5, C8, C11, C20, and C46, all of the H5N1 subtype) are shown. (B) The size of 25 randomly selected plaques per clone in infected MDCK cells was measured using ImageJ software. The plaque size of parent strain AR236 was considered 100%. *, statistically significant differences in relation to the parental virus (AR236) (P < 0.001).
FIG 3
FIG 3
Replication kinetics (multistep growth curve) of different reassortant avian influenza viruses (clones C5, C8, C11, C20, and C46) and the HPAIV H5N1 parent strain (A/chicken/Egypt/AR236/2015) in MDCK cells. Subconfluent monolayers of MDCK cells were infected with each of the reassortant clones (C5, C8, C11, C20, and C46) or the parental virus (HPAIV H5N1 A/chicken/Egypt/AR236/2015) at a multiplicity of infection of 0.001. Virus titers in the supernatants were measured in triplicate at 1, 8, 24, 48, and 72 h. Statistically significant differences from the titers of the parental virus (AR236) were observed for C5, C8, and C11 at 8 h after infection and for C46 at 24 h and at 48 h after infection (P < 0.01).
FIG 4
FIG 4
Survival of chickens after intravenous (A) or oculonasal (B) inoculation with reassortant clones C5, C8, C11, C20, and C46 or HPAIV H5N1 parent strain A/chicken/Egypt/AR236/2015. Statistically significant differences compared to the survival of chickens inoculated with the parental virus (AR236) were observed for C5, C20, and C46 for both routes of inoculation (P < 0.01).
FIG 5
FIG 5
Oropharyngeal and cloacal shedding of different reassortant clones (C5, C8, C11, C20, and C46) and of the HPAIV H5N1 parent strain (A/chicken/Egypt/AR236/2015) in 8-week-old White Leghorn specific-pathogen-free chickens at day 2 postinfection. Six groups of 8 week-old SPF chickens were inoculated oculonasally with 105 TCID50 of five reassortant clones (C5, C8, C11, C20, and C46) or with the H5N1 parent strain (AR236). Viral shedding in oropharyngeal and cloacal swab specimens at 2 dpi was determined. The y axis represents the calculated TCID50 milliliter−1 equivalents. The x axis indicates the different reassortant clones. Within each box plot, the center line represents the median, while the top and bottom borders mark the 75th and 25th percentiles, respectively. Whiskers represent the minimum and maximum values. Extreme outliers are indicated by circles.
FIG 6
FIG 6
Viral shedding in nasal wash fluids of ferrets infected with reassortant clone C11 or C46 or with the HPAIV H5N1 parent strain (A/chicken/Egypt/AR236/2015). The y axis represents the calculated TCID50 milliliter−1 equivalents extrapolated from the RT-qPCR Cq values obtained from a titration of viral infectivity of the parental virus (AR236) in MDCK cells. The x axis indicates the results for the different reassortant clones on 5 different days. Each bar represents a single ferret.
FIG 7
FIG 7
(A, B) C11-infected ferret lung at 4 dpi. (A) Mild, acute, catarrhal bronchiolitis with protein-rich fluid, desquamated and degenerating epithelia (arrow), and scant neutrophils within the lumen. (B) Focal spot of influenza A virus nucleoprotein-immunoreactive cellular debris, epithelial cells, and alveolar macrophages interpreted as remnants of a necrotic bronchiolus. (C, D) AR236-infected ferret lung at 4 dpi. (C) Moderate, acute, catarrhal and suppurative (broncho-)pneumonia with intra-alveolar neutrophils (arrow), macrophages (arrowhead), and protein-rich edema. (D) Multifocal accumulation of influenza A virus nucleoprotein-immunoreactive cellular debris, alveolar macrophages, and/or type II pneumocytes. (E, F) C46-infected ferret nasal conchae at 4 dpi. (E) Mild, acute, focal degeneration of epithelial cells (arrow) within the respiratory mucosa. (F) Multiple influenza A virus nucleoprotein-immunoreactive epithelial cells within superficial and deeper layers of the respiratory mucosa. (G, H) C46-infected ferret lung at 4 dpi. (G) Moderate, subacute, coalescing, proliferative pneumonia with hyperplastic type II pneumocytes (arrow) and alveolar histiocytosis. (H) A discrete, large round cell with an influenza A virus nucleoprotein-immunoreactive nucleus and cytoplasm interpreted to be an alveolar macrophage or desquamated type II pneumocyte. (A, C, E, G) Hematoxylin and eosin stain. (B, D, F, G) Influenza A virus nucleoprotein immunohistochemistry by the avidin-biotin-peroxidase complex method with 3-amino-9-ethylcarbazol as the chromogen and hematoxylin counterstain. Bars = 20 μm.
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