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. 2022 Jan 12;96(1):e0136621.
doi: 10.1128/JVI.01366-21. Epub 2021 Oct 6.

Opposite Outcomes of the Within-Host Competition between High- and Low-Pathogenic H5N8 Avian Influenza Viruses in Chickens Compared to Ducks

Pierre Bessière #  1 , Thomas Figueroa #  1 , Amelia Coggon  1 , Charlotte Foret-Lucas  1 , Alexandre Houffschmitt  1 , Maxime Fusade-Boyer  1 , Gabriel Dupré  1 , Jean-Luc Guérin  1 , Maxence Delverdier  1 , Romain Volmer  1
Affiliations

Opposite Outcomes of the Within-Host Competition between High- and Low-Pathogenic H5N8 Avian Influenza Viruses in Chickens Compared to Ducks

Pierre Bessière et al. J Virol. .

Abstract

Highly pathogenic avian influenza viruses (HPAIV) emerge from low-pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse genetics-engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. In contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variant interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between high- and low-pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention, and they underscore the importance of within-host viral variant interactions in virus evolution.

Keywords: avian viruses; emergence; evolution; highly pathogenic; influenza; low pathogenic.

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Figures

FIG 1
FIG 1
Characterization of H5N8HP and H5N8LP viruses in cell culture. (A) Sequence alignment of H5N8HP and H5N8LP HA cleavage sites. (B) H5N8HP and H5N8LP growth kinetics in the absence or presence of exogenous trypsin in Madin-Darby canine kidney (MDCK) cells, DF-1 cells, and CCL-141 cells. Cells were infected at an MOI of 10−5 TCID50. HA RNA load was analyzed by RT-qPCR using primers common to H5N8HP and H5N8LP. The dotted line represents the limit of detection for each experiment. Results are representative of an experiment performed three times.
FIG 2
FIG 2
H5N8HP has a stronger advantage over H5N8LP in embryonated chicken eggs than in embryonated duck eggs. (A) EID50 doses used in the in ovo experiments. (B and C) Chicken (B) and duck (C) embryonated eggs were infected in the allantoic cavity either with H5N8HP or H5N8LP alone or with a combination of both. Following a 24-h incubation at 37°C, viral RNA was extracted from the allantoic fluid and HA levels were determined by RT-qPCR using H5N8HP- or H5N8LP-specific primers. Statistical analysis with one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test from two independent experiments performed with a total of 9 chicken embryonated eggs per experimental point; from three independent experiments performed with 19 to 27 duck embryonated eggs per experimental point (detailed in Table S1). Results are expressed as means ± standard errors of the means (SEM). The dotted line represents the limit of detection for each experiment.
FIG 3
FIG 3
H5N8HP and H5N8LP coinfection in chickens results in increased pathogenesis. Chickens were inoculated via the choanal route with H5N8LP alone (L7), H5N8HP alone (H3 and H4), or with a combination of H5N8LP and H5N8HP (L7H3 and L7H4). (A) EID50 doses used in the in vivo chicken experiments. (B) Survival curves. Statistical analysis used log rank (Mantel-Cox) test. (C to E) Viral shedding was analyzed by quantifying HA RNA levels by RT-qPCR using H5N8HP-specific primers from RNA extracted from oropharyngeal swabs (C) and cloacal swabs (D) or by RT-qPCR using H5N8LP-specific primers from RNA extracted from oropharyngeal swabs (E). Numbers of H5N8HP or H5N8HP swab-positive animals are indicated below each time point. Statistical analysis used two-tailed Mann-Whitney test. Results are expressed as means ± SEM. The dotted line represents the limit of detection. (F and G) H5N8HP load was analyzed from total RNA extracted from the lungs (F) and the brain (G) using H5N8HP-specific primers. HA RNA levels were normalized using the 2−ΔCT method. Statistical analysis used one-way ANOVA with Tukey’s multiple-comparison test. Results are expressed as means ± SEM. The dotted line represents the limit of detection. dpi, days postinfection.
FIG 4
FIG 4
Immunohistochemical anti-NP staining of hematoxylin-counterstained chickens lung or brain sections. Analysis was performed on noninfected (NI) and moribund chickens. Scale bar, 50 μm.
FIG 5
FIG 5
Immune markers mRNA expression following chickens infection. (A to D) mRNA expression levels of IFN-β (A), IFN-α (B), Mx (C), and IFN-γ (D) determined by RT-qPCR performed on lung total RNA. (E) mRNA expression levels of Mx determined by RT-qPCR performed on RNA extracted from oropharyngeal swabs. mRNA levels were normalized using the 2−ΔCT method. Statistical analysis used one-way ANOVA with Tukey’s multiple-comparison test. Results are expressed as means ± SEM. #, P < 0.05 compared to noninfected (NI) animals.
FIG 6
FIG 6
H5N8HP and H5N8LP coinfection in chickens results in higher H5N8HP growth. (A) Individual H5N8HP oropharyngeal shedding curves in H4 chickens (blue lines) and L7H4 chickens (red lines). Each curve corresponds to a single animal from the onset of H5N8HP excretion to death. (B) The slopes of individual H5N8HP excretion curves were calculated. (C) H5N8HP shedding duration, i.e., number of days where chickens were H5N8HP oropharyngeal swab positive. Statistical analysis used two-tailed Mann-Whitney test. Results are expressed as means ± SEM. The dotted line corresponds to the limit of detection. dpi, days postinfection.
FIG 7
FIG 7
H5N8HP and H5N8LP coinfection in ducks results in decreased pathogenesis. Ducks were inoculated via the choanal route with H5N8LP alone (L7), H5N8HP alone (H4), or a combination of H5N8LP and H5N8HP (L7H4). (A) EID50 doses used in the in vivo duck experiments. (B) Survival curves. Statistical analysis used log rank (Mantel-Cox) test. (C to E) Viral shedding was analyzed by quantifying HA RNA levels by RT-qPCR using H5N8HP-specific primers from RNA extracted from oropharyngeal swabs (C) and cloacal swabs (D) or by RT-qPCR using H5N8LP-specific primers from RNA extracted from oropharyngeal (E) and cloacal swabs (F). Numbers of H5N8HP or H5N8HP swab-positive animals are indicated below each time point. Statistical analysis used two-tailed Mann-Whitney test. Results are expressed as means ± SEM. The dotted line represents the limit of detection. (G and H) H5N8HP load was analyzed from total RNA extracted from the lungs (G) and the brain (H) using H5N8HP-specific primers. HA RNA levels were normalized using the 2−ΔCT method. Statistical analysis used one-way ANOVA with Tukey’s multiple-comparison test. Results are expressed as means ± SEM. The dotted line represents the limit of detection. dpi, days postinfection.
FIG 8
FIG 8
Immunohistochemical anti-NP staining of hematoxylin-counterstained ducks lung or brain sections. Analysis was performed on ducks necropsied at 1 (D1) and 3 days (D3) postinfection, moribund ducks, and noninfected (NI) ducks. (A) Lung. (B) Brain. Scale bar, 50 μm.
FIG 9
FIG 9
Immune markers mRNA expression following infection in ducks. (A to D) mRNA expression levels of IFN-β (A), IFN-α (B), Mx (C), and IFN-γ (D) determined by RT-qPCR performed on lung total RNA. (E) mRNA expression levels of Mx determined by RT-qPCR performed on RNA extracted from oropharyngeal swabs. mRNA levels were normalized using the 2−ΔCT method. Statistical analysis used one-way ANOVA with Tukey’s multiple-comparison test. Results are expressed as means ± SEM. #, P < 0.05 compared to noninfected (NI) animals.
FIG 10
FIG 10
Ducks mount a more potent antiviral innate immune response compared to chickens. (A) Correlation between Mx mRNA levels and viral RNA levels in the lungs. (B) Mean Mx/HA ratios. Mx mRNA and HA RNA levels were normalized using the 2−ΔCT method. Statistical analysis used two-tailed Mann-Whitney test. Results are expressed as means ± SEM.
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