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. 2023 Feb 14;11(1):e0422922.
doi: 10.1128/spectrum.04229-22. Epub 2023 Jan 10.

In Vitro and In Vivo Characterization of H5N8 High-Pathogenicity Avian Influenza Virus Neurotropism in Ducks and Chickens

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

In Vitro and In Vivo Characterization of H5N8 High-Pathogenicity Avian Influenza Virus Neurotropism in Ducks and Chickens

Charlotte Foret-Lucas et al. Microbiol Spectr. .

Abstract

H5N8 high-pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4B, which circulated during the 2016 epizootics in Europe, was notable for causing different clinical signs in ducks and chickens. The clinical signs preceding death were predominantly neurological in ducks versus respiratory in chickens. To investigate the determinants for the predominant neurological signs observed in ducks, we infected duck and chicken primary cortical neurons. Viral replication was identical in neuronal cultures from both species. In addition, we did not detect any major difference in the immune and inflammatory responses. These results suggest that the predominant neurological involvement of H5N8 HPAIV infection in ducks could not be recapitulated in primary neuronal cultures. In vivo, H5N8 HPAIV replication in ducks peaked soon after infection and led to an early colonization of the central nervous system. In contrast, viral replication was delayed in chickens but ultimately burst in the lungs of chickens, and the chickens died of respiratory distress before brain damage became significant. Consequently, the immune and inflammatory responses in the brain were significantly higher in duck brains than those in chickens. Our study thus suggests that early colonization of the central nervous system associated with prolonged survival after the onset of virus replication is the likely primary cause of the sustained inflammatory response and subsequent neurological disorders observed in H5N8 HPAIV-infected ducks. IMPORTANCE The severity of high-pathogenicity avian influenza virus (HPAIV) infection has been linked to its ability to replicate systemically and cause lesions in a variety of tissues. However, the symptomatology depends on the host species. The H5N8 virus of clade 2.3.4.4B had a pronounced neurotropism in ducks, leading to severe neurological disorders. In contrast, neurological signs were rarely observed in chickens, which suffered mostly from respiratory distress. Here, we investigated the determinants of H5N8 HPAIV neurotropism. We provide evidence that the difference in clinical signs was not due to a difference in neurotropism. Our results rather indicate that chickens died of respiratory distress due to intense viral replication in the lungs before viral replication in the brain could produce significant lesions. In contrast, ducks better controlled virus replication in the lungs, thus allowing the virus to replicate for a sufficient duration in the brain, to reach high levels, and to cause significant lesions.

Keywords: chicken; duck; highly pathogenic avian influenza; influenza; neuron; neurotropism; primary.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
H5N8 HPAIV infections in primary neurons. (A and B) Assessment of chicken and duck cortical neuron morphology and purity. (A) Immunofluorescence staining of neurons after 4 days of culture with βIII-tubulin (red) and DAPI (blue). Scale bar, 50 μm. (B) Percentage of neurons, calculated as the ratio of the number of βIII-tubulin-stained neurons to the total number of DAPI-stained nuclei, from 10 randomly selected microscope fields for each species. (C) Immunofluorescence staining of chicken and duck neurons infected at an MOI of 2 and fixed 8 h postinfection. NS1, green; βIII-tubulin, red; DAPI, blue. Scale bar, 25 μm. (D) Viral replication kinetics. Neurons from both species were infected at an MOI of 10−4 TCID50. HA RNA load was analyzed by RT-qPCR. Data are displayed as mean ± standard error of the mean (SEM) from three independent experiments. Statistical analysis was performed used two-tailed Mann-Whitney test. The dotted line represents the limit of detection. (E) Immune and inflammatory marker mRNA expression following chicken and duck neuron infection. Neurons from both species were infected at an MOI of 2. mRNA expression levels of IFN-β, Mx, OAS, CCL5, and IL-8 were determined by RT-qPCR performed on chicken and duck neuron total RNA. mRNA levels were normalized using the 2ΔΔ−CT method. Data are displayed as mean ± standard error of the mean (SEM) from three independent experiments. Statistical analysis used the two-tailed Mann-Whitney test. The dotted line represents mRNA expression levels of noninfected (NI) neurons. Results are expressed as means ±SEM. *, P < 0.05; **, P < 0.01; #, P < 0.05 compared to noninfected neurons.
FIG 2
FIG 2
Mortality and viral replication following chicken and duck H5N8 HPAIV infection. (A) Survival curves of H5N8-infected chickens and ducks. (B) Viral shedding was analyzed by quantifying HA RNA levels by RT-qPCR from RNA extracted from oropharyngeal swabs. Statistical analysis was performed with the two-tailed Mann-Whitney test. Results are expressed as means ± SEM. The dotted line represents the limit of detection. (C) Shedding duration, i.e., number of days for which oropharyngeal swabs were positive for HA RNA by RT-qPCR. Statistical analysis was performed with the two-tailed Mann-Whitney test. Results are expressed as means ± SEM. (D and E) Viral loads were analyzed from total RNA extracted from lungs (D) and brain (E). HA RNA levels were normalized using the 2DD-CT method. Ch, chicken; Du: duck; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Statistical analysis was performed with the two-tailed Mann-Whitney test. Results are expressed as means ± SEM. The dotted line represents the limit of detection. dpi, days postinfection.
FIG 3
FIG 3
Histopathological analysis of brain samples following H5N8 HPAIV infection. (A) Immunohistochemical anti-NP staining of hematoxylin-counterstained chicken and duck brain sections from moribund animals. Scale bars, 100 μm. (B) Hematoxylin and eosin staining on brain sections from moribund animals. Lymphocytic meningoencephalitis is observed only in the brains of H5N8 HPAIV-infected ducks. The star symbol indicates sites of lymphocytic infiltration of perivascular meningeal spaces, and the triangle indicates neuronal necrosis and the losange focal gliosis. Scale bars, 50 μm.
FIG 4
FIG 4
Immune and inflammatory response markers in chicken and duck lungs and brains. mRNA expression levels of IFN-β, IFN-α, OAS, Mx, CCL5, and IL-8 in brain (A) and lung (B) samples, determined by RT-qPCR performed on total RNA. mRNA levels were normalized using the 2DD-CT method and expressed as fold changes over noninfected animals. Statistical analysis was performed using the two-tailed Mann-Whitney test. Results are expressed as means ± SEM. Ch, chicken; Du, duck; D1, day 1; D3, day 3; Mor, moribund; NI, noninfected; #, P < 0.05 compared with noninfected animals. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
Mean Mx/HA and OAS/HA ratios in the lungs and brain. Mx and OAS mRNA and HA RNA levels from lung (A) and brain (B) samples were normalized using the 2DD-CT method. Statistical analysis used the two-tailed Mann-Whitney test. Results are expressed as means ± SEM. *, P < 0.05; **, P < 0.01.
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