Host gene expression profiles in ferrets infected with genetically distinct henipavirus strains - PubMed (original) (raw)

Host gene expression profiles in ferrets infected with genetically distinct henipavirus strains

Alberto J Leon et al. PLoS Negl Trop Dis. 2018.

Abstract

Henipavirus infection causes severe respiratory and neurological disease in humans that can be fatal. To characterize the pathogenic mechanisms of henipavirus infection in vivo, we performed experimental infections in ferrets followed by genome-wide gene expression analysis of lung and brain tissues. The Hendra, Nipah-Bangladesh, and Nipah-Malaysia strains caused severe respiratory and neurological disease with animals succumbing around 7 days post infection. Despite the presence of abundant viral shedding, animal-to-animal transmission did not occur. The host gene expression profiles of the lung tissue showed early activation of interferon responses and subsequent expression of inflammation-related genes that coincided with the clinical deterioration. Additionally, the lung tissue showed unchanged levels of lymphocyte markers and progressive downregulation of cell cycle genes and extracellular matrix components. Infection in the brain resulted in a limited breadth of the host responses, which is in accordance with the immunoprivileged status of this organ. Finally, we propose a model of the pathogenic mechanisms of henipavirus infection that integrates multiple components of the host responses.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Differences in minimal infectious dose of HeV, NiV-M and NiV-B in ferrets.

A first study using different infective doses of the three viruses was performed to establish the LD50 of each virus in ferrets: A-C) Survival rates are shown for 6-week old ferrets (n = 5 per group) that were infected with different doses (101, 102, 103 and 105 TCID50) of NiV-M, NiV-B and HeV, respectively. D-F) A comparative study using a dose of 5,000 TCID50 for each of the three viruses was performed in ferrets: D) cumulative clinical scores, calculated as described in the methods section; E) % lung weight per body weight; F) average number of histological lesions in the lung tissue per field of view. Tissues were harvested from 6-week old ferrets infected with NiV-B (grey), NiV-M (black solid) and HeV (black dashed) on various days post infection as described in Materials and Methods. Samples from 5 animals per group were analyzed at each time point. * p<0.01, two-way ANOVA, Bonferroni’s multiple comparison test. Error bars represent standard deviations.

Fig 2

Fig 2. Viral spread across body compartments in henipavirus infected ferrets.

A comparative study using a dose of 5,000 TCID50 for each of the three viruses was performed in ferrets: viral growth in the ferret tissue, as measured by quantitative RT-PCR in the trachea (A), lung (B), olfactory bulb (C), frontal brain (D), liver (E), kidney (F), spleen (G), and blood(H); tissues were harvested from 6-week old ferrets infected with NiV-B (grey), NiV-M (black solid) and HeV (black dashed) on various days post infection as described in Materials and Methods. Samples from 5 animals per group were analyzed at each time point. * p<0.01, two-way ANOVA, Bonferroni’s multiple comparison test. Error bars represent standard deviations.

Fig 3

Fig 3. Gene expression profiles in the lung tissue of ferrets infected with HeV and NiV-B.

Ferrets were infected with 5,000 TCID50 of HeV and NiV-B and euthanized at 3 and 5 d.p.i. and together with an uninfected control group (n = 3 per group). Transcriptomic analysis of the lung tissue was performed by RNA-seq. A) Number of significantly up- and down-regulated genes with respect to the control group (fold-variation>±1.5 and FDR<0.05) at 3 and 5 dpi. B) Functional gene classification of differentially expressed genes. For each time-point, up- and down-regulated genes were subjected to functional classification and a selected group of gene categories from 1Gene Ontology and 2KEGG Pathways that are related with the immune responses are shown. For each functional category, the number of up- and down-regulated genes and the level of significance of the enrichment (Fisher’s exact test p-value) are indicated. C) Heatmap showing the level of activation of genes belonging to the Response to Type I Interferon category, which encompasses antiviral response genes, during the course of HeV and NiV-B infection. D) Functional enrichment of up-regulated genes that belong to protein-protein interaction (PPI) hubs of transcription factors. E-F) Evolution in the mRNA expression levels of selected macrophage markers and lymphocyte markers, respectively, during HeV and NiV-B infection. These sets of markers were selected from the literature according to their association with these two cell populations, and their coordinated upregulation reflects an increase of the associated cell population in the tissue. Dark grey shaded areas indicate no variation with respect to the control group (<±1.5 fold-change).

Fig 4

Fig 4. Decreased expression of genes related with cell cycle and growth signaling in the lung tissue during infection with HeV and NiV-B.

A) Evolution of gene enrichment score (Fisher’s exact test p-value) of the Cell Cycle KEGG Pathway throughout the infection. B) Functional gene classification of differentially expressed genes in the lung tissue. Regulated genes were subjected to functional classification and selected 1Gene Ontology categories and 2KEGG Pathways that are related to the immune responses and cell growth signaling are shown. For each functional category, the number of up- and down-regulated genes and the level of significance of the enrichment (Fisher’s exact test p-value) are indicated. C) Functional enrichment of down-regulated genes that belong to protein-protein interaction (PPI) hubs of transcription factors.

Fig 5

Fig 5. Gene expression profiles in the brain of ferrets infected with HeV and NiV-B.

Ferrets were infected with 5,000 TCID50 of HeV and NiV-B and euthanized at 3 and 5 d.p.i. (n = 3 per group, except for HeV_D5 with n = 2) and together with an uninfected control group (n = 2). Transcriptomic analysis of frontal lobe tissue was performed by RNA-seq. A) Number of significantly up- and down-regulated genes with respect to the control group (fold-variation>±1.5 and FDR<0.05) at 3 and 5 d.p.i. B) Evolution of gene enrichment score (Fisher’s exact test p-value) of the Innate Immune Response category (Gene Ontology) throughout the infection. C) Functional gene classification of differentially expressed genes in the brain tissue. Regulated genes were subjected to functional classification and a selected 1Gene Ontology categories and 2KEGG Pathways that are related to the immune responses and cell growth signaling are shown. For each functional category, the number of up- and down-regulated genes and the level of significance of the enrichment (Fisher’s exact test p-value) are indicated. D-E) Evolution in the mRNA expression levels of selected macrophage markers and lymphocyte markers, respectively, during HeV and NiV-B infection in the brain tissue. Dark grey shaded areas indicate no variation with respect to the control group (<±1.5 fold-change).

Fig 6

Fig 6. Sequential activation of host responses during Henipavirus infection.

A) Upon infection of the upper and lower respiratory tract, the virus quickly spreads to a myriad of organs, including the central nervous system, liver, spleen, heart, kidney, bladder and blood (schematic representation of the data from HeV infection). B) In the lung tissue, the virus presents high growth rates and only after the inflammatory responses become fully active, at around day 4, the levels of virus stabilize but without significantly decreasing. Signaling and effector molecules of the innate immunity become expressed, innate immune cells migrate to the lung tissue, but, interestingly, gene expression data strongly suggest that lymphocytes are not migrating and expanding in the affected tissues. Additionally, the expression of genes related to the cell cycle, growth factors and growth factor signaling decline through the infectious process, possibly related to the degradation of the physiological functions of the lung tissue. C) In the brain tissue, the virus grows at a slower rate as compared to the lung tissue. The brain mounts a restricted immune response in accordance with the immunoprivileged status of this tissue that includes infiltration and/or local expansion of macrophages, and possibly of lymphocytes, but without leading to a noticeable alteration of the viral growth kinetics.

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