Type I interferon signaling protects mice from lethal henipavirus infection

Abstract

Hendra virus (HeV) and Nipah virus (NiV) are closely related, recently emerged paramyxoviruses that form Henipavirus genus and are capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. However, in contrast to many other species and despite expression of functional virus entry receptors, mice are resistant to henipavirus infection. We report here the susceptibility of mice deleted for the type I interferon receptor (IFNAR-KO) to both HeV and NiV. Intraperitoneally infected mice developed fatal encephalitis, with pathology and immunohistochemical features similar to what was found in humans. Viral RNA was found in the majority of analyzed organs, and sublethally infected animals developed virus-specific neutralizing antibodies. Altogether, these results reveal IFNAR-KO mice as a new small animal model to study HeV and NiV pathogenesis, prophylaxis, and treatment and suggest the critical role of type I interferon signaling in the control of henipavirus infection.

Figure 1.

Permissiveness of primary murine brain glial cell cultures to NiV infection. Brain cultures from either wild-type (A, C, E) or IFNAR-KO mice (B, D, F) were mock-infected (A and B), or infected with NiV-EGFP (MOI = 1) and observed 48 h later under light (A–D) and fluorescent (E and F) microscope (×100). Formation of large multinuclear syncytia is indicated by arrows and presented in insert at higher (×300) magnification (C–F). Supernatants (G and H) and cells (I and J) from cultures obtained after NiV infection at MOI = 0.02 (G and I) or MOI = 1 (H and J) were taken at 24 and 48 h postinfection and titrated on Vero cells (G and H), and RNA was analyzed by RT-qPCR for the expression of IFN-I (I and J). Results are presented as average viral titers from triplicate cultures ± SD (G and H; *P < .01, Mann–Whitney U test) or no. of copies/μg of IFNß-specific RNA ± SD (I and J, **P < .01, ***P < .001; 2-way ANOVA test followed with Bonferroni post-test).

Figure 2.

Survival of mice infected by henipavirus. C57BL/6 wt (A) and IFNAR-KO (B–D) mice were infected with either NiV (A, B, D) or HeV (C). Intraperitoneal (ip; A–D) and intranasal (in; B and C) infections were performed with 10⁶ PFU and intracerebral (ic) infections (A, B) with 10⁵ PFU of virus. (D) LD₅₀ was established in 9–10-week-old IFNAR-KO mice using increasing doses of NiV from 10² to 10⁶ PFU injected intraperitoneally. Animals were monitored daily for the development of clinical symptoms over 3 weeks.

Figure 3.

Analysis of henipavirus replication in different murine organs by RT-qPCR. (A) 4-week-old IFNAR-KO mice were inoculated either intranasally (in) or intraperitoneally (ip) with 10⁶ PFU of HeV. (B) IFNAR-KO and C57Bl/6 mice were infected intraperitoneally with indicated doses of NiV. Organ samples were analyzed for the expression of either HeV (A) or NiV N gene (B) by RT-qPCR, either from mice succumbing to the infection (5–10 days postinfection) or mice surviving infection at the end of experiment (21 days postinfection). Results are expressed in no. of copies/μg of RNA extracted from organs collected at necropsy, and horizontal lines correspond to the average of each group. Open symbols represent animals that survived to the infection and were analysed 21 days postinfection, and filled symbols represent those that succumbed to the infection. All RT-qPCR experiments were performed in duplicates. The difference between the 2 routes of HeV infection was established statistically for all tested organs (P < .005, Mann–Whitney U test).

Figure 4.

Pathology of henipavirus infection in IFNAR-KO mice. Animals were inoculated intraperitoneally with either mock preparation (A–C), or 10⁶ PFU of either NiV (D–F, J and K) or HeV (G–I, L). Brain (A, D, G, J, L), lungs (B, E, H, K), or liver (C, F, I) were analyzed after hematoxylin and eosin staining (A–I) or by immunohistochemistry (J–L) 5–7 days postinfection. (D) Severe vasculitis, hemorrhage, and leukocyte infiltration in NiV-infected brain. (G) Perivascular cuffing (arrow head) and eosinophilic neurons (arrow) following HeV infection. (J) Staining of NiV antigens in neurons. (L) Staining of HeV antigens in brain ependymal cells (arrow). Vasculitis, alveolar necrosis and inflammation following either NiV (E) or HeV infection (H). (K) Staining of NiV antigens in pneumocytes. (F) Severe hepatitis lesions, leukocytes infiltration (arrow) and necrotizing plaques (arrow head) in NiV-infected mouse. (I) No significant lesions were observed in the liver of HeV-infected animal.

Figure 5.

HeV infection in neurons of IFNAR-KO mice. Animals were inoculated intraperitoneally with 10⁶ PFU of HeV. Brains were analyzed by immunofluorescence at 6 days postinfection and stained with a rabbit polyclonal anti-NiV N antibody (green) and NeuroTrace, specific for neuronal Nissl substance (red). (A) Axonal labeling of HeV-infected neurons. Magnification ×20. (B) Typical viral inclusions in the cytoplasm of infected neurons. Magnification ×63.

Figure 6.

Production of neutralizing antibodies in mice surviving henipavirus infection. Mice were infected with 10⁶ PFU of either HeV (A) or NiV-specific (B) and virus-specific neutralizing antibodies were assessed in the serum at 21 days postinfection Horizontal lines correspond to the average titer in each group. HeV-infected mice were 4, 11 and 12 weeks-old in indicated groups (4 w, 11 w, and 12 w) and NiV-infected mice were either 4 weeks old (intranasal [in]) or 10 weeks old (intraperitoneal [ip]).