Feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine

Abstract

Nipah virus (NiV) and Hendra virus (HeV) are paramyxoviruses capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. Case reports from outbreaks and previous challenge experiments have suggested that cats were highly susceptible to NiV infection, responding with a severe respiratory disease and systemic infection. Here we have assessed the cat as a model of experimental NiV infection and use it in the evaluation of a subunit vaccine comprised of soluble G glycoprotein (sG). Two groups of two adult cats each were inoculated subcutaneously with either 500 or 5,000 50% tissue culture infective dose(s) (TCID(50)) of NiV. Animals were monitored closely for disease onset, and extensive analysis was conducted on samples and tissues taken during infection and at necropsy to determine viral load and tissue tropism. All animals developed clinical disease 6 to 9 days postinfection, a finding consistent with previous observations. In a subsequent experiment, two cats were immunized with HeV sG and two were immunized with NiV sG. Homologous serum neutralizing titers were greater than 1:20,000, and heterologous titers were greater than 1:20,000 to 16-fold lower. Immunized animals and two additional naive controls were then challenged subcutaneously with 500 TCID(50) of NiV. Naive animals developed clinical disease 6 to 13 days postinfection, whereas none of the immunized animals showed any sign of disease. TaqMan PCR analysis of samples from naive animals revealed considerable levels of NiV genome in a wide range of tissues, whereas the genome was evident in only two immunized cats in only four samples and well below the limit of accurate detection. These results indicate that the cat provides a consistent model for acute NiV infection and associated pathogenesis and an effective subunit vaccine strategy appears achievable.

FIG. 1.

NiV susceptibility study body temperatures and respiration rates. (A) Rectal temperatures obtained at various days postinfection in cats inoculated with either 500 TCID₅₀ of NiV (circles) or 5,000 TCID₅₀ of NiV (squares). All cats became febrile 6 to 8 days postinfection. (B) Respiration rates determined for cats infected with either 500 TCID₅₀ NiV (circles) or 5,000 TCID₅₀ NiV (squares). Immediately prior to euthanasia, all cats exhibited a marked increase in respiration rate.

FIG. 2.

Histopathology and immunohistopathology associated with NiV infection in cats. (A) Necrotizing alveolitis (arrows) in NiV-infected cat and endothelial syncytial cell (arrowhead) (HE). (B) Broncho-alveolitis in NiV-infected cat, with bronchiolar epithelial syncytial cells (arrows) and intraluminal debris (arrowhead) (HE). (C) Positive staining for NiV antigen in regions of alveolitis, including endothelial syncytial cells (arrows) (anti-NiV polyclonal antibody). (D) Positive staining for NiV antigen in bronchiolar epithelial cells (arrows) and intraluminal debris (arrowhead) (anti-NiV polyclonal antibody). Scale bar for all images = 50 μm.

FIG. 3.

NiV genome in cats detected by TaqMan PCR. Normalized, relative NiV genome levels in samples collecting during NiV infection in cats and at necropsy. TaqMan PCR C_T values were determined in triplicate for the NiV genome and normalized by dividing this value by the 18S rRNA C_T values for each sample. The relative NiV genome was determined by linear regression of NiV cDNA standard curves for each assay. Values are expressed as the average of all replicates. The adrenal gland, liver, lung, spleen, and lymph nodes consistently displayed the highest relative NiV genome levels, while the brain and heart frequently revealed the lowest. The genome was detectable in the blood in all cats 1 day prior to euthanasia but only detectable in the urine from cats infected with 5,000 TCID₅₀ of NiV.

FIG. 4.

Temperature recordings of naive and sG-immunized cats infected with NiV. (A) Two cats in the vaccine efficacy study were implanted with radiotelemetry temperature transmitters prior to NiV inoculation (arrow). Cat 10 became febrile (>39°C) 7 days postinfection, while cat 9 remained clinically normal until day 11, becoming febrile on day 12, and was euthanized on day 13. Rectal temperatures were recorded during sampling periods and are indicated for each cat (circles). (B) Three cats showed gradual but slight increases in temperature after NiV infection (arrow), while the fourth showed a gradual decrease over the course of the study. All immunized cats remained within normal physiological parameters up to 24 days p.i.