Large-particle aerosol exposure to the Bangladesh or Malaysia strain of Nipah virus results in markedly divergent disease presentation in African Green Monkeys

Abstract

Nipah virus (NiV), a highly pathogenic zoonotic paramyxovirus, causes severe respiratory and neurological disease in humans, with a case-fatality rate around 60%. Descriptions of cases in the clinical setting suggest that the two primary lineages of NiV cause disease with different presentations and outcomes. To define strain-specific differences in disease progression and host responses, African green monkeys were exposed to either the Malaysia (NiV-M) or Bangladesh (NiV-B) strain using a large-particle aerosol exposure. NiV-M infection resulted in a fatality rate of 27%, while NiV-B infection led to a 75% fatality rate characterized by rapid respiratory decline and systemic viral dissemination. Among survivors, NiV-M-infected animals mounted robust immunoglobulin M, immunoglobulin G, and neutralizing antibody responses, whereas NiV-B survivors exhibited weaker and delayed humoral responses. Non-survivors of both strains showed elevated pro-inflammatory cytokines, thrombocytopenia, and multi-organ dysfunction. Imaging showed that NiV-M infection was associated with neuroinflammation and systemic vasculitis, while NiV-B infection caused progressive pulmonary pathology. Histopathological analysis confirmed widespread vasculitis and encephalitis in animals with NiV-M infection and diffuse pulmonary hemorrhage and fibrin thrombi, consistent with vascular injury and coagulopathy, in animals with NiV-B infection. Cytokine profiling and flow cytometry showed a more intense and dysregulated immune response to NiV-B infection. Fatal outcomes in both groups were associated with thrombocytopenia, elevated pro-inflammatory cytokines, and multi-organ dysfunction. This study highlights fundamental differences in virulence, immune evasion, and pathogenesis between NiV strains and underscores the value of the African green monkey aerosol model for evaluating medical countermeasures under conditions that closely mimic natural human exposure.

Fig 1. Study design for comparative pathogenesis of NiV-M and NiV-B in AGMs.

Adult AGMs were exposed to either NiV-M or NiV-B via large-particle aerosol (≈500 PFU) to assess strain-specific disease progression. Two groups of 12 animals (two cohorts NiV-M; and two cohorts for NiV-B) were used; Two later cohorts repeated the initial exposures to both strains to increase statistical power. Clinical monitoring and scheduled sampling (blood, swabs, CSF) were conducted at baseline and multiple time points post-exposure through the end of the study. Whole-body CT and brain MRI were performed longitudinally, with early-phase MRI omitted in later cohorts due to anesthesia-related complications. Imaging and clinical data were aligned by study week after the acute phase for consistency across cohorts. BAL, bronchoalveolar lavage; CSF, cerebral spinal fluid; CT, computed tomography; AGMs, African green monkeys; MRI, magnetic resonance imaging; NiV, Nipah virus; NiV-B, Nipah virus Bangladesh strain; NiV-M, Nipah virus Malaysia strain.

Fig 2. Comparative summary of clinical, virological, and imaging findings in cynomolgus and rhesus macaques following NiV-M or NiV-B exposure.

Summary data for all animals included in this study including dose given, particle size at exposure, clinical scores and day post exposure at euthanasia, comparative change in platelets and various clinical assessments.

Fig 3. Clinical progression in AGMs following NiV-M or NiV-B aerosol exposure.

(A) Kaplan-Meier curves show survival of AGMs exposed to NiV-M (n = 12) or NiV-B (n = 12) via large-particle aerosol. NiV-B led to a shorter disease course (Median survival time = 12 d) compared to NiV-M (Median survival time = 17 d). (B) Animals were grouped as uninfected survivors (circles), non-survivors (triangles), or seroconverted survivors (squares) based on virological and clinical outcomes. Clinical scores indicate rapid disease progression after onset. Neurologic signs were seen only in animals with NiV-M infection. (C) Percent weight changes relative to baseline. (D) Temperature profiles. Animals with NiV-M infection showed transient fevers (10–14 d) and hypothermia at euthanasia. NiV-B animals showed no fever but had a sharper terminal temperature drop. (E) Respiratory rates increased significantly in non-survivors exposed to either strain, consistent with pulmonary pathology. Rates remained unchanged in survivors and uninfected animals. Data (except survival) are presented as group means. (F) Longitudinal clinical scores and outcome timeline of cynomolgus and rhesus macaques following NiV-M or NiV-B infection.

Fig 4. Hematologic, biochemical, and coagulation changes in AGMs following NiV-M and NiV-B exposure.

Animals were grouped as uninfected survivors (circles), non-survivors (triangles), or seroconverted survivors (squares) based on virological and clinical outcomes. (A) Time course of hematologic parameters (white blood cells, red blood cells, hemoglobin, hematocrit, and platelets) in NiV-M, NiV-B, and uninfected animals. Non-survivors exposed to NiV-M and NiV-B showed significant thrombocytopenia at terminal time points compared to their own baselines or uninfected survivors. Uninfected animals remained stable. (B) Longitudinal serum chemistry profiles, including glucose, BUN, creatinine, ALP, amylase, and albumin. NiV-B-exposed animals with fatal outcomes exhibited marked elevations in BUN, creatinine, and liver enzymes consistent with multi-organ dysfunction. Survivors and uninfected animals showed minimal changes. (C) Coagulation parameters: Partial Thromboplastin Time (PTT), thrombin time (TT), fibrinogen, and VWF antigen. Animals with NiV-B infection exhibited prolonged PTT/TT and elevated VWF antigen levels compared to those with NiV-M infection and uninfected groups, indicating greater endothelial dysfunction and coagulopathy. Data are presented as group means.

Fig 5. Viral RNA load and tissue dissemination in AGMs following NiV-M and NiV-B exposure.

Animals were grouped as uninfected survivors (circles), non-survivors (triangles), or seroconverted survivors (squares) based on virological and clinical outcomes. Quantification of viral RNA (vRNA) in whole blood (A), serum (B), nasal swabs (C), and oral swabs (D) over time. Survivors had undetectable vRNA in all sample types. (E) Detection of vRNA by RT-qPCR and (F) viable virus in tissues by plaque assay. NiV viral RNA and live virus were detected only in non-survivors, with widespread distribution across multiple organs. All survivors were negative by both assays, consistent with complete viral clearance at necropsy. Data are presented as group means with values of each animal.

Fig 6. Host immune responses to NiV-M and NiV-B infection in AGMs.

Animals were grouped as uninfected survivors (circles), non-survivors (triangles), or seroconverted survivors (squares) based on virological and clinical outcomes. (A) Serological responses to NiV-M and NiV-B exposure. Animals with NiV-M infection developed IgM, IgG, and neutralizing antibodies; survivors maintained robust titers through study end. Two survivors lacked detectable antibodies, indicating no infection. In NiV-M non-survivors, IgM responses were present, but IgG and neutralizing responses were delayed or insufficient. In contrast, no antibodies were detected in NiV-B-exposed animals, indicating disease progression outpaced humoral immunity. Seroconversion rates were significantly higher in NiV-M compared to NiV-B. (B) Longitudinal immune cell dynamics measured by flow cytometry. NiV-M non-survivors showed early lymphocyte depletion (CD4 + , CD8 + , B, and NK cells) with terminal-phase rebound; survivors showed similar trends, while uninfected animals remained stable. In NiV-B non-survivors, CD4 + T cells increased at terminal time points, with suppressed CD8 + T cell and B-cell populations earlier in the disease. No significant changes were observed in uninfected animals. Due to the small sample size and inter-animal variation, statistical comparisons were not performed. (C) Plasma cytokine profiles. Non-survivors exposed to either NiV strain showed sharp increases in IFN-γ, IL-15, and IL-1RA. NiV-B non-survivors also had elevated IL-6, IL-10, G-CSF, MCP-1 and MIP-1β. Survivors of NiV-M infection showed transient cytokine elevations returning to baseline by 10–14 d and sustained increases in G-CSF. Data are presented as group means.

Fig 7. Quantitative CT analysis of lung pathology in AGMs following NiV exposure.

Percent change in lung hyperdensity (PCLH) was quantified over time using serial chest CT imaging to assess pulmonary disease progression in AGMs exposed to NiV-M or NiV-B. Animals were grouped as uninfected survivors (circles), non-survivors (triangles), or seroconverted survivors (squares) based on virological and clinical outcomes. NiV-B non-survivors (n = 7) showed a significant increase in PCLH, with peak values reaching over 350%, compared to NiV-M non-survivors (n = 3), which showed peak PCLH around 200%. In contrast, both uninfected animals (NiV-M: n = 2; NiV-B: n = 5) and seroconverted survivors (NiV-M: n = 6) showed minimal changes in PCLH throughout the study period. A significant difference in peak PCLH was observed between NiV-B non-survivors and all other groups. These findings indicate that severe lung hyperdensity changes detected by CT imaging are associated with fatal disease progression in NiV-B–exposed animals. Data are presented as group means.

Fig 8. Histopathologic lesions and NiV antigen distribution in multiple tissues of AGMs with NiV infection.

(A: a–d) Lymphoid tissues (animal 09230): (a, b) Spleen: white pulp germinal center depletion, hemorrhage, and syncytial cell formation (hematoxylin and eosin stain (HE), 200X [a] and 400X [b]). (c) Tracheobronchial lymph node: follicular lymphoid depletion with necrosis and hemorrhage (HE, 200X). (d) NiV antigen in affected follicles (IHC, 400X). (B: e–h) Lung tissues (animals 08112 and 9501): (e) Necrotizing interstitial pneumonia with hemorrhage and edema (HE, 100X). (f) NiV antigen within pneumocytes and syncytial cells (IHC, 400X). (g) Interstitial pneumonia with fibrin and hyaline membrane formation (HE, 200X). (h) Hyaline membranes highlighted using Carstair’s fibrin stain (400X). (C: i–n) Brain tissues (animals 08078, 08233 and 8671): (i, j) Putamen: encephalomalacia and vessel occlusion by fibrin thrombus (HE, 100X and 400X). (k) Neuronophagia and neuropil vacuolation. (l) Brainstem from 8671 showing moderate necrotizing, lymphohistiocytic multifocal encephalitis with hemorrhage. (m) Cerebral cortex: Mild-to-moderate non-suppurative encephalitis and meningitis (HE, 100X). Lesions were multifocal and were the most severe in and surrounding the olfactory tubercle. (n) NiV antigen in neurons and glial cells (IHC, 200X). (D: o–p) Urinary bladder (animal 09501): (o) Submucosal hemorrhage, edema, and lymphocytic inflammation (HE, 40X). (p) Vessel occlusion with fibrin thrombus (HE, 100X).