Establishment and application of a surrogate model for human Ebola virus disease in BSL-2 laboratory

Abstract

The Ebola virus (EBOV) is a member of the Orthoebolavirus genus, Filoviridae family, which causes severe hemorrhagic diseases in humans and non-human primates (NHPs), with a case fatality rate of up to 90%. The development of countermeasures against EBOV has been hindered by the lack of ideal animal models, as EBOV requires handling in biosafety level (BSL)-4 facilities. Therefore, accessible and convenient animal models are urgently needed to promote prophylactic and therapeutic approaches against EBOV. In this study, a recombinant vesicular stomatitis virus expressing Ebola virus glycoprotein (VSV-EBOV/GP) was constructed and applied as a surrogate virus, establishing a lethal infection in hamsters. Following infection with VSV-EBOV/GP, 3-week-old female Syrian hamsters exhibited disease signs such as weight loss, multi-organ failure, severe uveitis, high viral loads, and developed severe systemic diseases similar to those observed in human EBOV patients. All animals succumbed at 2-3 days post-infection (dpi). Histopathological changes indicated that VSV-EBOV/GP targeted liver cells, suggesting that the tissue tropism of VSV-EBOV/GP was comparable to wild-type EBOV (WT EBOV). Notably, the pathogenicity of the VSV-EBOV/GP was found to be species-specific, age-related, gender-associated, and challenge route-dependent. Subsequently, equine anti-EBOV immunoglobulins and a subunit vaccine were validated using this model. Overall, this surrogate model represents a safe, effective, and economical tool for rapid preclinical evaluation of medical countermeasures against EBOV under BSL-2 conditions, which would accelerate technological advances and breakthroughs in confronting Ebola virus disease.

Keywords: Ebola virus (EBOV); Pathogenicity; Recombinant vesicular stomatitis virus; Surrogate models; Syrian hamster; Vaccine evaluation and drug screening.

Fig. 1

A Scheme diagram of the generation of recombinant vesicular stomatitis virus VSV-EBOV/GP. The recombinant full-length plasmid (p3.1-VSVΔG-EBOV GP) and four helper plasmids (pcDNA3.1-VSV-N, pcDNA3.1-VSV-P, pcDNA3.1-VSV-L, and pcDNA3.1-VSV-G) were co-transfected into BSR/T7 cells and obtained a recombinant virus bearing the glycoprotein of EBOV. B One-step growth curves of WT VSV, VSV-EBOV/GP, VSV-SUDV/GP, and VSV-LASV/GP. WT VSV, VSV-EBOV/GP, VSV-SUDV/GP, and VSV-LASV/GP were inoculated into Vero E6 cells at an MOI of 0.1. Viruses were collected at 12 ​h intervals to measure titers.

Fig. 2

Weight change and percentage of survival in rodents of VSV-EBOV/GP infection. Sprague-Dawley (SD) rats (A), Hartley guinea pigs (B), BALB/c mice (C), Syrian hamsters (D) were inoculated with VSV-EBOV/GP (10⁷ TCID₅₀) via intraperitoneal (i.p.) route. Weight change and percentage of survival were monitored. E Weight change and percentage of survival were monitored in Syrian hamsters inoculated with WT-VSV, VSV-SUDV/GP, VSV-LASV/GP, and VSV-EBOV/GP via i.p. route.

Fig. 3

Characterization of VSV-EBOV/GP infection in 3-week-old Syrian hamsters. A Three-week-old Syrian hamsters were intraperitoneally infected with VSV-EBOV/GP (10⁷ TCID₅₀). B Animals were monitored for weight changes, temperature changes, and survival. C, D Blood biochemistry and blood cell count were analyzed at 1.5 days post-infection. E Viral loads including hearts, livers, spleens, lungs, kidneys, stomachs, intestines, and brains were determined at 1.5 days post-infection. Data presented as mean ​± ​SEM. Statistical analyses were performed using One-way ANOVA. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001. F Severe uveitis was observed in the eyes of VSV-EBOV/GP infected animals. G, H Histopathological and immunohistochemistry assays of the liver, spleen, lung and kidney at 1.5 days post-infection. Scale bar ​= ​100 ​μm. Hepatic lesions including hepatocellular necrosis, nuclear fragmentation (black arrows), lymphocytic infiltration (blue arrows), granulocytic infiltration (green arrows), and hepatocellular steatosis (yellow arrows) were observed. Splenic lesions including lymphocytic necrosis with nuclear fragmentation (black arrows), cellular necrosis with nuclear fragmentation (blue arrows), neutrophilic infiltrate (green arrows), and bruising (yellow arrows) were observed. Lung tissue showed diffuse mild thickening of the alveolar wall, narrowing or loss of the alveolar lumen with inflammatory cell infiltration (black arrows); bronchial epithelial cells were detached (blue arrows). Kidney lesions were seen as hydropic degeneration of renal tubular epithelial cells (black arrows) and dilatation of renal tubular interstitial vessels seen as stasis (blue arrows).

Fig. 4

Comparison of age-related and gender-associated of Syrian hamster infected with VSV-EBOV/GP. 3-week-old (A), 3-month-old (B) and 1-year-old (C) Syrian hamsters were infected with VSV-EBOV/GP (10⁷ TCID₅₀) via intraperitoneal route. Weight change and percentage survival were monitored. Viral loads were evaluated by TCID₅₀ in the liver and spleen at 3 days post-infection (D).

Fig. 5

Comparison of challenge route-dependent of Syrian hamster infected with VSV-EBOV/GP (10⁷ TCID₅₀) via intraperitoneal (i.p.), subcutaneous (s.c.), intramuscular (i.m.), and intranasal (i.n.) route. Animals were monitored for weight changes and survival (A). B Blood biochemistry and blood cell count were analyzed at 1.5 days post-infection. C Viral loads including liver, spleen, lung, and kidney were determined at 1.5 days post-infection. D, E Histopathological changes of the liver, spleen, lung and kidney, and pathological scores at 1.5 days post-infection. Scale bar ​= ​100 ​μm. Hepatic lesions including hepatocellular necrosis, nuclear fragmentation (black arrows), lymphocytic infiltration (blue arrows), granulocytic infiltration (green arrows), and hepatocellular steatosis (yellow arrows) were observed. Splenic lesions including cellular necrosis with nuclear fragmentation (blue arrows), neutrophilic infiltrate (green arrows), and bruising (yellow arrows) were observed. Lung lesions including alveolar hemorrhage (red arrows), granulocytic infiltration (green arrows), vascular stasis (yellow arrows) and moderate thickening of the alveolar wall (black arrows) were observed. Kidney lesions including necrosis of tubular epithelial cells (black arrows), eosinophilic material in the lumen of the tubules (yellow arrows), glomerular capillary stasis (red arrows), and interstitial capillary stasis (blue arrows) were observed. F, G Immunohistochemistry assays of the liver, spleen, lungs and kidneys, and histochemistry scores. Data presented as mean ​± ​SEM. Statistical analyses were performed using One-way ANOVA. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001.

Fig. 6

Equine anti-EBOV immunoglobulins protect Syrian hamsters from lethal challenge of VSV-EBOV/GP. A Schematic representation of the preventive and therapeutic effects of antibodies evaluated in the Syrian hamster model. B Weight changes and percentage of survival in treatment and prevention groups. C Viral loads were evaluated by TCID₅₀ in the liver at 3 days post-infection (dpi) and 5 dpi in treatment group, prevention group, and attack/control group. D The neutralizing antibody titer of Equine anti-EBOV immunoglobulins (starting concentration of 50 ​μg/mL, serially diluted two-fold from 1:3). E Pathology was examined, and representative hematoxylin and eosin staining images of liver are showed. Scale bar ​= ​100 ​μm. Hepatic lesions including hepatocellular necrosis, nuclear fragmentation (black arrows), lymphocytic infiltration (blue arrows), granulocytic infiltration (green arrows), hepatocyte hydropic degeneration (red arrows), and a small amount of hepatocellular steatosis (yellow arrows) were observed. F Viral antigen in the liver of VSV-EBOV/GP-infected Syrian hamster in treatment group, prevention group, and attack group at 3 dpi and 5 dpi.

Fig. 7

Evaluation of the protective efficacy of EBOV GP Δmuc in lethal Syrian hamster model. A Schematic representation of the preventive effects of vaccination evaluated in the Syrian hamster model. B Weight change and percentage of survival after VSV-EBOV/GP infection. C Viral loads were evaluated by TCID₅₀ in the liver at 3 days post-infection (dpi) and 5 dpi. D Serum neutralizing antibody following second and third immunization. E Representative hematoxylin and eosin stains of liver at 3 dpi and 5 dpi. Scale bar ​= ​100 ​μm. Hepatic lesions including hepatocellular necrosis, nuclear fragmentation (black arrows), lymphocytic infiltration (blue arrows), and granulocytic infiltration (green arrows) were observed. F Viral antigen in the liver of VSV-EBOV/GP-infected Syrian hamster at 3 dpi and 5 dpi.