A Surrogate Animal Model for Screening of Ebola and Marburg Glycoprotein-Targeting Drugs Using Pseudotyped Vesicular Stomatitis Viruses

Abstract

Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans. In this study, we aimed to establish a surrogate animal model that can be used for anti-EBOV and -MARV drug screening under BSL-2 conditions by focusing on the replication-competent recombinant vesicular stomatitis virus (rVSV) pseudotyped with the envelope glycoprotein (GP) of EBOV (rVSV/EBOV) and MARV (rVSV/MARV), which has been investigated as vaccine candidates and thus widely used in BSL-2 laboratories. We first inoculated mice, rats, and hamsters intraperitoneally with rVSV/EBOV and found that only hamsters showed disease signs and succumbed within 4 days post-infection. Infection with rVSV/MARV also caused lethal infection in hamsters. Both rVSV/EBOV and rVSV/MARV were detected at high titers in multiple organs including the liver, spleen, kidney, and lungs of infected hamsters, indicating acute and systemic infection resulting in fatal outcomes. Therapeutic effects of passive immunization with an anti-EBOV neutralizing antibody were specifically observed in rVSV/EBOV-infected hamsters. Thus, this animal model is expected to be a useful tool to facilitate in vivo screening of anti-filovirus drugs targeting the GP molecule.

Keywords: Ebola virus; Filovirus; Marburg virus; Syrian hamster; animal model; drug screening; recombinant vesicular stomatitis virus.

Figure 1

Body weight changes and survival curves of animals after rVSV/EBOV infection. (A) F344/N rats, (B) BALB/c mice, and (C) Syrian hamsters (3 animals for each group) were injected intraperitoneally (i.p.) with 10⁷, 10^6.5, and 10^7.2 PFU of rVSV/EBOV, respectively. The symbols represent mean group weights and the bars represent standard errors.

Figure 2

Body weight changes and survival curves of Syrian hamsters infected with rVSV/EBOV, rVSV/MARV, or rVSV/VSV. (A) Four hamsters were infected i.p. with 10⁶, 10⁵, 10⁴, or 10³ PFU of rVSV/EBOV. (B) Three hamsters were infected i.p. with 10^7.5, 10^6.5, or 10^5.5 PFU of rVSV/MARV. (C) Four hamsters were infected i.p. with 10⁶, 10⁵, 10⁴, or 10³ PFU of rVSV/VSV. All animals were monitored for body weight and survival for 7 days post-infection (dpi). The symbols represent mean group weights and the bars represent standard errors (A, B, and C upper panel).

Figure 3

Virus titers in hamsters infected with rVSVs. Four hamsters in each group were infected i.p. with 10⁷ PFU of (A) rVSV/EBOV, (B) rVSV/MARV, or (C) rVSV/VSV. Thirty-six hours after virus inoculation, tissue samples were collected and used for virus titration in plaque assays. Two of four rVSV/EBOV-infected, three of four rVSV/MARV-infected, and three of four rVSV/VSV-infected animals already succumbed at the sampling time point. Each symbol represents the value of an individual hamster. The bars represent the means for the infected hamsters. The broken lines indicate the detection limit (<2.0 log₁₀ PFU/g).

Figure 4

Histological and immunohistochemical analyses of liver, spleen, and brain (cerebral cortex). (A) Tissue sections were stained with hematoxylin and eosin (HE) and a rabbit anti-VSV N polyclonal antibody. (B) Enlarged images of the spleen sections stained with the anti-VSV N antibody are also shown. Scale bars represent 100 μm. Brown-stained cells represent viral antigen-positive cells.

Figure 4

Histological and immunohistochemical analyses of liver, spleen, and brain (cerebral cortex). (A) Tissue sections were stained with hematoxylin and eosin (HE) and a rabbit anti-VSV N polyclonal antibody. (B) Enlarged images of the spleen sections stained with the anti-VSV N antibody are also shown. Scale bars represent 100 μm. Brown-stained cells represent viral antigen-positive cells.

Figure 5

(A) Blood cell counts and (B) liver parameters in hamsters infected with rVSVs. Four hamsters in each group were infected i.p. with 10⁷ PFU of rVSV/EBOV, rVSV/MARV, or rVSV/VSV. Twelve hours after virus inoculation, blood and serum samples were collected and used for cell counts and blood biochemistry. Each symbol represents the value for an individual hamster. The bars represent the means of the infected hamsters. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005.

Figure 6

Effects of prophylactic treatment with the neutralizing antibody in rVSV/EBOV-infected hamsters. Three hamsters in each group were treated i.p. with 100 μg of MAb ch6D6 or negative control MAb chDNP one day before (A) rVSV/EBOV, (B) rVSV/MARV, or (C) rVSV/VSV i.p. challenge (10⁷ PFU), or (D) treated with saline alone. Symbols represent percentages of body weight for each hamster (upper and middle panels). Open and solid symbols represent survival curves of 6D6- and chDNP-treated animals, respectively (lower panel).

Figure 7

Therapeutic effect of post-exposure treatment with the neutralizing antibody in rVSV/EBOV-infected hamsters. Three hamsters in each group were treated i.p. with 100 μg of MAb ch6D6 or negative control MAb chDNP one hour after (A) rVSV/EBOV, (B) rVSV/MARV, or (C) rVSV/VSV i.p. challenge (10⁷ PFU), or (D) treated with saline alone. Symbols represent percentages of weight for each hamster (upper and middle panels). Open and solid symbols represent survival curves of 6D6- and chDNP-treated animals, respectively (lower panel).