Interferon Receptor-deficient Mice Research Articles

A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease

The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

CCL2 expression is mediated by type I IFN receptor and recruits NK and T cells to the lung during MVA infection.

Migration of leukocytes to the site of microbial infection is important for the development of effective host immunity. Recombinant modified vaccinia virus Ankara is frequently used as a viral vector vaccine in preclinical and clinical studies. In comparison to other vaccinia virus strains, modified vaccinia virus Ankara robustly induces chemokine expression and rapid attraction of leukocytes. In particular, chemokine (C-C motif) ligand 2 (CCL2) has been shown to be critical for leukocyte recruitment to the lung. In this study, MVA-induced CCL2 expression in murine macrophages was dependent on type I interferon receptor and not Toll-like receptor-2. The critical role of type I interferon receptor signaling for CCL2 production in the lung was confirmed in type I interferon receptor-deficient mice (Ifnar1(-/-)). In addition, comparing Ifnar1(-/-) and Ccl2(-/-) mice with wild-type mice, we observed a similar impairment in the recruitment of natural killer and T cells to the lung after intranasal infection with modified vaccinia virus Ankara. Conversely, neutrophil recruitment was not affected in Ifnar1(-/-) and Ccl2(-/-) mice. We conclude that type I interferons, besides their known antiviral properties, can initiate the recruitment and activation of leukocytes via induction of chemokine expression including CCL2.

Potential application of silver nanoparticles to control the infectivity of Rift Valley fever virus in vitro and in vivo

In this work we have tested the potential antiviral activity of silver nanoparticles formulated as Argovit™ against Rift Valley fever virus (RVFV). The antiviral activity of Argovit was tested on Vero cell cultures and in type-I interferon receptor deficient mice (IFNAR −/− mice) by two different approaches: (i) different dilutions of Argovit were added to previously infected cells or administrated to animals infected with a lethal dose of virus; (ii) virus was pre-incubated with different dilutions of Argovit before inoculation in mice or cells. Though the ability of silver nanoparticles to control an ongoing RVFV infection in the conditions tested was limited, the incubation of virus with Argovit before the infection led to a reduction of the infectivity titers both in vitro and in vivo. These results reveal the potential application of silver nanoparticles to control the infectivity of RVFV, which is an important zoonotic pathogen.

Development of a recombinant, chimeric tetravalent dengue vaccine candidate

Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24–67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8+ T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.

Development and Characterization of Monoclonal Antibodies Directed Against the Nucleoprotein of Heartland Virus.

Heartland virus (HRTV), a phlebovirus first isolated from two Missouri farmers in 2009, has been proposed to be transmitted to humans by the bite of infected Amblyomma americanum ticks. It is closely related to severe fever with thrombocytopenia syndrome virus (SFTSV) from China, another previously unrecognized phlebovirus that has subsequently been associated with hundreds of cases of severe disease in humans. To expand diagnostic capacity to detect HRTV infections, 20 hybridoma clones secreting anti-HRTV murine monoclonal antibodies (MAbs) were developed using splenocytes from HRTV-inoculated AG129 alpha/beta and gamma interferon receptor-deficient mice. Nine of these MAbs were characterized herein for inclusion in future HRTV diagnostic assay development. All of the MAbs developed were found to be non-neutralizing and reactive to linear epitopes on HRTV nucleocapsid protein. MAb 2AF11 was found to be cross-reactive with SFTSV.

ID: 255: Autoimmunity due to constitutive activation of cytoplasmic viral RNA sensors

MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. Once MDA5 senses replicating viruses, it triggers signal to activate antiviral genes including those of types I and III IFN. Activation of IFN system is critical as antiviral innate immunity and promotes activation of acquired immunity. These immune responses orchestrate eradication of infecting viruses. On the other hand, MDA5 has been implicated in autoimmunity. The mechanisms of how MDA5 contribute to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, IPS-1, indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand, but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. In humans, mutations of MDA5 and RIG-I were found in patients of Aicardi-Goutieres syndrome and Singleton-Merton syndrome. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

94: A viral sensor MDA5 and autoimmune diseases

MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms and also progressive encephalopathy without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5.These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

A novel live-attenuated vaccine candidate for mayaro Fever.

Mayaro virus (MAYV) is an emerging, mosquito-borne alphavirus that causes a dengue-like illness in many regions of South America, and which has the potential to urbanize. Because no specific treatment or vaccine is available for MAYV infection, we capitalized on an IRES-based approach to develop a live-attenuated MAYV vaccine candidate. Testing in infant, immunocompetent as well as interferon receptor-deficient mice demonstrated a high degree of attenuation, strong induction of neutralizing antibodies, and efficacy against lethal challenge. This vaccine strain was also unable to infect mosquito cells, a major safety feature for a live vaccine derived from a mosquito-borne virus. Further preclinical development of this vaccine candidate is warranted to protect against this important emerging disease.

Rapid expansion of CD8+ T cells in wild-type and type I interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus Ankara.

Immunization with modified vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8(+) T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNAR(-/-)). We found a minimal dose of 10(5) PFU of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNAR(-/-) mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus-specific CD8(+) T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen-specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naive CD8(+) T cells by MVA immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses. We demonstrate that single-shot low-dose immunizations with vaccinia virus MVA can rapidly induce T cell-mediated protective immunity against lethal orthopoxvirus infections. Our data provide new evidence for an efficient protective capacity of vaccination with replication-deficient MVA. These data are of important practical relevance for public health, as the effectiveness of a safety-tested, next-generation smallpox vaccine based on MVA is still debated. Furthermore, producing sufficient amounts of vaccine is expected to be a major challenge should an outbreak occur. Moreover, prevention of other infections may require rapidly protective immunization; hence, MVA could be an extremely useful vaccine for delivering heterologous T cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination.

Autoimmune Disorders Associated with Gain of Function of the Intracellular Sensor MDA5

MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

Mouse models for dengue vaccines and antivirals

Dengue virus (DENV) has substantial global impact, with an estimated 390million people infected each year. In spite of this, there is currently no approved DENV-specific vaccine or antiviral. One reason for this is the difficulty involved with development of an adequate animal model. While non-human primates support viral replication, they do not exhibit signs of clinical disease. A mouse model is an ideal alternative; however, wild-type mice are resistant to DENV-induced disease. Infection of interferon receptor-deficient mice results in disease that recapitulates key features of severe dengue disease in humans. For the development of vaccines, interferon receptor-deficient mice provide a stringent model for testing vaccine-induced immune components from vaccinated wild-type mice.

Humanized monoclonal antibody 2C9-cIgG has enhanced efficacy for yellow fever prophylaxis and therapy in an immunocompetent animal model

Yellow fever virus (YFV) causes significant human disease and mortality in tropical regions of South and Central America and Africa, despite the availability of an effective vaccine. No specific therapy for YF is available. We previously showed that the humanized monoclonal antibody (MAb) 2C9-cIgG provided prophylactic and therapeutic protection from mortality in interferon receptor-deficient strain AG129 mice challenged with YF 17D-204 vaccine. In this study we tested the prophylactic and therapeutic efficacy of this MAb against virulent YFV infection in an immunocompetent hamster model. Intraperitoneal (ip) administration of a single dose of MAb 2C9-cIgG 24h prior to YFV challenge resulted in significantly improved survival rates in animals treated with 380 or 38μg of MAb compared to untreated animals. Treatment with the higher dose also resulted in significantly improved weight gain and reductions in serum alanine aminotransferase (ALT) and virus titers in serum and liver. Prophylactic treatment with 2C9-cIgG 24h prior to virus challenge prevented the development of a virus-neutralizing antibody (vnAb) response in hamsters. Administration of a single ip dose of 380μg of 2C9-cIgG as late as 72h post-YFV challenge also resulted in significant improvement in survival rates. Hamsters treated at 4–72h post-virus challenge developed a robust vnAb response. Enhanced survival and improvement of various disease parameters in the hamster model when MAb 2C9-cIgG is administered up to 3days after virus challenge demonstrate the clinical potential of specific antibody therapy for YF.

Infection of Type I Interferon Receptor-Deficient Mice with Various Old World Arenaviruses: A Model for Studying Virulence and Host Species Barriers

Lassa virus causes hemorrhagic Lassa fever in humans, while the related Old World arenaviruses Mopeia, Morogoro, and Mobala are supposedly apathogenic to humans and cause only inapparent infection in non-human primates. Here, we studied whether the virulence of Old World arenaviruses in humans and non-human primates is reflected in type I interferon receptor deficient (IFNAR-/-) mice by testing several strains of Lassa virus vs. the apathogenic viruses Mopeia, Morogoro, and Mobala. All Lassa virus strains tested—Josiah, AV, BA366, and Nig04-10—replicated to high titers in blood, lung, kidney, heart, spleen, brain, and liver and caused disease as evidenced by weight loss and elevation of aspartate and alanine aminotransferase (AST and ALT) levels with a high AST/ALT ratio. Lassa fever-like pathology included acute hepatitis, interstitial pneumonia, and pronounced disturbance of splenic cytoarchitecture. Infiltrations of activated monocytes/macrophages expressing inducible nitric oxide synthase and T cells were found in liver and lung. In contrast, Mopeia, Morogoro, and Mobala virus replicated poorly in the animals and acute inflammatory alterations were not noted. Depletion of CD4+ and CD8+ T cells strongly enhanced susceptibility of IFNAR-/- mice to the apathogenic viruses. In conclusion, the virulence of Old World arenaviruses in IFNAR-/- mice correlates with their virulence in humans and non-human primates. In addition to the type I interferon system, T cells seem to regulate whether or not an arenavirus can productively infect non-host rodent species. The observation that Lassa virus overcomes the species barrier without artificial depletion of T cells suggests it is able to impair T cell functionality in a way that corresponds to depletion.

Neuromyelitis optica-like pathology is dependent on type I interferon response

Neuromyelitis optica is an antibody-mediated autoimmune inflammatory disease of the central nervous system. Reports have suggested that interferon beta which is beneficial for multiple sclerosis, exacerbates neuromyelitis optica. Our aim was to determine whether type I interferon plays a role in the formation of neuromyelitis optica lesions. Immunoglobulin G from a neuromyelitis optica patient was injected intracerebrally with human complement to type I interferon receptor deficient and wildtype mice. Loss of aquaporin-4 and glial fibrillary acidic protein was reduced in type I interferon receptor deficient mice brain. Our findings suggest that type I interferon signaling contributes to neuromyelitis optica pathogenesis.

Combined action of type I and type III interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread

STAT1-deficient mice are more susceptible to infection with severe acute respiratory syndrome coronavirus (SARS-CoV) than type I interferon (IFN) receptor-deficient mice. We used mice lacking functional receptors for both type I and type III IFN (double knockout, dKO) to evaluate the possibility that type III IFN plays a decisive role in SARS-CoV protection. We found that viral peak titres in lungs of dKO and STAT1-deficient mice were similar, but significantly higher than in wild-type mice. The kinetics of viral clearance from the lung were also comparable in dKO and STAT1-deficient mice. Surprisingly, however, infected dKO mice remained healthy, whereas infected STAT1-deficient mice developed liver pathology and eventually succumbed to neurological disease. Our data suggest that the failure of STAT1-deficient mice to control initial SARS-CoV replication efficiently in the lung is due to impaired type I and type III IFN signalling, whereas the failure to control subsequent systemic viral spread is due to unrelated defects in STAT1-deficient mice.

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery.

Interleukin-22

Ischemia-reperfusion injury (IRI) is common in general surgery and organ transplantation, and in the case of liver, it triggers proinflammatory innate immune cascade and hepatic necrosis, leading to increased incidence of early and late organ rejection. Interleukin (IL)-22, an inducible cytokine of T-cell origin and a member of the IL-10 superfamily, acts on target tissues through IL-22 receptor (IL-22R1). Partial hepatic warm ischemia was induced in C57Bl/6 wild-type (WT) and type 1 interferon receptor-deficient (KO) mice for 90 min followed by 6 to 24 hr of reperfusion. WT mice were treated at 30 min before the ischemia insult with recombinant IL-22 or anti-IL-22 neutralizing antibody; phosphate-buffered saline and IgG served as respective controls. IL-22 was detected at 24 hr but not 6 hr of liver IRI. The expression of IL-22R1 was increased by 6 hr of reperfusion in WT but not type 1 interferon receptor KO mice that were protected from IRI. Treatment of WT mice with recombinant IL-22 decreased serum aspartate aminotransferase levels, ameliorated cardinal histological features of IR damage (Suzuki's score) and diminished leukocyte sequestration, along with the expression of IL-22R1 and pro-inflammatory cytokines. IL-22 antibody did not appreciably affect IRI but increased IL-22R1 transcription in the liver. Administration of IL-22 protein exerted hepatoprotection by STAT3 activation. This is the first report investigating immune modulation by T-cell-derived IL-22 in liver injury caused by warm ischemia and reperfusion. Treatment with IL-22 protein may represent a novel therapeutic strategy to prevent liver IRI in transplant recipients.

A small animal peripheral challenge model of yellow fever using interferon-receptor deficient mice and the 17D-204 vaccine strain

Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne pathogen that requires wild-type (wt), virulent strains to be handled at biosafety level (BSL) 3, with HEPA-filtration of room air exhaust (BSL3+). YFV is found in tropical regions of Africa and South America and causes severe hepatic disease and death in humans. Despite the availability of effective vaccines (17D-204 or 17DD), YFV is still responsible for an estimated 200,000 cases of illness and 30,000 deaths annually. Besides vaccination, there are no other prophylactic or therapeutic strategies approved for use in human YF. Current small animal models of YF require either intra-cranial inoculation of YF vaccine to establish infection, or use of wt strains (e.g., Asibi) in order to achieve pathology. We have developed and characterized a BSL2, adult mouse peripheral challenge model for YFV infection in mice lacking receptors for interferons α, β, and γ (strain AG129). Intraperitoneal challenge of AG129 mice with 17D-204 is a uniformly lethal in a dose-dependent manner, and 17D-204-infected AG129 mice exhibit high viral titers in both brain and liver suggesting this infection is both neurotropic and viscerotropic. Furthermore the use of a mouse model permitted the construction of a 59-biomarker multi-analyte profile (MAP) using samples of brain, liver, and serum taken at multiple time points over the course of infection. This MAP serves as a baseline for evaluating novel therapeutics and their effect on disease progression. Changes (4-fold or greater) in serum and tissue levels of pro- and anti-inflammatory mediators as well as other factors associated with tissue damage were noted in AG129 mice infected with 17D-204 as compared to mock-infected control animals.

Epizootic hemorrhagic disease virus infection of type I interferon receptor deficient mice

Type I interferon receptor deficient (IFNAR−/−) mice were infected with an Israeli isolate of serotype 7 of epizootic hemorrhagic disease virus (EHDV; Orbivirus, Reoviridae). Two out of two mice that received 5×105 50% tissue culture infectious doses (TCID50) by intraperitoneal injection died or were euthanized in a moribund state on day 5 after infection. One mouse out of three that had been inoculated with 5×102 TCID50 died on day 7 while the remaining mice did not show any clinical signs and survived until the end of the experiment. Spleens of all dead mice were highly positive in an EHDV real-time RT-PCR (quantification cycle values ≤15) and contained ≥105.8 TCID50 of virus per ml of homogenate. The viral RNA content and virus titer in the spleens of the two surviving mice, on the other hand, were over 100-fold lower. Different from data reported for BTV, the outcome of EHDV infection of IFNAR−/− mice is dose-dependent and subclinical infections can occur.

Lethality and pathogenesis of airborne infection with filoviruses in A129 α/β −/− interferon receptor-deficient mice

Normal immunocompetent mice are not susceptible to non-adapted filoviruses. There are therefore two strategies available to establish a murine model of filovirus infection: adaptation of the virus to the host or the use of genetically modified mice that are susceptible to the virus. A number of knockout (KO) strains of mice with defects in either their adaptive or innate immunity are susceptible to non-adapted filoviruses. In this study, A129 α/β -/- interferon receptor-deficient KO mice, strain A129 IFN-α/β -/-, were used to determine the lethality of a range of filoviruses, including Lake Victoria marburgvirus (MARV), Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV) and Côte d'Ivoire ebolavirus (CIEBOV), administered by using intraperitoneal (IP) or aerosol routes of infection. One hundred percent mortality was observed in all groups of KO mice that were administered with a range of challenge doses of MARV and ZEBOV by either IP or aerosol routes. Mean time to death for both routes was dose-dependent and ranged from 5.4 to 7.4 days in the IP injection challenge, and from 10.2 to 13 days in the aerosol challenge. The lethal dose (50 % tissue culture infective dose, TCID(50)) of ZEBOV for KO mice was <1 TCID(50) ml(-1) when administered by either the IP or aerosol route of infection; for MARV the lethal dose was <1 TCID(50) ml(-1) by the IP route of infection and <10 TCID(50) ml(-1) by the aerosol route. In contrast, there was no mortality after infection with SEBOV or REBOV by either IP or aerosol routes of infection; all the mice lost weight (~15 % loss of group mean body weight with SEBOV and ~7 % with REBOV) but recovered to their original weights by day 14 post-challenge. There was no mortality in mice administered with CIEBOV via the IP route of infection and no clinical signs of infection were observed. The progression of disease was faster following infection with ZEBOV than with MARV but ultimately both viruses caused widespread infection with high titres of the infectious viruses in multiple organs. Histopathological observations were consistent with other animal models and showed widespread organ damage. This study suggests that MARV and ZEBOV are more virulent when administered via the IP route rather than by aerosol infection, although both are highly virulent by either route. The KO mouse may provide a useful model to test potential antiviral therapeutics against wild-type filoviruses.