Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

Viral hemorrhagic fevers caused by the arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15-35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC(50)=500-800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC(50) of 200-350 nm). Although our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.     

Envelope exchange for the generation of live-attenuated arenavirus vaccines

Arenaviruses such as Lassa fever virus cause significant mortality in endemic areas and represent potential bioterrorist weapons. The occurrence of arenaviral hemorrhagic fevers is largely confined to Third World countries with a limited medical infrastructure, and therefore live-attenuated vaccines have long been sought as a method of choice for prevention. Yet their rational design and engineering have been thwarted by technical limitations. In addition, viral genes had not been identified that are needed to cause disease but can be deleted or substituted to generate live-attenuated vaccine strains. Lymphocytic choriomeningitis virus, the prototype arenavirus, induces cell-mediated immunity against Lassa fever virus, but its safety for humans is unclear and untested. Using this virus model, we have developed the necessary methodology to efficiently modify arenavirus genomes and have exploited these techniques to identify an arenaviral Achilles' heel suitable for targeting in vaccine design. Reverse genetic exchange of the viral glycoprotein for foreign glycoproteins created attenuated vaccine strains that remained viable although unable to cause disease in infected mice. This phenotype remained stable even after extensive propagation in immunodeficient hosts. Nevertheless, the engineered viruses induced T cell-mediated immunity protecting against overwhelming systemic infection and severe liver disease upon wild-type virus challenge. Protection was established within 3 to 7 d after immunization and lasted for approximately 300 d. The identification of an arenaviral Achilles' heel demonstrates that the reverse genetic engineering of live-attenuated arenavirus vaccines is feasible. Moreover, our findings offer lymphocytic choriomeningitis virus or other arenaviruses expressing foreign glycoproteins as promising live-attenuated arenavirus vaccine candidates.     

SAR studies of 4-acyl-1,6-dialkylpiperazin-2-one arenavirus cell entry inhibitors

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R¹ and R⁴ scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R⁶ substituents and iterative R¹, R⁴ and R⁶ substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.     

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8⁺ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8⁺ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8⁺ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.     

An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses

Five New World (NW) arenaviruses cause human hemorrhagic fevers. Four of these arenaviruses are known to enter cells by binding human transferrin receptor 1 (hTfR1). Here we show that the fifth arenavirus, Chapare virus, similarly uses hTfR1. We also identify an anti-hTfR1 antibody, ch128.1, which efficiently inhibits entry mediated by the glycoproteins of all five viruses, as well as replication of infectious Junín virus. Our data indicate that all NW hemorrhagic fever arenaviruses utilize a common hTfR1 apical-domain epitope and suggest that therapeutic agents targeting this epitope, including ch128.1 itself, can be broadly effective in treating South American hemorrhagic fevers.     

Transferrin receptor 1 in the zoonosis and pathogenesis of New World hemorrhagic fever arenaviruses

At least five New World arenaviruses cause severe human hemorrhagic fevers. These viruses are transmitted to humans through contact with their respective South American rodent hosts. Each uses human transferrin receptor 1 (TfR1) as its obligate receptor. Accidental similarities between human TfR1 and TfR1 orthologs of arenaviral host species enable zoonoses, whereas mice and rats are not infectable because they lack these TfR1 determinants of infection. All pathogenic New World arenaviruses bind to a common region of the apical domain of TfR1. The ability of a New World arenavirus to use human TfR1 is absolutely predictive of its ability to cause hemorrhagic fevers in humans. Nonpathogenic arenaviruses, closely related to hemorrhagic fever arenaviruses, cannot utilize human TfR1 but efficiently enter cells through TfR1 orthologs of their native rodent hosts. Mutagenesis studies suggest that minor changes in the entry glycoproteins of these nonpathogenic viruses may allow human transmission. TfR1 is upregulated as a result of iron sequestration during the acute-phase response to infection, and the severity of disease may result from amplification of viral replication during this response.     

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Background

Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4⁺ T cells are needed for optimal CD8⁺ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4⁺ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4⁺ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy.

Results

By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4⁺ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4⁺ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4⁺ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies.

Conclusions

The identification of CD4⁺ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.     

New world clade B arenaviruses can use transferrin receptor 1 (TfR1)-dependent and -independent entry pathways, and glycoproteins from human pathogenic strains are associated with the use of TfR1

Arenaviruses are rodent-borne viruses, with five members of the family capable of causing severe hemorrhagic fevers if transmitted to humans. To date, two distinct cellular receptors have been identified that are used by different pathogenic viruses, α-dystroglycan by Lassa fever virus and transferrin receptor 1 (TfR1) by certain New World clade B viruses. Our previous studies have suggested that other, as-yet-unknown receptors are involved in arenavirus entry. In the present study, we examined the use of TfR1 by the glycoproteins (GPs) from a panel of New World clade B arenaviruses comprising three pathogenic and two nonpathogenic strains. Interestingly, we found that TfR1 was only used by the GPs from the pathogenic viruses, with entry of the nonpathogenic strains being TfR1 independent. The pathogenic GPs could also direct entry into cells by TfR1-independent pathways, albeit less efficiently. A comparison of the abilities of TfR1 orthologs from different species to support arenavirus entry found that the human and feline receptors were able to enhance entry of the pathogenic strains, but that neither the murine or canine forms were functional. Since the ability to use TfR1 is a characteristic feature of the human pathogens, this interaction may represent an important target in the treatment of New World hemorrhagic fevers. In addition, the ability to use TfR1 may be a useful tool to predict the likelihood that any existing or newly discovered viruses in this family could infect humans.     

Failure to prove arenavirus infection among the small mammals from an endemic area of Korean hemorrhagic nephrosonephritis.

In the light of recent knowledge on a complex of diseases caused by a new group of viruses, arenaviruses, virological studies largely directed toward small field mammals were undertaken during 1973-1974 aiming at etiological clarification of Korean hemorrhagic nephrosonephritis (KHNN). Specimens were collected in an endemic area of KHNN located north to northeast of Seoul. Virus isolation tests with 299 urine specimens and 131 mite pools recovered from small mammals and 14 acute stage sera from typical cases yielded negative results. Complement-fixation (CF) tests failed to detect antibodies against the antigens of Congo, lymphocytic choriomeningitis (LCM), Tacaribe, and Pichinde viruses among 366 small mammal sera. In addition, CF tests of 59 of the above sera against Apoi and Lassa virus antigens were negative. The results do not support the likelihood of an arenavirus being transmitted among Korean small field mammals, the overwhelming majority of which were Apodemus agrarius. A hypothesis that KHNN is caused by a virus of small field mammal origin was not proved within the technical limit of relatively unsophisticated methods employed herein.     

Inhibition of Innate Immune Responses Is Key to Pathogenesis by Arenaviruses

Mammalian arenaviruses are zoonotic viruses that cause asymptomatic, persistent infections in their rodent hosts but can lead to severe and lethal hemorrhagic fever with bleeding and multiorgan failure in human patients. Lassa virus (LASV), for example, is endemic in several West African countries, where it is responsible for an estimated 500,000 infections and 5,000 deaths annually. There are currently no FDA-licensed therapeutics or vaccines available to combat arenavirus infection. A hallmark of arenavirus infection (e.g., LASV) is general immunosuppression that contributes to high viremia. Here, we discuss the early host immune responses to arenavirus infection and the recently discovered molecular mechanisms that enable pathogenic viruses to suppress host immune recognition and to contribute to the high degree of virulence. We also directly compare the innate immune evasion mechanisms between arenaviruses and other hemorrhagic fever-causing viruses, such as Ebola, Marburg, Dengue, and hantaviruses. A better understanding of the immunosuppression and immune evasion strategies of these deadly viruses may guide the development of novel preventative and therapeutic options.     

Cell entry by human pathogenic arenaviruses

The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15–35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ α-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.     

A Recombinant Vesicular Stomatitis Virus-Based Lassa Fever Vaccine Protects Guinea Pigs and Macaques against Challenge with Geographically and Genetically Distinct Lassa Viruses

Background

Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a “universal” LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models.

Methodologies/principle findings

Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever.

Conclusions/significance

Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever.     

In silico characterization of immunogenic epitopes presented by HLA-Cw*0401

Background

Arenaviruses are a family of rodent-borne viruses that cause several hemorrhagic fevers. These diseases can be devastating and are often lethal. Herein, to aid in the design and development of diagnostics, treatments and vaccines for arenavirus infections, we have developed a database containing protein sequences from the seven pathogenic arenaviruses (Junin, Guanarito, Sabia, Machupo, Whitewater Arroyo, Lassa and LCMV).

Results

The database currently contains a non-redundant set of 333 protein sequences which were manually annotated. All entries were linked to NCBI and cited PubMed references. The database has a convenient query interface including BLAST search. Sequence variability analyses were also performed and the results are hosted in the database.

Conclusion

The database is available at http://epitope.liai.org:8080/projects/arena and can be used to aid in studies that require proteomic information from pathogenic arenaviruses.     

Coverage of Related Pathogenic Species by Multivalent and Cross-Protective Vaccine Design: Arenaviruses as a Model System

Summary: The arenaviruses are a family of negative-sense RNA viruses that cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. There are currently no FDA-approved vaccines for the prevention of arenavirus disease, and therapeutic treatment is limited to the use of ribavirin and/or immune plasma for a subset of the pathogenic arenaviruses. The considerable genetic variability observed among the seven arenaviruses that are pathogenic for humans illustrates one of the major challenges for vaccine development today, namely, to overcome pathogen heterogeneity. Over the past 5 years, our group has tested several strategies to overcome pathogen heterogeneity, utilizing the pathogenic arenaviruses as a model system. Because T cells play a prominent role in protective immunity following arenavirus infection, we specifically focused on the development of human vaccines that would induce multivalent and cross-protective cell-mediated immune responses. To facilitate our vaccine development and testing, we conducted large-scale major histocompatibility complex (MHC) class I and class II epitope discovery on murine, nonhuman primate, and human backgrounds for each of the pathogenic arenaviruses, including the identification of protective HLA-restricted epitopes. Finally, using the murine model of lymphocytic choriomeningitis virus infection, we studied the phenotypic characteristics associated with immunodominant and protective T cell epitopes. This review summarizes the findings from our studies and discusses their application to future vaccine design.     

Genetic diversity among Lassa virus strains

The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a "molecular clock" was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation.     

Individual and Bivalent Vaccines Based on Alphavirus Replicons Protect Guinea Pigs against Infection with Lassa and Ebola Viruses

Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.     

pH-induced activation of arenavirus membrane fusion is antagonized by small-molecule inhibitors

The arenavirus envelope glycoprotein (GPC) mediates viral entry through pH-induced membrane fusion in the endosome. This crucial process in the viral life cycle can be specifically inhibited in the New World arenaviruses by the small-molecule compound ST-294. Here, we show that ST-294 interferes with GPC-mediated membrane fusion by targeting the interaction of the G2 fusion subunit with the stable signal peptide (SSP). We demonstrate that amino acid substitutions at lysine-33 of the Junín virus SSP confer resistance to ST-294 and engender de novo sensitivity to ST-161, a chemically distinct inhibitor of the Old World Lassa fever virus. These compounds, as well as a broadly active inhibitor, ST-193, likely share a molecular target at the SSP-G2 interface. We also show that both ST-294 and ST-193 inhibit pH-induced dissociation of the G1 receptor-binding subunit from GPC, a process concomitant with fusion activation. Interestingly, the inhibitory activity of these molecules can in some cases be overcome by further lowering the pH used for activation. Our results suggest that these small molecules act to stabilize the prefusion GPC complex against acidic pH. The pH-sensitive interaction between SSP and G2 in GPC represents a robust molecular target for the development of antiviral compounds for the treatment of arenavirus hemorrhagic fevers.     

Identification of a broad-spectrum arenavirus entry inhibitor

Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC(50)) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC(50)s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 microM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.     

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.