Mice Lacking Functional STAT1 Are Highly Susceptible to Lethal Infection with Lassa Virus

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Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 ( A renaviruses are enveloped viruses with a bisegmented negative-strand RNA genome. Each genome segment, large (L; ϳ7.3 kb) and small (S; ϳ3.5 kb) uses an ambisense coding strategy to direct synthesis of two polypeptides in opposite direction, separated by a noncoding intergenic region (IGR) (1, 2). The S segment encodes for the viral nucleoprotein (NP) and the glycoprotein precursor (GPC), which is co-and posttranslationally processed by the signal peptidase and SKI-1/S1P cellular proteases, respectively, to produce the mature surface virion glycoproteins GP1 and GP2 and the stable signal peptide (SSP) that together form the GP1/GP2/SSP complex present at the surface of mature virions and that mediate receptor recognition and cell entry (1, 3). The L segment encodes the viral RNA-dependent RNA polymerase (L polymerase) and the matrix Z protein. Arenaviruses cause chronic infections of rodents with worldwide distribution, where human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose important public health problems in regions where these viruses are endemic (1, 4-8). Thus, Lassa virus (LASV) infects several hundred thousand people yearly in West Africa, resulting in a high number of Lassa fever cases that are associated with high morbidity and mortality. Likewise, Junin virus (JUNV) causes Argentine HF, a severe illness endemic to Pampas in Argentina, which is characterized by hemorrhagic and neurological manifestations and a fatality rate of 15 to 30% (8). Moreover, mounting evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance (9-12). In addition, several arenaviruses, including LASV, JUNV, and LCMV, pose a credible biodefense threat (13). Concerns about arenavirus infections of humans are exacerbated due to the lack of U.S. Food and Drug Administrationlicensed vaccines and current antiarenaviral therapy being limited to an off-label use of ribavirin (Rib) that is only partially effective (14-16). Therefore, there is an unmet need to iden...

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confidence: 99%

Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication

Ngo

Cubitt

Iwasaki

et al. 2015

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“…A functional IFN system has been shown to be required for arenavirus clearance in several mouse models (37)(38)(39)(40)(41). We showed in this study that the NP RNase-defective rPICVs induced early and strong IFN responses in guinea pigs that limited virus replication in various organs (Fig.…”

Section: Discussionmentioning

confidence: 65%

In Vitro and In Vivo Characterizations of Pichinde Viral Nucleoprotein Exoribonuclease Functions

Huang

Shao

Lan

et al. 2015

Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFNdeficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication. IMPORTANCEArenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

“…We infected Stat1 Ϫ/Ϫ mice (n ϭ 5/group) via the intraperitoneal (i.p.) route with 10 4 PFU of recent LASV isolates that were obtained directly from serum samples from fatal (LV2384, lethal isolate; we have previously evaluated the virulence of this virus for Stat1 Ϫ/Ϫ mice [16]) and nonfatal (LV2350, nonlethal isolate) LF cases that were collected during a recent outbreak in Sierra Leone in 2012. Four out of five mice infected with the lethal isolate reached the monitoring criteria that required euthanasia between days 7 and 8 p.i.…”

Section: Lethal and Nonlethal Human Lasv Isolates Induce Either Lethamentioning

confidence: 99%

“…Previously, we published that mice lacking a functional STAT1 pathway (Stat1 Ϫ/Ϫ ) are highly susceptible to infection with LASV and develop lethal disease with pathology similar to that reported for humans (16). With these findings and knowledge of LCMV-related hearing loss studies (12), we aimed to develop a reproducible murine model of hearing loss resulting from LASV infection.…”

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confidence: 97%

Animal Model of Sensorineural Hearing Loss Associated with Lassa Virus Infection

Yun

Ronca

Tamura

et al. 2016

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Approximately one-third of Lassa virus (LASV)-infected patients develop sensorineural hearing loss (SNHL) in the late stages of acute disease or in early convalescence. With 500,000 annual cases of Lassa fever (LF), LASV is a major cause of hearing loss in regions of West Africa where LF is endemic. To date, no animal models exist that depict the human pathology of LF with associated hearing loss. Here, we aimed to develop an animal model to study LASV-induced hearing loss using human isolates from a 2012 Sierra Leone outbreak. We have recently established a murine model for LF that closely mimics many features of human disease. In this model, LASV isolated from a lethal human case was highly virulent, while the virus isolated from a nonlethal case elicited mostly mild disease with moderate mortality. More importantly, both viruses were able to induce SNHL in surviving animals. However, utilization of the nonlethal, human LASV isolate allowed us to consistently produce large numbers of survivors with hearing loss. Surviving mice developed permanent hearing loss associated with mild damage to the cochlear hair cells and, strikingly, significant degeneration of the spiral ganglion cells of the auditory nerve. Therefore, the pathological changes in the inner ear of the mice with SNHL supported the phenotypic loss of hearing and provided further insights into the mechanistic cause of LF-associated hearing loss. IMPORTANCESensorineural hearing loss is a major complication for LF survivors. The development of a small-animal model of LASV infection that replicates hearing loss and the clinical and pathological features of LF will significantly increase knowledge of pathogenesis and vaccine studies. In addition, such a model will permit detailed characterization of the hearing loss mechanism and allow for the development of appropriate diagnostic approaches and medical care for LF patients with hearing impairment.