Comparison of Routes of Administration, Frequency, and Duration of Favipiravir Treatment in Mouse and Guinea Pig Models of Ebola Virus Disease.

Favipiravir is a ribonucleoside analogue that has been explored as a therapeutic for the treatment of Ebola Virus Disease (EVD). Promising data from rodent models has informed nonhuman primate trials, as well as evaluation in patients during the 2013-2016 West African EVD outbreak of favipiravir treatment. However, mixed results from these studies hindered regulatory approval of favipiravir for the indication of EVD. This study examined the influence of route of administration, duration of treatment, and treatment schedule of favipiravir in immune competent mouse and guinea pig models using rodent-adapted Zaire ebolavirus (EBOV). A dose of 300 mg/kg/day of favipiravir with an 8-day treatment was found to be fully effective at preventing lethal EVD-like disease in BALB/c mice regardless of route of administration (oral, intraperitoneal, or subcutaneous) or whether it was provided as a once-daily dose or a twice-daily split dose. Preclinical data generated in guinea pigs demonstrates that an 8-day treatment of 300 mg/kg/day of favipiravir reduces mortality following EBOV challenge regardless of route of treatment or duration of treatments for 8, 11, or 15 days. This work supports the future translational development of favipiravir as an EVD therapeutic.

Characterization of Ebola Virus Mucosal Challenge Routes in Cynomolgus Macaques.

Zaïre ebolavirus (EBOV) causes Ebola virus disease (EVD), a devastating viral hemorrhagic fever in humans. Nonhuman primate (NHP) models of EVD traditionally use intramuscular infection with higher case fatality rates and reduced mean time-to-death compared to contact transmission typical of human cases of EVD. A cynomolgus macaque model of oral and conjunctival EBOV was used to further characterize the more clinically relevant contact transmission of EVD. NHPs challenged via the oral route had an overall 50% survival rate. NHPs challenged with a target dose of 1 × 102 PFU or 1 × 104 PFU of EBOV via the conjunctival route had 40% and 100% mortality, respectively. Classic signs of lethal EVD-like disease were observed in all NHPs that succumbed to EBOV infection including viremia, hematological abnormalities, clinical chemistries indicative of hepatic and renal disease, and histopathological findings. Evidence of EBOV viral persistence in the eye was observed in NHPs challenged via the conjunctival route. IMPORTANCE This study is the first to examine the Kikwit strain of EBOV, the most commonly used strain, in the gold-standard macaque model of infection. Additionally, this is the first description of the detection of virus in the vitreous fluid, an immune privileged site that has been proposed as a viral reservoir, following conjunctival challenge. The oral and conjunctival macaque challenge model of EVD described here more faithfully recapitulates the prodrome that has been reported for human EVD. This work paves the way for more advanced studies to model contact transmission of EVD, including early events in mucosal infection and immunity, as well as the establishment of persistent viral infection and the emergence from these reservoirs.

Evaluation of molnupiravir (EIDD-2801) efficacy against SARS-CoV-2 in the rhesus macaque model.

Molnupiravir (EIDD-2801) is a prodrug of a ribonucleoside analogue that is currently being used under a US FDA emergency use authorization for the treatment of mild to moderate COVID-19. We evaluated molnupiravir for efficacy as an oral treatment in the rhesus macaque model of SARS-CoV-2 infection. Twenty non-human primates (NHPs) were challenged with SARS-CoV-2 and treated with 75 mg/kg (n = 8) or 250 mg/kg (n = 8) of molnupiravir twice daily by oral gavage for 7 days. The NHPs were observed for 14 days post-challenge and monitored for clinical signs of disease. After challenge, all groups showed a trend toward increased respiration rates. Treatment with molnupiravir significantly reduced viral RNA levels in bronchoalveolar lavage (BAL) samples at Days 7 and 10. Considering the mild to moderate nature of SARS-CoV-2 infection in the rhesus macaque model, this study highlights the importance of monitoring the viral load in the lung as an indicator of pharmaceutical efficacy for COVID-19 treatments. Additionally, this study provides evidence of the efficacy of molnupiravir which supplements the current ongoing clinical trials of this drug.

Machupo Virus with Mutations in the Transmembrane Domain and Glycosylation Sites of the Glycoprotein Is Attenuated and Immunogenic in Animal Models of Bolivian Hemorrhagic Fever.

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuates MACV in an interferon alpha/beta and gamma (IFN-α/ß and -γ) receptor knockout (R-/-) mouse model. However, some capability to induce neurological pathology still remained. The highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. An F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in the GPC TMD. The MACV mutant is fully attenuated in IFN-α/ß and -γ R-/- mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV completely protected guinea pigs from wild-type MACV lethal challenge. Last, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which provide insight into the rational design of potential vaccine candidates for highly pathogenic arenaviruses. IMPORTANCE For arenaviruses, the only vaccine available is the live attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.

STAT-1 Knockout Mice as a Model for Wild-Type Sudan Virus (SUDV).

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014-2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Natural history of disease in cynomolgus monkeys exposed to Ebola virus Kikwit strain demonstrates the reliability of this non-human primate model for Ebola virus disease.

Filoviruses (Family Filoviridae genera Ebolavirus and Marburgvirus) are negative-stranded RNA viruses that cause severe health effects in humans and non-human primates, including death. Except in outbreak settings, vaccines and other medical countermeasures against Ebola virus (EBOV) will require testing under the FDA Animal Rule. Multiple vaccine candidates have been evaluated using cynomolgus monkeys (CM) exposed to EBOV Kikwit strain. To the best of our knowledge, however, animal model development data supporting the use of CM in vaccine research have not been submitted to the FDA. This study describes a large CM database (122 CM, 62 female and 60 male, age 2 to 9 years) and demonstrates the consistency of the CM model through time to death models and descriptive statistics. CMs were exposed to EBOV doses of 0.1 to 100,000 PFU in 33 studies conducted at three Animal Biosafety Level 4 facilities, by three exposure routes. Time to death was modeled using Cox proportional hazards models with a frailty term that incorporated study-to-study variability. Despite significant differences attributed to exposure variables, all CMs exposed to the 100 to 1,000 pfu doses commonly used in vaccine studies died or met euthanasia criteria within 21 days of exposure, median 7 days, 93% between 5 and 12 days of exposure. Moderate clinical signs were observed 4 to 5 days after exposure and preceded death or euthanasia by approximately one day. Viremia was detected within a few days of infection. Hematology indices were indicative of viremia and the propensity for hemorrhage with progression of Ebola viremia. Changes associated with coagulation parameters and platelets were consistent with coagulation disruption. Changes in leukocyte profiles were indicative of an acute inflammatory response. Increased liver enzymes were observed shortly after exposure. Taken together, these factors suggest that the cynomolgus monkey is a reliable animal model for human disease.

Mucosal Challenge Ferret Models of Ebola Virus Disease.

Recent studies have shown the domestic ferret (Mustela putorius furo) to be a promising small animal model for the study of Ebola virus (EBOV) disease and medical countermeasure evaluation. To date, most studies have focused on traditional challenge routes, predominantly intramuscular and intranasal administration. Here, we present results from a non-clinical pathogenicity study examining oronasal, oral, and ocular mucosal challenge routes in ferrets. Animals were challenged with 1, 10, or 100 plaque forming units EBOV followed by monitoring of disease progression and biosampling. Ferrets administered virus via oronasal and oral routes met euthanasia criteria due to advanced disease 5-10 days post-challenge. Conversely, all ferrets dosed via the ocular route survived until the scheduled study termination 28-day post-challenge. In animals that succumbed to disease, a dose/route response was not observed; increases in disease severity, febrile responses, serum and tissue viral load, alterations in clinical pathology, and gross/histopathology findings were similar between subjects. Disease progression in ferrets challenged via ocular administration was unremarkable throughout the study period. Results from this study further support the ferret as a model for EBOV disease following oral and nasal mucosa exposure.

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity.

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

Filovirus Virulence in Interferon α/ß and γ Double Knockout Mice, and Treatment with Favipiravir.

The 2014 Ebolavirus outbreak in West Africa highlighted the need for vaccines and therapeutics to prevent and treat filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would facilitate the screening of anti-filovirus agents. To that end, we characterized knockout mice lacking α/ß and γ interferon receptors (IFNAGR KO) as a model for wild-type filovirus infection. Intraperitoneal challenge of IFNAGR KO mice with several known human pathogenic species from the genus Ebolavirus and Marburgvirus, except Bundibugyo ebolavirus and Taï Forest ebolavirus, caused variable mortality rate. Further characterization of the prototype Ebola virus Kikwit isolate infection in this KO mouse model showed 100% lethality down to a dilution equivalent to 1.0 × 10-1 pfu with all deaths occurring between 7 and 9 days post-challenge. Viral RNA was detectable in serum after challenge with 1.0 × 10² pfu as early as one day after infection. Changes in hematology and serum chemistry became pronounced as the disease progressed and mirrored the histological changes in the spleen and liver that were also consistent with those described for patients with Ebola virus disease. In a proof-of-principle study, treatment of Ebola virus infected IFNAGR KO mice with favipiravir resulted in 83% protection. Taken together, the data suggest that IFNAGR KO mice may be a useful model for early screening of anti-filovirus medical countermeasures.

Zika virus infection elicits auto-antibodies to C1q.

Zika virus (ZIKV) causes mostly asymptomatic infection or mild febrile illness. However, with an increasing number of patients, various clinical features such as microcephaly, Guillain-Barré syndrome and thrombocytopenia have also been reported. To determine which host factors are related to pathogenesis, the E protein of ZIKV was analyzed with the Informational Spectrum Method, which identifies common information encoded by primary structures of the virus and the respective host protein. The data showed that the ZIKV E protein and the complement component C1q cross-spectra are characterized by a single dominant peak at the frequency F = 0.338, suggesting similar biological properties. Indeed, C1q-specific antibodies were detected in sera obtained from mice and monkeys infected with ZIKV. As C1q has been known to be involved not only in immunity, but also in synaptic organization and different autoimmune diseases, a ZIKV-induced anti-C1q antibody response may contribute to the neurological complications. These findings might also be exploited for the design of safe and efficacious vaccines in the future.

Merimepodib, an IMPDH inhibitor, suppresses replication of Zika virus and other emerging viral pathogens.

Zika virus (ZIKV), a member of the Flaviviridae family, has recently been linked to abnormal pregnancies, fetal death, microcephaly, and Guillain-Barré syndrome in humans. Merimepodib (MMPD, VX-497), a potent inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH), has shown antiviral activity against HCV and a variety of DNA and RNA viruses in vitro. In this report, we expand the antiviral spectrum of MMPD, and demonstrate that MMPD inhibits ZIKV RNA replication with an EC50 of 0.6 µM. Furthermore, MMPD reduces the virus production of ZIKV as well as several other important emerging viral pathogens such as Ebola, Lassa, Chikungunya, and Junin viruses. The inhibition can be reversed by addition of exogenous guanosine to culture media, consistent with the mechanism of action of MMPD as an IMPDH inhibitor. We also provide evidence that MMPD can be used in combination with other antivirals such as ribavirin and T-705 (favipiravir) to enhance suppression of virus production.

Mouse Model of Neurological Complications Resulting from Encephalitic Alphavirus Infection.

Long-term neurological complications, termed sequelae, can result from viral encephalitis, which are not well understood. In human survivors, alphavirus encephalitis can cause severe neurobehavioral changes, in the most extreme cases, a schizophrenic-like syndrome. In the present study, we aimed to adapt an animal model of alphavirus infection survival to study the development of these long-term neurological complications. Upon low-dose infection of wild-type C57B/6 mice, asymptomatic and symptomatic groups were established and compared to mock-infected mice to measure general health and baseline neurological function, including the acoustic startle response and prepulse inhibition paradigm. Prepulse inhibition is a robust operational measure of sensorimotor gating, a fundamental form of information processing. Deficits in prepulse inhibition manifest as the inability to filter out extraneous sensory stimuli. Sensory gating is disrupted in schizophrenia and other mental disorders, as well as neurodegenerative diseases. Symptomatic mice developed deficits in prepulse inhibition that lasted through 6 months post infection; these deficits were absent in asymptomatic or mock-infected groups. Accompanying prepulse inhibition deficits, symptomatic animals exhibited thalamus damage as visualized with H&E staining, as well as increased GFAP expression in the posterior complex of the thalamus and dentate gyrus of the hippocampus. These histological changes and increased GFAP expression were absent in the asymptomatic and mock-infected animals, indicating that glial scarring could have contributed to the prepulse inhibition phenotype observed in the symptomatic animals. This model provides a tool to test mechanisms of and treatments for the neurological sequelae of viral encephalitis and begins to delineate potential explanations for the development of such sequelae post infection.

The Ectodomain of Glycoprotein from the Candid#1 Vaccine Strain of Junin Virus Rendered Machupo Virus Partially Attenuated in Mice Lacking IFN-αß/γ Receptor.

Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC.

Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic.

UNLABELLED: Machupo virus (MACV) is the causative agent of Bolivian hemorrhagic fever. Our previous study demonstrated that a MACV strain with a single amino acid substitution (F438I) in the transmembrane domain of glycoprotein is attenuated but genetically unstable in mice. MACV is closely related to Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. Others and our group have identified the glycoprotein to be the major viral factor determining JUNV attenuation. In this study, we tested the compatibility of the glycoprotein of the Candid#1 live-attenuated vaccine strain of JUNV in MACV replication and its ability to attenuate MACV in vivo. Recombinant MACV with the Candid#1 glycoprotein (rMACV/Cd#1-GPC) exhibited growth properties similar to those of Candid#1 and was genetically stable in vitro. In a mouse model of lethal infection, rMACV/Cd#1-GPC was fully attenuated, more immunogenic than Candid#1, and fully protective against MACV infection. Therefore, the MACV strain expressing the glycoprotein of Candid#1 is safe, genetically stable, and highly protective against MACV infection in a mouse model. IMPORTANCE: Currently, there are no FDA-approved vaccines and/or treatments for Bolivian hemorrhagic fever, which is a fatal human disease caused by MACV. The development of antiviral strategies to combat viral hemorrhagic fevers, including Bolivian hemorrhagic fever, is one of the top priorities of the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Here, we demonstrate for the first time that MACV expressing glycoprotein of Candid#1 is a safe, genetically stable, highly immunogenic, and protective vaccine candidate against Bolivian hemorrhagic fever.

The glycoprotein precursor gene of Junin virus determines the virulence of the Romero strain and the attenuation of the Candid #1 strain in a representative animal model of Argentine hemorrhagic fever.

UNLABELLED: The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 (Can) strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective, and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV (rJUNV) strains encoding different gene combinations of the pathogenic Romero (Rom) and attenuated Can strains of JUNV. All strains of rJUNV exhibited in vitro growth kinetics similar to those of their parental counterparts. Analysis of virulence of the rJUNV in a guinea pig model of lethal infection that closely reproduces the features of AHF identified the envelope glycoproteins (GPs) as the major determinants of pathogenesis and attenuation of JUNV. Accordingly, rJUNV strains expressing the full-length GPs of Rom and Can exhibited virulent and attenuated phenotypes, respectively, in guinea pigs. Mutation F427I in the transmembrane region of JUNV envelope glycoprotein GP2 has been shown to attenuate the neurovirulence of JUNV in suckling mice. We document that in the guinea pig model of AHF, mutation F427I in GP2 is also highly attenuating but insufficient to prevent virus dissemination and development of mild clinical and pathological symptoms, indicating that complete attenuation of JUNV requires additional mutations present in Can glycoprotein precursor (GPC). IMPORTANCE: Development of antiviral strategies against viral hemorrhagic fevers, including AHF, is one of the top priorities within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Live-attenuated Candid #1 strain, derived from the 44th mouse brain passage of the prototype XJ strain of JUNV, has been demonstrated to be safe, immunogenic, and highly protective and is currently licensed for human use in Argentina. However, the bases for the attenuated phenotype of Candid #1 have not been established. Therefore, the identification and functional characterization of viral genetic factors implicated in JUNV pathogenesis and attenuation would significantly improve the understanding of the molecular mechanisms underlying AHF and facilitate the development of novel antiviral strategies.

Animal Model of Sensorineural Hearing Loss Associated with Lassa Virus Infection.

UNLABELLED: 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. IMPORTANCE: Sensorineural 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.

A substitution in the transmembrane region of the glycoprotein leads to an unstable attenuation of Machupo virus.

Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype.

Rescue of a recombinant Machupo virus from cloned cDNAs and in vivo characterization in interferon (αß/γ) receptor double knockout mice.

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.

Mice lacking functional STAT1 are highly susceptible to lethal infection with Lassa virus.

Lassa fever (LF) is a potentially lethal human disease that is caused by the arenavirus Lassa virus (LASV). Annually, around 300,000 infections with up to 10,000 deaths occur in regions of Lassa fever endemicity in West Africa. Here we demonstrate that mice lacking a functional STAT1 pathway are highly susceptible to infection with LASV and develop lethal disease with pathology similar to that reported in humans.

In vivo imaging systems (IVIS) detection of a neuro-invasive encephalitic virus.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.