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1.  Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain 
Infection and Immunity  2014;82(5):2098-2105.
Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.
PMCID: PMC3993426  PMID: 24614653
2.  Aerosolized Rift Valley Fever Virus Causes Fatal Encephalitis in African Green Monkeys and Common Marmosets 
Journal of Virology  2013;88(4):2235-2245.
Rift Valley fever (RVF) is a veterinary and human disease in Africa and the Middle East. The causative agent, RVF virus (RVFV), can be naturally transmitted by mosquito, direct contact, or aerosol. We sought to develop a nonhuman primate (NHP) model of severe RVF in humans to better understand the pathogenesis of RVF and to use for evaluation of medical countermeasures. NHP from four different species were exposed to aerosols containing RVFV. Both cynomolgus and rhesus macaques developed mild fevers after inhalation of RVFV, but no other clinical signs were noted and no macaque succumbed to RVFV infection. In contrast, both marmosets and African green monkeys (AGM) proved susceptible to aerosolized RVF virus. Fever onset was earlier with the marmosets and had a biphasic pattern similar to what has been reported in humans. Beginning around day 8 to day 10 postexposure, clinical signs consistent with encephalitis were noted in both AGM and marmosets; animals of both species succumbed between days 9 and 11 postexposure. Marmosets were susceptible to lower doses of RVFV than AGM. Histological examination confirmed viral meningoencephalitis in both species. Hematological analyses indicated a drop in platelet counts in both AGM and marmosets suggestive of thrombosis, as well as leukocytosis that consisted mostly of granulocytes. Both AGM and marmosets would serve as useful models of aerosol infection with RVFV.
PMCID: PMC3911574  PMID: 24335307
3.  Broad Spectrum Antiviral Activity of Favipiravir (T-705): Protection from Highly Lethal Inhalational Rift Valley Fever 
Development of antiviral drugs that have broad-spectrum activity against a number of viral infections would be of significant benefit. Due to the evolution of resistance to currently licensed antiviral drugs, development of novel anti-influenza drugs is in progress, including Favipiravir (T-705), which is currently in human clinical trials. T-705 displays broad-spectrum in vitro activity against a number of viruses, including Rift Valley Fever virus (RVFV). RVF is an important neglected tropical disease that causes human, agricultural, and economic losses in endemic regions. RVF has the capacity to emerge in new locations and also presents a potential bioterrorism threat. In the current study, the in vivo efficacy of T-705 was evaluated in Wistar-Furth rats infected with the virulent ZH501 strain of RVFV by the aerosol route.
Methodology/Principal Findings
Wistar-Furth rats are highly susceptible to a rapidly lethal disease after parenteral or inhalational exposure to the pathogenic ZH501 strain of RVFV. In the current study, two experiments were performed: a dose-determination study and a delayed-treatment study. In both experiments, all untreated control rats succumbed to disease. Out of 72 total rats infected with RVFV and treated with T-705, only 6 succumbed to disease. The remaining 66 rats (92%) survived lethal infection with no significant weight loss or fever. The 6 treated rats that succumbed survived significantly longer before succumbing to encephalitic disease.
Currently, there are no licensed antiviral drugs for treating RVF. Here, T-705 showed remarkable efficacy in a highly lethal rat model of Rift Valley Fever, even when given up to 48 hours post-infection. This is the first study to show protection of rats infected with the pathogenic ZH501 strain of RVFV. Our data suggest that T-705 has potential to be a broad-spectrum antiviral drug.
Author Summary
Broad-spectrum antiviral drugs are preferred because they have the capacity to treat a range of viral illnesses rather than just one. Food and Drug Administration (FDA) approval of antiviral drugs to treat neglected tropical diseases is difficult to obtain due to ethical and logistical considerations when conducting human clinical trials. Rift Valley Fever (RVF) is an endemic tropical disease that causes human morbidity and mortality, as well as economic damage to the livestock industry. There are no licensed antiviral drugs to treat RVF. In this study, we found that a novel anti-influenza drug, Favipiravir (T-705), is able to prevent lethal RVF in rats, and therefore shows promise as a broad-spectrum antiviral treatment.
PMCID: PMC3983105  PMID: 24722586
4.  Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with Ebola and Marburg viruses 
Vaccine  2008;26(52):6894-6900.
Considerable progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against Ebola and Marburg viruses. A vaccine based on recombinant vesicular stomatitis virus (VSV) seems to be particularly robust as it can also confer protection when administered as a postexposure treatment. While filoviruses are not thought to be transmitted by aerosol in nature the inhalation route is among the most likely portals of entry in the setting of a bioterrorist event. At present, all candidate filoviral vaccines have been evaluated against parenteral challenges but none have been tested against an aerosol exposure. Here, we evaluated our recombinant VSV-based Zaire ebolavirus (ZEBOV) and Marburg virus (MARV) vaccines against aerosol challenge in cynomolgus macaques. All monkeys vaccinated with a VSV vector expressing the glycoprotein of ZEBOV were completely protected against an aerosol exposure of ZEBOV. Likewise, all monkeys vaccinated with a VSV vector expressing the glycoprotein of MARV were completely protected against an aerosol exposure of MARV. All control animals challenged by the aerosol route with either ZEBOV or MARV succumbed. Interestingly, disease in control animals appeared to progress slower than previously seen in macaques exposed to comparable doses by intramuscular injection.
PMCID: PMC3398796  PMID: 18930776
Ebola virus; Marburg virus; Filovirus; Nonhuman primates; Aerosol; Vaccines
5.  Choice of inbred rat strain impacts lethality and disease course after respiratory infection with Rift Valley Fever Virus 
Humans infected with Rift Valley Fever Virus (RVFV) generally recover after a febrile illness; however, a proportion of patients progress to a more severe clinical outcome such as hemorrhagic fever or meningoencephalitis. RVFV is naturally transmitted to livestock and humans by mosquito bites, but it is also infectious through inhalational exposure, making it a potential bioterror weapon. To better understand the disease caused by inhalation of RVFV, Wistar-Furth, ACI, or Lewis rats were exposed to experimental aerosols containing virulent RVFV. Wistar-Furth rats developed a rapidly progressing lethal hepatic disease after inhalational exposure; ACI rats were 100-fold less susceptible and developed fatal encephalitis after infection. Lewis rats, which do not succumb to parenteral inoculation with RVFV, developed fatal encephalitis after aerosol infection. RVFV was found in the liver, lung, spleen, heart, kidney and brain of Wistar Furth rats that succumbed after aerosol exposure. In contrast, RVFV was found only in the brains of ACI or Lewis rats that succumbed after aerosol exposure. Lewis rats that survived s.c. infection were not protected against subsequent re-challenge by aerosol exposure to the homologous virus. This is the first side-by-side comparison of the lethality and pathogenesis of RVFV in three rat strains after aerosol exposure and the first step toward developing a rodent model suitable for use under the FDA Animal Rule to test potential vaccines and therapeutics for aerosol exposure to RVFV.
PMCID: PMC3417668  PMID: 22919694
Rift Valley Fever Virus; aerosol exposure; respiratory infection; LD50; inbred rat strain
6.  Growth conditions and environmental factors impact aerosolization but not virulence of Francisella tularensis infection in mice 
In refining methodology to develop a mouse model for inhalation of Francisella tularensis, it was noted that both relative humidity and growth media impacted the aerosol concentration of the live vaccine strain (LVS) of F. tularensis. A relative humidity of less than 55% had a negative impact on the spray factor, the ratio between the concentration of LVS in the aerosol and the nebulizer. The spray factor was significantly higher for LVS grown in brain heart infusion (BHI) broth than LVS grown in Mueller–Hinton broth (MHb) or Chamberlain's chemically defined medium (CCDM). The variability between aerosol exposures was also considerably less with BHI. LVS grown in BHI survived desiccation far longer than MHb-grown or CCDM-grown LVS (~70% at 20 min for BHI compared to <50% for MHb and CCDM). Removal of the capsule by hypertonic treatment impacted the spray factor for CCDM-grown LVS or MHb-grown LVS but not BHI-grown LVS, suggesting the choice of culture media altered the adherence of the capsule to the cell membrane. The choice of growth media did not impact the LD50 of LVS but the LD99 of BHI-grown LVS was 1 log lower than that for MHb-grown LVS or CCDM-grown LVS. Splenomegaly was prominent in mice that succumbed to MHb- and BHI-grown LVS but not CCDM-grown LVS. Environmental factors and growth conditions should be evaluated when developing new animal models for aerosol infection, particularly for vegetative bacterial pathogens.
PMCID: PMC3468843  PMID: 23087911
tularemia; Francisella tularensis; respiratory infection; aerosol exposure; mice
7.  Pneumonic Tularemia in Rabbits Resembles the Human Disease as Illustrated by Radiographic and Hematological Changes after Infection 
PLoS ONE  2011;6(9):e24654.
Pneumonic tularemia is caused by inhalation of the gram negative bacterium, Francisella tularensis. Because of concerns that tularemia could be used as a bioterrorism agent, vaccines and therapeutics are urgently needed. Animal models of pneumonic tularemia with a pathophysiology similar to the human disease are needed to evaluate the efficacy of these potential medical countermeasures.
Principal Findings
Rabbits exposed to aerosols containing Francisella tularensis strain SCHU S4 developed a rapidly progressive fatal pneumonic disease. Clinical signs became evident on the third day after exposure with development of a fever (>40.5°C) and a sharp decline in both food and water intake. Blood samples collected on day 4 found lymphopenia and a decrease in platelet counts coupled with elevations in erythrocyte sedimentation rate, alanine aminotransferase, cholesterol, granulocytes and monocytes. Radiographs demonstrated the development of pneumonia and abnormalities of intestinal gas consistent with ileus. On average, rabbits were moribund 5.1 days after exposure; no rabbits survived exposure at any dose (190–54,000 cfu). Gross evaluation of tissues taken at necropsy showed evidence of pathology in the lungs, spleen, liver, kidney and intestines. Bacterial counts confirmed bacterial dissemination from the lungs to the liver and spleen.
The pathophysiology of pneumonic tularemia in rabbits resembles what has been reported for humans. Rabbits therefore are a relevant model of the human disease caused by type A strains of F. tularensis.
PMCID: PMC3172242  PMID: 21931798
8.  Telemetric analysis to detect febrile responses in mice following vaccination with a live-attenuated virus vaccine 
Vaccine  2009;27(49):6814-6823.
Nonhuman primates (NHP) are considered to be the most appropriate model for predicting how humans will respond to many infectious diseases. Due to ethical and monetary concerns associated with the use of NHP, rodent models that are as predictive of responses likely to be seen in human vaccine recipients are warranted. Using implanted telemetry devices, body temperature and activity were monitored in inbred and outbred mouse strains following administration of the live-attenuated vaccine for Venezuelan equine encephalitis virus (VEEV), V3526. Following analysis of individual mouse data, only outbred mouse strains showed changes in diurnal temperature and activity profiles following vaccination. Similar changes were observed following VEEV challenge of vaccinated outbred mice. From these studies, we conclude, outbred mouse strains implanted with telemeters are a sensitive model for predicting responses in humans following vaccination.
PMCID: PMC2783281  PMID: 19761841
vaccine; mouse; telemetry
9.  Pathogenesis of aerosolized Eastern Equine Encephalitis virus infection in guinea pigs 
Virology Journal  2009;6:170.
Mice and guinea pigs were experimentally exposed to aerosols containing regionally-distinct strains (NJ1959 or ArgM) of eastern equine encephalitis virus (EEEV) at two exclusive particle size distributions. Mice were more susceptible to either strain of aerosolized EEEV than were guinea pigs; however, clinical signs indicating encephalitis were more readily observed in the guinea pigs. Lower lethality was observed in both species when EEEV was presented at the larger aerosol distribution (> 6 μm), although the differences in the median lethal dose (LD50) were not significant. Virus isolation and immunohistochemistry indicated that virus invaded the brains of guinea pigs within one day postexposure, regardless of viral strain or particle size distribution. Immunohistochemistry further demonstrated that neuroinvasion occurred through the olfactory system, followed by transneuronal spread to all regions of the brain. Olfactory bipolar neurons and neurons throughout the brain were the key viral targets. The main microscopic lesions in infected guinea pigs were neuronal necrosis, inflammation of the meninges and neuropil of the brain, and vasculitis in the brain. These results indicate that guinea pigs experimentally infected by aerosolized EEEV recapitulate several key features of fatal human infection and thus should serve as a suitable animal model for aerosol exposure to EEEV.
PMCID: PMC2770496  PMID: 19852817
10.  Identification of a Surrogate Marker for Infection in the African Green Monkey Model of Inhalation Anthrax▿  
Infection and Immunity  2008;76(12):5790-5801.
In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.
PMCID: PMC2583550  PMID: 18852240
11.  The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever 
Genome Biology  2007;8(8):R174.
Primate blood cells were analysed for changes in global gene expression patterns at several time points following infection with Ebola virus, providing insights into potential mechanisms of viral pathogenesis and host defense.
Infection with Ebola virus (EBOV) causes a fulminant and often fatal hemorrhagic fever. In order to improve our understanding of EBOV pathogenesis and EBOV-host interactions, we examined the molecular features of EBOV infection in vivo.
Using high-density cDNA microarrays, we analyzed genome-wide host expression patterns in sequential blood samples from nonhuman primates infected with EBOV. The temporal program of gene expression was strikingly similar between animals. Of particular interest were features of the data that reflect the interferon response, cytokine signaling, and apoptosis. Transcript levels for tumor necrosis factor-α converting enzyme (TACE)/α-disintegrin and metalloproteinase (ADAM)-17 increased during days 4 to 6 after infection. In addition, the serum concentration of cleaved Ebola glycoprotein (GP2 delta) was elevated in late-stage EBOV infected animals. Of note, we were able to detect changes in gene expression of more than 300 genes before symptoms appeared.
These results provide the first genome-wide ex vivo analysis of the host response to systemic filovirus infection and disease. These data may elucidate mechanisms of viral pathogenesis and host defense, and may suggest targets for diagnostic and therapeutic development.
PMCID: PMC2375004  PMID: 17725815

Results 1-11 (11)