Cell-specific image-guided transcriptomics identifies complex injuries caused by ischemic acute kidney injury in mice.

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.

The efficacy of poly-ICLC against Ebola-Zaire virus (EBOV) infection in mice and cynomolgus monkeys.

The potential protection of poly-ICLC (Hiltonol®) a double stranded RNA (dsRNA) against EBOV infection was assessed with prophylactic and therapeutic administration to wild type and TLR3-negative mice, and in non-human primates (NHPs) by measuring EBOL serum titers, survival extension, and serum liver and kidney function markers. Various doses of aqueous and liposomal poly-ICLC monotherapy provided robust protection in otherwise lethal murine EBOV challenge models, when treatment is started on the day 0 or one day after virus challenge. There was no advantage of liposomal vs. the aqueous poly-ICLC form. Protection appeared to be independent of TLR-3. NHPs treated with poly-ICLC and challenged with EBOV survived longer but eventually succumbed to Ebola infection. Nevertheless, the liver and kidney serum markers were markedly reduced in the infected and treated NHPs. In the two longest surviving poly-ICLC- treated NHPs, the day 10 serum EBOV titer was reduced 2.1 and 30 fold respectively.

Systems biology for organotypic cell cultures.

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, "organotypic" cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

The cyanobacterial lectin scytovirin displays potent in vitro and in vivo activity against Zaire Ebola virus.

The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein's mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC50) of 50 nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC50. Injected subcutaneously in mice, SVN reached a peak plasma level of 100 nm in 45 min, but was cleared within 4h. When ZEBOV-infected mice were given 30 mg/kg/day of SVN by subcutaneous injection every 6h, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70-90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections.

Interferon-ß therapy prolongs survival in rhesus macaque models of Ebola and Marburg hemorrhagic fever.

There is a clear need for novel, effective therapeutic approaches to hemorrhagic fever due to filoviruses. Ebola virus hemorrhagic fever is associated with robust interferon (IFN)-α production, with plasma concentrations of IFN-α that greatly (60- to 100-fold) exceed those seen in other viral infections, but little IFN-ß production. While all of the type I IFNs signal through the same receptor complex, both quantitative and qualitative differences in biological activity are observed after stimulation of the receptor complex with different type I IFNs. Taken together, this suggested potential for IFN-ß therapy in filovirus infection. Here we show that early postexposure treatment with IFN-ß significantly increased survival time of rhesus macaques infected with a lethal dose of Ebola virus, although it failed to alter mortality. Early treatment with IFN-ß also significantly increased survival time after Marburg virus infection. IFN-ß may have promise as an adjunctive postexposure therapy in filovirus infection.

Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone.

Mitoxantrone, an FDA-approved therapeutic for the treatment of cancer and multiple sclerosis, was previously reported to exhibit antiviral activity against vaccinia virus. To determine whether this activity extends to other orthopoxviruses, mitoxantrone was tested against cowpox and monkeypox. Mitoxantrone demonstrated an EC(50) of 0.25 µM against cowpox and 0.8 µM against monkeypox. Intraperitoneal treatment of cowpox virus-challenged C57Bl/6 mice with 0.5 mg/kg mitoxantrone resulted in 25% survival and a significant increase in survival time. In an effort to improve its efficacy, mitoxantrone was tested for synergistic activity with cidofovir. In vitro tests demonstrated significant synergy between the two drugs against cowpox; however, no synergistic effect on animal survival or median time-to-death was seen in intranasally-infected BALB/c mice. Significantly fewer animals survived when treated with a combination of 0.5 mg/kg mitoxantrone and 100 mg/kg cidofovir than with 100 mg/kg cidofovir alone. This is, to our knowledge, the first report of limited anti-orthopoxvirus activity by mitoxantrone in an animal model.

Intrabronchial inoculation of cynomolgus macaques with cowpox virus.

The public health threat of orthopoxviruses from bioterrorist attacks has prompted researchers to develop suitable animal models for increasing our understanding of viral pathogenesis and evaluation of medical countermeasures (MCMs) in compliance with the FDA Animal Efficacy Rule. We present an accessible intrabronchial cowpox virus (CPXV) model that can be evaluated under biosafety level-2 laboratory conditions. In this dose-ranging study, utilizing cynomolgus macaques, signs of typical orthopoxvirus disease were observed with the lymphoid organs, liver, skin (generally mild) and respiratory tract as target tissues. Clinical and histopathological evaluation suggests that intrabronchial CPXV recapitulated many of the features of monkeypox and variola virus, the causative agent of smallpox, infections in cynomolgus macaque models. These similarities suggest that CPXV infection in non-human primates should be pursued further as an alternative model of smallpox. Further development of the CPXV primate model, unimpeded by select agent and biocontainment restrictions, should facilitate the development of MCMs for smallpox.

Evaluation of monkeypox disease progression by molecular imaging.

Infection of nonhuman primates (NHPs) with monkeypox virus (MPXV) is currently being developed as an animal model of variola infection in humans. We used positron emission tomography and computed tomography (PET/CT) to identify inflammatory patterns as predictors for the outcome of MPXV disease in NHPs. Two NHPs were sublethally inoculated by the intravenous (IV) or intrabronchial (IB) routes and imaged sequentially using fluorine-18 fluorodeoxyglucose ((18)FDG) uptake as a nonspecific marker of inflammation/immune activation. Inflammation was observed in the lungs of IB-infected NHPs, and bilobular involvement was associated with morbidity. Lymphadenopathy and immune activation in the axillary lymph nodes were evident in IV- and IB-infected NHPs. Interestingly, the surviving NHPs had significant (18)FDG uptake in the axillary lymph nodes at the time of MPXV challenge with no clinical signs of illness, suggesting an association between preexisting immune activation and survival. Molecular imaging identified patterns of inflammation/immune activation that may allow risk assessment of monkeypox disease.

Simian hemorrhagic fever virus infection of rhesus macaques as a model of viral hemorrhagic fever: clinical characterization and risk factors for severe disease.

Simian Hemorrhagic Fever Virus (SHFV) has caused sporadic outbreaks of hemorrhagic fevers in macaques at primate research facilities. SHFV is a BSL-2 pathogen that has not been linked to human disease; as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model for hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. Here we describe the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64% with signs of hemorrhagic fever and multiple organ system involvement. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggest that SHFV may serve as a suitable model of BSL-4 pathogens.

Cowpox virus infection of cynomolgus macaques as a model of hemorrhagic smallpox.

Hemorrhagic smallpox was a rare but severe manifestation of variola virus infection that resulted in nearly 100% mortality. Here we describe intravenous (IV) inoculation of cowpox virus Brighton Red strain in cynomolgus macaques (Macaca fascicularis) which resulted in disease similar in presentation to hemorrhagic smallpox in humans. IV inoculation of macaques resulted in a uniformly lethal disease within 12 days post-inoculation in two independent experiments. Clinical observations and hematological and histopathological findings support hemorrhagic disease. Cowpox virus replicated to high levels in blood (8.0-9.0 log(10) gene copies/mL) and tissues including lymph nodes, thymus, spleen, bone marrow, and lungs. This unique model of hemorrhagic orthopoxvirus infection provides an accessible means to further study orthopoxvirus pathogenesis and to identify virus-specific and nonspecific therapies. Such studies will serve to complement the existing nonhuman primate models of more classical poxviral disease.

Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses.

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.

Infection of cynomolgus macaques with a recombinant monkeypox virus encoding green fluorescent protein.

Monkeypox virus (MPXV) causes a vesiculopustular rash illness resembling smallpox in humans and produces a similar disease in nonhuman primates. To enhance the ability of researchers to study experimental MPXV infections, we inserted a gene encoding green fluorescent protein (GFP) into Monkeypox virus Zaire-79. Wild-type and MPXV-GFP replicated with similar kinetics in cell culture and caused a similar disease when injected intravenously into cynomolgus macaques. In MPXV-GFP-infected animals, examination under fluorescent light facilitated the identification of skin lesions during disease development and internal sites of replication at necropsy. MPXV-GFP could improve the quantitative assessment of antiviral therapy and vaccine efficacy.

A novel respiratory model of infection with monkeypox virus in cynomolgus macaques.

Variola, the causative agent of smallpox, and the related monkeypox virus are both select agents that, if purposefully released, would cause public panic and social disruption. For this reason research continues in the areas of animal model and therapeutic development. Orthopoxviruses show a widely varying degree of host specificity, making development of accurate animal models difficult. In this paper, we demonstrate a novel respiratory infection technique that resulted in "classic" orthopox disease in nonhuman primates and takes the field of research one step closer to a better animal model.

Molecular imaging of influenza and other emerging respiratory viral infections.

Research on the pathogenesis and therapy of influenza and other emerging respiratory viral infections would be aided by methods that directly visualize pathophysiologic processes in patients and laboratory animals. At present, imaging of diseases, such as swine-origin H1N1 influenza, is largely restricted to chest radiograph and computed tomography (CT), which can detect pulmonary structural changes in severely ill patients but are more limited in characterizing the early stages of illness, differentiating inflammation from infection or tracking immune responses. In contrast, imaging modalities, such as positron emission tomography, single photon emission CT, magnetic resonance imaging, and bioluminescence imaging, which have become useful tools for investigating the pathogenesis of a range of disease processes, could be used to advance in vivo studies of respiratory viral infections in patients and animals. Molecular techniques might also be used to identify novel biomarkers of disease progression and to evaluate new therapies.

Comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route.

Monkeypox virus (MPXV) infection has recently expanded in geographic distribution and can be fatal in up to 10% of cases. The intravenous (i.v.) inoculation of nonhuman primates (NHPs) results in an accelerated fulminant disease course compared to that of naturally occurring MPXV infection in humans. Alternative routes of inoculation are being investigated to define an NHP model of infection that more closely resembles natural disease progression. Our goal was to determine if the intrabronchial (i.b.) exposure of NHPs to MPXV results in a systemic disease that better resembles the progression of human MPXV infection. Here, we compared the disease course following an i.v. or i.b. inoculation of NHPs with 10-fold serial doses of MPXV Zaire. Classical pox-like disease was observed in NHPs administered a high virus dose by either route. Several key events were delayed in the highest doses tested of the i.b. model compared to the timing of the i.v. model, including the onset of fever, lesion appearance, peak viremia, viral shedding in nasal and oral swabs, peak cytokine levels, and time to reach endpoint criteria. Virus distribution across 19 tissues was largely unaffected by the inoculation route at the highest doses tested. The NHPs inoculated by the i.b. route developed a viral pneumonia that likely exacerbated disease progression. Based on the observations of the delayed onset of clinical and virological parameters and endpoint criteria that may more closely resemble those of human MPXV infection, the i.b. MPXV model should be considered for the further investigation of viral pathogenesis and countermeasures.

Vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates.

Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVDeltaG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVDeltaG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.