[Show abstract][Hide abstract] ABSTRACT: Background
Crimean Congo hemorrhagic fever (CCHF) is a tick-borne hemorrhagic zoonosis associated with high mortality. Pathogenesis studies and the development of vaccines and antivirals against CCHF have been severely hampered by the lack of suitable animal model. We recently developed and characterized a mature mouse model for CCHF using mice carrying STAT1 knockout (KO).
Given the importance of interferons in controlling viral infections, we investigated the expression of interferon pathway-associated genes in KO and wild-type (WT) mice challenged with CCHF virus. We expected that the absence of the STAT1 protein would result in minimal expression of IFN-related genes. Surprisingly, the KO mice showed high levels of IFN-stimulated gene expression, beginning on day 2 post-infection, while in WT mice challenged with virus the same genes were expressed at similar levels on day 1.
We conclude that CCHF virus induces similar type I IFN responses in STAT1 KO and WT mice, but the delayed response in the KO mice permits rapid viral dissemination and fatal illness.
[Show abstract][Hide abstract] ABSTRACT: The continuing use of high-throughput assays to investigate cellular responses to infection is providing a large repository of information. Due to the large number of differentially expressed transcripts, often running into the thousands, the majority of these data have not been thoroughly investigated. Advances in techniques for the downstream analysis of high-throughput datasets are providing additional methods for the generation of additional hypotheses for further investigation. The large number of experimental observations, combined with databases that correlate particular genes and proteins with canonical pathways, functions and diseases, allows for the bioinformatic exploration of functional networks that may be implicated in replication or pathogenesis. Herein, we provide an example of how analysis of published high-throughput datasets of cellular responses to hemorrhagic fever virus infection can generate additional functional data. We describe enrichment of genes involved in metabolism, post-translational modification and cardiac damage; potential roles for specific transcription factors and a conserved involvement of a pathway based around cyclooxygenase-2. We believe that these types of analyses can provide virologists with additional hypotheses for continued investigation.
[Show abstract][Hide abstract] ABSTRACT: Vaccination is currently the most effective strategy to medically control viral diseases. However, developing vaccines is a long and expensive process, and traditional methods, such as attenuating wild-type viruses by serial passage, may not be suitable for all viruses and may lead to vaccine safety considerations, particularly in the case of the vaccination of particular patient groups, such as the immunocompromised and the elderly. In particular, developing vaccines against emerging viral pathogens adds a further level of complexity, as they may only be administered to small groups of people or only in response to a specific event or threat, limiting our ability to study and evaluate responses. In this commentary, we discuss how novel techniques may be used to engineer a new generation of vaccine candidates as we move toward a more targeted vaccine design strategy, driven by our understanding of the mechanisms of viral pathogenesis, attenuation and the signaling events which are required to develop a lasting, protective immunity. We will also briefly discuss the potential future role of vaccine adjuvants, which could be used to bridge the gap between vaccine safety, and lasting immunity from a single vaccination.
[Show abstract][Hide abstract] ABSTRACT: Developing vaccines to biothreat agents presents a number of challenges for discovery, preclinical development, and licensure. The need for high containment to work with live agents limits the
amount and types of research that can be done using complete pathogens, and small markets reduce potential returns for industry. However, a number of tools, from comparative pathogenesis of viral strains at the molecular level to novel computational approaches, are being used to understand the basis of viral attenuation and characterize protective immune responses. As the amount of basic molecular knowledge grows, we will be able to take advantage of these tools not only to rationally attenuate virus strains for candidate vaccines, but also to assess immunogenicity and safety in silico. This review discusses how a basic understanding of pathogenesis, allied with systems biology and machine learning methods, can impact biodefense vaccinology.
BioMed Research International 06/2010; 2010(1110-7243):236528. DOI:10.1155/2010/236528 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The arenaviruses include a number of important pathogens including Lassa virus and Junin virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaviruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection, and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-alpha. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease.
BioMed Research International 04/2010; 2010. DOI:10.1155/2010/956823 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the last 10 years new concerns have arisen about safety of the live, attenuated yellow fever (YF) 17D vaccine, in particular viscerotropic adverse events, which have a case-fatality rate of 64%. A non-replicating cell culture-based vaccine would not cause these adverse events, and potentially could be used in persons with precautions or contraindications to use of the live vaccine, including age <9 months and >60 years, egg allergy, immune suppression, and pregnancy. We developed a whole virion vaccine from the 17D strain inactivated with beta-propiolactone, and adsorbed to aluminum hydroxide. The inactivated vaccine was highly immunogenic in mice, hamsters, and cynomolgus macaques. After a single dose in hamsters and macaques, neutralizing antibody titers were similar to those elicited by the live 17D vaccine (YF-VAX, Sanofi Pasteur). After two doses of inactivated vaccine, neutralizing antibody titers in hamsters were significantly higher than after a single dose of YF-VAX [geometric mean titer (GMT) 20,480 vs. 1940, respectively (P<0.001, ANOVA)]. Hamsters given a single dose or two doses of inactivated vaccine or a single dose of YF-VAX were fully protected against hepatitis, viremia, weight loss and death after challenge with YF virus (Jimenez strain). A clinical trial of the inactivated vaccine (XRX-001) has been initiated.
[Show abstract][Hide abstract] ABSTRACT: Lassa virus pathogenesis is believed to involve dysregulation of cytokines. We have previously shown nuclear factor-kappaB (NF-kappaB) inhibition using a BSL-2 model for Lassa fever. Here we further define the potential mechanism for NF-kappaB inhibition as involving increased levels of repressive p50/p50 homodimers, and suggest a novel therapeutic strategy that acts via modulation of host signaling.
[Show abstract][Hide abstract] ABSTRACT: Immunosuppression following infection with HIV-1 predisposes patients to a myriad of opportunistic pathogens, one of the most important of which is Mtb. Granulysin, expressed by NK cells and CTL, exhibits potent antimicrobial activity against Mtb and several other opportunistic pathogens associated with HIV-1 infection. The immune signals that promote granulysin expression in human CTL are not fully understood. Using primary human CD8+ T cells, in this study, we identify IL-21 as a strong inducer of granulysin, demonstrate that IL-21 and IL-15 activate granulysin expression within CD8+ CD45RO+ T cells, and establish a role for Jak/STAT signaling in the regulation of granulysin within CD8+ T cells. We show that infection of PBMC from healthy donors in vitro with HIV-1 suppresses granulysin expression by CD8+ T cells, concomitant with reduced p-STAT3 and p-STAT5, following activation with IL-15 and IL-21. Of note, simultaneous signaling through IL-15 and IL-21 could partially overcome the immunosuppressive effects of HIV-1 on granulysin expression by CD8+ T cells. These results suggest that HIV-1 infection of PBMC may reduce the antimicrobial profile of activated CD8+ T cells by disrupting signaling events that are critical for the induction of granulysin. Understanding the effects of HIV-1 on CD8+ T cell activation is essential to understanding the physiological basis for inadequate cytotoxic lymphocyte activity in HIV+ patients and for informed guidance of cytokine-based therapy to restore T cell function.
[Show abstract][Hide abstract] ABSTRACT: Arenaviruses are important emerging pathogens and include a number of hemorrhagic fever viruses classified as NIAID category A priority pathogens and CDC potential biothreat agents. Infection of guinea pigs with the New World arenavirus Pichindé virus (PICV) has been used as a biosafety level 2 model for the Lassa virus. Despite continuing research, little is known about the molecular basis of pathogenesis, and this has hindered the design of novel antiviral therapeutics. Modulation of the host response is a potential strategy for the treatment of infectious diseases. We have previously investigated the global host response to attenuated and lethal arenavirus infections by using high-throughput immunoblotting and kinomics approaches. In this report, we describe the differential nuclear proteomes of a murine cell line induced by mock infection and infection with attenuated and lethal variants of PICV, investigated by using two-dimensional gel electrophoresis. Spot identification using tandem mass spectrometry revealed the involvement of a number of proteins that regulate inflammation via potential modulation of NF-kappaB activity and of several heterogeneous nuclear ribonuclear proteins. Pathway analysis revealed a potential role for transcription factor XBP-1, a transcription factor involved in major histocompatibility complex II (MHC-II) expression; differential DNA-binding activity was revealed by electrophoretic mobility shift assay, and differences in surface MHC-II expression were seen following PICV infection. These data are consistent with the results of several previous studies and highlight potential differences between transcriptional and translational regulation. This study provides a number of differentially expressed targets for further research and suggests that key events in pathogenesis may be established early in infection.
Journal of Virology 12/2008; 83(2):687-700. DOI:10.1128/JVI.01281-08 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arenaviruses are rodent-borne RNA viruses, and some have the capacity to cause hemorrhagic fever and death in infected individuals and thus have been identified as a potential bioterrorism threat. Ribavirin and supportive care are currently the approved therapeutic options for individuals suffering from arenavirus-induced hemorrhagic fever. However, new research has suggested that immune plasma treatment or kinase inhibitors may provide a therapeutic option for treating arenavirus infections in humans. This article puts forth a perspective as to the potential use of kinase inhibitors as an antiviral therapeutic for arenavirus infections.
[Show abstract][Hide abstract] ABSTRACT: Arenaviridae is a family of enveloped viruses some of which are capable of causing hemorrhagic fever syndromes in humans. In this report, we demonstrate that treatment of host cells with the tyrosine kinase inhibitor genistein inhibits infection of cells with the New World arenavirus Pichindé (PICV). The greatest degree of inhibition was observed in pre-treated target cells, but modest inhibition of infection was also seen when drug was added to cultures up to 48h after infection. We show that PICV-induced phosphorylation of the activating transcription factor-2 protein (ATF-2) and cyclic adenosine monophosphate response element binding protein (CREB) is inhibited following genistein treatment. Lastly, genistein treatment also inhibited transduction of cells with pseudotyped retrovirus particles expressing envelope proteins of the Old World arenavirus Lassa virus. These results demonstrate that kinase activity is required for arenavirus infection and that therapeutics designed to inhibit kinase activity should be explored.
Antiviral Research 03/2008; 77(2):153-6. DOI:10.1016/j.antiviral.2007.09.005 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study examined nuclear and cytoplasmic shuttling of the African swine fever virus (ASFV) A238L protein, which is an inhibitor of NF-kappaB and of calcineurin phosphatase. The results showed that the protein was present in both the nucleus and the cytoplasm in ASFV-infected cells and that the higher molecular mass 32 kDa form of the A238L protein was the predominant nuclear form, which accumulated later in infection. In contrast, both the 28 and 32 kDa forms of the A238L protein were present in the cytoplasm. The A238L protein was actively imported into the nucleus and exported by a CRM1-mediated pathway, although a pool of the protein remained in the cytoplasm and did not enter the nucleus. By using a recombinant ASFV from which the A238L gene had been deleted, it was shown that expression of A238L did not inhibit nuclear import of the NF-kappaB p50 or p65 subunit and did not inhibit nuclear export of p65 by a CRM1-mediated pathway. The results were consistent with a model in which A238L functions within both the nucleus and the cytoplasm.
Journal of General Virology 03/2007; 88(Pt 2):411-9. DOI:10.1099/vir.0.82358-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphorylation plays a key role in regulating many signaling pathways. Although studies investigating the phosphorylated forms of signaling pathways are now commonplace, global analysis of protein phosphorylation and kinase activity has lagged behind genomics and proteomics. We have used a kinomics approach to study the effect of virus infection on host cell signaling in infected guinea pigs. Delineating the host responses which lead to clearance of a pathogen requires the use of a matched, comparative model system. We have used two passage variants of the arenavirus Pichinde, used as a biosafety level 2 model of Lassa fever virus as it produces similar pathologies in guinea pigs and humans, to compare the host cell responses between infections which lead to either a mild, self-limiting infection or lethal disease. Using this model, we can begin to understand the differences in signaling events which give rise to these markedly different outcomes. By contextualizing these data using pathway analysis, we have identified key differences in cellular signaling matrices. By comparing these differentially involved networks, we have identified a number of key signaling "nodes" which show differential phosphorylations between mild and lethal infections. We believe that these nodes provide potential targets for the development of antiviral therapies by acting at the level of the host response rather than by directly targeting viral proteins.
Journal of Virology 03/2007; 81(4):1923-33. DOI:10.1128/JVI.02199-06 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic
fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new
antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants
of Pichinde virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead
to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified
key intermediates which may be targets for therapeutic intervention.
Journal of Virology 11/2006; 80(20):10248-52. DOI:10.1128/JVI.01384-06 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific "on-chip" captured proteins have been obtained that were pure enough to do "on-chip" digestion and the subsequent identification of the "on-chip" bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays.
Biochemical and Biophysical Research Communications 10/2006; 347(3):586-93. DOI:10.1016/j.bbrc.2006.06.132 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: African swine fever virus (ASFV) can cause an acutely fatal haemorrhagic fever in domestic pigs although in its natural hosts, warthogs, bushpigs and the soft tick vector, Ornithodoros moubata, ASFV causes inapparent persistent infections. The virus is a large, cytoplasmic, double-stranded DNA virus which has a tropism for macrophages. As it is the only member of the Asfarviridae family, ASFV encodes many novel genes not encoded by other virus families. The ability of the virus to persist in its natural hosts and in domestic pigs, which recover from infection with less virulent isolates, shows that the virus has effective mechanisms to evade host defence systems. This review focuses on recent progress made in understanding the function of ASFV-encoded proteins, which are involved in modulating the host response to infection. Growing evidence suggests that a major strategy used by the virus is to modulate signalling pathways in infected macrophages, thus interfering with the expression of a large number of immunomodulatory genes. One potent immunomodulatory protein, A238L, inhibits both activation of the host NFkappaB transcription factor and inhibits calcineurin phosphatase activity. Calcineurin-dependent pathways, including activation of the NFAT transcription factor, are therefore inhibited. Another ASFV-encoded protein, CD2v, resembles the host CD2 protein, which is expressed on T cells and NK cells. This virus protein causes the adsorption of red blood cells around virus-infected cells and extracellular virus particles. Expression of the CD2v protein aids virus dissemination in pigs and the protein also has a role in impairing bystander lymphocyte function. This may be mediated either by a direct interaction of CD2v extracellular domain with ligands on lymphocytes or by an indirect mechanism involving interaction of the CD2v cytoplasmic tail with host proteins involved in signalling or trafficking pathways. Two ASFV proteins, an IAP and a Bcl2 homologue, inhibit apoptosis in infected cells and thus facilitate production of progeny virions. The prediction is that half to two-thirds of the approximately 150 genes encoded by ASFV are not essential for replication in cells but have an important role for virus survival and transmission in its hosts. These genes provide an untapped repository, and will be valuable tools for deciphering not only how the virus manipulates the host response to infection to avoid elimination, but also useful for understanding important host anti-viral mechanisms. In addition, they may provide leads for discovery of novel immunomodulatory drugs.