ARBOVIRUSES

Arboviral diagnoses and strain variability

Aaron Brault, UC Davis

Abstract:
The introduction of West Nile virus into North America in 1999 has demonstrated the ability of an exotic arthropod-borne virus to become established and rapidly spread in a new environment. Importation of additional exotic arthropod-borne viruses from the Pacific Rim is a distinct possibility, yet diagnostic reagents for many of these agents are not readily available. We propose the development of new diagnostic technologies for the rapid and simultaneous testing of specimens for Category A & B list arthropod-borne viruses. These rapid and sensitive assays will allow for simultaneous screening of mosquito and vertebrate tissue samples for a panel of potentially introduced mosquito-borne viruses. Sequence diversity of RNA viruses can be problematic for the identification of viral agents by contemporary nucleotide amplification methodologies that rely on sequence specificity. In order to alleviate the potential need for sequence specific reagents, we additionally propose the development of replication defective replicon particles that express fluorescent markers based on conserved replication determinants for pathogen identification. The following specific aims will be developed in order to increase the sensitivity, specificity and to increase the sample throughput in order to deal with a potential arboviral introduction into the western United States:

1. Determine the genetic identity of exotic, zoonotic arboviruses that potentially could be introduced into the U.S. Complete genome sequences of exotic Pacific Rim arboviruses (Togaviridae, Flaviviridae, Bunyaviridae) will be determined to identify conserved sequence targets for the development of family-specific and virus-specific primer/probe sets.
2. Develop multiplexed fluorescence probe-based real-time RT-PCR assays for the rapid detection of introduced arboviruses. Multiplex formatted nucleotide amplification methodologies will be developed for robotic TaqMan workstations. Comparisons of the specificity, sensitivity and accuracy of new diagnostic assays to standard plaque assays and single virus primer sets by double-blinded laboratory assessments and evaluations using field specimens collected from mosquito control districts will be performed.
3. Develop novel replicon-based diagnostic assays that utilize the conserved replication components of genotypically divergent viruses for the identification of novel agents. Trans-complementation of nonstructural viral components will be investigated for the generation of viral constructs with family and virus-specific reactivity. The development of replication-dependent fluorescence will be used to assess the presence of human pathogens.
4. Develop rapid Western blot assays for serological identification of antibodies to arboviruses of bioterrorism importance. Western blot strips will be prepared from viral lysates. Banding patterns resulting from incubation with homologous and heterologous anti-viral immunoglobulin will be interpreted to specifically identify agents causing arboviral infections. Virus-specific recombinant proteins will be incorporated into the blots to increase the specificity for virus identification. Rapid enzyme immunoassays using the recombinant antigens may then be developed as a separate subaward project under Dr. Carl Hanson, CA DHS

Arboviral immunoserology

Carl V. Hanson, (Brault - subcontract), California Department of Health Services
Applicant Organization: Public Health Foundation Enterprises, Inc.

Abstract:
Arboviral Immunoserology is a project of the PSWRCE closely coordinated with the ?Arboviral Diagnosis and Strain Variability? project of Investigator Aaron Brault at the University of California at Davis. Arboviruses are highly diverse and adaptable arthropod-borne infectious agents of bioterrorism importance. The goal of these integrated projects is to determine complete genome sequences of exotic Pacific Rim arboviruses and to use this information to develop rapid, multiplexed nucleic acid diagnostic assays along with immunoserologic assays. The latter assays will also take advantage of incorporating recombinant proteins based upon the new sequence information. The role of immunoserology in detecting BT agents should ?ideally? be very minor (assay of nucleic acids or proteins should provide earliest detection), but in reality serology may be very significant due to BT victims being initially recognized when symptomatic -- at a stage when antibodies are being produced, and even when viral nucleic acids and proteins may no longer be present. Thus, serologic studies are synergistic with genomic studies, both in order to obtain complete diagnostic coverage and because of the way in which genomics can influence the design of serologic tests. Serologic Western blots in particular help identify those viral components that elicit a detectable humoral immune response, and will thus influence the development of other, simpler serologic assays. (The progress of both projects during Year 1 is described in the Renewal Application of the ?Arboviral Diagnostics and Strain Variability? project.)

In proposed Year 2 immunoserology studies:

1. Many of the preliminary results from Year 1 studies using viral antibody Western blots will undergo confirmation with the use of more numerous positive and negative control sera;
2. Optimization of the Western blots will be undertaken by varying the multiplicity of infection, the time in culture and the extent of cytopathic effect at the time of harvest to optimize antigenicity of the viral lysates;
3. Many of the viruses will be grown in the C6/36 mosquito cell line to determine the effect on antigenicity of this more natural virus production. Preliminary experiments have already shown higher titers of WNV, SLE and dengue viruses in mosquito cells;
4. Use of temperature and protease inhibitors (such as ammonium chloride to inhibit furin, which cleaves flavivirus prM protein) will be explored to optimize viral antigen production;
5. Studies will be extended to additional arboviruses in coordination with nucleic acid sequencing activities in Dr. Brault?s laboratory; and
6. finally, we will negotiate to collaborate in the use of high-throughput protein cloning and expression, such as that available in the laboratory of Phil Felgner at UC Irvine, in order to develop high-resolution Western blots based on protein microarrays.

Cellular tropism of Dengue virus in vivo

Eva Harris, UC Berkeley

Abstract:
Epidemiologic studies imply that sequential infections with distinct dengue virus (DEN) serotypes are associated with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Understanding the basis for this phenomenon is crucial for developing therapies and a safe dengue vaccine. The lack of a small animal model has greatly hampered the elucidation of the mechanism of severe dengue. Our preliminary data suggest that DEN tropism early in infection may be similar in mice and humans. Thus, we propose to use a murine model to study primary DEN infection, using cellular tropism and viral load/kinetics as endpoints. Since the cellular tropism of DEN in humans is not well defined, we will apply techniques developed to study murine DEN infection to detect the cellular localization of replicating virus in humans through autopsy studies, as well as through flow cytometric studies of human peripheral blood mononuclear cells (PBMCs) obtained early in the course of DEN infection. The goal of Specific Aim 1 is to identify the cellular targets of DEN infection in mice and evaluate parameters that influence cellular tropism and viral load. We will define the cellular tropism of DEN in mice and determine how cellular tropism, viral load, and kinetics of DEN infection are affected by virus serotype and strain and age and strain of mice during primary DEN infection. In vitro and en vivo studies of DEN infection of primary mouse cells will complement in vivo experiments. This knowledge will provide an alternative endpoint in the mouse model and will be used to assess alterations in secondary DEN infections in additional studies. Cellular targets of DEN infection in fatal human DHF/DSS cases will be identified via autopsy studies in Specific Aim 2. In collaboration with the Ecuadorian Ministry of Health, techniques developed in Aim 1 will be applied to determine the cellular localization of DEN antigen and RNA in human autopsies. Immunohistochemistry will be performed on various tissues to identify cell type and presence of replicating DEN. Lastly, cells that are infected with DEN will be phenotyped via flow cytometry in human PBMCs obtained from viremic Nicaraguan patients to identify targets of DEN early in infection. Together, these investigations will provide useful tools and critical data regarding the in vivo cellular tropism of DEN, a category A Priority Pathogen, in both mice and human cases. They will also generate a model system for studying the effects of primary and ultimately sequential infections of DEN and potentially other flaviviruses.

Neuropathogenesis of West Nile virus infection

Howard S. Fox, The Scripps Research Institute

Abstract:
The recent and continuing West Nile virus (WNV) epidemic in the US contributes to increased concerns about emerging virus infections, and in the current era, the potential use of pathogens such as virus of the Flaviviridae family in terrorism. Infection with WNV is asymptomatic in many individuals, but can cause severe neurologic symptomatology and death in others. Additionally, there are increasing numbers of reports of persistent neurological symptom in individuals who survive WNV infection. Understanding the factors that lead to and/or protect from viral pathogenesis is crucial in this and similar infections. The role of the innate immune response, the first line of defense critical to initiating viral clearance and promoting a robust adaptive T cell response, has not been investigated in WNV. Furthermore, in addition to its protective role, it is possible that the innate immune response may contribute to pathology, especially in the brain. An understanding of this front-line innate response will allow insights into both immunity to WNV as well as potential therapies by manipulation of innate immunity. Here, we will focus our attention on natural killer (NK) cells and the innate immune response to WNV. We will utilize a mouse model involving infection with the WNV to study the mechanism of viral pathogenesis and the NK response

Factors influencing the epidemic potential of Dengue

Shannon Bennett, University of Hawaii
Project period: May 2007 - April 2009

Abstract:
Mosquito-borne viruses such as dengue (DENV) are prominent on Category A, B and C bioterrorism agent lists, and are among the most important emergent/resurgent infectious diseases at the dawn of the 21st century, causing major public health problems. DENV in particular has demonstrated an amazing capacity to invade and become established in new geographic regions. The 20th century pandemic of dengue/dengue hemorrhagic fever (DEN/DHF), which currently plagues 50 to 100 million people annually, has intensified in the past 25 years, with a dramatic increase in disease incidence and geographic expansion, including to parts of the US. Effective laboratory-based surveillance and analysis is paramount for the detection of emergent viruses and assessment of their epidemic potential based on their genetic properties. Genetic factors appear to be involved in DENV expansion and increasing pathogenesis based on phylogenetic analyses paired with epidemiological patterns. Partial sequence analysis of the phylodynamics of emerging DENV in Puerto Rico indicates sporadic nonrandom genetic changes associated with significant outbreaks, but it is unclear whether all emergent DENV would share similar patterns of genetic change. Furthermore, the phenotypic effects of these changes, with respect to disease causing potential, remains untested. We are in the process of investigating these important questions in multiple populations with a history of dengue outbreaks to determine a), whether DENV strains circulating before and during an epidemic differ consistently in their genetic composition and/or pattern of evolution (e.g., whether evolutionary change was effected by random genetic drift or non-random adaptive processes); and b), the potentially-disease-causing characteristics exhibited by these epidemic variants in experimentally infected mosquitoes.

The interface between the host immune response and dengue virus (DENV) pathogenesis

Sujan Shresta, La Jolla Institute for Allergy and Immunology
Project period: May 2006 - April 2008

Abstract:
Dengue virus (DENV) causes dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), the most prevalent arthropod-borne viral illnesses in humans worldwide. Studies suggest that DHF/DSS is an immunopathogenic disease, in which the interferon (IFN) system may be dysregulated. However, the precise role of the IFN-dependent immunity in response to DENV infection in vivo is not fully defined, due to the lack of an adequate animal model. Our long-term goals are to determine how the immune system interacts with DENV in vivo and to develop a better small animal model for DHF/DSS. We will test the hypothesis that the IFN-dependent, signal transducer and activator of transcription 1 (STAT1)-independent pathway signals via IFN-? receptors to control primary DENV infection in mice. Using genetically-deficient mouse strains and our recently generated DENV strains that cause a more relevant disease in mice, the research will examine the role of various IFN-dependent, STAT1-independent pathways in controlling primary DENV infection in vivoand at the cellular level in bone marrow-derived dendritic cells . Furthermore, it will identify viral components that modulate the various IFN-dependent pathways in vivo . Overall, this research will elucidate the immune response to DENV infection in vivo and provide novel insights into STAT1-dependent versus STAT1-independent responses. By identifying the molecular basis of the primary immune response to DENV infection in vivo, this project will set the foundation for investigating sequential DENV infection in vivo, and it will provide new opportunities for optimizing a mouse model of DENV disease through immunologic manipulation.

The role of human monocytes in Dengue virus infection

P'ng Loke, University of California San Francisco
Project period: May 2007 - April 2008

Abstract:
Despite the fact that dengue is the most important vector borne viral disease in the world, there are substantial gaps in our knowledge of its pathogenesis. Most importantly from the clinical perspective, there is no way to predict and identify the 500,000 people who suffer from the severe form of dengue Haemorrhagic Fever (DHF) or dengue Shock Syndrome (DSS) from the majority of the 50 million people who are infected with the virus every year. Monocytes/macrophages are one of the primary target cells for dengue virus infection, yet little is known about their responses during this disease. We will test our primary hypothesis that aberrant innate immune responses by monocytes during dengue infection distinguish patients that suffer the severe clinical symptoms of DHF/DSS from patients with the milder symptoms of dengue fever (DF). Our long term goal is to develop diagnostic tools to prospectively identify the subset of patients who will develop DHF/DSS. Our secondary hypothesis is that a subset of the inflammatory genes differentially expressed in patients that suffer from DHF/DSS can be used as biomarkers to identify the subset of DHF/DSS patients in a diagnostic system.

Development of in vitro assays to study flavivirus RNA replication

Bert L. Semler

Abstract:
Flaviviruses like dengue virus and West Nile virus have potential for use as bioterrorism weapons. Although efforts into vaccine development against these agents are ongoing, the ultimate success of this approach remains to be demonstrated. Thus, there is a compelling need for novel anti-viral therapies against flaviviruses. Currently, drugs with activity against these viruses are either non-existent or are of limited efficacy. For these reasons, there is a need to identify novel targets for antiviral chemotherapy. The experiments outlined in this proposal are aimed at identifying flavivirus replication protein interfaces as potential targets for new anti-viral agents. The specific viruses targeted will be dengue and West Nile viruses, Category A and Category B agents, respectively. The first aim of this project will be to develop an in vitro RNA replication assay for dengue virus and West Nile virus. Successful completion of this aim will provide a platform for genetic and biochemical studies that will dissect the functions of the individual flavivirus non-structural proteins during initiation complex formation and subsequent elongation of newly-initiated RNA chains. The second aim of the research will involve developing a quantitative, reverse transcriptase polymerase chain reaction (qRT-PCR) assay to quantify flavivirus RNA replication intermediates. Such a sensitive, strand-specific readout of RNA replication will provide a powerful tool for assessing the levels of RNA synthesis following in vitro manipulation of viral proteins predicted to have specific roles in flavivirus replication. The final aim of the proposal is devoted to the identification of protein-protein interfaces for dengue virus replication proteins. Data from this latter aim will provide information about contact sites between viral replication proteins, providing potential novel targets for future development of small molecules that would disrupt such interactions and thereby inhibit dengue virus replication.

Structure aided drug discovery against the Dengue viral protease.

Peter Kuhn

Abstract:
This developmental project capitalizes on existing infrastructure of the PSWRCE and drug development expertise at the Novartis Institute of Tropical Disease (NITD) in Singapore. It aims at delivering small molecule compounds that will effectively inhibit the Dengue protease. It uses as a starting point existing peptidic inhibitors and knowledge about the crystallization of the protease. The proposed endpoints of this developmental project are potent tool compounds that specifically inhibit the Dengue protease. Success in this area would substantially advance our ability for effective development of therapeutic intervention of Dengue fever worldwide. Dengue is a mosquito-borne infection, which is a major public health concern worldwide. The WHO estimates that approximately 40% of the world’s population is at risk. This compares to only nine countries having experienced a dengue epidemic before 1970. Dengue fever shows severe flu-like symptoms but rarely causes death. Dengue hemorrhagic fever is a potentially deadly complication.