Browsing by Subject "West Nile virus"
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Item Characterization of single-cycle flavivirus particles for use as a vaccine to prevent West Nile disease and to examine immune responses to flavivirus infection(2009-08-28) Douglas Gregory Widman; Peter W. Mason; Stanley M. Lemon; Nigel Bourne; Mark Heise; Gustavo ValbuenaWest Nile virus (WNV) is responsible for the largest outbreak of viral encephalitis in the history of North America, yet there are no vaccines available to prevent this disease. To address these needs we have developed RepliVAX WN, a single-cycle flavivirus (SCFV)-based vaccine to prevent West Nile disease. RepliVAX WN contains a C-deleted WNV genome, and is produced in trans-complementing cell lines that express WNV C. When used for vaccination, RepliVAX WN infects a single cell where the genome replicates and drives the production of highly antigenic subviral particles (SVPs) and NS1 without producing infectious virions. Thus, RepliVAX is expected to be highly potent yet exhibit a safety profile superior to traditional live-attenuated viral vaccines.\r\nHere we demonstrate that RepliVAX WN can be safely passaged in C-expressing cell lines and that this blind passage selected for mutations used to engineer a second-generation RepliVAX WN with an enhanced in vitro growth phenotype. When evaluated in mouse and hamster models of WN disease, this second-generation RepliVAX was safe, exhibited 100% protective efficacy, and induced significantly higher antibody levels than the parental virus. Furthermore, we observed that RepliVAX WN-induced antibody levels remain steadily at high levels for at least 6 months after vaccination of hamsters, and all animals were protected from lethal WNV challenge at this time. Evaluation in non-human primates indicated that one or two doses of RepliVAX WN was safe, induced WNV-specific antibody responses, and protected animals from WNV viremia. \r\nHaving demonstrated the usefulness of RepliVAX WN as a vaccine to prevent WN disease, we were interested in the immunological mechanisms underlying vaccine immunity. We observed that although RepliVAX WN vaccination induces high levels of interferon (IFN) alpha, the ability to respond to either type-I or type-II IFNs was not required for the development of activated B cells, IgG, IgM, or neutralizing antibody titers. Type-I IFN signaling did, however, play a role in viral gene expression, as in vivo imaging of animals inoculated with luciferase-expressing SCFVs revealed 1000-fold greater bioluminescence in the absence of a type-I IFN response. The affect of this IFN response on gene expression was dramatic, but short lived and did not appear to play a role in SCFV persistence, as SCFV gene expression was detectable for at least 18 days after SCFV inoculation. Taken together these results demonstrate the usefulness of SCFVs like RepliVAX WN as vaccines to prevent flavivirus disease, and tools with which to examine immune responses to viral infection.Item Epidemiology and clinical investigation of West Nile Virus encephalitis in Houston, Texas(2006-08-17) Kristy O. Murray; Robert Tesh; R. Palmer Beasley; Laura Rudkin; Karl Anderson; Billy Philips; Alan BarrettIn 2002, West Nile virus (WNV) appeared in Houston. Retrospective medical chart reviews were completed for 172 WNV cases hospitalized in the Houston area between 2002 and 2004; 113 had encephalitis (including 17 fatal cases), 47 had meningitis, and 12 were uncomplicated fever cases. Risk factors associated with progression from encephalitis to death were renal insufficiency, requiring intubation and mechanical ventilation, presence of myoclonis or tremors, and loss of consciousness. Pleocytosis in the cerebrospinal fluid was considered protective. A nested case-control study identified advanced age (odds ratio [OR]=1.1; p<0.001), history of hypertension, including those cases taking drugs which can induce hypertension (OR=2.9; p=0.012), and history of cardiovascular disease (OR=3.5; p=0.061) as independent risk factors for developing encephalitis from WNV infection. After adjusting for age, race/ethnicity (being black) (OR=12.0; p<0.001), chronic renal disease (OR=10.6; p<0.001), being positive for hepatitis C virus (OR=23.1; p=0.0013), and immunosuppression (OR=3.9; p=0.033) were identified as risk factors for death. Patient interviews were used to examine demographics and medical histories; age of 55 years or greater (Odds Ratio [OR]=5.7; 95% CI 1.8, 17.9; p=0.003) and history of hypertension (OR=3.7; 95% CI 1.2, 12.0; p=0.027) were independently associated with encephalitis, supporting the findings from the nested case-control study. At three years post-onset, 42% of patients were still reporting symptoms related to their WNV illness. Objective measurements revealed significant differences in both the mini-mental status exam and Barthel Index between encephalitis patients and those who presented with meningitis or fever. New onset depression was reported by 31% of cases, with 75% having CES-D scores indicative of mild to severe depression. Serial blood draws were tested by ELISA, and persistence of anti-WNV IgM antibodies were found in 24% and 18.4% of patients approximately two and three years post-onset, respectively. Persistence of IgM from previous transmission seasons could potentially result in false interpretation of positive test results. This study provides us with a better understanding of the clinical aspects of a virus that is likely to continue to circulate in the United States, and allows us to identify populations at risk for encephalitis and death.Item Identification and characterization of cell-adapted mutations in West Nile Virus and replicons(2007-12-19) Shannan Lee Rossi; Peter W. Mason; Scott Weaver; Robert Tesh; Ilya Frolov; Charles RiceFlavivirus persistence in cell culture is achieved by altering the interaction between the viral genome and the host cell. To identify some of the factors that contribute to a persistent West Nile virus (WNV) infection, WNV subgenomic replicon (WNR) genomes were modified to contain neomycin phosphotransferase (antibiotic-resistance) gene and cells that persistently harbored this WNR were selected for using G418. There were many changes to the genomes harvested from WNR-bearing cell cultures compared to the parental genome, perhaps the most striking was the prevalence of NS2A mutations. WNR and WNV genomes harboring these mutations replicated more poorly than wt WNR or WNV genomes in vitro. These NS2A mutant genomes, as well as a genome with a large deletion in the 3’ UTR, were highly attenuated Swiss-Webster outbred mice. Low levels of IFN were produced from cells infected with NS2A or 3’ UTR deletion WNR genomes compared to wt WNR genomes. Since the WNR and WNV cannot efficiently replicate, little stress is placed on the cell during replication, resulting in minimal engagement of the cell’s stress and apoptotic responses and a noncytopathic infection. As a result, little cytopathic effect and apoptotic death were observed in cells infected with NS2A or 3’ UTR deletion WNR mutants compared to cells infected with wt WNR genomes. This lack of death may have been attributed to low levels of CHOP, a pro-apoptotic protein that is induced during endoplasmic reticulum stress. Taken together, these data support the hypothesis that WNR and WNV genomes that replicate poorly do not efficiently produce signs of their replication that can be recognized by the cell and place little stress upon the cell, resulting in an attenuated noncytopathic, persistent infection.Item Insights into Virus-Host Interactions: Regulation of Cytokine Signaling Is a Virulence Determinant of West Nile Virus(2007-08-08) Keller, Brian Christopher; Gale, Michael JrWest Nile virus (WNV) has rapidly become a pathogen of global importance over the past two decades. Its recent association with severe neurological disease and emergence in the Western Hemisphere suggest that the virus has acquired the ability to effectively evade host immune defenses. One of the earliest steps in controlling viral infection occurs through the action of interferon (IFN) and its downstream activation of an antiviral state within the infected cell and neighboring tissue. To begin to understand how WNV evades host defenses, studies were initiated to examine the interaction of virulent (TX02) and avirulent (MAD78) WNV strains with the host IFN system. Compared to TX02, MAD78 replicated at lower levels in cultured human cells, was highly sensitive to the antiviral actions of IFN in vitro and demonstrated a completely avirulent phenotype in wild-type mice. In contrast to TX02 and other pathogenic forms of WNV, MAD78 was defective in its ability to disrupt IFN-induced JAK-STAT signaling. However, replication of MAD78 was rescued in cells lacking a functional IFNalpha /beta receptor (IFNAR). Consistent with this, MAD78 virulence was unmasked upon infection of mice lacking IFNAR. The regulation of IFN signaling was multifactorial involving a combination of viral and host factors. In particular, overexpression of various proteins from the pathogenic TX02 strain and the nonpathogenic MAD78 strain attenuated signaling to an IFN-responsive promoter, suggesting that viral products from both strains are capable of contributing to the IFN signal block. Differences between TX02 and MAD78 were identified, however, when host suppressors of cytokine signaling (SOCS) proteins were shown to be differentially upregulated by the two strains. Furthermore, expression of a dominant-negative form of SOCS1 partially restored IFN signaling during TX02 infection indicating that SOCS proteins do in fact participate in virus-induced signaling suppression. Importantly, WNV regulation of signaling was not restricted to IFNalpha /beta but included IFNgamma and IL-6, cytokines that similarly utilize JAK-STAT. These studies demonstrate novel insights into the complex interactions that occur between a virus and its infected host cell and may allow for the identification of viral and cellular targets for the development of improved therapeutics and new vaccine strategies.Item Mathematical Modeling the Zoonotic and Vector Transmission Dynamics of West Nile virus as They Relate to Human Morbidity and Mortality(2014-12-04) Laine, Christopher GlenWest Nile virus (WNV), an arthropod-borne flavivirus, naturally circulates between passeriform birds and mosquitoes. Other vertebrates, such as humans, may become infected during the bloodmeal of infectious mosquitoes. WNV initially invaded New York in 1999, rapidly swept west across the North American continent, and is now endemic across the continental United States. The focus of this study was to use mathematical modeling, for improving current public health understanding on how infectious cycles of birds and mosquitoes, infection and cross-infection, and environmental dynamics of WNV, along with human pathology, influences human morbidity and mortality in the Dallas, Tarrant, and Denton counties of Texas. During a comprehensive literature review of WNV, avian pathophysiology, public health entomology, human pathophysiology, and epidemiology, we proposed a novel mathematical model. Subsequently, we developed an epidemic model of the WNV dynamics, in the avian host (American crow), mosquito vector (Culex), and two age classifications of humans (?39 & ?40). The bifurcation of human age was conducted due to the risk of humans developing neuroinvasive disease increases 1.5X for every decade of life. We also divided human infected classes into asymptomatic, West Nile Fever (WNF), and West Nile neuroinvasive disease (WNND), as WNND is the only fatal form. The model was then calibrated to observed data, from the endemic years between 2003-1012. A sensitivity analysis of each individual variable and parameter was conducted to test influence on human morbidity and mortality. Focusing on the most sensitive variables, we conducted a multivariate analysis, in which we formulated situations such as drought, avian concentration and population fragmentation, insecticide usage, larvae side usage, and habitat modification through the reduction of standing water. We were able to successfully simulate the endemic years, and outbreaks, between 2003 and 2012, but underestimated the outbreak year of 2012. This model illustrates the observed link between infected mosquito densities to human health outcomes. Climate changes that effect the mosquito population and their interaction with humans have been shown to be important factors influencing human morbidity and mortality. In the future this model may also be useful in predicting the effect of various disease control strategies.Item The Role of Climatic and Environmental Variability on West Nile Virus in Harris County, Texas, 2006-2007(2010-07-14) Berhane, StephenBetween the years 2006-2007, Harris County, located at the heart of the Houston metropolitan area, experienced a nearly 90% decline in the number of female mosquitoes which tested positive for the West Nile virus. Different theories exist as to why such a precipitous drop occurred and this study attempts to determine the extent to which climatic variability between the two years played a role. The Mosquito Control Division of Harris County Public Health and Environmental Services gathered the data on vectors and reservoirs. Then using GIS, spatial analysis, and geostatistical tools the vector and reservoir data was compared to climatic data to investigate any changes in viral distribution. Previous studies of the area until now have used a limited amount of climatic data; this study seeks to improve the resolution of climatic data analyzed. A higher resolution of data was achieved by including as-of-yet unused data from a network of over 150 gauges maintained by various state and local agencies in addition to previously used data from NOAA COOP stations. Using this dense network of station's values for precipitation, temperature and other climatic variables were interpolated for all of Harris County and used in the analysis. Based on results, water availability was the most likely out of all the climatic variables to the precipitous drop of West Nile virus positive female mosquitoes from 2006-2007. Correlations between all climatic variables and mosquito abundance and West Nile virus positives showed mixed results compared to a previous study in the same area.Item West Nile virus versus the host cell: Identification of host factors that modulate infection(2008-08-27) Felicia Gilfoy Santa Maria Guerra; Peter W. Mason; Robert Davey; Nigel Bourne; Gregg Milligan; Frank Scholle; Don M. EstesThere are two competing aspects of virus-host interactions: (i) those that enhance virus transmission and (ii) those that reduce or prevent viral replication and transmission. Each of these types of interactions is critical and both must be investigated to fully understand viral pathogenesis. Cells encode a variety of molecules which are critical for recognizing viral infections. One such molecule, the dsRNA sensor PKR, has been shown to be important for IFN-beta induction. Therefore, to determine whether PKR was involved in the recognition of WNV infection, cells lacking PKR were infected with WNV and assayed for IFN production. Interestingly, PKR-null cells demonstrated dramatically lower levels of WNV-induced IFN compared to wild type cells. Additionally, chemical inhibition of PKR activity or post-translational gene silencing of PKR expression severely impaired WNV-induced IFN production, suggesting that PKR is critical for the induction of IFN following WNV infection. Further analysis suggested that PKR may be important for the activation of NF-kappaB, suggesting a possible mechanism of IFN-beta induction. Consistent with cell line data, PKR was shown to be critical for WNV-induced IFN production in primary mouse bone marrow-derived dendritic cells.\r\nThe recognition of WNV is an important aspect of controlling infection; however, it is only one side of the story. Host factors which WNV utilizes to facilitate its infection and replication are also key to understanding viral pathogenesis. The presence or absence of specific factors may control the level of viral replication, host tropisms and, ultimately, viral pathogenesis. To identify host co-factors that are essential for WNV infection and/or replication, small interfering RNAs (siRNAs) were used to systematically knockdown host gene products and levels of WNV infection and/or replication was assayed in the absence of these factors. A siRNA library screen identified ten cellular proteins which are essential for WNV infection and/or replication. Two of these genes encoded subunits of the proteasome. Chemical inhibitors of proteasome activity confirmed that the proteasome is critical for efficient WNV replication.Item West Nile wake-up call : Texas responds to unfamiliar insect-borne diseases(2013-12) Nicklas, Margaret Mary, 1964-; Dahlby, TracyWest Nile virus affected nearly 2,000 people in Texas in 2012, killing 89. Neither state and local public health institutions nor the medical community were well prepared for the unprecedented outbreak. The virus is carried by mosquitoes and can cause paralysis and other neurological damage. Other diseases carried by insect vectors, like dengue fever, are poised to resurge or emerge in Texas. Suspected effects of climate change, like warmer winters, may contribute to the prevalence of such diseases and frequency of outbreaks. Adequate surveillance of human cases of these diseases is crucial to the public health response, but is hampered by a low level of diagnosis and reporting throughout the state and spotty vector surveillance by local entities.