Western equine encephalitis virus (WEEV) is an arboviral pathogen naturally found in North America. The primary disease phenotype associated with WEEV infection in susceptible hosts is a relatively long prodromal period followed by viral encephalitis. By contrast, in the current work, experimental inoculation of WEEV into the peritoneum of Syrian golden hamsters produced rapid death within approximately 96 h. It was determined that direct virus killing of lymphoid cells leads to death in WEEV-infected Syrian golden hamsters, and that inflammatory cytokines have the potential to enhance virus-induced lymphoid cell destruction. It was further concluded that WEEV retains its ability to cause encephalitis in Syrian golden hamsters, if hamsters survive the early stages of virus infection or if virus is introduced directly into the CNS. Death in WEEV-infected hamsters is associated with lymphonecrotic lesions in the absence of pathological lesions in the central nervous system (CNS). Few clinical parameters were altered by WEEV infection, with the exception of circulating lymphocyte numbers. Circulating lymphocyte numbers decreased dramatically during WEEV infection, and lymphopenia was identified as a consistent indicator of eventual death. Virus infection also increased serum concentrations of the cytokines interferon and tumor necrosis factor-alpha (TNF-alpha). Hamster peritoneal macrophages exposed to WEEV expressed TNF-alpha in a dose-responsive manner. Macrophage expression of TNF-alpha could be significantly inhibited by treatment of cells with anti-inflammatory agents flunixin meglumine (FM) or dexamethasone (Dex). Anti-inflammatory treatment also protected macrophages from cytotoxicity associated with exposure to WEEV. Treatment of WEEV-infected hamsters with either FM or Dex significantly improved survival compared to placebo-treated controls. WEEV induced cytotoxicity in hamster splenocytes exposed to WEEV in a virus dose-responsive manner. Supernatant from WEEV-exposed macrophages significantly enhanced WEEV killing of splenocytes. Hamsters that survived the early stages of WEEV infection occasionally developed signs of neurological disease and died approximately 6 to 9 d after virus inoculation. These animals had histopathological lesions in the CNS consistent with alphavirus-induced encephalitis. Inoculation of WEEV directly into the CNS caused apparent encephalitic disease. Death following CNS inoculation of WEEV was rapid and concurrent with histopathological lesions in the CNS similar to lesions seen in encephalitic hamsters following peripheral inoculation.

Within the state of Florida, there are three arboviruses of public health importance that can cause neuroinvasive disease in humans: West Nile Virus, Saint Louis Encephalitis Virus, and Eastern Equine Encephalitis Virus. Mosquitoes (Diptera: Culicidae) within the genus Culex are known and suspected vectors of these diseases. The vectors of these diseases can be present in urban wetland habitats that allow for exposure to residential communities. Vector ecology must be investigated in order to understand the dynamics of disease transmission. In Hillsborough County, Florida the spatial and temporal distribution of these vectors are not well established. An ecological study was conducted in the University of South Florida's Ecopreserve using trapping methodologies to sample the adult and gravid females as well as collect the egg population. Collections were made at three spatial points for the duration of July through December 2013 and compared to meteorological variables. Culex erraticus, a proposed bridge vector of Eastern Equine Encephalitis, was the most abundant adult species and gravid female captured. Culex nigripalpus, primary Floridian vector of Saint Louis Encephalitis and bridge vector of West Nile Virus, was the second most abundant adult species caught as well as the majority of eggs collected. Based on the results collected, the presence of Culex erraticus and Culex nigripalpus was confirmed. The majority of Culex erraticus adults were collected in September and October and Culex nigripalpus adults were the highest in July and August. The results of the gravid and egg collection generated crucial insight regarding methodology for studying vector ecology within this urban wetland habitat. However, modeling at spatial points based on meteorological variables yielded inconsistent results that illicit further investigation regarding these arboviral vectors of disease.

Thesis (Ph. D.)--University of Tennessee, Knoxville, 2009. / Title from title page screen (viewed on Nov. 13, 2009). Thesis advisor: Reid R. Gerhardt. Vita. Includes bibliographical references.

Thesis (D.P.H.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on July 7, 2010). Includes bibliographical references.

Our study was focused on the effect of tick-borne encephalitis virus infection on global gene expression in two human neural cell lines (neuroblastoma and glioblastoma). Changes of gene expression were determined using microarray approach. We identified several genes with up-or down-regulated expression in neural cells following the infection. The changes in expression of some of them were similar in both cell lines,other exhibited different pattern.

Tick-borne encephalitis virus (strain Hypr) was serially subcultured in PS cells and tick cell line IRE/CTVM19, producing four different viral variants. Biological properties of these new variants were investigated in mouse model. Possible determinants of virulence were found by full-genome sequencing. The role of glycosylation for tick-borne encephalitis virus was evaluated.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Natalia Cernicchiaro / Japanese encephalitis virus (JEV) is a virus of the Flavivirus genus that may result in encephalitis in vertebrate hosts. This vector-borne zoonosis occurs in Eastern and Southeastern Asia and an intentional or inadvertent introduction into the United States (US) would lead to important public health and economic consequences. The objective of this study was to gather, appraise, and synthesize primary research literature to identify and quantify vector and host competence for JEV, using a systematic review-metaanalysis (SR-MA) approach. After defining the research question, we performed a search in selected electronic databases. The title and abstract of the identified articles were screened for relevance using a defined set of exclusion and inclusion criteria, and relevant articles were subjected to a risk of bias assessment followed by data extraction. Random-effects subgroup meta-analysis models were fitted by species (mosquito or vertebrate host species) to estimate pooled summary measures as well as to compute the variance between studies. Meta-regression models were fitted to assess the association between different predictors and the outcomes of interest and to identify sources of heterogeneity among studies. Data were extracted from 171 peer-reviewed articles. Most studies were observational (59.06%) and reported vector competence (60.2%). The outcome measures reported pertained to transmission efficiency, host preference, and vector susceptibility to infection within vector competence; and susceptibility to infection within host competence. All outcome measures (JEV proportion of infection in vectors and hosts from observational studies; and JEV infection, dissemination, and transmission rates in vectors from experimental studies) had high heterogeneity. Mosquito species, diagnostic method, country, and capture method represented important sources of heterogeneity associated with the proportion of JEV infection in vectors; host species and region were considered sources of heterogeneity associated with the proportion of JEV infection in hosts; and diagnostic and mosquito capture methods were deemed important contributors of heterogeneity for the minimum infection rate (MIR) outcome. Mosquito species and administration route represented the main sources of heterogeneity associated with JEV infection rate in vectors. Quantitative estimates resulting from this SR-MA will be inputted into risk assessment models to evaluate risks associated with the introduction of JEV in the US.

Japanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis (JE), the leading cause of vaccine-preventable neurological disease. JEV is a flavivirus that is primarily transmitted through the bite of infected mosquitoes, similar to dengue virus (DENV), St. Louis encephalitis virus (SLEV), West Nile virus (WNV), and Zika virus (ZIKV). The two viral characteristics that dictate virulence are (1) neuroinvasiveness, the ability of the virus to invade the central nervous system(CNS), and (2) neurovirulence, the capacity of the virus to kill resident cells in the CNS. The clinically proven live-attenuated JEV vaccine, SA14-14-2, lacks both pathogenic characteristics unlike its virulent parental virus, SA14. Previous work has revealed the viral E gene as the main determinant of these two pathogenic properties, though the molecular mechanisms behind their attenuation remain unclear. The E gene encodes for the viral envelope glycoprotein that is involved in viral entry into susceptible host cells. The E protein of SA14-14-2 differs from SA14 by nine amino acids. To investigate the role of these mutations in JEV virulence, we created a series of SA14E mutants using infectious cDNA technology. Here, we report the independent function of domains I (DI) and II (DII) of the viral E protein in JEV neurotropism. We reveal that an individual mutation in DI, E138K,and synergism between two mutations in DII, E244G and K279M,are independently sufficient for the attenuation of JEV neuroinvasion. Also, we report that multiple E mutations are required for full attenuation of JEV neurovirulence. Overall, our findings show the direct relationship between genetic factors and JEV neuroinvasion. These results provide a solid foundational base for the logical development of other, currently non-existing, live-attenuated neurotropic flavivirus vaccines and antivirals.

The Caprine Arthritis Encephalitis Virus (CAEV) genome encodes three structural (gag, pol, and env) and three accessory (rev, tat, and vif) genes. The Rev-C protein regulates Gag, Pol and Env expression by transporting their mRNAs to the cytoplasm. Rev trans-activation requires binding of Rev to an RNA structure called the Rev Response Element (RRE-C). Previous mutational analyses have shown that two domains of Rev are required for its function. The basic domain mediates RRE binding and multimer formation, and the nuclear export signal (NES) mediates trans-activation. Preliminary experiments demonstrate that Rev-C N-terminal deletion mutants bind the RRE less avidly than does wildtype Rev. As a result, it was hypothesized that an additional domain located in the N-terminal exon of Rev-C was required for optimal RRE binding. To test this hypothesis, Rev-C alanine scanning mutants were generated and in vitro RRE binding assays were performed. Alteration of Rev-C amino acids K13, E14, N15, V19, T20, M21 and R27 dramatically decreased affinity for RRE-C. These data demonstrate that Rev-C N-terminal amino acids are required for optimal RRE-C binding and suggest that a third functional domain exists within the N-terminus of Rev-C.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus, Human Immunodeficiency Virus type 1 (HIV-1). The CAEV genome contains several small open reading frames (ORFs) that encode viral regulatory proteins. One of these non-structural proteins, Rev-C, is required for cytoplasmic transport of viral un/incompletely spliced mRNAs and efficient viral replication. In HIV-1 and visna virus, Rev is responsible for the temporal shift from non-structural protein synthesis to synthesis of structural proteins that is observed during the viral infectious cycle. Since it encodes a Rev protein, CAEV would be predicted to exhibit a similar temporal shift in gene expression during its replicative cycle. Immunoprecipitation analysis of 35S-pulse labeled, CAEV-infected goat synovial membrane (GSM) cells indicates that Rev-C is more abundant than is Gag at 12 h post-infection (PI); at later times PI Gag predominates. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) experiments using nuclear and cytoplasmic RNA from CAEV-infected GSM cells indicates that the viral unspliced gag mRNA accumulates significantly in the cytoplasm only after Rev is detected. These data indicate that a temporal shift from viral non-structural to structural gene expression occurs in CAEV infected GSM cells.