The Vector borne viruses from different virus families account for many medically significant viral pathogens. More specifically, the vector borne flaviviruses, which belong to the Family Flaviviridae, genus Flavivirus, comprise some of the most important emerging and re-emerging viral pathogens. The tick borne flaviviruses (TBFV) include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Powassan/deer tick virus (POWV/DTV) and Langat virus (LGTV). TBFV are generally transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. The mosquito borne flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Our current research is focused on the TBFV, but studying the biology of TBFV will reveal insight into the biology of other vector borne viruses. The research in our laboratory employs virology, immunology, advanced imaging techniques, genomics, cell biology, molecular biology, and vector biology. We study LGTV, a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. LGTV can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. Studies using LGTV will form the basis for work on the more virulent BSL-3 POWV/DTV and BSL-4 TBFV. Analysis of virus interactions with the invertebrate host. Ixodid ticks represent the natural reservoir of TBFV, are critical for virus persistence in nature, and are the major vector for infection of humans. Transmission of flaviviruses to humans occurs during tick feeding. We have developed a first generation microarray to investigate salivary gland transcriptional changes in Ixodes scapularis nymphs during feeding or after infection with LGTV. The immediate goal of this work is to identify tick salivary gland transcripts that play a role during feeding or for the replication or transmission of TBFV. The long-term goal of this work is to identify novel tick salivary gland genes that could be targeted for the development of vaccines that have the potential to disrupt tick feeding and/or flavivirus transmission. In 2013, , we expressed several recombinant tick salivary gland proteins that were differentially expressed in LGTV-infected ticks in a baculovirus system and tested for their effect on flavivirus replication in vitro on relevant cell types such as tick and mammalian cells. There was no apparent effect on viral replication by any of the proteins in either tick or mammalian cells. Also in 2012, we utilized a novel in vivo model for LGTV infection that mimics a tick bite by inoculating virus into the ear pinna. We injected LGTV in the presence of the same recombinant tick salivary gland proteins to see if infection were affected. No effect was observed, but genetic analysis of the virus revealed the presence of the attenuating E:D308A mutation. Comparison of TBFV biology in mammalian and tick cells. A key difference between TBFV infection of vertebrate and arthropod host systems is that infection of ticks is persistent and non-cytolytic, whereas infection of mammalian hosts is typically acute and cytopathic. We are investigating the nature of this difference to identify responsible host and viral factors. We published a study comparing LGTV virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal microscopy, electron microscopy, and electron tomography. Flavivirus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. Electron tomography revealed virus-induced spherical vesicles thought to protect replicative intermediates from intracellular antiviral systems. In contrast to mammalian cells, TBFV-infection in tick cells shows delayed and decreased membrane proliferation. Additionally, electron tomography of infected tick cells shows a shift from spherical vesicles to tubular profiles, especially in the context of persistently infected cells. In 2013, we extended these structural studies to cultures of primary embryonic brain. LGTV infection was restricted to cells of neuronal origin, and ultrastructural studies revealed results similar to those observed in mammalian cell lines, although the frequency of the tubular profiles appeared greater than in permanent cell lines. IThe viral non-structural protein 4A (NS4A), which has been implicated in the membrane rearrangements, was cloned and expressed in mammalian cells. This protein by itself was unable to induce membrane rearrangements. To facilitate expression and study of viral and cellular genes in tick cells, we modified a method of transfection and demonstrated that vectors bearing the chicken beta-actin promoter increased expression of target genes by at least 10-fold. Viral determinants of pathogenicity. Previous data collected in this lab demonstrated that upon passaging of the tick-borne flavivirus, Langat virus (LGTV), in ISE-6 tick cells, three coding mutations (M:K115E, NS3:F604L, and NS4A:A81V) arose within the LGTV genome. This virus was subsequently demonstrated to have reduced neuroinvasiveness as examined by intraperitoneal infection of three week old laboratory mice. Efforts to study the role of these mutations were confounded by difficulties inserting these mutated residues in a full length molecular clone of the TP21 strain of LGTVIn order to circumvent this problem, we developed a full length TP21 clone that contained a number of unique restriction sites in the viral sequence. This has enabled us to excise short segments of the virus, manipulate the sequence, and re-insert the mutated fragment back into the full length clone.