The tick borne flaviviruses (TBFV) includes Tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus and Powassan virus. These viruses are listed among the NIAID category B and C lists of priority for research into pathogenesis, treatment and vaccine development. As their name suggests, the TBFV viruses are transmitted by ticks, and following infection of humans, cause encephalitis, meningitis or hemorrhagic fevers resulting in approximately 10 000 hospitalizations annually with mortality rates as high as 40%. The TBFV belong to the Family Flaviviridae, genus Flavivirus, which comprise some of the most serious emerging and re-emerging pathogens. Other members include the mosquito-borne dengue virus (DEN), West Nile virus (WNV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLE) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses. Our laboratory has been studying Langat virus (LGTV), a member of the TBFV. LGTV (biosafety level 2; BSL2) is naturally attenuated compared to the more virulent TBFV, and shares approximately 80% identity with TBEV (BSL4) at the amino acid level. Therefore, LGTV is an excellent model to study various aspects of pathogenesis and replication of the TBFV. Specifically, our laboratory has used LGTV to study two areas of TBFV research which are outlined below. 1. Interactions between TBFV and interferon signaling. Interferons (IFNs), type I (IFN?? and IFN?O) and type II (IFN??), are crucial elements of the innate immune response to flavivirus infection, restricting virus replication, dissemination and lethality in mouse models. Type I IFN treatment of humans is a leading therapeutic candidate for flavivirus infection. However, such treatment often fails. We have shown that LGTV interferes with IFN signaling by directly inhibiting Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction. In this manner, LGTV is similar to the mosquito-borne flaviviruses DEN, WNV, JEV and YFV, which can also inhibit IFN-mediated JAK-STAT signaling. Our laboratory demonstrated that LGTV suppresses JAK-STAT signaling in response to both type I and type II IFN. The block in signaling was due to a failure of phosphorylation of the JAKs. Examination of the ability of each individual nonstructural (NS) protein to prevent JAK-STAT signaling revealed that LGTV NS5 was primarily responsible for inhibiting signal transduction, via binding the IFN receptor complexes. The finding that NS5 functions as an interferon antagonist was unexpected because NS4B was previously demonstrated as the primary JAK-STAT antagonist of DEN. Thus, LGTV NS5 was identified as a novel flavivirus inhibitor of IFN responses. Furthermore, NS5 has a crucial role in viral RNA replication via its two enzymatic domains, the RNA-dependent RNA polymerase (RdRP) and the methyltransferase. By expressing a series of N- and C-terminal truncation mutants, we determined that the minimal sequence of NS5 required for its IFN-inhibitory function mapped to amino acids 355-735. This region overlaps entirely with the RdRP. By performing functional assays with approximately 350 mutagenized NS5 constructs, we identified a unique functional site on the RdRP responsible for inhibition of JAK-STAT signaling. These are remarkable findings because they indicate at least three functions for the TBFV NS5 !V a methyltransferase, the RdRP and the JAK-STAT inhibitory function, with the domains responsible for the latter two overlaping. It has recently been demonstrated that IFN-evasion by JEV is also achieved by NS5. Thus, our work has significant implications for understanding the pathogenesis of both TBFV and of JEV. 2. Study of virus replication in two hosts of TBFV: the tick and mouse. Current animal models of virus replication and pathogenesis for LGTV are limited to outbred lines of neonatal mice or to SCID mice. Neither of these models lend themselves to rigorous study of virus pathogenesis and immunity. We have recently demonstrated that C57BL/6 mice are susceptible to LGTV infection by intraperitoneal (IP) and intracerebral (IC) routes of inoculation as well as following the bite of an infected tick. This is significant as this is the genetic background for many of the mouse knock-out strains. Thus, we can utilize this mouse model to dissect viral mechanisms of pathogenesis as well as immune responses to infection. In addition to the mouse model of infection, we have developed a novel method of infecting tick larvae with LGTV by immersion. This method does not require feeding on viremic animals or microinjection. This extremely versatile method permits synchronous infection of large numbers of ticks with a defined virus inoculum, and without a requirement for that virus to establish an infection and viremia in mice. This method not only enables study of virus replication within the initial infected tick larvae, but also results in efficient trans-stadial transmission to tick nymphs as well as horizontal transmission to C57BL/6 mice enabling study of virus replication in these hosts. The immersion method of infection will be a very powerful tool to study viral and host determinants for pathogenesis in both ticks and in the mammalian host. In addition, it will also be possible to examine early events in virus replication in the mouse, an event that has not been very well characterized. In contrast to virus infection in laboratory mouse models and in humans, infection of TBFV in tick vectors is persistent without obvious cytopathic effect. We are interested in virus determinants that confer a selective replication advantage in either tick or mammalian systems. To examine this question, we derived a number of LGTV variants by repeatedly passaging the virus in tick or mammalian cell culture, followed by sequencing of the virus genome. We anticipated that virus adaptation to replication in these two cells types would be associated with sequence changes that either alter efficiency of virus replication in other cell types or perhaps alter virulence following inoculation of 14 day old C57Bl/6 mice. To summarize this work, we identified two clusters of coding changes in the LGTV genome associated with host adaptation. The first was in the envelope (E) protein while the second was in the region encompassing NS3, NS4A and NS4B. We plan to use the two animal models of infection, both tick and mouse, in combination with reverse genetics to determine the relative contribution of these genetic changes to virus transmission and virulence.
