The tick borne encephalitis (TBE) complex of viruses (genus: Flaviviridae), which includes Tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus and Powassan virus, 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 TBE viruses are transmitted by ticks, and following infection of humans, cause encephalitis, meningitis or hemorrhagic fevers with mortality rates as high as 40%. The TBE viruses belong to the Family Flaviviridae, genus flavivirus, which comprise some of the most serious and vexing viral pathogens. Other members include the mosquito-borne viruses yellow fever virus (YFV), dengue virus (DEN), West Nile Virus (WNV), Japanese encephalitis virus (JEV) and St. Louis encephalitis virus (SLE). Another medically important member of the flaviviridae is Hepatitis C virus. Our laboratory has been studying Langat virus (LGTV), a member of the TBE serocomplex of viruses. LGTV (biosafety level 2; BSL2) is naturally attenuated compared to the more virulent TBE viruses, 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 TBE viruses. Specifically, our laboratory has used LGTV to study three areas of TBE virus research which are outlined below. 1. Interactions between TBE viruses and interferon signaling. There is currently no specific treatment for infection with TBE viruses. Type I interferon treatment of humans is a leading therapeutic candidate for flavivirus infection. However, such treatment often fails. The mosquito-borne flaviviruses DEN, WNV, JEV and YFV are all known to interfere with interferon signaling by directly inhibiting interferon-mediated janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction. However, very little is known about the interactions between the tick-borne viruses and IFN signaling pathways. Our laboratory has demonstrated that LGTV inhibits JAK-STAT signaling in response to both type I (IFNalpha and IFNbeta) and type II (IFNgamma) interferons. The block in signaling was due to a block in the phosphorylation of the JAKs. Examination of the ability of each individual nonstructural protein to prevent JAK-STAT signaling revealed that LGTV NS5 was primarily responsible for inhibiting signal transduction, via binding the IFN receptor complexes. This finding was unique among the flaviviruses and thus represents a novel mechanism of inhibition in this important group of viruses. 2. Study of virus replication in two hosts of TBE viruses: 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 vigorous study of virus pathogenesis and immunity. We have recently found that C57BL/6 mice are susceptible to infection by multiple routes of inoculation. 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. Thus, it is extremely versatile, enabling infection of large numbers of ticks with the same genotype of virus, regardless of the ability of that virus to establish an infection and viremia in mice. In the past year, we derived a number of LGTV variants by repeatedly passaging the virus in tick or mammalian cell culture. We are currently utilizing the two models of infection to examine virus replication, pathogenesis and transmission (both between tick and mammal, and between tick life stages) of different virus variants. The ultimate goal of this work is to identify viral sequences involved in transmission and/or virulence. 3. Design of DNA vaccines to protect against infection. Current TBEV vaccines are live-attenuated or killed-virus vaccines, making them expensive to produce and have a number of safety issues associated with them. To abate these concerns, we have identified viral cDNA sequences for use in a DNA expression vector. The sequences correspond to the structural proteins preM and E which when expressed together form a subviral particle that elicits a good antibody response in vaccinated mice. A second sequence corresponding to the nonstructural protein NS1 also elicits a good antibody response in vaccinated mice. However only expression of the subviral particle elicited a neutralizing antibody response. We are currently working to determine if these responses are protective and the basis for that protection.
