Pathogenesis of Tick-Borne Flavivirus Infections

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

In the US, the cases of all tick-borne infections are increasing dramatically and case reports of serious illness and death from Tick-borne flaviruses (TBFV) are also up. TBFV POWV/DTV is transmitted by Ixodes scapularis ticks, the vector for Lyme disease and several other emerging agents. Research in our laboratory employs virology, immunology, entomology, advanced imaging techniques, genomics, proteomics, cell biology, molecular biology, and vector biology. We study LGTV at BSL-2 and POWV/DTV at BSL-3.. TBFV biology in ex vivo cultures of I. scapularis organs. Infection in ticks is a critical, but understudied, feature of TBFV biology. In the past year, we continued studies utilizing ex vivo organ cultures to look for genes associated with POWV replication. Four proviral transcripts were identified via RNA interference assays. In situ hybridization localized several of these transcripts within granular acini. The initial barrier to TBFV infection in ticks is the midgut. Therefore, the biology of virus in midgut is also crucial. Dr. Ochwoto is culturing adult ixodid midguts. To date he has used microscopy to characterize basic structure of the midgut, and has shown that LGTV can infect the cultures. The specific cell types and nature of replication is as yet undetermined. Finally, pathogen-tick interaction studies need to be confirmed in intact ticks, and examined in both replete and partially fed ticks. One convenient avenue is the use of small capsules with membrane submerged in blood. Microbial interactions within the microbiome of the tick Ixodes scapularis Studies indicate that B. burgdorferi can infect over 70% of I. scapularis ticks in some regions of the U.S., while POWV/DTV infects less than 5%. One intriguing possibility is that ticks infected with one microbe may be less susceptible to infection by a second. A new project studies microbial interactions in the tick, and the techniques are now being used by other intramural labs. Although severely impaired by COVID-19, the specific aims of this project are: 1.) Establish new techniques to study potential interactions between microbial symbionts of the tick I. scapularis using the artificial membrane system for blood feeding. Using this system we can sequentially introduce specific microbial species and directly assess their impact on the ability of a second agent to infect, persist in and be transmitted by the tick. 2.) Characterize interactions between microbes. These studies will highlight vulnerabilities in the microbial physiology that can inform countermeasure development. 3.) We are also using the artificial membrane system to study mutants of the relapsing fever bacterium Borrelia hermsii in the soft tick Ornithodoros hermsi. This work is in collaboration with Dr. Frank Gherardini and Sandra Stewart in the Laboratory of Bacteriology. Pathogenesis of TBFV infections. In the past year, Dr. Dylan Flather has continued work on understanding the host factors that are required for tick-borne flavivirus replication in human cells. It has become clear that the protein requirements for POWV and DTV differ from the closely related but non-pathogenic LGTV and that unique proviral proteins are utilized by these viruses depending on the infected cell type. Describing the effect of candidate genes on virus replication is near complete. Of particular interest is whether any of the target proteins act as virus receptors. Flavivirus infection is associated with the hijacking of cellular host machinery to synthesize large amounts of viral proteins resulting in tremendous expansion of the endoplasmic reticulum (ER). Activation of the liver X receptor (LXR) pathway leads to increased efflux of cholesterol and perturbations of membrane biogenesis within the cell. This year we published a manuscript demonstrating LXR agonists impaired replication of POWV and ZIKV in a human neuroblastoma cell line. We plan to continue evaluating the role of the LXR pathway and modulators of this pathway throughout virus infection. Additionally, this year we published our work on the pro-survival AKT pathway and its role in TBFV infection. Utilizing CRISPR knockout cell lines, we demonstrated the decrease of total phosphorylated AKT, AKT1, and AKT2 over the course of acute infection and asimultaneous increase in AKT3. The lack of AKT1 or AKT2 resulted in a level of protection from LGTV-induced cell death. Evaluation of FDA-approved drugs to suppress replication of tick-borne flaviviruses. Based on the PhD research of Dr. Stacey Scroggs and a thorough literature search, several drugs were selected for antiviral evaluation to suppress tick-borne flaviviruses in cell culture. Previously, we reported that the antiviral potency of enoxacin and ciprofloxacin against LGTV in HEK293 and SK-N-SH cells is low. Recently, we found that the potency for these drugs against DTV was also low (effective concentration 50 value over 200M in SK-N-SH cells). Niclosamide and remdesivir were also tested for anti-DTV potency and cytotoxicity. The cellular toxicity of niclosamide was high in HEK293 and Vero cells, and will not be pursued further. The anti-viral potency of remdesivir to suppress DTV replication needs further evaluation. Development of an animal model for persistent POWV and DTV infection. Severe TBFV infections are often followed by chronic neurologic disease, and there is some evidence that viral persistence may be a factor. Earlier work in BVBV demonstrates that TBFV can initiate and maintain persistent infection in cell culture systems. Other earlier work shows that mortality following Powassan (POWV) infection is not 100% in C57BL/6 mice and that viral RNA persists in infected brains. We are using 6-week old C57BL/6 mice infected with DTV to model persistent TBFV infection.