Pathogenesis of Tick-Borne Flavivirus Infections

National Institute Of Allergy And Infectious Diseases

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Abstract

Research in our laboratory employs virology, immunology, entomology, advanced imaging techniques, genomics, proteomics, cell biology, molecular biology, and vector biology. We primarily study LGTV at BSL2 and POWV/DTV at BSL3. TBFV biology in I. scapularis. Infection in ticks is a critical, but understudied, feature of TBFV biology. Ticks are ectoparasites and important vectors of pathogens affecting humans, livestock, wildlife, and companion animals. With the increasing burden of tick-borne diseases, it is crucial to deepen our understanding of tick-pathogen interactions. Histology is often used to examine individual tick organs; however, whole-tick histological sections offer a more comprehensive view of internal anatomy and allow for simultaneous observation of multiple organs. Building on last year’s findings, Dr Weck focused on the application whole-tick histology to analyze anatomical changes during the molt from nymph to adult in Ixodes scapularis, and made progress in several areas. First, in situ hybridization (ISH) for LGTV was optimized for histological sections of whole ticks and successfully performed. Preliminary results indicate that the synganglion may play a role in viral persistence during developmental stages (transstadial transmission). Next, a standard curve was established for quantification of LGTV in tick organs, using RT-qPCR. Preliminary analyses of dissected organs from infected ticks revealed that the synganglion showed the highest viral loads, suggesting it may serve as a reservoir for viral maintenance. Furthermore, tracking the observed remodeling of salivary glands during molting, we have now collected salivary glands from all experimental groups for scanning electron microscopy (SEM), which has allowed us to observe in detail the external morphological changes occurring throughout the molting process. In parallel, additional samples were collected for further histological and comparative analysis, which will provide insights into cellular and structural alterations across developmental stages. These analyses are currently ongoing. Finally, we optimized an improved artificial feeding method for both nymphs and larvae, allowing us to incorporate larval stages into the study. This addition enables us to examine the transition from larvae to nymphs, expanding our understanding of LGTV transmission dynamics. We are completing a second round of experiments using ticks exclusively fed via the artificial system, which provides enhanced environmental control and reproducibility. Basic tick biology studies The study of basic tick biology helps to inform many questions about tick borne diseases. Thus, Dr Stewart is pursuing 2 basic tick projects; one probing the possible expanding range of Ixodes scapularis and the other studying the argasid tick, Argas monolakensis. Ixodes scapularis range expansion The deer tick, Ixodes scapularis, is the leading vector of disease in the United States, transmitting seven different human pathogens. Among these are POWV, a protozoan, and several bacteria most importantly Borrelia burgdorferi, a causative agent of Lyme disease. The range of I. scapularis has been expanding westward from the Atlantic coastal states to the eastern half of North Dakota. Historically, this tick species was not found in Montana, but recently three adult specimens were captured in the eastern edge of the state. This suggests that the range of I. scapularis may have expanded into Montana, potentially bringing new pathogens of public health concern into the state. This year we conducted field collections and established a community outreach program to map the potential range of I. scapularis in Montana. In this program we have established informal partners at veterinary clinics, hospitals, state parks, county extension offices and at local colleges. Finally, we have arranged to staff hunting check stations in eastern Montana alongside state wildlife biologists and search deer carcasses for attached ticks. Employing these methods will allow us to cover larger areas and deploy more people to look for ticks than we can on our own. These efforts will help public health officials alert citizens and medical health professionals of the risks posed by ticks in Montana. Argas monolakensis project A geographically isolated tick species, Argas monolakensis, is indigenous and localized to the islands in Mono Lake, CA. These ticks feed primarily on nesting California Gulls (Larus californicus) but will feed on humans if presented the opportunity. The bites of other Argas species can cause allergic reactions in humans and transmit Borrelia anserina, causative agent of avian spirochetosis, in poultry. A. monolakensis is understudied but has the potential to transmit pathogens to California Gulls, which in turn may disperse these microbes throughout the western U.S. Tick specimens field collected by Dr. Stewart generated a draft genome and transcriptome of A. monolakensis. This is the only genome to be generated from the Argas genus. Importantly, the data was also used to characterize the constituents of the microbiome of this tick, identifying a potential new Borrelia species, a variety of viral families, and a novel Coxiella endosymbiont. We will characterize the pathogenic potential of these microbes for mammals and a collaboration with Dr. Scott A. Shaffer, San Jose State University, will compare the microbes we identified in the ticks with those identified in the microbiome of the California Gull. Finally, comparative genomic analysis of existing tick sequences will provide insight into tick evolution. Identification of genes essential for TBFV infections and antivirals. Ms. Offerdahl completed the initial screen of two commercially available antiviral libraries for efficacy against LGTV. From the results, we have chosen 20 of the most promising to further characterize. Most of this subset have not yet been tested against TBFV and several have not been reported as potential antivirals against any flaviviruses. We are continuing these studies utilizing additional cell lines as well as the more pathogenic POWV. The use of single cell technology to study TBFV infections Dr. Aspinwall is using single cell analysis to study ISE-6 cells infected by LGTV. The purpose of this project is to identify transcriptional changes in tick cells cause directly or indirectly by LGTV infection. The project will later expand into tick tissues, but initially we are using cell culture models to modify existing procedures to study both viral and host RNA using commercially available systems. Once these initial studies are completed, the work will be expanded to the study of BSL2 LGTV and BSL3 pathogen POWV. We are also optimizing tissue dissociation protocols to isolate individual cells which will facilitate single cell analysis of infected ticks. Single cell sequencing of LGTV infected Ixodes scapularis cell line (ISE-6) We have successfully modified the procedures to detect LGTV RNA in single cell sequencing of an infected tick cell line. While we still have plans to expand this research to include tick tissue, novel insights are apparent in this data both regarding the cellular composition of ISE6 cells and the effect of virus infection on their tick vectors. To validate these findings, we have generated sequence from multiple time points during LGTV infection of tick cells and will publish the findings. Single cell sequencing of LGTV infected Ixodes scapularis tissues To perform single cell sequencing analysis on infected ticks, it is necessary to have readily available a large cohort of LGTV infected ticks. Developing this key component has been challenging. We are employing multiple infection strategies to secure a cohort of ticks with 90% or greater infection rate. Tick infections are planned, and infection rates will be assessed after molting. We continue our collaborations with the Tirloni lab on tissue dissociation and RNA scope analysis in association with this project. Effect of tick pathogen on I. scapularis behavior The final analysis and publication of work conducted under Dr Aspinwall’s previous supervisor, Dr Tais Saito was completed. This work combined behavioral and proteomic findings on the shift in tick motility and feeding behavior induces by a pathogenic bacterium. The biology of segmented tick-borne orthoflavivirus-like infection The goal of this project is to establish cell culture models for gene expression and infection with the segmented orthoflavivirus-like virus, Jingmen tick virus (JMTV). The results obtained from these experiments will improve our understanding of the relationship between the newly emerging jingmenviruses and orthoflaviviruses. The rescue and characterization of JMTV infection in animal cell lines will improve our understanding of JMTV biology. In the past year, cell culture models for (JMTV) gene expression and infection were developed. Immunofluorescent assays and computational prediction results revealed JMTV non-structural (NS3) and structural (VP1) proteins distribution in the cytoplasm, VP1 and NS3 co-localized with the endoplasmic reticulum and plasma membrane. Importantly, our preliminary experiments in establishing a reverse genetics system for the rescue of jingmenviruses indicated that it will be possible to rescue JMTV from direct DNA plasmid transfection and that the best cells for JMTV rescue experiments is human embryonic kidney (HEK 293T) cells. Finally, by means of collaborations with San Diego Zoo Wildlife Alliance (SDZWA), we obtained two tortoise cell lines; Desert tortoise (Gopherus agassizii) and Galapagos tortoise (Chelonoidis nigra) cells. In this project, we are collaborating with SDZWA to study biology of orthoflaviviruses in tortoise cells. This will include characterizing replicating viruses in tortoise cell lines and investigating the integration of endogenous viral elements in the tortoise genome.