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 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 characterize the cytoarchitecture and biology of virus infection. Midgut cultures were viable for at least 8 days. Dr. Ochwoto observed two clearly defined cell layers separated by a basement membrane: an exterior network of smooth muscle cells, the actin filaments of which could be identified by staining with the phallotoxin phalloidin, and an internal lining layer composed of digestive, generative, or stem cells bearing a sparse microvillous surface. The smooth muscle cells were arranged in an annular circumferential pattern at regular intervals along the long axis of midgut diverticula. When the cultures were infected with the TBFV Langat virus (LGTV), viral production increased until 96 hours. Infected cells were readily identified by immunohistochemical staining for viral envelope (E) protein, nonstructural protein 3 (NS3) and dsRNA; confocal microscopy of the stained cultures suggested that digestive cells were the target for virus infection. Infected cells also exhibited an expansion of membranes derived from the endoplasmic reticulum (ER) as evidenced by staining with the ER marker PDI, a finding reminiscent of infected cell cultures. Electron microscopy of infected cultures revealed virus-like particles in the basolateral region between generative cells and infected cells exhibited membrane rearrangements similar to TBFV infected cell cultures. Dr. Ochwotos results demonstrated that TBFV can replicate in midgut cells and suggested a mechanism for virus dissemination from tick midguts. This work will be submitted for publication this FY. The studies must be confirmed in both replete and partially fed intact ticks. The artificial membrane system (QV) will likely prove useful. 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. This project studies microbial interactions in the tick, and develops new techniques to study several different viral and bacterial infections in ticks. Dr. Stewart has perfected a membrane based artificial blood feeding system for adult I. scapularis as well as nymphs and adults of Ornithodorus hermsii. The method also permits controlled, quantitative studies with B. hermsii mutants through ingestion during the blood meal, and thus more closely recapitulates pathogen acquisition in nature than other artificial systems. Working with another group, he applied the system to tick borne relapsing fever, a severe and sometimes fatal disease. By inoculating the blood with B. hermsii, they were able to obtain infected ticks for quantitative studies on the acquisition of this pathogen. After molting, these ticks transmitted the spirochetes to mice. Using a genetically engineered deletion mutant incapable of persisting in mice, we showed that this strain is acquired by O. hermsi during blood feeding at equivalent densities as the wild-type and confirmed that Vmp is not required for infection of the tick or through the molt. This work was published in the current FY. Characterization of cellular mechanisms essential for TBFV infections. In the past year, Dr. Flather has continued work on understanding the host factors that are required for tick-borne flavivirus replication in human cells. Understanding the host factor requirements for POWV replication has been an ongoing interest of the lab for the past several years. Genetic screening strategies performed in human cell lines revealed putative proviral host factors in the POWV infectious cycle. Two independent approaches, CRISPR screens and haploid mutagenesis screens, suggest that genes of the ER membrane protein complex, dystrophin-glycoprotein complex, activating signal co-integrator complex, and heparan sulfate biosynthesis are targeted for functionalities that promote POWV replication. To understand whether these factors and pathways are generally shared by TBFVs, validation studies were performed using three different TBFVs (POWV, DTV, and LGTV) and with two human cell lines (Huh7.5.1 and Hap1). Dr Falther has found that specific factors that were identified through his genetic screens fall into three distinct categories: those that are broadly shared by the TBFVs, those that are cell line specific factors, and those that are virus specific factors. For example, when the transmembrane protein dystroglycan (DAG1) is knocked out of cells, he observed a reduction in POWV replication, but not DTV or LGTV replication, suggesting that DAG1 is a POWV specific factor. In contrast, High Density Lipoprotein Binding Protein (HDLBP) is functionally important only in Hap1 cells, but for all TBFVs tested. Work to characterize the role that putative proviral host factors play in the infectious cycles of TBFVs is underway, particularly for those that may act as a receptor for TBFVs. In the future we also hope to describe whether there are homologous factors in Ixodid ticks that are necessary for replication of these viruses in the vector species. Development of an animal model for persistent POWV and DTV infection. Understanding how these viruses cause long-term symptoms as well as the possible role of viral persistence is important for developing therapies to treat chronic infection. However, there are not good animal models to study chronic disease. We inoculated six-week-old C57BL/6 mice with DTV and assayed for infectious virus, viral RNA, and inflammation during the acute infection, 21, 56, and 84 days post infection (dpi) in central nervous system (CNS) and non-CNS tissues. Only 21% of the mice were symptomatic and 83% recovered, but most mice (86%) were viremic 3 dpi. Infectious virus was only detected in the brains of mice sampled during the acute infection. Viral RNA was detected in the brain and spinal cord until 21 dpi but was more widespread in mice sampled during the acute infection. Meningitis and encephalitis were visible in acute mice and from mice sampled at 21 dpi but were significantly more severe in the former. Long-term inflammation was detected until 56 dpi in the brain and 84 dpi in the spinal cord, albeit at low levels. This will be a good model of persistent Powassan disease that mimics disease in humans.