Pathogenesis of Tick-Borne Flavivirus Infections

National Institute Of Allergy And Infectious Diseases

Investigators

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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 I. scapularis. Infection in ticks is a critical, but understudied, feature of TBFV biology. Our ex vivo organ culture work has yielded considerable information about TBFV infection in midgut and salivary glands of adult ticks, and is presently under review for publication. In the past year, Dr. Ochwoto and Dr. Stewart employed artificial membrane blood feeding and demonstrated that adult I. scapularis ticks can be infected with relatively high efficiency by feeding them on LGTV infected blood. Studies on the midgut of fed adults revealed the expected differentiation and proliferation of the intestinal epithelium into mature digestive cells, and the results indicated that these cells support virus replication. Initial studies suggest that the salivary glands also become infected while the ticks are feeding and that prior infection of the midgut and subsequent systemic dissemination of virus are not required for infection of the salivary glands. The nymphal stage of the ticks is the most important for human infection, so in the past year we have started to study infection in nymphs. However, when efforts were made to infect them using the artificial membrane system, the results were disappointing and inconsistent. As a result, Dr. Weck is planning to infect nymphs by feeding them on LGTV infected mice. Salivary glands have at least 3 types of acini and each acini contains multiple cell types. It is unclear if all acinar types or all cell types are permissive for virus infection. One way to approach this interesting question would be to infect ex vivo salivary gland cultures, dissociate the infected cultures and subject the cells to single cell analysis. In the past year, Dr. Ochwoto and Ms. Theriault applied cell dissociation methods to dissected salivary glands from adult ticks. The acini were refractory to dissociation using simple enzymatic digestion and we have not yet obtained single cell suspensions satisfactory for analysis. Microbial interactions in Ixodes scapularis ticks. Ixodes scapularis ticks harbor multiple infectious agents, including TBFV, B. burgdorferi and Anaplasma phagocytophilum, but polymicrobial human infections are rare. A possible explanation is that infection with one microbe interferes with infection by a second. In the past year, Dr. Stewart established a collaboration with Dr Eastwood from Virginia Tech to examine co-infections with anaplasma and TBFV. Dr Stewart is also developing tools to identify the TBFV receptor in both mammalian and arthropod systems. The approach is to create virus-like particles with a fluourescent tag that can be used in various binding assays. The expression constructs have been developed and protocols to purify the VLP are being tested. Identification of genes essential for TBFV infections and antivirals. In the past year, our work on genome wide screens for host factors regulating TBFV infection has undergone analysis and should be ready for publication in the coming FY. Also in the past year, we began testing several libraries of small molecules against LGTV. Several promising compounds have been identified and will be evaluated to see if they might be useful antivirals. Development of an animal model for chronic POWV/DTV disease. 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. In the past year, we published our mouse model of chronic POWV/DTV disease. The results indicated that the continued presence of detectable virus infection was not required for chronic CNS disease, suggesting that host responses play an important role. In an interesting side project, we supplied a vial of 1940's vintage rocky mountain spotted fever vaccine, prepared from infected ticks, to Dr Taubenberger and his colleagues. Deep sequencing of the material revealed the the presence of Rickettsia ricketsii, demonstrating that that agent against which the vaccine designed was present. In addition, however, sequence reads of Coxiella burnetii, the agent of Q fever, were also identified. This likely indicated that some of the ticks were co-infected with both agents.