The Vector borne viruses from different virus families account for many medically significant viral pathogens. More specifically, the vector borne flaviviruses, which belong to the Family Flaviviridae, genus Flavivirus, comprise some of the most important emerging and re-emerging viral pathogens. The tick borne flaviviruses (TBFV) include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Powassan/deer tick virus (POWV/DTV) and Langat virus (LGTV). TBFV are generally transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. The mosquito borne flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Our current research is focused on the TBFV, but studying the biology of TBFV will elucidate the biology of other vector borne viruses. The research in our laboratory employs virology, immunology, advanced imaging techniques, genomics, cell biology, molecular biology, and vector biology. We study LGTV, a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. LGTV can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. Studies have also been initiated on the more virulent autochthonous BSL-3 POWV/DTV. Comparison of TBFV biology in mammalian and tick cells. A key difference between TBFV infection of vertebrate and arthropod host systems is that infection of ticks is persistent and non-cytolytic, whereas infection of mammalian hosts is typically acute and cytopathic. We are investigating the nature of this difference to identify responsible host and viral factors. We published a study comparing LGTV virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal microscopy, electron microscopy, and electron tomography. Flavivirus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. Electron tomography revealed virus-induced spherical vesicles thought to protect replicative intermediates from intracellular antiviral systems. In contrast to mammalian cells, TBFV-infection in tick cells shows delayed and decreased membrane proliferation. Additionally, electron tomography of infected tick cells shows a shift from spherical vesicles to tubular profiles, especially in the context of persistently infected cells. In 2014, we expanded these structural studies to cultures of primary embryonic brain. LGTV infection was largely restricted to cells of neuronal origin, and ultrastructural studies revealed results similar to those observed in mammalian cell lines, although the frequency of the tubular profiles appeared greater than in permanent cell lines. Cellular structures associated with viral replication and virions were observed all along the processes of neurons, suggesting that these components are transported to actual nerve endings. Also in 2014, we have developed molecular clones expressing the nonstructural proteins, either singly or in clusters, that should prove suitable for evaluating the role of specific elements in the rearrangement of the cellular membranes. The vectors backbones should allow for good expression in both mammalian and tick cell lines. Molecular biology and molecular pathogenesis of acute and persistent TBFV infection. Persistent infection plays a crucial role in natural life cycle of TBFV in rodent and arthropod hosts, and may also be responsible for prolonged debilitating sequelae observed in survivors of acute TBFV infection. However, this aspect of TBFV biology has been little studied. We successfully established a model of persistent infection of 293T cells with LGTV derived from a full-length molecular clone. Infection resulted in an acute cytopathic phase, in which >90% cell death was observed by day 5. However, if the cultures are re-fed and carefully maintained, the sparse cells surviving the lytic crisis repopulate the culture. These 293T cell cultures can be serially passaged for longer than 30 weeks. Throughout this time, greater than 90% of the cells are positive for LGTV E protein and the cultures produce infectious virus. Although the persistently infected 293T cell cultures produced virus throughout the period, the titer declined with continued passage from near 108 ffu/ml, to approximately 104 ffu/ml. Interestingly, the copy numbers of (+) and (-) viral RNA strands did not change, suggesting a dramatically increasing particle:PFU ratio, consistent with the presence of defective interfering (DI) particles. We examined this hypothesis using specifically designed primers, and found evidence of internal deletions in the structural protein portion of the genome. We are intensively investigating these findings using a deep sequencing strategy specifically designed to capture and characterize viral genomes. Thus, we have successfully established a model of persistent LGTV infection that will prove useful for studying virus biology as well as both the virus and the cell transcriptome. DI particles have been previously identified in persistent flavivirus infections, but an essential role in either initiation or maintenance of persistence is not established. We have also studied changes in the cellular transcriptome, during acute infection as well as the initiation and maintenance of persistent LGTV infection in 293T cells. As noted, LGTV infection of 293T or other mammalian cells leads to an acute cytopathic lytic crisis in which most cells die. The primary mechanism of cell death is thought to be apoptosis. The remaining cells are somehow able to maintain persistent virus replication while either evading or down-modulating the relevant cell death response. Although it seems probable that the apoptosis pathway may be involved, our agnostic approach is to examine the entire cellular response by employing deep sequencing and network analysis of the cellular transcriptome. This unbiased approach enables us to identify the various pathways involved in both acute and persistent LGTV virus infection. We anticipate the implication of novel pathways not previously associated with viral infection. When the cellular transcriptome of 293T cells infected for 48 hours with LGTV (acute) was compared to that from mock infected cells, the expression level of 800 genes was perturbed. However, a similar comparison between persistently infected and mock infected cells indicated that expression levels of 3000 genes were affected .The initial pathway analysis has so far suggested that certain genes, such as IFN-β, may be switched off in the persistent phase, a possible mechanism modulating the antiviral state observed in the acute phase. Evaluation of the results revealed that a set of genes was uniquely perturbed in the persistent phase, but not in the acute phase. This data has enabled us to identify the top 10 pathways specifically associated with viral persistence. Specifically, transcriptome analyses allowed the enrollment of 150 genes into several pathways. Analysis to date implicates pathways important for cell survival, including apoptosis, cell cycle, and immune responses, but also metabolic processes and transcription regulation. More detailed definition of the pathways and elucidation of the roles of specific genes are ongoing. We are presently extending the viral and cellular transcriptome studies of persistent infection to the more virulent BSL3 Powassan/Deer tick virus.
| Mlera, Luwanika; Meade-White, Kimberly; Dahlstrom, Eric et al. (2018) Peromyscus leucopus mouse brain transcriptome response to Powassan virus infection. J Neurovirol 24:75-87 |
| Kellman, Eliza M; Offerdahl, Danielle K; Melik, Wessam et al. (2018) Viral Determinants of Virulence in Tick-Borne Flaviviruses. Viruses 10: |
| Grabowski, Jeffrey M; Offerdahl, Danielle K; Bloom, Marshall E (2018) The Use of Ex Vivo Organ Cultures in Tick-Borne Virus Research. ACS Infect Dis 4:247-256 |
| Paules, Catharine I; Marston, Hilary D; Bloom, Marshall E et al. (2018) Tickborne Diseases - Confronting a Growing Threat. N Engl J Med 379:701-703 |
| Offerdahl, Danielle K; Dorward, David W; Hansen, Bryan T et al. (2017) Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines. Virology 501:54-62 |
| Lewy, Tyler G; Grabowski, Jeffrey M; Bloom, Marshall E (2017) BiP: Master Regulator of the Unfolded Protein Response and Crucial Factor in Flavivirus Biology?. Yale J Biol Med 90:291-300 |
| Grabowski, Jeffrey M; Tsetsarkin, Konstantin A; Long, Dan et al. (2017) Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control. MBio 8: |
| Mlera, Luwanika; Meade-White, Kimberly; Saturday, Greg et al. (2017) Modeling Powassan virus infection in Peromyscus leucopus, a natural host. PLoS Negl Trop Dis 11:e0005346 |
| Mlera, Luwanika; Lam, Jennifer; Offerdahl, Danielle K et al. (2016) Transcriptome Analysis Reveals a Signature Profile for Tick-Borne Flavivirus Persistence in HEK 293T Cells. MBio 7: |
| Offerdahl, Danielle K; Clancy, Niall G; Bloom, Marshall E (2016) Stability of a Tick-Borne Flavivirus in Milk. Front Bioeng Biotechnol 4:40 |
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