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 primarily 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. In addition, in the past year we have initiated lab and animal studies using 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 previously 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 2015, we continued 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. The viral proteins and cellular partners responsible for the membrane rearrangements are a topic of interest in BVBV. Therefore, we have developed molecular clones expressing the nonstructural proteins, either singly or in clusters, that should prove suitable for evaluating the role of specific proteins in the rearrangement of the cellular membranes. It is hoped that the vector backbones should allow for good expression in both mammalian and tick cell lines. We have determined that expression in mammalian cells is higher using vectors with the chicken beta-actin promoter, and that Effectene is most efficacious transfetin reagent. Initial results implicate that NS4a in the ER expansion and membrane rearrangement associated with TBFV infection. 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. Published a model of initiation and maintenance of persistent infection of 293T cells with LGTV derived from a full-length molecular clone. Infection resulted in an acute lytic crisis, in which >90% cell death was observed by day 5. However, the sparse cells surviving the lytic crisis repopulate the culture, which 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. By intensively investigating these findings with segmental RT-PCR and a deep sequencing strategy we determined that DI particles are not present at the initiation of persistence, but that once persistence is maintained, approximately 35% of the viral genomes contain a specific in-frame deletion affecting E and NS1. The deleted genomes code for a hybrid protein comprised of the N-terminal portion of E and the C-terminal portion of NS1, a moiety never previously reported nor predicted. DI particles have been previously identified in persistent flavivirus infections, but ours is the first to demonstrate that DI particles do not play a role in the initiation of persistence. A role for DI particles, and the hybrid protein, in the maintenance of persistence is under study. We have also studied changes in the cellular transcriptome, during acute and persistent infection. As noted, LGTV infection causes an acute lytic crisis of most cells, and we have shown the mechanism of cell death is apoptosis. The surviving cells somehow evade apoptosis and go on to establish persistent infection. Thus, the apoptosis pathway is implicated and our agnostic deep sequencing approach confirms that gene pathways associated with cell survival and apoptosis avoidance are involved. Intensive bioinformatics perusal is underway using Ingenuity Pathway Analysis and other similar software packages, and this work is being prepared for publication. Peromyscus leucopus is the identified reservoir species for that autochthonous pathogenic POWV. We have begun experiments to characterize POWV/DTV infection in the P. leucopus mice. An intraperitoneal dose of POWV that is lethal to age-matched Balb/C or C57Bl, does not produce a detectable response in P. leucopus. However, when the same dose is administered intra-cerebrally, the P. leucopus mice develop an asymptomatic infection restricted to the brain and characterized by low levels of virus and viral RNA in the brain, as well as neutralizing antibody. A mild meningoencephalitis is evident by 15 days after infection, but resolves. Virus can also be identified by in situ hybridization. Thus, the natural rodent reservoir host for POWV is somehow able to restrict and control the infection. This has interesting implications for the role of the mouse in the POWV biological cycle.
| 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 |
| 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 |
| 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 |
| 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; 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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