Flaviviruses including dengue virus (DENV) and Zika virus (ZIKV) cause significant human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum (ER) is a shared replication strategy, despite different clinical outcomes. While the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs.LGTV-and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTVand POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3= untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission.IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications,

In nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. These viruses account for considerable human morbidity and mortality worldwide. Increasing and substantial evidence of viral persistence in humans, which includes the isolation of RNA by RT-PCR and infectious virus by culture, continues to be reported. Viral persistence can also be established in vitro in various human, animal, arachnid and insect cell lines in culture. Although some research has focused on the potential roles of defective virus particles, evasion of the immune response through the manipulation of autophagy and/or apoptosis, the precise mechanism of flavivirus persistence is still not well understood. We propose additional research for further understanding of how viral persistence is established in different systems. Avenues for additional studies include determining if the multifunctional flavivirus protein NS5 has a role in viral persistence, the development of relevant animal models of viral persistence as well as investigating the host responses that allow vector borne flavivirus replication without detrimental effects on infected cells. Such studies might shed more light on the viral-host relationships, and could be used to unravel the mechanisms for establishment of persistence.

The tick-borne flavivirus, Powassan virus (POWV) causes life-threatening encephalitis in humans in North America and Europe. POWV is transmitted by ixodid tick vectors that feed on small to medium-sized mammals, such as Peromyscus leucopus mice, which may serve as either reservoir, bridge or amplification hosts. Intraperitoneal and intracranial inoculation of 4-week old Peromyscus leucopus mice with 103 PFU of POWV did not result in overt clinical signs of disease. However, following intracranial inoculation, infected mice seroconverted to POWV and histopathological examinations revealed that the mice uniformly developed mild lymphocytic perivascular cuffing and microgliosis in the brain and spinal cord from 5 to 15 days post infection (dpi), suggesting an early inflammatory response. In contrast, intracranial inoculation of 4-week old C57BL/6 and BALB/c mice was lethal by 5 dpi. Intraperitoneal inoculation was lethal in BALB/c mice, but 40% (2/5) of C57BL/6 mice survived. We concluded that Peromyscus leucopus mice infected i.c. with a lethal dose of POWV support a limited infection, restricted to the central nervous system and mount an antibody response to the virus. However, they fail to develop clinical signs of disease and are able to control the infection. These results suggest the involvement of restriction factors, and the mechanism by which Peromyscus leucopus mice restrict POWV infection remains under study.