Abstract
ABSTRACTIxodes 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. LGTV- and 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.
Highlights
Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs)
The growth of recombinant flaviviruses may differ from parental viruses [50, 51]; the potential difference in growth between LGTV expressing green fluorescent protein (LGTVGFP) and its wild-type Langat virus (LGTV) TP21 strain in these organs has not been determined
We demonstrated that dissected organs from female I. scapularis were viable in culture and that the cultures supported replication of the low-neurovirulence LGTV and the pathogenic Powassan virus (POWV), indicating that the organ cultures were suitable for the detailed study of TBFV biology in a highly controlled arthropod system
Summary
Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). 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, July/August 2017 Volume 8 Issue 4 e01255-17. The tick-borne encephalitis viruses (TBEVs) and Powassan virus (POWV lineage I [LB strain] and lineage II [Deer tick virus strain; DTV]) cause severe meningoencephalitis, whereas Langat virus (LGTV) has low neurovirulence in humans [7, 9,10,11,12]. The diseases caused by these viruses are well characterized, and the experimental biology and pathogenesis of TBFV infections in mammalian hosts have been reasonably well studied
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