Flavivirus Infection ofIxodes scapularis(Black-Legged Tick)Ex VivoOrganotypic Cultures and Applications for Disease Control

Abstract

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,

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

“…Four recent developments provide the groundworks for rapidly advancing our understanding of I. scapularis biology and microbial interactions. These are: (1) application of RNA interference (RNAi) gene silencing methods for functional analysis of tick proteins; (2) the release of the annotated I. scapularis genome (Gulia-Nuss et al, 2016); (3) a contained, artificial feeding system for nymphal and adult I. scapularis (Oliver et al, 2016); and (4) ex vivo organ culture of midgut and salivary glands (Grabowski et al, 2017(Grabowski et al, , 2019.…”

PERK-Mediated Unfolded Protein Response Signaling Restricts Replication of the Tick-Borne Flavivirus Langat Virus

“…At 0, 24, 48, and 72 h post culture, supernatant was removed and replaced with new media containing alamarBlue reagent (AbD Serotec, Kidlington, UK) at a 1:10 dilution [45]. Cells were then incubated for 2 h at 37 • C. Absorbance was measured at 570 nm using a Molecular Devices SpectraMax Plus 384 plate reader and SoftMax Pro v6.5 software [46]. Data shown are results of two biological replicates with three technical replicates each.…”

Powassan virus, a scoping review of the global evidence

“…However, two other potential antiflaviviral agents, the NS5 capping enzyme inhibitor BG‐323 (Bullard et al., ) and adenosine analog NITD008 (Yin et al., ), significantly reduced POWV titres in vitro in their respective studies. An I. scapularis organ culture model was developed to study the effects of POWV localization and replication to ultimately enhance screening for potential vaccine candidates and proteins for therapeutic use (Grabowski et al., ).…”