The Use of Ex Vivo Organ Cultures in Tick-Borne Virus Research

Abstract

Each year there are more than 15 000 cases of human disease caused by infections with tick-borne viruses (TBVs). These illnesses occur worldwide and can range from very mild illness to severe encephalitis and hemorrhagic fever. Although TBVs are currently identified as neglected vector-borne pathogens and receive less attention than mosquito-borne viruses, TBVs are expanding into new regions, and infection rates are increasing. Furthermore, effective vaccines, diagnostic tools, and other countermeasures are limited. The application of contemporary technologies to TBV infections presents an excellent opportunity to develop improved, effective countermeasures. Experimental tick and mammal models of infection can be used to characterize determinants of infection, transmission, and virulence and to test candidate countermeasures. The use of ex vivo tick cultures in TBV research provides a unique way to look at infection in specific tick organs. Mammal ex vivo organ slice and, more recently, organoid cultures are additional models that can be used to elucidate direct tissue-specific responses to infection. These ex vivo model systems are convenient for testing methods involving transcript knockdown and small molecules under tightly controlled conditions. They can also be combined with in vitro and in vivo studies to tease out possible host factors and potential vaccine or therapeutic candidates. In this brief perspective, we describe how ex vivo cultures can be combined with modern technologies to advance research on TBV infections.

Keywords: flavivirus; organ; therapeutic; tick; vaccine; virus.

Figure 1.. Ex vivo tick culture options.

The backless tick culture involves removal of legs, mouthparts and the dorsal back of the tick before placing into culture. Metamorphosing tick culture focuses on the molting adult stage of the tick, which is removed from an engorged nymph before being placed into culture. Specific tick organs can be dissected from fed (engorged) or unfed ticks and placed directly into culture. These cultures can then be infected with virus in the culture vessel to provide infected cultures.

Figure 2.. Ex vivo mammal culture options.

(A) Sectioned organ slices are cultured on membrane inserts allowing efficient gas exchange and long-term culture maintenance. (B) Dissociated stem cells are allowed to aggregate within the culture vessel then extracellular matrix gel (blue) is added to support the developing organoids. Organoid culture is continued in tissue culture dishes or spinning bioreactors.

Figure 3.. Standard experimental setup for dsRNA/siRNA/small molecule soaking and recombinant-live virus growth assays in ex vivo cultures.

Several approaches to use (A) small molecule and (B) siRNA/dsRNA-mediated RNA interference transcript knockdown assays with ex vivo tick or mammal cultures exist. Identifying viability of cultures can be completed alone with the small molecule/siRNA/dsRNA treatments. Depending on experimental setup, options to pretreatment or posttreatment with small molecule/siRNA/dsRNA before or after tick-borne virus (TBV) infection exist. In addition, (C) ex vivo cultures can be infected with recombinant TBVs that may serve as live-virus vaccine candidates. Virus-infected, ex vivo cultures can be analyzed at different timepoints post infection where multiple parameters of virus infection can be measured (genome replication, replication, antigen presence and genome presence).

Figure 4.. Use of dsRNA-mediated RNA interference assays in ex vivo tick cultures to help identify possible countermeasures.

Tick organ cultures can be used for dsRNA-mediated, RNA interference transcript knockdown studies in tick-borne virus (TBV)-infected tick organs to identify effect on virus infection. Organ viability can also be obtained post dsRNA-mediated transcript knockdown. dsRNA-mediated transcript knockdowns can also be completed in TBV-infected ticks to identify 1) biological phenotypes in ticks and 2) effect on virus infection/transmission. Select tick genes with transcript knockdowns resulting in decreased virus infection, and ideally virus transmission, may possibly be considered as candidate targets for antigen characterization and/or small molecule screening studies.