Tick-borne flaviviruses are recognized as one of the major global concerns to human health. These viruses are mostly transmitted through the bite of an infected Ixodes tick that results in meningitis, encephalitis, biphasic fever, febrile illness, or hemorrhagic fever in humans (Kellman et al., 2018; Madison-Antenucci et al., 2020). Tick-borne flaviviruses including Tick-borne encephalitis virus (TBEV), Powassan virus (POWV), Langat virus (LGTV), Kyasanur forest disease virus (KFDV), Louping ill virus, and Omsk hemorrhagic fever virus (OHFV) are both genetically and antigenically related to each other (Hermance and Thangamani, 2018; Kellman et al., 2018; Hermance et al., 2019; Pierson and Diamond, 2020). Several studies, including our own, have primarily focused on studying tick-bacteria interactions (Khanal et al., 2017, 2018; Taank et al., 2018, 2020; Dahmani et al., 2020; Ramasamy et al., 2020; Regmi et al., 2020). In contrast, a limited number of studies have focused on understanding interactions between tick-borne flaviviruses and their arthropod vectors at the molecular level (Neelakanta and Sultana, 2015; Grabowski and Hill, 2017; Wolf et al., 2020). Therefore, a detailed understanding of the connection between tick-borne flaviviruses and their vector host is essential to design preventive strategies and to disrupt the pathogen transmission.

Langat virus is a naturally attenuated flavivirus with non-pathogenic/low-pathogenic concerns to human health. Therefore, our laboratory used it as a model pathogen to understand the interactions of virulent tick-borne flaviviral infections in ticks. In addition, work with LGTV can be safely performed at the Biosafety level 2 (BSL-2) (Zhou et al., 2018). Like other tick-borne flaviviruses, LGTV has a lipid-envelope and a positive-sense single-stranded RNA genome of approximately 11 kb in length (Radzi et al., 2015). Arthropods play key role(s) in the acquisition and transmission of numerous viruses in different geographic regions (Nuttall and Labuda, 2003; Shaw and Catteruccia, 2019). Ticks have developed paramount strategies to indulge as successful vectors for pathogen acquisition and transmission. They have an ability to efficiently take a blood meal through biting a host and allowing viral survival and replication within them for an extensive period of time (Nuttall et al., 2000; Mitzel et al., 2007; Hart and Thangamani, 2020; Boulanger and Wikel, 2021). Ixodid ticks are reported to be the most prominent vectors that play key role(s) in the flavivirus acquisition and transmission cycle (Mitzel et al., 2007). Flaviviruses can infect Ixodid ticks at any developmental stage (larvae, nymphs, or adults), and the infection can be maintained throughout the tick’s lifespan (Mitzel et al., 2007; Bowman and Nuttall, 2008; Hart and Thangamani, 2020). Tick-borne flaviviruses usually spend 95% of their life cycle in Ixodes tick vectors rather in the vertebrate host including the accidental human host (Maffioli et al., 2014).

Regardless of the significance of Ixodid ticks as key vectors for bacterial acquisition and transmission, little is known regarding the flavivirus-vector host interactions. Laboratory models mimicking natural routes of infection of animals and ticks with tick-borne viruses are urgently needed to study molecular mechanisms of virus transmission. An understanding of the molecular mechanism(s) is essential to gain in-depth knowledge on the acquisition of the virus from the vertebrate host to ticks, pathogen replication in the tick midgut, crossing of the mid-gut barrier, persistent survival in the salivary glands, and then transmission to the vertebrate host. Technical challenges faced in a research laboratory have limited the number of studies conducted on the flavivirus-tick host interactions. The two common methods used for infecting ticks in a laboratory condition are parenteral inoculation by microinjection (Talactac et al., 2017) and synchronous infection of ticks by immersion method (Mitzel et al., 2007). There are several limitations for these two methods, as majority of the flaviviruses need high biocontainment level facilities (such as BSL-3 or BSL-4), which add to the technical issues on working with these pathogens. Microinjection method to generate LGTV-infected ticks is an artificial system. The success in generating LGTV-infected ticks by microinjection is facilitated by the evasion of the midgut escape barrier to allow direct access into the hemolymph. This process also ensures a relatively quicker progression into the tick-salivary gland, however, generating cohorts of infected ticks with equal pathogen burden is a major limitation. This factor is a critical determinant to assess vector competency in these artificially infected ticks (Talactac et al., 2017; Sun et al., 2020). Tick salivary glands enhance virus replication that can be detected in females at least upon 120 days post-infection (Nuttall and Labuda, 2003; Slovák et al., 2014). For synchronous LGTV-infection of ticks, large volumes (1–2 ml) of concentrated virus stocks (such as 10⁷ or 10⁸ pfu/ml) and longer incubations (17 days) are required for generation of small batch of infected ticks. Also, only 60–70% of ticks were capable of acquiring pathogen loads by body absorbance or bathing in the virus stocks (Taank et al., 2018; Regmi et al., 2020).

Our previous studies have demonstrated the role of arthropod-derived extracellular vesicles (EVs or exosomes) in the transmission of flaviviruses such as LGTV, and DENV2/3, which perhaps are mediated by the CD63 ortholog Tsp29Fb (Vora et al., 2018; Zhou et al., 2018, 2020). I. scapularis ISE6 cells have shown that LGTV might utilize the tick-derived molecules to facilitate its replication in the host cells (Grabowski et al., 2016, 2017). In addition, we noted that LGTV infection suppress Ixodes scapularis IsSMase gene expression, a molecule involved in exosome biogenesis (Regmi et al., 2020). IsSMase (Sphingomyelinase D or SMase D, a venomous protein ortholog of spiders) is a tick saliva component that regulates the cytokine expression to modulate the programming of vertebrate immune response via arthropod exosomes (Wikel, 2017; Regmi et al., 2020). IsSMase neutralizes the Th1 cytokine response toward a Th2-induced cytokine response and modulates CD4 + T cells in order to express interleukin 4 (IL-4) (Wikel, 2017; Regmi et al., 2020). Our recent studies focusing on understanding the molecular mechanisms during pathogen-vector-host interactions have revealed different survival strategies used by microbes (Taank et al., 2018, 2020; Vora et al., 2018; Zhou et al., 2018, 2020; Dahmani et al., 2020; Ramasamy et al., 2020; Regmi et al., 2020). Until this study, evaluating vector competence of I. scapularis for LGTV upon blood feeding has never been investigated. In the current study, utilizing LGTV as a model pathogen, we established a method to infect naïve I. scapularis by feeding these ticks on LGTV-infected blood from murine host. This study not only gives a novel method to generate LGTV-infected I. scapularis ticks but also provides an efficient tool to study acquisition dynamics of this important vector.

The developmental cycle of an arthropod vector from an egg through larva and/or nymph to the adult stage could take years (Mitzel et al., 2007; Stewart and Bloom, 2020). Arthropod vectors such as I. scapularis ticks feed on the vertebrate host for about 3–6 days to acquire a complete blood meal (or full engorgement) and this extended event allows successful acquisition and/or transmission of pathogens (Heinze et al., 2012; McNally et al., 2012; Stewart and Bloom, 2020). The ability of ticks to transstadially transmit flaviviruses provide greater possibilities for the evolutionary changes in the virus at the phenotype and/or genotype level in the vector (Kellman et al., 2018). Consequently, thoughtful investigations to study the interactions between an arthropod vector and the tick-borne flaviviruses are essential to understand an in-depth mechanism of viral pathogenesis.

To combat vector-borne viral diseases, novel methods need to be developed to address prevention and control strategies. Development of simple approaches to infect ticks would rapidly advance investigations in learning vector-host interactions. Several studies have reported the acquisition and subsequent transmission of bacterial pathogens from the vertebrate host to ticks such as I. scapularis, the vector of Borrelia burgdorferi (Levin and Fish, 2000; Bockenstedt et al., 2006), human granulocytic ehrlichiosis (Hodzic et al., 1998), and transmission of Anaplasma phagocytophilum (Levin and Ross, 2004). Michael Levin and colleagues infected the mice with A. phagocytophilum, and upon feeding of larval ticks they demonstrated the pathogen loads in ticks. The infected larval ticks were allowed to molt, and the nymphs were found to have maintained the bacterial loads (Levin and Ross, 2004). So far, we believe that no acquisition studies have been recorded and shown for tick-borne viruses such as LGTV. Our current study provides evidence for the acquisition of tick-borne LGTV by both nymphal and larval ticks upon feeding on infected mice. In this study, we fed the nymphal and larval ticks on LGTV-infected mice and observed that ticks acquired a significant level of viral burden that was detectable in both nymphal (partially or fully fed) and larval ticks. Furthermore, arthropod vector such as, Amblyomma tigrinum, A. ovale, and A. tonelliae larvae effectively acquired the flaviviruses after feeding on viremic chicks (Flores et al., 2019). The technical difficulties of the previously used methods for infecting ticks, such as parenteral inoculation (Talactac et al., 2017) and synchronous infection of ticks by immersion method, restrict key studies of the tick-virus interactions at the higher containment levels (Mitzel et al., 2007). Our previous studies (Taank et al., 2018; Regmi et al., 2020) have shown that synchronous infection of ticks with LGTV or A. phagocytophilum is possible in 60–70% of ticks; however, it would take 17 days of incubation. Also, the immersion method is arduous to assess with high priority pathogens such as POWV and TBEV due to the requirement of higher containments/facilities and the use of larger volumes of concentrated viruses (at the doses of 10⁷–10⁹ pfu/ml or perhaps much higher dose).

Studies that determine tropism of LGTV in rodents are limited. In the current study, we reported a simple method of acquisition of LGTV infection by a large number of larval or nymphal ticks feeding on murine blood. Mice administered intraperitoneally with 5 × 10⁴ pfu of LGTV dose showed replicative viral loads on day 3 p.i., however, the viral infection persisted until day 6 p.i., in blood and peripheral tissues such as spleen, liver, and skin (Figure 2). Also, we found that LGTV disseminated to the mice brain tissue, suggesting compromised blood-brain barrier (BBB) and neuroinvasion of the virus. This data suggested that inoculation with viral dose of 5 × 10⁴ pfu is required for LGTV replication and dissemination into mice tissues. Also, detection of viral loads in mice blood and skin indicated the LGTV spread in the murine host. The detection of higher viral loads in the peripheral tissues (such as spleen and liver) suggested enhanced replication at these sites. The dissemination of viral loads into the brain further suggests that high viremia in blood and peripheral tissues could lead to the breach of the BBB and neuroinvasion. In addition, detection of LGTV in partially fed nymphal ticks also demonstrated higher viral loads suggesting an early and successful acquisition of virus into ticks. The detection of LGTV loads at 120 h PF (or day 6 p.i. for mice) of nymphal or larval ticks further suggested successful acquisition, replication, and persistence of virus in blood-fed ticks collected at later time point of mice infection. Transstadial transmission of LGTV from infected larval ticks to molted nymphs further suggested that acquisition of pathogen loads by larval ticks is transmitted to the next tick stage and is perhaps maintained in the tick body. Our recent study showed that LGTV suppress IsSMase transcripts for its survival and replication in unfed and partially fed (24 h DF) nymphal ticks (Regmi et al., 2020). Down-regulation of IsSMase expression in LGTV-infected nymphs (partially or fully fed), larvae, and molted nymphs suggested the pathogen influence of gene expression in all stages of ticks. This study has also shown that IsSMase reduced levels correlates with down regulation of its enzymatic activity and accumulation of sphingomyelin (SM) lipid levels that may support membrane associated viral replication and exosome biogenesis upon LGTV infection in tick cells. We report an appropriate, fast, and efficient method to generate LGTV infected blood fed ticks (as large batches). Compared to the other approaches such as microinjections or synchronous infections, we believe that this method is a natural way to generate large batches of LGTV-infected ticks.

In summary, viral loads were detected both in murine tissues and in nymphal/larval ticks fed on LGTV-infected mice suggesting viral-replication in mice blood and dissemination into tissues including skin and, furthermore, acquisition into the nymphal/larval tick’s body from the infected murine host. We have also provided evidence for the transstadial transmission of LGTV from fed larval ticks to molted nymphs. Our study provides a method not only to generate LGTV-infected ticks but also provides a tool to study acquisition and transmission dynamics of this virus and perhaps other high priority tick-borne flaviviruses, such as TBEV and POWV. This study would considerably boost investigation, not only in understanding the viral acquisition and transmission and possibly other vector-host interactions, but could also accelerate anti-vector/transmission-blocking vaccine research in the field of tick-borne viral diseases.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by University of Tennessee, College of Veterinary Medicine, Institutional Animal Care and Use Committee (IACUC).

GN infected mice and generated fed I. scapularis uninfected or LGTV-infected ticks. WA and KR performed all molecular analysis and wrote the first manuscript draft. HS modified, revised, and finalized the manuscript, collected all required materials and reagents, designed and coordinated the entire study, organized all the data, and compiled and supervised overall investigations. All authors performed experiments, discussed, analyzed, and interpreted the data in several settings, read and edited the manuscript, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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