TRIpartite Motif (TRIM) protein 5 alpha (TRIM5α) is a well-characterized cellular inhibitor of lentivirus replication that limits transmission of related viruses between primates. We previously reported that TRIM5α derived from humans and rhesus macaques inhibits replication of orthoflaviviruses belonging to the tick-borne encephalitis virus (TBEV) serocomplex, including TBEV, Kyasanur forest disease virus and Langat virus (LGTV), but interestingly not the tick-borne Powassan virus (POWV). To further characterize the primate TRIM5α and orthoflavivirus interface, we screened TRIM5α variants from representative old- and new-world primates for restriction capacity. TRIM5α from old-world African green monkey, De Brazza's monkey and chimpanzee demonstrated virus-specific restriction of tick-borne orthoflaviviruses. Efforts to determine why TRIM5α fails to inhibit POWV revealed that our lab stock had acquired a non-synonymous mutation in NS3 that, when introduced into a POWV molecular clone, facilitated virus replication in the presence of all inhibitory primate TRIM5α proteins. Infection of human dendritic cells with TRIM5α-resistant POWV resulted in high early replication and strong induction of interferon responses that limited replication compared with the wild-type virus. Thus, primate TRIM5α functions as a potent cellular barrier to infection with tick-borne orthoflaviviruses that restrains replication to a level that may help avoid early innate immune recognition.

The detailed mechanism and sequence by which tick-borne flaviviruses (TBFVs), such as Langat virus (LGTV), infect and disseminate in arthropod hosts remain undefined. To begin characterizing these processes, we used artificial membrane feeding chambers to feed adult Ixodes scapularis ticks with blood containing LGTV. At 24, 48, 72, and 96 hours (h) after attachment, we removed and dissected the partially fed ticks to obtain the midgut and salivary glands. Histology confirmed infection in cells of the digestive epithelium lineage; infection was noted in midgut generative cells and the more differentiated functional digestive cells over the course of feeding. The viral envelope (E) protein, nonstructural protein 3 (NS3), and double-stranded RNA (dsRNA) were readily detected in these cells by 48 h after infection. Parallel analysis indicated that cells in salivary gland acini were also infected by 48 h, where virus target cells appeared to be the granular cells in acini types II and III. Thus, both salivary glands and midgut showed direct evidence of infection by 48 h. Although viral staining was not observed at 24 h, when organs were removed at 24 h and individually cultured ex vivo, the virus was detected. Taken together, our results provide evidence of LGTV infection in both the salivary glands and midgut within the first 24 h of a blood meal. The findings should prompt a reevaluation of the systemic dissemination of TBFV in infected ticks.

Arthropod - and vertebrate-associated jingmenviruses (arbo-JMV) have segmented positive-strand RNA genomes and are provisional members of the genus Orthoflavivirus (family Flaviviridae). Current investigations have described arbo-JMV infection in vertebrate hosts in proximity to humans. This raises concerns about the virus host range and public health implications. This review explores the genomic and evolutionary relationship between arbo-JMV and orthoflaviviruses and evaluates the potential of arbo-JMV to pose a public health threat.

In October 2023, a partially engorged female Ixodes tick was removed from a dog in Bozeman, Montana, USA, that had recently spent time in eastern Montana. The tick was identified as I. scapularis according to morphologic characteristics and genomic sequencing, suggesting an expanded geographic distribution requiring continued public health surveillance.

Sandfly-borne phleboviruses (SBPs) are an important cause of febrile diseases and neuroinvasive infections in humans, especially in endemic regions. They have been described in Central Asia, Africa, the Middle East, and the Mediterranean regions. Current investigations suggest that SBPs originated from Africa, albeit with little zoonotic threat information. Particularly pertinent is the recent identification of genetically diverse SBPs associated with human infection circulating in North and East Africa. Spread of these viruses to new regions may pose a significant risk to the local populations with little or no pre-existing immunity. Additionally, the lack of SBP detection methods at the point of care may lead to an incorrect diagnosis of malaria and influenza, inappropriate treatment, and an underestimated disease burden. Despite the availability of a wide range of analytic approaches that include cell culture, electron microscopy, and serological screening, diagnosis remains a challenge. Application of new molecular techniques such as next-generation sequencing (NGS) would enable description of new SBPs; however, correlation with additional field clinical data is needed to evaluate the zoonotic significance of any new SBPs. In this review, we provide a summary of the disease ecology of SBPs in Africa to concatenate the existing knowledge on transmission dynamics. The review also highlights the limited surveillance of SBPs in Africa, thus confirming the need for enhanced virus characterization incorporating advanced approaches such as capture-based target enrichment NGS, allowing for the detection of existing and novel SBPs, in addition to epidemiologic data on their clinical relevance.

This clinical insights article examines reasons behind the increase in tick-borne diseases and what clinicians should know about diagnosis and mitigation.

A bite from an infected tick is the primary means of transmission for tick-borne flaviviruses (TBFV). Ticks ingest the virus while feeding on infected blood. The traditional view is that the virus first replicates in and transits the tick midgut prior to dissemination to other organs, including salivary glands. Thus, understanding TBFV infection in the tick midgut is a key first step in identifying potential countermeasures against infection. Ex vivo midgut cultures prepared from unfed adult female Ixodes scapularis ticks were viable and remained morphologically intact for more than 8 days. The midgut consisted of two clearly defined cell layers separated by a basement membrane: an exterior network of smooth muscle cells and an internal epithelium composed of digestive generative cells. The smooth muscle cells were arranged in a stellate circumferential pattern spaced at regular intervals along the long axis of midgut diverticula. When the cultures were infected with the TBFV Langat virus (LGTV), virus production increased by two logs with a peak at 96 hours post-infection. Infected cells were readily identified by immunofluorescence staining for the viral envelope protein, nonstructural protein 3 (NS3) and dsRNA. Microscopy of the stained cultures suggested that generative cells were the primary target for virus infection in the midgut. Infected cells exhibited an expansion of membranes derived from the endoplasmic reticulum; a finding consistent with TBFV infected cell cultures. Electron microscopy of infected cultures revealed virus particles in the basolateral region between epithelial cells. These results demonstrated LGTV replication in midgut generative cells of artificially infected, ex vivo cultures of unfed adult female I. scapularis ticks.

Rocky Mountain spotted fever (RMSF) is a rapidly progressive and often fatal tick-borne disease caused by Rickettsia rickettsii. Its discovery and characterization by Howard Ricketts has been hailed as a remarkable historical example of detection and control of an emerging infectious disease, and subsequently led to the establishment of the Rocky Mountain Laboratories (RML). Here, we examined an unopened bottle of a vaccine, labeled as containing RMSF inactivated by phenol-formalin of infected ticks, developed prior to 1944 at RML by DNA analysis using Illumina high throughput sequencing technology. We found that it contains DNA from the Rocky Mountain wood tick (Dermacentor andersoni), the vector of RMSF, the complete genome of Rickettsia rickettsii, the pathogen of RMSF, as well as the complete genome of Coxiella burnetii, the pathogen of Q-fever. In addition to genomic reads of Rickettsia rickettsii and Coxiella burnetii, smaller percentages of the reads are from Rickettsia rhipicephali and Arsenophonus nasoniae, suggesting that the infected ticks used to prepare the vaccine carried more than one pathogen. Together, these findings suggest that this early vaccine was likely a bivalent vaccine for RMSF and Q-fever. This study is the among the first molecular level examinations of an historically important vaccine.

Half of Powassan infection survivors experience long-term, mild to severe neurological symptoms. The progression from acute to chronic Powassan disease is not well understood, severely limiting treatment and prevention options.

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae.Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM).TBEV virions are small enveloped spherical particles about 50 nm in diameter.The TBEV genome consists of a single-stranded positive sense RNA molecule.The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs).The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity.The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encepha-litis. The primary function of its capsid protein (TBEVC) is to Tick-borne pathogen idae family, (TBFVs),

The relapsing fever agent Borrelia hermsii is transmitted by the tick Ornithodoros hermsi. To study the B. hermsii-tick interactions required for pathogen acquisition and transmission we developed an artificial membrane feeding system for O. hermsi nymphs and adults that results in a high percentage of engorgement. This system provides the nutritional requirements necessary for the tick to develop, mate, and produce viable eggs. By inoculating the blood with B. hermsii, we were able to obtain infected ticks for quantitative studies on pathogen acquisition and persistence. These ticks subsequently transmitted the spirochetes to mice, validating this system for both acquisition and transmission studies. Using this feeding method, a mutant of the antigenic variation locus of B. hermsii (Vmp-) that is incapable of persisting in mice was acquired by ticks at equivalent densities as the wild-type. Furthermore, Vmp is not required for persistence in the tick, as the mutant and wild-type strains are maintained at similar numbers after ecdysis and subsequent feeding. These results support the theory that Vmp is an adaptation for mammalian infection but unnecessary for survival within the tick. Interestingly, B. hermsii numbers severely declined after acquisition, though these ticks still transmitted the infection to mice. This procedure reduces animal use and provides a safe, highly controlled and well-contained alternative method for feeding and maintaining O. hermsi colonies. Importantly, this system permits quantitative studies with B. hermsii strains through ingestion during the blood meal, and thus more closely recapitulates pathogen acquisition in nature than other artificial systems.

Repurposing FDA-approved drugs that treat respiratory infections caused by coronaviruses, such as SARS-CoV-2 and MERS-CoV, could quickly provide much needed antiviral therapies. In the current study, the potency and cellular toxicity of four fluoroquinolones (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) were assessed in Vero cells and A549 cells engineered to overexpress ACE2, the SARS-CoV-2 entry receptor. All four fluoroquinolones suppressed SARS-CoV-2 replication at high micromolar concentrations in both cell types, with enoxacin demonstrating the lowest effective concentration 50 value (EC50) of 126.4 μM in Vero cells. Enoxacin also suppressed the replication of MERS-CoV-2 in Vero cells at high micromolar concentrations. Cellular toxicity of levofloxacin was not found in either cell type. In Vero cells, minimal toxicity was observed following treatment with ≥37.5 μM enoxacin and 600 μM ciprofloxacin. Toxicity in both cell types was detected after moxifloxacin treatment of ≥300 μM. In summary, these results suggest that the ability of fluoroquinolones to suppress SARS-CoV-2 and MERS-CoV replication in cultured cells is limited.

The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1β. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.

The testicle surprisingly emerged as important for Zika virus (ZIKV) infection, viral persistence, and sexual transmission. Systemic infection traffics ZIKV to the cells of the testes via the hematogenous or lymphogenous routes. Studies in mice and humans have shown that Sertoli cells, spermatogonia, and primary spermatocytes are preferred targets of ZIKV infection, but Leydig cells in the interstitium are infected sparingly. ZIKV-infected testicular cells may harbor virus/viral RNA for months, suggesting viral persistence. ZIKV destroys infected testicular cells leading to atrophy, which is associated with low testosterone and inhibins levels, and may thus negatively impact fertility in men. Testicular atrophy appears to be a slow process because ZIKV infection of the testis does not cause a dramatic induction of genes that cause apoptotic cell death. Nevertheless, ZIKV infection of the testis is important for persistence and sexual transmission. The ZIKV-host cell relationship is intriguingly complex and requires further study.

Tick-borne flaviviruses (TBFVs) are reemerging public health threats. To develop therapeutics against these pathogens, increased understanding of their interactions with the mammalian host is required. The PI3K-AKT pathway has been implicated in TBFV persistence, but its role during acute virus infection remains poorly understood. Previously, we showed that Langat virus (LGTV)-infected HEK 293T cells undergo a lytic crisis with a few surviving cells that become persistently infected. We also observed that AKT2 mRNA is upregulated in cells persistently infected with TBFV. Here, we investigated the virus-induced effects on AKT expression over the course of acute LGTV infection and found that total phosphorylated AKT (pAKT), AKT1, and AKT2 decrease over time, but AKT3 increases dramatically. Furthermore, cells lacking AKT1 or AKT2 were more resistant to LGTV-induced cell death than wild-type cells because they expressed higher levels of pAKT and antiapoptotic proteins, such as XIAP and survivin. The differential modulation of AKT by LGTV may be a mechanism by which viral persistence is initiated, and our results demonstrate a complicated manipulation of host pathways by TBFVs.

Infected Ixodes scapularis (black-legged tick) transmit a host of serious pathogens via their bites, including Borrelia burgdorferi, Babesia microti, and tick-borne flaviviruses (TBFVs), such as Powassan virus (POWV). Although the role of female I. scapularis ticks in disease transmission is well characterized, the role of male ticks is poorly understood. Because the pathogens are delivered in tick saliva, we studied the capacity of male salivary glands (SGs) to support virus replication. Ex vivo cultures of SGs from unfed male I. scapularis were viable for more than a week and maintained the characteristic tissue architecture of lobular ducts and acini. When SG cultures were infected with the TBFVs Langat virus (LGTV) or POWV lineage II (deer tick virus), the production of infectious virus was demonstrated. Using a green fluorescent protein-tagged LGTV and confocal microscopy, we demonstrated LGTV infection within SG acinus types II and III. The presence of LGTV in the acini and lobular ducts of the cultures was also shown via immunohistochemistry. Furthermore, the identification by in situ hybridization of both positive and negative strand LGTV RNA confirmed that the virus was indeed replicating. Finally, transmission electron microscopy of infected SGs revealed virus particles packaged in vesicles or vacuoles adjacent to acinar lumina. These studies support the concept that SGs of male I. scapularis ticks support replication of TBFVs and may play a role in virus transmission, and further refine a useful model system for developing countermeasures against this important group of pathogens.

The unfolded protein response (UPR) maintains protein-folding homeostasis in the endoplasmic reticulum (ER) and has been implicated as both beneficial and detrimental to flavivirus infection. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a sensor of the UPR, is commonly associated with antiviral effects during mosquito-borne flavivirus (MBFV) infection, but its relation to tick-borne flavivirus (TBFV) infection remains largely unexplored. In this study, we identified changes in UPR and autophagic activity during Langat virus (LGTV) infection. LGTV robustly activated UPR and altered autophagic flux. Knockdown of endogenous PERK in human cells resulted in increased LGTV replication, but not that of closely related Powassan virus (POWV). Finally, on examining changes in protein levels of components associated with UPR and autophagy in the absence of PERK, we could show that LGTV-infected cells induced UPR but did not lead to expression of C/EBP homologous protein (CHOP), an important downstream transcription factor of multiple stress pathways. From these data, we hypothesize that LGTV can antagonize other kinases that target eukaryotic initiation factor 2α (eIF2α), but not PERK, implicating PERK as a potential mediator of intrinsic immunity. This effect was not apparent for POWV, a more pathogenic TBFV, suggesting it may be better equipped to mitigate the antiviral effects of PERK.

The deer tick Ixodes scapularis transmits a variety of disease agents in the United States, spreading the bacteria that causes Lyme borreliosis, the protozoan agent of babesiosis, and viruses such as Powassan. However, a variety of other organisms have also evolved symbiotic relationships with this tick species, and it seems likely that some of these microbes have simultaneously coevolved mechanisms to impact each other and their tick host. The number of organisms identified as I. scapularis symbionts has increased seemingly exponentially with the advent of PCR and next generation sequencing technologies, but convincing arguments have proposed that some of these are of environmental origin, unadapted to surviving the physiological conditions of the tick or that they are artifacts of ultrasensitive detection methods. In this review, we examine the diversity of the known microbes occurring within the I. scapularis microbiome, the evidence for interactions between microbes, and discuss whether some organisms reported to be symbionts of I. scapularis are experimental artifacts.