Deer Tick Virus Research Articles

Prevalence and Genetic Diversity of Deer Tick Virus (Powassan Virus, Lineage II) in Ixodes scapularis Ticks in Five Habitats at a Nature Reserve in Southern Maine, United States.

Deer tick virus (DTV), also known as Powassan virus lineage II, is a rising health concern due to increased recognition as a cause of human encephalitis. Since European tick-borne encephalitis virus persists in nature in enzootic foci (i.e., higher prevalence rates in small pockets in nature), we sought to determine whether DTV is also focally maintained in relation to habitat type, to better understand factors leading to human risk of exposure. From 2018 to 2021, questing Ixodes scapularis ticks were collected from five habitats at the Wells National Estuarine Research Reserve (WNERR) in Wells, ME: forest with invasive vegetation in the understory, edge, shrub, forest with native vegetation in the understory, and open field. Deer tick virus prevalence was greater in adult ticks (2.0%) than in nymphs (0.5%). Deer tick virus prevalence in adult ticks collected from forest with invasive vegetation was 3.2% compared to 0 to 1.7% in other habitat types. A hot spot analysis revealed a higher number of infected adults collected per hour on one of the transects located in forest with invasive vegetation. Phylogenetic analysis of 37 full-length DTV genomes sequenced in this study revealed four major clades from the WNERR, and there was high genetic diversity within each transect, suggesting frequent, short-range dispersal between habitats. Analysis of DTV sequences from other New England counties and states also indicated long-distance dispersal to and/or from the WNERR. This study provides preliminary evidence that DTV is focal and that the risk of encountering DTV-infected ticks in forest with invasive vegetation may be greater than in other habitat types.

Adjusting susceptibilities of C57BL/6 mice to orthoflaviviruses for evaluation of antiviral drugs by altering the levels of interferon alpha/beta receptor function

The purpose of this study was to optimize the infectivity of four different orthoflaviviruses in mice for evaluating antiviral drugs by using wild-type mice with intact interferon responses, type 1 interferon alpha/beta receptor knockout mice, or by injecting wild type C57BL/6 mice with varying doses of anti-type 1 interferon receptor antibody (MAR1-5A3) to optimize the infectivity and lethality. West Nile virus productively infected wild-type C57BL/6 mice to cause lethality, whereas Usutu virus required a complete absence of type 1 interferon receptor function. Deer tick virus (lineage 2 Powassan virus) and Japanese encephalitis viruses required a dampening of type 1 interferon responses by adjusting the doses of MAR1-5A3 antibody injections. Challenge dose-responsive mortality, weight loss, and viral titers of these two viruses were observed if the type 1 interferon responses were dampened with MAR1-5A3. Conversely, without MAR1-5A3 injections, these disease phenotypes were not viral challenge dose-responsive. From these different interferon-responsive models, the appropriate lethality was identified to determine that 7-deaza-2'-C-methyladenosine has high efficacy for West Nile and Usutu viruses, and low efficacy for deer tick and Japanese encephalitis viruses.

Selection Pressure Profile Suggests Species Criteria among Tick-Borne Orthoflaviviruses.

Orthoflaviviruses are arthropod-borne viruses that are transmitted by mosquitoes or ticks and cause a range of significant human diseases. Among the most important tick-borne orthoflaviviruses (TBFVs) is tick-borne encephalitis virus (TBEV), which is endemic in Eurasia, and Powassan virus, which is endemic in Asia and North America. There is a significant controversy regarding species assignment in the tick-borne encephalitis virus complex due to the complex phylogenetic, serological, ecological, and pathogenetic properties of viruses. Comparing the rate of non-synonymous to synonymous substitutions (dN/dS) over the course of tick-borne orthoflavivirus diversification suggests that there is a very strong stabilizing selection (Nei-Gojobori dN/dS < 0.1) among tick-borne orthoflaviviruses that differ by less than 13.5% amino acid/21.4% nucleotide sequences, and discretely more rapid accumulation of non-synonymous substitutions (dN/dS > 0.13) among more divergent viruses that belong to distinct species. This pattern was similarly observed in genome regions encoding structural (E) and non-structural (NS3) proteins. Below this distance threshold, viruses appear fit and strongly tied to their ecological niche, whereas above the threshold, a greater degree of adaptation appears necessary. This species criterion suggests that all subtypes of TBEV, all related ovine/caprine encephalomyelitis viruses, and Omsk hemorrhagic fever virus (OHFV) together correspond to a single species. Within this species, viruses make up 11 subtypes that are reliably segregated by a 10% nucleotide distance cut-off suggested earlier for TBEV. The same 10% subtype cut-off suggests that Powassan virus includes two subtypes, Powassan and Deer Tick virus.

Features of Consistent Powassan Virus Lineage II Focus in Southern Maine, United States.

Powassan virus lineage II or deer tick virus (DTV) is a rare but increasingly reported human infection in the United States transmitted by Ixodes scapularis ticks. The virus is thought to be maintained in environmental foci that are optimal for tick and vertebrate reservoirs, but details on DTV ecology are poorly understood. We investigated DTV tick infection rates and reservoir host abundance in a focus of consistent DTV activity in Maine, USA. Host and tick abundance, vegetation, and microclimate conditions were measured in three forest sites representing increasing invasive understory infestation. Sites were selected representing native understory, mixed vegetation with some invasive Japanese barberry (Berberis thunbergii), and a highly invasive site dominated by Japanese barberry. Japanese barberry in the mixed vegetation site averaged 1 m in height with space between plants, whereas the highly invasive site had impenetrable Japanese barberry over 1.5 m. The DTV infection rate was greater in the highly invasive site. Density of I. scapularis ticks were significantly lower in the native forest site, and no DTV was found. Another feature of the DTV focus was more stable humid microclimate throughout the year compared with the other sites and a nearby continuous section of forest, consistent with reports from Connecticut, USA. We conclude that invasive Japanese barberry stands provide favorable and consistent microclimate conditions to maintain high DTV infection rates annually among questing I. scapularis ticks. Understanding environmental and landscape features that support high infection rates could lead to the identification of high-risk habitats for contracting DTV.

Interspecies co-feeding transmission of Powassan virus between a native tick, Ixodes scapularis, and the invasive East Asian tick, Haemaphysalis longicornis

BackgroundPowassan virus, a North American tick-borne flavivirus, can cause severe neuroinvasive disease in humans. While Ixodes scapularis are the primary vectors of Powassan virus lineage II (POWV II), also known as deer tick virus, recent laboratory vector competence studies showed that other genera of ticks can horizontally and vertically transmit POWV II. One such tick is the Haemaphysalis longicornis, an invasive species from East Asia that recently established populations in the eastern USA and already shares overlapping geographic range with native vector species such as I. scapularis. Reports of invasive H. longicornis feeding concurrently with native I. scapularis on multiple sampled hosts highlight the potential for interspecies co-feeding transmission of POWV II. Given the absence of a clearly defined vertebrate reservoir host for POWV II, it is possible that this virus is sustained in transmission foci via nonviremic transmission between ticks co-feeding on the same vertebrate host. The objective of this study was to evaluate whether uninfected H. longicornis co-feeding in close proximity to POWV II-infected I. scapularis can acquire POWV independent of host viremia.MethodsUsing an in vivo tick transmission model, I. scapularis females infected with POWV II (“donors”) were co-fed on mice with uninfected H. longicornis larvae and nymphs (“recipients”). The donor and recipient ticks were infested on mice in various sequences, and mouse infection status was monitored by temporal screening of blood for POWV II RNA via quantitative reverse transcription polymerase chain reaction (q-RT-PCR).ResultsThe prevalence of POWV II RNA was highest in recipient H. longicornis that fed on viremic mice. However, nonviremic mice were also able to support co-feeding transmission of POWV, as demonstrated by the detection of viral RNA in multiple H. longicornis dispersed across different mice. Detection of viral RNA at the skin site of tick feeding but not at distal skin sites indicates that a localized skin infection facilitates transmission of POWV between donor and recipient ticks co-feeding in close proximity.ConclusionsThis is the first report examining transmission of POWV between co-feeding ticks. Against the backdrop of multiple unknowns related to POWV ecology, findings from this study provide insight on possible mechanisms by which POWV could be maintained in nature.Graphical

Comparative Pathogenesis of Two Lineages of Powassan Virus Reveals Distinct Clinical Outcome, Neuropathology, and Inflammation.

Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that the infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlates with unique clinical presentations for each lineage. Comparative transcriptomics of infected brains demonstrates the activation of different immune pathways and downstream host responses. Ultimately, the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that the different disease presentations occur in clinical cases due to the inherent differences in the two lineages of Powassan virus.

Active Surveillance of Powassan Virus in Massachusetts Ixodes scapularis Ticks, Comparing Detection Using a New Triplex Real-Time PCR Assay with a Luminex Vector-Borne Panel.

Powassan virus is an emerging tick-borne pathogen capable of causing severe neuroinvasive disease. As the incidence of human Powassan virus grows both in magnitude and geographical range, the development of sensitive detection methods for diagnostics and surveillance is critical. In this study, a Taqman-based triplex real-time PCR assay was developed for the simultaneous and quantitative detection of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis ticks. An exon-exon junction internal control was built-in to allow for accurate detection of RNA quality and the failure of RNA extraction. The newly developed assay was also applied to survey deer tick virus in tick populations at 13 sites on Cape Cod and Martha's Vineyard Island in Massachusetts. The assay's performance was compared with the Luminex xMAP MultiFLEX Vector-borne Panel 2. The results suggested that the real-time PCR method was more sensitive. Powassan virus infection rates among ticks collected from these highly endemic tick areas ranged from 0.0 to 10.4%, highlighting the fine-scale geographic variations in deer tick virus presence in this region. Looking forward, our PCR assay could be adopted in other Powassan virus surveillance systems.

Development of a colorimetric loop-mediated isothermal amplification diagnostic assay for deer tick virus

Deer tick virus (DTV) is an emerging pathogen in North America. This virus can cause nervous system complications such as encephalitis in humans. Further, no data are available regarding long-term effects of infection from DTV patients across variable age groups. Diagnostic tools of DTV used by government laboratories are based on RT-PCR using patient serum or ticks. This paper explores the feasibility of a colorimetric loop-mediated isothermal amplification (LAMP) assay to create a point-of-care diagnostic methodology for use in field and in primary care. LAMP consists of six primers that bind to target DNA and amplify variable length nucleotide strands that can be visualized through side reactions or via electrophoresis. First, a viable LAMP primer set, and a primer set that/ dimerizes and amplifies DNA regardless of compatibility were created in silico and validated in vitro. Then, a specific LAMP assay was developed. Our findings showed this method can be performed within 30 min and can measure with limits of detection comparable to PCR.

Of Murines and Humans: Modeling Persistent Powassan Disease in C57BL/6 Mice.

Powassan infection is caused by two closely related, tick-transmitted viruses of the genus Flavivirus (family Flaviviridae): Powassan virus lineage I (POWV) and lineage II (known as deer tick virus [DTV]). Infection is typically asymptomatic or mild but can progress to neuroinvasive disease. Approximately 10% of neuroinvasive cases are fatal, and half of the survivors experience long-term neurological sequelae. Understanding how these viruses cause long-term symptoms as well as the possible role of viral persistence is important for developing therapies. We intraperitoneally inoculated 6-week-old C57BL/6 mice (50% female) with 103 focus-forming units (FFU) DTV and assayed for infectious virus, viral RNA, and inflammation during acute infection and 21, 56, and 84 days postinfection (dpi). Although most mice (86%) were viremic 3 dpi, only 21% of the mice were symptomatic and 83% recovered. Infectious virus was detected only in the brains of mice sampled during the acute infection. Viral RNA was detected in the brain until 84 dpi, but the magnitude decreased over time. Meningitis and encephalitis were visible in acute mice and from mice sampled at 21 dpi. Inflammation was observed until 56 dpi in the brain and 84 dpi in the spinal cord, albeit at low levels. These results suggest that the long-term neurological symptoms associated with Powassan disease are likely caused by lingering viral RNA and chronic inflammation in the central nervous system rather than by a persistent, active viral infection. The C57BL/6 model of persistent Powassan mimics illness in humans and can be used to study the mechanisms of chronic disease. IMPORTANCE 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. Infection of C57BL/6 mice with DTV mimics clinical disease in humans, and the mice exhibit CNS inflammation and viral RNA persistence until at least 86 dpi, while infectious virus is undetectable after 12 dpi. These findings suggest that the long-term neurological symptoms of chronic Powassan disease are in part due the persistence of viral RNA and the corresponding long-term inflammation of the brain and spinal cord. Our work demonstrates that C57BL/6 mice can be used to study the pathogenesis of chronic Powassan disease.

Phylodynamics of deer tick virus in North America.

The burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV; Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of POWV encephalitis. Here, we use a multifaceted approach to evaluate the emergence of the II POWV lineage, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the Northeast USA with an average infection rate of 1.4 per cent. High-depth, whole-genome sequencing of eighty-four POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the Northeast USA and patterns of geographic dispersal within and between regions. A Bayesian skyline analysis demonstrated DTV population expansion over the last 50 years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests an increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus.

Identification and characterization of novel lineage 1 Powassan virus strains in New York State

ABSTRACT Powassan virus (POWV, family Flaviviridae) is a reemerging tick-borne virus endemic in North America and Russia. In 1997, a POWV-like agent was isolated from Ixodes scapularis in New England and determined to be genetically distinct from the original POWV isolate. This revealed the existence of two lineages: lineage 1, prototype Powassan virus (POWV-1) and lineage 2, deer tick virus (DTV). POWV-1 is thought to be primarily maintained in a cycle between I. cookei and woodchucks and I. marxi and squirrels, while DTV is primarily maintained in a cycle between I. scapularis and small mammal hosts. Recent tick, mammalian, and human isolates from New York State (NYS) have been identified as DTV, but for the first time in 45 years, we detected four POWV-1 isolates, including the first reported isolation of POWV-1 from I. scapularis. We aimed to investigate genotypic and phenotypic characteristics of recent NYS isolates through sequence analysis and evaluation of replication kinetics in vitro and in vivo. Our sequencing revealed genetic divergence between NYS POWV-1 isolates, with two distinct foci. We found that POWV-1 isolates displayed variable replication kinetics in nymphal ticks but not in cell culture. POWV-1 isolated from I. scapularis displayed increased fitness in experimentally infected I. scapularis as compared to historic and recent POWV-1 isolates from I. cookei. These data suggest the emergence of divergent POWV-1 strains in alternate tick hosts and maintenance of genetically and phenotypically discrete POWV-1 foci.

Tick-Borne Pathogens in Questing Blacklegged Ticks (Acari: Ixodidae) From Pike County, Pennsylvania.

Active surveillance was conducted by collecting questing ticks from vegetation through a 2-yr survey in Pike County, Pennsylvania. Over a thousand blacklegged ticks (Ixodes scapularis Say) and American dog ticks (Dermacentor variabilis Say) were collected. A single specimen of the following species was collected: lone star tick (Amblyomma americanum L.), rabbit tick (Haemaphysalis leporispalustris Packard), and an Asian longhorned tick (Haemaphysalis longicornis Neumann). This study represents the largest county-wide study in Pennsylvania, surveying 988 questing I. scapularis adult and nymphs. Molecular detection of five distinct tick-borne pathogens was screened through real-time PCR at a single tick resolution. Respectively, the overall 2-yr adult and nymph prevalence were highest with Borrelia burgdorferi (Spirochaetales: Spirochaetacceae) (45.99%, 18.94%), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) (12.29%, 7.95%) where the variant-ha (8.29%, 3.03%) was overall more prevalent than the variant-v1 (2.49%, 4.17%), Babesia microti (Piroplasmida: Babesiidae) (4.97%, 5.30%), Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) (1.38%, 1.89%), and Powassan virus lineage II [POWV]/deer tick virus (DTV) (2.07%, 0.76%). Adult and nymph coinfection prevalence of B. burgdorferi and B. microti (3.03%, 4.97%) and adult coinfection of B. burgdorferi and A. phagocytophilum or A. phagocytophilum and B. microti were significantly higher than the independent infection rate expected naturally. This study highlights the urgency to conduct diverse surveillance studies with large sample sizes to better understand the human risk for tick-borne diseases within small geographical areas.

Detection of Borrelia miyamotoi and Powassan Virus Lineage II (Deer Tick Virus) from Odocoileus virginianus Harvested Ixodes scapularis in Oklahoma

Odocoileus virginianus (white-tailed deer) is the primary host of adult Ixodes scapularis (deer tick). Most of the research into I. scapularis has been geographically restricted to the northeastern United States, with limited interest in Oklahoma until recently as the I. scapularis populations spread due to climate change. Ticks serve as a vector for pathogenic bacteria, protozoans, and viruses that pose a significant human health risk. To date, there has been limited research to determine what potential tick-borne pathogens are present in I. scapularis in central Oklahoma. Using a one-step multiplex real-time reverse transcription-PCR, I. scapularis collected from white-tailed deer was screened for Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, and deer tick virus (DTV). Ticks (n = 394) were pooled by gender and life stage into 117 samples. Three pooled samples were positive for B. miyamotoi and five pooled samples were positive for DTV. This represents a minimum infection rate of 0.8% and 1.2%, respectively. A. phagocytophilum, B. burgdorferi, and B. microti were not detected in any samples. This is the first report of B. miyamotoi and DTV detection in Oklahoma I. scapularis ticks. This demonstrates that I. scapularis pathogens are present in Oklahoma and that further surveillance of I. scapularis is warranted.

Incrimination of shrews as a reservoir for Powassan virus

Powassan virus lineage 2 (deer tick virus) is an emergent threat to American public health, causing severe neurologic disease. Its life cycle in nature remains poorly understood. We use a host-specific retrotransposon-targeted real time PCR assay to test the hypothesis that white-footed mice, considered the main eastern U.S. reservoir of the coinfecting agent of Lyme disease, is the reservoir for deer tick virus. Of 20 virus-infected host-seeking nymphal black-legged ticks 65% fed on shrews and none on mice. The proportion of ticks feeding on shrews at a site is positively associated with prevalence of viral infection, but not the Lyme disease agent. Viral RNA is detected in the brain of one shrew. We conclude that shrews are a likely reservoir host for deer tick virus and that host bloodmeal analysis can provide direct evidence to incriminate reservoir hosts, thereby promoting our understanding of the ecology of tick-borne infections.

Using Loops to Jump Through Hoops in Flaviviridae

Recent advances in diagnostic testing measures are crucial in determining infection rates in large populations. For Deer Tick Virus (DTV), a member of Flaviviridae, there has been minimal amounts of testing since its prevalence in recent decades. Here we examine recently developed loop-mediated isothermal amplification (LAMP) primers that target the RNA-dependent RNA-polymerase at the terminal end of the polycistron. This sequence is highly conserved across the genus and can provide addition diagnostic potential when considering testing where multiple viruses are endemic to a region. The benefits of using LAMP are increasingly useful in the diagnostic setting where rapid results are needed, and low-cost care is required without use of a thermocycler. We also investigate the possibility of a new type of multiplex LAMP assay that could advance upon our current findings of sequence homology within the viral family. We intend to report upon the progress to implement this technology as a bioinformatics program that can be utilized for further multiplex LAMP testing in vitro.

Powassan Virus Experimental Infections in Three Wild Mammal Species.

Powassan virus (POWV) is a tick-borne virus maintained in sylvatic cycles between mammalian wildlife hosts and ticks (primarily Ixodes spp.). There are two currently recognized lineages, POWV-lineage 1 (POWV-L1) and deer tick virus (DTV; lineage 2), both of which can cause fatal neurologic disease in humans. Increased numbers of human case reports in the northeastern and north central United States in recent years have fueled questions into POWV epidemiology. We inoculated three candidate wildlife POWV reservoir hosts, groundhogs (Marmota monax), striped skunks (Mephitis mephitis), and fox squirrels (Sciurus niger), with either POWV-L1 or DTV. Resulting viremia, tissue tropism, and pathology were minimal in most inoculated individuals of all three species, with low (peak titer range, 101.7-103.3 plaque-forming units/mL serum) or undetectable viremia titers, lack of detection in tissues except for low titers in spleen, and seroconversion in most individuals by 21 days postinoculation (DPI). Pathology was limited and most commonly consisted of mild inflammation in the brain of POWV-L1- and DTV-inoculated skunks on four and 21 DPI, respectively. These results reveal variation in virulence and host competence among wild mammalian species, and a likely limited duration of host infectiousness to ticks during enzootic transmission cycles. However, POWV can transmit rapidly from tick to host, and tick co-feeding may be an additional transmission mechanism. The rare and low-level detections of viremia in these three, common, wild mammal species suggest that vector-host dynamics should continue to be explored, along with eco-epidemiological aspects of enzootic POWV transmission in different regions and virus lineages.

Ticks of genus Ixodidae: bioecological mechanisms of adaptation to environmental conditions, medical and epidemiological significance

Considering the geographical position of Ukraine, which is located on the border of several physical and geographical zones, the country has a significant variety of parasitic invertebrates and requires constant monitoring in the current integration conditions. Nowadays climatic changes and anthropogenic effect have an impact on ecosystems organization and cause the incidence of transmissible diseases, including tick-borne infections. In order to prevent the spread of pathogens, there is a necessity to analyze modern data of Ixodidae genus ticks, as components of natural and focal humans and animals diseases, their ecological features, behavioral responses, features of biotopic spread, biological patterns, their epidemiological significance. Our review analysis revealed that all stages of tick development depend on three major natural factors: temperature, humidity, duration of the photoperiod, and the presence of a host. Particularly important is the combination of the first two factors that determines the occurrence of two peaks of the number of ticks in April - May and in August – September, the last is less extended. At this time of year the nymphs and imago density increases and their infectious agents rate is increased too. The study of the epidemiological condition proves the fact that nymphs and imago of I. ricinus are equally infected with the most common pathogens of infections: Borrelia burgdorferi sensu lato (sl) and Borrelia miyamotoi, rickettsiae bacteria (Anaplasma phagocytophilum), flaviviruses (tick-borne encephalitis virus and deer tick virus) and the protozoans (Babesia microti, Babesia duncani, Babesia divergens and Babesia venatorum). Often there is a different combination of these pathogens in the one tick. The infectious agents rate have an impact on the ticks behavior differences in the feeding duration, increasing ticks bites frequency, decreasing of blood feeding, and a prolongation of the life cycle. Therefore within the transboundary territories the urgent control of agents and vectors of infectious and invasive animals and humans diseases is required, studying changes of migrant thermophilic species habitats geography, and systematic measures for the prevention of parasites implementation is needed.

Characterization of flavivirus infection in salivary gland cultures from male Ixodes scapularis ticks.

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.

Powassan Virus Neuropathology and Genomic Diversity in Patients With Fatal Encephalitis.

BackgroundPowassan virus (POWV) is an emerging cause of severe encephalitis; very little is known about human pathogenicity due to challenges in diagnosis and viral RNA recovery. We present 3 patients with fatal encephalitis due to POWV lineage II (deer tick virus). MethodsWe obtained 27 unique samples, including from brain biopsy and autopsy, and used metagenomic sequencing, quantitative reverse transcriptase polymerase chain reaction, and a newly developed CRISPR-based diagnostic assay to perform the first detailed characterization of POWV compartmentalization and genomics between and within human subjects. ResultsIn all 3 patients, imaging and histopathology findings were notable for profound cerebellar involvement. All patients were initially diagnosed with POWV by metagenomic sequencing, and 2 of the 3 had negative clinical testing by serology. We detected POWV RNA in 13 clinical samples; levels were highest in the cerebellum, and there was very little involvement of peripheral tissue. We assembled complete POWV genomes from 8 samples, providing unique information about the strains of POWV lineage II (deer tick virus) that infect humans. ConclusionsWe demonstrate the utility of molecular assays for detecting POWV infection, including in seronegative patients, and nominate viral genomic features that may relate to human infection and neuropathogenicity. The cerebellum was identified as a key target POWV in fatal infection, by radiological and histopathological findings as well as molecular testing.

Development of a small animal model for deer tick virus pathogenesis mimicking human clinical outcome.

Powassan virus (POWV) is a tick-borne flavivirus that encompasses two genetic lineages, POWV (Lineage I) and deer tick virus (DTV, Lineage II). In recent years, the incidence of reported POWV disease cases has increased, coupled with an expanded geographic range of the DTV tick vector, Ixodes scapularis. POWV and DTV are serologically indistinguishable, and it is not known whether clinical manifestations, pathology, or disease outcome differ between the two viruses. Six-week-old male and female BALB/c mice were footpad-inoculated with DTV doses ranging from 101 to 105 FFU. Dose-independent mortality, morbidity, and organ viral loads were observed for mice inoculated with sequentially increasing doses of DTV. By study completion, all surviving mice had cleared their viremias but detectable levels of negative-sense DTV RNA were present in the brain, indicating viral persistence of infectious DTV in the central nervous system. For mice that succumbed to disease, neuropathology revealed meningoencephalitis characterized by microscopic lesions with widespread distribution of viral RNA in the brain. These findings, coupled with the rapid onset of neurological signs of disease and high viral titers in nervous tissue, highlight the neurotropism of DTV in this mouse model. Additionally, disease outcome for DTV-infected mice was not affected by sex, as males and females were equally susceptible to disease. This is the first study to comprehensively characterize the clinical disease outcome in a small animal model across a spectrum of POWV/DTV infection doses. Here, we developed a small animal model for DTV pathogenesis that mimics the manifestations of POWV disease in humans. Since it is currently not known whether DTV and POWV differ in their capacity to cause human disease, the animal model detailed in our study could be utilized in future comparative pathogenesis studies, or as a platform for testing the efficacy of vaccines, and anti-virals.