Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Identification of reactive Borrelia burgdorferi peptides associated with Lyme disease.

Borrelia burgdorferi, the agent of Lyme disease, is estimated to cause >400,000 annual infections in the United States. Serology is the primary laboratory method to support the diagnosis of Lyme disease, but current methods have intrinsic limitations that require alternative approaches or targets. We used a high-density peptide array that contains >90,000 short overlapping peptides to catalog immunoreactive linear epitopes from >60 primary antigens of B. burgdorferi. We then pursued a machine learning approach to identify immunoreactive peptide panels that provide optimal Lyme disease serodiagnosis and can differentiate antibody responses at various stages of disease. We examined 226 serum samples from the Lyme Biobank and the National Institutes of Health, which included sera from 110 individuals diagnosed with Lyme disease, 31 probable cases from symptomatic individuals, and 85 healthy controls. Cases were grouped based on disease stage and presentation and included individuals with early localized, early disseminated, and late Lyme disease. We identified a peptide panel originating from 14 different epitopes that differentiated cases versus controls, whereas another peptide panel built from 12 unique epitopes differentiated subjects with various disease manifestations. Our method demonstrated an improvement in B. burgdorferi antibody detection over the current two-tiered testing approach and confirmed the key diagnostic role of VlsE and FlaB antigens at all stages of Lyme disease. We also uncovered epitopes that triggered a temporal antibody response that was useful for differentiation of early and late disease. Our findings can be used to streamline serologic targets and improve antibody-based diagnosis of Lyme disease. IMPORTANCE: Serology is the primary method of Lyme disease diagnosis, but this approach has limitations, particularly early in disease. Currently employed antibody detection assays can be improved by the identification of alternative immunodominant epitopes and the selection of optimal diagnostic targets. We employed high-density peptide arrays that enabled precise epitope mapping for a wide range of B. burgdorferi antigens. In combination with machine learning, this approach facilitated the selection of serologic targets early in disease and the identification of serological indicators associated with different manifestations of Lyme disease. This study provides insights into differential antibody responses during infection and outlines a new approach for improved serologic diagnosis of Lyme disease.

Tick-Borne Co-Infections: Challenges in Molecular and Serologic Diagnoses.

Co-infections are a poorly understood aspect of tick-borne diseases. In the United States alone, nineteen different tick-borne pathogens have been identified. The majority of these agents are transmitted by only two tick species, Ixodes scapularis and Amblyomma americanum. Surveillance studies have demonstrated the presence of multiple pathogens in individual ticks suggesting a risk of polymicrobial transmission to humans. However, relatively few studies have explored this relationship and its impact on human disease. One of the key factors for this deficiency are the intrinsic limitations associated with molecular and serologic assays employed for the diagnosis of tick-borne diseases. Limitations in the sensitivity, specificity and most importantly, the capacity for inclusion of multiple agents within a single assay represent the primary challenges for the accurate detection of polymicrobial tick-borne infections. This review will focus on outlining these limitations and discuss potential solutions for the enhanced diagnosis of tick-borne co-infections.

Morphologic and genetic characterization of Pterygodermatites (Mesopectines) valladaresi n. sp. (Nematoda, Rictulariidae), a parasite of the mouse Mus musculus (Rodentia, Muridae) from the Canary Islands (Spain).

A new rictulariid nematode Pterygodermatites (Mesopectines) valladaresi n. sp., parasite of the house mouse Mus musculus (Rodentia: Muridae) in the Canary Islands (Spain) is described by means of light and scanning electron microscopy. The new species belongs to the subgenus Mesopectines characterized by a more or less dorsal orientation of the buccal capsule, the presence of three oesophageal teeth, the morphology of the oral denticles and the Spirurida type of arrangement of caudal papillae in males. The most discriminant characteristics between the new species and the existing species in the subgenus Mesopectines are (a) the number of cuticular projection pairs (62-64), (b) the size of right and left spicules (respectively, 62-90 µm and 123-139 µm), (c) the number of midventral fans in males (3-4), (d) the number of prevulvar/total cuticular projection pairs (38-42/63-71), (e) the posterior differentiation of combs into spines in relation to the position of the vulva and (f) the anterior position of the vulva in relation to the oesophagus-intestine junction in females. Parasitized hosts and geographical distribution are also useful criteria to distinguish P. (Me.) valladaresi n. sp. from the remaining species of the subgenus. In addition, the cox1 sequence of the new species is provided and compared with available data of related species.

Title: Caractérisation morphologique et génétique de Pterygodermatites (Mesopectines) valladaresi n. sp. (Nematoda, Rictulariidae), un parasite de la souris Mus musculus (Rodentia, Muridae) des îles Canaries (Espagne). Abstract: Un nouveau nématode rictulaire, Pterygodermatites (Mesopectines) valladaresi n. sp., parasite de la souris domestique Mus musculus (Rodentia, Muridae) aux îles Canaries (Espagne) est décrit par microscopie optique et électronique à balayage. La nouvelle espèce appartient au sous-genre Mesopectines caractérisé par une orientation plus ou moins dorsale de la capsule buccale, la présence de trois dents œsophagiennes, la morphologie des denticules buccaux et le type Spirurida d'arrangement des papilles caudales chez les mâles. Les caractéristiques les plus discriminantes entre la nouvelle espèce et les espèces existantes du sous-genre Mesopectines sont (a) le nombre de paires de projections cuticulaires (62­64), (b) la taille des spicules droit et gauche (respectivement, 62­90 µm et 123­139 µm), (c) le nombre de bourrelets cuticulaires médio-ventraux chez les mâles (3­4), (d) le nombre de peignes prévulvaires par rapport au nombre total de peignes (38­42/63­71), (e) la transformation en épines des peignes après la vulve et (f) la position antérieure de la vulve par rapport à la jonction œsophage-intestin chez les femelles. Les hôtes parasités et la répartition géographique sont également des critères utiles pour distinguer P. (Me.) valladaresi n. sp. des autres espèces du sous-genre. De plus, la séquence du cox1 de la nouvelle espèce est fournie et comparée aux données disponibles sur les espèces apparentées.

Myiasis by Cordylobia anthropophaga (Calliphoridae) in rodents from Cape Verde.

PURPOSE: The tumbu fly, Cordylobia anthropophaga (Diptera: Calliphoridae), is widely distributed in continental tropical and subtropical Africa, being the most common cause of furuncular myiasis in Sub-Saharan Africa. The aim of the present work was to analyze the role of rodents as possible reservoirs of C. anthropophaga in Cape Verde, considering the zoonotic character of this fly species. MATERIALS AND METHODS: A total of 150 peridomestic rodents were studied in Santiago island. For the obtained larvae, morphological and molecular characters were analyzed. RESULTS: Cordylobia anthropophaga was found in 6.4% of the peridomestic Rattus rattus analyzed. The present work unveils the presence of C. anthropophaga in rodents of the African archipelago of Cape Verde, introduced probably with West African humans and/or animals. CONCLUSION: The presence in peridomestic animals, and the wide range of species that this fly can affect, entails a zoonotic risk of myiasis by tumbu fly.

Capture Sequencing Enables Sensitive Detection of Tick-Borne Agents in Human Blood.

Assay sensitivity can be a limiting factor in the use of PCR as a tool for the detection of tick-borne pathogens in blood. We evaluated the performance of Tick-borne disease Capture Sequencing Assay (TBDCapSeq), a capture sequencing assay targeting tick-borne agents, to test 158 whole blood specimens obtained from the Lyme Disease Biobank. These included samples from 98 individuals with signs and symptoms of acute Lyme disease, 25 healthy individuals residing in Lyme disease endemic areas, and 35 samples collected from patients admitted to the Massachusetts General Hospital or referred to the infectious disease clinic. Compared to PCR, TBDCapSeq had better sensitivity and could identify infections with a wider range of tick-borne agents. TBDCapSeq identified a higher rate of samples positive for Borrelia burgdorferi (8 vs. 1 by PCR) and Babesia microti (26 vs. 15 by PCR). TBDCapSeq also identified previously unknown infections with Borrelia miyamotoi, Ehrlichia, and Rickettsia species. Overall, TBDCapSeq identified a pathogen in 43 samples vs. 23 using PCR, with four co-infections detected versus zero by PCR. We conclude that capture sequencing enables superior detection of tick-borne agents relative to PCR.

Detection of antibodies to Anaplasma phagocytophilum and Babesia microti using linear peptides.

Anaplasma phagocytophilum and Babesia microti are emerging tick-borne pathogens in the United States. Although active infection is typically diagnosed by direct diagnostic tests, such as blood smear or polymerase chain reaction assay, serologic assays can be helpful to identify past infections, and the use of acute plus convalescent testing can potentially identify recent infections. We employed a peptide array to select sets of linear peptides for serologic diagnosis of infections with A. phagocytophilum and B. microti. Three optimal peptides were selected for each agent based on their performance with clinical specimens. All three A. phagocytophilum peptides were located within the conserved fragments of the MSP2 antigen. Two B. microti peptides were located in the N terminus of the SA-1 antigen; the third was in the BMN 1-17 antigen. We found that these peptides can be a useful tool for detection of antibody reactivity to both of these pathogens.

Virulence potential of Rickettsia amblyommatis for spotted fever pathogenesis in mice.

Rickettsia amblyommatis belongs to the spotted fever group of Rickettsia and infects Amblyomma americanum (Lone Star ticks) for transmission to offspring and mammals. Historically, the geographic range of A. americanum was restricted to the southeastern USA. However, recent tick surveys identified the progressive northward invasion of A. americanum, contributing to the increased number of patients with febrile illnesses of unknown etiology after a tick bite in the northeastern USA. While serological evidence strongly suggests that patients are infected with R. amblyommatis, the virulence potential of R. amblyommatis is not well established. Here, we performed a bioinformatic analysis of three genome sequences of R. amblyommatis and identified the presence of multiple putative virulence genes whose products are implicated for spotted fever pathogenesis. Similar to other pathogenic spotted fever rickettsiae, R. amblyommatis replicated intracellularly within the cytoplasm of tissue culture cells. Interestingly, R. amblyommatis displayed defective attachment to microvascular endothelial cells. The attachment defect and slow growth rate of R. amblyommatis required relatively high intravenous infectious doses to produce dose-dependent morbidity and mortality in C3H mice. In summary, our results corroborate clinical evidence that R. amblyommatis can cause mild disease manifestation in some patients.

Development of a capture sequencing assay for enhanced detection and genotyping of tick-borne pathogens.

Inadequate sensitivity has been the primary limitation for implementing high-throughput sequencing for studies of tick-borne agents. Here we describe the development of TBDCapSeq, a sequencing assay that uses hybridization capture probes that cover the complete genomes of the eleven most common tick-borne agents found in the United States. The probes are used for solution-based capture and enrichment of pathogen nucleic acid followed by high-throughput sequencing. We evaluated the performance of TBDCapSeq to surveil samples that included human whole blood, mouse tissues, and field-collected ticks. For Borrelia burgdorferi and Babesia microti, the sensitivity of TBDCapSeq was comparable and occasionally exceeded the performance of agent-specific quantitative PCR and resulted in 25 to > 10,000-fold increase in pathogen reads when compared to standard unbiased sequencing. TBDCapSeq also enabled genome analyses directly within vertebrate and tick hosts. The implementation of TBDCapSeq could have major impact in studies of tick-borne pathogens by improving detection and facilitating genomic research that was previously unachievable with standard sequencing approaches.

Polymicrobial Nature of Tick-Borne Diseases.

Tick-borne diseases have doubled in the last 12 years, and their geographic distribution has spread as well. The clinical spectrum of tick-borne diseases can range from asymptomatic to fatal infections, with a disproportionate incidence in children and the elderly. In the last few years, new agents have been discovered, and genetic changes have helped in the spread of pathogens and ticks. Polymicrobial infections, mostly in Ixodes scapularis, can complicate diagnostics and augment disease severity. Amblyomma americanum ticks have expanded their range, resulting in a dynamic and complex situation, possibly fueled by climate change. To document these changes, using molecular biology strategies for pathogen detection, an assessment of 12 microbes (9 pathogens and 3 symbionts) in three species of ticks was done in Suffolk County, New York. At least one agent was detected in 63% of I. scapularis ticksBorrelia burgdorferi was the most prevalent pathogen (57% in adults; 27% in nymphs), followed by Babesia microti (14% in adults; 15% in nymphs), Anaplasma phagocytophilum (14% in adults; 2% in nymphs), Borrelia miyamotoi (3% in adults), and Powassan virus (2% in adults). Polymicrobial infections were detected in 22% of I. scapularis ticks, with coinfections of B. burgdorferi and B. microti (9%) and of B. burgdorferi and A. phagocytophilum (7%). Three Ehrlichia species were detected in 4% of A. americanum ticks. The rickettsiae constituted the largest prokaryotic biomass of all the ticks tested and included Rickettsia amblyommatis, Rickettsia buchneri, and Rickettsia montanensis The high rates of polymicrobial infection in ticks present an opportunity to study the biological interrelationships of pathogens and their vectors.IMPORTANCE Tick-borne diseases have increased in prevalence in the United States and abroad. The reasons for these increases are multifactorial, but climate change is likely to be a major factor. One of the main features of the increase is the geographic expansion of tick vectors, notably Amblyomma americanum, which has brought new pathogens to new areas. The clinical spectrum of tick-borne diseases can range from asymptomatic to fatal infections, with a disproportionate incidence in children and the elderly. In addition, new pathogens that are cotransmitted by Ixodes scapularis have been discovered and have led to difficult diagnoses and to disease severity. Of these, Borrelia burgdorferi, the agent of Lyme disease, continues to be the most frequently transmitted pathogen. However, Babesia microti, Borrelia miyamotoi (another spirochete), Anaplasma phagocytophilum, and Powassan virus are frequent cotransmitted agents. Polymicrobial infection has important consequences for the diagnosis and management of tick-borne diseases.