Tick-borne encephalitis virus capsid protein induces translational shutoff as revealed by its structural-biological analysis.

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 encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

Genomic Confirmation of Borrelia garinii, United States.

Lyme disease is a multisystem disorder primarily caused by Borrelia burgdorferi sensu lato. However, B. garinii, which has been identified on islands off the coast of Newfoundland and Labrador, Canada, is a cause of Lyme disease in Eurasia. We report isolation and whole-genome nucleotide sequencing of a B. garinii isolate from a cotton mouse (Peromyscus gossypinus) in South Carolina, USA. We identified a second B. garinii isolate from the same repository. Phylogenetic analysis does not associate these isolates with the previously described isolates of B. garinii from Canada.

Natural Clerodendrum-derived tick repellent: learning from Nepali culture.

Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80-100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20-60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides.

Proteome Profiling of PMJ2-R and Primary Peritoneal Macrophages.

In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-R cells as a model of peritoneal macrophages should be carefully validated. All MS data are deposited in ProteomeXchange with the identifier PXD022133.

Novel targets and strategies to combat borreliosis.

Lyme borreliosis is a bacterial infection that can be spread to humans by infected ticks and may severely affect many organs and tissues. Nearly four decades have elapsed since the discovery of the disease agent called Borrelia burgdorferi. Although there is a plethora of knowledge on the infectious agent and thousands of scientific publications, an effective way on how to combat and prevent Lyme borreliosis has not been found yet. There is no vaccine for humans available, and only one active vaccine program in clinical development is currently running. A spirited search for possible disease interventions is of high public interest as surveillance data indicates that the number of cases of Lyme borreliosis is steadily increasing in Europe and North America. This review provides a condensed digest of the history of vaccine development up to new promising vaccine candidates and strategies that are targeted against Lyme borreliosis, including elements of the tick vector, the reservoir hosts, and the Borrelia pathogen itself.

Analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection.

Tick-borne encephalitis virus (TBEV) is a member of the genus Flavivirus. It can cause serious infections in humans that may result in encephalitis/meningoencephalitis. Although several studies have described the involvement of specific genes in the host response to TBEV infection in the central nervous system (CNS), the overall network remains poorly characterized. Therefore, we investigated the response of DAOY cells (human medulloblastoma cells derived from cerebellar neurons) to TBEV (Neudoerfl strain, Western subtype) infection to characterize differentially expressed genes by transcriptome analysis. Our results revealed a wide panel of interferon-stimulated genes (ISGs) and pro-inflammatory cytokines, including type III but not type I (or II) interferons (IFNs), which are activated upon TBEV infection, as well as a number of non-coding RNAs, including long non-coding RNAs. To obtain a broader view of the pathways responsible for eliciting an antiviral state in DAOY cells we examined the effect of type I and III IFNs and found that only type I IFN pre-treatment inhibited TBEV production. The cellular response to TBEV showed only partial overlap with gene expression changes induced by IFN-ß treatment - suggesting a virus-specific signature - and we identified a group of ISGs that were highly up-regulated following IFN-ß treatment. Moreover, a high rate of down-regulation was observed for a wide panel of pro-inflammatory cytokines upon IFN-ß treatment. These data can serve as the basis for further studies of host-TBEV interactions and the identification of ISGs and/or lncRNAs with potent antiviral effects in cases of TBEV infection in human neuronal cells.

Europe-Wide Meta-Analysis of Borrelia burgdorferi Sensu Lato Prevalence in Questing Ixodes ricinus Ticks.

Lyme borreliosis is the most common zoonotic disease transmitted by ticks in Europe and North America. Despite having multiple tick vectors, the causative agent, Borrelia burgdorferisensu lato, is vectored mainly by Ixodes ricinus in Europe. In the present study, we aimed to review and summarize the existing data published from 2010 to 2016 concerning the prevalence of B. burgdorferi sensu lato spirochetes in questing I. ricinus ticks. The primary focus was to evaluate the infection rate of these bacteria in ticks, accounting for tick stage, adult tick gender, region, and detection method, as well as to investigate any changes in prevalence over time. The data obtained were compared to the findings of a previous metastudy. The literature search identified data from 23 countries, with 115,028 ticks, in total, inspected for infection with B. burgdorferi sensu lato We showed that the infection rate was significantly higher in adults than in nymphs and in females than in males. We found significant differences between European regions, with the highest infection rates in Central Europe. The most common genospecies were B. afzelii and B. garinii, despite a negative correlation of their prevalence rates. No statistically significant differences were found among the prevalence rates determined by conventional PCR, nested PCR, and real-time PCR.IMPORTANCEBorrelia burgdorferisensu lato is a pathogenic bacterium whose clinical manifestations are associated with Lyme borreliosis. This vector-borne disease is a major public health concern in Europe and North America and may lead to severe arthritic, cardiovascular, and neurological complications if left untreated. Although pathogen prevalence is considered an important predictor of infection risk, solitary isolated data have only limited value. Here we provide summarized information about the prevalence of B. burgdorferi sensu lato spirochetes among host-seeking Ixodes ricinus ticks, the principal tick vector of borreliae in Europe. We compare the new results with previously published data in order to evaluate any changing trends in tick infection.

Ehrlichia minasensis sp. nov., isolated from the tick Rhipicephalus microplus.

Recently, we obtained a rickettsial isolate (Ehrlichia sp. UFMG-EVT) from the haemolymph of engorged Rhipicephalus microplus tick females. On the basis of maximum-likelihood phylogenetic analysis using 16S rRNA gene, groEL, dsb, gltA and trp36 sequences we showed that Ehrlichia sp. UFMG-EVT belongs to the α-Proteobacteria, family Anaplasmataceae, genus Ehrlichia. Ehrlichia sp. UFMG-EVT is a sister taxon of Ehrlichia canis with 16S rRNA gene, groEL, dsb, gltA and trp36 sequence similarities of 98.3 %, 97.2 %, 94.7 %, 94.3 % and 49.1 %, respectively. Ehrlichia sp. UFMG-EVT has been maintained in the laboratory by continuous passage in the IDE8 tick cell line where the ultrastructure was characterized using electron microscopy and was found to resemble that of E. canis, Ehrlichia muris and Ehrlichia chaffeensis, but not Ehrlichia ruminantium and Ehrlichia ewingii. We propose the name Ehrlichia minasensis sp. nov. for this bacterium to acknowledge the place from where it was initially isolated, Minas Gerais, Brazil. The type strain is strain Ehrlichia sp. UFMG-EVT ( = DSM 100393T = TCB-TBB-0018T).

Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells.

A short upstream open reading frame (uORF) was recently identified in the 5' untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle.

Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses.

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

Prevalence of Borrelia miyamotoi in Ixodes ticks in Europe and the United States.

Borrelia miyamotoi, a relapsing fever-related spirochete transmitted by Ixodes ticks, has been recently shown to be a human pathogen. To characterize the prevalence of this organism in questing Ixodes ticks, we tested 2,754 ticks for a variety of tickborne pathogens by PCR and electrospray-ionization mass spectrometry. Ticks were collected from California, New York, Connecticut, Pennsylvania, and Indiana in the United States and from Germany and the Czech Republic in Europe from 2008 through 2012. In addition, an isolate from Japan was characterized. We found 3 distinct genotypes, 1 for North America, 1 for Europe, and 1 for Japan. We found B. miyamotoi infection in ticks in 16 of the 26 sites surveyed, with infection prevalence as high as 15.4%. These results show the widespread distribution of the pathogen, indicating an exposure risk to humans in areas where Ixodes ticks reside.

A First Look at the Relationship Between Large Herbivore-Induced Landscape Modifications and Ixodes ricinus Tick Abundance in Rewilding Sites.

Background: While the influence of landscape and microclimatic conditions on tick populations is well-documented, there remains a gap in more specific data regarding their relationship to rewilding efforts with large herbivore activity. Objective: This pilot study, spanning from 2019 to 2021, explores the effects of naturalistic grazing by large semi-wild ungulates on tick abundance in the Milovice Reserve, Czechia. Methods: Tick collection was observed using flagging techniques at two distinct sites of rewilding area: one grazed, actively utilized by animals involved in the rewilding project, and one ungrazed, left fallow in neighboring areas utilized only by wild animals. Transects, each measuring 150 m in length and 5 m in width (750 m2), were established at these two sampling locations from March to September between 2019 and 2021. To minimize potential bias resulting from tick movement, a 300 m buffer zone separated the two sites. Data analysis employed a generalized estimating equations (GEE) model with negative binomial regression. The study assessed potential variations in tick abundance between selected transects, considering factors such as plant cover seasonality, temperature, and humidity. Results: During the collection periods, we gathered 586 live ticks, with 20% found in grazed areas and 80% in ungrazed areas. Notably, tick abundance was significantly higher in ungrazed areas. Peaks in tick abundance occurred in both grazed and ungrazed areas during spring, particularly in April. However, tick numbers declined more rapidly in grazed areas. Microclimatic variables like temperature and humidity did not significantly impact tick abundance compared to landscape management and seasonal factors. Conclusion: Rewilding efforts, particularly natural grazing by large ungulates, influence tick abundance and distribution. This study provides empirical data on tick ecology in rewilded areas, highlighting the importance of landscape management and environmental factors in tick management and conservation. Trophic rewilding plays a crucial role in shaping ecosystems and tick population dynamics in transformed landscapes.

Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system.

BACKGROUND: The clinical course of tick-borne encephalitis (TBE), a disease caused by TBE virus, ranges from asymptomatic or mild influenza-like infection to severe debilitating encephalitis or encephalomyelitis. Despite the medical importance of this disease, some crucial steps in the development of encephalitis remain poorly understood. In particular, the basis of the disease severity is largely unknown. METHODS: TBE virus growth, neutralizing antibody response, key cytokine and chemokine mRNA production and changes in mRNA levels of cell surface markers of immunocompetent cells in brain were measured in mice with different susceptibilities to TBE virus infection. RESULTS: An animal model of TBE based on BALB/c-c-STS/A (CcS/Dem) recombinant congenic mouse strains showing different severities of the infection in relation to the host genetic background was developed. After subcutaneous inoculation of TBE virus, BALB/c mice showed medium susceptibility to the infection, STS mice were resistant, and CcS-11 mice were highly susceptible. The resistant STS mice showed lower and delayed viremia, lower virus production in the brain and low cytokine/chemokine mRNA production, but had a strong neutralizing antibody response. The most sensitive strain (CcS-11) failed in production of neutralizing antibodies, but exhibited strong cytokine/chemokine mRNA production in the brain. After intracerebral inoculation, all mouse strains were sensitive to the infection and had similar virus production in the brain, but STS mice survived significantly longer than CcS-11 mice. These two strains also differed in the expression of key cytokines/chemokines, particularly interferon gamma-induced protein 10 (IP-10/CXCL10) and monocyte chemotactic protein-1 (MCP-1/CCL2) in the brain. CONCLUSIONS: Our data indicate that the genetic control is an important factor influencing the clinical course of TBE. High neutralizing antibody response might be crucial for preventing host fatality, but high expression of various cytokines/chemokines during TBE can mediate immunopathology and be associated with more severe course of the infection and increased fatality.

The rare ospC allele L of Borrelia burgdorferi sensu stricto, commonly found among samples collected in a coastal plain area of the southeastern United States, is associated with ixodes affinis ticks and local rodent hosts Peromyscus gossypinus and Sigmodon hispidus.

The rare ospC allele L was detected in 30% of Borrelia burgdorferi sensu stricto strains cultured from a tick species, Ixodes affinis, and two rodent host species, Peromyscus gossypinus and Sigmodon hispidus, collected in a coastal plain area of Georgia and South Carolina, in the southeastern United States.

Detection of Borrelia burgdorferi sensu stricto ospC alleles associated with human lyme borreliosis worldwide in non-human-biting tick Ixodes affinis and rodent hosts in Southeastern United States.

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in European and North American regions where Lyme disease is endemic and where it is not endemic revealed a close relatedness of geographically distinct populations. ospC alleles A, B, and L were detected on both continents in vectors and hosts, including humans. Six ospC alleles, A, B, L, Q, R, and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3, and M, were identified in the far-western United States. Four ospC alleles, B, G, H, and L, were abundant in the southeastern United States. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern United States with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in European and North American regions where the disease is endemic were detected in B. burgdorferi strains isolated from the non-human-biting tick Ixodes affinis and rodent hosts in the southeastern United States. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern United States, a finding that confirms the hypothesis of transoceanic migration of Borrelia species.

Knockdown of proteins involved in iron metabolism limits tick reproduction and development.

Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus, and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1, fer2, and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Borrelia spirochetes in European exotic farm animals.

Ticks transmit a broad spectrum of pathogens, threatening both animal and human health. Tick survival and proliferation are strongly dependent on host selection and suitability. The hard tick Ixodes ricinus, which is widespread throughout most of Europe, is a host generalist capable of feeding on many different vertebrate species. Pasture-kept exotic farm animals may be at a high risk for tick and tick-borne pathogens infestations but research characterizing this is currently lacking. This study focused on the detection of Borrelia spirochetes (including Borrelia miyamotoi) in exotic farm animals. Using nested-PCR with Borrelia-specific primers, 121 serum samples from 54 exotic farm animals of several species bred in four different farms in Bohemia and Moravia (Czechia) were tested. Positive samples were sequenced for the identification of Borrelia species. The prevalence of Borrelia DNA in the samples ranged from 13 to 67%, depending on the sampling site. The sequencing results confirmed the DNA presence of multiple spirochete species from the Borrelia burgdorferi sensu lato complex. Only one sample from an ostrich (Struthio camelus) was found to be positive for Borrelia myiamotoi. The results show that exotic farm animals can serve as hosts for hard ticks and can be infected by Borrelia spirochetes, transmitted by hard ticks. Therefore, these animals could play a relevant role in maintaining Borrelia spirochetes in nature.

Borrelia carolinensis sp. nov., a novel species of the Borrelia burgdorferi sensu lato complex isolated from rodents and a tick from the south-eastern USA.

A group of 16 isolates with genotypic characteristics different from those of known species of the Borrelia burgdorferi sensu lato complex were cultured from ear biopsies of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina, USA, and from the tick Ixodes minor feeding on N. floridana. Multilocus sequence analysis of members of the novel species, involving the 16S rRNA gene, the 5S-23S (rrf-rrl) intergenic spacer region and the flagellin, ospA and p66 genes, was conducted and published previously and was used to clarify the taxonomic status of the novel group of B. burgdorferi sensu lato isolates. Phylogenetic analysis based on concatenated sequences of the five analysed genomic loci showed that the 16 isolates clustered together but separately from other species in the B. burgdorferi sensu lato complex. The analysed group therefore represents a novel species, formally described here as Borrelia carolinensis sp. nov., with the type strain SCW-22(T) (=ATCC BAA-1773(T) =DSM 22119(T)).