Fatal tick-borne encephalitis virus infection in Dalmatian puppy-dogs after putative vector independent transmission.

In a retrospective metatranscriptomics study, we identified tick-borne encephalitis virus (TBEV) to be the causative agent for a fatal non-suppurative meningoencephalitis in a three-week-old Dalmatian puppy in Switzerland. Further investigations showed that the two other littermates with similar signs and pathological lesions were also positive for TBEV. By using an unbiased approach of combining high-throughput sequencing (HTS) and bioinformatics we were able to solve the etiology and discover an unusual case of TBEV in three young puppies. Based on our findings, we suggest that a vector-independent transmission of TBEV occurred and that most likely an intrauterine infection led to the severe and fulminant disease of the entire litter. We were able to demonstrate the presence of TBEV RNA by in situ hybridization (ISH) in the brain of all three puppies. Furthermore, we were able to detect TBEV by RT-qPCR in total RNA extracted from formalin-fixed and paraffin embedded (FFPE) blocks containing multiple peripheral organs. Overall, our findings shed light on alternative vector-independent transmission routes of TBEV infections in dogs and encourage veterinary practitioners to consider TBEV as an important differential diagnosis in neurological cases in dogs.

Identification of New Microfoci and Genetic Characterization of Tick-Borne Encephalitis Virus Isolates from Eastern Germany and Western Poland.

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.

Studies on the sequential pathology of Kyasanur Forest Disease (KFD) in Mouse brain: KFD virus induces apoptosis of neurons in cerebrum and hippocampus.

The sequential pathology of Kyasanur forest disease (KFD) in mouse brain was assessed in this study. Kyasanur forest disease virus (KFDV) strain P9605 used in this study was confirmed by real-time reverse transcriptase-polymerase chain reaction targeting the NS5 gene. Mouse Lethal Dose 50 (MLD50) of the virus was determined by in-vivo mice inoculation test. One MLD50 of the KFDV was inoculated intra-cerebrally into 36 mice aged 2-3 weeks. Another group of 36 age-matched mice that served as control group were inoculated with plain media. Six mice each from infected and control groups were euthanized every 24 hrs intervals for six days. Brain tissues were collected in 10% NBF. The collected brain tissues were processed and subjected to histopathological studies by Hematoxylin and Eosin staining. Grossly, the infected mice showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indication of hind quarter paralysis on day four post-infection. These symptoms got aggravated with complete paralysis of the hind quarters, inability to move and death on 5th and 6th day post-infection. Microscopically, the brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. The real-time RT-PCR on hippocampus of the KFDV-infected mouse brain showed three-fold higher expression levels of Caspase 3, a crucial mediator of apoptosis. The cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected, possibly correlating with the gross symptoms of hind quarter paralysis, ataxia, and other motor neuron dysfunctions noticed. Taken together, these findings reveal that KFDV induces apoptosis of neurons in the cerebrum and hippocampus of KFDV infected mice. Further studies are needed to confirm if the lesions noticed in mice brain simulate the brain lesions in humans since gross motor-neuron symptoms are similar in mice as well as humans.

Differential susceptibility of geographically distinct Ixodes ricinus populations to tick-borne encephalitis virus and louping ill virus.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa, ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

Direct Evidence of Powassan Virus Vertical Transmission in Ixodes scapularis in Nature.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.

Recapitulation of the Powassan virus life cycle in cell culture.

Powassan virus (POWV) is a tick-borne flavivirus known for causing fatal neuroinvasive diseases in humans. Recently, there has been a noticeable increase in POWV infections, emphasizing the urgency of understanding viral replication, pathogenesis, and developing interventions. Notably, there are no approved vaccines or therapeutics for POWV, and its classification as a biosafety level-3 (BSL-3) agent hampers research. To overcome these obstacles, we developed a replicon system, a self-replicating RNA lacking structural proteins, making it safe to operate in a BSL-2 environment. We constructed a POWV replicon carrying the Gaussia luciferase (Gluc) reporter gene and blasticidin (BSD) selectable marker. Continuous BSD selection led to obtain a stable POWV replicon-carrying Huh7 cell lines. We identified cell culture adaptive mutations G4079A, G4944T and G6256A, resulting in NS2AR195K, NS3G122G, and NS3V560M, enhancing RNA replication. We demonstrated the utility of the POWV replicon system for high-throughput screening (HTS) assay to identify promising antivirals against POWV replication. We further explored the applications of the POWV replicon system, generating single-round infectious particles (SRIPs) by transfecting Huh7-POWV replicon cells with plasmids encoding viral capsid (C), premembrane (prM), and envelope (E) proteins, and revealed the distinct antigenic profiles of POWV with ZIKV. In summary, the POWV replicon and SRIP systems represent crucial platforms for genetic and functional analysis of the POWV life cycle and facilitating the discovery of antiviral drugs.IMPORTANCEIn light of the recent surge in human infections caused by POWV, a biosafety level-3 (BSL-3) classified virus, there is a pressing need to understand the viral life cycle and the development of effective countermeasures. To address this, we have pioneered the establishment of a POWV RNA replicon system and a replicon-based POWV SRIP system. Importantly, these systems are operable in BSL-2 laboratories, enabling comprehensive investigations into the viral life cycle and facilitating antiviral screening. In summary, these useful tools are poised to advance our understanding of the POWV life cycle and expedite the development of antiviral interventions.

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.

The Vector Competence of Asian Longhorned Ticks in Langat Virus Transmission.

Haemaphysalis longicornis (the longhorned tick), the predominant tick species in China, serves as a vector for a variety of pathogens, and is capable of transmitting the tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis. However, it is unclear how these ticks transmit TBEV. Langat virus (LGTV), which has a reduced pathogenicity in humans, has been used as a surrogate for TBEV. In this study, we aimed to investigate the vector competence of H. longicornis to transmit LGTV and demonstrate the efficient acquisition and transmission of LGTV between this tick species and mice. LGTV localization was detected in several tick tissues, such as the midgut, salivary glands, and synganglion, using quantitative PCR and immunohistochemical staining with a polyclonal antibody targeting the LGTV envelope protein. We demonstrated the horizontal transmission of LGTV to different developmental stages within the same generation but did not see evidence of vertical transmission. It was interesting to note that we observed mice acting as a bridge, facilitating the transmission of LGTV to neighboring naïve ticks during blood feeding. In conclusion, the virus-vector-host model employed in this study provides valuable insights into the replication and transmission of LGTV throughout its life cycle.

Proteomic and metabolomic analysis of the serum of patients with tick-borne encephalitis.

Tick-borne encephalitis virus (TBEV) is a common virus in Europe and Asia, causing around 10,000 to 10,500 infections annually. It affects the central nervous system and poses threats to public health. However, the exact molecular mechanisms of TBE pathogenesis are not yet fully understood due to the complex interactions between the virus and its host. In this study, a comprehensive analysis was conducted to characterize the serum metabolome and proteome of adult patients infected with TBEV, in comparison to a control group of healthy individuals. Liquid chromatography tandem mass spectrometry (LC-MS) was employed to monitor metabolic and proteomic alternations throughout the progression of the disease, significant physiological changes associated with different stages of the disease were identified. A total of 44 proteins and 115 metabolites exhibited significantly alternations in the sera of patients diagnosed with TBE. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of these metabolites and proteins revealed differential enrichment of genes associated with the extracellular matrix, complement binding, hemostasis, lipid metabolism, and amino acid metabolism between TBE patients and healthy controls. We gained valuable understanding of the specific metabolites implicated in the host's responses to TBE, establishing a basis for further research on TBE disease. SIGNIFICANCE: The current investigation revealed a comprehensive and systematic differences on TBE using LC-MS platform from human serum samples of TBE patients and healthy individuals providing the immune response to the invasion of TBE.

Molecular Detection and Phylogenetic Analysis of Tick-Borne Encephalitis Virus from Ticks Collected from Cattle in Kyrgyzstan, 2023.

Ticks are important vectors of the tick-borne encephalitis virus (TBEV). In Kyrgyzstan, the livestock farming trade and nomadic lifestyle enable tick-borne diseases to be imported from neighboring countries, but there are few relevant studies. In this study, we collected 40 ticks from cattle in Kyrgyzstan. Molecular marker analysis identified the ticks as Ixodes persulcatus (97.5%; n = 39) and Haemaphysalis punctata (2.5%; n = 1). Real-time PCR screening revealed two ticks to be positive for TBEV, but only one tick was amplified using nested PCR targeting the TBEV envelope (E) and non-structure 5 (NS5) gene. The obtained sequences belonged to the TBEV Siberian subtype and phylogenetic tree analysis results confirmed that the virus was related to the Bosnia strain. We also performed next-generation sequencing, which confirmed the TBEV Siberian subtype. Continuous research and surveillance of TBEV in Kyrgyzstan are required to provide further information on tick-borne diseases.

Structural diversity of tick-borne encephalitis virus particles in the inactivated vaccine based on strain Sofjin.

The main approach to preventing tick-borne encephalitis (TBE) is vaccination. Formaldehyde-inactivated TBE vaccines have a proven record of safety and efficiency but have never been characterized structurally with atomic resolution. We report a cryoelectron microscopy (cryo-EM) structure of the formaldehyde-inactivated TBE virus (TBEV) of Sofjin-Chumakov strain representing the Far-Eastern subtype. A 3.8 Å resolution reconstruction reveals the structural integrity of the envelope E proteins, specifically the E protein ectodomains. The comparative study shows a high structural similarity to the previously published structures of the TBEV European subtype strains Hypr and Kuutsalo-14. A fraction of inactivated virions exhibits asymmetric features including the deformations of the membrane profile. We propose that the heterogeneity is caused by inactivation and perform a local variability analysis on the small parts of the envelope protein shell to reveal membrane curvature features possibly induced by the inactivation. The results of this study will have implications for the design of novel vaccines against diseases caused by flaviviruses.

Antiviral activity of porphyrins and porphyrin-like compounds against tick-borne encephalitis virus: Blockage of the viral entry/fusion machinery by photosensitization-mediated destruction of the viral envelope.

Tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis (TBE), is a medically important flavivirus endemic to the European-Asian continent. Although more than 12,000 clinical cases are reported annually worldwide, there is no anti-TBEV therapy available to treat patients with TBE. Porphyrins are macrocyclic molecules consisting of a planar tetrapyrrolic ring that can coordinate a metal cation. In this study, we investigated the cytotoxicity and anti-TBEV activity of a large series of alkyl- or (het)aryl-substituted porphyrins, metalloporphyrins, and chlorins and characterized their molecular interactions with the viral envelope in detail. Our structure-activity relationship study showed that the tetrapyrrole ring is an essential structural element for anti-TBEV activity, but that the presence of different structurally distinct side chains with different lengths, charges, and rigidity or metal cation coordination can significantly alter the antiviral potency of porphyrin scaffolds. Porphyrins were demonstrated to interact with the TBEV lipid membrane and envelope protein E, disrupt the TBEV envelope and inhibit the TBEV entry/fusion machinery. The crucial mechanism of the anti-TBEV activity of porphyrins is based on photosensitization and the formation of highly reactive singlet oxygen. In addition to blocking viral entry and fusion, porphyrins were also observed to interact with RNA oligonucleotides derived from TBEV genomic RNA, indicating that these compounds could target multiple viral/cellular structures. Furthermore, immunization of mice with porphyrin-inactivated TBEV resulted in the formation of TBEV-neutralizing antibodies and protected the mice from TBEV infection. Porphyrins can thus be used to inactivate TBEV while retaining the immunogenic properties of the virus and could be useful for producing new inactivated TBEV vaccines.

Kyasanur Forest disease virus NS3 helicase: Insights into structure, activity, and inhibitors.

Kyasanur Forest disease virus (KFDV), a tick-borne flavivirus prevalent in India, presents a serious threat to human health. KFDV NS3 helicase (NS3hel) is considered a potential drug target due to its involvement in the viral replication complex. Here, we resolved the crystal structures of KFDV NS3hel apo and its complex with three phosphate molecules, which indicates a conformational switch during ATP hydrolysis. Our data revealed that KFDV NS3hel has a higher binding affinity for dsRNA, and its intrinsic ATPase activity was enhanced by dsRNA while being inhibited by DNA. Through mutagenesis analysis, several residues within motifs I, Ia, III, V, and VI were identified to be crucial for NS3hel ATPase activity. Notably, the M419A mutation drastically reduced NS3hel ATPase activity. We propose that the methionine-aromatic interaction between residues M419 and W294, located on the surface of the RNA-binding channel, could be a target for the design of efficient inhibitor probes. Moreover, epigallocatechin gallate (EGCG), a tea-derived polyphenol, strongly inhibited NS3hel ATPase activity with an IC50 value of 0.8 µM. Our computational docking data show that EGCG binds at the predicted druggable hotspots of NS3hel. Overall, these findings contribute to the development and design of more effective and specific inhibitors.

Cytoarchitecture of ex vivo midgut cultures of unfed Ixodes scapularis infected with a tick-borne flavivirus.

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.

Cellular stress is triggered by tick-borne encephalitis virus and limits the virus replication in PMJ2-R mouse macrophage cell line.

Viral infection may represent a stress condition to the host cell. Cells react to it by triggering the defence programme to restore homeostasis and these events may in turn impact the viral replication. The knowledge about tick-borne encephalitis virus (TBEV) infection-associated stress is limited. Here we investigated the interplay between TBEV infection and stress pathways in PMJ2-R mouse macrophage cell line, as macrophages are the target cells in early phases of TBEV infection. First, to determine how stress influences TBEV replication, the effect of stress inducers H2O2 and tunicamycin (TM) was tested. Viral multiplication was decreased in the presence of both stress inducers suggesting that the stress and cellular stress responses restrict the virus replication. Second, we investigated the induction of oxidative stress and endoplasmic reticulum (ER) stress upon TBEV infection. The level of oxidative stress was interrogated by measuring the reactive oxygen species (ROS). ROS were intermittently increased in infected cells at 12 hpi and at 72 hpi. As mitochondrial dysfunction may result in increased ROS level, we evaluated the mitochondrial homeostasis by measuring the mitochondrial membrane potential (MMP) and found that TBEV infection induced the hyperpolarization of MMP. Moreover, a transient increase of gene expression of stress-induced antioxidative enzymes, like p62, Gclm and Hmox1, was detected. Next, we evaluated the ER stress upon TBEV infection by analysing unfolded protein responses (UPR). We found that infection induced gene expression of two general sensors BiP and CHOP and activated the IRE1 pathway of UPR. Finally, since the natural transmission route of TBEV from its tick vector to the host is mediated via tick saliva, the impact of tick saliva from Ixodes ricinus on stress pathways in TBEV-infected cells was tested. We observed only marginal potentiation of UPR pathway. In conclusion, we found that TBEV infection of PMJ2-R cells elicits the changes in redox balance and triggers cellular stress defences, including antioxidant responses and the IRE1 pathway of UPR. Importantly, our results revealed the negative effect of stress-evoked events on TBEV replication and only marginal impact of tick saliva on stress cellular pathways.

DNA Vaccine Encoding the Artificial T-Cell Polyepitope Immunogen of Tick-Borne Encephalitis Virus.

A promising approach to the development of new means for preventing infection caused by tick-borne encephalitis virus can be DNA vaccines encoding polyepitope T-cell immunogens. A DNA vaccine pVAX-AG4-ub encoding an artificial polyepitope immunogen that includes cytotoxic and T-helper epitopes from the NS1, NS3, NS5, and E proteins of the tick-borne encephalitis virus has been obtained. The developed construct ensured the synthesis of the corresponding mRNAs in transfected eukaryotic cells. Immunization of mice with pVAX-AG4-ub induced the formation of a virus-specific T-cell response providing 50% protection from lethal infection with the virus.

Infection of wild-caught wood mice (Apodemus sylvaticus) and yellow-necked mice (A. flavicollis) with tick-borne encephalitis virus.

The distribution of tick-borne encephalitis virus (TBEV) is expanding to Western European countries, including the Netherlands, but the contribution of different rodent species to the transmission of TBEV is poorly understood. We investigated whether two species of wild rodents native to the Netherlands, the wood mouse Apodemus sylvaticus and the yellow-necked mouse Apodemus flavicollis, differ in their relative susceptibility to experimental infection with TBEV. Wild-caught individuals were inoculated subcutaneously with the classical European subtype of TBEV (Neudoerfl) or with TBEV-NL, a genetically divergent TBEV strain from the Netherlands. Mice were euthanised and necropsied between 3 and 21 days post-inoculation. None of the mice showed clinical signs or died during the experimental period. Nevertheless, TBEV RNA was detected up to 21 days in the blood of both mouse species and TBEV was also isolated from the brain of some mice. Moreover, no differences in infection rates between virus strains and mouse species were found in blood, spleen, or liver samples. Our results suggest that the wood mouse and the yellow-necked mouse may equally contribute to the transmission cycle of TBEV in the Netherlands. Future experimental infection studies that include feeding ticks will help elucidate the relative importance of viraemic transmission in the epidemiology of TBEV.

[Investigation of oncolytic potential of vaccine strains of yellow fever and tick-borne encephalitis viruses against glioblastoma and pancreatic carcinoma cell lines].

INTRODUCTION: Flaviviruses, possessing natural neurotropicity could be used in glioblastoma therapy using attenuated strains or as a delivery system for antitumor agents in an inactivated form. OBJECTIVE: To investigate the sensitivity of glioblastoma and pancreatic carcinoma cell lines to vaccine strains of yellow fever and tick-borne encephalitis viruses. MATERIALS AND METHODS: Cell lines: glioblastoma GL-6, T98G, LN-229, pancreatic carcinoma MIA RaCa-2 and human pancreatic ductal carcinoma PANC-1. Viral strains: 17D yellow fever virus (YF), Sofjin tick-borne encephalitis virus (TBEV). Virus concentration were determined by plaque assay and quantitative PCR. Determination of cell sensitivity to viruses by MTT assay. RESULTS: 17D YF was effective only against pancreatic carcinoma tumor cells MIA Paca-2 and had a limited effect against PANC-1. In glioblastoma cell lines (LN229, GL6, T98G), virus had no oncolytic effect and the viral RNA concentration fell in the culture medium. Sofjin TBEV showed CPE50 against MIA Paca-2 and a very limited cytotoxic effect against PANC-1. However, it had no oncolytic effect against glioblastoma cell lines (LN229, T98G and GL6), although virus reproduction continued in these cultures. For the GL6 glioblastoma cell line, the viral RNA concentration at the level with the infection dose was determined within 13 days, despite medium replacement, while in the case of the LN229 cell line, the virus concentration increased from 1 × 109 to 1 × 1010 copies/ml. CONCLUSION: Tumor behavior in organism is more complex and is determined by different microenvironmental factors and immune status. In the future, it is advisable to continue studying the antitumor oncolytic and immunomodulatory effects of viral strains 17D YF and Sofjin TBEV using in vivo models.

Hazard potential of Swiss Ixodes ricinus ticks: Virome composition and presence of selected bacterial and protozoan pathogens.

Ticks play an important role in transmitting many different emerging zoonotic pathogens that pose a significant threat to human and animal health. In Switzerland and abroad, the number of tick-borne diseases, in particular tick-borne encephalitis (TBE), has been increasing over the last few years. Thus, it remains essential to investigate the pathogen spectrum of ticks to rapidly detect emerging pathogens and initiate the necessary measures. To assess the risk of tick-borne diseases in different regions of Switzerland, we collected a total of 10'286 ticks from rural and urban areas in ten cantons in 2021 and 2022. Ticks were pooled according to species, developmental stage, gender, and collection site, and analyzed using next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR). The metagenomic analysis revealed for the first time the presence of Alongshan virus (ALSV) in Swiss ticks. Interestingly, the pool-prevalence of ALSV was higher than that of tick-borne encephalitis virus (TBEV). Furthermore, several TBEV foci have been identified and pool prevalence of selected non-viral pathogens determined.