Laboratory Testing for Powassan Virus: Past, Present, and Future.

Powassan virus is a tick-borne flavivirus that can cause severe neuroinvasive disease, with areas of endemicity in the Northeast and Midwest United States, Canada, and Russia. Diagnosis is challenging and relies on a high index of suspicion and choosing the right test based on duration of infection and the patient's immune status. This review covers laboratory testing for Powassan virus, including historical considerations, modern options, and methods being developed in the research space.

A reporter virus particle seroneutralization assay for tick-borne encephalitis virus overcomes ELISA limitations.

Tick-borne encephalitis (TBE) virus is the most prevalent tick-transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)-based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. The RVP-based SNT for TBEV exhibited a highly significant correlation with the traditional virus-based SNT (R2 = 0.8637, p < 0.0001). The RVP-based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%-97.4%) and specificity of 100% (95% CI: 81.6%-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV-positive by ELISA but negative by RVP-based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP-based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.

Changes in cerebrospinal fluid proteome of patients with tick-borne encephalitis.

Tick-borne encephalitis (TBE) is one of the main diseases transmitted by ticks, the incidence of which is increasing. Moreover, its diagnosis and therapy are often long and difficult according to nonspecific symptoms and complex etiology. This study aimed to observe changes in the proteome of cerebrospinal fluid from TBE patients. Cerebrospinal fluid (CSF) of TBE patients (n = 20) and healthy individuals (n = 10) was analyzed using a proteomic approach (QExactiveHF-Orbitrap mass spectrometer) and zymography. Obtained results show that in CSF of TBE patients, the top-upregulated proteins are involved in pro-inflammatory reaction (interleukins), as well as antioxidant/protective response (peroxiredoxins, heat shock proteins). Moreover, changes in the proteome of CSF are not only the result of this disease development, but they can also be an indicator of its course. This mainly applies to proteins involved in proteolysis including serpins and metalloproteinases, whose activity is proportional to the length of patients' convalescence. The obtained proteomic data strongly direct attention to the changes caused by the development of TBE to antioxidant, pro-inflammatory, and proteolytic proteins, knowledge about which can significantly contribute to faster and more accurate diagnosis of various clinical forms of TBE.

A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick.

Tick-borne encephalitis virus (TBEV), a zoonotic pathogen, can cause severe neurological complications and fatal outcomes in humans. Early diagnosis of TBEV infection is crucial for clinical practice. Although serological assays are frequently employed for detection, the lack of antibodies in the early stages of infection and the cross-reactivity of antibodies limit their efficacy. Conventional molecular diagnostic methods such as RT-qPCR can achieve early and accurate identification but require specialized instrumentation and professionals, hindering their application in resource-limited areas. Our study developed a rapid and visual TBEV molecular detection method by combining RT-recombinase-aided amplification, the CRISPR/Cas13a system, and lateral flow dipsticks. The diagnostic sensitivity of this method is 50 CFU/ml, with no cross-reactivity with a variety of viruses. The detection can be carried out within 1 h at a temperature between 37 and 42 °C, and the results can be visually determined without the need for complex instruments and professionals. Subsequently, this assay was used to analyze clinical samples from 15 patients suspected of TBEV infection and 10 healthy volunteers, and its sensitivity and specificity reached 100 %, which was consistent with the results of RT-qPCR. These results indicate that this new method can be a promising point-of-care test for the diagnosis of tick-borne encephalitis.

No detection of tick-borne encephalitis virus RNA in blood, urine or saliva of hospitalised immunocompetent tick-borne encephalitis patients.

Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by vaccination or cross-reactivity to previous flavivirus infections may result in false positive TBEV serology. Detection of TBEV RNA may be an alternative diagnostic approach to detect viral presence and circumvent the diagnostic difficulties present when using serology. Viral RNA in blood is commonly detectable only in the first viremic phase usually lasting up to two weeks, and not in the second neurologic phase, when the patients contact the health care system and undergo diagnostic work-up. TBEV RNA has previously been detected in urine in a few retrospective TBE cases in the neurologic phase, and furthermore RNA of other flaviviruses has been detected in patient saliva. In this study, blood, saliva and urine were collected from 31 hospitalised immunocompetent patients with pleocytosis and symptoms of aseptic meningitis and/or encephalitis, suspected to have TBE. We wanted to pursue if molecular testing of TBEV RNA in these patient materials may be useful in the diagnostics. Eleven of the 31 study patients were diagnosed with TBE based on ELISA detection of TBEV specific IgG and IgM antibodies. None of the study patients had TBEV RNA detectable in any of the collected patient material.

Clinical characteristics and factors affecting disease severity in hospitalized tick-borne encephalitis patients in Norway from 2018 to 2022.

PURPOSE: To describe the clinical characteristics and factors associated with disease severity in a Norwegian cohort of hospitalized patients with tick-borne encephalitis (TBE). METHODS: This observational multicenter study included hospitalized patients with TBE in the endemic area in the southeastern region of Norway from 2018 to 2022. Clinical signs and findings from laboratory tests, EEG, CT and MRI scans were recorded. Patient characteristics were compared among those with mild, moderate, and severe TBE, and factors associated with disease severity were identified. RESULTS: Nearly all eligible patients were included in the final cohort (153/189 participants, 81%). The median age was 56 years, 63% were men, and 7% were vaccinated against TBE; no participants were fully vaccinated. TBE presented as mild (meningeal) disease in 31% of patients and as moderate or severe (encephalitic) disease in 54% and 14% of patients, respectively. We found that 46% of the patients had a monophasic course, 64% had hyponatremia, and 7% presented with central nervous system (CNS) symptoms without pleocytosis in cerebrospinal fluid (CSF). Dysesthesia, a symptom previously not described, was reported in 10% of the patients. Most objective findings were related to the CNS. Preexisting comorbidities, CRP and CSF protein levels were predictors of more severe disease. CONCLUSION: This novel presentation of a large Norwegian cohort supports TBE as a serious disease in the southeastern region of Norway. The majority of hospitalized patients presented with encephalitis, and fewer presented with meningitis. Comorbidities, CRP and CSF protein levels were associated with more severe disease. TRIAL REGISTRATION: Prosjekt #2,296,959 - The Norwegian Tick-borne Encephalitis Study - NOTES. Acute phase characteristics and long-term outcomes. - Cristin.

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.

Severe Tick-Borne Encephalitis (TBE) in a Patient with X-Linked Agammaglobulinemia; Treatment with TBE Virus IgG Positive Plasma, Clinical Outcome and T Cell Responses.

PURPOSE: A patient with X-linked agammaglobulinemia (XLA) and severe tick-borne encephalitis (TBE) was treated with TBE virus (TBEV) IgG positive plasma. The patient's clinical response, humoral and cellular immune responses were characterized pre- and post-infection. METHODS: ELISA and neutralisation assays were performed on sera and TBEV PCR assay on sera and cerebrospinal fluid. T cell assays were conducted on peripheral blood the patient and five healthy vaccinated controls. RESULTS: The patient was admitted to the hospital with headache and fever. He was not vaccinated against TBE but receiving subcutaneous IgG-replacement therapy (IGRT). TBEV IgG antibodies were low-level positive (due to scIGRT), but the TBEV IgM and TBEV neutralisation tests were negative. During hospitalisation his clinical condition deteriorated (Glasgow coma scale 3/15) and he was treated in the ICU with corticosteroids and external ventricular drainage. He was then treated with plasma containing TBEV IgG without apparent side effects. His symptoms improved within a few days and the TBEV neutralisation test converted to positive. Robust CD8+ T cell responses were observed at three and 18-months post-infection, in the absence of B cells. This was confirmed by tetramers specific for TBEV. CONCLUSION: TBEV IgG-positive plasma given to an XLA patient with TBE without evident adverse reactions may have contributed to a positive clinical outcome. Similar approaches could offer a promising foundation for researching therapeutic options for patients with humoral immunodeficiencies. Importantly, a robust CD8+ T cell response was observed after infection despite the lack of B cells and indicates that these patients can clear acute viral infections and could benefit from future vaccination programs.

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.

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.

[Etiological diagnostic methods and research progress of forest encephalitis].

Forest encephalitis is a natural focal disease transmitted through the bite of hard ticks, and its pathogen is the tick-borne encephalitis virus from the Flaviviridae family. The mortality rate of forest encephalitis is relatively high, making laboratory testing significant in diagnosing this disease. This article elaborates on the etiological diagnostic methods and recent research progress in forest encephalitis. Laboratory tests for forest encephalitis mainly include routine examinations, serological tests, virus isolation, and molecular biological testing. The detection of serum-specific IgM antibodies against the forest encephalitis virus is of great importance for early diagnosis, and specific IgG antibodies serve as a "gold standard" for differentiation from other diseases. Techniques such as enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay for detecting specific IgM antibodies in serum and/or cerebrospinal fluid, the serum hemagglutination inhibition test or serum complement fixation test, and the double serum hemagglutination inhibition test or complement fixation test all contribute to the early diagnosis. The development of molecular testing methods is rapid, and techniques such as metabolomics, digital PCR, and matrix metalloproteinases are also applied in the early diagnosis of forest encephalitis.

Serological and molecular screening of arenaviruses in suspected tick-borne encephalitis cases in Finland.

Lymphocytic choriomeningitis virus (LCMV) is one of the arenaviruses infecting humans. LCMV infections have been reported worldwide in humans with varying levels of severity. To detect arenavirus RNA and LCMV-reactive antibodies in different geographical regions of Finland, we screened human serum and cerebrospinal fluid (CSF) samples, taken from suspected tick-borne encephalitis (TBE) cases, using reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). No arenavirus nucleic acids were detected, and the overall LCMV seroprevalence was 4.5%. No seroconversions were detected in paired serum samples. The highest seroprevalence (5.2%) was detected among individuals of age group III (40-59 years), followed by age group I (under-20-year-olds, 4.9%), while the lowest seroprevalence (3.8%) was found in age group IV (60 years or older). A lower LCMV seroprevalence in older age groups may suggest waning of immunity over time. The observation of a higher seroprevalence in the younger age group and the decreasing population size of the main reservoir host, the house mouse, may suggest exposure to another LCMV-like virus in Finland.

Suspected tick-borne flavivirus meningoencephalomyelitis in dogs from the UK: six cases (2021).

OBJECTIVES: Tick-borne encephalitis virus and louping ill virus are neurotropic flaviviruses transmitted by ticks. Epidemiologically, tick-borne encephalitis is endemic in Europe whereas louping ill's predominant geographical distribution is the UK. Rarely, these flaviviruses affect dogs causing neurological signs. This case series aimed to describe the clinical, clinicopathological, and imaging findings, as well as the outcomes in six dogs with meningoencephalitis and/or meningomyelitis caused by a flavivirus in the UK in 2021. MATERIALS AND METHODS: Observational retrospective case-series study. Clinical data were retrieved from medical records of dogs with positive serological or immunohistochemical results from three different institutions from spring to winter 2021. RESULTS: Six dogs were included in the study. All dogs presented an initial phase of pyrexia and/or lethargy followed by progressive signs of spinal cord and/or intracranial disease. Magnetic resonance imaging showed bilateral and symmetrical lesions affecting the grey matter of the thalamus, pons, medulla oblongata, and thoracic or lumbar intumescences with none or mild parenchymal and meningeal contrast enhancement. Serology for tick-borne encephalitis virus was positive in five dogs with the presence of seroconversion in two dogs. The viral distinction between flaviviruses was not achieved. One dog with negative serology presented positive immunohistochemistry at post-mortem examination. Three dogs survived but presented neurological sequelae. Three dogs were euthanased due to the rapid progression of the clinical signs or static neurological signs. CLINICAL SIGNIFICANCE: These cases raise awareness of the presence of tick-borne encephalitis as an emergent disease or the increased prevalence of louping ill virus affecting dogs in the UK.

Powassan Virus Encephalitis: A Tertiary Center Experience.

BACKGROUND: Powassan virus (POWV) is an emerging arthropod-borne flavivirus, transmitted by Ixodes spp. ticks, which has been associated with neuroinvasive disease and poor outcomes. METHODS: A retrospective study was conducted at Mayo Clinic from 2013 to 2022. We included clinical and epidemiologic data of probable and confirmed neuroinvasive POWV cases. RESULTS: Sixteen patients with neuroinvasive POWV were identified; their median age was 63.2 years, and 62.5% were male. Six patients presented with rhombencephalitis, 4 with isolated meningitis, 3 with meningoencephalitis, 2 with meningoencephalomyelitis, and 1 with opsoclonus myoclonus syndrome. A median time of 18 days was observed between symptom onset and diagnosis. Cerebrospinal fluid analysis showed lymphocytic pleocytosis with elevated protein and normal glucose in the majority of patients. Death occurred within 90 days in 3 patients (18.8%), and residual neurologic deficits were seen in 8 survivors (72.7%). CONCLUSIONS: To our knowledge, this is the largest case series of patients with neuroinvasive POWV infection. We highlight the importance of a high clinical suspicion among patients who live in or travel to high-risk areas during the spring to fall months. Our data show high morbidity and mortality rates among patients with neuroinvasive disease.

Paretic complications of tick-borne encephalitis and Lyme neuroborreliosis in the Czech Republic: Characteristics and clinical outcome.

Tick-borne encephalitis (TBE) and Lyme neuroborreliosis (LNB), the most common tick-borne diseases of the central nervous system in Central Europe, are frequently associated with pareses. The aim of this study was to characterise paretic complications in patients with TBE and LNB, including their severity, persistence and impact on the patients' quality of life. Our retrospective observational study included patients with aseptic CNS infection due to TBE virus or Borrelia burgdorferi sensu lato. Paretic complications were evaluated in the acute phase and the patients were followed up until complete regression or long-term stabilisation of any neurological deficit. The severity of the neurological deficit was graded according to the modified Rankin Scale (mRS). A total of 823 patients (582 with TBE, 241 with LNB) was included. Paretic complications were diagnosed in 63 TBE patients (10.8 %) and in 147 LNB patients (61.0 %). In TBE, the most common neurological deficit was brachial plexus paresis in 21 patients (33 %) and bulbar symptoms in 18 patients (29 %). In LNB patients, facial nerve palsy was the most frequent neurological deficit (117patients; 79.6 %), followed by lower limb paresis in 23 patients (15.6 %). Forty-nine TBE patients and 134 LNB paretic patients completed follow-up. Paresis resolved within 3 weeks in 16 TBE patients (33 %) and 53 LNB patients (39.5 %), but the proportion of patients with paresis persisting for more than 12 months was significantly higher in TBE (34.7 vs. 3.7 %, p < 0.001). The mean mRS was significantly higher in TBE paretic patients compared to LNB (p < 0.001). Paretic complications are significantly more common in LNB than in TBE but pareses associated with TBE last longer than in LNB and considerably reduce the quality of life of patients. Prevention remains the only way to influence the long-term motor deficits of TBE.

Results of Tick-Borne Encephalitis Virus (TBEV) Diagnostics in an Endemic Area in Southern Germany, 2007 to 2022.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted neurotropic flavivirus in Europe and Asia. Our analysis aimed to investigate the contribution of TBEV-specific antibody detection by serological assays and TBEV RNA detection by real-time PCR to the diagnosis of tick-borne encephalitis (TBE). We analyzed data from 3713 patients from 16 years of laboratory TBEV diagnostics in an endemic area in Southern Germany. During this period, 126 cases of TBE were diagnosed. TBEV-specific IgM ELISA tests showed a high clinical sensitivity (96.8%) and a very high clinical specificity (99.7%). In immunocompetent patients, TBE was reliably diagnosed by detection of TBEV IgM antibodies in serum. Intrathecal TBEV IgG antibody synthesis was detected in 46 of 84 (55%) cases by analysis of paired serum and cerebrospinal fluid (CSF) samples. None of the 87 immunocompetent TBE patients tested had detectable TBEV RNA in serum or CSF. In contrast, in two TBE patients without TBEV-specific antibodies, diagnosis could only be made by the detection of TBEV RNA in CSF. Both patients had previously been treated with the B cell-depleting antibody rituximab. Therefore, in patients with CNS infection and humoral immunodeficiency, it is necessary to include TBEV PCR in the diagnostic approach.

Tick-borne encephalitis.

Tick-borne encephalitis (TBE) is a viral tick-borne infection occurring in many parts of Europe and Asia as described in this review. Increasing TBE case numbers have been reported over recent decades. In Denmark the infection is rare (1-14 annual cases). The rise in TBE in Denmark is mainly driven by microfoci outside of Bornholm, primarily North Zealand. Clinical illness has a bi-phasic presentation: "summer-flu" which may be followed by a neuroinfection. No specific treatment exists, and mortality is less-than 1%. A considerable percentage of patients may experience neurological sequelae. TBE is preventable through vaccination.

A typical case of tick-borne encephalitis.

Tick-borne encephalitis is a rare infection in Denmark. The infection has been endemic to the island of Bornholm for many years. During the past 10 years, transmission has occurred in other parts of Denmark, particularly North Zealand. This case report describes a typical tick-borne encephalitis case diagnosed in the first viraemic phase of the disease course. Clinical presentation and diagnostic criteria are described.

[Difficulties in diagnosing the chronic course of tick-borne encephalitis in children]. / Trudnosti diagnostiki khronicheskogo techeniya kleshchevogo entsefalita u detei.

Infections transmitted by ixodic ticks in childhood account for about 20%, and tick-borne encephalitis accounts for up to 25% of cases. A feature of the course of tick-borne encephalitis is unpredictability and the possibility of chronization in 1-3% of cases with late diagnosis and inadequate therapy. The article presents a clinical observation of the disease in a child whose chronically progressive course of amyotrophic form was diagnosed 1 year and 8 months after the onset of the disease. The presence of anamnestic data, features of clinical manifestations in the dynamics and typical neurovirualization picture of the disease, even with negative results of serological and molecular genetic studies of blood and cerebrospinal fluid, allowed to make a diagnosis, prescribe etio-pathogenetic therapy and thereby stabilize the patient's condition. The use of complex drugs in the treatment, such as Cytoflavin, allows to optimize the course of the disease.

[Small Tick - Big Danger].

INTRODUCTION: We report a 23-year-old patient who presented to the general practitioner due to persisting headache, fever, and vomiting. In the further course, a tetraparesis dominated on the right side, dysphagia and dysarthria occurred, and a general tonic-clonic seizure. Further examinations confirmed tick-borne encephalomyelitis as well as polyradiculitis. After two months of rehabilitation, neuropsychological as well as focal-neurological deficits persisted in the unvaccinated patient.