Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology.

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

Targeting the NS2B-NS3 protease of tick-borne encephalitis virus with pan-flaviviral protease inhibitors.

Tick-borne encephalitis (TBE) is a severe neurological disorder caused by tick-borne encephalitis virus (TBEV), a member of the Flavivirus genus. Currently, two vaccines are available in Europe against TBEV. However, TBE cases have been rising in Sweden for the past twenty years, and thousands of cases are reported in Europe, emphasizing the need for antiviral treatments against this virus. The NS2B-NS3 protease is essential for flaviviral life cycle and has been studied as a target for the design of inhibitors against several well-known flaviviruses, but not TBEV. In the present study, Compound 86, a known tripeptidic inhibitor of dengue (DENV), West Nile (WNV) and Zika (ZIKV) proteases, was predicted to be active against TBEV protease using a combination of in silico techniques. Further, Compound 86 was found to inhibit recombinant TBEV protease with an IC50 = 0.92 µM in the in vitro enzymatic assay. Additionally, two more peptidic analogues were synthetized and they displayed inhibitory activities against both TBEV and ZIKV proteases. In particular, Compound 104 inhibited ZIKV protease with an IC50 = 0.25 µM. These compounds represent the first reported inhibitors of TBEV protease to date and provides valuable information for the further development of TBEV as well as pan-flavivirus protease inhibitors.

Unusual cause at an unusual time-Powassan virus rhombencephalitis.

Powassan virus lineage II (POWV) is an emerging tick-borne neurotropic pathogen, transmitted to humans by the bite of infected Ixodes scapularis ticks. In the United States, the disease is most prevalent in the Northeast and the upper Midwest and occurs mostly during the spring and summer months when tick activity is the highest. Some patients infected with POWV develop severe encephalitis, with high mortality. We report the case of a 42-year-old healthy man who developed progressive diplopia and dysarthria in December following a deer hunting trip. Routine blood work was unrevealing and MRI was normal. Extensive work-up for infectious, autoimmune, and paraneoplastic causes was positive only for POWV. The patient was treated with supportive care and intravenous corticosteroids, with an excellent outcome. We present a rare clinical presentation of a potentially fatal emerging disease that responded favorably to corticosteroids.

Unexpected complete recovery of a patient with severe tick-borne encephalitis treated with favipiravir.

We report a case of tick-borne encephalitis (TBE) in a 22-year-old man, who was admitted to the Medical University of Vienna hospital with severe meningoencephalitis, unresponsive and dependent on a respirator. He had given a history of a recent tick bite, but because he had previously received a full course of vaccination against TBE, West Nile virus infection was suspected. Because the antiviral drug favipiravir has been reported to be active against WNV, therapy was initiated, and continued even after a diagnosis of TBE was confirmed, due to significant improvement of symptoms. Within days, the patient's symptoms resolved, and he was discharged after complete recovery at 15 days after onset. Although this single case does not permit any conclusion as to the role of favipiravir in the favorable outcome, it suggests that the drug should be further evaluated in laboratory animal models and in appropriate clinical settings.

Comparison of tick-borne encephalitis between children and adults-analysis of 669 patients.

The aim of our study was to compare the course of TBE in children and adults. A retrospective analysis of the medical records of 669 patients was performed. The patients were categorized into 2 groups: Group I with 68 children and group II with 601 adults. TBE symptoms in children were milder compared with adults, with meningitis in 97% of cases. In adults, meningoencephalitis and meningoencephalomyelitis made up 49.26% of cases. Nausea and vomiting are more frequent in children, while neurological manifestations are more frequent in adults. There were no differences in CSF pleocytosis at the onset of disease in both groups, while CSF protein concentration was higher in adults. Children treated with corticosteroids over 7 days had higher checkup pleocytosis than pleocytosis at the onset of disease compared with adults. Corticosteroid use prolongs the disease duration but does not influence the development of TBE sequelae. Children had more favourable outcomes than adult patients.

Powassan Virus in a Hunter Returning from a Trip in the Adirondack Park.

Powassan virus is a rare flavivirus that may be transmitted by tick bite and is associated with encephalitis. Infections have been described in the northern United States, Canada, and Russia. We present the case of a 56-y-old man who presented to our hospital with symptoms of confusion, altered behavior, and headache. The patient developed fever and status epilepticus despite supportive care and required endotracheal intubation. Six days before presentation, the patient had returned from a hunting trip in the Adirondack region of New York State.

Suspected Neuro-invasive Powassan Virus Infection in a Pediatric Patient.

Powassan virus lineage II (POWV), also known as deer tick virus, is an emerging tick-borne pathogen transmitted by Ixodes scapularis, the natural vector for the organisms that causes Lyme disease, babesiosis, and anaplasmosis. POWV is the only tick-borne flavivirus in North America known to cause disease in humans. We present a suspected pediatric case of POWV infection in northern Wisconsin.

Case report: Tick-borne encephalitis virus infection in beneficiaries of the U.S. military healthcare system in southern Germany.

Tick-borne encephalitis (TBE) is caused by a flavivirus usually transmitted to humans via the bite of an infected Ixodes ricinus tick. The disease is endemic to central Europe, including Germany where it is a potential threat to U.S. service members and other beneficiaries. This report describes 3 cases of TBE in persons living during 2017 and 2018 in the region of Germany with the highest incidence of TBE: a 36-year-old active duty service member and 2 non-service member beneficiaries aged 17 and 7 years. Each patient presented with debilitating symptoms and, following recovery from their acute illnesses, experienced troubling sequelae for months afterward. The nature of their initial illnesses varied from one another, as did the length and nature of their sequelae. The criteria for diagnosing TBE based upon clinical symptoms and laboratory test results are described. Preventive strategies for protecting residents in Germany from TBE include measures to avoid tick bites. The potential for use of the TBE virus vaccine, not Food and Drug Administration- approved in the U.S. but available in Europe, is discussed.

[The efficacy of anti-viral therapy and serotherapy of tick-borne encephalitis in children]. / Effektivnost' protivovirusnoi terapii i seroterapii kleshchevogo entsefalita u detei.

OBJECTIVE: To evaluate the efficacy of different methods of antivirus therapy of tick-borne encephalitis (TBE) in children in the acute period and during chronic infections. MATERIAL AND METHODS: During 1 year, 130 children, aged 7-17 years, with TBEV received therapy in the acute period (an average in 3.5±1.3 days) in groups 1 (n=84) and 2 (n=20), and in the chronic infection in groups 3 (n=15) and 4 (n=11). Ribavirin orally, recombinant interferon-α2 (IFN-α2) i/m or in suppositories and anaferon orally were prescribed to children of groups 1 and 3. Children of groups 2 and 4 received tick-borne immunoglobulin (IgG)i/m and ribonuclease i/m. At admission, all patients received infusions of cytoflavin in the drip at the rate of 0.6 ml/kg per day. Etiological diagnosis included ELISA (IgM, G, viral antigen), and virus RNA by PCR in the blood and CSF. MRI of the brain and cervical spinal cord using standard programs was performed. All studies were performed prior to and in the course of treatment. RESULTS AND CONCLUSION: In patients of group 1, the period of increase in symptoms was reduced by ~ 4 days, and the duration of impairment of consciousness and pleocytosis in CSF ~ by 5 days, which was accompanied by a faster clearance of the virus in CSF, compared with group 2. In group 1, recovery without neurological deficit was observed in 83.3% (n=70), all patients had no progression of infection. In group 2, 30% of children (n=6) acquired TBEV chronic infection, and in 55% (n=11) there was a neurological deficit without progression. In patients of group 3 with chronic TBEV, the improvement was observed in 86.7% of cases, and complete regression of symptoms occurred in 1 patient, and replication of the virus was arrested in all of them. In group 4, symptoms increased in 72.7%, while virus replication was preserved and atrophic changes in the CNS increased on MRI. Antiviral therapy (ribavirin, IFN-α2 and release of active antibodies to gamma interferon -anaferon children) has the highest efficacy when prescribed for the first 5 days, while IgG and ribonuclease have insufficient efficacy in TBEV.

Tick-borne Encephalitis: Stroke-like Presentation.

Tick-borne encephalitis, caused by the tick-borne virus (TBEV), is endemic in central, eastern, and northern Europe eastwards through Russian Siberia and China. For the year 2009, the highest incidence in Scandinavian countries was in Sweden. The clinical symptoms have a wide spectrum. We report a unique case of clinical symptoms and radiological findings compatible with a stroke-like inflammatory lesion in the thalamus, suggesting microangiopathy from TBEV. Our case shows that TBEV could be a possible cause of stroke-like lesions.

Polio-Like Manifestation of Powassan Virus Infection with Anterior Horn Cell Involvement, Canada.

Evidence of spinal cord involvement in Powassan virus infection is largely limited to mouse models. We report a case of a polio-like illness caused by Powassan virus infection in a 62-year-old man in Canada. Magnetic resonance imaging showed T2 hyperintensities in the anterior horns of the cervical spinal cord.

An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus for Mice.

The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine, and 4'-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

Assessment of the tau protein concentration in patients with tick-borne encephalitis.

There have been suggestions that tick-borne encephalitis (TBE) may cause neurodenenerative changes in the brain. The aim of this study was the assessment of the tau protein concentration in cerebrospinal fluid (CSF) of patients with different clinical forms of TBE. The concentration of tau protein in CSF was determined using Fujirebio tests (Ghent, Belgium) in 35 patients with TBE: group I-patients with meningitis (n = 16); group II-patients with meningoencephalitis (n = 19). None of the patients reported any neurodegenerative disorder that could affect the results of the study. The control group (CG) consisted of 10 patients in whom inflammatory process in central nervous system was excluded. Tau protein concentration in CSF before treatment did not differ significantly between the examined groups, while its concentration was significantly higher in encephalitis group than in CG after 14 days of treatment. Significant increase in tau protein concentration after treatment was observed in both examined groups. The comparison between the group of patients who fully recovered and patients who presented with persistent symptoms on discharge showed significant differences in tau protein concentration before and after treatment. ROC curve analysis indicates that CSF tau protein concentration before treatment may predict complicated course of the disease with 90.9% specificity and 80% sensitivity, while after treatment, specificity became 72.7% and 71.4% for sensitivity. Correlation analysis showed that in TBE patients (both meningoencephalitis and meningitis groups), CSF pleocytosis before treatment correlated negatively with tau protein concentration in CSF. (1) Neurodegeneration process is present in TBE encephalitis. (2) Tau protein concentration may be used as a predictor of complicated course of TBE.

Tick-borne encephalitis in Europe and Russia: Review of pathogenesis, clinical features, therapy, and vaccines.

Tick-borne encephalitis (TBE) is an illness caused by tick-borne encephalitis virus (TBEV) infection which is often limited to a febrile illness, but may lead to very aggressive downstream neurological manifestations. The disease is prevalent in forested areas of Europe and northeastern Asia, and is typically caused by infection involving one of three TBEV subtypes, namely the European (TBEV-Eu), the Siberian (TBEV-Sib), or the Far Eastern (TBEV-FE) subtypes. In addition to the three main TBEV subtypes, two other subtypes; i.e., the Baikalian (TBEV-Bkl) and the Himalayan subtype (TBEV-Him), have been described recently. In Europe, TBEV-Eu infection usually results in only mild TBE associated with a mortality rate of <2%. TBEV-Sib infection also results in a generally mild TBE associated with a non-paralytic febrile form of encephalitis, although there is a tendency towards persistent TBE caused by chronic viral infection. TBE-FE infection is considered to induce the most severe forms of TBE. Importantly though, viral subtype is not the sole determinant of TBE severity; both mild and severe cases of TBE are in fact associated with infection by any of the subtypes. In keeping with this observation, the overall TBE mortality rate in Russia is ∼2%, in spite of the fact that TBEV-Sib and TBEV-FE subtypes appear to be inducers of more severe TBE than TBEV-Eu. On the other hand, TBEV-Sib and TBEV-FE subtype infections in Russia are associated with essentially unique forms of TBE rarely seen elsewhere if at all, such as the hemorrhagic and chronic (progressive) forms of the disease. For post-exposure prophylaxis and TBE treatment in Russia and Kazakhstan, a specific anti-TBEV immunoglobulin is currently used with well-documented efficacy, but the use of specific TBEV immunoglobulins has been discontinued in Europe due to concerns regarding antibody-enhanced disease in naïve individuals. Therefore, new treatments are essential. This review summarizes available data on the pathogenesis and clinical features of TBE, plus different vaccine preparations available in Europe and Russia. In addition, new treatment possibilities, including small molecule drugs and experimental immunotherapies are reviewed. The authors caution that their descriptions of approved or experimental therapies should not be considered to be recommendations for patient care.

Impact of pre-existing treatment with statins on the course and outcome of tick-borne encephalitis.

OBJECTIVES: Although statins have anti-inflammatory and potentially also antimicrobial (including antiviral) activity, their therapeutic impact on infectious diseases is controversial. In this study, we evaluated whether pre-existing statin use influenced the course and outcome of tick-borne encephalitis. METHODS: To assess the influence of statin usage on the severity of acute illness and the outcome of tick-borne encephalitis, univariate and multivariable analyses were performed for 700 adult patients with tick-borne encephalitis of whom 77 (11%) were being treated with statins, and for 410 patients of whom 53 (13%) were receiving statins, respectively. RESULTS: Multivariable analyses found no statistically significant association between statin usage and having a milder acute illness. There was also no statistically significant benefit with respect to a favorable outcome defined by the absence of post-encephalitic syndrome (ORs for a favorable outcome at 6 months was 0.96, 95% CI: 0.46-2.04, P = 0.926; at 12 months 0.29, 95% CI: 0.06-1.33, P = 0.111; at 2-7 years after acute illness 0.44, 95% CI: 0.09-2.22, P = 0.321), by a reduction in the frequency of six nonspecific symptoms (fatigue, myalgia/arthralgia memory disturbances, headache, concentration disturbances, irritability) occurring during the 4 week period before the last examination, or by higher SF-36 scores in any of the eight separate domains of health as well as in the physical and mental global overall component. Furthermore, there were no significant differences between patients receiving statins and those who were not in the cerebrospinal fluid or serum levels for any of the 24 cytokines/chemokines measured. CONCLUSIONS: In this observational study, we could not prove that pre-existing use of statins affected either the severity of the acute illness or the long-term outcome of tick-borne encephalitis.

Effect of a single dose of mannitol on hydration status and electrolyte concentrations in patients with tick-borne encephalitis.

OBJECTIVE: This study was performed to assess the effect of a single dose of 15% mannitol on the hydration status and electrolyte balance in patients with tick-borne encephalitis (TBE). METHODS: Forty-one patients with TBE were treated with 0.25 g/kg of 15% mannitol. The electrolyte concentrations (Na, K, and Cl), creatinine concentration, and hydration status were measured before and after mannitol infusion. RESULTS: After mannitol administration, 7 patients had hyponatremia, 3 had hypokalemia, 1 had hyperkalemia, and 17 had hypochloremia. The total body water volume (TBW) changed by 0.44% ± 0.55%, the external body water volume (EBW) changed by 0.12% ± 0.15%, and the internal body water volume (IBW) changed by 0.19% ± 0.40%. The mean ECW/ICW ratio was 0.7694 ± 0.07 before treatment and 0.7699 ± 0.07 after treatment. Age was correlated with the TBW change in men (R = 0.42, p < 0.05) and with the potassium change in women (R = 0.66, p < 0.05). CONCLUSIONS: Patients with TBE should receive mannitol two to four times daily depending on the clinical manifestation. Administration of a single dose of mannitol (0.25 g/kg) requires at least 300 mL of fluid supplementation. Bioimpedance might be useful for individual evaluation of dehydration. Additionally, patients require monitoring for potential hyponatremia. Older men may be more prone to dehydration after receiving mannitol.

Probing chemical space of tick-borne encephalitis virus reproduction inhibitors with organoselenium compounds.

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is the leading cause of arboviral neuroinfections in Europe. Only a few classes of the nucleoside and non-nucleoside inhibitors were investigated against TBEV reproduction. Paving the way to previously unexplored areas of anti-TBEV chemical space, we assessed the inhibition of TBEV reproduction in the plaque reduction assay by various compounds derived from cyanothioacetamide and cyanoselenoacetamide. Compounds from seven classes, including 4-(alkylthio)-2-aryl-3-azaspiro[5.5]undec-4-ene-1,1,5-tricarbonitriles, 3-arylamino-2-(selenazol-2-yl)acrylonitriles, ethyl 6-(alkylseleno)-5-cyano-2-oxo-1,2-dihydropyridine-3-carboxylates, 6-(alkylseleno)-2-oxo-1,4,5,6-tetrahydropyridine-3-carbonitriles, 2-(alkylseleno)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carbonitriles, 8-selenoxo-3,5,7,11-tetraazatricyclo[7.3.1.02,7 ]tridec-2-ene-1,9-dicarbonitriles, and selenolo[2,3-b]quinolines, inhibited TBEV reproduction with EC50 values in the micromolar range while showing moderate cytotoxicity and no inhibition of enterovirus reproduction. Thus, new scaffolds with promising anti-TBEV activity were found.

Fatal Powassan Encephalitis (Deer Tick Virus, Lineage II) in a Patient With Fever and Orchitis Receiving Rituximab.

Importance: Powassan virus is a rare but increasingly recognized cause of severe neurological disease. Objective: To highlight the diagnostic challenges and neuropathological findings in a fatal case of Powassan encephalitis caused by deer tick virus (lineage II) in a patient with follicular lymphoma receiving rituximab, with nonspecific anti-GAD65 antibodies, who was initially seen with fever and orchiepididymitis. Design, Setting, and Participants: Comparison of clinical, radiological, histological, and laboratory findings, including immunohistochemistry, real-time polymerase chain reaction, antibody detection, and unbiased sequencing assays, in a single case report (first seen in December 2016) at an academic medical center. Exposure: Infection with Powassan virus. Main Outcomes and Measures: Results of individual assays compared retrospectively. Results: In a 63-year-old man with fatal Powassan encephalitis, serum and cerebrospinal fluid IgM antibodies were not detected via standard methods, likely because of rituximab exposure. Neuropathological findings were extensive, including diffuse leptomeningeal and parenchymal lymphohistiocytic infiltration, microglial proliferation, marked neuronal loss, and white matter microinfarctions most severely involving the cerebellum, thalamus, and basal ganglia. Diagnosis was made after death by 3 independent methods, including demonstration of Powassan virus antigen in brain biopsy and autopsy tissue, detection of viral RNA in serum and cerebrospinal fluid by targeted real-time polymerase chain reaction, and detection of viral RNA in cerebrospinal fluid by unbiased sequencing. Extensive testing for other etiologies yielded negative results, including mumps virus owing to prodromal orchiepididymitis. Low-titer anti-GAD65 antibodies identified in serum, suggestive of limbic encephalitis, were not detected in cerebrospinal fluid. Conclusions and Relevance: Owing to the rarity of Powassan encephalitis, a high degree of suspicion is required to make the diagnosis, particularly in an immunocompromised patient, in whom antibody-based assays may be falsely negative. Unbiased sequencing assays have the potential to detect uncommon infectious agents and may prove useful in similar scenarios.