Age-dependent Powassan virus lethality is linked to glial cell activation and divergent neuroinflammatory cytokine responses in a murine model.

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10-20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5-15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2-4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFß, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE: Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.

Tick-borne encephalitis in Europe, 2012 to 2016.

Since 2012, tick-borne encephalitis (TBE) is a notifiable in the European Union. The European Centre for Disease Prevention and Control annually collects data from 28 countries plus Iceland and Norway, based on the EU case definition. Between 2012 and 2016, 23 countries reported 12,500 TBE cases (Ireland and Spain reported none), of which 11,623 (93.0%) were confirmed cases and 878 (7.0%) probable cases. Two countries (Czech Republic and Lithuania) accounted for 38.6% of all reported cases, although their combined population represented only 2.7% of the population under surveillance. The annual notification rate fluctuated between 0.41 cases per 100,000 population in 2015 and 0.65 in 2013 with no significant trend over the period. Lithuania, Latvia and Estonia had the highest notification rates with 15.6, 9.5 and 8.7 cases per 100,000 population, respectively. At the subnational level, six regions had mean annual notification rates above 15 cases per 100,000 population, of which five were in the Baltic countries. Approximately 95% of cases were hospitalised and the overall case fatality ratio was 0.5%. Of the 11,663 cases reported with information on importation status, 156 (1.3%) were reported as imported. Less than 2% of cases had received two or more doses of TBE vaccine.

Tick Saliva Enhances Powassan Virus Transmission to the Host, Influencing Its Dissemination and the Course of Disease.

UNLABELLED: Powassan virus (POWV) is an encephalitic tick-borne flavivirus which can result in serious neuroinvasive disease with up to a 10% case fatality rate. The study objective was to determine whether the salivary gland extract (SGE) from Ixodes scapularis ticks facilitates the transmission and dissemination of POWV in a process known as saliva-activated transmission. Groups of BALB/c mice were footpad inoculated with either a high dose of POWV with and without SGE or a low dose of POWV with and without SGE. Mice from each group were sacrificed daily. Organ viral loads and gene expression profiles were evaluated by quantitative real-time PCR. Both groups of mice infected with high-dose POWV showed severe neurological signs of disease preceding death. The presence of SGE did not affect POWV transmission or disease outcome for mice infected with the high dose of POWV. Neuroinvasion, paralysis, and death occurred for all mice infected with the low dose of POWV plus SGE; however, for mice infected with the low dose of POWV in the absence of SGE, there were no clinical signs of infection and no mice succumbed to disease. Although this group displayed low-level viremias, all mice were completely healthy, and it was the only group in which POWV was cleared from the lymph nodes. We conclude that saliva-activated transmission occurs in mice infected with a low dose of POWV. Our study is the first to demonstrate virus dose-dependent saliva-activated transmission, warranting further investigation of the specific salivary factors responsible for enhancing POWV transmission. IMPORTANCE: Powassan virus (POWV) is a tick-borne flavivirus that continues to emerge in the United States, as is evident by the surge in number and expanding geographic range of confirmed cases in the past decade. This neuroinvasive virus is transmitted to humans by infected tick bites. Successful tick feeding is facilitated by a collection of pharmacologically active factors in tick saliva. In a process known as saliva-activated transmission, tick bioactive salivary molecules are thought to modulate the host environment, making it more favorable for the transmission and establishment of a pathogen. This phenomenon has been demonstrated for several tick-borne pathogens; however, a systematic investigation of the role of tick saliva on dissemination and pathogenesis of a tick-borne viral disease has never been attempted before. This study will fill that gap by systematically examining whether the presence of tick saliva contributes to the transmission and dissemination of POWV in mice.

[POPULATION MORBIDITY BY INFECTIONS TRANSMITIED VIA IXODES PER- SULCATUS IN THE NORTH AND SOUTH OF IRKUTSK REGION].

AIM: Evaluate the degree of epidemic risk of emergence of tick-borne encephalitis (TBE) and ixodes ticks' borreliosis (ITB) in municipalities (MPs) of Irkutsk region with various natural- climate conditions. MATERIALS AND METHODS: Morbidity was compared for TBE and ITB during 2001 - 2015 in MPs of Irkutsk region located, to the north or south of the 55th parallel, i.e. in the con- ditions of different severity of sharply continental climate. 5-year average data were analyzed. RESULTS: ITB morbidity was 2 - 3 times higher than TBE for all the 5-year periods in the north of the region, whereas in the south -differences were not present. Moreover, in MPs located to the north of the 55th parallel in 2001 - 2015 a decrease of TBE and ITB morbidity did not occur, as in the south of the region. CONCLUSION: The lack of population morbidity reduction by TBE and ITB in the north and prevalence of the latter nosoform requires enhancement of a complex of prophylaxis measures in these MPs with an accent in development of non-specific means, includ- ing acaricidic treatment, enhancement of operations of centers of express diagnostics of transmissible infections, familiarizing of the population with the use of protective suits during periods of high activity of ticks.

Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses.

UNLABELLED: Vector-borne flaviviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus, and dengue virus, cause millions of infections in humans. TBEV causes a broad range of pathological symptoms, ranging from meningitis to severe encephalitis or even hemorrhagic fever, with high mortality. Despite the availability of an effective vaccine, the incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuroinflammation in LGTV-infected mice, characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. IMPORTANCE: The type I interferon (IFN) system is important to control viral infections; however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system are poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system, is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection. The first phase is in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). In the second phase, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.

[Investigation of Therapeutic Efficacy of Triazavirin Against Experimental Forest-Spring Encephalitis on Albino Mice].

The comparative study of the therapeutic efficacy of Triazavirin against experimental Forest-Spring encephalitis on albino mice vs. the active drug Ribavirin® showed that in high doses (200-400 mg/kg) Triazavirin moderately protected the infected animals. A significant increase of the animal lifespan in the test groups (from 4.1 to 4.8 days) and a statistically (p ≤ 0.05) valid decrease of the virus accumulation in the target organ (the brain) were observed.

Tick-borne encephalitis: Clinical findings and prognosis in adults.

In middle and eastern European countries, tick-borne encephalitis (TBE) is one of the most important human infections of the central nervous system. TBE virus (TBEV) is mainly transmitted by tick bites and in very rare cases by unpasteurized milk. The incubation period is on an average 7-10 days. A biphasic course of illness with a prodromal period occurs in about two third of patients. In European countries, TBE presents as meningitis in about 50 % of patients, as meningoencephalitis in 40 %, and as meningoencephalomyelitis in 10 %. The severity of TBE increases with age, in children and adolescents, meningitis is the predominant form of the disease. Long-term prognosis is unfavorable in about 40-50 % of patients who suffer from sequelae for months to years, mainly in terms of pareses, ataxia, and other gait disturbances. No specific treatment for TBE is known so far, but TBE can be successfully prevented by active immunization.

Phylogenetic and virulence analysis of tick-borne encephalitis virus field isolates from Switzerland.

Tick-borne encephalitis (TBE) is an endemic disease in Switzerland, with about 110-120 reported human cases each year. Endemic areas are found throughout the country. However, the viruses circulating in Switzerland have not been characterized so far. In this study, the complete envelope (E) protein sequences and phylogenetic classification of 72 TBE viruses found in Ixodes ricinus ticks sampled at 39 foci throughout Switzerland were analyzed. All isolates belonged to the European subtype and were highly related (mean pairwise sequence identity of 97.8% at the nucleotide and 99.6% at the amino acid level of the E protein). Sixty-four isolates were characterized in vitro with respect to their plaque phenotype. More than half (57.8%) of isolates produced a mixture of plaques of different sizes, reflecting a heterogeneous population of virus variants. Isolates consistently forming plaques of small size were associated with recently detected endemic foci with no or only sporadic reports of clinical cases. All of six virus isolates investigated in an in vivo mouse model were highly neurovirulent (100% mortality) but exhibited a relatively low level of neuroinvasiveness, with mouse survival rates ranging from 50% to 100%. Therefore, TBE viruses circulating in Switzerland belong to the European subtype and are closely related. In vitro and in vivo surrogates suggest a high proportion of isolates with a relatively low level of virulence, which is in agreement with a hypothesized high proportion of subclinical or mild TBE infections.

Epidemiology of a tick-borne viral infection: theoretical insights and practical implications for public health.

The morbidity of tick-borne encephalitis (TBE) varies yearly by as much as 10-fold among the people of Western Siberia. This long-term variation is dependent on many factors such as the density of the tick populations, the prevalence of TBE virus (TBEV) among sub-adult ticks, the yearly virulence of the TBEV, and prophylactic measures. Here we highlight the role of small mammal hosts in the circulation of TBEV through the ecosystem. Refining classical models of non-viremic horizontal transmission, we emphasize the recently understood fact that the physiological and immunological status of the small mammal hosts affects the tick and virus-host interactions. In addition to its theoretical interest, our approach may lead to some practical improvements in the precision of epidemiological forecasts and perhaps in forestalling the severity of outbreaks of TBE, or, at least, in forewarning medical authorities and the general public of impending TBE outbreaks.

Procalcitonin in hantavirus infections.

In hantavirus infections levels of serum leukocytes or C-reactive protein are usually elevated to levels found in serious bacterial infections. However, procalcitonin in patients infected with hantavirus has not yet been discussed in the literature. A total of 29 adult patients with hantavirus infection, 30 with sepsis, and 19 with tick-borne encephalitis were included in this observational retrospective study. The median procalcitonin level in patients with hantavirus infection was 0.53 µg/L (range 0.09-11.71 µg/L), in the group with sepsis 4.33 µg/L (range 0.08-161.1 µg/L) and in patients with viral meningitis 0.08 µg/L (range 0.05-0.12 µg/L). The difference between all three groups was statistically significant (p < 0.001). A higher procalcitonin level was found in patients with hemorrhagic fever with renal syndrome caused by Dobrava virus (0.74 µg/L; range 0.09-2.83 µg/L) than in those with Puumala virus infections (0.50 µg/L; range 0.10-11.71 µg/L). However, the difference was not statistically significant (p = 0.895). This study confirmed previous findings demonstrating the association of elevated procalcitonin with bacterial infection. However, increased procalcitonin serum level was also found in hantavirus infections with overlapping results between viral and severe bacterial infections.

[Long-term prognosis of patients with primary myelitic manifestation of tick-borne encephalitis: a trend analysis covering 10 years]. / Langzeitprognose bei primär myelitischer Manifestation der FSME : Eine Verlaufsanalyse über 10 Jahre.

BACKGROUND: While some studies have been published about the prognosis of the meningitic and encephalitic course of tick-borne encephalitis (TBE), only few data exist about the long-term prognosis of TBE myelitis. The aim of the present prospective study therefore was to investigate such patients over a period of 10 years. METHOD: In Baden-Württemberg between 1994 and 1999, 731 patients fell ill with TBE. Of them 81 (11%) suffered from encephalomyelitis. All patients were asked to participate in this study, 57 of whom agreed. Individual impairments were measured by allocating single scores for the paresis of the extremities or cranial nerves, ataxia, impaired consciousness, respiratory paralysis and defective hearing. The total impairment was measured at follow-up investigations at 1, 3, 5 and 10 years. RESULTS: A total of 11 patients (19%) recovered, 29 (51%) suffered from persisting pareses or other impairments and 17 (30%) died 1-10 years after the acute disease. The most important ameliorations occurred during the first year after the acute disease; thereafter, improvements were lesser and more seldom. The clinical findings after 5 and 10 years correlated well with the status of the acute disease (r=0.8, p<0.01) allowing one to hazard a prognosis at the first presentation. The best restitution was seen for ataxia, impairment of consciousness, double vision, urinary retention and mild paresis of only one extremity (4/5). Patients with tetraparesis and simultaneous occurrence of respiratory paralysis and/or dysphagia, dysarthria or paresis of the neck muscles had the worst prognosis. CONCLUSION: A myelitic course of TBE is associated with the chance to recover in only about 20% of patients. Clinical deficits do not always correlate to findings in magnetic resonance tomography but to observations in postmortem studies.

[Comparative study of antiviral activity of luteolin and 7,3'-disulfate luteolin].

Antiviral activity of 7,3'-disulfate luteolin, extracted from Zostera marina was studied on an experimental model of tick-borne encephalitis (TBE) in vivo and in vitro. The drug increased the survival of the experimental mice infected with TBE virus and prolonged their lifespan. It was shown that 7,3'-disulfate luteolin reduced the virus accumulation in the SPEV cells by 2.0-4.0 lg TCID50/ml.

Alkhurma haemorrhagic fever virus causes lethal disease in IFNAR-/- mice.

ABSTRACTAlkhurma haemorrhagic fever virus (AHFV), a tick-borne flavivirus closely related to Kyasanur Forest disease virus, is the causative agent of a severe, sometimes fatal haemorrhagic/encephalitic disease in humans. To date, there are no specific treatments or vaccines available to combat AHFV infections. A challenge for the development of countermeasures is the absence of a reliable AHFV animal disease model for efficacy testing. Here, we used mice lacking the type I interferon (IFN) receptor (IFNAR-/-). AHFV strains Zaki-2 and 2003 both caused uniform lethality in these mice after intraperitoneal injection, but strain 2003 seemed more virulent with a median lethal dose of 0.4 median tissue culture infectious doses (TCID50). Disease manifestation in this animal model was similar to case reports of severe human AHFV infections with early generalized signs leading to haemorrhagic and neurologic complications. AHFV infection resulted in early high viremia followed by high viral loads (<108 TCID50/g tissue) in all analyzed organs. Despite systemic viral replication, virus-induced pathology was mainly found in the spleen, lymph nodes, liver and heart. This uniformly lethal AHFV disease model will be instrumental for pathogenesis studies and countermeasure development against this neglected zoonotic pathogen.

Early mortality following intracerebral infection with the Oshima strain of tick-borne encephalitis virus in a mouse model.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent that causes acute central nervous system (CNS) disease in humans. In this study, we examined the pathogenic process following intracerebral infection with the Oshima strain of TBEV in a mouse model. Intracerebral infection resulted in dose-dependent mortality, and all mice died following challenge with 10(2) PFU or more of the virus within 10 days. Acutely necrotic neurons and widespread inflammation were observed throughout the CNS. We therefore conclude that mortality following intracerebral infection results from a direct CNS pathology.

Development of a small animal model for deer tick virus pathogenesis mimicking human clinical outcome.

Powassan virus (POWV) is a tick-borne flavivirus that encompasses two genetic lineages, POWV (Lineage I) and deer tick virus (DTV, Lineage II). In recent years, the incidence of reported POWV disease cases has increased, coupled with an expanded geographic range of the DTV tick vector, Ixodes scapularis. POWV and DTV are serologically indistinguishable, and it is not known whether clinical manifestations, pathology, or disease outcome differ between the two viruses. Six-week-old male and female BALB/c mice were footpad-inoculated with DTV doses ranging from 101 to 105 FFU. Dose-independent mortality, morbidity, and organ viral loads were observed for mice inoculated with sequentially increasing doses of DTV. By study completion, all surviving mice had cleared their viremias but detectable levels of negative-sense DTV RNA were present in the brain, indicating viral persistence of infectious DTV in the central nervous system. For mice that succumbed to disease, neuropathology revealed meningoencephalitis characterized by microscopic lesions with widespread distribution of viral RNA in the brain. These findings, coupled with the rapid onset of neurological signs of disease and high viral titers in nervous tissue, highlight the neurotropism of DTV in this mouse model. Additionally, disease outcome for DTV-infected mice was not affected by sex, as males and females were equally susceptible to disease. This is the first study to comprehensively characterize the clinical disease outcome in a small animal model across a spectrum of POWV/DTV infection doses. Here, we developed a small animal model for DTV pathogenesis that mimics the manifestations of POWV disease in humans. Since it is currently not known whether DTV and POWV differ in their capacity to cause human disease, the animal model detailed in our study could be utilized in future comparative pathogenesis studies, or as a platform for testing the efficacy of vaccines, and anti-virals.

A case series of fatal meningoencephalitis in Mongolia: epidemiological and molecular characteristics of tick-borne encephalitis virus.

In Mongolia, the incidence and fatality rates of tick-borne encephalitis (TBE) have been increasing. We aimed to identify the epidemiological and molecular characteristics of tick-borne encephalitis virus (TBEV) associated with fatal meningoencephalitis in Mongolia. We conducted a descriptive study of 14 fatal cases of TBE that occurred between 2008 and 2017 in Mongolia. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect viral RNA in brain tissue. RT-PCR products from six patients who died from TBE between 2013 and 2017 were directly sequenced and analysed phylogenetically. Ticks collected from Selenge and Bulgan provinces were also tested for TBEV by RT-PCR. Between 2008 and 2017, there were 14 fatal TBE cases in hospitals in Mongolia. The 14 patients who died reported receiving tick bites in Bulgan or Selenge province; 71.4% of deaths resulted from tick bites in Bulgan province. The TBE case fatality rate was 28.6% for patients in Bulgan province and 2.7% for those in Selenge province. All of the fatalities were men; the median age was 45 ± 12.6 years. Tick bites occurred between April and June in forested areas. In 2013, a 388 base pair fragment of the envelope (E) gene was obtained from a hospitalized patient. The closest relatives of this virus are Far-Eastern TBEV isolates. The case fatality rate differed between two provinces where tick bites occurred. A higher number of TBE cases and the virulent Far-Eastern subtype occurred in patients in Bulgan province. This province should increase vaccination coverage, training, education and investigations.

Fatal Meningoencephalomyelitis due to the Tick-borne Encephalitis Virus: The First Detailed Neurological Observation in a Japanese Patient from the Central Part of Hokkaido Island.

To date, the only instance of tick-borne encephalitis (TBE) in Japan was reported from the southern part of Hokkaido Island in 1993; no other cases have been reported since then. We herein report the first case of TBE reported in the central part of Hokkaido Island, and describe the fatal clinical course of a patient who presented with meningoencephalomyelitis, which partly involved the nerve root. Magnetic resonance imaging (MRI) of the patient's cranium and spine revealed characteristic central nervous system involvement. Our case report is extremely relevant to efforts to protect public health and for precautions against TBE pandemics.

Tick-borne encephalitis in Slovenia from 2000 to 2004: comparison of the course in adult and elderly patients.

OBJECTIVE: To present epidemiological data and clinical characteristics of tick-borne encephalitis (TBE) in adult patients, and to compare findings in the subgroup over the age of 60 years with those aged 60 or under. METHODS: The information for this retrospective study was obtained by review of medical documentation. All patients over 15 years of age hospitalized at the Department of Infectious Diseases, University Medical Center Ljubljana, Slovenia, between 2000 and 2004 with pleocytosis (cerebrospinal fluid leukocyte count >5 x 10(6) cells/l) and the presence of serum IgM antibodies against TBE virus qualified for inclusion in this report. Patients were divided into two groups according to their age (patients over the age of 60 were classified as seniors and those aged 60 or under as adults); the findings in the two groups were compared. RESULTS: Of 448 patients with TBE, 318 were in the adult group and 130 in the senior group. Males predominated in both groups. A biphasic course of the illness was reported by 56% of patients. There were no significant differences in the majority of clinical parameters in the initial phase of TBE but several distinctions between adults and seniors were found in the second phase of the illness. Adults more often presented with fever, headache, stiff neck and photophobia, whereas seniors more frequently reported fatigue, altered consciousness and decreased muscle strength, these differences indicating a more classic course of TBE in the younger group and a somehow different and more severe acute disease in the older group. More severe acute disease and less favorable outcome in seniors was further corroborated by the occurrence of urine retention (18/318, 5.7% versus 27/130, 20.8%; P < 0.001), frequency of pareses (10/318, 3.1% versus 7/130, 5.4%; P = 0.002) and the need for antiedematous treatment (103/318, 32.4% versus 61/130, 46.9%; P = 0.005), as well as by the duration of treatment, duration of hospital stay and the death rate (0/318 versus 3/130, 2.3%; P = 0.024). Several distinctions were present also in laboratory findings, including higher cerebrospinal fluid leukocyte count in the adults than in the seniors (107 x 10(6) cells/l versus 47 x 10(6) cells/l; P < 0.001). CONCLUSION: Direct comparison of the course and outcome of TBE revealed several distinctions between patients over 60 years of age and those aged 60 or under and corroborates previous assumptions that TBE is a more serious illness in the elderly population.

Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

Predictors, Neuroimaging Characteristics and Long-Term Outcome of Severe European Tick-Borne Encephalitis: A Prospective Cohort Study.

BACKGROUND AND OBJECTIVES: Tick-borne encephalitis (TBE) still represents a considerable medical and health economic problem in Europe and entails a potential threat to travellers. The aim of this study was to characterise the conditions of severe TBE by precisely recording its clinical variants, the related neuroimaging features, and the variant-specific long-term outcome and by identifying predictors for severe courses. METHODS: A cohort of 111 TBE patients (median age 51, range 17-75 years; 42% females) was analysed prospectively. Data were acquired from the department of neurology, University Hospital Heidelberg, and the infectious diseases registry of the Robert-Koch institute Berlin. Neurological status was ascertained by protocol at admission and discharge and the degree of disability was scored using the modified RANKIN Scale (mRS; clinical score addressing neurological disability, range from 0, healthy to 6, dead) at admission and at follow-up. Follow-up examination was conducted by means of a telephone interview. To identify independent predictors for severe TBE and functional outcome, modelled logistic regression was performed. MRI changes were correlated with infection variants. To assess alpha-motor neuron injury patterns, we used high-resolution magnetic resonance neurography (hrMRN). Analyses were performed at the Department of Neurology, University Hospital, University of Heidelberg from April 2004 through September 2014. RESULTS: Acute course: 3.6% of patients died during the acute infection. All patients with a lethal course suffered from meningoencephaloradiculitis (MER, 14.4% of the cohort), which is associated with a significantly higher risk of requiring intensive care (p = 0.004) and mechanical ventilation (p<0.001) than menigoencephalitis (ME, 27.9% of the cohort). At admission, both MER and ME groups were severely affected, with the MER group having a statistically higher mRS score (median of 5 in the MER groups versus 4 in the ME group; p<0.001). Long-term outcome: outcome for MER was considerably worse (median mRS = 4) than for ME (mRS = 1, p<0.0001) and meningitis (mRS = 0, 57.7% of the cohort). RISK FACTORS: advanced age (p<0.001) and male gender (p = 0.043) are independent risk factors for a severe infection course. Furthermore, we identified pre-existing diabetes mellitus (p = 0.024) as an independent risk factor for MER. In MER, alpha-motor neuron injury accounts for the poor prognosis confirmed by hrMRN. CONCLUSION AND RELEVANCE: These data provide critical information for neurologists and other health professionals to use in evaluating TBEV patients who live in or travel to endemic areas. This information can be used to classify clinical presentation and estimate infection-associated complications and individual prognosis. Furthermore, the risk for severe, disabling infections in older patients should prompt general practitioners to recommend and encourage vaccination to those patients living in or travelling to endemic areas.