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.

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.

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.

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.

Utilisation of ISA Reverse Genetics and Large-Scale Random Codon Re-Encoding to Produce Attenuated Strains of Tick-Borne Encephalitis Virus within Days.

Large-scale codon re-encoding is a new method of attenuating RNA viruses. However, the use of infectious clones to generate attenuated viruses has inherent technical problems. We previously developed a bacterium-free reverse genetics protocol, designated ISA, and now combined it with large-scale random codon-re-encoding method to produce attenuated tick-borne encephalitis virus (TBEV), a pathogenic flavivirus which causes febrile illness and encephalitis in humans. We produced wild-type (WT) and two re-encoded TBEVs, containing 273 or 273+284 synonymous mutations in the NS5 and NS5+NS3 coding regions respectively. Both re-encoded viruses were attenuated when compared with WT virus using a laboratory mouse model and the relative level of attenuation increased with the degree of re-encoding. Moreover, all infected animals produced neutralizing antibodies. This novel, rapid and efficient approach to engineering attenuated viruses could potentially expedite the development of safe and effective new-generation live attenuated vaccines.

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.

[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.

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.

[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.

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.

Protective role of TNF-α, IL-10 and IL-2 in mice infected with the Oshima strain of Tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) causes acute central nervous system disease. Here, we investigated the roles of the TNF-α, IL-10 and other cytokines in appropriate KO mice following infection with Oshima and Sofjin strains of TBEV. Following infection with the Oshima strain, mortality rates were significantly increased in TNF-α KO and IL-10 KO mice compared with wild type (WT) mice. These results suggested that TNF-α and IL-10 play protective roles against fatal infection due to Oshima strain infection. However, viral loads and proinflammatory cytokine levels in the brain of TNF-α KO andIL-10 KO mice were not significantly different compared with those of WT mice. On the other hand, all WT, TNF-α KO and IL-10 KO mice died following infection with Sofjin strain. Interestingly, Sofjin-infected mice did not exhibit an up-regulated mRNA level of IL-2 in the spleen in all groups of mice, whereas Oshima-infected mice showed significantly increased level of IL-2 compared with mock-infected mice. From these results, we suggest that TNF-α, IL-10 and IL-2 are key factors for disease remission from fatal encephalitis due to infection with Oshima strain of TBEV.

Kyasanur Forest disease virus infection in mice is associated with higher morbidity and mortality than infection with the closely related Alkhurma hemorrhagic fever virus.

BACKGROUND: Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic fever virus (AHFV) are closely related members of the Flavivirus genus and are important causes of human disease in India and the Arabian Peninsula, respectively. Despite high genetic similarity, the viruses have distinctly different host ranges and ecologies. Human cases of KFDV or AHFV develop a spectrum of disease syndromes ranging from liver pathology to neurologic disease. Case reports suggest KFDV is more commonly associated with hepatic and gastrointestinal manifestations whereas AHFV is more commonly associated with neurologic disease. METHODOLOGY/PRINCIPAL FINDINGS: Inoculation of three immunocompetent laboratory mouse strains revealed that KFDV was consistently more lethal than AHFV. In subsequent studies utilizing C57BL/6J mice, we demonstrated that KFDV infection was associated with higher viral loads and significantly higher mortality. KFDV-infected mice rapidly developed more severe disease than AHFV-infected mice, as evidenced by significant abnormalities on clinical chemistry panels and more severe pathology in the brain and gastrointestinal tract. CONCLUSIONS/SIGNIFICANCE: Infections of C57BL/6J mice with KFDV or AHFV resulted in clinical disease syndromes that closely approximate the diseases seen in human cases. Despite high genetic similarity, there were clear differences in survival, viral kinetics, clinical chemistry data and histology. These results suggest that distinct mouse models for AHFV and KFDV are necessary in order to gain a better understanding of the unique pathogenesis of each virus, as well as to provide platforms for testing promising vaccines and therapeutics.

Elevation of matrix metalloproteinase-9 level in cerebrospinal fluid of tick-borne encephalitis patients is associated with IgG extravassation and disease severity.

BACKGROUND: Tick-borne encephalitis (TBE), caused by tick-borne encephalitis virus (TBEV), is an infectious disease involving the central nervous system (CNS). The pathogenesis of CNS injury has not been clearly demonstrated. Matrix metalloproteinase-9 (MMP-9) and some cytokines, such as interleukin 6 (IL-6), may play important roles in the disruption of the blood-brain barrier (BBB) and the pathogenesis of TBE. METHODS: 72 cerebrospinal fluid (CSF) samples were collected from TBE patients in north eastern China. IgG levels in CSF and serum were compared and MMP-9 and IL-6 levels were evaluated by ELISA. The correlation between the elevated MMP-9 levels and IgG extravasation, disease severity, and neuroinflammation was analyzed. RESULTS: Increased concentration of MMP-9 was detected in some of the CSF samples, and the elevation was found to be closely related to CSF TBEV IgG extravasation and enhancement of IL-6 expression. Moreover, elevated levels of MMP-9 were found to be correlated with IL-6 enhancement. Four of the 72 patients, the ones who died, presented with high CSF MMP-9 levels. CONCLUSIONS: In TBE patients, elevated CSF MMP-9 levels were associated with brain inflammatory reaction, disruption of the blood-brain barrier, and disease severity.

Epidemiology of tick-borne encephalitis in the Czech Republic 1970-2008.

This article presents major epidemiologic features of tick-borne encephalitis (TBE) in the Czech Republic, using data of laboratory-confirmed cases since 1970. A total of 17,053 cases of TBE were reported in the Czech Republic (population 10 million) in 1970-2008. The data show several important features. First, the pattern of TBE incidence changed over time. Until the end of the 1970s, TBE was characterized by periods of alternately higher and lower incidence (between 180 and 595 cases per year); the 1980s were a period of low incidence with minimum variability; since the beginning of the 1990 s, there has been a steep rise in incidence, with marked year-to-year variation (e.g., 745 cases were registered in 1995, and a maximum of 1029 cases were registered in 2006). Second, the age distribution of TBE incidence has changed. Until the end of 1990 s, incidence peaked among those 15-19 years of age, with a gradual decline with age. In the 2000s, however, TBE incidence has been rising in those aged 60-64 years, with a sharp decline in those older than 65 years. Third, the seasonal pattern of TBE has changed markedly over time. In the earlier period, incidence had a clear peak in July/August; since the 1990 s, more cases have occurred in earlier and later months of the year. The proportion of cases occurring in April, May, October, and November increased from 9% in the 1970s to 23% in 2000-2008. Fourth, the geographical distribution of TBE also changed over time, with TBE increasingly occurring in the mountainous districts at higher altitudes. These changes in incidence patterns appear to be linked with changes in climatic and meteorological conditions. The link between climate change and TBE incidence is plausible, since TBE is a recreation-related infection associated with outdoor activities, and since climatic changes affect the life cycle of the vector.

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.

T-cell clones expressing different T-cell receptors accumulate in the brains of dying and surviving mice after peripheral infection with far eastern strain of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV), a representative acute central nervous system disease-inducible virus, is known to elicit dose-independent mortality in a mouse model. We previously reported that subcutaneous infection with a wide range of TBEV Oshima strain challenge doses (10(2)-10(6) PFU) produced an approximately 50% mortality rate. However, the factors playing critical roles in mortality and severity remain unclear. In this study, we distinguished surviving and dying mice by their degree of weight loss after TBEV infection, and investigated qualitative differences in brain-infiltrating T cells between each group by analyzing T-cell receptor (TCR) repertoire and complementary determining region 3 (CDR3) sequences. TCR repertoire analysis revealed that the expression levels of VA8-1, VA15-1, and VB8-2 families were increased in brains derived from both surviving and dying mice. CDR3 amino acid sequence characteristics differed between each group. In dying mice, high frequencies of VA15-1/AJ12 and VB8-2/BJ1.1 gene usage were observed. While in surviving mice, high frequencies of VA8-1/AJ15 or VA8-1/AJ23 gene usage were observed. VB8-2/BJ2.7 gene usage and short CDR3 were observed frequently in both surviving and dying mice. However, no differences in T-cell activation markers and apoptosis-related genes were observed between these groups using quantitative real-time PCR analysis. These results suggest that TBEV-infection severity may be involved in antigen specificity, but not in the number or activation level of brain-infiltrating T cells.

Breakdown of the blood-brain barrier during tick-borne encephalitis in mice is not dependent on CD8+ T-cells.

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. The disease is characterized by fever and debilitating encephalitis that can progress to chronic illness or fatal infection. In this study, changes in permeability of the blood-brain barrier (BBB) in two susceptible animal models (BALB/c, and C57Bl/6 mice) infected with TBE virus were investigated at various days after infection by measuring fluorescence in brain homogenates after intraperitoneal injection of sodium fluorescein, a compound that is normally excluded from the central nervous system. We demonstrate here that TBE virus infection, in addition to causing fatal encephalitis in mice, induces considerable breakdown of the BBB. The permeability of the BBB increased at later stages of TBE infection when high virus load was present in the brain (i.e., BBB breakdown was not necessary for TBE virus entry into the brain), and at the onset of the first severe clinical symptoms of the disease, which included neurological signs associated with sharp declines in body weight and temperature. The increased BBB permeability was in association with dramatic upregulation of proinflammatory cytokine/chemokine mRNA expression in the brain. Breakdown of the BBB was also observed in mice deficient in CD8(+) T-cells, indicating that these cells are not necessary for the increase in BBB permeability that occurs during TBE. These novel findings are highly relevant to the development of future therapies designed to control this important human infectious disease.

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.