Transcriptomic Studies Suggest a Coincident Role for Apoptosis and Pyroptosis but Not for Autophagic Neuronal Death in TBEV-Infected Human Neuronal/Glial Cells

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, Flavivirus genus, is responsible for neurological symptoms that may cause permanent disability or death. With an incidence on the rise, it is the major arbovirus affecting humans in Central/Northern Europe and North-Eastern Asia. Neuronal death is a critical feature of TBEV infection, yet little is known about the type of death and the molecular mechanisms involved. In this study, we used a recently established pathological model of TBEV infection based on human neuronal/glial cells differentiated from fetal neural progenitors and transcriptomic approaches to tackle this question. We confirmed the occurrence of apoptotic death in these cultures and further showed that genes involved in pyroptotic death were up-regulated, suggesting that this type of death also occurs in TBEV-infected human brain cells. On the contrary, no up-regulation of major autophagic genes was found. Furthermore, we demonstrated an up-regulation of a cluster of genes belonging to the extrinsic apoptotic pathway and revealed the cellular types expressing them. Our results suggest that neuronal death occurs by multiple mechanisms in TBEV-infected human neuronal/glial cells, thus providing a first insight into the molecular pathways that may be involved in neuronal death when the human brain is infected by TBEV.

The role of IFITM proteins in tick-borne encephalitis virus infection

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in endemic regions of northern Asia and central and northern Europe. Interferon induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as the West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show the most significant role being that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR–Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of co-culture assays suggest that TBEV might partially escape IFN- and IFITM-mediated suppression during high-density co-culture infection when the virus enters naïve cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. Importance: TBEV infection may result in encephalitis, chronic illness or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new endemic centers arise. Although effective TBEV vaccines have been approved, vaccination coverage is low, and, due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. The IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies regarding the role of IFITM proteins in the TBEV infection have been published so far. Understanding of antiviral innate immune responses is crucial for future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both IFN- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

Analysis of laboratory methods for monitoring of tick-borne encephalitis natural foci

Introduction. Ixodid ticks simultaneously are hosts and vectors of tick-borne encephalitis virus (TBEV), presenting a high risk to humans. Monitoring of the vectors part of TBEV population is usually held by means of express analysis methods (ELISA and PCR), but only isolation and identification of infectious virus is reliable evidence of TBEV circulation in the natural foci. Objectives — to demonstrate the TBEV infection rates of Ixodid ticks from natural TBE foci of Baikal Region, based on comprehensive study, including ELISA, PCR and isolation of virus on laboratory mice (LM) model. Methods. Questing adult Ixodid ticks (n = 20 111, mainly — Ixodes persulcatus Schulze, 1930), were collected in TBE natural foci of Baikal Region during 2013–2020. The suspension on saline solution was prepared from the each tick and analyzed by ELISA first. The samples with positive ELISA results were verified in PCR-RT. Furthermore, randomly selected samples with negative ELISA results were analyzed by PCR. Suspensions with positive ELISA and PCR results have been inoculated to suckling LM intracerebrally. Results. The samples with positive PCR results have been divided into two groups: group 1 — all suspensions with positive ELISA results, group 2 — randomly selected samples with negative ELISA results. The positive PCR results in group 1 made up 70.5 % with average Ct rate 24.9. The positive PCR results in group 2 have been obtained in 2.2 % of cases with average Ct rate 30.7. The isolation on LM model was more successful in group 1 (25.8 vs 13.0 %; р < 0.01; df = 69). Conclusion. ELISA is more useful for study of large amounts of ticks during monitoring of natural TBE foci, offering insight into the epidemically important vectors rate. To get the more full assessment of the ticks’ infection rate one must use ELISA and PCR simultaneously, and sum the results into general rate. For high strains isolation results the LM should be inoculated with the suspensions, which had shown positive both ELISA and PCR results.

Clinical Presentation and Laboratory Diagnostic Work-Up of a Horse with Tick-Borne Encephalitis in Switzerland

Tick-borne encephalitis is an important viral tick-borne zoonosis in Europe and Asia. The disease is induced by tick-borne encephalitis virus (TBEV). This report describes a 16-year-old Warmblood gelding presenting with sudden onset of lethargy, ataxia, and muscle fasciculations on the nostrils, the lips, and the eye lids as the most important clinical findings. The horse further had a mild facial nerve paralysis with drooping of the right upper and lower lip. Diagnosis was based on paired serum samples using TBEV-ELISAs revealing high serum IgM in the first sample with normal IgM in the second sample and an increase in serum IgG and neutralizing antibodies, indicating acute and recent infection. TBEV was confirmed by a virus-neutralization test, revealing a fivefold increase in antibodies 32 days after of the onset of clinical signs. Although the specific PCR on cerebrospinal fluid (CSF) was negative, TBEV-specific IgG and IgM were identified in the CSF of the horse. Treatment consisted of anti-inflammatory and anti-oxidative treatment and the horse recovered with a mild drooping of the right nostril as the only remaining clinical sign. TBEV infection is a potential differential diagnosis of neurological disease in horses living in endemic areas and this is the first report to describe the diagnostic criteria in a horse as recommended in humans with suspected TBEV infection.

TBE in Austria

Since 1972, the documentation of human cases of tick-borne encephalitis (TBE) in Austria has been performed by the Center for Virology, Medical University of Vienna, which acts as the National Reference Laboratory for TBE and other flavivirus infections. Only hospitalized patients with a recent tick-borne encephalitis virus (TBEV) infection confirmed by laboratory diagnosis are counted as cases. Confirmation is usually based on immunoglobulin (Ig) serology (namely enzyme-linked immunosorbent assay [ELISA] for IgM and IgG). However, this confirmation may be supplemented by virus neutralization and polymerase chain reaction (PCR) analyses if needed.

TBE in France

The first human case of tick-borne encephalitis virus (TBEV) infection in France was reported in 1968 in Alsace, an eastern region next to the German border: a gamekeeper working in a forest near Strasbourg.1 Between 1970 and 1974, an extensive research survey confirmed the presence of TBEV in ticks and rodents in this French region. Eight percent of adult tick batches collected were infected (Ixodes ricinus) by the TBEV. Tick collection occurred in a forest near Strasbourg, the main city in the region. Nymphs were more rarely infected (1.6% of the collected lots).1 These data were confirmed in 2011 in Alsace in Guebwiller’s Valley, a middle altitude forest, with identification of western (European) subtype TBEV (TBEV-EU). The infection rate still remains low: TBEV was detected only in the I. ricinus nymphs (2.48%) that were collected during May; however, not in those collected during the other spring or summer months. In a more recent study, Bestehorn et al., collected ticks (953 male, 856 female adult ticks and 2,255 nymphs) in endemic foci in the upper Rhine region in France and Germany between 2016, 2017 and 2018 by flagging2. The minimal infection rate (MIR) of the collected ticks in the Foret de la Robertsau (France) was estimated to 0,11% (1 nymph/944 ticks). The isolated and sequenced TBEV strain from Foret de la Robertsau (F) is related to circulating TBEV isolates from eastern Bavaria and the Czech Republic. In the French department Alsace, there are today at least two independent TBEV strains circulating: the historical Alsace strain isolated in 1971 and the newly identified strain from Foret de la Robertsau. Other wooded regions (Ardennes) were explored for TBEV in ticks, but without evidence of virus infection.

Chapter 8: TBE in animals

TBE can cause clinical symptomatic disease in dogs and horses. Diagnosis of TBEV infection in animals is similar to diagnosis in humans. Animals can be used as sentinels for human exposure.

Chapter 10: Diagnosis

TBE appears with non-characteristic clinical symptoms, which cannot be distinguished from other forms of viral encephalitis or other diseases. Cerebrospinal fluid and neuro-imaging may give some evidence of TBE, but ultimately cannot confirm the diagnosis. Thus, proving the diagnosis “TBE” necessarily requires confirmation of TBEV-infection by detection of the virus or by demonstration of specific antibodies from serum and/or cerebrospinal fluid. During the phase of clinic symptoms from the CNS, the TBEV can only rarely be detected in the cerebrospinal fluid of patients. Most routinely used serological tests for diagnosing TBE (ELISA, HI, IFA) show cross reactions resulting from either infection with other flaviviruses or with other flavivirus vaccines.

Enteric Ganglioneuritis, a Common Feature in a Subcutaneous TBEV Murine Infection Model

Tick-borne encephalitis (TBE) is a severe neurologic disease in Europe and Asia. Disease expression ranges from asymptomatic to severe neurological clinical pictures, involving meningitis, encephalitis, meningoencephalitis and potentially fatal outcome. Humans mostly become infected with TBE virus (TBEV) by the bite of an infected tick. Gastrointestinal (GI) symptoms in humans are mainly attributed to the first viremic phase of TBEV infection with unspecific symptoms and/or resulting from severe neurological impairment of the central nervous system (CNS). We used the subcutaneous TBEV-infection of C57BL/6 mice as a model to analyze GI complications of TBE. We observed the acute distension and segmental dilation of the intestinal tract in 10 of 22 subcutaneously infected mice. Histological analysis revealed an intramural enteric ganglioneuritis in the myenteric and submucosal plexus of the small and large intestine. The numbers of infiltrating macrophages and CD3+ T lymphocytes correlated with the severity of ganglioneuritis, indicating an immune-mediated pathogenesis due to TBEV-infection of the enteric plexus. Our study demonstrates that the inflammation of enteric intramural ganglia presents to be a common feature in TBEV-infected mice. Accordingly, the results of this mouse model emphasize that GI disease manifestation and consequences for long-term sequelae should not be neglected for TBEV-infections in humans and require further investigation.

Identification of host antiviral genes differentially induced by clinically diverse strains of Tick-Borne Encephalitis Virus

Tick Borne Encephalitis Virus (TBEV) is an important human arthropod-borne virus, which causes tick-borne encephalitis (TBE), an acute viral infection of the central nervous system (CNS) that causes neurological symptoms of varying severity. TBEV is prevalent in large parts of central- and northern-Europe as well as Northern Asia, and strains of varying pathogenicity have been described. Both host and viral specific characteristics have been postulated to determine the outcome of TBEV infection, but the exact basis of their clinical variability remains undefined.Here, we report the generation of Spinach RNA aptamer labelled TBEV replicons of high (Hypr) and low (Vs) pathogenicity isolates and perform the first direct comparison of both strains in cell culture. We show that pathogenic Hypr replicates to higher levels than Vs in mammalian cells, but not in arthropod cells, and that the basis of this difference maps to the NS5 region, encoding the methyltransferase and RNA polymerase. For both Hypr and Vs strains, NS5 and the viral genome localized to defined intracellular structures typical of positive strand RNA viruses, but Hypr was associated with significant activation of IRF-3, caspase-3 and caspase-8, whilst Vs activated Akt, affording protection against caspase-mediated apoptosis. Activation of TIAR and the formation of cytoplasmic stress granules were an additional early feature of Vs but not Hypr replication. Taken together, these findings highlight NS5 and novel host cell responses as key underling factors for the differential clinical characteristics of TBEV strains.ImportanceTick-borne encephalitis virus (TBEV) is an emerging virus of the flavivirus family spread by ticks. Tick bite can transfer the virus and lead to a febrile infection, Tick-borne encephalitis, of varying severity. There is no specific therapeutic treatment and control in endemic areas is by vaccination. The basis of the different pathologies shown following TBEV infection, from mild to fatal, is not clear although the virus genotype clearly has a role. Mapping the basis of their differential effects would allow focus on the stages of the replication cycle responsible, which might guide the development of therapeutic interventions or the creation of purposefully attenuated strains as candidate vaccines.