Tick-borne Encephalitis Virus Strains Research Articles

Applied and Theoretical Aspects of the Ecology of Pathogens of Hemorrhagic Fever with Renal Syndrome and Tick-Borne Encephalitis

Relevance. Hemorrhagic fever with renal syndrome (HFRS) and tick-borne encephalitis (TBE) are the most common natural focal diseases of viral etiology In Russia. The medical and social significance of these two infections is determined by the extensive fociof their spread, high annual morbidity rates in the population, and the possibility of developing severe forms of the disease leading to permanent disability and death. The causative agents of HFRS In Russia are 6 types of hantaviruses Hantaan, Amur, Seoul, Puumala, Kurkino and Sochi, which, immunologically and genetically significantly different from each other, support their existence in nature through six different species of small mammals, which are sources of infection in humans. TBE virus strains that cause disease in humans belong to five phylogenetically distinct subtypes, including: Far Eastern, European, Siberian, “178-79” and Baikal. Aim. Analysis of the role of small mammals and ticks in the ecology of HFRS and TBE pathogens. Conclusions. Small mammals retain and transmit hantaviruses to uninfected animals and ticks. At the same time, ticks retain and transmit to ticks and mammals not only the TBE virus, but also hantaviruses. Despite the taxonomic difference between the viruses that cause HFRS and TBE, some of their ecological properties are similar.

A candidate tick-borne encephalitis virus vaccine based on virus-like particles induces specific cellular and humoral immunity in mice1

Tick-borne encephalitis (TBE) is an important zoonotic viral disease transmitted by ticks. In recent decades, global climate change has increased human exposure to ticks, and mortality rates have gradually risen. Effective vaccines are essential for controlling TBE as specific antiviral treatment is unavailable. Vaccine candidates based on virus-like particles (VLPs) have previously been demonstrated to be efficient in eliciting excellent immune responses against influenza virus and SARS-CoV-2. Here, we constructed TBE virus (TBEV) VLPs containing the envelope and membrane proteins derived from the Far Eastern TBEV strain (WH2012) using an insect cell-baculovirus expression system. Induction of immune responses was investigated in mice following intramuscular injection with the TBEV VLPs vaccine candidates formulated of Poly(I:C) & Montanide ISA 201VG combination adjuvants. Mice produced memory T-cells and serum-specific IgG antibodies that averaged up to 1:104.6 and remained at 1:104 (mean) for 24 weeks after three immunizations. TBEV VLPs vaccine was able to provide long-term antibody protection against TBEV, making it a promising subunit vaccine candidate for this disease.

Identification and characterization of new Siberian subtype of tick-borne encephalitis virus isolates revealed genetic variations of the Chinese strains

Tick-borne encephalitis virus (TBEV) is a pathogen that causes febrile infectious diseases and neurological damage to humans. TBEVs are prevalent from Europe to Far Eastern Asia, including Northeastern China. The understanding of TBEV phylogeny in China has been limited owing to insufficient genomic data on Chinese TBEV strains. Here, six TBEV strains were isolated from ticks collected in Inner Mongolia. The transmission electron microscopy revealed spherical particles with an enveloped structure of 50–60 nm in diameter. Phylogenetic analysis showed that, two strains were classified as the Siberian subtype, while the remaining four were identified as the Far Eastern subtype. Migration analyses based on TBEV ORF and envelope (E) protein sequences revealed that Chinese TBEV strains were migrated from Russia and/or Kazakhstan into China. Hulun Buir and Mudanjiang, the northeastern region of China, are considered hotspots with multiple import and export routes of Chinese TBEV strains. These results promote the understanding of TBEV genetic variations and phylogeny in China and suggest the importance of improving investigation of TBEV prevalence, which would instrumental for vaccine design strategies and better preparation for controlling TBEV infection in humans.

Unraveling the role of human microglia in tick-borne encephalitis virus infection: insights into neuroinflammation and viral pathogenesis

Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus responsible for severe infections of the central nervous system. Although neurons are predominantly targeted, specific involvement of microglia in pathogenesis of TBE is not yet fully understood. In this study, the susceptibility of human microglia to TBEV is investigated, focusing on productive infection and different immune responses of different viral strains. We investigated primary human microglia and two immortalized microglial cell lines exposed to three TBEV strains (Hypr, Neudörfl and 280), each differing in virulence. Our results show that all microglia cultures tested support long-term productive infections, regardless of the viral strain. In particular, immune response varied significantly with the viral strain, as shown by the differential secretion of cytokines and chemokines such as IP-10, MCP-1, IL-8 and IL-6, quantified using a Luminex 48-plex assay. The most virulent strain triggered the highest cytokine induction. Electron tomography revealed substantial ultrastructural changes in the infected microglia, despite the absence of cytopathic effects. These findings underscore the susceptibility of human microglia to TBEV and reveal strain-dependent variations in viral replication and immune responses, highlighting the complex role of microglia in TBEV-induced neuropathology and contribute to a deeper understanding of TBE pathogenesis and neuroinflammation.

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.3

TBE In Bulgaria

First cases of probable tick-borne encephalitis (TBE) were reported in 1961 by Andonov et al. in eastern regions of Bulgaria.1 Possible TBE cases with the typical two-wave fever, originating from consumption of raw goat milk, were described back in 1953 by Vaptzarov et al. in southern Bulgaria.2 Investigations in the 1960s were able to isolate 3 tick-borne encephalitis virus (TBEV) strains from Haemaphysalis punctata and 1 from Dermacentor marginatus ticks from goats and sheep in the district of Plovdiv.3 The antigenic properties of these 4 virus strains were identical to the highly virulent strain “Hypr” of the European subtype of TBEV (TBEV-EU).3

In vivo rescue of arboviruses directly from subgenomic DNA fragments

ABSTRACT Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the “Infectious Subgenomic Amplicons” (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo. Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo. It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

Differential susceptibility of geographically distinct Ixodes ricinus populations to tick-borne encephalitis virus and louping ill virus.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa, ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

The structure of inactivated mature tick-borne encephalitis virus at 3.0 Å resolution

ABSTRACT Tick-borne encephalitis virus (TBEV) causes a severe disease, tick-borne encephalitis (TBE), that has a substantial epidemiological importance for Northern Eurasia. Between 10,000 and 15,000 TBE cases are registered annually despite the availability of effective formaldehyde-inactivated full-virion vaccines due to insufficient vaccination coverage, as well as sporadic cases of vaccine breakthrough. The development of improved vaccines would benefit from the atomic resolution structure of the antigen. Here we report the refined single-particle cryo-electron microscopy (cryo-EM) structure of the inactivated mature TBEV vaccine strain Sofjin–Chumakov (Far-Eastern subtype) at a resolution of 3.0 Å. The increase of the resolution with respect to the previously published structures of TBEV strains Hypr and Kuutsalo-14 (European subtype) was reached due to improvement of the virus sample quality achieved by the optimized preparation methods. All the surface epitopes of TBEV were structurally conserved in the inactivated virions. ELISA studies with monoclonal antibodies supported the hypothesis of TBEV protein shell cross-linking upon inactivation with formaldehyde.

Phylogenetic characterisation of tick-borne encephalitis virus from Lithuania.

The Baltic states are the region in Europe where tick-borne encephalitis (TBE) is most endemic. The highest notification rate of TBE cases is reported in Lithuania, where the incidence of TBE has significantly increased since 1992. A recent study reported 0.4% prevalence of TBE virus (TBEV) in the two most common tick species distributed in Lithuania, Ixodes ricinus and Dermacentor reticulatus, with the existence of endemic foci confirmed in seven out of Lithuania's ten counties. However, until now, no comprehensive data on molecular characterisation and phylogenetic analysis have been available for the circulating TBEV strains. The aim of this study was to analyse TBEV strains derived from I. ricinus and D. reticulatus ticks collected from Lithuania and provide a genotypic characterisation of viruses based on sequence analysis of partial E protein and NS3 genes. The 54 nucleotide sequences obtained were compared with 81 TBEV strains selected from the NCBI database. Phylogenetic analysis of the partial E and NS3 gene sequences derived from 34 Lithuanian TBEV isolates revealed that these were specific to Lithuania, and all belonged to the European subtype, with a maximum identity to the Neudoerfl reference strain (GenBank accession no. U27495) of 98.7% and 97.4%, respectively. The TBEV strains showed significant regional genetic diversity. The detected TBEV genotypes were not specific to the tick species. However, genetic differences were observed between strains from different locations, while strains from the same location showed a high similarity.

Changes in the Genome of the Tick-Borne Encephalitis Virus during Cultivation

The tick-borne encephalitis virus (TBEV) strain C11-13 (GenBank acc. no. OQ565596) of the Siberian genotype was previously isolated from the brain of a deceased person. TBEV C11-13 variants obtained at passages 3 and 8 in SPEV cells were inoculated into the brains of white mice for subsequent passages. Full genome sequences of all virus variants were analyzed by high-throughput sequencing. A total of 41 single nucleotide substitutions were found to occur mainly in the genes for the nonstructural proteins NS3 and NS5 (GenBank MF043953, OP902894, and OP902895), and 12 amino acid substitutions were identified in the deduced protein sequences. Reverse nucleotide and amino acid substitutions were detected after three passages through mouse brains. The substitutions restored the primary structures that were characteristic of the isolate C11-13 from a human patient and changed during the eight subsequent passages in SPEV cells. In addition, the 3'-untranslated region (3'-UTR) of the viral genome increased by 306 nt. The Y3 and Y2 3'-UTR elements were found to contain imperfect L and R repeats, which were probably associated with inhibition of cellular XRN1 RNase and thus involved in the formation of subgenomic flaviviral RNAs (sfRNAs). All TBEV variants showed high-level reproduction in both cell cultures and mouse brains. The genomic changes that occurred during successive passages of TBEV are most likely due to its significant genetic variability, which ensures its efficient reproduction in various hosts and its broad distribution in various climatic zones.

Immunity to Tick-Borne Encephalitis Virus NS3 Protein Induced with a Recombinant Modified Vaccinia Virus Ankara Fails to Afford Mice Protection against TBEV Infection.

Tick-borne encephalitis (TBE) is a serious neurological disease caused by TBE virus (TBEV). Because antiviral treatment options are not available, vaccination is the key prophylactic measure against TBEV infections. Despite the availability of effective vaccines, cases of vaccination breakthrough infections have been reported. The multienzymatic non-structural protein 3 (NS3) of orthoflaviviruses plays an important role in polyprotein processing and virus replication. In the present study, we evaluated NS3 of TBEV as a potential vaccine target for the induction of protective immunity. To this end, a recombinant modified vaccinia virus Ankara that drives the expression of the TBEV NS3 gene (MVA-NS3) was constructed. MVA-NS3 was used to immunize C57BL/6 mice. It induced NS3-specific immune responses, in particular T cell responses, especially against the helicase domain of NS3. However, MVA-NS3-immunized mice were not protected from subsequent challenge infection with a lethal dose of the TBEV strain Neudoerfl, indicating that in contrast to immunity to prME and NS1, NS3-specific immunity is not an independent correlate of protection against TBEV in this mouse model.

Infection of wild-caught wood mice (Apodemus sylvaticus) and yellow-necked mice (A. flavicollis) with tick-borne encephalitis virus

The distribution of tick-borne encephalitis virus (TBEV) is expanding to Western European countries, including the Netherlands, but the contribution of different rodent species to the transmission of TBEV is poorly understood. We investigated whether two species of wild rodents native to the Netherlands, the wood mouse Apodemus sylvaticus and the yellow-necked mouse Apodemus flavicollis, differ in their relative susceptibility to experimental infection with TBEV. Wild-caught individuals were inoculated subcutaneously with the classical European subtype of TBEV (Neudoerfl) or with TBEV-NL, a genetically divergent TBEV strain from the Netherlands. Mice were euthanised and necropsied between 3 and 21 days post-inoculation. None of the mice showed clinical signs or died during the experimental period. Nevertheless, TBEV RNA was detected up to 21 days in the blood of both mouse species and TBEV was also isolated from the brain of some mice. Moreover, no differences in infection rates between virus strains and mouse species were found in blood, spleen, or liver samples. Our results suggest that the wood mouse and the yellow-necked mouse may equally contribute to the transmission cycle of TBEV in the Netherlands. Future experimental infection studies that include feeding ticks will help elucidate the relative importance of viraemic transmission in the epidemiology of TBEV.

Recombinant TBEV Protein E of the Siberian Subtype Is a Candidate Antigen in the ELISA Test System for Differential Diagnosis.

The tick-borne encephalitis virus (TBEV) is one of the most common members of the Orthoflavivirus genus, which comprises the causative agents of severe diseases in humans and animals. Due to the expanding areas of orthoflavivirus infection, its differential diagnosis is highly demanded. Commercial test kits based on inactivated TBEV may not provide reliable differentiation between flaviviruses because of serological crossover in this genus. Application of recombinant domains (sE and dIII) of the TBEV Sukhar-strain protein E as antigens in an ELISA test system allowed us to identify a wide range of antibodies specific to different TBEV strains. We tested 53 sera from human patients with confirmed TBE diagnosis (the efficacy of our test system based on sE protein was 98%) and 56 sera from patients with other orthoflavivirus infections in which no positive ones were detected using our ELISA test system, thus being indicative of its 100% specificity. We also tested mouse and rabbit sera containing antibodies specific to 17 TBEV strains belonging to different subtypes; this assay exhibited high efficacy and differentiation ability in detecting antibodies against TBEV from other orthoflaviviruses such as Omsk hemorrhagic fever, Powassan, yellow fever, dengue, West Nile, Zika, and Japanese encephalitis viruses.

TBE in Bulgaria

First cases of probable tick-borne encephalitis (TBE) were reported in 1961 by Andonov et al. in eastern regions of Bulgaria. Possible TBE cases with the typical two-wave fever, originating from consumption of raw goat milk, were described back in 1953 by Vaptzarov et al. in southern Bulgaria. Investigations in the 1960s were able to isolate 3 tick-borne encephalitis virus (TBEV) strains from Haemaphysalis punctata and 1 from Dermacentor marginatus ticks from goats and sheep in the district of Plovdiv. The antigenic properties of these 4 virus strains were identical to the highly virulent strain “Hypr” of the European subtype of TBEV (TBEV-EU).

TBE in Sweden

Tick-borne encephalitis virus (TBEV) was isolated for the first time in Sweden in 1958 (from ticks and from 1 tick-borne encephalitis [TBE] patient). In 2003, Haglund and colleagues reported the isolation and antigenic and genetic characterization of 14 TBEV strains from Swedish patients (samples collected 1991–1994). The first serum sample, from which TBEV was isolated, was obtained 2–10 days after onset of disease and found to be negative for anti-TBEV immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA), whereas TBEV-specific IgM (and TBEV-specific immunoglobulin G/cerebrospinal fluid [IgG/CSF] activity) was demonstrated in later serum samples taken during the second phase of the disease.

Development and characterization of chimera of yellow fever virus vaccine strain and Tick-Borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is one of the most threatening pathogens which affects the human central nervous system (CNS). TBEV circulates widely in Northern Eurasia. According to ECDC, the number of TBE cases increase annually. There is no specific treatment for the TBEV infection, thus vaccination is the main preventive measure. Despite the existence of several inactivated vaccines currently being licensed, the development of new TBEV vaccines remains a leading priority in countries endemic to this pathogen. Here we report new recombinant virus made by infectious subgenomic amplicon (ISA) approach using TBEV and yellow fever virus vaccine strain (YF17DD-UN) as a genetic backbone. The recombinant virus is capable of effective replication in mammalian cells and induce TBEV-neutralizing antibodies in mice. Unlike the original vector based on the yellow fever vaccine strain, chimeric virus became neuroinvasive in doses of 107-106 PFU and can be used as a model of flavivirus neuroinvasiveness, neurotropism and neurovirulence. These properties of hybrid structures are the main factors limiting their practical use as vaccines platforms.

High neutralizing antibody mismatch as a possible reason for vaccine failure in two children with severe tick-borne encephalitis

We describe two adolescents (13 and 16 years old) with severe tick-borne encephalitis (TBE) and vaccination breakthrough (VBT). Both suffer from severe persistent neurologic sequelae. Both patients had high TBE-IgG-titers after vaccination at the beginning of the infection and a low or missing TBE-IgM response (Type 2 vaccine failure). Neutralization tests show low titers against the respective infecting TBE virus strain and higher titers against the vaccine strain at the beginning of the infection implying an individual weak or impaired immune response to the respective virus as possible cause of TBE vaccine failure.We do not know of any similar observation or explanation for the phenomenon and at the moment can only speculate of a severe course correlated to highly mismatched IgG. This constellation of high TBE IgGs, the lack of immune response and a severe course strongly resembles the severe TBE courses that occurred in the past after TBE immunoglobulin administration.To our knowledge differentiation between structural and functional antibodies by neutralization tests with a) the affecting TBE virus strain and b) the vaccine virus strain in TBE vaccine failures has never been described before. We conclude (1) to consider a TBE virus infection also in vaccinated children presenting with meningoencephalitis, (2) to perform a broad immunological work-up in severe TBE especially after VBT, (3) to further study if high mismatch IgG's are a possible reason for vaccine failure.

Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.

Identifying New Clusterons: Application of TBEV Analyzer 3.0.

Early knowledge about novel emerging viruses and rapid determination of their characteristics are crucial for public health. In this context, development of theoretical approaches to model viral evolution are important. The clusteron approach is a recent bioinformatics tool which analyzes genetic patterns of a specific E protein fragment and provides a hierarchical network structure of the viral population at three levels: subtype, lineage, and clusteron. A clusteron is a group of strains with identical amino acid (E protein fragment) signatures; members are phylogenetically closely related and feature a particular territorial distribution. This paper announces TBEV Analyzer 3.0, an analytical platform for rapidly characterizing tick-borne encephalitis virus (TBEV) strains based on the clusteron approach, workflow optimizations, and simplified parameter settings. Compared with earlier versions of TBEV Analyzer, we provide theoretical and practical enhancements to the platform. Regarding the theoretical aspect, the model of the clusteron structure, which is the core of platform analysis, has been updated by analyzing all suitable TBEV strains available in GenBank, while the practical enhancements aim at improving the platform's functionality. Here, in addition to expanding the strain sets of prior clusterons, we introduce eleven novel clusterons through our experimental results, predominantly of the European subtype. The obtained results suggest effective application of the proposed platform as an analytical and exploratory tool in TBEV surveillance.