Charge-changing point mutations in the E protein of tick-borne encephalitis virus.

Introduction of point mutations is one of the forces enabling arboviruses to rapidly adapt in a changing environment. The influence of these mutations on the properties of the virus is not always obvious. In this study, we attempted to clarify this influence using an in silico approach. Using molecular dynamics (MD) simulations, we investigated how the position of charge-changing point mutations influences the structure and conformational stability of the E protein for a set of variants of a single TBEV strain. The computational findings were supported by experimental evaluation of relevant properties of virions, such as binding to heparan sulfate, thermostability, and susceptibility of the viral hemagglutinating activity to detergents. Our results also point to relationships between E protein dynamics and viral neuroinvasiveness.

Renaming of the genus Flavivirus to Orthoflavivirus and extension of binomial species names within the family Flaviviridae.

This review provides a summary of the recently ratified changes to genus and species nomenclature within the virus family Flaviviridae along with reasons for these changes. First, it was considered that the vernacular terms "flaviviral", "flavivirus", and "flaviviruses" could under certain circumstances be ambiguous due to the same word stem "flavi" in the taxon names Flaviviridae and Flavivirus; these terms could either have referred to all viruses classified in the family Flaviviridae or only to viruses classified in the included genus Flavivirus. To remove this ambiguity, the genus name Flavivirus was changed to Orthoflavivirus by the International Committee on Taxonomy of Viruses (ICTV). Second, all species names in the family were changed to adhere to a newly ICTV-mandated binomial format (e.g., Orthoflavivirus zikaense, Hepacivirus hominis) similar to nomenclature conventions used for species elsewhere in biology. It is important to note, however, that virus names remain unchanged. Here we outline the revised taxonomy of the family Flaviviridae as approved by the ICTV in April 2023.

Effect of immature tick-borne encephalitis virus particles on antiviral activity of 5-aminoisoxazole-3-carboxylic acid adamantylmethyl esters.

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is common in Europe and Asia and causes a severe disease of the central nervous system. A promising approach in the development of therapy for TBEV infection is the search for small molecule antivirals targeting the flavivirus envelope protein E, particularly its ß-n-octyl-d-glucoside binding pocket (ß-OG pocket). However, experimental studies of candidate antivirals may be complicated by varying amounts and different forms of the protein E in the virus samples. Viral particles with different conformations and arrangements of the protein E are produced during the replication cycle of flaviviruses, including mature, partially mature, and immature forms, as well as subviral particles lacking genomic RNA. The immature forms are known to be abundant in the viral population. We obtained immature virion preparations of TBEV, characterized them by RT-qPCR, and assessed in vivo and in vitro infectivity of the residual mature virions in the immature virus samples. Analysis of the ß-OG pocket structure on the immature virions confirmed the possibility of binding of adamantylmethyl esters of 5-aminoisoxazole-3-carboxylic acid in the pocket. We demonstrated that the antiviral activity of these compounds in plaque reduction assay is significantly reduced in the presence of immature TBEV particles.

Precise tracking of vaccine-responding T cell clones reveals convergent and personalized response in identical twins.

T cell receptor (TCR) repertoire data contain information about infections that could be used in disease diagnostics and vaccine development, but extracting that information remains a major challenge. Here we developed a statistical framework to detect TCR clone proliferation and contraction from longitudinal repertoire data. We applied this framework to data from three pairs of identical twins immunized with the yellow fever vaccine. We identified 600 to 1,700 responding TCRs in each donor and validated them using three independent assays. While the responding TCRs were mostly private, albeit with higher overlap between twins, they could be well-predicted using a classifier based on sequence similarity. Our method can also be applied to samples obtained postinfection, making it suitable for systematic discovery of new infection-specific TCRs in the clinic.

Ability of inactivated vaccines based on far-eastern tick-borne encephalitis virus strains to induce humoral immune response in originally seropositive and seronegative recipients.

Tick-borne encephalitis (TBE) remains one of the major public health concerns in northern Eurasia, and its' area is expanding. TBE virus (TBEV) includes three subtypes and several monophyletic groups, cocirculating in Russia. Five inactivated vaccines are used for TBE prophylaxis. The rising number of people subjected to vaccination brings up the issue of the impact of individual recipient characteristics on vaccination efficacy. The present work studies correlations among the vaccination scheme, sex, age, body mass index (BMI), chronic diseases, postvaccinal reaction, pre-existing anti-TBEV antibodies, and postvaccinal humoral immunity development. Sera were collected during clinical trials in the TBEV Siberian subtype endemic area. Adult recipients were vaccinated with Tick-E-Vac and EnceVir vaccines based on Far-Eastern TBEV strains. Vaccine ability to induce humoral immunity in different categories of recipients was estimated by seroconversion rates and the percentage of recipients with high neutralizing antibody titers (≥1:500). High immunogenicity of vaccines based on Far-Eastern TBEV strains in the TBEV Siberian subtype endemic area in all groups of recipients was demonstrated. Impact of pre-existing contact with the virus and high BMI on humoral immune response development 14 days after the first immunization was evidenced. Nevertheless, the difference was significantly less pronounced 30 days after the first vaccination and undetectable after the second one.

Tick-borne flavivirus reproduction inhibitors based on isoxazole core linked with adamantane.

Infections caused by flaviviruses pose a huge threat for public health all over the world. The search for therapeutically relevant compounds targeting tick-borne flaviviruses requires the exploration of novel chemotypes. In the present work a large series of novel polyfunctionalized isoxazole derivatives bearing substituents with various steric and electronic effects was obtained by our unique versatile synthetic procedure and their antiviral activity against tick-borne encephalitis, Omsk hemorrhagic fever, and Powassan viruses was studied in vitro. The majority of studied isoxazoles showed activity in low micromolar range. No appreciable cytotoxicity was observed for tested compounds. The lead compounds, 5-aminoisoxazole derivatives containing adamantyl moiety, exhibited strong antiviral activity and excellent therapeutic index.

Evaluation of the population heterogeneity of TBEV laboratory variants using high-throughput sequencing.

We studied minor variants within two tick-borne encephalitis virus (TBEV) populations with a common ancestor: the mouse brain-adapted variant EK-328c and the tick-adapted variant M. High-throughput sequencing with custom amplicons from RT-PCR viral RNA was performed on Illumina MiSeq 2*250 paired-end v2 chemistry. Using the LowFreq program (default settings) and Sanger-sequenced consensus as a reference, variants with an abundance of 1 % and above within the studied populations were identified. Using the obtained data in the context of our previous studies, we concluded that TBEV variants, which are different from the major population phenotype and can become a major part of the viral population under favourable environmental conditions, can exist at abundances of less than 1 % in the long-term. The comparison of our data with the literature allowed us to conclude that the laboratory variant EK-328c and variant M have similar SNV counts to TBEV variants from natural populations and some fast-evolving RNA viruses.

Intracellular degradation and localization of NS1 of tick-borne encephalitis virus affect its protective properties.

Currently, many DNA vaccines against infectious diseases are in clinical trials; however, their efficacy needs to be improved. The potency of DNA immunogen can be optimized by targeting technologies. In the current study, to increase the efficacy of NS1 encoded by plasmid, proteasome targeting was applied. NS1 variants with or without translocation sequence and with ornithine decarboxylase as a signal of proteasomal degradation were tested for expression, localization, protein turnover, proteasomal degradation and protection properties. Deletion of translocation signal abrogated presentation of NS1 on the cell surface and increased proteasomal processing of NS1. Fusion with ornithine decarboxylase led to an increase of protein turnover and the proteasome degradation rate of NS1. Immunization with NS1 variants with increased proteasome processing protected mice from viral challenge only partially; however, the survival time of infected mice was prolonged in these groups. These data can give a presupposition for formulation of specific immune therapy for infected individuals.

New tools in nucleoside toolbox of tick-borne encephalitis virus reproduction inhibitors.

Design and development of nucleoside analogs is an established strategy in the antiviral drug discovery field. Nevertheless, for many viruses the coverage of structure-activity relationships (SAR) in the nucleoside chemical space is not sufficient. Here we present the nucleoside SAR exploration for tick-borne encephalitis virus (TBEV), a member of Flavivirus genus. Promising antiviral activity may be achieved by introduction of large hydrophobic substituents in the position 6 of adenosine or bulky silyl groups to the position 5'. Introduction of methyls to the ribose moiety does not lead to inhibition of TBEV reproduction. Possible mechanisms of action of these nucleosides include the inhibition of viral entry or interaction with TBEV non-structural protein 5 methyltransferase or RNA-dependent RNA polymerase domains.

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

BACKGROUND: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. METHODS: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. RESULTS: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. CONCLUSIONS: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.

Morphological features of Ixodes persulcatus and I. ricinus hybrids: nymphs and adults.

Our aim was to reveal morphological features of first-generation Ixodes persulcatus and I. ricinus hybrids (nymphs and adults) obtained under laboratory conditions for further study of natural populations of these species in sympatry foci. In 65 nymphs of three groups I. ricinus (23 specimens), I. persulcatus (21 specimens), and hybrids (21 specimens), 16 parameters were evaluated (length/width of the scutum and capitulum, length of the hypostome, palp, tarsus I, coxa I, sternal setae, and various scutal and alloscutal setae) and discrimination analysis was performed allowing differentiation of hybrid nymphs from original species. General effectiveness of classification of I. ricinus, I. persulcatus, and hybrids was >95 %. Discriminant functions are presented allowing classification of I. persulcatus, I. ricinus, and hybrid nymphs. For description of morphology, 27 adult hybrids (13 males and 14 females) were examined under a stereo microscope at 14-28× (without preparation of permanent mounts). The following morphological distinctions of hybrids from original species were described: posterior marginal groove is not clear (as in I. ricinus) and absence of syncoxa on coxa I (as in I persulcatus). In hybrid males, simultaneous absence of syncoxa on coxa I (as in I. persulcatus) and a long internal spur on coxa I (as in I. ricinus) can be used as a diagnostic feature. Based on the detected characteristics, 10 of 157 ticks collected in Karelia in I. ricinus and I. persulcatus sympatry area were classified as hybrids.

Synthesis and assessment of 4-aminotetrahydroquinazoline derivatives as tick-borne encephalitis virus reproduction inhibitors.

Tick-borne encephalitis virus (TBEV) belonging to Flavivirus genus causes severe infection in humans. The search for therapeutically relevant compounds targeting TBEV requires the exploration of novel chemotypes. A versatile synthesis of previously unknown 4-aminopyrimidines and 4-aminopyrimidine N-oxides based on a fluorosubstituted heterocyclic core is described. A representative series of 4-aminotetrahydroquinazoline derivatives, containing aliphatic and aromatic substituents as well as the adamantane framework, was obtained and their activity against tick-borne encephalitis virus reproduction was studied. Nine compounds were found to inhibit TBEV entry into the host cells. A bulky hydrophobic adamantyl group was identified to be important for the antiviral activity. The developed synthetic route allowed an easy access to a consistent compound library for further structure-activity relationship studies.

Identification of soil-dwelling predators of tick nymphs (Acari: Ixodidae) by stable isotope labeling.

Ticks (Family Ixodidae) spend most of their life cycle as immature stages in the soil and litter, and as any other soil invertebrates, are likely to be controlled top-down by soil-dwelling predators. To date, the ability of soil invertebrate predators to control ixodid tick population remains little known, partly due to methodological difficulties. In the current study, we developed and successfully tested a novel method of labeling live Ixodes ricinus (L., 1758) (Ixodida: Ixodidae) nymphs with a 15N isotope label. Labeled ticks were used in a small-scale 8-day-long microcosm experiment to reveal soil predators attacking nymphs. Only a small fraction (4.1% of all samples) of soil generalist predators preyed upon nymphs. A strong 15N label was found in 5 predator species, namely 2 spiders (Pachygnatha listeri Sundevall, 1830, Tetragnathidae and Ozyptila sp., Theridiidae), 2 gamasid mites (Pergamasus beklemischevi Sellnick, 1929 and Pergamasus quisquiliarum [Canestrini, 1882], Parasitidae), and 1 staphylinid beetle (Geostiba circellaris [Gravenhorst, 1806], Staphylinidae). The isotopic labeling can be a useful tool in revealing a range of invertebrate predators that can control tick populations in soil.

Orthoflavivirus Lammi in Russia: Possible Transovarial Transmission and Trans-Stadial Survival in Aedes cinereus (Diptera, Culicidae).

In the last few years, there has been a dramatic increase in the number of discovered viruses that are transmitted by arthropods. Some of them are pathogenic for humans and mammals, and the pathogenic potential of others is unknown. The genus Orthoflavivirus belongs to the family Flaviviridae and includes arboviruses that cause severe human diseases with damage to the central nervous system and hemorrhagic fevers, as well as viruses with unknown vectors and viruses specific only to insects. The latter group includes Lammi virus, first isolated from a mosquito pool in Finland. It is known that Lammi virus successfully replicates in mosquito cell lines but not in mammalian cell cultures or mice. Lammi virus reduces the reproduction of West Nile virus during superinfection and thus has the potential to reduce the spread of West Nile virus in areas where Lammi virus is already circulating. In this work, we isolated Lammi virus from a pool of adult Aedes cinereus mosquitoes that hatched from larvae/pupae collected in Saint Petersburg, Russia. This fact may indicate transovarial transmission and trans-stadial survival of the virus.

High-Throughput Sequencing Reveals Three Rhabdoviruses Persisting in the IRE/CTVM19 Cell Line.

Cell cultures derived from ticks have become a commonly used tool for the isolation and study of tick-borne pathogens and tick biology. The IRE/CTVM19 cell line, originating from embryos of Ixodes ricinus, is one such line. Previously, reovirus-like particles, as well as sequences with similarity to rhabdoviruses and iflaviruses, were detected in the IRE/CTVM19 cell line, suggesting the presence of multiple persisting viruses. Subsequently, the full genome of an IRE/CTVM19-associated rhabdovirus was recovered from a cell culture during the isolation of the Alongshan virus. In the current work, we used high-throughput sequencing to describe a virome of the IRE/CTVM19 cell line. In addition to the previously detected IRE/CTVM19-associated rhabdovirus, two rhabdoviruses were detected: Chimay rhabdovirus and Norway mononegavirus 1. In the follow-up experiments, we were able to detect both positive and negative RNA strands of the IRE/CTVM19-associated rhabdovirus and Norway mononegavirus 1 in the IRE/CTVM19 cells, suggesting their active replication in the cell line. Passaging attempts in cell lines of mammalian origin failed for all three discovered rhabdoviruses.

Phenotypic assessment of antiviral activity for spiro-annulated oxepanes and azepenes.

Evolutionary potential of viruses can result in outbreaks of well-known viruses and emergence of novel ones. Pharmacological methods of intervening the reproduction of various less popular, but not less important viruses are not available, as well as the spectrum of antiviral activity for most known compounds. In the framework of chemical biology paradigm, characterization of antiviral activity spectrum of new compounds allows to extend the antiviral chemical space and provides new important structure-activity relationships for data-driven drug discovery. Here we present a primary assessment of antiviral activity of spiro-annulated derivatives of seven-membered heterocycles, oxepane and azepane, in phenotypic assays against viruses with different genomes, virion structures, and genome realization schemes: orthoflavivirus (tick-borne encephalitis virus, TBEV), enteroviruses (poliovirus, enterovirus A71, echovirus 30), adenovirus (human adenovirus C5), hantavirus (Puumala virus). Hit compounds inhibited reproduction of adenovirus C5, the only DNA virus in the studied set, in the yield reduction assay, and did not inhibit reproduction of RNA viruses.

Hybrids of Ixodes ricinus and Ixodes persulcatus ticks effectively acquire and transmit tick-borne encephalitis virus.

Ixodes rici nus and Ixodes persulcatus ticks are the main vectors of tick-borne encephalitis virus (TBEV), which has three main subtypes connected with certain tick species: the European subtype, associated with I. ricinus, and the Siberian and Far-Eastern subtypes, associated with I. persulcatus. Distribution ranges of these species overlap and form large sympatric areas in the East European Plain and Baltic countries. It has previously been shown that crossing of I. ricinus and I. persulcatus is possible, with the appearance of sterile hybrids. Hybridization of ticks can affect not only the spread of ticks but also the properties of natural foci of arbovirus infections, in particular TBEV. In the present study, we analyzed the effectiveness of virus transmission from infected mice to larvae and nymphs and trans-stadial transmission (from larvae to nymph and adult) in I. ricinus, I. persulcatus, and hybrids. For this purpose, we bred a hybrid generation from the crossing of I. persulcatus females and I. ricinus males, and we used the Siberian and European subtypes of TBEV. We showed that after feeding on infected mice, virus prevalence in engorged ticks decreased over time, and after molting, the opposite was true. In hybrids we observed the highest acquisition effectiveness and RNA copy numbers during Siberian TBEV subtype transmission. The efficiency of trans-stadial transmission of both TBEV subtypes was similar in hybrids and parental species. After the second trans-stadial TBEV transmission, a significant increase in ticks' infection rates was observed only in specific subtype-tick combination. Our data demonstrate the possible features of TBEV circulation in the I. ricinus and I. persulcatus sympatry area.

Development Features of Ixodes ricinus × I. persulcatus Hybrids under Laboratory Conditions.

Widely distributed Ixodes ricinus and Ixodes persulcatus ticks transmit many pathogens of both medical and veterinary significance. The ranges of these tick species overlap and form large sympatric areas in the East European Plain and Baltic countries. It has previously been shown that crossing I. ricinus and I. persulcatus is possible, resulting in the appearance of sterile hybrids. In the present study, we analyzed the features of this hybrid's life cycle under laboratory conditions. For this purpose, virgin females of I. ricinus and I. persulcatus ticks were obtained in the laboratory, and hybrid generations of ticks were bred from the reciprocal crossings of these two tick species. According to our data, mating the females of I. ricinus and I. persulcatus with the males of another species leads to a decrease in the engorgement success of the females, a decrease in the number of hatched larvae, and the appearance of a hybrid generation in which both females and males are sterile. Under laboratory conditions at a constant room temperature and under natural daylight, the morphogenetic diapause of the engorged I. persulcatus larvae began in September. For I. persulcatus nymphs, it occurred earlier than for I. ricinus, in October and November, respectively. The hybrids generally repeated the features of the life cycle of the mother species.

Conserved Sequences in the 5' and 3' Untranslated Regions of Jingmenvirus Group Representatives.

The Jingmenvirus group (JVG), with members such as Jingmen tick virus (JMTV), Alongshan virus (ALSV), Yanggou tick virus (YGTV), and Takachi virus (TAKV), is drawing attention due to evidence of it causing disease in humans and its unique genome architecture. In the current work, complete untranslated regions (UTRs) of four strains of ALSV and eight strains of YGTV were obtained. An analysis of these sequences, as well as JVG sequences from GenBank, uncovered several regions within viral UTRs that were highly conserved for all the segments and viruses. Bioinformatics predictions suggested that the UTRs of all the segments of YGTV, ALSV, and JMTV could form similar RNA structures. The most notable feature of these structures was a stable stem-loop with one (5' UTR) or two (3' UTR) AAGU tetraloops on the end of a hairpin.

Quantitative Polymerase Chain Reaction System for Alongshan Virus Detection.

The recently discovered Jingmenvirus group includes viruses with a segmented genome, RNA of a positive polarity, and several proteins with distant homology to the proteins of the members of the genus Orthoflavivirus. Some Jingmenvirus group members, namely the Alongshan virus (ALSV) and Jingmen tick virus, are reported to be tick-borne human pathogens that can cause a wide variety of symptoms. The ALSV is widely distributed in Eurasia, yet no reliable assay that can detect it exists. We describe a qPCR system for ALSV detection. Our data showed that this system can detect as little as 104 copies of the ALSV in a sample. The system showed no amplification of the common tick-borne viruses circulating in Eurasia, i.e., the Yanggou tick virus-which is another Jingmenvirus group member-or some known members of the genus Orthoflavivirus. The qPCR system was tested and had no nonspecific signal for the Ixodes ricinus, I. persulcatus, Dermacentor reticulatus, D. marginatus, Haemaphysalis concinna, and H. japonica ticks. The qPCR system had no nonspecific signal for human and sheep serum as well. Overall, the qPCR system described here can be used for reliable and quantitative ALSV detection.