Conservation of Major Satellite DNAs in Snake Heterochromatin.

Repetitive DNA sequences constitute a sizeable portion of animal genomes, and tandemly organized satellite DNAs are a major part of them. They are usually located in constitutive heterochromatin clusters in or near the centromeres or telomeres, and less frequently in the interstitial parts of chromosome arms. They are also frequently accumulated in sex chromosomes. The function of these clusters is to sustain the architecture of the chromosomes and the nucleus, and to regulate chromosome behavior during mitosis and meiosis. The study of satellite DNA diversity is important for understanding sex chromosome evolution, interspecific hybridization, and speciation. In this work, we identified four satellite DNA families in the genomes of two snakes from different families: Daboia russelii (Viperidae) and Pantherophis guttatus (Colubridae) and determine their chromosomal localization. We found that one family is localized in the centromeres of both species, whereas the others form clusters in certain chromosomes or subsets of chromosomes. BLAST with snake genome assemblies showed the conservation of such clusters, as well as a subtle presence of the satellites in the interspersed manner outside the clusters. Overall, our results show high conservation of satellite DNA in snakes and confirm the "library" model of satellite DNA evolution.

RBD-Protein/Peptide Vaccine UB-612 Elicits Mucosal and Fc-Mediated Antibody Responses against SARS-CoV-2 in Cynomolgus Macaques.

Antibodies provide critical protective immunity against COVID-19, and the Fc-mediated effector functions and mucosal antibodies also contribute to the protection. To expand the characterization of humoral immunity stimulated by subunit protein-peptide COVID-19 vaccine UB-612, preclinical studies in non-human primates were undertaken to investigate mucosal secretion and the effector functionality of vaccine-induced antibodies in antibody-dependent monocyte phagocytosis (ADMP) and antibody-dependent NK cell activation (ADNKA) assays. In cynomolgus macaques, UB-612 induced potent serum-neutralizing, RBD-specific IgG binding, ACE2 binding-inhibition antibodies, and antibodies with Fc-mediated effector functions in ADMP and ADNKA assays. Additionally, immunized animals developed mucosal antibodies in bronchoalveolar lavage fluids (BAL). The level of mucosal or serum ADMP and ADNKA antibodies was found to be UB-612 dose-dependent. Our results highlight that the novel subunit UB-612 vaccine is a potent B-cell immunogen inducing polyfunctional antibody responses contributing to anti-viral immunity and vaccine efficacy.

Identification of Iguania Ancestral Syntenic Blocks and Putative Sex Chromosomes in the Veiled Chameleon (Chamaeleo calyptratus, Chamaeleonidae, Iguania).

The veiled chameleon (Chamaeleo calyptratus) is a typical member of the family Chamaeleonidae and a promising object for comparative cytogenetics and genomics. The karyotype of C. calyptratus differs from the putative ancestral chameleon karyotype (2n = 36) due to a smaller chromosome number (2n = 24) resulting from multiple chromosome fusions. The homomorphic sex chromosomes of an XX/XY system were described recently using male-specific RADseq markers. However, the chromosomal pair carrying these markers was not identified. Here we obtained chromosome-specific DNA libraries of C. calyptratus by chromosome flow sorting that were assigned by FISH and sequenced. Sequence comparison with three squamate reptiles reference genomes revealed the ancestral syntenic regions in the C. calyptratus chromosomes. We demonstrated that reducing the chromosome number in the C. calyptratus karyotype occurred through two fusions between microchromosomes and four fusions between micro-and macrochromosomes. PCR-assisted mapping of a previously described Y-specific marker indicates that chromosome 5 may be the sex chromosome pair. One of the chromosome 5 conserved synteny blocks shares homology with the ancestral pleurodont X chromosome, assuming parallelism in the evolution of sex chromosomes from two basal Iguania clades (pleurodonts and acrodonts). The comparative chromosome map produced here can serve as the foundation for future genome assembly of chameleons and vertebrate-wide comparative genomic studies.

A novel RBD-protein/peptide vaccine elicits broadly neutralizing antibodies and protects mice and macaques against SARS-CoV-2.

The development of safe and effective vaccines to respond to COVID-19 pandemic/endemic remains a priority. We developed a novel subunit protein-peptide COVID-19 vaccine candidate (UB-612) composed of: (i) receptor binding domain of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc; (ii) five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins (three from S2 subunit, one from membrane and one from nucleocapsid), and one universal Th peptide; (iii) aluminum phosphate as adjuvant. The immunogenicity and protective immunity induced by UB-612 vaccine were evaluated in four animal models: Sprague-Dawley rats, AAV-hACE2 transduced BALB/c mice, rhesus and cynomolgus macaques. UB-612 vaccine induced high levels of neutralizing antibody and T-cell responses, in all animals. The immune sera from vaccinated animals neutralized the SARS-CoV-2 original wild-type strains and multiple variants of concern, including Delta and Omicron. The vaccination significantly reduced viral loads, lung pathology scores, and disease progression after intranasal and intratracheal challenge with SARS-CoV-2 in mice, rhesus and cynomolgus macaques. UB-612 has been tested in primary regimens in Phase 1 and Phase 2 clinical studies and is currently being evaluated in a global pivotal Phase 3 clinical study as a single dose heterologous booster.

Autoimmune Effect of Antibodies against the SARS-CoV-2 Nucleoprotein.

COVID-19 caused by SARS-CoV-2 is continuing to spread around the world and drastically affect our daily life. New strains appear, and the severity of the course of the disease itself seems to be decreasing, but even people who have been ill on an outpatient basis suffer post-COVID consequences. Partly, it is associated with the autoimmune reactions, so debates about the development of new vaccines and the need for vaccination/revaccination continue. In this study we performed an analysis of the antibody response of patients with COVID-19 to linear and conformational epitopes of viral proteins using ELISA, chip array and western blot with analysis of correlations between antibody titer, disease severity, and complications. We have shown that the presence of IgG antibodies to the nucleoprotein can deteriorate the course of the disease, induce multiple direct COVID-19 symptoms, and contribute to long-term post-covid symptoms. We analyzed the cross reactivity of antibodies to SARS-CoV-2 with own human proteins and showed that antibodies to the nucleocapsid protein can bind to human proteins. In accordance with the possibility of HLA presentation, the main possible targets of the autoantibodies were identified. People with HLA alleles A01:01; A26:01; B39:01; B15:01 are most susceptible to the development of autoimmune processes after COVID-19.

Filterability of Erythrocytes in Patients with COVID-19.

For the first time, the influence of COVID-19 on blood microrheology was studied. For this, the method of filtering erythrocytes through filters with pores of 3.5 µm was used. Filterability was shown to significantly decrease with the increasing severity of the patient's condition, as well as with a decrease in the ratio of hemoglobin oxygen saturation to the oxygen fraction in the inhaled air (SpO2/FiO2). The filterability of ≤ 0.65, or its fast decrease during treatment, were indicators of a poor prognosis. Filterability increased significantly with an increase in erythrocyte count, hematocrit and blood concentrations of hemoglobin, albumin, and total protein. The effect of these parameters on the erythrocyte filterability is directly opposite to their effect on blood macrorheology, where they all increase blood viscosity, worsening the erythrocyte deformability. The erythrocyte filterability decreased with increasing oxygen supply rate, especially in patients on mechanical ventilation, apparently not due to the oxygen supplied, but to the deterioration of the patients' condition. Filterability significantly correlates with the C-reactive protein, which indicates that inflammation affects the blood microrheology in the capillaries. Thus, the filterability of erythrocytes is a good tool for studying the severity of the patient's condition and his prognosis in COVID-19.

High Neutralizing Antibody Levels Against Severe Acute Respiratory Syndrome Coronavirus 2 Omicron BA.1 and BA.2 After UB-612 Vaccine Booster.

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has caused high rates of breakthrough infections in those previously vaccinated with ancestral strain coronavirus disease 2019 (COVID-19) vaccines. Here, we demonstrate that a booster dose of UB-612 vaccine candidate delivered 7-9 months after primary vaccination increased neutralizing antibody levels by 131-, 61-, and 49-fold against ancestral SARS-CoV-2 and the Omicron BA.1 and BA.2 variants, respectively. Based on the receptor-binding domain protein binding antibody responses, the UB-612 third-dose booster may lead to an estimated approximately 95% efficacy against symptomatic COVID-19 caused by the ancestral strain. Our results support UB-612 as a potential potent booster against current and emerging SARS-CoV-2 variants.

Longitudinal multiparametric characterization of platelet dysfunction in COVID-19: Effects of disease severity, anticoagulation therapy and inflammatory status.

INTRODUCTION: Defects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19. MATERIALS AND METHODS: Analysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed. RESULTS AND CONCLUSIONS: All patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber. We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus.

16S rRNA gene sequencing data of the upper respiratory tract microbiome in the SARS-CoV-2 infected patients.

The SARS-CoV-2 pandemic is a big challenge for humanity. The COVID-19 severity differs significantly from patient to patient, and it is important to study the factors protecting from severe forms of the disease. Respiratory microbiota may influence the patient's susceptibility to infection and disease severity due to its ability to modulate the immune system response of the host organism. This data article describes the microbiome dataset from the upper respiratory tract of SARS-CoV-2 positive patients from Russia. This dataset reports the microbial community profile of 335 human nasopharyngeal swabs collected between 2020-05 and 2021-03 during the first and the second epidemic waves. Samples were collected from both inpatients and outpatients in 4 cities of the Russian Federation (Moscow, Kazan, Irkutsk, Nizhny Novgorod) and sequenced using the 16S rRNA gene amplicon sequencing of V3-V4 region. Data contains information about the patient such as age, sex, hospitalization status, percent of damaged lung tissue, oxygen saturation (SpO2), respiratory rate, need for supplemental oxygen, chest computer tomography severity score, SARS-CoV-2 lineage, and also information about smoking and comorbidities. The amplicon sequencing data were deposited at NCBI SRA as BioProject PRJNA751478.

TREC/KREC Levels in Young COVID-19 Patients.

COVID-19 patients with acute respiratory distress syndrome (ARDS) have an immune imbalance when systemic inflammation and dysfunction of circulating T and B cells lead to a more severe disease. Using TREC/KREC analysis, we studied the level of mature naive T and B cells in peripheral blood of COVID-19 patients and its relationship with clinical and laboratory data. TREC/KREC analysis was performed by multiplex real-time quantitative PCR on a sample of 36 patients aged 45 years or younger. The reduced TREC/KREC level was observed in ARDS patients compared with non-ARDS patients, and similar results were found for the deceased patients. During days 6 to 20 of hospitalization, a higher neutrophil-to-lymphocyte ratio (NLR) was detected in ARDS patients compared with non-ARDS patients. TREC/KREC negatively correlated with NLR; the highest correlation was recorded for TREC per 100,000 cells with the coefficient of determination R2 = 0.527. Thus, TREC/KREC analysis is a potential prognostic marker for assessing the severity and outcome in COVID-19.

Analysis of a Queueing Model with Batch Markovian Arrival Process and General Distribution for Group Clearance.

In this paper we consider a single server queueing model with under general bulk service rule with infinite upper bound on the batch size which we call group clearance. The arrivals occur according to a batch Markovian point process and the services are generally distributed. The customers arriving after the service initiation cannot enter the ongoing service. The service time is independent on the batch size. First, we employ the classical embedded Markov renewal process approach to study the model. Secondly, under the assumption that the services are of phase type, we study the model as a continuous-time Markov chain whose generator has a very special structure. Using matrix-analytic methods we study the model in steady-state and discuss some special cases of the model as well as representative numerical examples covering a wide range of service time distributions such as constant, uniform, Weibull, and phase type.

Magnetic resonance imaging of changes in the brain of children cured of acute lymphoblastic leukemia.

This study was aimed to systematize magnetic resonance imaging (MRI) presentation of toxic leukoencephalopathy, to find the correlation between method of central nervous system (CNS) leukemia prevention and changes on MRI, to find relationship between existence leukoencephalopathy on imaging and neurocognitive deficits in pediatric patients after anti-leukemic therapy. Brain MRI data of 48 children, who underwent a therapy course according to the ALL-MB intermediate risk protocol, was evaluated. In accordance with two arms of this protocol, they received either radiation therapy, or additional intrathecal administration of chemotherapeutic agents as a prevention of CNS leukemia. Also, neurocognitive tests were performed. According to the results of the performed investigation, 10 (50%) out of 20 children, who received cranial irradiation and 18 (66.6%) out of 27 patients, who received only intrathecal therapy demonstrated abnormal brain changes (leukoencephalopathy) according to MRI data. Leukoencephalopathy was mostly presented by diffuse zones and localized predominantly in the frontal and temporal lobes. There was no correlation between method of CNS prevention and the existence of leukoencephalopathy on MRI. The analysis of our data did not show significant differences in brain damage and severity of cognitive impairment depending on the type of prevention of CNS leukemia. Moreover, in this study no statistical correlation was found between leukoencephalopathy on MRI and neurocognitive impairment according to clinical tests data. Further long-term prospective studies and examinations should be performed to assess late neurotoxic effects.

αß T Cell-Depleted Haploidentical Hematopoietic Stem Cell Transplantation without Antithymocyte Globulin in Children with Chemorefractory Acute Myelogenous Leukemia.

We evaluated the outcome of αß T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT) in a cohort of children with chemorefractory acute myelogenous leukemia (AML). Twenty-two patients with either primary refractory (n = 10) or relapsed refractory (n = 12) AML in active disease status received a transplant from haploidentical donors. The preparative regimen included cytoreduction with fludarabine and cytarabine and subsequent myeloablative conditioning with treosulfan and thiotepa. Antithymocyte globulin was substituted with tocilizumab in all patients and also with abatacept in 10 patients. Grafts were peripheral blood stem cells engineered by αß T cell and CD19 depletion. Post-transplantation prophylactic therapy included infusion of donor lymphocytes, composed of a CD45RA-depleted fraction with or without a hypomethylating agent. Complete remission was achieved in 21 patients (95%). The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) was 18%, and the cumulative incidence of chronic GVHD was 23%. At 2 years, transplantation-related mortality was 9%, relapse rate was 42%, event-free survival was 49%, and overall survival was 53%. Our data suggest that αß T cell-depleted haploidentical HSCT provides a reasonable chance of long-term survival in a cohort of children with chemorefractory AML and creates a solid basis for further improvement.

PPP3CB contributes to poor prognosis through activating nuclear factor of activated T-cells signaling in neuroblastoma.

We previously identified a gain-of-function mutation in PPP3CB in a neuroblastoma (NB) with MYCN amplification. Here we investigated the functional and clinical role of PPP3CB in NB. High PPP3CB expression was an independent indicator predicting poor prognosis of NB. Overexpression of wildtype or mutated PPP3CB (PPP3CBmut) promoted cell growth, but PPP3CB knockdown decreased cell growth in NB cells. Forced expressions of PPP3CB and PPP3CBmut activated NFAT2 and NFAT4 transcription factors and inhibited GSK3ß activity, resulting in the increase in the expressions of c-Myc, MYCN, and ß-catenin, which were downregulated in response to PPP3CB knockdown. Treatment with calcineurin inhibitor cyclosporin A (CsA) or FK506 suppressed cell proliferation and induced apoptotic cell death in both MYCN-amplified and MYCN-non-amplified NB cell lines. Expression of PPP3CB protein was decreased in response to two calcineurin inhibitors. c-Myc, MYCN, and ß-catenin were downregulated at the mRNA and protein levels in CsA or FK506-treated NB cells. Our data indicate that elevated expression of PPP3CB and the expression of its constitutively active mutant contribute to the aggressive behavior of NB tumors and therefore suggest that inhibition of calcineurin activity might have therapeutic potential for high-risk NB.

A novel approach to a rabies vaccine based on a recombinant single-cycle flavivirus vector.

The RepliVax® vaccine (RV) platform is based on flavivirus genomes that are rationally attenuated by deletion. These single-cycle RV vaccine candidates targeting flavivirus pathogens have been demonstrated to be safe, highly immunogenic, and efficacious in animal models, including non-human primates. Here we show utility of the technology for delivery of a non-flavivirus immunogen by engineering several West Nile-based RV vectors to express full-length rabies virus G protein. The rabies virus G protein gene was incorporated in place of different West Nile structural protein gene deletions. The resulting RV-RabG constructs were demonstrated to replicate to high titers (8 log10 infectious particles/ml) in complementing helper cells. Following infection of normal cells, they provided efficient rabies virus G protein expression, but did not spread to surrounding cells. Expression of rabies virus G protein was stable and maintained through multiple rounds of in vitro passaging. A sensitive neurovirulence test in 2-3 day old neonatal mice demonstrated that RV-RabG candidates were completely avirulent indicative of high safety. We evaluated the RV-RabG variants in several animal models (mice, dogs, and pigs) and demonstrated that a single dose elicited high titers of rabies virus-neutralizing antibodies and protected animals from live rabies virus challenge (mice and dogs). Importantly, dogs were protected at both one and two years post-immunization, demonstrating durable protective immunity. The data demonstrates the potential of the RepliVax® technology as a potent vector delivery platform for developing vaccine candidates against non-flavivirus targets.

Fermented Milk Consumption and Common Infections in Children Attending Day-Care Centers: A Randomized Trial.

OBJECTIVES: This multicenter, double-blind, randomized, placebo-controlled clinical trial investigated the effect of a fermented milk product containing the Lactobacillus casei National Collection of Microorganisms and Cell Cultures (CNCM) I-1518 strain on respiratory and gastrointestinal common infectious diseases (CIDs) in children attending day-care centers in Russia. METHODS: Children ages 3 to 6 years received 100 g of a fermented milk product (n = 300) or a control product (n = 299) twice daily for 3 months, followed by a 1-month observation period. The primary outcome was the incidence of CIDs during the product consumption period. RESULTS: There was no significant difference in the incidence of CIDs between the groups (N = 98 with fermented milk product vs N = 93 with control product). The overall number of CIDs (and no severe cases at all) in both study groups and in all 12 centers, however, was unexpectedly low resulting in underpowering of the study. No differences were found between the groups in the duration or severity of disease, duration of sick leave from day-care centers, parental missed working days, or in quality-of-life dimensions on the PedsQL questionnaire (P > 0.05).There was, however, a significantly lower incidence of the most frequently observed CID, rhinopharyngitis, in children consuming the fermented milk product compared with those consuming the control product (N = 81 vs N = 100, relative risk 0.82, 95% confidence interval 0.69-0.96, P = 0.017) when considering the entire study period. CONCLUSIONS: Although no other significant differences were shown between the fermented milk and control product groups in this study, lower incidence of rhinopharyngitis may indicate a beneficial effect of this fermented milk product.

Therapy of advanced-stage mature B-cell lymphoma and leukemia in children and adolescents with rituximab and reduced intensity induction chemotherapy (B-NHL 2004M protocol): the results of a multicenter study.

Pediatric mature B-cell non-Hodgkin lymphomas (B-NHLs) are highly aggressive malignant tumors that are curable with chemotherapy (ChT). High-dose methotrexate (MTX) is considered indispensable for successful treatment, but this therapy frequently induces severe mucositis and infectious complications, especially in induction, which can cause treatment failure. A prospective multicenter trial of combined immunochemotherapy for advanced-stage B-NHL with rituximab and the modified NHL-BFM-90 protocol was conducted. The major differences from the original protocol were a decrease in the dose of MTX from 5000 to 1000 mg/m/24 h in the first 2 ChT blocks and the addition of rituximab at 375 mg/m to each of the first 4 blocks of ChT. Eighty-three newly diagnosed patients with a median age of 8.84 years with Burkitt lymphoma/leukemia and diffuse large B-cell lymphomas stage III to IV were included. Four patients died during induction ChT due to tumor lysis syndrome and infection. Two additional patients died subsequently due to tumor resistance. Complete remission was achieved in 77 (92.8%) patients; 2 patients relapsed at 1 and 3 months, and 2 developed secondary malignancies at 1 and 6.5 years, respectively, after the completion of therapy. The overall survival probability was 82%±8% with a median follow-up of 65.2 months. Combined therapy with rituximab and intensive ChT with a reduced MTX dose of 1 g/m in the 2 induction courses was feasible and produced high cure rates in patients with pediatric advanced-stage mature B-NHL.

Single-dose vaccine against tick-borne encephalitis.

Tick-borne encephalitis (TBE) virus is the most important human pathogen transmitted by ticks in Eurasia. Inactivated vaccines are available but require multiple doses and frequent boosters to induce and maintain immunity. Thus far, the goal of developing a safe, live attenuated vaccine effective after a single dose has remained elusive. Here we used a replication-defective (single-cycle) flavivirus platform, RepliVax, to generate a safe, single-dose TBE vaccine. Several RepliVax-TBE candidates attenuated by a deletion in the capsid gene were constructed using different flavivirus backbones containing the envelope genes of TBE virus. RepliVax-TBE based on a West Nile virus backbone (RV-WN/TBE) grew more efficiently in helper cells than candidates based on Langat E5, TBE, and yellow fever 17D backbones, and was found to be highly immunogenic and efficacious in mice. Live chimeric yellow fever 17D/TBE, Dengue 2/TBE, and Langat E5/TBE candidates were also constructed but were found to be underattenuated. RV-WN/TBE was demonstrated to be highly immunogenic in Rhesus macaques after a single dose, inducing a significantly more durable humoral immune response compared with three doses of a licensed, adjuvanted human inactivated vaccine. Its immunogenicity was not significantly affected by preexisting immunity against WN. Immunized monkeys were protected from a stringent surrogate challenge. These results support the identification of a single-cycle TBE vaccine with a superior product profile to existing inactivated vaccines, which could lead to improved vaccine coverage and control of the disease.

Characterization of the RepliVax platform for replication-defective flavivirus vaccines.

RepliVax, a novel replication-defective vaccine platform has recently been described as a suitable means of generating potent vaccines targeting flaviviruses. In this study, we directly compared attenuation, immunogenicity and efficacy of several prototype RepliVax constructs to available, well characterized live attenuated (LAV) and inactivated (INV) flavivirus vaccine controls in mice and hamsters. Other important aspects of general mechanisms and properties of RepliVax vaccines were also studied. The prototypes were found to be nonpathogenic in sensitive suckling mouse neurovirulence tests, and highly immunogenic and efficacious in mice and hamsters, with evidence that immunogenicity can be comparable to LAV controls in terms of both magnitude and durability of response. Our data also suggest that choice of inoculation route can be beneficial for maximizing RepliVax immunogenicity. Additionally, different vaccine constructs can be administered as cocktail formulations without compromising immunogenicity of individual components. RepliVax constructs were determined to induce a Th1 biased immune response, similar to LAVs, and different from INV inducing a Th2 type response. The results presented validate the utility of the RepliVax platform for development of novel flavivirus vaccines.

The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome.

Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E(315)) and NS5 (NS5(654,655)) proteins, and into the 3' non-coding region (Delta30) of TBEV/DEN4. The variant that contained all three mutations (vDelta30/E(315)/NS5(654,655)) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vDelta30/E(315)/NS5(654,655) should be further evaluated as a TBEV vaccine.