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Objective : We evaluate the immunotherapeutic potential of the yellow fever virus vaccine strain 17D (YFV 17D) for intratumoral therapy of pancreatic cancer in mice. Methods : The cytopathic effect of YFV 17D on mouse syngeneic pancreatic cancers cells were studied both in vitro and in vivo and on human pancreatic cancers cells in vitro. Results : YFV 17D demonstrated a strong cytopathic effect against human cancer cells in vitro. Although YFV 17D did not exhibit a lytic effect against Pan02 mouse cells in vitro, a single intratumoral administration of 17D caused a delay in tumor growth and an increase in median survival by 30%. Multiple injections of 17D did not further improve the effect on tumor growth; however, it notably extended the median survival. Furthermore, preliminary immunization with 17D enhanced its oncotherapeutic effect. Conclusions : Intratumoral administration of yellow fever virus vaccine strain 17D delayed tumor in a murine model of pancreatic cancer. The fact that YFV 17D in vitro affected human cancer cells much more strongly than mouse cancer cells appears promising. Hence, we anticipate that the in vivo efficacy of YFV-17D-based oncolytic therapy will also be higher against human pancreatic carcinomas compared to its effect on the mouse pancreatic tumor.

Oncolytic viruses represent a promising class of immunotherapeutic agents for the treatment of malignant tumors. The proposed mechanism of action of various oncolytic viruses has initially been explained by the ability of such viruses to selectively lyse tumor cells without damaging healthy ones. Recently, there have emerged more studies determining the effect of the antiviral immunostimulating mechanisms on the effectiveness of treatment in cancer patients. Stimulation of innate immune cells by an oncolytic virus can initiate an adaptive antitumor immune response, yet at the same time, the antiviral mechanisms of the immune system can limit the spread of the virus, thereby reducing its effectiveness. Thus, the success of the clinical application of the oncolytic viruses directly depends on the three key components: tumor immunosuppression, antiviral responses, and antitumor immune responses. The review presents current data on the influence of pattern recognition receptors on the effectiveness of oncolytic viruses.

Combined viral and photodynamic therapy for oncological diseases has great potential to treat aggressive tumors such as glioblastomas. A conjugate of vesicular stomatitis virus (VSV) with protoporphyrin IX was prepared, and its oncolytic effects were studied and compared to the effects of the individual components. The VSV showed an oncolytic effect on glioblastoma cell lines T98G and LN229 at a virus titer of 10 5 TCID 50 /mL. A VSV titer of 10 4 TCID 50 /mL was sufficient for neuroblastoma cell death. A study of the effect of VSV in tumor 3D cell modeling found that VSV had a clear viral cytopathic effect on spheroids of T98G and LN229 cells. Conjugation with the porphyrin significantly reduced the viral titer, but when irradiated, lysis of cells was observed. Photodynamic treatment of T98G and LN229 cells and spheroids with protoporphyrin IX as a photosensitizer also had a cytotoxic effect on cells and, to a lesser extent, on the tumoroids, as complete cell death was not achieved for the tumoroids. The combination therapy, which involved sequential photodynamic therapy using protoporphyrin IX as a photosensitizer and treatment with VSV, was shown to significantly enhance efficacy, resulting in complete cell death of both T98G and LN229 cells and tumoroids. The combination treatment allowed for the use of a lower viral titer (10 3 -10 4 TCID 50 /mL) and a lower porphyrin concentration (0.5 μg/mL) to achieve a significant cytotoxic effect. As a result, the implementation of this combination therapy would likely lead to fewer side effects from the treatment. This study clearly demonstrated the excellent perspectives of combination therapy for the treatment of highly aggressive tumors such as glioblastomas.

Introduction: Flaviviruses, possessing natural neurotropicity could be used in glioblastoma therapy using attenuated strains or as a delivery system for antitumor agents in an inactivated form., Objective: To investigate the sensitivity of glioblastoma and pancreatic carcinoma cell lines to vaccine strains of yellow fever and tick-borne encephalitis viruses., Materials and Methods: Cell lines: glioblastoma GL-6, T98G, LN-229, pancreatic carcinoma MIA RaCa-2 and human pancreatic ductal carcinoma PANC-1. Viral strains: 17D yellow fever virus (YF), Sofjin tick-borne encephalitis virus (TBEV). Virus concentration were determined by plaque assay and quantitative PCR. Determination of cell sensitivity to viruses by MTT assay., Results: 17D YF was effective only against pancreatic carcinoma tumor cells MIA Paca-2 and had a limited effect against PANC-1. In glioblastoma cell lines (LN229, GL6, T98G), virus had no oncolytic effect and the viral RNA concentration fell in the culture medium. Sofjin TBEV showed CPE 50 against MIA Paca-2 and a very limited cytotoxic effect against PANC-1. However, it had no oncolytic effect against glioblastoma cell lines (LN229, T98G and GL6), although virus reproduction continued in these cultures. For the GL6 glioblastoma cell line, the viral RNA concentration at the level with the infection dose was determined within 13 days, despite medium replacement, while in the case of the LN229 cell line, the virus concentration increased from 1 × 10 9 to 1 × 10 10 copies/ml., Conclusion: Tumor behavior in organism is more complex and is determined by different microenvironmental factors and immune status. In the future, it is advisable to continue studying the antitumor oncolytic and immunomodulatory effects of viral strains 17D YF and Sofjin TBEV using in vivo models.

Oncolytic viruses offer a promising approach to tumor treatment. These viruses not only have a direct lytic effect on tumor cells but can also modify the tumor microenvironment and activate antitumor immunity. Due to their high pathogenicity, flaviviruses have often been overlooked as potential antitumor agents. However, with recent advancements in genetic engineering techniques, an extensive history with vaccine strains, and the development of new attenuated vaccine strains, there has been a renewed interest in the Flavivirus genus. Flaviviruses can be genetically modified to express transgenes at acceptable levels, and the stability of such constructs has been greatly improving over the years. The key advantages of flaviviruses include their reproduction cycle occurring entirely within the cytoplasm (avoiding genome integration) and their ability to cross the blood-brain barrier, facilitating the systemic delivery of oncolytics against brain tumors. So far, the direct lytic effects and immunomodulatory activities of many flaviviruses have been widely studied in experimental animal models across various types of tumors. In this review, we delve into the findings of these studies and contemplate the promising potential of flaviviruses in oncolytic therapies.

The idea of using the lytic power of viruses against malignant cells has been entertained for many decades. However, oncolytic viruses gained broad attention as an emerging anti-cancer therapy only recently with the successful implementation of several oncolytic viruses to treat advanced melanoma. Here we review the history of oncolytic viruses in the Russian Federation and recent biotechnological advances in connection with the perspectives of their practical use against aggressive tumors such as glioblastoma or pancreatic cancer. A particular emphasis is made on novel applications of safe non-lytic virus-derived vectors armed with prodrug-converting enzyme transgenes. Rational improvement of oncotropism by conjugation with biopolymers and nanoformulations is also discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Current serological tests for the emerging tick-borne pathogen Borrelia miyamotoi lack diagnostic accuracy. To improve serodiagnosis, we investigated a protein array simultaneously screening for IgM and IgG reactivity against multiple recombinant B. miyamotoi antigens. The array included six B. miyamotoi antigens: glycerophosphodiester phosphodiesterase (GlpQ), multiple variable major proteins (Vmps), and flagellin. Sera included samples from cases of PCR-proven Borrelia miyamotoi disease (BMD), multiple potentially cross-reactive control groups (including patients with culture-proven Lyme borreliosis, confirmed Epstein-Barr virus, cytomegalovirus, or other spirochetal infections), and several healthy control groups from regions where Ixodes is endemic and regions where it is nonendemic. Based on receiver operating characteristic (ROC) analyses, the cutoff for reactivity per antigen was set at 5 μg/mL for IgM and IgG. The individual antigens demonstrated high sensitivity but relatively low specificity for both IgM and IgG. The best-performing single antigen (GlpQ) showed a sensitivity of 88.0% (95% confidence interval [CI], 78.9 to 93.5) and a specificity of 94.2% (95% CI, 92.7 to 95.6) for IgM/IgG. Applying the previous published diagnostic algorithm-defining seroreactivity as reactivity against GlpQ and any Vmp-revealed a significantly higher specificity of 98.5% (95% CI, 97.6 to 99.2) but a significantly lower sensitivity of 79.5% (95% CI, 69.3 to 87.0) for IgM/IgG compared to GlpQ alone. Therefore, we propose to define seroreactivity as reactivity against GlpQ and any Vmp or flagellin which resulted in a comparable sensitivity of 84.3% (95% CI, 74.7 to 90.8) and a significantly higher specificity of 97.9% (95% CI, 96.9 to 98.7) for IgM/IgG compared to GlpQ alone. In conclusion, we have developed and validated a novel serological tool to diagnose BMD that could be implemented in clinical practice and epidemiological studies. IMPORTANCE This paper describes the protein array as a novel serological test for the diagnosis of Borrelia miyamotoi disease (BMD), by reporting the methodology, the development of a diagnostic algorithm, and its extensive validation. With rising numbers of ticks and tick bites, tick-borne diseases, such as BMD, urgently deserve further societal and medical attention. B. miyamotoi is prevalent in Ixodes ticks across the northern hemisphere. Humans are exposed to, and infected by, B. miyamotoi and develop BMD in Asia, in North America, and to a lesser extent in Europe. However, the burden of infection and disease remains largely unknown, due to the noncharacteristic clinical presentation, together with the lack of awareness and availability of diagnostic tools. With this paper, we offer a novel diagnostic tool which will assist in assessing the burden of disease and could be implemented in clinical care.

The review provides a brief historical outline of the discovery and study of tick-borne encephalitis (TBE) during three expeditions to the Far East (19371939). As a result of the Far Eastern expeditions, the TBE virus was discovered, numerous strains were isolated, a vector-borne transmission pathway was established, the main features of epidemiology, clinic and pathomorphology of the disease were described, serotherapy was tested, first inactivated "brain" vaccine against TBE was developed and its effectiveness was proved. The history of the discovery and study of TBE is marked by the heroism of researchers and tragic events illness of some participants, death during the development of the first vaccine, arrest of L.A. Zilber and two performers.

Background: HIV infection is a major health problem in Russia. We aimed to assess HIV prevalence in different population groups and to compare the characteristics of 4th generation immunoassays from Abbott, Bio-Rad, Vector-Best, Diagnostic Systems, and Medical Biological Unit., Methods: The study included 4452 individuals from the general population (GP), 391 subjects at high risk of HIV infection (HR) and 699 with potentially interfering conditions. HIV positivity was confirmed by immunoblot and by HIV RNA, seroconversion and virus diversity panels were also used. HIV avidity was employed to assess recent infections., Results: The prevalence in GP was 0.40%, higher in males (0.62%) and in people aged < 40 years (0.58%). Patients attending dermo-venereal centers and drug users had a high prevalence (34.1 and 58.8%). Recent infections were diagnosed in 20% of GP and in 4.2% of HR. Assay sensitivity was 100% except for one false negative (99,54%, MBU). Specificity was 99.58-99.89% overall, but as low as 93.26% on HR (Vector-Best). Small differences on early seroconversion were recorded. Only the Abbott assay detected all samples on the viral diversity panel., Conclusion: HIV infection rate in the high-risk groups suggests that awareness and screening campaigns should be enhanced. Fourth generation assays are adequate but performance differences must be considered.

Background: The genus Borrelia comprises spirochaetal bacteria maintained in natural transmission cycles by tick vectors and vertebrate reservoir hosts. The main groups are represented by a species complex including the causative agents of Lyme borreliosis and relapsing fever group Borrelia. Borrelia miyamotoi belongs to the relapsing fever group of spirochetes and forms distinct populations in North America, Asia, and Europe. As all Borrelia species B. miyamotoi possess an unusual and complex genome consisting of a linear chromosome and a number of linear and circular plasmids. The species is considered an emerging human pathogen and an increasing number of human cases are being described in the Northern hemisphere. The aim of this study was to produce a high quality reference genome that will facilitate future studies into genetic differences between different populations and the genome plasticity of B. miyamotoi., Results: We used multiple available sequencing methods, including Pacific Bioscience single-molecule real-time technology (SMRT) and Oxford Nanopore technology (ONT) supplemented with highly accurate Illumina sequences, to explore the suitability for whole genome assembly of the Russian B. miyamotoi isolate, Izh-4. Plasmids were typed according to their potential plasmid partitioning genes (PF32, 49, 50, 57/62). Comparing and combining results of both long-read (SMRT and ONT) and short-read methods (Illumina), we determined that the genome of the isolate Izh-4 consisted of one linear chromosome, 12 linear and two circular plasmids. Whilst the majority of plasmids had corresponding contigs in the Asian B. miyamotoi isolate FR64b, there were only four that matched plasmids of the North American isolate CT13-2396, indicating differences between B. miyamotoi populations. Several plasmids, e.g. lp41, lp29, lp23, and lp24, were found to carry variable major proteins. Amongst those were variable large proteins (Vlp) subtype Vlp-α, Vlp-γ, Vlp-δ and also Vlp-β. Phylogenetic analysis of common plasmids types showed the uniqueness in Russian/Asian isolates of B. miyamotoi compared to other isolates., Conclusions: We here describe the genome of a Russian B. miyamotoi clinical isolate, providing a solid basis for future comparative genomics of B. miyamotoi isolates. This will be a great impetus for further basic, molecular and epidemiological research on this emerging tick-borne pathogen.

Objectives: Borrelia miyamotoi disease (BMD) is an emerging tick-borne disease in the Northern hemisphere. Serodiagnosis by measuring antibodies against glycerophosphodiester-phosphodiesterase (GlpQ) has been performed experimentally but has not been extensively clinically validated. Because we had previously shown the differential expression of antigenic variable major proteins (Vmps) in B. miyamotoi, our aim was to study antibody responses against GlpQ and Vmps in PCR-proven BMD patients and controls., Methods: We assessed seroreactivity against GlpQ and four Vmps in a well-described, longitudinal cohort of sera from BMD patients (n=182), healthy blood donors (n=136) and controls (n=68). All samples were tested by ELISA and positive sera were tested by western blot, and antibody dynamics and diagnostic value were assessed., Results: IgM antibodies against GlpQ and Vmps peaked between 11 and 20 days, and IgG between 21 and 50 days, after disease onset. Various combinations of GlpQ and Vmps increased sensitivity and/or specificity compared to single antigens. Notably, the GlpQ or variable large protein (Vlp)-15/16 combination yielded a sensitivity of 94.7% (95% CI: 75.4-99.7) 11-20 days after disease onset and a specificity of 96.6% (92.7-98.4) for IgM. A specificity of 100% (97.8-100) for IgM, and 98.3% for IgG (95.2-100), was found when positivity was defined as reactivity to GlpQ and any Vmp, with maximum sensitivities of 79% (56.7-91.5) for IgM and 86.7% (62.1-97.6) for IgG., Conclusions: We clearly demonstrate here the diagnostic potential of these seromarkers. Our findings will facilitate future epidemiological and clinical studies on BMD and lead to the development of a serologic test to be used in clinical practice., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Borrelia miyamotoi is an emerging relapsing fever (RF) Borrelia species that is reported to cause human disease in regions in which Lyme borreliosis is endemic. We recently showed that B. miyamotoi tick isolates are resistant to amoxicillin in vitro ; however, clinical isolates have not been studied. Therefore, our aim was to show the antimicrobial susceptibility of recently obtained clinical isolates of B. miyamotoi A dilution series of various antibiotics was made in modified Kelly-Pettenkofer medium with 10% fetal calf serum. The susceptibilities of different B. miyamotoi clinical, B. miyamotoi tick, RF Borrelia , and Borrelia burgdorferi sensu lato isolates were tested by measuring MICs through colorimetric changes and by counting motile spirochetes by dark-field microscopy after 72 h of incubation. The ceftriaxone and azithromycin MIC ranges of the six B. miyamotoi clinical isolates tested were 0.03 to 0.06 mg/liter and 0.0016 to 0.0032 mg/liter, respectively. These values are similar to MICs for RF Borrelia strains and B. miyamotoi tick isolates. All tested RF Borrelia strains were susceptible to doxycycline (microscopic MIC range, 0.0625 to 0.25 mg/liter). In contrast to the MICs of the tested B. burgdorferi sensu lato strains and in line with our previous findings, the amoxicillin MICs (range, 8 to 32 mg/liter) of all RF Borrelia strains, including B. miyamotoi clinical isolates, were above the clinical breakpoint for resistance (≤4 mg/liter). Clinical isolates of B. miyamotoi are highly susceptible to doxycycline, azithromycin, and ceftriaxone in vitro Interestingly, as described previously for tick isolates, amoxicillin shows poor in vitro activity against B. miyamotoi clinical isolates., (Copyright © 2018 American Society for Microbiology.)

Here, we report the whole-genome sequence of six clinical Borrelia miyamotoi isolates from the Russian Federation. Using two independent next-generation sequencing platforms, we determined the complete sequence of the chromosome and several plasmids. All strains have an Asian genotype with 99.8% chromosome nucleotide similarity with B. miyamotoi strain FR64b., (Copyright © 2018 Kuleshov et al.)

Objectives: Borrelia miyamotoi has been shown to infect humans in Eurasia and North America causing hard tick-borne relapsing fever (HTBRF). In vitro cultivation of B. miyamotoi was described recently; but clinical isolation of relapsing fever Borrelia is cumbersome. Our aim was to develop a straightforward protocol enabling B. miyamotoi isolation directly from the blood of patients., Methods: Modified Kelly-Pettenkorfer (MKP-F) medium, with or without anticoagulants, or blood from healthy human volunteers, was spiked with B. miyamotoi spirochaetes in vitro. Subsequently, either media or plasma was used for cultivation directly, or after an additional centrifugation step. This isolation protocol was tested in a clinical setting on patients suspected of HTBRF., Results: Dipotassium-EDTA, trisodium citrate and lithium heparin inhibited growth of B. miyamotoi at concentrations ≥250 μg/mL, 2.5 mM and 1 IU/mL, respectively. However, when plasma originating from human blood containing B. miyamotoi spirochaetes was subjected to an additional centrifugation step at 8000 g, suspended and inoculated into fresh MKP-F media, positive cultures were observed within 2 weeks. Of importance, this straightforward protocol allowed for isolation of B. miyamotoi from six out of nine patients with confirmed HTBRF., Conclusions: Direct culture from K 2 -EDTA, trisodium citrate and lithium heparin plasma containing B. miyamotoi is hampered due to anticoagulants. Using a simple centrifugation protocol we were able to circumvent this detrimental effect, allowing for the first clinical isolation of B. miyamotoi. This will be of value for future research on the pathogenesis, genetics, diagnosis, therapy and epidemiology of HTBRF and other tick-borne relapsing fevers., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Borrelia miyamotoi causes systemic febrile illness and is transmitted by the same tick species that transmits Borrelia burgdorferi sensu lato and tick-borne encephalitis virus. We describe a serological test using a fragment of glycerophosphodiester phosphodiesterase (GlpQ) as an antigen, and determined its performance in well-defined patient categories., Methods: Serum of patients with PCR-confirmed Borrelia miyamotoi disease (BMD), Lyme borreliosis (LB), tick-borne encephalitis (TBE), and healthy blood donors (HBD) were collected in Udmurt Republic, Russia. Sera of BMD and LB patients were collected at hospital admission, one week, one month and one year after admission., Results: The levels of IgM and IgG anti-GlpQ antibodies, determined as optical density values in Luminex bead-based assays, were significantly higher in the BMD patient group than in LB patients, TBE patients or HBD group (all p<0.05)., Conclusions: By using a strict cut-off value, it was possible to exclude B. miyamotoi infection in LB and TBE patients and to serologically confirm B. miyamotoi infection in 44% to 94% of the PCR-positive BMD patients (95% confidence interval). Thus, sensitive serological assays should not solely rely on rGlpQ, to support the diagnosis of acute BMD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ixodes tick-borne borreliosis caused by Borrelia miyamotoi (ITBB-BM) is a previously unknown infectious disease discovered in Russia., Aim: The present study continues the investigation of the clinical features of ITBB-BM in the context of an immune system-pathogen interaction., Subjects and Methods: The study enrolled 117 patients with ITBB-BM and a comparison group of 71 patients with Lyme disease (LD) that is ITBB with erythema migrans. All the patients were treated at the New Hospital, Yekateringburg. More than 100 clinical, epidemiological and laboratory parameters were obtained from each patient's medical history and included in the general database. A subset of patients hospitalized in 2015 and 2016 underwent additional laboratory examinations. Namely, the levels of B. miyamotoi-specific IgM and IgG antibodies were measured by the protein microarray containing GlpQ protein and four variable major proteins (VMPs): Vlp15/16, Vlp18, Vsp1, and Vlp5. The blood concentration of Borrelia was estimated by quantitative real-time PCR., Results: In contrast to LD, first of all (p<0.001) the following clinical features were typical for ITBB-BM: the absence of erythema migrans (in 95% of patients), fever (93%), fatigue (96%), headache (82%), chill (41%), nausea (28%), lymphopenia (56%), thrombocytopenia (46%), the abnormal levels of alanine aminotransferase (54%) and C-reactive protein (98%), proteinuria (61%). Given the set of these indicators, the course of ITBB-BM was more severe in approximately 70% of patients. At admission, only 13% and 38% of patients had antibodies to GlpQ and VMPs, respectively; at discharge, antibodies to GlpQ and VMPs were detected in 88% of patients. There was no statistically significant association of the antibody response with individual clinical manifestations and laboratory parameters of the disease. However, patients with more severe ITBB-BM produced less IgM antibodies to VMPs and GlpQ at the time of discharge., Conclusion: ITBB-BM is a moderate systemic disease accompanied by the production of specific antibodies in virtually all patients.

Since 1999, human cases of West Nile fever/neuroinvasive disease (WND) have been reported annually in Russia. The highest incidence has been recorded in three provinces of southern European Russia (Volgograd, Astrakhan and Rostov Provinces), yet in 2010-2012 the distribution of human cases expanded northwards considerably. From year to year, the number of WND cases varied widely, with major WND outbreaks in 1999, 2007, 2010, and 2012. The present study was aimed at identifying the most important climatic and environmental factors potentially affecting WND incidence in the three above-mentioned provinces and at building simple prognostic models, using those factors, by the decision trees method. The effects of 96 variables, including mean monthly temperature, relative humidity, precipitation, Normalized Difference Vegetation Index, etc. were taken into account. The findings of this analysis show that an increase of human WND incidence, compared to the previous year, was mostly driven by higher temperatures in May and/or in June, as well as (to a lesser extent) by high August-September temperatures. Declining incidence was associated with cold winters (December and/or January, depending on the region and type of model). WND incidence also tended to decrease during year following major WND outbreaks. Combining this information, the future trend of WND may be, to some extent, predicted, in accordance with the climatic conditions observed before the summer peak of WND incidence.

Borrelia miyamotoi is distantly related to B. burgdorferi and transmitted by the same hard-body tick species. We report 46 cases of B. miyamotoi infection in humans and compare the frequency and clinical manifestations of this infection with those caused by B. garinii and B. burgdorferi infection. All 46 patients lived in Russia and had influenza-like illness with fever as high as 39.5°C; relapsing febrile illness occurred in 5 (11%) and erythema migrans in 4 (9%). In Russia, the rate of B. miyamotoi infection in Ixodes persulcatus ticks was 1%-16%, similar to rates in I. ricinus ticks in western Europe and I. scapularis ticks in the United States. B. miyamotoi infection may cause relapsing fever and Lyme disease-like symptoms throughout the Holarctic region of the world because of the widespread prevalence of this pathogen in its ixodid tick vectors.

Polytypic strains containing the fragments of genes of Siberian and Far Eastern tick-borne encephalitis (TBE) virus subtypes were isolated from the brain of fatal TBE patients, the blood of TBE patients, and Ixodes persulcatus ticks in the foci of concomitant circulation of the two subtypes. The interaction of the Siberian and Far Eastern TBE virus subtypes was studied in the neural phase of the infection of albino mice and Syrian hamsters in order to understand conditions for formation of these strains and their role in the etiology of acute TBE. Their viral progeny was genotyped by reverse transcription-polymerase chain reaction and fluorescence hybridization assay with genotype-specific probes. Mixed infection showed an effect of synergism, independent reproduction of the two subtypes in the brain and spleen, competitive exclusion of one subtype from the viral population. The type of the Interaction depended on the species of animals, the properties of partner strains, and the target organ.