[Investigation of oncolytic potential of vaccine strains of yellow fever and tick-borne encephalitis viruses against glioblastoma and pancreatic carcinoma cell lines].

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 CPE50 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 × 109 to 1 × 1010 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.

Serological diagnosis and prevalence of HIV-1 infection in Russian metropolitan areas.

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.

[History of the discovery and study of tick-borne encephalitis in Russia: three Far Eastern expeditions (1937-1939)].

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.

Serodiagnosis of Borrelia miyamotoi disease by measuring antibodies against GlpQ and variable major proteins.

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.

[Clinical presentation of Ixodes tick-borne borreliosis caused by Borrelia miyamotoi in the context of an immune response to the pathogen]. / Klinicheskie proiavleniia iksodovogo kleshchevogo borrelioza, vyzvannogo Borrelia miyamotoi, v kontekste immunnogo otveta na vozbuditel'.

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.

Development and optimization of an in vitro cultivation protocol allows for isolation of Borrelia miyamotoi from patients with hard tick-borne relapsing fever.

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 K2-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.

[Interaction of the Siberian and Far Eastern subtypes of tick-borne encephalitis virus in mammals with mixed infection. I. Factors influencing the type of interaction].

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.