Introduction. Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS. Objective. Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent. Materials and methods. Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA. Results and discussion. For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays. Conclusion. The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.

Relevance. Hemorrhagic fever with renal syndrome (HFRS) and tick-borne encephalitis (TBE) are the most common natural focal diseases of viral etiology in Russia. The medical and social significance of these two infections is determined by the extensive foci of their spread, high annual morbidity rates, and the presence of severe forms of the disease can lead to permanent disability and even death. Aim. To assess the current epidemiological situation of HFRS and TBE in the Russian Federation. Conclusions. Over 23 years (from 2000 to 2022), 164,582 cases of HFRS were identified in Russia with an average annual rate of 4.9 cases per 100 thousand population, as well as 71,579 cases of TBE with an average annual rate of 2.5 cases per 100 thousand population; 668 (0.4%) and 1136 (1.6%) deaths from HFRS and TBE, respectively; 4030 (2.5%) and 9414 (13%) children under the age of 14 years among patients with HFRS and TBE, respectively. The incidence of HFRS and TBE per 100 thousand population of Russia was higher among rural residents than among urban residents. Most cases of HFRS were registered in the autumn-winter period, and TBE – at the end of June – the first half of July. Of the 85 administrative regions of Russia, cases of HFRS and TBE are registered in 42, in 18 – only HFRS, in 13 – only TBE, and in 12 regions no clinically diagnosed cases of HFRS and TBE have been identified.

Relevance. Hemorrhagic fever with renal syndrome (HFRS) is leading among natural focal human diseases in Russia, the causative agents of which - orthohantaviruses - belong to the order Bunyavirales, family Hantaviridae. More than 98% of HFRS cases in Russia are caused by the Puumala virus. It is a serious zoonosis for which there is still no specific treatment. The WHO has not approved a vaccine. The aim of this study was to investigate the effect of formaldehyde, β-propiolactone, hydrogen peroxide, ultraviolet rays, gamma irradiation and thermal inactivation on the immunogenic activity of inactivated vaccine preparations against HFRS Materials and methods. To achieve this aim, experimental vaccine preparations based on the PUU-TKD/VERO strain of Puumala virus were prepared and inactivated using the methods described above. The time intervals required for complete inactivation of the virus were determined, and the effects of the inactivators on viral RNA and immunogenic activity of the vaccine preparations were evaluated in BALB/c mouse and Syrian hamster models. Results. According to our results, vaccine preparations inactivated by different chemical and physical methods, which differ significantly in the mechanism of the mechanism of interaction with the virus, show no significant differences in immunogenic activity, except for thermal inactivation. Conclusion. A certain advantage of β-propiolactone is the short virus inactivation time, its complete degradation into non-toxic compounds within a few hours, and the reduction of total protein content after sterilization filtration, which is probably due to less aggregation of virus particles and cellular proteins

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

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

We report the effects of storage conditions and human serum albumin addition to the vaccine composition on the immunogenic properties of an experimental vaccine against hemorrhagic fever with renal syndrome. To obtain an experimental vaccine, the Puumala virus, propagated in Vero cells culture, was concentrated, inactivated with beta-propiolactone, and purified by chromatography on the CaptoCore 700 multimodal sorbent (GE Healthcare). The target component in the vaccine was 20.2 106 of viral RNA copies/ml. The humoral immune response to the vaccine was determined by measuring the neutralizing antibodies in the blood serum of immunized Syrian hamsters (Mesocricetus auratus). It was revealed that the Puumala virus inactivated with beta-propiolactone induces a pronounced humoral immune response, which indicates preservation of relevant immunogenic epitopes. Аccording to our study, viral RNA is more stable during storage of the inactivated vs. intact virus. It was confirmed that full virus inactivation occurred after 24 hours of storage at 37C. A significant decrease in the virus immunogenicity during the thermal inactivation was noted, which confirms the need for careful selection of storage conditions for thermolabile Hantaviruses. A direct correlation was observed between viral titer and viral RNA copy number/ml. Adding human serum albumin stabilized viral infectivity during storage: Puumala virus titer remained at the same level for 3 months of storage at 62C, whereas without albumin, the titer decreased to undetectable level. The addition of 0.1% human serum albumin to the inactivated virus stabilized its immunogenic properties during long-term storage. It was shown that vaccine preparations with human serum albumin (0.11%) show higher dose-dependent stability upon repeated freezing assessed by detecting viral RNA level and their immunogenicity. It can be assumed that human serum albumin provides better preservation of of viral genetic material during storage, as well the protein epitopes responsible for the protective immune response in the form of neutralizing antibodies induction, undergo less pronounced conformational changes in the presence of albumin. These data support the addition of human serum albumin to the vaccine formulation.

We worked out a method of hantavirus Puumala cultivation in Vero cells on a micro-carrier in small volumes of pseudosuspension. The results obtained demonstrate the effectiveness of this cultivation method in terms of the amount of viral harvest and the effort expended and will serve as a starting point for further scale-up of the vaccine against hemorrhagic fever with renal syndrome in the bioreactor.

Human securin (PTTG1) is a protooncogene whose expression is elevated in many types of malignant cells. We previously discovered a minor short isoform of securin lacking exons 3 and 4. The missing exons encode the main recognition site (D-box) of the anaphase-promoting complex (APC/C). We show that these two PTTG1 isoforms have different effects on transcription. Here, we have studied the effects of overexpression and selective knockdown of the short and complete securin isoforms on cell proliferation using the xCELLigence system. Notably, selective knockdown of the short isoform mRNA led to a dramatic decrease in cell growth, while overexpression of both isoforms accelerated cell growth. To search for genes with alternative isoforms similar to securin, we analyzed the GENCODE database and found that 54 of 128 genes with a PTTG1-like set of APC/C recognition sites have known isoforms without the D-box. Overall, the data obtained indicate the existence of a new class of alternative isoforms and reinstates the importance of minor isoforms.

In the Russian Federation, the vaccine against hemorrhagic fever with renal syndrome is at the stage of preclinical and clinical trials. The aim of the study was to analyze an effect of vaccine schedule on neutralizing antibodies (nAB) dynamics in guinea pig models applied with experimental Puumala virus based hantavirus vaccine (HV). Quantitative evaluation of neutralizing antibodies was presented as antibody titer geometric mean expressed in binary logarithms (log2) by the 50% reduction of focus-forming units (FRNT50) in Vero cell in the focus reduction neutralization test. The HV dual inoculation to guinea pigs was carried out in 14 day intervals, booster injection was applied on day 182 after the onset, in the thigh muscle tissue by using 0.3 ml undiluted (HV-u/d) and in the 1/10 dilution (HV-1/10). nAB titer on day 14 after the first HV-u/d and HV-1/10 injection was measured to be at titer of 5.50.3 and 4.80.3, respectively. After the second injection, the nAB peak was as high as 90.2 on day 42 after the first HV-u/f injection, and 6.50.2 on day 14 after the HV-1/10 injection. nAB decreased down to 6.20.3 and 50.3, respectively, on day 364 after the first injection. The booster HV-u/d and HV-1/10 injection induced increase in nAB up to 9.50.3 and 6.50.3, respectively. After the booster injection, it induced significantly higher nAB observed on day 238 after the first HV-u/d injection and delayed up to the 294 day for the HV-1/10. The results of the study indicated the early formation of the immune response, long-term nAB persistence and significantly enhanced immune response after the booster injection on day 182, which indicated a potential for the booster injection a year later. The immunological efficacy and protective activity of the vaccine schedule may be finally assessed according to the results of clinical trials.

Human securin (PTTG1) is a protooncogene whose expression is elevated in many types of malignant cells. We previously discovered a minor short isoform of securin lacking exons 3 and 4. The missing exons encode the main recognition site (D-box) of the anaphase-promoting complex (APC/C). We show that these two PTTG1 isoforms have different effects on transcription. Here, we have studied the effects of overexpression and selective knockdown of the short and complete securin isoforms on cell proliferation using the xCELLigence system. Notably, selective knockdown of the short isoform mRNA led to a dramatic decrease in cell growth, while overexpression of both isoforms accelerated cell growth. To search for genes with alternative isoforms similar to securin, we analyzed the GENCODE database and found that 54 of 128 genes with a PTTG1-like set of APC/C recognition sites have known isoforms without the D-box. Overall, the data obtained indicate the existence of a new class of alternative isoforms and reinstates the importance of minor isoforms.

Relevance. Hemorrhagic fever with renal syndrome (HFRS) is an acute viral zoonosis. Being widespread in Eurasia, it holds a leading place in Russia among natural focal human diseases. The vast majority of HFRS cases in Russia, about 98%, are associated with Puumala virus. The disease is characterized by a wide range of clinical manifestations. Early specific diagnostics appears to be of a great importance for starting timely pathogenic therapy. The aim of the study was to clarify the diagnostic value of detecting hantavirus antibodies in the HFRS suspected patient urine. Materials and methods. Blood sera and urine samples from 68 patients at the Infectious Diseases Hospital in the city of Ufa, obtained with a 2-day interval, as well as urine and blood serum samples from 15 convalescents 1, 2, 3 and 6 months after disease onset were examined for hantavirus antibodies. 53 blood sera and urine samples from patients residing in Moscow, Moscow and Samara regions collected at different time points during the disease course were investigated in parallel. Antibodies were detected by the indirect immunofluorescence method. Results. On day 3, 4, 5 and 6 of disease, while specific antibodies were detected in the blood serum, antibodies in the urine were found in 85.7%, 89.4%, 93.1% and 100% of patients, respectively. The peak quantity of antibodies was excreted in the urine from days 5 to 11, which corresponds to the oliguric stage of the disease. In the convalescent period, antibodies were still detected in urine 1, 2 and 3 months afterwards in 86.7%, 46% and 20% of cases, respectively, but not detected 6 months later, which probably reflects the process of long-term restoration of the kidneys function. A moderate positive correlation between specific antibodies in serum and urine was observed only in the oliguric period of the disease. Conclusions. Detection of hantavirus antibodies simultaneously in blood serum and urine of febrile patients instead of paired blood sera allows to conduct HFRS diagnostics within the very first days of hospitalization and prevent severe complications due to timely pathogenic therapy.

INTRODUCTION:Hemorrhagic fever with renal syndrome (HFRS) holds a leading place among natural focal human diseases in Russian Federation. There is no etiotropic therapy for the disease now. The vaccine prophylaxis is the most effective method to control this infection. The main criteria for inactivated vaccines evaluation are its immunogenicity and specific activity.The study purposes were to develop a sensitive and specific real-time PCR method for viral RNA quantification in the inactivated vaccine and to study the correlation between the viral RNA amount and vaccine immunogenicity.MATERIAL AND METHODS:L-segment fragments of the Puumala, Hantaan, and Sochi vaccine strains were selected as diagnostic targets for oligonucleotides and fluorescent probes. The immunogenicity of experimental vaccines was determined by the induction of neutralizing antibodies in BALB/c mice.RESULTS:A highly specific, sensitive and reproducible real-time PCR method has been developed. The analytical sensitivity was 1.24 ± 1.5 x 102 copies/ml for Puumala virus; 1.16 ± 1.4 * 102 copies/ml for Hantaan; 1.32 ± 1.8 * 102 copies/ ml for Sochi, with a virus content of 1.5 ± 0.5 lg FFU/ml; 1.8 ± 0.5 lg FFU/ml and 2.2 ± 0.5 lg FFU/ml, respectively. The viral RNA amount in experimental vaccine preparations inactivated with β-propiolactone was proportional to the neutralizing antibodies titer observed in mice following the immunization.DISCUSSION:It was found that different virus inactivators differently affects the detected viral RNA amount, but not the vaccine immunogenicity, which indicates the same degree of the immunogenic proteins damage. The direct relationship between the viral RNA copy number and vaccine immunogenicity makes it possible to use this criterion for vaccine dosage preparation.CONCLUSION:The developed method for viral RNA quantification is a promising tool for the specific activity control of the HFRS vaccine.

Приведены результаты многолетнего мониторинга эндемичной по ГЛПС территории Оренбургской области, где заболевание вызывает хантавирус Пуумала. Показано, что очаги ГЛПС–Пуумала как в степном, так и лесостепном ландшафте приурочены к интразональным пойменным лесам в бассейнах рек Урал, Самара, Сакмара и других, которые благоприятны для обитания основного хозяина вируса Пуумала – европейской рыжей полевки (Myodes glareolus). Заболеваемость ГЛПС и площадь очагов резко выросла за последние два десятилетия, и интенсивный показатель на 100 тыс. населения приблизился к средним значениям, отмеченным на активной очаговой территории лесной зоны. Продемонстрирована тесная связь уровня заболеваемости с численностью инфицированных рыжих полевок. Определены и закартированы места с разным уровнем риска заражения людей; выявлены населенные пункты и их окрестности, где заражается большая часть местного и приезжего населения. Показано, что на некоторых участках активной очаговой территории число заражений может значительно повышаться за счет большого притока горожан. The results of long-term monitoring of the Orenburg region territory, which is endemic for HFRS caused by Puumala hantavirus, are presented. It has been established that HFRS-Puumala foci, both in the steppe and forest-steppe landscapes, are confined to interzonal floodplain forests in the basins of the Ural, Samara, Sakmara and others rivers, which are favorable for the habitation of the main host of the Puumala virus – the European bank vole (Myodes glareolus). The incidence of HFRS and the foci area have been increased sharply over the past two decades and the average incidence rate per 10E5 approached the average values in the active foci of the forest zone. A close relationship between the incidence rate and the number of infected bank voles has been demonstrated. Locations with different levels of human infection risk were identified and mapped; identified settlements and their surroundings, where most of the local and visiting population are infected. It has been established that in some areas of the active focal area, the risk of infection can significantly increase due to a large influx of citizens.

Effects of different classes of adjuvants (aluminum hydroxide, spherical protein particles, thermolabile В protein, low-endotoxic pyrogen-free lipopolysaccharide) have been studied in order to enhance the immunogenicity of inactivated vaccines against hemorrhagic fever with renal syndrome. The Puumala virus-based monovaccine and multivalent vaccine based on Puumala, Hantaan and Sochi viruses were analyzed, and BALB/c mice were used as an animal model. It was shown that low-endotoxic pyrogen-free lipopolysaccharide stimulated the production of virus-neutralizing antibodies and increased the vaccine stability during storage, which allows to reduce the antigenic load of the vaccine. Aluminum hydroxide slightly increased the production of T-cells immune response mediators and did not affect the neutralizing antibodies induction and vaccine stability. Despite the adjuvant effect, it was shown that spherical protein particles and thermolabile В protein were unacceptable for vaccines administered to humans due to the high protein load and toxic effects, respectively. hantavirases, hemorrhagic fever with renal syndrome, inactivated vaccines, adjuvants, immune response The authors are grateful to Dr. O.V. Karpova (Department of Virology, Moscow State University) for providing the preparation of spherical particles, and to Dr. A.N. Noskov (Gamaleya Research Institute of Epidemiology and Microbiology, Moscow) for providing the preparation of the thermolabile enterotoxin B-subunit.

Hemorrhagic fever with renal syndrome (HFRS) is an acute zoonotic disease caused by orthohantavirases Puumala, Dobrava-Belgrad (4 genotypes), Hantaan (genotype Amur) and Seoul.. In the Russian Federation, HFRS takes a leading place among all natural focal human diseases. Formalin, β-propiolactone, and ultraviolet radiation were tested to select the optimal method of virus inactivation during the development of the whole-virion vaccine against HFRS. The specific activity and the immunogenicity of the vaccine were determined, respectively, by the number of copies of viral RNA/mL and by the titer of neutralizing antibodies in the BALB/c mice blood serum in response to immunization. An analysis of the immunogenicity of vaccines inactivated with formalin, β-propiolactone or UV radiation did not reveal significant differences in the level of neutralizing antibodies they induced. At the same time, β-propiolactone has obvious technological advantages compared to formalin and UV radiation: the time of inactivation of hantavirus is reduced tenfold, neutralization of the inactivator and control of its content in the final vaccine are not required, and the amount of total protein in the vaccine decreases. hemorrhagic fever with renal syndrome, hantavirases, inactivated vaccines, neutralizing antibodies, formalin, β-propiolactone, ultraviolet

Relevance. Hemorrhagic fever with renal syndrome (HFRS) is a non-transmissible viral zoonosis widespread in Eurasia, and in Russia it occupies a leading position among zoonotic viral infections and one of the first places among all natural focal human diseases. Aim. Obtaining evidence of the safety, quality and efficacy of a polyvalent vaccine against HFRS as a result of its preclinical studies using scientific assessment methods that meet the requirements and rules of good laboratory practice. Materials and methods. For preclinical studies of the polyvalent vaccine against HFRS, the materials and methods were used in strict accordance with the requirements of the regulatory documents, as well as previously described methods used to control the vaccine at the technological stages of its manufacture. Results. The data obtained as a result of preclinical studies of the polyvalent vaccine agains HFRS indicate a high immunogenicity and stability of the vaccine, the absence of: acute and chronic toxicity, allergenic, immunotoxic and mutagenic, as well as toxic effects on the reproductive organs of animals, embryo development and offspring, born to females who received the vaccine within 20 days of gestation. Conclusion. The obtained results of preclinical studies comply with the requirements for immunobiological medical preparations designed for humans, and are the basis for conducting the 1st phase of clinical trials the polyvalent vaccine against HFRS.

BACKGROUND:Hemorrhagic fever with renal syndrome (HFRS) occupies a leading place among natural focal human diseases in the Russian Federation. Sporadic incidence of HFRS-Sochi has been annually recorded in the Krasnodar Territory since 2000. The group outbreak of the HFRS-Sochi was first registered in Gelendzhik in the fall of 2013. METHODS:Serological methods were used: indirect immunofluorescence, enzyme immunoassay, FRNT in Vero cells, and methods for the viral RNA detection: PCR and RT-PCR. RESULTS:Data of clinical, epidemiological, immunological and molecular studies of 3 out of 4 cases in HFRS-Sochi outbreak are presented. Severity of the disease correlated with early gastrointestinal disorders appearance. Patient MA gastrointestinal disorders were joined on day 3 of a fever. Clinical and laboratory studies revealed signs of kidneys, liver, pancreas damage, bilateral hydrothorax, bilateral polysegmental pneumonia and polyneuropathy. As a result of long-term treatment, the patient recovered. Patient AA had gastrointestinal disturbances the next day after fever onset. The patient was not saved, despite early hospitalization. Hantavirus antigen and RNA were detected in the lung tissues 2 out of 10 Black-Sea field mice captured in the affected area, as well as in the organs of deceased patient. The most severe clinical course of the disease in close relatives, son and father, with a fatal outcome in the latter case may be the result of genetic features. The severity and outcome of the disease was not depend on day of hospitalization and correlated with the early manifestations of gastrointestinal disorders. CONCLUSIONS:Presented data confirm high virulence and pantropism of the Sochi virus, as well as the epidemiological role of Black-Sea field mouse (Apodemus ponticus) as the host of the Sochi virus and the source of human infection.

Hantaviruses are highly pathogenic causative agents of hantaviral fevers, including hemorrhagic fever with renal syndrome, registered among people in countries of the Eurasian continent and a disease called «hantavirus pulmonary syndrome» – in the countries of North and South America. More recently, the spread of hantaviral diseases has been detected in Africa. There are still no drugs for specific antiviral therapy. The most promising method of dealing with hantaviral fevers is specific prophylaxis, that is, vaccination of the population against hantaviruses, which determine the endemicity of different territories. This review summarizes current data on existing and developed vaccines against hantaviral fevers.

The results of various methods of Puumala virus inactivation, including ultraviolet rays (UV), β -propiolactone (BPL) and formalin are presented. Immunogenicity of vaccine preparations obtained using these virus inactivation methods did not differ significantly in the experiments on BALB/c mice. Essential advantage of UV and BPL in relation to formaldehyde is the short time of virus inactivation.

Addition of adjuvants is a generally recognized method of enhancing the immunogenicity of vaccines. There is a large variety of adjuvants, therefore the choice of an adjuvant is based on the comparison of adjuvants efficacy in animal models, as well as assessment of their safety and tolerability. There are many different groups of substances that may have adjuvant properties, but relatively little attention is paid to carbohydrate-based adjuvants, despite the fact that they are compatible with live vector vaccines, safe, well tolerated, and their production is not laborious. Thanks to these advantages the use of these particular adjuvants in combination with any type of vaccine, including vector vaccines and DNA vaccines, may be most appropriate and promising. This review examines various types of carbohydrate adjuvants and the mechanisms that determine their efficacy.