[Real-time PCR assay development for the control of vaccine against hemorrhagic fever with renal syndrome].

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 ß-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.

[Hemorrhagic fever with renal syndrome group outbreak caused by Sochi virus.]

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.