[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.

[Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

[Actual problems of hemorrhagic fever with renal syndrome].

From 2000 to 2011 85 600 cases of hemorrhagic fever with renal syndrome (HFRS) were registered in Russian Federation. Epidemically active foci of HFRS infection are located generally in temperate latitudes of the European part and the Far East. In the Far East regions whose fraction of all the HFRS disease cases in Russia is around 2%, the causative agents of the infection are Hantaan, Amur, Seoul hantaviruses, the natural reservoir for those are striped field mouse, Korean field mouse and brown rat. In the European part of Russia the causative agent of the infection are Puumala hantavirus as well as 2 genetic subtypes of Dobrava virus, the main reservoirs of those in the nature are bank vole, striped field mouse and Black Sea field mouse, respectively. 9 strain of Puumala and 10 strains of Dobrava virus were isolated. Based on sequencing of Dobrava virus strains significant differences were detected between Dobrava virus strains isolated from Black Sea field mouse from Sochi and striped field mouse from Lipetsk Region. Cultural inactivated vaccine against HFRS was developed and completed preclinical trials.

[Spatial structure of natural foci of hantavirus on the territory of Northwestern Caucasus].

For the period from 2001 to 2011 zoological and epizootological studies in more than 100 points of Northwestern Caucasus including territories of Krasnodar Region and Republic of Adygea were carried out. 8723 specimens of small mammals represented by 20 rodent species and 7 insectivorous species were captured and examined. Organs and blood from 5057 specimens of creatures were studied for hantavirus infection. The presence of natural foci of circulation of 2 species of hantavirus--Dobrava/Belgrade and Tula--was established. Sochi viruses and presumably Kurkin with main natural hosts--Caucasian wood and field mice belong to the first species. Tula and Adler viruses with the main host--Microtus genus vole belong to the second species. Quantitative characteristics of infection of small mammals of various species during different seasons and years on the examined territories were obtained, that allowed to create a map of allocation of foci of hantavirus circulation that differ by structure.

[The epidemiological, epizootological, and etiological characteristics of the 2006-2007 outbreak of hemorrhagic fever with renal syndrome in the Tambov Region].

The findings suggest that there are natural foci of hantavirus infection in the Tambov Region. There is evidence that Dobrava/Belgrade hantavirus (DOB-Aa) was a leading etiological agent in the outbreak of the disease in the winter of 2006-2007. Epidemiological analysis showed that the outbreak of hemorrhagic fever with renal syndrome (HFRS) afflicted the region during November to April, by reaching its peak in January (52.2%). Among the patients with HFRS, rural dwellers were 91%. People were infected with the virus mainly by taking care of domestic animals (97.2%). The reservoir of the virus and the source of its human infection in the outbreak was a field mouse, its western subspecies Apodemus agrarius agrarius, which was absolutely dominated among all the virus carriers.

[Discovery, clinical and etiological characteristic of hemorrhagic fever with renal syndrome in the subtropical zone of Krasnodar region].

Twenty-six patients with hemorrhagic fever with renal syndrome (HFRS) were revealed as a result of serological examination of 582 patients with fever living around Sochi town. Etiologic role of Dobrava virus subtype as the cause of HFRS was assessed by immunofluorescent and ELISA assays, and neutralization test. The principal host of this virus and source of infection for humans is Caucasian forest mouse Apodemus ponticus. HFRS morbidity was sporadic and not dependent from patients' occupation and season. Comparative analysis of clinical and laboratory data from HFRS cases caused by DOB/Sochi and DOB/Lipetsk subspecies, as well as Puumala virus showed higher proportion of severe forms of disease in patients with HFRS from Sochi.

[The epizootological and virological characteristics of a natural hantavirus infection focus in the subtropic zone of the Krasnodarsk Territory].

A natural focus of hantavirus infection was detected and examined during the studies conducted in 2000-2002 around the Sochi (the western spurs of the Great Caucasus Ridge, which descended to the Black Sea (the Krasnodar Territory of Russia). At least 4 rodent species, such as Microtus majori, A. (S.) ponticus, A. agrarius, A. (S.) ciscaucasicus, were shown to participate in the circulation of hantaviruses. A comparative analysis of the nucleotide sequences of genomic S- and M-segments of hantaviruses has provided evidence that 13 viral RNA isolates from the A. (S.) ciscaucasicus belong to the Dobrava/Belgrade virus clade; however the RNA isolate from the Microtus majori belong to the Tula virus clade.

[Nervous system involvement in hemorrhagic fever with renal syndrome].

The authors of the article describe two cases of hemorrhagic fever with renal syndrome (HFRS) with prevalence of signs of nervous system involvement. The first case was a 40-year-old woman with moderate HFRS, who developed Guillain-Barre syndrome of axonal-demyelinating polyneuropathy. An important observation was the absence of hemorrhagic or renal syndrome; combined therapy including plasmapheresis was successful. The second case demonstrated polymorphism of HFRS clinical manifestations with prevalence of neurological symptoms, which consisted in encephalopathy and no renal failure signs; hemorrhagic syndrome was moderate. In both cases the diagnosis was confirmed by elevated titer of antibodies to HFRS virus, belonging to the group of hantaviruses.

[The spread of hantaviruses in western Siberia]. / O rasprostranenii khantavirusov v Zapadnoi Sibiri.

Studies made in west Siberia established the existence of at least 4 hantavirus types: Puumala, Tula, Topografov, and Dobrava/Belgrade. The authors detected Puumala virus genovariants in voles, Tula virus in sagebrush and narrow-skulled voles, and Topografov virus in Siberian lemmings. The etiological role of the hantavirus Dobrada/Belgrade was defined in the structure of morbidity of hemorrhagic fever with the renal syndrome. In Russia, hantaviruses were first detected in mites: a hantavirus antigen was found in one of the enzyme immunoassayed pools of the mole mites (Haemogamasus ambulans) collected from the Siberian lemming brooding burrow in the near-floodplain habitats of the transitional Yenisei tundras in southwest Taimyr.

[Outbreak of hemorrhagic fever with kidney syndrome in Egor'ev district of Moscow region]. / Analiz vspyshki gemorragicheskoi likhoradki s pochechnym sindromom v Egor'evskom raione Moskovskoi oblasti.

Epizootological, serological, and molecular virological analysis of an outbreak of hemorrhagic fever with renal syndrome (HFRS) in the Egoryevsk district of Moscow region (September 1995-January 1996) has been carried out. Hantavirus (Puumala) antigen and virus-specific antibodies were isolated from bank voles captured in the endemic focus. Anti-Puumala antibodies were detected in the sera of all HFRS patients and in 2% healthy residents of the endemic focus. Analysis of nucleotide sequence (RNA from hantavirus-positive lung of a bank vole) showed that the studied hantavirus is a distinct genotype of Puumala virus. Hence, a new highly active natural focus of HFRS associated with Puumala virus, dangerous for the population, has been revealed in Moscow region.

[Adaptive property of hantaviruses during their isolation from cultured VERO-E6 cells in relation to the type of the ecological host]. / Adaptatsionnaia sposobnost' khantavirusov pri izoliatsii na kul'ture kletok VERO-E6 v zavisimosti ot vida ékologicheskogo khoziaina.

As a result of virological studies, 185 lung tissue specimens from 4 rodent species caught near Khabarovsk were isolated and fixed in the passages of cultured Vero-6 cells of 68 hantavirus strains. The capacity of the strains to adapt to the cells was assessed by using the adaptive index involving the mean rates of successful isolation, its duration, hantavirus antigen titer in the material used for infection. The strains of hantavirus serotypes were noted for the highest adaptive properties, which are ecologically associated with rodents of the family Mus, such as field and East-Asiatic mice. Lower adaptive capacities were established for the strains of hantavirus serotypes, which are ecologically related to rodents of the family Cricetidae, such as large and large-toothed redback voles. The differences found in the adaptive capacities of hantavirus strains cultured in Vero-E6 cells reflect the degree of specialization of some hantavirus serotypes to particular host rodent species during their long-term coevolution.

[An epidemiological analysis of hemorrhagic fever with renal syndrome morbidity in the Republic of Bashkortostan in 1997]. / Epidemiologicheskii analiz zabolevaemosti gemorragicheskoi likhoradkoi s pochechnym sindromom v Respublike Bashkortostan v 1997 g.

The outbreak of hemorrhagic fever with renal syndrome (HFRS) in the Republic of Bashkortostan, resulting in 10,057 registered cases of the disease (287 cases per 100,000 of the population), was analyzed. HFRS cases among the population were registered in 52 out of 54 regions of Bashkortostan. 31% of the total number of patients were the inhabitants of rural regions (170 cases per 100,000) and 69% were urban dwellers (295 cases per 100,000), mainly in Ufa (512 cases per 100,000). HFRS morbidity among males was fourfold higher than among females. In 70% of cases persons aged 20-49 years were affected. 5% of the total number of patients were children aged up to 14 years. In 34 cases (0.4%) the severe clinical course of the disease had a fatal outcome. Cases of HFRS were registered from April 1997 till March 1998 with the highest morbidity rate observed during the period of August-December. In most cases (46.8%) both urban and rural dwellers contacted infection during a short-term stay in the forest. As the result of the serological examination of the patients, all HFRS cases were etiologically attributed to hantavirus, serotype Puumala. The main natural reservoir of this virus and the source of human infection in Bashkortostan were bank voles (Clethrionomys glareolus), the domination species among small mammals in this region.

[A system of immunoenzyme analysis using biotinylated monoclonal antibodies for typing hantavirus antigens]. / Sistema immunofermentnogo analiza s ispol'zovaniem biotinilirovannykh monoklonal'nykh antitel dlia tipirovaniia antigenov khantavirusov.

Enzyme immunoassay with biotin-labeled monoclonal antibodies to Puumala virus was used for typing hantavirus antigens. The system is type-specific, that is, it detects Puumala virus only, which was demonstrated by typing 9 hantavirus antigens in infected Vero E5 cells and by epizootological analysis of an HFRS outbreak in the Yegoryevsk district of the Moscow region in November-December 1995. The suggested system may be used as an element of EIA screening of natural samples in analysis of the screening results.

[The antigenic differentiation of hantaviruses by using monoclonal antibodies]. / Antigennaia differentsiatsiia khantavirusov s pomoshch'iu monoklonal'nykh antitel.

85 hantavirus (HV) strains from Russia and other counties were examined by indirect fluorescent antibody test using monoclonal antibodies (MAbs) prepared against hantaviruses: Hantaan (Hantaan 76-118, A-9 strains), Seoul (SR-1, R-22 strains) and Puumala (Hallnas 83-223 strain). 68 HV strains were differentiated into five virus type groups: Hantaan, Puumala, Seoul, Prospect Hill and Belgrade-Dobrava. 17 strains were found to be antigenically closely related but distinct from Hantaan type (4 strains), Puumala (6 strains) and Prospect Hill (7 strains). The antigenic characteristics of these 17 strains suggested two supplementary antigenic subgroups of the Hantaan, two of Puumala and two of Prospect Hill.

[Polyclonal immunoenzyme system for determining hantaviral antigens: assessment of specificity using monoclonal antibodies and the polymerase chain reaction]. / Poliklonal'naia immunfermentnaia sistema dlia opredeleniia antigenov khantavirusov: otsenka spetsifichnosti s pomoshch'iu monoklonal'nykh antitel i polimeraznoi tsepnoi reaktsii.

Genus-specific monoclonal antibodies to nucleocapsid protein of Pumala virus and polymerase chain reaction were used to assess the specificity of a previously described polyclonal (based on human IgG to Pumala hantavirus) enzyme immunoassay (EIA) system for detecting hantavirus antigens. Parallel testing of crude lung suspensions from wild rodents trapped in different regions of Russia for hantavirus antigens and RNA showed 100% coincidence of the results of three tests. These data indicate a high specificity of polyclonal EIA system and its efficacy for screening of natural samples.

Khabarovsk virus: a phylogenetically and serologically distinct hantavirus isolated from Microtus fortis trapped in far-east Russia.

Two hantavirus strains, MF43 and MF113, isolated from Microtus fortis trapped in the Khabarovsk region of far-eastern Russia, were analysed by direct nucleotide sequencing of PCR generated fragments of the M and S segments, by immunofluorescence and by focus reduction neutralization tests (FRNT). The nucleotide sequences revealed that the two isolates were closely related to each other but distinct from all other hantaviruses. Phylogenetic analysis of the M and S segments showed that the MF strains form a separate branch in the Hantavirus tree, positioned between the branches of Prospect Hill and Puumala viruses. The strains were shown to be serologically distinct from the other hantavirus serotypes by FRNT using immune rabbit sera. Puumala virus was the closest relative, both genetically and serologically. We propose that this new hantavirus serotype should be named Khabarovsk (KBR).

[Genetic differentiation of hantaviruses using the polymerase chain reaction and sequencing]. / Geneticheskaia differentsiatsiia khantavirusov s pomoshch'iu polimeraznoi tsepnoi reaktsii i sekvenirovaniia.

Thirty-two hantavirus strains and 8 samples of lung tissue from rodents collected in different regions of Russia have been examined by molecular biological methods. Two methodological approaches have been employed for the study of genetic relationships between the viruses: nested PCR assay and common RT-PCR with subsequent direct sequencing of 200 and 365 base pair of G2 protein encoding regions of M-segment, respectively, and the resultant sequences were compared with those of the prototype hantavirus. The study revealed a mosaic pattern of distribution of different hantavirus genotypes on the territory of Russia.

[Dot-blot analysis in laboratory diagnosis of hemorrhagic fever with renal syndrome]. / Dot-blot analiz v laboratornoi diagnostike gemorragicheskoi likhoradki s pochechnym sindromom.

A sensitive micro EIA utilizing antigen or antibody dotted onto nitrocellulose filters (Dot-ELISA, Dot-blot) has been developed for laboratory diagnosis of hemorrhagic fever with the renal syndrome (HFRS) by detecting the virus specific IgM and IgG in the sera of HFRS patients and the antigen in crude lung suspensions of wild rodents. Measurements of specific IgM and IgG in paired HFRS sera collected during the first month of the disease showed clear-cut seroconversion and 100% correlation with the results of immunofluorescent test. The results of antigen detection in lung suspensions of 605 wild rodents trapped in various regions of Russia were identical to standard ELISA results. This rapid and inexpensive test may be useful in early serologic diagnosis of HFRS and in field serological and epizootological studies.

Detection and subsequent sequencing of Puumala virus from human specimens by PCR.

A sensitive method based on PCR was developed for the detection of Puumala virus (PUU) in human samples. The assay was found to be specific for PUU-like strains and distinguished between these and hantaviruses of other serotypes. The detection limit was found to be 10(-5) focus-forming units. Clinical samples were collected from patients with nephropathia epidemica in Sweden and western Russia. Five whole blood samples collected from patients in Russia with the acute phase of disease were found to be positive by the PCR. All samples were negative for PUU antigen when examined by enzyme-linked immunosorbent assay. Virus isolation on Vero E6 cells from several of the acute-phase samples, including the 5 PCR-positive samples, was not successful. The amplified samples were subjected to direct nucleic acid sequencing for confirmation of identity. The sequences differed from each other and were closely related to the Russian bank vole isolate CG-1820, thereby indicating the origin of nephropathia epidemica. The PCR was used for amplification and subsequent nucleotide sequencing of eight PUU-like isolates with different geographic origins. The Swedish strains were more closely related to the Finnish PUU prototype strain, Sotkamo, than to the Russian isolates. Interestingly, a Belgian isolate, CG-13891, differed markedly from all other PUU strains.