Hemorrhagic Fever with Renal Syndrome, Russia.

In Russia, 131,590 cases of hemorrhagic fever with renal syndrome caused by 6 different hantaviruses were reported during 2000-2017. Most cases, 98.4%, were reported in western Russia. The average case-fatality rate was 0.4%, and strong regional differences were seen, depending on the predominant virus type.

Life-Threatening Sochi Virus Infections, Russia.

Sochi virus was recently identified as a new hantavirus genotype carried by the Black Sea field mouse, Apodemus ponticus. We evaluated 62 patients in Russia with Sochi virus infection. Most clinical cases were severe, and the case-fatality rate was as high as 14.5%.

Oral prolonged release beclomethasone dipropionate and prednisone in the treatment of active ulcerative colitis: results from a double-blind, randomized, parallel group study.

OBJECTIVES: Double-blind study comparing efficacy and safety of the topically acting corticosteroid beclomethasone dipropionate (BDP) to prednisone (PD) in patients with active, mild-to-moderate ulcerative colitis (UC). METHODS: Overall, 282 patients were randomized to receive BDP-prolonged release tablets 5 mg once daily for 4 weeks and then every other day for an additional 4 weeks or oral PD 40 mg once daily for the initial 2 weeks tapered of 10 mg every 2 weeks during the 8-week study period. Efficacy end point was the non-inferiority of BDP vs. PD in terms of Disease Activity Index (DAI) score <3 or reduction by at least 3 points for patients with a baseline DAI ≥7 at week 4. Safety end point was the proportion of patients with steroid-related adverse events (AEs) and cortisol <150 nmol/l at week 4. RESULTS: DAI response rates at week 4 were 64.6% and 66.2% with BDP and PD, respectively, demonstrating non-inferiority of BDP vs. PD (delta: -1.56; 95% confidence interval (CI) -13.00-9.88, P=0.78). Patients with steroid-related AEs and cortisol <150 nmol/l at week 4 were 38.7% in the BDP group and 46.9% in the PD group (P=0.17 between groups). No safety signals were observed in both the groups. CONCLUSIONS: BDP was non-inferior to PD in the treatment of active UC, with a good safety profile in both the groups.

Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics.

Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes - Dobrava, Kurkino, Saaremaa, and Sochi - that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans.

Cases of Hemorrhagic Fever with Renal Syndrome in Russia during 2000-2022.

During 2000-2022, a total of 69 of Russia's 85 administrative regions reported 164,580 hemorrhagic fever with renal syndrome (HFRS) cases, with an annual average rate of 4.9 cases/100,000 population (105 popul.). European Russia reported 162,045 (98.5%) cases in 53/60 regions with 9.7 cases/105 popul. Asian Russia reported 2535 (1.5%) cases in 16/25 regions with 0.6 cases/105 popul. In the same period, Russia reported 668 (0.4%) fatal HFRS cases, and 4030 (2.4%) cases among children under the age of 14 years. Most HFRS cases occurred during autumn and winter. The incidence among rural residents was 6.7 per 105 popul., higher than the urban 4.4 per 105 popul.; however, among HFRS patients, rural and urban residents account for 35% and 65%, respectively. Six hantaviruses, causing HFRS of different clinical severity, were recognized as pathogens: Hantaan (HTNV) and Amur (AMUV) of Orthohantavirus hantanense species, Seoul (SEOV) of Orthohantavirus seoulense species, Puumala (PUUV) of Orthohantavirus puumalaense species, and Kurkino (KURV) and Sochi (SOCV) of Orthohantavirus dobravaense species, with the principal hosts Apodemus agrarius coreae, Apodemus peninsulae, Rattus norvegicus, Myodes glareolus, Apodemus agrarius agrarius, and Sylvaemus ponticus, respectively. It was found that 97.7% of HFRS cases are caused by PUUV, therefore, this virus plays the main role in the HFRS morbidity structure in Russia.

Isolation of sochi virus from a fatal case of hantavirus disease with fulminant clinical course.

Sochi virus, a novel genetic variant of Dobrava-Belgrade virus, was isolated in cell culture from a fulminant lethal case of hantavirus disease presenting with shock and combined kidney and lung failure. Sochi virus is transmitted to humans from host reservoir Apodemus ponticus and must be considered a life-threatening emerging agent.

Various Adjuvants Effect on Immunogenicity of Puumala Virus Vaccine.

Various adjuvant effects on the immunogenicity of the candidate inactivated Puumala virus vaccine were detected in BALB/c mice. Adjuvants under study were: aluminum hydroxide, spherical particles of Tobacco mosaic virus coat protein, B subunit of heat-labile enterotoxin of Escherichia coli, and low endotoxic lipopolysaccharide of Shigella sonnei. Aluminum hydroxide (1 mg/ml) did not affect neutralizing antibodies' induction and vaccine stability during storage compared to immunization with the vaccine without adjuvant. B subunit of heat-labile enterotoxin (0.2 µg/ml), low endotoxic lipopolysaccharide (50 µg/ml), and plant virus-based spherical particles (300 µg/ml) significantly enhance the humoral immune response of vaccine (p < 0.0001). Pronounced stimulation of IL-12 and IFN-É£ was observed when mice were immunized with vaccines both with adjuvants (except of aluminum hydroxide) and without adjuvants. It has been shown that low endotoxic lipopolysaccharide contributes not only to enhance the immune response but also to stabilize vaccine immunogenicity during at least 1 year storage.

Molecular diagnostics of hemorrhagic fever with renal syndrome during a Dobrava virus infection outbreak in the European part of Russia.

A large outbreak of hemorrhagic fever with renal syndrome (HFRS) occurred in the winter of 2006-2007 in a region southeast of Moscow in Central European Russia. Of the 422 patients with HFRS investigated in this study, 58 patients were found to be infected by Puumala virus, whereas as many as 364 were infected by Dobrava-Belgrade virus (DOBV). Early serum samples from 10 DOBV-infected patients were used for nucleic acid amplification, which was successful for 5 patients. Molecular analyses demonstrated that the causative hantavirus belongs to the DOBV-Aa genetic lineage, which is carried by the striped field mouse (Apodemus agrarius) as the natural reservoir host. Neutralization assays with convalescent-phase sera from these patients confirmed infection by DOBV-Aa; related viruses, such as the Dobrava-Slovenia virus (DOBV-Af) and the Dobrava-Sochi virus (DOBV-Ap), were neutralized at lower efficiencies. The clinical courses of the 205 patients enrolled in the study were found to be mostly mild to moderate; however, an unexpectedly high fraction (27%) of patients exhibited severe illness. One patient died from kidney failure and showed symptoms of generalized subcutaneous hemorrhage. The results provide molecular, serodiagnostic, and clinical evidence that DOBV-Aa is a common pathogen in East Europe that causes large outbreaks of HFRS.

Epidemiological study of hantavirus infection in the Samara Region of European Russia.

European Russia is a highly endemic area of hemorrhagic fever with renal syndrome (HFRS), a rodent-borne zoonotic disease, caused by hantaviruses. In total, 145 small mammals of four species (Myodes glareolus, Apodemus flavicollis, A. agrarius, and A. uralensis) were trapped in the Samara region of European Russia in August 2005 and examined for the presence of hantavirus (HV). Anti-HV antibodies were found in six of 68 (8.8%) M. glareolus and in one of 19 (5.3%) A. flavicollis by indirect immunofluorescent antibody assay (IFA). The Puumala virus (PUUV), which is one of the hantavirus species, was detected in the lungs of seven M. glareolus by RT-PCR. The virus S-segment was extremely similar (96.2% to 99.3%) to the sequence found in a fatal case of HFRS in the Samara region. Phylogenetic analyses of S and M segments showed that the Samara PUUVs form a cluster within the Russian Volga lineage and apparently differ from other European PUUVs. Anti-PUUV antibodies were found in blood sera from seven HFRS patients and from one undiagnosed patient from the Samara region, using IFA and an enzyme-linked immunosorbent assay (ELISA). These data suggest that the bank vole M. glareolus is a primary natural reservoir and vector for PUUV, which is the main causative agent of HFRS in humans in the Samara region.

Hemorrhagic fever with renal syndrome caused by 2 lineages of Dobrava hantavirus, Russia.

Dobrava-Belgrade virus (DOBV) is a European hantavirus that causes hemorrhagic fever with renal syndrome (HFRS); case-fatality rates in Balkan countries are as high as 12%. To determine causative agents, we examined 126 cases of DOBV-associated HFRS in central and southern European Russia. In central Russia (Lipetsk, Voronezh, Orel regions), outbreaks were caused by a DOBV variant (DOBV-Aa) carried by Apodemus agrarius. In southern Russia (Sochi district), where HFRS is endemic, HFRS cases were caused by a new DOBV variant (DOBV-Ap), found in A. ponticus, a novel hantavirus natural host. Both viruses, DOBV-Aa/Lipetsk and DOBV-Ap/Sochi, were isolated through Vero E6 cells, genetically characterized, and used for serotyping of the HFRS patients' serum. The clinical severity of HFRS caused by DOBV-Aa resembles that of HFRS caused by Puumala virus (mild to moderate); clinical severity of disease caused by DOBV-Ap infections is more often moderate to severe.

Genetic and antigenic analyses of a Puumala virus isolate as a potential vaccine strain.

Puumala virus (PUUV), a causative agent of hemorrhagic fever with renal syndrome (HFRS), is prevalent in Europe and European Russia. No vaccine has been developed for PUUV-associated HFRS, primarily because of the low viral yield in cultured cells. A PUUV strain known as DTK/Ufa-97 was isolated in Russia and adapted for growth in Vero E6 cells maintained in serum-free medium. The DTK/Ufa-97 strain produced a higher viral titer in serum-free medium, suggesting that it may prove useful in the development of an HFRS vaccine. When PUUV-infected Vero E6 cells were grown in serum-free medium, the DTK/Ufa-97 strain yielded more copies of intracellular viral RNA and a higher viral titer in the culture fluid than did the Sotkamo strain. Phylogenetic analysis revealed that PUUVs can be classified into multiple lineages according to geographical origin, and that the DTK/Ufa-97 strain is a member of the Bashkiria-Saratov lineage. The deduced amino acid sequences of the small, medium, and large segments of the DTK/Ufa-97 strain were 99.2% to 100%, 99.3% to 99.8%, and 99.8% identical, respectively, to those of the Bashkirian PUUV strains and 96.9%, 92.6%, and 97.4% identical, respectively, to those of the Sotkamo strain, indicating that the PUUVs are genetically diverse. However, DTK/Ufa-97 and other strains of PUUV exhibited similar patterns of binding to a panel of monoclonal antibodies against Hantaan virus. In addition, diluted antisera (i.e., ranging from 1:160 to 1:640) specific to three strains of PUUV neutralized both homologous and heterologous viruses. These results suggest that the DTK/Ufa-97 strain is capable of extensive growth and is antigenically similar to genetically distant strains of PUUV.

Severe hantavirus disease in children.

BACKGROUND: Very recently, a novel European hantavirus, Sochi virus, has been discovered which causes severe courses of hantavirus disease with a case fatality rate of about 15 percent. OBJECTIVES: We aimed to study to which extent and with which clinical severity children were affected by Sochi virus infection. STUDY DESIGN: Sochi virus infection of patients was confirmed by molecular, serological, and epizoonotic studies. Clinical and laboratory parameters were analyzed for the age group of up to 15 years (n = 6) in comparison to all older patients (n = 56). RESULTS: 9.7 percent of patients with hantavirus disease studied (6/62) were up to 15 years old. The children showed moderate to severe clinical courses similarly to the situation in adults. CONCLUSIONS: While children are in general considered to be less affected by hantavirus infections than adults, in case of highly pathogenic hantaviruses, such as Sochi virus, frequency of clinical cases as well as their clinical course are comparable between children and adults. Therefore, hantavirus disease, particularly in regions endemic to highly pathogenic hantaviruses, should be considered in cases of unclear fever and kidney/pulmonary failure in children.

Adler hantavirus, a new genetic variant of Tula virus identified in Major's pine voles (Microtus majori) sampled in southern European Russia.

Although at least 30 novel hantaviruses have been recently discovered in novel hosts such as shrews, moles and even bats, hantaviruses (family Bunyaviridae, genus Hantavirus) are primarily known as rodent-borne human pathogens. Here we report on identification of a novel hantavirus variant associated with a rodent host, Major's pine vole (Microtus majori). Altogether 36 hantavirus PCR-positive Major's pine voles were identified in the Krasnodar region of southern European Russia within the years 2008-2011. Initial partial L-segment sequence analysis revealed novel hantavirus sequences. Moreover, we found a single common vole (Microtusarvalis) infected with Tula virus (TULV). Complete S- and M-segment coding sequences were determined from 11 Major's pine voles originating from 8 trapping sites and subjected to phylogenetic analyses. The data obtained show that Major's pine vole is a newly recognized hantavirus reservoir host. The newfound virus, provisionally called Adler hantavirus (ADLV), is closely related to TULV. Based on amino acid differences to TULV (5.6-8.2% for nucleocapsid protein, 9.4-9.5% for glycoprotein precursor) we propose to consider ADLV as a genotype of TULV. Occurrence of ADLV and TULV in the same region suggests that ADLV is not only a geographical variant of TULV but a host-specific genotype. High intra-cluster nucleotide sequence variability (up to 18%) and geographic clustering indicate long-term presence of the virus in this region.

Genetic similarity of Puumala viruses found in Finland and western Siberia and of the mitochondrial DNA of their rodent hosts suggests a common evolutionary origin.

A total of 678 small mammals representing eight species were trapped in western Siberia in 1999-2000 and assayed for the presence of hantaviruses. Eighteen animals, all Clethrionomys species, were antigen positive by enzyme-linked immunosorbent assay (ELISA). Small and medium genome segments were recovered by RT-PCR from six samples from Clethrionomys glareolus and three from Clethrionomys rufocanus. Sequence comparison and phylogenetic analysis revealed that these hantaviruses were Puumala virus and were similar to hantavirus strains from Finland. To confirm these data, partial nucleotide sequences of the rodent hosts' cytochrome b genes were obtained, as well as several sequences from genes from rodents trapped at different localities of European Russia and western Siberia. The cytochrome b sequences of Siberian bank voles were similar to sequences of C. glareolus, trapped in Finland. These data suggest that the Puumala hantaviruses, as well as their rodent hosts, share a common evolutionary history. We propose that these rodents and viruses may be descendents of a population of bank voles that expanded northward from southern refugia during one of the interglacial periods.

The effects of tensile-compressive loading mode and microarchitecture on microdamage in human vertebral cancellous bone.

The amount of microdamage in bone tissue impairs mechanical performance and may act as a stimulus for bone remodeling. Here we determine how loading mode (tension vs. compression) and microstructure (trabecular microarchitecture, local trabecular thickness, and presence of resorption cavities) influence the number and volume of microdamage sites generated in cancellous bone following a single overload. Twenty paired cylindrical specimens of human vertebral cancellous bone from 10 donors (47­78 years) were mechanically loaded to apparent yield in either compression or tension, and imaged in three dimensions for microarchitecture and microdamage (voxel size 0.7×0.7×5.0 µm3). We found that the overall proportion of damaged tissue was greater (p=0.01) for apparent tension loading (3.9±2.4%, mean±SD) than for apparent compression loading (1.9±1.3%). Individual microdamage sites generated in tension were larger in volume (p<0.001) but not more numerous (p=0.64) than sites in compression. For both loading modes, the proportion of damaged tissue varied more across donors than with bone volume fraction, traditional measures of microarchitecture (trabecular thickness, trabecular separation, etc.), apparent Young׳s modulus, or strength. Microdamage tended to occur in regions of greater trabecular thickness but not near observable resorption cavities. Taken together, these findings indicate that, regardless of loading mode, accumulation of microdamage in cancellous bone after monotonic loading to yield is influenced by donor characteristics other than traditional measures of microarchitecture, suggesting a possible role for tissue material properties.

Three-dimensional dynamic bone histomorphometry.

Dynamic bone histomorphometry is the standard method for measuring bone remodeling at the level of individual events. Although dynamic bone histomorphometry is an invaluable tool for understanding osteoporosis and other metabolic bone diseases, the technique's two-dimensional nature requires the use of stereology and prevents measures of individual remodeling event number and size. Here, we used a novel three-dimensional fluorescence imaging technique to achieve measures of individual resorption cavities and formation events. We performed this three-dimensional histomorphometry approach using a common model of postmenopausal osteoporosis, the ovariectomized rat. The three-dimensional images demonstrated the spatial relationship between resorption cavities and formation events consistent with the hemiosteonal model of cancellous bone remodeling. Established ovariectomy was associated with significant increases in the number of resorption cavities per unit bone surface (2.38 ± 0.24 mm⁻² sham surgery versus 3.86 ± 0.35 mm⁻² bilateral ovariectomy [OVX], mean ± SD, p < 0.05) and total volume occupied by cavities per unit bone volume (0.38% ± 0.06% sham versus 1.12% ± 0.18% OVX, p < 0.001), but there was no difference in surface area per resorption cavity, maximum cavity depth, or cavity volume. In addition, we found that established ovariectomy is associated with increased size of bone formation events because of the merging of formation events (23,700 ± 6,890 µm² sham verusus 33,300 ± 7,950 µm² OVX). No differences in mineral apposition rate (determined in 3D) were associated with established ovariectomy. That established estrogen depletion is associated with increased number of remodeling events with only subtle changes in remodeling event size suggests that circulating estrogens may have their primary effect on the origination of new basic multicellular units with relatively little effect on the progression and termination of active remodeling events.

Development of a diagnostic method applicable to various serotypes of hantavirus infection in rodents.

Antigenic diversity among different hantaviruses requires a variety of reagents for diagnosis of hantavirus infection. To develop a diagnostic method applicable to various hantavirus infections with a single set of reagents, we developed an enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid proteins of three hantaviruses, Amur, Hokkaido, and Sin Nombre viruses. This novel cocktail antigen-based ELISA enabled detection of antibodies against Hantaan, Seoul, Amur, Puumala, and Sin Nombre viruses in immunized laboratory animals. In wild rodent species, including Apodemus, Rattus, and Myodes, our ELISA detected antibodies against hantaviruses with high sensitivity and specificity. These data suggest that our novel diagnostic ELISA is a useful tool for screening hantavirus infections and could be effectively utilized for serological surveillance and quarantine purposes.

An efficient in vivo method for the isolation of Puumala virus in Syrian hamsters and the characterization of the isolates from Russia.

Puumala virus (PUUV) and other Arvicolinae-borne hantaviruses are difficult to cultivate in cell culture. To isolate these hantaviruses efficiently, hantavirus nucleocapsid protein (NP)-positive but seronegative wild rodents were selected by NP-detection ELISA. Three of 68 Myodes glareolus captured in Samara, Russia, were NP-positive and seronegative. Syrian hamsters were inoculated with lung homogenates from NP-positive rodents for virus propagation. Virus isolation in vitro was carried out by inoculation of lung homogenates of NP-positive hamsters to Vero E6 cell monolayers. Two PUUV strains (Samara49/CG/2005 and Samara94/CG/2005) from M. glareolus were isolated in Vero E6 cells. Nucleotide and amino acid sequence identities of the S segment of these isolates to those of PUUV F-s808 from a fatal HFRS patient in Samara region were 96.7-99.3% and 99.3-100.0%, respectively. Morphologic features of Vero E6 cells infected with PUUV strain Samara49/CG/2005 were quite similar to those of Hantaan virus-infected cells. Isolation of Hokkaido virus from Myodes rufocanus captured in Hokkaido, Japan, was also performed. Hokkaido virus NP and RNA were recovered and maintained in hamsters. These results suggest that inoculation of Syrian hamsters with rodent samples is an efficient method for the isolation and maintenance of PUUV and other Arvicolinae-borne hantaviruses.

Voxel size and measures of individual resorption cavities in three-dimensional images of cancellous bone.

Cavities formed by osteoclasts on the surface of cancellous bone during bone remodeling (resorption cavities) are believed to act as stress risers and impair cancellous bone strength and stiffness. Although resorption cavities are readily detected as eroded surfaces in histology sections, identification of resorption cavities in three-dimensional images of cancellous bone has been rare. Here we use sub-micrometer resolution images of rat lumbar vertebral cancellous bone obtained through serial milling (n=5) to determine how measures of the number and surface area of resorption cavities are influenced by image resolution. Three-dimensional images of a 1 mm cube of cancellous bone were collected at 0.7x0.7x5.0 microm/voxel using fluorescence based serial milling and uniformly coarsened to four other resolutions ranging from 1.4x1.4x5.0 to 11.2x11.2x10 microm/voxel. Cavities were identified in the three-dimensional image as an indentation on the cancellous bone surface and were confirmed as eroded surfaces by viewing two-dimensional cross-sections (mimicking histology techniques). The number of cavities observed in the 0.7x0.7x5.0 microm/voxel images (22.0+/-1.43, mean+/-SD) was not significantly different from that in the 1.4x1.4x5.0 microm/voxel images (19.2+/-2.59) and an average of 79% of the cavities observed at both of these resolutions were coincident. However, at lower resolutions, cavity detection was confounded by low sensitivity (<20%) and high false positive rates (>40%). Our results demonstrate that when image voxel size exceeds 1.4x1.4x5.0 microm/voxel identification of resorption cavities by bone surface morphology is highly inaccurate. Experimental and computational studies of resorption cavities in three-dimensional images of cancellous bone may therefore require images to be collected at resolutions of 1.4 microm/pixel in-plane or better to ensure consistent identification of resorption cavities.