The Specificity of Epizootic and Epidemiological Processes in Natural Foci of Hemorrhagic Fever with Renal Syndrome and Tick-Borne Encephalitis in Russia, as the Basis for the Prospects of Creating a Combined Vaccine for the Prevention of These Infections.

Hemorrhagic fever with renal syndrome (HFRS) and tick-borne encephalitis (TBE) are the most common viral diseases in Russia. HFRS is caused by six different types of hantaviruses: Hantaan, Amur, Seoul, Puumala, Kurkino, and Sochi, which are transmitted to humans through small mammals of the Muridae and Cricetidae families. TBE is caused by viruses belonging to five different phylogenetic subtypes. The similarities in the ecology of HFRS and TBE pathogens is presented here. Hantavirus-infected small mammals can transmit the virus to uninfected animals, and ticks can also transmit hantavirus to other ticks and mammals. Hantavirus transmission from ticks to humans is possible only hypothetically based on indirect data. Over the past 23 years, 164,582 cases of HFRS (4.9 per 105 people) and 71,579 cases of TBE (2.5 per 105 people) were registered in Russia. The mortality rate was 0.4% (668 cases) in HFRS and 1.6% deaths (1136 cases) in TBE. There were 4030 HFRS (2.5%) and 9414 TBE (13%) cases in children under 14 years old. HFRS and TBE cases were registered in 42 out of 85 Russian regions; in 18-only HFRS, in 13-only TBE, and 12 had no reported cases. The prospects of applying a combined vaccine for HFRS and TBE prevention are shown in this paper.

Phenotypic assessment of antiviral activity for spiro-annulated oxepanes and azepenes.

Evolutionary potential of viruses can result in outbreaks of well-known viruses and emergence of novel ones. Pharmacological methods of intervening the reproduction of various less popular, but not less important viruses are not available, as well as the spectrum of antiviral activity for most known compounds. In the framework of chemical biology paradigm, characterization of antiviral activity spectrum of new compounds allows to extend the antiviral chemical space and provides new important structure-activity relationships for data-driven drug discovery. Here we present a primary assessment of antiviral activity of spiro-annulated derivatives of seven-membered heterocycles, oxepane and azepane, in phenotypic assays against viruses with different genomes, virion structures, and genome realization schemes: orthoflavivirus (tick-borne encephalitis virus, TBEV), enteroviruses (poliovirus, enterovirus A71, echovirus 30), adenovirus (human adenovirus C5), hantavirus (Puumala virus). Hit compounds inhibited reproduction of adenovirus C5, the only DNA virus in the studied set, in the yield reduction assay, and did not inhibit reproduction of RNA viruses.

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.

Evolutionary Formation and Distribution of Puumala Virus Genome Variants, Russia.

We analyzed Puumala virus (PUUV) sequences collected from bank voles from different regions of Russia. Phylogenetic analysis revealed PUUV reassortments in areas with the highest hemorrhagic fever with renal syndrome incidence, indicating reassortment might contribute to pathogenic properties of PUUV. Continued surveillance is needed to assess PUUV pathogenicity in Russia.

A fatal case of haemorrhagic fever with renal syndrome in Kursk Region, Russia, caused by a novel Puumala virus clade.

Haemorrhagic fever with renal syndrome (HFRS) is the most widespread natural-focal human disease in the Russian Federation. In this study, we report virological assessment of a fatal case of HFRS-PUUV (Puumala virus) in the Kursk Region. The infection caused severe multiorgan failure and the maximum viral load was detected in the tissue of the spleen. Viral sequences were obtained from the patient's autopsy material and lung tissues of bank voles captured in the region. These sequences formed a new clade in the PUUV phylogenetic tree, an outgroup to all known Russian (RUS) lineage sequences. On the other hand viruses collected in the Kursk Region grouped with the RUS lineage and are separated from all other PUUV linages. We propose to nominate this novel group as W-RUS as the identified viruses were collected near the western Russian boundary. The recombination signals between their ancestors and RUS lineage representatives from the Volga region were revealed. The strain Samara_94/CG/2005 suggestively emerged as the result of reassortment between the ancestors of W-RUS and DTK-Ufa-97.

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.

Pre-Clinical Studies of Inactivated Polyvalent HFRS Vaccine.

Hemorrhagic fever with renal syndrome (HFRS) is the most common natural focal disease in the Russian Federation with about 6-12 thousand cases annually. 97.7% of all HFRS cases in Russia are caused by the Puumala virus, 1.5%-by the Hantaan, Amur, Seoul viruses, and about 0.8% by the Kurkino and Sochi viruses. There are no licensed vaccines for the prevention of HFRS in the European Region; there are no specific therapeutic to treat orthohantavirus infections. Here we report the results of candidate polyvalent HFRS vaccine preclinical studies. The vaccine was produced on the basis of three viruses: Puumala, strain PUU-TKD/VERO, Hantaan, strain HTN-P88/VERO, and Sochi, strain DOB-SOCHI/VERO. These viruses were inactivated with ß-propiolacton, purified by gel filtration and aluminum hydroxide adsorbed. 18-20 g female BALB/c mice were immunized intramuscularly 2 or 3 times with a 2-week intervals and blood was taken 2 weeks after immunization. FRNT50 performed for virus specific antibodies determination. ELISA kits (Bender MedSystems, Cusabio) were used for detection of cytokines IL-1ß, IL-12, INF-É£. Neutralizing antibodies geometric mean titers to the Puumala, Hantaan, and Sochi viruses were: 9.22 ± 0.31, 9.17 ± 0.26, 8.96 ± 0.34 log2/ml. Up to 1/32 vaccine dilution neutralizing antibodies were identified in 10/10 immunized mice with titers ≥ 3,32 log2/ml. IL-12 and INF-É£ increased after immunization in average 5.5 and 2.8 times respectively, that reflects the Th1 type immunity stimulation. IL-1ß slightly increased, that may suggest vaccine low reactogenicity. According to our preclinical investigations, the candidate polyvalent HFRS vaccine elicits balanced immune response to the Puumala, Hantaan and Sochi viruses.

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.

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.

Phylogeography of Crimean Congo Hemorrhagic Fever Virus.

Crimean Congo hemorrhagic fever virus (CCHFV) is one of the most severe viral zoonozes. It is prevalent throughout Africa, Asia and southern Europe. Limited availability of sequence data has hindered phylogeographic studies. The complete genomic sequence of all three segments of 14 Crimean Congo hemorrhagic fever virus strains isolated from 1958-2000 in Russia, Central Asia and Africa was identified. Each genomic segment was independently subjected to continuous Bayesian phylogeographic analysis. The origin of each genomic segment was traced to Africa about 1,000-5,000 years ago. The virus was first introduced to South and Central Asia in the Middle Ages, and then spread to China, India and Russia. Reverse transfers of genomic segments from Asia to Africa were also observed. The European CCHFV genotype V was introduced to Europe via the Astrakhan region in South Russia 280-400 years ago and subsequently gradually spread westward in Russia, to Turkey and the Balkans less than 150 years ago. Only a few recombination events could be suggested in S and L genomic segments, while segment reassortment was very common. The median height of a non-reassortant phylogenetic tree node was 68-156 years. There were reassortment events within the European CCHFV lineage, but not with viruses from other locations. Therefore, CCHFV in Europe is a recently emerged zoonosis that represents a spillover from the global gene pool.

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%.

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.

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.

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.

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.

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 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.