The first identification of Orthohantavirus dobravaense, Kurkino virus (Hantaviridae: Orthohantavirus) in the Volga Federal District

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Introduction

Orthohantaviruses, members of the family Hantaviridae and genus Orthohantavirus, are widespread across all continents except Antarctica. Pathogenic orthohantaviruses cause human diseases such as hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, which are registered in the Old and New World, respectively [1].

HFRS is an acute zoonotic infection that is widespread in Eurasia. The clinical picture of the disease is characterized by intoxication, fever, and kidney damage, but the severity, intensity of symptoms, and presence of pathognomonic signs can vary significantly depending on the species and genotype of the pathogenic virus [2].

Representatives of different species and genotypes of orthohantaviruses are associated with specific natural reservoirs, which are small mammals, mainly rodents. The main route of human infection is aerogenic: viral particles enter the respiratory tract when aerosols containing particles of excrement, dried urine, or saliva from an infected animal are inhaled [3].

In Russia, HFRS ranks among the top natural focal infections in terms of incidence: over a 10-year period from 2015 to 2024, 64,954 cases were registered in the Russian Federation, more than 82% of which were in the Volga Federal District (VFD)¹. In the Asian part of the Russian Federation, the main pathogens of HFRS are Orthohantavirus hantanense (formerly Hantaan orthohantavirus – HNTV) and O. seoulense (formerly Seoul orthohantavirus – SEOV), while in the European part, they are O. puumalaense (formerly Puumala orthohantavirus – PUUV), which causes the vast majority of cases in the country, and O. dobravaense (ODOB, formerly Dobrava-Belgrade orthohantavirus – DOBV) – the Kurkino and Sochi viruses [2].

The orthohantavirus genome consists of three segments of single-stranded negative-sense RNA: small (S), medium (M), and large (L), encoding, respectively, the nucleocapsid protein (N), the precursor of membrane glycoproteins Gn and Gc (GPC), and RNA-dependent RNA polymerase (RdRp) [4].

Among the orthohantaviruses circulating in Europe, ODOB poses the greatest threat to human life, characterized by a particularly severe course of the disease and a relatively high mortality rate of up to 12% [5]. Currently, a number of ODOB genetic groups are distinguished, associated with certain species of mice of the genus Apodemus, which are their natural reservoirs. However, cases of possible virus transfer from one genetic group to a mouse species associated with another group have also been identified [6].

The Dobrava virus genetic group (also referred to as DOBV-Af) is associated with the yellow-necked mouse (A. flavicollis) and circulates mainly in Southeast and Central Europe [7, 8]. Representatives of this genetic group are associated with a severe clinical course of the disease and are characterized by the highest virulence and lethality among all ODOB genotypes. The main natural reservoir of the Kurkino virus group (DOBV-Aa) is the field mouse (A. agrarius), which inhabits mainly Central Europe, European Russia and Western Siberia. The associated form of HFRS usually occurs in mild and moderately severe forms [9–13]. The Sochi virus genetic group (DOBV-Ap) is associated with the Black See field mouse (A. ponticus) and is characterized by a clinical course of HFRS of moderate to severe severity. The isolates of this group studied to date have been detected in rodents caught in the south of European Russia [11, 14].

The main pathogen of HFRS in the Russian Federation is PUUV, but in a number of regions there are also cases and outbreaks of HFRS associated with ODOB, which pose a significant threat to public health [11, 13, 15]. Searching for ODOB in unexplored regions of the country appears necessary to identify new potential foci of HFRS. Studying the genome of the detected isolates may be useful for determining the patterns of ODOB distribution in the populations of their natural reservoirs. However, a significant part of Russia, including the Volga Federal District, has remained unexplored in terms of circulating ODOB genetic variants.

The aim of the study is to identify ODOB in a number of regions of the Russian Federation and to analyze their genome.

Materials and methods

Frozen samples of lung tissue from rodents (natural reservoirs of ODOB) caught between 2015 and 2023 (provided by the Center of Hygiene and Epidemiology in the Republic of Tatarstan) were used as biomaterial for the study. The biomaterial samples were stored at −70 °C.

Total RNA was extracted from rodent tissues using the ExtractRNA reagent (Eurogen, Russia) in accordance with the manufacturer’s recommended protocol. The extracted RNA drugs were stored at −70 °C. Primary screening of samples for viral RNA detection was performed using reverse transcription polymerase chain reaction (RT-PCR) using the BioMaster RT-PCR Premium (2×) kit (Biolabmix, Russia) and specific primers of our own design, complementary to various regions of the ODOB genome. Complementary DNA (cDNA) synthesis was performed using RNAscribe RT reverse transcriptase (Biolabmix, Russia) according to the manufacturer’s instructions. The synthesized cDNA was stored at −70 °C. Individual regions of the viral genome were amplified using the 5×Screen Mix reaction mixture (Eurogen, Russia) and a set of specific primers. The PCR products obtained were separated in agarose gel and purified using the Cleanup S-Cap kit (Eurogen, Russia) according to the manufacturer’s instructions and sequenced by Sanger sequencing using the ABI PRISM 310 Big Dye Terminator 3.1 Sequencing Kit (ABI, USA). Nucleotide sequences were aligned using the ClustalW method with the MegAlign program (DNASTAR, USA). Comparative analysis of nucleotide and amino acid sequences was also performed using MegAlign (DNASTAR, USA), using the sequences of individual ODOB segments (Table 1) stored in the GenBank database as references². For phylogenetic analysis, we used the MEGA11 program [16], the Maximum Likelihood method and the Tamura-Nei model [17]. The sequences of the S and M segments of the O. hantanense HV004 strain were used as an outgroup.

 

Table 1. List of reference sequences from the GenBank database used for comparative and phylogenetic analysis

Таблица 1. Список референсных последовательностей из базы данных GenBank, использованных для проведения сравнительного и филогенетического анализа

Region Регион	Isolate Изолят	GenBank accession number № в GenBank
		S segment S-сегмент	M segment M-сегмент
Slovakia / Словакия	SK/Aa	AY961615	AY961616
Eastern Slovakia / Восточная Словакия	East Slovakia/400Af/98	AY168576	AY168577
Slovakia, Košice / Словакия, Кошице	East Slovakia/862Aa/97	AJ269550	AY168578
	East Slovakia-856-Aa	AJ269549
Greece, Ano-Poroia / Греция, Ано-Поройя	DOBV/Ano-Poroia/Afl9/1999	AJ410615	AJ410616
Turkey, Igneada / Турция, Игнеада	Turkey/Igneada/13Af/2009	MW055919	MW055918
Slovenia / Словения	Slo/Af-BER	GU904029	GU904035
Germany / Германия	GER/07/293/Aa	GQ205401	GQ205409
	GER/07/607/Af	GQ205402	GQ205410
	GER/05/477/Af	GQ205406	GQ205411
	GER/08/118/Aa	GQ205407	GQ205412
	GER/08/131/Af	GQ205408	GQ205413
Lithuania, Rusne / Литва, Русне	LT17/RUS_56	MT580917
	LT18/RUS_2	MT580918	MT580914
Lithuania, Lukstas / Литва, Лукштас	LT18/LK_11	MT580919	MT580916
	LT18/LK_57	MT580920
Lithuania, Tytuvenai / Литва, Титувенай	LT18/TYT_44	MT580921	OK422868
Lithuania, Birzai / Литва, Биржай	LT16/BR_42	MT580922	OK422867
Austria, Lake Neusiedl / Австрия, Нойзидлер-Зе	KS18/1812	MN657233	MW091492
	KS12/1827	MW151008	MW091491
Italy, Udine / Италия, Удине	21RS2853-23		OM677635
Croatia, Gerovo / Хорватия, Герово	DOBV/Croatia_Gerovo/Af966/2008	KC676598
Croatia, Zutica / Хорватия, Зутика	DOBV/Croatia_Zutica/Af813/2007	KC676602
Russia, Sochi / Россия, Сочи	Ap1584/Sochi-01	EU188449	EU188450
	Ap/Sochi/43	JF920151
	10636/Ap	KP878311
	10645/Ap	KP878312
Russia, Goryachiy Klyuch / Россия, Горячий Ключ	Ap-1/Goryachiy Klyuch-2000	AF442622
Russia, Krasnodar / Россия, Краснодар	P-s1223/Krasnodar-2000	AF442623
Russia, Lipetsk / Россия, Липецк	Aa1854/Lipetsk-02	EU188452	EU188453
Russia, Kurkino / Россия, Куркино	Kurkino/44Aa/98	AJ131672
	Kurkino/Aa38/97		GQ303581
	Kurkino/Aa44/97		GQ303582
China, Hubei / Китай, Хубэй	HV004	JQ083395	JQ083394

 

Authors confirm compliance with institutional and national standards for the use of laboratory animals in accordance with «Consensus Author Guidelines for Animal Use» (IAVES, July 23, 2010). The research protocol was approved by the Ethics Committee of the institution (Protocol No 23 dated June 30, 2020).

Results

A total of 275 samples of lung tissue from A. agrarius and A. flavicollis caught between 2016 and 2023 in the territories of several regions of the Volga, Central and Ural Federal Districts were examined (Table 2).

 

Table 2. Trapping regions and rodent species

Таблица 2. Регионы отлова и видовой состав грызунов

Federal distict / Федеральный округ	Region / Регион	Rodents number / Количество грызунов	A. agrarius	A. flavicollis
Volga Приволжский	Republic of Tatarstan Республика Татарстан	131	29	102
	Republic of Bashkortostan Республика Башкортостан	12	2	10
	Udmurt Republic Удмуртская Республика	18	18	0
	Chuvash Republic Чувашская Республика	13	0	13
	Kirov Oblast Кировская область	3	3	0
	Nizhny Novgorod Oblast Нижегородская область	5	0	5
	Ulyanovsk Oblast Ульяновская область	18	7	11
Central Центральный	Kostoma Oblast Костромская область	4	4	0
	Tula Oblast Тульская область	53	53	0
Ural Уральский	Chelyabinsk Oblast Челябинская область	18	0	18
In total Всего		275	116	159

 

As a result of the study, orthohantavirus RNA was detected in only one sample obtained from A. flavicollis caught near the “Sozidatel” allotment in the Cherdaklinsky district of the Ulyanovsk region of the VFD in 2019. The detected RNA isolate was designated ML1779. No orthohantavirus RNA was detected in A. agrarius. Thus, the average infection rate of A. flavicollis samples was very low, at only 0.63%. The results obtained are consistent with the data from ODOB in Poland, where the virus was detected in only one of 194 samples tested [8], and in Germany, where 7 virus strains were isolated from 366 samples [6].

Nucleotide sequences of the S and M segments of the genome were obtained for the detected RNA isolate. The length of the sequences and their location within the segment were 629 nucleotides (positions 36–664) and 441 nucleotides (1718–2158) for the S and M segments, respectively (hereinafter, the localization of nucleotide sequences within the genome segments is indicated relative to the S and M segments of the genome of isolate Aa1854/Lipetsk-02 from Russia, GenBank numbers EU188452 and EU188453, respectively). Based on the obtained nucleotide sequences, the amino acid sequences of the N and GPC protein regions with a length of 147 and 209 amino acid residues, respectively, were calculated.

A comparative analysis showed that the nucleotide sequence similarity values of the S- and M-segments of the ML1779 RNA isolate and the ODOB reference isolates ranged from 85.1% to 94.0% and 79.8% to 92.1%, respectively, which allows it to be identified as ODOB (Fig. 1).

 

Fig. 1. The similarity values obtained by comparing the sequences of S and M segment fragments, N and GPC proteins of the identified (ML1779) and reference ODOB isolates/strains.

Рис. 1. Значения идентичности, полученные при сравнении последовательностей участков S- и M-сегментов, белков N и GPC выявленного (ML1779) и референсных изолятов/штаммов ODOB.

 

The most similar to ML1779 are the partial S-segment of strains from Russia: AJ131672 (Kurkino) and EU188452 (Lipetsk), with sequence similarity values of 94.0 and 93.8%, respectively. For the partial M-segment of ML1779, the highest values (92.1%) were also observed when compared with strains from Russia: EU188453 (Lipetsk), GQ303581, and GQ303582 (Kurkino).

The amino acid sequence similarity values of the N and GPC ML1779 proteins and reference isolates ranged from 98.1–100.0 and 93.2–99.3%, respectively (Fig. 1). The highest sequence similarity values were found when comparing strains from Russia (Lipetsk and Kurkino), Lithuania, Austria and Slovakia (Fig. 1).

The vast majority of ODOB reference strains, whose nucleotide and amino acid sequence similarity values were highest when compared to isolate ML1779, were isolated from A. agrarius, with the exception of three strains from Germany, which were isolated from A. flavicollis. At the same time, ODOB reference strains isolated from A. flavicollis, which were caught in Slovenia, Croatia, Greece and Turkey, were characterized by lower nucleotide and amino acid sequence similarity values when compared to the studied isolate.

Based on the results of phylogenetic analysis of nucleotide sequences of sequenced regions of the S and M segments, all reference strains were grouped into three large clades (Figs. 2, 3). The DOBV-Ap clade combines strains of the Sochi virus genetic group circulating in the Southern Federal District, carried by A. ponticus, while the DOBV-Af clade includes strains of the Dobrava virus genetic group detected in A. flavicollis, which were caught in Turkey, Greece, Croatia, Slovenia and eastern Slovakia. At the same time, the DOBV-Aa clade includes strains of the Kurkino virus genetic group from Germany, Austria, Slovakia, Lithuania, and central European Russia – from the Lipetsk and Tula regions (Lipetsk and Kurkino), as well as a studied isolate from the Ulyanovsk region, with the identified isolate ML1779 included in the “Russia, center” subclade together with reference strains from the Lipetsk and Tula regions.

 

Fig. 2. A phylogenetic tree constructed for the nucleotide sequences of the partial S segment (pos. 36–664).

The isolate identified in the Volga Federal District is designated as ML1779. Isolates obtained from A. flavicollis are indicated in blue, from A. agrarius – in green, from A. ponticus – in brown. Bootstrap values below 70 are not shown.

Рис. 2. Филогенетическое древо, построенное для нуклеотидных последовательностей участка S-сегмента (позиция 36–664).

Изолят, выявленный в ПФО, обозначен как ML1779. Изоляты, полученные от A. flavicollis, обозначены синим, от A. agrarius – зеленым, от A. ponticus – коричневым цветом. Значения бутстрепов ниже 70 не показаны.

 

Fig. 3. A phylogenetic tree constructed for the nucleotide sequences of the partial M segment (pos. 1718–2158).

The isolate identified in the Volga Federal District is designated as ML1779. Isolates obtained from A. flavicollis are indicated in blue, from A. agrarius – in green, from A. ponticus – in brown. Bootstrap values below 70 are not shown.

Рис. 3. Филогенетическое древо, построенное для нуклеотидных последовательностей участка M-сегмента (позиции 1718–2158).

Изолят, выявленный в ПФО, обозначен как ML1779. Изоляты, полученные от A. flavicollis, обозначены синим, от A. agrarius – зеленым, от A. ponticus – коричневым цветом. Значения бутстрепов ниже 70 не показаны.

 

Thus, the results of comparative and phylogenetic analysis indicate that isolate ML1779 belongs to the Kurkino virus genetic group. It should be noted that the DOBV-Aa clade includes ODOB strains identified in both A. flavicollis (ML1779 and three strains from Germany) and A. agrarius (the remaining strains).

Discussion

The main pathogen of HFRS in European Russia is PUUV, but isolated cases and outbreaks caused by ODOB have been reported in a number of regions [18]. As a rule, HFRS caused by ODOB is more often moderate to severe than that caused by PUUV, and therefore it is necessary to study the prevalence of different genetic variants of ODOB in order to improve epidemiological safety in potentially dangerous regions. Although ODOB was identified more than 30 years ago [19], currently, only limited information is available in the literature on the areas of circulation of strains of different genetic variants of the virus. Thus, in Russia, ODOB (Kurkino and Sochi viruses) has been found only in a number of locations in central European Russia, Astrakhan region, western Siberia [13], and Krasnodar Krai [11]. The ML1779 isolate we identified in the Ulyanovsk region is the first case of ODOB detection in the VFD, which allows us to conclude that the Kurkino virus genetic variants are more widespread in European Russia (Fig. 4).

 

Fig. 4. The location of the studied (ML1779) and reference ODOB strains.

Representatives of the Dobrava virus genetic group are indicated in gray, Kurkino virus in blue, and Sochi virus in yellow. Isolates obtained from A. flavicollis are indicated by squares, isolates from A. agrarius – by circles, isolates from A. ponticus – by triangles. The map was modified from https://snazzymaps.com/style/253658/only-borders, accessed on September 23, 2025.

Рис. 4. Локация исследованного (ML1779) и референсных штаммов ODOB.

Представители генетической группы вируса Добрава обозначены серым, вируса Куркино – голубым, вируса Сочи – желтым цветом. Изоляты, полученные от A. flavicollis, обозначены квадратами, от A. agrarius – кругами, от A. ponticus – треугольниками. Измененная карта с сайта https://snazzymaps.com/style/253658/only-borders, дата доступа 23.09.2025.

 

The localization of isolate ML1779 provides grounds for expanding the presumed area of virus circulation to the left bank of the Volga River. The nucleotide and amino acid sequences of the ML1779 isolate genome showed high levels of similarity with pathogenic reference strains of the Kurkino virus from the Lipetsk and Tula regions, which allows us to conclude that there is a real threat of HFRS cases due to human infection with this virus at least in part of the VFD.

It is also important to note that the reference strains of the Kurkino virus from Lipetsk and Kurkino were isolated from A. agrarius, and most of the identified strains of the Kurkino virus group, whose nucleotide sequences are available in GenBank, were also detected in A. agrarius. However, strains of this genetic group were also found in A. flavicollis from Germany [6]. The ML1779 isolate identified in this study was found in A. flavicollis, which is most likely a case of spillover, as previously described by M. Schlegel et al. [6] (multiple spillover of DOBV-Aa in A. flavicollis). Currently, there is insufficient data to conclude whether the yellow-necked mouse is a natural reservoir for any of the genetic variants of the Kurkino virus. It is also possible that the transfer of the Kurkino virus genetic group contributes to an increase in its genetic diversity. Therefore, it seems appropriate to conduct further research to identify ODOB genetic variants in both A. agrarius and A. flavicollis populations in European Russia.

It should be noted that earlier in the Ulyanovsk region, PUUV isolates were detected in samples of biomaterial from bank voles caught in 2019 near the villages of Kurlan and Mullovka, located 40–60 km from the location of isolate ML1779 [20]. Thus, co-circulation of two orthohantavirus species, ODOB and PUUV, has been recorded in the relatively small territory of the Ulyanovsk region. Previously, a similar case of co-circulation of two orthohantavirus species (PUUV and ODOB) was detected in Belarus [9], and in the Republic of Bashkortostan, co-circulation of three orthohantavirus species was detected: PUUV, O. tulaense, and O. seewisense [21]. Thus, co-circulation of two or more orthohantaviruses in one territory appears to be a fairly common phenomenon, including in the VFD. Therefore, it seems important to study the possibility of genetic material exchange between co-circulating orthohantavirus species and the emergence of hybrid genetic variants, which requires further research.

Conclusion

This study conducted the first large-scale search for ODOB in populations of A. agrarius and A. flavicollis in several regions of the VFD and neighboring regions. It was found that the infection rate of the studied small rodents with orthohantaviruses was close to zero. Only one orthohantavirus isolate, identified as belonging to the Kurkino virus genetic group, was detected in A. flavicollis from the Ulyanovsk region. The most closely related strains to the identified strain were Kurkino virus strains previously found in A. agrarius in the Lipetsk and Tula regions. Thus, it has been proven for the first time that: 1) the range of Orthohantavirus dobravaense (Kurkino virus) extends to part of the Volga Federal District; 2) Orthohantavirus dobravaense (Kurkino virus) and Orthohantavirus puumalaense (Puumala virus) co-circulate in the Ulyanovsk region.

¹ EMISS (2025). Available at: https://www.fedstat.ru/indicator/38208# (accessed: 22.09.2025).

² National Center for Biotechnology Information (NCBI), GenBank (2025). Available at: https://www.ncbi.nlm.nih.gov/genbank/

About the authors

Timur R. Nasyrov

Institute of Fundamental Medicine and Biology Federal State Autonomous Educational Institution of Higher Education «Kazan (Volga Region) Federal University»

Email: nasyrovtimur1@mail.ru
ORCID iD: 0009-0000-4116-0471

laborant researcher, OpenLab ‘Gene and Cell Technologies’

Russian Federation, 420008, Kazan

Polina I. Elboeva

Institute of Fundamental Medicine and Biology Federal State Autonomous Educational Institution of Higher Education «Kazan (Volga Region) Federal University»

Email: polinaelboeva@mail.ru
ORCID iD: 0009-0001-2949-3289

junior researcher, OpenLab ‘Gene and Cell Technologies’

Russian Federation, 420008, Kazan

Ekaterina V. Martynova

Institute of Fundamental Medicine and Biology Federal State Autonomous Educational Institution of Higher Education «Kazan (Volga Region) Federal University»

Email: ignietferro.venivedivici@gmail.com
ORCID iD: 0000-0003-1537-3099

Cand. Sci. (Med.), leading researcher, OpenLab ‘Gene and Cell Technologies’

Russian Federation, 420008, Kazan

Olesia V. Okhlopkova

Research Institute of Virology, Federal Research Center for Fundamental and Translational Medicine

Author for correspondence.
Email: ohlopkova.lesia@yandex.ru
ORCID iD: 0000-0002-8214-7828

Cand. Sci. (Biol.), senior researcher, Research Institute of Virology

Russian Federation, 630060, Novosibirsk

Yuriy A. Tyurin

Kazan State Medical University of the Ministry of Health of the Russian Federation

Email: tyurin.yurii@yandex.ru
ORCID iD: 0000-0002-2536-3604

D. Sci. (Med.), assistant professor, assistant professor of Department of Biochemistry and Clinical Laboratory Diagnostics

Russian Federation, 420012, Kazan

Emmanuel Kabwe

Institute of Fundamental Medicine and Biology Federal State Autonomous Educational Institution of Higher Education «Kazan (Volga Region) Federal University»

Email: emmanuelkabwe@ymail.com
ORCID iD: 0000-0003-4328-8190

Cand. Sci. (Biol.), senior researcher, OpenLab ‘Gene and Cell Technologies’

Russian Federation, 420008, Kazan

Yuriy N. Davidyuk

Institute of Fundamental Medicine and Biology Federal State Autonomous Educational Institution of Higher Education «Kazan (Volga Region) Federal University»

Email: davi.djuk@mail.ru
ORCID iD: 0000-0002-4409-2942

Cand. Sci. (Biol.), assistant professor, senior researcher, OpenLab ‘Gene and Cell Technologies’

Russian Federation, 420008, Kazan

References

Supplementary files