Hemorrhagic fever with renal syndrome (HFRS) is one of the widespread viral natural focal infections in the world [1][2]. More than 200,000 cases are registered annually worldwide [3].

In the territory of the Russian Federation, the incidence rate of HFRS occupies one of the leading places among all natural focal infections. Circulation of the hantavirus pathogen is registered in all federal districts of Russia. The most epidemically active natural foci are found in the European part of Russia, they account for up to 98% of confirmed cases of HFRS (of the total number of cases in the country), the Siberian and Far Eastern regions – 2% of cases [4][5][6]. In the European part of Russia, morbidity is mainly associated with Puumala and Dobrava-Belgrade viruses, while in the Far East – with Hantaan, Amur, and Seul [7][8].

Various species of viruses, causative agents of HFRS, have close ties with their natural carriers, turning them into natural reservoirs representing a source of infection for humans. The species composition of animals infected with hantaviruses in different regions of the world today totals 84 species from 14 families of mammals. In the Russian Federation, the red vole (the main carrier of hantaviruses) accounts for more than half of all positive samples detected, while the share of infected samples from the field mouse (Apodemus agrarius) is 8.5%. Single infected individuals were detected in the study of material from the Caucasian and East Asian mouse (Apodemus ponticus, Apodemus peninsulae) and gray rat (Rattus norvegicus) [6]. In addition, hantavirus antigen was detected in 16 species of birds [9][10].

Recently, there has been a tendency to expand the boundaries of natural foci with the involvement of new subjects previously considered free from this infection. Circulation of hantaviruses was confirmed in the south of the European part of Russia, in the Volgograd and Astrakhan Regions, Stavropol and Krasnodar Territories, the Republics of Adygea, Kalmykia, and Crimea [11][12][13], most of which are bordering with the Rostov Region (RR). The first laboratory-confirmed ELISA case of the disease in the region was registered in the Peschanokopsky District in 2018; subsequently, one patient was detected in the Salsky District in 2019 [14]. In the period of 2020–2021, there were no official data on cases of HFRS in the RR [4][15]. In 2022, two cases of the disease were registered in the cities of Rostov-on-Don and Taganrog. The reference center for monitoring of HFRS categorized the RR as the second group, with a low incidence rate (with a range of incidence rates from 0.05 to 0.91 per 100 thousand population) [16].

Examination of convalescents with a disease duration from 5 to 25 years revealed the presence of antibodies to hantavirus in 98% of the examined persons in the Republic of Bashkiria and in 95% – in the Primorye Territory, which indicates a long-term, lifelong immunity to this infection [8]. Serologic studies of the state of population immunity to HFRS pathogens were conducted in a number of subjects of the Russian Federation. The highest level of seropositive serum was observed in the Primorye Territory (28.6%), Samara Region (27.0%), Komi Republic (13.4%), Ulyanovsk Region (10.6%), Sverdlovsk Region (10%), Chelyabinsk Region (9.1%), Jewish Autonomous Region (8.2%), and Tula Region (8.07%) [13].

There was a close correlation in the detection of small mammals (SMs) infected with hantavirus and individuals seropositive to this virus among the population of administrative territories where parallel studies of SMs and humans were conducted. Due to the serologic examination of people with diseases similar in some symptoms to HFRS, patients with this infection were identified in a number of Russian regions where HFRS had not been previously registered [17][18]. The diversity of clinical manifestations determines the high importance of the specific diagnostics of HFRS. Immunologic methods are the main ones in laboratory diagnostics [19][20].

The aim of the study was to assess the epizootological and epidemiological situation of HFRS in the RR.

Field material and formation of samples from SMs for laboratory research were conducted in accordance with the current regulatory documents SP 3.1.7.2614-10; SanPiN 3.3686-21 during 2020– 2022 within the boundaries of 35 administrative districts of the RR and in the vicinity of Rostov-on-Don (SP 3.1.7.2614-10. Prevention of hemorrhagic fever with renal syndrome: Sanitary and epidemiological rules. Moscow: Federal Center of Hygiene and Epidemiology of Rospotrebnadzor, 2010 of April 26, 2010. No. 38. 15 p.; SanPiN 3.3686-21 “Sanitary and Epidemiologic Requirements for the Prevention of Infectious Diseases”).

In 2020, 356 mammal specimens of seven species were captured and studied: common vole (Microtus arvalis), little wood mouse (Sylvaemus uralensis), house mouse (Mus musculus), social vole (Microtus socialis), yellow-throated mouse (Sylvaemus flavicollis), little white-toothed mouse (Crocidura suaveolens), and European wood mouse (Sylvaemus sylvaticus).

In 2021, 422 specimens of rodents, insectivores, and hares of 14 species were examined: house mouse, European wood mouse, little wood mouse, Eastern European vole (Microtus rossiaemeridionalis), little white-toothed vole, mound mouse (Mus spicilegus), social vole, common rodent (Sorex araneus), gray hamster (Cricetulus migratorius), russak hare (Lepus europaeus), yellow-bellied mouse, yellow-bellied mouse (Sylvaemus fulvipectus), common vole, and red-bellied vole (Myodes glareolus).

In 2022, 1,379 individuals of 14 species were examined: lesser white-toothed shrew (Crocidura suaveolens), lesser shrew (Sorex minutus), common shrew (Sorex araneus), house mouse (Mus musculus), yellow-necked mouse (Apodemus flavicollis), steppe mouse (Mus spicilegus), European wood mouse (Apodemus sylvaticus), pygmy field mouse (Sylvaemus uralensis), common vole (Microtus arvalis), Eastern European vole (Microtus mystacinus), red-backed vole (Clethrionomys glareolus), grey hamster (Cricetulus migratorius), yellow-bellied mouse (Sylvaticus fulvipectus), and social vole (Microtus socialis).

The objects of epizootologic monitoring were combined into samples taking into account the species of animal, place of capture, and date of collection. All stages of the study were conducted in accordance with MR 3.1.7.0250-21 “Epidemiology. Prevention of infectious diseases. Infections common to humans and animals tactics and volumes of zoological works in natural foci of infectious diseases”, international ethical norms, and normative documents on bioethics. The presence of hantavirus antigens in SM samples was determined by the ELISA method, using the ELISA test system “HANTAGNOST” (production of FGUP “Enterprise for Production of Bacterial and Viral Preparations of the Institute of Poliomyelitis and Viral Encephalitis named after M.P. Chumakov”) in accordance with the manufacturer’s instructions.

A monitoring study of the immune layer of healthy donors in RR residents during 2020–2022 was carried out by the ELISA method. Biomaterial was obtained in accordance with the principles of legality, compliance with ethical standards, and openness. A total of 1062 blood samples from residents of different districts of the region were tested.

Immunoglobulins of class G (IgG) to hantaviruses were determined in blood serum. Samples in which IgG was detected were additionally analyzed for the presence of immunoglobulins of class M (IgM). Vector-Best (Novosibirsk), “VectoHanta-IgG”, and “VectoHanta-IgM” test systems were used according to the manufacturer’s instructions. Only samples with optical density (OD) of the sample ≥ critical OD (positivity coefficient PC ≥ 1) were considered positive. The test systems are specific to the major epidemically significant hantaviruses of the HANV, SEUV, PUUV, and DOBV subtypes. The reaction results were recorded on an Infinite F50 recording photometer (TECAN, Austria).

Confidence intervals for the proportion of positive samples were determined by Wilson’s method at a confidence level of p³0.95 using software tools provided at https://epitools.ausvet.com.au.

To assess the current state of activity of natural foci of HFRS in the RR, the authors analyzed the data of epizootological monitoring carried out by the staff of the Rostov Anti-Plague Institute in 2020–2022; the results are presented in Table 1. A total of 2157 specimens of SMs (15 species) were collected, 35 administrative districts of the region and the vicinity of Rostov-on-Don were surveyed.

In 2020, 356 specimens of mammals of seven species were captured and analyzed in 13 administrative territories, combined into 155 samples. The antigen of HFRS pathogens was detected in 11 samples (Table 1), virus infection of small rodents amounted to 7.1% [ 4.0–12.3]. Positive findings were found in six samples of house mouse in the Azovsky, Proletarsky, Myasnikovsky, and Neklinovsky Districts, two samples of common vole in the Salsky District, two samples of European wood mouse in the Azovsky and Neklinovsky Districts, and one sample of pygmy field mouse in the Azovsky District.

In 2021, epizootologic monitoring was conducted in 25 administrative territories of the RR (Table 1). During the study period, 422 specimens (215 samples) of SMs of 14 species were captured. Positive findings were detected in six samples in four districts of the region: Salsky (common vole – two samples, yellow-bellied mouse – one sample), Oblivsky (small wood mouse – one sample), Morozovsky (common vole – one sample), and Neklinovsky (European wood mouse – one sample). According to the ELISA data, SM infestation reaches 2.8% [ 1.3–6.0] (six positive findings) in three species: common vole, yellow-bellied mouse, and European wood mouse.

In 2022, monitoring was conducted in 31 administrative territories of the RR (Table 1). A total of 1379 specimens (307 samples) of SMs of 14 species were captured. Markers of HFRS pathogens were detected in three districts of the region: Neklinovsky (Eastern European vole), Kasharsky (small wood mouse, common vole), and Matveevo-Kurgan (common vole). Spontaneous infection in 2022 was detected in 1.3% [ 0.7–3.8] of samples (5 positive findings) of three SM species: Eastern European vole, lesser wood mouse, and common vole.

To assess the spread of hantaviruses on the territory of the RR in 2020–2022, the results of serological monitoring of the state of population immunity to hantaviruses were analyzed. The results of the studies are reflected in Table 2.

In 2020, class G antibodies in healthy donors (n = 186) were detected in 7.0% [ 4.1–11.6] of samples. Among seropositive individuals, residents of rural areas and small towns, living in private households with homestead farms prevailed. Positive samples were found in the Remontnensky (13.5%) and Salsky (12.2%) Districts and in Volgodonsk (2.1%). The median age of seropositive donors was 50 years, among which women predominated (57.1%). None of the samples showed simultaneous presence of IgM and IgG to HFRS.

In 2021, 424 samples of blood serum from residents living in three administrative districts (Salsky, Neklinovsky, and Azovsky) and seven urban districts (Rostov-on-Don, Shakhty, Taganrog, Kamensk-Shakhtinsky, Volgodonsk, Zernograd, and Morozovsk), whose population makes up 52.7% of the total population of the RR, were tested. The proportion of seropositive results amounted to 4.5% [ 2.9–6.9]. The presence of serologic markers to HFRS pathogens was detected in different territories with a frequency from 1.9% to 12.2% (Table 2). Class G antibodies to hantaviruses were detected in the following cities: Taganrog – 12.2%, Morozovsk – 11.8%, Shakhty – 5.4%, and Zernograd – 5.0%. Single cases were registered in the cities of Rostov-on-Don, Kamensk-Shakhtinsky, and Volgodonsk. A total of 19 positive samples were detected. The median age of seropositive donors was 54 years, of which 63.2% were men. In two samples, antibodies of classes M and G were simultaneously present (Zernograd city – a woman of 20 years old, Morozovsk city – a man of 60 years old), which may indicate an acute period of the disease.

In 2022, 452 samples of blood serum obtained from a number of districts of the region and the city of Rostov-on-Don were examined. Seropositive results were found in 7.1% [ 5.1–9.8] of donors living in: Remontnensky – 15.7%, Neklinovsky – 14.3%, Salsky – 12.9%, and Rostov-on-Don – 4.3%. Single positive samples were registered in the Veselovsky and Tselinsky Districts. The median age of seropositive donors was 51.5 years, of which 59.4% were men.

In a parallel study of IgG-positive samples for class M immunoglobulins, the results coincided in 46.9% (15 cases), which may indicate a high proportion of people who may have had a mild form of the disease. Such matches were found in the Neklinovsky, Salsky, and Remontnensky Districts and the city of Rostov-on-Don. The average age of donors suspected of contact with hantavirus was 44.5 years. Unfortunately, this study was conducted retrospectively, which does not allow the authors to establish with certainty the diagnosis of hantavirus infection.

Screening of the healthy population for the presence of specific antibodies to hantaviruses and epizootological monitoring of SMs for the presence of hantavirus antigen showed the spread of HFRS pathogens on the territory of the RR (Figure 1).

In the course of the research, antibodies to hantaviruses were detected in residents of 14 administrative territories. Detection of carriers with antigens of hantaviruses in most of these territories makes it possible to assume infection of residents with hantaviruses. Visualization of the results showed the coincidence of seropositive samples and positive findings in SMs in the Azovsky, Kamensky, Neklinovsky, Neklinovsky, Salsky, and Morozovsky Districts. The above-mentioned factors indicate the risks of infection of the population and complication of the epidemiological situation on HFRS in the RR.

As a result of epizootological monitoring, markers of HFRS pathogens were detected in the Azovsky, Kamensky, Kasharsky, Matveevo-Kurgan, Morozovsky, Myasnikovsky, Neklinovsky, Oblivsky, Proletarsky, and Salsky Districts. Six species of mammals (house mouse, common vole, yellow-bellied mouse, European wood mouse, Eastern European vole, and small wood mouse) were infected with hantaviruses. The proportion of SM-infected animals captured in the period of 2020–2022 amounted to 7.1%, 2.8%, and 1.3%, respectively. In the Neklinovsky District, positive samples in SMs were detected during three years of observation, in the Salsky District – in 2020 and 2021. Detection of hantavirus markers in rodent populations suggests the circulation of the infectious agent in the RR.

The population of rural areas and cities of the RR has contact with hantaviruses, which is confirmed as a result of serologic monitoring by IgG detection in healthy donors in 2020–2022 in 7.0%, 4.5%, and 7.1% of samples, respectively. Positively reacting donors belong to the age group of persons of active working age from 41 years and older.

The coincidence of positive findings of the hantavirus antigen in SM samples and seropositive donor samples in the Azovsky, Kamensky, Neklinovsky, Salsky, and Morozovsky Districts suggests the presence of a natural focus of HFRS.

Registration of HFRS cases in humans in 2018, 2019, and 2022, analysis of the results of studies of the immune layer of the population in relation to HFRS causative viruses, and data on the detection of virus markers in carrier samples suggest the presence on the territory of the RR of the natural focus of HFRS, the boundaries and activity of which need to be clarified, as well as the real danger of infection of the population living in these areas.

Further studies of the HFRS biocenotic and spatial structure, isolation and characterization of circulating hantaviruses, as well as a study of the dynamics of immune response in febrile patients with suspected hantavirus infection, are necessary to conclude about the degree of activity of the natural focus.