Circulation of West Nile virus (WNV) was first confirmed in Russia in 1963 by specialists from the Institute of Poliomyelitis and Viral Encephalitis of the USSR Academy of Medical Sciences and the Astrakhan Regional Sanitary and Epidemiological Station [1]. Up to the end of the 20th century, the incidence of this new for Russia natural focal infection was periodically registered only in the Astrakhan Region. The 1999 outbreak, which affected some territories of southern Russia with a severe course and high (up to 10%) lethality, became a turning point in the study of WNV and the organization of a system for monitoring the circulation of the pathogen [2]. In subsequent years, the incidence of WNV steadily increased, as well as the number of territories where cases were diagnosed. Thus, in 2000, the incidence of WNV was officially registered for the first time in the Rostov Region, in 2006 – in the Ulyanovsk Region, in 2010 – in the Voronezh Region, the Chelyabinsk Region, the Republic of Kalmykia, and the Krasnodar Territory (according to the published data – since 1999), in 2011 – in the Republic of Tatarstan, in 2012 – in the Republic of Adygea, Stavropol Territory, in the Belgorod, Lipetsk, Kursk, Samara, Saratov, Omsk, and Novosibirsk Regions, in 2013 – in the Kaluga Region, in 2018 – in the Republics of Crimea and Dagestan, in 2021 – in the Tula Region and the city of Moscow, in 2022 – in the Tambov, Vladimir, Tver Regions, the Karachayevo-Circassian Republic and the Khanty-Mansi Autonomous Area – Yugra [2–5].

Based on the presented data, the territorial distribution of WNV in Russia is fairly wide. However, the area of the disease remains poorly studied. In the territories adjacent to the regions with registered disease incidence, in similar climatogeographic conditions, there is no convincing evidence of WNV circulation. In obtaining new and clarifying the existing data on the nature of epizootic and epidemic manifestations of the disease, targeted prospecting studies are critical, since routine monitoring, conducted in Russia for more than 10 years, does not allow for reliable assessment of the activity of WNV foci.

The aim of the study was to investigate the intensity of contact with the WNV pathogen among residents of some central and southern territories of Russia.

Blood samples collected in July-August 2023 from the population living in 11 subjects of the Russian Federation (Republics of Ingushetia, North Ossetia-Alania, Mari El, Mordovia, Chechen, Kabardino-Balkarian, and Chuvash Republics, Nizhny Novgorod, Ivanovo, Yaroslavl, and Kostroma Regions) were used for the study. These territories were selected due to the lack of reliable data on the circulation of WNV over the years (since 2009) of monitoring studies or the presence of evidence indirectly indicating the endemicity of the territory (the presence of an immunized layer among the population in the absence of registered cases).

Patients of somatic and infectious hospitals, including intensive care units, as well as outpatient clinics, with a fever of unclear etiology at the time of seeking medical care or occurring after hospitalization were included. The age of the examined patients ranged from 1 to 97 years old.

The study was conducted with voluntary informed consent of the patients. The study protocol was approved by the Bioethics Commission of the Federal State Institution “Volgograd Research Institute for Plague Control” of Rospotrebnadzor (protocol No. 3 dated April 25, 2022).

Blood samples were collected by the personnel of medical organizations in vacutainers with EDTA in the volume of 5 ml. A total of 1,230 blood samples were collected. Blood serum samples obtained as a result of centrifugation were analyzed at the stationary laboratory of FCUZ “Volgograd Research Institute of Plague Control” of Rospotrebnadzor by the Enzyme Immunoassay (EIA) for the presence of IgM and IgG antibodies to WNV using VectoNil-IgG and VectoNil-IgM reagent kits (Vector-Best, Russia).

In samples positive for the presence of class IgG antibodies to WNV, the avidity index was determined using the VectoNil-IgG-avidity kit (Vector-Best CJSC, Russia).

In areas endemic for tick-borne viral encephalitis, all seropositive samples to WNV were tested for the presence of antibodies to tick-borne encephalitis virus (TBE) using VectoVKE-IgM and VectoVKE-IgG reagent kits (Vector-Best CJSC, Russia). A significant excess of antibody titers directed to WNV antigens compared to antibodies to TBE (4-fold or more) was considered positive for the presence of antibodies to WNV. To confirm the specificity of the obtained results, serum samples containing IgM class antibodies were additionally tested in the neutralization reaction with WNV Volgograd 601/18 strain, deposited in the state collection of viral infectious agents and rickettsioses of the Federal Budgetary Institution “Vector” of Rospotrebnadzor.

Given the relatively short period of viremia in the patient’s body [6], not all blood samples were tested for the presence of WNV RNA, but selective samples taken from patients whose fever duration at the time of collection did not exceed 7 days. The PCR method was used for the detection of WNV RNA with the AmpliSense®WNV-FL reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia); genotyping of WNV was performed using Ampligen-WNV-genotype-1/2/4 (Volgograd Research Anti-Plague Institute of Rospotrebnadzor, Russia).

The sources of information on the registered incidence of WNV included statistical monitoring forms No. 2 “Information on Infectious and Parasitic Diseases” in the Russian Federation for 2009–2022, the structure of morbidity, and the amount of the immunized layer to WNV among the population of the surveyed territories (report data submitted by the Rospotrebnadzor offices in the constituent entities of the Russian Federation to the Reference Center for Monitoring of the WNV Pathogen, and materials of scientific publications).

Statistical processing of the data was done in Microsoft Excel 2016.

Detection of WNV cases. IgM antibodies to WNV, a marker of the acute infectious process, were detected in serum samples of 10 people (0.8%) living in 6 subjects of the Russian Federation (Table 1). Sera from residents of the Republics of Mari El, Mordovia, Nizhny Novgorod, Ivanovo, and Kostroma Regions containing IgM antibodies to WNV were tested in parallel for the presence of IgM antibodies to TBE with negative results. The specificity of the detected antibodies to WNV was also verified by the neutralization reaction. All patients with WNV did not leave their places of permanent residence during the possible incubation period.

The presence of IgM in the sera of all 10 detected patients was found in the period from Day 2 to Day 18 of the disease and agrees with the published data on the timing of appearance and duration of IgM circulation [7][8]. IgG was not detected in 9 out of 10 serum samples, indicating an early period of the disease. The absence of IgG to WNV on Day 18 from the onset of the disease was noted in one patient diagnosed with COVID-19. This observation could be associated with a delay or functional insufficiency of the patient’s immune response and required repeated studies in dynamics. In 1 sample from a patient from the Ivanovo Region, along with the registered humoral immune response (antibodies of IgM and IgG classes), RNA of WNV was detected. The data of the patient’s medical history indicated that the blood was collected on Day 7 from the appearance of clinical symptoms and fit the pattern of IgG dynamics [9].

WNV RNA was detected in blood samples from 8 people (1.8%) out of 6 subjects (Table 1). In 7 samples (87.5%), the threshold cycle (Ct) value was between 30 and 32, and in 1 sample (12.5%), it was within 27, indicating a low viral load. According to the results of typing of positive samples, the isolated WNV RNA fragments belonged to genotype 2 in a blood sample from a patient from the Ivanovo Region, genotype 1 in a blood sample from 1 patient from the Republic of Mordovia, and 2 patients from the Republic of Ingushetia. In the remaining samples, it was not possible to determine the WNV genotype because the concentration of WNV RNA in the tested samples was below the sensitivity threshold of the reagent kit used (1×10⁴ copies/mL). All clinical material positive for WNV RNA was taken for a virologic study in order to obtain virus isolates and study their properties. The results obtained up to date indicate that WNV isolates from the Chuvash and Kabardino-Balkarian Republics and the Nizhny Novgorod Region belong to genotype 2.

It should be mentioned that in 2023, there was a confirmed case of the circulation of WNV genotype 1, the presence of which was not registered on the territory of the Russian Federation since 2020. According to retrospective data, the known area of WNV genotype 1 distribution includes some territories in the south of European Russia (Astrakhan, Volgograd Regions, Stavropol Territory), Western and Eastern Siberia (Omsk, Novosibirsk, and Tomsk Regions), and the Far East (Primorye Territory) [3][10][11]. The circulation of WNV genotype 1 in the central part of Russia was not described.

Summarizing the results of the molecular-genetic and immunological methods of research, the total share of detection of WNV markers indicative of the acute phase of the disease was 1.4%.

All subjects with laboratory-confirmed cases of WNV in 2023 had not previously submitted data on registered cases of this infectious disease to the statistical reporting forms. At the same time, the confirmation of local cases of WNV in the territories adjacent to the surveyed areas suggests active but undiagnosed contact of the population with WNV. Thus, in the North Caucasus Federal District, the sporadic incidence of WNV was noted in the Stavropol Territory in 2012, 2018, 2019, and 2022, in the Republic of Dagestan – in 2018, 2021, and 2022, and in the Karachayevo-Circassian Republic – in 2022 [4][5]. Imported cases from the Chechen Republic in the absence of officially registered morbidity were repeatedly detected in other subjects of the Russian Federation (in 2011, 2013, and 2018 in the Astrakhan Region, in 2016 in the Saratov Region) [12].

In the territory of the Volga Federal District, manifestations of the epidemic process of WNV were observed in the Republic of Tatarstan (2011 – 4 cases, 2012 – 3, 2018 – 1) [13], adjacent to the Republic of Mari El and the Chuvash Republic, as well as in the Ulyanovsk Region (2009 – 1 case, 2012 – 4, 2022 – 2), bordering the Republic of Mordovia and the Chuvash Republic. In the central part of Russia, WNV incidence was verified in the Tver Region (2022 – 7 cases), bordering the Yaroslavl Region, and the Vladimir Region (2022 – 1 case), bordering the Yaroslavl and Ivanovo Regions [5].

In 7 out of 17 (41.2%) WNV patients, medical specialists made a preliminary diagnosis of “acute respiratory viral infection”, in 5 (29.4%) – other diseases of infectious genesis (measles, COVID-19, gastroenterocolitis, community-acquired pneumonia, fever of unclear etiology). In other cases, WNV was diagnosed among patients with somatic, including surgical, pathology.

In all diagnosed cases, WNV did not affect the central nervous system. Mild forms of the disease accounted for 88.2% of cases, and moderately severe – 11.8%. This distribution of cases by the severity of clinical course of WNV is not typical for Russia, since 71.4% of cases registered in 2009–2022 had a moderately severe course of the disease, 17.9% – mild, and 10.7% – severe.

The analysis of the structure of the detected cases revealed the predominance of women among the diseased, who accounted for 70.6% of all cases (the Russian average was 43.3%). WNV cases were detected in all age groups except for children under 1 year of age, with the predominance of infections in the category “60 years and older” (35.3%, 31.9% overall in Russia). The share of children under 14 years of age among WNV cases was 5.9% (6% overall in Russia). A case of WNV disease in a child was laboratory-confirmed only in the Nizhny Novgorod Region.

The occupational composition of the patients was quite heterogeneous, but one can note the dominance of such groups as non-employed persons of working age and pensioners, who accounted for 35.3% and 23.5%, respectively (20.5% and 27% overall in Russia).

WNV patients were predominantly detected among the urban population – 58.8% (overall in Russia – 76.1%). Based on the results of the epidemiological investigation of WNV cases, it was found that the proportion of infections in urban areas was 47.1% (overall in Russia – 28.9%), in rural areas – 29.4% (25.1%), and when traveling to natural recreation areas – 23.5% (21.2%). No cases of infection were detected after visits to countryside (dacha) plots.

Study of the immunized layer to WNV. The presence of a WNV immunized layer was found in the population of all surveyed territories (Table 2). The overall level of seroprevalence to WNV among residents of the central and southern regions of Russia was 4.5%, with the highest rates in the Chechen Republic (10%) and the Republic of North Ossetia-Alania (14%).

The rate of antibody detection in men and women did not differ significantly (Table 2). The distribution of positive findings by age groups showed that IgG to WNV was detected in all studied groups. The highest share of positive findings was observed in patients aged 20–29 years old and 30–39 years old (Table 3), but the differences were statistically insignificant (p>0.05).

When comparing the results of the present study and the size of the immunized layer detected as a result of routine serologic monitoring, similar values of seroprevalence to WNV were found for the population of the Kabardino-Balkarian Republic (average for the period 2010–2022 – 4.5%), Kostroma (3%), Ivanovo (3.1%), Nizhny Novgorod (4.2%), and Yaroslavl Regions (1.5%).

In the Republic of Mari El, antibodies to WNV were previously detected once among the sample group “livestock breeders” (10% in 2011). The higher share of positive findings compared to the results of the present study is probably associated with the cross-reactivity of sera to tick-borne encephalitis virus circulating in the territory of the Republic, in the absence of differentiating diagnostic tests, as well as belonging of livestock breeders to the group of high risk of WNV infection.

In the Republic of Mordovia, the rate of antibodies to WNV in this study was higher compared to the reported data (2020 – 0.7%, 2021 – 1.5%), which may be associated with the choice of different “indicator” population groups for the study of population immunity.

There was no information on the presence of an immunized layer to WNV among the population of the Republics of North Ossetia-Alania and Ingushetia, Chechen and Chuvash Republics during a routine serologic examination.

There are data on the detection of low-avid IgG to WNV in the population of 8 subjects, indicating recent (not more than 3 months ago) disease. In the Republics of Ingushetia, Mari El and Chuvash Republic, all detected IgG antibodies to WNV were categorized as low-avid, while in the Nizhny Novgorod Region and the Republic of North Ossetia-Alania, 86% of positive findings confirmed the active epidemic process of WNV in these territories.

Based on the results of these studies, local cases of WNV were laboratory-confirmed for the first time in 9 regions of central Russia and the North Caucasus, which added to the understanding of the area of this infectious disease. It should be noted that the confirmation of local transmission of WNV to humans is not regarded by the authors as an ongoing territorial spread of WNV in Russia, since the area of the disease remained within the previously established boundary at the level of the Tver Region [5]. The lack of registration of WNV cases among the population of the studied territories in the period of 2009–2022 may be a consequence of insufficiently effective (non-systematic) work on active detection of WNV among patients with fever, etiologically unidentified meningitis, meningoencephalitis, and severe forms of “respiratory” infections.

WNV was most often verified in the group of patients with a provisional diagnosis of acute respiratory viral infections, which indicates low vigilance of medical specialists toward WNV among patients with respiratory and influenza-like manifestations. At the same time, the flu-like variant of the course of WNV accounts for up to 90% of all clinically expressed cases of the disease [2][3]. The occurrence of fever in patients with somatic pathology requires the attention of medical specialists, which does not exclude the course of the infectious process.

The obtained data on the distribution of diagnosed cases by sex, age, social status, clinical manifestations of the disease, and places of infection differ from those obtained in Russia during the long-term period of observation, since a certain group of the population (mainly patients of inpatient medical institutions of infectious and somatic profiles) was subjected to a screening examination. No cases of severe (neuroinvasive) course were confirmed among patients with WNV, which can be explained by the low share of patients diagnosed with meningitis, meningoencephalitis, and encephalitis who underwent screening (3.2%).

The results of the genotyping of nucleotide sequences of WNV in samples of clinical material allowed concluding that WNV genotype 1 is more widespread in Russia. Further in-depth molecular genetic studies are required to investigate the probable routes of migration of this genovariant (from “old” foci in the Astrakhan Region or endemic regions of the world).

For the first time, the authors obtained up-to-date data on the level of the immunized layer to WNV among residents of 4 areas: the Republics of North Ossetia-Alania, Ingushetia, Chechen and Chuvash Republics. The lack of positive results during routine monitoring among the population of the Republics of North Ossetia-Alania and Chuvash Republic is probably related to the quality of organization of collection and delivery of material and laboratory tests. In the Chechen Republic and the Republic of Ingushetia, no serologic screening of healthy population sample groups for the presence of antibodies to WNV was conducted during 2009–2022. The only data in available publications, obtained more than 30 years ago (1991), indicate that the proportion of positive findings in blood serum samples from the population of the Chechen Republic was 0.3% (1 out of 288 samples) [14]. The data of the present study, which revealed 14% of seropositive samples, suggested that the intensity of WNV circulation in the Chechen Republic had significantly increased over the past period.

No significant differences were found between the results of the serum examination of men and women. The age structure of humoral immunity to WNV demonstrated that the most active latent immunization occurs among young people due to their high mobility and the possibility of contact with natural focal complexes. At the same time, the low specific weight of positive findings in persons in the groups “60–69 years old” and “70 and more years old” can probably be explained by age-related changes in the immune response.

The presence of low-avidity IgG antibodies in blood serum samples, together with data on the laboratory confirmation of acute cases of the disease, allows the authors to conclude about the intensive frequency of contact of the population with the causative agent of WNV in most of the studied territories.

Thus, the obtained data indicate the presence of active circulation of WPV in the central regions of European Russia and the North Caucasus and the need to significantly improve the quality of monitoring for the WNV pathogen in these areas. In connection with the first-time incidence of the disease in a number of territories, the primary task is to develop a comprehensive plan of organizational, anti-epidemic, and preventive measures against WNV, to organize examinations of febrile patients with undetermined diagnosis, manifestations of meningitis and meningoencephalitis, to train medical specialists in the clinic, epidemiology, and diagnosis of WNV, and to provide the laboratory base of medical organizations and institutions of Rospotrebnadzor with the necessary means of etiological, epidemiological, and preventive measures against WNV.