Characterization of non-polio enteroviruses (Picornaviridae, Enterovirus) isolated from clinical specimens and wastewater samples collected in Saint Petersburg and the Leningrad Region

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Introduction

Enteroviruses (EVs), belonging to the genus Enterovirus of the family Picornaviridae, are one of the leading causes of infectious diseases with neurological and dermatological symptoms in children. Monitoring of the circulation of these viruses is carried out as part of the program “Epidemiological Surveillance and Prevention of Enterovirus (Non-Polio) Infection for 2023–2027,” which is considered an integral part of the National Action Plan to Maintain the Russian Federation’s Polio-Free Status¹,². Enterovirus infection (EVI) is characterized by a variety of clinical presentations, manifesting as involvement of various organs and systems. The most common diseases include: viral stomatitis and hand-foot-mouth disease, herpangina (vesicular pharyngitis), respiratory syndrome, and neuropathies. In severe cases, this infection leads to acute flaccid paralysis syndrome, meningitis, encephalitis, or meningoencephalitis, which requires urgent hospitalization [1]. At the same time, it is known that 80% of EVI cases manifest as asymptomatic viral carriage with a prolonged period of viral shedding and transmission to susceptible individuals, typically through household contact, which causes disease outbreaks and the widespread transmission of the infection². It is precisely this characteristic that contributes to the persistent circulation of EV in the population, despite the presence of immune individuals [2]. According to data from the I.N. Blokhina Research Institute of Epidemiology and Microbiology, the incidence rate of EVI per 100,000 people shows constant fluctuations and reflects regular outbreaks: 2018 – 9.9; 2019 – 12.6; 2020 – 0.8; 2021 – 4.2; 2022 – 7.5; 2023 – 12.56. At the same time, the post-COVID period saw a rise in the incidence of enterovirus meningitis (EVM): 2021 – 0.1; 2022 – 1.0; 2023 – 1.9 [3]. When interpreting the incidence rates for 2020–2021, it is important to note that this period coincided with the COVID-19 pandemic and the implementation of anti-epidemic restrictions, which could have influenced the reported incidence of EVI.

Furthermore, EVI is characterized by polymorphism: a specific virus type can lead to forms of infection with different clinical presentations, but at the same time, different EV types can cause similar nosological forms, which is primarily due to the characteristics of the viral genome [4]. The EV nucleic acid consists of a linear, single-stranded, positive-sense RNA 7.5–8.0 kb in length, which has non-coding regions at the 5’ and 3’ ends, flanking an open reading frame, the translation of which results in a polyprotein consisting of approximately 2,100 amino acids [5]. The polyprotein is subsequently cleaved by proteases into three protein precursors (P1–P3), which are the precursors of 11 virus-specific proteins: 4 structural proteins (VP1–VP4) and 7 non-structural proteins (2A–2C, 3A–3D) [6]. The structural proteins VP1 and VP3, which are the major antigens of EV, are of the greatest significance in genotyping, pathogenesis, and, consequently, clinical manifestations. The high rate of molecular evolution characteristic of RNA viruses can lead to changes in the amino acid composition and conformation of these proteins. Such changes, including point mutations and frequent recombination events among circulating strains, influence the mechanism by which viral antigens interact with cellular receptors and the host’s antibodies [7]. Genetic variability is the primary factor determining viral serological diversity and evolutionary patterns [8]. The large number of variations in the epitopes of the VP1 and VP3 proteins means that antibodies that bind to one type of EV are unable to interact with others; this, along with asymptomatic viral carriage, is one of the main reasons for the persistent, long-term circulation of EV in the population [9].

In addition to comprehensive laboratory monitoring of EV, which includes the initial detection of the virus in clinical or environmental samples using molecular genetic methods and/or the isolation of the virus in cell cultures followed by taxonomic identification, further genetic and phylogenetic characterization of the identified isolates is necessary for the molecular epidemiological analysis of the viruses. A detailed study of the genetic diversity of EV is key to understanding pathogenesis, predicting epidemic outbreaks, and identifying cross-border introductions of new types and genotypes into the territory of the Russian Federation.

The aim of the study was to examine the diversity of non-polio enterovirus (NPEVs) types isolated from clinical specimens and wastewater samples collected in St. Petersburg and the Leningrad Region, and to provide a molecular-epidemiological characterization of Coxsackievirus B5 (CVB5) isolates.

Materials and methods

The study material consisted of stool samples collected in St. Petersburg and the Leningrad region from children residing in the Russian Federation who presented with clinical symptoms of EVI (serous meningitis, enterovirus vesicular stomatitis with exanthema) and who had been in contact with patients, as well as healthy migrant children examined as part of the National Action Plan to Maintain the Russian Federation’s Polio-Free Status, and wastewater samples collected from sewer collectors in St. Petersburg and the Leningrad Region. In total, from 2018 to 2023, sequencing of the VP3/VP1 variable region of the viral genome was performed on viruses isolated from 71 samples: 9 isolates–from children residing in the Russian Federation who exhibited clinical symptoms of EVI or had been in contact with EVI patients; 59 isolates–from healthy migrant children under 5 years of age screened as part of the aforementioned National Action Plan; and 3 isolates–from wastewater samples. Among the 59 isolates obtained from migrant children, 57 (96.6%) were isolated from children who had arrived from Tajikistan, and 2 (3.4%) were isolated from children who had arrived from Uzbekistan. The study was conducted with the informed consent of the minor patients and their legal representatives. The study protocol was approved by the Ethics Committee of the Pasteur Research Institute of Epidemiology and Microbiology (Protocol No. 88 dated October 3, 2023).

The initial isolation of EV from fecal and wastewater samples was performed by incubating fecal suspensions and wastewater concentrates with RD (human rhabdomyosarcoma cells), HER-2 (human laryngeal squamous cell carcinoma cells), and L20-B (cells from a mouse mammary gland tumor with a vector genome expressing a specific cell receptor for three types of poliovirus) in accordance with the guidelines of the World Health Organization (WHO) and MUK 4.2. 4063-24 (2024)³,⁴. All cell lines were obtained from the WHO National Reference Laboratory for Poliomyelitis Diagnosis at the M.P. Chumakov Institute of Poliomyelitis and Viral Encephalitis. Infected cell cultures were incubated in a thermostat at 36.5 °C. The initial observation period lasted 5–7 days, while the total duration of virological observation, including repeated passages, was up to 10–14 days. Counting was performed under an inverted microscope by assessing the cytopathic effect of the virus on the cell culture, based on morphological changes in the cells and degradation of the culture monolayer.

Nucleic acid extraction from samples passaged on RD, HEp-2, and L20-B cell cultures was performed using the Ampli-Prime Riboprep kit (Central Research Institute of Epidemiology). Residual DNA background from the cell cultures was removed using the DNase E enzyme (Eurogen) to prevent false-positive results at low viral titers. Reverse transcription of the viral genome was performed using the MMLV RT kit (Eurogen) with random primers to ensure complete coverage of the virus-specific nucleic acid and to compensate for low viral titers in clinical specimens and wastewater samples. Amplification of the hypervariable region of the VP3/VP1 viral genome was performed using degenerate serotype-specific primers (amplicon length ≈ 700 bp) and a commercial PCR Master Mix (2×) kit (ThermoScientific). PCR protocol: 95 °C – 3 min; 95 °C – 30 s, 48.5 °C – 30 s, 72 °C – 1 min (39 cycles); 72 °C – 10 min. Analysis of the sequencing reaction products was performed using an ABI Prism 3500 genetic analyzer (Applied Biosystems, USA).

Chromatograms were visualized using Chromas software, and genetic sequences were aligned using the R programming language (the Biostrings, pwalign, and DECIPHER packages–the main packages within the Bioconductor repository). Initial analysis of the obtained nucleotide sequences, followed by genotyping, was performed using the NCBI BLAST algorithm and the Enterovirus Genotyping Tool by comparing them with nucleotide sequences uploaded to the international GenBank database. Phylogenetic analysis of sequenced EV strains using the maximum likelihood method (ML) was performed using the R programming language (the Biostrings and phangorn packages) based on the SYM+G(4)+I evolutionary model. Reference genetic sequences selected from the international GenBank database are listed in the Table. The selection criterion was an amino acid similarity threshold of over 70%, based on the recommendations of the International Committee on Taxonomy of Viruses (ICTV)⁵. To assess the reliability of the phylogenetic tree’s branch topology, we used the bootstrap method with 1,000 post-start iterations. The phylogenetic tree was visualized and edited using FigTree v. 1.4.4. A heatmap of nucleotide similarity between strains was generated using SDTv 1.3. A dated phylogenetic tree and a root-to-tip regression plot were constructed using TreeTime [10].

 

Table. Accession numbers and metadata for reference genetic sequences of the CVB5 virus from the international GenBank database, used in the phylogenetic analysis and in constructing a heatmap of nucleotide sequence similarity among strains

Таблица. Идентификационные номера и метаданные референтных генетических последовательностей вируса CVB5 из международной базы данных GenBank, использованных в филогенетическом анализе и при построении тепловой карты уровня нуклеотидного соответствия между штаммами

GenBank accession number Номер последовательности из GenBank	Region of virus isolation Регион выделения вируса	Date of virus isolation Дата выделения вируса
PV822004	Russian Federation: Yekaterinburg Российская Федерация: Екатеринбург	01.09.2022
OR678400	Kazakhstan: Astana Казахстан: Астана	03.06.2022
OR678426	Kazakhstan: Astana Казахстан: Астана	06.09.2022
OP441390	China Китай	08.07.2014
OQ919261	China Китай	10.09.2018
OQ842459	USA США	10.2017
OQ842456	USA США	07.2014
OQ842457	USA США	02.2015
OQ633095	China Китай	2018
MW762688	China Китай	2016
OQ408283	China Китай	06.2022
ON152867	China Китай	09.2021
OP672300	China Китай	01.05.2010
OP672301	China Китай	13.07.2013
OP672302	China Китай	16.09.2014
OP672303	China Китай	04.06.2015
OP672304	China Китай	14.07.2016
OP672305	China Китай	01.01.2017
OP672308	China Китай	31.05.2018
OM417125	Thailand Таиланд	14.08.2011
MZ229657	China Китай	14.06.2019
MZ826338	China Китай	04.2017
OL955504	Guatemala Гватемала	2019
MW015045	Japan: Toyama Япония: Тояма	02.12.2006
MK791149	China Китай	27.08.2018
MK000156	Haiti: Port-au-Prince Гаити: Порт-о-Пренс	17.03.2016
MF574099	China Китай	03.06.2010
MH201081	China Китай	08.2015
MH752988	USA: Michigan США: Мичиган	2009
KY744347	China Китай	2013
MF678315	Australia Австралия	2007
KY352499	China Китай	30.04.2014
KY303900	China Китай	2013
KU574625	Thailand: Bangkok Таиланд: Бангкок	29.06.2011
KX139461	Germany Германия	30.11.2010
KP289438	China Китай	2013
KP266578	China Китай	23.05.2009
JX017380	China Китай	2011
JN695050	China: Changchun Китай: Чанчунь	2010
GU376747	China Китай	2008
HQ998851	China Китай	2010
AY875692	South Korea Южная Корея	2000
AF114383	USA/strain «Faulkner» США/штамм «Faulkner»	–
LS451290	Romania Румыния	1970
KT285013	Taiwan Тайвань	1983
KT285014	Pakistan Пакистан	1991

For the continuation of the table, see page 362.

 

Metadata processing for typed EV isolates, visualization, and statistical calculations were performed using the R programming language (ggplot2, dplyr, tidyr, and readxl packages). Variables are presented as absolute values (abs.) and percentages (%). The frequency of individual types and species of NPEVs was calculated as the ratio of the number of isolates of the corresponding type or species to the total number of typed isolates. For each proportion, a 95% confidence interval (CI) was calculated using the exact Clopper–Pearson binomial method. The values of the proportions and CIs are presented as percentages.

Results

At the St. Petersburg Regional Center for Surveillance of Poliomyelitis and Acute Flaccid Paralysis (Pasteur Research Institute of Epidemiology and Microbiology), genetic sequences of the hypervariable region of the VP3/VP1 genome were obtained from 71 AFP isolates. The sample collection covered the epidemiologically significant period from 2018 to 2023. All isolates were typed based on the sequenced VP3/VP1 genomic region, which allowed for the identification of 25 enterovirus serotypes. It was found that, within the studied sample from St. Petersburg and the Leningrad Region, viruses belonging to the Enterovirus betacoxsackie group were most frequently isolated: CVB5 (12.7%; 95% CI 6.0–22.7%), ECHO 11 (11.3%; 95% CI 5.0–21.0%), ECHO 13 (8.5%; 95% CI 3.2–17.5%), ECHO 14 (8.5%; 95% CI 3.2–17.5%), and ECHO 6 (8.5%; 95% CI 3.2–17.5%). The spread of ECHO 25 (5.6%; 95% CI 1.6–13.8%) and ECHO 29 (5.6%; 95% CI 1.6–13.8%) viruses was also recorded. Collectively, these 7 virus types accounted for 60.6% of the total number of typed strains. Less frequently isolated were alpha-coxsackie enteroviruses: CVA8 (4.2%; 95% CI 0.9–11.9%), CVA10 (2.8%; 95% CI 0.3–9.8%), CVA3 (2.8%; 95% CI 0.3–9.8%), and CVA4 (2.8%; 95% CI 0.3–9.8%). At the species level, Enterovirus betacoxsackie predominated–53/71 (74.6%; 95% CI 62.9–84.2); Enterovirus alphacoxsackie accounted for 17/71 (23.9%; 95% CI 14.6–35.5), and Enterovirus coxsackiepol accounted for 1/71 (1.4%; 95% CI 0.04–7.6).

An analysis of the typical EV structure revealed differences among the three groups (residents, migrants, and wastewater) (Fig. 1). Among migrant children, the most frequently detected EV serotypes were EV 11 (11.9%), EV 14 (10.2%), EV 13 (8.5%), EV 6 (8.5%), and CVB5 (5.1%). In contrast, samples collected from resident children tested positive for CVB5 (66.7%), CVA16 (11.1%), CVA6 (11.1%), and ECHO 6 (11.1%).

 

Fig. 1. Distribution of enterovirus types among the three groups of samples.

Рис. 1. Распределение типов энтеровирусов между тремя группами образцов.

 

In three wastewater samples collected from central collection systems, the following viruses were isolated: ECHO 11 (33.3%), ECHO 13 (33.3%), and ECHO 24 (33.3%).

An analysis of the serotype distribution of the isolated viruses over a 6-year period demonstrates an uneven representation of various viral serotypes in the study material. CVB5 and ECHO 11 were the most frequently detected among the isolates studied, whereas a number of other serotypes (e.g., CVA6 or ECHO 14) were less common. Since the CVB5 virus was one of the predominant types of enteroviruses in the sample studied, a molecular genetic analysis of this type was conducted in addition to the epidemiological characterization.

A detailed analysis of the topology of the CVB5 virus phylogenetic tree, constructed using the ML method, made it possible to characterize the degree of relatedness among Russian strains isolated in St. Petersburg and the Leningrad Region (GenBank accession numbers: PZ291072–PZ291080), and foreign reference strains from the international GenBank database (Fig. 2). The results of the analysis clearly show the phylogenetic positioning of the six CVB5 strains isolated in 2018 from children residing in St. Petersburg (6716/RUS_SPb/2018, 6717/RUS_SPb/2018, 6722/RUS_SPb/2018, 6723/RUS_SPb/2018, 6724/RUS_SPb/2018, 6725/RUS_SPb/2018), are located within Genetic Cluster II, which consists of strains isolated in China, Kazakhstan, and the Russian Federation. In contrast, three CVB5 strains isolated in 2021 from migrant children arriving from Tajikistan (8022/RUS_LO/2021, 8637/RUS_SPb/2021, and 8638/RUS_SPb/2021), form a distinct subgroup within cluster I, closely associated with a Pakistani strain (KT285014), forming a separate paraphyletic group with it. Their degree of genetic similarity is confirmed both by the low number of genetic differences as shown by the heatmap (Fig. 3) and by the high bootstrap support for the internal node (99.3%), which significantly exceeds the generally accepted 95% confidence threshold, indicating the group’s robustness to variations in the dataset [11]. This reliable grouping is the main argument in favor of their probable common route of introduction into the territory of St. Petersburg and the Leningrad Region.

 

Fig. 2. Phylogenetic tree of CVB5 strains, constructed using the maximum likelihood method based on the nucleotide sequences of the VP3/VP1 region. The green branches correspond to taxa forming genetic cluster I; the blue branches correspond to taxa forming genetic cluster II; the pink branch marks the outgroup. Taxa marked in red are strains isolated in the territories of St. Petersburg and Leningrad Region.

Рис. 2. Филогенетическое дерево штаммов СVВ5, построенное методом максимального правдоподобия на основе нуклеотидных последовательностей региона генома VP3/VP1. Ветви зеленого цвета относятся к таксонам, образующим I генетический кластер; ветви голубого цвета относятся к таксонам, образующим II генетический кластер; ветвь розового цвета маркирует аутгруппу. Таксоны, отмеченные красным цветом, являются штаммами, выделенными на территориях Санкт-Петербурга и Ленинградской области.

 

Fig. 3. A heatmap showing the similarities and differences in nucleotide sequences between the studied Coxsackievirus B5 virus strains (marked with blue ovals) and reference Coxsackievirus B5 virus strains from the GenBank database. The level of nucleotide similarity between the strains is represented by a color gradient on the scale on the right side of the graph.

Рис. 3. Тепловая карта сходств и различий нуклеотидов между исследуемыми штаммами вируса СVВ5 (отмечены синими овалами) и референтными штаммами вируса СVВ5 из базы данных GenBank. Уровень нуклеотидного соответствия штаммов отражен в виде цветового градиента на шкале в правой части графика.

 

Thus, the fact that the 2018 and 2021 CVB5 strains belonging to separate clusters indirectly indicates that the detection of these EV strains in St. Petersburg and the Leningrad Region is not the result of a single introduction followed by local circulation; rather, it points to multiple and independent routes of introduction.

A heat map based on nucleotide sequences allows for a visual assessment of the degree of genetic similarity between CVB5 strains isolated in the region and reference strains from an international database (Fig. 3). Analysis of the map confirms the significant genetic proximity of the regional strains to one another and their relationship to viruses from Central, East, and South Asia. For example, the group of CVB5 viruses isolated in 2021 exhibits 87% nucleotide similarity to a Pakistani strain (KT285014.1), which is consistent with the phylogenetic reconstruction data (Fig. 2). CVB5 viruses circulating in St. Petersburg and the Leningrad Region in 2018 show significant (> 96%) similarity to Chinese and Kazakhstani strains.

Of particular interest is the analysis of a dated phylogenetic tree of CVB5 strains, which allows us to estimate the time to most recent common ancestor (tMRCA) for specific viruses. Thus, according to the data presented in Fig. 4, the date of the tMRCA for EV strains isolated in St. Petersburg and the Leningrad Region in 2021 (the upper node marked by an arrow) is estimated to be approximately 2015. For the monophyletic group consisting of strains isolated in 2018 (the lower node marked by an arrow), the tMRCA is dated to approximately the second half of 2016. These estimates are indirectly supported by the root-to-tip regression plot, which visualizes an upward-sloping linear regression line between the date of virus isolation and the genetic distance of the strains from the root of the tree. This graph and the dated phylogenetic tree, together with the analysis of the cladogram (Fig. 2), provide indirect support for the hypothesis that the various CVB5 strains isolated in the Russian Federation in 2018 and 2021 represent independent introductions, which casts doubt on the long-term circulation of identical CVB5 strains in St. Petersburg and the Leningrad Region, followed by molecular evolution.

 

Fig. 4. A time-scaled phylogenetic tree of CVB5 strains (a) based on the nucleotide sequences of the VP3/VP1 region and a root-to-tip regression plot (b) demonstrating the rate of molecular evolution, the coefficient of determination, and the linear regression between the date of virus isolation and genetic distance, generated using TreeTime. To the left of the tree, a color gradient indicates the date of strain isolation; black arrows point to the internal nodes of CVB5 strains isolated in St. Petersburg and the Leningrad Region.

Рис. 4. Датированное филогенетическое дерево штаммов CVB5 (а), построенное на основе нуклеотидных последовательностей региона генома VP3/VP1 и график root-to-tip regression (б), демонстрирующий скорость молекулярной эволюции, коэффициент детерминации и линейную регрессию между датой выделения вируса и генетическим расстоянием, созданные при помощи TreeTime. Слева от дерева обозначена цветовая градация по дате выделения штаммов; черные стрелки указывают на внутренние узлы штаммов CVB5, выделенных на территориях Санкт-Петербурга и Ленинградской области.

 

Discussion

The identified viral profile, with a notable contribution from Enterovirus betacoxsackie–including CVB5, ECHO 11, and 13–is consistent with data from other studies showing that members of this genus often account for a significant proportion of circulating non-polio enteroviruses and are associated with neuroinfections. For example, according to the results of a molecular epidemiological characterization of enterovirus neuroinfections in children in Greece (2017–2020), CVB5 and ECHO 11 were among the leading viral types (along with ECHO 30 and 16), indicating that the co-circulation of ECHO 11 and CVB5 is typical for the European region [12]. On the other hand, the significant contribution of ECHO 13 to the landscape of circulating enteroviruses in the sample of this study is also consistent with international epidemiological surveillance findings: ECHO 13 was responsible for epidemic surges of aseptic meningitis in Spain in 2000, and in Belgium during the summer of 2000, this virus was among the most frequently isolated types alongside ECHO 30, ECHO 6, and CVB5, illustrating the ability of ECHO 13 to become the predominant type during certain epidemic seasons [13]. An epidemiological analysis of EV circulating in the Russian Federation also indicates that the dominance or high prevalence of CVB5, ECHO 11, and 13 are not unique to the Northwestern Federal District: for example, long-term monitoring of non-structural enteroviruses (NSEVs) in the Krasnodar Krai has recorded years with a high proportion of ECHO 13 alongside the simultaneous circulation of CVB5 and ECHO 11 [14]. According to epidemiological monitoring data from the Russian Federation for 2024, 46 types of enteroviruses were identified in patients with acute viral infections, with the species composition consisting predominantly of Enterovirus alphacoxsackie and Enterovirus betacoxsackie: 64.67% and 33.72%, respectively, while Enterovirus coxsackiepol and Enterovirus deconjuncti accounted for 1.47% and 0.13%. The same surveillance study noted regional differences in the viral spectrum: EV-A71 was more frequently detected in the constituent entities of the Central, Northwestern, Southern, and Volga Federal Districts; ECHO 25 was more frequently detected in the constituent entities of the Ural, Siberian, and Far Eastern Federal Districts; and Coxsackievirus A10 was among the dominant enteroviruses across Russia [3].

The spectrum of typified NPEVs in the resident and migrant groups underscores that migration flows constitute a distinct source of viral circulation. A similar conclusion was previously reached for northwestern Russia: a comparison of NPEVs isolated from children in migrant families and from residents revealed a significant divergence in the genotypic spectrum and the frequent detection of unique genotypes among migrants, which was interpreted as an argument in favor of systematic screening of at-risk groups to prevent the local circulation of imported EV [15]. Current epidemiological surveillance data also confirm the marked typological diversity of NPEVs among migrants: in 2024, 85 NPEV strains belonging to 28 types were identified among migrants from Tajikistan in the Russian Federation. Among them were types also present in the current study collection–including ECHO 11, ECHO 13, ECHO 14, ECHO 6, ECHO 25, ECHO 29, and CVB5, as well as types that highlight the difference in the spectrum of enteroviruses among migrants compared to that recorded among residents of the Russian Federation. Thus, according to the data from this monitoring, ECHO 29 was not detected among residents of the Russian Federation in 2024, whereas it was detected among migrants from Tajikistan; furthermore, ECHO 12, Coxsackievirus A13, and Enterovirus C99 were detected relatively frequently among migrants, whereas they were rarely recorded among residents–one case of each [3]. From an epidemiological standpoint, the heightened attention given to outbreaks of enterovirus infections (EVI) caused by non-endemic virus types is justified, since the infection is asymptomatic in 80% of cases, and viral shedding into the environment—which leads to the infection of susceptible populations—can last for weeks: in pediatric studies, the duration of enterovirus shedding in stool reached 7–11 weeks⁶. Consequently, monitoring population groups with an increased likelihood of infection with a rare viral strain is of particular interest for molecular epidemiological surveillance of NPEV circulation and the detection of non-endemic or previously unidentified genetic variants. A conceptually similar approach is used in European wastewater surveillance practices, which describe targeted environmental monitoring schemes for the early detection of outbreaks in groups associated with population movement (e.g., evacuation shelters and refugee camps) [16]. In the present study, viruses isolated from migrant children constituted the majority of the collection; therefore, the results of the typological analysis should be interpreted as a characterization of the studied collection of isolates obtained within the surveillance system, rather than as a direct assessment of the structure of circulating NPEVs among the entire population of St. Petersburg and the Leningrad Region.

The phylogenetic analysis of CVB5 demonstrates that the strains isolated from resident children in 2018 and strains isolated from migrant children in 2021 belong to different genetic lineages, which is consistent with international data on the complex molecular-epidemiological characteristics of this type. A large-scale analysis has shown that the CVB5 virus forms several global genotypes that can co-circulate in the same region over an extended period of time, creating conditions for repeated introductions and the parallel circulation of multiple genotypes of the same virus [17]. As part of the surveillance of acute viral infections in the Russian Federation, scenarios of independent introductions have previously been described for other types of enteroviruses: for example, for ECHO 30 genotype h in the Northwest, an association between local circulation and repeated introductions from Southeast Asia was discussed, demonstrating a sequential link between virus importation, local circulation, and subsequent genetic divergence of enteroviruses within the Russian Federation [18].

Furthermore, the obtained data are consistent with current understanding of the molecular evolution of enteroviruses. In an early study by D. Rezig et al., based on an analysis of nucleotide sequences from the VP1 region of the CVB5 virus genome, four genetic clusters were identified that potentially correspond to distinct genotypes [19]. Subsequently, in a study by H. Shen, four distinct genotypes (A–D) were identified, for which frequent recombination events and the possible co-circulation of multiple genotypes within a specific region were described [20]. In a study by H. Huang et al. on the genotyping and evolutionary dynamics of human EV, it was demonstrated that CVB5 is characterized by a high rate of nucleotide substitutions and frequent turnover of circulating viruses belonging to different genetic lineages, accompanied by the formation of regionally restricted clusters [21]. The authors showed that the molecular evolution of EV is uneven, and the emergence of new phylogenetic groups is often associated with cross-border migration of viruses and their subsequent local adaptation. These patterns correlate with the results of the present study, in which the 2018 and 2021 CVB5 strains formed genetically distinct clusters, which may represent distinct genotypes, with different tMRCA values and high levels of nucleotide similarity, indicating independent pathways of introduction and the absence of continuous local circulation of a single viral lineage. Thus, the identified structure of phylogenetic clusters is consistent with the globally described model of EV evolutionary dynamics, which includes the possible co-circulation of CVB5 viruses belonging to different genetic lineages, regular introductions, and subsequent divergence of viral populations.

The results confirm the high epidemiological significance of molecular typing of the EV, particularly sequencing of the VP3/VP1 region, as a tool for assessing the circulation and cross-border transmission of the EV in St. Petersburg and the Leningrad Region. A detailed analysis of the virus types circulating among the population allows for the identification of critical risk groups and sources of potential epidemic threats. The difference in the viral composition between healthy migrant children and resident children indicates the heterogeneity of the epidemiological situation. At the same time, a comprehensive analysis of the phylogenetic tree, the nucleotide similarity heatmap, and root-to-tip regression allowed for a more in-depth characterization of the molecular-epidemiological properties of CVB5 viruses isolated in St. Petersburg and the Leningrad Region. The identification of two distinct genetic lineages confirms the complexity of viral circulation and the need for continuous monitoring to ensure the timely detection of new genetic variants. These data highlight the significance of molecular epidemiology in the prevention and control of enterovirus infection, underscoring the importance of integrating molecular methods with classical epidemiological approaches.

Conclusions

1. In the studied collection, consisting of 71 isolates of non-poliomyelitis enteroviruses isolated from clinical specimens and wastewater samples collected in St. Petersburg and the Leningrad Region between 2018 and 2023, 25 EV types were identified. At the species level, Enterovirus betacoxsackie predominated (74.6%), with CVB5, ECHO 11, ECHO 13, ECHO 14, and ECHO 6 being the most frequently detected viruses.
2. The typical structure of NPEV varied among the groups of samples studied. The observed differences between isolates obtained from migrant children and resident children confirm the epidemiological importance of screening various population groups as part of enterovirus infection surveillance.
3. Phylogenetic analysis showed that the CVB5 virus strains isolated from resident children in 2018 and from migrant children in 2021 belong to two distinct genetic lineages. The combined data from phylogenetic reconstruction, nucleotide similarity analysis, and tMRCA dates indicate independent pathways of introduction of the studied CVB5 strains into the territory of the Russian Federation.
4. The results confirm the epidemiological significance of molecular typing of EV, particularly sequencing of the VP3/VP1 region, for characterizing the genetic diversity of viruses and identifying possible cross-border introductions. The complex application of phylogenetic analysis, nucleotide similarity assessment, and molecular dating allows for a more detailed characterization of the molecular epidemiological features of EV circulation.

 

¹ National Action Plan for Maintaining the Polio-Free Status of the Russian Federation. Federal Budgetary Institution of Health «Federal Center for Hygiene and Epidemiology» of Rospotrebnadzor. Available at: https://fcgie.ru/page,3,koord_tsentr.html

² Epidemiological surveillance of enteroviral (non-polio) infection. Methodological Guidelines MU 3.1.1.4015-24, 2024

³ World Health Organization. Polio laboratory manual. 4th ed. Geneva: World Health Organization; 2004. WHO/IVB/04.10

⁴ Organization and conduct of testing of environmental samples for polioviruses and non-polio enteroviruses. Methodological guidelines for control methods MUK 4.2.4063-24, 2024.

⁵ Taxonomic proposal to the ICTV Executive Committee: 2008.084V.HRV-C-Sp. Creation of a new human rhinovirus C species in the genus Enterovirus. Available at: https://ictv.global/system/files/proposals/approved/Vertebrate_Viruses_(through_2014)/2008.084V.HRV-C-Sp.pdf

⁶ European Centre for Disease Prevention and Control. Rapid Risk Assessment – Enterovirus detections associated with severe neurological symptoms in children and adults in European countries, 8 August 2016. Stockholm: ECDC; 2016. Available at: https://www.ecdc.europa.eu/en/publications-data/rapid-risk-assessment-enterovirus-detections-associated-severe-neurological

About the authors

Kirill A. Antonenkov

St. Petersburg Pasteur Institute

Author for correspondence.
Email: antonenkovkirill3877@gmail.com
ORCID iD: 0009-0006-5232-3188

Junior Researcher, Laboratory of Etiology and Control of Viral Infections

Russian Federation, 197101, St. Petersburg

Natalia I. Romanenkova

St. Petersburg Pasteur Institute

Email: romanenkova@pasteurorg.ru
ORCID iD: 0000-0001-6970-6368

PhD in medicine, Leading Researcher, Laboratory of Etiology and Control of Viral Infections

Russian Federation, 197101, St. Petersburg

Yulia V. Ostankova

St. Petersburg Pasteur Institute

Email: shenna1@yandex.ru
ORCID iD: 0000-0003-2270-8897

PhD in biology, Head of the Laboratory of Virology and Immunology of HIV Infection

Russian Federation, 197101, St. Petersburg

Veronika A. Evseeva

St. Petersburg Pasteur Institute

Email: evseeva@pasteurorg.ru
ORCID iD: 0009-0006-7642-7272

Virologist, Laboratory of Etiology and Control of Viral Infections

Russian Federation, 197101, St. Petersburg

Maria A. Blokhinova

St. Petersburg Pasteur Institute

Email: mblokhinova@gmail.com
ORCID iD: 0009-0005-2509-2065

Junior Researcher, Laboratory of Etiology and Control of Viral Infections

Russian Federation, 197101, St. Petersburg

Olga I. Kanaeva

St. Petersburg Pasteur Institute

Email: olkanaeva@gmail.com
ORCID iD: 0000-0002-7699-5097

PhD in biology, Head of the Laboratory of Etiology and Control of Viral Infections

Russian Federation, 197101, St. Petersburg

Tatiana V. Arbuzova

St. Petersburg Pasteur Institute

Email: arbuzowa95@yandex.ru
ORCID iD: 0000-0002-3074-8656

Junior Researcher, Laboratory of Molecular Genetic Monitoring

Russian Federation, 197101, St. Petersburg

References

Supplementary files