Genetic diversity of parechoviruses ( Picornaviridae: Paavivirinae: Parechovirus: Parechovirus ahumpari ) circulating in Nizhny Novgorod in 2021–2024

Vladimir V. Zverev; Зверев Владимир Владимирович; Svetlana G. Selivanova; Селиванова Светлана Григорьевна; Natalia V. Epifanova; Епифанова Наталия Владимировна; Alexander Y. Kashnikov; Кашников Александр Юрьевич; Natalia V. Ponomareva; Пономарева Наталья Вячеславовна; Lyudmila N. Golitsyna; Голицына Людмила Николаевна; Nadezhda A. Novikova; Новикова Надежда Алексеевна

doi:10.36233/0507-4088-351

Genetic diversity of parechoviruses (Picornaviridae: Paavivirinae: Parechovirus: Parechovirus ahumpari) circulating in Nizhny Novgorod in 2021–2024

Authors: Zverev V.V.¹, Selivanova S.G.¹, Epifanova N.V.¹, Kashnikov A.Y.¹, Ponomareva N.V.¹, Golitsyna L.N.¹, Novikova N.A.¹
Affiliations:
1. Academician I.N. Blokhina Nizhny Novgorod Research Institute of Epidemiology and Microbiology, Rospotrebnadzor
Issue: Vol 70, No 6 (2025)
Pages: 581-588
Section: ORIGINAL RESEARCHES
URL: https://virusjour.crie.ru/jour/article/view/16813
DOI: https://doi.org/10.36233/0507-4088-351
ID: 16813

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Abstract

Introduction. Parechoviruses of the Parechovirus ahumpari (PeV-A) species, pathogenic to humans, are widespread and genetically diverse infectious agents. Infections caused by these viruses are characterized by a wide variety of clinical manifestations ranging from mild intestinal or respiratory diseases to severe CNS lesions. The high-risk group for the disease are newborns and infants. PeV-A species are classified in 19 types that have a varying distribution in different territories. In Russia, the type composition of territorial parechovirus populations has not been sufficiently studied, which determines the relevance of monitoring the circulation of these viruses using genotyping.

The aim of the study was to identify and investigate the genetic diversity of parechoviruses that circulated in Nizhny Novgorod in the period 2021–2024.

Materials and methods. 5,073 stool samples from children hospitalized in an infectious hospital with a diagnosis of acute gastroenteritis were examined for the presence of human parechoviruses. The detection of parechoviruses was carried out by RT-PCR. Viral types were determined by Sanger sequencing of VP1 genome fragment. The nucleotide sequences were analyzed using MEGA X and Beast v1.8.4 software.

Results. Parechoviruses were detected in children aged 3 months to 17 years with a frequency of 0.06–2.08% in different years, an average of 1.46 ± 0.16%. Viral type has been identified for 52 strains. Six types of PeV-A parechoviruses have been identified. The PeV-A1 was a predominant type (80.4%). Types PeV-A2 to PeV-A6 have been found in isolated cases. Heterogeneity of the PeV-A1 population in Nizhny Novgorod was represented by virus genotypes 1A and 1B, with an absolute predominance of genotype 1B, which included 16 genetic variants.

Conclusion. The data obtained expand information on the type and genetic diversity of pathogenic for humans parechoviruses circulating among the population of central Russia (based the example of the Nizhny Novgorod region).

Keywords

parechovirus, molecular diagnostics, monitoring, genetic diversity

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Introduction

Parechoviruses (PeV), pathogenic to humans (family Picornaviridae, genus Parechovirus, species Parechovirus ahumpari) are small (30 nm in diameter) non-enveloped viruses with icosahedral (pseudo-T3) symmetry of the capsid surrounding a single-stranded positive-sense RNA genome [1]. PeVs were first isolated by R. Wigand and A. Sabin in 1956 and initially classified as ECNO22 and ECNO23 viruses within the genus Enterovirus [2]. In the late 1990s, based on a number of molecular biological and genetic properties that distinguish PeVs from other enteroviruses (type of cytopathic effect, absence of suppression of protein synthesis in the cell), ESNO22 and ESNO23 viruses were renamed and separated into the independent genus Parechovirus [3]. Currently, the genus Parechovirus includes six species (A–F). PeVs that are pathogenic to humans belong to the species Parechovirus ahumpari (PeV-A) and are represented by 19 virus types (PeV-A1 to PeV-A19) [4]. The hosts of the remaining five PeV species are various species of mammals, birds and reptiles.

Similar to enteroviruses, PeVs cause a variety of human diseases, such as acute respiratory and intestinal diseases, serous meningitis, encephalitis, myocarditis, exanthema, as well as neonatal sepsis, but the ratio of leading clinical forms for these viruses varies [5]. Diseases caused by PeV vary from asymptomatic and mild forms of respiratory and gastrointestinal infections to severe neurological diseases accompanied by paralysis [6]. It is impossible to differentiate between enterovirus and PeV infections based on clinical symptoms, therefore an etiological diagnosis can only be made based on laboratory test results. The main risk group for PeV infection is newborns and children under 5 years of age [7]. PeV also contributes to primary infectious pathology in adults [8]. PeV infection can be more severe in older people and those with chronic diseases [9].

PeVs circulate worldwide, but their actual prevalence is difficult to estimate because most countries do not have comprehensive active surveillance programs for the identification, diagnosis and typing of PeVs, and serological surveillance data are limited [10]. In a number of studies conducted as part of national surveillance programs for enterovirus infections in Japan (2010), Denmark, Finland, the Netherlands (2008, 2013, 2014) and the United States (2016), the average frequency of PeV detection in samples collected from children with clinically suspected viral infections was 2% [18]. Epidemiological surveillance of PeV circulation in the Russian Federation is not regulated. The first report on the detection of PeV in the Russian Federation was published in 2006 [11], and previously isolated PeV were classified as E22 or E23. A large number of Russian PeV strains were detected as a result of virus monitoring in children with acute intestinal infection in Nizhny Novgorod in 2006–2010. In 2012, a similar study was conducted in Novosibirsk. To date, PeV types A1, A3, A4, A5 and A6 have been identified in Russia [12, 13].

The widespread prevalence and significance of PeV in infectious diseases in infants determine the importance of monitoring the circulation, studying the genetic diversity and variability of these viruses.

The aim of the study is to identify and investigate the genetic diversity of PeV that circulated in Nizhny Novgorod during the period 2021–2024.

Materials and methods

The study used stool samples from 5,073 children hospitalized with a diagnosis of acute intestinal infection (A08.4 – unspecified viral intestinal infection, ICD-10) at the children’s infectious diseases hospital in Nizhny Novgorod between 2021 and 2024. The study was conducted with the voluntary informed consent of patients’ legal representatives. The study protocol was approved by the Local Ethics Committee of the I.N. Blokhina Nizhny Novgorod Research Institute of Epidemiology and Microbiology of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor) (protocol No. 5 of March 24, 2020).

Total RNA was isolated using the Ribo-Prep reagent kit from a 10% fecal suspension prepared in saline solution (0.9% sodium chloride solution in water). Reverse transcription was performed using the Reverta-L kit (Central Research Institute of Epidemiology for Rospotrebnadzor, Russia) in accordance with the instructions. PeV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR) with hybridization-fluorescence detection of amplification products using the AmpliSens Enterovirus/Parechovirus-FL test system (Central Research Institute for Epidemiology of Rospotrebnadzor, Russia).

The type of PeV was determined using the method proposed by W. Nix et al. [14]. Reagents manufactured by Eurogen, Sintol and Biolabmix LLC (Russia) were used for complementary DNA (cDNA) amplification. cDNA fragments were sequenced automatically on a Beckman Coulter CEQ 8000 genetic analyzer (Beckman Coulter, USA) using the Dye Terminator Cycle Sequencing (DTCS) QuickStartKit (Beckman Coulter, USA).

PeV-A1 sequences deposited in the GenBank international database were used for phylogenetic analysis. Nucleotide sequence alignment (Clustal W algorithm) and genetic difference assessment (Tamura Nei model) were performed using MEGA X software [15]. Reconstruction of phylogenetic relationships and construction of dendrograms were performed using the MCMC algorithm in the Beast v. 1.8.4 software package [16]. Groups of sequences with a posterior probability of less than 0.95 were not taken into account in the analysis. Genotypes and subgenotypes were designated in accordance with the data presented in the literature [17].

Results

When fecal samples from 5,073 children with suspected viral acute intestinal infection were examined using RT-PCR, PeV was detected in 74 cases. The average detection rate over 4 years was 1.46 ± 0.16% (Table).

Table. Detection of parechoviruses in children hospitalized with acute gastroenteritis in 2021–2024

Таблица. Обнаружение парэховирусов у детей, госпитализированных с диагнозом ОКИ в 2021–2024 гг.

Years Год	Number of samples examined Количество исследованных образцов	Detected PeV Выявлено ПЭВ
Years Год		Quantity Количество	% ± m
2021	1737	42	2,42 ± 0,3
2022	1492	1	0,06
2023	913	19	2,08 ± 0,47
2024	931	12	1,3 ± 0,4
Всего Total	5073	74	1,46 ± 0,16

During the study period, PeV was most frequently detected in September–October (64.86%; 48 out of 74 cases), with a significant number of cases occurring in August, November, and December (10.81% in August, 9.46% in November and December; 8 and 7 cases, respectively). two cases were recorded in July, and one case each in April and July.

PeV was detected in children aged 3 months to 17 years (average age 1 year 10 months). PeV was most frequently detected (59.46%; 44/74) in children under 3 years of age. Children aged 3 to 7 years accounted for 31.08% (23/74) of cases.

The type of PeV was determined for 52 of the 74 (70.27%) strains detected. A total of 6 types of PeV were identified (Fig. 1).

Fig. 1. Distribution of PeV-A types detected in Nizhny Novgorod in 2021–2024.

Рис. 1. Распределение типов PeV-A, обнаруженных в Нижнем Новгороде в 2021–2024 гг.

The dominant type each year was PeV-A1, which was identified in 78.4% (41/52) of the typed strains. Other PeV types were also identified: PeV-A4 in 7.8% (4/52) of cases, PeV-A2 in 3.9% (2/52), PeV-A5 in 3.9% (2/52), PeV-A6 – in 3.9% (2/52) and PeV-A3 – in 1.9% (1/52). PeV was represented by 3 types in 2021, by 4 types in 2023, and by 5 types in 2024.

A phylogenetic analysis was performed based on 85 nucleotide sequences of the VP1 region of the PeV-A1 strains genome. Fig. 2 shows a phylogenetic tree illustrating the genetic diversity of PeV-A1 identified in this study and previously in the Russian Federation (in 2008–2012) [12, 13, 18]. The Nizhny Novgorod strains of PeV-A1 are represented by two genotypes: 1A and 1B.

Fig. 2. Phylogenetic tree constructed using the MCMC algorithm based on the nucleotide sequence analysis of a 267-nt fragment of the VP1 region of PeV-A1 strains.

* – nodes with posterior probability greater than 0.95 are indicated.

Рис. 2. Филогенетическое древо, построенное по алгоритму MCMC на основе анализа нуклеотидной последовательности фрагмента (267 н.о.) области VP1 генома штаммов PeV-A1.

* – отмечены узлы с апостериорной вероятностью выше 0,95.

In the Russian Federation, PeV-A1 genotype 1A was detected in 2008–2010 (Nizhny Novgorod) and in 2012 (Novosibirsk). One of the strains detected in 2024 (No. 601/2024) belonged to genotype 1A, which includes the prototype Harris strain. The homology of the nucleotide sequences of the Nizhny Novgorod strain 601/2024 and the prototype Harris strain was 79.6%, and with the sequences of Russian strains that had circulated earlier, it varied between 86.1% and 95.7%, with the most significant differences being with strains identified in Novosibirsk in 2012 [13]. Foreign PeV-A1 genotype 1A viruses in databases are represented by viruses identified in different regions of the world in the 2000s and early 2010s, and the homology of their sequences and strain 601/2024 did not exceed 86.6%.

The vast majority of PeV-A1 strains identified in this study, much like during 2006–2010, belonged to the currently dominant genotype 1B. Among these viruses, based on differences in nucleotide sequences, at least 5.0% can be differentiated into 16 genetic variants. At the same time, 6 genetic variants (2, 9, 12, 13, 15, and 16) are represented by only one strain each. Among the strains identified in 2021, 11 genetic variants were differentiated, in 2023 – 5 genetic variants, and in 2024 – 3 genetic variants. Genetic variants 1 and 7 were represented by viruses identified in 2021 and 2023, and genetic variant 10 was represented by viruses identified in 2023 and 2024.

The sequences of modern Nizhny Novgorod strains belonging to genetic variants 1–3 formed a monophyletic cluster with the sequences of PeV-A1 strains identified in Nizhny Novgorod in 2008 and 2009 and previously assigned to subgenotype 1B₁ [18]. However, the divergence of nucleotide sequences between modern and “old” strains was 11.3–17.5%.

The strains of genetic variant 1 are most genetically similar (97.3–98.5% homology of nucleotide sequences) to PeV-A1, which circulated in the Republic of Belarus in 2018 (MK167994). The virus identified in Iran in 2019 (MK792785, homology with genetic variant 2 was 37.3%, with genetic variant 3 – 92.9–93.1%) is most closely related to strains of genetic variants 2 and 3. Strains of PeV-A1, closely related to PeV genetic variants 1–3, were also detected in patients with acute respiratory infection in Japan and diarrhea in China in 2019–2022 [19, 20].

For strains belonging to genetic variants 4 and 5, the virus identified in the United Kingdom in 2013 (KU885005) showed the highest similarity (89.7–93.2% homology) among foreign PeV-A1 strains.

For strains belonging to genetic variants 7–12, the greatest similarity (89.7–93.2% homology) was shown by viruses detected in respiratory samples in Japan in 2019 [19] and in the US in 2021 (OR728261) and in children with diarrhea in China in 2019 [20].

The only Nizhny Novgorod strain belonging to genetic variant 13 showed close relatedness to PeV-A1 detected in 2017–2018 in Germany in children with various clinical forms of infection [21].

No strains with nucleotide sequence homology above 90.0% were found in international databases for PeV-A1 genetic variants 6, 14, 15, and 16.

Discussion

Human-infecting PeV (PeV-A) are genetically diverse pathogens that are widespread throughout the world. The detection rate of PeV-A could vary significantly (from 1.1 to 55.0%) depending on the region and the current epidemiological situation [18]. In this study, the average detection rate of PeV-A over 4 years was 1.46%, which was lower than in a previous study conducted in 2006–2010, when the detection rate of PeV was 6.16% [12].

The seasonality of PeV infection worldwide shows considerable variability and appears to depend on the predominant genotype, geographic region, and current epidemiological situation. For example, PeV-A1 can be detected throughout the year, with an increase in detection rates in the summer and fall months [18]. Similar circulation was established in a previous study from 2006 to 2010, when PeV was detected relatively evenly throughout the year [12]. In this study, the maximum number of PeV detection occurred in September–October.

PeV infection and related diseases are mainly reported in young children. Monitoring of PeV circulation in Europe in 2015–2021 showed that the age structure of infected individuals may depend on the predominant virus type: PeV-A3 – PeV-A5 were detected mainly in children under 3 months of age, PeV-A1 and PeV-A6 were more often detected in children aged 1 to 5 years [22]. In this study, most PeVs were detected in children older than 1 year, which is typical for a situation where PeV-A1 predominates in the typical structure.

Currently, P. ahumpari is represented by 19 virus types. In this study, six PeV-A types were detected: PeV-A1 to PeV-A6; the dominant type (78.85% of typed isolates) was PeV-A1. In a previous study, PeV-A1 was identified in 91.55% of typed strains [12]. There has been a slight decrease in the proportion of the dominant type PeV-A1 in the etiological structure of the infection and an increase in the total proportion of PeV-A2–PeV-A6. At the same time, PeV-A2–PeV-A6 were detected in isolated cases. Data collected during surveillance of non-polio enteroviruses showed that PeV types PeV-A1 and PeV-A3–PeV-A6 are the most common worldwide, with PeV-A1 predominating [19–24]. It has been noted that PeV-A1 and PeV-A6 mainly cause acute respiratory and intestinal disorders, while PeV-A3–PeV-A5 cause febrile illnesses and are more common than other PeVs in patients with symptoms of CNS involvement. Other types of PeV, including PeV-A2 and PeV-A7–PeV-A19, are rare in Europe, the US, and China, and are most commonly found in India, Pakistan, Africa, and Latin America [10, 25].

Each type of PeV is characterized by genetic heterogeneity. Among PeV-A1, two genotypes are differentiated: 1A and 1B [17]. In this study, the vast majority (97.56%; 40/41) of PeV-A1 strains belonged to genotype 1B, which is globally dominant. We found extreme genetic diversity among the PeV-A1 strains of this genotype that we studied, as evidenced by the existence of 16 genetic variants. The established level of genetic differences between viruses of different genetic variants indicates the absence of direct epidemiological links between cases of the diseases caused. Every year, viruses of new genetic variants were identified, with a significant level of genetic differences from those identified in previous years. The established genetic heterogeneity of the territorial population of PeV appears to be a consequence of the dynamic evolution of PeV and intensive population migration, as a result of which viruses of new genetic variants are introduced into the region annually.

Conclusion

This study presents the results of the research carried out between 2021 and 2024, during which data on the circulation of human PeV in Nizhny Novgorod was collected. Using the example of monitoring PeV in children hospitalized with a diagnosis of acute intestinal infection, the typical diversity of Nizhny Novgorod PeV strains is shown, represented by six types of viruses, which, according to published data, can cause diseases with other clinical manifestations, including severe forms. The established genetic heterogeneity of strains of the dominant PeVA1 genotype 1B confirms the dynamic evolution of parechoviruses, which may lead to the formation of strains with altered pathogenic properties and virulence. These facts indicate the importance of monitoring the circulation and diagnosis of PeV in patients with various clinical manifestations of infection, including neuroinfections.