1. Introduction

Human enteroviruses (HEVs) are small single stranded RNA viruses of the family Picornaviridae (https://www.picornaviridae.com) that cause different types of infectious illnesses in humans affecting millions of people every year in the world. Symptoms can vary from mild to severe and can result in syndromes like respiratory diseases, herpangina, hand-foot-and-mouth disease, aseptic meningitis, acute hemorrhagic conjunctivitis, and myocarditis. [1, 2]. Herpangina and meningitis in children are significant illnesses caused by enteroviruses because of the potential severity and impact on children’s health. Herpangina is highly contagious and spread very quickly, and it mostly affects younger children and is characterized by fever, sore throat, and small ulcers in the mouth and at the back of the throat [3, 4]. The ulcers can be painful and cause discomfort for children when drink and eat, which increases the risk of dehydration [3]. Symptoms can be confused with other illnesses; therefore, accurate diagnosis is significant for proper management and treatment [1, 4]. Herpangina complications are rare; however, in some cases, they can cause brainstem encephalitis and acute flaccid paralysis.

Based on serological tests, enteroviruses (EVs) were originally divided into coxsackieviruses A and B, echoviruses, and polioviruses subgroups [5]. Species of the genus Enterovirus are divided based on their pathogenicity, to human (Enterovirus A–D and Rhinovirus A–C) and animal species (Enterovirus E–J) [6]. However, with the development of molecular biology methods, classification is based on genetic characteristics and phylogenetic relationships between strains [7], where each EV species includes different serotype groups. Molecular typing of HEV serotypes is important for many reasons, such as in case of severe clinical disorders (some serotypes can cause fatal neurological disease), for the discrimination of polio and nonpolio enteroviruses, as well as for epidemiological and phylogenetic studies (discovering newly occurring serotypes) [8].

The EVs structure comprises nonenveloped positive stranded RNA molecule (approximately, 7400 nt long), which is surrounded by the structural proteins VP1 to VP4 (each in sixty copies) that form icosahedral capsid structure of the virus [9]. The genome of enterovirus is a single RNA molecule with small virus-encoded protein VPg at the 5′ end and untranslated region (UTR) from both 5′ and 3′ ends [10]. Many research studies have shown that VP1 capsid protein is a good marker for enterovirus serotypes’ differentiation and is widely used for PCR-based typing [10–12]. Recently known detection and classification methods are based on reverse-transcription polymerase chain reaction (RT-PCR) [13].

The HEV-B species consists of the vast majority of EV serotypes, and according to King’s [14] virus taxonomy, it includes coxsackievirus A9 (CVA9), coxsackieviruses B (CVB) 1–6, echoviruses (EV) 1–7, 9, 11–21, 24–27, 29–33, and enterovirus 69. Moreover, only coxsackievirus A9 from all coxsackieviruses belongs to HEV-B, while others belong to HEV-A and HEV-C [14].

Enteroviruses can be transmitted among humans in different ways: direct contact (fecal-oral way) and indirect contact (contaminated equipment) or drinking water from infected reservoirs. Infections predominate in the summer and autumn seasons, however, can occur sporadically during the year. According to previous research, younger children are known to be affected more often comparing to adults [15, 16]. Although, in most cases, infections caused by EVs occur with mild symptoms as respiratory or gastrointestinal illnesses, some of them can cause severe complications for patients, from central nervous system invasion to meningitis, encephalitis, and paralysis [17].

Understanding the molecular signatures of HEVs is essential for effective diagnosis and treatment protocols. The situation with HEV infection molecular typing in Kazakhstan is not yet fully studied, and there is no whole genome data available yet. This research study describes clinical diseases and associated HEV serotypes, as well as whole genome sequencing of 65 EV isolates collected from children in central infectious diseases hospital, located in Astana in 2022. The aim of the study is to investigate the molecular epidemiology of occurring HEV infection and the prevalence of circulating serotypes in Kazakhstan. In this study, we analyzed 65 sequences of HEV and compared with the publicly available NCBI database to understand important evolutionary dynamics and infection spread.

2. Materials and Methods

3. Results

3.1. Samples’ Overview

During the period from May 14 to October 12, 2022, 152 patients with a preliminary diagnosis of enterovirus infection were admitted to the Children’s Infectious Diseases Hospital No. 3 in Astana. Schematic representation of the samples collected is shown in Figure 1.

The largest number of patient visits occurred during the summer period, as shown in Figure 2; 67% of the studied patients were hospitalized in June-July. There was a decrease in the number of requests during August, followed by a peak increase in September (23.7%). The number of children under 10 years of age predominates and equals 74.3% (n = 113) of the total number of admitted patients, compared with the number of children aged over 10 years of 25.7% (n = 39) (average age of patients: 8.1 years). Moreover, among patients under the age of 10 years, more than 59% of patients were male (n = 67). Around 91.5% (139/152) of children were hospitalized with a diagnosis of aseptic meningitis and with a diagnosis of herpangina 8.5% (13/152).

Laboratory cytology results were available for 131 of 152 patients. The median value of blood leukocytes was 10.36 cells/L, with 25th and 75th percentile values of 7.99 cells/L and 13.27 cells/L, respectively. The median percentage of lymphocytes and neutrophils in CSF samples were 53% and 42%, respectively, with the values of the 25th and 75th percentiles being 20% and 77% for lymphocytes, and 22.5% and 79% for neutrophils, respectively.

4. Results of Whole-Genome Sequencing and Identification of Serotypes

Nearly complete whole-genome sequences of 65 enterovirus isolates were amplified and assembled from RNA samples collected from 58 patients. From all patients, 50 were diagnosed with aseptic meningitis and 8 with enterovirus infection herpangina. From 50 meningitis patients, 32 CSF samples and 24 NSW samples were selected for whole genome sequencing analysis. Nine sequences were obtained from NSW samples from 8 patients with enterovirus herpangina (Table 1).

Consensus phylogenetic tree was built using the ML algorithm for the VP1 sequence region (Figures 3(a) and 3(b)). Phylogenetic clades were mostly separated by enterovirus serotypes, which is consistent with previous enterovirus research studies [34].

These strains were labeled using the following format: “GenBank accession number”/“Serotype”/“Country of origin”/“Year of isolation” (strains with partially available metadata were also included). The prototype Enterovirus B69 was used as an outgroup. Bootstrap values >50% are shown as blue circles.

Based on phylogenetic analysis of the VP1 gene, 32 sequences obtained from CSF samples of 29 patients with meningitis were clustered into 6 serotypes: CVA9, CVB5, E6, E9, E11, and E21 (Figure 4). 24 sequences obtained from NSW samples of 21 patients with meningitis were clustered into 7 serotypes: CVA9, CVB5, CVB3, E6, E9, E21, and E25. Two CSF samples were found to be infected with both E21/CVB5 and E11/CVB5, respectively. In one patient, CVA9 was identified in a collected CSF sample and two serotypes were identified in the NSW sample (E9/CVA9) (Table 1).

Of the 9 sequences from 8 patients with enterovirus infection, 6 were identified as CVA9, 2 as CVB5, and 1 as E6, with one sample found to be coinfected with CVB5/E6 (Table 1, Figure 4).

4.2. Phylogenetic Analysis by Complete Coding Sequence

Based on phylogenetic analysis of the complete coding sequence, 65 sequences were clustered according to specific serotypes (Figure 5).

The 25 CVA9 sequences formed three separate clusters, and the third cluster was represented by a single sequence. We did not find any features in the distribution of sequences in the clades in comparison with the sampling time. For example, sequences collected in June are evenly represented across all contributions, including a third clade represented by a single sequence from NSW from a patient with meningitis that is most genetically distant from all of them by over 3.7% (Table S3).

The CVA9 sequences of EV-H1-vir2 and EV-38 isolated from the same patient with CSF and NSW were 100% identical. The 14 CVB5 sequences formed two clusters, representing sequences collected from patients that were hospitalized in different periods. All 9 E9 sequences were collected in June and formed two clusters, differing from each other by 8% (Table S4).

The E6 sequences are the most genetically homogeneous, with less than 2% variability among themselves (Table S5).

5. Discussion

In Kazakhstan, cases of enterovirus infection are recorded annually, with peak values in the summer-autumn period [37]. In our study, more than 90% of hospitalized cases of enterovirus infection were due to aseptic meningitis, and only 8.5% were due to enterovirus herpangina. These data do not reflect the true picture of the spread of enterovirus HA in Kazakhstan since the classic mild course of herpangina may remain unreported due to home or local hospital treatment. The high number of cases of aseptic meningitis in our study correlates with previous study data, in which enteroviruses were identified as etiological agents in 73% of cases of encephalitis and meningitis in Kazakhstan, with an average incidence of 14 per 100,000 population, and for children under 15 years of age, the incidence is 35.9 per 100,000 children [38]. Despite the high incidence rate, information on circulating serotypes is limited. This is the first study to describe the genetic features of enteroviruses circulating in Kazakhstan based on nearly complete whole-genome data.

The highest incidence was recorded in the summer months, and more than 67% of patients were hospitalized in June-July months, followed by a decrease in hospitalization cases in August and a rise in incidence in September. Peaks of incidence in the summer-autumn period are typical for the northern hemisphere [39]. Of the 152 patients, an almost complete genome sequence of enteroviruses was obtained in 38% (58 patients), while real-time PCR confirmed the presence of enterovirus infection in all patients. The lower efficiency of genome-wide amplification in comparison with the original article [18] may be due to the lack of information about the viral load in the studied samples, RNA degradation, and differences in the amplification reagents used. The assembly revealed coinfection in 6 of the 58 patients. In patients with aseptic meningitis, the presence of two serotypes was established in 2 samples from CSF (E21/CVB5 and E11/CVB5), 2 samples from NSW (E6/CVA9), and in one patient, 3 serotypes were obtained (CSF - Coxsackievirus A9 and Echovirus E9/Coxsackievirus A9 in a sample from NSW), while the complete coding sequences of Coxsackievirus A9 from NSW and CSF were completely identical. The patient with HA was found to be coinfected with NSW CVB5/E6. The introduction of molecular genetic tests and sequencing schemes for genotyping enteroviruses has expanded our understanding of the epidemiology of enteroviruses and led to the discovery of coinfection. Coinfection is most often detected in patients with HFMD syndrome and can reach 34%, while simultaneous persistence of different serotypes can aggravate the severity of the infection and lead to atypical cases involving the respiratory and cardiovascular systems [40–42]. In the current study, we did not record any specific clinical course in patients with coinfections, which may be due to the limited sample size.

The most prevalent enterovirus genotype circulating in Astana during the seasonal rise of enterovirus infection in 2022 is CVA9, which was detected in 18 of 50 patients with aseptic meningitis and 6 of 8 patients with herpangina. Coxsackievirus A9, like many enteroviruses, exhibits a wide range of clinical manifestations, including aseptic meningitis (AM), hand, foot, and mouth disease (HFMD), acute flaccid paralysis (AFP), and persistent diarrhoea. Generalized febrile exanthema can cause pathologies in the placenta in pregnant women and severe generalized infections in newborns [43–46]. In some outbreaks of aseptic meningitis, CVA9 was the main serovariant in China [47], Canada [48], and South Africa [49]. CVA9 belongs to the virus serovars that were most often isolated in serous meningitis in Russia, while in 2022, there was an increase in the incidence of meningitis associated with CVA9 [50, 51]. CVA9 isolated in our study belongs to genotype I, which is mainly represented by Russian strains [23]. Kazakhstan and Russia have the longest land border in the world and close economic and cultural ties, which may facilitate the circulation of identical genotypes.

Coxsackievirus B5 in our study was the second most abundant enterovirus identified in 12 people with aseptic meningitis and in 2 patients with enterovirus herpangina. In global distribution, CVB5 is not a common serovariant in severe neurological complications, including acute flaccid paralysis, encephalitis, and aseptic meningitis [52]. However, in Poland [53] and China [24, 54, 55], CVB5 has been the predominant serovariant in several outbreaks in recent years. As shown in Farshadpour and Taherkhani [56], enterovirus was identified as a main cause of aseptic meningitis among studied population in Iran. Echovirus 30 was emerged as a predominant strain that caused aseptic meningitis in children in their first year of life [56]. Genotyping clustered the sequences of the current study into genotype D, which is predominantly represented by sequences from China. Given the migration pattern of CVB5 from Northeast China to Northwest China and East China to Northwest China [24], it is tempting to hypothesize a shift in the CVB5 circulation boundaries from China to Kazakhstan.

Echoviruses that were predominant in our study were E6 and E9. Echovirus 6 is one of the five enteroviruses that were diagnosed as the etiological agent of neurological complications, including acute flaccid paralysis (AFP), encephalitis, and aseptic meningitis [52, 57, 58]. In our case, E6 was detected in 8 patients with aseptic meningitis, of which only one in CSF and one case with herpangina, while coinfection with other enteroviruses was identified in 4 patients. High levels of E6 coinfection may promote recombination changes, requiring monitoring of the serovariant at the genetic level and tracking changes in virulence [59].

Nevertheless, official statistical data indicate a seasonal increase in enterovirus infections in 2022. Regular genotype studies in enterovirus infections will improve the control and differentiation of outbreaks from seasonal increases and assess the prevalence of dominant genotypes and the association of the clinical course with enterovirus genotypes in Kazakhstan. Analysis of global data shows that echovirus 30, echovirus 6, and echovirus 13 are most commonly detected in enterovirus meningitis [52]. Echoviruses also tend to predominate in meningitis cases recorded during endemic periods. A four-year follow-up in Poland shows the predominance of echovirus 6 and echovirus 30 in enterovirus meningitis cases [60]. In China, E6, E18, E11, E9, E30, and CVB5 were identified as the most common genotypes in aseptic meningitis between 2009 and 2018 [61–63]. In Latin American countries, aseptic meningitis is also usually dominated by echoviruses E30 and E6 [64]. In our case, the predominance of the CVA9 genotype is observed in aseptic meningitis and herpangina, which is usually characteristic of outbreaks [47–49]. Nevertheless, official statistical data indicate a seasonal increase in enterovirus infections in 2022. Regular genotype studies in enterovirus infections will improve the control and differentiation of outbreaks from seasonal increases and assess the prevalence of dominant genotypes and the association of the clinical course with enterovirus genotypes in Kazakhstan.

Current study had several limitations, such as the strategy of laboratory diagnosis protocols in hospital laboratories requires sending the samples for molecular detection analysis using RT-PCR, which in some cases is very time consuming, and doctors might have to wait for the results before the treatment. Reliable rapid diagnosis of enterovirus infections is essential in children; therefore, the use of sensitive screening tests for EV infection detection is important in clinical settings and hospitals. However, in most cases, symptoms are mild in patients’ with enteroviral infections and require only supportive medications aimed at relieving the symptoms because it is challenging to distinguish between viral infections with similar symptoms based only on clinical representation. Given the prevalence of asymptomatic EV infections, merely detecting EV in a specimen does not necessarily indicate the cause of disease.

Identifying enterovirus serotypes is challenging and requires sequencing of the strains or sequencing of the VP1 gene, which is not available in some regions of the country. Genotyping can be more informative compared to serotyping, and utilizing the nucleotide sequence of VP1 can serve as a proxy for antigenic typing, aiding in distinguishing between enterovirus serotypes. This study aimed to explore the genetic diversity of enterovirus circulating in the area and to give a broader understanding of the processes underlying this transmission of enteroviral infection among children in Kazakhstan.

6. Conclusions

The reason for the seasonal rise in enterovirus infection in Astana in 2022 is eight serovars of group B enteroviruses; regular monitoring with genotyping is essential to understand any changes in their population structure. The central Eurasian location of Kazakhstan facilitates the circulation of both European and Asian genotypes of enteroviruses. The detection of coinfection in CSF and NSW, as well as the identification of different serotypes in the patients’ CSF and NSW samples, suggests the possibility of misidentification of the etiological agent of aseptic meningitis during virological examination of NSW and stool samples. The introduction of NGS genotyping will expand knowledge about the possible coinfection of different enteroviruses and, consequently, recombination patterns in them.

Ethical Approval

The clinical research described in the article was approved by the local bioethical committee of Astana Medical University under approval number 6, dated January 31, 2022. The study was conducted in accordance with the principles outlined in the Helsinki Declaration of 1975 and its amendments from 2005, which ensure ethical guidelines for medical research involving human subjects.

Consent

Written informed consent was obtained from the parents or guardians of the children participated in the study. Laboratory tests for hospitalized patients were conducted within the framework of standard protocol of diagnostics and treatment approved by the Joint Committee on the quality of medical services of the Ministry of Health of the Republic of Kazakhstan from “16” October 2020, Protocol no. 117.

Disclosure

Our study is also aligned with these goals. In our comprehensive study, we concluded all available high-quality clinical studies, and the presented research is the first study of the genetic variation and genotypic diversity of the main pathogen of viral meningitis in Central Eurasia. Our results will influence future diagnostics and supposedly request more specific clinical testing for coxsackieviruses, which appeared to be the predominant causes of aseptic meningitis in children in Kazakhstan. We confirm that this work is original and has not been published elsewhere, nor is it currently under consideration for publication elsewhere. A preprint has previously been published [https://www.researchsquare.com/article/rs-3580377/v1] [DOI: 10.21203/rs.3.rs-3580377/v1] [65].

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Dinara Kamalova conducted formal analysis and validation of the data, writing, reviewing, editing, and contribution to the final version of the manuscript. Akhmetova Assel performed conceptualization, data curation, writing of the original draft, writing, reviewing, editing, and contribution to the final version of the manuscript. Amirgazin Asylulan conducted conceptualization, formal analysis, validation of the data, data curation, writing, reviewing, editing, and contribution to the final version of the manuscript. Igor Sytnik conducted conceptualization, data curation, writing of the original draft, and contribution to the final version of the manuscript. Rudenko Viktoriya conducted data collection and processing of patient data and interpretation of results. Yessimkhanova Gulzhan conducted data collection and processing of patient data and interpretation of results. Bayesheva Dinagul conducted data collection and processing of patient data and interpretation of results. Yegorov Sergey conducted writing, reviewing, editing, and contribution to the final version of the manuscript. Alexander Shustov conducted writing of the original draft, formal analysis, validation of the data, data curation, writing, reviewing, editing, and contribution to the final version of the manuscript. Dinara Kamalova and Akhmetova Assel are the co-first authors.