Genetic diversity of enteroviral meningitis, herpangina and hand, foot, and mouth disease in Kazakhstan

Genetic diversity of enteroviral meningitis, herpangina and hand, foot, and mouth disease in Kazakhstan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Genetic diversity of enteroviral meningitis, herpangina and hand, foot, and mouth disease in Kazakhstan Kamalova Dinara, Akhmetova Assel, Amirgazin Asylulan, Sytnik Igor, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3580377/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Aug, 2024 Read the published version in International Journal of Microbiology → Version 1 posted You are reading this latest preprint version Abstract Enteroviral infection is a common cause of aseptic meningitis, herpangina, and hand, foot, and mouth disease in children. Limited data are available on the enteroviral subtypes associated with hospitalization for these conditions in Kazakhstan. We collected cerebrospinal fluid (CSF) and nasopharyngeal swabs (NSW) from children (N=152, median age=8 years) hospitalized with symptoms of aseptic meningitis (AM, N=139) or herpangina (HA, N=13) disease. We then genotyped enteroviral subtypes associated with AM (n=50) and HA (n=9) using next-generation sequencing (NGS) on the viral protein 1 (VP1), followed up by whole-genome sequencing of the isolated viral species. All identified EVs were species B EV, consisting of five echovirus (E6, E9, E11, E21 and E25) and three coxsackievirus (CVA9, CVB3 and CVB5) serotypes within the cohort. The most abundant EVs were CVA9 (38.5%), CVB5 (21.5%) and E6 (13.8%). Most HA samples (6/9) were genotyped with coxsackievirus CVA9, while AM was associated with a variety of both echovirus and coxsackievirus serotypes. The results suggest that coxsackievirus CVA9 may be the dominant serotype circulating in the HA population, while AM is more diverse in terms of circulating echovirus and coxsackievirus serotypes. Further studies are needed to determine the clinical implications of these findings and to investigate potential differences in disease severity or outcomes associated with different EV serotypes. Molecular Epidemiology Aseptic meningitis Enterovirus children coxsackievirus serotypes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Human enteroviruses (HEVs) are small single stranded RNA viruses of the family Picornaviridae (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, myocarditis etc. (Sawyer, 2002; Park et al., 2012). Based on serological tests, enteroviruses (EVs) were originally divided to coxsackieviruses A and B, echoviruses and polioviruses subgroups (Hyypia et al., 1997). 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) (de Crom et al., 2016). However, with the development of molecular biology methods classification is based on genetic characteristics and phylogenetic relationships between strains (Pöyry et al., 1996), where each EV species include 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 non-polio enteroviruses, as well as for epidemiological and phylogenetic studies (discovering newly occurring serotypes) (Nasri et al., 2007). The EVs structure comprises of non-enveloped 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 (Laitinen et al., 2016). The genome of enterovirus is single RNA molecule with small virus-encoded protein VPg at the 5’ end, and untranslated region (UTR) from both 5’ and 3’ ends (Joffret et al., 2018). Many research studies have shown that VP1 capsid protein is a good marker for the enteroviruses serotypes differentiation and was widely used for PCR-based typing (Oberste, Maher, Kilpatrick, Flemister, et al., 1999; Caro et al., 2001; Joffret et al., 2018). Recently known detection and classification methods are based on reverse-transcription polymerase chain reaction (RT-PCR) (Oberste, Maher, Kilpatrick, & Pallansch, 1999). The HEV-B species consist of the vast majority of EV serotypes and according to the King A. M (2000) 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 (King A. M, 2000). Enteroviruses can be transmitted among humans through different ways, direct contact (fecal-oral way), indirect contact (contaminated equipments) or through 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 (Stellrecht et al., 2002; Bubba et al., 2017). 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 (Majer et al., 2020). Understanding the molecular signatures of HEVs is essential for the effective diagnosis and treatment protocols. The situation with HEV infections molecular typing in Kazakhstan is not yet fully studied and 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 infections and the prevalence of circulating serotypes in Kazakhstan. In this study, we analyzed 65 sequences of HEV and compared with publicly available NCBI database to understand important evolutionary dynamics and infection spread. Materials and methods Patients and selection criteria This study provides a retrospective analysis of 152 cases of hospitalization of children in the 3rd Children's Infectious Diseases Hospital in Astana from May 14 to October 12, 2022 with a preliminary diagnosis of enterovirus infection. Children's Infectious Diseases Hospital Astana is the only specialized institution providing inpatient treatment for childhood infections in Astana (population 1.35 million people). Enterovirus infections were classified by clinical phenotypes, using the following definitions: herpangina (HA) and aseptic meningitis (AM) caused by enterovirus. Herpangina and hand-foot-mouth diseases (HFMD) cause similar symptoms with ulcers in or around mouth, with HFMD causing vesicular lesions on the arms, legs, knees, or buttocks. A nasopharyngeal swabs (NSW) was taken from patients using a sterile cotton swab and placed in a transport medium (DMEM with 50 µg/ml of gentamicin) and stored at minus 70ºС until RNA extraction. Aseptic meningitis was accompanied by an increase in body temperature up to 38–40ºС, the development of neck rigidity, headaches, photophobia was observed, and some patients experienced vomiting, loss of appetite, diarrhea, rash, pharyngitis, and myalgia. For patients with signs of meningitis, according to the standard protocol, a cerebrospinal fluid (CSF) puncture was prescribed, which was divided into 3 equal aliquots by 200 μl. According to a standard diagnostic protocol two CSF sample aliquots were used for enterovirus diagnosis. One sample was analyzed using bacteriology and cytology methods at the hospital laboratory. And the second aliquot was sent for laboratory analysis using RT-PCR in the RSE "Center for Sanitary and Epidemiological Expertise" Medical Centre Hospital of President's affairs Administration of the Republic of Kazakhstan. The third part of the CSF was stored at minus 80ºС for additional studies and genotyping. In case of presence of vesicular lesions of the oral cavity, a nasopharyngeal swab was also collected from patients. In total, for the specified period, 139 children were hospitalized with aseptic meningitis diagnosis confirmed and the presence of enterovirus RNA in cerebrospinal fluid samples detected. Herpangina was diagnosed in 13 children patients, and enterovirus RNA detected from NSW samples. RNA extraction Viral RNA was extracted from 200 μl CSF or NSW samples in transport medium using GeneJET kit Viral DNA and RNA Purification Kit (ThermoScientific, Lithuania) according to the manufacturer's instructions. Whole genome sequencing and assembly of genomes Almost full-length genomes of enteroviruses were amplified as described previously by Isaacs et al. (2018), except that the readily-made reaction mix from BioMaster LR HS-PCR 2x (Biolabmix, Russia) was used for amplification step. Second-stage PCR products were purified with AMPure XP magnetic beads (Beckman Coulter, USA) in a 1:1 ratio, with elution in 20 µl of DNase/RNase-Free Distilled Water. Libraries were prepared using Illumina® DNA Prep kit, (M) Tagmentation (96 Samples) (Illumina, Catalog #20018705) with double barcoding. Libraries were sequenced on Illumina MiSeq using MiSeq Reagent Kit v3 (600-cycle) (Catalog #LMS -102-3003). Reagents were used according to the manufacturers' instructions. The quality control of the obtained reads was carried out using the FastQC program (Andrews, 2017). Before denovo assembly, reads were trimmed using SeqTK (with options: - b 20 -e3) and Sickle (with options: - t sanger - q 30 - l 200 - g) followed by deduplication in the FastP program (with options: -- dedup -- dup _ calc _ accuracy 6 -- disable _ quality _ filtering). To assemble the genomes, Megahit v1.2.9 was used with varying the length of κ- mers individually for each sample, selecting the assembly with the longest lengths for subsequent contigs analysis. The resulting contigs were then used as corresponding references to obtain consensus sequences using BWA. Variants were determined using FreeBayes, consensuses were generated by BCFtools consensus. The ends of assemblies with less than 30x coverage were removed. Determination of serotypes Serotypes were determined by constructing a phylogenetic tree from the database generated with VP1 sequences of enteroviruses from GenBank (www.ncbi.nlm.nih.gov/genbank/). The database was selected using keywords such as "enterovirus", "VP1", "human enterovirus B". In total, approximately 26,000 genome sequence data with VP1 regions were downloaded. Using the generated database and our sequences, a phylogenetic tree was reconstructed, which made it possible to filter the 81 isolates most closely related to our sequences from the database. Further analysis was performed with 146 enterovirus isolates, including 65 obtained within the current study and Enterovirus 69 selected as outgroup (GenBank: AY302560.1) (Oberste et al., 2004). Statistical selection of nucleotide substitution model for phylogenetic analysis was performed using ModelTest -NG (v0.1.7) (Darriba et al., 2020) and the best fitting model was selected for the reconstruction of the phylogenies. Phylogenetic tree was built using the maximum likelihood (ML) method with the bootstrap random sampling method, n=1000. Estimated bootstrap (bs) values greater than 50% were shown for the tree nodes. Interactive Tree Of Life (iTOL) (Letunic & Bork, 2007) online tool was used for interpretation, visualization and annotation of the phylogenetic tree. Genotyping by VP1 region Representative datasets were downloaded separately for each serotype group to describe the molecular epidemiology of HEV-B serotypes in Kazakhstan. Phylogenetic relatedness of enterovirus isolates collected within current study with the most recent genotyping and sub-genotyping data was compared. Phylogenetic dendrograms were reconstructed based on entire VP1 sequences of the international strains downloaded from GenBank. Genotyping of Coxsackievirus A9 (CVA9) was based on Zhao et al. (2022) algorithm, that included 110 international sequences and 25 obtained within current study. Coxsackievirus B5 (CVB5) genotyping of 134 isolates, including 14 Kazakhstani was performed using the same algorithm as described in He et al. (2022). Coxsackievirus B3 (CVB3) strains were analyzed based on Yang et al. (2022), including 250 worldwide strains and 1 isolate from Kazakhstan. Genetic diversity of Echoviruses group worldwide datasets and the relatedness of Kazakhstani isolates were reconstructed according to Cheng et al. (2021) for Echovirus 6 (E6)(n=73); Zhang et al. (2022) for Echovirus 9 (E9)(n=57); Li et al. (2019) and Grapin et al. (2023) for Echovirus 11 (E11) (n=140). However, there are no currently available genotyping schemes for Echovirus 21 (E21) and 25 (E25) groups. Therefore, entire VP1 coding sequences of available E21 and E25 serotypes were downloaded from GenBank (for 15 th of August 2023) and phylogenetic dendrograms reconstructed, including sequences obtained within current study. Genotypes were distinguished based on estimated divergence between/within groups, calculating pairwise genetic distances in MEGA X (Kumar et al., 2018). Genotypes determined with >15% difference between, and <15% within groups. The pairwise genetic distances for E21 and E25 serotypes were estimated within each serotype group using the Kimura 2-parameter model, with gamma distribution model (shape parameter = 5) in MEGA X (Kumar et al., 2018). Enterovirus sequences were classified into so-called genotypes within each serotype (shown in Figures S7-8) if they shared >85% sequence identity based within the VP1 capsid gene. Sequences were aligned in Mafft (v7.520) (Katoh & Standley, 2013) and best-fitting substitution model was selected (for each serotype group) using ModelTest-NG (v0.1.7) (Darriba et al., 2020) program. Maximum likelihood tree reconstructed for VP1 sequences with bootstrapping of replicates 1000 times using RAxML-NG (v1.2.0). The ML tree was reconstructed to demonstrate the genetic diversity of the worldwide strains and the allocation of sequenced Kazakhstani enterovirus strains to particular genotype and sub-genotype group. Interactive Tree Of Life (iTOL) (v1.0) (Letunic & Bork, 2007) was used for illustration and annotation of the ML tree. Analysis of whole genome sequencing data Phylogenetic analysis of 65 whole genome sequences from 58 infected patients in Kazakhstan was performed. Nucleotide substitution model for evolutionary analysis was performed in ModelTest-NG (v0.1.7) (Darriba et al., 2020) and the best fitting model was selected as “GTR+I+G”. Phylogenetic tree was reconstructed using the maximum likelihood (ML) method with the bootstrap random sampling method in RAxML-NG (v1.2.0), with n=1000 bootstraps. Statistically significant bootstrap (bs) values greater than 50% were shown for the tree nodes. To detect if sufficient “temporal signal” was present in the data TempEst (v1.5.3) (Rambaut et al., 2016) software was used. Phylogenetic tree was visualized and annotated in Interactive Tree Of Life (iTOL) (Letunic & Bork, 2007) online tool. Pairwise genetic distances were estimated using ape package in R (R Core Team, 2022). Results 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 shown in Figure 1. The largest number of patient visits occurred during 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) from the total number of admitted patients, comparing with the number of children aged over 10 years 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, with a diagnosis of herpangina 8.5% (13/152). 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). Table 1. Enterovirus results obtained in current study with metadata from patients. Consensus phylogenetic tree was built using ML algorithm for the VP1 sequence region (Figure 3). Phylogenetic clades were mostly separated by enterovirus serotypes, which is consistent with previous enterovirus research studies (Oberste, Maher, Kilpatrick, & Pallansch, 1999). Figure 3 (a, b). Phylogenetic dendrograms reconstructed by using the whole VP1 gene sequence of 65 Human enterovirus B sequences collected from infected patients in Kazakhstan and 81 sequences downloaded from available worldwide Human enterovirus B strains of serotypes CVA9, CVB3, CVB5 (3a) and E6, E9, E11, E21 and E25 (3b). These strains were labelled 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% 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, 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, E25. Two CSF samples were found to be infected with both E21/CVB5 and E11/CVB5, respectively. In one patient, CVA9 was identified in collected CSF sample and two serotypes in 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 co-infected with CVB5/E6 (Table 1, Figure 4). Genotyping of strains by the VP1 gene Subsequently, we analyzed genotypes based on VP1 gene among certain serotypes. The obtained sequences were grouped with Asian and European genotype lineages (Appendix 1, Figure S1-S8). The 25 Coxsackievirus A9 samples clustered as a separate clade into genotype I and are genetically closest to the 2013 Russian sequence. 14 Coxsackievirus B5 sequences formed a cluster in genotype D and are genetically close to isolates circulating in China in 2015-2018. 9 Echovirus 6 sequences clustered into genotype E and are genetically close to European and American lineages. 7 Echovirus 9 sequences were clustered into a separate clade within genotype F, together with European lineages. 3 Echovirus 11 sequences were included in the D5 sub-genotype. This sub-genotype also includes lineage 1 strains circulating in France and Spain in 2022 and 2023 and associated with neonatal infection with liver failure (Grapin et al., 2023; Piralla et al., 2023; World Health Organization, 2023). This lineage was clustered within a separate clade in sub-genotype D5. A single Coxsackievirus B3 sequence was clustered into the most prevalent genotype E. For the analysis of Echovirus 21 genotypes, 31 complete VP1 sequences were downloaded from GenBank (sequences deposited until August 15, 2023). Current analysis included 3 sequences from Kazakhstan obtained as part of this study collected from CSF and NSW samples from patients with aseptic meningitis. Based on the phylogenetic dendrogram and percentage of identity between strains, E21 serotype can be divided into 10 genotypes A-J, the maximum difference in nucleotide sequences within one genotype was 12%, and the minimum average difference in nucleotide sequences between genotypes was 17% (Appendix 1, Table S1). Genotype A is represented by one echovirus 21 prototype strain Farina. Genotypes B, C, and F are represented by single sequences from Madagascar, China and France. For genotypes D, E and I, geographic relationships with strains from China, Central African Republic and India are monitored. Genotype J combines strains from Asia and Europe. 3 sequences from Kazakhstan were included in the H genotype together with strains from China and the United Kingdom. For the analysis of Echovirus 25 genotypes, 91 complete VP1 sequences were obtained from GenBank (sequences deposited until August 15, 2023). The analysis included 3 sequences from Kazakhstan obtained as part of this study. E25 can be divided into 8 genotypes A-H, the identity of nucleotide sequences in the genotypes exceeded 85%. Kazakhstani strains are included in genotype A, and are located in a separate clade, which may subsequently become a sub-genotype. Genotypes B and C include strains isolated from China and the USA, respectively. Genotypes D and H are the largest and combine strains circulating on the Eurasian and American continents. Genotype E combines two strains from the USA and Nigeria. Genotypes F and G include strains isolated in Europe. 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). Figure 5. Phylogenetic analysis of the 65 complete coding sequence obtained in current study. The 25 CVA9 sequences formed three separate clusters, 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 time 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). Discussion In Kazakhstan, cases of enterovirus infection are recorded annually, with peak values in the summer-autumn period (Begaidarova et al., 2015). In our study, more than 90% of hospitalized cases of enterovirus infection were due to aseptic meningitis and only 8.5% 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 agent in 73% of cases of encephalitis and meningitis in Kazakhstan, with the 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 (Bumburidi et al., 2021). 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, 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 (Brouwer et al., 2021). 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 (Isaacs et al., 2018) 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 co-infection in 6 of 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 HFMD was found to be coinfected in 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 co-infection. Co-infection 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 (Ooi et al., 2007; Yang et al., 2011; de Sousa et al., 2021; Guo et al., 2022). In current study, we did not record any specific clinical course in patients with co-infections, 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 new-borns (Batcup et al., 1985; Johnson & Vogt, 2010; Huang et al., 2013; Shabani et al., 2018). In some outbreaks of aseptic meningitis, CVA9 was the main serovariant in China (Cui et al., 2010), Canada (Pabbaraju et al., 2013), South Africa (Smuts et al., 2018). 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 (Novikova et al., 2013; Itani et al., 2023). CVA9 isolated in our study belongs to genotype I, which is mainly represented by Russian strains (Zhao et al., 2022). 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 (Suresh et al., 2020). However, in Poland (Toczylowski et al., 2020) and China (Wang et al., 2010; He et al., 2022; Liu et al., 2022) CVB5 has been the predominant serovariant in a number of outbreaks in recent years. 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 (He et al., 2022), 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) and encephalitis, aseptic meningitis (Centers for Disease Control and Prevention, 2010; Khetsuriani et al., 2010; Suresh et al., 2020). In our case, E6 was detected in 8 patients with aseptic meningitis, of which only one in CSF and one case with herpangina, while co-infection with other enteroviruses was identified in 4 patients. High levels of E6 co-infection may promote recombination changes, requiring monitoring of the serovariant at the genetic level and tracking changes in virulence (Cabrerizo et al., 2014). Conclusions The reason for seasonal rise in enterovirus infection in Astana in 2022 is 8 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 co-infection 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 co-infection of different enteroviruses and, consequently, recombination patterns in them. Declarations Competing interests: The authors declare no competing interests. Data availability statement Sequences obtained within current study were submitted to GenBank under OR678399-OR678463 accession numbers. Ethics Statement 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. 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 quality of medical services of Ministry of Health of the Republic of Kazakhstan from "16" October 2020, Protocol №117. References Batcup, G., Holt, P., Hambling, M. H., Gerlis, L. 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Sci Rep, 12 (1), 2293. https://doi.org/10.1038/s41598-022-06309-1 Zhao, H., Wang, J., Chen, J., Huang, R., Zhang, Y., Xiao, J., Song, Y., Ji, T., Yang, Q., Zhu, S., Wang, D., Lu, H., Han, Z., Zhang, G., Li, J., & Yan, D. (2022, Apr 15). Molecular Epidemiology and Evolution of Coxsackievirus A9. Viruses, 14 (4). https://doi.org/10.3390/v14040822 Table Table 1 is available in the Supplementary Files section. Supplementary Files 3196760table7250506s30x4h.pdf Table 1. Enterovirus results obtained in current study with metadata from patients. JVI0166823FiguresS1toS8.pdf JVI0166823TablesS1toS5.pdf Cite Share Download PDF Status: Published Journal Publication published 25 Aug, 2024 Read the published version in International Journal of Microbiology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3580377","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":247332769,"identity":"acae8c9b-2b59-4a06-80ed-95349f708a39","order_by":0,"name":"Kamalova Dinara","email":"","orcid":"https://orcid.org/0000-0002-8444-3305","institution":"L.N. Gumilyov Eurasian National University, Astana, Kazakhstan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kamalova","middleName":"","lastName":"Dinara","suffix":""},{"id":247332770,"identity":"87c038f3-f9e8-4120-bc1c-38d70b1027fd","order_by":1,"name":"Akhmetova Assel","email":"","orcid":"https://orcid.org/0000-0003-2206-7890","institution":"National Center for Biotechnology, Astana, Kazakhstan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akhmetova","middleName":"","lastName":"Assel","suffix":""},{"id":247332771,"identity":"683d47ef-3aed-4ed8-b1e7-6e706b9f5b06","order_by":2,"name":"Amirgazin Asylulan","email":"","orcid":"https://orcid.org/0000-0001-9418-7758","institution":"National Center for Biotechnology, Astana, Kazakhstan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amirgazin","middleName":"","lastName":"Asylulan","suffix":""},{"id":247332772,"identity":"27da3f37-8933-46fd-bf69-e37cda07ee15","order_by":3,"name":"Sytnik Igor","email":"","orcid":"https://orcid.org/0000-0003-1464-4387","institution":"National Center for Biotechnology, Astana, Kazakhstan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sytnik","middleName":"","lastName":"Igor","suffix":""},{"id":247337463,"identity":"c52b9a96-60b0-4a98-bbc3-18c8c6d18531","order_by":4,"name":"Shevtsov Alexander","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie3QMQrCMBSA4UggXRTXCNJe4ZVAu/QwlYIudZVuFoRMule8RMFB3RK65gAOHiLiJCioVZwk7eiQfwmEfCQvCNls/xh9Ly7g7xZpQQRC7EOgPRmVqC3xNkt20Vk12TlY6vP+5oY5SbSJwEkFVKhqeliQZLBWwIaCCDASmgZI8vG0rLoB7nEYFcjJY+PDipTpJ5nAi9w5zF9EGIc5pkAlj+KadDjEFBGZGx92HM+oUpFfz7LizC8wSYyXeEWy1VlGvbBfSX3lrkcdzow/9iPcfMRms9lsDT0AqhBE/hTBr/EAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0307-1053","institution":"National Center for Biotechnology, Astana, Kazakhstan","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shevtsov","middleName":"","lastName":"Alexander","suffix":""}],"badges":[],"createdAt":"2023-11-08 16:55:57","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-3580377/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3580377/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1155/2024/7796913","type":"published","date":"2024-08-26T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":46369486,"identity":"fc33e04d-b07e-4171-aae3-a162d2a46d27","added_by":"auto","created_at":"2023-11-14 00:46:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267737,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart showing the sampling scheme of the EV strains collected from infected patients in May-October 2022.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/a7290dea0b624e7cf0735bbe.jpg"},{"id":46369487,"identity":"36b055ca-8001-4c59-bc58-1b7d2b03aa56","added_by":"auto","created_at":"2023-11-14 00:46:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143194,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of samples collected from patients for EV diagnosis.\u003c/p\u003e\n\u003cp\u003eX-axis represent percentage of samples collected from male/female patients from total number of samples (n=152), y-axis show month of sampling. Numbers on histogram bars show exact number of samples collected.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/7d71be49c9c7d82186b54910.jpg"},{"id":46370347,"identity":"34ff7eda-9196-4bce-ab01-115ded9631c7","added_by":"auto","created_at":"2023-11-14 00:54:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1806072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a, b).\u003c/strong\u003e Phylogenetic dendrograms reconstructed by using the whole VP1 gene sequence of 65 Human enterovirus B sequences collected from infected patients in Kazakhstan and 81 sequences downloaded from available worldwide Human enterovirus B strains of serotypes CVA9, CVB3, CVB5 (3a) and E6, E9, E11, E21 and E25 (3b). These strains were labelled 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 \u0026gt;50% shown as blue circles.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/46f0bc49630dce7613c689da.jpg"},{"id":46369488,"identity":"6b8e6a97-9870-4023-b0b5-de545eb07b1f","added_by":"auto","created_at":"2023-11-14 00:46:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125698,"visible":true,"origin":"","legend":"\u003cp\u003eEV serotypes identified in patients with diagnosed aseptic meningitis disease and herpangina by sample types.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/1b2c0465292768ccb4c8d618.jpg"},{"id":46369492,"identity":"29b92f3c-57ae-4c6a-a4bb-e6f5b20d68be","added_by":"auto","created_at":"2023-11-14 00:46:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":618312,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of the 65 complete coding sequence obtained in current study.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/da19458ba2be24fff99acab6.jpg"},{"id":65722954,"identity":"f9a4ea7d-b086-4976-88c9-d729c6eeb458","added_by":"auto","created_at":"2024-10-01 17:20:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3409634,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/2da5e454-3dc0-4ee9-9c7f-c23aa72a322b.pdf"},{"id":46369493,"identity":"1fcf3c51-cd4a-4364-a8ab-a759fc82ba35","added_by":"auto","created_at":"2023-11-14 00:46:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":178340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. \u003c/strong\u003eEnterovirus results obtained in current study with metadata from patients.\u003c/p\u003e","description":"","filename":"3196760table7250506s30x4h.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/24a3f0973ff8fc92bb462eee.pdf"},{"id":46369490,"identity":"1fa3cc23-7f11-47a4-96a6-6bc3ebf217e0","added_by":"auto","created_at":"2023-11-14 00:46:12","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":866052,"visible":true,"origin":"","legend":"","description":"","filename":"JVI0166823FiguresS1toS8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/10868a1eae4905d9d2e843da.pdf"},{"id":46369489,"identity":"b6d2ab4e-74da-4f9b-a114-1e439fe825d1","added_by":"auto","created_at":"2023-11-14 00:46:12","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":366596,"visible":true,"origin":"","legend":"","description":"","filename":"JVI0166823TablesS1toS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3580377/v1/05d809428f428ae7e83b27e7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eGenetic diversity of enteroviral meningitis, herpangina and hand, foot, and mouth disease in Kazakhstan\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman enteroviruses (HEVs) are small single stranded RNA viruses of the family \u003cem\u003ePicornaviridae \u003c/em\u003e(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, myocarditis etc. (Sawyer, 2002; Park et al., 2012).\u003c/p\u003e\n\u003cp\u003eBased on serological tests, enteroviruses (EVs) were originally divided to coxsackieviruses A and B, echoviruses and polioviruses subgroups (Hyypia et al., 1997). 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) (de Crom et al., 2016). However, with the development of molecular biology methods classification is based on genetic characteristics and phylogenetic relationships between strains (P\u0026ouml;yry et al., 1996), where each EV species include 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 non-polio enteroviruses, as well as for epidemiological and phylogenetic studies (discovering newly occurring serotypes) (Nasri et al., 2007).\u003c/p\u003e\n\u003cp\u003eThe EVs structure comprises of non-enveloped 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 (Laitinen et al., 2016). The genome of enterovirus is single RNA molecule with small virus-encoded protein VPg at the 5\u0026rsquo; end, and untranslated region (UTR) from both 5\u0026rsquo; and 3\u0026rsquo; ends (Joffret et al., 2018). Many research studies have shown that VP1 capsid protein is a good marker for the enteroviruses serotypes differentiation and was widely used for PCR-based typing (Oberste, Maher, Kilpatrick, Flemister, et al., 1999; Caro et al., 2001; Joffret et al., 2018). Recently known detection and classification methods are based on reverse-transcription polymerase chain reaction (RT-PCR) (Oberste, Maher, Kilpatrick, \u0026amp; Pallansch, 1999).\u003c/p\u003e\n\u003cp\u003eThe HEV-B species consist of the vast majority of EV serotypes and according to the King A. M (2000) 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 (King A. M, 2000).\u003c/p\u003e\n\u003cp\u003eEnteroviruses can be transmitted among humans through different ways, direct contact (fecal-oral way), indirect contact (contaminated equipments) or through 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 (Stellrecht et al., 2002; Bubba et al., 2017). 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 (Majer et al., 2020). \u003c/p\u003e\n\u003cp\u003eUnderstanding the molecular signatures of HEVs is essential for the effective diagnosis and treatment protocols. The situation with HEV infections molecular typing in Kazakhstan is not yet fully studied and 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 infections and the prevalence of circulating serotypes in Kazakhstan. In this study, we analyzed 65 sequences of HEV and compared with publicly available NCBI database to understand important evolutionary dynamics and infection spread.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and selection criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study provides a retrospective analysis of 152 cases of hospitalization of children in the 3rd Children\u0026apos;s Infectious Diseases Hospital in Astana from May 14 to October 12, 2022 with a preliminary diagnosis of enterovirus infection. Children\u0026apos;s Infectious Diseases Hospital Astana is the only specialized institution providing inpatient treatment for childhood infections in Astana (population 1.35 million people).\u003c/p\u003e\n\u003cp\u003eEnterovirus infections were classified by clinical phenotypes, using the following definitions: herpangina (HA) and aseptic meningitis (AM) caused by enterovirus.\u003c/p\u003e\n\u003cp\u003eHerpangina and hand-foot-mouth diseases (HFMD) cause similar symptoms with ulcers in or around mouth, with HFMD causing vesicular lesions on the arms, legs, knees, or buttocks. A nasopharyngeal swabs (NSW) was taken from patients using a sterile cotton swab and placed in a transport medium (DMEM with 50 \u0026micro;g/ml of gentamicin) and stored at minus 70\u0026ordm;С until RNA extraction.\u003c/p\u003e\n\u003cp\u003eAseptic meningitis was accompanied by an increase in body temperature up to 38\u0026ndash;40\u0026ordm;С, the development of neck rigidity, headaches, photophobia was observed, and some patients experienced vomiting, loss of appetite, diarrhea, rash, pharyngitis, and myalgia. For patients with signs of meningitis, according to the standard protocol, a cerebrospinal fluid (CSF) puncture was prescribed, which was divided into 3 equal aliquots by 200 \u0026mu;l. According to a standard diagnostic protocol two CSF sample aliquots were used for enterovirus diagnosis. One sample was analyzed using bacteriology and cytology methods at the hospital laboratory. And the second aliquot was sent for laboratory analysis using RT-PCR in the RSE \u0026quot;Center for Sanitary and Epidemiological Expertise\u0026quot; Medical Centre Hospital of President\u0026apos;s affairs Administration of the Republic of Kazakhstan. The third part of the CSF was stored at minus 80\u0026ordm;С for additional studies and genotyping. In case of presence of vesicular lesions of the oral cavity, a nasopharyngeal swab was also collected from patients.\u003c/p\u003e\n\u003cp\u003eIn total, for the specified period, 139 children were hospitalized with aseptic meningitis diagnosis confirmed and the presence of enterovirus RNA in cerebrospinal fluid samples detected. Herpangina was diagnosed in 13 children patients, and enterovirus RNA detected from NSW samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral RNA was extracted from 200 \u0026mu;l CSF or NSW samples in transport medium using GeneJET kit Viral DNA and RNA Purification Kit (ThermoScientific, Lithuania) according to the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome sequencing and assembly of genomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlmost full-length genomes of enteroviruses were amplified as described previously by Isaacs et al. (2018), except that the readily-made reaction mix from BioMaster LR HS-PCR 2x (Biolabmix, Russia) was used for amplification step. Second-stage PCR products were purified with AMPure XP magnetic beads (Beckman Coulter, USA) in a 1:1 ratio, with elution in 20 \u0026micro;l of DNase/RNase-Free Distilled Water. Libraries were prepared using Illumina\u0026reg; DNA Prep kit, (M) Tagmentation (96 Samples) (Illumina, Catalog #20018705) with double barcoding. Libraries were sequenced on Illumina MiSeq using MiSeq Reagent Kit v3 (600-cycle) (Catalog #LMS -102-3003). Reagents were used according to the manufacturers\u0026apos; instructions.\u003c/p\u003e\n\u003cp\u003eThe quality control of the obtained reads was carried out using the FastQC program (Andrews, 2017). Before \u003cem\u003edenovo\u003c/em\u003e assembly, reads were trimmed using SeqTK (with options: - b 20 -e3) and Sickle (with options: - t sanger - q 30 - l 200 - g) followed by deduplication in the FastP program (with options: -- dedup -- dup _ calc _ accuracy 6 -- disable _ quality _ filtering). To assemble the genomes, Megahit v1.2.9 was used with varying the length of \u0026kappa;- mers individually for each sample, selecting the assembly with the longest lengths for subsequent contigs analysis. The resulting contigs were then used as corresponding references to obtain consensus sequences using BWA. Variants were determined using FreeBayes, consensuses were generated by BCFtools consensus. The ends of assemblies with less than 30x coverage were removed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of serotypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerotypes were determined by constructing a phylogenetic tree from the database generated with VP1 sequences of enteroviruses from GenBank (www.ncbi.nlm.nih.gov/genbank/). The database was selected using keywords such as \u0026quot;enterovirus\u0026quot;, \u0026quot;VP1\u0026quot;, \u0026quot;human enterovirus B\u0026quot;. In total, approximately 26,000 genome sequence data with VP1 regions were downloaded. Using the generated database and our sequences, a phylogenetic tree was reconstructed, which made it possible to filter the 81 isolates most closely related to our sequences from the database. Further analysis was performed with 146 enterovirus isolates, including 65 obtained within the current study and Enterovirus 69 selected as outgroup (GenBank: AY302560.1) (Oberste et al., 2004). Statistical selection of nucleotide substitution model for phylogenetic analysis was performed using ModelTest -NG (v0.1.7) (Darriba et al., 2020) and the best fitting model was selected for the reconstruction of the phylogenies. Phylogenetic tree was built using the maximum likelihood (ML) method with the bootstrap random sampling method, n=1000. Estimated bootstrap (bs) values greater than 50% were shown for the tree nodes. Interactive Tree Of Life (iTOL) (Letunic \u0026amp; Bork, 2007) online tool was used for interpretation, visualization and annotation of the phylogenetic tree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping by VP1 region\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative datasets were downloaded separately for each serotype group to describe the molecular epidemiology of HEV-B serotypes in Kazakhstan. Phylogenetic relatedness of enterovirus isolates collected within current study with the most recent genotyping and sub-genotyping data was compared. Phylogenetic dendrograms were reconstructed based on entire VP1 sequences of the international strains downloaded from GenBank. \u003c/p\u003e\n\u003cp\u003eGenotyping of Coxsackievirus A9 (CVA9) was based on Zhao et al. (2022) algorithm, that included 110 international sequences and 25 obtained within current study. Coxsackievirus B5 (CVB5) genotyping of 134 isolates, including 14 Kazakhstani was performed using the same algorithm as described in He et al. (2022). Coxsackievirus B3 (CVB3) strains were analyzed based on Yang et al. (2022), including 250 worldwide strains and 1 isolate from Kazakhstan. Genetic diversity of Echoviruses group worldwide datasets and the relatedness of Kazakhstani isolates were reconstructed according to Cheng et al. (2021) for Echovirus 6 (E6)(n=73); Zhang et al. (2022) for Echovirus 9 (E9)(n=57); Li et al. (2019) and Grapin et al. (2023) for Echovirus 11 (E11) (n=140). However, there are no currently available genotyping schemes for Echovirus 21 (E21) and 25 (E25) groups. Therefore, entire VP1 coding sequences of available E21 and E25 serotypes were downloaded from GenBank (for 15\u003csup\u003eth\u003c/sup\u003e of August 2023) and phylogenetic dendrograms reconstructed, including sequences obtained within current study. Genotypes were distinguished based on estimated divergence between/within groups, calculating pairwise genetic distances in MEGA X (Kumar et al., 2018). Genotypes determined with \u0026gt;15% difference between, and \u0026lt;15% within groups.\u003c/p\u003e\n\u003cp\u003eThe pairwise genetic distances for E21 and E25 serotypes were estimated within each serotype group using the Kimura 2-parameter model, with gamma distribution model (shape parameter = 5) in MEGA X (Kumar et al., 2018). Enterovirus sequences were classified into so-called genotypes within each serotype (shown in Figures S7-8) if they shared \u0026gt;85% sequence identity based within the VP1 capsid gene.\u003c/p\u003e\n\u003cp\u003eSequences were aligned in Mafft (v7.520) (Katoh \u0026amp; Standley, 2013) and best-fitting substitution model was selected (for each serotype group) using ModelTest-NG (v0.1.7) (Darriba et al., 2020) program. Maximum likelihood tree reconstructed for VP1 sequences with bootstrapping of replicates 1000 times using RAxML-NG (v1.2.0). The ML tree was reconstructed to demonstrate the genetic diversity of the worldwide strains and the allocation of sequenced Kazakhstani enterovirus strains to particular genotype and sub-genotype group. Interactive Tree Of Life (iTOL) (v1.0) (Letunic \u0026amp; Bork, 2007) was used for illustration and annotation of the ML tree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of whole genome sequencing data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis of 65 whole genome sequences from 58 infected patients in Kazakhstan was performed. Nucleotide substitution model for evolutionary analysis was performed in ModelTest-NG (v0.1.7) (Darriba et al., 2020) and the best fitting model was selected as \u0026ldquo;GTR+I+G\u0026rdquo;. Phylogenetic tree was reconstructed using the maximum likelihood (ML) method with the bootstrap random sampling method in RAxML-NG (v1.2.0), with n=1000 bootstraps. Statistically significant bootstrap (bs) values greater than 50% were shown for the tree nodes. To detect if sufficient \u0026ldquo;temporal signal\u0026rdquo; was present in the data TempEst (v1.5.3) (Rambaut et al., 2016) software was used. Phylogenetic tree was visualized and annotated in Interactive Tree Of Life (iTOL) (Letunic \u0026amp; Bork, 2007) online tool. Pairwise genetic distances were estimated using \u003cem\u003eape\u003c/em\u003e package in R (R Core Team, 2022). \u003c/p\u003e"},{"header":"Results ","content":"\u003cp\u003e\u003cstrong\u003eSamples overview\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the period from May 14 to October 12, 2022, 152 patients with a preliminary diagnosis of enterovirus infection were admitted to the children\u0026apos;s infectious diseases hospital No. 3 in Astana. Schematic representation of the samples collected shown in Figure 1.\u003c/p\u003e\n\u003cp\u003eThe largest number of patient visits occurred during 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) from the total number of admitted patients, comparing with the number of children aged over 10 years 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, with a diagnosis of herpangina 8.5% (13/152).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults of whole-genome sequencing and identification of serotypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNearly 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eEnterovirus results obtained in current study with metadata from patients.\u003c/p\u003e\n\u003cp\u003eConsensus phylogenetic tree was built using ML algorithm for the VP1 sequence region (Figure 3). Phylogenetic clades were mostly separated by enterovirus serotypes, which is consistent with previous enterovirus research studies (Oberste, Maher, Kilpatrick, \u0026amp; Pallansch, 1999).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3 (a, b).\u003c/strong\u003e Phylogenetic dendrograms reconstructed by using the whole VP1 gene sequence of 65 Human enterovirus B sequences collected from infected patients in Kazakhstan and 81 sequences downloaded from available worldwide Human enterovirus B strains of serotypes CVA9, CVB3, CVB5 (3a) and E6, E9, E11, E21 and E25 (3b). These strains were labelled using the following format: \u0026lsquo;GenBank accession number\u0026rsquo;/\u0026lsquo;Serotype\u0026rsquo;/\u0026lsquo;Country of origin\u0026rsquo;/\u0026lsquo;Year of isolation\u0026rsquo; (strains with partially available metadata were also included). The prototype Enterovirus B69 was used as an outgroup. Bootstrap values \u0026gt;50% shown as blue circles.\u003c/p\u003e\n\u003cp\u003eBased 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, 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, E25. Two CSF samples were found to be infected with both E21/CVB5 and E11/CVB5, respectively. In one patient, CVA9 was identified in collected CSF sample and two serotypes in NSW sample (E9/CVA9) (Table 1).\u003c/p\u003e\n\u003cp\u003eOf 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 co-infected with CVB5/E6 (Table 1, Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping of strains by the VP1 gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, we analyzed genotypes based on VP1 gene among certain serotypes. The obtained sequences were grouped with Asian and European genotype lineages (Appendix 1, Figure S1-S8). The 25 Coxsackievirus A9 samples clustered as a separate clade into genotype I and are genetically closest to the 2013 Russian sequence. 14 Coxsackievirus B5 sequences formed a cluster in genotype D and are genetically close to isolates circulating in China in 2015-2018. 9 Echovirus 6 sequences clustered into genotype E and are genetically close to European and American lineages. 7 Echovirus 9 sequences were clustered into a separate clade within genotype F, together with European lineages. 3 Echovirus 11 sequences were included in the D5 sub-genotype. This sub-genotype also includes lineage 1 strains circulating in France and Spain in 2022 and 2023 and associated with neonatal infection with liver failure (Grapin et al., 2023; Piralla et al., 2023; World Health Organization, 2023). This lineage was clustered within a separate clade in sub-genotype D5. A single Coxsackievirus B3 sequence was clustered into the most prevalent genotype E.\u003c/p\u003e\n\u003cp\u003eFor the analysis of Echovirus 21 genotypes, 31 complete VP1 sequences were downloaded from GenBank (sequences deposited until August 15, 2023). Current analysis included 3 sequences from Kazakhstan obtained as part of this study collected from CSF and NSW samples from patients with aseptic meningitis. Based on the phylogenetic dendrogram and percentage of identity between strains, E21 serotype can be divided into 10 genotypes A-J, the maximum difference in nucleotide sequences within one genotype was 12%, and the minimum average difference in nucleotide sequences between genotypes was 17% (Appendix 1, Table S1). Genotype A is represented by one echovirus 21 prototype strain Farina. Genotypes B, C, and F are represented by single sequences from Madagascar, China and France. For genotypes D, E and I, geographic relationships with strains from China, Central African Republic and India are monitored. Genotype J combines strains from Asia and Europe. 3 sequences from Kazakhstan were included in the H genotype together with strains from China and the United Kingdom.\u003c/p\u003e\n\u003cp\u003eFor the analysis of Echovirus 25 genotypes, 91 complete VP1 sequences were obtained from GenBank (sequences deposited until August 15, 2023). The analysis included 3 sequences from Kazakhstan obtained as part of this study. E25 can be divided into 8 genotypes A-H, the identity of nucleotide sequences in the genotypes exceeded 85%. Kazakhstani strains are included in genotype A, and are located in a separate clade, which may subsequently become a sub-genotype. Genotypes B and C include strains isolated from China and the USA, respectively. Genotypes D and H are the largest and combine strains circulating on the Eurasian and American continents. Genotype E combines two strains from the USA and Nigeria. Genotypes F and G include strains isolated in Europe.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis by complete coding sequence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on phylogenetic analysis of the complete coding sequence, 65 sequences were clustered according to specific serotypes (Figure 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 5.\u0026nbsp;\u003c/strong\u003ePhylogenetic analysis of the 65 complete coding sequence obtained in current study.\u003c/p\u003e\n\u003cp\u003eThe 25 CVA9 sequences formed three separate clusters, 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 time 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).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn Kazakhstan, cases of enterovirus infection are recorded annually, with peak values in the summer-autumn period (Begaidarova et al., 2015). In our study, more than 90% of hospitalized cases of enterovirus infection were due to aseptic meningitis and only 8.5% 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 agent in 73% of cases of encephalitis and meningitis in Kazakhstan, with the 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 (Bumburidi et al., 2021). 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.\u003c/p\u003e\n\u003cp\u003eThe highest incidence was recorded in the summer months, 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 (Brouwer et al., 2021). 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 (Isaacs et al., 2018) 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 co-infection in 6 of 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 HFMD was found to be coinfected in 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 co-infection. Co-infection 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 (Ooi et al., 2007; Yang et al., 2011; de Sousa et al., 2021; Guo et al., 2022). In current study, we did not record any specific clinical course in patients with co-infections, which may be due to the limited sample size.\u003c/p\u003e\n\u003cp\u003eThe 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 new-borns (Batcup et al., 1985; Johnson \u0026amp; Vogt, 2010; Huang et al., 2013; Shabani et al., 2018). In some outbreaks of aseptic meningitis, CVA9 was the main serovariant in China (Cui et al., 2010), Canada (Pabbaraju et al., 2013), South Africa (Smuts et al., 2018). 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 (Novikova et al., 2013; Itani et al., 2023). CVA9 isolated in our study belongs to genotype I, which is mainly represented by Russian strains (Zhao et al., 2022). 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.\u003c/p\u003e\n\u003cp\u003eCoxsackievirus 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 (Suresh et al., 2020). However, in Poland (Toczylowski et al., 2020) and China (Wang et al., 2010; He et al., 2022; Liu et al., 2022) CVB5 has been the predominant serovariant in a number of outbreaks in recent years. 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 (He et al., 2022), it is tempting to hypothesize a shift in the CVB5 circulation boundaries from China to Kazakhstan.\u003c/p\u003e\n\u003cp\u003eEchoviruses 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) and encephalitis, aseptic meningitis (Centers for Disease Control and Prevention, 2010; Khetsuriani et al., 2010; Suresh et al., 2020). In our case, E6 was detected in 8 patients with aseptic meningitis, of which only one in CSF and one case with herpangina, while co-infection with other enteroviruses was identified in 4 patients. High levels of E6 co-infection may promote recombination changes, requiring monitoring of the serovariant at the genetic level and tracking changes in virulence (Cabrerizo et al., 2014).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe reason for seasonal rise in enterovirus infection in Astana in 2022 is 8 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 co-infection in CSF and NSW, as well as the identification of different serotypes in the patients\u0026rsquo; 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 co-infection of different enteroviruses and, consequently, recombination patterns in them.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCompeting interests: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequences obtained within current study were submitted to GenBank under OR678399-OR678463 accession numbers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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. 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 quality of medical services of Ministry of Health of the Republic of Kazakhstan from \u0026quot;16\u0026quot; October 2020, Protocol №117.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBatcup, G., Holt, P., Hambling, M. H., Gerlis, L. M., \u0026amp; Glass, M. R. (1985). 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Molecular Epidemiology and Evolution of Coxsackievirus A9. \u003cem\u003eViruses, 14\u003c/em\u003e(4). https://doi.org/10.3390/v14040822 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aseptic meningitis, Enterovirus, children, coxsackievirus, serotypes","lastPublishedDoi":"10.21203/rs.3.rs-3580377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3580377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnteroviral infection is a common cause of aseptic meningitis, herpangina, and hand, foot, and mouth disease in children. Limited data are available on the enteroviral subtypes associated with hospitalization for these conditions in Kazakhstan. We collected cerebrospinal fluid (CSF) and nasopharyngeal swabs (NSW) from children (N=152, median age=8 years) hospitalized with symptoms of aseptic meningitis (AM, N=139) or herpangina (HA, N=13) disease. We then genotyped enteroviral subtypes associated with AM (n=50) and HA (n=9) using next-generation sequencing (NGS) on the viral protein 1 (VP1), followed up by whole-genome sequencing of the isolated viral species. All identified EVs were species B EV, consisting of five echovirus (E6, E9, E11, E21 and E25) and three coxsackievirus (CVA9, CVB3 and CVB5) serotypes within the cohort. The most abundant EVs were CVA9 (38.5%), CVB5 (21.5%) and E6 (13.8%). Most HA samples (6/9) were genotyped with coxsackievirus CVA9, while AM was associated with a variety of both echovirus and coxsackievirus serotypes.\u003c/p\u003e\n\u003cp\u003eThe results suggest that coxsackievirus CVA9 may be the dominant serotype circulating in the HA population, while AM is more diverse in terms of circulating echovirus and coxsackievirus serotypes. Further studies are needed to determine the clinical implications of these findings and to investigate potential differences in disease severity or outcomes associated with different EV serotypes.\u003c/p\u003e","manuscriptTitle":"Genetic diversity of enteroviral meningitis, herpangina and hand, foot, and mouth disease in Kazakhstan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-14 00:46:07","doi":"10.21203/rs.3.rs-3580377/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60423bcf-7fab-4a2f-90fc-1efd48a89638","owner":[],"postedDate":"November 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":26252295,"name":"Molecular Epidemiology"}],"tags":[],"updatedAt":"2024-10-01T17:20:01+00:00","versionOfRecord":{"articleIdentity":"rs-3580377","link":"https://doi.org/10.1155/2024/7796913","journal":{"identity":"international-journal-of-microbiology","isVorOnly":true,"title":"International Journal of Microbiology"},"publishedOn":"2024-08-26 00:00:00","publishedOnDateReadable":"August 26th, 2024"},"versionCreatedAt":"2023-11-14 00:46:07","video":"","vorDoi":"10.1155/2024/7796913","vorDoiUrl":"https://doi.org/10.1155/2024/7796913","workflowStages":[]},"version":"v1","identity":"rs-3580377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3580377","identity":"rs-3580377","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}