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Enterovirus B types cause severe infection in infants aged 0–3 months



Enterovirus (EV) infections are being increasingly seen in younger infants, often being more severe than in older children. The risk factors of EV infection in infants have been inadequately investigated till date.


We conducted a retrospective study on hospitalized children with laboratory-confirmed EV infection (50 infants aged 0–3 months and 65 older than 3 months) at a tertiary care center in China. Prevalence, clinical characteristics, and genetic features of the virus were analyzed, and independent predictors for severe infection were assessed.


Clinical findings showed that severe infection was more common in infants aged 0–3 months than in older children (78.0% vs. 35.4%, p < 0.001), with higher morbidity of pneumonia, meningitis, and sepsis (p < 0.01). EV-B types were detected more frequently in infants aged 0–3 months than in older children (88.0% vs. 7.7%, p < 0.001). Echovirus 11 was the most identified EV-B, and it recombined with E6 in P2 and P3 regions. Risk factors for severe EV infection included EV-B types infection, age less than 3 months, elevated alanine aminotransferase level, abnormal platelet count, and abnormal cerebrospinal fluid characteristics.


Our data indicated that EV-B types mainly cause severe infection in infants aged 0–3 months. Therefore, knowledge about EV-B types could have implications in designing effective intervention and prevention strategies for young infants with severe EV infection.


Enterovirus (EV) are small non-enveloped RNA viruses belonging to the family Picornaviridae, with genomes approximately 7500 nucleotides in length. Currently, more than 100 EV types, assigned to four species, have been found to infect humans, namely enterovirus A (EV-A), EV-B, EV-C, and EV-D, based on genetic divergence [1, 2]. EV are distributed worldwide and have a seasonal incidence patterns in temperate regions during summer and fall and occur year-round in the tropics [3]. EV infection in children manifest as a spectrum of clinical disorders, including non-specific febrile illness, hand-foot-mouth disease (HFMD), meningitis, viral encephalitis, myocarditis, sepsis, and pulmonary edema. The severity and mortality of EV infection are generally inversely related to age, especially in newborns within the first few days of life [4, 5].

In recent decades, several EV-A-associated HFMD outbreaks, particularly those caused by EV-A71, coxsackievirus (CV) A6, CVA10, and CVA16 types, involving millions of children under 5 years of age, have been described in detail [6,7,8,9]. Unlike EV-A infections, EV-B infections (echovirus (E) 1–7, E9, E11–21, E24–27, E29–33, CVB1–6, and CVA9) are commonly identified in children less than 1 year old and are characterized by serious disease, with high mortality rate [10, 11]. The circulation of EV-B types has been associated with several EV outbreaks in Asia, Europe, and the USA in recent years [12,13,14]. However, the incidence of infant infections and circulating types varies markedly across countries and regions.

There is growing interest in the understanding of epidemiology of EV in infants, particularly due to their association with severe disorders. Awareness of the clinical features associated with severe conditions in infants, recognition of the risk factors, and monitoring of the infection types might help pediatricians diagnose severe cases promptly and treat them appropriately to reduce mortality. In this study, we described the epidemiological, clinical, and genetic characteristics of EV infections in a cohort of children admitted to a tertiary care center in Guangzhou, China. Furthermore, with the clinical and molecular epidemiological patterns of EV infection, we aimed to explore the clinical spectrum and relationship of individual EV types with the risk factors of severe infection in infants.


Study design and patients

Children hospitalized with laboratory-confirmed EV infection, between January and December 2019, at the Guangdong Women and Children Hospital, Guangzhou, China were included in this study. A confirmed case of EV infection was defined by positive EV-RNA findings in stool, plasma, and/or cerebrospinal fluid (CSF) specimens. Moreover, we extracted demographic data, clinical symptoms or signs, laboratory data, CSF parameters, and outcomes upon admission from the patients’ electronic medical records, and all laboratory tests were performed according to the clinical care needs of the children.

EV detection and type determination

Nucleic acids were extracted from stool, plasma, and/or CSF samples of suspected children using the Ex-DNA/RNA Virus Kits (Tianlong Bio-technology, Suzhou, China), and amplified using the commercial pan-enterovirus q-PCR kit (DAAN Gene, Guangzhou, China) for EV in the 7500 fast real-time PCR system (Applied Biosystems, Foster City, USA), according to the manufacturer’s instructions [15]. In addition, all EV-positive samples were amplified on the basis of partial VP1 sequences for further type identification, as described previously [15].

Full-length genome amplification

To understand the molecular epidemiology of EV in infants better, three long-distance PCR amplifications of the most common EV type in infants were obtained using the TaKaRa LA Taq® Kit (Dalian, China). The primers used for RT-PCR are listed in Additional file 1: Table S1, and the full-length genome was sequenced using the “primer-walking” strategy. Finally, complete nucleotide sequences of selected strains were obtained by assembling the sequenced fragments, which were eventually submitted to GenBank (accession numbers: MW883610 to MW883614).

Phylogenetic and recombination analysis

Multiple sequence alignments were performed using the ClustalX, and phylogenetic trees were drawn using the neighbor-joining method via MEGA X [16]. Bootstrap analyses with 1000 re-samplings were performed to determine the confidence values for groupings within the phylogenetic trees. The SimPlot and Bootscan analyses were performed using SimPlot 3.5.1 with a 500-nucleotide window moving in steps of 20 nucleotides [17]. Evolutionary divergence of the 5ʹ UTR, four viral capsid proteins (VP1–VP4), and seven non-structural proteins (2A–2C and 3A–3D) between the selected strains and 64 reference strains from the GenBank was estimated via MEGA X using the Kimura 2-parameter model [16]. Heatmap of the 5ʹ UTR, VP1–VP4, 2A–2C, and 3A–3D genes was created based on evolutionary divergences.

Statistical analysis

Descriptive statistical analysis for population characteristics in the study and clinical findings was performed using SPSS 22.0. Categorical variables are reported as frequencies and percentages after comparison using the chi-square test or Fisher’s exact test. The quantitative data are presented as the median (interquartile range, IQR) and were compared using the nonparametric rank-sum test (Kruskal–Wallis test). Odds ratio (OR) and 95% confidence intervals (95% CIs) were used to estimate the risk factors of severe infection using SPSS 22.0, a multiple logistic regression analysis.


Demographic characteristics and seasonal distribution

A total of 115 children, hospitalized with laboratory-confirmed EV infections between January and December 2019, were included in the study (Fig. 1). The median age was 10.0 months (IQR 0.7–19.0), and 75 (65.2%) participants were males. Fifty (43.5%) participants were infants aged 0–3 months [median age 21 days (IQR 12–27)], and 65 (56.5%) were children older than 3 months [median age 17.0 months (13.0–26.5)].

Fig. 1
figure 1

Flow diagram of children hospitalized with EV infections in Guangzhou, China during January–December 2019

Since the incidence and types of EV infection are influenced by seasonal cycles, we checked the monthly variation (Fig. 2a). A total of 109 (94.8%) hospitalizations occurred from March to October, and the number of cases peaked from April to June. However, there was a significant difference in seasonal changes in the occurrence of EV infection between infants aged 0–3 months and children older than 3 months. A peak (mainly caused by E11) was observed in the infant group from April to July, with no case thereafter. On the contrary, two peaks were observed in the number of cases in the children group. The first peak was unexpected, appearing in April; the second peak appeared in August.

Fig. 2
figure 2

Children hospitalized with EV infections, as per age group, month, and EV types. a Number of children hospitalized with EV infections, by age group and month. b Number of children hospitalized with EV infections by age group and EV types

Type distribution

Two EV species, comprising of 14 different types, accounted for all the 115 reports over this period. EV-A types, including CVA6 [43 (37.4%) of 115], CVA4 [14 (12.2%)], CVA16 [6 (5.2%)], CVA10 [2 (1.7%)], and CVA5 [1 (0.9%)], were identified in 66 (57.4%) cases. EV-B, accounting for the remaining 49 (43.6%) cases, included E11 [30 (26.1%)], E18 [6 (5.2%)], CVA9 [3 (2.6%)], E25 [2 (1.7%)], E9 [2 (1.7%)], CVB3 [2 (1.7%)], CVB2 [2 (1.7%)], E3 [1 (0.9%)], and E6 [1 (0.9%)]. The median age of EV-B infections was 21 days (IQR 12–27), which was significantly less than the 16.5 months (IQR 13.0–26.0) observed for EV-A infections (p < 0.001).

The species of EV infecting at different ages were significantly different (Fig. 2b). EV-B was detected more frequently in infants aged 0–3 months than in older children [44 (88.0%) of 50 vs. 5 (7.7%) of 65, p < 0.001]. However, EV-A was more common in children older than 3 months [60 (92.3%) of 65 vs. 6 (12.0%) of 50, p < 0.001], and only 5 (7.7%) were infected with EV-B. E11 was the predominant serotype identified in 29 (58.0%) of the 50 infants, with a noticeably higher proportion than that in older children [1 (1.5%) of 65] (p < 0.001). Additionally, E18 was detected in five (10.00%) infants, whereas only one such case (1.5%) was found in older children. However, CVA6 was the most commonly observed serotype in older children [42 (64.6%) of 65], followed by CVA4 [10 (15.4%)] and CVA16 [6 (9.2%)].

Clinical analysis

Clinical case files of the 115 patients, with symptoms registered by the pediatrician upon hospital admission, were carefully analyzed, according to age and EV species (Table 1 and Additional file 2: Table S2). The patients showed typical signs and symptoms, including fever [103 (89.6%) of 115], rash [60 (52.2%)], tachycardia [40 (34.8%)], coughing [37 (32.2%)], convulsions [28 (24.3%)], and diarrhea [23 (20.0%)]. However, presentation with fever, rash, tachycardia, coughing, vomiting, startle, and convulsions was significantly less common among infants aged 0–3 months than in children older than 3 months (all p < 0.01). Although the clinical features of infants aged 0–3 months were not typical, the median days of hospital stay were significantly longer than those of older children [13 (IQR7–17) vs. 5 (IQR 4–7), p < 0.001].

Table 1 Clinical characteristics of children with EV infection according to age, Guangzhou, China, 2019

The disease spectrum of patients with EV infection varied in this study. HFMD [59 (51.3%) of 115], pneumonia [44 (38.3%)], meningitis [36 (31.3%)], sepsis [19 (16.5%)], and gastrointestinal dysfunction [23 (20.0%)] were the main disorders frequently observed in 115 patients. Of the 59 patients who fulfilled the definition of HFMD, 55 (93.2%) cases were caused by EV-A, and four (6.8%) were caused by EV-B. Majority [58 (98.3%) of 59] of HFMD cases were children older than 3 months, and only one was an infant. However, the infants aged 0–3 months had significantly higher prevalence rates of pneumonia [34 (68.0%), 50 vs. 10 (15.4%) of 65, p < 0.01], meningitis [28 (56.0%) vs. 8 (12.3%), p < 0.001], and sepsis [14 (28.0%) vs. 5 (7.7%), p < 0.001] than older children. Hepatitis occurred in six (5.2%) infants and two (1.7%) children, and all the cases were associated with EV-B infections.

A total of 62 (53.9%) cases were classified as having severe disease, of whom 39 (62.9%) were infants. Thus, the incidence of severe EV-B-associated infection was 73.5% (36 of 49), which was significantly higher than the 39.4% (26 of 66) of EV-A infections (p < 0.001). Notably, two EV-B-associated individuals died after the onset of the disease; one 5-day-old infant was infected with CVB3, and died of pulmonary edema and subventricular hemorrhage after 28 days of ventilator support, and another (7-month-old) was infected by E18, and died of brain edema.

Laboratory parameters of blood and CSF

Blood test results, according to age and EV serotypes, are presented in Table 2. Compared to older children, infants aged 0–3 months had higher neutrophil counts, lymphocyte counts, and lactate dehydrogenase (LDH) levels (all p < 0.05). Additionally, the levels of inflammatory markers like C-reaction protein (CRP) in older children were significantly higher than those in the infant group [15.3 mg/L (IQR 4.7–31.5) vs. 3.5 mg/L (1.0–18.5), p = 0.004].

Table 2 Laboratory tests from the patients with EV infection, Guangzhou, China, 2019

CSF parameters according to EV type and age are shown in Table 2. In this study, a total of 60 CSF samples from 33 infants (aged 0–3 months) and 27 older children were included. CSF pleocytosis, elevated protein levels, and low CSF glucose were significantly more common in infants than in children (all p < 0.001). In addition, the positive rate of EV in CSF samples from young infants was 76.7% (23 of 30), which was significantly higher than the 20.0% (5 of 25) in the children group (p < 0.001).

Risk factors for severe infection

In this study, among the 115 patients with EV infections, 62 (53.9%) developed severe disease. Risk factors were analyzed between severe and non-severe EV infections by multiple logistic regression analysis using the forward stepwise logistic regression model. As shown in Table 3, severe EV infection was associated with the following factors: infection by EV-B (OR 4.260, 95% CI 1.907–9.517), young age, less than 3 months (OR 6.474, 95% CI 2.794–15.002), abnormal platelet count (OR 2.745, 95% CI 1.278–5.897), and ALT level > 40 U/L (OR 3.064, 95% CI 1.031–9.105). Additionally, abnormal CSF characteristics, including EV positivity (OR 12.071, 95% CI 2.379–61.261), elevated protein (OR 13.913, 95% CI 1.691–114.447), and pleocytosis (OR 9.481, 95% CI 5.633–27.413) were also independent predictors of severe infection.

Table 3 Multinomial logistic regression analysis of risk factors for severe EV infection

Phylogenetic analysis of EV-B types

To understand the molecular epidemiology of EV-B types better, a portion of the VP1 gene from all 49 viruses isolated from EV-B infections was amplified and selected for phylogenetic analysis (Fig. 3). The phylogenetic tree indicated that all 30 E11 strains were closely related to viruses detected in China and the USA in 2018. Additionally, all six E18 strains showed high homology with the strains isolated from the Yunnan, Jiangsu, Hebei, and Sichuan provinces of China in 2015 and 2016.

Fig. 3
figure 3

Neighbor-joining tree of the partial VP1 gene of the EV-B strains in Guangzhou, China. The 44 EV-B strains obtained from infants aged 0–3 months and 5 EV-B strains obtained from children older than 3 months, in this study, are shown in red. Scale bar indicates the number of nucleotide substitutions per site. Bootstrap values were calculated on 1000 replicates. Phylogenetic nodes with bootstrap values over 80 are marked as purple lines

Complete genome sequence analysis of E11

To help determine the evolution of predominant EV type in infants aged 0–3 months during the study year, we randomly performed full-length genome sequencing of E11 from five infants. Phylogenetic analysis revealed that all five E11 strains that circulated in Guangzhou city during 2019 clustered monophyletically with the E11 strains (MN597937 and MN597943) isolated from sewage samples in 2018 in Guangzhou city.

Based on the heatmap results of evolutionary divergence between E11 sequence and other closely related EV sequences, we found that the nucleotide sequences of 5ʹ UTR, VP1–VP4, 2A, and 2B regions of E11 strains displayed higher sequence identity with the strains (MN597927, MN597948, MN597926, and MN597950) isolated in Guangzhou city in 2018, whereas 2C and 3A–3D genes showed minimum evolutionary divergence with E6 strains from Jiangsu province (MK791151) and Zhejiang province (MN145871) of China in 2018 (Fig. 4a). The highest similarity of 2C and 3A–3D regions with E6 suggested possible recombination.

Fig. 4
figure 4

Recombination analysis of E11 genomes in Guangzhou, China during January–December 2019. a Heatmap representation of evolutionary divergences between 64 reference strains and 5 E11 strains (MW883610–MW883614) obtained. Scale bars indicate the number of nucleotide substitutions per site, and bootstrap values were calculated on 1000 replicates. Red squares show positive correlation, and blue squares show negative correlation across the different regions of E6 and E11. b Similarity plot analysis comparing the 5 E11 strains (MW883610–MW883614), obtained in this study, with other closely related EV-B reference strains using Simplot software version 3.5.1 with a window size of 500 nt and step size of 20 bp. c Bootscan analysis comparing 5 E11 strains (MW883610–MW883614) obtained in this study with other closely related EV-B reference strains, using a window size of 500 nt and step size of 20 bp, and genetic distance of the Kirmura 2-parameter model. The arbitrary recombinant threshold was 70%

Similarity plot and bootscanning were conducted between E11 and E6 strains to investigate the recombination phenomenon. Simplot analyses (Fig. 4b) showed that all five E11 strains in our study displayed the highest degree of identity with four E11 strains (MN597927, MN597948, MN597926, and MN597950) in the 5ʹ UTR and P1 (VP1–VP4) regions, and that similarity in the P2 (2B–2C) region decreased sharply. Thus, it was evident that recombination events with the E6 strain (MN597937 and MN597943) occurred partially in the P2 and P3 genomic regions. Furthermore, the Bootscan plot (Fig. 4c) indicated E11 strains in our study had a closer evolutionary relationship with E6 strains in the P2 and P3 genomic regions, with a sequence similarity of over 70.0%.


This study showed that seasonal variations in EV infections with respect to age were evident. Most infants aged 0–3 months had a seasonal pattern of infections. They presented during summer, with a peak from April to July, which was apparently different from the epidemic features in older children, and consistent with previous studies [4, 11, 18]. Our current study also showed that the circulating EV types, affecting each age range, differed substantially. The most common EV detected in children older than 3 months was EV-A, whereas EV-B, represented by the E11 type, was significantly more frequent in younger infants. In the USA and Europe, EV-B is the one most commonly reported in neonates [12, 13, 19, 20].

EV infection in neonates can present with clinical symptoms, ranging from asymptomatic, non-specific febrile illness to severe, life-threatening disease, and it is difficult to distinguish from non-EV infection based on the clinical signs only. In this study, fever was the most common symptom in both infants aged 0–3 months and older children. However, the other clinical symptoms, including rashes, tachycardia, coughing, vomiting, startle, and convulsions, frequently observed in children with HFMD, were not typical for infants, as suggested in earlier retrospective studies [13, 21]. Rashes are often suggested as a diagnostic basis for viral infection. However, only 6.0–27.3% of cases of infants with EV infection have been reported with rashes [4, 13, 22]. In this study, only 8% (4/50) of the infants developed cutaneous rashes. As an important indication of host inflammation in viral infection, less frequent rashes make the early clinical diagnosis of EV infection difficult for neonatologists.

Severe illnesses due to EV are commonly seen in neonates; mortality is exceptionally high when meningitis, myocarditis, and hepatitis occur [4, 13, 23]. More than three-quarters of neonates were diagnosed with severe infection in this study, particularly in the first two weeks of life. Meningitis, sepsis, pneumonia, myocarditis, and/or hepatitis are the most common clinical presentations. In contrast, only 35.4% of children older than 3 months showed severe infection. Meningitis is often associated with age-specific pleocytosis and/or neurological symptoms. Previous studies had shown that EVs can invade the central nervous system and disrupt the blood–brain barrier, resulting in more than 75% of meningitis cases, some of which are life-threatening [7, 13, 24]. In our study, out of 36 patients with meningitis, 28 (77.8%) were infants aged 0–3 months. It could be due to the immature brain of neonates and their imperfect blood–brain barrier. A higher detection rate of 76.7% (23 of 30) for EVs in CSF samples available from 30 neonates further substantiated this finding.

Pneumonia is the most common comorbidity in lower respiratory tract-EV-B infection, and can be rapidly progressive, leading to severe pneumonia [25]. In this study, more than one-third (44/115) cases were accompanied by pneumonia. EV-B types were the predominant pathogens in patients with pneumonia. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection associated with a wide range of pathogens, and pediatricians frequently encounter it in young infants [13, 19, 26]. Hepatitis is often associated with EV-B infections, and mortality is especially high when it occurs concomitantly with myocarditis [27]. In this study, a total of 8 cases (6 infants and 2 children) developed symptoms of hepatitis during hospitalization, and all were associated with EV-B infection.

Understanding the risk factors and monitoring the parameters associated with severe infection may lead to effective prophylaxis and prompt aggressive treatment to reduce morbidity and mortality. Infection with EV-B and younger age are considered the major risk factors for developing severe infections [5]. A retrospective study on 2356 infants with known EV types during 1983–2003 reported that CVB1–5 and E11 have an increased risk of neonatal infection, with CVB4 being associated with the highest mortality rate of 40% [28]. In our study, we found that EV-B infection and age less than 3 months increased the risk of EV infection 4.260-fold and 6.474-fold, respectively. Moreover, of the two deceased children, one was positive for CVB3 and the other was for E18, which illustrated that EV-B infection is a major risk factor. Notably, the mother of the 7-month-old child was also positive for E18 as per stool sample test, with 100% similarity in VP1 gene compared to that of the children, but showed no obvious clinical symptom. Previous studies had shown that transmission of EVs from mother to infant is relatively common, occurring in 30–50% of cases [23, 29, 30].

Xiao-Qing Lv et al. had previously reported that an abnormal platelet count could be an independent predictor of severe EV infection [31]. Our results also showed that if the platelet count was abnormal, the risk of severe infection was increased 2.745-fold. Furthermore, abnormal CSF test result, such as pleocytosis, constituted a significant risk factor for developing severe EV infection, as observed by previous investigators [32], and elevated protein levels and pleocytosis in CSF increased the risk of severe infection 13.913-fold and 9.481-fold, respectively. Moreover, we elucidated that the positive result of EV in CSF was an independent predictor of severe infection, and the OR increased to 12.071.

Genomic recombination is a major driving force in the evolution, diversification, and shaping of genetic architecture of EVs [33], and time-correlated recombination events of EV-B are more frequent than those of other human EV species [34]. However, until recently, only limited complete genome sequences of E11 strains were available in the public database, with most of them coming from non-clinical isolates. In this study, we obtained 5 full-length E11 genomes, analyzed their phylogenetic characteristics, and found homologous recombination events to have occurred with E6 strains in China in 2018. Multiple phylogenetic studies presented previously provided evidence that RNA recombination of EVs only occurred throughout the entire non-structural region, and recombination sites were mainly located in region P2 [33,34,35,36]; notably, the recombination site was in the junction between 2B and 2C, as per our study. These observations suggested that non-structural proteins may be functionally interchangeable with other variants within EVs. Furthermore, in the infected host, effective recombination events are critical for RNA viruses to overcome tissue-type specific antiviral selection, establish robust infection and virulence, and adapt rapidly to dynamic selective environments [37, 38]. However, the changes in phenotypic characteristics of E11 recombination, including their fitness and pathogenicity, need to be investigated further.


In summary, EV affects infants aged 0–3 months differently and more severely than in older children. Clinical manifestations in infants with EV mainly included meningitis, sepsis, pneumonia, and even death. EV-B types were the most common in neonatal EV infection, and recombination events were observed in the P2 and P3 regions of predominant type E11 with E6 from China. In addition, we identified independent predictors of severe EV infection, including EV-B infection, age less than 3 months, elevated ALT level, abnormal platelet count, and abnormal CSF characteristics. Taken together, EV-B infections should be routinely considered in neonates with meningitis, sepsis, and pneumonia, with or without a rash, particularly during EV season.

Availability of data and materials

All data supporting the conclusions of this article are included herein.









Hand-foot-mouth disease


Cerebrospinal fluid


Odds ratio


Confidence interval






White blood cell








C-reactive protein


Alanine aminotransferase


Aspartate aminotransferase


Uric acid


Lactate dehydrogenase


Creatine kinase




Viral capsid protein


Interquartile range


Odds ratio


Confidence interval


No significant


Not detected


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The authors would like to thank all clinicians and other medical staff who helped collect the samples and clinical data.


This work was supported by the Guangdong Natural Science Foundation [Grant Number 2022A1515012226].

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Authors and Affiliations



YX and LM designed the study. YX, DL, ZW, TY, LL, and XJ contributed to data collection, data management, and data extraction. YX and DL performed data analysis. DL, TY, and XJ participated in material preparation. YX drafted the original manuscript. LM, YX, and DL revised all subsequent versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Mingyong Luo.

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Ethics approval and consent to participate

The study protocol was approved by the ethics committee of the Guangdong Women and Children Hospital (ref. 202101324). Requirement of written informed consent was waived, since virological testing in patients who underwent regular medical examination at the hospital was a routine diagnostic procedure. All information collected from patients was delinked from individual patient identifiers. This study was performed in accordance with the Helsinki Declaration of 1964, and its later amendments.

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Not applicable.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1: Table S1.

Primers used for complete genome amplification of echovirus 11

Additional file 2: Table S2.

Clinical characteristics of children with EV infection according to EV species, Guangzhou, China, 2019

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Yang, X., Duan, L., Zhan, W. et al. Enterovirus B types cause severe infection in infants aged 0–3 months. Virol J 20, 5 (2023).

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