Enterovirus D68 infection in children with acute lower respiratory tract infection in China

doi:10.21203/rs.3.rs-1401303/v2

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Enterovirus D68 infection in children with acute lower respiratory tract infection in China

https://doi.org/10.21203/rs.3.rs-1401303/v2

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Background

EV-D68 infection-related acute flaccid paralysis (AFP) has attracted much attention since the outbreak in the USA in 2014. Notably, EV–D68 was detected in a child with acute flaccid paralysis for the first time in China in 2018.

Methods

Based on a multicentre study monitoring viral pathogens of acute lower respiratory tract infection (ALRTI) in children in China from May 2017 to December 2019, the infection of EV-D68 in children with ALRTI in China was analysed.

Results

In this study, 10 EV-D68 positive specimens were detected from 3,071 samples collected from patients with ALRTI. These 10 patients presented with mild diseases and no neurological symptoms and signs. The phylogenetic analysis of EV-D68 showed that all 10 EV-D68 sequences obtained in this study belonged to subclade B3 which circulates dominantly worldwide, and shared high identity with sequences of EV-D68 strains obtained from patients with AFP in the USA. Recombination analysis showed that no recombination event was found. Moreover, four EV-D68 strains were successfully isolated and the isolate in this study was able to infect "neuronal-like" cell line SH-SY5Y, which was used to identify the neurotropism of enteroviruses. Furthermore, potential amino acids and nucleotide sites related to EV-D68 neurovirulence were analysed, but according to genetic variation analysis, these reported sites in complete genome may not be enough to explain the neurovirulence of EV-D68.

Conclusions

EV-D68 infection was detected in a small number of children with ALRTI in China from 2017 to 2019. The genotype of EV-D68 in this study is subclade B3, which can infect the "neuronal-like" cell line SH-SY5Y.

Enterovirus D68

Genetic analysis

Acute lower respiratory tract infection

Children

China

Human enteroviruses (HEVs) belonging to the genus Enterovirus within the family Picornaviridae are single-stranded positive-sense RNA and non-enveloped viruses. HEVs comprise 116 enterovirus serotypes and are divided into 4 speciess (A–D) [1]. Enterovirus D68 (EV–D68) is a member of enterovirus species D and was first isolated from respiratory samples of children with pneumonia and bronchiolitis in California in 1962. EV–D68 had ever been classified as human rhinovirus 87 due to its similar growth environment requirements with rhinovirus [2] and it was re–identified as EV–D68 based on genotyping in 2002 [3].

The whole–genome (~ 7,400–nucleotide) of EV–D68 consists of a single open reading frame coding for a polyprotein. Two untranslated regions (UTR) at each end of the open reading frame coiding region. The 5’–end contains a highly structured internal ribosome entry site that initials the viral RNA translation and relates to the virulence [4, 5]. The 3’–end containing secondary structure relates to the RNA synthesis [6]. Protease 2A and 3C process the polyprotein into 11 proteins (4 structural proteins VP1–4 and 7 functional proteins 2A–C and 3A–D).

VP1 sequence is used as a target for EV dectection and classification [7]. According to VP1 sequences, EV–D68 could be divided into 4 clades (A–D). Clade B circulated dominantly worldwide and clade D was detected occasionally, while clade A and C were rarely detected [8]. Clade B is further divided into subclade B1/B2/B3. Subclade B1 and B3 dominated during the outbreak in the USA in 2014 and 2018 respectively [9, 10]. In China, subclade B1 was first reported in Hong Kong in 2011 and subclade B3 first appeared in Chinese mainland in 2014 [10, 11]. After 2014, B3 was the main detected subclade in China though there was an upsurge of a novel subclade D3 that emerged in China in 2016 [11].

EV–D68 infection had been received extensive attention since the outbreak in 2014 [12]. It was associated with severe lower respiratory tract infections among all age groups, predominantly in children. Several studies showed that nearly 50% of hospitalized children with EV–D68 infection needed intensive care during the outbreak of EV–D68 in the USA [12]. EV–D68 infections were reported in many countries, such as Spain [13], Japan [14], Netherlands [15], Canada [16], England [17], etc. Moreover, an increase of acute flaccid paralysis (AFP) cases was also observed during the outbreak of EV–D68 [18]. EV–D68 was detected as the only etiologic agent in some cases with AFP. Regrettably, EV–D68 was not detected in cerebrospinal fluid (CSF) from patients with AFP [8]. But EV–D68 was detected from the CSF of two cases with aseptic meningitis and meningoencephalitis in Spain [13]. Recently, SH–SY5Y as a neuroblastoma cell line was used to determine which EV–D68 strains are neurotropic and neuropathogenic. David et al. found that EV–D68 could replicate in the SH–SY5Y cell line [19]. What's more, Alison et al. found that sialic acid was not essential for EV–D68 strains after 2014 to target motor neurons by retrograde axonal transport [20].

There were few data about the prevalence of EV–D68 in China after 2017. Notably, EV–D68 was detected in a case for the first time with AFP in China in 2018 [21]. In this study, we explored EV–D68 infection in patients with acute lower respiratory tract infection (ALRTI) based on a multicenter study of a network monitoring viral pathogens of ALRTI from 7 hospitals in 6 provinces in China from 2017 to 2019. These data will help us understand the epidemiology of EV-D68 infections in hospitalized children with ALRTI in China.

Patients

A total of 3,071 cases hospitalized with ALRTI from a multicenter study including 7 hospitals in 6 provinces (Beijing, Ningxia, Zhejiang, Liaoning, Guizhou, Guangdong) were enrolled in this study, with 660 from Beijing, 500 from Ningxia, 450 from Zhejiang, 608 from Guangdong, 440 from Guizhou, and 304 from Liaoning. The diagnosis of ALRTI and PICU admmission are based on the criteria established by Pediatrics Society and Respiratory Society, Chinese Medical Association [22]. The specimens including nasopharyngeal swabs and sputum were collected.

EV–D68 detection

Viral nucleic acid was extracted by QIAamp MinElute Virus Spin Kit (QIAGEN, Germany). Respiratory viruses including Influenza A, Influenza B, Rhinovirus, Coronavirus NL63/229E/OC43/HKU1, Parainfluenza Virus Type I/II/III/IV, Human Metapneumovirus, Boca Virus, Respiratory Syncytial Virus, Adenovirus Virus, Enterovirus were first screened by Multiplex Real–time PCR Diagnostic Kit Rapid Detection of 16 Speciess of Respiratory Pathogens (Beijing Applied Biological Technologies Co.,Ltd., China). The enterovirus–positive specimens were further tested by EV–D68 nucleic acid detection real–time PCR kit (Bioperfectus technologies, China).

Virus isolation and infection assay

All EV–D68 positive specimens were cultured in Rhabdomyosarcoma (RD) cells. RD cells were maintained in high–glucose Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). The EV–D68 isolate further infects SH–SY5Y cells which were maintained in a 1:1 mixture of DMEM and F–12K (Gibco) medium supplemented with 10% FBS.

As for virus isolation,specimens were blindly passed in RD cells for 3 generations. The standard for successful virus isolation: Typical EV–D68 cytopathic effect appeared and 80% of the cells died in 3 generations.

Sequence amplification and sequencing

All EV–D68 positive specimens were performed on VP1 gene sequencing. The EV–D68 isolates were performed on the whole–genome sequencing. The strain Beijing–R0132 (Accession Number: KP240936) sequence was downloaded from Genbank to design primers (Table 1). SuperScript™ III One–Step RT–PCR System with Platinum™ Taq DNA Polymerase was used to amplify the sequences segmentally. The reaction system volume is 20 ml, including RNA template 2 ml, primer concentration 0.25 mM, and the amplification product were sequenced at SinoGenoMax Co., Ltd.

Bioinformatics analysis

One hundred and sixteen complete genomes of EV–D68 were downloaded from the GenBank database. Based on VP1 sequence typing, 116 VP1 sequences were obtained to construct the phylogenetic tree with 10 VP1 sequences in this study (Accession number: MZ824215–MZ824224).

To investigate whether the sequence differences are correlated with the neurotropism, the complete genome sequences of 9 EV–D68 strains obtained from AFP patients (AFP strain) in the USA were downloaded (including 6 subclade B1 strains and 3 subclade B3 strains) and used to analyze the genetic variation of amino acids (AA).

BioEdit v7.2.0 (http://www.mbio.ncsu.edu/BioEdit/page2.html) software was used for identity and diversity analysis of nucleotide and AA.

MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft/) software was used for multiple alignment. Phylogenetic trees were generated by MEGA v6.0 (Sudhir Kumar, Arizona State University, Tempe, AZ, USA) software with the neighbor–joining method and the Tajima–Nei model. The reliability of phylogenetic trees was estimated by the bootstrap method with 1,000 replicates.

Similarity plot and bootscan analysis were conducted using Simplot v3.5.1 software (window size of 200, a step size of 20 and Kimura (2–parameter) distance model with the Fermon strain as a query squence (Accession Number: NC038308).

Clinical characteristics of patients with EV–D68 infection

Among 3,071 specimens, 10 EV–D68 positive specimens were detected (4 cases from Zhejiang province and 6 cases from Ningxia province). The annual positive specimens of EV–D68 were 1/485 in 2017, 9/1,518 in 2018, and 0/1,068 in 2019. Four EV–D68 strains (YC17106/NX/CHN/2017, YC18116/NX/CHN/2018, YC18137/NX/CHN/2018, WZ17226/ZJ/CHN/2018) were isolated.

The demographic and clinical characteristics of 10 EV–D68 patients are shown in Table 2. Children aged <6 m, 6 m–1 y, 1–3 y, 4–6 y, 7–12 y accounted for 10% (1/10), 30% (3/10), 30% (3/10), 10% (1/10), 20% (2/10), respectively. Nine patients were male and one patient was female. All patients were admitted to the hospital between June and October.

Among 10 EV–D68 patients, 8 patients were diagnosed with pneumonia and 2 patients with bronchiolitis. No case was diagnosed with severe pneumonia or admitted to the ICU ward. No neurological symptoms and signs were found. All cases had no history of asthma or immunodeficiency. All patients were cured and fully recovered.

The patients had common clinical respiratory symptoms, such as cough (100%, 10 cases), dyspnea (30%, 3 cases), stuffy nose (10%, 1 case), and wheeze (50%, 5 cases). No patients had fever and sore throat. As for medical hisotory, no patients had a history of asthma and autoimmune disease. Two patients were co–infected with 2009H1N1 and respiratory syncytial virus, respectively.

Phylogenetic analysis

Ten VP1 sequences and 4 complete genome sequences were successfully obtained. Ten VP1 sequences from this study share 95.7–99.5% nucleotide identity and 98.0–100.0% AA identity with each other. According to VP1 sequences, all 10 isolates in this study belong to subclade B3 (Figure 1).

Four complete genome sequences in this study share 96.6–99.1% nucleotide identity and 99.3–99.6% AA identity in coding region. Compared with the EV–D68 Fermon prototype strain, the nucleotide identity of four complete genome sequences in this study ranges from 87.2 to 87.5%，andthe nucleotide and AA identities range from 87.3 to 87.5% and 95.9 to 96.1% in the coding region. The detailed results show that the nucleotide and AA identity of the structural protein region is more variable than that of the functional protein region. In the structural protein region, the identity of the VP1 sequence is the lowest showing the high variabilities of the VP1 sequence. The AAs of protein 2A are the most variable in the functional protein region and the AAs of protein 3B is conserved (AA identity:100%). To verify whether these findings are applicable among different subclades of EV–D68 and other serotypes of enterovirus species D, four complete genome sequences from this study compared with 8 subclades of EV–D68 and 4 serotypes of enterovirus species D. The results are consistent with the genetic analysis compared with EV–D68 Fermon prototype strain, except that the AA identity of protein 3B presents higher variabilities in different serotypes of enterovirus species D (Table 3).

Recombination analysis

To explore the recombination event, the phylogenetic trees of each protein (VP2–4, 2A–2C, 3A–3D) were constructed. The comparative analysis sequences comprised each subclade of EV–D68 and other serotypes of enterovirus species D, such as EV–D70/90/111/120. The results showed that 4 sequences from this study were always in the same cluster with EV–D68 subclade B3 (Figure 2A–J). To confirm this finding, the recombination analysis in Simplot was conducted. The similarity plot and bootscan analysis results also suggested there were no recombination events (Figure 2K–L).

Infection in cell line SH–SY5Y

David et al. found that EV–D68 subclade B1 AFP strains could infect “neuronal–like” cell line SH–SY5Y, but the Fermon prototype strain could not [19]. However, the subclade B3 strains circulated dominantly nowadays were not tested for the ability to infect cell line SH–SY5Y in their study. Four EV–D68 strains obtained in this study belonged to subclade B3 and shared high identities. So the strain WZ17226 was selected as a representative strain for the experiment. We used the strain WZ17226 to infect SH–SY5Y cells and 100% CPE was observed at 48 h post–infection (Figure 3A). Then, the replicate curve of WZ17226 in SH–SY5Y cells showed that it could replicate in SH–SY5Y cells and reach a peak at 48 h post–infection (Figure 3B). Finally, the titer of strain WZ17226 in SH–SY5Y cells was determined and was ~10^4.4TCID₅₀/ml by 72 h post–infection. These results indicated that the EV–D68 strain obtained in this study could infect SH–SY5Y cells and caused CPE.

Genetic variation analysis

The infection in SH–SY5Y cells indicated that the strains obtained in this study have the ability to infect neurocytes. But the patients in this study didn’t present with any neurological symptoms and signs. AA substitutions in the coding region may have a greater impact on the virulence of the virus. Firstly, the coding region results show 31 AA substitutions, including 15/862 AA substitutions in structural protein sequences and 16/1331 in functional protein regions. The AA substitutions in capsid proteins are mostly distributed in VP1 and VP2, while the functional proteins 2B and 3B have no AA substitution (Table 4). A study showed that 6 AA substitutions (M291T, V341A, T860N, D927N, S1108G, and R2005K) related to neurovirulence were found in subclade B1 AFP strains [23]. In this study, these 6 AA substitutions were found in subclade B1 AFP strains in 2014 but not in our four subclade B3 strains, three subclade B3 AFP strains in 2016 as well as one subclade B1 AFP strain in the USA in 2013. Secondly, the noncoding region was analysed. Three mutation positions in 5'UTR (127T, 262C, 339T) and an incomplete pseudoknot structure in 3'UTR were speculated to be related to neurotropis of EV–D68 strains. [6, 24]. But these three nucleotide substitutions are also not observed and a complete pseudoknot structure could be observed in our four strains and subclade B3 AFP strains.

Based on a multicenter study monitoring viral pathogens of ALRTI, we investigated the prevalence of EV–D68 infection in children with ALRTI in parts of China from May 2017 to December 2019. Ten cases of EV–D68 infection were identified among 3071 patients with ALRTI. In China and the USA, the peak of EV–D68 appeared every two years since 2014 [11]. In this study, the number of EV–D68 positive cases was 9 in 2018, 1 in 2017, and 0 in 2019. To a certain extent, it confirmed this trend though the number of EV–D68 positive cases was too small.

Moreover, we paid attention to different subclades of EV–D68 infection in different age groups. EV–D68 could infect all age groups, but most EV–D68 strains were predominantly from children and belonged to clade B[25]. All strains from children in this study also belonged to clade B. However, a study showed that all strains from patients (> 18 years old) belonged to clade D in Hong Kong [25]. In 2016 in China, a novel subclade D3 was detected, most of which (11/15) were from patients > 18 years old [11]. Unfortunately, this study did not involve adults, so further studies involving all age groups are needed.

Huang et al. performed a retrospective study of EV–D68–related diseases in Beijing, China from 2011 to 2015. Most of the patients (11/12) with EV–D68 infection were children. Of 12 patients, 10 patients (10/12) had a fever, and no patient presented with AFP [26]. The following surveillance of EV–D68 infection for ALRTI cases in Beijing from 2015 to 2017 showed that 71.43% of patients had a fever, no AFP was found among 42 EV–D68 cases, and only 2 patients were admitted to ICU [11]. Of 277 hospitalized children with EV–D68 infection in the USA, 50% of them were admitted to the ICU, and some cases had neurologic signs, including AFP and opsoclonus/myoclonus syndrome [9]. This result indicated that clinical characteristics of cases with EV–D68 infection in the USA differed from those in China. Some studies showed that patients with asthma or reactive airway disease tended to show severe illness with EV–D68 infection [27]. During the outbreak in the USA in 2018, a study found that the number of admissions for asthma and EV–D68 infection were positively correlated [9], and patients with asthma required intensive care more frequently [12]. A nation–wide study in Japan in 2015 also verified the association between EV–D68 and asthma hospitalizations/mechanical ventilation but didn't prove a direct relationship between EV–D68 and asthma exacerbation [28].

A study in China in 2015 showed that EV–D68 might be associated with a history of recurrent wheezing and asthma exacerbation but cases were too few and they didn't perform a statistical analysis [29]. All 10 patients with EV–D68 infection presented with mild disease in this study. No patients had a history of asthma, and 50% of patients presented with wheezing. Whether asthma or wheezing is related to the severity of EV–D68 infection in China still needs to be explored.

The replacement of the virus genotype is a major cause of new outbreaks [8]. A study showed that a novel clade of EV–D68 was found during the 2014 outbreak in the USA [24]. Furthermore, two other outbreaks of a novel subclade B3 happened in the USA in 2016 [30] and 2018 [9]. Xiang et al. analysed the genotypes of EV–D68 strains from China in 2014, and found a genotype replacement from 2006 to 2014 in China, but no outbreak of EV–D68 was reported in China. Then they further analysed the genome sequences of Chinese EV–D68 strains and American strains in 2014. They found that six coding polymorphisms (M291T, V341A, T860N, D927N, S1108G, and R2005K) related to neurovirulence did not appear in Chinese strains, and the X loop sequences of the 3’–UTR related to viral replication that should be complementary were not complementary in American strains in 2014. They speculated that these differences might contribute to the prevalence of EV–D68 between China and the USA [6]. But according to genetic variation analysis in this study, these differences in complete genome may not be enough to explain the neurovirulence of EV–D68. EV–D68 strains which were defined as clade B by Xiang in 2014 were reclassified as subclade B3 based on the latest genotyping [11].

Moreover, the strains during the outbreak in the USA in 2014 belonged to subclade B1 [10]. The differences in gene sequence found by Xiang exist between subclade B1 and B3 both from China and America. Additionally, subclade B1 and B3 were the predominant genotypes duiring the outbreak in the USA in 2014 and 2018 respectively [9], while subclade B1 and B3 were both detected in patients with AFP, which reminds us that both subclade B1 and B3 could both cause the outbreak and be associated with neurological complications.

It is worth noting that EV–D68 was the first time detected in a child with acute flaccid myelitis in China in 2018[21], suggesting that EV–D68 infection may be associated with neurological complication in China. SH–SY5Y is a "neuronal–like" cell line derived from neuroblastoma. David et al. successfully used it as a cell model to identity the neurotropism of EV–D68 strains [19]. The EV–D68 strain obtained (WZ17226) in our study could also infect SH–SY5Y and cause the CPE. Further genetic variations analysis showed that the sequence in this study was highly homologous to American subclade B3 AFP strains. Moreover, one study reported that a mouse AFP model was successfully established using the strain Beijing–R0132 which belongs to subclade B3 and was isolated from clinical sample in China [31]. It is very necessary to strengthen the surveillance of EV–D68 infection in China. But the cell experiment could not support the neurotropism of EV–D68 strains in vivo, and more experiments on animal models are required to determine the neurotropism of EV–D68.

In conclusion, a small number of pediatric cases of ALRTI were associated with EV–D68 infection in China. The patients in this study presented with mild diseases and no neurological symptoms and signs. All 10 EV–D68 strains still belonged to subclade B3. Additionally, the EV–D68 isolate in this study can infect the "neuronal–like" cell line SH–SY5Y. More studies about the pathogenesis of EV–D68 are needed.

Amino acid

AFP

Acute flaccid paralysis

ALRTI

Acute lower respiratory tract infection

CSF

Cerebrospinal fluid

EV–D68

Enterovirus D68

UTR

Untranslated regions

PCR

Polymerase chain reaction

RD cell

Rhabdomyosarcoma cell

TCID₅₀

50% tissue culture infective dose

Viral protein

PICU

Pediatric intensive care unit.

CPE

Cytopathic effect

Ethics approval and consent to participate

This study was approved by the Medical Ethics Committee of Beijing Children’s Hospital, Capital Medical University (No. 2017–k–15).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no conflict of interest.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Number

81971922), Beijing Natural Science Foundation (Grant Number L192014), National Science and

Technology Major Projects (Grant Number 2017ZX10104001–005–010, 2017ZX10103004–004), and

the CAMS Innovation Fund for Medical Sciences (CIFMS) (Grant Number 2019–I2M–5–026). These

sponsors had no role in the study design, data analysis, manuscript preparation, or publication decision.

Author Contributions

QL, XC and ZX proposed and designed this research. QL, JA, YZ, RW, YD, and MZ performed the detection experiments. QL, and XC organized and analysed the data. QL wrote the manuscript and made the Figures and Tables. ZX and XC checked and finalized the manuscript. LL and CL provided clinical sample resources. All authors read and approved the final manuscript.

Acknowledgments

Thanks to all the partners and teachers who helped me with this paper.

Simmonds P, Gorbalenya AE, Harvala H, Hovi T, Knowles NJ, Lindberg AM, Oberste MS, Palmenberg AC, Reuter G, Skern T et al: Recommendations for the nomenclature of enteroviruses and rhinoviruses. Archives of Virology 2020, 165(3):793–797.
Savolainen C, Blomqvist S, Mulders MN, Hovi T: Genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70. Journal of general virology 2002, 83(Pt 2):333.
Ishiko H, Miura R, Shimada Y, Hayashi A, Nakajima H, Yamazaki S, Takeda N: Human Rhinovirus 87 Identified as Human Enterovirus 68 by VP4–Based Molecular Diagnosis. Intervirology 2002, 45(3):136–141.
Li R, Zou Q, Chen L, Zhang H, Wang Y: Molecular analysis of virulent determinants of enterovirus 71. PLoS One 2011, 6(10):e26237.
Yeh MT, Wang SW, Yu CK, Lin KH, Lei HY, Su IJ, Wang JR: A single nucleotide in stem loop II of 5'–untranslated region contributes to virulence of enterovirus 71 in mice. PLoS One 2011, 6(11):e27082.
Xiang Z, Xie Z, Liu L, Ren L, Xiao Y, Paranhos–Baccalà G, Wang J: Genetic divergence of enterovirus D68 in China and the United States. Scientific reports 2016, 6(1):27800–27800.
Oberste MS, Maher K, Kilpatrick DR, Pallansch MA: Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 1999, 73(3):1941–1948.
Sun J, Hu X–Y, Yu X–F: Current Understanding of Human Enterovirus D68. Viruses 2019, 11(6):490.
Wang H, Diaz A, Moyer K, Mele–Casas M, Ara–Montojo MF, Torrus I, McCoy K, Mejias A, Leber AL: Molecular and Clinical Comparison of Enterovirus D68 Outbreaks among Hospitalized Children, Ohio, USA, 2014 and 2018. Emerging Infectious Diseases 2019, 25(11):2055–2063.
Huang W, Wang G, Zhuge J, Nolan SM, Dimitrova N, Fallon JT: Whole–Genome Sequence Analysis Reveals the Enterovirus D68 Isolates during the United States 2014 Outbreak Mainly Belong to a Novel Clade. Scientific Reports 2015, 5(1).
Shen L, Gong C, Xiang Z, Zhang T, Li M, Li A, Luo M, Huang F: Upsurge of Enterovirus D68 and Circulation of the New Subclade D3 and Subclade B3 in Beijing, China, 2016. Scientific reports 2019, 9(1):6073–6073.
Messacar K, Abzug MJ, Dominguez SR: 2014 outbreak of enterovirus D68 in North America. J Med Virol 2016, 88(5):739–745.
Gonzalez–Sanz R, Taravillo I, Reina J, Navascues A, Moreno–Docon A, Aranzamendi M, Romero MP, Del CM, Perez–Gonzalez C, Perez–Castro S et al: Enterovirus D68–associated respiratory and neurological illness in Spain, 2014–2018. Emerg Microbes Infect 2019, 8(1):1438–1444.
Funakoshi Y, Ito K, Morino S, Kinoshita K, Morikawa Y, Kono T, Doan YH, Shimizu H, Hanaoka N, Konagaya M et al: Enterovirus D68 respiratory infection in a children's hospital in Japan in 2015. Pediatr Int 2019, 61(8):768–776.
Knoester M, Scholvinck EH, Poelman R, Smit S, Vermont CL, Niesters HG, Van Leer–Buter CC: Upsurge of Enterovirus D68, the Netherlands, 2016. Emerg Infect Dis 2017, 23(1):140–143.
Drews SJ, Simmonds K, Usman HR, Yee K, Fathima S, Tipples G, Tellier R, Pabbaraju K, Wong S, Talbot J: Characterization of enterovirus activity, including that of enterovirus D68, in pediatric patients in Alberta, Canada, in 2014. J Clin Microbiol 2015, 53(3):1042–1045.
Kamau E, Harvala H, Blomqvist S, Nguyen D, Horby P, Pebody R, Simmonds P: Increase in Enterovirus D68 Infections in Young Children, United Kingdom, 2006–2016. Emerg Infect Dis 2019, 25(6):1200–1203.
Messacar K, Asturias EJ, Hixon AM, Van Leer–Buter C, Niesters HGM, Tyler KL, Abzug MJ, Dominguez SR: Enterovirus D68 and acute flaccid myelitis—evaluating the evidence for causality. The Lancet Infectious Diseases 2018, 18(8):e239–e247.
Brown DM, Hixon AM, Oldfield LM, Zhang Y, Novotny M, Wang W, Das SR, Shabman RS, Tyler KL, Scheuermann RH: Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells. mBio 2018, 9(5).
Hixon AM, Clarke P, Tyler KL: Contemporary Circulating Enterovirus D68 Strains Infect and Undergo Retrograde Axonal Transport in Spinal Motor Neurons Independent of Sialic Acid. J Virol 2019, 93(16).
Wang X, Zhang P, Li J, Chu Y, Li Z, Yang Y, Li F, Cui S, Huo D, Wang Y et al: Enterovirus D68 in a 6–year–old acute flaccid myelitis case in China, 2018: a case report. BMC Infectious Diseases 2020, 20(1).
China NHC: Guideline for diagnosis and treatment of community–acquired pneumonia in Children (2019 version). Chinese Journal of Clinical Infectious Diseases 2019, 12(01):6–13.
Greninger AL, Naccache SN, Messacar K, Clayton A, Yu G, Somasekar S, Federman S, Stryke D, Anderson C, Yagi S et al: A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012–14): a retrospective cohort study. Lancet Infect Dis 2015, 15(6):671–682.
Zhang Y, Cao J, Zhang S, Lee AJ, Sun G, Larsen CN, Zhao H, Gu Z, He S, Klem EB et al: Genetic changes found in a distinct clade of Enterovirus D68 associated with paralysis during the 2014 outbreak. Virus Evol 2016, 2(1):vew015.
Lau SK, Yip CC, Zhao PS, Chow WN, To KK, Wu AK, Yuen KY, Woo PC: Enterovirus D68 Infections Associated with Severe Respiratory Illness in Elderly Patients and Emergence of a Novel Clade in Hong Kong. Sci Rep 2016, 6:25147.
Zhang T, Li A, Chen M, Wu J, Huang F: Respiratory infections associated with enterovirus D68 from 2011 to 2015 in Beijing, China. J Med Virol 2016, 88(9):1529–1534.
Moss RB: Enterovirus 68 Infection––Association with Asthma. J Allergy Clin Immunol Pract 2016, 4(2):226–228.
Korematsu S, Nagashima K, Sato Y, Nagao M, Hasegawa S, Nakamura H, Sugiura S, Miura K, Okada K, Fujisawa T: "Spike" in acute asthma exacerbations during enterovirus D68 epidemic in Japan: A nation–wide survey. Allergol Int 2018, 67(1):55–60.
Xiao Q, Ren L, Zheng S, Wang L, Xie X, Deng Y, Zhao Y, Zhao X, Luo Z, Fu Z et al: Prevalence and molecular characterizations of enterovirus D68 among children with acute respiratory infection in China between 2012 and 2014. Sci Rep 2015, 5:16639.
Wang G, Zhuge J, Huang W, Nolan SM, Gilrane VL, Yin C, Dimitrova N, Fallon JT: Enterovirus D68 Subclade B3 Strain Circulating and Causing an Outbreak in the United States in 2016. Sci Rep 2017, 7(1):1242.
Sun S, Bian L, Gao F, Du R, Hu Y, Fu Y, Su Y, Wu X, Mao Q, Liang Z: A neonatal mouse model of Enterovirus D68 infection induces both interstitial pneumonia and acute flaccid myelitis. Antiviral Research 2019, 161:108–115.

Table 1. Primers for complete genome amplification of EV–D68
Primer	Sequence	Position
EV–D68 F1	5‘–TTAAAACAGCCTTGGGGTTGTT–3’	1–218
EV–D68 R1	5‘–AGCGGATAACGGATAGGTTGT–3’	1–218
EV–D68 F2	5‘–GGTTGTTCCCACTCCAAGG–3’	16–1143
EV–D68 R2	5’–ACCACCATCCTGTACTTCCA–3‘	16–1143
EV–D68 F3	5‘–ATGAAGCAGTAGCCATTGACAA–3’	1039–2228
EV–D68 R3	5‘–AGTGACATAGCCAACATTAGCA–3’	1039–2228
EV–D68 F4–VP1	5‘–TGTAACCCTGATAATACCTTGGAT–3’	2135–2902
EV–D68 R4–VP1	5‘–ACACTAGCATTACTACCTGACTG–3’	2135–2902
EV–D68 F5–VP1	5‘–AGATAAGAACTTTTTCAAATGGACAAT–3’	2648–3446
EV–D68 R5–VP1	5‘–GCACTTGCATCTTGCTATTTG–3’	2648–3446
EV–D68 F6	5‘–TCACTTAGCCACTACAGAGGAG–3’	3332–4499
EV–D68 R6	5‘–GTCTGGAGGCAGGGAATAAATG–3’	3332–4499
EV–D68 F7	5‘–ACATGGCTCTCCAGGAACT–3’	4400–5543
EV–D68 R7	5‘–ATCAGTCAAGTCTCTAAGTGCA–3’	4400–5543
EV–D68 F8	5‘–CACACGCATCTGTTGGAGAA–3’	5458–6590
EV–D68 R8	5‘–AGCATCATAGCCAGTGTAATCA–3’	5458–6590
EV–D68 F9	5‘–CCTTTACGCCACATTTCACAA–3’	6458–7263
EV–D68 R9	5‘–AGAACGAATCTAACCATTTCCG–3’	6458–7263
EV–D68 F10	5‘–CCAGTGATGCCAATGAAGGAG–3’	7050–7335
EV–D68 R10	5‘–GCCCCCAAGTGACCAAAATTT–3’	7050–7335

Table 2. Demographic and clinical characteristics of 10 patients with EV–D68 infection
Characteristics	n=10
Demographics
Sex (n, %)
Male	9 (90.00)
Female	1 (10.00)
Clinical presentation (n, %)
Cough	10 (100.00)
Fever	0 (0.00)
Dyspnea	3 (30.00)
Stuff nose	1 (10.00)
Diarrhea	1 (10.00)
Wheeze	5 (50.00)
Neurologic	0 (0.00)
Diagnosis (n, %)
Pneumonia	8 (80.00)
Bronchiolitis (with diarrhea)	2 (20.00)
Past History (n, %)
Asthma	0 (0.00)
Immunodeficiency	0 (0.00)
PICU admission (n, %)	0 (0.00)
Hospitalization [d, median (IQR)]	5.6 (4–8)
EV–D68, enterovirus D68; IQR, interquartile range; PICU, pediatric intensive care unit.

Table 3. Nucleotide and amino acid identities of four complete genomes in this study compared with different subclades of EV–D68 and four other serotypes of enteroviruses species D

Nucleotide identity (%) (Amino acid identity (%))

Region

WZ17226/ZJ/CHN/2018, YC17106/NX/CHN/2017,YC18116/NX/CHN/2018,YC18137/NX/CHN/2018

Fermon

EV–D111

EV–D94

EV70

EV–120

5'UTR

86.6–86.7

90.5–92.1

97.4–97.8

94.9–95.4

97.9–99

94.5–95.5

90.2–91.2

90.8–92.5

85.7–86.7

71.8–72.1

74.4–75.1

74.2–74.9

——

VP4

87.4–88.4(98.5–98.5)

88.8–89.3(98.5–98.5)

94.2–95.1(98.5–98.5)

91.3–92.2(97.1–97.1)

98.5–99.5(100–100)

90.3–91.3(98.5–98.5)

85.5–85.9(98.5–98.5)

87.4–88.8(98.5–98.5)

85–86.4(98.5–98.5)

77.2–78.2(94.2–94.2)

77.7–79.2(88.4–88.4)

75.3–76.3(92.7–92.7)

75.8–75.8(89.8–89.8)

VP2

86.8–87.5(95.1–95.5)

88.1–88.3(95.9–96.3)

94.4–95.9(97.1–97.5)

92.6–93.5(98.3–98.7)

96.9–99.1(99.5–100)

90.8–91.8(97.5–97.9)

88.1–8.8(96.3–96.7)

90–90.7(95.5–95.9)

87.5–88.1(96.7–97.1)

69.4–69.6(79.2–79.2)

74.1–74.3(86.2–86.6)

68.5–69.2(79.2–79.2)

——

VP3

85.2–85.8(94.0–94.8)

89.2–89.2(96.1–96.5)

94.8–95.4(98.7–99.1)

92.9–93.4(98.7–99.1)

97.1–97.7(99.1–100)

92.4–93.6(98.7–99.1)

89.2–89.5(95.3–95.7)

89.3–90.2(96.1–96.5)

88.3–89(95.3–95.7)

70.6–70.9(80–80.8)

70.9–72(80.8–81.7)

68.5–69.5(78.7–79.5)

——

VP1

85.2–85.4(90.9–91.2)

86.7–87.7(93.8–94.1)

94.8–95.6(97–97.4)

92.4–93(98–98.3)

97.4–98(99–99.3)

90–90.6(94.8–95.1)

85.4–86(92.6–92.9)

86.4–87.1(91.9–92.2)

85.6–86.3(92.2–92.6)

63–63.5(67.9–68.2)

66.8–67(68.9–69.2)

62.4–63.1(67.2–67.2)

62.9–63.1(65.7–65.7）

86.3–89.9(95.9–96.5)

88.2–88.8(96.5–97.9)

94.1–95.6(96.5–97.9)

92–92.2(95.9–97.2)

97.2–97.9(98.6–100)

91.1–92.2(97.2–98.6)

88.6–90.7(95.2–95.9)

89.5–90.7(97.2–97.9)

89.5–90.2(95.9–96.5)

73.4–73.9(83.6–85)

72.1–72.7(83.6–85)

72.1–72.5(78.2–79.5)

——

87.2–87.8(98.9–98.9)

90.5–91.2(100–100)

95.2–95.2(100–100)

92.9–93.2(97.9–97.9)

97.9–99.3(100–100)

89.5–90.2(98.9–98.9)

90.9–92.9(97.9–97.9)

92.2–92.9(100–100)

88.5–89.2(98.9–98.9)

74.4–75(86.8–86.8)

73.4–74.4(86.8–86.8)

69.3–70.3(85.8–85.8)

——

87.6–87.9(96.9–97.5)

90.5–91.1(97.5–98.1)

94.6–95(98.1–98.7)

92.9–93.2(98.1–98.7)

97.8–98.3(99–99.6)

92.4–93.2(98.1–98.7)

89.2–89.7(96.9–97.5)

88.5–90(97.2–97.8)

89–89.7(96.6–97.2)

78–78.6(88.5–89.5)

76.8–77.1(87.9–88.5)

77–77.5(86.7–87.3)

——

89.1–89.8(97.7–97.7)

91.7–92.5(98.8–98.8)

98.1–98.5(100–100)

95.5–95.8(100–100)

98.1–99.2(100–100)

93.2–94.3(100–100)

91–92.5(98.8–98.8)

91.3–92.8(98.8–98.8)

88.7–90.2(96.6–96.6)

79.7–80.5(89.8–89.8)

80.1–80.5(89.8–89.8)

74.9–75.6(88.7–88.7)

——

86.3–87.8(100–100)

89.3–90.9(100–100)

92.4–96.9(100–100)

92.4–93.9(100–100)

95.4–100(100–100)

90.9–92.4(100–100)

86.3–87.8(100–100)

87.8–89.3(100–100)

89.3–90.3(100–100)

71.2–75.7(81.8–81.8)

72.7–75.7(86.3–86.3)

74.2–75.7(86.3–86.3)

——

86.5–86.8(98.3–98.3)

90.5–91(97.8–97.8)

95.9–97.4(99.4–99.4)

93.8–94.5(98.3–98.3)

96.3–98.3(100–100)

91.4–93.6(98..3–98.3)

89–91.2(97.8–97.8)

89.2–90.3(96.7–96.7)

89.9–91.6(98.3–98.3)

74.6–75.2(87.9–87.9)

73.5–74.3(88.5–88.5)

72.8–73.2(83–83)

——

89.4–89.7(97.1–97.8)

91.2–91.8(98–98.2)

96.4–96.8(98.4–99.1)

93.8–94.3(98.9–99.5)

98.1–98.8(99.3–100)

92.5–93.1(98–98.6)

91–91.3(98.2–98.4)

90.8–91.4(97.8–98)

89.7–90.2(97.5–98.2)

77.6–77.8(90.8–91.4)

77.9–78.1(90.3–91)

77–77.8(88.6–89.2)

——

3'UTR

89.2–93.8

90.7–95.3

93.8–98.4

90.7–95.3

95.3–100

90.1–94.3

90.7–95.3

83–97.6

60–62.5

61.2–63.7

51.2–53.6

——

Complete genome

87.2–87.5

89.9–90.1

95.7–96

93.4–93.6

97.6–98.5

92.1–92.4

89.2–89.5

89.7–90.3

88.2–88.6

73.1–73.2

74.2–74.2

72–72.3

——

Coding region

87.3–87.5(95.9–96.1)

89.7–89.9(93–97.3)

95.5–95.8(98.3–98.5)

93.3–93.5(98.4–98.6)

97.5–98.2(99.4–99.7)

91.8–92(97.8–98.1)

89–89.3(96.5–96.8)

89.6–90.1(96.7–96.9)

88.5–88.9(96.3–96.6)

73.4–73.5(83.8–84)

74.2–74.4(84.7–84.8)

72–72.4(82–82.2)

——

Structural protein region

86–86.2(93.7–97.8)

88.1–88.3(95.4–95.8)

95.1–95.4(97.6–98)

92.7–93.1(98.2–98.6)

97.4–98.6(99.4–99.6)

91–91.2(97.1–97.4)

87.2–87.6(94.9–95.2)

88.5–88.9(94.6–95)

87.1–87.5(94.9–95.2)

68–68.3(76.1–76.3)

70.8–71.1(78.4–78.8)

67–67.4(75.6–75.8)

——

Fuctional protein region

88.1–88.4(97.3–97.6)

90.7–91(98–98.1)

95.7–96.1(98.6–98.9)

93.4–93.7(98.4–98.7)

97.6–98.5(99.4–99.7)

92.2–92.6(98.4–98.6)

90.2–90.5(97.5–97.8)

90.3–91(97.9–98.2)

89.4–89.8(97.1–97.5)

76.8–76.9(88.7–89.2)

76.3–76.7(88.6–89)

75.2–75.7(86.1–86.5)

——

Table 4. The amino acid substitutions of four strains in this study compared with AFP strains in the USA

Strains

Acssion Number

Subclade

VP4

VP2

VP3

VP1

138

151

222*

163

194

218

277

290

308

102

273

277

139

161

274

345

360

US/CA/14

–6092

KP100792

US/CO/14

–94

KP100793

US/CA/14

–6100

KP100796

US/CO/14–93

KP126911

CA/AFP/11

–1767

KM892501

US/CO/13–60

KP100794

USA/FL/2016

–19504

KX675261

USA/NY/2016

–19505

KX675262

USA/TX/2016–19506

KX675263

WZ17226/ZJ

/CHN/2018

MZ824221

YC18116/NX

/CHN/2018

MZ824223

YC18137/NX

/CHN/2018

MZ824224

YC17106/NX

/CHN/2017

MZ824222

*Red represents the speculated sites associated with neurovirulence of EV–D68.

No competing interests reported.

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Table S1. The information of uploaded sequences.
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Table S2. Specific information on enrolled patients.

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Enterovirus D68 infection in children with acute lower respiratory tract infection in China

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