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Outbreak of Enterovirus D68 Among Children in Japan—Worldwide Circulation of Enterovirus D68 Clade B3 in 2018

Ikuse, Tatsuki MD*; Aizawa, Yuta MD, PhD*; Yamanaka, Takayuki MD; Habuka, Rie MD*; Watanabe, Kanako PhD; Otsuka, Taketo MD, PhD*; Saitoh, Akihiko MD, PhD*

Author Information
The Pediatric Infectious Disease Journal: January 2021 - Volume 40 - Issue 1 - p 6-10
doi: 10.1097/INF.0000000000002889


Enteroviruses (EVs) belong to the family Picornaviridae and genus Enterovirus and are classified into 7 species, including EV-A to EV-D and rhinoviruses A–C.1 Among EV-D species, EV-D68 is important in severe respiratory infection and acute flaccid paralysis.2 It is phylogenetically divided into 4 major groups—clade A through D—and each clade has subclades defined by genetic variants.

EV-D68 was first isolated in 1962 in the United States.3 Until 2014, EV-D68 was detected sporadically;1 however, after the large US outbreak in 2014, it is now recognized as a reemerging infection in many countries, and sporadic outbreaks have been reported around the world since 2014.4 During outbreaks of EV-D68, prominent increases in asthma-like respiratory illness have been reported. These illnesses tend to be severe and to respond poorly to standard treatments for asthma exacerbation.2

There was an upsurge of pediatric asthma-like respiratory illness in Japan in autumn 2015, and EV-D68 was eventually identified as the causative agent.5 In September 2018 in Niigata, Japan, we experienced a rapid increase in severe asthma-like respiratory illness in children and thus started active surveillance of EV-D68 among pediatric patients with such illnesses in the region from October to November 2018.

The objectives of this study were to determine whether EV-D68 was responsible for the outbreak of asthma-like respiratory illness in children in Niigata, Japan, in 2018, and to identify and compare the EV-D68 clade with other clades reported in outbreaks around the world.



In October and November 2018, we obtained nasopharyngeal swabs from children (<15 years of age) who presented with wheezing or were diagnosed as having asthma exacerbation6 during hospitalization in 1 of 8 pediatric hospitals affiliated with Niigata University, in Niigata, Japan. Written informed consent was obtained from the patients or their caregivers. This study was approved by the ethics committee of Niigata University (approval number: 2018-0428).

Clinical Data

We collected clinical data from the medical records of EV-D68-positive patients, including age, sex, past medical history of allergic diseases (asthma, atopic dermatitis, food allergy, and allergic rhinitis), and clinical symptoms, signs, results of rapid antigen test for respiratory microorganisms (if they were performed) and treatment. To document the upsurge of patients with wheezing episodes, the monthly numbers of inpatients with a diagnosis of asthma, or related diseases causing wheezing episodes, in the 8 hospitals in 2018 were reported. For comparison, we collected data for the same clinical variables from a group of patients with a negative test result for EV-D68.

Sample Collection

We collected respiratory samples from patients by using a nasopharyngeal swab. All samples (47 samples) were obtained within 1 week of admission and 39 samples (83%) were collected within 3 days of admission. They were stored at −80°C and sent to the laboratory of Niigata University.

EV-D68 Detection

RNA was extracted from samples with a QIAamp MinElute Virus Spin Kit (Qiagen, Valencia, CA), in accordance with the manufacturer’s instructions. EV-D68 infection was diagnosed by EV-D68–specific real-time polymerase chain reaction (RT-PCR) targeting of the viral protein (VP) 1 region of EV-D68, as previously described.7

Molecular and Phylogenetic Analyses

After viral RNA was converted to cDNA by using SuperScript VILO MasterMix (Invitrogen, Carlsbad, CA), nested PCR for the partial VP1 region was performed for EV-D68-positive samples, using primers provided by Dr Allan Nix at the US Centers for Disease Control and Prevention (see Table, Supplemental Digital Content 1, For the first PCR, the 18-μL PCR mixture contained 2 μL of cDNA, 0.5 μM of each primer (AN1019 and AN1014), and 10 μL of iQ Supermix (Bio-Rad Laboratories, Hercules, CA). The cycling conditions were 95°C for 3 minutes, followed by 40 cycles at 95°C for 30 seconds, 42°C for 30 seconds (0.4°C/second), and 60°C for 90 seconds. For the second PCR, the 18-μL PCR mixture contained 2 μL of the first PCR product, 0.5 μM of each primer (AN1021 and AN1022), and 10 μL of iQ Supermix. The cycling conditions were 95°C for 3 minutes, followed by 40 cycles at 95°C for 30 seconds, 52°C for 20 seconds, and 72°C for 60 seconds for the second PCR. Amplicon size was approximately 590 bp. The samples were analyzed by sequencing and genotyping by using BLAST analysis (

EV-D68 Identification

Using Molecular Evolutionary Genetics Analysis software, version 6,8 after multiple alignments with the Clustal W program, we identified the best substitution model, as indicated by the lowest Bayesian Information Criterion scores. The phylogenetic tree was constructed by using the maximum likelihood method with Tamura 3-parameter and discrete Gamma distribution with 6 rate categories and 1000 bootstrap replicates, and partial VP1 region sequences for EV-D68 strains detected in the reports published during the period from 2014 through 2018.1,9–18 The partial VP1 regions of the current 11 EV-D68 strains have been deposited to the NCBI GenBank database (accession number: LC515238–LC515248).

Detection of Worldwide EV-D68 Outbreaks

To identify worldwide EV-D68 outbreaks and clarify the clade circulated after 2014, we searched PubMed from September 2018 to November 2019 with the terms “enterovirus D68” and “outbreak.”

Statistical Analysis

Descriptive statistics are reported as medians with interquartile ranges (IQR) or percentages, as appropriate. We compared clinical data, such as sex, age, symptoms, and treatment, between EV-D68 RT-PCR-positive and -negative patients by using the Fisher Exact Test or Mann-Whitney U test, in accordance with the data distribution. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).19 A P value of <0.05 was considered to indicate statistical significance.


EV-D68-Positive Cases

In 2018, 368 children with asthma or illness related to wheezing episodes were admitted to 1 of 8 hospitals in Niigata, Japan, and 176 (48%) of these patients were admitted during the 3-month period from September through November 2018 (Figure 1). Among the 176 patients, 47 patients were able to participate in the study with available samples between October and November 2018, EV-D68 was positive in 22 (47%) and negative in 25 (53%) (see Figure, Supplemental Digital Content 2, Among the 22 positive cases, 11 (50%) patients were male, and their median age was 4.6 years. Most patients (77%) in the outbreak had a history of allergic disease. Common symptoms included low-grade fever (86%) and rhinorrhea (68%). In addition, respiratory symptoms such as wheezing (100%), tachypnea (77%), and retraction (73%) were frequently observed. Among the 22 EV-D68-positive patients, the tests were performed in 11 (50%) for respiratory syncytial virus (RSV), 10 (45%) for human metapneumovirus (hMPV), 2 (9%) for Mycoplasma pneumoniae, and 1 (5%) for influenza virus (Flu); however, all these tests were negative. Among the 25 EV-D68-negative patients, the tests were performed in 11 (44%) for RSV, 11 (44%) for hMPV, 4 (16%) for Mycoplasma pneumoniae, 2 (8%) for Flu, and 1 (4%) for adenovirus; however, again, all tests were negative. There was no significant difference between EV-D68-positive and EV-D68-negative patients in any clinical characteristic (Table 1). No patient was intubated, exhibited limb weakness, or died.

TABLE 1. - Characteristics of Patients Positive and Negative for Enterovirus D68 on PCR Analysis
EV-D68 RT-PCR Positive (n = 22) EV-D68 RT-PCR Negative (n = 25) P
Median age, yr (IQR) 4.6 (2.9–6.4) 3.8 (2.3–4.7) 0.44
Male, n (%) 11 (50%) 11 (40%) 0.77
Past medical history of allergic disease*, n (%) 17 (77%) 22 (88%) 0.45
Admitted in October, n (%) 16 (73%) 20 (80%) 0.73
Admitted in November, n (%) 6 (27%) 5 (20%)
High fever (>38.5°C), n (%) 3 (14%) 5 (20%) 0.71
Rhinorrhea, n (%) 15 (68%) 20 (80%) 0.51
Cough, n (%) 22 (100%) 25 (100%) NA
Wheezing, n (%) 21 (95%) 21 (84%) 0.36
Retraction, n (%) 16 (73%) 16 (64%) 0.55
Tachycardia, n (%) 13 (59%) 14 (56%) 1.0
Tachypnea, n (%) 17 (77%) 15 (60%) 0.08
Hypoxia (SpO2 <95 %), n (%) 13 (59%) 14 (56%) 1.0
Muscle weakness, n (%) 0 (0%) 0 (0%) NA
Systemic steroids, n (%) 21 (95%) 24 (96%) 0.59
 Days of systemic steroids, median (IQR) 5.0 (4.0–6.0) 5.0 (4.0–6.0) 0.69
Supplemental oxygen, n (%) 17 (77%) 20 (80%) 1.0
 Days of oxygen, median (IQR) 4.5 (3.3–6.0) 4.5 (3.0–5.0) 0.98
Length of hospital stay, d, median (IQR) 6.5 (5.3–7.8) 6.0 (5.0–7.0) 0.47
*Allergic disease includes asthma, atopic dermatitis, food allergies, and allergic rhinitis.
Greater than the upper limit of normal for age.20
EV-D68 indicates Enterovirus D68; IQR, interquartile range; SpO2, oxygen saturation; NA, not available.

Monthly numbers of pediatric patients with wheezing episodes were admitted to 8 hospitals in Niigata, Japan, in 2018. The number of pediatric patients with wheezing episodes significantly increased from September to November 2018.

Phylogenetic and Molecular Analysis

VP1 sequences were successfully obtained for 50% (11/22) of EV-D68 samples (see Figure, Supplemental Digital Content 2, The phylogenetic tree revealed that all sequences belonged to subclade B3 branch, which included other strains identified in the United States4 and China1 in 2014; in Osaka, Japan,21 in 2015; in the United States1 and Europe15,16 in 2016; and in the United States22 and France9 in 2018 (Figure 2).

Phylogenetic tree based on VP1 region sequences of enterovirus D68 detected in October and November 2018 in Niigata, Japan. The nucleotide sequences of the VP1 region were analyzed, and the phylogenetic tree was constructed by using the maximum likelihood method with Tamura 3-parameter and discrete Gamma distribution with 6 rate categories and 1000 bootstrap replicates. Bootstrap values above 70% are shown. Scale bars show nucleotide substitutions per site. Nucleotide sequence data for other enterovirus D68 strains were downloaded from the GenBank databases. Circles indicate detections of the current outbreak. Each strain was described as “accession number_clade_detected region_detected year.”

Summary of World EV-D68 Outbreaks in 2016–2018

In 2014, multiple clades (B1, B2, B3, and D1) caused outbreaks in North America,1,14,17 Europe,15,23 and China.17,18 In contrast, only 1 outbreak was reported in 2015. This outbreak occurred in Japan and was caused by clade B3.21 In 2016, there were multiple outbreaks, in the United States,1 Europe,15,16 and China,12 and the dominant clade was B3. No outbreaks were reported in 2017. In 2018, outbreaks of clade B3 and D1 were reported in the United States,22 Europe,9,11 and Japan (Table 2).

TABLE 2. - Reported Enterovirus D68 Clades in Outbreaks During 2014–2018, by Country/Region
Country/Region Year
2014 2015 2016 2017 2018
United States1,13,22 B1, B2, D1 B3 B3
Canada14 B2
France9,15 B1, B2 B3 B3, D1
Italy11,16 B3 D1
Spain24 B3, D1
Sweden25 B3
United Kingdom10 B3
Netherlands26 B3
China12,17,18 B3 B3, D3
Japan21 B3 B3
Each EV-D68 clade (A to D) has numbered subclades categorized by the phylogenetic analysis.


In the current study, we detected an EV-D68 outbreak among children with asthma-like respiratory illness in Niigata, Japan, during autumn 2018. EV-D68 was detected in almost half the cases during this period.

EV-D68 infections may cause a wide range of respiratory infections among children, from pharyngitis and rhinitis to more-severe lower respiratory infections and asthma-like respiratory illness.2 Severe respiratory symptoms and asthma-like respiratory illness are associated to underlying diseases; however, precise mechanisms related to asthma-like illness have been unknown. EV-D68-positive patients had more-severe respiratory symptoms and required longer oxygen treatment and longer treatment with drugs, such as systemic steroids, magnesium, or β-stimulant inhalation, than did patients infected with other virus respiratory infections.27,28 However, there were no differences in clinical characteristics between these patient groups in the current study (Table 1). Importantly, our study population only included patients with wheezing episodes or asthma exacerbation, not those with only acute viral infection, even although the latter were included in previous studies.27,28 A future study should investigate viral diseases that cause similar symptoms and evaluate the detailed clinical manifestations in a larger study population.

EV-D68 outbreaks have occurred around the world, especially since the US outbreak in 2014 (Table 2). Outbreaks and the clade strain responsible were reported in some countries. Clade B3 was responsible for worldwide outbreaks in 2015 (only in Japan), 2016, and 2018. Clade B3 has been detected more frequently than other clades (B1, B2, and D1) since 2014 and has caused simultaneous outbreaks in different areas of the world.1,9–18 During September through November 2018, clade B3 was reported in France and the United States, and clade D1 was detected simultaneously in France and Italy.9,11,22 Children are more likely than adults to be affected in clade B3 outbreaks.9 Previous studies1,9,12,22,26 reported that, outside Japan, clade B3 caused outbreaks every 2 years, starting in 2014. Although the clinical difference between each clade has not been known yet, clade B3 appears to have greater potential than other clades to periodically circulate around the world.

Using a new protocol, we were able to sequence the VP1 region in half the present samples. We also used the CODEHOP approach29 for sequencing the same samples; however, only 3/22 (14%) samples were successfully sequenced, as there were 12 mismatches (46%) in the binding site of the forward primer (26 base pairs) and 18 mismatches (67%) in the binding site of the reverse primer (27 base pairs). We believe the current protocol is more sensitive than the CODEHOP approach for analyzing the VP1 region of EV-D68, as there are fewer variations in primer binding sites in the current protocol. Recent studies reported varied success rates for VP1 sequencing of EV-D68 (44%–95%),9,11,16 perhaps because base substitutions of the VP1 region in different strains affect sequencing success rates. To increase detection rates, alternative methods, such as those that target fewer variable sites, need to be investigated.

Our study was a case-based surveillance, and we were unable to determine the start of the EV-D68 outbreak with precision. In addition, our data were collected in 1 prefecture in Japan and may not reflect conditions nationwide; however, local health laboratories in the other prefectures had reported EV-D68 detections in 2018 on the monthly reports of the National Institute of Infectious Diseases.30 Based on the reports, we had rarely detected EV-D68 cases before August 2018; however, accumulation of EV-D68 cases (104 cases) was observed between September and November 2018 in Japan.30 With these national reports combined with our current data, we were able to estimate that EV-D68 disseminated simultaneously over Japan from September to November 2018.

There were some study limitations. First, we did not exclude other respiratory microorganisms in EV-D68-positive and EV-D68-negative patients. A part of EV-D68-positive and -negative patients were tested for rapid antigen tests for common viral infections, although they were all negative. Additionally, the incidence of other respiratory microorganisms, which may cause severe respiratory infections (eg, RSV, hMPV, Flu, and rhinovirus), did not increase significantly during the study period based on the National Institute of Infectious Diseases reports.30 Therefore, we speculated that cocirculation of other respiratory viruses was not happening during the study period. Second, we only analyzed data from children in the current study. Recent studies reported that EV-D68 clade D1 is more likely to cause respiratory infections in adults than in children.9,11,24 To better understand the clinical characteristics of EV-D68 infection in relation to clade, EV-D68 surveillance of both children and adults is needed.

We detected EV-D68 clade B3, the dominant clade responsible for outbreaks, in the half of patients during an outbreak of asthma-like respiratory illness in children in Niigata, Japan, in 2018. Detection and detailed virologic analyses of EV-D68 is an important aspect of worldwide surveillance and will increase understanding of the epidemiologic characteristics of EVD-68 infection around the world.


We thank Dr. Allan Nix at the US Centers for Disease Control and Prevention for providing information regarding primers for detecting EV-D68. We are grateful to the patients, their parents and guardians, family members, and the physicians who participated in this study. The hospitals that participated in this study were Niigata University Medical and Dental Hospital, Niigata City General Hospital, Niigata Prefectural Central Hospital, Niigata Prefectural Shibata Hospital, Niigata Prefectural Tokamachi Hospital, Uonuma Kikan Hospital, Kashiwazaki General Hospital and Medical Center, and JA Niigata Kouseiren Ojiya General Hospital. We acknowledge David Kipler for editing the article.


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    enterovirus D68; Japan; clade B3; outbreak; asthma

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