Enterovirus 68 (EV-D68) is associated with respiratory diseases, such as acute upper respiratory tract infections (URTIs), lower respiratory tract infections (LRTIs), pneumonia, neurological diseases, and acute ﬂaccid myelitis (AFM). In recent years, there have been global outbreaks of EV-D68 epidemics. However, there is no effective vaccine against EV-D68, and the understanding of the seroprevalence characteristics of EV-D68 is limited. To evaluate the epidemiological features of this emerging infection in mainland China, serum samples from 20 pairs of pregnant women and their neonates, 405 infants and children (ages 1 month–15 years), and 50 adults were collected to measure EV-D68 neutralizing antibodies (NtAbs). The results showed that the geometric mean titers (GMTs) of pregnant women and their neonates were 168 (95%CI: 93.6–301.7) and 162.3 (95%CI: 89.9–293.1), respectively. The seroprevalence rate of EV- D68 antibodies was negatively correlated with age in 1-month-old to 12-month-old infants (84% for 1-month-old infants vs 10% for 1-year-old infants), whereas it was positively correlated with age for 1-year-old to 15-year-old children (10% for 1-year-old children vs 92% for 15-year-old children). This study evaluated maternal antibodies against EV-D68 in neonates. Our results showed that if mothers had high levels of anti-EV-D68 NtAbs, the NtAbs titers in their neonates were also high. The GMTs and seroprevalence rates of each age group indicated that EV-D68 infection was very common in China. Periodical EV-D68 seroprevalence surveys and vaccination campaigns should be the top priority for preventing EV-D68 infection. Introduction EV-D68 was ﬁrst isolated in California from children Human enteroviruses (HEVs) are classiﬁed into 12 spe- with pneumonia and bronchiolitis in 1962 . Since then, cies, including enteroviruses A through J and rhinoviruses EV-D68 infections have been identiﬁed only sporadically A through C . EV-D68 belongs to enterovirus D. As a around the world. For example, there were only 26 cases non-enveloped, positive-sense, single-stranded RNA of documented EV-D68 respiratory disease in the United virus, EV-D68 has a genome that contains a single open States from 1970 to 2005 . However, the upsurge of EV- reading frame coding for a poly-protein (P1), the pre- D68 cases in the past few years showed clusters of cursor of four viral capsid proteins, VP1, VP2, VP3, and infections in Europe, the Americas, Asia, Oceania and 6–8 VP4, and seven non-structural proteins, 2A, 2B, 2C, 3A, Africa . In particular, more than 1000 cases, including 3B, 3C, and 3D. VP1 and VP3 are the major antigenic 14 deaths, were reported during the epidemic of EV-D68 2, 3 9 epitopes . infection in 2014 in the United States , resulting in strong public attention toward this virus. During August 2006–April 2010 in Beijing, China, coxsackievirus A21 and enterovirus 68 were detected in enterovirus-positive Correspondence: Qunying Mao (email@example.com)or Zhenglun Liang (firstname.lastname@example.org) adults with acute respiratory tract infections, and the EV- National Institute for Food and Drug Control, Beijing, China D68 positive rate was 10% (13/130) . EV-D68 was also Hualan Biological Engineering Inc, Xinxiang, China 11–14 reported in other areas of China . More cases were Full list of author information is available at the end of the article. These authors contributed equally: Shiyang Sun and Fan Gao © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; Sun et al. Emerging Microbes & Infections (2018) 7:99 Page 2 of 7 Fig. 1 Cytopathic effects observed in RD cells (×10). a Control. b Cells infected with the synthetic virus. c Western blotting using anti-EV-D68 VP1 antibodies. Lane1: marker; lane2: cell control; lane3: Fermon virus; lane4: synthetic virus reported in different geographical locations in the fol- determined. The virus titers of the synthetic virus and 11, 15 8 7.5 lowing years . EV-D68 infection was predominantly the Fermon strain were 10 and 10 TCID /ml, found in pediatric patients and caused a wide range of respectively. Western blotting showed that the prepared symptoms: fever, runny nose, sneezing, cough, skin rash, virus contained a special band of EV-D68 VP1 with the and body/muscle aches. It could lead to severe acute Fermon strain as the positive control (Fig. 1c), indicating upper respiratory tract infections (URTIs), lower that the synthetic virus produced by the reverse genetics respiratory tract infections (LRTIs), pneumonia, neuro- method could cause CPE in RD cells with high titers and logical illness, acute ﬂaccid myelitis (AFM), or even could be used as a challenge virus in the EV-D68 NtAb 6, 15–17 death . There is no effective vaccine, medicine or assay. treatment that can prevent this virus from spreading. In humans, humoral immunity with neutralizing anti- NtAbs assay for EV-D68 using the synthetic virus strain or bodies is crucial for protection against EV-D68 infec- the Fermon strain tion . Unfortunately, immunogenicity in maternal sera To conﬁrm the speciﬁcity of the EV-D68 synthetic virus and the pattern of immune responses against EV-D68 strain, the NtAbs of the anti-serum of other enteroviruses, have not been well studied in mainland China. Further such as hepatitis A, enterovirus 71, coxsackievirus A16, study is necessary to understand the distribution of coxsackievirus A6, coxsackievirus A10, coxsackievirus B3, immunogenicity against EV-D68 infection. To investigate and coxsackievirus B5 (stored in our laboratory), were the seroprevalence of EV-D68 infection in Jiangsu pro- tested with the synthetic virus. The results showed that vince, China, we conducted a cross-sectional study. the synthetic virus could not be neutralized by any anti- Trans-placental serum from prenatal women and serum serum for other enteroviruses (data not shown). This from their neonates were collected and analyzed to result demonstrated that the synthetic virus strain had identify the age-speciﬁc seroprevalence rate of EV-D68 good speciﬁcity. infections in infants/children (ages 1 month to 15 years). The difference between NtAbs assays using the syn- All serum samples were collected in August 2010 in thetic virus strain and the Fermon strain was investi- Donghai County in Jiangsu Province, China . In addition, gated. The EV-D68 NtAbs for 50 healthy adult sera were the seroprevalence of EV-D68 infection in adults was measured using both the synthetic virus strain and the investigated using adult serum provided by Hualan Bio- Fermon strain. The results showed that a 100% ser- logical (Fengqiu) single-mining Plasma Co., Ltd, in 2015. oprevalence rate (50/50) was found for both challenge viruses. The NtAbs GMTs were 166.3 (95% CI Results 126.8–218.0) and 88.6 (95% CI 61.9–128.3) against the EV-D68 Virus preparation synthetic virus strain and the Fermon strain, respec- Reverse genetics was used to produce the EV-D68 tively (Fig. 2a). The synthetic virus strain from China virus. Two plasmids were transfected into 293T cells showed higher NtAbs GMTs than the Fermon strain with Lipo 2000. After 3–4 days of incubation, the cells from America (p = 0.0378), which was in accordance were frozen and thawed. The supernatant was harvested with the epidemiology. The statistical analysis showed and used to infect the RD cells. The infected RD cells that there was good correlation between NtAb titers were cultured for ﬁve to seven days and periodically against the synthetic virus strain and against the Fermon examined for viral cytopathogenic effect (CPE) (Fig. 1a, strain (r = 0.444) (Fig. 2b). Therefore, the synthetic virus b). The viruses were harvested from the cultured cells, was chosen as the challenge strain in the subsequent and the tissue culture infective dose of 50% (TCID )was research. 50 Sun et al. Emerging Microbes & Infections (2018) 7:99 Page 3 of 7 Fig. 2 Comparison of the synthetic virus and the Fermon strain in NtAbs using 50 adult serum samples. a Comparison of the NtAbs of anti- EV-D68 against the synthetic virus and Fermon (p = 0.0378). b The correlation of NtAbs against EV-D68 between the synthetic virus and Fermon (r = 0.444) Table1 Seroprevalence rates and GMTs of NtAbs from Table2 Relationship of EV-D68 NtAbs in blood samples the blood samples of 20 pairs of prenatal women and their collected from prenatal women and their neonates neonates The titer of prenatal The titer of neonates Total titer Subjects Seroprevalence rates GMTs 95% CI women Prenatal women 100% (20/20) 168.0 93.6–301.7 1:8–1:64 1:96–1:512 >1:512 Neonates 100% (20/20) 162.3 89.9–293.1 1:8–1:64 8(20%) 2(5%) – 10(25%) 1:96–1:512 2(5%) 20(50%) 2(5%) 24(60%) >1:512 – 2(5%) 4(10%) 6(15%) Seroprevalence rate and GMTs of anti-EV-D68 in blood samples from prenatal women and their neonates Total 10(25%) 24(60%) 6(15%) 40(100%) To determine the maternal antibodies, serum samples from 20 pairs of women and their neonates were col- lected. The EV-D68 NtAbs were tested using the synthetic Seroprevalence rate and GMTs of EV-D68 NtAb in 0-year- viruses (Table 1). The results showed that the ser- old to 15-year-old children oprevalence rates of EV-D68 NtAbs were 100% (20/20) in To understand the EV-D68 NtAbs distribution among both prenatal women and their neonates. The GMTs of newborns and juveniles, 16 cross-sectional serum samples EV-D68 NtAbs were 168.0 and 162.3 in prenatal women from children ranging in age from 1 month to 11 to 15 and their neonates, respectively. The GMT difference years old were collected and detected against the synthetic between prenatal women and their neonates was not viruses. Figure 3a shows that the titers were negatively statistically signiﬁcant (p = 0.817). correlated with age among neonates younger than 1 year To further analyze the relationship of antibody titers old. The titers were positively correlated with age among between prenatal women and their neonates, we classiﬁed children 1 to 15 years old, reaching a peak for 11-year-old NtAbs titers into four groups: negative (<8), low (8–64), to 15-year-old children, although that value remained moderate (96–512) and high (>512). The percentages of lower than that of the neonates. The NtAbs titer was infants in each group are shown in Table 2. The results below 1:64 for all infants 6 months to 1 year old, making showed that the NtAbs titers of eight pairs (20%) of those infants susceptible to EV-D68 infection. For the 11- mothers and their neonates were in the range of 1:8 to year-old to 15-year-old children, the NtAbs titers were 1:64, the NtAbs titers of 20 pairs (50%) were in the range even higher than 1:512, which may be caused by super- of 1:96 to 1:512, and the NtAbs titers of four pairs (10%) infection in this age group. were above 1:512. There was a positive correlation To further understand the NtAbs against EV-D68 in between EV-D68 NtAbs in neonates and those in their those newborns and children, the seroprevalence rates mothers (r = 0.76, p < 0.01). The results demonstrated and GMTs (positive serum) were calculated (Fig. 3a, b). that prenatal women with high levels of anti-EV-D68 The seroprevalence rates of EV-D68 (NtAb ≥ 1:8) gradu- could transfer EV-68 NtAbs to neonates. ally decreased from 100% for newborns to 10% for infants Sun et al. Emerging Microbes & Infections (2018) 7:99 Page 4 of 7 Fig. 3 Seroprevalence rate and GMTs of anti-EV-D68 in blood samples of 0-year-old to 15-year-old children. a EV-D68 NtAb prevalence (100%) by age group; “n” was the number of the serum samples. b The GMT (positive serum) and seroprevalence rates were evaluated in different age groups younger than 1 year old (Fig. 3a). The seroprevalence rates year old, 2 to 5 years old, and 6 to 11 years old), the gradually increased from 10% for children 1 year old to seroprevalence rates were 79% (96/121), 20% (33/109), 92% for children 11 to 15 years old (Fig. 3a). Similar to the 44% (34/77), and 83% (65/78), respectively, whereas the seroprevalence rates, the GMTs also followed the same GMTs were 25.2 (21.0–30.2), 15.0 (12.0–19.0), 24.7 trend (Fig. 3b), with 8-month-old infants having the (17.15–35.6), and 71.9(50.8–101.8), respectively. The lowest GMT. The GMTs decreased gradually from 162.3 seroprevalence rates and GMTs of anti-EV-D68 anti- for newborns (95 CI: 89.9–293.1) to 10.1 for 8-month-old bodies were lower in the 6-month-old to 12-month-old infants (95 CI: 8.3–12.3). Then, the GMTs increased infants than in the other age groups. The seroprevalence gradually from 10.1 for 8-month-old infants (95 CI: rates and GMTs of anti-EV-D68 antibodies in the 6-year- 8.3–12.3) to 126.0 for 11-year-old to 15-year-old children old to 15-year-old children were higher than those in 1- (95 CI: 78.0–203.6). Since the NtAb trend was slightly year-old to 5-year-old children. different for 8-month-old to 12-month-old infants, EV- D68 infection might occur in infants older than 8 months. Discussion The overall seroprevalence rate of anti-EV-D68 anti- As the pathogen of HEV-D species, EV-D68 causes bodies was 59% (228/385, Table 3). For the four age serious infectious diseases worldwide. Amino acid groups in this study (1 to 5 months old, 6 months to 1 sequence sites on the surface of the viral capsid may Sun et al. Emerging Microbes & Infections (2018) 7:99 Page 5 of 7 Table3 Seroprevalence rates and GMTs of EV-D68 neutralizing antibodies among age groups 1mo–5mo 6mo–1yr 2yr–5yr 6yr–15yr a b c GMT 25.2(21.0–30.2) 15.0 (12.0–19.0) 24.7(17.15–35.6) 71.9(50.8–101.8) Seroprevalence rates (%) 79(96/121) 20(33/109) 44 (34/77) 83(65/78) The seropositive rate of anti-EV-D68 in 6-month-old to 12-month-old infants was obviously lower than that in 1-month-old to 5-month-old infants, p = 0.0084 The seropositive rate of anti-EV-D68 in 6-month-old to 12-month-old infants was obviously lower than that in 2-year-old to 5-year-old children, p = 0.0097 The seropositive rate of anti-EV-D68 in 6-month-old to 12-month-old infants was obviously lower than that in 6-year-old to 15-year-old children, p = 0.0038 explain differences of NtAb assays using different their mothers. The situation was different for EV71 and strains .To analyze EV-D68 NtAbs for mothers, infants CVA16. The GMTs of anti-EV-D68, anti-EV71, and anti- and children in China, the epidemic strain of China CVA16 were 162.3, 20.0, and 4.6 in neonates, respec- (KP240936, Beijing-R0132) was developed via reverse tively , which might be due to the different sample sizes genetics, and the prototype strain (AY426531, Fermon) and ability to induce antibodies to these viruses. was purchased from ATCC. Our research showed that As the neonates grew (from 0 to 8 months), the NtAbs adult serum had higher NtAb GMTs against the synthetic seroprevalence and GMT decreased gradually. For the 8- strain than against the Fermon strain (p = 0.0378), month-old to 12-month-old infants, the GMT increased although there was a high correlation between the two slowly while the seroprevalence decreased further. This strains (r = 0.444,). Therefore, the synthetic strain was inverse pattern of GMT and seroprevalence was caused by chosen as the challenge strain for the NtAb assay in this the decrease of maternally derived antibodies being par- study. tially offset by the acquired antibodies from infection. Our In addition, a 100% seroprevalence rate was found for research showed that early infection might begin at the Chinese population in 2015, similar to that in Finland 8 months in infants. The same pattern was also reported in 2002 and in Beijing of mainland China in 2004 and in in another EV-D68 NtAb study for 6-month-old to 35- 2009. The GMTs were 166.3, 40–98, and 44.5 in China in month-old infants, who were previously enrolled in a 2015, Finland in 2002, and Beijing in 2004/2009, respec- clinical trial to assess the immunogenicity of an enter- 18, 21 tively . The results demonstrated that adults were ovirus 71 (EV-A71) vaccine in Jiangsu Province (clinical trial No. NCT01508247) and who were included in a two- widely infected with EV-D68 in both China and Finland. The difference in GMTs may be caused by several factors, year follow-up study (January 2012–January 2014) in our including epidemic virus, strength, region and even the laboratory. The lowest GMT was also found among 4- 24, 25 detection method of the virus strain. Therefore, the month-old to 8-month-old infants . standard study of the EV-D68 NtAb assay must be per- The titer distribution of these infants showed that the formed to conﬁrm the comparability of results from dif- lowest NtAb titers were 80% negative and 100% lower ferent regions at different times. than 1:64 in 6-month-old to 12-month-old infants. For Maternal antibodies are the major immune mechanism children 1 year old and older, the NtAb indexes increased that protects neonates against pathogens, but they can as the children grew older. Therefore, infants 6 months 22, 23 also interfere with the effects of immunization . The old and older should be treated as a susceptible popula- weakened rate in neonates was different in different tion. Because of the declining protection from maternally regions because of the different levels of infection rates derived antibodies, the risk of infection increases at this and titers. For example, based on epidemiology and the age with increasing exposure to crowds. A study in weakened rates of maternal antibodies, the World Health mainland China also showed that the infection primarily Organization (WHO) recommends that 2, 9, and occurred among pre-school and school-aged children. In 12 months are the appropriate prime time-points for that study, GMTs were higher in the group aged 6.1–15 immunizations of polio, measles, and hepatitis A virus years old than in the group aged 0.5–6 years . Our study vaccines, respectively. For HEV-A species, the maternal had similar results. Most 2-year-old to 5-year-old children anti-EV71 and anti-CVA16 antibodies were evaluated , received pre-school education in kindergarten and and the EV71 vaccine was approved in China in 2015 showed a high seroprevalence rate of 44% and GMTs of (https://clinicaltrials.gov/). However, there have been no 24.7. This ﬁnding was further supported by the high studies on mothers and their neonates regarding anti-EV- incidence of EV-D68 in pre-school children who were D68 antibodies. In our study, we described the maternally younger than 5 years. Hence, strengthening health derived EV-D68 NtAbs in mothers and neonates. The improvement measures in kindergartens is necessary to GMT results demonstrated that newborn babies could effectively prevent infection. As the children grew, the obtain a high proportion and titer of EV-D68 NtAbs from seroprevalence rate increased to 83% and the GMT Sun et al. Emerging Microbes & Infections (2018) 7:99 Page 6 of 7 increased to 71.9 for 6-year-old to 15-year-old children collected. To further investigate the age-related ser- (school-aged children). Such data demonstrate that oprevalence of NtAbs against EV-D68 in infants and immunity against EV-D68 was relatively high in children young children, serum specimens were also collected at this age, suggesting that EV-D68 infection was highly from different age groups: 1, 2, 3, 4, 5, 6,7, 8, and 9 to prevalent in this age group. The above results show that 11 months, and 1, 2, 3, 4, 5, 6 to 10, and 11 to 15 years, EV-D68 infection was age-dependent and that there respectively. All 405 serum samples were collected ran- domly in Donghai County in August 2010. might be multiple infections during the period of 6-year- old to 15-year-old. To evaluate the seroprevalence of EV-D68 infection In summary, this study demonstrated that a high level of among adults, 50 adult serum samples were collected in maternal antibodies against EV-D68 was common in Henan province, China, in 2015. Informed consent was infants in China. From neonates to juveniles, the maternal obtained from either the participants or their guardians. antibodies decreased, but acquired antibodies from This study was approved by the ethical review committee infection increased gradually. The lowest NtAb level of of the National Institutes for Food and Drug Control. The EV-D68 was found in 6-month-old to 12-month-old serum was separated immediately after collection and infants. Therefore, an early infection might occur in stored at −80 °C. infants older than 8 months. A prime immunization course for vaccination must be established that can help Measurement of neutralizing antibodies protect this susceptible population. Because research on In our previous study, we had established Standard EV-D68 is limited in mainland China, surveillance on EV- Operating Procedures (SOPs) for the determination of 26, 27 D68 must be strengthened, and further study on EV-D68 enterovirus A71 and coxsackievirus A16 NtAbs . EV- virological characteristics, diagnostic method and vaccine D68 NtAbs were detected in this study based on this development are necessary. previously reported method. Brieﬂy, blood samples were diluted to a ratio of 1:8 and then inactivated at 56℃ for Methods and materials 30 min. The inactivated serum was serially diluted from Cells and virus 1:8 to 1:1024 and placed in a 96-microtiter plate. After The EV-D68 prototype strain, Fermon (GenBank 100 TCID of EV-D68 was added to the above plate, the accession no.AY426531), was purchased from ATCC. The mixture was incubated for 2 h at 33 °C in a CO incubator complete genome (GenBank accession no.KP240936, to allow the antibodies to bind to the virus. After the Beijing-R0132) was constructed under the pBluescriptII incubation, the RD cell suspension (2–3× 10 cells/ml) SK-vector (Tai he gene, Beijing, China). 5′UTR and 3′ was added. The cell control, serum control, virus control UTR were added to the T7 promoter of the ribozyme and and virus back titration (if the result of the back drop was the T7 terminator, respectively. The RNA polymerase 32~320 TCID /well, the test was considered a success) gene sequence was constructed under the CMV promoter were included in each plate, and the plate was placed in a (pcDNA6.1 vector). The virus was grown in the mono- CO incubator at 33 °C for 7 days. Cytopathogenicity was layer Rhuman Embryonic Kidney (293T cells) and loaded observed using microscopy to identify the titers of NtAbs, in a 6-well plate (Corning) with Dulbecco’s modiﬁed Eagle deﬁned as the inhibition of 50% CPE. medium (DMEM) and 10% fetal bovine serum (FBS). Lipo 2000 reagent (Invitrogen) was used to co-transfect Statistical analysis 293T cells with a plasmid of the synthetic genome and The titers of the neutralizing antibodies were log- the RNA polymerase gene according to the manu- transformed to calculate the GMTs with 95% conﬁdence facturer’s procedure. The resulting cells were cultured for intervals. All statistical analyses were performed using the 3–4 days before the cell suspensions were harvested. The GraphPad Prism software package. Group comparisons collected cells were frozen and thawed 3 times, and the were performed using the Student’s t test, and a p value < genomes were collected with centrifugation at 12,000 rpm 0.05 was considered statistically signiﬁcant. The titers for 10 min. The resulting viral genomes were mixed with a above 1:1024 were designated as 1:1024. monolayer of human rhabdomyoma cells (RD cells) with DMEM in 2% FBS. The synthetic virus was harvested, and Acknowledgements the tissue culture infective dose of 50% (TCID ) was We thank the Jiangsu Provincial Center for Disease Control and Prevention for their help with sample collection. We thank the Major Special Projects Funding determined. Program (No. 2016ZX09101120) from the Ministry of Science and Technology of the People’s Republic of China for their support. The current study was Human subjects and serum samples sponsored by the Major Special Projects Funding Program (No. 2016ZX09101120) from the Ministry of Science and Technology of the People’s Serum specimens were collected from prenatal women Republic of China. The funders had no role in the study design, data collection and their neonates at birth to determine the titer of NtAbs and analysis, data interpretation, decision to publish, or preparation of the against EV-D68. A total of 20 pairs of serum samples were manuscript. Sun et al. Emerging Microbes & Infections (2018) 7:99 Page 7 of 7 Author details 10. Xiang, Z. et al. Coxsackievirus A21, enterovirus 68, and acute respiratory tract 1 2 National Institute for Food and Drug Control, Beijing, China. Hualan Biological infection, China. Emerg. Infect. Dis. 18, 821–824 (2012). Engineering Inc, Xinxiang, China. Jiangsu Provincial Center for Disease Control 11. Zhang, T. et al. Respiratory infections associated with enterovirus D68 from and Prevention, Nanjing, China 2011 to 2015 in Beijing, China. J. Med Virol. 88,1529–1534 (2016). 12. Xiao, Q. et al. Prevalence and molecular characterizations of enterovirus D68 among children with acute respiratory infection in China between 2012 and Conﬂict of interest 2014. Sci. Rep. 5, 16639 (2015). The authors declare that they have no conﬂict of interest. 13. Lau,S.K.etal. EnterovirusD68 infections associated with severe respiratory illness in elderly patients and emergence of a novel clade in Hong Kong. Sci. Ethics approval Rep. 6, 25147 (2016). This study was approved by the institutional review board of the Jiangsu 14. Huang, Y. P. et al. Molecular and epidemiological study of enterovirus D68 in Provincial Center of Disease Control and Prevention and was conducted in Taiwan. J. Microbiol Immunol. Infect. 50, 411–417 (2017). accordance with the Declaration of Helsinki, Good Clinical Practice, and 15. Zhang, T. et al. Enterovirus D68-associated severe pneumonia, China, 2014. Chinese regulatory requirements. Emerg. Infect. Dis. 21,916–918 (2015). 16. Imamura, T. et al. Enterovirus 68 among children with severe acute respiratory Informed consent infection, the Philippines. Emerg. Infect. Dis. 17,1430–1435 (2011). All guardians of the participants provided written informed consent. 17. Meijer A., et al. Continued seasonal circulation of enterovirus D68 in the Netherlands, 2011-2014. Euro Surveill. 19, 20935 (2014). 18. Xiang, Z. et al. Seroepidemiology of enterovirus D68 infection in China. Emerg. Received: 7 February 2018 Revised: 8 April 2018 Accepted: 29 April 2018 Infect. Dis. 6,e32 (2017). 19. Ji, H. et al. Seroepidemiology of human enterovirus71 and coxsackievirusA16 in Jiangsu province, China. Virol. J. 9, 248 (2012). 20. Zhang, Y. et al. Neutralization of Enterovirus D68 isolated from the 2014 US outbreak by commercial intravenous immune globulin products. J. Clin. Virol. 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