A Spatially Encoded Fluorescent Biosensor for Multiplexed and High-Throughput Detection of HFMD Viruses via Parallel Entropy-Driven Circuits and Combinatorial Photonic Crystal Arrays.

To the Editor: Hand, foot, and mouth disease (HFMD) is a common illness in children and is mainly caused by coxsackievirus A16 (CA16) and enterovirus 71 (EV71). Although its clinical course is usually uneventful and most patients experience a full recovery, serious neurologic complications, including encephalitis, can occur secondarily to HFMD caused by EV71. Such neurological complications occurred during an epidemic in Taiwan in 1998 (1). Encephalitis caused by EV71 is characterized by rhombencephalitis, which is a combination of brainstem encephalitis and cerebellitis. Signs and symptoms of rhombencephalitis are irritability, myoclonus, ataxia, and cranial nerve involvement (1). In contrast to EV71, HFMD caused by CA16 is associated with few neurologic complications with the exception of infrequent aseptic meningitis (2). We report a case of rhombencephalitis that developed in an infant as a complication of HFMD caused by CA16. HFMD was diagnosed in a 23-month-old girl on the basis of high fever (>40°C, 3 d duration), stomatitis, and multiple papules on her palms, soles, and buttocks. Her illness occurred in the summer of 2007, when sentinel surveillance in the region indicated an epidemic of HFMD caused by both CA16 and EV71. She was admitted to our hospital in Fukoka, Japan, on day 4 of illness because of abnormal eye movement, irritability, and inability to stand. She had intermittent to-and-fro, horizontal oscillations of the eyes (ocular flutter). She also had truncal and limb ataxia and myoclonus in her head and limbs. Brain magnetic resonance imaging (MRI) showed T1-low and T2-high bulbopontine and cerebellar lesions around the fourth ventricle (Figure). Peripheral blood showed a mild leukocytosis (13.13 × 109/L) and a C-reactive protein level within reference range (0.9 mg/L). Blood chemistry results were unremarkable. Cerebrospinal fluid (CSF) examination showed mononuclear pleocytosis (74/µL) with normal protein and glucose levels. CA16 was isolated from her stool specimen on day 4 of illness. Based on reverse transcription–PCR, CSF was negative for enterovirus RNA. Figure Axial T2-weighted slice of brain by magnetic resonance imaging, showing hyperintensity lesions in the pons and cerebellum around the fourth ventricle. Without specific treatment, our patient’s fever resolved on day 5 of illness. The myoclonus, ocular flutter, and irritability subsided by day 16, when MRI findings returned to normal. Ataxia disappeared gradually ≈1 month after onset, and no neurologic sequelae occurred. Neutralizing antibody titers against CA16 and EV71 on day 21 of illness were 32 and <8, respectively. Based on the sequence analysis of the partial VP1 region (876 bp), we classified the patient’s CA16 strain phylogenetically as genetic lineage C (3). This lineage was identical to lineage 2 (4), which became the dominant circulating strain in Asia, including Japan, after the late 1990s (98.2% identical to the 1018T/VNM/05 strain isolated in Vietnam in 2005 [GenBank accession no. {type:entrez-nucleotide,attrs:{text:AM292441,term_id:148287177}}AM292441]) (4,5). The patient’s symptoms of irritability, ataxia, myoclonus, and ocular flutter 3 days after the onset of typical HFMD manifestations, along with CSF mononuclear pleocytosis and the lesions around the fourth ventricle shown on MRI, led to the diagnosis of rhombencephalitis associated with HFMD. Virologic examination, including virus isolation and antibody assay, suggested that HFMD was caused by CA16 but not by EV71, although the possibility that CA16 infection was coincidental to the rhombencephalitis could not be excluded. Although rhombencephalitis can be related to various infectious agents (6), HFMD complicated by this condition has been exclusively caused by EV71 (1,7,8). In Japan, CA16 and EV71 are consistently the 2 major causative agents of HFMD (9). EV71 infection is much more frequently associated with serious neurologic complications and fatalities than is CA16 (2). Since 1997, several HFMD outbreaks with multiple cases of severe neurologic pathologies have occurred in the Asia-Pacific region including Malaysia, Taiwan, and Western Australia (1,7,8). These complications were associated exclusively with EV71. Why rhombencephalitis developed in our patient with CA16-related HFMD is unclear. One possibility is that the CA16 strain might have acquired neurovirulence by genetic recombination with EV71; phylogenetic evidence supports the possible occurrence of intertypic recombination involving EV71 and CA16 (10). Through phylogenetic analysis of the VP1 sequences, we classified the CA16 strain isolated from the patient’s stool phylogenetically as genetic lineage C (3), a lineage which was identical to lineage 2 (4). Genetic recombinations among enteroviruses occur mainly in noncapsid regions (10). We did not conduct phylogenetic analysis of the noncapsid regions of the patient’s CA16 strain because sequence data on the regions were very limited. Besides the viral factors, host factors, such as immune status and environmental factors, could confer susceptibility to neurologic complications of enteroviral infections. Rhomobencephalitis associated with HFMD developed in this patient and was caused by CA16. Therefore, neurologic complications, including rhombencephalitis, should be considered even when CA16 is the prevalent virus causing HFMD.

Objective To explore the epidemiological and pathogenic characteristics of the hand, foot and mouth disease (HFMD) in Yangzhou city from 2012 to 2016, so as to provide evidences for prevention and control of the disease. Methods Information of the cases was collected by the Chinese national HFMD surveillance system from 2012 to 2016, and was analyzed by descriptive epidemiological methods. Meanwhile, samples of HFMD cases were collected for viral nucleic acid detection from Sentinel hospitals. Results 35 720 cases of HFMD were reported in Yangzhou from 2012 to 2016 and the average annual incidence was 155.30/ 100 000 (35 720/23 000 000), the male-to-female ratio of ordinary cases was 1.54 :1 and the percentage of severe cases was 1.60‰ (56/35 720). There were 2 fatal cases. Children of ≤5 years old were the most susceptible to HFMD (91.83%, 32 800/35 720). Most of the patients were the scatter children (60.18%, 21 498/35 720) and the children in kindergartens and nursery schools (35.93%, 12 834/35 720). There were two peaks in May-July and October-December. The incidences were high in 2012, 2014 and 2016, and were low in 2013 and 2015. Coxsackievirus A16 (Cox A16) (33.28%, 432/1 298) and enterovirus 71 (EV71) (28.58%, 371/1 298) were the major pathogens for HFMD in Yangzhou from 2012 to 2016. EV71 accounted for most of the severe cases (76.79%, 43/56). The positive rate of EV71 among severe cases (1.05%, 13/1 242) was significantly higher than that of ordinary cases (x2=66.62, P<0.01). Conclusions There were significant seasonal and gender, age, occupation and regional differences in the occurrence of HFMD in Yangzhou. The cases showed an alternating trend of high and low incidence peaks in different years. Meanwhile, Cox A16 and EV71 were the major pathogens for HFMD, and EV71 was more common among severe cases. Epidemiology and etiological surveillance of HFMD should be enhanced according to the rules above, especially for the monitoring of EV71. Key words: Hand, foot, and mouth disease; Epidemiology; Severe cases; Pathogenic features

To investigate the etiology of hand, foot and mouth disease (HFMD) in Beijing during 2013, and study the clinical characteristics of HFMD caused by the main serotypes of enterovirus in the study. Clinical data and 128 stool samples were collected from 128 hospitalized children with HFMD in Beijing Ditan Hospital during 2013. One step RT-PCR method was used for enterovirus genotyping to investigate the etiology of HFMD. Clinical characteristics of HFMD caused by the main serotypes of enterovirus were analyzed. And VP1 segments of the main virus were amplified to construct phylogenetic tree for the phylogenetic analysis. A total of 128 hospitalized children with HFMD were included. HFMD was more likely developed in children under 2 years of age (81.6%, 102/125); 11 different enteroviruses were genotyped, with a total enterovirus positive rate of 76.6% (98/128); the positive rate of coxsackievirus A6 (CA6), 43.0% ( 55/128), was the highest, followed by enterovirus 71 (EV71), accounting for 14.8% (19/128). HFMD caused by CA6 was atypical, the rashes of which involved the perioral, trunk, limbs, face and neck (47%, 26/55), besides the common parts. Of the 55 cases caused by CA6, 6 children had clinical manifestations of nervous system involvement, one of whom even displayed type 2 respiratory failure. Mental status change more likely to occur in EV71-infected children than in CA6-infected ones (42% (8/19) vs. 11% (6/55) (χ(2)=7.041, P=0.008)); 13 children displayed onychomadesis, including 12 CA6 cases (23%, 12/53) and 1 CA10 cases (17%, 1/6), in the convalescence of hand, foot and mouth disease, and the correlation between onychomadesis and CA6 infection was significant (χ(2)=9.297, P=0.002). Phylogenetic analysis of 33 CA6 VP1 showed that the CA6 isolates of this study were highly similar to that of Taiwan and the nucleotide similarity was 95.91%-98.89%. CA6 was the major pathogen of hospitalized children with hand, foot and mouth disease in Beijing during 2013, followed by EV71. The rashes caused by CA6 involved a wide range of skin sites and patients with CA6 infection displayed manifestations of neurological involvement or pulmonary edema similar to EV71 infection. Mental status change more likely occurred in EV71-infected children when neurological system was involved..

Analysis and solution of false-positives when testing CVA16 sera using an antibody assay against the EV71 virus

Objective To investigate the etiology of hand, foot and mouth disease (HFMD) and to analyze the genetic characteristics of three prevalent enteroviruses in Qingdao in 2015. Methods Citywide surveillance data in 2015 were used to analyze the epidemiological characteristics of HFMD in Qingdao. RNA samples extracted from throat swab of HFMD cases were examined for general enteroviruses (EVs) such as enterovirus 71(EV71), coxsackievirus A16 (CA16) and CA6 by real-time RT-PCR. Full-length VP1 genes of EV-positive specimens were amplified and sequenced. Sequencing results were phylogenetically analyzed using MEGA 7.0 software package. Results A total of 804 patients with HFMD were identified from 1 176 patients in 2015. CA6 (41.4%), EV71 (31.6%) and CA16 (15.3%) were the predominant EVs causing HFMD. Children 5 years of age and under accounted for 80.3% of the 804 HFMD cases. CA6 was responsible for 48.9% of HFMD cases in children under 3 years old. The epidemic subtypes of EV71 and CA16 in Qingdao were C4a and B1b, respectively. Twenty-eight randomly selected CA6 strains were all classified into type A genome. Conclusion CA6, EV71 and CA16 were the prevalent pathogens causing HFMD in Qingdao in 2015. CA6 became the most predominant pathogen, mainly targeting children under 3 years old. C4a remained the prevailing subtype of EV71, while different from the co-prevalence of B1a and B1b subtypes in the past, B1b became the predominant subtype of CA16. CA6 strains circulating in Qingdao in 2015 mainly belonged to type A genome and evolved into multiple smaller branches. However, CA6 strains isolated in Qingdao in 2015 and 2013 located in different branches. Key words: Hand, foot and mouth disease; Enterovirus; Phylogenetic analysis; VP1 gene

Objective To analyze the etiologic spectrum of hand, foot and mouth disease （HFMD） and the molecular characteristics of coxsackievirus A10 （CVA10） strains isolated in Qingdao from year 2010 to 2012.Methods Throat swab specimens were collected from patients with HFMD to detect to-tal enteroviruses （ EVs） , EV71 and CVA16 strains by multiplex real time RT-PCR.The EV-positive speci-mens were further detected by a semi-nested RT-PCR to amplify the sequence of viral genes encoding VP1. The serotypes of EVs were identified based on the sequences of genes encoding VP1.The full-length gene se-quences encoding VP1 of CVA10 isolates were amplified and sequenced. The phylogenetic analysis was con-ducted by using MEGA5.0 software package.Results A total of 1919 outpatients with mild HFMD and 1336inpatients with serious HFMD were recruited in this study .CVA16 strains were the predominant patho-gen for outpatients in year 2010 （ prevalence rate of 53%） and 2012 （ prevalence rate of 73%） .EV71 strains were the predominant pathogen for outpatients in year 2011 （ prevalence rate of 78%） and inpatients in year 2010 （ prevalence rate of 70%） and 2011 （ prevalence rate of 86%） . Some serotypes other than CVA16 or EV71 were the predominant pathogens for inpatients in year 2012 （ prevalence rate 44%） . CVA10 strains were identified in 12 patients with HFMD in year 2010and 17 patients with HFMD in 2012. The full-length gene sequences encoding VP1 of 23 CVA10 isolates were successfully amplified and se-quenced.The phylogenetic analysis showed that the 23 CVA10 isolates all belonged to genotype C and could be further divided into six clades.The genes encoding VP1 of the 23 CVA10 strains shared 97.3% to 1000.%homologies in amino acid sequences.The CVA10 isolates were also similar to their counterparts isolated from other regions during the same period.Conclusion CVA16 and EV71 strains were the preva-lent pathogens of HFMD in Qingdao from year 2010 to 2012, co-circulated with some other serotypes of EVs, especially CVA10 strains.The CVA10 strains belonged to genotype C and shared high homologies among them and their counterparts circulated in other regions during the same period. Key words: Hand, foot and mouth disease; Enterovirus; Coxsackievims AIO; Phylogeneticanalysis

Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD) in children. The host defense mechanisms against CVA16 infection remain almost entirely unknown. Unlike previous observations with enterovirus 71 (EV71) infection, here we show that gamma interferon (IFN-γ) or invariant NK T cell deficiency does not affect disease development or the survival of CVA16-infected mice. In contrast, type I interferon receptor deficiency resulted in the development of more severe disease in mice, and the mice had a lower survival rate than wild-type mice. Similarly, a deficiency of Toll-like receptor 3 (TLR3) and TRIF, but not other pattern recognition receptors, led to the decreased survival of CVA16-infected mice. TLR3-TRIF signaling was indispensable for the induction of type I interferons during CVA16 infection in mice and protected young mice from disease caused by the infection. In particular, TRIF-mediated immunity was critical for preventing CVA16 replication in the neuronal system before disease occurred. IFN-β treatment was also found to compensate for TRIF deficiency in mice and decreased the disease severity in and mortality of CVA16-infected mice. Altogether, type I interferons induced by TLR3-TRIF signaling mediate protective immunity against CVA16 infection. These findings may shed light on therapeutic strategies to combat HFMD caused by CVA16 infection. IMPORTANCE Hand, foot, and mouth disease (HFMD) is a major threat to public health in the Asia-Pacific region. Both CVA16 and EV71 are major pathogens that are responsible for HFMD. The majority of research efforts have focused on the more virulent EV71, but little has been done with CVA16. Thus far, host immune responses to CVA16 infection have not yet been elucidated. The present study discovered an initial molecular mechanism underlying host protective immunity against CVA16 infection, providing the first explanation for why CVA16 and EV71 cause different clinical outcomes upon infection of humans. Therefore, different therapeutic strategies should be developed to treat HFMD cases caused by these two viruses.

Objective To discuss the change trends of pathogen of severe hand, foot and mouth disease(HFMD) in Chaoshan area during 2011 to 2015. Methods All 1 410 throat swabs of cases who were diagnosed as HFMD were collected from children hospitalized in our hospital during May 2011 to August 2015.Enterovirus were detected by nest RT-PCR, and the results of these positive cases diagnosed as severe HFMD were analyzed. Results (1) There were 216 positive cases(67.29%, 216/321) diagnosed as severe HFMD, including 53.70%(116/216)enterovirus 71(EV71), 19.91%(43/216)coxsackievirus A16(CA16), 12.04%(26/216)CA6, 8.80%(19/216)CA10, 3.24%(7/216)CA4, 0.93%(2/216)coxsackievirus B5, 0.46%(1/216)enteric cytopathogenic human orphan virus and 0.93%(2/216)unclassified samples were unclassified to species.(2)Five cases of critical HFMD were all caused by EV71.(3)The EV71 positive samples were given priority to severe cases(51.79%, 116/224)and the non EV71 positive samples were given priority to mild cases(82.08%, 458/558), the difference was statistically significant(χ2=91.68, P<0.001). (4)The change trends of severe HFMD year by year were consistent with the change trends of EV71 composition, and were highly correlated(Rs=0.9, P=0.037). (5) Severe HFMD caused by non EV71 virus gradually increased. Conclusion Severe HFMD in Chaoshan area during 2011 to 2015 were mainly caused by EV71, non EV71 viruses including CA16, CA6, CA10, CA4, coxsackievirus B5, enteric cytopathogenic human orphan virus 6 could also develop to severe HFMD.The composition ratio of severe HFMD increased accordingly in the year of EV71 as the dominant pathogen.The proportion of severe HFMD caused by non EV71 virus gradually increased after 2013 year. Key words: Severe hand, foot and mouth disease; Pathogen; Enterovirus 71; Non enterovirus 71 virus

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) have caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and young children. This disease has become a serious public health problem, as no vaccines or antiviral drugs have been approved for EV71 and CA16 infections. In this study, we compared four monovalent vaccines, including formalin-inactivated EV71 virus (iEV71), EV71 virus-like particles (VLPs) (vEV71), formalin-inactivated CVA16 virus (iCVA16) and CVA16 VLPs (vCVA16), along with two bivalent vaccines, including equivalent doses of formalin-inactivated EV71+CVA16 virus (iEV71+iCVA16) and EV71+CVA16 VLPs (vEV71+vCVA16). The IgG titers and neutralization antibodies titers demonstrated that there are no immune interference exists between the two immunogens of EV71 and CVA16. IgG subclass isotyping revealed that IgG1 and IgG2b were induced primarily in all vaccine groups. Furthermore, cross-neutralization antibodies were elicited in mouse sera against other sub-genotypes of EV71 and CVA16. In vivo challenge experiments showed that the immune sera from vaccinated animals could confer passive protection to newborn mice against lethal challenge with 14 LD50 of EV71 and 50 LD50 of CVA16. Our results indicated that bivalent vaccination is promising for HFMD vaccine development. With the advantage of having a better safety profile than inactivated virus vaccines, VLPs should be used to combine both EV71 and CVA16 antigens as a candidate vaccine for prevention of HFMD virus transmission.

To understand etiological types and distribution features of hand-foot-mouth disease (HFMD) in Henan province between 2008 and 2011. A total of 30 486 specimens of feces, rectal swabs or throat swabs from HFMD patients were collected by each Municipal CDC in Henan from 2008 to 2011. The enterovirus 71 (EV71), coxsackie virus A16 (CA16) and other enterovirus (EV) were detected by RT-PCR or real time RT-PCR. The VP1 gene of EV71 was amplified and the sequences were analyzed by bioinformatics software. A genetic evolution tree of the sequence was constructed as well. The positive rates of EV71, CA16 and other EV were 62.70% (11 209/17 876), 12.03% (2150/17 876), 25.27% (4517/17 876) in 17 876 laboratory diagnosed cases, respectively. The differences were statistically significant (χ(2) = 157.17, P < 0.05). The positive rates of EV71, CA16 and other EV were 63.40% (7370/11 624), 11.58% (1346/11 624) and 25.02% (2908/11 624) in male patients and 61.40% (3839/6252), 12.86% (804/6252) and 25.74% (1609/6252) in female patients, respectively. The differences were statistically significant (χ(2) = 4.06, P < 0.05). The children under 5 years old were high-risk population of HFMD, accounting to 97.67% (17 459/17 876) of the laboratory-diagnosed patients.86.92% (15 537/17 876) cases were children between 1 to 3 years old. Constituent ratio of EV71 changed seasonally during a year, there was a high infection ratio of EV71 between April and June, especially in May, the infection ratio reached 69.34% (2384/3438). The positive rates of EV71, CA16 and other EV were 82.48% (5715/6929), 1.76% (122/6929) and 15.76% (1092/6929) among the 6929 laboratory-diagnosed severe cases, respectively. The positive rates of EV71 was higher than CA16 and other EV (χ(2) = 9259.17, 6170.81, P < 0.05, respectively). There were 117 deaths because of severe HFMD, 55 (47.01%) of which were laboratory confirmed. 50 death cases were infected by EV71, and according to the genetic evolution analysis, the VP1 gene of EV71 strain was belonged to subtype C4 of gene C. The EV71 and CA16 were the main pathogens which caused HFMD in Henan province, and EV71 virus was the dominant strain, belonging to C4 subtype of gene C.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by several genotypes of human enterovirus species A and represent a significant global public health concern. Enterovirus 71 (EV71) infections cause a deadly neurological disease for which no specific anti-viral drugs or vaccines has received regulatory approval. Published reports have described a dramatically increased incidence of coxsackievirus A6 (CV-A6) infections associated with HFMD since their occurrence in 2008 in Finland. The aim of this study was to evaluate the burden of human enteroviruses associated to HFMD and herpangina in patients and establish epidemiological profiles of these viruses in Thailand in 2012. Detection and genotype determination of enteroviruses were accomplished by reverse transcription-polymerase chain reaction and sequencing of the VP1 region. Enterovirus-positive samples were differentiated into 17 genotypes (CV-A 4, A5, A6, A8, A9, A10, A12, A16, A21, B1, B2, B4, B5, echovirus 7, 16, 25 and EV71). The result showed CV-A6 (33.5%), followed by CV-A 16 (9.4%) and EV71 (8.8%) as the most frequent genotypes in HFMD, CV-A 8 (19.3%) in herpangina and CAV6 (1.5%) in influenza like illness. Enterovirus infections were most prevalent during July with 34.4% in HFMD, 39.8% in herpangina and 1.6% in ILI. The higher enterovirus infection associated with HFMD and herpangina occurred in infants over one year-old. This represents the first report describing the circulation of multiple enteroviruses in Thailand. Furthermore, multiplex real-time PCR using TaqMan probes for broad detection of EVs and differentiation of EV71, CV-A6 and CV-A16 were developed and validated. The multiplex real-time PCR system which was run in two separate tubes was capable of screening and specific detection of the three selected enteroviruses, without cross-reactions with the other examined RNA viruses. The detection limit of the assays was 10 copies/µl for EV71, CV-A6, CV-A16 and panenterovirus. The overall diagnostic sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the multiplex rRT-PCR assay were greater than those of conventional RT-PCRs. The multiplex rRT-PCR assays provide the highly sensitive detection and rapid simultaneous typing of EV71, CV-A6 and CV-A16 thus rendering it feasible and attractive for large scale surveillance of EVs associated with HFMD outbreaks. Moreover, this study also investigated the relationship between these disease outbreaks with the evolutionary dynamics of CV-A6 and the appearance of novel recombinant forms (RFs) of the virus. Based on the analysis of the VP1 gene, the substitution rates of CV-A6 was estimated at 8.1 × 10-3 substitutions/site/year. There was an increasing likelihood between CV-A6 genome recombination and VP1 sequence divergence, with an estimated half-life of the RFs of 3.1 years. Bayesian phylogenetic analysis of the data showed that recently occurring recombination groups (RF-E, -F, -H, -J and -K) shared a common ancestor (RF-A). Recombination breakpoints were frequently observed between the 2A-2C gene and the 5´ untranslated region. This study revealed the potential for new CV-A6 variants to emerge and potentially modify disease outcomes of this major etiologic agent for HFMD affecting children worldwide.

Objective To investigate the etiology spectrum of hand foot and mouth disease (HFMD) and to analyze the molecular characteristics of Coxsackievirus A10 and A6 in Qingdao in 2014. Methods Throat swabs of HFMD cases were tested for total enteroviruses (EVs), EV-A71, CV-A16, CV-A10 and CV-A6 by multiplex real time RT-PCR. Other EV serotypes were identified through the sequences of partial VP1 gene. The full-length of VP1 gene of CV-A10 and CV-A6 were amplified and sequenced. The sequences were phylogenetically analyzed using MEGA 5.0 software package. Results A total of 1727 HFMD patients were detected in 2014 and 11serotypes of enteroviruses were identified. EV-A71(38.0%, 410/1078), CV-A16(28.8%, 311/1078), CV-A10(14.1%, 152/1078)and CV-A6(3.2%, 34/1078)were the most dominant pathogen in 2014 in Qingdao. Proportions of CV-A10 in enterovirus infected children varied dramatically in different ages(χ2=15.19, P=0.001), showing a downtrend with age. Proportion of CV-A10 in inpatients was much higher than that in outpatients(χ2=49.1, P <0.001), while 60% of those inpatients showed nervous system damage symptoms, with pathological reflex or disappearance of physiological reflex. Phylogenetic analysis of complete VP1 sequences showed that all CV-A10 strains identied in this study belonged to genotype C, 71.7% of which located in branch C5; all CV-A6 strains belonged to genotype D while 83.3% of which located in branch D5. Conclusions EV-A71, CV-A16, CV-A10 and CV-A6 were the prevalent pathogens of HFMD in Qingdao in 2014. CV-A10 was more frequently detected in lower age group with HFMD, which can cause damage to nervous system. Strains of branch C5 in genotype C were the dominant strains of CV-A10 circulated in Qingdao in 2014 while strains of branch D5 in genotype D were the major strains of CV-A6. Key words: Hand, foot and mouth disease; Coxsackievirus A10; Coxsackievirus A6; Phylogenetic analysis

Aside from enterovirus 71 (EV71) and coxsackie virus A16 (CV-A16), viruses that are known to cause hand-foot-and-mouth disease (HFMD), epidemiological profiles of other enteroviruses that induce HFMD are limited. We collected 9949 laboratory surveillance HFMD cases and 1230 serum samples from infants and children in Shanghai from 2012–2016. Since 2013, CV-A6 has displaced EV71 and CV-A16 to become the predominant serotype. Interestingly, novel epidemiological patterns in EV71 and CV-A16 infections were observed, with one large peak in both 2012 and 2014, followed by two smaller peaks in the respective following years (2013 and 2015). Through sequencing, we found that C4a, B1b, D-Cluster-1 and B constituted the major subgenotypes of EV71, CV-A16, CV-A6 and CV-A10, respectively. Among healthy individuals, 50.49% and 54.23% had positive neutralising antibodies (NtAbs) against EV71 and CV-A16, respectively, indicating that EV71 and CV-A16 silent infections were common. These populations may be an important potential source of infection. The overall seropositive rate of EV71 NtAbs showed a fluctuating, markedly downward trend, indicating the potential risk of a future EV71 epidemic. High CV-A16 NtAb seroprevalence corroborated a documented CV-A16 ‘silent’ epidemic. Children aged 1–5 years had the lowest EV71 NtAb seropositive rate, whereas those aged 1–2 years exhibited the lowest CV-A16 NtAb seropositive rate. This is the first comprehensive investigation of the epidemiology and aetiology, as well as the seroprevalence, of HFMD in Shanghai between 2012 and 2016. This study provides the latest insights into developing a more efficient HMFD vaccination programme.

Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.

To understand the epidemiological profiles of hand, foot and mouth disease (HFMD) and the major enteroviruses causing the epidemics of HFMD in Shanghai from 2010 to 2014. The city-wide surveillance data between 2010 and 2014 were used to analyze the epidemiologic characteristics of the HFMD outbreaks in Shanghai. The annual incidence of HFMD was estimated based on the 2010 Shanghai Census data. From 2010 to 2014, the reported HFMD cases were 41 080, 37 323, 51 172, 42 198, and 65 018, respectively; the severe cases (case-severity ratio) were 469 (1.14%), 456 (1.22%), 318 (0.62%), 104 (0.25%) and 248 (0.38%), respectively. Based on Shanghai census data by the end of 2010, the attack rates of HFMD in Shanghai were 0.16%-0.28% in the entire population. In terms of the proportion of HFMD cases and severe cases in the specific population, male accounted for 59.62%-61.48% and 62.26%-73.08%, migrant population accounted for 51.86%-62.40% and 72.01%-80.38%; children aged 1.0-1.9 years comprised the highest proportion, up to 22.70%-27.00% and 32.08%-36.40%. HFMD peaked from April to July, in parallel with the peak circulation of enterovirus (EV) 71, and a small peak usually occurred in autumn and winter. All the critically severe and fatal cases were caused by EV71. The detection rates of EV71 and Coxsackievirus A (CA) 16 were 73.08%-88.09% and 1.12%-2.90% in severe HFMD cases, 19.75%-48.74% and 2.02%-23.69% in uncomplicated inpatients, and 16.78%-40.08% and 8.36%-33.39% in mild community cases, respectively. The detection rates of CA6 and CA10 in the mild community cases in 2014 were 18.38% and 1.43%, respectively. In 2013 non-EV71 and non-CA16 enteroviruses comprised 74.86% in the community cases. The annual HFMD outbreaks occurred in Shanghai during 2010-2014. Children under 5 years of age, migrant population and male were the major susceptible population. EV71 and CA16 were the predominant pathogens causing the epidemics of HFMD except in 2013, and CA6 was prevalent in the community cases in 2014. The major peak season of HFMD usually overlapped with the peak of EV71 circulation and the majority of severe HFMD cases were associated with EV71 infection.