Coxsackievirus B: The important agent of hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a common pediatric infectious illness caused by enteroviruses (EVs). EV-A serotypes are the main pathogens associated with HFMD. In this study, 213 stool samples from 213 children with severe HFMD in Yunnan, China in 2013, 2015, and 2016 were further analyzed retrospectively for EV-B infection. A total of 70.0% of the specimens tested positive for EV.20 EV serotypes were detected. The predominant serotype was enterovirus A71 (EV-A71, 27.7%), followed by coxsackievirus B4 (CV-B4, 16.4%), CV-A16 (9.9%), CV-B5 (6.6%), and Echovirus 9 (E-9,4.7%). EV-A and EV-B accounted for 45.1% and 41.3%, respectively. Among the positive specimens, 28.6% were CV-Bs. Co-infection was present in 19.3% of these cases. In the study, CV-B5 and the majority of CV-B4 isolates belonged to genotypes VI and C3, respectively. This result indicates that EV-B, especially CV-Bs, might be the important agents associated with HFMD and this knowledge will contribute to the prevention and treatment of the disease.

Molecular characterization of a novel clade echovirus 3 isolated from patients with hand-foot-and-mouth disease in southwest China.

Echovirus 3 (E3) belongs to the species Enterovirus B. Currently, three nearly whole-genome sequences of E3 are available in GenBank in China. In this study, we determined the whole genomic sequences of six E3 strains isolated from the stools of patients with hand-foot-and-mouth disease in Southwest China in 2022. Their nucleotide and amino acid sequences shared 82.1%-86.4% and 96.6%-97.2% identity with the prototype Morrisey strain, respectively, and showed 87.1% and 97.2% mutual identity. The six E3 strains are not clustered with other Chinese strains and formed a novel subgenotype (C6) with the recent American and British strains. Recombination analyses revealed that intertype recombination had occurred in the 2 C and 3D regions of the six E3 strains with coxsackieviruses B5 and B4, respectively. This study augments the nearly whole-genome sequences of E3 in the GenBank database and extends the molecular characterization of this virus in China.

Complete genome analysis of echovirus 30 strains isolated from hand-foot-and-mouth disease in Yunnan province, China.

BACKGROUND: Echovirus 30 is prone to cause hand-foot-and-mouth disease in infants and children. However, molecular epidemiologic information on the spread of E30 in southwestern China remains limited. In this study, we determined and analyzed the whole genomic sequences of E30 strains isolated from the stools of patients with hand-foot-and-mouth disease in Yunnan Province, China, in 2019. METHODS: E30 isolates were obtained from fecal samples of HFMD patients. The whole genomes were sequenced by segmented PCR and analyzed for phylogeny, mutation and recombination. MEGA and DNAStar were used to align the present isolates with the reference strains. The VP1 sequence of the isolates were analyzed for selection pressure using datamonkey server. RESULTS: The complete genome sequences of four E30 were obtained from this virus isolation. Significant homologous recombination signals in the P2-3'UTR region were found in all four isolates with other serotypes. Phylogenetic analysis showed that the four E30 isolates belonged to lineage H. Comparison of the VP1 sequences of these four isolates with other E30 reference strains using three selection pressure analysis models FUBAR, FEL, and MEME, revealed a positive selection site at 133rd position. CONCLUSIONS: This study extends the whole genome sequence of E30 in GenBank, in which mutations and recombinations have driven the evolution of E30 and further improved and enriched the genetic characteristics of E30, providing fundamental data for the prevention and control of diseases caused by E30. Furthermore, we demonstrated the value of continuous and extensive surveillance of enterovirus serotypes other than the major HFMD-causing viruses.

Isolation and characterization of a novel clade of coxsackievirus B2 associated with hand, foot, and mouth disease in Southwest China.

Coxsackievirus B2 (CVB2) is an enterovirus B (EV-B) species and can cause aseptic meningitis, myocarditis and hand, foot, and mouth disease (HFMD). We characterized a novel CVB2 (YN31V3) associated with HFMD in Yunnan, Southwest China, in 2019. Although YN31V3 and other Mainland China epidemic strains mainly belonged to genotype C, YN31V3 formed an independent branch. The genome sequence of the strain YN31V3 from this study showed a 12.91% nucleotide difference to its closest strain RW41-2/YN/CHN/2012. Recombination analyses showed that the newly isolated YN31V3 was probably a recombinant, which was closely related to CVB2 strains in the genomic P1 region and other EV-B strains in the P2 and P3 regions, respectively. YN31V3 strain had a temperature-sensitive phenotype. The challenge of suckling BALB/c mice with YN31V3 could cause symptoms of disease and severe pathological lesions.

Characterization of coxsackievirus A10 strains isolated from children with hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a contagious disease that threatens the health of children under 5 years of age. Coxsackievirus A10 (CV-A10) is one of the main pathogens of HFMD. Currently, preventive vaccines and specific therapeutic drugs are not available for CV-A10. In this study, a total of 327 stool specimens were collected from pediatric patients from 2009 to 2017 during HFMD surveillance, among which 14 CV-A10 strains could only be isolated from rhabdomyosarcoma cells, but not from KMB17 and Vero cells. Through adaptive culture, 2 and 11 CV-A10 strains were recovered from Vero and KMB17 cell cultures, respectively. The growth of CV-A10 strains in Vero cells was better than that in KMB17 cells. The 14 CV-A10 strains belonged to the F genotype, and the nucleotides and amino acids of their complete genomes shared 92.6%-96.3% and 98.4%-98.9% identities, respectively. The different CV-A10 strains exhibited varying virulence in vivo, but had similar effects on tissue injury, with the hind limb muscles, kidneys, and lungs being severely affected. Additionally, the hind limb muscles had the highest viral loads. CV-A10 was found to exhibit a strong tropism to muscle tissue. The results of this study are critical to developing vaccines against CV-A10 infections.

Co-infection and enterovirus B: post EV-A71 mass vaccination scenario in China.

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common child infectious disease caused by more than 20 enterovirus (EV) serotypes. In recent years, enterovirus A71 (EV-A71) has been replaced by Coxsackievirus A6 (CV-A6) to become the predominant serotype. Multiple EV serotypes co-circulate in HFMD epidemics, and this study aimed to investigate the etiological epidemic characteristics of an HFMD outbreak in Kunming, China in 2019. METHODS: The clinical samples of 459 EV-associated HFMD patients in 2019 were used to amplify the VP1 gene region by the three sets of primers and identify serotypes using the molecular biology method. Phylogenetic analyses were performed based on the VP1 gene. RESULTS: Three hundred and forty-eight cases out of 459 HFMD patients were confirmed as EV infection. Of these 191 (41.61%) were single EV infections and 34.20% had co-infections. The EVs were assigned to 18 EV serotypes, of which CV-A6 was predominant (11.33%), followed by CV-B1 (8.93%), CV-A4 (5.23%), CV-A9 (4.58%), CV-A 16 (3.49%) and CV-A10 and CVA5 both 1.96%. Co-infection of CV-A6 with other EVs was present in 15.25% of these cases, followed by co-infection with CV-A16 and other EVs. The VP1 sequences used in the phylogenetic analyses showed that the CV-A6, CV-B1 and CV-A4 sequences belonged to the sub-genogroup D3 and genogroups F and E, respectively. CONCLUSION: Co-circulation and co-infection of multiple serotypes were the etiological characteristic of the HFMD epidemic in Kunming China in 2019 with CV-A-6, CV-B1 and CV-A4 as the predominant serotypes. This is the first report of CV-B1 as a predominant serotype in China and may provide valuable information for the diagnosis, prevention and control of HFMD.

A 5-year molecular epidemiology survey of human enterovirus 71 before vaccine application in Yunnan Province, China.

Enterovirus A71 (EV-A71) infection is known to cause hand, foot, and mouth disease (HFMD). Last year, an inactivated EV-A71 whole virus vaccine was used to prevent this disease in Yunnan, China. To obtain a viral genetic background for evaluating vaccine protection and monitor the adaptive evolution of the virus after the vaccination, a 5-year molecular epidemiology survey was performed before the vaccination. Twenty-six EV-A71 strains were separated from 561 stool specimens of patients with serious HFMD. The whole-genomic sequences of these strains were sequenced. Phylogenetic trees were constructed, and the mutation spectra were analyzed based on these viral sequences. There was no obvious mutation for the circular EV-A71 strains of the same year. Pathogenic EV-A71 strains may arise from a "subgroup" randomly each year. Whole-genomic analyses showed that a hotspot nonsynonymous substitution potentially affecting the immunogenicity of vaccines was found in the 2A gene, but not in genes of the viral capsid proteins, and the genetic diversity of whole viral genomes associated with the incidence of HFMD. Therefore, it will be valuable to monitor the genome-wide changes of EV-A71 to detect the adaptive mutations affecting immunogenicity or perform investigations using genetic diversity as a parameter.

Molecular characterization of Coxsackievirus A16 strains isolated from children with severe hand, foot, and mouth disease in Yunnan, Southwest China, during 2009-2015.

Coxsackievirus A16 (CV-A16) commonly causes mild symptoms, but severe diseases, such as aseptic meningitis, encephalitis, and even fatal cases, have been reported. Thirteen CV-A16 strains were isolated from patients with severe hand, foot, and mouth disease in Yunnan, Southwest China, from 2009 to 2015. Subgenotype B1a and B1b of CV-A16 were predominantly circulating the region with B1b the predominant strain in recent years. The mean rate of nucleotide substitution based on the VP1 gene sequence was 4.545 × 10 -3 substitution per site per year from 2009 to 2015. These results may help in understanding the genetic diversity of CV-A16 and develop a CV-A16 vaccine.

Ongoing change of severe hand, foot, and mouth disease pathogens in Yunnan, China, 2012 to 2016.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enteroviruses (EVs). In this study, a total of 341 children with serious HFMD were admitted to a pediatric hospital in Yunnan, China in 2012 to 2016. EVs were detected in 283 specimens (83.0%) and were assigned to 17 EV types. Enterovirus A71 (EV-A71) was predominant, accounting for 41.6%, and was followed by coxsackievirus A16 (CV-A16; 18.8%), CV-A6 (9.1%), CV-A10 and E-9 (2.9%), CV-B5 (1.8%), CV-A9 (1.2%), E-30 (0.9%), E-18, CV-A4, C-B3, and CV-A2 (0.6%) and other EV types such as CV-A8, CV-A14, E-14, E-11, and CV-B4 (0.3%). All of the EV-A71 isolates belonged to C4a; the CV-A16 belonged to B1b or B1a, although the B1b strains were predominant; and CV-A6 belonged to D3. In 2012 to 2014, E-9 was the third most frequent serotype (8.2%, 5.0%, and 6.5%, respectively). E-9 was not detected in 2015 and 2016. CV-A6 was not detected in 2012 but was the second most frequent serotype (25.3%) in 2015. Active etiological surveillance of HFMD makes it necessary to be aware of these emerging pathogens.

Identification of a new recombinant strain of echovirus 33 from children with hand, foot, and mouth disease complicated by meningitis in Yunnan, China.

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common childhood disease, which is usually caused by enterovirus A (EV-A) serotypes. Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the main etiologic agents. Multiple serotypes of enterovirus B serotypes (EV-B) have been detected in outbreaks or sporadic cases of HFMD. RESULTS: During HFMD surveillance in Yunnan, China in 2013, two echovirus 33 (E-33) isolates were recovered in cell culture and typed by molecular methods from the cerebrospinal fluid (CSF) and feces of two sporadic cases of HFMD complicated by meningitis. Sequence analysis indicated that the study isolates, YNK35 and YNA12, formed an independent branch, and belonged to E-33 genotype H. Recombination analysis indicated multiple recombination events in the genomic sequence of isolate YNK35. The recombination mainly occurred in the non-structural coding region of P2 and P3, and involved intra-species recombination of species B. CONCLUSION: In this study, the complete sequences of two E-33 isolates were determined. This is the first report of severe HFMD associated with E-33 in Yunnan China, and it enriches the number of full-length genome sequences of E-33 in the GenBank database.

A comparative study of multiple clinical enterovirus 71 isolates and evaluation of cross protection of inactivated vaccine strain FY-23 K-B in vitro.

BACKGROUND: Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease, which mostly affects infants and children and leads to severe neurological diseases. Vaccination offers the best option for disease control. We have screened the virus strain FY-23 K-B, which is used as an inactivated vaccine strain. An important issue in the development of vaccines is whether they provide cross protection against all other strains. METHODS: We collected and identified 19 clinical EV71 isolates from mainland China, which all belong to the C4 genotype. We established growth curves of the strains in Vero cells, performed genetic analysis, and evaluated the cross protection efficacy through neutralizing assays using antisera from a rabbit, monkey and adult human immunized with the FY-23 K-B vaccine strain. RESULTS: The antisera showed broad cross protection among the C4 subgroup strains and homotype strain. Neutralizing indexes (NIs) among the isolates and homotype strain of antisera varied between 56.2-1995.3 for rabbit, 17.8-42,169.7 for monkey and 31.6-17,782.8 for human, whereas NIs against Coxsackievirus A16 or other enteroviruses were below 10. CONCLUSIONS: These results suggested that FY-23 K-B used as an antigen could elicit broad spectrum neutralizing antibodies with cross protective efficacy among C4 genotype strains.

Molecular characterization of a new human coxsackievirus B2 associated with severe hand-foot-mouth disease in Yunnan Province of China in 2012.

Human coxsackievirus B2 (CVB2) belongs to the species Human enterovirus B and can cause aseptic meningitis, myocarditis and hand-foot-mouth disease (HFMD). We first determined the complete genome of the RW41-2/YN/CHN/2012 strain, isolated from a patient with HFMD and aseptic meningitis in the Yunnan Province, China in 2012. The strain shared 83.5 % and 82.2 % nucleotide similarity with CVB2 prototype strain Ohio-1, in the complete VP1 gene and the complete genome, respectively. Using phylogenetic and homogeneity analyses for the complete VP1 gene, CVB2 strains could be divided into four genogroups (A-D); the RW41-2/YN/CHN/2012 strain belonging to genogroup D. The amino acid sequence of VP1 is highly conserved. Recombination analyses showed the newly isolated RW41-2/YN/CHN/2012 strain was probably a recombinant, which was closely related to strain CVB2 (KM386639) in the genomic P1 and P2 regions and strains of other human enterovirus B (HEV-B) viruses (KT353721, JX644073, and KP262053) in the P3 region.

Molecular characterization of a new human echovirus 11 isolate associated with severe hand, foot and mouth disease in Yunnan, China, in 2010.

Human echovirus 11 (E-11), a member of the species Enterovirus B, frequently causes aseptic meningitis and hand, foot and mouth disease (HFMD). We determined the complete genome sequence of strain 520K/YN/CHN/2010, isolated from a subject with HFMD and aseptic meningitis in Yunnan Province, China, in 2010. The strain shared 78.8% and 81.1% nucleotide sequence similarity with prototype strain Gregory in the complete VP1 gene and the complete genome, respectively. Only the VP2-VP3-VP1 genome region of 520K/YN/CHN/2010 was similar to that of the E-11 strain; the other genome regions were most similar to those of other members of the species Enterovirus B. Using phylogenetic analysis and sequence comparisons of the complete VP1 gene, E-11 strains could be divided into five genogroups, and the 520K/YN/CHN/2010 strain was found to belong to genogroup A. Recombination analysis showed evidence of recombination with other member of the species Enterovirus B, especially the E-9 strain MSH/KM812/2010. Persistent surveillance of HFMD pathogens might help predict potential emerging viruses and related disease outbreaks.

Coxsackievirus B3 HFMD animal models in Syrian hamster and rhesus monkey.

Coxsackievirus B3 (CVB3) is the pathogen causing hand, foot and mouth disease (HFMD), which manifests across a spectrum of clinical severity from mild to severe. However, CVB3-infected mouse models mainly demonstrate viral myocarditis and pancreatitis, failing to replicate human HFMD symptoms. Although several enteroviruses have been evaluated in Syrian hamsters and rhesus monkeys, there is no comprehensive data on CVB3. In this study, we have first tested the susceptibility of Syrian hamsters to CVB3 infection via different routes. The results showed that Syrian hamsters were successfully infected with CVB3 by intraperitoneal injection or nasal drip, leading to nasopharyngeal colonization, acute severe pathological injury, and typical HFMD symptoms. Notably, the nasal drip group exhibited a longer viral excretion cycle and more severe pathological damage. In the subsequent study, rhesus monkeys infected with CVB3 through nasal drips also presented signs of HFMD symptoms, viral excretion, serum antibody conversion, viral nucleic acids and antigens, and the specific organ damages, particularly in the heart. Surprisingly, there were no significant differences in myocardial enzyme levels, and the clinical symptoms resembled those often associated with common, mild infections. In summary, the study successfully developed severe Syrian hamsters and mild rhesus monkey models for CVB3-induced HFMD. These models could serve as a basis for understanding the disease pathogenesis, conducting pre-trial prevention and evaluation, and implementing post-exposure intervention.

Screening of a new candidate coxsackievirus B1 vaccine strain based on its biological characteristics.

Coxsackievirus B1 (CVB1) is one of the significant pathogens causing viral myocarditis, hand, foot, and mouth disease (HFMD), and aseptic meningitis, and it has been associated with type 1 diabetes (T1DM). No effective antiviral drugs against CVB1 infection or preventive vaccines are available. Due to the success of two inactivated vaccines against enterovirus 71 and poliovirus, an inactivated Vero cell-based CVB1 vaccine could be developed. In this study, we isolated a high-growth CVB1 virus strain KM7 in Vero cells and developed a Vero-adapted vaccine candidate strain KM7-X29 via three rounds of plaque purification and serial passages. The KM7-X29 strain was grouped into the GII sub-genotype, which belonged to the Chinese epidemic strain and grew to a titer of more than 107 CCID50/ml in Vero cells. The inactivated CVB1 vaccine produced by the KM7-X29 strain induced an effective neutralizing antibody response in BALB/c mice, and maternal antibodies were able to provide a 100% protective effect against lethal challenges with a CVB1 strain in suckling BALB/c mice. Thus, the KM7-X29 strain might be used as a new candidate coxsackievirus B1 vaccine strain. The neonatal murine model of CVB1 infection will contribute to the development of the CVB1 vaccine.

Molecular characterization of echovirus 9 strains isolated from hand-foot-and-mouth disease in Kunming, Yunnan Province, China.

Echovirus 9 (E9) belongs to the species Enterovirus B. So far, 12 whole genome sequences of E9 are available in GenBank. In this study, we determined the whole genomic sequences of five E9 strains isolated from the stools of patients with hand-foot-and-mouth disease in Kunming, Yunnan Province, China, in 2019. Their nucleotide and amino acid sequences shared 80.8-80.9% and 96.4-96.8% identity with the prototype Hill strain, respectively, and shared 99.3-99.9% and 99.1-99.8% mutual identity, respectively. Recombination analyses revealed that intertype recombination had occurred in the 2C and 3D regions of the five Yunnan E9 strains with coxsackieviruses B5 and B4, respectively. This study augmented the whole genome sequences of E9 in the GenBank database and extended the molecular characterization of this virus in China.

Pathogenic analysis of coxsackievirus A10 in rhesus macaques.

Coxsackievirus A10 (CV-A10) is one of the etiological agents associated with hand, foot and mouth disease (HFMD) and also causes a variety of illnesses in humans, including pneumonia, and myocarditis. Different people, particularly young children, may have different immunological responses to infection. Current CV-A10 infection animal models provide only a rudimentary understanding of the pathogenesis and effects of this virus. The characteristics of CV-A10 infection, replication, and shedding in humans remain unknown. In this study, rhesus macaques were infected by CV-A10 via respiratory or digestive route to mimic the HFMD in humans. The clinical symptoms, viral shedding, inflammatory response and pathologic changes were investigated in acute infection (1-11 day post infection) and recovery period (12-180 day post infection). All infected rhesus macaques during acute infection showed obvious viremia and clinical symptoms which were comparable to those observed in humans. Substantial inflammatory pathological damages were observed in multi-organs, including the lung, heart, liver, and kidney. During the acute period, all rhesus macaques displayed clinical signs, viral shedding, normalization of serum cytokines, and increased serum neutralizing antibodies, whereas inflammatory factors caused some animals to develop severe hyperglycemia during the recovery period. In addition, there were no significant differences between respiratory and digestive tract infected animals. Overall, all data presented suggest that the rhesus macaques provide the first non-human primate animal model for investigating CV-A10 pathophysiology and assessing the development of potential human therapies.

Characterization of Coxsackievirus A6 Strains Isolated From Children With Hand, Foot, and Mouth Disease.

Coxsackievirus A6 (CVA6) is a key pathogen causing hand, foot and mouth disease (HFMD). However, there are currently no specific antiviral drugs or vaccines for treating infections caused by CVA6. In this study, human rhabdomyosarcoma (RD), African green monkey kidney (Vero), and human embryonic lung diploid fibroblast (KMB17) cells were used to isolate CVA6 from 327 anal swab and fecal samples obtained during HFMD monitoring between 2009 and 2017. The VP1 genes of the isolates were sequenced and genotyped, and the biological characteristics of the representative CVA6 strains were analyzed. A total of 37 CVA6 strains of the D3 gene subtypes were isolated from RD cells, all of which belonged to the epidemic strains in mainland China. Using the adaptive culture method, 10 KMB17 cell-adapted strains were obtained; however, no Vero cell-adapted strains were acquired. Among the KMB17 cell-adapted strains, only KYN-A1205 caused disease or partial death in suckling mice, and its virulence was stronger than its RD cell-adapted strain. The pathogenic KYN-A1205 strain caused strong tropism to the muscle tissue and led to pathological changes, including muscle necrosis and nuclear fragmentation in the forelimb and hindlimb. Sequence analysis demonstrated that the KYN-A1205 strain exhibited multiple amino acid mutations after KMB17 cell adaptation. Moreover, it showed strong pathogenicity, good immunogenicity and genetic stability, and could be used as an experimental CVA6 vaccine candidate.

Inhibition of both endplate nutritional pathways results in intervertebral disc degeneration in a goat model.

BACKGROUND: The vertebral endplate route was demonstrated to be the main pathway for nutrition to the intervertebral disc. However, it is still a controversial issue on whether the blocking of the endplate nutritional pathway could result in intervertebral disc degeneration (IDD) in animal models. The aim was therefore to investigate the effect of the inhibition of both endplate nutritional pathways by bone cement injection on the IDD in a goat model. METHODS: Two lumbar intervertebral discs (L2-3 and L3-4) in eight 24-month-old goats were blocked in both endplate nutritional pathways by cement injection, and the other two lumbar intervertebral discs (L1-2 and L4-5) remained intact as normal controls. Effective blocking area percentage in nucleus pulposus (NP) was calculated, and X-rays, magnetic resonance imaging (MRI), and histology studies were performed at 4, 12, 24, and 48 weeks after operation. RESULTS: The mean effective blocking area percentage was 60.7 ± 5.3%. Imaging examinations at the time of 48 weeks after blocking the endplate nutritional pathways showed obvious IDD, with larger disc height reduction and higher degrees of disc degeneration grading compared with the normal controls. Histological examinations including HE, Masson's trichrome, Sirius Red, and proteoglycan stainings also confirmed the degenerative changes of the blocked discs. CONCLUSIONS: The endplate nutritional route could be inhibited by blocking both endplate pathways with cement injection in a goat model. The severe inhibition in the endplate nutritional pathways may result in IDD.

Molecular characteristics of hand, foot, and mouth disease for hospitalized pediatric patients in Yunnan, China.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by multiple enteroviruses (EVs) in China. To better define the etiologic agents and clinical characteristics of HFMD, we conducted this study in Yunnan, China.In this study, 1280 stool specimens were collected from pediatric patients hospitalized for treatment of HFMD in 2010. EV was detected with nested reverse transcription polymerase chain reaction and directly genotyped by gene sequencing of the viral protein 1 (VP1) region. Phylogenetic analysis was performed based on the VP1 partial gene and the clinical characteristics were analyzed using SPSS Software.Of 1280 specimens, 1115 (87.1%) tested positive for EV. Seventeen different EV serotypes were detected. Coxsackievirus A16 (CA16) was the most frequently detected serotype (615/1115 cases, 55.1%), followed by enterovirus 71 (EV71; 392/1115, 35.2%), CA10 (45/1115, 4.0%), and CA4 (23/1115, 2.1%). Among the 709 severe cases, CA16, EV71, CA10, and CA4 accounted for 48.0%, 42.0%, 3.5%, and 2.3%, respectively. Of the 26 critical cases, 13 were caused by EV71, 9 by CA16, 2 by CA4, and 1 each were the result of CA10 and E9, respectively. All EV71, CA16, CA10, and CA4 isolates were highly homologous to the strains isolated from mainland China, and belonged to the C4a, B1a, G, and C genotypes, respectively.Our study showed that EV71 and CA16 were the main causative agents for severe and critical HFMD, but other serotypes can also cause severe and critical cases.