A novel subgenotype C6 Enterovirus A71 originating from the recombination between subgenotypes C4 and C2 strains in mainland China.

Recombination plays important roles in the genetic diversity and evolution of Enterovirus A71 (EV-A71). The phylogenetics of EV-A71 in mainland China found that one strain DL71 formed a new subgenotype C6 with unknown origin. This study investigated the detailed genetic characteristics of the new variant. DL71 formed a distinct cluster within genotype C based on the genome and individual genes (5'UTR, VP4, VP1, 2A, 2B, 2C, 3D, and 3'UTR). The average genetic distances of the genome and individual genes (VP3, 2A, 2B, 2C, 3A, 3C, and 3D) between DL71 and reference strains were greater than 0.1. Nine recombination events involving smaller fragments along DL71 genome were detected. The strains Fuyang-0805a (C4) and Tainan/5746/98 (C2) were identified as the parental strains of DL71. In the non-recombination regions, DL71 had higher identities with Fuyang-0805a than Tainan/5746/98, and located in the cluster with C4 strains. However, in the recombination regions, DL71 had higher identities with Tainan/5746/98 than Fuyang-0805a, and located in the cluster with C2 strains. Thus, DL71 was a novel multiple inter-subgenotype recombinant derived from the dominant subgenotype C4 and the sporadic subgenotype C2 strains. Monitoring the emergence of new variants by the whole-genome sequencing remains essential for preventing disease outbreaks and developing new vaccines.

Susceptibility and cytokine responses of human neuronal cells to multiple circulating EV-A71 genotypes in India.

Enterovirus-A71 (EV-A71) associated Hand, foot and mouth disease (HFMD) is a highly contagious viral infection affecting children in Asia-Pacific region and has become a major threat to public health. Although several EV-A71 genotypes (C, D, and G) were isolated in India in recent years, no recognizable outbreak of EV-A71 caused HFMD, Acute Flaccid paralysis (AFP) or encephalitis have been reported so far. It is essential to study the pathogenicity or cell tropism of these Indian isolates in order to understand their tendency to cause disease. We investigated the susceptibility and cytokine responses of indigenous EV-A71 genotypes (D and G) isolated from cases of AFP and genotype C viruses isolated from cases of HFMD and encephalitis, in human cells in-vitro. Although all three EV-A71 genotypes could infect and replicate in human muscle and neuronal cells, the genotype D virus showed a delayed response in human neuronal cells. Quantification of cytokine secretion in response to these isolates followed by confirmation with gene expression assays in human neuronal cells revealed significantly higher secretion of pro-inflammatory cytokines TNF-α IL-8, IL-6, IP-10 (p < 0.001) in G genotype infected cells as compared to pathogenic C genotypes whereas the genotype D virus could not induce any of the inflammatory cytokines. These findings will help to better understand the host response to indigenous EV-A71 genotypes for management of future EV-A71 outbreaks in India, if any.

Adaptation and Virulence of Enterovirus-A71.

Outbreaks of hand, foot, and mouth disease caused by enterovirus-A71 (EV-A71) can result in many deaths, due to central nervous system complications. Outbreaks with many fatalities have occurred sporadically in the Asia-Pacific region and have become a serious public health concern. It is hypothesized that virulent mutations in the EV-A71 genome cause these occasional outbreaks. Analysis of EV-A71 neurovirulence determinants is important, but there are no virulence determinants that are widely accepted among researchers. This is because most studies have been done in artificially infected mouse models and because EV-A71 mutates very quickly to adapt to the artificial host environment. Although EV-A71 uses multiple receptors for infection, it is clear that adaptation-related mutations alter the binding specificity of the receptors and allow the virus to adopt the best entry route for each environment. Such mutations have confused interpretations of virulence in animal models. This article will discuss how environment-adapted mutations in EV-A71 occur, how they affect virulence, and how such mutations can be avoided. We also discuss future perspectives for EV-A71 virulence research.

First evidence of enterovirus A71 and echovirus 30 in Uruguay and genetic relationship with strains circulating in the South American region.

Human enteroviruses (EVs) comprise more than 100 types of coxsackievirus, echovirus, poliovirus and numbered enteroviruses, which are mainly transmitted by the faecal-oral route leading to diverse diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, among others. Since enteroviruses are excreted in faeces, wastewater-based epidemiology approaches are useful to describe EV diversity in a community. In Uruguay, knowledge about enteroviruses is extremely limited. This study assessed the diversity of enteroviruses through Illumina next-generation sequencing of VP1-amplicons obtained by RT-PCR directly applied to viral concentrates of 84 wastewater samples collected in Uruguay during 2011-2012 and 2017-2018. Fifty out of the 84 samples were positive for enteroviruses. There were detected 27 different types belonging to Enterovirus A species (CVA2-A6, A10, A16, EV-A71, A90), Enterovirus B species (CVA9, B1-B5, E1, E6, E11, E14, E21, E30) and Enterovirus C species (CVA1, A13, A19, A22, A24, EV-C99). Enterovirus A71 (EV-A71) and echovirus 30 (E30) strains were studied more in depth through phylogenetic analysis, together with some strains previously detected by us in Argentina. Results unveiled that EV-A71 sub-genogroup C2 circulates in both countries at least since 2011-2012, and that the C1-like emerging variant recently entered in Argentina. We also confirmed the circulation of echovirus 30 genotypes E and F in Argentina, and reported the detection of genotype E in Uruguay. To the best of our knowledge this is the first report of the EV-A71 C1-like emerging variant in South-America, and the first report of EV-A71 and E30 in Uruguay.

Genetic diversity and evolution of enterovirus A71 subgenogroup C1 from children with hand, foot, and mouth disease in Thailand.

Enterovirus A71 (EV-A71) can cause hand, foot, and mouth disease (HFMD) in children and may be associated with severe neurological complications. There have been numerous reports of increased incidence of EV-A71 subgenogroup C1 (EV-A71 C1) infections associated with neurological diseases since the first occurrence in Germany in 2015. Here, we describe 11 full-length genome sequences of 2019 EV-A71 C1 strains isolated from HFMD patients in Thailand from 2019 to early 2020. The genetic evolution of 2019 EV-A71 C1 was traced in the outbreaks, and the emergence of multiple lineages was detected. Our results demonstrated that 2019 EV-A71 C1 from Thailand emerged through recombination between its nonstructural protein gene and those of other EV-A genotypes. Bayesian-based phylogenetic analysis showed that the 2019 EV-A71 C1 Thai strains share a common ancestor with variants in Europe (Denmark and France). The substitution rate for the 2019 EV-A71 C1 genome was estimated to be 4.38 × 10-3 substitutions/(site∙year-1) (95% highest posterior density interval: 3.84-4.94 × 10-3 substitutions/[site∙year-1]), approximating that observed between previous EV-A71 C1 outbreaks. These data are essential for understanding the evolution of EV-A C1 during the ongoing HFMD outbreak and may be relevant to disease outcomes in children worldwide.

Sporadic hand, foot, and mouth disease cases associated with non-C4 enterovirus 71 strains in Xiamen, China, from 2009 to 2018.

Enterovirus 71 (EV71) has caused large hand, foot, and mouth disease (HFMD) epidemics among young children, and EV71 infection is the leading cause of severe HFMD cases and deaths. In mainland China, the prevalence and risk factors of non-C4 EV71 strains are still unclear. In this study, we monitored non-C4 strains over a 10-year HFMD epidemiological surveillance period in Xiamen. The 5'UTR and VP1 coding region of EV71 strains were amplified by RT-nested PCR and sequenced. Thirty-two non-C4 EV71 strains were identified during 2009-2018. This study provides important information about the prevalence of EV71 in China that will be applicable for development of vaccines and diagnostic reagents as well as establishment of policies for HFMD prevention and control.

The epidemiological characteristics of enterovirus infection before and after the use of enterovirus 71 inactivated vaccine in Kunming, China.

Enterovirus A71 (EV-A71) inactivated vaccines have been widely inoculated among children in Kunming City after it was approved. However, there was a large-scale outbreak of Enteroviruses (EVs) infection in Kunming, 2018. The epidemiological characteristics of HFMD and EVs were analysed during 2008-2018, which are before and three years after EV-A71 vaccine starting to use. The changes in infection spectrum were also investigated, especially for severe HFMD in 2018. The incidence of EV-A71 decreased dramatically after the EV-A71 vaccine starting use. The proportion of non-CV-A16/EV-A71 EVs positive patients raised to 77.17-85.82%, while, EV-A71 and CV-A16 only accounted for 3.41-7.24% and 6.94-19.42% in 2017 and 2018, respectively. CV-A6 was the most important causative agent in all clinical symptoms (severe HFMD, HFMD, Herpangina and fever), accounting from 42.13% to 62.33%. EV-A71 only account for 0.36-2.05%. In severe HFMD, CV-A6 (62.33%), CV-A10 (11.64%), and CV-A16 (10.96%) were the major causative agent in 2018. EV-A71 inactivated vaccine has a good protective effect against EV-A71 and induced EVs infection spectrum changefully. EV-A71 vaccine has no or insignificant cross-protection effect on CV-A6, CV-A10, and CV-A16. Herein, developing 4-valent combined vaccines is urgently needed.

Circulation of enterovirus A71 during 2019-2020, Marseille, France.

Enteroviruses A71 (EVs-A71) are known to cause serious neurological infections, especially in the pediatric population. We report here eight cases of EV-A71 infection diagnosed in Marseille over the past 2 years (seven cases in 2019 and one case in 2020). Only children under 5 years of age were affected, including one case of acute flaccid paralysis. Viral RNA was detected by RT-PCR in peripheral samples for all cases (feces and upper respiratory samples). Phylogenetic analyses based on VP1 and 2C3C coding regions revealed that all these cases of EV-A71 infection were caused by viruses belonging to the subgenogroup C1 that currently circulates in Europe and that these viruses are genetically closed to other EVs-A71 recently detected in European countries. These data therefore reinforce the usefulness of the enterovirus surveillance network and the need for systematic screening for EV-A71 in case of an enteroviral infection. This study therefore suggests that the systematic screening for EV-A71 in case of enteroviral infection could provide additional data for enterovirus surveillance networks.

Natural intertypic and intratypic recombinants of enterovirus 71 from mainland China during 2009-2018: a complete genome analysis.

Surveillance of recombinant enterovirus 71 (EV71) and subgenotype replacement is vital for preventing and controlling hand, foot, and mouth disease (HFMD) outbreaks. Despite this, data on recombinant variants and phylogeny of circulating EV71 strains in mainland China are limited. In this study, recombinant variants of EV71 were identified in mainland China from 2009 to 2018. Phylogenetic analysis indicated that except for individual strains (CQ2014-86/CQ/CHN/2014 and EV71/Xiamen/2009 (B5)), almost all of the EV71 strains in mainland China belonged to the subgenotype C4a. Analysing complete genome sequences of 196 EV71 isolates, 3 intertypic recombination strains (VR1432, 30-2/2015/BJ, and Guangdong-2009) and 5 intratypic recombination strains (EV71/P1034/2013, VR1432, Henan-ZMD/CHN/2012, Hubei-WH/CHN/2012, and EV71/P868/2013/China) were identified among naturally circulating EV71. The breakpoints of these recombinant strains were located within the P1, P2, and P3 encoding regions. Notably, a double recombinant (VR1432) resulting from recombination between EV71 subgenotype C4a and C4b strain SHZH98 and a CA8 strain Donovan was identified. This study reports these specific intertypic and intratypic recombination events for the first time highlighting the importance of genetic recombination in the emergence of new enterovirus variants.

Genetic Diversity Analysis of Coxsackievirus A8 Circulating in China and Worldwide Reveals a Highly Divergent Genotype.

Coxsackievirus A8 (CV-A8) is one of the pathogens associated with hand, foot and mouth disease (HFMD) and herpangina (HA), occasionally leading to severe neurological disorders such as acute flaccid paralysis (AFP). Only one study aimed at CV-A8 has been published to date, and only 12 whole-genome sequences are publicly available. In this study, complete genome sequences from 11 CV-A8 strains isolated from HFMD patients in extensive regions from China between 2013 and 2018 were determined, and all sequences from GenBank were retrieved. A phylogenetic analysis based on a total of 34 complete VP1 sequences of CV-A8 revealed five genotypes: A, B, C, D and E. The newly emerging genotype E presented a highly phylogenetic divergence compared with the other genotypes and was composed of the majority of the strains sequenced in this study. Markov chain Monte Carlo (MCMC) analysis revealed that genotype E has been evolving for nearly a century and somehow arose in approximately 2010. The Bayesian skyline plot showed that the population size of CV-A8 has experienced three dynamic fluctuations since 2001. Amino acid residues of VP1100N, 103Y, 240T and 241V, which were embedded in the potential capsid loops of genotype E, might enhance genotype E adaption to the human hosts. The CV-A8 whole genomes displayed significant intra-genotypic genetic diversity in the non-capsid region, and a total of six recombinant lineages were detected. The Chinese viruses from genotype E might have emerged recently from recombining with European CV-A6 strains. CV-A8 is a less important HFMD pathogen, and the capsid gene diversity and non-capsid recombination variety observed in CV-A8 strains indicated that the constant generation of deleterious genomes and a constant selection pressure against these deleterious mutations is still ongoing within CV-A8 quasispecies. It is possible that CV-A8 could become an important pathogen in the HFMD spectrum in the future. Further surveillance of CV-A8 is greatly needed.

Rules governing genetic exchanges among viral types from different Enterovirus A clusters.

The species Enterovirus A (EV-A) consists of two conventional clusters and one unconventional cluster. At present, sequence analysis shows no evidence of recombination between conventional and unconventional EV-A types. However, the factors underlying this genetic barrier are unclear. Here, we systematically dissected the genome components linked to these peculiar phenomena, using the viral reverse genetic tools. We reported that viral capsids of the unconventional EV-A types expressed poorly in human cells. The trans-encapsidation outputs across conventional and unconventional EV-A types were also with low efficiency. However, replicons of conventional types bearing exchanged 5'-untranslated region (UTR) or non-structural regions from the unconventional types were replication-competent. Furthermore, we created a viable recombinant EVA71 (conventional type) with its P3 region replaced by that from EVA89 (unconventional type). Thus, our data for the first time reveal the potential for fertile genetic exchanges between conventional and unconventional EV-A types. It also discloses that the mysterious recombination barriers may lie in uncoordinated capsid expression and particle assembly by different EV-A clusters.

Characterization of Plaque Variants and the Involvement of Quasi-Species in a Population of EV-A71.

Viral plaque morphologies in human cell lines are markers for growth capability and they have been used to assess the viral fitness and selection of attenuated mutants for live-attenuated vaccine development. In this study, we investigate whether the naturally occurring plaque size variation reflects the virulence of the variants of EV-A71. Variants of two different plaque sizes (big and small) from EV-A71 sub-genotype B4 strain 41 were characterized. The plaque variants displayed different in vitro growth kinetics compared to the parental wild type. The plaque variants showed specific mutations being present in each variant strain. The big plaque variants showed four mutations I97L, N104S, S246P and N282D in the VP1 while the small plaque variants showed I97T, N237T and T292A in the VP1. No other mutations were detected in the whole genome of the two variants. The variants showed stable homogenous small plaques and big plaques, respectively, when re-infected in rhabdomyosarcoma (RD) and Vero cells. The parental strain showed faster growth kinetics and had higher viral RNA copy number than both the big and small plaque variants. Homology modelling shows that both plaque variants have differences in the structure of the VP1 protein due to the presence of unique spontaneous mutations found in each plaque variant This study suggests that the EV-A71 sub-genotype B4 strain 41 has at least two variants with different plaque morphologies. These differences were likely due to the presence of spontaneous mutations that are unique to each of the plaque variants. The ability to maintain the respective plaque morphology upon passaging indicates the presence of quasi-species in the parental population.

Mosaic genome of Human Coxsackievirus A4 associated with herpangina and HFMD in Yancheng, China, 2016 and 2018.

OBJECTIVES: To better understand the spectrums of pathogens causing herpangina and circulation of Coxsackievirus A4 in Yancheng, China. METHODS: Stool samples from herpangina and HFMD cases were collected. Real Time PCR Kits was used to identify Enterovirus 71, CV-A16 and CV-A6, and nested reverse transcription PCR (nRT-PCR) to detect the other enterovirus types. Complete VP1 and genome sequence of CV-A4 were amplified by using nRT-PCR. Genetic, phylogenetic and recombination analysis were performed. RESULTS: Co-circulation of three recombinant CV-A4 groups, including one novel (C2 lineage), was identified in Yancheng, China, 2016 and 2018. One was the major causative agent of herpangina, and another two were responsible for HFMD. Phylogenetic and recombination analysis indicated that the non-structural region of their genome originated from the same ancestry and subsequently adaptation. C2 lineage of CV-A4 group may be introduced from countries outside China and its genome occurred recombination in China. CONCLUSION: Novel recombinant CV-A4 was mainly associated with herpanginain in Yancheng, 2018, China. C2 lineage of CV-A4 group with recombinant non-structural region was also identified in HFMD patients.

Co-circulation of coxsackieviruses A-6, A-10, and A-16 causes hand, foot, and mouth disease in Guangzhou city, China.

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common infectious disease occurring in children under 5 years of age worldwide, and Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CVA-16) are identified as the predominant pathogens. In recent years, Coxsackievirus A6 (CVA-6) and Coxsackievirus A10 (CVA-10) have played more and more important role in a series of HFMD outbreaks. This study aimed to understand the epidemic characteristics associated with HFMD outbreak in Guangzhou, 2018. METHODS: The clinical and laboratory data of 1220 enterovirus-associated HFMD patients in 2018 were analysed in this study. Molecular diagnostic methods were performed to identify its serotypes. Phylogenetic analyses were depicted based on the complete VP1 gene. RESULTS: There were 21 enterovirus serotypes detected in Guangzhou in 2018. Three serotypes of enterovirus, CVA-6 (364/1220, 29.8%), CVA-10 (305/1220, 25.0%), and CVA-16 (397/1220, 32.5%), were identified as the causative pathogens and accounted for 87.3% among all 1220 HFMD patients. In different seasons, CVA-6 was the predominant pathogen of HFMD during autumn, and CVA-10 as well as CVA-16 were more prevalent in summer. Patients infected by CVA-6, CVA-10 or CVA-16 showed similar clinical features and laboratory characteristics, and the ratios of severe HFMD were 5.8, 5.9, and 1.5% in the three serotypes. Phylogenetic analyses of VP1 sequences showed that the CVA-6, CVA-10, and CVA-16 sequences belonged to the sub-genogroup E2, genogroup E, and genogroup B1, respectively. CONCLUSIONS: CVA-6, CVA-10, and CVA-16 were the predominant and co-circulated serotypes in Guangzhou China, 2018, which should be the new target for prevention and control of HFMD. Our findings provide useful information for diagnosis, treatment, and prevention of HFMD.

Genotipificación del virus de la enfermedad mano-pie-boca atípica en un hospital pediátrico de la Ciudad de Buenos Aires, Argentina / Atypical hand-foot-mouth disease virus genotyping in a pediatric hospital in Buenos Aires city, Argentin

La enfermedad mano-pie-boca (EMPB) típica es exantemática, con sintomatología clásica de fiebre, exantema papulovesicular en las manos y los pies, asociada o no a herpangina. Es causada, principalmente, por enterovirus 71 y virus Coxsackie A16, miembros del género Enterovirus. En los últimos años, se han descrito brotes mundiales de EMPB con manifestaciones atípicas causadas, sobre todo, por el virus Coxsackie A6. La EMPB atípica se considera emergente con características clínicas y epidemiológicas peculiares: la afección de adultos, el predominio en invierno y un amplio espectro de manifestaciones clínicas en la extensión y la distribución de las lesiones. Las características morfológicas de las lesiones son muy variables: pueden simular varicela, impétigo o vasculitis.Se describe el caso de un niño de 4 años con EMPB atípica. Se detalla su forma de presentación, evolución clínica, metodología diagnóstica y terapéutica empleada.

Typical hand-foot-mouth disease (HFMD) is an exanthematous viral disease with a classic symptomatology of fever, papulovesicular rash on the hands and feet with or without herpangina. It is usually caused by enterovirus 71 and Coxsackievirus A16, members of the genus Enterovirus. Recently, worldwide outbreaks of HFMD with atypical manifestations caused by Coxsackievirus A6 have been described. Atypical HFMD is considered an emerging disease due to its peculiar clinical and epidemiological characteristics: it affects adults, has a wide spectrum of clinical manifestations in the extension and distribution of the lesions and occurs in winter. The morphological characteristics of the lesions are very variable and can be misdiagnosed as chickenpox, impetigo or vasculitis. Here we describe the symptoms, clinical evolution, diagnostic methodology and treatment employed on a 4-year-old male patient with atypical HFMD.

[Atypical hand-foot-mouth disease virus genotyping in a pediatric hospital in Buenos Aires city, Argentina]. / Genotipificación del virus de la enfermedad mano-pie-boca atípica en un hospital pediátrico de la Ciudad de Buenos Aires, Argentina.

Typical hand-foot-mouth disease (HFMD) is an exanthematous viral disease with a classic symptomatology of fever, papulovesicular rash on the hands and feet with or without herpangina. It is usually caused by enterovirus 71 and Coxsackievirus A16, members of the genus Enterovirus. Recently, worldwide outbreaks of HFMD with atypical manifestations caused by Coxsackievirus A6 have been described. Atypical HFMD is considered an emerging disease due to its peculiar clinical and epidemiological characteristics: it affects adults, has a wide spectrum of clinical manifestations in the extension and distribution of the lesions and occurs in winter. The morphological characteristics of the lesions are very variable and can be misdiagnosed as chickenpox, impetigo or vasculitis. Here we describe the symptoms, clinical evolution, diagnostic methodology and treatment employed on a 4-yearold male patient with atypical HFMD.

La enfermedad mano-pie-boca (EMPB) típica es exantemática, con sintomatología clásica de fiebre, exantema papulovesicular en las manos y los pies, asociada o no a herpangina. Es causada, principalmente, por enterovirus 71 y virus Coxsackie A16, miembros del género Enterovirus. En los últimos años, se han descrito brotes mundiales de EMPB con manifestaciones atípicas causadas, sobre todo, por el virus Coxsackie A6. La EMPB atípica se considera emergente con características clínicas y epidemiológicas peculiares: la afección de adultos, el predominio en invierno y un amplio espectro de manifestaciones clínicas en la extensión y la distribución de las lesiones. Las características morfológicas de las lesiones son muy variables: pueden simular varicela, impétigo o vasculitis. Se describe el caso de un niño de 4 años con EMPB atípica. Se detalla su forma de presentación, evolución clínica, metodología diagnóstica y terapéutica empleada.

Sequence analysis of the first B5 subgenogroup strain of enterovirus 71 isolated in Korea.

Enterovirus A71 (EV71), the main etiological agent of handfoot- mouth disease (HFMD), circulates in many areas of the world and has caused large epidemics since 1997, especially in the Asia-Pacific region. In this study, we determined the full-genome sequence of CMC718, a newly isolated EV71 strain in Korea. The CMC718 genome was 7,415 nucleotides in length and was confirmed by whole-genome phylogenetic analysis to belong to the B5 genotype. In particular, CMC718 demonstrated maximum identity with strain M988 of the B5 genotype and numerous amino acid variants were detected in the 3D domain of the viral protein P3, which is consistent with the mutation pattern of a B5 strain isolated in 2012-2013. Comparison of the CMC718 sequence with other EV71 reference strains confirmed the relationship and genetic variation of CMC718. Our study was a full-genome sequence analysis of the first EV71 strain of the B5 genotype isolated in South Korea. This information will be a valuable reference for the development of methods for the detection of recombinant viruses, the tracking of infections, and the diagnosis of EV71.

Coxsackievirus A10 causing hand-foot-and-mouth disease in Uruguay.

We report for the first time in South America an HFMD case associated with Coxsackievirus A10. The viral strain belongs to a lineage involved in important European outbreaks and probably entered Uruguay after 2017 with a Greek origin. These findings call for strengthening the regional surveillance of HFMD.

Genetic characterization of VP1 of coxsackieviruses A2, A4, and A10 associated with hand, foot, and mouth disease in Vietnam in 2012-2017: endemic circulation and emergence of new HFMD-causing lineages.

While conducting sentinel surveillance of hand, foot, and mouth disease (HFMD) in Vietnam, we found a sudden increase in the prevalence of coxsackievirus A10 (CV-A10) in 2016 and CV-A2 and CV-A4 in 2017, the emergence of which has been reported recently to be associated with various clinical manifestations in other countries. However, there have been only a limited number of molecular studies on those serotypes, with none being conducted in Vietnam. Therefore, we sequenced the entire VP1 genes of CV-A10, CV-A4, and CV-A2 strains associated with HFMD in Vietnam between 2012 and 2017. Phylogenetic analysis revealed a trend of endemic circulation of Vietnamese CV-A10, CV-A4, and CV-A2 strains and the emergence of thus-far undescribed HFMD-causing lineages of CV-A4 and CV-A2. The Vietnamese CV-A10 strains belonged to a genotype comprising isolates from patients with HFMD from several other countries; however, most of the Vietnamese strains were grouped into a local lineage. Recently, emerging CV-A4 strains in Vietnam were grouped into a unique lineage within a genotype comprising strains isolated from patients with acute flaccid paralysis from various countries. New substitutions were detected in the putative BC and HI loops in the Vietnamese CV-A4 strains. Except for one strain, Vietnamese CV-A2 isolates were grouped into a unique lineage of a genotype that includes strains from various countries that are associated with other clinical manifestations. Enhanced surveillance is required to monitor their spread and to specify their roles as etiological agents of HFMD or "HFMD-like" diseases, especially for CV-A4 and CV-A2. Further studies including whole-genome sequencing should be conducted to fully understand the evolutionary changes occurring in these newly emerging strains.