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Coxsackievirus A16 (CV-A16) is a significant etiologic agent of hand, foot, and mouth disease (HFMD) and herpangina (HA), with the capacity to progress to severe complications, including encephalitis, aseptic meningitis, acute flaccid paralysis, myocarditis, and other critical conditions. Beijing's epidemiological surveillance system, established in 2008, encompasses 29 hospitals and 16 district disease control centers. From 2019 to 2021, the circulation of CV-A16 was characterized by the co-circulation of B1a and B1b clades. Multiple cases of HFMD linked to clade B1c has not been reported in Beijing until 2022. This study enrolled 400 HFMD and 493 HA cases. Employing real-time RT-PCR, 368 enterovirus-positive cases were identified, with 180 selected for sequencing. CV-A16 was detected in 18.89% (34/180) of the cases, second only to CV-A6, identified in 63.33% (114/180). Full-length VP1 gene sequences were successfully amplified and sequenced in 22 cases, revealing the presence of clades B1a, B1b, and B1c in 14, 3, and 5 cases, respectively. A cluster of five B1c clade cases occurred between June 29 and July 17, 2022, within a 7-km diameter region in Shunyi District. Phylogenetic analysis of five complete VP1 gene sequences and two full-genome sequences revealed close clustering with the 2018 Indian strain (GenBank accession: MH780757.1) within the B1c India branch, with NCBI BLAST results showing over 98% similarity. Comparative sequence analysis identified three unique amino acid variations (P3S, V25A, and I235V). The 2022 Shunyi District HFMD cases represent the first instances of spatiotemporally correlated CV-A16 B1c clade infections in Beijing, underscoring the necessity for heightened surveillance of B1c clade CV-A16 in HFMD and HA in this region.
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Enfermedad de Boca, Mano y Pie , Filogenia , Humanos , Beijing/epidemiología , Enfermedad de Boca, Mano y Pie/virología , Enfermedad de Boca, Mano y Pie/epidemiología , Masculino , Femenino , Preescolar , Lactante , Niño , Genotipo , Enterovirus/genética , Enterovirus/clasificación , Enterovirus/aislamiento & purificación , Proteínas de la Cápside/genética , Adolescente , Monitoreo EpidemiológicoRESUMEN
Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5'UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/µl, for the 5' UTR was 50.1187 copies/µl and for the RNaseP gene was 158.49 copies/µl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100â¯% (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94â¯% (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95â¯% (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5â¯hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.
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Enterovirus , Enfermedad de Boca, Mano y Pie , Reacción en Cadena de la Polimerasa Multiplex , Sensibilidad y Especificidad , Enfermedad de Boca, Mano y Pie/diagnóstico , Enfermedad de Boca, Mano y Pie/virología , Humanos , Enterovirus/genética , Enterovirus/aislamiento & purificación , Enterovirus/clasificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Diagnóstico Diferencial , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Límite de Detección , Preescolar , Regiones no Traducidas 5'/genética , ARN Viral/genética , Fluorescencia , LactanteRESUMEN
Coxsackievirus A16 (CV-A16) and coxsackievirus A10 (CV-A10), more commonly etiological agents of hand, foot and mouth disease (HFMD), are capable of causing severe neurological syndromes with high fatalities, but their neuropathogenesis has rarely been studied. Mounting evidence indicated that pyroptosis is an inflammatory form of cell death that might be widely involved in the pathogenic mechanisms of neurotropic viruses. Our study was designed to examine the effects of NLRP3-mediated pyroptosis in CV-A16- and CV-A10-induced inflammatory neuropathologic formation. In this work, it was showed that SH-SY5Y cells were susceptible to CV-A16 and CV-A10, and meanwhile their infections could result in a decreasing cell viability and an increasing LDH release as well as Caspase1 activation. Moreover, CV-A16 and CV-A10 infections triggered NLRP3-mediated pyroptosis and promoted the release of inflammatory cytokines. Additionally, activated NLRP3 accelerated the pyroptosis formation and aggravated the inflammatory response, but inhibited NLRP3 had a dampening effect on the above situation. Finally, it was further revealed that NLRP3 agonist enhanced the viral replication, but NLRP3 inhibitor suppressed the viral replication, suggesting that NLRP3-driven pyroptosis might support CV-A16 and CV-A10 production in SH-SY5Y cells. Together, our findings demonstrated a mechanism by which CV-A16 and CV-A10 induce inflammatory responses by evoking NLRP3 inflammasome-regulated pyroptosis, which in turn further stimulated the viral replication, providing novel insights into the pathogenesis of CV-A16 and CV-A10 infections.
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Proteína con Dominio Pirina 3 de la Familia NLR , Piroptosis , Replicación Viral , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Citocinas/metabolismo , Citocinas/genética , Inflamación/virología , Enterovirus/fisiología , Enterovirus/patogenicidad , Línea Celular Tumoral , Inflamasomas/metabolismo , Enterovirus Humano A/fisiología , Enterovirus Humano A/patogenicidad , Supervivencia CelularRESUMEN
BACKGROUND: Hand, foot, and mouth disease (HFMD) is an enteroviral disease that occurs as outbreaks and sporadic cases in India. In this study, we investigated and characterized the aetiology of HFMD cases that occurred in Karnataka, South India from April to October 2022. METHODS: Throat swabs, vesicular swabs, urine, and blood samples from suspected cases were analysed by reverse transcription polymerase chain reaction (RT-PCR) for the detection of enteroviruses. Molecular typing of the enterovirus-positive samples was carried out by amplifying the partial virion protein 1(VP1) gene sequence, followed by sequencing and phylogenetic analysis. RESULTS: Out of the 187 samples received from 82 cases, 93 (50%) tested positive (55/82 cases, 67%) for enteroviruses, with the majority of the HFMD cases reported in paediatric population of less than 5 years (36/55, 65.4%), while 3 cases (3/55, 5.4%) were adults. Out of the 55 enterovirus-positive cases, 31 showed partial VP1 region amplification and 19 of these cases were typed as coxsackievirus A16 (CV-A16) (13/19, 68.4%) and CV-A6 (6/19, 31.6%). The CV-A16 strains identified belonged to subclade B1c while two CV-A6 strains belonged to subclade E2. On molecular testing for other viruses causing fever-rash symptoms, 4/27 (15%) enterovirus-negative cases were detected as herpes simplex virus (1 case) and varicella zoster virus (3 cases) positive. CONCLUSION: The main causative agent of HFMD in Karnataka in 2022 was CV-A16, followed by CV-A6. Apart from the common paediatric HFMD cases, adult cases were also reported during this period. Further studies involving laboratory and clinical investigations are essential for monitoring and managing HFMD in the community.
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Infecciones por Enterovirus , Enterovirus , Enfermedad de Boca, Mano y Pie , Adulto , Niño , Humanos , Enfermedad de Boca, Mano y Pie/epidemiología , Epidemiología Molecular , Filogenia , India/epidemiología , Enterovirus/genética , Infecciones por Enterovirus/epidemiología , Brotes de Enfermedades , Antígenos Virales/genética , China/epidemiologíaRESUMEN
Coxsackievirus A16 (CV-A16) is still an important pathogen that causes hand, foot and mouth disease (HFMD) in young children and infants worldwide. Previous studies indicated that CV-A16 infection is usually mild or self-limiting, but it was also found that CV-A16 infection can trigger severe neurological complications and even death. However, there are currently no vaccines or antiviral compounds available to either prevent or treat CV-A16 infection. Therefore, investigation of the virusâhost interaction and identification of host proteins that play a crucial regulatory role in the pathogenesis of CV-A16 infection may provide a novel strategy to develop antiviral drugs. Here, to increase our understanding of the interaction of CV-A16 with the host cell, we analyzed changes in the proteome of 16HBE cells in response to CV-A16 using tandem mass tag (TMT) in combination with LCâMS/MS. There were 6615 proteins quantified, and 172 proteins showed a significant alteration during CV-A16 infection. These differentially regulated proteins were involved in fundamental biological processes and signaling pathways, including metabolic processes, cytokineâcytokine receptor interactions, B-cell receptor signaling pathways, and neuroactive ligandâreceptor interactions. Further bioinformatics analysis revealed the characteristics of the protein domains and subcellular localization of these differentially expressed proteins. Then, to validate the proteomics data, 3 randomly selected proteins exhibited consistent changes in protein expression with the TMT results using Western blotting and immunofluorescence methods. Finally, among these differentially regulated proteins, we primarily focused on HMGB1 based on its potential effects on viral replication and virus infection-induced inflammatory responses. It was demonstrated that overexpression of HMGB1 could decrease viral replication and upregulate the release of inflammatory cytokines, but deletion of HMGB1 increased viral replication and downregulated the release of inflammatory cytokines. In conclusion, the results from this study have helped further elucidate the potential molecular pathogenesis of CV-A16 based on numerous protein changes and the functions of HMGB1 Found to be involved in the processes of viral replication and inflammatory response, which may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.
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Enterovirus , Proteína HMGB1 , Replicación Viral , Humanos , Cromatografía Liquida , Citocinas/metabolismo , Enterovirus/fisiología , Enfermedad de Boca, Mano y Pie , Proteína HMGB1/metabolismo , Proteómica , Espectrometría de Masas en Tándem , Línea CelularRESUMEN
Coxsackievirus A16 (CV-A16) is a significant pathogen responsible for causing hand foot and mouth disease (HFMD) and herpangina (HA). This study aimed to investigate the recent evolution and spread of CV-A16 by monitoring HFMD and HA cases in 29 hospitals across 16 districts in Beijing from 2019 to 2021. The first five cases of HFMD and the first five cases of HA each month in each hospital were included in the study. Real-time reverse transcription polymerase chain reaction was used to identify CV-A16, CV-A6, and EV-A71. From each district, two to four CV-A16 positive samples with a relatively long sampling time interval every month were selected for sequencing. A total of 3344 HFMD cases and 2704 HA cases were enrolled in this study, with 76.0% (2541/3344) of HFMD and 45.4% (1227/2704) of HA cases confirmed to be infected by enterovirus. Among the EV-positive samples, CV-A16 virus was detected in 33.61% (854/2541) of HFMD cases and 13.4% (165/1227) of HA cases, with the predominant cluster being B1a. Both B1a and B1b had a co-circulation of local and imported strains, with different origin time (1993 vs. 1995), different global distribution (14 countries vs. 10 countries), and different transmission centers but mainly distributed in the southern and eastern regions of Beijing. Strengthening surveillance of HFMD in southern and eastern regions will improve the prevention and control efficiency of enterovirus infections.
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Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Enfermedad de Boca, Mano y Pie , Herpangina , Humanos , Enfermedad de Boca, Mano y Pie/epidemiología , Herpangina/epidemiología , Filogenia , Beijing/epidemiología , Enterovirus/genética , Enterovirus Humano A/genética , China/epidemiologíaRESUMEN
Hand, foot and mouth disease, a childhood disorder caused by enteroviruses, is intermittently endemic in the Asia-Pacific region and endangers the lives of many infants and young children. Coxsackievirus A16 (CV-A16) is one of the major pathogens causing hand, foot, and mouth disease on occasion, resulting in catastrophic neurological sequelae and patient death. Currently, no clinical interventions are available that completely block the CV-A16 infection. Therefore, research on anti-CV-A16 treatment continues to be a significant focus of interest. This report provides a detailed background on and an introduction to CV-A16; a description of the viral gene and protein structures and a summary of the current advances in pharmaceutical targets, drug research and other related areas.
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Infecciones por Enterovirus , Enterovirus , Enfermedad de Boca, Mano y Pie , Humanos , Antígenos Virales , Infecciones por Enterovirus/tratamiento farmacológico , Enfermedad de Boca, Mano y Pie/tratamiento farmacológico , ProteínasRESUMEN
PURPOSE: Hand, Foot and Mouth disease (HFMD) is a contagious pediatric viral disease caused due to enteroviruses (EV) of the family Picornaviridae. Cases of HFMD were reported from a tertiary care health centre, Udhampur, (Jammu and Kashmir), Northern India. The present study highlights the clinical and molecular virological aspects of HFMD cases. MATERIAL AND METHODS: Cases reported during August 2016-September 2017, and clinically diagnosed as HFMD of all age groups were included. Clinical, Biochemical and molecular virology aspects were compared. Clinical samples (n â= â50) such as vesicle swab, buccal and throat swabs were collected for enterovirus detection. EV-RNA was detected by 5'NCR based RT-PCR and genotyping by VP1 gene amplification and cycle sequencing. RESULTS: Of the cases of HFMD enrolled (n â= â50), highest (84%) were of children aged <5 years, presented either or both anathemas and exanthemas with prodromal symptoms (fever, irritability). Clinical presentations involved mainly oral ulcers on lips and tongue (48%). Oral erosions were either single or multiple in numbers. Exanthemas were seen on hand and palm, widely spread up to buttocks, legs, arms and trunk. Of these, six patients were found anemic. Complete blood count (CBC) indicated lymphocytosis and C-reactive protein (n â= â10) in children aged <5 years. EV-RNA was detected in 78% (39/50) of the clinical samples. VP1 gene based typing indicated the presence of CV-A16, CVA6 and EV-A71 types. CONCLUSIONS: The study highlights association of EVs in HFMD cases in the reported region. CV-A16, CV-A6 and EV-A71 types were reported for the first time from Udhampur (J&K), Northern India. No differences were observed in the clinical profile of EV strains detected. Circulation of the strains warrant and alarm outbreaks. More focused studies on HFMD and monitoring of viral strains is mandatory.
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Infecciones por Enterovirus , Enterovirus , Enfermedad de Boca, Mano y Pie , Niño , Humanos , Lactante , Enterovirus/genética , Tipificación Molecular , Antígenos Virales/genética , India/epidemiología , ARN , China/epidemiologíaRESUMEN
Hand, foot and mouth disease (HFMD) is a common infectious disease in western Asia area and the full range of the long-term sequelae of HFMD remains poorly described. We conducted a retrospective hospital-based cohort study of HFMD patients with central nervous system (CNS) complications caused by EV-A71 or CV-A16 between 2010 and 2016. Patients were classified into three groups, including CNS only, autonomic nervous system (ANS) dysregulation, and cardiorespiratory failure. Neurologic examination, neurodevelopmental assessments, Magnetic Resonance Imaging (MRI) and lung function, were performed at follow up. Of the 176 patients followed up, 24 suffered CNS only, 133 ANS dysregulation, and 19 cardiorespiratory failure. Median follow-up period was 4.3 years (range [1.4-8.3]). The rate of neurological abnormalities was 25% (43 of 171) at discharge and 10% (17 of 171) at follow-up. The rates of poor outcome were significantly different between the three groups of complications in motor (28%, 38%, 71%) domain (p=0.020), but not for cognitive (20%, 24%, 35%), language (25%, 36%, 41%) and adaptive (24%, 16%, 26%) domains (p = 0.537, p = 0.551, p = 0.403). For children with ventilated during hospitalization, 41% patients (14 of 34) had an obstructive ventilatory defect, and one patient with scoliosis had mixed ventilatory dysfunction. Persistent abnormalities on brain MRI were 0% (0 of 7), 9% (2 of 23) and 57% (4 of 7) in CNS, ANS and cardiorespiratory failure group separately. Patients with HFMD may have abnormalities in neurological, motor, language, cognition, adaptive behaviour and respiratory function. Long-term follow-up programmes for children's neurodevelopmental and respiratory function may be warranted.
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Infecciones por Enterovirus/epidemiología , Enterovirus/aislamiento & purificación , Enfermedad de Boca, Mano y Pie/epidemiología , Insuficiencia Cardíaca/epidemiología , Trastornos del Neurodesarrollo/epidemiología , Insuficiencia Respiratoria/epidemiología , Sistema Nervioso Autónomo/virología , Capacidad Cardiovascular , Sistema Nervioso Central/virología , Niño , Preescolar , China/epidemiología , Enterovirus/genética , Infecciones por Enterovirus/virología , Femenino , Estudios de Seguimiento , Enfermedad de Boca, Mano y Pie/virología , Insuficiencia Cardíaca/virología , Hospitalización , Humanos , Lactante , Recién Nacido , Pacientes Internos , Imagen por Resonancia Magnética , Masculino , Trastornos del Neurodesarrollo/virología , Reacción en Cadena de la Polimerasa , Insuficiencia Respiratoria/virología , Estudios RetrospectivosRESUMEN
Coxsackievirus A16 (CV-A16), one of major etiological agents of hand, foot and mouth disease (HFMD), causes outbreaks of the disease in young children all over the world. In order to promote the prevention and control of HFMD, the research and development of CV-A16 vaccine have been carried out in China. However, due to lacking of a recognized CV-A16 antigen detection method, the evaluation and quality control (QC) of vaccine effectiveness are greatly limited. In this study, we established a quantitative enzyme-linked immunosorbent assay (Q-ELISA) to determine the antigen concentration in CV-A16 vaccines that can be applied in manufacturing in China. A neutralizing antibody 16E1 was used as a capture antibody that can bind to various CV-A16 antigens of different subgenotypes, and an antiserum from CV-A16-immunized rabbit conjugated by HRP was suitable for detecting and quantifying CV-A16 antigens. The Q-ELISA was validated for specificity, linearity, accuracy, precision and robustness by using the CV-A16 antigen national standard (NS). Furthermore, we utilized the Q-ELISA to quantify antigen contents of vaccine bulks from six manufacturers and other intermediate products from one manufacturer. The results indicated that the Q-ELISA can satisfy the requirements of QC for all manufacturers involved.
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Enterovirus Humano A , Enterovirus , Enfermedad de Boca, Mano y Pie , Vacunas , Animales , China , Ensayo de Inmunoadsorción Enzimática , Enfermedad de Boca, Mano y Pie/diagnóstico , Enfermedad de Boca, Mano y Pie/prevención & control , ConejosRESUMEN
Enteroviruses (EVs) are positive-sense RNA viruses, with over 50,000 nucleotide sequences publicly available. While most human infections are typically associated with mild respiratory symptoms, several different EV types have also been associated with severe human disease, especially acute flaccid paralysis (AFP), particularly with endemic members of the EV-B species and two pandemic types-EV-A71 and EV-D68-that appear to be responsible for recent widespread outbreaks. Here we review the recent literature on the prevalence, characteristics, and circulation dynamics of different enterovirus types and combine this with an analysis of the sequence coverage of different EV types in public databases (e.g., the Virus Pathogen Resource). This evaluation reveals temporal and geographic differences in EV circulation and sequence distribution, highlighting recent EV outbreaks and revealing gaps in sequence coverage. Phylogenetic analysis of the EV genus shows the relatedness of different EV types. Recombination analysis of the EV-A species provides evidence for recombination as a mechanism of genomic diversification. The absence of broadly protective vaccines and effective antivirals makes human enteroviruses important pathogens of public health concern.
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Human enteroviruses (HEVs) pose an ongoing threat to global public health. Particularly, enterovirus-A71 (EV-A71), the main pathogen causing hand-foot-and-mouth disease (HFMD), has caused ongoing outbreaks globally in recent years associated with severe neurological manifestations and several deaths. Currently, no effective antivirals are available for the prevention or treatment of EV-A71 infection. In this study, we found that sodium copper chlorophyllin (CHL), a health food additive and an over-the-counter anticancer medicine or treatment to reduce the odor of urine or feces, exhibited potent inhibitory activity against infection by divergent EV-A71 and coxsackievirus-A16 (CV-A16) isolates at a low micromolar concentration with excellent safety. The antiviral activity of each was confirmed by colorimetric viral infection and qRT-PCR assays. A series of mechanistic studies showed that CHL did not target the host cell but blocked the entry of EV-A71 and CV-A16 into the host cell at the postattachment stage. In the mouse model, CHL could significantly reduce the viral titer in the lungs and muscles. Since CHL has been used in clinics for many years with excellent safety, it has the potential to be further developed into a prophylactic or therapeutic to prevent or treat HFMD caused by EV-A71 or CV-A16 infection.
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Antivirales/farmacología , Clorofilidas/farmacología , Enterovirus Humano A/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Animales , Infecciones por Enterovirus/tratamiento farmacológico , Enfermedad de Boca, Mano y PieRESUMEN
Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) remain the predominant etiological agents of hand, foot, and mouth disease (HFMD). The clinical manifestations caused by the two viruses are obviously different. CV-A16 usually triggers a repeated infection, and airway epithelial integrity is often the potential causative factor of respiratory repeated infections. Our previous studies have demonstrated that there were some differentially expressed miRNAs involved in the regulation of adhesion function of epithelial barrier in EV-A71 and CV-A16 infections. In this study, we compared the differences between EV-A71 and CV-A16 infections on the airway epithelial barrier function in human bronchial epithelial (16HBE) cells and further screened the key miRNA which leaded to the formation of these differences. Our results showed that more rapid proliferation, more serious destruction of 16HBE cells permeability, more apoptosis and disruption of intercellular adhesion-associated molecules were found in CV-A16 infection as compared to EV-A71 infection. Furthermore, we also identified that microRNA-4516 (miR-4516), which presented down-regulation in EV-A71 infection and up-regulation in CV-A16 infection was an important regulator of intercellular junctions by targeting Poliovirus receptor related protein 1(PVRL1). The expressions of PVRL1, claudin4, ZO-1 and E-cadherin in CV-A16-infected cells were significantly less than those in EV-A71-infected cells, while the expressions of these proteins were subverted when pre-treated with miR-4516-overexpression plasmid in EV-A71 infected and miR-4516-knockdown plasmid in CV-A16 infected 16HBE cells. Thus, these data suggested that the opposite expression of miR-4516 in EV-A71 and CV-A16 infections might be the initial steps leading to different epithelial impairments of 16HBE cells by destroying intercellular adhesion, which finally resulted in different outcomes of EV-A71 and CV-A16 infections.
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Adhesión Celular/genética , Enterovirus Humano A/genética , Enfermedad de Boca, Mano y Pie/patología , MicroARNs/genética , Nectinas/metabolismo , Mucosa Respiratoria/fisiología , Uniones Estrechas/metabolismo , Animales , Apoptosis/genética , Cadherinas/biosíntesis , Línea Celular , Proliferación Celular/fisiología , Chlorocebus aethiops , Claudina-4/biosíntesis , Células Epiteliales/fisiología , Células Epiteliales/virología , Epitelio/fisiología , Epitelio/virología , Células HEK293 , Enfermedad de Boca, Mano y Pie/virología , Humanos , Nectinas/biosíntesis , Permeabilidad , Mucosa Respiratoria/virología , Células Vero , Carga Viral , Proteína de la Zonula Occludens-1/biosíntesisRESUMEN
Using China's national surveillance data on hand, foot and mouth disease (HFMD) for 2008-2015, we described the epidemiologic and virologic features of recurrent HFMD. A total of 398,010 patients had HFMD recurrence; 1,767 patients had 1,814 cases of recurrent laboratory-confirmed HFMD: 99 reinfections of enterovirus A71 (EV-A71) with EV-A71, 45 of coxsackievirus A16 (CV-A16) with CV-A16, 364 of other enteroviruses with other enteroviruses, 383 of EV-A71 with CV-A16 and CV-A16 with EV-A71, and 923 of EV-A71 or CV-A16 with other enteroviruses and other enteroviruses with EV-A71 or CV-A16. The probability of HFMD recurrence was 1.9% at 12 months, 3.3% at 24 months, 3.9% at 36 months, and 4.0% at 38.8 months after the primary episode. HFMD severity was not associated with recurrent episodes or time interval between episodes. Elucidation of the mechanism underlying HFMD recurrence with the same enterovirus serotype and confirmation that HFMD recurrence is not associated with disease severity is needed.
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Enfermedad de Boca, Mano y Pie/epidemiología , Enfermedad de Boca, Mano y Pie/virología , Adolescente , Niño , Preescolar , China/epidemiología , Enterovirus/clasificación , Epidemias , Femenino , Geografía Médica , Enfermedad de Boca, Mano y Pie/historia , Historia del Siglo XXI , Humanos , Lactante , Estimación de Kaplan-Meier , Masculino , Probabilidad , Vigilancia en Salud Pública , Recurrencia , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Human enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. However, we observed chlorpromazine (CPZ) or dynasore (DNS), which inhibit clathrin and dynamin mediated endocytosis, did not suppress EV71 cell entry in particular cell types. So the current knowledge of entry mechanisms by EV71 is not complete. METHODS: Viral infection was examined by flow cytometry or end-point dilution assays. Viral entry was monitored by immunofluorescence or pseudoviral infections. Various inhibitors were utilized for manipulating endocytic pathways. Cellular proteins were knockdown by siRNA. RESULTS: CPZ and DNS did not inhibit but rather enhance viral infection in A549 cells, while they inhibited infections in other cells tested. We further found CPZ did not affect EV71 binding to target cells and failed to affect viral translation and replication, but enhanced viral entry in A549 cells. Immunofluorescence microscopy further confirmed this increased entry. Using siRNA experiment, we found that the enhancement of EV71 infection by CPZ did not require the components of clathrin mediated endocytosis. Finally, CPZ also enhanced infection by Coxackivirus A16 in A549 cells. CONCLUSIONS: CPZ and DNS, previously reported as EV71 entry inhibitors, may rather lead to increased viral infection in particular cell types. CPZ and DNS increased viral entry and not other steps of viral life cycles. Therefore, our study indicated an unknown dynamin-independent entry pathway utilized by enteroviruses that cause Hand-Foot-and-Mouth Diseases.
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Endocitosis/efectos de los fármacos , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/fisiología , Infecciones por Enterovirus/virología , Enfermedad de Boca, Mano y Pie/virología , Internalización del Virus/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Clorpromazina/farmacología , Clatrina/metabolismo , Dinaminas/metabolismo , Infecciones por Enterovirus/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Enfermedad de Boca, Mano y Pie/metabolismo , Humanos , Hidrazonas/farmacología , Acoplamiento Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Objective To study the epidemiology of hand,foot,and mouth disease (HFMD) and the spectrum of serotypes in the other enterovirus (EV) (non-EV-A71 and non-Coxsaekievirus group A 16,CV-A 16) from 2016 to 2017 in Guangzhou,to provide the basis for its treatment,prevention and control.Methods Enteroviruses universal type,EV-A71 and CV-A16 were detected by real time reverse transeription-polymerase chain reaction in the specimens from HFMD suspected patients from 2016 to 2017.The positive specimens of non-EV-A71 and non-CV-A16 were amplified and sequenced based on 5'-untranslated region (UTR) region.The spectrum of serotypes was analyzed with BLAST in NCBI on the basis of 5'-UTR region.Results A total of 25779 specimens from HFMD patients were collected during 2016-2017,16 300 (63.23 ％) of which were positive.The positive rates of EV-A71,CV-A16,non-EV-A71 and non-CV-A16 were 4.57％ (1 178/25 779),12.70％ (3 274/25 779) and 45.96％ (11 848/25779),respectively.The average positive rate of non-EV-A71 and non-CV-A16 in 2017 was 55.68％,which was higher than that in 2016.Sequence analysis showed that there were 16 genotypes in 95 non-EV-A71 and non-CV-A16 positive specimen,including CV-A6,CV-A10,CV-A4,CV-A2,CV-A8,CV-A12,CV-A9,Coxsakievirus B5 (CV-B5),CV-B2,CV-B4,CV-B3,Echovirus 1 (E1),E16,E30,E2 and E18.CV-A6 (26.32％),and CV-A10 (15.79％) were the most common genotypes,followed by CV-A4 (6.32％)、CV-A8(4.21％),and CV-A2 (4.21％).Conclusions The infection rate of EV-A71 is very low during 2016-2017.From April to July 2016,there is a small peak of CV-A16 infection.The non-EV-A71 and non-CV-A16 enterovirus becomes the main causative agent of HFMD during 2016 to 2017.CV-A6 and CV-A10 are the most prevalent pathogens of non-EV-A71 and non-CV-A16 enterovirus.Research and monitoring of CV-A6,CV-A10 as the main non-EV-A71and non-CV-A16 virus should be strengthened.
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Hand foot and mouth disease (HFMD) is a relatively unreported disease in India. This study was undertaken to characterize the enterovirus type/s associated with two unexpectedly-massive epidemics that occurred in Bangalore, India in 2013 and 2015. Stool samples of 229 children with HFMD living in Northern and Southern areas of Bangalore were tested by RT-PCR; 189 (82.5%) were enterovirus positive. The Indian CV-A16 strains exhibited 98-99% sequence identity with those reported in France and China in the 5' untranslated region. BLAST and phylogenetic analyses of complete genomes of representative Indian isolates revealed that the 2015 epidemic was predominated by an inter-species recombinant between CV-A16 and coxsackievirus B5. The 2013 epidemic was primarily caused by nonrecombinant strains. The CV-A16 strains circulated in India since 2007 and phylogeographic analyses indicated imported cases in France and China. In conclusion, CV-A16-associated HFMD epidemics should be recognized as an emerging public health problem in India.
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Brotes de Enfermedades , Enterovirus , Enfermedad de Boca, Mano y Pie/epidemiología , Enfermedad de Boca, Mano y Pie/virología , Regiones no Traducidas 5' , Teorema de Bayes , Epidemias , Evolución Molecular , Genoma Viral , Enfermedad de Boca, Mano y Pie/historia , Historia del Siglo XXI , Humanos , India/epidemiología , Filogenia , Vigilancia de la Población , ARN Viral , Recombinación Genética , Análisis de Secuencia de ARN , Análisis Espacial , Secuenciación Completa del GenomaRESUMEN
This study aimed to investigate the epidemiological characterizations and pathogen spectrum of hand, foot, and mouth disease (HFMD) in Yantai City, Shandong Province, China, during 2011-2015, and to study the nucleotide evolution and amino acid variation of coxsackievirus A16 (CV-A16) epidemic strains that caused HFMD. The HFMD epidemic began to rise in March, and became prevalent from May to August, reached its peak in June, and then declined in September every year, children aged one to 5 years-old had the highest incidence rate whereas the incidence in children under 6 months was very low, and there were more males than females. Enterovirus nucleic acid detection using real-time reverse transcription polymerase chain reaction was performed on 2130 clinical specimens collected from patients with HFMD between 2011 and 2015, and 2012 enterovirus positive samples were detected, including 678 CV-A16, 639 EV-A71, and 695 other enteroviruses. In total, 60 CV-A16 isolates were randomly selected each year for virus isolation, of which 33 CV-A16 strains were randomly selected for further characterization because CV-A16 is the predominant serotype that caused HFMD in Yantai City, and a phylogenetic tree based on the VP1 region was constructed. All 33 CV-A16 strains belonged to the Bla and B1b genotypes, with a nucleotide similarity of 87.9-100% and deduced amino acid similarity of 98.6-100%. Compared with the reference strain Tainan/5079/98 (AF177911), amino acid mutations were identified at positions 11, 23, 25, 31, 99, 145, and 289, where differences were observed among 33 strains, indicating a unique mutation map of CV-A16 in Yantai City. Our findings demonstrate the etiologic characteristics of HFMD, provide supporting evidence for the prevention and control of HFMD, and open a promising avenue for vaccine development against HFMD, by targeting CV-A16.
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Enterovirus Humano A/genética , Enfermedad de Boca, Mano y Pie/epidemiología , Enfermedad de Boca, Mano y Pie/virología , Niño , Preescolar , China/epidemiología , Enterovirus Humano A/aislamiento & purificación , Epidemias , Femenino , Genotipo , Enfermedad de Boca, Mano y Pie/prevención & control , Humanos , Incidencia , Lactante , Masculino , Filogenia , Prevalencia , Reacción en Cadena en Tiempo Real de la Polimerasa , SerogrupoRESUMEN
Coxsackievirus A16 (CV-A16), CV-A6, and enterovirus D68 (EV-D68) belong to the Picornaviridae family and are major causes of hand, foot, and mouth disease (HFMD) and pediatric respiratory disease worldwide. The biological characteristics of these viruses, especially their interplay with the host innate immune system, have not been well investigated. In this study, we discovered that the 3Cpro proteins from CV-A16, CV-A6, and EV-D68 bind melanoma differentiation-associated gene 5 (MDA5) and inhibit its interaction with MAVS. Consequently, MDA5-triggered type I interferon (IFN) signaling in the retinoic acid-inducible gene I-like receptor (RLR) pathway was blocked by the CV-A16, CV-A6, and EV-D68 3Cpro proteins. Furthermore, the CV-A16, CV-A6, and EV-D68 3Cpro proteins all cleave transforming growth factor ß-activated kinase 1 (TAK1), resulting in the inhibition of NF-κB activation, a host response also critical for Toll-like receptor (TLR)-mediated signaling. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed novel mechanisms to subvert host innate immune responses by targeting key factors in the RLR and TLR pathways. Blocking the ability of 3Cpro proteins from diverse enteroviruses and coxsackieviruses to interfere with type I IFN induction should restore IFN antiviral function, offering a potential novel antiviral strategy.IMPORTANCE CV-A16, CV-A6, and EV-D68 are emerging pathogens associated with hand, foot, and mouth disease and pediatric respiratory disease worldwide. The pathogenic mechanisms of these viruses are largely unknown. Here we demonstrate that the CV-A16, CV-A6, and EV-D68 3Cpro proteins block MDA5-triggered type I IFN induction. The 3Cpro proteins of these viruses bind MDA5 and inhibit its interaction with MAVS. In addition, the CV-A16, CV-A6, and EV-D68 3Cpro proteins cleave TAK1 to inhibit the NF-κB response. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed a mechanism to subvert host innate immune responses by simultaneously targeting key factors in the RLR and TLR pathways. These findings indicate the potential merit of targeting the CV-A16, CV-A6, and EV-D68 3Cpro proteins as an antiviral strategy.
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Cisteína Endopeptidasas/metabolismo , Enterovirus/inmunología , Enterovirus/patogenicidad , Interacciones Huésped-Patógeno , Evasión Inmune , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/antagonistas & inhibidores , Proteínas Virales/metabolismo , Proteasas Virales 3C , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteolisis , Transducción de SeñalRESUMEN
Coxsackievirus A16 (CV-A16) causes human hand, foot and mouth disease, but its pathogenesis is unclear. In rhesus macaques, CV-A16 infection causes characteristic vesicles in the oral mucosa and limbs as well as viremia and positive viral loads in the tissues, suggesting that these animals reflect the pathologic process of the infection. An immunologic analysis indicated a defective immune response, which included undetectable neutralizing antibodies and IFN-γ-specific memory T-cells in macaques infected with CV-A16. Furthermore, existing neutralizing antibodies in macaques immunized with the inactivated vaccine were surprisingly unable to protect against a viral challenge despite the presence of a positive T-cell memory response against viral antigens. The virus was capable of infecting pre-conventional dendritic cells and replicating within them, which may correlate with the immunological characteristics observed in the animals.