Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor

Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-D-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor

Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes

BACKGROUND: Human enterovirus 71 (EV-71) is a common causative agent of hand, foot and mouth disease (HFMD). In recent years, the virus has caused several outbreaks with high numbers of deaths and severe neurological complications. Several new EV-71 subgenotypes were identified from these outbreaks. The mechanisms that contributed to the emergence of these subgenotypes are unknown. RESULTS: Six EV-71 isolates from an outbreak in Malaysia, in 1997, were sequenced completely. These isolates were identified as EV-71 subgenotypes, B3, B4 and C2. A phylogenetic tree that correlated well with the present enterovirus classification scheme was established using these full genome sequences and all other available full genome sequences of EV-71 and human enterovirus A (HEV-A). Using the 5' UTR, P2 and P3 genomic regions, however, isolates of EV-71 subgenotypes B3 and C4 segregated away from other EV-71 subgenotypes into a cluster together with coxsackievirus A16 (CV-A16/G10) and EV-71 subgenotype C2 clustered with CV-A8. Results from the similarity plot analyses supported the clustering of these isolates with other HEV-A. In contrast, at the same genomic regions, a CV-A16 isolate, Tainan5079, clustered with EV-71. This suggests that amongst EV-71 and CV-A16, only the structural genes were conserved. The 3' end of the virus genome varied and consisted of sequences highly similar to various HEV-A viruses. Numerous recombination crossover breakpoints were identified within the non-structural genes of some of these newer EV-71 subgenotypes. CONCLUSION: Phylogenetic evidence obtained from analyses of the full genome sequence supports the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the most common causal agent of HFMD. It is suggested that these recombination events played important roles in the emergence of the various EV-71 subgenotypes

Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC50 values ranging from 6–9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71

Recent developments in antiviral agents against enterovirus 71 infection

Enterovirus 71 (EV-71) is the main etiological agent of hand, foot and mouth disease (HFMD). Recent EV-71 outbreaks in Asia-Pacific were not limited to mild HFMD, but were associated with severe neurological complications such as aseptic meningitis and brainstem encephalitis, which may lead to cardiopulmonary failure and death. The absence of licensed therapeutics for clinical use has intensified research into anti-EV-71 development. This review highlights the potential antiviral agents targeting EV-71 attachment, entry, uncoating, translation, polyprotein processing, virus-induced formation of membranous RNA replication complexes, and RNA-dependent RNA polymerase. The strategies for antiviral development include target-based synthetic compounds, anti-rhinovirus and poliovirus libraries screening, and natural compound libraries screening. Growing knowledge of the EV-71 life cycle will lead to successful development of antivirals. The continued effort to develop antiviral agents for treatment is crucial in the absence of a vaccine. The coupling of antivirals with an effective vaccine will accelerate eradication of the disease

Estudos soroepidemiológico e molecular de infecção por EV-71 no Estado do Pará, Brasil

In many countries, the Enterovirus 71 (EV-71) Picornaviridae family is associated to hand, foot and mouth disease in addition to acute neurological diseases while in Brazil these viruses are more closely associated to the latter group. The aim of this research was to use the first EV-71 isolate of the Northern region of Brazil in molecular and seroepidemiologic studies. Two (2.2%) out of 88 stool samples (44 cases of AFP), collected from January 1998 to December 2000 were positive for EV-71 isolation (73442/PA/99). Nucleotide sequence of the gen that codifies the VP1 protein showed that isolate 73442/PA/99 was similar to the EV-71 strains belonging to genotype B - more closely identified with EV-71 from North America. Neutralization test with 389 sera samples collected from January 1998 to November 2001, from individuals ranging from 0 to 15 years of age living in the city of Belém, State of Pará showed the following results in relation to isolate 73442/PA/99 and prototype BrCr: a total of 207 individuals (53.2%) had neutralization antibodies to both viruses, 167 (42.9%) had no antibodies and 15 showed the presence of neutralizing antibodies to one of the two viruses. Only 20.2% of the children aged 0 to 3 had neutralizing antibodies to EV-71, indicating that these children were more susceptible to the infection. Both the seroprevalence study and VP1 sequencing were important to demonstrate the spread and the molecular pattern of the EV-71 circulating in the Northern Region of Brazil.Em muitos países, o Enterovírus 71 (EV-71) família Picornaviridae é associado a doença de pé-mão e boca e doenças neurológicas agudas enquanto que no Brasil esse vírus está mais associado às últimas. O objetivo desta pesquisa foi utilizar em estudos moleculares e soroepidemiológicos, o primeiro isolamento de EV-71 obtido na região norte do Brasil. No período de janeiro de 1998 a dezembro de 2000 foram coletadas 88 amostras (44 casos de PFA) de fezes das quais, duas (2,2%) foram positivas para EV-71 (73442/PA/99). A seqüência de nucleotídeos do gen que codifica a proteína VP1 mostrou que o isolado 73442/PA/99 foi similar às cepas de EV-71 pertencentes ao grupo B- mais próxima das norte americanas. Teste de neutralização com 389 amostras de soro colhidas no período de janeiro de 1998 a novembro de 2001, de indivíduos com idade de 0 a 15 anos residentes na cidade de Belém, Estado do Pará mostrou os seguintes resultados em relação ao isolado 73442/PA/99 e ao protótipo BrCr: 207 indivíduos (53,2%) tinham anticorpos neutralizantes para ambos os vírus, 167 (42,9%) não tinham anticorpos e 15 tinham anticorpos para um dos dois vírus. Somente 20,2% das crianças com idade de 0 a 3 anos tinham anticorpos neutralizantes para EV-71, indicando que essas crianças estavam mais suscetíveis à infecção. Tanto o estudo de soroprevalência quanto o de sequenciamento da VP1 foram importantes para demonstrar a propagação e o padrão molecular do EV-71 circulante na região norte do Brasil

PLoS One

Different subgenogroups of enterovirus 71 (EV-71) have caused numerous outbreaks of hand, foot, and mouth disease worldwide, especially in the Asia-Pacific region. During the development of a vaccine against EV-71, the genetic and antigenic diversities of EV-71 isolates from Taiwan were analyzed by phylogenetic analyses and neutralization tests. The results showed that the dominant genogroups had changed twice, from B to C and from C to B, between 2009 and 2012. The subgenogroup B5 (B5b cluster) was dominant in 2008-2009 but was replaced by subgenogroup C4 in 2010-2011. From the end of 2011 to 2012, the re-emerging subgenogroup B5 (B5c cluster) was identified as the dominant subgenogroup of EV-71 outbreaks, and subgenogroups C2 and C4 were detected in sporadic cases. Interestingly, the amino acid substitution at position 145 in the VP1 gene was observed in some strains isolated from patients with acute flaccid paralysis. Furthermore, thirty-five strains and their corresponding serum samples were used to analyze the cross-protections and antigenic diversities among different subgenogroups (C4a, C5, B4, B5b, B5c, and C2-like) of EV-71. Evident antigenic diversity existed only for the C2-like subgenogroup, which was not effectively neutralized by other serum samples. In contrast, the anti-C2-like serum sample showed broad cross-reactivity against all other subgenogroups. Therefore, these results may provide valuable information for the selection of EV-71 vaccine candidates and the evolution of EV-71 subgenogroups in Taiwan from 2009 to 2012

Genetic diversity and C2-like subgenogroup strains of enterovirus 71, Taiwan, 2008

<p>Abstract</p> <p>Background</p> <p>Human enterovirus 71 (EV-71) is known of having caused numerous outbreaks of hand-foot-mouth disease, and other clinical manifestations globally. In 2008, 989 EV-71 strains were isolated in Taiwan.</p> <p>Results</p> <p>In this study, the genetic and antigenic properties of these strains were analyzed and the genetic diversity of EV-71 subgenogroups surfacing in Taiwan was depicted, which includes 3 previously reported subgenogroups of C5, B5, and C4, and one C2-like subgenogroup. Based on the phylogenetic analyses using their complete genome nucleotide sequences and neutralization tests, the C2-like subgenogroup forms a genetically distinct cluster from other subgenogroups, and the antisera show a maximum of 128-fold decrease of neutralization titer against this subgenogroup. In addition, the subgenogroup C4 isolates of 2008 were found quite similar genetically to the Chinese strains that caused outbreaks in recent years and thus they should be carefully watched.</p> <p>Conclusions</p> <p>Other than to be the first report describing the existence of C2-like subgenogroup of EV-71 in Taiwan, this article also foresees a potential of subgenogroup C4 outbreaks in Taiwan in the near future.</p

Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain

Producción CientíficaHand, foot and mouth disease (HFMD) is a childhood illness frequently caused by genotypes belonging to the enterovirus A species, including coxsackievirus (CV)-A16 and enterovirus (EV)-71. Between 2010 and 2012, several outbreaks and sporadic cases of HFMD occurred in different regions of Spain. The objective of the present study was to describe the enterovirus epidemiology associated with HFMD in the country. A total of 80 patients with HFMD or atypical rash were included. Detection and typing of the enteroviruses were performed directly in clinical samples using molecular methods. Enteroviruses were detected in 53 of the patients (66%). CV-A6 was the most frequent genotype, followed by CV-A16 and EV-71, but other minority types were also identified. Interestingly, during almost all of 2010, CV-A16 was the only causative agent of HFMD but by the end of the year and during 2011, CV-A6 became predominant, while CV-A16 was not detected. In 2012, however, both CV-A6 and CV-A16 circulated. EV-71 was associated with HFMD symptoms only in three cases during 2012. All Spanish CV-A6 sequences segregated into one major genetic cluster together with other European and Asian strains isolated between 2008 and 2011, most forming a particular clade. Spanish EV-71 strains belonged to subgenogroup C2, as did most of the European sequences circulated. In conclusion, the recent increase of HFMD cases in Spain and other European countries has been due to a larger incidence of circulating species A enteroviruses, mainly CV-A6 and CV-A16, and the emergence of new genetic variants of these viruses

Virus Res

Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11-21 aa contain EV-71-specific antigenic sites, whereas positions 1-5 and 51-100 contain epitopes shared with human coxsackievirus A16 (CV-A16) and human echovirus 6 (E-6). The N-terminal truncated protein of VP1, VP\ue2\u201a?\ue2\u201a\u2020\ue2\u201a\u2039\ue2\u201a\u201e\ue2\u201a\u192, exhibited good stability and was recognized by anti-EV-71 specific rabbit sera. Alignment analysis showed that VP\ue2\u201a?\ue2\u201a\u2020\ue2\u201a\u2039\ue2\u201a\u201e\ue2\u201a\u192 is highly conserved among EV-71 strains from different genotypes but was heterologous among other enteroviruses. When the GST-VP\ue2\u201a?\ue2\u201a\u2020\ue2\u201a\u2039\ue2\u201a\u201e\ue2\u201a\u192 fusion protein was incorporated as antibody-capture agent in a WB assay and an ELISA for detecting anti-EV-71 IgM in human sera, sensitivities of 91.7% and 77.8% were achieved, respectively, with 100% specificity for both. The characterized EV-71 VP1 protein truncated to positions 6-43 aa has potential as an antigen for detection of anti-EV-71 IgM for early diagnosis of EV-71 infection in a WB format.CC999999/Intramural CDC HHS/United States2016-01-04T00:00:00Z24952304PMC469880