Echovirus Research Articles

Enterovirus Infection of Human β-Cells Activates Dendritic Cells and Triggers Innate Antiviral Responses: Are Enteroviruses Convicted Now?

Despite a large number of epidemiologic associations, clinical studies, and animal models, a direct involvement of pathogens in the etiology of human type 1 diabetes is still a matter of critical debate. Human enteroviruses (HEV), such as coxsackievirus B (CVB) and echoviruses (EV), have been associated with type 1 diabetes since the late 1960s (1) and have been found in pancreatic isolates of type 1 diabetic patients (2–4) (rev. in 5). However, there are several difficulties hampering the conviction of HEV as inducers of type 1 diabetes: 1 ) pancreatic biopsies are not done on a regular basis in type 1 diabetic patients; 2 ) HEV infections are frequently found in nondiabetic individuals and often remain asymptomatic; 3 ) the clinical manifestation of type 1 diabetes may occur years to decades after infection (virus infection as a “hit-and-run” event); 4 ) type 1 diabetic patients as well as healthy individuals undergo multiple virus infections throughout their lifetime, some of which might even protect them from rather than induce autoimmune disease; and 5 ) genetic predispositions might cover possible environmental (i.e., virus) factors. In this issue of Diabetes , Schulte et al. (6) address the mechanism by which HEV infection of human islets might influence type 1 diabetes onset and/or progression. They found that monocyte-derived dendritic cells (DCs) efficiently phagocytosed CVB3-infected human and porcine islets. Phagocytosis of CVB3-infected islets induced the …

An aseptic meningitis outbreak caused by echovirus 6 in Anhui province, China

An outbreak of aseptic meningitis (AM) occurred in Jinzhai County in Anhui province from April to July in 2005. Totally, 97 children aged 3-15 years were hospitalized. To identify the etiologic agent, 77 cerebrospinal fluid specimens (CSF) and 5 fecal specimens were collected from the patients and cultured by human rhabdomyosarcoma (RD) cell line. Thirty isolates of human echovirus 6 (E6) from 27 CSF and 3 fecal specimens were confirmed by neutralization assay and sequencing analysis of the VP1 gene. The homology of VP1 gene among Anhui isolates was 99.7-100.0% and it indicated that this AM outbreak probable caused by a single transmission link of E6. Phylogenetic analysis based on all the available complete VP1 sequences indicated that E6 could be divided into clusters A, B, and C with at least 15% diversity between clusters and the C cluster could be further divided into C1, C2, C3, and C4. The Anhui isolates most resembled a 2005 strain from Russia (25465 Tambov) and belong to C4. This is the first report that E6 was responsible for an outbreak of AM in China. J. Med. Virol. 82:441-445, 2010. (c) 2010 Wiley-Liss, Inc.

Genetic Diversity of Echovirus 6 Strains Circulating in Korea

Echovirus 6 (ECV6) is the prevalent serotype detected in aseptic meningitis cases in Korea. To analyze the genetic variation of ECV6 isolates recently circulating in Korea, we determined the partial sequence of the VP1 capsid gene from 22 Korean ECV6 isolates and performed pairwise analysis against 42 reference strains from the GenBank database using MegAlign. The 22 Korean ECV6 isolates formed 3 distinct genetic clusters: Kor-lineage Ⅰ, Ⅱ, and Ⅲ. The Korean ECV6 strains showed significant genetic diversity with 14.8~22.8% nucleotide divergence among the 3 different lineages. These ECV6 Kor-lineages were demonstrated to belong to different genetic clusters using VP1 sequence-based phylogenetic analysis, implying that the recently circulating Korean ECV6 strains have potential antigenic variation.

Activités antivirale et antioxydante in vitro d'huiles essentielles de Thymus capitatus (L.) Hoffmans. & Link de Tunisie

Three essential oil samples of Thymus capitatus (L.) Hoffmans. & Link were tested for cytotoxicity by MTT assay in Vero cells and antiviral activity against type 1 Herpes simplex virus (HSV1), Echovirus 11 (ECV11) and Adenovirus (ADV) using cytopathic effect (CPE) reduction essay. In order to determine the mode of antiviral action of the essential oil, thyme was added at different times to the cells or viruses during infection. Both HSV1 and ECV11 were significantly inhibited when virus were pretreated with the essential oil prior to adsorption, while ADV was affected after penetration into the host cell. In vitro antioxidant properties of the essential oils, obtained by DPPH and ABTS assays, were interesting compared to the synthetic antioxidant Trolox.

Vitronectin receptors, v integrins, are recognized by several non-RGD-containing echoviruses in a continuous laboratory cell line and also in primary human Langerhans' islets and endothelial cells

Previously published data suggest that the RGD-recognizing integrin, alphavbeta3, known as the vitronectin receptor, acts as a cellular receptor for RGD-containing enteroviruses, coxsackievirus A9 (CAV-9) and echovirus 9 (E-9), in several continuous cell lines as well as in primary human Langerhans' islets. As this receptor is also capable of binding the ligands by a non-RGD-dependent mechanism, we investigated whether vitronectin receptors, alpha v integrins, might act as receptors for other echoviruses that do not have the RGD motif. Blocking experiments with polyclonal anti-alphavbeta3 antibody showed that both primary human islets and a continuous laboratory cell line of green monkey kidney origin (GMK) are protected similarly from the adverse effects of several non-RGD-containing echovirus (E-7, -11, -25, -30, -32) infections. In contrast, corresponding studies on primary human endothelial cells showed that the receptor works only for E-25, E-30, E-32 and CAV-9. The inhibitory effect of the antibody was not restricted to prototype strains of echoviruses, as GMK cells infected with several field isolates of the corresponding serotypes were also protected from virus-induced cytopathic effects. Co-localization of virus particles with the receptor molecules in both GMK and primary human endothelial cells was demonstrated by live-cell stainings and confocal microscopy. Remarkably, in spite of similar virus-receptor co-localization and a comparable protective effect of the alphavbeta3 antibody, the entry pathways of the studied virus strains seemed to be divergent.

Two clusters of mutations map distinct receptor-binding sites of echovirus 11 for the decay-accelerating factor (CD55) and for canyon-binding receptors

In this study we present the comparative sequence analysis of the parental haemagglutinating ( daf+) and mutant non-haemagglutinating ( daf−) clones of echovirus 11 (EV11) isolated from the prototype strain Gregory. The sequence comparison revealed only a single amino acid substitution in the capsid protein VP2 of each mutant clone. These substitutions were located in the area of viral receptor-binding site for DAF. Since daf− mutants of EV11 did not interact with DAF, they used an alternative receptor for the cell entry. To elucidate the nature of the alternative receptor we used subvariant clones of EV11 adapted to human rhabdomyosarcoma (RD), human carcinoma (HEp-2) and African Green monkey kidney (BGM) cell lines. The usage of the subvariant clones with altered host range and the cell cultures of human and simian origin allowed us to map the amino acid substitutions associated with the adaptation of EV11 to the alternative cellular receptors. These amino acid substitutions were located on the surface of the virion in the canyon area. Hence the virus canyon may serve as the receptor-binding site for the alternative (in respect to DAF) cellular receptor(s).

Variability of Virus Antibodies in Intravenous Immune Globulins: West Nile Virus, Echo Viruses 9, 11, 13, 30

RATIONALE: Individual lots of IVIG are produced from thousands of plasma donations, and thus reflect the serostatus of a large donor population. The titers against emerging viruses (e.g. West Nile Virus) or viruses with fluctuating epidemiology (e.g. Echo viruses) may still vary in IVIG, depending on where/from which donor community plasma was collected. METHODS: Neutralizing antibodies were determined for • West Nile Virus • Echo virus serotypes 9, 11, 13, 30 in IVIG lots produced from plasma collected in different • geographies, i.e. Europe and the US • donor communities, i.e. plasmapheresis vs. recovered from blood donations RESULTS: Testing IVIG lots produced from plasma collected prior to and after the 1999 introduction of WNV into the US, the seroconversion of parts of the US population (approx. 1%) is demonstrated. The WNV neutralization titers of currently produced IVIG lots still differ significantly, ranging from undetectable to comparable with individuals with past WNV infection. Echovirus neutralization titers in IVIG lots vary widely, for different serotypes as well as different geographies and plasma donor communities. The information may be relevant in the rare situations when Echovirus infections occur even in IVIG substituted individuals. CONCLUSIONS: IVIG lots reflect the virus epidemiology of specific donor communities, and some of the differences observed may potentially be of relevance in certain clinical circumstances.

The incidence and aetiology of central nervous system infections in Helsinki in 1980.

The aetiology of central nervous system infections was surveyed in a study at Aurora Hospital, Helsinki, in 1980. Of the 146 patients with central nervous system infections, 113 had aseptic meningitis, 23 bacterial and one tuberculous meningitis, and nine meningoencephalitis or encephalitis. The probable aetiology of aseptic meningitis was established in 67% of the 106 patients properly tested, the commonest agents being mumps (27%), Coxsackie (24%) and ECHO (9%) viruses. Haemophilus influenza type b was the most frequent cause of bacterial meningitis (39%), occurring solely among infants and young children. There were no cases due to Neisseria meningitidis group A, which used earlier to be epidemic in Finland. The incidences of aseptic, bacterial and tuberculous meningitis in Helsinki in 1980 (based on a total of 174 patients treated in the three hospitals admitting patients with central nervous system infections) were 26.7, 5.2 and 0.2 cases per 100,100 annually, and those of encephalitides and myelitis 3.5 and 0.6 cases per 100,000 annually.

Acute peripheral facial palsy: CSF findings and etiology

CSF and serum were examined in acute and convalescence phase from 56 patients with acute idiopathic peripheral facial palsy. CSF protein analysis, viral and borrelia serology were performed. Borrelia infection was found in 9/56 cases and was often associated with inflammatory CSF findings. One patient each had serological evidence for a recent or ongoing infection with herpes simplex, varicella zoster, adeno, influenza B, echo and Epstein-Barr virus, but none had specific intrathecal antibody synthesis; 11 patients had a serological pattern compatible with a reactivated Epstein-Barr virus infection. Eleven patients displayed mononuclear CSF pleocytosis. Four of them had a borrelia infection. A disturbed blood-brain barrier was observed in 19 patients. Intrathecal immunoglobulin synthesis as indicated by elevated IgM-indices was found in 16 patients and by IgG indices in three. Nine patients had oligoclonal IgG bands in serum and CSF, three exclusively in CSF. It is concluded that patients with facial palsy often have inflammatory CSF findings, indicating a generalised central nervous system affection, and not only a mononeuritis. The importance of viral infections in the pathogenesis is still obscure. Borrelia is the most common infectious cause of facial palsy.

Molecular epidemiology of Echovirus 6 in Greece

The objective was to investigate the genetic relationships among Echovirus 6 (E6) strains circulating in Greece and to compare them with the respective strains from other geographic regions. Cerebrospinal fluid samples collected during the period 2006-2007 from 84 patients with aseptic meningitis or encephalitis were tested for a probable enteroviral infection. Two RT-PCRs amplifying overlapping regions of the VP1 gene were performed, while isolation procedures were applied in one third of cases. All PCR products were sequenced, and further phylogenetic analysis was performed for E6 strains. Enteroviruses were detected in 27 out of 84 cases (32.14%) and E6 was the predominant serotype (11 out of 27, 40.74%). Three distinct clades of Greek E6 sequences were seen in the phylogenetic tree: sequences of the present study were placed in clades A and B, while sequences of a former study in Greece were clustered in clade C. Sequences of clades A and C presented high genetic homology (>95%) with sequences from other countries, while sequences of clade B were unique, differing by more than 15% from all known E6 sequences. The most prevalent enterovirus in Greece during the period 2006-2007 was E6, and was associated with aseptic meningitis. A high degree of heterogeneity was observed among Greek E6 strains.

Molecular identification and clinical features of enteroviral infection in children of central Korea: An overview of enteroviral epidemiology between spring 2005 and autumn 2006

= Abstract = Purpose : Enteroviruses (EVs) are commonly known to cause infection, especially in infants and children. This report presents an overview of enterovirus epidemiology in central Korea. Methods : From the spring of 2005 to the autumn of 2006, we collected the cerebrospinal fluid (CSF) and stool samples from the pediatric patients with a febrile illness or suspected meningitis who were admitted to hospitals in central Korea. In order to test for EVs, cell lines were derived from pretreated susceptible specimen, and the cytopathic effects were observed. Seminested real time-polymerase chain reaction (RT-PCR) and direct sequencing were performed for genotypic and phylogenetic analyses. Results : Of the 305 patients examined, 51 (16.7%) tested positive for EV. Of these 51 patients, 44 showed the following serotypes: Echovirus (ECV) 18 (18 cases, 35.2%), Coxsackievirus B (CVB) 5 (13 cases, 25.4%), ECV25 (5 cases, 9.8%), ECV9 (4 cases, 7.8%), ECV5 (3 cases, 5.8%), and EV74 (1 case, 1.9%). In 2005, between June and August, ECV18 and CVB5 were mostly responsible for the enteroviral infections among the patients in central Korea. In 2006, between July and August, ECV25 was mostly the cause of enteroviral infection. Conclusions : There is a need for continuous surveillance of enteroviral infection and its clinical manifestations, particularly for EV74, which was first identified in Korea. (Korean J Pediatr 2009;52:1234-1240)

Echovírus 6 associado à doença exantemática

Exantema viral é considerado problema comum em regiões tropicais, afetando principalmente crianças. Diversos exantemas cutâneos estão associados a infecções por Enterovirus. Amostras biológicas provenientes de uma criança apresentando exantema generalizado foram enviadas ao Laboratório de Vírus Entéricos do Instituto Adolfo Lutz para a realização do diagnóstico laboratorial. Amostra viral isolada em RD (human rhabdomyosarcoma cells) foi submetida à reação em cadeia pela polimerase apresentando um produto de 437 pares de base, característico de gênero Enterovirus. O sorotipo echovirus 6 (E-6) foi identificado por ensaio de imunofluorescência indireta. Em adição, as amostras pareadas de soro apresentaram soroconversão para E-6. Até o momento, não há relatos do envolvimento de E-6 associado a doenças exantemáticas no Brasil, enfatizando a importância da vigilância epidemiológica para essas doenças e suas complicações.

Génotypage direct rapide des entérovirus dans le liquide céphalorachidien : étude prospective de deux ans au sein d’un laboratoire de virologie clinique

Enterovirus (EV - 68 serotypes) infections comprise a wide spectrum of clinical presentations including infections of the central nervous system. In severe clinical presentation or epidemics, the precise identification of the involved serotype is necessary. To perform enterovirus genotyping directly in cerebrospinal fluid (CSF) samples, and to assess its feasibility in a laboratory setting. Enterovirus genotyping was carried out directly with CSF specimens tested for the diagnostic procedure by amplifying the complete 1D gene encoding the VP1 protein of the HEV-B serotypes (the most frequent) - providing results in two days. Secondly, sequences 1A/1B encoding the VP4/VP2 capsid proteins, respectively, were analysed (results in five days). Direct enterovirus genotyping allowed the identification of enterovirus involved in 77 out of 81 (95%) meningitis cases between January 2006 and December 2007. In combination with the indirect genotyping of enterovirus isolates, identification of the type was achieved in 94 out of 97 (96.9%) patients included in the study. The most frequent serotypes were echovirus 6 (E6) and 13 in 2006, coxsackievirus B2 and E30 in 2007. Four children presented an EV71 associated meningitis. When prospectively applied in a laboratory setting, direct enterovirus genotyping in CSF samples allows the identification of the involved enterovirus in two to five days. This time frame is relevant for an optimal patient management, the rapid identification of a new enterovirus variant or in the context of an epidemic alert.

Synthesis and antiviral activity of 4-quinolinecarboxylic acid hydrazides

Antiviral agents were sought by using ethyl esters of 2-R-4-quinolinecarboxylic acids to synthesize the corresponding aryl (heteryl) hydrazides. 2-Methyl-4-quinolinecarboxylic acid hydrazide was used to synthesize vinylogs containing a nitro group in the substituent. The resulting compounds were tested for antiviral activity against ECHO virus type 6.

Role of class I human leukocyte antigen molecules in early steps of echovirus infection of rhabdomyosarcoma cells

Several echoviruses use decay accelerating factor (DAF) as a cell surface receptor. However, most of them require additional cell surface coreceptors. We investigated the respective roles of DAF and class I human leukocyte antigen (HLA) molecules in the early steps of the echovirus 11 (EV11) lifecycle in rhabdomyosarcoma (RD) cells. EV11 infection was inhibited at an early stage by anti-β2-microglobulin (β2m) and anti-HLA monoclonal antibodies and by a soluble monochain HLA class I molecule. Expression of class I HLA molecules restored the early steps of the EV11 lifecycle, but its expression was not sufficient for EV11 replication and particle production. Expression of HLA class I molecules was associated with leukocyte cell line permissiveness to EV11 infection. In conclusion, HLA class I molecules are involved in the early steps of EV11 infection of RD cells and appear to participate in a complex interplay of surface molecules acting as coreceptors, including DAF.

Monoclonal antibody specific for the cellular receptor of echoviruses.

Cell lines of primate origin carry membrane receptors which are specific for echoviruses (EV). The present report describes isolation and characterization of a monoclonal antibody (Mab 143) reacting with the membrane of KB cells. The Mab was selected for its protection of different cell lines from primate origin against the CPE of EV-11. This protection was found to extend to most EV serotypes and to coxsackievirus A9, while the replication of several other picornaviruses was not affected. The fluorecein isothiocyanate labelled Mab did not react with cell lines from bovine, canine, or rabbit origin, but bound specifically to the cell lines from human or simian origin. These results suggest that a unique receptor site is used by most EV serotypes for binding onto and penetration into susceptible cells.

Laboratory testing of specimens from patients with viral encephalitis from some regions of China

To understand the viral etiology of viral encephalitis in China by detecting IgM antibody and viral RNA in the clinical samples of patients from some provinces of China by enzyme-linked immunosorbent assay and polymerase chain reaction. Serum and cerebrospinal fluid samples of 771 patients with viral encephalitis or meningitis were collected from six provinces of China and were stored at -20 degrees C or -70 degrees C. Enzyme-linked immunosorbent assays were used for detection of IgM antibody to Japanese encephalitis virus, coxsackievirus, echovirus, herpes simplex virus, measles virus, varicella-zoster virus, mumps virus, cytomegalovirus and Epstein-Barr virus. Polymerase chain reaction was applied for the detection of viral RNA of enteroviruses and seadornavirus. IgM antibody was detected in 567 of 771 (73.5%) cases. The most common pathogen was Japanese encephalitis virus (47.0%, 362/771), followed by mumps virus (10.6%, 82/771), enteroviruses (8.8%, 68/771), herpes simplex virus (5.7%, 44/771), measles virus (0.4%, 3/771), varicella-zoster virus (0.4%, 3/771), Epstein-Barr virus (0.4%, 3/771), and cytomegalovirus (0.3%, 2/771). Enterovirus was positive in 8 cases, seadornavirus was negative in all the cases by PCR. According to the study, Japanese encephalitis was the most important encephalitis in China. Mumps virus was another important pathogen. Enteroviruses and herpes simplex virus were also important pathogens.

Phagocytosis of Picornavirus-Infected Cells Induces an RNA-Dependent Antiviral State in Human Dendritic Cells

Dendritic cells (DCs) play a central role in instructing antiviral immune responses. DCs, however, can become targeted by different viruses themselves. We recently demonstrated that human DCs can be productively infected with echoviruses (EVs), but not coxsackie B viruses (CVBs), both of which are RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. We now show that phagocytosis of CVB-infected, type I interferon-deficient cells induces an antiviral state in human DCs. Uptake of infected cells increased the expression of the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 as well as other interferon-stimulated genes and protected DCs against subsequent infection with EV9. These effects depended on recognition of viral RNA and could be mimicked by exposure to the synthetic double-stranded RNA analogue poly(I:C) but not other Toll-like receptor (TLR) ligands. Blocking endosomal acidification abrogated protection, suggesting a role for TLRs in the acquisition of an antiviral state in DCs. In conclusion, recognition of viral RNA rapidly induces an antiviral state in human DCs. This might provide a mechanism by which DCs protect themselves against viruses when attracted to an environment with ongoing infection.

Molecular phylogeny and genetic variability of echovirus 30 based on the analysis of the 3' end of the VP1 gene

Echovirus 30 represent one of the most frequently isolated enterovirus serotype, incriminated in various pathologies, essentially aseptic meningitis. Several works studied the molecular epidemiology of these viruses. By analysing a region of 260 nucleotides situated in the end of the VP1 gene (region regrouping the majority of the sequences of the Echovirus 30), we proposed to realise a synthesis work which regroup the main epidemiological studies on the Echovirus 30. We established a phylogenetic profile of 87 Echovirus strains geographically distinct and isolated during a half a century (1957-2003). The phylogentic tree permitted to distinguish 2 genogroups which the nucleotide divergence exceeds 20%. The 2 genogroups also present internal subdivisions named genotypes which the nucleotide divergence is more than 15%. Finally, we noted phylogenetic regroupings within a same genotype. The general profile of the phylogenetic tree is characterised by a distribution of the Echovirus 30 strains in the time independently of their geographically isolation, which reveals a genetic evolution of these viruses related to their high genetic plasticity and the rapid circulation from a geographic area to another.

Echovirus 6 strains derived from a clinical isolate show differences in haemagglutination ability and cell entry pathway

Two echovirus 6 (EV6) strains were isolated from a clinical sample after successive sub-cultures in PLC (human hepatocellular carcinoma) and HeLa (human cervical adenocarcinoma) cells. The first strain retained its haemagglutinating capacity (HAEV6) while the second became non-haemagglutinating (NHAEV6). Virus binding assay showed that HAEV6 was capable of binding to DAF-expressing cells but not NHAEV6 confirming the role of DAF in EV6 haemagglutination. The lack of competition between the two viral strains during coinfections suggested that each strain used a different cell entry pathway. We provide evidence showing that HAEV6 used preferentially the lipid raft-dependent caveolae pathway, whereas NHAEV6 followed the clathrin-mediated pathway. Comparison of the sequences of HAEV6 and NHAEV6 revealed five amino acid changes in the VP1, VP2 and VP3 capsid proteins distributed in domains which are known to be highly immunogenic or suggested to be involved in receptor binding, virion stability and pathogenicity.