Human Coxsackievirus Research Articles

Redistribution of accumulated 2,3,7,8-tetrachlorodibenzo- p-dioxin during coxsackievirus B3 infection in the mouse

The tissue redistribution of accumulated 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) during infection was studied in adult male A/J mice using a common human virus coxsackievirus B3. Before infection (day 1), all mice were injected intraperitoneally (i.p.) with 1 μCi 3H-TCDD, corresponding to 0.5 μg TCDD kg −1. One group was sacrificed before virus inoculation (day 0). Of the remaining mice, one subgroup was inoculated i.p. with CB3 virus while the other subgroup served as a noninfected control. On days 0, 4 and 7, the spleen, thymus, heart, pancreas, liver, white and brown adipose tissue, skeletal muscle, lung, kidney, brain, adrenals, thyroid, testes, epididymis and blood were sampled from infected and noninfected groups. Liquid scintillation was used to determine the 3H-TCDD-content of the tissues. The results showed that the accumulated TCDD was redistributed due to infection. The major changes occurred in the organs involved in the infectious process. In the target organs for coxsackievirus B3 (the pancreas and heart), the TCDD concentration peaked in relation to noninfected control values, concurrent with the development of inflammatory lesions ( P<0.001 and 0.01, respectively for the heart and pancreas). The TCDD levels in the thymus increased three-fold during the infection to an estimated 0.5 pmol g −1 tissue on day 7 of the infection, whereas the levels in noninfected mice did not change markedly ( P<0.001). In the brain of infected mice, the TCDD concentration increased significantly with time, at day 7 reaching two-fold levels in comparison with noninfected controls ( P<0.001). The findings suggest that a common infection causes redistribution of a previously accumulated environmental pollutant, resulting in increased concentrations and potentially increased toxicity in selected target tissues.

The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10.5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Genomic organization and chromosomal localization of the human Coxsackievirus B-adenovirus receptor gene

Genomic organization and chromosomal localization of the human Coxsackievirus B-adenovirus receptor gene

The complete consensus sequence of coxsackievirus B6 and generation of infectious clones by long RT-PCR

The full length sequence for the human pathogen coxsackievirus B6 (CVB6, Schmitt strain) has been determined. We used long RT-PCR to generate full length DNA amplicon of CVB6, and then directly sequenced the amplicons. One-step cloning of the full length amplicon enabled us to obtain an infectious clone of CVB6. RNA generated from CVB6 amplicon DNA or CVB6 clones, by transcription with T7 RNA polymerase, was demonstrated to be infectious upon transfection into HeLa cells in vitro. The CVB6 genome is characteristic of enteroviruses, with a 5′-non-translated region (743 nucleotides) followed by an open reading frame (encoding a 2184 amino acid polyprotein) and a 3′-non-translated region (100 nucleotides) and polyadenylated tail. The predicted amino acid sequence of CVB6 clustered with the other CVB serotypes and swine vesicular disease virus (SVDV).

Genomic organization and chromosomal localization of the human Coxsackievirus B-adenovirus receptor gene.

Myocarditis and dilated cardiomyopathy (DCM) are common causes of morbidity and mortality in children. Many studies have implicated the enteroviruses and, particularly, the Coxsackievirus-B family as etiologic agents of the acquired forms of these diseases. However, we have shown the group-C adenoviruses to be as commonly detected as enteroviruses in the myocardium of children and adults with these diseases. It has remained something of a conundrum why two such divergent virus families cause these diseases. The recent description of the common human Coxsackievirus B-adenovirus receptor (CAR) offers at least a partial explanation. In order to characterize the CAR gene, we screened a bacterial artificial chromosomal (BAC) library (RPCI11) using a polymerase chain reaction (PCR) product derived from the 3' end of the CAR cDNA sequence. This identified 13 BACs that were further characterized by PCR amplification of seven contiguous regions of the entire cDNA sequence. Eleven of the BACs were determined to encode pseudogenes while the other two BACs (131J5 and 246M1) encoded the presumed functional gene. PCR amplification of a monochromosomal hybrid panel indicated the presence of pseudogenes on chromosomes 15, 18, and 21 while the functional gene is encoded on chromosome 21. Fluorescence in situ hybridization analysis indicated that the gene is located at 21q11.2. DNA sequencing of BACs 131J5 and 246M1 revealed the presence of seven exons. The DNA sequences have been determined for each exon-intron boundary, and putative promoter sequences and transcription initiation sites identified. No consensus polyadenylation signal was identified.

Expression of the coxsackievirus and adenovirus receptor in cultured human umbilical vein endothelial cells: regulation in response to cell density.

Primary cultures of human umbilical vein endothelial cells (HUVEC) express the human coxsackievirus and adenovirus receptor (HCAR). Whereas HCAR expression in HeLa cells was constant with respect to cell density, HCAR expression in HUVEC increased with culture confluence. HCAR expression in HUVEC was not quantitatively altered by infection with coxsackievirus B.

Genomic and phylogenetic characterization of coxsackievirus B2 prototype strain Ohio-1

The human picornavirus coxsackievirus B2 (CVB2) is often linked to several infections, from mild respiratory diseases to more severe illnesses such as myocarditis. In this study, we report the complete genome sequence of CVB2 prototype strain Ohio-1. The genome sequence was determined from reverse transcribed viral RNA, amplified with long distance PCR and used for non-radioactive sequencing. The full length PCR amplicons were used for in vitro transcription and the obtained cRNA was lipofected onto green monkey kidney cells, in order to confirm that the PCR generated sequence reflects a viable virus RNA. The CVB2 genome sequence shows a typical enterovirus genome organization with a total length of 7411 nucleotides. Phylogenetic analysis, using the CVB2 polyprotein in comparison with other enterovirus polyproteins, clearly shows that CVB2 clusters with the coxsackievirus B-like enteroviruses and is more related to coxsackievirus B4 (CVB4) than any other published CVB serotype. The grouping of CVB2 and CVB4 as one subgroup has earlier been reported in connection with receptor usage and ability to replicate in different cell lines. The exposed viral capsid proteins of CVB2 (VP1–VP3) show high similarity to other CVB proteins, except in regions that are likely to be surface epitopes.

The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures.

Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry. AdV-mediated IL-12 expression was measured using a commercial ELISA. Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50. Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.

The action of three antiseptics/disinfectants against enveloped and non-enveloped viruses

The antiviral action of chloroxylenol, benzalkonium chloride and cetrimide/chlorhexidine was assessed against a range of enveloped and non-enveloped human viruses using a suspension test method. Viral suspensions of 10 6–10 7 pfu/TCID 50 or sfu were prepared in each of the antiseptic/ disinfectant solutions in the presence of a bovine serum/yeast extract mixture to simulate ‘dirty conditions’. During incubation, aliquots were removed at predetermined timepoints up to 10 min to assess the kinetics of inactivation. Results indicate that all products were effective in inactivating the enveloped viruses herpes simplex virus type 1 and human immunodeficiency virus type 1, whilst being ineffective in inactivating human coronavirus, also enveloped, and the non-enveloped viruses. The exception to this was the benzalkonium chloride-based product (Dettol Hospital Concentrate) which was active against the non-enveloped human coxsackie virus. Four antiseptic/disinfectant solutions with chloroxylenol, benzalkonium chloride, cetrimide/ chlorhexidme and povidone-iodine were also assessed for antiviral effect against human immunodeficiency virus in the presence of whole human blood. All four solutions proved to be effective within l min despite the cytotoxic nature of the compounds to the detection system.

The murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses.

Complementary DNA clones encoding the murine homolog (mCAR) of the human coxsackievirus and adenovirus receptor (CAR) were isolated. Nonpermissive CHO cells transfected with mCAR cDNA became susceptible to infection by coxsackieviruses B3 and B4 and showed increased susceptibility to adenovirus-mediated gene transfer. These results indicate that the same receptor is responsible for virus interactions with both murine and human cells. Analysis of receptor expression in human and murine tissues should be useful in defining factors governing virus tropism in vivo.

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.

A single amino acid change in protein synthesis initiation factor 4G renders cap-dependent translation resistant to picornaviral 2A proteases.

Infection of cells with picornaviruses of the rhino-, aphtho-, and enterovirus groups causes a shut-off in cap-dependent translation of cellular mRNAs but permits cap-independent viral RNA translation to proceed. This shut-off is thought to be mediated in part by the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G), although there is evidence to the contrary. Cleavage of eIF4G results in the separation of the eIF4E-binding domain from the ribosome- and helicase-binding domains of the factor, thereby limiting the ability of eIF4G to function in cap-dependent recruitment of mRNAs. Previously we determined the cleavage site within eIF4G targeted by the 2A proteases from human coxsackievirus serotype B4 and human rhinovirus serotype 2 using highly purified eIF4F and recombinant proteases. To examine further the role proteolysis of eIF4G plays in shut-off of translation, we altered the 2A cleavage site in human eIF4G by site-directed mutagenesis. Strikingly, the replacement of one amino acid at the 2A cleavage site resulted in a protein that is approximately 100-fold resistant to cleavage by coxsackievirus 2A protease and 10-50-fold for rhinovirus 2A. Alteration of the cleavage site had no effect on factor activity since the variant was just as active as wild-type eIF4G in restoring cap-dependent translation to an in vitro translation system depleted of endogenous eIF4G. Furthermore, the presence of the variant form of eIF4G rendered in vitro translation reactions resistant to the 2A protease-mediated inhibition of cap-dependent translation initiation. These results support the model that 2A proteases inhibit cap-dependent translation through direct proteolysis of eIF4G.

Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus and rhinovirus.

The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by initiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4 gamma. Infection of cells with picornaviruses of the rhino- and enterovirus groups causes a shut-off in translation of cellular mRNAs but permits viral RNA translation to proceed. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4 gamma, which is catalyzed, directly or indirectly, by the picornaviral 2A protease. In this report we have used highly purified recombinant 2A protease from either human Coxsackievirus serotype B4 or rhinovirus serotype 2 to cleave eIF-4 gamma in vitro in the eIF-4 complex purified from rabbit reticulocytes. Neither the rate of cleavage nor fragment sizes were affected by addition of eIF-3. The NH2- and COOH-terminal fragments of eIF-4 gamma were separated by reverse phase HPLC and identified with specific antibodies, and the NH2-terminal sequence of the COOH-terminal fragment was determined by automated Edman degradation. The cleavage site for both proteases is 479GRPALSSR decreases GPPRGGPG494 in rabbit eIF-4 gamma, corresponding to 478GRTTLSTR decreases GPPRGGPG493 in human eIF-4 gamma.

Chiral discrimination and antipicornavirus activity of 6‐oxazolinylisoflavan

Racemic 6-oxazolinylisoflavan, a highly effective inhibitor of rhinovirus serotype 1B in vitro, was resolved by high-performance liquid chromatography on a chiral stationary phase in order to study the activity of the enantiomers against picornaviruses. The absolute configuration of the two isomers was determined by circular dichroism curves. The antipicornavirus activity of each isomer, separately collected, was evaluated in vitro against human rhinovirus serotype 1B, enterovirus 71, echovirus 6, coxsackievirus B4, and poliovirus type 2 by means of the plaque reduction assay. Both enantiomers were inhibitors of picornavirus replication with the degree of their activity depending on virus and isomer tested.

Prophylactic efficacy of WIN 54954 in prevention of experimental human coxsackievirus A21 infection and illness : G.M.Schiff, J.R. Sherwood, E.C. Young, L.J. Mason, James N. Gamble Inst Med Res, Cincinnati., OH USA

Prophylactic efficacy of WIN 54954 in prevention of experimental human coxsackievirus A21 infection and illness : G.M.Schiff, J.R. Sherwood, E.C. Young, L.J. Mason, James N. Gamble Inst Med Res, Cincinnati., OH USA

Thermal and water source effects upon the stability of enteroviruses in surface freshwaters

The long-term survival of three human enterovirus serotypes, Coxsackievirus B3, echovirus 7, and poliovirus 1 was examined in samples of surface freshwater collected from five sites of physically different character. These were an artificial lake created by damming a creek, a small groundwater outlet pond, both a large- and a medium-sized river, and a small suburban creek. Survival was studied at temperatures of -20, 1, and 22 degrees C. The average amount of viral inactivation was 6.5-7.0 log10 units over 8 weeks at 22 degrees C, 4-5 log10 units over 12 weeks at 1 degree C, and 0.4-0.8 log10 units over 12 weeks at -20 degrees C. The effect of incubation temperature upon viral inactivation rate was statistically significant (p less than 0.00001). As determined by pairing tests, survival was also significantly related to both viral serotype and water source at each of the three incubation temperatures (p less than or equal to 0.05). Efforts were made to determine whether the rate of viral inactivation observed at the different incubation temperatures was related to characteristics inherent to the water that was collected from the different locations. The characteristics examined included physical and chemical parameters, indigenous bacterial counts, and the amount of bacterial growth that the waters would support (measured as the maximum number of generations which seeded bacteria could undergo after being placed into either pasteurized or sterile-filtered water samples). Analysis of viral inactivation rate versus these characteristics revealed three apparent effectors of viral persistence.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine, porcine and ovine picornaviruses: identification of viruses with properties similar to human coxsackieviruses.

Eleven bovine, 19 porcine, and 3 ovine picornaviruses were tested for their ability to grow in the presence of the viral inhibitors 2-(alpha-hydroxy-benzyl)- benzimidazole (HBB) and guanidine-HC1 (GHC1). The nature of the lesions produced by inoculation of newborn mice with these viruses was also investigated. Nine bovine viruses were inhibited by both compounds, and produced skeletal myonecrosis in mice, suggesting similarities to the human coxsackie group B viruses and indicating potential pathogenicity for bovine species. One bovine virus (VH7) was inhibited by GHC1 but not by HBB and caused widespread skeletal muscle damage in mice typical of coxsackie group A viruses. Another bovine virus (F266a) was inhibited only by HBB. None of the porcine or ovine viruses showed significant inhibition by either compound nor produced lesions in mice.

Use of the nitrocellulose-enzyme immunosorbent assay for rapid, sensitive and quantitative detection of human enteroviruses

A modified enzyme immunosorbent assay (EIA) employing nitrocellulose (NC) membrane as a high-capacity solid phase was successfully employed for the sensitive and rapid detection of human enteric viruses, poliovirus and Coxsackievirus B-5. The sensitivity of the NC-EIA ranged from 7 to 70 pg of viral antigen diluted in phosphate-buffered saline. When virus was added to crude supernatants of mollusc tissue homogenates prepared by the standard procedure for the recovery of viruses in molluscs, the sensitivity was reduced by approximately 10-fold. The sensitivity of the NC-EIA for the detection of viral antigens was 10- to 100-fold higher than conventional EIA using polystyrene microtiter plates as solid phase supports. This simple, rapid and sensitive assay using minimal amounts of antibodies should prove to be a useful and practical diagnostic tool to augment infectivity assays currently employed by various virus monitoring procedures. The method also may be applicable for the detection of difficult to grow and/or non-cultivatable enteric viruses which may be present in sewage-contaminated environments.

Specificity of IgM antibodies in acute human coxsackievirus B infections, analysed by indirect solid phase enzyme immunoassay and immunoblot technique.

The specificity of the IgM response in acute human coxsackievirus B infections was examined by indirect solid phase enzyme immunoassay and immunoblot techniques. IgM antibodies detected by ELISA were either strictly type-specific, type-predominant or group-reactive to coxsackieviruses B-1 to B-5. Homotypic and type-dominant responses were clearly correlated with the serotype isolated from the patient. Analysis of ELISA antigens by SDS-gel electrophoresis, protein transfer and subsequent immunodetection of individual virus polypeptides, revealed VP1 as the major antigen; it was detected by homotypic as well as heterotypic serum specimens.

The role of host genetics in the pathogenesis of coxsackievirus infection in the pancreas of mice.

The susceptibility of mice to the diabetogenic effect of the M variant of encephalomyocarditis (MEMC) virus is probably inherited as a recissive trait. Three strains of mice that were susceptible to MEMC virus, three strains that were not susceptible to MEMC virus, and three types of F1 hybrid strains were infected with the common human coxsackie virus, group B, type 4 (CB4). They were examined to determine the titer of virus and histochemistry in the pancreas, levels of blood glucose, urinalysis, glucose tolerance, and levels of plasma amylase and insulin. There was a positive correlation between MEMC virus-susceptibility (diabetes-prone) and CB4-induced beta cell degranulation with concurrent hyperinsulemia and hypoglycemia. Also, as for MEMC virus, the titer of CB4 in the pancreas was not genotype-dependent. However, there was an inverse relation between destruction of the exocrine pancreas and the effect on the endocrine pancreas. These results suggest that the genetic factors responsible for the effect of MEMC virus on the endocrine pancreas are also responsive to CB4, and the proposed recessive nature of these factors is maintained for both viruses. In contrast, these factors, which are operatively recessive traits for the effect of CB4 on the endocrine pancreas, appear to be expressed as dominant traits with respect to the exocrine pancreas.