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The promising potential of RNA interference-based antiviral therapies has been well established. However, the antiviral efficacy is largely limited by genomic diversity and genetic instability of various viruses, including human enterovirus B (HEB). In this work, the first evidence supporting the anti-HEB activity of the small interfering RNA (siRNA) targeting the highly conserved cis-acting replication element (CRE) within virus coding region 2C is presented. HeLa cells pre-treated with siRNA complementary to the conserved sequence of the loop region of CRE(2C) were effectively rescued from the cytopathic effects of HEBs. Downregulation of virus replication and attenuation of cytotoxicity were consistently observed in various reference strains and clinical isolates. Cells treated with this siRNA were resistant to the emergence of viable escape mutants and showed sustained antiviral ability. Collectively, the data suggest that the siRNA based on the disordered structure within the highly conserved cis-acting coding region has potential as a universal, persistent anti-HEB agent. The same strategy can be successfully applied to the development of siRNA with consistent antiviral effects in other virus groups possessing similar RNA elements.

Objective: To understand coxsackievirus B3 (CVB3) virulence at the molecular level., Method: A mutation library was generated from noncardiovirulent CVB3/0. Highly virulent mutation variants were recovered and characterized both phenotypically and genotypically., Results: The variants consistently caused destruction of multiple tissues together with active virus production and induced severe mortality in vivo. The extent of infectious virus generation was directly correlated with that of histopathological aberration. Genotypic analysis of the entire genome indicated that the virulent viruses encode nucleotide substitutions in the 5'-nontranslated region, which have previously been identified in other virulent CVB3s., Conclusion: The present study provides evidence that particular nucleotide substitutions in the 5'-nontranslated region of nonvirulent CVB3 can lead to active virus replication, which is sufficient to induce virulence., ((c) 2008 S. Karger AG, Basel)

Objectives: We investigated the clinical features, pathologic changes, and viral RNA kinetics in the course of acute and subacute experimental coxsackievirus B3 (CVB3) infection in a murine model., Methods: Five-week-old A/J inbred male mice were divided into 5 groups. Four of those groups were inoculated intraperitoneally with 5 x 10(4) (group 1), 1 x 10(5) (group 2), 5 x 10(5) (group 3), or 1 x 10(6) (group 4) PFU of CVB3. Control mice were inoculated with uninfected Vero cell lysate in DMEM. Mice from each group were sacrificed on days 7 or 14 after inoculation., Results: Bloody diarrhea, earlier weight loss, perianal swelling, and death were correlated with higher viral load. One of ten mice in group 3 and 5 of 10 mice in group 4 died spontaneously between days 4 and 12 after inoculation. All of the remaining 34 mice of infected groups demonstrated extensive pancreatic inflammation. Focal myocarditis developed in only 4 (11.8%) of those 34 subjects. Amylase and creatine kinase activities in the serum were increased in the mice of infected groups. CVB3 RNA was detected in the heart and pancreatic tissue in all subjects. The CVB3 RNA copy number in pancreatic tissue was not correlated with the severity of inflammation., Conclusions: In the murine model, viral loading dose determines the clinical features of CVB3-induced infection, and the severity of pancreatitis is not correlated with the viral loading dose or tissue level of viral RNA. ., (Copyright 2006 S. Karger AG, Basel.)

Coxsackievirus B3 (CVB3) has been identified as a major causative agent of acute and chronic myocarditis, but the involvement of other CVB serotypes in myocarditis has not been investigated. To dissect the pathological properties of different CVB serotypes toward primary cardiomyocytes, we tested their effects on primary cardiomyocyte cultures from neonatal rats. Morphological abnormalities were examined by both light and fluorescence microscopy after Hoechst 33342 staining, and loss of cell viability was estimated by MTT assay. All six CVB serotypes showed a similar degree of severe toxicity toward primary cardiomyocytes. CVB clinical isolates had cytopathic effects (CPEs) similar to those of their respective CVB reference strains. Within 1-2 days of infection with multiplicities of infection MOI 50, the cells began to experience morphological changes including cell shrinkage, rounding-up, and slight nuclear condensation. The irreversible loss of cell viability was readily observed within 3-5 days following virus infection. These results suggest that all six CVB serotypes induce direct, irreversible toxicity towards cardiomyocytes, which eventually leads to the death of infected cells. These findings indicate that the variations in CVB serotype are not the limiting factor determining the susceptibility of cardiomyocytes to CVB infection.

Objective: To investigate histopathological changes and distribution of coxsackievirus B5 (CVB5) RNA in mouse heart, liver, and pancreas during the acute phase of infection., Methods: C3H/HeJ male mice, aged 3-4 weeks, were inoculated intraperitoneally with 5 x 10(5) plaque-forming units of CVB5 and sacrificed at 1, 2, 3, 4, 7 and 10 days postinfection (p.i.). Inflammation of the heart, liver, and pancreatic tissue sections was evaluated by hematoxylin and eosin staining, and virus was detected using antibody to viral coat protein VP1. A quantitative real-time RT-PCR method, using primers and probe targeted to the highly conserved sequences in the 5'-untranslated region of the virus, was used to evaluate the kinetics of CVB5 RNA during the development of myocarditis or pancreatitis., Results: Marginal inflammatory changes were observed in the heart tissues although viral RNA was constantly present between 1 and 10 days p.i., peaking at 4 days p.i. The pancreatic tissues displayed massive lymphocyte infiltration and loss of acinar cells at day 4 p.i. and viral RNA was detected between 1 and 10 days p.i., peaking at 2-3 days p.i. In the liver, viral RNA was detected between 1 and 7 days. No mortality was observed., Conclusions: CVB5 induced acute pancreatitis without subsequent development of myocarditis. Clearance of CVB5 RNA from the pancreas and heart was slower than clearance from the liver. Our real-time RT-PCR method, which is more sensitive than conventional plaque assay, may provide valuable insight into viral RNA kinetics during CVB5 infection., (Copyright (c) 2005 S. Karger AG, Basel.)

Objectives: We attempted to characterize nonpolio enteroviruses recovered from Korean patients with aseptic meningitis., Methods: We performed RT-PCR on the 5'-nontranslated region using clinical specimens. Infectious clinical isolates were amplified by infecting Vero cells with RT-PCR-positive clinical specimens. We then investigated the direct effect in primary neuronal cells or cardiomyocytes following virus infection., Results: Total 12 clinical isolates were subtypically analyzed by both RT-PCR/sequencing comparison of the VP-1 region and neutralization assay. 43-2, 43-2S, 57 and 58 were found to be coxsackievirus B1 (CVB1), 312 to be CVB5, 14-2S and 327 to be echovirus 6, 165 to be echovirus 9, 337 to be echovirus 11, and 270 to be echovirus 30. All the clinical isolates tested showed profound cytotoxicity to various degrees in the primary neuronal cells within 24 h postinfection at 10 MOI. By contrast, a significant cytopathic effect was observed in the primary cardiomyocytes at 3-5 days postinfection at 50 MOI., Conclusions: The present study suggests that the clinical isolates recovered from Korean patients belonged to different CVB or echovirus serotypes and that these viruses showed diversities in their virulence in primary neuronal cells and cardiomyocytes., (Copyright (c) 2005 S. Karger AG, Basel.)

Coxsackievirus B (CVB) is often associated with aseptic meningitis and encephalitis, but the six serotypes of CVB vary in their relative disease severity. To elucidate the detailed mechanisms of CVB-induced cytopathological effects, the morphological and biochemical characteristics caused by the CVB serotypes in mouse primary cortical neuronal cells were investigated. By 24 h post-infection, all CVB serotypes except CVB2 induced severe cytotoxic alterations, including a loss of neurites. Both fluorescence and transmission electron microscopy revealed CVB-induced morphological changes indicative of apoptosis, including heavily condensed nuclei, subsequent chromatin condensation into the periphery of the nuclei and oligonucleosomal DNA fragmentation. It was also found that infection with all six CVB serotypes led to productive virus replication, which was completed prior to an apoptotic signal. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone significantly inhibited nuclear changes associated with virus-induced apoptosis, but had less effect on virus-associated cytopathic effects and no effect on virus production. In contrast, the transcription inhibitor actinomycin D profoundly inhibited all three virus-induced events. Taken together, these findings demonstrate that all six CVB serotypes can efficiently replicate in mouse cortical neuronal cells and that productive replication of these CVBs, except for CVB2, induces multiple cytopathological effects, including apoptotic alterations.

Objective: To investigate the relationship between enteroviral infection and myocardial tissue apoptosis during the development of viral myocarditis in a murine model., Methods: C3H/HeJ mice were inoculated with two strains of coxsackievirus B3, specifically CVB3 (cardiovirulent Nancy strain) and CVB3/0 (noncardiovirulent strain). Mice were sacrificed at 4, 7 and 10 days postinfection (p.i.). Hearts were removed, and plaque assays and RT-PCR were performed to detect the presence of viruses. Myocardial tissue sections were additionally evaluated by hematoxylin and eosin staining for inflammation, VP1 and Bax immunohistochemical staining for detection of virus and Bax expression, and TUNEL and Apostain for localization of apoptosis., Results: CVB3 replicated to significantly higher titers than CVB3/0 at all time points. Histopathological analyses revealed significant inflammatory changes at all time points in CVB3-infected mice, in contrast to minimal changes in CVB3/0-infected mice. TUNEL and Apostain assays of myocardial tissues from mice infected with CVB3 disclosed maximum apoptotic lesions at 4 days p.i. and to a lesser extent at 7 and 10 days p.i. Moreover, CVB3-infected myocardial tissues displayed significantly enhanced Bax expression at 4 days p.i., and lesions overlapped with VP1-stained areas., Conclusions: These data indicate that (1) the cardiovirulent strain CVB3 induces more severe inflammation and apoptosis than the noncardiovirulent CVB3/0 strain, (2) viral replication is localized in inflammatory and apoptotic lesions in myocardial tissues, (3) apoptotic changes are observed in the early stages of myocarditis and (4) Bax may be associated with the apoptosis process in CVB3-induced myocarditis., (Copyright 2003 S. Karger AG, Basel)

Coxsackievirus B (CVB) causes a wide spectrum of human diseases which are closely associated with direct destruction of infected cells. We investigated the morphological and biochemical characteristics of CPEs in permissive Vero cells caused by different CVB serotypes. Regardless of serotype, the infected cells experienced similar degrees of CPEs within 24 h postinfection (p.i.). Using both Hoechst 33342 staining and transmission electron microscopy, we consistently observed morphological properties of apoptosis, heavily condensed nuclei and subsequent chromatin condensation into the periphery of the nuclei within 12 h p.i. Moreover, we noticed typical oligonucleosomal DNA fragmentation, while productive CVB multiplication was accomplished within 6 h p.i. prior to an apoptotic signal. Caspase inhibitor significantly prohibited nuclear changes due to apoptosis with no influence on virus production and cell death, demonstrating that all the CVBs induced more than one type of pathological effect, including apoptotic alteration in permissive Vero cells., (Copyright 2003 S. Karger AG, Basel)

Enterovirus infections of the central nervous system (CNS) are common and important causes of morbidity in immunocompromised children and adults. In this study we identify and characterize coxsackievirus B4-induced neuronal death. To investigate the CNS pathophysiology resulting from this viral infection, cultured rat neurons were infected with coxsackievirus B4 (CVB4) and nuclear morphology, phosphatidylserine (PS) externalization, and the effects of Actinomycin D or cycloheximide (CHX) were examined. CVB4 induced neuronal cell death within 24 h while PS externalization was apparent in cell bodies 16 h after CVB4 infection. Actinomycin D or CHX significantly reduced CVB4 induced-neuronal cell death in a dose-dependent manner. Pretreatment with CHX or actinomycin D also inhibited nuclear condensation, which occurred after CVB4 infection. However, the changes were relatively unresponsive to zVAD-fmk. These results suggest that CVB4 induces CHX- and actinomycin D-sensitive, but zVAD-fmk-insensitive neuronal apoptosis.