Yoo Kyum Kim

A Small Interfering RNA Targeting Coxsackievirus B3 Protects Permissive HeLa Cells from Viral Challenge

ABSTRACT We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal antienterovirus strategy.

Coxsackievirus B3 induces apoptosis in the early phase of murine myocarditis: a comparative analysis of cardiovirulent and noncardiovirulent strains.

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.

Characteristics of Apoptotic Cell Death Induced by Coxsackievirus B in Permissive Vero Cells

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.

Antiviral potency of a siRNA targeting a conserved region of coxsackievirus A24

Coxsackievirus A24 (CVA24) is responsible for acute hemorrhagic conjunctivitis, a highly contagious eye disease for which no prevention or treatment is currently available. We thus assessed the antiviral potential of a small interfering RNA (siRNA) targeting CVA24. HeLa cells with or without four different siRNAs complementary to 2C or 3D genome region, were challenged with various CVA24s. Among several siRNAs, a siRNA targeting the highly conserved genome region called the cis-acting replication element (CVA24-CRE), was the only siRNA that decreased virus replication and subsequent cytotoxicity by both CVA24 variant and clinical isolates. Furthermore, CVA24-CRE had effective antiviral activity against CVA24 in primary human conjunctival cells. In addition, CVA24-CRE was highly resistant to the emergence of genetically altered escape mutants. Collectively, the present study provides evidence that CVA24-CRE targeting a conserved viral genome region had universal, prolonged anti-CVA24 activity. This siRNA may thus hold a potential to act clinically as a novel anti-CVA24 agent.

Coxsackievirus B4-induced neuronal apoptosis in rat cortical cultures

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.

A novel program to design siRNAs simultaneously effective to highly variable virus genomes

A major concern of antiviral therapy using small interfering RNAs (siRNAs) targeting RNA viral genome is high sequence diversity and mutation rate due to genetic instability. To overcome this problem, it is indispensable to design siRNAs targeting highly conserved regions. We thus designed CAPSID (Convenient Application Program for siRNA Design), a novel bioinformatics program to identify siRNAs targeting highly conserved regions within RNA viral genomes. From a set of input RNAs of diverse sequences, CAPSID rapidly searches conserved patterns and suggests highly potent siRNA candidates in a hierarchical manner. To validate the usefulness of this novel program, we investigated the antiviral potency of universal siRNA for various Human enterovirus B (HEB) serotypes. Assessment of antiviral efficacy using Hela cells, clearly demonstrates that HEB-specific siRNAs exhibit protective effects against all HEBs examined. These findings strongly indicate that CAPSID can be applied to select universal antiviral siRNAs against highly divergent viral genomes.

Primary neurons become less susceptible to coxsackievirus B5 following maturation: the correlation with the decreased level of CAR expression on cell surface.

Coxsackievirus B (CVB) is one of the major pathogens of aseptic meningitis and meningioencephalitis, particularly in newborn infants. To analyze the influence of neural maturation on susceptibility to CVB infection, we prepared immature and mature neurons from 16‐day‐old BALB/c embryonic cortex. In contrast to immature neurons, mature neurons were less susceptible to CVB5 infection, as indicated by the decrease of cytopathic features. In mature neurons, progeny virus production was significantly hindered, and virus capsid protein VP1 synthesis and virus genome amplification were concomitantly reduced. In addition, the expression of coxsackievirus and adenovirus receptor (CAR), the major receptor of CVB5, was down‐regulated in mature neurons. The antibody treatment specific to CAR significantly attenuated CVB5 susceptibility of immature neurons. These findings demonstrate that mature neurons become less susceptible to CVB by the decrease of CAR level. Thus, the data strongly support the idea that the level of virus receptor in neurons is one of the crucial determinants in the age‐dependency of CVB virulence in central nervous system. J. Med. Virol. 80:434–440, 2008.

Distribution of Viral RNA in Mouse Tissues during Acute Phase of Coxsackievirus B5 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 × 105 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.

Characterization of nonpolio enteroviruses recovered from patients with aseptic meningitis in Korea.

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.

An antiviral small-interfering RNA simultaneously effective against the most prevalent enteroviruses causing acute hemorrhagic conjunctivitis.

PURPOSE Acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease, is caused primarily by either enterovirus 70 (EV70) or coxsackievirus A24 (CVA24) infection. Yet methods to prevent or cure AHC are not available. Recent evidence has shown that small-interfering RNAs (siRNAs), mediators of posttranscriptional gene knockdown, can act as effective antiviral agents. Thus, the authors attempted to develop a novel siRNA-based anti-AHC agent effective against both EV70 and CVA24. METHODS Concurrent screening of the entire viral genome sequences of EV70 and CVA24 using the CAPSID program identified five different siRNA candidates complementary to genome regions of both viruses. The antiviral potentials of these siRNAs were assessed by treating MRC5 and primary human conjunctival cells with the siRNAs and following this with viral challenge. RESULTS Among the five siRNAs, AHCe-3D-3 siRNA showed excellent cytoprotective effects and dramatic decreases in virus replication and virus protein synthesis. This siRNA, targeting the virus polymerase 3D gene, also induced similar antiviral effects in primary human conjunctival cells. CONCLUSIONS These findings strongly suggest that the AHCe-3D-3 siRNA, homologous to two different AHC-associated enteroviruses, can provide equivalent antiviral activities against both AHC-causing enteroviruses. Such an siRNA may be developed as a clinically valuable AHC control agent.