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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.

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.)

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.)

A major problem with reverse transcription-polymerase chain reaction (RT-PCR) is that the products amplified from RNA and DNA are not distinguishable. The use of a primer set targeted for an intron-containing sequence or RNA extract without contaminating DNA has been established to overcome this problem. However, an intron sequence does not always exist in the target region and complete removal of DNA during RNA extraction is not practical. For these reasons, we developed differential amplifying RT-PCR (DART-PCR) based on differences in reaction temperatures between RT and PCR, as well as in architectures between RNA and DNA. DART-PCR was designed to differentiate products amplified from RNA and DNA regardless of the presence of introns. DART-PCR can be readily applied for diagnosis of active infection of human cytomegalovirus by detection of glycoprotein B mRNA, which gene lacks introns. Other advantages of DART-PCR were as follows: there is no need for separation of RNA from DNA, simultaneous/differential detection in a single tube and possibility of determination of relative amounts of RNA and DNA.