RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.RNA interference (RNAi) is a biological process in which double-stranded RNA within the cell induces specific degradation of mRNA with homologous sequences. Protein components of the RNA interference machinery first cleave the long double-stranded RNA into 19-to 21-base pair short interfering RNA (siRNA) and then use the short RNA molecules as guides to target homologous RNA species (reviewed in references 7 and 30). The introduction or expression of siRNA in mammalian cells to target disease-causing genes or virus-specific sequences for degradation represents a potential new therapeutic strategy. Previous reports have shown that RNAi induction has promising antiviral activity against positive-and negative-stranded RNA viruses and DNA viruses in model systems (12, 23). A major concern regarding the use of RNA interference activity against virus infections, particularly RNA viruses, is the development of resistance. Many RNA viruses encode polymerase enzymes that lack proofreading abilities and as a result have high rates of mutation. Thus, there is a high probability that viruses with resistance to RNA interference induced by a particular siRNA will evolve during virus replication through the incorporation of nucleotide mutations within the target sequence of the siRNA. Previous reports have described the selection of human immunodeficiency virus and poliovirus escape mutants in response to prolonged RNA interference activity (2, 4).We have analyzed the potential of hepatitis C virus (HCV) to escape siRNA treatments. HCV infections can lead to the development of liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment ava...

Hepatitis C virus (HCV) is an important human pathogen that is estimated to infect over 180 million people worldwide. Chronic HCV infection causes liver failure and hepatocellular carcinoma and in North America is currently the primary indication for liver transplantation (2). Therapy for HCV infection is limited to a combination of interferon (IFN) and ribavirin. However, therapy is not only difficult to tolerate due to severe side effects, but is successful in only about 50% of all cases. Clearly, there is a need for the development of more effective antiviral therapies. The focus currently is on studying specific interactions with the host that are required for HCV replication. A more complete understanding of viral replication will allow researchers to identify molecular targets for antiviral drug development.

In nature, nuclear polyhedrosis viruses (NPV) are transmitted when susceptible insect larvae ingest viral occlusion bodies (OB). These dissociate in the alkaline environment of the midgut and release encapsulated virions (PDV) which bind to midgut epithelial cells and initiate an infection. A previous study showed that expression of the Autographa californica NPV (AcMNPV) p74 gene during replication is essential for the production of infectious OB. A set of p74 deletion and overexpression recombinants was used for the production and screening of monoclonal antibodies, and for an investigation of gross cytopathology and localization of p74. No differences in virus structure or morphogenesis were observed in infected cells when the p74 gene of AcMNPV was deleted, even though the infectivity of OB harvested from the cells was abolished when they were fed to Trichoplusia ni

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5′ UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5′ untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5′ UTR RNA folding, and mutant 5′ UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5′ UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5′ end annealing or 3′ overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5′ UTR, in conjunction with any Ago isoform, modifies the 5′ UTR structure to stabilize the viral genome and promote HCV RNA accumulation.

Cervical auscultation is experiencing a renaissance as an adjunct to the clinical swallowing assessment. It is a controversial technique with a small evidence base. We have aimed to establish whether cervical auscultation interpretation is based on the actual sounds heard or, in practice, influenced by information gleaned from other aspects of the clinical assessment, medical notes, or previous knowledge. We sought to determine (a) rater reliability and its impact on the clinical value of cervical auscultation and (b) how judgments compare with the "gold standard": videofluoroscopy. Swallow sounds were computer recorded via a Littmann stethoscope. Sounds were sampled from 10 healthy control swallows with no aspiration/penetration and 10 patient swallows with aspiration/penetration, all recorded during simultaneous videofluoroscopy. The system generated sound quality similar to "live" bedside listening, a feature rarely seen in cervical auscultation studies. The 20 sound clips were classified as "normal" or "abnormal" by 19 volunteer speech-language pathologists with experience in cervical auscultation. After at least four weeks, 11 of these judges rated the sounds rerandomized on a new CD. Intrarater reliability kappa ranged from -0.12 to 0.71. Individual reliability did not correlate with years of experience, practice pattern, or frequency of use. Interrater reliability kappa = 0.17. Comparison with radiologically defined aspiration/penetration yielded 66% specificity, 62% sensitivity, and majority consensus gave 90% specificity, 80% sensitivity. There was a significant relationship between individual reliability and true positive rate (r(s) = 0.623, p = 0.040). The reliability of individual judges varied widely and thus, inevitably, agreement between judges was poor. Validity is dependent upon reliability: Improving the poor raters would improve the overall accuracy of this technique in predicting abnormality in swallowing. The group consensus correctly identified 17 of the 20 clips so we may speculate that the swallow sound contains audible cues that should in principle permit reliable classification.

miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5= untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound fulllength RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5=-to-3= exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122. Hepatitis C virus (HCV) is a hepatotropic virus that infects an estimated 150 million humans worldwide, a significant portion of whom do not know their status due to the largely asymptomatic nature of the infection (1). The virus is transmitted by blood-to-blood contact, and humans are the only known reservoir. Chronic infection occurs in approximately 70% of cases and can lead to sequelae such as metabolic disease, steatosis, hepatocellular carcinoma, and decompensated liver disease late in infection (2).One of the major determinants of the virus' hepatotropism is its requirement for the liver-specific, liver-abundant miR-122 microRNA (miRNA) (3, 4). miR-122 binds to two sites at the 5= end of the virus' positive-sense RNA genome and has been shown to directly enhance viral RNA accumulation, since mutation of the miR-122 binding sites abolishes RNA accumulation, and the provision of exogenous miR-122 sequences that have compensatory mutations to restore binding also reinstates RNA accumulation (4-10). Argonaute-2, one of the key effector proteins in the microRNA pathway and a component of the RNA-induced silencing complex (RISC), binds in association with miR-122 and is required to increase HCV replication, while several other proteins in the microRNA pathway and RISC have been implicated in either the biogenesis or activity of miR-122 (5, 11-14). Although miR-122 uses canonical microRNA se...

The study of Hepatitis C Virus (HCV) has benefitted from the use of the Huh7 cell culture system, but until recently there were no other widely used alternatives to this cell line. Here we render another human hepatoma cell line, Hep3B, permissive to the complete virus life cycle by supplementation with the liver-specific microRNA miR-122, known to aid HCV RNA accumulation. When supplemented, Hep3B cells produce J6/JFH-1 virus titres indistinguishable from those produced by Huh7.5 cells. Interestingly, we were able to detect and characterize miR-122-independent replication of di-cistronic replicons in Hep3B cells. Further, we show that Argonaute-2 (Ago2) is required for miR-122-dependent replication, but dispensable for miR-122-independent replication, confirming Ago2's role in mediating the activity of miR-122. Thus Hep3B cells are a model system for the study of HCV, and miR-122 independent replication is a model to identify proteins involved in the function of miR-122.