Recent advances in reverse genetics of hepatitis C virus (HCV) made it possible to determine the properties and biochemical compositions of HCV virions. Sedimentation analysis and characterization of HCV RNAcontaining particles produced in the cultured cells revealed that HCV virions cover a large range of heterogeneous densities in sucrose gradient. The fractions of low densities are infectious, while the higher-density fractions containing the majority of HCV virion RNA are not. HCV core protein and apolipoprotein B and apolipoprotein E (apoE) were detected in the infectious HCV virions. The level of apoE correlates very well with HCV infectivity. Both apoE-and HCV E2-specific monoclonal antibodies precipitated HCV, demonstrating that HCV virions contain apoE and E2 proteins. apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity in a dose-dependent manner, resulting in a reduction of infectious HCV by nearly 4 orders of magnitude. The knockdown of apoE expression by specific small interfering RNAs (siRNAs) remarkably reduced the levels of intracellular as well as secreted HCV virions. The apoE siRNA suppressed HCV production by more than 100-fold at 50 nM. These findings demonstrate that apoE is required for HCV virion infectivity and production, suggesting that HCV virions are assembled as apoE-enriched lipoprotein particles. Our findings also identified apoE as a novel target for discovery and development of antiviral drugs and monoclonal antibodies to suppress HCV virion formation and infection.Hepatitis C virus (HCV) is a major cause of liver diseases, affecting approximately 170 million people worldwide (59). Most (ϳ85%) acutely HCV-infected individuals become chronic carriers that can develop cirrhosis and hepatocellular carcinoma (50). HCV is an enveloped RNA virus with a singlestrand and positive-sense RNA genome and is classified as Hepacivirus in the Flaviviridae family (47). The genomic RNA consists of a long open reading frame and relatively short untranslated regions (UTR) at the 5Ј and 3Ј ends (11,32,36,46,53). The 5Ј and 3Ј UTR contain cis-acting RNA elements important for HCV polyprotein translation and RNA replication (16-18, 38, 39, 61, 62). The translation of HCV polyprotein is mediated by the internal ribosomal entry site within the 5Ј UTR (46, 58). Upon translation, the HCV polyprotein is cleaved by cellular peptidases and viral proteases into different viral proteins in the order of C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (36, 38). A number of studies demonstrated that the NS3 to NS5B proteins are sufficient for HCV RNA replication (4, 6, 37), which occurs in the membranebound replication complex consisting of HCV RNA and proteins as well as cellular proteins (13,14,42,57). The core and NS5B coding regions also contain cis-acting RNA elements important for HCV RNA replication and regulation (40,63). Last, the newly synthesized HCV proteins and genomic RNA are packaged to form progeny virus particles. However, the molecular aspects underlying HCV virion assembly, matur...

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive-and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10 8 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.Discovered in 1989 by molecular cloning (10), hepatitis C virus (HCV) has been recognized as a major cause of viral hepatitis in humans. HCV infection is characterized by the establishment of chronic infection in the majority (up to 85%) of individuals exposed to HCV. It is estimated that approximately 4 million people in the United States and 170 million people worldwide are persistently infected (9, 38). The chronic HCV infection carries an increased risk of developing fatal liver diseases such as cirrhosis, liver failure, and hepatocellular carcinoma. HCV is a single-stranded positive-sense RNA virus belonging to the Hepacivirus genus of the family Flaviviridae (30). The 9.6-kb RNA genome encodes a single polyprotein that is cleaved by cellular and viral proteases into at least 10 structural (C, E1, E2, and probably p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins that play important roles in virus entry, replication, assembly, and pathogenesis (24, 29). The sequence and structures of the untranslated regions (UTR) at both the 5Ј and 3Ј ends of the HCV RNA genome, which contain cis-acting RNA elements required for HCV RNA translation and replication, are highly conserved (7,13,20,24,25,35,40,41).A great deal of progress has been made with respect to the HCV genome organization, properties and roles of viral proteins and conserved RNA sequence/structures, virus-host interactions, and mechanisms of HCV replication since the dis...

Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN-␣Hepatitis C virus (HCV) is a member of the Flaviviridae family, with a single-stranded and positive-sense RNA genome of approximately 9.6 kb in length (12, 51). The viral RNA genome contains a single open reading frame flanked by untranslated regions (UTR) at both the 5Ј and 3Ј ends. A large viral polyprotein precursor of about 3,010 amino acids is translated and is subsequently cleaved by both cellular and viral proteases into at least 10 different structural (C, E1, E2, and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (42, 50). Replication of HCV RNA occurs in the cytoplasmic membrane-bound multiple-protein complex containing the HCV RNA and nonstructural proteins NS3 to NS5B (15, 42). Owing to the lack of a proofreading activity, the virally encoded RNA-dependent RNA polymerase (RdRp) catalyzes an error-prone RNA replication and thereby results in a mixture of HCV variants (quasispecies) in the infected individual (6). Based on genome sequence similarity, HCV is grouped into six major genotypes and numerous subtypes (55).HCV is a common cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (9, 68). The vast majority (ϳ85%) of individuals exposed to HCV become chronically infected and carry a higher risk of developing cirrhosis and hepatocellular carcinoma (9, 13). The pegylated alpha interferon (PEG-IFN-␣) in combination with ribavirin is currently the optimal therapy to treat HCV infection (13). However, the sustained virologic response (SVR) to IFNbased therapy varied significantly among patients infected with different HCV genotypes (19,30,44). In general, HCV genotypes 2 and 3 are sensitive to PEG-IFN-␣ and ribavirin combination therapy, with an SVR of more than 80%. By contrast, genotypes 1 and 4 are refractory to IFN-based therapy, with an SVR of only about 40 to 50%. The underlying mechanisms of chronic HCV infection, IFN-induced antiviral state against HCV replication, and IFN resistance have not been defined.Many studies have demonstrated that virus infection, replication, and double-stranded RNA (dsRNA) can activate cellular antiviral pathways and induce IFN production (53, 54). IFN stimulates a large number of cellular genes (IFN-stimulated genes [ISGs]) (14, 25), many of which possess direct or indirect antiviral activities, including the well-characterized protein kinase R (PKR), 2Ј,5Ј-oligoadenylate synthetase/RNase L, and Mx (53,54,67). PKR is implicated in controlling HCV replication and mediating the IFN-induced antiviral state against HCV replication (21,52). Several independent studies have found that PKR was induced by HCV RNA replication or

In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 x 10(2) plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T(m)s): 89.23 +/- 0.27 degrees C for velogenic strains, 90.17 +/- 0.35 degrees C for pigeon mesogenic strains, 91.25 +/- 0.14 degrees C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.

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