Bovine viral diarrhea virus as a surrogate model of hepatitis C virus for the evaluation of antiviral agents

Abstract

The identification and development of new antiviral agents that can be used to combat hepatitis C virus (HCV) infection has been complicated by both technical and logistic issues. There are few, if any, robust methods by which HCV virions can be grown in vitro. The development of HCV RNA replicons has been a great breakthrough that has allowed for the undertaking of significant screening efforts to identify inhibitors of HCV intracellular replication. However, since replicons do not undergo a complete replication cycle, drug screening programs and mechanism of action studies based solely on these assays will not identify compounds targeting either early (virion attachment, entry, uncoating) or late (virion assembly, egress) stages of the viral replication cycle. Drugs that negatively affect the infectivity of new virions will also not be identified using HCV RNA replicons. Bovine viral diarrhea virus (BVDV) shares a similar structural organization with HCV, and both viruses generally cause chronic long-term infections in their respective hosts. The BVDV surrogate model is attractive, since it is a virus-based system. It is easy to culture the virus in vitro, molecular clones are available for genetic studies, and the virus undergoes a complete replication cycle. Like HCV, BVDV utilizes the LDL receptor to enter cells, uses a functionally similar internal ribosome entry site (IRES) for translation, uses an NS4A cofactor with its homologous NS3 protease, has a similar NS3 helicase/NTPase, a mechanistically similar NS5B RNA-dependent RNA polymerase, and a seemingly equivalent mechanism of virion maturation, assembly and egress. While the concordance between drugs active in either BVDV or HCV is largely unknown at this time, BVDV remains a popular model system with which drugs can be evaluated for potential antiviral activity against HCV and in studies of drug mechanism of action.

Introduction

The hepatitis C virus (HCV) is an enveloped virus with a positive-sense RNA genome of approximately 9.6 kb (Choo et al., 1991, Kato, 2000, Rosenberg, 2001). HCV is the major etiologic agent of non-A, non-B hepatitis, and over 170 million people are infected with HCV worldwide (WHO, 2002). These infections are often asymptomatic; however, HCV infection frequently causes chronic hepatitis, which can progress to liver cirrhosis, end-stage liver disease, and hepatocellular carcinoma (Liang et al., 2000). Approximately 3.9 million Americans are infected with HCV, making HCV the most common blood-borne infection in the United States (Kim et al., 2002b). Infection with HCV is also the leading cause for liver transplantation in this country. No vaccine is available for HCV, and the current treatment for HCV infection, ribavirin and interferon-α (IFN-α), is only effective in a subset of patients. Both of these antiviral agents are broadly acting antiviral compounds that are non-specific for HCV. Also, this therapy is not tolerated well by patients. Therefore, new antiviral agents to treat HCV infection are desperately needed.

Technically, we have been retarded in our abilities to identify new antiviral drugs for HCV due to our inability to propagate the virus reproducibly in vitro (reviewed in Bartenschlager and Lohmann, 2001). The invention of a self-replicating subgenomic HCV RNA replicon (Lohmann et al., 1999a) was a great breakthrough in HCV research. These bicistronic replicons are based on constructs containing the authentic 5′ and 3′ non-translated regions (NTR) of the virus genome, a neomycin phosphotransferase (Neo) gene to allow exclusive survival of cells that continue to produce the transgene in the presence of G418, and the internal ribosome entry site (IRES) element of EMCV mediating the translation of the downstream HCV non-structural (NS) proteins involved in polyprotein processing and RNA replication (reviewed in Bartenschlager and Lohmann, 2001). HCV RNA replicons are robust systems that have shown utility in the evaluation of antiviral agents affecting HCV translation, RNA replication, and polyprotein proteolytic processing (Frese et al., 2001, Guo et al., 2001, Cheney et al., 2002, Lanford et al., 2003, Zhou et al., 2003). However, HCV RNA replicons do not produce infectious virions. Therefore, they cannot be used for the identification and characterization of antiviral agents acting at early steps of the viral replication cycle such as virus attachment to and entry into cells, or the subsequent uncoating process following entry; nor do they allow for the identification of compounds affecting late stages of the replication cycle, such as virion maturation and egress from cells. Similarly, antiviral agents that act through their ability to reduce the infectivity of newly formed virions cannot be identified or characterized using HCV RNA replicons. Since HCV RNA replicons do not faithfully reproduce all steps of the HCV replication cycle, and due to the relatively high costs associated with the use of this technology for the development of antiviral agents, surrogate viruses are still widely utilized for the identification and characterization of anti-HCV antiviral agents.

Other viruses have only been used rarely as surrogate viruses in the study of HCV. The yellow fever virus (YFV) is the type member of the Flavivirus genus of the Flaviviridae family of viruses. This virus has been employed as a surrogate model of HCV replication for the evaluation of antiviral agents (Neyts et al., 1996). However, since its genomic structure is further removed phylogenetically from that of HCV than is bovine viral diarrhea virus (BVDV) and other members of the Flaviviridae, and because it uses a cap-dependent rather than an IRES-mediated mechanism of translation initiation, it is seldom employed as an in vitro surrogate model of HCV. Rather, the YFV is used in the evaluation of antiviral compounds in a range of action studies. These studies are typically performed to see if compounds act specifically against HCV or BVDV, or non-specifically against multiple viruses in order to ascertain whether drugs have a broad spectrum of antiviral activity against all Flaviviridae viruses. This is the case with both of the broadly acting antiviral compounds IFN-α and ribavirin, and the use of YFV has been informative in confirmatory studies on the mechanism of action of these compounds (Buckwold et al., 2003).

The GB virus B (GBV-B) is also an interesting candidate for use as a surrogate model of HCV replication for the evaluation of antiviral agents (Beames et al., 2001). This hepatotrophic virus can undergo a complete replication cycle in tissue culture, making it possible to identify compounds affecting all stages of the viral life cycle. GBV-B has both a relatively high degree of sequence identity with HCV (27–33% amino acid identity with HCV across the polyprotein (Muerhoff et al., 1995)) compared to other members of the Flaviviridae family and a genomic RNA structure very similar to that of HCV. Unfortunately, since the virus can only be grown in the primary hepatocytes of certain non-human primates, this model has not been widely utilized. Clearly it is not possible today to use GBV-B in high-throughput screening (HTS) efforts for drug discovery.

Due to these limitations inherent in the field of HCV drug discovery and development, there has been great interest in the use of BVDV as a surrogate model system. BVDV is the type member of the Pestivirus genus of the Flaviviridae family of viruses (Colett et al., 1988). Other members of this group include classical swine fever virus (formerly known as hog colera virus) and border disease virus of sheep. BVDV is easy to grow in tissue culture, and molecular clones are available for genetic studies (Meyers et al., 1996, Vassilev et al., 1997. Common strains of BVDV such as NADL, which is available from the American Type Culture Collection (ATCC), are cytopathic in tissue culture, allowing for facile assessment of antiviral activity using a cytoprotection assay. The validation of the utility of the BVDV surrogate virus model is incomplete, since most of the few drugs that are known to be active against HCV have not been tested against BVDV; however, its replication in cell culture is inhibited by the drugs currently used to treat HCV infections. Ribavirin and IFN-α are effective antiviral agents against BVDV (Markland et al., 2000, Ouzounov et al., 2002, Buckwold et al., 2003, Stuyver et al., 2003), HCV RNA replicons (Frese et al., 2001, Guo et al., 2001, Cheney et al., 2002, Lanford et al., 2003, Zhou et al., 2003), and related in vitro HCV infection and expression systems (Castet et al., 2002, Contreras et al., 2002). BVDV has been the most widely utilized in vitro HCV surrogate model system for the identification and characterization of antiviral agents for use against HCV. The strengths and weaknesses of the use of the BVDV model system for this indication, as well as examples of its use in this regard, are reviewed below.