Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di-and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2. The discovery of safe and effective antiviral drugs continues to present considerable challenges. The rapid emergence of resistance to antiviral drugs is a major problem, and combinations of a limited repertoire of antiviral drugs often need to be employed as a therapeutic strategy. The major stumbling block in antiviral drug development is the limited structural, mechanistic, and functional information on many virus-specific molecular targets. Consequently, the design of target-specific compounds that interfere with viral life cycles is a difficult challenge. There continues to be a substantial unmet clinical need for antiviral drugs with different structures and unique mechanisms of action, other than those conferred by conventional nucleoside analogs (2).Given a lack of sufficient structural information on new targets, an approach that seems appropriate for antiviral drug discovery is the screening of structurally diverse compounds, generated using combinatorial approaches that would modulate biological pathways without regard to specific molecular targets. In theory, this approach would allow simultaneous functional validation of a target, as well as the discovery of a lead structure that modulates the function of the target. This strategy has been variously referred to as "diversity-oriented organic synthesis for therapeutic target validation" or "combinatorial target-guided ligand assembly" (6, 20).We describe here the application of this concept for the discovery of anti-hepatitis B virus (HBV) agents. In recent publications, we have reported methods of assembling various classes of di-and trinucleotide libraries (7,8,18). Through screening, as well as lead optimization work, and in conjunction with cell-based assays, we have discovered that certain molecules show very promising anti-HBV activity. Furthermore, some of these molecules display synergistic activity when used in combination with lamivudine (3TC) and adefovir dipivoxil (ADV), the two nucleoside analogs currently licensed for the treatment of chronic HBV infection. This work summarizes the in vitro analysis and preliminary toxicity evaluation of these compounds.(A preliminary report of this work was presented at the 14th International Conference for Antiviral Research, Seattle, Washington, 8 to 12 April 2001.) MATERIALS AND METHODSTest compounds. Nucleotide libraries were synthesized ...

ORI-9020, a novel dinucleotide, evaluated in transgenic mice expressing hepatitis B virus (HBV), significantly reduced liver HBV DNA (P < 0.001). Levels of HBeAg and HBsAg in serum and of HBcAg in liver were not affected by treatment. A minimal effective dosage was determined to be between 1.6 and 0.5 mg/kg of body weight/day, which was similar to that observed for adefovir dipivoxil.The discovery of safe and effective antiviral drugs continues to present considerable challenges for hepatitis B virus (HBV) therapies. The novel dinucleotides ORI-9020, ORI-7246, and ORI-7170 have been developed and identified to be active against HBV in cell culture (2a). The mode of antiviral action of ORI-9020 and ORI-7246 appears to be direct interference of HBV replication at the level of HBV reverse transcriptase and/or DNA polymerase at an early step, either at, or prior to, the production of the first strand of HBV DNA. These experiments show that these compounds have antiviral activities comparable with adefovir dipivoxil (ADV). Furthermore, when used in combination with lamivudine (3TC) possible synergistic antiviral effects were apparent.Given some of the favorable characteristics of ORI-9020, the anti-HBV activity was evaluated in a previously developed transgenic HBV mouse model (2). Experiments with transgenic mice expressing HBV have demonstrated the model's utility for evaluating potential anti-HBV compounds such as interleukin-12 (1), 3TC (5), alpha interferon (6), ADV (4), and entecavir (3). The results of the evaluation of ORI-9020 in the transgenic mouse model are reported here.Transgenic HBV mice were originally obtained from Frank Chisari (Scripps Research Institute, La Jolla, Calif.) (2). Animal use and care was in compliance with the Utah State University Institutional Animal Care and Use Committee.For the first animal experiment, ORI-9020 was prepared fresh daily at a dosage of 100 mg/kg of body weight /day, which was equal to 170 mol/kg/day, and was injected intraperitoneally (i.p.) using cremaphor-ethanol-saline (CES) (10:10:80) or physiological saline as vehicles. ADV (Gilead, Foster City, Calif.), the positive control, was prepared using the CES vehicle. A dosage of 10 mg/kg/day (19.9 mol/kg/day) was used. In the second experiment to determine the minimal effective concentration, ORI-9020 was prepared in sterile saline in onehalf-log dilutions from 50 to 0.05 mg/kg/day. The drug was delivered i.p. in a volume of 0.1 ml.Liver samples were analyzed for HBV DNA, HBV RNA, and HBcAg, and serum samples were processed for HBV DNA, HBeAg, and HBsAg according to previously published methods (4). HBV DNA and RNA were detected in liver by Southern and Northern blot analysis, respectively. A competitive quantitative PCR was used to detect HBV DNA in the serum. HBeAg and HBsAg were detected using an in-house assay.For the first experiment, male transgenic mice with HBeAg titers in the upper 85% of the range were block randomized across treatment groups according to HBeAg titers. Mice received i.p. injections once da...

No abstract

African horse sickness is a major infectious disease of equids and is caused by African horse sickness virus (AHSV), a dsRNA virus with 10 genome segments encoding for 7 structural and 4/5 non-structural proteins. Here, we focused on the characterisation of the AHSV NS4, the latest protein found to be expressed by this virus. In silico analysis of available sequences confirmed the existence of two phylogenetically distinct AHSV clades: NS4-I and NS4-II. NS4-II is further divided into three subtypes (a, b and g). Confocal microscopy demonstrated that all AHSV NS4 types localised in the cytoplasm of infected cells, unlike the BTV NS4 which, has a strong nucleolar localisation. The replication kinetics of reverse genetics derived AHSV NS4 deletion mutants (AHSVDNS4) were similar to their wild type counterparts in insect (Kc) or interferon incompetent (BSR) cells. However, replication of AHSVDNS4 mutants in primary horse endothelial cells was restricted, in comparison to wild-type viruses. Importantly, primary cells restriction to AHSV replication was dependent on the JAK/STAT pathway. Furthermore, AHSVDNS4 mutants were not able to efficiently suppress the secretion of anti-viral cytokines from primary cells, while the wild-type viruses suppressed this response to varying degrees. Importantly, AHSVDNS4 mutants were less virulent than their wild type counterparts in a murine model of AHSV infection. These results indicate that AHSV NS4 has a role in interferon IFN antagonism and a determinant of viral virulence. We are currently carrying out mass spectrometry analyses to identify the cellular proteins interacting with the AHSV NS4.