Evaluation of a novel antiviral compound effective against multiple pestivirus species

Abstract

Bovine viral diarrhea virus (BVDV) is a viral pathogen of cattle and other wild and domestic ungulates that is capable of affecting multiple body systems, most notably the gastrointestinal, reproductive, immune and respiratory systems. Aside from causing clinical concern, BVDV is a significant contaminant in laboratory systems and biological products due to its often noncytopathic nature. The virus is also widely used as a surrogate viral model to study Hepatitis C infection in humans. Despite its significance to multiple clinical and research fields, practical methods of BVDV control are limited largely to management protocols and biosecurity safeguards. Specific therapeutics or decontaminants for BVDV are not currently available for use in the farm or the laboratory. Previous research identified a family of antiviral compounds that were safe and effective when used to treat or prevent BVDV infection in various cell lines. This research continued exploration of the identified compound with the highest therapeutic index (DB772) and its potential to be used as an antiviral preventive or therapeutic in vivo as well as it effectiveness against other viruses closely related to BVDV. In the initial study, calves persistently infected with BVDV were treated intravenously with either the antiviral compound or the drug diluent every eight hours over a six day period. In addition to other parameters, viral titers in white blood cells were compared to pre-treatment levels. Treatment with DB772 was associated with a rapid drop in viral titers. However, with one exception, the titers rebounded to near pre-treatment levels during or immediately following the treatment period. Further study revealed that virus isolated from the treated calves with rebound titers was resistant to DB772 whereas pre-treatment isolates were susceptible to the compound. Full genome sequencing was performed on the isolates and revealed single or multiple mutations in the region encoding NS5b, which forms the RNA-dependent RNA polymerase. These mutations have not been described in wild-type isolates and consequently the growth curves of wild-type and mutant isolates were studied but significant changes were not detected. In a second study, calves naïve to BVDV were again treated with either the compound or diluent intravenously before intranasal challenge with BVDV. Treatment with DB772 successfully prevented infection in calves although these same calves were shown to be susceptible to BVDV following treatment after the serum concentration of DB772 was calculated to have fallen below in vitro protective levels. Thirdly, the in vitro efficacy of DB772 against BVDV and closely related viruses was examined by infection of cells with BVDV, border disease virus, HoBi virus, pronghorn virus and Bungowannah virus. Infected cells were incubated in in medium containing 0, 0.006, 0.01, 0.02, 0.05, 0.1, 0.2, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 or 25 μM DB772 and subsequently assayed for the presence of virus by virus isolation and titration or polymerase chain reaction. Cytotoxicity of the compound for different cell types used in the study was evaluated using a commercially available cell counting kit. The compound was shown to effectively inhibit all pestiviruses studied at concentrations > 0.20 μM. Cytotoxicity was not evident until DB772 concentrations exceeded 25 μM.