Abstract
BackgroundThe emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV) is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. One mode of action for CDV has been demonstrated to be the inhibition of the viral DNA polymerase.ResultsWe have isolated several CDV resistant (CDVR) vaccinia viruses through a one step process, two of which have unique single mutations within the DNA polymerase. An additional resistant virus isolate provides evidence of a second site mutation within the genome involved in CDV resistance. The CDVR viruses were 3–7 fold more resistant to the drug than the parental viruses. The virulence of the CDVR viruses was tested in mice inoculated intranasally and all were found to be attenuated.ConclusionResistance to CDV in vaccinia virus can be conferred individually by at least two different mutations within the DNA polymerase gene. Additional genes may be involved. This one step approach for isolating resistant viruses without serial passage and in the presence of low doses of drug minimizes unintended secondary mutations and is applicable to other potential antiviral agents.
Highlights
The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds
CDV cytotoxicity, effective concentration for abolishing vaccinia virus (VV) plaque formation We first established the concentration of CDV that would effectively eliminate wt VV plaques without having significant cytotoxic effects on the BSC40 cells (Figure 1)
The presence of GFP made the identification of small plaques much easier; this parental virus is thymidine kinase (TK) negative attenuating the virus and rendering it an unsuitable backbone to later assess the CDV resistant (CDVR) virus phenotype in animals [10]
Summary
The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV) is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. Outbreaks of monkeypox, a virus indigenous to equatorial Africa, have occurred recently in both the US and Western Africa in human populations and demonstrate the potential of viruses to be rapidly transmitted throughout the world [1]. Cidofovir (CDV) has been approved under an investigational new drug application for the emergency treatment of certain orthopoxvirus infections, it is not orally bioavailable and is nephrotoxic. A lipophilic derivative of CDV has been shown to have increased bioavailability while retaining effectiveness against orthopoxvirus infections in vitro and in vivo and is currently in phase I/II clinical studies [2,3,4]. Drug concentrations of as low as 50 μM are effective at abolishing plaque formation
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