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Cidofovir Resistance in Vaccinia Virus Is Linked to Diminished Virulence in Mice

Graciela Andrei,^1, Don B. Gammon,^2, Pierre Fiten,³ Erik De Clercq,¹ Ghislain Opdenakker,³ Robert Snoeck,¹ and David H. Evans^2*

Laboratory of Virology,¹ Laboratory of Immunobiology, Rega Institute for Medical Research, Minderbroedersstraat 10, Katholieke Universiteit Leuven, Leuven B-3000, Belgium,³ Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton AB T6G 2H7, Canada²

Received 24 March 2006/ Accepted 14 July 2006

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3'-5' exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.

G. Andrei and D. B. Gammon contributed equally to this work.

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