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Antimicrobial Agents and Chemotherapy, December 2006, p. 4038-4043, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00380-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mutations in the E9L Polymerase Gene of Cidofovir-Resistant Vaccinia Virus Strain WR Are Associated with the Drug Resistance Phenotype{triangledown}

Richard S. Kornbluth,1 Donald F. Smee,2 Robert W. Sidwell,2 Victoria Snarsky,1 David H. Evans,3 and Karl Y. Hostetler1*

Department of Medicine, Division of Infectious Diseases, University of California, San Diego, and the Veterans Medical Research Foundation, San Diego, California,1 Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah,2 Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, The University of Alberta, Edmonton, Alberta T6G 2H7, Canada3

Received 28 March 2006/ Returned for modification 8 May 2006/ Accepted 7 September 2006

Cidofovir (CDV) is an effective drug against viruses of the Orthopoxviridae family and is active in vitro against variola virus, the cause of smallpox. However, CDV-resistant poxviruses can be generated by repeated in vitro passage in the presence of suboptimal concentrations of CDV. To determine if mutations in the E9L polymerase gene could confer resistance to this nucleoside analog, this gene was sequenced from CDV-resistant vaccinia virus and found to encode five amino acid changes, centered on an N-terminal region associated with 3'->5' exonuclease activity. Transfer of this mutant E9L gene into wild-type vaccinia virus by marker rescue sufficed to confer the resistance phenotype. E9L polymerase mutations occurred sequentially during passage in CDV, and an H296Y/S338F double mutant that conferred an intermediate CDV resistance phenotype was identified. In vitro, the marker-rescued CDV-resistant vaccinia virus containing all five mutations grew nearly as well as wild-type vaccinia virus. However, the virulence of this virus for mice was reduced, as 10- to 30-fold more CDV-resistant virus than wild-type virus was required for lethality following intranasal challenge. Cidofovir and hexadecyloxypropyl-cidofovir gave partial protection to mice infected with the virus when used at 50 and 100 mg/kg of body weight given as single treatments 24 h after virus exposure, whereas 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) was completely protective at 25, 50, and 100 mg/kg/day when given daily for 5 days. These findings suggest that drug therapy for poxviruses may be complicated by drug resistance but that treatment of the infection with currently known compounds is possible.

* Corresponding author. Mailing address: Department of Medicine - 0676, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0676. Phone: (858) 552-8585, ext. 2616. Fax: (858) 534-6133. E-mail: khostetl{at}ucsd.edu.

{triangledown} Published ahead of print on 18 September 2006.

Antimicrobial Agents and Chemotherapy, December 2006, p. 4038-4043, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00380-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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