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Antimicrobial Agents and Chemotherapy, July 2005, p. 2710-2715, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2710-2715.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene

Sunwen Chou,* Laura C. Van Wechel, Heather M. Lichy, and Gail I. Marousek

Medical and Research Services, VA Medical Center and Oregon Health & Science University, Portland, Oregon

Received 30 November 2004/ Returned for modification 8 February 2005/ Accepted 3 March 2005

A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.

* Corresponding author. Mailing address: VA Medical Center, P3ID 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239. Phone: (503) 273-5115. Fax: (503) 273-5116. E-mail: chous{at}ohsu.edu.

Antimicrobial Agents and Chemotherapy, July 2005, p. 2710-2715, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2710-2715.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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