The ability to distinguish morphologically identical strains of pathogens is critical to proper diagnosis and treatment of opportunistic infections such as cytomegalovirus in AIDS patients. The investigators propose to use microsatellites to investigate important questions of transmission, virulence and drug resistance in the herpes-like virus, cytomegalovirus. While relations between genotype and virulence have been detected for other genetic markers, microsatellites may provide a large number of new markers to aid in the understanding of CMV virulence. Microsatellite sequences are short arrays of direct repeats which accumulate length mutations due to frequent replication slippage errors. The investigators have found that these loci occur frequently enough even in small genomes (8000 bp to 50 Mbp) to make them a very useful, and largely untapped, source of genetic markers in microorganisms. DNA sequencing of such loci often detects alleles that have the same length but a different sequence, allowing them to distinguish among various strains with high certainty. In a preliminary survey, four such repeats have been located in the cytomegalovirus genome, and the investigators have also identified suitable loci for study in Cryptosporidium parvum, Pneumocystis, Cryptococcus, and Mycobacterium. The development of microsatellite markers for such organisms represents a novel approach to the characterization of opportunistic infections, and may allow them to follow outbreaks in significant detail. The applicants intend to use this approach to examine relationships among genotype, virulence and drug resistance in cytomegalovirus. This information may aid in therapy and prevention. The investigators' data from C. albicans show the feasibility of microsatellite typing for distinguishing among strains and for studying the evolution of resistance to fungistatic agents in strains isolated from HIV-positive individuals. Similar approaches will be taken to the study of strains of cytomegalovirus, which have a great range of effects on host cells and which readily give rise to mutants resistant to antiviral agents.
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