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Diverse Herpes Simplex Virus Type 1 Thymidine Kinase Mutants in Individual Human Neurons and Ganglia ^,

Kening Wang,^1* Gowtham Mahalingam,¹ Susan E. Hoover,^1, Erik K. Mont,^1, Steven M. Holland,² Jeffrey I. Cohen,¹ and Stephen E. Straus¹

Medical Virology Section,¹ Immunopathogenesis Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland²

Received 25 January 2007/ Accepted 18 April 2007

Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations of tk mutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSV tk mutants in animal neurons is impaired. Here we report the detection of mutated HSV tk sequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1 tk open reading frames from ganglia were amplified by PCR, cloned, and sequenced. tk mutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1 tk mutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSV tk mutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected with tk mutant virus alone. These data suggest that diverse tk mutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.

Published ahead of print on 25 April 2007.

This paper is dedicated to the memory of Stephen E. Straus, who was our mentor and inspiration for this study.

Present address: Section of Infectious Diseases, Arizona Health Sciences Center, 1501 N. Campbell Ave., P.O. Box 245039, Tucson, AZ 85724.

Present address: Miami-Dade County Medical Examiner Department, Miami, FL 33136.