MUTATION OF THE THYMIDINE KINASE GENE IN A GANCICLOVIR-RESISTANT STRAIN OF HERPES-SIMPLEX VIRUS TYPE-1 (HSV-1) DETECTED BY PCR-SSCP AND PCR-DIRECT SEQUENCING

A combination of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-direct sequencing was applied for rapi d determination of the thymidine kinase (TK) gene mutation in a newly established ganciclovir-resistant strain of herpes simplex virus type 1 (HSV-1), named PH-DHPG(r). For PCR-SSCP analysis of the whole coding region of the TK gene, we designed seven pairs of primers which ampli fied 7 fragments of DNA with overlapping ends. Results showed that the second part of the TK gene of PH-DHPG (base No. 121 to 318 from the 5 ' end of the coding region) contained a mutation. PCR-direct sequencin g of this part revealed an amber mutation at codon 95 [Glu (GAG) --> s top (TAG)] which lies between the fourth and fifth methionine codons o f the TK polypeptide. This mutation resulted in complete loss of TK ac tivity and drug resistance without temperature-dependence. The present method should be useful for detecting TK gene mutations in other drug -resistant HSV-1 strains. It could also be used for sequential monitor ing of TK gene mutations of HSV-1 in immunocompromized patients and cl one-analysis of TK gene mutants in HSV-1 samples.