Typing of human cytomegalovirus gB genotypes (gB) and UL97 mutation from clinical specimens at King Chulalongkorn Memorial Hospital

Abstract

Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in immunocompromised patients. Although HCMV is believed to have only one serotype, genomic variants among clinical strains occurred throughout entire genome. Recently, sequence polymorphism of its envelope glycoproteins especially gB as commonly used to do genotyping of HCMV. There were at least 4 different gB genotypes. For treatment of HCMV associated diseases and preemptive treatment of infection, ganciclovir (GCV) is the most widely used. Acquisition of mutation in HCMV UL97 phosphotransferase appears to be a crucial step in the selection of GCV resistant strain. In Thailand, HCMV genotyping and HCMV UL97 mutation have never been studied before, therefore these studies are interesting. HCMV gB genotyping by nested PCR-RFLP was successfully done in 113 of 161 specimens (113/161, 70.19 %) from 96 patients. The infection of mixed genotypes was the most prevalent (39/113, 34.51 %), followed by gB1 (37/113, 32.74 %), gB3 (17/113, 15.04 %), gB2 (12/113, 10.62 %), and untype, designated as UT (8/113, 7.08 %). None of gB4 has been detected, although we attempted to find gB4 from mixed genotypes by cloning method. In addition, 2 new gB restriction patterns (UT1 and UT2) were identified. Both of them were clustered nearly branch of the same origin to gB1 with approximately 97 % homology. Accordingly, sequences among clinical strains within each group had more similarly than 97 % and about 71 %-75 % between strains of the differing group. Based on sequencing, 5 clinical strains were showed distinct gB genotype determining by RFLP, 1 gB1 and 4 gB2 sequences were gB3 instead. Intragenotypic variation of HCMV gB genotype was shown among clinical samples as well as within the same clinical sample by using cloning method. Study UL97 mutation related to GCV resistance by sequencing demonstrated 8 out of 10 amino acids substitutions were novel positions. Only 2 positions have been reported to associate with HCMV GCV resistance, Ala substitution by Thr (Ala 590 Thr) and Met substitution by Thr (Thr 601 Met). In our study, they were substituted with Glu (Ala 590 Glu) and Lys (Thr 601 Lys). Therefore, all of 10 UL97 mutations in this present work were not known whether those would confer to GCV resistance. In conclusion, mixed HCMV genotypes were found predominantly in this study, suggesting the mutation of HCMV within a patient or the possibility of reinfection of new type / strain. Moreover, sequencing method is still the best method for HCMV gB genotyping. Besides sequencing, the study of UL97 mutation conferring GCV resistance needs the drug resistant phenotypic effect