High genotypic diversity and a novel variant of human cytomegalovirus revealed by combined UL33/UL55 genotyping with broad-range PCR

Merlin Deckers,Sebastian Voigt,Hartmut Hengel,Abigail Edubio,Jörg Hofmann,Karl-Anton Kreuzer,Henrike Reinhard,Bernhard Ehlers

doi:10.1186/1743-422x-6-210

Abstract

The known strains of human cytomegalovirus (HCMV) represent genotypic variants of a single species, and HCMV genotypic variability has been studied in order to reveal correlations between different disease patterns and the presence of certain HCMV genotypes, either as single or as multiple infections. The methods used for the detection of HCMV genotypes have not always been sophisticated enough to achieve complete comprehensiveness, mainly because only one genotype is usually detected in a certain specimen, due to primer specificity and genome copy number. To improve detection of variant HCMV genotypes in mixed infections, we developed PCR assays with degenerate primers targeting two variable HCMV genes, glycoprotein B (gB, UL55) and the G-protein-coupled receptor gene UL33. Primers were designed to bind conserved sites in the genomes of HCMV variants and great ape CMVs. To analyse if samples contained one or more HCMV genotypic variants, PCR assays were supplemented with oligonucleotides containing locked nucleic acids. This broad-range PCR methodology and subsequent sequence analysis detected all gB/UL55 and UL33 genotypic variants known to date in primary clinical specimens, but also revealed that many samples contained genotype mixtures. Importantly, a novel UL33 genotypic variant could be discovered in several specimens, and one HCMV isolate was plaque-purified containing the novel UL33 genotype and a so far undescribed variant of gB.

Highlights

The existence of genotypic variants of human cytomegalovirus (HCMV) was first reported by Chou & Dennison [1] by analysing glycoprotein B gene sequences spanning the gB cleavage site (CLS) of different HCMV isolates.Four distinct CLS genotypes were reported
To improve detection of variant HCMV genotypes in mixed infections, we developed PCR assays with degenerate primers targeting two variable HCMV genes, glycoprotein B and the Gprotein-coupled receptor gene UL33
Using oligonucleotides substituted with locked nucleic acids (LNA), we recently developed a variant of the universal herpesvirus PCR and applied it to the detection of unknown herpesviruses in Old World monkeys

Summary

Introduction

Four distinct CLS genotypes (gBCLS-1 to -4) were reported. Further studies provided evidence for homologous recombination between gBCLS, gBN and gBC (page number not for citation purposes). A fifth gBCLS genotype was detected in several AIDS patients [4]. Additional distinct gBN sequences were described in AIDS patients, but not assigned to a genotype numbering system [5,6]. Several laboratories investigated HCMV genotype variability using PCR-based methods to unravel possible relationships between certain HCMV CLS genotypes and disease outcome, and results have been conflicting at times, the occurrence of infections with more than one HCMV genotype and its impact on severity of HCMV disease has been considered crucial [reviewed in [7,8,9,10,11,12,13,14]]

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Results