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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.

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