Research

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

Merlin Deckers¹ , Jörg Hofmann² , Karl-Anton Kreuzer³ , Henrike Reinhard⁴ , Abigail Edubio⁵ , Hartmut Hengel⁴ , Sebastian Voigt⁶ and Bernhard Ehlers¹

¹  P14 Molekulare Genetik und Epidemiologie von Herpesviren, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany

²  Institut für Medizinische Virologie, Charitéplatz 1, 10117 Berlin, Germany

³  Klinik I für Innere Medizin, Joseph-Stelzmann-Straße 9, 50924 Köln, Germany

⁴  Institut für Virologie, Heinrich-Heine-Universität Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany

⁵  P11 HIV-Variabilität und molekulare Epidemiologie, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany

⁶  FG12 Virale Infektionen, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany

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Virology Journal 2009, 6:210doi:10.1186/1743-422X-6-210

Published:	26 November 2009

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.