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

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.

Human Cytomegalovirus Genomes Sequenced Directly from Clinical Material: Variation, Multiple-Strain Infection, Recombination and Mutation

“…Hypervariation in UL55, which encodes glycoprotein B (gB), is located in two regions (UL55N, near the N terminus, and UL55X, encompassing the proteolytic cleavage site) [23, 39]. Five genotypes (G1-G5) have been assigned to each region [23, 39-41], which are separated by 927 bp that are 80% identical in all strains. All genomes had one of the recognised UL55X genotypes ( Supplementary Table 5 ).…”

A Novel Bat Herpesvirus Encodes Homologues of Major Histocompatibility Complex Classes I and II, C-Type Lectin, and a Unique Family of Immune-Related Genes

“…MsHV B33 and B78 belong to the G-protein coupled receptor (GCR) family, which is characterized by seven transmembrane domains. While HCMV UL33 is genetically diverse and UL78 is highly conserved among HCMV clinical isolates, both are dispensable for replication in fibroblasts (22,54). MsHV b132 is another protein with a GCR signature but with only one transmembrane domain instead of seven, and it is not homologous to any known proteins.…”

Antiviral function and efficacy of polyvalent immunoglobulin products against CMV isolates in different human cell lines

“…The lysate was stored at -80°C in aliquots. The glycoprotein B (gB) genotype was determined as described elsewhere [16]. The fibroblast-adapted strain AD169 and the endothelial strain TB40 were kindly provided by G. Jahn (University of Tübingen, Germany).…”

“…Human embryonal lung fibroblasts (HELF, passage [10][11][12][13][14][15][16][17][18][19][20], derived from a cell strain established in our laboratory, and retinal pigmented epithelial cells (RPE), purchased from ATCC, were grown in complete MEM with Earle's salts, 25 mM HEPES (BioWhittaker, Apen, Germany), supplemented with up to 10% foetal calf serum (FCS), glutamine (20 mM), non-essential amino acids, sodium pyruvate (Biochrom, Berlin, Germany) and 50 lg/ml gentamycinsulfate (BioWhittaker, Apen, Germany). Telomerase-immortalized microvascular endothelial (TIME) cells (kindly provided by B. Adler, Max von Pettenkofer Institut, München, Germany) were grown in EGM-MV2-medium (BioWhittaker, Apen, Germany) and passaged up to ten times.…”