Background: Human cytomegalovirus (hCMV) is an important agent of serious viral infection in the solid organ transplant and allogeneic hematopoietic stem cell transplant populations. Severe hCMV infections are associated with graft rejection and other comorbidities. The hCMV gB is a major envelope glycoprotein and is associated with host cell entry and cell-to-cell viral transmission. The gB genotypes (gB1-gB4) have been shown to have varied cytopathic effects on culture cell lines influencing pathogenicity and severity of CMV-associated disease. Four distinct hCMV genotypes have been described based on sequence variations within a region of the UL55 gene using PCR and restriction analysis. Here we describe a sequencing-based method for determining the gB genotype of clinical hCMV isolates.

Methods: End-point PCR with hCMV specific primers to amplify a portion of the UL55 gene in CMV-positive plasma samples followed by dideoxy-terminator sequencing to collect bidirectional nucleotide sequences between codons 337 and 525 was used. Sequence data collected were aligned with gB genotype-specific consensus sequences constructed from type-strains and sequences published in peer-reviewed journals. Phylogenetic analysis was performed by the Tamura-Nei genetic distance model using the neighbor-joining method with 1,000 bootstrap replicates. The resulting dendrogram and percent identity matrix were used to assign gB genotypes to the samples tested.

Results: The gB genotype distribution in 67 clinical hCMV samples was found to be: 29 gB1 (43.3%), 10 gB2 (14.9%), 15 gB3 (22.4%), 8 gB3 variant (11.9%), 4 gB4 (6.0%), and 1 mixed infection containing gB1 and gB4 genotypes (1.5%). All samples shared ≥98% identity with their respective gB consensus sequence with the exception of 1 gB3 sample (96.3% identity to gB3 consensus) and the mixed sample (gB1 and gB4 identities 95.4% and 95.6%, respectively).

Conclusions: The prognostic benefits associated with confirming the gB genotype in CMV-positive patients undergoing immunosuppressive therapy is currently unclear but recent reports have shown the potential importance of this information. We describe here an accurate and sensitive means to detect and determine hCMV gB genotype(s) in clinical plasma specimens.

CITATION INFORMATION: Cowden S, Grantham K, Caton I, Hester J, Altrich M, Kleiboeker S. Development of a Sequencing-Based Cytomegalovirus UL55 Glycoprotein B (gB) Genotyping Assay. Am J Transplant. 2016;16 (suppl 3).

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