Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus

Abstract

Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.

Fig. 1.

G+C content of the CyCMV genome. The base composition across the CyCMV genome is represented by percent GC (blue) and AT (green) contents. Window size, 50 bp.

Fig. 2.

Restriction enzyme digestion of CyCMV genome. To assess gross viral genome structure, a restriction digest analysis was performed. CyCMV viral DNA was digested with the HindIII and BamHI restriction enzymes. DNA fragments (900 ng) were separated by electrophoresis on a 0.8% agarose gel (a). The digested CyCMV DNA has an additional BamHI band (*) at approximately 2.7 kbp. A map of the CyCMV genome digested with HindIII (31 sites) and BamHI (49 sites) was generated using CLC Main Workbench (v 6.1) (b). Lane MW, 10-kbp ladder.

Fig. 3.

Map of ORFs in the CyCMV genome. CyCMV encodes 262 putative ORFs that are annotated by gene name and color coded based on gene families. The genomic organization of CyCMV is largely colinear with that of RhCMV. The CyCMV genes with an HCMV homologue are annotated by “cy” followed by the HCMV name, and the arrowheads indicate the directions of the ORFs. Core genes are herpesvirus core genes.

Fig. 4.

CyCMV gene similarities between RhCMV strains and HCMV. A bit score was calculated for each CyCMV gene versus its putative homologue in RhCMV or HCMV. The data points would be expected to be distributed along the line x = y (gray dashed line), where CyCMV is no more closely related to HCMV than to RhCMV 68.1 or 180.92, respectively. The graphs represent comparisons of the CyCMV gene homologue bit scores versus the two RhCMV strains (a), RhCMV 68.1 versus HCMV (b), and RhCMV 180.92 versus HCMV (c). Outlier genes are annotated according to their CyCMV names and putative functions. MTMP, multiple-transmembrane protein; MGP, membrane glycoprotein.

Fig. 5.

Phylogenetic analysis of CyCMV genes. Unrooted phylogenetic trees were created using Geneious Tree Builder with the protein sequences of various CMV strains. The relationships between strains are shown as cladograms, and the number of substitutions per site is listed on each branch. The CMV strains include CyCMV (bold), HCMV AD169, CCMV, BaCMV (baboon CMV), GoCMV (gorilla CMV), ColCMV (colobus guereza CMV), and RhCMV 68.1 and 180.92. SSDB, single-stranded DNA-binding protein.