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.

INTRODUCTION

Human cytomegalovirus (HCMV), also known as human herpesvirus 5 (HHV-5), is a member of the Betaherpesvirus family (including HHV-6 and HHV-7). Cytomegalovirus (CMV) is a double-stranded DNA virus with the largest genome of any herpesvirus. The virus is transmitted horizontally through bodily secretions and can cross the placental barrier to facilitate vertical transmission (reviewed in reference 44). CMV results in a lifelong infection characterized by the establishment of latency in myeloid progenitor cells, followed by periodic reactivation. CMV elicits a strong cellular immune response, and the CMV-specific T cells of some individuals can account for greater than 10% of the total T-cell population (16, 22, 64). In immunocompetent individuals, CMV infection is generally asymptomatic and controlled by the cell-mediated immune response; however, in immunocompromised individuals (i.e., neonates, transplant patients, and AIDS patients), it can cause severe diseases, such as congenital disorders, CMV retinitis, and a variety of opportunistic infections.

Various lab-adapted and clinical strains of HCMV have been isolated and sequenced; most notable are AD169 (13), Toledo (46), Towne (17), and Merlin (15). Furthermore, there are a number of clinical strains that have been cloned as bacterial artificial chromosomes, such as TB40/E (62), TR, PH, and FIX (VR1814) (46). The full-length genomes of CMVs from a number of different animal species, including mice (54), rats (68), guinea pigs (33, 59), and tree shrews (6), have been isolated and sequenced. Given their high degree of genetic relatedness to humans, nonhuman primates (NHPs) likely represent the best animal model to study HCMV biology. A variety of CMVs from Old and New World primates have also been described (37), including chimpanzee CMV (14, 63), rhesus CMV strains 68.1 and 180.92 (28, 57), cercopithecine herpesvirus 5 (CeHV-5) strains GR2715 and Colburn (accession no. FJ483968 and FJ483969, respectively), squirrel monkey CMV (SsciCMV-1; accession no. FJ483967), and owl monkey CMV (AtriCMV-1; accession no. FJ483970). CMVs are highly species-specific viruses (32, 44) and are consequently incapable of infecting even closely related species (A. P. N. Ambagala et al., unpublished data). This specificity restricts the study of CMV to its target species and reiterates the importance of developing animal models that are closely related to humans in an effort to study HCMV pathogenesis.

Animal models to study CMV biology have been largely limited to mice, guinea pigs, and rhesus macaques. As an alternative, cynomolgus macaques (Macaca fascicularis) are a species of Old World monkeys that have the potential to serve as a novel NHP model to study CMV pathogenesis. Cynomolgus macaques are extensively used as an animal model for infectious disease research (12, 23, 36, 67) and transplant research (21, 34, 60) and are becoming an increasingly popular NHP model for human immunodeficiency virus (HIV) vaccine development (7, 47, 66). Within the field of HIV vaccine design, there is a real need to diversify the pool of vectors undergoing testing. Recent studies using rhesus macaque CMV (RhCMV) as a simian immunodeficiency virus (SIV) vaccine vector have shown much promise in the ability of the vaccine to mount a robust effector memory response, thus providing vaccinated macaques with long-term protection from SIV disease progression (26, 29). CMV strains are not conserved even between closely related NHPs, and our recent experience suggests that cynomolgus macaques are not readily infected with RhCMV (Ambagala et al., unpublished). In order to overcome this strong host restriction and evaluate CMV as an HIV vector in a cynomolgus macaque-SIV model, we must use a cynomolgus macaque CMV (CyCMV). We have recently isolated and characterized a novel CyCMV (Ottawa strain) (3). Here we describe the complete genomic sequence and organization of the CyCMV genome for its use as an alternative NHP model to evaluate CMV pathogenesis and vaccine strategies. We compare and contrast the structural and functional genes of CyCMV with those of HCMV and RhCMV with respect to pathogenesis, immune evasion, and species specificity.

MATERIALS AND METHODS

CyCMV viral DNA isolation.

Ottawa strain CyCMV was isolated from catheter-derived urine samples from a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin as described previously (3). A cynomolgus macaque fibroblast cell line was not available at the time of isolation. Initial attempts were made to grow CyCMV in telomerase-immortalized rhesus macaque fibroblast cells (Telo-RF) (35); however, given the rapid growth properties of Telo-RF cells, this cell line could not support the slow growth kinetics of the CyCMV clinical isolate (3). To circumvent this, we propagated the virus in human fetal lung fibroblast cells (MRC-5) (30), which have slower growth properties and have been used extensively to propagate CMVs (9, 18, 63). CyCMV was passaged 16 times in MRC-5 cells to obtain high-titer virus stocks. The virus was not plaque purified, and thus, the sequence likely represents a consensus of one or more strain variants. In order to isolate viral DNA, CyCMV-infected cells were lysed with Hirt extraction buffer (2× Tris-EDTA, 1.2% SDS) for 20 min at room temperature, treated with 1 M sodium chloride overnight at 4°C, and subsequently centrifuged at 27,000 × g for 35 min at 4°C to precipitate the cellular DNA and proteins. The supernatant containing viral DNA was treated with an RNase cocktail (60 μg/ml RNase A and 160 U/ml RNase T1; Fermentas) for 2 h at 37°C and with pronase (1 mg/ml; Roche) for 2 h at 37°C. The supernatant was deproteinized with three phenol-chloroform extractions, and the viral DNA was precipitated with 0.3 M sodium acetate and 2 volumes of absolute ethanol overnight at −20°C. The sample was centrifuged at 17,000 × g for 30 min at 4°C, and the viral DNA was overlaid on a discontinuous 5 to 20% sucrose gradient containing ethidium bromide (2 μg/ml). Following centrifugation at 200,000 × g for 2.5 h at 4°C, the viral DNA was visualized by UV illumination, collected and diluted in 1.5 volumes of water, and precipitated with 0.3 M sodium acetate and 2.5 volumes of absolute ethanol overnight at −20°C. The sample was centrifuged at 15,000 × g for 30 min at 4°C and washed once with 70% ethanol, and the viral DNA was resuspended in water. CyCMV viral DNA (1 μg) was digested with 20 U of HindIII or BamHI at 37°C overnight and fractionated by gel electrophoresis on a 0.8% agarose gel.

Next-generation DNA sequencing.

Using 9.4 μg of CyCMV DNA, a paired-end library with a 500-bp insert size was prepared to generate read lengths of 72 bp. To sequence the complete CyCMV genome, high-throughput Illumina Genome Analyzer II paired-end sequencing was performed at The Centre of Applied Genomics, Toronto, Ontario, Canada.

Bioinformatic assembly.

The CyCMV genome was assembled de novo from 18,205,114 paired 72-bp reads (∼6,000-fold coverage) derived from a run of the Illumina Genome Analyzer II platform. Isolated paired ends were filtered to match the barcode (3,391,350 paired reads, ∼1,120-fold coverage) and were assembled using Velvet (version 0.7.55) (73). The best results were obtained using a kmer length of 39, the shortPaired mode, an insert length of 500 bp, and an expected coverage of 242 to yield a single large contig of 220 kbp.

Gap closing.

The resulting Velvet assembly had 11 gaps (runs of Ns), with lengths of 9 to 124. We implemented a simple greedy assembly program that started from a seed sequence, identifying all possible overlapping reads and extended the seed until no further extension was possible. This process is analogous to those described previously (40, 72). By providing the Velvet program with the areas close to gaps as seeds, we were able to generate sequence and close 10 of the 11 gaps in the initial Velvet assembly.

PCR sequencing.

To confirm the integrity of the sequence, areas of low coverage from the next-generation sequencing data were verified by Sanger sequencing. PCR amplicons were gel purified with GeneClean II (MP Biomedicals) if necessary, cloned into the pCR-Blunt II-TOPO vector (Invitrogen), and transformed with chemically competent cells (Invitrogen). The full-length gene inserts were confirmed by Sanger sequencing using standard sequencing primers [M13 For (UP), 5′-CACGACGTTGTAAAACGAC-3′; M13 Rev (−27), 5′-CAGGAAACAGCTATGAC-3′ (Invitrogen)]. Any remaining sequence was determined by primer walking. Sequences were aligned by ClustalW using Geneious Pro 5.1.7 (Biomatters Ltd., Auckland, New Zealand).

Error correction.

In order to identify additional assembly errors, we aligned all of the Illumina reads with the finished assembly and corrected 64 positions (out of a total assembly size of 220 kbp) where the base pair present in the reference genome occurred 7 times less frequently than an alternative base and replaced each such base with the alternative. Furthermore, for regions of the assembly with low coverage or with conflicting base calls (including all 11 of the gaps initially identified as described above), we generated 71 Sanger sequences. By analyzing the Sanger sequences together with the base qualities in the aligned Illumina data, we were able to correct 24 additional assembly errors (14 single base pair modifications and 10 insertions/deletions of 1 to 23 bp). Finally, the CyCMV sequence was aligned with that of RhCMV 68.1 (accession no. AY186194) to confirm the orientation of the sequence.

ORF assignment.

Open reading frames (ORFs) were identified on both strands with Geneious Pro 5.1.7 (Biomatters Ltd., Auckland, New Zealand) by using the following criteria: (i) the ORF began with a start codon (ATG) and ended with a stop codon, (ii) the ORF was a minimum of 100 amino acids (aa) long (including the start and stop codons), and (iii) the ORF did not overlap another ORF on the same strand within the same reading frame (13). The majority (83%) of the genes were annotated by using these criteria, and the remaining genes were identified using a smaller size criterion of ≥30 aa in an effort to identify all possible ORFs. Homology for each ORF was determined using the BLASTP program (NCBI) with >20% identity. BLAST scores (2) and homology details were obtained from NCBI.

The genome homology of CyCMV with other CMVs was determined by Geneious alignment using a global alignment with free and end gaps and a cost matrix of 65% similarity (5.0/−4.0), a gap open penalty of 12, and a gap extension penalty of 3 (Geneious Pro 5.1.7; Biomatters Ltd., Auckland, New Zealand).

Nucleotide sequence accession number.

The fully annotated and complete nucleotide sequence of the Ottawa strain of CyCMV has been submitted to GenBank and assigned accession number JN227533.

RESULTS

Genomic analysis of CyCMV.

CyCMV was isolated from a cynomolgus macaque of Philippine origin and sequenced using paired-end sequencing (Illumina) to an average of 516-fold coverage/nucleotide. The CyCMV genome is 218,041 bp in length with a 49.5% G+C content and 84% protein-coding density. CyCMV is shorter and less GC rich than HCMV AD169 (229,354 bp, 57.2%) (13) or chimpanzee CMV (CCMV) (241,087 bp, 61.7%) (14) and more comparable to both strains of RhCMV (68.1, 221,459 bp; 180.92, 215,678 bp, 49% G+C content) (28, 57) (Fig. 1). Similar to other CMV genomes, CyCMV has a low G+C content at the beginning of the genome (bp 4559 to 17305), in various regions across the genome (bp 72074 to 74174, 91077 to 94827, 137006 to 140096, 161230 to 163989, and 172919 to 175284), and at the end of the genome (bp 205859 to 211243). Consistent with other CMV genomes, CyCMV is organized with a unique long region followed by a unique short region. When the genome sequence and gene products of CyCMV are compared to those of other CMVs, CyCMV most closely resembles RhCMV. The CyCMV genome is 54.8% identical to that of HCMV AD169 (13), 53.6% identical to that of CCMV (14), 54.2% identical to that of AtriCMV-1 (accession no. FJ483970), 57.5% identical to that of SsciCMV-1 (accession no. FJ483967), 67.5% identical to that of CeHV-5 strain GR2715 (accession no. FJ483968), 67.8% identical to that of CeHV-5 strain Colburn (accession no. FJ483969), 89.8% identical to that of RhCMV 68.1 (28), and 88.2% identical to that of RhCMV 180.92 (57) at the nucleotide level. The first base call of CyCMV corresponds to bp 3996 of HCMV (AD169), is −490 bp from the first nucleotide of the CCMV genome, and is −50 bp from beginning of the RhCMV genome.

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.

Restriction digestion.

To assess gross viral genome structure, a restriction digest analysis was performed and digested bands were confirmed based on predicted fragment sizes (Geneious Pro 5.1.7; Biomatters Ltd., Auckland, New Zealand). The CyCMV genome was digested with restriction enzymes HindIII and BamHI and fractionated on an agarose gel (Fig. 2a). The digested CyCMV fragments were compared with the predicted fragments generated from the sequence data using bioinformatic software (Fig. 2b). All fragments were present at the expected size, with the exception of an additional band running at approximately 2.7 kbp upon digestion with BamHI (Fig. 2a). It is possible that there is an extra BamHI site located between cy92 and cyUL69 that is not accurately represented in the final sequence data. This region encompasses the putative origin of lytic replication, a region known for structural complexity (10). This region is inherently challenging to sequence due the presence of inverted and repeated sequence motifs (10) and proved difficult to sequence in our study by both next-generation and Sanger sequencing. The RhCMV genome does indeed contain a BamHI restriction site in the origin of lytic replication. We predict that the additional CyCMV band is the result of a missing restriction site in the sequence, although the restriction site may be present in the viral DNA.

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.

Gene assignment.

We have identified 262 putative ORFs (Table 1) with a mean coding length of 789 bp. The genes were numbered starting at the left of the genome and continuing to the right with nomenclature similar to that used in annotating the ORFs in other NHP CMVs. The genomic organization of CyCMV is largely colinear with that of RhCMV. The CyCMV gene arrangement with color-coded herpesvirus core genes and gene families is shown in Fig. 3. All alpha-, beta-, and gammaherpesviruses have 40 conserved genes known as core genes (44). CyCMV contains 39 of the 40 core genes with no homologue to HCMV UL108. The function of UL108 is not known, and its deletion from the HCMV genome results in only moderate growth defects (17).

Table 1.

CyCMV gene products

ORFa	Translation		Strand	Size (aa)	Mol mass (kDa)	Function/gene family(ies)b	HCMVc homologue (%)d	RhCMV homologue (%)d
	Start	Stop						68.1	180.92
cyTRL1	1040	2776	+	579	63.6	RL1	TRL1 (37)	Rh01 (84.1)	rhRL1 (84.9)
cy02	1693	1953	+	87	9.9			rh02 (76.8)	rh2 (78)
cy03	2097	2831	+	245	26.2			rh2.1 (62)	rh2.1 (65.1)
cy04	2828	3325	+	166	18.2			rh03 (92.7)	rh3 (92)
cy05	2913	3611	−	233	30.0			rh04 (88.4)	rh4 (87.9)
cy06	2961	3263	+	101	11.5			rh3.1 (97)	rh3.1 (96)
cy07	3298	3783	+	162	18.2			rh3.2 (89)	rh3.2 (89.6)
cyRL11	3738	4559	+	274	30.1	IgG Fc-binding membrane glycoprotein/RL11	RL11 (31.3)	Rh05 (94.9)	rh5 (95.2)
cy09	4873	5358	+	162	18.6			rh06 (47.6)	rh6 (46.8)
cy10	5475	6074	+	200	22.7			rh07 (91.5)	rh7 (91.5)
cy11	6049	6690	+	214	24.3			rh08 (88.4)	rh8 (82.4)
cy12	8067	9140	+	358	39.9			Rh17 (85.2)	rh17 (86.3)
cy13	8075	8437	−	121	13.1			rh18 (71.9)	rh18 (72.5)
cyUL7	9222	10166	+	315	35.6	Putative membrane glycoprotein/RL11	UL7 (34.7)	Rh19 (87.6)	rhUL7 (87.6)
cyUL6	10224	10817	+	198	22.1	Putative membrane glycoprotein/RL11	UL6 (27.9)	Rh20 (84.8)	rhUL6 (84.1)
cyUL9a	10844	11527	+	228	26.4	Putative membrane glycoprotein/RL11	UL9 (28.5)	Rh21 (62.2)	rh21 (66)
cy17	11527	11772	+	82	10.0			Rh22 (42.9)	rh22 (56.8)
cyUL11	11724	12404	+	227	25.4	Membrane glycoprotein/RL11	UL11 (33.3)	Rh23 (80.6)	rhUL11 (81)
cyUL9b	12455	12817	+	121	13.7	Putative membrane glycoprotein/RL11	UL9 (25.3)	rh24 (87.5)	rh24 (87.5)
cyUL9c	12896	13570	+	225	24.7	Putative membrane glycoprotein/RL11	UL9 (28.5)	Rh25 (92.9)	rh26 (29.5)
cyUL9d	13590	14408	+	273	31.0	Putative membrane glycoprotein/RL11	UL9e (31.9)	Rh26 (77.7)	rh26 (78.1)
cy22	14556	15164	+	203	23.0			rh27 (94.1)	rh27 (90.1)
cy23	15166	15789	+	208	23.8			rh28 (73.4)	rh28 (77.3)
cy24	15865	17235	+	457	49.6			Rh29 (85.5)	rh29 (91)
cy25	17311	17634	−	108	11.5			rh30 (94.4)	rh30 (93.2)
cyUL13	17351	18658	+	436	50.3	Putative secreted protein	UL13 (34.2)	Rh31 (92.9)	rh31 (93.6)
cy27	17573	17743	−	57	6.3			rh32 (89.3)	rh32 (87.5)
cy28	17798	18025	−	76	8.7			rh32 (85.3)	rh32 (86.7)
cyUL14	18934	19845	+	304	35.3	Putative membrane glycoprotein/UL14	UL14 (32.8)	Rh33 (97.7)	rhUL14 (97.4)
cy30	20786	21109	+	108	12.7			Rh35 (95.4)
cyUL19	21438	21725	+	96	10.9		UL19 (46.2)	rhUL19 (97)	rhUL19 (95.8)
cyUL20	21834	23189	+	452	51.0	T-cell receptor γ chain homologue	UL20 (36.7)	Rh36 (94.5)	rhUL20 (94.7)
cyUL21A	23294	23656	−	121	13.9	CC chemokine-binding protein	UL21A (41.6)	rh37 (97.5)	rhUL21a (98.3)
cy34	23932	24195	−	88	9.2			rh39 (56)
cyUL23	24752	25690	−	313	35.9	TP/US22	UL23 (44.4)	Rh40 (95.2)
cy36	25437	25817	+	127	14.7			rh41 (95.2)	rh41 (94.4)
cyUL24	25747	26673	−	309	35.1	TP/US22	UL24 (53.3)	Rh42 (98.1)	rhUS22 (28)
cyUL25	26740	28509	+	590	67.4	Tegument phosphoprotein/UL25	UL25 (40.4)	Rh43 (95.1)	rhUL25 (95.2)
cyUL26	28573	29328	−	252	28.2	TP; transcriptional activator of major immediate-early promoter/US22	UL26 (46.7)	Rh44 (97.1)	rhUL26 (97.1)
cyUL27	29282	31015	−	578	65.7		UL27 (55.8)	Rh46 (97.8)	rhUL27 (97.8)
cyUL28	31100	32113	−	338	38.7	US22	UL28 (67.1)	Rh47 (97.6)	rhUL28 (97.9)
cy42	31514	31786	+	91	10.2			rh48 (94.4)
cy43	32224	32388	+	55	6.3			Rh49 (88.7)	rh49 (90.6)
cyUL29	32242	33252	−	337	38.9	US22	UL29 (62.1)	Rh50 (99.1)	rhUL29 (98.8)
cy45	32385	32789	−	135	15.2			rh51 (93.3)	rh51 (94)
cy46	32638	33150	+	171	19.3			rh52 (93.7)
cy47	33304	33537	+	78	9.0			rh52 (100)
cyUL30	33326	33592	−	89	10.3		UL30 (37.7)
cy49	33582	34016	−	145	17.4			rh53 (95.5)
cyUL31	33899	35524	+	542	61.0	dUTPase	UL31 (58.6)	Rh54 (98.2)
cyUL32	35535	37667	−	711	79.5	Major TP (pp150)	UL32 (54.1)	Rh55 (93)	rhUL32 (93.1)
cy52 ex1	37621	37773	+	51	6.0		UL33 (94)
cyUL33 ex2	38035	39024	+	330	37.2	Virion envelope protein/GPCR	UL33 (56.2)	Rh56 (92.4)	rhUL33 ex2 (95.7)
cyUL34	39229	40086	+	286	32.9	Repression of US3 transcription	UL34 (64.4)	Rh57 (96.6)	rhUL34 (98.9)
cy54	39543	39929	+	129	13.9			rh58 (92.2)	rh58 (93)
cyUL35	40149	41921	+	591	66.9	Tegument phosphoprotein; interaction with UL82 protein/UL25	UL35 (43.9)	Rh59 (98.5)	rhUL25 (22.3)
cyUL36 ex1	42038	43201	−	388	44.7	Immediate-early TP; inhibitor of caspase-8-induced apoptosis (vICA)/US22	UL36 (44)	rhUL36 (96.9)	rhUL36 (97.2)
cy57	42308	42607	+	100	11.5			rh59.1 (92)	rh59.1 (91.9)
cyUL36 ex2	43248	43529	−	94	10.5	Immediate-early TP; inhibitor of caspase-8-induced apoptosis (vICA)/US22	UL36 (58.4)	rhUL36 (96.6)	rhUL36 (97.4)
cyUL37 ex1	43627	44445	−	273	31.2	Immediate-early glycoprotein; mitochondrial inhibitor of apoptosis (vMIA)	UL37 (30.5)	rhUL37 (96)	rhUL37 (96)
cy59	44646	45029	+	128	13.5			rh63 (96.9)
cyUL38	44750	45631	−	294	33.2	Virion envelope glycoprotein	UL38 (54.7)	Rh64 (96.6)	rhUL38 (96.9)
cy61	44816	45181	+	122	13.8			rh65 (93.4)	rh65 (92.6)
cy62	45377	45676	+	100	11.3			rh65.1 (89)	rh65.1 (87.9)
cyUL37 ex2	45673	45963	−	97	11.0	Immediate-early glycoprotein	UL37 (36.6)	rhUL37 (90.6)	rhUL37 (90.3)
cy63	46313	46843	−	177	19.0			rh67 (93.2)
cyUL41A	46921	47160	−	80	9.4	Virion envelope protein	UL41Af (36.7)	rhUL41a (99)	rhUL41a (98.7)
cyUL42	47291	47680	−	130	14.3	Putative MP	UL42 (45.9)	Rh68 (96.8)	rhUL42 (96.1)
cyUL43	47664	48665	−	334	38.5	TP/US22	UL43 (46.3)	Rh69 (97.9)	rhUS22 (26.1)
cyUL44	48784	49956	−	391	44.0	DNA polymerase processivity factor	UL44 (69.2)	Rh70 (99)
cy68	49186	49662	+	159	18.0			rh71 (92.4)
cyUL45	50197	52749	−	851	97.0	TP; ribonucleotide reductase subunit 1	UL45 (61.2)	Rh72 (98.5)
cy70	50458	50817	+	120	13.4			rh73 (92.4)
cy71	52235	52537	+	101	11.3			rh74 (100)
cyUL46	52768	53640	−	291	33.1	Capsid triplex subunit 1	UL46f (72.1)	Rh75 (98.6)
cyUL47	53639	56515	+	959	110.6	TP	UL47 (41.8)	Rh76 (97.3)	rhUL47 (97.2)
cyUL48	56536	63069	+	2,178	246.9	Large TP	UL48 (41.3)	Rh78 (98.3)	rhUL48 (98.4)
cy75	57035	57427	+	131	15.1			rh78.1 (89)	rh78.1 (89.2)
cyUL48a	63141	63362	−	74	8.4	Small capsid protein	UL48a (65.3)	rhUL48a (100)	rhUL48a (100)
cy77	63241	63906	+	222	23.8			rh79 (98.6)
cyUL49	63355	64824	−	490	56.2		UL49 (71.5)	Rh80 (99.2)	rhUL49 (99.4)
cyUL50	64814	65695	−	294	32.4	Inner nuclear MP; nuclear egress MP	UL50 (80.6)		rhUL50 (99.3)
cyUL51	65721	66056	−	112	12.4	DNA-packaging protein; terminase component	UL51 (83.1)	Rh82 (99.1)	rhUL51 (97.3)
cyUL52	66116	67774	+	553	62.5		UL52 (55.9)	Rh83 (98.2)	rhUL52 (98.4)
cy82	66226	66534	−	103	10.8			rh84 (97.6)	rh84 (97.6)
cyUL53	67767	68630	+	288	32.9	TP; nuclear matrix protein; nuclear egress lamina protein	UL53 (75.4)	Rh85 (98.3)
cy84	68175	68606	−	144	16.0			rh86 (100)
cyUL54	68608	71715	−	1,036	116.6	DNA polymerase	UL54 (62.2)	Rh87 (99)	rhUL54 (98.9)
cy86	71536	71913	+	126	14.1			Rh88 (97.6)	rh88 (98.4)
cyUL55	71734	74283	−	850	97.1	Glycoprotein B	UL55 (58.4)	Rh89 (79.5)	rhUL55 (76.8)
cyUL56	74249	76558	−	770	88.3	DNA-packaging terminase component	UL56 (73.2)	Rh91 (98.6)	rhUL56 (98.7)
cy89	75252	76622	−	457	48.4			rh91.1 (97)	rh91.1 (97.6)
cyUL57	76705	80196	−	1,164	129.3	Single-stranded DNA-binding protein	UL57 (74.8)	Rh92 (99.7)	rhUL57 (99.6)
cy91	81413	81661	−	83	9.3			rh93 (86.6)	rh93 (87.8)
cy92	81579	81770	−	64	6.7			rh93 (50)
cy93	82075	82446	−	124	12.6			rh95 (97.4)	rh95 (98.3)
cyUL69	84231	86564	−	778	87.4	TP; multiple regulatory protein	UL69f (49.5)	Rh97 (96)	rhUL69 (96.1)
cy95	85222	85530	+	103	11.8			rh98 (99)
cy96	85773	86561	+	263	28.4			rh99 (94.7)	rh99 (95)
cyUL70	86498	89236	−	913	105.5	DNA helicase primase subunit	UL70 (65.3)	Rh100 (98.5)	rhUL70 (98.5)
cy98	87773	88096	+	108	11.5			rh99.1 (94)	rh99.1 (94.4)
cyUL71	89249	89968	+	240	26.4	TP	UL71 (59.5)	rhUL71 (95)	rhUL71 (95.4)
cyUL72	90036	91067	−	344	39.2	dUTPase/dUTPase	UL72 (58.7)	Rh101 (98)	rhUL72 (97.7)
cyUL73	91062	91373	+	104	11.8	Glycoprotein N	UL73 (60.8)	Rh102 (97.1)
cyUL74	91354	92535	−	394	45.9	Glycoprotein O	UL74 (43.7)	Rh103 (94.7)	rhUL74 (94.4)
cyUL74A	92534	92704	+	57	6.4	Envelope glycoprotein 24	UL74A (55.3)
cyUL75	92761	94923	−	721	81.6	Glycoprotein H	UL75 (49.1)	Rh104 (98.3)	rhUL75 (98.2)
cyUL76	95056	95940	+	295	32.8	Virion-associated regulatory protein	UL76 (55.4)	Rh105 (98.6)	rhUL76 (98.3)
cyUL77	95609	97396	+	596	67.4	Portal-capping protein; DNA packaging	UL77 (64.5)	Rh106 (99.5)	rhUL77 (99.3)
cy107	95705	96088	−	128	14.4				rh106.1 (96.1)
cyUL78	97523	98662	+	380	41.9	Putative chemokine receptor/GPCR	UL78 (30.4)	Rh107 (91.6)	rhUL78 (91.6)
cyUL79	98758	99558	−	267	30.5		UL79 (71)	Rh108 (98.9)	rhUL79 (99.2)
cyUL80	99557	101392	+	612	66.4	Capsid maturation protease	UL80 (44.1)	Rh109 (97.9)	rhUL80 (97.9)
cyUL82	101507	103156	−	550	61.6	Tegument phosphoprotein pp71 (upper matrix protein)/UL82, dUTPase	UL82 (42.3)	Rh110 (95.3)	rhUL83b (26)
cyUL83a	103286	104911	−	542	62.2	Major tegument phosphoprotein pp65 (lower matrix protein)/UL82, dUTPase	UL83 (35.3)	Rh111 (95.6)	rhUL83a (95.4)
cy113	103486	103737	−	84	9.2			R83a (89.5)
cyUL83b	104980	106050	−	357	40.4	Major tegument phosphoprotein pp65 (lower matrix protein)/UL82, dUTPase	UL83 (38.9)	Rh112 (94.7)	rhUL83b (94.4)
cy115	105787	106218	+	144	15.8			rh113 (87.6)	rh113 (86.9)
cyUL83c	106169	106594	−	142	15.9	Major tegument phosphoprotein pp65 (lower matrix protein)/UL82, dUTPase	UL83 (45.2)	Rh112 (97.1)	rhUL83b (97.1)
cyUL84	106714	108258	−	515	57.6	Role in organizing DNA replication/UL82, dUTPase	UL84 (50)	Rh114 (98.8)	rhUL84 (98.6)
cy118	106775	107149	+	125	13.5			rh115 (96)	rh115 (95.2)
cy119	106850	107209	−	120	13.3				rh115.1 (90.8)
cy120	108000	108458	+	153	16.1			rh116 (97.4)
cyUL85	108173	109099	−	309	34.7	Capsid triplex subunit 2	UL85f (74)	Rh117 (99.4)
cyUL86	109160	113191	−	1,344	151.3	Major capsid protein	UL86 (76)	Rh118 (98.9)	rhUL86 (98.8)
cy123	109725	110039	+	105	11.8			rh119 (98.1)
cy124	110652	111386	−	245	28.7			rh120 (97.1)
cyUL87	113206	115758	+	851	96.5		UL87 (67.7)	Rh122 (99.4)
cyUL88	115771	116973	+	401	45.5	TP	UL88g (53.7)	Rh123 (98)
cyUL89 ex1	116970	117917	−	316	35.8	DNA-packaging terminase component	UL89 (84.8)	UL89 (100)	rhUL89 (100)
cy128	117633	118202	+	190	20.8			rh125 (94.7)	rh125 (94.2)
cyUL91	118234	118545	+	104	10.9		UL91 (56.3)	Rh126 (92.3)	rhUL91 (94.2)
cyUL92	118430	119143	+	238	26.5		UL92h (90.5)	UL92 (100)	rhUL92 (98.7)
cyUL93	119109	120671	+	521	59.7	DNA-packaging; TP	UL93 (48.2)	Rh128 (96.5)	rhUL93 (96.5)
cy132	119270	119590	+	107	11.1			rh128.1 (94)	rh128.1 (93.4)
cyUL94	120547	121587	+	347	37.8	TP; binds single-stranded DNA	UL94 (58.8)	Rh129 (98.5)	rhUL94 (96.2)
cyUL89 ex2	121576	122508	−	311	36.2	DNA-packaging terminase component	UL89 (86.5)	UL89 (99.7)	rhUL89 (100)
cyUL95	122507	123802	+	432	47.1		UL95 (66.3)	Rh130 (99.5)	rhUL95 (99.3)
cyUL96	123799	124188	+	130	14.8	TP	UL96 (59.8)	Rh131 (97.7)	rhUL96 (96.9)
cyUL97	124245	126071	+	609	67.9	TP; viral serine-threonine protein kinase	UL97 (66.5)	Rh132 (96.4)	UL97 (96.9)
cyUL98	126122	127792	+	557	63.4	DNase	UL98 (66)	Rh134 (99.5)
cy138	126634	127035	−	134	14.6			rh135 (98.5)
cy139	127073	127522	−	150	16.9			Rh136 (99.3)	rh136 (98.7)
cyUL99	127729	128181	+	151	16.5	Myristylated tegument phosphoprotein (pp28)	UL99 (57.1)	rh137 (94)
cyUL100	128354	129424	−	357	41.0	Glycoprotein M	UL100 (58.1)	Rh138 (98.3)
cyUL102	129613	131790	+	726	80.5	DNA helicase primase subunit	UL102 (67)	Rh139 (97.8)	rhUL102 (97.7)
cyUL103	131812	132567	−	252	28.9	TP	UL103f (55.1)	Rh140 (97.2)
cyUL104	132494	134464	−	657	75.5	Capsid portal protein	UL104 (68.3)	Rh141 (99.4)	rhUL104 (99.2)
cyUL105	134301	136880	+	860	97.6	DNA helicase primase subunit	UL105 (71.9)	Rh142 (99.2)
cy146	134450	134929	+	160	18.0			rh142.1 (92)	rh142.1 (93)
cy147	137750	137881	+	44	5.2				rh142.3 (97.6)
cy148 ex1	140183	140380	+	66	7.1	Interleukin-10-like protein precursor		Ul11A (90.6)	rhUL111a (93.5)
cyUL111.5A ex2	140268	140729	+	154	17.7	Latency-associated viral interleukin-10	UL111.5A (37)	Rh143 (92.8)	rhUL111a (91)
cy148 ex3	141097	141186	+	30	3.3	Interleukin-10-like protein precursor		Ul11A (91.3)	rhUL111a (91.3)
cyUL112 ex1	141591	142385	+	265	28.3	Early phosphoprotein (p50)	UL112 (54.3)		rhUL112 (91.4)
cyUL112/UL113 ex2	142484	143374	+	297	30.7	Early phosphoprotein (p84)	UL112/UL113 (34.8)	rhUL112 (94.6)	rhUL112 (94.6)
cyUL114	143485	144228	−	248	28.3	Uracil-DNA glycosylase	UL114 (69)	Rh146 (99.6)
cyUL115	144191	144967	−	259	29.2	Glycoprotein L	UL115 (50.6)	Rh147 (98.8)	rhUL115 (98.4)
cyUL116	144978	146060	−	361	38.2	Putative membrane glycoprotein	UL116 (26.7)	Rh148 (92.5)	rhUL116 (82.5)
cy153	145585	146082	+	166	16.8			rh147.1 (97)	rh147.1 (86.7)
cy154	145714	146199	−	162	20.1			rh149 (92.9)	rh149 (90.7)
cyUL117	146042	147190	−	383	42.6		UL117 (47.8)	Rh150 (99.7)	rhUL117 (99.5)
cy156	147026	147328	+	101	11.1			rh149.1 (97)	rh149.1 (98)
cyUL119 ex1	147215	147946	−	244	28.4	Virion envelope glycoprotein; IgG Fc-binding glycoprotein	UL119 (35.1)	rhUL119 (96.5)	rhUL119 (96.1)
cy157 ex2	147894	148532	−	213	21.3			rhUL119 (79.8)	rhUL119 (65.8)
cy158	148043	148195	−	51	5.4			rh153 (76.9)	rh153 (87.5)
cyUL120	148581	149177	−	199	22.6	Putative membrane glycoprotein/UL120	UL120 (44.1)	Rh154 (92.4)
cyUL121	149179	149727	−	183	21.0	Putative membrane glycoprotein/UL120	UL121 (26.9)	Rh155 (96.7)	rh155 (96.2)
cyUL122 ex1	149990	151474	−	495	53.6	Immediate-early 2 transactivator	UL122 (58.7)	IE (89.9)	rhUL122 (88.7)
cyUL123 ex2	151977	153122	−	382	43.1	Major immediate-early 1 cotransactivator	UL123 (23.7)	IE 1 (59.4)	rhUL123 (60.7)
cy161 ex3	153182	153559	−	126	14.4	Immediate-early protein		IE (63.3)	rhUL122 (63.3)
cy161 ex4	153665	153784	−	40	4.1	Immediate-early protein		IE 1 (87.2)	rhUL123 (84.6)
cy162	153674	153937	+	88	10.4			rh156.1 (92)	rh156.1 (90.8)
cy163	153783	154238	+	152	15.8			rh156.2 (93)	rh156.2 (94)
cyUL126	154779	154925	−	49	5.7		UL126 (55.8)
cy165	155301	155795	−	165	18.6			rh157.1 (87)	rh157.1 (87.8)
cy166	155342	155491	+	50	5.8			rh157.1 (61)	rh157.1 (61.4)
cy167	155476	155679	+	68	7.1				rh157.3 (55.1)
cy168	155547	155780	+	78	8.4			rh157.1 (47)	rh157.1 (53.6)
cy169	155555	155704	−	50	5.2				rh157.3 (91.8)
cy170	155717	156094	−	126	14.8			rh157 (70.2)	rh157.3 (79.6)
cy171	155719	156183	+	155	18.3			rh157 (75.8)	rh157 (80.9)
cy172	155823	156128	+	102	11.6			rh157.2 (82)	rh157.2 (82)
cyUL128 ex1	156700	157182	−	161	18.3	Putative secreted protein; putative CC chemokine	UL128 (37.1)		rhUL128 (96.7)
cyUL128 ex2	157420	157608	−	63	7.1	Putative secreted protein; putative CC chemokine	UL128 (55.2)		rhUL128 (93.6)
cy174	157610	158317	−	236	25.5				rh157.4 (76.2)
cyUL130	158438	158731	−	98	11.5	Putative secreted protein	UL130 (40.7)		rhUL130 (85.6)
cyUL131A	159065	159322	−	86	9.7	Putative secreted protein	UL131A (35.3)		rh131a (96.5)
cyUL132	159366	160025	−	220	24.1	Envelope glycoprotein	UL132 (32.1)	Rh160 (94.9)	rhUL132 (94.4)
cyUL148	160091	161071	−	327	37.0	Putative membrane glycoprotein	UL148 (30.5)	Rh159 (89)	rhUL148 (89.1)
cyUL147	161273	161707	−	145	16.5	Chemokine vCXCL2/UL146	UL147 (43.3)	Rh158 (88.2)
cyUL146	161786	162148	−	121	13.5	Chemokine vCXCL1/UL146	UL146i (28.1)
cy181	162372	162590	−	73	8.2	Alpha-chemokine-like protein
cy182	162689	163015	−	109	12.1	Alpha-chemokine-like protein
cy183	163138	163470	−	111	12.5	Alpha-chemokine-like protein		rh161 (35.2)
cy184	163554	164072	−	173	19.3	Alpha-chemokine-like protein		rh161 (97.3)
cyUL145	164157	164462	−	102	11.3		UL145 (65.6)	Rh162 (99)
cyUL144	164880	165395	−	172	18.7	Membrane glycoprotein; tumor necrosis factor receptor homologue	UL144f (29.6)	Rh163 (98.8)
cyUL141	165611	166909	−	433	49.1	Membrane glycoprotein/UL14	UL141 (39.4)	Rh164 (97)
cy188	167408	167854	−	149	17.1			rh165 (94.6)
cy189	167899	168426	−	176	19.4			Rh166 (96)
cy190	168557	169060	−	168	18.2			rh167 (95.2)	rh167 (95.9)
cy191	169318	169977	−	220	24.7			rh168 (94.1)	rh168 (93.6)
cy192	170074	170505	+	144	16.3			rh168.1 (79)	rh168.1 (79.7)
cy193	170080	170643	−	188	20.8			rh169 (90.4)	rh169 (88.2)
cy194	170777	171343	−	189	21.2			rh170 (96.8)	rh170 (97.3)
cy195	171346	172185	−	280	30.7			Rh171 (91.4)	rh171 (90.7)
cy196	172385	172915	−	177	19.9			rh172 (96)	rh172 (97.2)
cy197	172418	172792	+	125	13.9			rh171.1 (90)	rh171.1 (91.9)
cyUL153	172967	174088	−	374	40.6	MP RL13/RL11	UL153h (32.8)	rh173 (57.3)	rh173 (93)
cy199	175240	176325	−	362	39.9			rh174 (92.2)	rh174 (92.2)
cy200	177022	177480	+	153	16.5			rh175 (92.8)	rh175 (93.4)
cy201	177171	177824	−	218	23.7			rh176 (92.2)	rh176 (93.1)
cy202	177790	178263	−	158	17.8			rh177 (78.8)	rh177 (78.2)
cy203	177884	178657	−	258	28.2			rh178 (86.1)	rh178 (84.9)
cy204	178734	178931	−	66	7.0				rh178.2 (82.8)
cy205	178882	179079	+	66	6.9			rh178.1 (51)	rh178.1 (47.8)
cy206	179072	179575	+	168	18.2			rh178.3 (64.1)	rh178.3 (71.7)
cy207	179212	179535	+	108	11.8			rh178.3 (78)	rh178.3 (80.8)
cy208	179835	180350	+	172	18.6			rh179 (92.4)	rh179 (95.3)
cy209	179951	180157	−	69	6.8			rh180 (91.2)	rh180 (95.6)
cyUS1	180377	180883	−	169	19.3	US1	US1 (49.4)	Rh181 (97.6)	rhUS1 (98.8)
cy211	180732	180965	+	78	8.3			rh180.1 (91)	rh180.1 (96.1)
cyUS2	181118	181708	−	197	23.3	Membrane glycoprotein/US2	US2 (21)	Rh182 (76.9)	rh182 (78.5)
cyUS3	182241	182786	−	182	20.7	Immediate-early glycoprotein/US2	US3 (26.4)	Rh184 (92.7)	rh184 (92.7)
cy214	182286	182579	+	98	10.8			Rh183 (90.7)	rh183 (90.7)
cy215	183413	183712	−	100	11.1			rh184.1 (96)	rh184.1 (96)
cy216	183794	184366	−	191	21.4			Rh185 (95.3)	rh185 (94.7)
cy217	184596	185300	−	235	27.8			rh186 (82.9)	rh186 (82.5)
cyUS11a	185545	186228	−	228	25.7	Membrane glycoprotein/US6	US11 (24)	Rh187 (92.1)	rh187 (91.6)
cy219	186324	186698	−	125	14.6			rh188 (94.4)
cyUS11b	186981	187823	−	281	32.8	Membrane glycoprotein/US6	US11 (29.1)	Rh189 (87.6)	rhUS11 (87.6)
cyUS12	188025	188807	−	261	30.0	Putative multiple-transmembrane protein/US12	US12 (37.2)	Rh190 (98.5)
cy222	188182	188334	−	51	5.4			rh191 (84)
cy223	188395	188724	−	110	12.0			rh191 (87.2)
cyUS13	188865	189629	−	255	29.7	Putative multiple-transmembrane protein/US12	US13f (25.9)	Rh192 (99.6)	rhUS13 (99.2)
cyUS14a	189742	190575	−	278	31.4	Putative multiple-transmembrane protein/US12	US14 (25.3)	Rh194 (97.8)	rh194 (98.2)
cyUS14b	190706	191434	−	243	27.4	Putative multiple-transmembrane protein/US12	US14 (25.3)	Rh195 (98.8)	rhUS14 (26.4)
cyUS14c	191527	192285	−	253	29.5	Putative multiple-transmembrane protein/US12	US14 (29.7)	Rh196 (98.4)	rhUS14 (98.8)
cy228	192391	193116	−	242	28.0			Rh197 (96.3)	rh197 (96.3)
cy229	192716	192985	−	90	10.8				rh196.1 (92.1)
cyUS17	193094	193918	−	275	30.4	Putative multiple-transmembrane protein/US12	US17 (42.9)	Rh198 (98.2)	rhUS17 (97.4)
cyUS18	194024	194824	−	267	30.1	Putative multiple-transmembrane protein/US12	US18f (28.6)		rhUS18 (98.1)
cyUS19	194944	195729	−	262	30.0	Putative multiple-transmembrane protein/US12	US19 (27.8)	Rh200 (95)	rh200 (95)
cyUS20	195790	196551	−	254	28.6	Putative multiple-transmembrane protein/US12	US20 (43.9)	Rh201 (99.6)	rhUS13 (27)
cyUS21	196599	197285	−	229	26.1	Putative multiple-transmembrane protein/US12	US21 (61.1)	Rh202 (98.2)
cyUS22	197407	199131	−	575	65.8	TP/US22	US22 (46.3)	Rh203 (96.9)	rhUS22 (96.7)
cyUS23	199290	201161	−	624	72.7	TP/US22	US23 (51.8)	Rh204 (98.2)	rhUS23 (97.9)
cy237	199589	200002	−	138	16.5			rh206 (90.5)	rh206 (89.7)
cy238	199788	200117	+	110	12.9			rh207 (92.5)	rh205 (92.5)
cy239	200946	201263	+	106	11.5			rh208 (89.5)	rh208 (90.5)
cyUS24	201185	202615	−	477	56.6	TP/US22	US24 (66.2)	rh209 (98.7)
cy241	202022	202207	+	62	7.0			rh210 (90)
cyUS26	202978	204759	−	594	67.3	US22	US26 (46.8)	rh211 (96.8)	rhUS26 (96.6)
cy243	203421	203735	−	105	11.6			rh212 (98.1)
cy244	203512	204027	+	172	19.0			rh213 (94.7)
cyUS28a	204931	205917	+	329	37.5	MP; CC and CX3C chemokine receptor; mediates cellular activation and migration; virion envelope glycoprotein/GPCR	US28 (29.1)	Rh214 (97.9)	rh214 (98.2)
cyUS28b	206261	207274	+	338	38.7	MP; CC and CX3C chemokine receptor; mediates cellular activation and migration; virion envelope glycoprotein/GPCR	US28 (25.4)	Rh215 (93.5)	rh218 (38.1)
cyUS28c	207400	208401	+	334	38.1	MP; CC and CX3C chemokine receptor; mediates cellular activation and migration; virion envelope glycoprotein/GPCR	US28 (24.5)	rhUS28.2 (97.3)	rh218 (37.8)
cy248	208296	208568	−	91	10.2			Rh217 (88.9)	rh217 (91.1)
cyUS28d	208474	209496	+	341	39.1	MP; CC and CX3C chemokine receptor; mediates cellular activation and migration; virion envelope glycoprotein/GPCR	US28 (26.2)	Rh218 (96.2)	rh218 (97.1)
cy250	209266	209571	−	102	11.4			rh219 (94.1)	rh219 (93.1)
cyUS28e	209641	211104	+	488	54.0	MP; CC and CX3C chemokine receptor; mediates cellular activation and migration; virion envelope glycoprotein/GPCR	US28 (40)	Rh220 (85.3)	rhUS28 (89.2)
cyUS29	211265	212581	+	439	49.3	Putative membrane glycoprotein	US29 (38.1)	Rh221 (95)	rhUS29 (94.8)
cy253	211728	212051	+	108	12.7			rh222 (97.2)
cyUS30	212499	213320	+	274	30.8	Putative membrane glycoprotein	US30 (22.2)	Rh223 (96)	rh223 (96)
cy255	213182	213544	−	121	12.6			rh224 (95.8)	rh224 (95.8)
cyUS31	213396	213881	+	162	18.5	US1	US31 (42.7)	Rh225 (94.4)	rhUS31 (93.2)
cy257	213572	213799	−	76	8.5			rh224 (95.2)	rh224 (93.7)
cyUS32	214008	214568	+	187	22.2	US1	US32 (44.7)	Rh226 (96.8)	rhUS32 (96.2)
cy259	214288	214482	−	65	7.2			rh227 (93.7)
cy260	214712	215017	+	102	10.9			rh228 (93.1)	rh228 (88.3)
cy261	215664	216176	−	171	18.6			rh229 (86.2)	rh229 (86.8)
cyTRS1	215743	217818	−	692	77.3	TP; immediate-early protein/US22	TRS1 (37.4)	Rh230 (92.4)	rhTRS1 (92.2)

^aThe CyCMV genes with an HCMV homologue are annotated as “cy” followed by the HCMV name.

^bFunctions and gene families were assigned based on studies of HCMV (44).

^cHCMV homologues are from strains AD169 and Toledo unless otherwise specified.

^dPercent identity based on a BLASTP search conducted in June 2011.

^eHCMV strain 3301.

^fHCMV strain Merlin.

^gHCMV strains CINCY and Towne.

^hHCMV strain Towne.

ⁱHCMV strain NT.

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.

Of the 262 CyCMV genes, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1 genes, and 200 are homologous to RhCMV 180.92 genes. With respect to the RhCMV genomes, CyCMV encodes homologues for 230 (89%) of the 260 RhCMV 68.1 genes and 180 (70%) of the 258 RhCMV 180.92 genes. CyCMV gene homologues were compared between HCMV and both strains of RhCMV based on their alignment bit scores to determine if a particular CyCMV gene is more closely related to a particular strain of CMV (Fig. 4). The majority of the CyCMV genes are biased toward the RhCMV genomes; however, there are some exceptions in which the HCMV gene homologues have the higher bit scores. The outliers are mainly membrane proteins (MP) and tegument proteins (TP).

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.

Genes missing from other macaque CMVs.

In the 262 CyCMV ORFs, four genes show homology to HCMV genes that are not present in either strain of the RhCMV sequenced genomes (68.1 or 180.92). These genes include UL30, UL74A, UL126, and UL146. It should be noted that a wild-type isolate of RhCMV (RhCMV_CNPRC) does contain the HCMV homologue of UL146 (48) and cyUL146 has 76.1% identity with its RhCMV_CNPRC counterpart. The functions of UL30 (cyUL30) and UL126 (cyUL126) have yet to be elucidated; however, it is known that UL74A (cyUL74A) encodes an envelope glycoprotein and UL146 (cyUL146) contains an alpha-chemokine homologue, vCXCL1, belonging to the UL146 gene family (50). UL146 exhibits a high degree of sequence variability between HCMV strains and among species (5). Notably, cyUL146 has retained the chemokine motif, ELRCXC (not shown), that is required for alpha-chemokines to recruit neutrophils (5). During CMV infection, vCXCL1 plays a role in neutrophil attraction and degranulation, resulting in increased viral dissemination both within and between hosts (50).

Tropism genes.

CyCMV encodes a number of HCMV homologues for tropism genes that have been shown to be essential for HCMV propagation in various cell types. The HCMV homologues (UL128, UL130, and UL131A) that have been shown to be associated with endothelial cell, macrophage, and dendritic cell tropism (25, 61) have been retained in CyCMV. The CyCMV genes that show homology with the HCMV UL128-131 region include cyUL128 ex1 (37.1%) and ex2 (55.2%), cyUL130 (40.7%), and cyUL131A (35.3%). An additional HCMV gene known to be required for viral replication in human microvascular endothelial cells (HMVEC) is UL24 (17), for which CyCMV encodes cyUL24 as a homologue with 53.3% identity. Similarly, HCMV UL64 and US29 were shown to be required for growth in human retinal pigment epithelial (RPE) cells (17). CyCMV encodes a US29 homologue (cyUS29) with 38.1% identity but does not encode a UL64 homologue. Functional studies are required to determine if CyCMV can replicate in epithelial cells in the absence of a UL64 homologue. Furthermore, in the functional profiling of HCMV, it was determined that the deletion of UL10 and UL16 increases the viral titer in RPE cells and the deletion of US16 and US19 also results in a higher viral titers in HMVEC (17). CyCMV does not encode the above-listed HCMV homologues, with the exception of cyUS19, which is an HCMV homologue of US19. With respect to RhCMV-specific tropism genes, four genes (Rh01, Rh159, Rh160, and Rh203) have been shown to be tropism determinants for RhCMV (strain 68.1) replication in rhesus RPE cells (38). The CyCMV homologues of these RhCMV tropism genes include cyTRL1 (84.1%), cyUL148 (89%), cyUL132 (94.9%), and cyUS22 (96.9%). CyCMV deletion studies are required to determine if CyCMV exhibits the same impaired viral replication in epithelial cells. The CyCMV tropism genes (cyUL24, cyUL131A, cyUL148, and cyUS22) encode full-length homologues of their respective HCMV and/or RhCMV counterparts. However, the homologues for cyTRL1, cyUL128 ex1/ex2, cyUL130, cyUL132, cyUS19, and cyUS29 represent only partial alignments with the intact CyCMV ORF due to N- and/or C-terminal truncations.

Functional genes.

Although CyCMV has homology with a number of HCMV MP and TP, the genes that have a functional role in DNA replication, packaging, and egress are the most conserved. These proteins include DNA-packaging terminase components (cyUL89 ex1, 84.8%; cyUL89 ex2, 86.5%; cyUL56, 73.2%), a DNA-packaging protein (cyUL51, 83.1%), nuclear egress membrane and lamina proteins (cyUL50, 80.6%; cyUL53, 75.4%), a major capsid protein (cyUL86, 76%), a single-stranded DNA-binding protein (cyUL57, 74.8%), capsid triplex subunits 1 and 2 (cyUL46, 72.1%; cyUL85, 74%), a DNA polymerase processivity factor (cyUL44, 69.2%), a uracil-DNA glycosylase (cyUL114, 69%), DNA helicase primase subunits (cyUL70, 65.3%; cyUL102, 67%; cyUL105, 71.9%), a capsid portal protein (cyUL104, 68.3%), a viral serine-threonine protein kinase (cyUL97, 66.5%), a DNase (cyUL98, 66%), a DNA polymerase (cyUL54, 62.2%), a small capsid protein (cyUL48a, 65.3%), a portal-capping/DNA-packaging protein (cyUL77, 64.5%), and ribonucleotide reductase subunit 1 (cyUL45, 61.2%). The percent identities to HCMV genes are described, and it should be noted that the RhCMV complements for these same genes are even more conserved, with an average of 99% identity to their CyCMV counterparts.

Surface glycoproteins.

CMV surface glycoproteins are commonly used for identification and classification purposes and to assess phylogenetic relationships between CMVs (3). CyCMV encodes HCMV homologues for glycoproteins B (cyUL55), N (cyUL73), O (cyUL74), H (cyUL75), M (cyUL100), and L (cyUL115). Glycoprotein N (UL73) is a highly variable HCMV glycoprotein (51); however, its CyCMV homologue (cyUL73) exhibits the highest degree of homology (60.8%) compared to the remaining HCMV glycoprotein homologues. Another highly polymorphic HCMV glycoprotein is glycoprotein O (UL74) (51), which is the least conserved of the glycoprotein homologues in CyCMV, with 43.7% identity.

Viral homologues of chemokine receptor and GPCR proteins.

Chemokine receptor (CXCL) and G protein-coupled receptor (GPCR) gene homologues are carried by CMVs from various species. These receptor homologues are organized in gene clusters, and the number of repeated genes in a cluster differs between species and between isolates, given that these genes are dispensable for growth in fibroblast cells (1). CyCMV has retained six alpha-chemokine receptor homologues that are clustered together in a 3.98-kbp coding region encompassing cyUL147 to cy184. Likewise, CyCMV contains a cluster of five GPCR homologues (cyUS28a, cyUS28b, cyUS28c, cyUS28d, and cyUS28e) that encode the HCMV homologue of US28, a GPCR known to bind chemokines (69). CyCMV encodes seven genes (cyUL33 ex2, cyUL78, and cyUS28a to cyUS28e) that are homologous to three of the four GPCR family genes (UL33, UL78, and US28). The only GPCR homologue absent from the CyCMV genome is US27, a virion envelope glycoprotein (44).

Immunomodulatory genes.

CMVs contain a number of genes that function to evade the immune response of the infected host. CyCMV encodes HCMV homologues for major histocompatibility complex class I (MHC-I) downregulation genes (US2, US3, and US11), viral interleukin-10 (UL111.5A), a tumor necrosis factor receptor homologue (UL144), and antiapoptotic genes (UL36, UL37 ex1, and UL38). In addition to the HCMV genes, cy203 carries a homologue of the RhCMV-specific gene (rh178) involved in MHC-I downregulation by interference with the translation of the heavy chain portion of the MHC-I molecule (53, 56). Although it was originally thought to be unique to RhCMV (53), it appears that this immunomodulatory gene may in fact be an NHP-specific immunevasin.

The HCMV immunomodulatory genes β2.7, UL16, UL18, UL142, US6, US8, and US10 are not present in CyCMV. Although these genes are important for evading the immune system, their deletion does not have any effect on viral growth in vitro (17, 42). According to the criterion (>20% identity) used to assign CyCMV homologues, CyCMV does not contain a homologue of the HCMV US6 gene. The RhCMV gene Rh185 has a low degree of sequence homology with US6; however, it has been shown to be functionally similar to US6 and therefore has been assigned as a putative homologue (49). Given that cy216 shows significant homology with Rh185 (95.3%) from the RhCMV 68.1 genome, we propose that this CyCMV gene may also function to downregulate MHC-I and may be considered a US6 homologue. We have previously shown that CyCMV downregulates MHC-I expression on the surface of CyCMV-infected cells (3). Further functional studies are needed to determine if CyCMV has equivalent or uncharacterized homologues of the missing immunomodulatory genes to evaluate the effects of these deletions on immunomodulation.

Antiapoptotic genes.

The antiapoptotic genes carried by HCMV include a 2.7-kbp viral RNA (β2.7) and the UL36 to UL38 genes (41). It is likely that CyCMV does not transcribe a β2.7 gene equivalent, given than CyCMV does not encode an HCMV homologue of the predicted ORF (RL4) from which the β2.7 transcript is derived (42). The absence of the β2.7 gene does not affect HCMV growth kinetics in vitro (42). CyCMV contains homologues for the UL36 to UL38 genes (cyUL36 ex1 and ex2, cyUL37 ex1, and cyUL38, respectively). The function of UL36 is to inhibit caspase-8-induced apoptosis (vICA), and cyUL36 ex1 and ex2 have 44% and 58.4% identity with UL36. Similarly, UL37 is a mitochondrial inhibitor of apoptosis (vMIA) and cyUL37 ex1 has 30.5% identity with this HCMV gene. Human CMV UL38 (54.7% identity to cyUL38) is an antiapoptotic gene that blocks the cellular response pathway induced by stress, thus preventing cellular apoptosis; alternatively, when it is deleted from the genome, the target cells undergo apoptosis and HCMV exhibits viral replication defects (45, 65). It remains to be determined if these CyCMV genes have the same antiapoptotic roles as their HCMV homologues.

Latency genes.

Of the known CMV latency transcripts, CyCMV encodes only an HCMV homologue of UL111.5A (cyUL111.5A). The second exon of cy148 (cyUL111.5A ex2) has 37% identity with the latency-associated UL111.5A HCMV gene that encodes the viral interleukin-10 protein. Like HCMV, cy148 carries a spliced transcript with 3 exons, which is analogous to UL111.5A during productive infection (31) and differs from the RhCMV 180.92 homologue (RhUL111a), which contains 4 exons (57). Comparable to RhCMV, CyCMV does not contain an HCMV homologue of UL81 and thus does not contain the UL81-82 antisense transcript (LUNA) that is involved in latency (8, 55). Although CyCMV has retained a number of gene homologues from the HCMV ULb′ region, it is missing the HCMV homologue of UL138, a known latency gene that is also absent from the RhCMV genomes. Substitution studies have shown that removing UL138 from HCMV does not affect the in vitro growth kinetics of the virus when propagated in fibroblast cells, although they suggest that it could be cell type specific (24).

Spliced transcripts.

CyCMV encodes at least eight genes that are the products of spliced mRNA transcripts, and these include the commonly spliced CMV genes. The spliced transcripts that have two exons include a virion envelope protein/GPCR family protein (cy52 ex1 and cyUL33 ex2), tegument protein vICA (cyUL36), immediate-early glycoprotein vMIA (cyUL37), a DNA-packaging terminase component (cyUL89), an early phosphoprotein (cyUL112), IgG Fc-binding glycoprotein (cyUL119 ex1 and cy57 ex2), and a putative CC chemokine (cyUL128). Furthermore, cy148 contains 3 exons (cy148 ex1, cyUL111.5A ex2, and cy148 ex3) to produce the viral interleukin-10 protein. The cy161 ORF is spliced into 4 exons, where cyUL122 ex1 and cyUL123 ex2 produce immediate-early proteins 2 and 1, respectively, and cy161 ex3 and cy161 ex4 encode immediate-early proteins.

Missing genes.

Similar to HCMV, CyCMV does not contain the RhCMV-specific viral homologue of cyclooxygenase-2 (COX-2) (28). CyCMV lacks an approximately 6.7-kbp coding region equivalent to that of RhCMV rh9 to rh16, which encompasses the COX-2 gene homologue of rh10. In comparison to HCMV, CyCMV is lacking full complements for 84 HCMV genes, although the vast majority of these genes are uncharacterized and their deletion from the HCMV genome does not affect viral growth kinetics (Table 2). At the left terminus of the genome, CyCMV is lacking all of the RL genes except TRL1 and RL11 (cyTRL1 and cyRL11, respectively). These genes are generally present only in clinical isolates of CMV, as they are dispensable for growth in vitro (17). The RL11 family of genes is not present in mouse or rat CMV (54, 68). Of the absent HCMV genes, the only ones that have been reported to be required for viral replication are UL60 and UL90, both of which encode functionally uncharacterized proteins. It appears that CyCMV is lacking the HCMV homologues (UL58 to UL68) spanning the origin of lytic replication (oriLyt) that resides between cy92 and cyUL69. These HCMV genes (UL58 to UL68) are present in the AD169 strain; however, they are not present in the Toledo strain of HCMV or in either of the RhCMV strains. The only RhCMV homologues missing from the oriLyt area are rh94 and rh96, suggesting that CyCMV is not lacking any crucial genes in this region and has a gene allocation similar to that of HCMV (Toledo) and RhCMV.

Table 2.

HCMV genes not present in CyCMV

Missing HCMV genea	Function/gene familyb	Effect of deletion on viral growth kineticsc
RL2		−
RL3		ND
RL4		−
RL5		ND
RL6	RL11 family	−
RL7		ND
RL8		ND
RL9		−
RL10	Envelope glycoprotein	−
RL12	Putative membrane glycoprotein/RL11 family	−
RL13	Putative membrane glycoprotein/RL11 family	−
RL14	RL11 family	ND
UL1	RL11 family	ND
UL2	Putative MP	+
UL3		−
UL4	Transcriptionally regulated envelope	−
	glycoprotein/RL11 family
UL5	RL11 family	−
UL8	RL11 family	−
UL10	Putative membrane glycoprotein/RL11 family	−
UL12		+
UL15	Putative MP	−
UL16	Membrane glycoprotein	−
UL17	Seven-TM membrane glycoprotein	−
UL18	Putative membrane glycoprotein; MHC-I	−
	homologue/UL18 family
UL22	Envelope glycoprotein; secreted glycoprotein	ND
UL39		−
UL40	Membrane glycoprotein	ND
UL58		ND
UL59		−
UL60		+++
UL61		ND
UL62		−
UL63		ND
UL64		−
UL65		+
UL66		ND
UL67		−
UL68		ND
UL80.5	Capsid scaffold protein	ND
UL81		ND
UL90		+++
UL101		ND
UL106		ND
UL107		ND
UL108		+
UL109		−
UL110		−
UL118		ND
UL124	Putative membrane glycoprotein	++d
UL125		ND
UL127		−
UL129		+
UL147A	Putative MP	ND
UL143		ND
UL142	Putative membrane glycoprotein; MHC-I	ND
	homologue/UL18 family
UL140	Putative MP	ND
UL139	Putative membrane glycoprotein	ND
UL138	Putative MP	ND
UL137		ND
UL136	Putative MP	ND
UL135	Putative secreted protein	ND
UL134		ND
UL133	Putative MP	ND
UL148A	Putative MP	ND
UL148B	Putative MP	ND
UL148C	Putative MP	ND
UL148D	Putative MP	ND
UL149		ND
UL150	Putative secreted protein	ND
IRS1	Immediate-early protein; TP/US22 family	−
US4		ND
US5		ND
US6	Putative membrane glycoprotein/US6 family	−
US7	Membrane glycoprotein/US6 family	−
US8	Membrane glycoprotein/US6 family	−
US9	Membrane glycoprotein/US6 family	−
US10	Membrane glycoprotein/US6 family	−
US15	Putative multiple-transmembrane	−
	protein/US12 family
US16	Putative multiple-transmembrane	−
	protein/US12 family
US25		−
US27	Virion envelope glycoprotein/GPCR family	−
US33		−
US34	Putative secreted protein	−
US34A	Putative MP	ND

^aAccording to ORFs of HCMV strains AD169 (X17403) and Toledo (GU937742).

^bFunctions and gene families were assigned based on studies of HCMV (44).

^cND, not determined; −, no effect on viral replication; +, modest effect on viral replication; ++, critical effect on viral replication; +++, required for viral replication.

^dEffects of deletion differ between studies (17).

Furthermore, CyCMV is also lacking complements of the UL2, UL12, UL65, UL108, and UL129 genes, all of which do not have a known function, with the exception of UL2 (putative MP). Only a modest effect on viral replication has been observed when these genes are deleted from the HCMV genome (17). There is ambiguity in the literature regarding the effect of knocking out the MP UL124 (17). The remaining HCMV gene deletions have yet to be examined for their effects on viral growth kinetics.

Phylogenetic analysis.

Phylogenetic trees have been generated to depict the evolutionary relatedness of genes common to different CMVs (Fig. 5). Genes that had the greatest number of sequenced strains were included in this analysis. As expected, these genes uniformly group more closely with RhCMV than with the other CMV strains. The cy216 gene was included in this analysis to clarify the discrepancy regarding the HCMV US6 homologue. For this gene, the number of substitutions per site for the branches separating CyCMV and RhCMV from HCMV is relatively high (0.994) in comparison to those of other genes represented by the phylogenetic trees. Furthermore of interest is the cy216 gene, in which the CyCMV complement is more closely related to RhCMV 68.1 than is RhCMV 180.92.

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.

DISCUSSION

The newly characterized CyCMV (Ottawa strain) is 218,041 bp in length, encodes 262 ORFs, and is most closely related to the two published genomes of RhCMV (strains 68.1 and 180.92). Although we have predicted 262 ORFs, we acknowledge that this may not be a complete representation of the CyCMV genome and that there may be additional ORFs carried in the genome that have yet to be elucidated. The virus was not plaque purified, and thus, the sequence likely represents a consensus of one or more strain variants. Our particular Illumina sequencing run had a calculated error frequency of 1% and acted as a baseline for substitution frequencies. In this manner, we determined that sequence calls other than the consensus represent, on average, only 1.9%. This determination does not allow us to calculate how many deviances from the consensus are contained simultaneously in a single genome sequence but does suggest that our current sequence likely represents a mixed population with only a diminutive portion of variants. With respect to interhost variability, we have previously examined the amino acid sequence of glycoprotein B (cyUL55) and have observed 99% identity between animals from the same geographic origin (3).

As the Ottawa strain of CyCMV is a multiply tissue culture-passaged virus, in vitro passage may have resulted in deletions impairing its coding capacity. However, in comparison to the multiple passages required to generate HCMV strains AD169 (54 passages) (19) and Towne (125 passages) (52), CyCMV (16 passages) would be considered only a moderately passaged isolate. Potential gene deletions could be further investigated by sequencing and characterizing a different isolate of CyCMV, specifically, a low-passage strain. The CyCMV genome is unique in that it contains four HCMV homologues (UL30, UL74A, UL126, and UL146) that are not present in either of the published RhCMV genomes (68.1 or 180.92), although an HCMV homologue of UL146 is present in a wild-type strain of RhCMV (RhCMV_CNPRC) (48). There is no putative function for UL30 and UL126; however, it is known that UL74A is an envelope glycoprotein and UL146 is an alpha-chemokine homologue (vCXCL1). It has been suggested that vCXCL1 may act as a virulence determinant of CMV disease in individuals with a compromised adaptive immune system (43). Although CyCMV is a multipassaged derivative, with respect to the chemokine and GPCR gene clusters, CyCMV resembles a minimally passaged virus in that it has retained the clusters of six alpha-chemokine receptor homologues and five GPCR homologues. The wild-type isolates of RhCMV contain six CXCL and five GPCR gene clusters; however, in annotated RhCMV strains 68.1 and 180.92, half or all of the CXCL genes are deleted while all of the GPCR genes in the clusters are retained (1). CyCMV does not appear to have lost these genes in the same way that the RhCMV genomes have.

CyCMV does not contain a viral COX-2 gene that appears to be unique to RhCMV (28). Cellular COX-2 expression is induced upon HCMV infection and has been shown to play an important role in HCMV replication (74). Unlike HCMV infection, RhCMV infection does not induce cellular COX-2 expression in the presence of the viral COX-2 isoform in the RhCMV genome (rh10) (58). Further studies are required to determine if CyCMV infection induces cellular COX-2 expression in the same way as HCMV infection.

Given the importance of cynomolgus macaques as a widely utilized animal model for infectious disease and transplant research, the isolation and characterization of this highly prevalent endogenous virus may have a variety of applications. The seroprevalence of CyCMV in the cynomolgus macaque colony at the Public Health Agency of Canada in Ottawa, Ontario, is estimated to be 100% as measured by a CyCMV-specific enzyme-linked immunosorbent assay (3). In other studies, it has been observed that greater than 95% of NHPs bred in captivity are CMV seropositive (4). Fortunately, it has been shown that CMV-seropositive rhesus macaques can be superinfected with RhCMV (27). This has yet to be examined in cynomolgus macaques, although we believe that, as with RhCMV and HCMV, it would be possible achieve superinfection with CyCMV.

CMVs have evolved with their hosts over millions of years and have contained CMV-specific genes that are related to each host species. We have preliminary data suggesting that it may be inherently difficult to cross the species-specific barrier and infect cynomolgus macaques with RhCMV (Ambagala et al., unpublished). Although the CyCMV genome is nearly 90% identical to that of RhCMV (at the nucleotide level) and the genes are largely colinear with those of RhCMV, clearly there are factors influencing the host range specificity of the virus. The mechanism by which a host cell restricts viral replication from a different species has not been well elucidated. However, it is known that this restriction does not occur exclusively during the entry phase of CMV infection, as it has been shown that CMV has the capacity to infect a host cell from a distant species (20). One possible mechanism by which the host cell inhibits CMV replication from other species may be mediated by apoptosis, suggesting that the foreign virus cannot overcome the cellular innate immune defense of the host (32). CMVs contain antiapoptotic genes (β2.7, UL36, UL37 ex1, and UL38) that function to overcome the apoptosis response induced by the host innate immune response following CMV infection (44). The HCMV homologues of the UL36 to UL38 antiapoptotic genes are encoded by the CyCMV genome (cyUL36 ex1 and ex2, cyUL37 ex1, and cyUL38), and these CyCMV genes show high homology (∼96 to 97% identity) with their RhCMV counterparts (Table 1). Although it has been suggested that these antiapoptotic genes are important for host restriction in vivo, this does not appear to be the situation in vitro, where the species specificity is less restricted. CyCMV productively infects and replicates in human (MRC-5), rhesus macaque (Telo-RF), and cynomolgus macaque (MSFT) fibroblast cell lines (unpublished data). It is known that when CMV strains are grown in fibroblast cell lines, they classically eliminate the tropism genes required for replication in different cell types, most notably, endothelial cells (71). Although CyCMV has been propagated in fibroblast cells prior to sequencing, the genes that are required for endothelial cell tropism (UL128, UL130, and UL131A) have been retained in CyCMV (cyUL128 ex1/ex2, cyUL130, and cyUL131A, respectively). Furthermore, we have preliminary evidence demonstrating that CyCMV infects and efficiently replicates in human umbilical vein endothelial cells (data not shown). Endothelial cell tropism plays an important role in natural infection and viral transmission (70); thus, CyCMV may have utility for examining viral dissemination and pathogenesis in endothelial cells.

Congenital CMV remains the most common viral cause of birth defects in newborns, and yet there is still no vaccine (11). The burden of CMV disease is apparent not only in children but also in adults, specifically, those receiving solid organ or bone marrow transplants and those suffering from an immunocompromising disease such as HIV/AIDS. We have reason to be hopeful regarding the ability to make a CMV vaccine, given the success attained with another herpesvirus, varicella-zoster virus, in which the licensed vaccine has been highly effective in reducing the mortality associated with varicella infections in the United States (39). We hope this newly sequenced and characterized CyCMV genome will provide the necessary groundwork for further studies evaluating the utility of cynomolgus macaques as an alternative NHP model in which to study CMV biology, pathogenesis, and vaccine design.