Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus

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.     

Reevaluation of the Coding Potential and Proteomic Analysis of the BAC-Derived Rhesus Cytomegalovirus Strain 68-1

Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown, and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several related Old World monkey (OWM) CMVs, we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs could be detected. Taken together, these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes, and OWMs than previously assumed.     

Functional genetic analysis of rhesus cytomegalovirus: Rh01 is an epithelial cell tropism factor

Rhesus cytomegalovirus (RhCMV) is an emerging model for human cytomegalovirus (HCMV) pathogenesis that facilitates experimental CMV infection of a natural primate host closely related to humans. We have generated a library of RhCMV mutants with lesions in genes whose HCMV orthologues have been characterized as nonessential for replication in human fibroblasts, and we characterized their replication in rhesus fibroblasts and epithelial cells. The RhCMV mutants grew well in fibroblasts, as predicted by earlier studies with HCMV. However, mutations in four genes caused replication defects in rhesus retinal pigment epithelial cells: Rh01 (an HCMV TRL1 orthologue), Rh159 (HCMV UL148), Rh160 (HCMV UL132), and Rh203 (HCMV US22). Growth of the Rh01-deficient mutant was examined in detail. After entry into epithelial cells, the mutant expressed representative viral proteins, accumulated viral DNA, and generated infectious virus, but it failed to spread efficiently. We conclude that Rh01 is a cell tropism determinant that has the potential to dramatically affect virus spread and pathogenesis.     

Use of specific-pathogen-free (SPF) rhesus macaques to better model oral pediatric cytomegalovirus infection

Background Congenital human cytomegalovirus (HCMV) infection can result in lifelong neurological deficits. Seronegative pregnant woman often acquire primary HCMV from clinically asymptomatic, but HCMV‐shedding children. Methods Potential age‐related differences in viral and immune parameters of primary RhCMV infection were examined in an oral rhesus CMV infection model in specific pathogen free macaques. RhCMV shedding was measured by real time PCR in plasma, saliva and urine. Immune parameters, including neutralizing and binding antibodies and RhCMV‐specific T cell responses, were assessed in longitudinally collected blood samples. Results The oral RhCMV infection model in infant SPF rhesus macaques demonstrated that (i) the susceptibility to oral RhCMV infection declines with age, and (ii) infant macaques shed RhCMV more persistently and at higher titers compared to adult macaques. (iii) Conclusions The oral infant RhCMV infection model appears to reflect viral pathogenesis in human HCMV‐infected children. Larger studies are needed to define immune parameters associated with better control of RhCMV in adult compared to young animals.     

Complete DNA sequence of the rat cytomegalovirus genome

We have determined the complete genome sequence of the Maastricht strain of rat cytomegalovirus (RCMV). The RCMV genome has a length of 229,896 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. RCMV was found to have counterparts of all but one of the open reading frames (ORFs) that are conserved between murine CMV (MCMV) and human CMV (HCMV). Like HCMV, RCMV lacks homologs of the genes belonging to the MCMV m02 glycoprotein gene family. However, RCMV contains 15 ORFs with homology to members of the MCMV m145 glycoprotein gene family. Four ORFs are predicted to encode homologs of host proteins; R33 and R78 both putatively encode G protein-coupled receptors, whereas r144 and r131 encode homologs of major histocompatibility class I heavy chains and CC chemokines, respectively. An intriguing feature of the RCMV genome is the presence of an ORF, r127, with similarity to the rep gene of parvoviruses as well as ORF U94 of human herpesvirus 6A (HHV-6A) and HHV-6B. Counterparts of these ORFs have not been found in the other sequenced herpesviruses.     

Rhesus cytomegalovirus particles prevent activation of interferon regulatory factor 3

One of the most important innate host defense mechanisms against viral infection is the induction of interferon (IFN)-stimulated genes (ISGs). Immediately upon entry, viruses activate interferon-regulatory factor 3 (IRF3), as well as nuclear factor kappaB (NF-kappaB), which transactivate a subset of ISGs, proinflammatory genes, as well as IFN genes. Most large DNA viruses exhibit countermeasures against induction of this response. However, whereas human cytomegalovirus (HCMV) inhibits IFN-dependent induction of ISGs, IFN-independent induction of ISGs is observed both in the presence and, even moreso, in the absence of viral gene expression. Rhesus CMV (RhCMV) is an emerging animal model for HCMV sharing important similarities in primary structure, epidemiology, and pathogenesis. To determine whether RhCMV would similarly induce ISGs, we performed DNA microarray and quantitative PCR analysis of ISG expression in rhesus fibroblasts infected with RhCMV or HCMV. In contrast to HCMV, however, RhCMV did not induce expression of ISGs or proinflammatory genes at any time after infection. Moreover, dimerization and nuclear accumulation of IRF3, readily observed in HCMV-infected cells, was absent from RhCMV-infected cells, whereas neither virus seemed to activate NFkappaB. RhCMV also blocked IRF3 activation by live or UV-inactivated HCMV, suggesting that RhCMV inhibits viral IRF3 activation and the resultant ISG induction with extraordinary efficiency. Since infection during inhibition of protein expression by cycloheximide or inactivation of viral gene expression by UV treatment did not trigger IRF3 activation or ISG expression by RhCMV, we conclude that RhCMV virions contain a novel inhibitor of IFN-independent viral induction of ISG expression by IRF3.     

Vaccination against a Virus-Encoded Cytokine Significantly Restricts Viral Challenge

Identification of immune correlates of protection for viral vaccines is complicated by multiple factors, but there is general consensus on the importance of antibodies that neutralize viral attachment to susceptible cells. Development of new viral vaccines has mostly followed this neutralizing antibody paradigm, but as a recent clinical trial of human cytomegalovirus (HCMV) vaccination demonstrated, this singular approach can yield limited protective efficacy. Since HCMV devotes >50% of its coding capacity to proteins that modulate host immunity, it is hypothesized that expansion of vaccine targets to include this part of the viral proteome will disrupt viral natural history. HCMV and rhesus cytomegalovirus (RhCMV) each encode an ortholog to the cellular interleukin-10 (cIL-10) cytokine: cmvIL-10 and rhcmvIL10, respectively. Despite extensive sequence divergence from their host''s cIL-10, each viral IL-10 retains nearly identical functionality to cIL-10. Uninfected rhesus macaques were immunized with engineered, nonfunctional rhcmvIL-10 variants, which were constructed by site-directed mutagenesis to abolish binding to the cIL-10 receptor. Vaccinees developed antibodies that neutralized rhcmvIL-10 function with no cross-neutralization of cIL-10. Following subcutaneous RhCMV challenge, the vaccinees exhibited both reduced RhCMV replication locally at the inoculation site and systemically and significantly reduced RhCMV shedding in bodily fluids compared to controls. Attenuation of RhCMV infection by rhcmvIL-10 vaccination argues that neutralization of viral immunomodulation may be a new vaccine paradigm for HCMV by expanding potential vaccine targets.     

Vaccine-induced control of viral shedding following rhesus cytomegalovirus challenge in rhesus macaques

The use of animal models of human cytomegalovirus (HCMV) infection is critical to refine HCMV vaccine candidates. Previous reports have demonstrated that immunization of rhesus monkeys against rhesus cytomegalovirus (RhCMV) can reduce both local and systemic replication of RhCMV following experimental RhCMV challenge. These studies used prime/boost combinations of DNA expression plasmids alone or DNA priming and boosting with either inactivated virion particles or modified vaccinia virus Ankara (MVA) expressing the same antigens. Viral outcomes included reduced RhCMV replication at the site of subcutaneous inoculation and RhCMV viremia following intravenous inoculation. Since shedding of cytomegalovirus from mucosal surfaces is critical for horizontal transmission of the virus, DNA priming/MVA boosting was evaluated for the ability to reduce oral shedding of RhCMV following subcutaneous challenge. Of six rhesus monkeys vaccinated exclusively against RhCMV glycoprotein B (gB), phosphoprotein 65 (pp65), and immediate-early 1 (IE1), half showed viral loads in saliva that were lower than those of control monkeys by 1 to 3 orders of magnitude. Further, there was a strong association of memory pp65 T cell responses postchallenge in animals exhibiting the greatest reduction in oral shedding. These results highlight the fact that a DNA/MVA vaccination regimen can achieve a notable reduction in a critical parameter of viral replication postchallenge. The recently completed clinical trial of a gB subunit vaccine in which the rate of HCMV infection was reduced by 50% in the individuals receiving the vaccine is consistent with the results of this study suggesting that additional immunogens are likely essential for maximum protection in an outbred human population.     

Immunogenicity and protective efficacy of DNA vaccines expressing rhesus cytomegalovirus glycoprotein B, phosphoprotein 65-2, and viral interleukin-10 in rhesus macaques

Rhesus cytomegalovirus (RhCMV) infection of macaques exhibits strong similarities to human CMV (HCMV) persistence and pathogenesis. The immunogenicity of DNA vaccines encoding three RhCMV proteins (a truncated version of glycoprotein B lacking the transmembrane region and endodomain [gBDeltaTM], phosphoprotein 65-2 [pp65-2], and viral interleukin-10 [vIL-10]) was evaluated in rhesus macaques. Two groups of monkeys (four per group) were genetically immunized four times with a mixture of either pp65-2 and gBDeltaTM or pp65-2, vIL-10, and gBDeltaTM. The vaccinees developed anti-gB and anti-pp65-2 antibodies in addition to pp65-2 cellular responses after the second booster immunization, with rapid responses observed with subsequent DNA injections. Weak vIL-10 immune responses were detected in two of the four immunized animals. Neutralizing antibodies were detected in seven monkeys, although titers were weak compared to those observed in naturally infected animals. The immunized monkeys and na?ve controls were challenged intravenously with 10(5) PFU of RhCMV. Anamnestic binding and neutralizing antibody responses were observed 1 week postchallenge in the vaccinees. DNA vaccination-induced immune responses significantly decreased peak viral loads in the immunized animals compared to those in the controls. No difference in peak viral loads was observed between the pp65-2/gBDeltaTM DNA- and pp65-2/vIL-10/gBDeltaTM-vaccinated groups. Antibody responses to nonvaccine antigens were lower postchallenge in both vaccine groups than in the controls, suggesting long-term control of RhCMV protein expression. These data demonstrated that DNA vaccines targeting the RhCMV homologues of HCMV gB and pp65 altered the course of acute and persistent RhCMV infection in a primate host.     

Pathogenesis of Experimental Rhesus Cytomegalovirus Infection

Human cytomegalovirus (HCMV) establishes and maintains a lifelong persistence following infection in an immunocompetent host. The determinants of a stable virus-host relationship are poorly defined. A nonhuman primate model for HCMV was used to investigate virological and host parameters of infection in a healthy host. Juvenile rhesus macaques (Macaca mulatta) were inoculated with rhesus cytomegalovirus (RhCMV), either orally or intravenously (i.v. ), and longitudinally necropsied. None of the animals displayed clinical signs of disease, although hematologic abnormalities were observed intermittently in i.v. inoculated animals. RhCMV DNA was detected transiently in the plasma of all animals at 1 to 2 weeks postinfection (wpi) and in multiple tissues beginning at 2 to 4 wpi. Splenic tissue was the only organ positive for RhCMV DNA in all animals. The location of splenic cells expressing RhCMV immediate-early protein 1 (IE1) in i.v. inoculated animals changed following inoculation. At 4 to 5 wpi, most IE1-positive cells were perifollicular, and at 25 wpi, the majority were located within the red pulp. All animals developed anti-RhCMV immunoglobulin M (IgM) antibodies within 1 to 2 wpi and IgG antibodies within 2 to 4 wpi against a limited number of viral proteins. Host reactivity to RhCMV proteins increased in titer (total and neutralizing) and avidity with time. These results demonstrate that while antiviral immune responses were able to protect from disease, they were insufficient to eliminate reservoirs of persistent viral gene expression.     

Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys

Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b' region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b' region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b' region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one na?ve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.     

Analysis and characterization of the complete genome of tupaia (tree shrew) herpesvirus

The tupaia herpesvirus (THV) was isolated from spontaneously degenerating tissue cultures of malignant lymphoma, lung, and spleen cell cultures of tree shrews (Tupaia spp.). The determination of the complete nucleotide sequence of the THV strain 2 genome resulted in a 195,857-bp-long, linear DNA molecule with a G+C content of 66.5%. The terminal regions of the THV genome and the loci of conserved viral genes were found to be G+C richer. Furthermore, no large repetitive DNA sequences could be identified. This is in agreement with the previous classification of THV as the prototype species of herpesvirus genome group F. The search for potential coding regions resulted in the identification of 158 open reading frames (ORFs) regularly distributed on both DNA strands. Seventy-six out of the 158 ORFs code for proteins that are significantly homologous to known herpesvirus proteins. The highest homologies found were to primate and rodent cytomegaloviruses. Biological properties, protein homologies, the arrangement of conserved viral genes, and phylogenetic analysis revealed that THV is a member of the subfamily Betaherpesvirinae. The evolutionary lineages of THV and the cytomegaloviruses seem to have branched off from a common ancestor. In addition, it was found that the arrangements of conserved genes of THV and murine cytomegalovirus strain Smith, both of which are not able to form genomic isomers, are colinear with two different human cytomegalovirus (HCMV) strain AD169 genomic isomers that differ from each other in the orientation of the long unique region. The biological properties and the high degree of relatedness of THV to the mammalian cytomegaloviruses allow the consideration of THV as a model system for investigation of HCMV pathogenicity.     

猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。     

Experimental coinfection of rhesus macaques with rhesus cytomegalovirus and simian immunodeficiency virus: pathogenesis

Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.     

A cyclooxygenase-2 homologue encoded by rhesus cytomegalovirus is a determinant for endothelial cell tropism

Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulators of a number of cellular processes, including the acute and chronic inflammatory response, hemostasis, and the innate immune response. Human cytomegalovirus (HCMV), which does not encode a viral COX-2 isoform, has been shown to induce cellular COX-2 expression. Importantly, although the precise role of COX-2 in CMV replication is unknown, COX-2 induction was shown to be critical for normal HCMV replication. In an earlier study, we identified an open reading frame (Rh10) within the rhesus cytomegalovirus (RhCMV) genome that encoded a putative protein (designated vCOX-2) with high homology to cellular COX-2. In the current study, we show that vCOX-2 is expressed with early-gene kinetics during RhCMV infection, resulting in production of a 70-kDa protein. Consistent with the expression of a viral COX-2 isoform, cellular COX-2 expression was not induced during RhCMV infection. Finally, analysis of growth of recombinant RhCMV with vCOX-2 deleted identified vCOX-2 as a critical determinant for replication in endothelial cells.     

A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques

To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.     

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.     

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.     

Development of Breeding Populations of Rhesus Macaques (Macaca mulatta) That Are Specific Pathogen-free for Rhesus Cytomegalovirus

Development of breeding colonies of rhesus macaques (Macaca mulatta) that are specific pathogen-free (SPF) for rhesus cytomegalovirus (RhCMV) is relatively straightforward and requires few modifications from current SPF programs. Infants separated from the dam at or within a few days of birth and cohoused with similarly treated animals remain RhCMV seronegative indefinitely, provided they are never directly or indirectly exposed to a RhCMV-infected monkey. By systematically cohousing seronegative animals into larger social cohorts, breeding populations of animals SPF for RhCMV can be established. The additional costs involved in expanding the current definition of SPF status to include RhCMV are incremental compared with the money already being spent on existing SPF efforts. Moreover, the large increase in research opportunities available for RhCMV-free animals arguably would far exceed the development costs. Potential new areas of research and further expansion of existing research efforts involving these newly defined SPF animals would have direct implications for improvements in human health.Abbreviations: HCMV, human cytomegalovirus; NHP, nonhuman primate; RhCMV, rhesus cytomegalovirus; SPF, specific pathogen-freeThe impetus for expanding the current SPF definition to include RhCMV is 2-fold. The first is the increasing number of studies involving infection of rhesus macaques with RhCMV as a nonhuman primate (NHP) model of human infection with human cytomegalovirus (HCMV). The second is the recognition that the current SPF protocols result in animals that are also uninfected with RhCMV, such that a relatively minor change in derivation can generate monkeys that meet the current SPF definition and that are uninfected with other endemic viruses, including RhCMV and simian foamy virus.