|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 394-398
Peripheral Blood CD14+ Cells From Healthy Subjects
Carry a Circular Conformation of Latent Cytomegalovirus
Genome
By
Cynthia A. Bolovan-Fritts,
Edward S. Mocarski, and
Jean A. Wiedeman
From the Department of Pediatrics, Section of Infectious Diseases,
University of California Davis; and the Department of Microbiology and
Immunology, Stanford University School of Medicine, Stanford, CA.
 |
ABSTRACT |
The majority of the human population harbors latent cytomegalovirus.
Although CD14+ peripheral blood mononuclear cells have
been implicated as sites of latency, the conformation of the latent
viral genome in these cells is unknown. In this study, the conformation
of viral genomic DNA was assessed in CD14+ cells from
healthy virus seropositive carriers using an electrophoretic separation
on native agarose gels in combination with polymerase chain reaction
detection. Here we show that the viral genome migrates as a circular
plasmid with a mobility equivalent to a circular 230-kb Shigella
flexneri megaplasmid marker. Neither linear nor complex or
integrated forms of the viral genome were detected. This report
provides further evidence that the CD14+ cell population
is an important site of viral latency in the naturally infected human
host. Detection of the viral genome as a circular plasmid during
latency suggests that this virus maintains its genome in a manner
analogous to other herpesviruses where latent viral genome conformation
has been studied.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HUMAN CYTOMEGALOVIRUS (CMV), a large 230- to 240-kb dsDNA member of the betaherpesvirus subfamily, is harbored
latently in 50% to 100% of healthy adults.1,2 The virus
can infect the human host directly through mucous membrane contact, or
by infected cells present in blood transfusions or tissue transplants. Infections caused by CMV are a leading infectious disease cause of
morbidity in allograft transplant recipients as well as other immunocompromised hosts (eg, human immunodeficiency virus [HIV] infected).3 They are also the predominant cause of
congenital infection in the Western world.4 CMV infection
is a significant risk after blood transfusion, because circulating
mononuclear cells of the peripheral blood (PB) provide a reservoir for
the latent virus.5-7 Reactivation of latent virus occurs in
the face of diminished immune surveillance and contributes more to the incidence of serious disease in immunocompromised hosts than primary infection. Despite the important role latency and reactivation play in
the pathogenesis of CMV disease, knowledge of the underlying mechanisms
controlling these processes remains limited.
Although viral DNA has been detected in circulating PB mononuclear
cells (PBMCs) from healthy seropositive individuals,8,9 the
conformation of this DNA has not been investigated. Identification of
the CMV genome form in blood cells can provide insight into understanding mechanisms of viral persistence and dissemination in the
human host. For herpesviruses where this has been studied, latent
infections are characteristically associated with a circular plasmid
form of the viral genome.10-14 In contrast, productive herpesviral infection is associated predominantly with linear viral
genome forms.12,15 It has long been known that the latent Epstein-Barr virus (EBV) is a circular extrachromasomal plasmid in
cultured B-lymphoblastoid cells.10,11 The latent herpes simplex virus-1 genome exists in an endless state based on DNA blot
analysis16 and an endless state has also been predicted for
the latent varicella-zoster virus genome.17 While these latter findings are consistent with a circular genome conformation analogous to EBV, tandemly integrated genomes or concatemers remain a
possibility. To analyze genome conformation specifically, a native
agarose gel system has been used to characterize large bacterial
plasmids18 and circular or linear forms of herpesvirus saimiri, herpesvirus ateles, EBV, and human herpesvirus 8 genomes.12-14 Rare cells in the PB CD14+
population, which can only be detected after polymerase chain reaction
(PCR) amplification, are known to carry CMV without evidence of
productive infection8,9,19 and can reactivate CMV after allogeneic stimulation.7 The focus of our study was to
apply native agarose gel electrophoresis, combined with PCR
amplification,13 to determine the CMV genome conformation
in PB mononuclear CD14+ cells from healthy seropositive
carriers. The results of this study show that the CMV genome migrates
as a circular plasmid form, suggesting that this virus maintains its
genome in the PB CD14+ cell population in a manner
analogous to other herpesviruses where latent viral genome conformation
has been studied.
| |
MATERIALS AND METHODS |
Isolation of CD14+ cells.
Individuals were identified as CMV seropositive using a CMV IgG ELISA
(Sigma, St Louis, MO). Healthy adult CMV-seropositive volunteers donated according to institutional review board guidelines. Fifty to 100 mL of blood was obtained by percutaneous venepuncture and
anticoagulated with EDTA (final concentrations, 5 mmol/L). A volume of 250 to 300 mL of pooled blood was prepared for each sample
set. PBMCs were isolated on Lymphoprep gradients (Nycomed, Oslo,
Norway). CD14+ cells were selected using
magnetic cell sorting with microbeads (Miltenyi Biotec, Auburn,
CA) according to manufacturer's instructions, using two
successive column purifications for each preparation. Purity analysis
was performed using fluorescein isothiocyanate (FITC) anti-CD14 clone
M P9 (Becton Dickinson, San Jose, CA) followed by flow
cytometry.
Cells and viruses used in control samples.
Primary human fibroblasts were infected at a multiplicity of infection
(MOI) of 5 plaque forming units (PFU) using CMV strain Toledo. Cells were obtained 7 days after infection, for preparation of
positive controls. The bacteria Shigella flexneri (gift from Stanley Falkow, Stanford University) contains a circular
megaplasmid of approximately 230 kb, and served as a circular plasmid
size marker. Bacteria were cultured in Luria broth, using appropriate biohazard precautions.
Preparation of agarose blocks for gel analysis.
Cell samples were prepared in agarose blocks using previously described
methods,20 and the CD14+ cells were prepared at
2.5 × 106 cells per 100 µL volume in each block.
Two agarose blocks (stacked front to back) per lane were cast directly
into gels.
Electrophoretic gel analysis.
Horizontal agarose gels were prepared according to methods previously
described.12 Low melting point (LMP) agarose
(0.75%) in 0.5× Tris-Borate-EDTA buffer was used to cast 25 × 20-cm gels. After the gel had solidified, a section of the gel
at the origin (3 × 15 cm) was excised, and the comb placed in the
cutout section. The section was filled with 0.8% LMP agarose
containing 2% sodium dodecyl sulfate (SDS) and 1 mg/mL of Proteinase K
added after gel temperature had cooled to 50°C. Bacteria samples
were loaded live onto gels using 108 cells per lane with
previously described methods.12 Gels were run at 4°C
for 3 hours at 0.8 V/cm, followed by 24 hours at 4.5 V/cm.
After electrophoresis, portions of the gel containing the controls were
stained in ethidium bromide solution (1 µg/mL) and photographed. For
PCR amplification, sample lanes were scored into sequential cores
starting from the origin, with the location of each recorded on a gel
template. Each agarose core was melted at 65°C for 15 minutes, then
digested with beta-agarase (FMC Bioproducts, Rockland,
ME) according to the manufacturer's instructions. DNA was precipitated with glycogen (10 µg per sample), ammonium acetate (Sigma) added to a final concentration of 2.5 mol/L, and 2.5 vol of
100% ethanol added. The resulting pellets were resuspended in 10 µL
of TE buffer (10 mmol/L TRIS-HCl pH 8, 1 mmol/L EDTA), and the entire volume was added to each PCR reaction.
PCR detection assays.
To reduce the risk of losing PCR amplification due to viral sequence
heterogeneity among different isolates, primer pairs directed to the
major early beta 2.7 gene were selected. This primer set recognizes a
conserved region of the CMV genome, and has been applied successfully
to detection of CMV isolates from eight different individuals and one
laboratory strain (AD 169) as previously reported.21
Forward primer, reverse primer, and probe oligonucleotides are as
previously reported.21 PCR amplifications (50-µL vol)
used reaction mix as reported with the following exceptions: Tris-HCl
pH 8.3, 3 mmol/L MgCl2, 1.25 U of Amplitaq Gold (Perkin Elmer, Norwalk, CT), and 1.25 U of Taq Extender
(Stratagene, La Jolla, CA). The primary PCR amplification
product was 315 bp. PCR reactions were performed in a Perkin Elmer 2400 thermocycler under the following conditions: 10 minutes at 94°C was
used to activate the Amplitaq polymerase, followed by 50 cycles of 20 seconds at 94°C, 30 seconds at 55°C, and 30 seconds at
70°C. After the cycles were completed, a final extension at
70°C for 7 minutes was performed. For nested PCR conditions, the
forward primer 5 CCG GTC GGC TTC TGT TTT AT 3 and reverse
primer 5 TCT CTT GTT GGG AAT CGT CG 3 were used. PCR
amplifications (30 cycles, 20-µL vol) used reaction mix as reported
above with the following exceptions: Tris-HCl pH 8.7, 0.1% Triton
X-100, and 0.1 U of native Taq polymerase (Promega, Madison,
WI). One microliter of the primary PCR reaction was added
as template. PCR conditions were 5 minutes at 94°C, followed by 30 cycles of 20 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. A final extension at 72°C for 7 minutes was
then performed. Nested PCR yielded a 202-bp product. The internal oligonucleotide probe used was the same as above. CD14+
cell samples were confirmed to contain CMV DNA before gel analysis, using total cell DNA prepared and PCR amplified as described above. When using limiting dilutions of positive control viral DNA as template, PCR reactions were sensitive enough to detect two template copies, without requiring a nested amplification.
The detection limits of the combined gel resolution and PCR approach
were determined, using limiting dilutions of CMV infected cells in a
background of negative cells. Viral DNA was detected down to a dilution
of  3 infected cells in a background of 106 uninfected
PBMCs collected from a seronegative donor (see Fig 2E).
This amount of viral DNA is expected to be in the range of 3 × 103 to 3 × 104 copies, well below the
detection limits for direct blot hybridization.22 To
further establish the limit of detection by PCR after recovery of DNA
after gel electrophoresis and beta agarase digestion, known copy
numbers of CMV cosmid clones were used as template. It was determined
that the level of sensitivity was 50 copies (data not shown). Detection
was lost below this level.
No inhibition of the PCR reactions was observed when low copy numbers
of CMV DNA were tested in the presence of melted agarose gels collected
from linear regions of uninfected cell DNA. Cores collected adjacent to
low copy positive sample lanes were also routinely negative. No
inhibition of the PCR reactions was observed, when tested using low
copy positive controls in the presence of beta agarase digested agarose
cores (data not shown).
DNA blot analysis of PCR products.
Ten percent of each PCR reaction volume was analyzed on 6%
polyacrylamide gels, with a 1-kb ladder marker (GIBCO-BRL,
Gaithersburg, MD). DNA was transferred to Zetaprobe
membrane (Boehringer Mannheim, Indianapolis, IN), by
capillary transfer in 0.4 N sodium hydroxide. The oligonucleotide probe
(above) was prepared using previously described methods.9
| |
RESULTS |
Resolution of viral genome forms using native agarose gels.
The Southern blot in Fig 1 shows that
circular forms of DNA are clearly resolved from those of unit-length
linear structures. The results obtained from a megaplasmid of
approximately 230 kb (similar in size to the CMV genome) carried in
S flexneri bacteria are shown in duplicate lanes 1 and 2. Probe-positive linear and circular forms of the megaplasmid are
observed. The linearized form seen is caused by the harsh lysozyme
treatment and shearing of some of the megaplasmid DNA.18
Duplicate lanes 3 and 4 show the distinct separation of probe-positive
linear and circular forms for EBV, a herpesvirus that is closely
related in size to CMV (170 kb). For EBV analyses, the B95-8 cell
line was used. In these cells, both circular and linear forms of the
EBV viral genome are present, because 5% of the cells are known to be
producing virus with the remaining number carrying latent circular
viral genome.13,23 Finally, in lane 5, CMV productively
infected cells (collected 7 days postinfection) show only a distinct
probe-positive unit-length linear 230-kb band and no circular form, as
would be predicted in a productive infection. In addition, some viral DNA is retained at the well origin for all lanes. The linearized megaplasmid form (lanes 1 and 2) migrates with a similar mobility as
the unit length linear form of the CMV genome and closely with the
linear EBV form. Overall, the clear resolution of circular versus
linearized genome forms is shown using the native agarose gel system.
View larger version (130K):
[in this window]
[in a new window]
| Fig 1.
Southern blot showing resolution of DNA forms. Lanes
loaded as the following: lanes 1 and 2, duplicate samples of
108 cells S flexneri bacteria carrying 230-kb
circular megaplasmid, loaded live in each lane; lanes 3 and 4, duplicate samples of EBV cell line B95-8 with 106 cells
loaded per lane; lane 5, 106 CMV lytically infected cells.
Southern blot was sectioned, and each section hybridized with probe
specific for those particular samples. The blots were then realigned
for figure. The (*) symbol marks the lower
edge of the proteinase K/SDS/agarose trough.
|
|
Purity of isolated CD14+ cell populations and
detection of CMV DNA before native agarose gel analysis.
CD14+ cells were isolated from PBMCs using positive
selection as described in Materials and Methods. The purity of the
CD14+ cells was routinely 95% or higher, based on flow
cytometry analysis (data not shown). Typical yields ranged from 10% to
20% of the original PBMC numbers. The cells displayed a monocyte type
morphology, and were able to adhere to glass or plastic.
Demonstration of free episomal form of CMV DNA from
CD14+ cell populations in healthy carriers.
To characterize the conformation of the CMV genome in PB mononuclear
CD14+ cells, we analyzed three independent sets of pooled
cells from healthy seropositive individuals.
Figure 2 shows the results obtained after
selection of CD14+ cells, electrophoretic separation, and
PCR amplification of DNA recovered from individual gel cores followed
by blot hybridization. A clear CMV probe-positive signal was observed
in each of three analyses, sets 1 through 3 (panels B through D), from
respective single cores with similar mobility to that of the circular
230-kb megaplasmid marker control (panel A). In all three sets, a CMV DNA positive signal was detected in only one discrete agarose core.
View larger version (48K):
[in this window]
[in a new window]
| Fig 2.
CMV genome conformation in PB CD14+ cells.
For all panels the top (Origin) of the gel is represented on the right
and the bottom of the gel is depicted on the left. Symbols + and  indicate positive and negative controls for PCR. Numbers 8, 5, and 4 label the probe-positive cores for panels (B), (C), and (D),
respectively. Size markers (315 bp and 202 bp) for the primary and
nested PCR products, respectively, are represented. (A) Ethidium
bromide-stained gel showing a representative lane containing
108 cells of S flexneri bacteria carrying the
230-kb circular megaplasmid marker. Ethidium bromide staining of
bacterial chromosomal DNA is associated with the region near the well
origin (Origin). The lower edge of the proteinase K/SDS/agarose trough
is seen as a brightly stained region midway between the circular marker
and the well origin. The circular megaplasmid control migrated at core
8 for all gels shown in panels (B), (C), (D), and (E). The apparent
linear megaplasmid band (Linear) invariably comigrated with the CMV
linear band as exemplified in the positive cores after PCR
amplification of low copy CMV-infected cells (E). (B) DNA blot analysis
of nested PCR products, from an inclusive series of sequential agarose
cores collected from the gel lane prepared from set 1 of pooled
CD14+ cells from five donors. (C) DNA blot analysis
similar to (B), from set 2 (3 donors). (D) DNA blot analysis of primary
PCR products, from set 3 (5 donors) and using only 10% of the
proteinase K normally used in block preparation. (E) Ethidium
bromide-stained gel of PCR amplification of low copy CMV-infected
cells.
|
|
A clear CMV probe-positive signal was observed in sample set 1 (panel
B; core 8), at a region comigrating with the circular 230-kb
megaplasmid marker (panel A). In addition, DNA blot analyses typically
showed both the primary PCR amplification product (315 bp) as well as
the nested amplification product (202 bp). Neither the region where
linear DNA would migrate nor the well origin showed positive signal.
For comparison, the relative mobility of the linear CMV genome on this
type of gel system is represented in Fig 2E, starting with 3
CMV-infected fibroblasts in a background of 106
CMV-negative PB cells.
Set 2 (panel C) exhibited a slightly slower migrating CMV
probe-positive core than that of the circular megaplasmid marker or the
migration of the CMV DNA probe positive core in sample set 1. For set
2, core 5 (panel C) migrated approximately 1 cm slower than core 8 (panel B), which was the core migrating with similar mobility to the
circular megaplasmid. Once again, there was no evidence of a
PCR-positive signal in the region of the gel representing linear 230-kb
CMV genome and none seen at the well origin, even after nested PCR
amplification and DNA blot analysis. For both sets 1 and 2 (panels B
and C) the primary PCR amplification showed CMV DNA migrating at a
mobility similar to the circular megaplasmid marker DNA and nested PCR
was not necessary to see this signal (see panel D where only primary
PCR was undertaken). Nested PCR was used to increase the sensitivity of
the assay for viral DNA in other regions of the gel. Our results
suggest that viral DNA was not present in the linear region of the gel
or near the well origin.
One explanation for the mobility differences between set 1 and set 2 might have been varying efficiency of proteinase K digestion due to the
fact that the blocks were prepared from such large numbers of
cells.12 To address this possibility, an additional pooled
set of CD14+ cells was prepared from seropositive
individuals similar in average age, gender, and serostatus as those of
the first pooled set. The cells were prepared in agarose blocks at the
same density (2.5 × 106 cells each) as in the first
two sets, but only 10% of the proteinase K was used in the digestion
step. As in previous gels, two agarose cell blocks (total of 5 × 106 cells) were loaded per lane. The results (panel D) show
the probe-positive signal was detected at the slowest observed
mobility, core 4 as compared with that of the circular megaplasmid
(core 8) and both previous CMV sample sets, but still in the region of
the gel where a circular form would be expected to migrate. Only
primary amplification was undertaken in this analysis (panel D),
because it had previously been established that a single round of PCR
was sufficient to amplify viral DNA in these samples. These results
suggest that incomplete proteolysis may contribute to reduced mobility
of circular CMV DNA, although these differences in migration may also
have other explanations, such as variation in the electrophoretic
conditions. Taken together, all three pooled sets of CD14+
cells showed probe-positive signal from cores in the region where a
circular form would migrate. Given the fact that these gels separate
primarily on the basis of structure,12,18 the slight mobility differences do not detract from the overall conclusion.
| |
DISCUSSION |
Using a native agarose gel electrophoresis system in conjunction with
PCR amplification, we were able to show that CMV genome in
CD14+ cells of healthy seropositive adult carriers is
detected only as a circular form without evidence of linear or
integrated CMV genome. The detection of circular CMV in the
CD14+ cell population from PB of healthy hosts indicates
that this is the form in which the latent viral genome persists in this population of cells. Because the predicted viral DNA copy number present in healthy subjects lies below the detection limits of direct
blot hybridization analysis,8 PCR amplification was used as
a means to extend the sensitivity of the detection limits. In our
results, the circular form of CMV DNA was consistently detected after
only one round of initial PCR amplification. Viral DNA was not detected
in the linear region of the gels or in the well origins in any of these
analyses, despite the fact that nested PCR conditions and DNA blot
hybridization analyses were used in a majority of analyses.
Viral DNA from productive or persistent infections is associated with
both large complex structures, such as concatemers or branched forms,
as well as linear forms of the viral genome, the products of
concatemers cleaved into unit-length linear viral DNA during the
packaging and assembly process. In the type of gel analysis used here,
branched, highly structured or very large DNA forms are retained at the
well origin of the gel, while linear forms of 50 to 700 kb migrate
with a mobility similar to linear CMV DNA.12,24,25 Both
large complex and/or concatemeric viral DNA and unit-length
linear forms were detected at the well origin and in the linear region
of the gel, respectively, for high copy and low copy CMV productively
infected cell controls (Fig 1 and Fig 2, respectively). These linear,
large concatemeric or integrated viral DNA do not migrate in the same
region of the gel with circular forms. If other forms exist in the
viral DNA reservoir during latency, the copy number was below our
detection limits.
Studies in healthy carriers have shown the presence of CMV DNA
throughout the myeloid lineage.8,26,27 Viral DNA has been detected in CD34+ cell populations that include early bone
marrow hematopoietic progenitors27 and CMV DNA and RNA from
bone marrow myeloid-committed progenitors coexpressing
CD33/15.26 In addition, CD14+ cells from PB,
but generally not CD14 cell types in PB, are the
predominant site of CMV viral genome.9,21 CMV virus can
also be reactivated from CD14+ PB cells from healthy
subjects after allogeneic stimulation and prolonged in vitro
culture.7 Our results have extended these observations and
indicate that latent viral DNA is maintained in CD14+ PB
cells as an extrachromosomal circular plasmid.
Finally, a circular plasmid conformation for the CMV genome is
consistent with that of other latent herpesviral genomes, and supports
studies suggesting the CMV genome is harbored in a latent state in
immature and more differentiated myeloid cells.26-29 Future studies should be undertaken to extend the analyses of the CMV genome
conformation to include these progressively earlier stages of the
myeloid cell lineage.
Taken together, these results are consistent with the proposed model
that the latent CMV genome resides in early CD34+ and
CD33+ progenitor cells in the bone marrow, and is
partitioned to the monocyte or dendritic lineage, where it can be
detected in CD14+ cells of the PB.30 In support
of this, we have reported the first successful characterization of the
CMV genome conformation in CD14+ cells from normal CMV
seropositive individuals and demonstrated that the genome persists as a
circular plasmid comigrating with a 230-kb circular megaplasmid marker.
| |
ACKNOWLEDGMENT |
We thank Mike McVoy, Lenore Pereira, Lucy Rasmussen, and Steve St. Jeor
for their critical review of this manuscript.
| |
FOOTNOTES |
Submitted July 13, 1998;
accepted September 4, 1998.
Supported by National Institutes of Health grants awarded to J.A.W.
(K08 AI01193) and E.S.M. (RO1 AI33852). J.A.W. is a recipient of UC
Davis Hibbard E. Williams Research Funds and UC Davis Medical Center
Children's Miracle Network Telethon Research Funds.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jean A. Wiedeman, Pediatric Infectious
Diseases, Neurosciences Bldg, 1515 Newton Ct, Room 600, Davis, CA,
95616; e-mail: jataylorwiedeman{at}ucdavis.edu.
| |
REFERENCES |
1.
Gerberding JL:
Incidence and prevalence of human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and cytomegalovirus among health care personnel at risk for blood exposure: Final report from a longitudinal study.
J Infect Dis
170:1410, 1994[Medline]
[Order article via Infotrieve]
2.
Bevan IS, Daw RA, Day JR, Ala FA, Walker MR:
Polymerase chain reaction for the detection of human cytomegalovirus in a blood donor population.
Br J Haematol
78:94, 1991[Medline]
[Order article via Infotrieve]
3.
Zaia JA, Forman SJ:
Cytomegalovirus infection in the bone marrow transplant recipient.
Infect Dis Clin North Am
9:879, 1995[Medline]
[Order article via Infotrieve]
4.
Dobbins JG, Stewart JA, Demmler GJ, and Collaborating registry group:
Surveillance of congenital cytomegalovirus disease, 1990-1991. Collaborating Registry Group.
MMWR Morb Mortal Wkly Rep
41:35, 1992
5.
Adler SP:
Transfusion-associated cytomegalovirus infections.
Rev Infect Dis
5:977, 1983[Medline]
[Order article via Infotrieve]
6.
Bowden RA:
Transfusion-transmitted cytomegalovirus infection.
Hematol Oncol Clin North Am
9:155, 1995[Medline]
[Order article via Infotrieve]
7.
Soderberg-Naucler C, Fish KN, Nelson JA:
Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors.
Cell
91:119, 1997[Medline]
[Order article via Infotrieve]
8.
Taylor-Wiedeman JA, Sissons JGP, Borysiewics LK, Sinclair JHG:
Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells.
J Gen Virol
72:2059, 1991[Abstract/Free Full Text]
9.
Taylor-Wiedeman JA, Sissons JGP, Sinclair JHG:
Induction of endogenous human cytomegalovirus gene expression after differentiation of monocytes from healthy carriers.
J Virol
68:1597, 1994[Abstract/Free Full Text]
10.
Nonoyama M, Pagano JS:
Separation of Epstein-Barr virus DNA from large chromosomal DNA in non-virus-producing cells.
Nature New Biol
238:169, 1972[Medline]
[Order article via Infotrieve]
11.
Adams A, Lindahl T:
Epstein-Barr virus genomes with properties of circular DNA molecules in carrier cells.
Proc Natl Acad Sci USA
72:1477, 1975[Abstract/Free Full Text]
12.
Gardella T, Medveczky P, Sairenji T, Mulder C:
Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis.
J Virol
50:248, 1984[Abstract/Free Full Text]
13.
Decker L, Klaman LD, Thorley-Lawson DA:
Detection of the latent form of Epstein-Barr virus DNA in the peripheral blood of healthy individuals.
J Virol
70:3286, 1996[Abstract]
14.
Decker LL, Shankar P, Khan G, Freeman RB, Dezube BJ, Lieberman J, Thorley-Lawson DA:
The Kaposi sarcoma-associated herpesvirus (KSHV) is present as an intact latent genome in KS tissue but replicates in the peripheral blood mononuclear cells of KS patients.
J Exp Med
184:283, 1996[Abstract/Free Full Text]
15.
Jean J, Yoshimura N, Furukawa T, Plotkin SA:
Intracellular forms of the parental human cytomegalovirus genome at early stages of the infective process.
Virology
86:281, 1978[Medline]
[Order article via Infotrieve]
16.
Efstathiou S, Minson AC, Field HJ, Anderson JR, Wildy P:
Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.
J Virol
57:446, 1986[Abstract/Free Full Text]
17.
Clarke P, Beer T, Cohrs R, Gilden DH:
Configuration of latent varicella-zoster virus DNA.
J Virol
69:8151, 1995[Abstract]
18.
Eckhardt T:
A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria.
Plasmid
1:584, 1978[Medline]
[Order article via Infotrieve]
19.
Jordan MC:
Latent infection and the elusive cytomegalovirus.
Rev Infect Dis
5:205, 1983[Medline]
[Order article via Infotrieve]
20.
Mcvoy MA, Adler SP:
Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within the concatemer.
J Virol
68:1040, 1994[Abstract/Free Full Text]
21.
Taylor-Wiedeman J, Hayhurst GP, Sissons JGP, Sinclair JH:
Polymorphonuclear cells are not sites of persistence of human cytomegalovirus in healthy individuals.
J Gen Virol
74:265, 1993[Abstract/Free Full Text]
22.
Mocarski ES, Stinski MF:
Persistence of the cytomegalovirus genome in human cells.
J Virol
31:761, 1979[Abstract/Free Full Text]
23.
Miller G, Lipman M:
Release of infectious Epstein-Barr virus by transformed marmoset leukocytes.
Proc Natl Acad Sci USA
70:190, 1973[Abstract/Free Full Text]
24.
Unger L, Ziegler SF, Huffman GA, Knauf VC, Peet R, Moore LW, Gordon MP, Nester EW:
New class of limited-host-range Agrobacterium mega-tumor-inducing plasmids lacking homology to the transferred DNA of a wide-host-range, tumor-inducing plasmid.
J Bacteriol
164:723, 1985[Abstract/Free Full Text]
25.
Rosenberg C, Casse-Delbart F, Dusha I, David M, Baucher C:
Megaplasmids in the plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum.
J Bacteriol
150:402, 1982[Abstract/Free Full Text]
26.
Hahn G, Jores R, Mocarski ES:
Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells.
Proc Natl Acad Sci USA
95:3937, 1998[Abstract/Free Full Text]
27.
Mendelson M, Monard S, Sissons JGP, Sinclair JHG:
Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors.
J Gen Virol
77:3099, 1996[Abstract/Free Full Text]
28.
Kondo K, Kaneshima H, Mocarski ES:
Human cytomegalovirus latent infection of granulocyte-macrophage progenitors.
Proc Natl Acad Sci USA
91:11879, 1994[Abstract/Free Full Text]
29.
Kondo K, Xu J, Mocarski ES:
Human cytomegalovirus latent gene expression in granulocyte-macrophage progenitors in culture and in seropositive individuals.
Proc Natl Acad Sci USA
93:11137, 1996[Abstract/Free Full Text]
30.
Sinclair J, Sissons P:
Latent and persistent infections of monocytes and macrophages.
Intervirology
39:293, 1996[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
A. K. L. Cheung, A. Abendroth, A. L. Cunningham, and B. Slobedman
Viral gene expression during the establishment of human cytomegalovirus latent infection in myeloid progenitor cells
Blood,
December 1, 2006;
108(12):
3691 - 3699.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. O. Simon, R. Holtappels, H.-M. Tervo, V. Bohm, T. Daubner, S. A. Oehrlein-Karpi, B. Kuhnapfel, A. Renzaho, D. Strand, J. Podlech, et al.
CD8 T Cells Control Cytomegalovirus Latency by Epitope-Specific Sensing of Transcriptional Reactivation
J. Virol.,
November 1, 2006;
80(21):
10436 - 10456.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Sinclair and P. Sissons
Latency and reactivation of human cytomegalovirus
J. Gen. Virol.,
July 1, 2006;
87(7):
1763 - 1779.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. O. Simon, C. K. Seckert, D. Dreis, M. J. Reddehase, and N. K. A. Grzimek
Role for Tumor Necrosis Factor Alpha in Murine Cytomegalovirus Transcriptional Reactivation in Latently Infected Lungs
J. Virol.,
January 1, 2005;
79(1):
326 - 340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Gredmark, T. Tilburgs, and C. Soderberg-Naucler
Human Cytomegalovirus Inhibits Cytokine-Induced Macrophage Differentiation
J. Virol.,
October 1, 2004;
78(19):
10378 - 10389.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Slobedman, J. L. Stern, A. L. Cunningham, A. Abendroth, D. A. Abate, and E. S. Mocarski
Impact of Human Cytomegalovirus Latent Infection on Myeloid Progenitor Cell Gene Expression
J. Virol.,
April 15, 2004;
78(8):
4054 - 4062.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. R. Visconti, J. Pennington, S. F. Garner, J.-P. Allain, and L. M. Williamson
Assessment of removal of human cytomegalovirus from blood components by leukocyte depletion filters using real-time quantitative PCR
Blood,
February 1, 2004;
103(3):
1137 - 1139.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Gredmark and C. Soderberg-Naucler
Human Cytomegalovirus Inhibits Differentiation of Monocytes into Dendritic Cells with the Consequence of Depressed Immunological Functions
J. Virol.,
October 15, 2003;
77(20):
10943 - 10956.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Hertel, V. G. Lacaille, H. Strobl, E. D. Mellins, and E. S. Mocarski
Susceptibility of Immature and Mature Langerhans Cell-Type Dendritic Cells to Infection and Immunomodulation by Human Cytomegalovirus
J. Virol.,
July 1, 2003;
77(13):
7563 - 7574.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Slobedman, E. S. Mocarski, A. M. Arvin, E. D. Mellins, and A. Abendroth
Latent cytomegalovirus down-regulates major histocompatibility complex class II expression on myeloid progenitors
Blood,
September 26, 2002;
100(8):
2867 - 2873.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. S. Beisser, L. Laurent, J.-L. Virelizier, and S. Michelson
Human Cytomegalovirus Chemokine Receptor Gene US28 Is Transcribed in Latently Infected THP-1 Monocytes
J. Virol.,
July 1, 2001;
75(13):
5949 - 5957.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction