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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2681-2687
RAPID COMMUNICATION
Molecular and Serological Examination of the Relationship of Human
Herpesvirus 8 to Multiple Myeloma: orf 26 Sequences in Bone Marrow
Stroma Are Not Restricted to Myeloma Patients and Other Regions of
the Genome Are Not Detected
By
John F. Tisdale,
A. Keith Stewart,
Bruce Dickstein,
Richard F. Little,
Ian Dubé,
D. Cappe,
Cynthia E. Dunbar, and
Kevin E. Brown
From the Hematology Branch, National Heart, Lung and Blood Institute,
Bethesda, MD; The Toronto Hospital, Toronto, Ontario, Canada; HIV and
AIDS Malignancy Branch, National Cancer Institute, Bethesda, MD; and
the Department of Laboratory Medicine, Sunnybrook Regional Health
Science Center, Toronto, Ontario, Canada.
 |
ABSTRACT |
Human herpesvirus 8 (HHV-8) genomic sequences were recently detected
by polymerase chain reaction (PCR) and in situ hybridization in bone
marrow stromal cells grown from multiple myeloma (MM) patients, but not
in cells from control subjects (Rettig et al, Science 276:1851,
1997). We sought to confirm these observations in our own
group of MM patients (n = 30). DNA was extracted from adherent
stromal cells grown under varying conditions and assayed for HHV-8
sequence using PCR to amplify the orf 26 (KS330) sequence (Chang et al,
Science 266:1865, 1997), as initially reported. Samples from
human control subjects (n = 25) were concurrently extracted and
analyzed. After 30 cycles of amplification, we did not detect any
positive samples. In a more sensitive nested PCR, samples from 18 of 30 (60%) MM patients were positive, at about the limit of detection, but
orf 26 sequence was also amplified from 11 of 25 (44%) human control
samples. However, PCR amplification from other regions of the viral
genome (orf 72 and orf 75) was uniformly negative for all MM and
control samples, despite equivalent sensitivity. Additionally, all sera
from MM patients were negative for HHV-8 IgG by immunofluorescence. Our
data do not support a role of HHV-8 in the etiology of MM but may
suggest the presence of a related (KS330-containing) virus in MM
patients and in some control subjects.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
HUMAN HERPESVIRUS 8 (HHV-8), also know as
Kaposi's sarcoma-associated herpesvirus, is a member of the
lymphotropic subgroup of herpesviruses. HHV-8 has been implicated in a
variety of disorders, including Kaposi's sarcoma
(KS),1 primary effusion
lymphomas,2 and multicentric Castleman's
disease.3 The virus is unusual in that the viral genome
encodes a large number of homologs of cellular genes, including genes
functioning in cell regulation (cyclin D, IRF-1), control of apoptosis
(bcl-2, DED domain proteins), cell-cell interaction (OX2),
immunoregulation (CD46/CR1, CR2), and cytokine signaling (CC
chemokines, CXC chemokine receptor, interleukin-6
[IL-6]).4 In particular, the viral encoded IL-6 (vIL-6)
has been shown to be functional in mouse B-cell proliferation assays,
preventing apoptosis in IL-6-dependent cells.5
IL-6 has been hypothesized as a major factor in the pathogenesis or
progression of multiple myeloma (MM) by a number of different animal
models, in vitro studies, and epidemiologic studies (reviewed in Klein
et al6). The most compelling human primary cell experiments showed that myeloma plasma cells induced paracrine production of IL-6
by marrow stromal cells.7,8 Both secretion of factors such
as IL-1 and/or tumor necrosis factor- (TNF-) by the tumor cells and direct adhesive interactions appeared to be important. Of
note, myeloma cells can stimulate excess IL-6 production from stromal
cells grown from both myeloma patients and normal donors,7 and the IL-6, in turn, stimulates the growth or prevents cell death of
the tumor cells. The in vivo role of these interactions is unclear, and
anti-IL-6 therapies have proved disappointing.9
Recently, it was reported that dendritic-like bone marrow stromal cells
grown from marrow mononuclear cells contained HHV-8 genomic sequences
detected by polymerase chain reaction (PCR) in 15 of 15 MM patients, a
lower percentage of patients with monoclonal gammopathy of undetermined
significance (MGUS), and no healthy controls.10 HHV-8
sequence was also detected in cultured cells by in situ hybridization
with an HHV-8-specific biotinylated probe. Although in the initial
report prolonged in vitro culture of the bone marrow cells was
required, a subsequent publication by the same group has described the
detection of HHV-8 sequences in multiple myeloma bone marrow core
biopsies by both PCR and in situ hybridization.11 The
possible involvement of HHV-8 viral IL-6 in myeloma progression has
been postulated, and viral IL-6 transcripts were detected in the marrow
stromal cells in the original report.10 We have sought to
confirm both these observations in myeloma patients and controls
studied at two collaborating institutions, using both PCR analysis for
multiple regions of the HHV-8 genome and serologic studies on the same
patient cohorts.
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MATERIALS AND METHODS |
Bone marrow samples.
All patients and volunteers gave their written informed consent before
participation in the study. The human studies were approved by the
Institutional Review Boards of both collaborating institutions and the
animal studies by the Animal Care and Use Committee.
Fresh bone marrow was collected from patients with MM before autologous
bone marrow transplantation (n = 15; Table
1) and mononuclear cells were fractionated on a ficoll-hypaque density gradient (Lymphocyte Separation Medium; Organon Teknika, Durham, NC).
Dexter-type long-term cultures were established as previously described
using complete long-term culture media (Stem Cell Technologies, Vancouver, British Columbia, Canada),12,13 with a weekly
half-volume media change (demidepopulation), and compared with parallel
cultures from normal human volunteers (n = 21), normal rhesus macaques (n = 7), and normal dogs (n = 5). At 21 days, the adherent layers were
harvested and an aliquot was cryopreserved for later DNA extraction.
Cell culture and DNA extraction were performed at a separate
institution from PCR analysis to reduce the risk of contamination with
PCR products.
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Table 1.
PCR Analysis of Myeloma Patient Stroma Grown in Dexter
Cultures and Concurrent Serological Testing for HHV-8 Antibody
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Additionally, stromal cell cultures grown under a variety of conditions
from patients with MM at various disease stages (n = 15;
Table 2) and control subjects (n = 4) were
analyzed for the presence of HHV-8 by PCR. Cryopreserved stromal cells
that were processed, grown, and harvested as previously described were initially analyzed and consisted of the adherent stromal cell fraction
grown from bone marrow mononuclear cells in Dulbecco's modified Eagles
medium supplemented with 10% fetal calf serum (D10).12
Parallel standard stromal cell cultures were established by plating
fresh bone marrow mononuclear cells or previously cryopreserved bone
marrow mononuclear cells at a density of 1 to 2 × 106
cells/mL in either Dulbecco's modified Eagles medium supplemented with
10% fetal calf serum, 2 mmol/L L-glutamine, 10 µg/mL gentamicin (D10), or Iscove's modified Dulbecco's medium, supplemented with 10%
fetal calf serum, 10% horse serum, 2 mmol/L L-glutamine, 10 µg/mL
gentamicin (long-term culture media [LTCM]; Stem Cell
Technologies) and incubated at 37°C in 5%
CO2, as previously described.12 The medium was
changed at 48 hours and then every 3 to 4 days until the adherent
stroma was confluent. The adherent cells were then harvested, washed,
and expanded. Finally, stromal cultures from additional subjects were
set up under all three conditions (D10, LTCM, and Dexter-type).
Cultures were harvested at 3 weeks, and DNA was extracted from the
fresh cells for HHV-8.
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Table 2.
PCR Analysis of Myeloma Patient Stroma Grown in Various
Culture Conditions and Concurrent Serological Testing for HHV-8
Antibody
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As a positive control, DNA was extracted from cell lines BCBL-1
(provided by Dr Robert Yarchoan, National Cancer Institute, Bethesda,
MD) and BCP-1 (ATCC, Rockville, MD), and Jurkat cell DNA was used as a
negative control.
PCR for HHV-8.
DNA was extracted from the stromal samples by proteinase K digestion
and phenol:chloroform precipitation or using the QIAamp Tissue kit
(Qiagen, Inc, Valencia, CA), according to the
manufacturer's protocol. DNA was extracted from approximately
106 cells and eluted into 200 µL of molecular biology
grade water. The concentration and purity of DNA were estimated by
measuring the optical densities at 260 and 280 nm (~1 µg of DNA was
obtained from 105 cells).
Amplification was performed using sets of primers from different parts
of the HHV-8 genome. Amplification from the orf 26 region (minor capsid
gene) was performed using the KS330223 primers KS1 and KS2,
as originally described, which amplify a 233-bp product.1 PCR reactions were performed with ExTaq DNA polymerase (Takara Biomedicals, Otsu, Japan) according to the manufacturer's
instructions (50 µL final volume, containing 100 to 500 ng of DNA, 80 µmol/L dNTPs, 0.5 mmol/L each primer, and 2 U ExTaq). Although PCR
was performed for 30 cycles in the initial experiments, this was
increased to 40 cycles of amplification for added sensitivity
(denaturation for 1 minute at 94°C, then 40 cycles of 40 seconds at
92°C, 40 seconds at 60°C, and 90 seconds at 75 °C, followed
by extension for 5 minutes at 75°C). The products (5 µL) were
analyzed by agarose electrophoresis, transferred to a membrane, and
hybridized using a specific oligonucleotide-radiolabeled
probe.1 A second round of PCR amplification was performed
on the (40 cycle) products using primers NS1 and NS2, which amplify a
160-bp product.14 Reactions were as described above (5 µL
of amplified product was amplified), with 30 cycles of amplification,
and products analyzed by agarose electrophoresis and Southern
hybridization.
In an attempt to develop more sensitive assays and to confirm the
results obtained using primers from the orf 26 (KS330) region, a second
nested PCR amplification was developed with primers from the orf 75 region (membrane antigen; KS631Bam2). The outside primers
(TAT-TCG-CGG-CCT-TGG-CAA-CC; AAG-ATG-CGC-ACC-GCG-TTG-TC) amplify a
408-bp product, and the internal primers (ACG-TAC-AGC-AGG-CCG-AGA-TG; GGA-GCT-GTC-GCG-ATA-GAG-GT) amplify a 245-bp product. PCR
reaction mixes and conditions were as described above, with 30 cycles
of amplification for both the outside and internal primer pairs. The
nested product was analyzed by Southern hybridization using a specific
oligonucleotide-radiolabeled probe
(CAG-TCT-GCT-CCA-TCT-CTA-CCA-CTA-CTT-CCA). A third one-stage PCR was
performed with primers from the orf 72 region (cyclin D homolog region)
that amplify a 106-bp product.15 Forty cycles of
amplification were analyzed using the probe previously described.15
To test for the integrity of the DNA and to exclude the presence of PCR
inhibitors, PCR amplification with  -actin primers was performed on
all samples, as previously described.12 In addition, some
repeatedly negative samples were spiked with 3 genome copies of HHV-8
and retested.
To reduce the risk of cross-contamination, all PCR reactions were
performed in a completely separate laboratory from the cell culture and
DNA extraction. Additional precautions included preparation of a master
mix from small working aliquots of commercial stock solutions and
molecular grade water, the use of aerosol barrier pipet tips (Molecular
Bioproducts, San Diego, CA), and autoclaved master mix and PCR tubes.
PCR reactions were set up by first aliquoting the master mix followed
by addition of the test DNA samples, the positive control dilutions,
Jurkat cell DNA, and finally a reagent control (water used to prepare
the master mix).
Sequencing of PCR products.
After amplification, PCR products were ligated into a cloning vector
(TA cloning; Invitrogen, Carlsbad, CA) and sequenced either by dideoxynucleotide chain termination using Sequinase II
(Amersham, Arlington Heights, IL) or automated cycle
sequencing with Taq polymerase (Perkin Elmer, Foster City,
CA). The sequences were aligned using DNAStar (DNAStar
Inc, Madison, WI).
Serology for HHV-8 antibody.
Sera were tested for HHV-8 IgG antibody by immunofluorescence using the
HHV-8 IgG IFA kit (Advanced Biotechnologies, Columbia, MD), according
to the manufacturer's protocol. Briefly, sera were tested at a 1:20
dilution and incubated with the HHV-8 infected (Epstein-Barr virus
[EBV] negative) cell line KS.16 Samples were considered
positive if there was either diffuse or restricted apple-green
fluorescence in the infected cells. Sera from children (provided by Dr
Naomi Luban, Children's National Medical Center, Washington,
DC), patients with breast cancer, and patients with Kaposi's sarcoma were tested in parallel.
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RESULTS |
Sensitivity of PCR.
The BCP-1 cell line contains 150 genome copies/cell of
HHV-8,17 and we used these cells to calculate the
sensitivity of the different PCR reactions. Jurkat cell DNA was spiked
with serial dilutions of DNA extracted from BCP-1 cells, and the DNA
was analyzed in the different assays. We calculated that the nested PCR
reactions (orf 26 region and orf 75 region) could detect less than 3 copies of HHV-8 in 200 ng of cellular DNA, as could the orf 26 (KS330) outside primers alone. The non-nested PCR using the orf 72 primers could detect less than 30 copies of HHV-8 DNA
(Fig 1).
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| Fig 1.
Sensitivity curves for three HHV-8 PCR assays using
primers from different regions of the genome followed by Southern
hybridization with specific internal probes. Jurkat cell DNA (J; 2 µg/reaction) was inoculated with a known copy number of BCP-1 HHV-8
DNA starting at 3 million (M) to 0.3 genome copies along with
amplification of BCBL-1, another HHV-8-containing cell line. Jurkat
cell DNA as well as water (H2O) were simultaneously
amplified. Non-nested amplification in the orf 26 and orf 72 region
were able to detect 30 genome copies per reaction. With longer
exposure, 3 genome copies could be reproducibly detected without
nesting using the orf 26 primers. Nested amplification in the orf 75 region could reproducibly detect 3 genome copies per reaction.
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To test that our primers were not specific for HHV-8 sequence in cell
lines only, DNA was extracted from fresh Kaposi sarcoma biopsies (3 individuals, 6 samples), 10-fold serial dilutions of the DNA were
prepared, and PCRs were performed. HHV-8 sequence was detected with all
three PCR reactions, with detection end points within 1 dilution of
each other (data not shown).
Detection of orf 26 (KS330) from stromal cultures.
Stromal samples grown under standard long-term culture conditions for 3 weeks and then frozen were examined from 15 MM patients and 20 normal
controls and 1 patient with breast cancer. Using the outside primers
from the orf 26 (KS330) region, initial experiments with only 30 cycles
of amplification were uniformly negative, but after 40 cycles of
amplification HHV-8 was initially detected in 9 of 15 myeloma patient
samples and 5 of 21 controls. Testing of separate aliquots of DNA at
two institutions showed that 2 of 15 myeloma samples were consistently
positive, and 13 of 15 samples were positive in at least one assay
(Table 1). Nested PCR in the orf 26 region uncovered no additional
positive patients (Fig 2). Similarly, after
testing separate aliquots of DNA from control patients at both
institutions, 2 were positive in both institutions, but a total of 10 of 21 were positive in at least one assay. (Insufficient DNA from
control subjects precluded testing the samples a third time.) orf 26 sequence was also detected in a total of 6 of of the rhesus samples,
but in none of 5 canine cultures.
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| Fig 2.
Ethidium bromide staining (upper panel) and Southern
hybridization (lower panel) of a representative PCR using the nested
orf 26 (KS330) primer set. The sensitivity curve (consisting of a known
copy number of HHV-8 from BCP-1 inoculated into 2 µg of Jurkat cell
DNA) shows the limit of detection to be 3 genome copies per reaction by
the presence of bands at the predicted 160-bp position (arrow) by both
ethidium staining and Southern hybridization. Numbers 1 through 14 represent patient samples and show scattered positives.
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In an attempt to enhance viral detection, stromal samples from an
additional 15 myeloma patients at differing disease stages were grown
in parallel under a variety of conditions, including those described in
the original report by Rettig et al,10 and the DNA was
analyzed (Table 2). orf 26 (KS330) HHV-8 sequence was detected in 5 of
15 patients under at least one culture condition. Samples
from no additional patients were positive when the PCR products were
amplified in the nested PCR. Stromal samples were also examined from 4 control patients (2 normal, 1 MGUS, and 1 breast cancer patient), and 1 normal donor was positive after nested PCR only.
The 160-bp product was sequenced from 2 patients, 2 control subjects,
and the two positive control cell lines BCP-1 and BCBL-1. Although
there was greater than 96% similarity, none of the products from
patients or controls was identical to the published or cell line
sequences and varied between 1 and 4 bp from the published HHV-8
sequence.
Detection of HHV-8 (orf 75, orf 72) from stromal cultures.
Every sample was also tested by the nested PCR from the orf 75 (KS651Bam) region. However, despite equal sensitivity and repetitive testing, no sample was ever positive (MM = 30, controls = 25). In
addition, samples from 26 patients and 2 controls were amplified using
primers from the orf 72 region. Again, no sample was positive.
Serology for HHV-8.
Serum samples were examined from 33 patients with MM (including 21 patients also analyzed by PCR [Table 1] and 12 additional patients),
6 patients with KS, 6 nontransfused children, and 10 patients with
breast cancer. All samples were coded and scored for fluorescence by
two independent observers before the code was broken. Serum from all KS
patients were positive with apple green fluorescence in both the
nucleus and cytoplasm (Fig 3). In contrast,
none of the 6 children, 10 breast cancer patients, or 33 myeloma
patients showed positive fluorescence.
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| Fig 3.
Representative serum indirect immunofluorescence assay.
Sera were tested in a 1:20 dilution by incubation on an
HHV-8-containing cell line (KS-1); approximately 30% of the KS-1
cells express HHV-8 proteins, whereas the remainder act as internal
controls. (A) represents a positive control, (B) a negative control, (C
and D) two MM patients, (E) a breast cancer patient, and (F) an AIDS
patient with KS. Positivity, green fluorescence restricted to
approximately 30% of the cells is seen in the positive control and the
AIDS patient only.
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DISCUSSION |
HHV-8 was first identified in acquired immunodeficiency syndrome
(AIDS)-associated KS lesions by Chang et al1 using
representational difference analysis. Since then, a great deal of work
has implicated the virus as critical in the development of KS; however,
investigators are not in complete agreement that the virus is
causative, and controversy exists as to whether HHV-8 is ubiquitous or
disease restricted.4 Seroprevalence studies support the
link between HHV-8 and KS and suggest that this human herpesvirus is
not ubiquitous in healthy adults.18 Antibodies to HHV-8
were infrequently found (1% to 2%) in the US or UK blood donor
population, hemophiliacs, and intravenous drug users and were more
common in human immunodeficiency virus (HIV)-infected homosexual men
without KS and in persons living in areas of Africa endemic for classic
KS. These results support the view that HHV-8 infection is largely
confined to those persons at risk for the development of KS.
Based on the presence of a biologically active human IL-6 homolog gene
encoded by HHV-8 and the previously recognized production of IL-6 from
the adherent cell fraction of bone marrow cultures from myeloma
patients, Rettig et al10 evaluated bone marrow stromal
cells grown from marrow mononuclear cells and detected HHV-8 genomic
sequences by PCR using primers amplifying in the orf 26 region in 15 of
15 MM patients, but not in normal control stromal cells. Reverse
transcription-PCR (RT-PCR) for viral IL-6 message was
positive in 3 of 3 myeloma patient-derived stromal cultures assayed but
in none of 2 controls. In situ hybridization for HHV-8-specific
sequences was also positive only in the cultured cells from myeloma
patients. Although in situ hybridization showed signal in 100% of the
cultured cells, PCR amplification for viral DNA (orf 26 region)
required 45 cycles, and no attempt was made to quantitate genome
number. Transmission electron microscopy of the cultured cells showed
ultrastructural characteristics of macrophages, yet viral structures
were not shown. Evaluation of core bone marrow biopsy samples by in
situ hybridization suggested that HHV-8 was present in the stromal cell
compartment.11 After the original report, preliminary
results in the form of "technical comments" appeared: a group in
France reported detection of orf 26 (KS330) HHV-8 sequences in 18 of 20 MM bone marrow biopsies after 60 cycles of PCR, but in 0 of 20 control
biopsies19; however, two other groups could not detect
HHV-8 sequences in any of 40 or 5 bone marrow samples of MM patients,
respectively.20,21 Additionally, clinical grade
functionally characterized dendritic cells derived from the blood of
patients with MM have been shown recently by two groups not to contain
HHV-8 sequences by sensitive PCR assays.22,23
However, our data using very sensitive nested PCR methodology do
support that sequence from the orf 26 region of HHV-8 is detectable in many myeloma patient-derived stromal cell cultures, but
at least 50% of both human and nonhuman primate control cultures also
contained orf 26 sequence. The inability to detect viral sequence in
any sample after amplification with 30 cycles of PCR despite
amplification of the control DNA dilutions suggests that the DNA exists
at a very low level in the cultured cells. Additionally, the different
results on repeat assays suggest that the orf 26 DNA exists at the
limit of detection by PCR that in our assay correlates with 3 genome
copies per 200,000 cells. To rule out an inhibitor of the PCR reaction
present in our samples, 2 persistently negative samples from MM
patients were amplified after the addition of 3 HHV-8 genome copies,
yielding positive results.
Unexpectedly, when other areas of the viral genome (orf 72, orf 75)
were amplified from the same DNA samples with an equally sensitive PCR
assay (limit of detection <3 genome copies per reaction), no
positives were detected in either the patient or control samples, even
with repetitive testing, despite the ability to detect HHV-8 sequences
in KS tissue. Furthermore, screening for serum antibodies to HHV-8 in
our myeloma patient cohort using an indirect immunofluorescence technique did not suggest a previous exposure to the virus. Whereas MM
patients (n = 33), breast cancer patients (n = 10), and normal children
(n = 6) were all negative by serology, HHV-8 antibodies were
demonstrated in all of 6 AIDS patients with KS. These results are in
agreement with those of several other groups have investigated the
seroprevalence of HHV-8 in MM patients and controls by assaying for
serum antibodies to HHV-8.20,21,24-26 All groups were
unable to confirm an association between HHV-8 and MM. The absence of antibodies to HHV-8 could not be explained by a generalized defective immunity to herpesviruses as antibody testing for other herpesviruses by two groups yielded their predicted freqencies.24,25
Additionally, epidemiologically, there is no increased incidence of
myeloma in regions of the world with a high seroprevalence for
HHV-8.26,27 Whereas strong epidemiologic, serologic,
virologic, and molecular (including sequence detection from various
regions of the genome) evidence supports the role of HHV-8 in KS,
multicentric Castleman's disease, and primary effusion lymphoma, the
finding of orf 26 sequence in both our patient cohort and controls is
not dissimilar to the published literature describing detection of
sequence in a variety of diseased and nondiseased tissues. orf 26 HHV-8
sequences have been reported to be present in a number of skin
conditions, including Bowen's disease, squamous cell carcinoma,
actinic keratosis, leukoplakia, as well as pemphigus vulgaris and
pemphigus foliaceous.28,29 Although not
restricted, HHV-8 sequences have also been detected by PCR in cerebral
tissue from patients with multiple sclerosis as well as cerebral tissue
from stillborn control samples.30 Additionally, HHV-8 has
been implicated in 3 patients with unexplained encephalitis who were
found to have viral sequence present by PCR in brain biopsy
specimens.31
The background prevalence of HHV-8 also remains unresolved. A group in
Italy assayed urogenital tract specimens by PCR (orf 26) and reported a
very high prevalence, with 30 of 33 or 91% of ejaculates from healthy
men undergoing varicocele correction positive for viral
sequences.14 In Sicily, where KS is endemic, only 13% of
semen from HIV-negative heterosexuals and 10% of AIDS patients without
KS could be found by PCR to contain HHV-8 sequences.32 Similarly, a group in the United States examined semen specimens by PCR
for HHV-8 in 99 HIV-infected men (95% of whom reported previous
homosexual contact), with only 1 of 99 positive. HHV-8 seropositivity
in this HIV-infected population was similar to other published reports
at 27%, and the only PCR-positive semen specimen was obtained from an
HHV-8 seropositive patient with active KS.33 Another group
was also unable to confirm the presence of HHV-8 sequences in
urogenital specimens obtained from both the United States and Italy
using a sensitive nested PCR capable of detecting less than 3 genome
copies per reaction.34
Even in healthy children and adults, a group in Japan reported a very
high prevalence of HHV-8 sequences, again orf 26, when PCR was
performed on peripheral blood mononuclear cells (35/56 [64%] and
12/15 [80%], respectively), although serologic studies were not
performed.35 Another group of investigators using a different primer set evaluated peripheral blood mononuclear cells from
children and adults with AIDS but without KS.36 None of 51 children weas positive, and 9 of 33 (27%) adults had detectable HHV-8
sequences in their peripheral blood. Serology for HHV-8 was also
performed in this study and correlated with the PCR results.
These reports have left the issue of disease restriction versus
ubiquitous distribution for HHV-8 largely unresolved. In general, when
highly sensitive assays for the orf 26 region have been used in both
diseased and nondiseased tissues, a high percentage of positive samples
have been reported; however, less sensitive PCR assays amplifying the
orf 26 region or sensitive PCR assays of other regions of the viral
genome have largely negative. Our results are in keeping with these
findings; the majority of both myeloma and control stromal cultures
contained detectable orf 26 sequences, albeit at low levels; HHV-8
sequence from other regions of the genome could not be amplified; and
antibodies to HHV-8 could not be detected in the serum of MM patients.
One possible explanation for these discrepancies is PCR contamination
with the orf 26 product. However, contamination is unlikely in our
study given the repeated negativity of the same samples on multiple
occasions in both the nonnested and nested assay in the orf 26 region,
similar results in assays run at two separate institutions, and the
fact that the number of positives remained constant throughout the
study period. Sequencing of several of the products yielded orf 26 HHV-8 sequence with greater than 96% similarity, yet none of the
products was identical to sequences derived from either of the two
positive control cell lines (nevertheless, the degree of sequence
variability is not sufficient to conclusively rule out contaminating
exogenous DNA given the error rate inherent to both the PCR and
sequencing procedures). Additionally, no samples were ever positive
using primers in the orf 72 or orf 75 region despite repetitive testing
with simultaneous amplification of the control BCP-1 DNA dilution in
every reaction, giving ample opportunity to contaminate the samples for
these regions in the same way.
Another possible explanation for these discrepancies is the presence of
a related human herpesvirus that retains sequence homology in the orf
26 region but not in the other areas assayed. Precedence for the
existence of a related virus exists in the nonhuman primate, because
limited DNA sequence for two herpesviruses closely related to HHV-8
have been reported in Macaca mulatta (rhesus)37,38 and a
third in Macaca nemestrina.38 A related rhesus virus almost
certainly explains the positive results seen in our rhesus derived
stromal cultures: in the immunofluorescence assay, 70% of our rhesus
sera were positive for HHV-8 antibodies (data not shown). Although we
were unable to confirm the recently proposed association between MM and
HHV-8 by either PCR for the viral genome or by seroprevalence in our
patient cohort, the presence of two HHV-8-like viruses within the same
nonhuman primate species, along with our data and those of others,
raises the possibility of a common related (KS330-containing) virus in
the human population more easily detected in the immunosuppressed host.
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FOOTNOTES |
Submitted March 3, 1998;
accepted July 30, 1998.
Address reprint requests to Kevin E. Brown, MD, Bldg
10/Room 7C218, National Institutes of Health, 9000 Rockville Pike,
Bethesda, MD 20892-1652; e-mail: brownk{at}gwgate.nhlbi.nih.gov.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract