Human herpesvirus 6A accelerates AIDS
progression in macaques
Paolo Lusso*†, Richard W. Crowley‡, Mauro S. Malnati*, Clelia Di Serio§, Maurilio Ponzoni¶, Angelique Biancotto?,
Phillip D. Markham**, and Robert C. Gallo†‡
Units of *Human Virology,§Biostatistics, and¶Pathology, San Raffaele Scientific Institute, 20132 Milan, Italy;‡Institute of Human Virology, University of
Maryland, Baltimore, MD 21201;?Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD 20892; and **Advanced Bioscience Laboratories, Kensington, MD 20894
Edited by Robert C. Gallo, University of Maryland, Baltimore, MD, and approved February 1, 2007 (received for review December 14, 2006)
Although HIV is the necessary and sufficient causative agent of AIDS,
genetic and environmental factors markedly influence the pace of
human herpesvirus 6A (HHV-6A), a cytopathic T-lymphotropic DNA
virus, fosters the progression to AIDS in synergy with HIV-1. In this
study, we investigated the effect of coinfection with HHV-6A on the
progression of simian immunodeficiency virus (SIV) disease in pig-
in a rapid appearance of plasma viremia associated with transient
clinical manifestations and followed by antibody seroconversion,
Whereas animals infected with HHV-6A alone did not show any
long-term clinical and immunological sequelae, a progressive loss of
However, progression to full-blown AIDS was dramatically acceler-
ated by coinfection with HHV-6A. Rapid disease development in
dually infected animals was heralded by an early depletion of both
CD4?and CD8?T cells. These results provide in vivo evidence that
HHV-6A may act as a promoting factor in AIDS progression.
simian immunodeficiency virus ? animal models ? herpesviruses
have been recognized, designated HHV-6A and HHV-6B, which
exhibit different genetic, immunologic, biological, and epidemio-
the epidemiology and disease associations of HHV-6A are less well
defined. Several lines of experimental and clinical evidence impli-
HHV-6A shares with HIV-1 a primary tropism for CD4?T
lymphocytes (5) and kills these cells in synergy with HIV-1 (6).
Moreover, HHV-6A can enhance the virulence and/or pathoge-
nicity of HIV-1 by several mechanisms, including activation of the
HIV-1 LTR (6–8), induction of CD4 expression and HIV-1
susceptibility in otherwise HIV-refractory cells such as CD8?T
cells (9) and NK cells (10), induction of HIV-enhancing cytokines
(11), and facilitation of the switch to CXCR4 usage (12). The
clinical evidence includes the frequent isolation of HHV-6 from
HIV-1-infected patients (1, 13–15), its frequent reactivation in
patients with progressive HIV-1 disease (16), its widespread dis-
semination in terminal AIDS patients (17, 18), its sustained repli-
cation in lymphoid tissue from HIV-infected subjects (19) associ-
ated with an increased HIV-1 load (20), as well as the correlation
between an early acquisition of HHV-6 in infancy and an acceler-
ated progression of HIV-1 disease (21). Strikingly, unlike typical
opportunistic infections, HHV-6 reactivation/reinfection tends to
occur at a relatively early stage during the progression of HIV-1
disease (16), as attested by still elevated numbers of circulating
CD4?T cells (22, 23) and preserved lymphoid tissue architecture
to the process of CD4?T cell destruction that leads to the
uman herpesvirus 6 (HHV-6) is a ?-herpesvirus that was
Despite the bulk of data hitherto accumulated, conclusive
evidence of the role played by HHV-6A in the progression of
HIV-1 disease is still lacking. In this study, we investigated the
effects of HHV-6A on AIDS progression by experimentally
coinfecting pig-tailed macaques (Macaca nemestrina) with
HHV-6A and simian immunodeficiency virus (SIV). A dramatic
acceleration of the immunological and clinical progression of
SIV disease was observed in animals coinfected with HHV-6A.
Study Design. To investigate the effects of HHV-6A on AIDS
progression, we experimentally coinfected pig-tailed macaques,
whose T cells are highly susceptible to HHV-6A infection in vitro
(24), with HHV-6A (strain GS) and a pathogenic SIV strain
(smE660) (25). Three groups of young adult animals, each com-
prising four randomly assigned animals, were infected by i.v.
inoculation with either SIV alone (group 1; animals 299, 301, 303,
and 307), HHV-6A alone (group 2; animals 309, 310, 311, and 312)
Dually infected animals were first inoculated with SIV and then
detectable antibodies to HHV-6A before inoculation, suggesting
that they were not naturally infected with HHV-6-related monkey
herpesviruses, as documented in drill monkeys and chimpanzees
(26). The animals were followed for up to 32 months after inocu-
lation, after which all surviving animals were euthanized.
Primary HHV-6A Infection. All macaques inoculated with HHV-6A
(groups 2 and 3) showed evidence of primary HHV-6A infection.
As seen in Fig. 1A, HHV-6A plasma viremia became detectable by
quantitative calibrated real-time PCR at week 1 after inoculation,
peaked between weeks 1 and 5, and then declined to disappear in
all animals by week 14. All of the animals had anti-HHV-6A IgG
seroconversion, which occurred after a mean of 3.0 ? 1.4 weeks in
group 2 and 2.2 ? 0.5 weeks in group 3 (Fig. 1B), even though in
two (312 and 316), the antibody reactivity was low and transient.
The establishment of a systemic infection and its productive nature
were confirmed by the detection of viral RNA transcripts by in situ
hybridization in lymph-node tissues obtained within 4 weeks of
inoculation (Table 1). Primary HHV-6A infection was associated
with clinical manifestations of mild to moderate intensity, such as
fever, nasal discharge, splenomegaly, and generalized lymphade-
performed research; M.S.M. contributed new reagents/analytic tools; P.L., M.S.M., C.D.S.,
M.P., A.B., and R.C.G. analyzed data; and P.L. and R.C.G. wrote the paper.
The authors declare no conflict of interest.
expressed and secreted; SIV, simian immunodeficiency virus.
†To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or gallo@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
March 20, 2007 ?
vol. 104 ?
no. 12 ?
appeared at week 2. Altogether, these findings indicated that M.
nemestrina is a susceptible animal model for HHV-6A infection.
Primary SIV Infection. All of the animals inoculated with SIV
(groups 1 and 3) showed evidence of primary SIV infection. Both
SIV plasma viremia (Fig. 1C) and SIV p27Gag antigenemia
[supporting information (SI) Fig. 4] became detectable and peaked
at week 2 after inoculation. No significant differences were ob-
served between groups 1 and 3 in the mean peak levels of
antigenemia (1.51 ? 0.81 vs. 1.74 ? 2.0 ng/ml) and plasma viremia
(6.56 ? 6.62 vs. 6.54 ? 6.75 log10genome equivalents/ml). Anti-
anti-HHV-6A IgG antibodies (B) during the acute phase of infection and the first year of follow-up in pig-tailed macaques singly infected with HHV-6A (group
viremia (C) and anti-SIV p27GagIgG antibodies (D) during the acute phase of infection and the first year of follow-up in pig-tailed macaques singly infected with
SIV (group 1, blue symbols) or coinfected with HHV-6A and SIV (group 3, green symbols). Time 0 corresponds to the time of SIV inoculation. Coinfected monkeys
were inoculated with HHV-6A at day 14 after SIV inoculation.
Virological markers in pig-tailed macaques singly or dually infected with SIVsmE660and HHV-6AGS. (A and B) Course of HHV-6A plasma viremia (A) and
Table 1. Expression of SIV and HHV-6A RNA in lymph nodes from singly and dually infected pig-tailed macaques
as detected by in situ hybridization
First biopsy (4 weeks after inoculation)
Second biopsy (6 months
SIV RNA on FDC HHV-6A RNASIV-RNA SIV RNA on FDC
Group 1: SIV only
Mean ? SD
822.2 ? 323.8
Group 2: HHV-6A only
Group 3: SIV ? HHV-6A
Mean ? SD
3,460.8 ? 5,187.5
???, very abundant. Deposition of SIV RNA on the surface of follicular dendritic cells (FDC) was graded as follows: ?, undetectable;
?, minimal; ?, low; ??, abundant; ???, very abundant. The amount of HHV-6A RNA transcripts was graded as follows: ?,
undetectable; ?, minimal; ?, low; ??, abundant.
www.pnas.org?cgi?doi?10.1073?pnas.0700929104Lusso et al.
genemia was transient, becoming undetectable at week 4 in all
animals except one (313, HHV-6A-coinfected); by contrast, SIV
viremia remained persistently positive in all animals. All animals
showed anti-p27Gagantibody seroconversion, which occurred after
a mean of 3.7 ? 1.5 weeks in group 1 vs. 4.5 ? 1.9 weeks in group
3 (Fig. 1D), with an inverse correlation between the time to
coefficient, ?0.725; P ? 0.042). Of note, the levels of anti-p27Gag
reactivity were persistently low in two animals (303 and 315), both
of which experienced a rapid progression to AIDS. The establish-
ment of systemic SIV infection was confirmed by in situ hybridiza-
tion in lymph nodes (Table 1) and by the repeated isolation of SIV
signs observed during primary SIV infection included fever, nasal
discharge, generalized lymphadenopathy and splenomegaly; the
fever was generally higher and of longer duration in animals
coinfected with SIV and HHV-6A. Immunologically, a transient
loss of circulating CD4?T cells was detected in two singly infected
and three coinfected animals.
Natural History of SIV Disease in Singly and Dually Infected Macaques.
After the acute phase, the animals were monitored at monthly
intervals for multiple clinical, virologic, and immunologic param-
eters. No long-term clinical and hematological alterations were
seen in animals singly infected with HHV-6A, despite the occa-
sional detection of low levels of plasma viremia (?100 genome
equivalents/ml). In particular, their CD4?and CD8?T cell counts
(Fig. 2B), associated with a marked reduction of lymphocyte
proliferation indices (data not shown). However, the progression
toward full-blown AIDS was dramatically accelerated in macaques
of the study, AIDS-defining clinical conditions developed in all 4
coinfected macaques, but in only one of 4 singly infected with SIV
(SI Table 2). Survival analysis showed that the difference in AIDS
progression between the two groups of SIV-infected animals was
close to statistical significance (P ? 0.076), although it failed to
reach it, most likely because of the low number of animals included
in each group.
The accelerated AIDS progression in HHV-6A-coinfected ma-
caques was heralded by a faster depletion of circulating CD4?T
cells (Fig. 2B), with a mean loss of 45.3 ? 18.3 cells per mm3/week
in HHV-6A-coinfected macaques vs. 23.9 ? 12.5 in singly infected
macaques over the first 14 weeks of infection. Strikingly, coinfected
animals also exhibited a faster depletion of circulating CD8?T
lymphocytes (Fig. 2C), with a mean loss of 35.7 ? 22.2 cells per
mm3/week (vs. 11.7 ? 28.4) over the first 32 weeks of infection.
Using an exponential mixed-effect model, there was a significant
difference both in CD4?and in CD8?T cell decline between the
two groups (P ? 0.01). Of note, the mean CD4?and CD8?T cell
counts did not adequately reflect the dramatic immunologic pro-
gression seen in three coinfected animals (315, 316, and 317)
because the fourth animal in this group (313) consistently showed
outlier values: for example, the time to reach CD4?T cell counts
?200/mm3was 63 weeks in animal 313 vs. a mean of 14.8 ? 2.9
counts ?800/mm3was 94 weeks vs. a mean of 9.8 ? 5.9 weeks.
Unlike the immunologic parameters, the levels of SIV plasma
viremia and antigenemia during follow-up were not significantly
different between singly and dually infected animals. Conversely,
disease progression in dually infected animals was accompanied by
mixed effect model, the levels of HHV-6A viremia were signifi-
0.01), suggesting that SIV infection exerted boosting effects on
HHV-6A replication. Dually infected animals also showed a sig-
nificantly faster decrease in anti-HHV-6A antibody reactivity over
time (P ? 0.01).
SIV Superinfection of Two HHV-6A-Infected Macaques. An opportu-
nity to further evaluate the effect of HHV-6A coinfection on the
natural course of SIV disease was offered by the accidental SIV
superinfection of two animals that were initially infected with
HHV-6A alone (310 and 312) during the course of the study. At
months 13 and 21, respectively, of HHV-6A inoculation, the
animals escaped from their cage and were involved in fighting with
SIV coinfected animals. As a result, both animals became super-
infected with SIV, as shown by a rapid appearance of p27Gag
antigenemia (SI Fig. 4). At that time, neither animal presented any
signs of clinical or immunological deterioration, with CD4?and
CD8?T cell counts stably within the normal range. Despite the
uncontrolled conditions of SIV superinfection and the different
timing and route of infection, these animals fitted the definition of
HHV-6A/SIV coinfection. After SIV acquisition, both animals
exhibited a very rapid decline of CD4?and CD8?T cells (Fig. 2 E
and F), developing AIDS-related conditions after 69 (310) and 15
(312) weeks of SIV superinfection. Upon inclusion of these two
additional macaques into the survival analysis, the difference in
statistically significant (P ? 0.032) (Fig. 2C).
Histopathology and Viral Replication in Lymphoid Tissue.Twolymph-
node biopsies were obtained from each animal, one during the
acute phase of infection and one 6 months after inoculation. In the
first set of biopsies, all of the animals showed evidence of follicular
or HHV-6A (data not shown), the nodal architecture was con-
served, whereas in dually infected animals it was largely effaced by
a florid follicular hyperplasia with confluent germinal centers (Fig.
3B). Coinfected lymph nodes showed higher levels of SIV RNA
deposited on the surface of follicular dendritic cells (Fig. 3D)
compared with those singly infected with SIV (Fig. 3C); likewise,
there was a higher number of SIV RNA-expressing cells, albeit not
statistically significant (Table 1). HHV-6A mRNA expression was
documented primarily in the extrafollicular area, with a higher
intensity in lymph nodes coinfected with SIV than in those singly
infected with HHV-6A (Table 1). Thus, HHV-6A and SIV were
simultaneously replicating in coinfected lymph nodes. In biopsies
obtained 6 months after inoculation, both the frequency of SIV-
infected cells and the levels of SIV-RNA deposition on follicular
dendritic cells were lower than in the first biopsies (Table 1);
animals with the most rapid disease progression showed an early
involution of the nodal architecture with germinal center atrophy
and effacement (data not shown).
A major hindrance to elucidating the role played by HHV-6A in
present study, we identified M. nemestrina as a suitable model for
the study of HHV-6A infection, as indicated by a rapid appearance
of plasma viremia after inoculation, accompanied by clinical man-
ifestations and followed by antibody seroconversion; moreover, as
typically seen in human subjects, HHV-6A was found to persist in
vivo after primary infection. Taking advantage of this new model,
we obtained the first conclusive in vivo evidence that HHV-6A
Although macaques coinfected with HHV-6A showed a signifi-
cantly faster depletion of CD4?T cells compared with macaques
singly infected with SIV, the difference in AIDS-free survival
between the two groups failed to reach statistical significance (P ?
0.076), most likely because of the relatively low number of animals,
which is a common limitation of studies with nonhuman primates.
accidental SIV superinfection of two animals originally infected
Lusso et al.
March 20, 2007 ?
vol. 104 ?
no. 12 ?
with HHV-6A alone, both of which experienced a very rapid
disease progression after acquiring SIV. A note of caution in
interpreting these results is mandatory because of the different
conditions in which SIV superinfection occurred (inoculation
HHV-6A infection), even though such conditions may in fact have
played against an optimal synergy between the two viruses. Re-
gardless, when these two additional animals were included in the
analysis, the difference in AIDS-free survival between singly and
dually infected animals reached statistical significance (P ? 0.032).
Various opportunistic agents have been suggested to accelerate
the course of HIV-1 disease. However, the case of HHV-6A is
unique because of the peculiar biological characteristics of this
herpesvirus, which shares with HIV/SIV a primary tropism for
CD4?T cells (5, 6). Thus, although other agents may foster the
documented in macaques coinfected with SIV and rhesus cyto-
megalovirus (27) or Mycobacterium bovis (28), HHV-6A has the
potential to directly affect the basic pathogenetic mechanism of the
Although the precise mechanism whereby HHV-6A triggered a
faster progression of SIV disease in our macaques is still at present
unknown, some indications emerged from our study. Using in situ
hybridization, we documented a simultaneous replication of
HHV-6A and SIV in coinfected lymphoid tissue, indicating that a
direct interaction between the two viruses could occur. A trend
toward higher levels of SIV RNA expression was evident in
HHV-6A-coinfected lymph nodes compared with those singly
infected with SIV, but the overall levels of SIV viremia and
antigenemia did not clearly distinguish the two groups of animals.
By contrast, in agreement with the frequency of HHV-6 reactiva-
tion/reinfection seen in HIV-1-infected patients (1, 13–19, 23), an
evident amplification of HHV-6A replication was seen in SIV-
coinfected monkeys, most likely favored by the immunologic dys-
regulation induced by SIV infection. Studies have demonstrated
(6–12, 24): one of the most peculiar is its ability to expand the
repertoire of HIV/SIV-susceptible cells by inducing de novo ex-
pression of the primary HIV/SIV receptor, CD4, in productively
infected CD8?T cells (9) or other cytotoxic effectors (10). Indeed,
our HHV-6A-coinfected macaques experienced a rapid depletion
of both CD4?and CD8?T cells. This early loss of CD8?T cells,
the in vivo spread of both SIV and HHV-6A, thereby fostering the
progression of immunodeficiency.
Another intriguing hypothesis to interpret the present results
stems from our recent observation that SIV isolates obtained from
curves for pig-tailed macaques singly infected with SIV (group 1, blue lines) or HHV-6A (group 2, red lines) or coinfected with SIV and HHV-6A (group 3, green
differences in survival between groups. (B and E) Depletion of peripheral blood CD4?T cells. (C and F) Depletion of peripheral blood CD8?T cells. A–C shows
Kaplan–Meier curves calculated for the original three groups of animals (group 3, n ? 4). D–F shows Kaplan–Meier curves calculated after inclusion in group 3
of two additional animals (310 and 312), which were accidentally superinfected with SIV at month 13 and 21, respectively, from the initial HHV-6A inoculation
because of drop-out or study termination. In group 2, only two animals (309 and 311) regularly completed the follow-up, because the remaining two animals
were reclassified as SIV/HHV-6A coinfected during the course of the study.
Clinical and immunological disease progression in pig-tailed macaques singly or dually infected with SIVsmE660and HHV-6AGS. Shown are Kaplan–Meier
www.pnas.org?cgi?doi?10.1073?pnas.0700929104Lusso et al.
HHV-6A-coinfected macaques after one year of infection had
acquired resistance to regulated on activation normal T cell ex-
pressed and secreted (RANTES) (A.B., J. C. Grivel, A. Lisco,
P.D.M., R.C.G., L. B. Margolis, and P.L., unpublished work).
RANTES is a CCR5-binding chemokine that blocks the entry of
SIV into cells (29). We reported that HHV-6A is a potent RAN-
TES inducer in lymphoid tissue (12), a property that may have
contributed to the initial containment of SIV replication in dually
infected macaques. In fact, no evident amplification of SIV was
observed in dually infected animals compared with those singly
subsequently evolved toward RANTES resistance, most likely
under the selective pressure of elevated RANTES levels. Resis-
tance to RANTES is increasingly recognized as a key virulence
factor in HIV-1 infection (30–32), which may allow the virus to
replicate in the high-RANTES milieu present within inflamed
lymphoid tissues (33), particularly on coinfection with other mi-
crobes (34). Thus, one of the possible mechanisms whereby
HHV-6A may foster the progression to AIDS is by facilitating an
early acquisition of RANTES resistance. Additional studies in
patients and nonhuman primates will be important to confirm the
relevance of these mechanisms to HIV/SIV disease progression. A
definitive elucidation of the role of HHV-6A in AIDS will have
implications not only for our understanding of AIDS pathogenesis,
measures for the control of HIV infection.
Animals. Twelve juvenile pig-tailed macaques (M. nemestrina)
were included in the study; their age at the time of enrollment
was 6–7 years. All were negative for SIV, STLV, herpes B virus,
filovirus, SRV-1 and -2, measles virues, and HHV-6A. The
animals were housed in individual cages at the animal facility of
Advanced Bioscience Laboratories (Rockville, MD). The study
protocol was approved by the National Cancer Institute Animal
Care and Use Committee. Transportation and housing condi-
tions, as well as all procedures including euthanasia were in
conformity to the Animal Welfare Act and the U.S. Government
Principles for the Utilization and Care of Vertebrate Animals
Used in Testing, Research and Training. After quarantine, the
animals were tested for baseline hematological and immunolog-
ical parameters before inoculation with SIV and/or HHV-6A.
Virus Strains and Inoculation Procedure. SIVsmE660is an uncloned
virus that was obtained at the time of AIDS-related death from the
derived from strain sm/F236 (25). SIVsmE660replicates in macaque
was expanded in vitro in CEMx174 cells. In vivo titration in
macaques showed an infectivity of 104.5half-maximal macaque
infectious doses (MID50)/ml (25). HHV-6AGS, isolated from an
expanded in vitro in activated HHV-6-negative human cord blood
mononuclear cells. The viral stock was derived from culture super-
natants clarified by centrifugation at 2,000 ? g and filtered through
a 0.4 ?M device; its in vitro infectivity was ?106half maximal cell
culture infection doses (CCID50)/ml. As a mock HHV-6A inocu-
lum, we used uninfected culture supernatants from the same cells
used for producing the HHV-6A stock prepared according to the
Each animal was randomly assigned to one study group and
inoculated intravenously. The SIV inoculum contained 500 MID50
diluted in 2 ml of sterile PBS. The HHV-6A inoculum contained
3 ? 106CCID50diluted in 3 ml of RPMI medium 1640. At time 0,
animals in groups 1 and 3 received SIV, and animals in group 2
received HHV-6A; 14 days later, animals in group 3 received
HHV-6A, whereas animals in group 1 were injected with the mock
Virologic and Serologic Assays. SIV antigenemia was measured by
using a commercial p27 antigen-capture assay (Abbott Laborato-
ries, Abbott Park, IL). Plasmatic levels of SIV RNA (SIV viremia)
were measured by NASBA as described (35). Plasmatic levels of
HHV-6A DNA (HHV-6A viremia) were measured by using a
quantitative calibrated real-time PCR (TaqMan), using specific
primers and probe as reported in ref. 36, with the addition of an
exogenous calibrator molecule to normalize for the DNA-recovery
Antibodies to SIV were assayed by ELISA, using a commercial
kit (Beckman Coulter, Fullerton, CA). Sera were tested at the
standard dilution of 1:40. Specific OD values were calculated by
subtracting from the average of triplicate wells incubated with
monkey serum the average of quadruplicate negative control wells
(incubated with all other reagents except monkey serum) plus two
times the standard deviation (SD) value.
Antibodies to HHV-6A were assayed by using an in-house
ELISA, modified from a method reported in ref. 38. Briefly, 30 ?
106HSB-2 cells infected with HHV-6AGSwere extensively washed
X-100 in PBS, pH 7.4; uninfected homologous cells were also
processed in parallel as a control. After clarification by centrifuga-
tion for 10 min at 9,000 ? g, 100 ?l of the HHV-6A antigen or
control cell lysate (each 100 ?g/ml) was used to coat 96-well
microtiter plates (Nunc, Naperville, IL) in 60 mM sodium carbon-
at the standard dilution of 1:40 for 2 h at room temperature. After
repeated washings, peroxidase-conjugated goat anti-human IgG
at 1:250 dilution for 2 h at room temperature. After additional
C); 317, coinfected with HHV-6A and SIV (B and D). A and B show Hematoxylin-
eosin staining. C and D show In situ hybridization for SIV RNA of successive
sections from the same lymph nodes as above . The arrows indicate enlarged
lymphoid follicles with reactive germinal centers (A and B) and specific SIV RNA
tissue from animal 303, the overall architecture is conserved, with lymphoid
throughout the parenchyma, with little, if any, specific signal within reactive
reactive germinal centers; an intense SIV RNA signal is visible in correspondence
to both subcapsular and deeper-located hyperplastic follicles.
Histopathology and in situ hybridization in lymph node tissues from
Lusso et al.
March 20, 2007 ?
vol. 104 ?
no. 12 ?
washings, the appropriate substrate was added for 30 min, after
which the reaction was halted. Specific OD values were calculated
by subtracting from the average of triplicate wells containing
HHV-6A antigen the average OD values (plus two times the SD
value) from triplicate negative-control wells incubated with the
In Situ Hybridization. In situ hybridization was performed by Dr.
Cecil Fox (Molecular Histology Laboratories, Gaithersburg, MD)
as described in refs. 39–41. Tissues were hybridized with antisense
35S-labeled RNA probes; sense probes were used in parallel as
negative controls. For SIV, both virus-expressing cells and virion-
associated viral RNA trapped on the surface of follicular dendritic
cells were evaluated. Determination of the number of SIV-
expressing cells and the amount of follicular dendritic cells-
sections, using a phosphorimager (Fuji Medical Systems, Burbank,
performed by using as a probe in vitro RNA transcripts from
molecular clone pZVH14, which encodes, among others, the large
tegument protein (U31) (42).
Statistical Analysis. Statistical analysis was performed by using the
S-statistical software, Version 2.1.1. Correlation analyses were
performed with Pearson and Spearman correlation coefficients to
performed nonparametrically by means of Kaplan–Meier curves.
Log–rank tests were used to investigate possible differences in
survival time between groups. Generalized mixed effect models
were used to assess differences in CD4 and CD8 slopes as well as
levels of viremia between groups. Because we focused on modeling
the trend components of quantitative outcomes and their evolution
over time, the response variables were addressed as time series
adding a random component to the fixed effects of the variable
‘‘group.’’ The random component accounts for unobservable het-
erogeneity among subjects, which is a critical issue in longitudinal
studies with a high source of random variation. A linear mixed-
effect model was used to compare the levels of HHV-6A and SIV
used to fit the decline of circulating CD4?and CD8?T cells over
time, which clearly showed an exponential trend; statistical evi-
dence in favor of the exponential model was obtained by Akaike
was ? ? 0.05.
We thank V. Hirsch (National Institute of Allergy and Infectious
Diseases, Bethesda, MD) for providing the SIVsmE660viral stock and for
technical advice; J. L. Southers, R. Woodward, and J. Treece for
veterinarian assistance; G. Franchini for technical advice; P. Farci
for reading the manuscript; A. Ambrosi for statistical advice; A. Nonis
for support in data analysis; P. Secchiero, G. Locatelli, S. Silva, T.
Rutigliano, and G. Faga ` for performing PCR assays; H. Miao for SIV
ELISA; and S. Orndorff for administrative management. The initial part
of this work was conducted when three of the authors (P.L., R.W.C.,
R.C.G.) were at the Laboratory of Tumor Cell Biology, National Cancer
Institute, Bethesda, MD. This work was supported by the National
Cancer Institute Intramural Research Program; the European Union
Biomed-2 Program (Brussels) Grant BMH4CT961301 (to P.L.); and the
Istituto Superiore di Sanita ` Italian AIDS Program (Rome) Grants
40B.57, 50C.17, 50D.17, and 50F.23 (to P.L.).
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