SIVmac251 Is Inefficiently Transmitted to Rhesus Macaques
by Penile Inoculation with a Single SIVenv Variant
Found in Ramp-up Phase Plasma
Zhong-Min Ma,1,2Brandon F. Keele,3Huma Qureshi,1,2Mars Stone,1,2Veronique DeSilva,1,2Linda Fritts,1,2
Jeffrey D. Lifson,3and Christopher J. Miller1,2
Despite the fact that approximately half of all HIV patients acquire infection through penile exposure, there have
been no recent studies of penile SIV transmission in rhesus macaques and the nature of the virus variants
transmitted, target cells, and pathways of virus dissemination to systemic lymphoid tissues are not known.
Single genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows
the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection.
Studies using the SGA strategy have shown that intrarectal and intravaginal SIV transmission to macaques
recapitulates key features of human HIV transmission. To date, no studies have used the SGA assay to identify
transmitted/founder virus(es) in macaques infected after penile SIV exposure. Here we report that SIV can be
transmitted by penile SIV exposure. However, similar exposure to a high-dose inoculum infects only about half
the animals, which is about 50% less efficient transmission than occurs after vaginal SIV challenge. In addition,
only a single SIV env variant established the systemic infection in all five animals that became infected after
penile exposure, a result that is consistent with low incidence and few transmitted HIV variants in heterosex-
ually infected men. Our results suggest that the penile transmission of SIVmac251 in rhesus macaques reca-
pitulates the key features of penile HIV-1 transmission and may provide insight into host or viral factors that
permit penile transmission and dissemination. Furthermore, this SIV challenge exposure route will be useful in
testing vaccines and other prophylactic approaches.
are infected with the virus worldwide. Numerous studies to
define the biology of HIV transmission have been undertaken
with the goal of identifying stages of the infection process,
from mucosal exposure through dissemination to distal lym-
phoid tissues establishment of systemic infection, to identify
targets for interventions. To a large extent this effort has fo-
cused on understanding rectal transmission in men and vag-
inal transmission in women,1–22but heterosexual men are
regularly infected through penile HIV exposure. Despite the
fact that approximately half of HIV patients acquire infection
through penile exposure,23–30there have been no preclinical
studies of AIDS vaccines using penile inoculation to challenge
immunized animals and nothing is known about the anti-HIV
IV is primarily transmitted by heterosexual contact
and approximately equal numbers of men and women
immune effector mechanisms that may be present on the
mucosal surfaces of the penis, which presumably serves as the
portal of entry for HIV transmission to males. The foreskin is
thought to be particularly important in HIV transmission to
males because the presence of an intact foreskin is associated
with an approximately 50% increased risk of HIV acquisi-
tion.30–36The foreskin and glans of the human penis have a
complete population of immune cells including potential HIV
tissue for HIV transmission, and circumcision can reduce HIV
acquisition,31–33other tissues of the penis must account for the
remaining transmissions in 50% of HIV-infected men.
Strategies to prevent penile HIV transmission should be di-
rected at the viral variants responsible for establishing pro-
ductive infection in men via this route of exposure. Identifying
1Center for Comparative Medicine and2California National Primate Research Center, University of California, Davis, California.
3SAIC-Frederick, Inc., National Cancer Institute, Frederick, Maryland.
AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 27, Number 12, 2011
ª Mary Ann Liebert, Inc.
(SGA) and direct sequencing, a technique that provides an ac-
curate characterization of the transmitted/founder viruses in-
volved in the initial establishment of systemic infection
following mucosal transmission.40,41Data generated using the
SGA approach and recent empirical data together with math-
ematical models have shown that immediately after transmis-
sion, in the absence of selective pressure, a founder HIV-1
variants in the HIV quasispecies with no or few shared muta-
the consensus of the sequences within that lineage represents
immune response or the onset of other selection pressures,
mutations occur randomly and thus the consensus sequence of
each low diversity lineage represents a transmitted or founder
Using the SGA sequencing approach and mathematical
modeling, it has now been reported in at least eight patient
cohorts representing HIV-1 subtypes A, B, C, and D that most
(60–90%) mucosal infections originate from single variant
transmissions.41,43–47The remaining 10–40% of infections are
initiated by a limited number of transmitted/founder HIV-1
variants. Therefore, for each individual infected, the potential
viral diversity in the period of acute infection is limited to a
single or few HIV-1 lineages. This genetic bottleneck is less
pronounced in individuals engaged in behaviors that may
plausibly facilitate transmission (anal-receptive intercourse or
intravenous drug use)43,48,49and in patients with sexually
transmitted infections.41,44–47Importantly, acute infection
with ’’heterogeneous’’ HIV populations has been linked to
more rapid disease progression.50–52The study of the number
of transmitted viral variants and their overall diversity can
thus have important implications for developing both pro-
phylactic vaccines and antiviral therapy.
Simian immunodeficiency virus (SIV) models of mucosal
HIV transmission in rhesus macaques are valuable for ex-
amining the transmitted or early founder populations that
establish productive infection because the genetic composi-
tion of the virus stock used for mucosal inoculation can be
readily defined and compared to the viruses transmitted fol-
lowing mucosal challenge. Furthermore, because the timing
of SIV transmission is known,4,6,9,53–55mathematical model-
ing is not needed to infer the timing of virus transmission in
macaques. SGA approaches can be used to define mucosal
challenge conditions for NHP models that recapitulate typical
HIV-1 transmissions in humans, resulting in transmission of
one, or only a few viral variants, providing animal models for
evaluation of vaccines and other prophylactic interventions
that recapitulate the virology of HIV transmission. Using the
SGA sequencing approach, one to five transmitted/founder
viruses were found in rhesus macaques infected intrarectally
(i.r.) either SIVmac251 or SIVsmE660.17In a separate titration
study, a dilution of challenge stock was found that repro-
ducibly infected animals with one or only a few viruses fol-
lowing intrarectal challenge.56Finally, rhesus macaques
vaginally challenged with SIVmac251 became systemically
infected with 1–10 founder viruses.11To date, no studies have
transmitted/founder viruses after penile SIV transmission.
Here we report that SIV can be transmitted to rhesus ma-
caques by penile SIV exposure. However, two exposures to a
high-dose inoculum on the same day infected only about half
the inoculated animals. In addition, only a single SIV env
variant established the systemic infection in all five animals
that became infected after penile inoculation, a result that is
consistent with low incidence and few transmitted HIV var-
iants in heterosexually infected men. Our results suggest that
the penile transmission of SIVmac251 in rhesus macaques
recapitulates the key features of penile HIV-1 transmis-
sion57,58andmayprovide insightintohostorviralfactors that
permit penile transmission and dissemination. Furthermore,
this inoculation strategy could be useful in testing vaccines
and other prophylactic approaches.
Materials and Methods
The mature male rhesus macaques (Macaca mulatta) used in
these studies were housed at the California National Primate
Research Center in accordance with the regulations of the
Care. These experiments were approved by the Institutional
Animal Use and Care Committee of the University of Cali-
fornia Davis.Allanimalswere negative for antibodiestoHIV-
2, SIV, type-D retrovirus, and simian T cell lymphotropic
virus type 1 at the time the study was initiated. When nec-
essary, animals were anesthetized with ketamine hydrochlo-
ride (10mg/kg; Parke-Davis, Morris Plains, NJ) or 0.7mg/kg
tiletamine HCl and zolazepan (Telazol, Fort Dodge Animal
Health, Fort Dodge, IA) injected intramuscularly.
Penile SIVmac251 inoculation
A cell-free stock of SIVmac251 (UCD-6/04) was produced
in Staphylococcus enterotoxin A-stimulated rhesus monkey
peripheral blood mononuclear cells (PBMCs) and used for
these studies. This SIVmac251 stock (UCD-6/04) contains
approximately 109viral RNA (vRNA) copies/ml and 10550%
tissue culture infection dose (TCID50)/ml when titered on
CEMX174 cells. On the day of challenge, serial 10-fold dilu-
tions of the SIV stock were made in phosphate-buffered saline
(PBS) (Table 1); 2ml of virus inoculum was used for all the
penile exposures in this study. Animals were exposed re-
peatedly, at weekly intervals with the more dilute inoculum,
same day. Animals that did not become infected after the first
inoculation with undiluted virus were inoculated using the
same procedure to more times at 4 weeks intervals. For the
challenge, a small cup was fashioned from a closed end of a
disposable 15-ml conical centrifuge tube and 2ml of virus
inoculum was placed into the cup. The penis was extended,
the foreskin was retracted, and the glans and shaft were in-
serted into the inoculum and held there for 5min. Then the
penis was allowed to retract into the foreskin and the animal
was placed into its cage in dorsal recumbency and allowed to
recover from anesthesia. Thus although no effort was made to
specifically inoculate the foreskin, we visually confirmed that
some of the inoculum did contact the area when the glans
retracted into the foreskin. Five of the 11 animals in this study
were repeatedly exposed with several doses of the virus, but
the inoculation series was stopped when vRNA was detected
in plasma collected 10–14 days after each challenge.
1260 MA ET AL.
Viral RNA isolation and cDNA synthesis
Plasma and virus stock were thawed at room temperature
and RNA was isolated from 0.5ml plasma using the QIAamp
Ultrasens Viral Kit (QIAGEN Inc., Valencia, CA) according to
the manufacturer’s protocol and eluted in 50ll. The vRNA
was reverse transcribed into cDNA using SuperscriptIII re-
agents (Invitrogen, Carlsbad, CA) with 2ll 50lM dT23VN (or
dNTPs, and 22ll viral RNA. This mixture was heated to 65?C
for 5min followed by incubation on ice for 2min. A master
mix of the following was then added: 8ll of 5·First-Strand
Buffer, 2ll of 0.1M DTT, 2ll of RNaseOUT Recombinant
RNase Inhibitor (40 units/ll), and 2ll of SuperScriptIII RT
(200 units/ll) and Incubated at 25?C for 5min, 50?C for
60min, and 70?C for 15min followed by adding 1ll Escher-
ichia coli RNase H (5U/ll) and incubated at 37?C for 20min.
cDNA was stored at -20?C to 80?C until amplification.
Quantitative viral RNA analysis
Reverse transcriptase polymerase chain reaction (RT-PCR)
samples. Samples were tested in replicates of four reactions
carried out in 96-well optical plates (Applied Biosystems,
Foster City, CA) in a 25ll reaction volume containing 5ll
cDNA and 20ll Mastermix (Applied Biosystems) using the
ABI 7900 robotic thermocycler. All sequences were amplified
for 2min at 50?C and 10min at 95?C followed by 50 cycles of
15s at 95?C and 1min at 60?C. The following primer pairs and
probes were used: SIVgag forward primer 1, 5¢-3¢ GGG AGA
TGG GCG TGA GAA A, reverse primer, CGT TGG GTC GTA
GCC TAA TTT T, and probe TCA TCT GCT TTC TTC CCT
GAC AAG ACG GA. The copy number of SIVgag was cal-
culated based on standard curves for a viral gene plasmid
spanning a concentration range from 0.1 to 108copies. Al-
though amplification from wells with 0.1 copy of SIVgag was
inconsistent, the assays consistently detected SIVgag in wells
containing one or more copies of the plasmid. The results
were analyzed with SDS 7900 system software version 2.3
(Applied Biosystems). The results for each sample are re-
ported as log10vRNA copies per ml of plasma RNA.
In a series of preliminary studies conducted separately to
evaluate the specificity and determine the background of the
PCR assay, RNA isolated from plasma from six rhesus ma-
caques that had never been exposed to SIV was subjected to
amplification. There was no amplification of SIVgag from any
of these samples. Thus, 50 SIVgag RNA copies/ml plasma (or
1.7 log10SIVgag copies/ml plasma) was used as the cutoff for
determining if a sample from an SIV-inoculated monkey was
Single genome amplification of SIVmac251 env
PCR so only one amplifiable molecule is present in each re-
action. cDNA was serially diluted and distributed among
independent PCR reactions to identify a dilution where am-
plification occurred in <30% of the total number of reactions.
PCR amplification was performed in the presence of 1·
buffer, 2mM MgSO4, 0.2mM of each deoxynucleoside tri-
phosphate, 0.2lM of each primer, and 0.025U/ll Platinum
Taq High Fidelity polymerase (Invitrogen) in a 20-ll reaction.
First round PCR was performed with sense primer SIVEnvF1
Table 1. Summary of Animals, Penile SIVmac251 Inoculations, and Virologic Outcomes
Day of first
No. of founder
No. of founder
aAge at first inoculation.
bAbove the cutoff of 1.7 log10copies/ml plasma.
c–gThese animals were inoculated with the least concentrated virus inoculum first and most concentrated virus inoculum last.
hAnimals were inoculated twice in 1 day with a 4h interval between inoculations.
iAnimals were inoculated twice in 1 day with a 4h interval between inoculations on 3 separate days, with 4 weeks between each of the
vRNA, viral RNA; SIV, simian immunodeficiency virus; SGA, single genome amplification.
PENILE SIV TRANSMISSION1261
SIVEnvR1 5¢-TGT AAT AAA TCC CTT CCA GTC CCC CC-3¢
under the following conditions: 1 cycle of 94?C for 2min, 35
cycles at 94?C for 15s, 55?C for 30s, and 68?C for 4min, fol-
lowed by a final extension of 68?C for 10min. Next, 1ll from
first-round PCR product was added to a second-round PCR
reaction that included the sense primer SIVEnvF2 5¢-TAT
AAT AGA CAT GGA GAC ACC CTT GAG GGA GC-3¢ and
CAA TTT GTA-3¢ performed under the same conditions used
for first-round PCR, but with a total of 45 cycles. Correct sized
amplicons were identified by agarose gel electrophoresis and
directly sequenced with second-round PCR primers and six
SIV-specific primers using BigDye Terminator technology. To
confirm PCR amplification from a single template, chro-
matograms were manually examined for multiple peaks,
indicative of the presence of amplicons resulting from PCR-
generated recombination events, Taq polymerase errors, or
multiple varianttemplates.Confirmation thatsequences were
derived from individual template molecules ensures propor-
tional representation of individual env sequences circulating
in vivo. Sequences containing two or more ambiguous sites
were excluded from analysis.
Sequences were aligned using Clustal W60and hand edited
using Jalview61or MacClade 4.08 to improve alignment
quality. All trees were constructed with Phylip62using the
neighbor-joining method63with the Kimura two-parameter
distance matrix.64Within-subject env diversity was analyzed
in three ways, as described in detail previously,43but fell only
into ‘‘homogeneous’’ diversity. Briefly, diversity was deter-
mined by visually inspecting sequences by using neighbor-
joining phylogenies and the Highlighter tool (www.hiv.
lanl.gov). Also, since the population grows exponentially
during the early phase of infection, distribution of pairwise
Hamming distances (HD) within each sample were examined
for star-like phylogeny and a single Poisson distribution of
changes indicating infection from a single viral variant.65
Lastly, mathematical modeling was used to test predictions of
expected maximum HD against assumptions of infection
variant phylogenies.65All sequences were deposited in Gen-
Bank with accession numbers JF298213–JF298241.
Outcomes of penile SIV inoculation
Since no infectivity data were available for penile SIV
transmission inrhesusmacaques, this study was performed by
sequentially escalating the concentration of the virus inoculum
after penile inoculation (Table 1, Fig. 1A). Neither of the two
animals inoculated seven times with 103TCID50of the SIV-
mac251 stock became systemically infected after penile inocu-
lation (Table 1,Fig.1B) andnoneofthetwo animalsinoculated
14 times with 103TCID50of the SIVmac251 stock became sys-
temically infected after penile inoculation (Table 1, Fig. 1B).
became systemically infected after one penile inoculation (Ta-
ble 1, Fig. 1C), but neither of the two animals inoculated seven
times with 104TCID50became systemically infected (Table 1,
Fig. 1C). Three of the five animals inoculated twice in a single
day (4h interval between inoculations) with the undiluted
SIVmac251 stock (105TCID50) became systemically infected
(Table 1, Fig. 1D). One of the two animals inoculated twice in a
single day (4h interval between inoculations) with 105TCID50
of the SIVmac251 stock every 4 weeks for 8 weeks became
systemically infected after penile inoculation with plasma
vRNA levels consistent with other infected animals (Table 1,
Fig. 1D). Once infected, the pattern of plasma viremia in ani-
mals infected by penile SIV inoculation was similar to plasma
vRNA in female rhesus macaques infected after vaginal SIV
7–10 days postinfection (PI), however, vRNA peaked at 14–21
days PI (Table 1, Fig. 1), which is delayed compared to vaginal
point plasma vRNA level is established at 6–8 weeks PI after
Diversity of viral env in the SIVmac251 stock
Phylogenetic relationships among the env sequences in the
SIVmac251 (UCD-2/02) stock that was produced from the
same seed stock that was used produce the SIVmac251 (UCD-
6/04) stock for this study have been described.11To charac-
terize the SIVmac251 (UCD-6/04) stock used in the present
study, a total of 36 env nucleotide sequences were derived by
SGA. Sequence diversity was analyzed by pairwise sequence
comparison, neighbor-joining phylogenetic tree reconstruc-
tion (Fig. 2A), and visualization with the Highlighter tool
(http:/ /www.hiv.lanl.gov) (Fig. 2B). Overall, the maximum
nucleotide diversity of the env sequences from the SIVmac251
stock (UCD-6/04) used for this study was 1.0%, which is
consistent with the (UCD-2/02) SIVmac251 stock11and other
SIVmac251 stocks.44It is worth noting, although perhaps not
stock are closely related to the env variants in the (UCD-2/02)
SIVmac251 stock (Fig. 2C).
Diversity and divergence of SIVenv in plasma of animals
after penile SIV inoculation
A total of 94 SGA-derived 2.7-kb env sequences were ob-
tained from the first vRNA+plasma sample of five rhesus
macaques (median of 20 sequences; range 16–20). SGAs were
derived from the first plasma sample that had vRNA levels
above the assay cutoff of 1.7 log10copies/ ml plasma. These
samples were collected 7 or 10 days after penile exposure to
SIVmac251 (Table 1). Although many of the animals were ex-
posed multiple times, we assumed that the exposure respon-
sible for transmitting the infection occurred 7–10 days before
phase of infection. This estimate of 7–10 days from the trans-
mitting exposure was confirmed using the Poisson diversity
tool (www.hiv.lanl.gov). The viral loads in these samples ran-
ged from 2.42 to 4.19 log10copies/ml of plasma (Table 1).
Although no sequences were overtly APOBEC hypermutated
as assessed using the Hypermut tool (www.hiv.lanl.gov),
several sequences were enriched for G-to-A mutations and
excluded from calculations of diversity and divergence from
the SIVmac251 consensus reference sequence. The mean se-
quence diversity of each animal during acute infection ranged
1262MA ET AL.
to 0.15% with an average maximum diversity of 0.09%. For
each infected animal, a single low-diversity lineage was
detected in the ramp-up phase plasma.
In addition to ramp-up sequences, 76 SGA-derived 2.7-kB
env sequences were obtained from set point vRNA+plasma
samples of the same five rhesus macaques (median of 17 se-
quences per animal; range 9–17). These sequences were de-
rived from plasma samples that had vRNA levels above 5.0
log10 copies/ml plasma. These samples were collected 12–19
weeks after the presumed transmitting penile SIVmac251
exposure (Fig. 1). The mean sequence diversity of in these
samples ranged from 0.1 to 0.2% with a mean of 0.18%. The
maximum sequence diversity for each animal in these sam-
ples ranged from 0.3 to 0.6% with an average maximum di-
versity of 0.4%. Overall, the total diversity correlates to the
amount of time since infection.
Enumeration of transmitted/founder populations
in animals after penile SIV inoculation
To enumerate the total number of transmitted/founder
variants, the number of distinct low-diversity viral env line-
ages present in each sample was estimated. Low-diversity
monophyletic lineages are defined as a unique series of
identical or nearly identical nucleotide sequences, differing
only by the number of individual polymorphisms that could
arise randomly through RT error within 7 days of transmis-
sion. In this study, we define a variant as an SGA-derived
genetic sequence with up to two individual nucleotide poly-
morphism differences from another variant assuming no re-
combination or selection pressure, and a gamma distributed
rate of mutation across sites. The maximum number of nu-
cleotide mutations likely to occur by random error within the
time frame from virus exposure has been determined math-
ematically. A 2.2- to 2.7-kb fragment of SIV env will accu-
mulate a maximum of two nucleotide polymorphisms in 1
week of SIV infection. Thus if a plasma SGA sequence had
more than two nucleotide substitutions relative to the con-
sensus sequence of each lineage, then the variant is assumed
to have been transmitted as distinct founder virus.
Basedonthesecriteria, a singleSIV env variantwas detected
in the ramp-up plasma of all five SIV-infected animals (Fig. 3).
A composite neighbor-joining tree, illustrating the phyloge-
netic relationshipofall SIV env sequences derived fromthe SIV
stock and all five animals, demonstrates that the SIV env
02468 101214 161820
Week Post Challenge
Log 10 vRNA copies /ml plasma
Week Post Challenge
Log 10 vRNA copies /ml plasma
Week Post Challenge
Log 10 vRNA copies /ml plasma
02468 1012141618 20
Week Post Challenge
Log 10 vRNA copies /ml plasma
animals inoculated 14 times became plasma vRNA+after penile inoculation. (B) At a dose of 103TCID50, none of the two
animals (36527, 36240) inoculated seven times became plasma vRNA+and none of the two animals (36317, 36364) inoculated 14
times became viremic. (C) One animal (36838) inoculated once with 104TCID50of the SIVmac251 stock became plasma vRNA+,
but neither of the two animals inoculated seven times became plasma vRNA+. (D) With a 105TCID50dose of the SIVmac251
stock, three (37349, 36336, 36193) of the five animals inoculated twice in a single day became plasma vRNA+and one of two
animals became plasma vRNA+after being inoculated twice in a single day a total of three times over an 8-week period. The
arrows under the x-axis indicate the timing of the inoculations. The double arrows in D indicate two inoculations with a 4-h
interval between inoculations. The individual animal’s symbols are noted in the key and data from animals are graphed up to
the day of their last inoculation if they remained uninfected and up to the day of necropsy if they became infected.
Plasma viral RNA (vRNA) levels after penile SIVmac251 inoculation. (A) With a 10 TCID50dose, none of the five
PENILE SIV TRANSMISSION1263
1264MA ET AL.
variants transmitted to the animals were widely distributed
among the env variants inthestock withnooverrepresentation
set point phase variants in plasma clearly arose from the
in the week 1 PI plasma. This result supports the conclusion
that each of the five animals was infected with a single SIV-
mac251 env variant that then gave rise to all subsequent viral
variants detected at the set point stage of infection.
This study demonstrates that SIV can be transmitted to
penis in a suspension of cell-free virions. Although penile
transmission of SIV is possible, it is not as efficient as vaginal
SIV transmission. For example, when exposed to an SIV-
mac251 inoculum containing 103TCID50, four males resisted
infection despite 7–14 penile exposures, while seven of eight
female macaques became infected after 14 vaginal exposures
of 21 female rhesus macaques became infected after two
vaginal challenges (separated by a 4h interval) in a single day
with 105TCID50of SIVmac251,9while only three of five male
macaques became infected after penile exposure to the same
inoculum and schedule (Table 1, Fig. 1). The relative ineffi-
ciency of penile SIV transmission is consistent with the epi-
demiologic studies in stable HIV-discordant couples that
concluded that women are more susceptible to acquiring HIV
per heterosexual act than men.27–29,66The reasons for the
difference in gender susceptibility remain to be determined,
but as shown herethese differences persist evenif thedoses of
virus to which the genders are exposed are the same. It seems
likely that differences in the relative surface area of the genital
mucosa (the surface area of the vaginal mucosa is much
greater than the surface area of the glans and foreskin), the
relative density of susceptible target cells in the genital mu-
cosa of men and women, and the time that the inoculum is in
contact with the genital mucosa contribute to the differences
in the efficiency of HIV transmission between the genders.
In heterosexual men, the presence of an intact fore-
skin is associated with an increased risk of HIV acquisi-
tion30,34–36,67,68and the efficiency of penile SIV transmission
would likely increase if an effort was made to directly inoc-
ulate the foreskin mucosa. In addition, HIV transmission to
men is dramatically increased by the presence of genital ul-
cers69; thus experimentally inducing penile inflammation
prior to SIV inoculation may be another strategy to enhance
penile SIV transmission efficiency while also modeling pop-
ulations at greatest risk of acquiring HIV.
Transmission of HIV is relatively inefficient compared to
other sexually transmitted infections (STI), which is reflected
in the fact that in most cases a single variant or only a few
variants from the viral quasispecies in an HIV-infected person
successfully establishes systemic infection.70–73As systemic
infection with a single variant occurred in five of five animals
after penile SIVmac251 transmission, this route of experi-
mental SIV inoculation accurately reflects the virology of HIV
transmission. In fact, when infection is transmitted, single SIV
env variants establish systemic infections more consistently
after penile SIVmac251 exposure compared to vaginal SIV-
mac251 challenge. After vaginal challenge of randomly se-
lected mature cycling multiparous rhesus macaques with a
complex SIVmac251 quasispecies11,17some animals became
infected with one variant, but in about half of the animals,
systemic infection was established by five or more unique
variants.11The transmission of multiple variants to some fe-
male rhesus may be due to the changes in physiology of the
female reproductive tract during the menstrual cycle or the
presence of vaginal inflammation at the time of inoculation.
Although ulcerative STIs have not been described in ma-
caques, female rhesus monkeys commonly have bacterial
vaginosis74,75that causes genital inflammation.76Alter-
natively, the same factors (discussed above) that may make
women relatively more susceptible to HIV infection may also
increase the number of variants that are transmitted by vag-
inal SIV inoculation. It remains to be seen if experimentally
induced inflammation alters the number of variants trans-
mitted by penile SIV inoculation.
The genetic bottleneck that results in a single viral variant
establishing an infection after HIV transmission43,52,70,71,77–79
could be due to a the presence of only a limited number of
viral variants in secretions of the infected partner, selective
transmission of viral variants, or selective amplification of
viral variants that cross the mucosal barrier. Although there
could be a reduction in overall diversity in mucosal secretions
compared to plasma, this penile infection study is not con-
sistent with first explanation since all five animals inoculated
with the genetically diverse SIVmac251 quasispecies became
infectedwith aunique, singlevirus variant. Itshould benoted
that analyzing 20 SGA-derived sequences provides a 95%
confidence estimate that variants representing greater than
15% of the population are not be missed.43Thus while the
most likely conclusion is that all five animals were infected
with a single variant we cannot exclude the possibility an
individual animal has more than one variant in the plasma
stock. (A) The neighbor-joining tree (the scale bar represents 0.001 nucleotide substitutions per site) and (B) highlighter plot.
Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and
cytosine in blue). (C) The neighbor-joining tree including all SGA-derived sequences from the SIVmac251 (UCD 6/04)
challenge stock used for the current study and the SIVmac251 (UCD 2/02) challenge stock used in a published study of
founder variants after vaginal inoculation.11Black stars indicate the variants that were amplified from the (UCD 2/02)
SIVmac251 stock and red stars indicate the variants that were amplified from the (UCD 6/04) SIVmac251 stock. (The scale bar
in A represents 0.0005 nucleotide substitutions per site and the scale bar in C represents 0.001 nucleotide substitutions per
site.) The APOBEC symbol indicates the location of APOBEC signatures in hypermutated sequences. The A/G symbol
indicates the location of sequences of G to A mutations.
Analysis of single genome amplification (SGA)-derived env sequences from the SIVmac251 (UCD 6/04) challenge
PENILE SIV TRANSMISSION1265
sequences from five male rhesus macaques infected after penile SIV inoculation. Variants from individual animals are
represented as different colors within the phylogenetic tree as denoted in the key. The sequences amplified from week 1
plasma (7 or 10 days PI) are denoted by an elliptical symbol and are lighter shaded whereas set point plasma sequences are
denoted by square symbols and are darker shaded. Black asterisk (*) indicates sequences from the (UCD 6/04) SIVmac251
stock. G-to-A mutations in the sequences are indicated with (^). The scale bar represents 0.001 nucleotide substitutions per
Composite neighbor-joining tree of SGA env sequences in the SIVmac251 virus stock and plasma SGA SIVenv
1266MA ET AL.
Despite thiscaveat,this isnow thefourthstudy toreachthe
conclusion that rhesus macaques became infected with a
single env variant after mucosal SIVmac251 exposure; the
other studies tested animals after vaginal and rectal SIVmac
251 inoculation.11,17,56Further, because all variants at set-
point were derived from the variants we identified at ramp-
up, all biologically relevant variants seem to have been found
with our approach. Determining the relative importance of
selective transmission versus selective amplification of a sin-
gle variant prior to systemic dissemination will be important
for understanding the challenges associated with preventing
HIV transmission through vaccination. In some HIV clades,
the transmitted HIV env variants share features that pre-
sumably enhance transmission to a seronegative host, but
they also make the transmitted variants relatively susceptible
to antibody neutralization.80In this case preexisting anti-
bodies may be able to efficiently block infection by the foun-
der viruses. On the other hand, if the neutralization-sensitive
variants appear as the founder population in blood by out-
days after transmission, then a neutralization-resistant vari-
ants may emerge as the primary founder viruses in the pres-
ence of established vaccine-induced neutralizing antibodies.
Finally, although infection is established relatively ineffi-
ciently by penile SIV inoculation, these data are consistent
with the epidemiology of HIV transmission in human popu-
Even after penile inoculations with a high virus dose the
pattern of viremia and the single variant founder SIV popu-
lations that establish infection accurately reflect the virology
of HIV infection. Given the consistency of infection, this route
of SIV inoculation can be used to model penile HIV trans-
mission to better understand virus transmission and dissem-
useful for accurately modeling HIV transmission in the pre-
clinical testing of HIV vaccines.
The authors thank the Primate Services Unit at the CNPRC
and Joseph Dutra, Ding Lu, and Lili Guo for excellent tech-
nical assistance. This work was supported by NIH Grants
RR00169 and AI8227, National Cancer Institute contract no.
HHSN266200400088C, and a gift from the James B. Pendleton
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Christopher J. Miller
CNPRC, UC Davis
One Shields Avenue
Davis, California 95616
PENILE SIV TRANSMISSION 1269
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