Despite the importance of rectal infection in the AIDS epidemic, current knowledge regarding HIV rectal entry is limited to in vitro work, which does not reproduce fully the complexity and specificity of the rectal milieu. An understanding of HIV mucosal entry would be of considerable help in the design of microbicides and vaccines. We addressed the question of rectal entry in the rhesus macaque infected by SIVmac251, the animal model closest to the human infection by HIV 
. In order to increase our chances to detect virus during early acute infection, we chose a viral dose ten times higher than in our previous work 
. This dose is in the low range of the doses used for studies of vaginal transmission 
. It is commensurate with the highest viral loads reported in the semen of HIV+
. This one hundred rAID50
dose does not affect the dissemination profile.
SIV entry is massive. If we consider our sampling to be representative of the entire rectum, one can estimate the viral DNA in the rectum of each macaque four hours pi to be on the order of 55,000 copies in lymphoid aggregates and on the order of 290,000 in the lamina propria (see Text S1
for details). As we could not find SIV DNA in mock-infected macaques, this suggests that approximately 3×105
viral particles were able to cross the rectal epithelia and undergo reverse transcription, representing close to 1% of input viral particles. Some of these copies must be integrated as spliced mRNA could be found in the rectum as early as four hours pi. The much lower number of cells positive for SIV antigens (on the order of 103
per macaque, Figure S2
) suggests that most copies of SIV DNA correspond to defective particles undergoing abortive infection. Cells positive for SIV antigens can be productively infected or have merely internalized viral antigens. As, in vivo, the average life cycle of SIV is under 10 hours 
, it is likely that the cells positive for SIV antigens observed sixteen hours pi and onwards are infected cells in which translation proceeds. The fraction of these cells which will go on to produce virus is currently not possible to estimate.
SIV entry is a very rapid process, as infection is established in the lamina propria, in lymphoid aggregates and in more distal sites at least at four hours pi, the earliest time point assessed. Viral entry after vaginal inoculation was shown, by in situ acidic inactivation, to be complete in less than 30 minutes 
, with reverse transcription completed as early as two hours pi 
. The reverse transcriptase inhibitor tenofovir applied rectally two hours after rectal infection protected only one third of inoculated macaques 
, indicating that reverse transcription can be completed in less than two hours. This is faster than expected from in vitro work. However, one should note that gut lymphocytes are inflammatory but hyporesponsive 
, and that this may accelerate the viral cycle. It was not possible to narrow down further the time necessary for virus to be imported from the inoculum as, in our hands, acidic inactivation was toxic by the rectal route.
SIV entry could involve trans-epithelial transport of SIV. Early reports of HIV in rectal epithelial cells of HIV+
have not been confirmed. Many now accept that infected epithelial cells are not found in vivo 
. In our previous work we had not observed infected rectocytes 
. In the present work we could never observe infected epithelial cells either by IHF or by ISH. We did find virus at distal sites as early as four hours pi. Trans-epithelial transport is a likely explanation for this rapid dissemination, as viral production in situ is unlikely over such a short period of time. Indeed, the estimated mean intracellular phase of the life cycle for HIV in vivo is 14.4 to 21.6 hours 
. It is shorter for SIV, with a mean life cycle of 9.4 hours 
. The trans-epithelial transport of SIV does not appear to involve capture by DC processes extending into the rectal lumen through tight junctions. Indeed, we never observed intraepithelial DCs at early time points of infection using classical DC markers (DC-SIGN, fascin). The presence of SIV in both lymphoid aggregates and digestive mucosa four hours pi argues in favor of transcytosis of virions by both the digestive epithelium and the follicle-associated epithelium. One should note that entry appears more efficient across the follicle-associated epithelium. However the surface of the digestive epithelium is much larger than that of the follicle-associated epithelium, and the amount of virus entering through this route could be greater.
SIV structural proteins are found intracellularly by IHF in intraepithelial cells (including T cells), in lamina propria cells (T cells and non-T cells), and in T cell-containing clusters in lymphoid aggregates. The non-T cells are neither macrophages nor mature DCs as they do not express CD68, DC-SIGN or fascin. The SIV antigen positive clusters probably correspond to the infection of a single cell by virus from the inoculum. Secondary diffusion of SIV antigen to neighboring cells could occur by infection (a possibility for macaques infected sixteen hours and onwards) or by cell-cell fusion. In favor of the former is the fact that lymphoid aggregates often contain several copies of SIV DNA (Table S2
cells are scarce and the detection of Env
singly spliced mRNA is rare. These observations suggest that SIV replication is initiated in very few cells of the rectal mucosa at early stages of infection. We have no evidence for viral production in the digestive part of the mucosa. In contrast, the presence of SIV+
cell clusters suggests that viral production occurs in lymphoid aggregates.
During the first day of infection at least, rectal DC-SIGN+
cells have an overall distribution pattern similar to healthy human and macaque rectum 
. They appear less abundant in the lamina propria, but more abundant in lymphoid aggregates than Jameson et al. observed in healthy rhesus macaque mucosa 
. They all express macrophage markers, as was described in healthy human rectum 
. In contrast to healthy mucosa 
, in early SIV infection rectal DC-SIGN+
cells do not all express MHC class II. DC-SIGN+
cells bind infectious virions, as infectious virus is enriched in the DC-SIGN+
cell fraction. The number of virions bound by each cell is presumably small as these cells are not positive by IHF.
SIV dissemination is rapid as cell-associated virus could be recovered from draining lymph nodes and from the peripheral blood as early as four hours pi. This indicates that virus-carrying cells have left the mucosa to percolate through the colic lymph node chain and reach the circulation in less than four hours. The very early presence of viral DNA in draining lymph nodes strongly suggests that infected cells are one means of viral dissemination from the mucosa. Virus could also travel as cell-bound virions. This is one explanation of the discrepancy observed between co-culture results and nested PCR results, the other one being a stochastic effect at very low viral loads. Finally, one cannot exclude transport as free virions in the lymph draining the rectum. To determine whether this occurs, one would have to cannulate lymph from the colon under prolonged anesthesia. This not only raises technical and ethical concerns, but could also modify exchanges between lymphoid tissues.
Rectal infection appears to involve rapid entry and reverse transcription, as has been noted for the vaginal 
and oral routes 
but there are clear differences with these entry pathways. Dissemination after rectal entry is possibly more rapid than after oral entry, where SIV DNA is found in many lymphoid tissues twenty-four hours pi 
. It is also more rapid than following vaginal entry, where SIV DNA could be found at very low-levels in draining lymph nodes eighteen hours pi and in some lymphoid tissues twenty-four hours pi 
. We found that a high proportion of virus crosses the epithelium upon rectal inoculation. This is similar to oral infection 
, but contrasts with vaginal entry where only a very small proportion of the total inoculum enters the vaginal mucosa 
. We show that T cells, but not fascin positive DCs or macrophages, are among the first targets of SIV during rectal transmission. This is not the case during vaginal transmission where fascin positive DCs are the first cells associated with SIV 
or during oral infection where macrophages are infected 
The rapid kinetics of rectal entry and dissemination has important consequences for the development of preventive strategies. Rhesus macaques can be protected from rectal infection by tenofovir applied locally prior to inoculation 
. Protection is not observed if the plasma concentration of tenofovir is below 75 ng/ml. This is in contrast with what is observed for vaginal infection, where protection is observed with plasma concentrations of tenofovir as low as 11 ng/ml 
. This suggests that protection from rectal infection (and possibly not from vaginal infection) requires inhibition of reverse transcription in distal sites such as lymph nodes and not exclusively in the rectum. One cannot exclude that routine use of reverse transcriptase inhibitors with such plasma concentrations will lead to side effects and present risks of selection for resistant reverse transcriptase mutants. Therefore, there is a use for microbicide preparations for rectal use aiming to prevent virion interaction with the epithelium (digestive as well as follicle-associated) and entry into cells. This would complement (or replace) preparations aiming to block reverse transcription.
In conclusion, we show that SIV crosses in less than four hours both the digestive epithelium and the follicle-associated epithelium. We propose that entry occurs by transcytosis at both sites, with rectocytes and M cells being the most likely candidates to carry out transcytosis of virions. Following entry, SIV infects T cells as well as non-T cells in the mucosa. SIV initiates replication locally in the rectal lymphoid aggregates, and to a lower extent in the lamina propria. Virus is rapidly transported to distal sites as infected cells, as virions associated with cells possibly expressing DC-SIGN or as free virions present in the lymph. Reverse transcription occurs in the rectal mucosa during the first hours of infection. Reverse transcription may also occur in draining lymph nodes. To be effective against rectal transmission of HIV, a vaccine will have to induce immunity at the rectal surface, but also in distal lymphoid sites.