To-date, data on the evolution of natural SIVcpz infection over time and on the impact of SIVcpz on chimpanzee populations are only available for the P. t. schweinfurthii subspecies from East-Central Africa and no data currently exist for representatives of the P. t. troglodytes subspecies, the natural reservoir of the ancestors of HIV-1 in humans. In this study, we describe for the first time the clinical observations and viral history over time in a naturally SIV infected P. t. troglodytes chimpanzee (Cam155/Ch-Go). The low CD4 counts observed in 2009, together with severe thrombocytopenia, weight loss and unusual frequent periods of infections with diverse pathogens, suggest a progressive SIV infection similar to HIV infection in humans, confirming previous observations that SIVcpz can be pathogenic in its natural host. Although, CD4 counts in 2004 and 2009 were measured with two different techniques, the observed decline in CD4 counts cannot be explained by potential different performances of the techniques on chimpanzee cells only. Moreover in 2009, CD4 counts of 1,256 CD4 cells/mm3 were observed on another SIV negative chimpanzee from the same sanctuary versus 283 CD4 cells/mm3 for Ch-Go with the same technique, and values reported in the literature for healthy SIV negative chimpanzees range also between 800 and 2,000 CD4 cells/mm3 .
When applying the CDC and WHO classification systems to the clinical and biological data available for Cam155/Ch-Go and reported in this study, the evolution of the SIV infection in this P. t. troglodytes chimpanzee currently corresponds to pre-AIDS in humans, CDC B2 or WHO stage III. The viral loads fluctuated between 4 and 5 log10 copies/ml. Nevertheless, different techniques were used to measure viral loads in Cam155/Ch-Go at the different time points, and it cannot be excluded that the commercial HIV-1 viral load assays used in this study underestimated values for SIVcpzPtt. The viral loads observed in Cam155/Ch-Go are in the range of the values observed for Ch-No (SIVcpzPts-Ant), the other naturally SIVcpz infected chimpanzee, although from the P. t. schweinfurthii subspecies and with an apparent non-progressive SIV infection [8, 28]. In the absence of a specific SIVcpz viral load test, comparisons over time or with other experimentally or naturally infected animals are difficult. Moreover, in Ch-No, viral loads fluctuated over time from 3.4 to 5.8 log10 copies/ml and could differ by more than 1 log according to the technique used . In addition to PCR, quantitative viral isolations have also been done from different plasma and PBMC dilutions for Ch-No, and important fluctuations have been observed over time, although there was no correlation at all between titres of infectious virus in plasma and viral load measured by PCR . No other data on viral load observed in natural SIVcpz infections are readily available for comparison but, for natural non-pathogenic SIVsm and SIVagm infections in mangabeys and African green monkeys, viral loads are also generally high [29, 30]. In contrast, in chimpanzees that were previously experimentally infected with HIV-1, plasma viral loads were undetectable or very low, except for the few animals that progressed to AIDS 4 to 18 years post-inoculation and for whom viral loads increased over time and could reach up to 6 log10 copies/ml . CD4 decline, severe thrombocytopenia, increased plasma viral loads and occurrence of opportunistic infections were also observed in the HIV-1 experimentally infected chimpanzees that developed AIDS in the Yerkes Primate Center . The animals that progressed faster to AIDS underwent superinfections with 2 or 3 strains, which was the case for the first animal (C499) that was reported with AIDS, or were infected with the pathogenic strain of this latter animal.
The naturally SIV infected P. t. schweinfurthii chimpanzee, Ch-No is still alive and in good health today, more than 20 years later, despite the relatively high plasma viral load and a severe and permanent thrombocytopenia that occurred approximately at age 7 . Thrombocytopenia was also seen in the experimentally infected chimpanzees with AIDS and is observed in humans and macaques with AIDS [12, 32]. Whether the asymptomatic period for natural SIVcpz is longer than for HIV in humans, or whether differences in incubation periods exist like in humans (i.e. rapid versus long-term progressors), is not known. Thus, it cannot be excluded that the SIVcpzPts-Ant infected chimpanzee may still develop a progressive infection.
Given the young age of Cam155 at seizure (1.5 years old), the chimpanzee was likely infected through vertical mother-to-child SIV transmission, as chimpanzees are not sexually active before the age of 8; however, horizontal transmission by blood contact (e.g. biting injuries) cannot be entirely excluded. In humans, in utero infected newborns develop AIDS more rapidly compared to those infected after birth , but survival rates and disease progressions in vertically HIV-1 infected infants can be variable . Recent studies on SIV pathogenicity in wild East African chimpanzees show a higher mortality rate among infants born to SIV positive mothers [13, 35]. The majority of the other known SIVcpz positive captive chimpanzees were most likely also infected through mother-to-child transmission, because they were all less than 3-4 years old at time of rescue (Additional file 1: Table S1) [1–3, 6, 36, 37]. Although they had no signs of AIDS at the time of diagnosis, some had chronic lymphadenopathy like Gab1 and cpz-US, or thrombocytopenia as Ch-No. Some died suddenly from acute infections (Cam5 and Cam13), as shown in Additional file 1: Table S1 summarizes the history of the previously reported SIVcpz positive captive animals; however, whether this was related to the SIV infection and an eventual degradation of the immune system is not known.
Phylogenetic analyses revealed that SIVcpzPtt-Cam155 fell within the radiation of the SIVcpzPtt group of viruses, as part of a clade including all other SIVcpzPtt strains, as well as HIV-1 groups M and N. However, SIVcpzPtt-Cam155 clustered most closely with SIVcpzPtt-Gab1 from northern Gabon and SIVcpzPtt-Cam13 from southwest Cameroon. We previously reported phylogeographic clustering of SIVcpzPtt strains in Cameroon, and observed high genetic diversity within small geographic areas. Although the geographic origin of this animal is not precisely known, it most likely originated around the Dja Reserve in south central Cameroon. The SIVcpzPtt-Cam155 sequence further illustrates the high genetic diversity among SIVcpzPtt strains in this area .
Our data demonstrate an important diversification and mutation rate of SIVcpzPtt-Cam155 over time, with nucleotide and amino acid diversity doubling in 5 years in the envelope, and an evolution of the putative envelope structure leading to escape mutants. Particularly, V1 and V4 loops were highly variable, as similarly observed in experimentally SIV infected macaques during progression to simian AIDS . Moreover, variability in V4 region is associated with modification of CD4 binding and plays a key role in the swarming nature of gp120 . V3 was modified in its crown and V2 was stable, in contrast to SIVcpzPts-Ant  or slow disease progressors [25, 26]. The progressive diversification of HIV in untreated infected humans underlies its ability to evade immunologic selective pressure, but this diversification may also be responsible for disease progression and destruction of immune system . Overall the evolutionary rate of HIV-1 slows down over time and seems to be correlated with the slope of the CD4 cell decline. Considering two time points, the SIVcpzPtt-Cam155 V1V4 nucleotide diversities five years apart (0.0237 in 2004 and 0.0491 in 2009) fit the trends of viral diversification across HIV-1 infected humans in diverse studies [41, 42], and the theoretical curve established by Lee et al. describing the evolution of C2V5 HIV diversity over time . In chimpanzees experimentally infected with HIV-1 , a higher viral diversity was seen in the progressor chimpanzees vs. the non-progressors; however, it has to be noted that these data originated from animals inoculated with two distinct HIV-1 strains, and recombination between the different strains could have biased the overall diversity observed over time. We also observed an increase of putative N-linked glycosylation sites over time in SIVcpzPtt-Cam155 envelope. Specific genetic modifications leading to the acquisition of PNGS were shown to result in an evolving protective glycan shield  and to be a characteristic of escape mutants since it reduces protein epitope exposure and thus facilitates viral evasion of antibody neutralization .