Detection and Partial Characterization of Simian I
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病菌学杂志 2005年第4期
Microbiology Division, Tulane National Primate Research Center, Covington
Tulane Health Sciences Center, Tulane School of Public Health and Tropical Medicine, Department of Tropical Medicine, New Orleans, Louisiana
School of Biological Sciences, University of Manchester, Manchester, United Kingdom
ABSTRACT
Human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that naturally infect sooty mangabeys (SMs; Cercocebus atys). In order to further investigate the relationship between HIV-2 and SIVsm, the SIV specific to the SM, we characterized seven new SIVsm strains from SMs sold in Sierra Leone markets as bush meat. The gag, pol, and env sequences showed that, while the viruses of all seven SMs belonged to the SIVsm-HIV-2 lineage, they were highly divergent viruses, in spite of the fact that most of the samples originated from the same geographical region. They clustered in three lineages, two of which have been previously reported. Two of the new SIVsm strains clustered differently in gag and env phylogenetic trees, suggesting SIVsm recombination that had occurred in the past. In spite of the fact that our study doubles the number of known SIVsm strains from wild SMs, none of the simian strains were close to the groups in which HIV-2 was epidemic (groups A and B).
TEXT
Despite an excellent understanding of the ancestry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 in African nonhuman primates (1, 2), nothing is known about the circumstances that led to the initial emergence and subsequent evolution of HIV to become significant human pathogen. The issue of pathogen emergence must be addressed if the potential for emergence of new infectious diseases is going to be understood. The simian counterparts of HIV constitute a highly diverse group of viruses that have been reported to infect more than 35 species of African nonhuman primates throughout sub-Saharan Africa (1). These viruses have a high prevalence in free-living monkeys, with infection rates of up to 60 to 80% in some primate species (10, 28, 35). Some simian immunodeficiency viruses (SIVs) have been reported to grow in vitro on human peripheral blood cells (7, 23), suggesting that they may represent a potential threat of infection for humans.
Lentiviruses have very probably been associated with nonhuman primates for thousands of years, whether or not their most recent common ancestor is ancient (6, 25) or recent (8). Recombination has contributed to the impressive picture of SIV diversity, with several simian species being infected by host-specific SIV types with diverse recombinant-strain ancestry (3, 5, 14, 22, 26, 27, 35). This history, involving host-dependent evolution, cross-species transmission events, and frequent recombination, explains the complex evolutionary history of the primate lentiviruses and accounts for the difficulties in analyzing SIV molecular data. The most notable cross-species transmission involved SIVcpz, which represents the ancestors of HIV-1 (12, 19, 34). Coincidently, in western Africa, a similar event occurred about the same time (within 30 years) and involved transmission of the SIV specific to the sooty mangabey (SIVsm) to humans, which reflects the ancestry of HIV-2 (10, 20, 21). Therefore, the ancestry is known, but the circumstances under which epidemic HIV-1 and HIV-2 viruses emerged are completely unknown (2).
The geographic distribution of SIVsm-infected sooty mangabeys (SMs; Cercocebus atys) overlaps the area of HIV-2 endemicity and includes Liberia, Sierra Leone, C?te d'Ivoire, Guinea-Bissau, and Guinea (38). SIVsm was apparently transmitted to humans in at least eight instances, forming groups A to H (9, 18, 21). These eight HIV-2 groups (previously called subtypes) are unevenly prevalent: groups A and B are endemic (17), while the other six correspond to single-person infections only (10, 18, 21, 39). Although SIVsm represents the ancestors of all HIV-2 viruses, no SIV counterpart has been found to cluster closely to groups A and B; in marked contrast, putative groups C to H cluster relatively closely to SIVsm strains in the SIVsm and HIV-2 phylogenies (10, 18, 21, 39), suggesting recent crossover infections. In this respect, their extremely low prevalence and lack of any known association with disease, apart from that of putative group H, may signify that these viruses are less well adapted to their human hosts and less pathogenic (21).
Our group has previously reported the characterization of six SIVsm strains from household pets and feral SMs in Sierra Leone (10). Although four of these viruses (SIVsmSL92a, -d, -e, and -f) originated from animals in the same troop, they were highly divergent and clustered into two different lineages within the HIV-2-SIVsm-SIVmac tree (10). The most significant phylogenetic relationship of these SIVsm isolates was that of SIVsmSL92b, from a household pet, which was closely related to HIV-2 subtype E strain PA (10). Although this patient was living in Los Angeles, Calif., he had emigrated from the same geographical region where SIVsmSL92b-infected SMs were found. Therefore, these results (10) have offered the most compelling evidence thus far for the simian origin of HIV-2.
In order to evaluate the risk of SIVsm exposure to humans in rural regions of Sierra Leone, we have investigated the prevalence of SIVsm infection in bush meat from monkeys sold in outdoor markets and collected from 1992 to 1993. We have also characterized the phylogenetic relationships of newly discovered SIVsm strains to further investigate the relationship between SIVsm and HIV-2 in this country of HIV-2 emergence. No previous bush meat studies had been conducted in Sierra Leone. This is the first study of SIV prevalence and diversity to use bush meat tissues because previous studies relied on blood collected through intracardiac punction (30).
Samples. During a survey carried out in rural Sierra Leone from 1992 to 1993, 29 spleen samples from indigenous monkey species were collected from carcasses in the market. Twelve samples were from SMs, five were from lesser spot-nosed monkeys (Cercopithecus petaurista), five were from Campbell's monkeys (Cercopithecus campbelli), four were from diana monkeys (Cercopithecus diana), two were from red colobus monkeys (Piliocolobus badius), and one was from a western red colobus monkey (Colobus polykomos) (Table 1).
DNA was isolated from 30 mg of tissue by using a DNeasy tissue kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions for use in PCR amplification of proviral DNA.
PCRs. Control PCR with primers 5'-CCA TCA CCA TCT TCC AGG AGC GAG-3' and 5'-CAC AGT CTT CTG GGT GGC AGT GAT-3' was done to show that the DNA extraction yielded amplifiable GAPDH (glyceraldehyde-3-phosphate dehydrogenase). All but one of the extracted samples gave a positive result, confirming the availability of DNA in the samples used subsequently for SIVsm PCR amplification.
Nested PCR was performed to obtain amplified fragments from the gag, env, long terminal repeat (LTR), and pol regions. A 793-bp gag fragment was obtained by a nested-PCR protocol using primers GagA-GagB and GagC-GagF, as described previously (21). Alternatively, primers GF1-GR1 and GF2A-GR3 (10) were used in a nested PCR to generate a 909-bp fragment in the gag region. These two fragments completely overlapped. Nested primers were used for sequencing. A 438-bp fragment in the gp36 env region was obtained by a previously described nested-PCR protocol with primers EF4-ER1 and EF5A-ER2A (9). A 130-bp LTR fragment was obtained by a nested-PCR protocol using primers LTRA-LTRB and LTRC-LTRD, as described previously (21). A 602-bp pol integrase fragment was obtained by using a slight variation of previously described primers used to amplify divergent SIVs (4, 13-16, 30). Polis4B (5'-CCA GCH CAY AAA GGW ATA GGW GGA AA-3') and PolORB (5'-ACT GCH CCT TCH CCT TTC CA-3') were used in the first round of amplification, and Polis4B was used again in a seminested reaction with Unipol2B (CCC CTA TTC CTC CCY TTC TTT TAA).
PCR products were purified using a QIAquick gel extraction kit (QIAGEN) and sequenced by direct sequencing and dye terminator methodologies (ABI PRISM BigDye terminator cycle sequencing ready reaction kit with AmpliTaq FS DNA polymerase [PE Biosystems, Warrington, United Kingdom] on an automated sequencer [ABI 373, stretch model; Applied Biosystems]).
Phylogenetic analysis. The gag, pol, and env nucleotide sequence alignments were obtained from the Los Alamos National Laboratory HIV sequence database (http://hiv-web.lanl.gov). Newly derived SIVsm sequences were aligned using the CLUSTALW (37) profile alignment option. The resulting alignments were adjusted manually where necessary. Regions of ambiguous alignment and all gap-containing sites were excluded.
Phylogenetic trees were inferred from the nucleotide sequence alignments by the neighbor-joining method (33) using the HKY85 model of nucleotide substitution (24) implemented with PAUP (36). The reliability of branching order was assessed by performing 1,000 bootstrap replicates, again by using neighbor joining and the HKY85 model. Phylogenetic trees were also inferred by maximum likelihood by using PAUP with models inferred from the alignment created by use of Modeltest (32). The neighbor-joining tree topology was used as the starting tree in a heuristic search using tree bisection-reconnection branch swapping.
SIV prevalence in bush meat samples from indigenous monkeys from Sierra Leone. The nature of the bush meat samples used in this study precluded a serological screening. Therefore, we screened the samples by PCR. Although this technique is less sensitive than serology in detecting divergent viruses, we used different sets of primers that have been previously shown to be very effective in detecting the SIVs that have been described to date (4, 10, 13-16, 21, 30). All samples but one were amplified by GAPDH primers, showing the existence of intact DNA in the extracted samples (Table 1).
Nested-PCR amplifications using different sets of SIV-specific primers were positive for 7 out of the 12 SM samples included in this study. In four cases, PCR yielded gag, pol, and env sequences; in two cases (SL93-119 and SL93-135), only gag and env sequences were obtained; for the remaining one (SL93-139), only pol and env sequences were obtained (Table 1). These results show a high prevalence of SIVsm infection in free-living SMs, 63.6% of the tested SMs being positive for SIV. Interestingly, SIVsm prevalence was not significantly different between adult and juvenile SMs. Moreover, the youngest SM included in this study was SIVsm infected, pointing to a potential vertical transmission of SIVsm in our study group. However, there is no known connection between SIVsm93SL080 and a potential mother. Therefore, it cannot be concluded that the infection resulted from vertical transmission. However, one should note that an SIVsm prevalence of up to 4% was reported in pet SMs in Sierra Leone (10). Since in most cases pet monkeys are captured when they are infants, after their mothers are killed, vertical transmission of SIV must be occurring and thus is inferred to be a significant mechanism of SIV transmission in the wild.
All other species were negative by PCR using all primer pairs. This result is surprising, since the prevalence of SIV was reported to be high in Central African Cercopithecus monkeys. The animals included in our study group were mainly adults; therefore, one should expect a relatively high prevalence of SIVs. The lack of positive results is probably not due to high divergence, since at least the pol sets of primers were frequently used repeatedly and were extremely effective in detecting new and divergent SIVs in past studies (13-16, 30). Therefore, these results suggest a lower prevalence in Cercopithecus monkeys in West Africa, at least for the species tested and for a limited number of samples. Analyses of larger groups of monkeys are necessary before conclusions concerning SIV prevalence in these species can be drawn.
Phylogenetic analyses. Analysis of the gag, pol, and env sequences showed that the newly characterized SIVsm strains clustered in the SIVsm-HIV-2 lineage (Fig. 1). Most of the bush meat SIVsm strains clustered with SIVsm strains from wild SMs from Sierra Leone. One strain from an infant SM (SIVsmSL93-080) did not cluster in the previously reported SIVsm SL clusters (10) but was more closely related to SIVsmLib1, a virus originating from the neighboring country of Liberia (Fig. 1). However, this relationship, although observed in all phylogenetic trees, was not supported by a high bootstrap value. For gag, SIVsmSL93-063 and SIVsmSL93-119 clustered relatively closely to the HIV-2 putative group E strain PA, a human virus from a patient who emigrated from the region where these two SIVs were found. To date, SIVsmSL93-063 and SIVsmSL93-119 are the most closely related simian counterparts of an HIV-2 lineage to be identified (Fig. 1A). SIVsmSL93-057 clusters in different phylogenetic positions in gag and pol trees (in which this strain is in the SIVsmSL92b cluster) from those in the env tree, where this virus clustered in the SIVsmSL92a cluster, indicating a possible recombinant-strain history. Note that SIVsmSL93-139 also exhibits the same discordant clustering that SIVsmSL93-057 does in the pol and env trees. Another strain, SIVsmSL93-135, also showed different phylogenetic relationships in gag and env trees, being grouped with SIVsmSL93a in the gag trees and with SIVsmSL92b in the env tree (Fig. 1A and C). This different clustering pattern in different genomic fragments is highly suggestive of recombination events that occurred in the wild.
Our study confirms the high prevalence of SIVsm in wild-living SMs. This prevalence is in the same range as those reported for other species of African nonhuman primates which are natural hosts of SIVs: African green monkeys (28) and mandrills (35). Our SIVsm infection rates in Sierra Leone bush meat samples from SMs is in the same range as that reported by our group previously when we used SM plasma samples (10) and more recently feces from SMs from the Tai Forest in C?te d'Ivoire (M. L. Santiago, F. Range, F. Bibollet-Ruche, C. Fruteau, R. Peho, J. F. Y. Brookfield, R. Noe, P. M. Sharp, G. M. Shaw, and B. H. Hahn, Abstr. 11th Conf. Retrovir. Oppor. Infect. 2004, abstr. 380, 2004). Therefore, our results may accurately reflect the real prevalence of SIVsm in the wild. These prevalence levels are significantly higher than that recently reported following the testing of blood from monkeys sold in bush meat markets in Cameroon, which was only 16% (30). This difference may be due to a greater stability of the virus in tissues such as spleen than in clotted blood sampled from dead monkeys in bush meat markets. An alternative explanation is that SIV prevalence varies with nonhuman primate species and geographical location, as already reported in Cameroon (30).
Previous studies have shown that HIV-2 prevalence in Sierra Leone is very low (0.1%) (9) in spite of apparently massive exposure to SIVsm in the markets. Also, cross-species transmission to humans is rare in West Africa, as reported to date (9), with only 1 person out of 9,314 tested having SIVsm-like infection (9). This finding raises the question of whether AIDS is really a straightforward zoonosis (2), i.e., a human disease in all cases resulting directly from cross-species SIV transmission events, or is more accurately deemed a transient infection of zoonotic origin which is only occasionally transmissible in the human population. This does not negate the overwhelming evidence that HIV originates from natural cross-species SIV transmission from African primates. Cross-species transmission of SIVsm (and SIVcpz) should be considered the proximate cause of the HIV-AIDS epidemics plaguing the world, while the ultimate cause of HIV and AIDS is related to both viral properties and the evolution of an HIV form readily transmissible in the human population. Cross-species transmission is fostered by different factors, such as human behavior, environmental changes through deforestation, and industrialization during the last century. Viral adaptation to the new host is necessary for the emergence of the new virus: of the eight groups of HIV-2, only the epidemic groups A and B were shown to have pathogenic potential, whereas groups C to G comprise nonepidemic strains that are weakly pathogenic, replicate poorly in infected humans, and are found only within the range of SMs and persons who emigrated from western Africa (9, 21). However, the finding of a minor HIV-2 viral lineage (putative group H) that is pathogenic (18) indicates that there is not necessarily a link between pathogenic and epidemic potential. To date, there is no evidence that SIV infection of other African primates can result in HIV or AIDS. However, SIV-infected monkeys may represent a source for new zoonotic events, given the propensity of SIV strains to recombine. Whereas the body of evidence seems to suggest that direct cross-species transmission may not result in the emergence of a successful virus, the outcome of a cross-species transmission may be successful if the infected individual is already infected with HIV. Such divergent recombinant events have been frequent in the evolutionary history of the primate lentiviruses (1, 3, 5, 13, 14, 26, 27, 35).
In spite of the fact that this study doubles the number of known SIVsm strains from wild SMs, there is still no evidence of simian counterparts to HIV-2 groups A and B. It is likely that the emergence of epidemic strains of HIV-2 did not occur in Sierra Leone but rather in a different region of the SMs' natural range. Epidemiologic data point to C?te d'Ivoire and Guinea-Bissau. Note that the oldest known strains of HIV-2 groups A and B were found in C?te d'Ivoire (11). Until recently, PCR fragments from only two SIVsm strains were available from C?te d'Ivoire, and they clustered closer to HIV-2 strains than to SIVsm strains from Sierra Leone (31). It was recently reported that SIVsm isolates from the Tai Forest show closer phylogenetic relationships with the epidemic groups of HIV-2, pointing to C?te d'Ivoire as their emergence area (Santiago et al., 11th Conf. Retrovir. Oppor. Infect.). Alternatively, as HIV-2 groups A and B have been estimated to have emerged about 70 years ago (29), it is possible that the SIVsm sources of the HIV-2 lineages in areas of endemicity may have become extinct. More virological surveys of SIVsm diversity in the area of HIV-2 endemicity are needed to investigate this further.
Nucleotide sequence accession numbers. The GenBank accession numbers for the sequences in this study are AY864786 to AY864798.
ACKNOWLEDGMENTS
This work was supported by grants RO1 AI-44596 and P51 RR000164 from the National Institute of Health.
We thank Tessa Williams, Meredith Hunter, and Nora Dillon for technical assistance and Theresa Secrist for administrative support.
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Tulane Health Sciences Center, Tulane School of Public Health and Tropical Medicine, Department of Tropical Medicine, New Orleans, Louisiana
School of Biological Sciences, University of Manchester, Manchester, United Kingdom
ABSTRACT
Human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that naturally infect sooty mangabeys (SMs; Cercocebus atys). In order to further investigate the relationship between HIV-2 and SIVsm, the SIV specific to the SM, we characterized seven new SIVsm strains from SMs sold in Sierra Leone markets as bush meat. The gag, pol, and env sequences showed that, while the viruses of all seven SMs belonged to the SIVsm-HIV-2 lineage, they were highly divergent viruses, in spite of the fact that most of the samples originated from the same geographical region. They clustered in three lineages, two of which have been previously reported. Two of the new SIVsm strains clustered differently in gag and env phylogenetic trees, suggesting SIVsm recombination that had occurred in the past. In spite of the fact that our study doubles the number of known SIVsm strains from wild SMs, none of the simian strains were close to the groups in which HIV-2 was epidemic (groups A and B).
TEXT
Despite an excellent understanding of the ancestry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 in African nonhuman primates (1, 2), nothing is known about the circumstances that led to the initial emergence and subsequent evolution of HIV to become significant human pathogen. The issue of pathogen emergence must be addressed if the potential for emergence of new infectious diseases is going to be understood. The simian counterparts of HIV constitute a highly diverse group of viruses that have been reported to infect more than 35 species of African nonhuman primates throughout sub-Saharan Africa (1). These viruses have a high prevalence in free-living monkeys, with infection rates of up to 60 to 80% in some primate species (10, 28, 35). Some simian immunodeficiency viruses (SIVs) have been reported to grow in vitro on human peripheral blood cells (7, 23), suggesting that they may represent a potential threat of infection for humans.
Lentiviruses have very probably been associated with nonhuman primates for thousands of years, whether or not their most recent common ancestor is ancient (6, 25) or recent (8). Recombination has contributed to the impressive picture of SIV diversity, with several simian species being infected by host-specific SIV types with diverse recombinant-strain ancestry (3, 5, 14, 22, 26, 27, 35). This history, involving host-dependent evolution, cross-species transmission events, and frequent recombination, explains the complex evolutionary history of the primate lentiviruses and accounts for the difficulties in analyzing SIV molecular data. The most notable cross-species transmission involved SIVcpz, which represents the ancestors of HIV-1 (12, 19, 34). Coincidently, in western Africa, a similar event occurred about the same time (within 30 years) and involved transmission of the SIV specific to the sooty mangabey (SIVsm) to humans, which reflects the ancestry of HIV-2 (10, 20, 21). Therefore, the ancestry is known, but the circumstances under which epidemic HIV-1 and HIV-2 viruses emerged are completely unknown (2).
The geographic distribution of SIVsm-infected sooty mangabeys (SMs; Cercocebus atys) overlaps the area of HIV-2 endemicity and includes Liberia, Sierra Leone, C?te d'Ivoire, Guinea-Bissau, and Guinea (38). SIVsm was apparently transmitted to humans in at least eight instances, forming groups A to H (9, 18, 21). These eight HIV-2 groups (previously called subtypes) are unevenly prevalent: groups A and B are endemic (17), while the other six correspond to single-person infections only (10, 18, 21, 39). Although SIVsm represents the ancestors of all HIV-2 viruses, no SIV counterpart has been found to cluster closely to groups A and B; in marked contrast, putative groups C to H cluster relatively closely to SIVsm strains in the SIVsm and HIV-2 phylogenies (10, 18, 21, 39), suggesting recent crossover infections. In this respect, their extremely low prevalence and lack of any known association with disease, apart from that of putative group H, may signify that these viruses are less well adapted to their human hosts and less pathogenic (21).
Our group has previously reported the characterization of six SIVsm strains from household pets and feral SMs in Sierra Leone (10). Although four of these viruses (SIVsmSL92a, -d, -e, and -f) originated from animals in the same troop, they were highly divergent and clustered into two different lineages within the HIV-2-SIVsm-SIVmac tree (10). The most significant phylogenetic relationship of these SIVsm isolates was that of SIVsmSL92b, from a household pet, which was closely related to HIV-2 subtype E strain PA (10). Although this patient was living in Los Angeles, Calif., he had emigrated from the same geographical region where SIVsmSL92b-infected SMs were found. Therefore, these results (10) have offered the most compelling evidence thus far for the simian origin of HIV-2.
In order to evaluate the risk of SIVsm exposure to humans in rural regions of Sierra Leone, we have investigated the prevalence of SIVsm infection in bush meat from monkeys sold in outdoor markets and collected from 1992 to 1993. We have also characterized the phylogenetic relationships of newly discovered SIVsm strains to further investigate the relationship between SIVsm and HIV-2 in this country of HIV-2 emergence. No previous bush meat studies had been conducted in Sierra Leone. This is the first study of SIV prevalence and diversity to use bush meat tissues because previous studies relied on blood collected through intracardiac punction (30).
Samples. During a survey carried out in rural Sierra Leone from 1992 to 1993, 29 spleen samples from indigenous monkey species were collected from carcasses in the market. Twelve samples were from SMs, five were from lesser spot-nosed monkeys (Cercopithecus petaurista), five were from Campbell's monkeys (Cercopithecus campbelli), four were from diana monkeys (Cercopithecus diana), two were from red colobus monkeys (Piliocolobus badius), and one was from a western red colobus monkey (Colobus polykomos) (Table 1).
DNA was isolated from 30 mg of tissue by using a DNeasy tissue kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions for use in PCR amplification of proviral DNA.
PCRs. Control PCR with primers 5'-CCA TCA CCA TCT TCC AGG AGC GAG-3' and 5'-CAC AGT CTT CTG GGT GGC AGT GAT-3' was done to show that the DNA extraction yielded amplifiable GAPDH (glyceraldehyde-3-phosphate dehydrogenase). All but one of the extracted samples gave a positive result, confirming the availability of DNA in the samples used subsequently for SIVsm PCR amplification.
Nested PCR was performed to obtain amplified fragments from the gag, env, long terminal repeat (LTR), and pol regions. A 793-bp gag fragment was obtained by a nested-PCR protocol using primers GagA-GagB and GagC-GagF, as described previously (21). Alternatively, primers GF1-GR1 and GF2A-GR3 (10) were used in a nested PCR to generate a 909-bp fragment in the gag region. These two fragments completely overlapped. Nested primers were used for sequencing. A 438-bp fragment in the gp36 env region was obtained by a previously described nested-PCR protocol with primers EF4-ER1 and EF5A-ER2A (9). A 130-bp LTR fragment was obtained by a nested-PCR protocol using primers LTRA-LTRB and LTRC-LTRD, as described previously (21). A 602-bp pol integrase fragment was obtained by using a slight variation of previously described primers used to amplify divergent SIVs (4, 13-16, 30). Polis4B (5'-CCA GCH CAY AAA GGW ATA GGW GGA AA-3') and PolORB (5'-ACT GCH CCT TCH CCT TTC CA-3') were used in the first round of amplification, and Polis4B was used again in a seminested reaction with Unipol2B (CCC CTA TTC CTC CCY TTC TTT TAA).
PCR products were purified using a QIAquick gel extraction kit (QIAGEN) and sequenced by direct sequencing and dye terminator methodologies (ABI PRISM BigDye terminator cycle sequencing ready reaction kit with AmpliTaq FS DNA polymerase [PE Biosystems, Warrington, United Kingdom] on an automated sequencer [ABI 373, stretch model; Applied Biosystems]).
Phylogenetic analysis. The gag, pol, and env nucleotide sequence alignments were obtained from the Los Alamos National Laboratory HIV sequence database (http://hiv-web.lanl.gov). Newly derived SIVsm sequences were aligned using the CLUSTALW (37) profile alignment option. The resulting alignments were adjusted manually where necessary. Regions of ambiguous alignment and all gap-containing sites were excluded.
Phylogenetic trees were inferred from the nucleotide sequence alignments by the neighbor-joining method (33) using the HKY85 model of nucleotide substitution (24) implemented with PAUP (36). The reliability of branching order was assessed by performing 1,000 bootstrap replicates, again by using neighbor joining and the HKY85 model. Phylogenetic trees were also inferred by maximum likelihood by using PAUP with models inferred from the alignment created by use of Modeltest (32). The neighbor-joining tree topology was used as the starting tree in a heuristic search using tree bisection-reconnection branch swapping.
SIV prevalence in bush meat samples from indigenous monkeys from Sierra Leone. The nature of the bush meat samples used in this study precluded a serological screening. Therefore, we screened the samples by PCR. Although this technique is less sensitive than serology in detecting divergent viruses, we used different sets of primers that have been previously shown to be very effective in detecting the SIVs that have been described to date (4, 10, 13-16, 21, 30). All samples but one were amplified by GAPDH primers, showing the existence of intact DNA in the extracted samples (Table 1).
Nested-PCR amplifications using different sets of SIV-specific primers were positive for 7 out of the 12 SM samples included in this study. In four cases, PCR yielded gag, pol, and env sequences; in two cases (SL93-119 and SL93-135), only gag and env sequences were obtained; for the remaining one (SL93-139), only pol and env sequences were obtained (Table 1). These results show a high prevalence of SIVsm infection in free-living SMs, 63.6% of the tested SMs being positive for SIV. Interestingly, SIVsm prevalence was not significantly different between adult and juvenile SMs. Moreover, the youngest SM included in this study was SIVsm infected, pointing to a potential vertical transmission of SIVsm in our study group. However, there is no known connection between SIVsm93SL080 and a potential mother. Therefore, it cannot be concluded that the infection resulted from vertical transmission. However, one should note that an SIVsm prevalence of up to 4% was reported in pet SMs in Sierra Leone (10). Since in most cases pet monkeys are captured when they are infants, after their mothers are killed, vertical transmission of SIV must be occurring and thus is inferred to be a significant mechanism of SIV transmission in the wild.
All other species were negative by PCR using all primer pairs. This result is surprising, since the prevalence of SIV was reported to be high in Central African Cercopithecus monkeys. The animals included in our study group were mainly adults; therefore, one should expect a relatively high prevalence of SIVs. The lack of positive results is probably not due to high divergence, since at least the pol sets of primers were frequently used repeatedly and were extremely effective in detecting new and divergent SIVs in past studies (13-16, 30). Therefore, these results suggest a lower prevalence in Cercopithecus monkeys in West Africa, at least for the species tested and for a limited number of samples. Analyses of larger groups of monkeys are necessary before conclusions concerning SIV prevalence in these species can be drawn.
Phylogenetic analyses. Analysis of the gag, pol, and env sequences showed that the newly characterized SIVsm strains clustered in the SIVsm-HIV-2 lineage (Fig. 1). Most of the bush meat SIVsm strains clustered with SIVsm strains from wild SMs from Sierra Leone. One strain from an infant SM (SIVsmSL93-080) did not cluster in the previously reported SIVsm SL clusters (10) but was more closely related to SIVsmLib1, a virus originating from the neighboring country of Liberia (Fig. 1). However, this relationship, although observed in all phylogenetic trees, was not supported by a high bootstrap value. For gag, SIVsmSL93-063 and SIVsmSL93-119 clustered relatively closely to the HIV-2 putative group E strain PA, a human virus from a patient who emigrated from the region where these two SIVs were found. To date, SIVsmSL93-063 and SIVsmSL93-119 are the most closely related simian counterparts of an HIV-2 lineage to be identified (Fig. 1A). SIVsmSL93-057 clusters in different phylogenetic positions in gag and pol trees (in which this strain is in the SIVsmSL92b cluster) from those in the env tree, where this virus clustered in the SIVsmSL92a cluster, indicating a possible recombinant-strain history. Note that SIVsmSL93-139 also exhibits the same discordant clustering that SIVsmSL93-057 does in the pol and env trees. Another strain, SIVsmSL93-135, also showed different phylogenetic relationships in gag and env trees, being grouped with SIVsmSL93a in the gag trees and with SIVsmSL92b in the env tree (Fig. 1A and C). This different clustering pattern in different genomic fragments is highly suggestive of recombination events that occurred in the wild.
Our study confirms the high prevalence of SIVsm in wild-living SMs. This prevalence is in the same range as those reported for other species of African nonhuman primates which are natural hosts of SIVs: African green monkeys (28) and mandrills (35). Our SIVsm infection rates in Sierra Leone bush meat samples from SMs is in the same range as that reported by our group previously when we used SM plasma samples (10) and more recently feces from SMs from the Tai Forest in C?te d'Ivoire (M. L. Santiago, F. Range, F. Bibollet-Ruche, C. Fruteau, R. Peho, J. F. Y. Brookfield, R. Noe, P. M. Sharp, G. M. Shaw, and B. H. Hahn, Abstr. 11th Conf. Retrovir. Oppor. Infect. 2004, abstr. 380, 2004). Therefore, our results may accurately reflect the real prevalence of SIVsm in the wild. These prevalence levels are significantly higher than that recently reported following the testing of blood from monkeys sold in bush meat markets in Cameroon, which was only 16% (30). This difference may be due to a greater stability of the virus in tissues such as spleen than in clotted blood sampled from dead monkeys in bush meat markets. An alternative explanation is that SIV prevalence varies with nonhuman primate species and geographical location, as already reported in Cameroon (30).
Previous studies have shown that HIV-2 prevalence in Sierra Leone is very low (0.1%) (9) in spite of apparently massive exposure to SIVsm in the markets. Also, cross-species transmission to humans is rare in West Africa, as reported to date (9), with only 1 person out of 9,314 tested having SIVsm-like infection (9). This finding raises the question of whether AIDS is really a straightforward zoonosis (2), i.e., a human disease in all cases resulting directly from cross-species SIV transmission events, or is more accurately deemed a transient infection of zoonotic origin which is only occasionally transmissible in the human population. This does not negate the overwhelming evidence that HIV originates from natural cross-species SIV transmission from African primates. Cross-species transmission of SIVsm (and SIVcpz) should be considered the proximate cause of the HIV-AIDS epidemics plaguing the world, while the ultimate cause of HIV and AIDS is related to both viral properties and the evolution of an HIV form readily transmissible in the human population. Cross-species transmission is fostered by different factors, such as human behavior, environmental changes through deforestation, and industrialization during the last century. Viral adaptation to the new host is necessary for the emergence of the new virus: of the eight groups of HIV-2, only the epidemic groups A and B were shown to have pathogenic potential, whereas groups C to G comprise nonepidemic strains that are weakly pathogenic, replicate poorly in infected humans, and are found only within the range of SMs and persons who emigrated from western Africa (9, 21). However, the finding of a minor HIV-2 viral lineage (putative group H) that is pathogenic (18) indicates that there is not necessarily a link between pathogenic and epidemic potential. To date, there is no evidence that SIV infection of other African primates can result in HIV or AIDS. However, SIV-infected monkeys may represent a source for new zoonotic events, given the propensity of SIV strains to recombine. Whereas the body of evidence seems to suggest that direct cross-species transmission may not result in the emergence of a successful virus, the outcome of a cross-species transmission may be successful if the infected individual is already infected with HIV. Such divergent recombinant events have been frequent in the evolutionary history of the primate lentiviruses (1, 3, 5, 13, 14, 26, 27, 35).
In spite of the fact that this study doubles the number of known SIVsm strains from wild SMs, there is still no evidence of simian counterparts to HIV-2 groups A and B. It is likely that the emergence of epidemic strains of HIV-2 did not occur in Sierra Leone but rather in a different region of the SMs' natural range. Epidemiologic data point to C?te d'Ivoire and Guinea-Bissau. Note that the oldest known strains of HIV-2 groups A and B were found in C?te d'Ivoire (11). Until recently, PCR fragments from only two SIVsm strains were available from C?te d'Ivoire, and they clustered closer to HIV-2 strains than to SIVsm strains from Sierra Leone (31). It was recently reported that SIVsm isolates from the Tai Forest show closer phylogenetic relationships with the epidemic groups of HIV-2, pointing to C?te d'Ivoire as their emergence area (Santiago et al., 11th Conf. Retrovir. Oppor. Infect.). Alternatively, as HIV-2 groups A and B have been estimated to have emerged about 70 years ago (29), it is possible that the SIVsm sources of the HIV-2 lineages in areas of endemicity may have become extinct. More virological surveys of SIVsm diversity in the area of HIV-2 endemicity are needed to investigate this further.
Nucleotide sequence accession numbers. The GenBank accession numbers for the sequences in this study are AY864786 to AY864798.
ACKNOWLEDGMENTS
This work was supported by grants RO1 AI-44596 and P51 RR000164 from the National Institute of Health.
We thank Tessa Williams, Meredith Hunter, and Nora Dillon for technical assistance and Theresa Secrist for administrative support.
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