Strain diversity of Treponema pallidum subsp. pertenue suggests rare interspecies transmission in African nonhuman primates.

In our most recent study, we found that in Tanzania infection with Treponema pallidum (TP) subsp. pertenue (TPE) is present in four different monkey species. In order to gain information on the diversity and epidemiological spread of the infection in Tanzanian nonhuman primates (NHP), we identified two suitable candidate genes for multi-locus sequence typing (MLST). We demonstrate the functionality of the MLST system in invasively and non-invasively collected samples. While we were not able to demonstrate frequent interspecies transmission of TPE in Tanzanian monkeys, our results show a clustering of TPE strains according to geography and not host species, which is suggestive for rare transmission events between different NHP species. In addition to the geographic stability, we describe the relative temporal stability of the strains infecting NHPs and identified multi-strain infection. Differences between TPE strains of NHP and human origin are highlighted. Our results show that antibiotic resistance does not occur in Tanzanian TPE strains of NHP origin.

Novel staphylococcal species that form part of a Staphylococcus aureus-related complex: the non-pigmented Staphylococcus argenteus sp. nov. and the non-human primate-associated Staphylococcus schweitzeri sp. nov.

We define two novel species of the genus Staphylococcus that are phenotypically similar to and have near identical 16S rRNA gene sequences to Staphylococcus aureus. However, compared to S. aureus and each other, the two species, Staphylococcus argenteus sp. nov. (type strain MSHR1132(T) = DSM 28299(T) = SSI 89.005(T)) and Staphylococcus schweitzeri sp. nov. (type strain FSA084(T) = DSM 28300(T) = SSI 89.004(T)), demonstrate: 1) at a whole-genome level considerable phylogenetic distance, lack of admixture, average nucleotide identity <95 %, and inferred DNA-DNA hybridization <70 %; 2) different profiles as determined by MALDI-TOF MS; 3) a non-pigmented phenotype for S. argenteus sp. nov.; 4) S. schweitzeri sp. nov. is not detected by standard nucA PCR; 5) distinct peptidoglycan types compared to S. aureus; 6) a separate ecological niche for S. schweitzeri sp. nov.; and 7) a distinct clinical disease profile for S. argenteus sp. nov. compared to S. aureus.

Fecal microbiomes of non-human primates in Western Uganda reveal species-specific communities largely resistant to habitat perturbation.

Primate gastrointestinal microbial communities are becoming increasingly appreciated for their relevance to comparative medicine and conservation, but the factors that structure primate "microbiomes" remain controversial. This study examined a community of primates in Kibale National Park, Uganda, to assess the relative importance of host species and location in structuring gastrointestinal microbiomes. Fecal samples were collected from primates in intact forest and from primates in highly disturbed forest fragments. People and livestock living nearby were also included, as was a geographically distant population of related red colobus in Kenya. A culture-free microbial community fingerprinting technique was used to analyze fecal microbiomes from 124 individual red colobus (Procolobus rufomitratus), 100 individual black-and-white colobus (Colobus guereza), 111 individual red-tailed guenons (Cercopithecus ascanius), 578 human volunteers, and 364 domestic animals, including cattle (Bos indicus and B. indicus × B. taurus crosses), goats (Caprus hircus), sheep (Ovis aries), and pigs (Sus scrofa). Microbiomes sorted strongly by host species, and forest fragmentation did not alter this pattern. Microbiomes of Kenyan red colobus sorted distinctly from microbiomes of Ugandan red colobus, but microbiomes from these two red colobus populations clustered more closely with each other than with any other species. Microbiomes from red colobus and black-and-white colobus were more differentiated than would be predicted by the phylogenetic relatedness of these two species, perhaps reflecting heretofore underappreciated differences in digestive physiology between the species. Within Kibale, social group membership influenced intra-specific variation among microbiomes. However, intra-specific variation was higher among primates in forest fragments than among primates in intact forest, perhaps reflecting the physical separation of fragments. These results suggest that, in this system, species-specific processes such as gastrointestinal physiology strongly structure microbial communities, and that primate microbiomes are relatively resistant to perturbation, even across large geographic distances or in the face of habitat disturbance.

Seroepidemiologic, molecular, and phylogenetic analyses of simian T-cell leukemia viruses (STLV-I) from various naturally infected monkey species from central and western Africa.

A study of simian T-cell lymphoma/leukemia virus infection, conducted on 747 nonhuman primates belonging to 14 different species in Central and Western Africa, indicated that 4 species (Cercopithecus aethiops, Erythrocebus patas, Papio doguera, and Cercopithecus mona pogonias) had a high prevalence of seropositivity to simian T-cell lymphoma/leukemia virus type I (STLV-I). The other nonhuman primate species, however, had negative or low levels of anti-HTLV-I antibodies. STLV-I pol and env DNA was detected in 12 of 12 different animals among the seropositive species. However, STLV-I pX DNA could be detected in only 10 of 12 animals. Comparative phylogenetic analyses based on 140 bp sequence of the pol gene indicate that these STLV-I isolates were 0-9% divergent from each other and were 3.5-7% divergent from the prototype related human retrovirus HTLV-I (ATK). The West African STLV-I isolates formed a unique phylogenetic cluster as did most of the Central African STLV-I isolates, save for STLV-I (Tan 90). The phylogenetic data indicate that cross species transmission of HTLV-I and STLV-I continued to occur long after their ancestral strain separated from the progenitor to HTLV-II. Comparative amino acid analyses indicated that there was marked conservation of the TAX protein regardless of host species, while the pol and REX proteins exhibited increasing levels of diversity.

A distinct African lentivirus from Sykes' monkeys.

Asymptomatic infection with simian immunodeficiency virus (SIV) has been demonstrated in African Sykes' monkeys (Cercopithecus mitis albogularis), and virus isolation confirmed infection with a novel SIV from Sykes' monkeys (SIVsyk). Macaques inoculated with SIVsyk became persistently infected but remained clinically healthy. We utilized polymerase chain reaction amplification to generate a full-length, infectious molecular clone of SIVsyk. The genome organization of SIVsyk is similar to that of the other primate lentiviruses, consisting of gag, pol, vif, vpr, tat, rev, env, and nef. A unique feature is the absence of the highly conserved NF-kappa B binding site in the long terminal repeat. SIVsyk is genetically equidistant from other primate lentiviruses. Thus, SIVsyk represents a new group that is distinct from the four previously recognized primate lentivirus groups: human immunodeficiency virus type 1 (HIV-1), SIV from sooty mangabeys (SIVsmm) and HIV-2, SIV from African green monkeys (SIVagm), and SIV from mandrills (SIVmnd). The genetic differences between SIVsyk and SIVagm, isolates derived from monkeys of the same genus, underscore the potential for other distinct SIVs which have yet to be isolated and characterized.

Simian immunodeficiency viruses from central and western Africa: evidence for a new species-specific lentivirus in tantalus monkeys.

Although up to 50% of African green monkeys (AGMs) are infected by simian immunodeficiency viruses (SIV) in their natural habitat, they remain asymptomatic carriers of these lentiviruses. They provide an attractive model to study not only the origin but also the link among genetic variation, host-virus adaptation, and pathogenicity of primate lentiviruses. SIVagm have been isolated from three species of AGM: the vervet (Cercopithecus pygerythrus), the grivet (Cercopithecus aethiops), and the sabaeus (Cercopithecus sabaeus) monkey. We studied four new SIVagm isolates from a fourth AGM species, the tantalus monkey (Cercopithecus tantalus), caught in the Central African Republic, and four new isolates from feral sabaeus monkeys from Senegal. Antigenic properties and partial env sequences were used to evaluate the diversity among these isolates. Alignment of env sequences in SIVagm isolated from tantalus and sabaeus monkeys permitted detailed mapping of the variable and conserved domains in the external glycoprotein. Genetic distances indicated that SIVagm isolates from tantalus monkeys are the most divergent among SIVagm in feral AGMs in Africa. The fact that AGMs are infected by four distinct lentiviruses, each specific for a single AGM species, supports the hypothesis of a coevolution of these viruses and their natural hosts and suggests that SIV transmission is a rare event among separated AGM species in the wild.

Sequence and phylogenetic analyses of a new STLV-I from a naturally infected tantalus monkey from Central Africa.

Simian T-cell leukemia virus (STLV-I) is an oncovirus highly related to human T-cell leukemia virus type I (HTLV-I). To further examine the extent of variability, dissemination patterns, phylogeny, and evolution of these viruses, we analyzed a new STLV-I variant from a naturally infected Cercopithecus aethiops var. tantalus from the Central African Republic. Sequence analyses of its LTR, gag, pol, env, and pX (OrfII) genes indicated that this isolate, STLV-I (Tan 90), is 6% divergent from the prototype HTLV-I (ATK) and is the most divergent African STLV-I characterized to date. Our phylogenetic data indicate that southeast Asian and African STLV-I and HTLV-I strains segregated from each other thousands of years ago and that Japanese HTLV-I strains represent a relatively recent introduction of African or New World isolates. The data also indicate that interspecies transmission occurred several times on different continents over prolonged periods of time.

Gut microflora of vervet and samango monkeys in relation to diet.

The microflora in the gastrointestinal tracts of wild vervet and samango monkeys (Cercopithecus aethiops and C. mitis, respectively) were studied, using fermentation acid analysis, electron microscopy, and culturing methods. The diets of the two species of monkey differ considerably, with that of the samango including a greater proportion of cellulose-rich leaf material, and this is reflected in the microflora. Volatile fatty acid measurements along the gut of both species showed that these end products of bacterial metabolism were concentrated in the cecum and colon. Electron microscopy indicated that morphologically similar bacteria were present in the cecum and colon of both species, but the samango possessed a distinct stomach microflora. Bacteria in the lumina of the four main regions of the gut of the monkeys (stomach, small intestine, cecum, and colon) were plated on a number of anaerobic media (Mann, Rogosa, and Sharp; clostridial basal; and complex media). The cecum and colon were found to contain higher numbers of microbes per gram (wet weight) of gut content than the stomach and small intestine. Microbial isolates were able to catabolize carboxymethyl cellulose and other polymers. This may aid the monkeys, particularly samangos, in the digestion of fibrous dietary components such as leaves.

Isolation and characterization of simian immunodeficiency viruses from two subspecies of African green monkeys.

Cercopithecus aethiops (African Green monkey), a nonhuman primate species distributed throughout subsaharan Africa, has been shown to have high seroprevalence rates of antibodies to simian immunodeficiency virus (SIV), and therefore, has been proposed as a natural reservoir for immunodeficiency viruses. Our laboratories have isolated SIV-like viruses from two East African subspecies of C. aethiops designated grivet and vervet monkeys. Analysis of the structural proteins based on the molecular weights and immunologic cross-reactivity to the prototypic SIV(MAC), HIV-1, and HIV-2 isolates suggests that these viruses are distinctly different. Heterogeneity was observed in the molecular weights of the gag, pol, and env gene products between SIV isolates from vervets [SIV(AGM(VER))] and grivets [SIV(AGM(GRI))]. Phenotypically, SIV(AGM(VER)) isolates were distinguishable from SIV(AGM(GRI)) isolates by the apparent size difference of the major core antigen p24. All SIV(AGM(GRI)) and SIV(AGM(VER)) isolates were found to encode a transmembrane protein of approximately 40 kD (gp40) in contrast to gp32 of SIV(MAC). Furthermore, the transmembrane protein was shown to be encoded by the entire env open reading frame, unlike gp32 of SIC(MAC) or gp36 of SIV(AGM(TYO-1)). These data indicate that viruses from C. aethiops share common features with SIV(MAC) and HIV-1, but represent diverse SIV-like viruses which may vary according to subspecies and geographic location.

Simian immunodeficiency viruses from African green monkeys display unusual genetic diversity.

African green monkeys are asymptomatic carriers of simian immunodeficiency viruses (SIV), commonly called SIVagm. As many as 50% of African green monkeys in the wild may be SIV seropositive. This high seroprevalence rate and the potential for genetic variation of lentiviruses suggested to us that African green monkeys may harbor widely differing genotypes of SIVagm. To investigate this hypothesis, we determined the entire nucleotide sequence of an infectious proviral molecular clone of SIVagm (155-4) and partial sequences (long terminal repeat and Gag) of three other distinct SIVagm isolates (90, gri-1, and ver-1). Comparisons among the SIVagm isolates revealed extreme diversity at the nucleotide and amino acid levels. Long terminal repeat nucleotide sequences varied up to 35% and Gag protein sequences varied up to 30%. The variability among SIVagm isolates exceeded the variability among any other group of primate lentiviruses. Our data suggest that SIVagm has been in the African green monkey population for a long time and may be the oldest primate lentivirus group in existence.

Partial purification and characterization of the reverse transcriptase of the simian immunodeficiency virus TYO-7 isolated from an African green monkey.

The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.

Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey.

We constructed a full-length molecular clone of simian immunodeficiency virus from an African green monkey. Upon transfection, this clone directed the production of virus particles cytopathic and infectious to human CD4+ leukemia cell lines. Mutations were introduced by recombinant DNA techniques into eight open reading frames of simian immunodeficiency virus from the African green monkey thus far identified. The phenotypes of mutant viruses, i.e., infectivity, cytopathogenicity, transactivation of gene expression controlled by a long terminal repeat, and viral RNA and protein syntheses, were examined by transfection and infection experiments. Three structural (gag, pol, and env) and two regulatory (tat and rev) gene mutants were not infectious, whereas vif, vpx, and nef were dispensable for infectivity and mutant viruses were highly cytopathic. In transient transfection assays, a rev mutant produced mainly small mRNA species and no detectable virus protein and particles. The transactivation potential of a tat mutant was about 10-fold less than that of wild-type DNA, generating small amounts of virus.

Efficiency of isolation of human rotavirus in primary African green monkey kidney cells.

Out of 212 human rotavirus (HRV) containing fecal specimens, 173 (81.6%) yielded virus on first passage in primary African Green monkey kidney cells (AGMK), while additional 34 specimens, did not yield virus on first passage. However, following blind passages, 18 of the 34 yielded virus in passage levels 2-8, thus raising the overall isolation rate to 90.1%. The isolation rate of HRV strains obtained in embryonic Rhesus monkey kidney cell line (MA-104), was only 41.4%. ELISA tests performed on fluids from infected cell cultures proved to be an efficient tool to measure virus replication. No differences were encountered in the isolation rates between subgroup I and II strains, while viruses lacking the antigenic determinants of both subgroups did not grow at all. However, one of those unusual group A strains was isolated and grew well in AGMK cells. Primary AGMK and MA-104 cells supported the growth of tissue culture adapted virus most efficiently when compared with six human and primate cell types.

Increased peripheral lymphocytes, lymphoid hepatitis and anaemia in African vervet monkeys seropositive to retroviruses.

Wild-caught African Vervet monkeys are commonly infected by Simian T-lymphotropic virus I (STLV1) and Simian immunodeficiency virus (SIV), yet the natural histories of these infections are largely unknown. Seropositivity was associated with increased total, T and atypical lymphocytes. In seropositive females there was mild, normocytic, normochromic anaemia. Lymphoid hepatitis was present in seven seropositive cases. African Vervets used in biomedical research, vaccine production and organ transplantation research are often infected by exogenous retroviruses which can be oncogenic and immunosuppressive in captive monkeys. Elimination of these infections may be possible by breeding Vervets in captivity.

Extensive genetic variability of simian immunodeficiency virus from African green monkeys.

Serological surveys have revealed that 30 to 50% of wild-caught African green monkeys have antibodies reactive to simian immunodeficiency virus (SIV), a retrovirus related to human immunodeficiency virus (HIV). Although the nucleotide sequence of one SIVagm isolate, Tyo1, was recently reported, the extent of genetic variability among SIVagm isolates remains to be determined. Restriction endonuclease mapping of infectious molecular clones of two SIVagm isolates (266 and 385), described in this note, revealed conservation of only 4 of 39 sites across the genome. Partial sequence analysis of the molecular clones revealed only 80% amino acid sequence conservation in the pol gene. Although the three Kenyan SIVagm isolates, Tyo1, 385, and 266, are more closely related to each other than to other primate lentiviruses, genetic variation among these three isolates is much greater than that observed previously among individual HIV type 1 (HIV-1), HIV-2, or SIVmac isolates. Less variability among HIV-1 and HIV-2 isolates could be explained by recent entry into the human population. The extensive genetic variation in these Kenyan SIVagm isolates should prompt continued examination of SIVagm variability from dispersed geographic regions; SIVagm strains much more closely related to HIV-1, HIV-2, or SIVmac which would be reasonable candidates for recent cross-species transmission may be found.

Variations in genome fragments coding for RNA polymerase in human and simian hepatitis A viruses.

The genome of hepatitis A virus (HAV) isolated from spontaneously infected African vervet monkey (Cercopithecus aethiops) has been cloned and partially sequenced. Comparison of genome fragments (1248 and 162 bp) from the 3D (RNA polymerase) region with the corresponding parts of human HAV genomes revealed a high degree of heterogeneity: there were altogether 257 nucleotide changes leading to 44 substitutions in predicted amino acid sequence, i.e. 89% amino acid identity. This divergence is considered to be significantly greater than genomic variations usually found among human HAV strains, where amino acid identity in the 3D region is over 98%.

Isolation of human immunodeficiency virus-related simian immunodeficiency viruses from African green monkeys.

We have isolated lentivirus strains that are related to the human immunodeficiency virus (HIV) from African green monkeys (Cercopithecus aethiops; AGM). Although immunologically related, these SIVagm are clearly distinct from other simian immunodeficiency virus (SIV) isolates, including isolates from Macaca mulatta (SIVmac) or even from other AGM. The SIVagm strains described in this communication grow well in a limited number of human T-lymphoma lines. Virus density, morphology, and reverse transcriptase activity are characteristic of the lentivirus group. SIVagm exhibits the following pattern of major virus proteins: p18, p28, gp45, p64, gp140. They appear to bind to the target cell via the CD4 or its primate analogue. Four virus isolates have already been molecularly cloned for detailed genomic analysis and within this SIV agm group they exhibit the genomic variability that is typical of lentiviruses. AGMs infected with this virus apparently remain healthy and therefore SIVagm not only provides a virus model for vaccine studies but also allows investigation of the defense mechanisms (immunological and others) that keep the AGMs healthy. Furthermore, precise genomic analysis of these and other SIV strains will lead to a better understanding of the evolution and pathogenicity of human lentiviruses like HIV.