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Since the development of the highly sensitive polymerase chain reaction, PCR has been increasingly used for the diagnosis of viral infections, including the detection of human immunodeficiency virus (HIV), the causative agent of AIDS. In our laboratory a diagnostic PCR is carried out on proviral HIV-1 DNA using a standardised algorithm based on three HIV-1 primer sets. The three primer sets, amplifying a fragment in the LTR-gag gene, in the pol gene and in the env gene, are situated within conserved regions of the HIV-1 genome. These primers allow us to detect not only HIV strains from Belgian patients but also from African patients, who are, for historical reasons, a substantial part of the HIV-positive patients in Belgium. We are able to detect 1-5 copies of proviral HIV-1 DNA with each of the three nested primer sets. A sensitivity and specificity of 92 and 100%, respectively, were achieved when testing 24 Belgian and African HIV-1 seropositive samples. In our lab, the same PCRs are also used for the detection of viral RNA in cases of a doubtful undetectable viral load when using a commercial HIV-1 viral load assay. This is because present-day commercial assays are not entirely reliable with divergent strains. Both our 'in-house' diagnostic DNA and RNA-PCR can also be used semiquantitatively with limiting dilutions.

We isolated a divergent simian T-lymphotropic virus (STLV) (strain PP1664) from a wild-caught African bonobo (pygmy chimpanzee, Pan paniscus). Molecular and phylogenetic characterization of this virus show that it reliably separates from the two well-established primate T-lymphotropic virus types, HTLV-I/STLV-I (PTLV-I) and PTLV-II, and from a third type isolated from an African-born Papio hamadryas and designated by us as PTLV-L. Four of eight bonobos kept at the Antwerp Zoo, Belgium, showed an aberrant PTLV serology. We amplified and sequenced a 709 bp PTLV proviral tax/rex fragment from one of the reactive bonobos. It differs by about 25 % from the homologous nucleotide sequences of PTLV-I and PTLV-L and by about 17 % from PTLV-II. This is comparable to the differences among the three known types. Including the most divergent STLV-I strains sequenced to date, for example, strain PHSu1 sequenced here, the divergence in this region within PTLV-I is less than 11 % and within PTLV-II less than 4%. Although very divergent, this new bonobo STLV is the closest well-characterized simian relative of HTLV-II, raising the possibility of very divergent new HTLV strains. Our results show that the number of PTLV types should be considered open and that the variety of indigenous viruses in the PTLV group is greatest in Africa. Thus, as for the other primate retroviruses HIV and SIV, PTLV most probably has its origins in Africa.

The presence of HIV-1 RNA in the plasma and serum of European and African patients was monitored using RNA-polymerase chain reaction (RNA-PCR) and the new isothermal NASBA nucleic acid amplification system encompassing a gel-based detection assay (ELGA). Identical RNA extraction procedures, provided by the NASBA amplification system, were used for both methods. The detection limit for HIV-1 RNA, measured on a 10-fold dilution series of spiked HIVIIIB in negative plasma, was about 0.05 CCID50 per test for both methods. Both NASBA and RNA-PCR were more sensitive than a p24 assay for the detection of circulating HIV-1 virus in blood: 17 of the 34 (50%) p24 antigen-tested seropositives were p24-positive while 32 (94%) were positive by NASBA and 30 (88%) by RNA-PCR. Among the 45 seropositives, 34 of which were tested for p24 antigen, 43 (96%) were positive by NASBA and 41 (91%) by RNA-PCR. Almost all seropositives had a detectable viral load in 100 microliters plasma. Lower viral loads were only encountered in some healthy seropositives with a higher CD4 count. There was no cross-reactivity with HIV-2 or HIV-I with both the RNA-PCR and NASBA. The extraction method used permitted the detection of HIV-1 RNA equally well in serum and in plasma with heparin or EDTA.

Due to the low evolutionary rate and the limited horizontal transmission of human T-lymphotrophic virus type I (HTLV-I), its phylogenetic analysis reveals the movements and contacts of ancient populations. Since simian strains cannot be distinguished from human strains by phylogenetic criteria, this virus has appropriately been called primate T-lymphotropic virus type I (PTLV-I). We sequenced the LTR of six PTLV-I strains: three HTLV-I strains from African patients with tropical spastic paraparesis (TSP) (Equateur, Zaire), two laboratory HTLV-I strains of Japanese origin, MT-2 and MT-4, and one STLV-I from a baboon of the primate center in Sukhumi, Georgia. We applied four phylogenetic inference methods: neighbor-joining (NJ), unweighted pair group method using arithmetic averages (UPGMA), Fitch and Wagner parsimony (pars), and maximum likelihood (ML), to these 6 LTR sequences and 18 published LTR sequences (cosmopolitan, African, and Melanesian HTLV-I strains and African and Asian STLV-I strains). Three major HTLV-I subtypes can be identified with all four methods: the cosmopolitan HTLV-Ia, the central African HTLV-Ib, clearly descendant from a STLV-I CH-like African ancestral simian strain, and the Melanesian HTLV-Ic, probably descendant from an Asian STLV-I strain. We observe a segregation of PTLV-I sequences according to their geographical origin and not according to host species. The Zairean strains form a cluster closely related to an STLV-I strain isolated from a chimpanzee (STLV-I CH) and distinct from western African strains, which belong to the cosmopolitan subtype of HTLV-I. The Sukhumi STLV-I strain found in a captive-born baboon was of Asian descent. We experienced rooting problems with UPGMA when using HTLV-II as an outgroup. Concordant results with all four methods were obtained by eliminating HTLV-II LTR sequence fragments with bad alignment to HTLV-I. This resulted in a HTLV-II root node on the African STLV-I TAN90 terminal branch (with bootstrap values above 92% for the NJ and pars methods) and not on the Asian STLV-I PtM3 branch, as has been derived by others based on their use of UPGMA. The results of the analyses also support a higher evolutionary rate of PTLV-I in Asia, implying that the trees obtained with the NJ and ML methods have a higher reliability. These results are more compatible with an ancient African origin of PTLV-I than with an Asian origin.