Department of Virology, Biomedical Primate Research Centre (BPRC), PO Box 3306, 2206 GH Rijswijk, The Netherlands
Correspondence
Ernst Verschoor
verschoor{at}bprc.nl
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ABSTRACT |
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Published ahead of print on 30 September 2003 as DOI 10.1099/vir.0.19427-0.
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MAIN TEXT |
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During a screening of serum samples from various nonhuman primates for antibodies against SIV, we detected a Schmidt's guenon that had serum antibodies that reacted strongly with SIVmac antigens used in a SIV antibody ELISA (Warren et al., 1998). The serum sample of this animal, Qu, was analysed further using the INNO-LIA HIV Confirmation assay (Innogenetics) and the diagnosis of infection with an immunodeficiency virus was confirmed (Fig. 1
, lane 1). In this assay, the serum sample cross-reacted strongly with the HIV-2 gp36 and HIV-1 p24 proteins and interacted weakly with HIV-1 gp41. This pattern of reactivity is comparable to that observed with sera of other African monkeys (Courgnaud et al., 2001
; Peeters et al., 2002
).
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Both amplification reactions were performed in a 50 µl volume using 10 µl DNA, 10 mM Tris/HCl (pH 8·3), 50 mM KCl, 0·01 % BSA, 50 pmol each primer, 0·2 mM each dNTP, 2 mM MgCl2 and 2·5 units AmpliTaqGold (PE Applied Biosystems). Samples were preheated for 15 min at 94 °C to activate the enzyme and then cycled for 20 s at 94 °C, 20 s at 55 °C and 2 min at 72 °C for 35 rounds of amplification. PCR products were isolated from agarose gel using the QIAquick Gel Extraction kit (Qiagen) and sequenced directly using the ABI PRISM BigDye Terminators V3.0 Cycle Sequencing kit on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems). Sequence analysis was performed using the Seqman II software package (DNASTAR).
In total, a 1895 nt partial pol sequence was amplified and sequenced. The fragment (EMBL accession no. AJ551401) was aligned to published SIV pol sequences using Se-Al Sequence Alignment Editor, version 2.0a11 (Rambaut, 2002) and all sites containing a gap were excluded from analysis. Genetic distances between the newly derived SIV and SIVs representing known lineages were calculated using Kimura's two-parameter method and a phylogenetic tree was constructed using the neighbour-joining method, as implemented in the PAUP, version 4.0b10, software package (Swofford, 2002
).
Genetically, the SIVschm pol sequence was highly divergent from other SIVs identified to date (Table 1). Overall genetic distances from other SIVs were comparable to those of SIVcol, from guereza colobus monkeys, and SIVolc, from olive colobus monkeys, which are among the most divergent SIVs described to date (Courgnaud et al., 2001
, 2003
). The most closely related to SIVschm was SIVgsn, from greater spot-nosed monkeys (Courgnaud et al., 2002
), which had 47·4 % genetic distance from the new virus. Direct comparison of the deduced amino acid sequences of the pol gene sequences confirmed its relatedness with SIVgsn (67 % identity) and showed an equidistant relationship with SIVcol and SIVolc (48 % identity) (data not shown).
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SIVgsn is particularly interesting as its genome contains a vpu gene, which was seen as a unique feature of the chimpanzee/HIV lineage. SIVgsn has a mosaic genome consisting of a gag/pol region, which is most related to that of SIVsyk (Fig. 2), and an env gene, which has the most similarity to SIVcpz env. Despite our efforts, we were not able to amplify the essential 3' end of the SIVschm genome applying multiple sets of primers designed on the basis of published SIV sequences and lineages. The difficulty of amplifying other regions of the SIVschm genome may reflect its distinction from known SIVs. The lack of high-quality DNA (due to the poor quality of the starting material) has prevented the use of the long PCR method, which targets unintegrated circular DNA, as described by Courgnaud et al. (2002)
. It thus remains to be seen whether the possession of a vpu gene is a general feature of the SIVgsn/SIVschm cluster and if a SIVcpz-like env is unique to SIVgsn. By solving this question, we may gain additional insight into the possible chimeric origin of SIVcpz in chimpanzees and, thus, also the origin of HIV-1.
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Received 13 June 2003;
accepted 24 September 2003.