1 Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
2 National Institute of Biomedical Research, Kinshasa, Democratic Republic of Congo
3 Cite Louis Pasteur-Laboratoire National de Sante Publique, Brazzaville 120, Republic of Congo
4 Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Japan
5 Department of Viral Infection and International Health, Graduate School of Medical Science, Kanazawa University, Japan
6 Kinshasa Zoo, Kinshasa, Democratic Republic of Congo
Correspondence
Tomoyuki Miura
tmiura{at}virus.kyoto-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence determined in this work is AY518534.
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MAIN TEXT |
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It is now widely accepted that HIV-1 originated from SIVcpz (Gao et al., 1999; Hahn et al., 2000
). Bailes et al. (2003)
suggested that SIVcpz could be a recombinant virus between SIVrcm from red-capped mangabeys (Cercocebus torquatus) and SIVgsn. Furthermore, viruses of the above-mentioned six major lineages may also have complex mosaic genomes (Salemi et al., 2003
). To understand better the evolutionary relationships among primate lentiviruses, a search for additional SIVs from other non-human primate species is essential. Thus, our research efforts have focused on non-human primates whose habitats are adjacent to or overlap the habitat of common chimpanzees in the forests of central Africa. In the present study, we genetically characterized a new SIV strain from a black mangabey (Lophocebus aterrimus) originally isolated in the Democratic Republic of Congo (DRC).
The animal was caught in the wild in 2001 in the Bas-Congo region, in the south-eastern part of the DRC, and kept in a separate cage for 1 year at the Kinshasa Zoo. Peripheral blood was collected in 2002 using acid citrate glucose anticoagulant solution. A plasma sample was initially tested for antibodies using a commercial particle agglutination kit (Genedia HIV-1/2, Fujirebio) and showed positive reactivity, with an antibody titre of 1 : 64. We then conducted a Western blot assay using commercial Western blot kits (HIV-1 and -2 LAV blot; Bio-Rad) and demonstrated strong reactivity against p18 and p25 (HIV-1 core antigens), p26 (HIV-2 core antigens) and gp105 (HIV-2 glycoprotein) (data not shown). These data suggested that this monkey was infected with an SIV strain related to HIV-1 or -2.
We carried out nested PCR to amplify the viral pol region using chromosomal DNA extracted from peripheral blood mononuclear cells. DNA was extracted using the QIAamp Blood DNA mini kit (Qiagen) according to the manufacturer's instructions. We first amplified a fragment of 150 bp in the pol region using oligonucleotide primers DR1 (5'-TRCAYACAGGRGCWGAYGA-3') and DR2 (5'-AIADRTCATCCATRTAYTG-3') for the first round and primers DR4 (5'-GGIATWCCICAYCCDGCAGG-3') and DR5 (5'-GGIGAYCCYTTCCAYCCYTGHGG-3') for the second round (Clewley et al., 1998; Courgnaud et al., 2002
). Amplification was carried out with an initial denaturation at 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, 45 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for 5 min. We succeeded in amplifying a 150 bp fragment from this seropositive sample. The PCR product purified from an agarose gel was subcloned into the pUC119 (Takara Bio) vector and sequenced using a cycle sequencing kit and automated sequencers (BigDye Terminator Cycle Sequencing Ready Reaction, ABI 373 and 3100; Applied Biosystems). We then performed a semi-nested PCR to amplify a 1800 bp fragment using the primers DR1 and Unipol2 (5'-CCCCTATTCCTCCCCTTCTTTTAAAA-3') for the first-round PCR and bkmpol1 (5'-GGATATGAAAGATGCTTACTATTCAG-3') and Unipol2 for the second-round PCR (Miura et al., 1990
). The primer bkmpol1 was specifically designed on the basis of the sequence of the 150 bp fragment. PCR was carried out as described above but with cycle conditions of 94 °C for 1 min, 45 °C for 1 min and 72 °C for 3 min. The sequence of the obtained fragment was determined as described above.
The SIV derived from the black mangabey was designated SIVbkm strain CDM201. We constructed a phylogenetic tree to compare the sequence of SIVbkm CDM201 with sequences of representative isolates of SIVs and HIVs. Sequences were aligned using CLUSTAL W with minor manual modifications and a phylogenetic tree was constructed using the maximum-likelihood method with the MOLPHY program (Higgins & Sharp, 1989; Adachi & Hasegawa, 1996
; Yamaguchi-Kabata & Gojobori, 2000
; Yamaguchi-Kabata et al., 2004
). A phylogenetic tree based on 1930 bp fragments of the pol region showed that SIVbkm was divergent from other SIV strains, but relatively close to SIVgsn and SIVsyk group isolates (Fig. 1
). Subsequently, we carried out a distance plot analysis to investigate the extent of sequence differences (Takehisa et al., 1999
). The reference strains used were HIV-1 groups M (strain HXB2), O (ANT70), SIVcpz (US), SIVsm (SL92L), SIVagm (TYO-1), SIVmnd-1 (GB1), SIVsyk (syk173), SIVcol (CGU-1), SIVgsn (99CM71), SIVrcm (Ngm), SIVmon (NG1) and SIVmus (01CM1085). The genetic distance (estimated using Kimura's two-parameter method) between each selected pair of sequences was determined by moving a window of 300 bp along the genome alignment in 10 bp increments and the distance was plotted at the midpoint. SIVmon exhibited a relatively close distance to SIVbkm throughout the pol reverse transcriptase (RT) to integrase (IN) region and, interestingly, HIV-1 group O strain ANT70 showed a close relationship to SIVbkm, with the shortest distance in the RNase H region (Fig. 2
). At the amino acid level, SIVbkm CDM201 showed moderate similarities with three other primate lentiviruses, SIVgsn (62·5 %), SIVmon (62·0 %) and SIVsyk (59·3 %) (Table 1
). However, in the RNase H region, SIVbkm CDM201 showed relatively high similarities to HIV-1 group O (ANT70) (68·6 %), SIVrcm (70·3 %) and SIVmnd-2 (68·6 %). In the RT and IN regions, the similarities between SIVbkm CDM201 and these three viruses were relatively low.
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ACKNOWLEDGEMENTS |
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Received 15 October 2004;
accepted 29 March 2005.
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