1
1
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First evidence of transmission of an HIV-1 M/O intergroup
recombinant virus.
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Paul Alain NGOUPO, Serge Alain SADEUH-MBA, Fabienne DE OLIVEIRA, Valérie
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Ngono, Laure NGONO, Patrice TCHENDJOU, Véronique PENLAP, Thomas
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MOUREZ, Richard NJOUOM, Anfumbom KFUTWAH, Jean-Christophe PLANTIER.
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SUPPLEMENTARY METHODS
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Estimates of evolutionary divergence
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The number of base substitutions per site between sequences obtained from intra- and
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inter-patients samples were estimated using the Kimura 2-parameter model [1]. The rate
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variation among sites was modeled with a gamma distribution (shape parameter = 1).
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The analysis involved 5 nucleotide sequences. Codon positions included were
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1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence
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pair. There were a total of 1033 and 543 positions for Pol and Env regions respectively,
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in the final dataset. Evolutionary analyses were conducted in MEGA 6.06 [2] .
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Phylogenetic analyses
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Sequences of three previously characterized HIV-1 M/O recombinants [3-5] and further
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HIV-1 nucleotide sequences representing HIV-1/M subtypes and HIV-1/O clades were
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downloaded from Los Alamos Database and aligned with the patient sequences by
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using CLUSTALW [6] with minor manual adjustments. All PROT-RT concatamer
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sequences (1033bp) in the pol gene and gp41 (543 bp) in the env gene were used for
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phylogenetic analyses. Results obtained with INT sequences were only informative
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because of the relative short length of the PCR fragment (239 bp). Phylogenetic trees
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were reconstructed in MEGA 6.06 software[2] by the neighbor-joining method with the
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Kimura two-parameter method for computing evolutionary distances [1]. All alignment
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gaps were removed from the analysis for each sequence pair. The reliability of the tree
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topologies was estimated by bootstrap analysis with 1,000 pseudo-replicate data sets.
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Near–full length genomes characterization
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Near–full length genomes of sample of October 2012 from REC003 and sample of
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March 2013 from REC024 were amplified using a strategy of amplification of 7
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overlapping fragments (supp figure 2) as follows:
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RNA was extracted from 200 µL of plasma sample. RT-PCR was performed using
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SuperScript™ III One-Step RT-PCR System with Platinum® Taq High Fidelity
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(Invitrogen) in a final volume of 50 µL containing 20 pmol of each primer (supp table
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1), 2 mM MgSO4 and 10 µL of RNA extract. We used a Perkin Elmer Gene Amp PCR
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System 9700 with the following cycling conditions: 50 °C for 30 min, 94 °C for 2 min,
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followed by 35 cycles of (94°C for 15 s, 55°C for 30 s, and 68 °C for 2min30s) and a
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final extension of 68°C for 10 min. 2 µL of RT-PCR products were then subjected to
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nested PCR reaction with HotStarTaq Master Mix (Qiagen) in a final volume of 50 µL
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containing 20 pmol of each primer (supp table 1) and 1.5 mM MgCl2. The cycling
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conditions consisted of 95°C for 15 min followed by 35 cycles (94 °C for 30 s, 50 °C
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for 30 s, and 72 °C for 2 min) and a final extension step of 72°C for 10 min.
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PCR amplicons were purified with the NucleoSpin Gel and PCR Clean-up (Macherey-
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Nagel, Düren, Germany) and subjected to sequencing with the CEQ Dye Terminator
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Cycle Sequencing with Quick Start kit (Beckman).HIV sequence BLAST search were
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performed
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(http://www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html)
from
the
LANL
database
and
3
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Genotyping Retrovirus Tool from the National Center for Biotechnology Information
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(http://www.ncbi.nlm.nih.gov/retroviruses/). Sequences of overlapping fragments were
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aligned and assembled using MEGA 6.06 software.
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Supplementary table 1: Primers used for near-full length genome characterization of the
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recombinant forms.
Genome region
LTR-Gag
Step
RT PCR
NESTED
RT PCR
NESTED
Gag-Pol
RT PCR
NESTED
RT PCR
NESTED
Pol
RT PCR
NESTED
RT PCR
Pol-Accessory
genes-Env
NESTED
RT PCR
NESTED
Accessory genesEnv
RT PCR
NESTED
RT PCR
Env
NESTED
Primers
LTRM514U25 (U)
G01 (L)
LTRO152 (U)
UNIL1 (L)
POLM2610U25 (U)
UNIL2 (L)
MW1 (U)
RT20 (L)
UNIU1 (U)
RTO1L (L)
PROT1 (U)
UNIL2 (L)
RTOXL1U (U)
POLU2 (L)
RTO1U (U)
INTO3L (L)
RTOXL1U (U)
POLORB (L)
RTO1U (U)
POLU2 (L)
VIF1 (U)
VPU1 (L)
POLM4920 (U)
ENVO6L (L)
ENVO6338U23 (U)
ENVO7402 (L)
ENVO7U ND2 (U)
V3DURR (L)
REVO6U (U)
UNIL5 (L)
ENVO7U (U)
UNIL3 (L)
V3DURA (U)
GP41NE 3’(L)
GP41NE 5’(U)
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55
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Fragment size (bp)
1768
1747
1174
466
1755
1750
1735
1594
1727
1650
1542
1521
1090
1078
1918
1254
1698
749
Supplementary table 1 (continued)
RT PCR
Env-LTR
Primer sequence (5’---------- 3’)
GCAAGCTTTATTGAGSSTTAAGCAG
AGGGGTCGTTGCCAAAGA
CTCAATAAAGCTTGCCTTGA
CCAAAGAGKGATYTGAGGG
GTTAAACARTGGCCATTRACAGARG
GAATCCAGGTRGCYTGCC
CCACARGGATGGAAAGGATCACC
CTGCCAGTTCTAGCTCTGCTTC
GGAAATGTGGAMAGGAAGG
AATTCCCATTCWGGAATCCA
TAATTTTTTAGGGAAGATCTGGCCTTCC
GAATCCAGGTRGCYTGCC
CTCCAYCCAGACAARTGGAC
GTATTACTACTGCCCCTTCACCTTTCCA
GAAARCTAAATTGGGCAAGTC
GGGTCTCTGCTRTCTCTGTAATA
CTCCAYCCAGACAARTGGAC
ACTGCHCCTTCHCCTTTCCA
GAAARCTAAATTGGGCAAGTC
GTATTACTACTGCCCCTTCACCTTTCCA
GGGTTTATTACAGGGACAGCAGAG
GGTTGGGGTCTGTGGGTACACAGG
AGAGAYCCWATTTGGAAAGGACC
TTGTGMTGCCCAAATATTATG
GGCTTTGMTAAYCCCATGTTTGA
TGTGTTACAATARAAGAAYTCTCCAT
TTTGMTAATCCCATGTTTGA
AAAGAATTCTCCATGACAGTTAAA
ATCTCCYATGGCAGGAAGAAG
YTGCTGTTGCACTATRCC
TTTGMTAAYCCCATGTTTGA
CCCATAGTGCTTCCTGCTGC
ATTCCAATACACTATTGTGCTCCA
TAAGTTGCTCAAGAGGTGGTA
TAAGTGCAGCAGGTAGCACTAT
NESTED
GP41NE3’ (L)
GP41NE 5’(U)
LTROL (L)
GP41OXL (U)
LTRM514L25 (L)
TAAGTTGCTCAAGAGGTGGTA
TAAGTGCAGCAGGTAGCACTAT
TCAAGGCAAGCTTTATTGAG
AACATTAGGCAGGGATATCAAC
GCAAGCTTTATTGAGSSTTAAGCAG
1922
1354
4
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Supplementary table 2. Genetic distances (intra- and inter-patients) between Pol and
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Env sequences obtained from the sequential samples of REC003 and REC024.
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Pol region
REC003
10/2012
REC003 REC003 REC003 REC024 REC024
10/2012 03/2013 09/2013 03/2013 09/2013
-
REC003
03/2013
0,004
REC003
09/2013
0,011
0,010
REC024
03/2013
0,037
0,036
0,039
REC024
09/2013
0,033
0,033
0,035
Envregion
REC003
10/2012
0,003
-
REC003 REC003 REC003 REC024 REC024
10/2012 03/2013 09/2013 03/2013 09/2013
-
REC003
03/2013
0,008
REC003
09/2013
0,004
0,006
REC024
03/2013
0,071
0,072
0,033
REC024
09/2013
0,063
0,066
0,034
0,020
-
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Supplementary figure 1: Algorithm used to detect dual HIV-1 M+O infections and HIV1 M/O recombinant forms at CPC laboratory
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Supplementary figure 2: PCR strategy for near-full length genome amplification
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Supplementary references
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1.
Kimura M. A simple method for estimating evolutionary rates of base substitutions
76
through comparative studies of nucleotide sequences. J Mol Evol. 1980
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Dec;16(2):111-20. 1980.
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2.
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Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular
Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 2013; 30:2725-2729.
3.
Vessiere A, Leoz M, Brodard V, Strady C, Lemee V, Depatureaux A, et al. First
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evidence of a HIV-1 M/O recombinant form circulating outside Cameroon. AIDS
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2010; 24:1079-1082.
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4.
Yamaguchi J, Bodelle P, Vallari AS, Coffey R, McArthur CP, Schochetman G, et al.
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HIV infections in northwestern Cameroon: identification of HIV type 1 group O and
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dual HIV type 1 group M and group O infections. AIDS Res Hum Retroviruses
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2004;20:944-957.
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5.
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al.Characterization of a highly replicative intergroup M/O human immunodeficiency
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virus type 1 recombinant isolated from a Cameroonian patient. J Virol 1999;73:7368-
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7375.
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6.
Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of
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progressive multiple sequence alignment through sequence weighting, position-
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specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673-
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