First evidence of transmission of an HIV-1 M/O intergroup recombinant virus.
Paul Alain NGOUPO, Serge Alain SADEUH-MBA, Fabienne DE OLIVEIRA, Valérie Ngono, Laure NGONO, Patrice TCHENDJOU, Véronique PENLAP, Thomas MOUREZ, Richard NJOUOM, Anfumbom KFUTWAH, Jean-Christophe PLANTIER.
SUPPLEMENTARY METHODS
Estimates of evolutionary divergence
The number of base substitutions per site between sequences obtained from intra- and inter-patients samples were estimated using the Kimura 2-parameter model [1]. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1).
The analysis involved 5 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 1033 and 543 positions for Pol and Env regions respectively, in the final dataset. Evolutionary analyses were conducted in MEGA 6.06 [2] .
Phylogenetic analyses
Sequences of three previously characterized HIV-1 M/O recombinants [3-5] and further HIV-1 nucleotide sequences representing HIV-1/M subtypes and HIV-1/O clades were downloaded from Los Alamos Database and aligned with the patient sequences by using CLUSTALW [6] with minor manual adjustments. All PROT-RT concatamer sequences (1033bp) in the pol gene and gp41 (543 bp) in the env gene were used for phylogenetic analyses. Results obtained with INT sequences were only informative because of the relative short length of the PCR fragment (239 bp). Phylogenetic trees were 2
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reconstructed in MEGA 6.06 software[2] by the neighbor-joining method with the Kimura two-parameter method for computing evolutionary distances [1]. All alignment gaps were removed from the analysis for each sequence pair. The reliability of the tree topologies was estimated by bootstrap analysis with 1,000 pseudo-replicate data sets.
Near–full length genomes characterization
Near–full length genomes of sample of October 2012 from REC003 and sample of March 2013 from REC024 were amplified using a strategy of amplification of 7 overlapping fragments (supp figure 2) as follows:
RNA was extracted from 200 µL of plasma sample. RT-PCR was performed using SuperScript™ III One-Step RT-PCR System with Platinum® Taq High Fidelity (Invitrogen) in a final volume of 50 µL containing 20 pmol of each primer (supp table 1), 2 mM MgSO4 and 10 µL of RNA extract. We used a Perkin Elmer Gene Amp PCR System 9700 with the following cycling conditions: 50 °C for 30 min, 94 °C for 2 min, followed by 35 cycles of (94°C for 15 s, 55°C for 30 s, and 68 °C for 2min30s) and a final extension of 68°C for 10 min. 2 µL of RT-PCR products were then subjected to nested PCR reaction with HotStarTaq Master Mix (Qiagen) in a final volume of 50 µL containing 20 pmol of each primer (supp table 1) and 1.5 mM MgCl2. The cycling conditions consisted of 95°C for 15 min followed by 35 cycles (94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min) and a final extension step of 72°C for 10 min.
PCR amplicons were purified with the NucleoSpin Gel and PCR Clean-up (Macherey- Nagel, Düren, Germany) and subjected to sequencing with the CEQ Dye Terminator Cycle Sequencing with Quick Start kit (Beckman).HIV sequence BLAST search were
performed from the LANL database
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Genotyping Retrovirus Tool from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/retroviruses/). Sequences of overlapping fragments were aligned and assembled using MEGA 6.06 software.
Supplementary table 1: Primers used for near-full length genome characterization of the recombinant forms.
Genome region Step Primers Primer sequence (5’--- 3’) Fragment size (bp)
LTR-Gag
RT PCR LTRM514U25 (U) GCAAGCTTTATTGAGSSTTAAGCAG 1768
G01 (L) AGGGGTCGTTGCCAAAGA
NESTED LTRO152 (U) CTCAATAAAGCTTGCCTTGA 1747
UNIL1 (L) CCAAAGAGKGATYTGAGGG
Gag-Pol
RT PCR POLM2610U25 (U) GTTAAACARTGGCCATTRACAGARG 1174
UNIL2 (L) GAATCCAGGTRGCYTGCC
NESTED MW1 (U) CCACARGGATGGAAAGGATCACC 466
RT20 (L) CTGCCAGTTCTAGCTCTGCTTC
RT PCR UNIU1 (U) GGAAATGTGGAMAGGAAGG 1755
RTO1L (L) AATTCCCATTCWGGAATCCA
NESTED PROT1 (U) TAATTTTTTAGGGAAGATCTGGCCTTCC 1750
UNIL2 (L) GAATCCAGGTRGCYTGCC
Pol
RT PCR RTOXL1U (U) CTCCAYCCAGACAARTGGAC 1735
POLU2 (L) GTATTACTACTGCCCCTTCACCTTTCCA
NESTED RTO1U (U) GAAARCTAAATTGGGCAAGTC 1594
INTO3L (L) GGGTCTCTGCTRTCTCTGTAATA
RT PCR RTOXL1U (U) CTCCAYCCAGACAARTGGAC 1727
POLORB (L) ACTGCHCCTTCHCCTTTCCA
NESTED RTO1U (U) GAAARCTAAATTGGGCAAGTC 1650
POLU2 (L) GTATTACTACTGCCCCTTCACCTTTCCA Pol-Accessory
genes-Env
RT PCR VIF1 (U) GGGTTTATTACAGGGACAGCAGAG 1542
VPU1 (L) GGTTGGGGTCTGTGGGTACACAGG
NESTED POLM4920 (U) AGAGAYCCWATTTGGAAAGGACC 1521
ENVO6L (L) TTGTGMTGCCCAAATATTATG
Accessory genes- Env
RT PCR ENVO6338U23 (U) GGCTTTGMTAAYCCCATGTTTGA 1090
ENVO7402 (L) TGTGTTACAATARAAGAAYTCTCCAT
NESTED ENVO7U ND2 (U) TTTGMTAATCCCATGTTTGA 1078
V3DURR (L) AAAGAATTCTCCATGACAGTTAAA
RT PCR REVO6U (U) ATCTCCYATGGCAGGAAGAAG 1918
UNIL5 (L) YTGCTGTTGCACTATRCC
NESTED ENVO7U (U) TTTGMTAAYCCCATGTTTGA 1254
UNIL3 (L) CCCATAGTGCTTCCTGCTGC Env
RT PCR V3DURA (U) ATTCCAATACACTATTGTGCTCCA 1698
GP41NE 3’(L) TAAGTTGCTCAAGAGGTGGTA
NESTED GP41NE 5’(U) TAAGTGCAGCAGGTAGCACTAT 749
Supplementary table 1 (continued)
GP41NE3’ (L) TAAGTTGCTCAAGAGGTGGTA Env-LTR
RT PCR GP41NE 5’(U) TAAGTGCAGCAGGTAGCACTAT 1922
LTROL (L) TCAAGGCAAGCTTTATTGAG
NESTED GP41OXL (U) AACATTAGGCAGGGATATCAAC 1354
LTRM514L25 (L) GCAAGCTTTATTGAGSSTTAAGCAG
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Supplementary table 2. Genetic distances (intra- and inter-patients) between Pol and Env sequences obtained from the sequential samples of REC003 and REC024.
Pol region REC003 10/2012
REC003 03/2013
REC003 09/2013
REC024 03/2013
REC024 09/2013 REC003
10/2012 -
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 0,003 -
Envregion REC003
10/2012 REC003
03/2013 REC003
09/2013 REC024
03/2013 REC024 09/2013 REC003
10/2012 -
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 -
Supplementary figure 1: Algorithm used to detect dual HIV-1 M+O infections and HIV-1 M/O recombinant forms at CPC laboratory 7
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Supplementary figure 2: PCR strategy for near-full length genome amplification
Supplementary references 10
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