Characterization of Partial and Near Full-Length Genomes of HIV1 Strains Sampled from Recently Infected Individuals in São Paulo, Brazil

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  Characterization of Partial and Near Full-Length Genomes of HIV1 Strains Sampled from Recently Infected Individuals in São Paulo, Brazil
  Characterization of Partial and Near Full-LengthGenomes of HIV-1 Strains Sampled from RecentlyInfected Individuals in Sa˜o Paulo, Brazil Sabri Saeed Sanabani 1,2,3 * , E´ velyn Regina de Souza Pastena 4 , Antonio Charlys da Costa 2,4 , VanessaPouza Martinez 4 , Walter Kleine-Neto 2,3 , Ana Carolina Soares de Oliveira 2,4 , Mariana Melillo Sauer 1 , KatiaCristina Bassichetto 5 , Solange Maria Santos Oliveira 5 , Helena Tomoko Iwashita Tomiyama 1 , EsterCerdeira Sabino 3,4 , Esper Georges Kallas 1 1 Division of Clinical Immunology and Allergy, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil,  2 Department of Translational Medicine, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil,  3 Fundac¸a˜o Pro-Sangue, Blood Center of Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil,  4 Department of Infectious Diseases, University of Sa˜o Paulo,Sa˜o Paulo, Brazil,  5 Public Health Department of Sa˜o Paulo, Sa˜o Paulo, Brazil Abstract Background:   Genetic variability is a major feature of human immunodeficiency virus type 1 (HIV-1) and is considered thekey factor frustrating efforts to halt the HIV epidemic. A proper understanding of HIV-1 genomic diversity is a fundamentalprerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines design. Here, we report on thepartial and near full-length genomic (NFLG) variability of HIV-1 isolates from a well-characterized cohort of recently infectedpatients in Sa˜o Paul, Brazil. Methodology:   HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 113 participants. The NFLGand partial fragments were determined by overlapping nested PCR and direct sequencing. The data were phylogeneticallyanalyzed. Results:   Of the 113 samples (90.3% male; median age 31 years; 79.6% homosexual men) studied, 77 (68.1%) NFLGs and 32(29.3%) partial fragments were successfully subtyped. Of the successfully subtyped sequences, 88 (80.7%) were subtype Bsequences, 12 (11%) BF1 recombinants, 3 (2.8%) subtype C sequences, 2 (1.8%) BC recombinants and subclade F1 each, 1(0.9%) CRF02 AG, and 1 (0.9%) CRF31 BC. Primary drug resistance mutations were observed in 14/101 (13.9%) of samples,with 5.9% being resistant to protease inhibitors and nucleoside reverse transcriptase inhibitors (NRTI) and 4.9% resistant tonon-NRTIs. Predictions of viral tropism were determined for 86 individuals. X4 or X4 dual or mixed-tropic viruses (X4/DM)were seen in 26 (30.2%) of subjects. The proportion of X4 viruses in homosexuals was detected in 19/69 (27.5%). Conclusions:   Our results confirm the existence of various HIV-1 subtypes circulating in Sa˜o Paulo, and indicate that subtypeB account for the majority of infections. Antiretroviral (ARV) drug resistance is relatively common among recently infectedpatients. The proportion of X4 viruses in homosexuals was significantly higher than the proportion seen in other studypopulations. Citation:  Sanabani SS, Pastena E´RdS, da Costa AC, Martinez VP, Kleine-Neto W, et al. (2011) Characterization of Partial and Near Full-Length Genomes of HIV-1Strains Sampled from Recently Infected Individuals in Sa˜o Paulo, Brazil. PLoS ONE 6(10): e25869. doi:10.1371/journal.pone.0025869 Editor:  Ronald H. Gray, Johns Hopkins University Bloomberg School of Public Health, United States of America Received  April 14, 2011;  Accepted  September 13, 2011;  Published  October 14, 2011 Copyright:    2011 Sanabani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  The study was supported by grants 04/15856-9 and 2006/50096-0 from the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sao˜ Paulo (FAPESP). Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: Introduction Human Immunodeficiency Virus (HIV) has several puzzling characteristics that separate it from other viruses. The immensegenetic variability of HIV-1, which results mainly from the error-prone nature of its reverse transcriptase (3 6 10 2 5 mutations pernucleotide per replication cycle) [1]. The high rate of mutationand the rapid turnover of HIV-1  in vivo  (10.3 6 10 9 particles perday) [2] allows for the accumulation and fixation of a variety of host-immune response selected advantageous genetic changes inthe virus population. These changes allow HIV-1 to resist theevolving defenses of the host. Recombination is another potentialevolutionary source that significantly contributes to the geneticdiversification of HIV and may produce more virulent viruses,drug resistant viruses, or viruses with altered cell tropism that maycompromise the effectiveness of antiretroviral therapy and presentmajor challenges for vaccine design [3].The striking variability of HIV has led researchers to classify the virus into four phylogenetic distantly related groups: Main group(M), Outlier group (O), non-M-non-O group (N) and P group,which most likely reflect four independent events of cross-speciestransmission from chimpanzees [4,5]. The M group, which PLoS ONE | 1 October 2011 | Volume 6 | Issue 10 | e25869  dominates the current AIDS pandemic, is subdivided into subtypes(A–D, F–H, J and K), sub-subtypes (A1, A2, F1 and F2), circu-lating recombinant forms (CRFs) and unique recombinantforms (URFs) [4]. The molecular delineation of HIV-1 is a usefulepidemiological instrument for tracking virus transmission andprovides information about the patterns of genetic divergence thatmay have occurred during viral evolution.HIV genetic variants are not geographically confined; however,there are circulating clades that are predominant in certain areas[6,7]. For example, in Central Africa the main reported subtype is A and D, whereas other countries in Europe, USA, Australia andThailand have reported subtype B as the main clade associatedwith their epidemic. Subtype C viruses are predominant inSouth Africa, Ethiopia and India, and CRF01_AE is the majorcirculating form in Southeast Asia [8,9,10]. The most prevalent HIV-1 subtypes in China, Japan’s largest neighbor, are circulating BC recombinant forms, CRF07_BC, and CRF08_BC, andaccount for 50% of the HIV infected population, with subtype BHIV-1 accounting for 32% [11].Brazil has the most populous nation in Latin America and theCaribbean and has the highest number of people living with HIVin the region (estimates vary from 460.000–810.000; UNAIDS/WHO. 2010 Report on the Global AIDS Epidemic; 2010). Asin European countries and North America, HIV-1 subtype Bis a major genetic clade circulating in Brazil, but the overallprevalence of non-B strains, particularly URF BF1, C and URFBC, has been increasing  [12,13,14]. Data from recent studies of  the near full-length genomes (NFLGs) of HIV-1 have providedevidence of the existence of Brazilian CRF strains designated asCRF28_BF, CRF29_BF, CRF39_BF, CRF40_BF, CRF46_BFand CRF31_BC ( studies have been conducted to develop and character-ize panels of well-defined NFLG HIV-1 strains to be used as aresource in the evaluation of vaccine candidates [15,16,17]. In oneof the largest studies conducted to date, Brown et al. [15] reportedthe complete genetic and biological characterization of a panel of 60 full-length sequenced HIV-1 isolates from 15 countries,including R5 and X4 viruses, representing clades A through Dand CRF01_AE.The present study involved the phylogenetic analysis of HIV-1partial and NFLGs, the evaluation of HIV drug resistance and theevaluation of viral co-receptor tropism in treatment-naı¨ ve recentlyinfected individuals from Sa˜o Paulo, one of the main cities in Brazil. Materials and Methods Study population One hundred and thirteen participants were selected from alarger cohort of 228 recently HIV-1 infected persons by order of entrance into the study. This sub-cohort was part of an ongoing project to investigate the host factors that contribute toimmunodeficiency progression and has been previously described[18]. Briefly, recent HIV-1 infection was determined by theSerologic Testing Algorithm for Recent HIV Seroconversion(STARHS), and individuals were included in the study when theyhad a negative desensitized ELISA HIV-1 test, as that couldindicate an incomplete antibody response as a consequence of recent HIV infection. For this study, patient PBMC samples werecollected using standard procedures during their first clinical visitbefore the beginning of therapy and stored at  2 80 u C until use.The patient data, including age, sexual orientation, number of CD4-positive T cells, and viral load were obtained from medicalrecords. All study participants signed an informed consent formand the project was approved by the ethics committee of thefederal University of Sa˜o Paulo. Amplification and sequencing of HIV-1 DNA ThegenomicDNAusedforthePCRanalyseswasextractedfrombuffy coat samples with a QIAamp DNA Blood Mini Kit (Qiagen,Hilden, Germany), according to the manufacturer’s instructions.Proviral DNA was used as the PCR template, as this allowedamplification of the NFLGs from five overlapping fragments aspreviously described [12,13]. All amplification reactions were done in duplicate to eliminate PCR artifacts such as a sequenced NFLGbeing assembled from heterogeneous DNA targets. The amplifiedfragments were purified by use of a QIAquick PCR Purification Kit(Qiagen, Hilden, Germany) and directly sequenced on both strandsusingavarietyofprimer-directedstrategiesand the PRISMBigDyeTerminator Cycle Sequencing Ready Reaction Kit (AppliedBiosystems/Perkin-Elmer, Foster City, CA) on an automatedsequencer (ABI 3130, Applied Biosystems). After excluding theprimer regions, the fragments for each amplicon were assembledinto contiguous sequences and edited with the Sequencher program4.7 (Gene Code Corp., Ann Arbor, MI). Screening for recombination events and identification of breakpoints  All sequences were screened for the presence of recombinationevents via the genotyping function of NCBI (NCBI Blast genotyping tool. Available at: and the jumping profile Hidden Markov Model(jpHMM) [19]. Recombination events were further confirmed bythe bootscanning method [20] implemented in SimPlot v. 3.5.1 forWindows [21] using the following parameters: window size 250 bp,step size 20 bp and the F84 model of evolution (Maximumlikelihood) as a model to estimate nucleotide substitution, thetransition\transversion ratio of 2.0, and a bootstrap of 100 trees.The significance threshold for the bootscan was set at 70%. Thealignment of multiple sequences, including reference sequencesrepresenting subtypes A–D, F–H, J and K (, were performed using the CLUSTAL X program [22] andthen followedbymanualeditinginthe BioEdit SequenceAlignmentEditor program [23]. Gaps and ambiguous positions were removedfrom the final alignment. The positions of crossover sites werepreliminarilydefinedbased onthedistribution ofinformative sites, amethod implemented in SimPlot v. 3.5.1, supporting the twoincongruent topologies which maximize the  x 2  value [24]. Phylogenetic tree analysis  All phylogenetic trees were estimated by use of a general timereversible model under maximum likelihood (ML) using PHYML v.2.4.4 [25]. Heuristic tree searches using the ML optimalitycriterion were created using the nearest-neighbor interchange(NNI) branch-swapping algorithm. The approximate likelihoodratio test (aLRT) based on a Shimodaira-Hasegawa-like procedurewas used as a statistical test to calculate branch support. All treeswere displayed using either the MEGA v.5 package or the freelyavailable Archaeopteryx Java software [26]. Nucleotide similaritieswere estimated using a maximum composite likelihood modelimplemented in MEGA version 5.0 software. The comparisons of tree topologies of the subtype B subgenomic regions wereperformed using an algorithm described by Nye et al. [27]. Genotypic tropism analysis For the predictions of HIV tropism, the  env   region identified inthe NFLGs and partial-length  env   sequences that would encompass Analysis of HIV-1 Strains in Sa˜o Paulo, BrazilPLoS ONE | 2 October 2011 | Volume 6 | Issue 10 | e25869  the V3 region were analyzed using a tropism prediction algorithmimplemented as the web-based service geno2pheno [coreceptor] To minimize the number of falsepredictions of CXC chemokine receptor 4 (CXCR4 or X4) tropicsequences as C–C chemokine receptor 5 (CCR5 or R5) tropic, aconservative false-positive rate (FPR) of 20% was used as a cutoff.Therefore, X4 or X4 dual/mixed-tropic viruses (X4/DM) werereported positive if their sequences had a prediction result FPRof  # 20% or the 11/25 rule predicted a X4 virus, otherwise, theywere considered R5-tropic viruses. Determination of drug-resistance mutations The proviral nucleotide sequences of the  pol   region weretranslated into amino acid sequences and subsequently screenedfor drug-resistance mutations using the most updated WHO listfor the surveillance of drug resistance mutations in antiretroviral-naive patients (SDRM version 2009, All nucleotide sequences obtained during our study werereported to GenBank (Accession numbers pending). Results Samples In total, 113 blood samples from recently HIV-1 infectedindividuals were subjected to NFLG sequencing. The medianbaseline HIV-1 viral load of 108 samples was 1.5 6 10 4 copies/ml(range, , 399 2 7 6 10 5  ). The median baseline CD4 cell countwas , 560 cells/mm3 (range, 83–2449 cells/mm3) in 98.2% of individuals. The participants’ ages ranged between 18 and 56 years, and the median age was 31 years. The majority of subjectswere males (  n =102; 90.3%) and a sizable proportion (  n =90;79.6%) reported transmission through homosexual contact. Allpatients were treatment-naive at the time of sample collection. Themain characteristics of the study population are given in Table 1.Virus variants with stop codons in different open reading frameswere identified in five samples. A gross deletion of 433 (Nucleotideposition from start of HXB2 genome; 5464–5896) and 487 bp(Nucleotide position from start of HXB2 genome; 1337–1823)were observed in isolate 04BR 1071 (characterized as subclade F1)and 04BR 1074 (characterized as subtype B), respectively. Asequence duplication of 322 bp (Nucleotide position from start of HXB2 genome; 8512–8826) was observed in isolate 03BR 2015(characterized as subtype B). We are currently assessing theaccuracy of the large deletions and insertions in the aforemen-tioned RNA isolates via ultra-deep 454 pyrosequencing. None of these sequences, including those with stop codons, were omittedfrom the analysis. HIV variants and sequences Sequences were obtained for all five overlapped fragments thatcover the NFLGs of 77 participants. Partial sequences wereobtained from at least one fragment derived from 32 samples asshown in Table 2. Only four samples did not amplify for anyfragment. The amplification failure in these samples was likely notbe due to subtype diversity, as our method is effective for theanalysis of diverse HIV-1 strains. The distribution of HIV-1 subtypes by NFLG The phylogenetic analysis of the 77 HIV-1 incident samples withNFLG sequences revealed 64 pure subtypes (83.1%) and 13recombinants subtypes (15.1%). A comparison of the NFLGsequences from 64 pure subtypes is presented in Figure 1. Withthe exception of isolates 04BR 2022 and 04BR 1071, which clusterwith high aLRT values within the reference isolate clusters forsubtype C and subclade F1, respectively, all samples clustered withsubtype B. This group contains sequences from both MSM (  n =57)and heterosexual individuals (  n =7; 5 males and 2 females). In thetopological phylogenetic tree of NFLG pure subtype B depicted inFigure 1; various samples are positioned in various well-definedclusters supported by high aLRT values. The stability and order of these clusters werefurther investigatedwith ML trees independentlymade with the  gag-pol   and  env   sequences using the same multiplegenome alignment (Figures 2a&2b). The phylogenetic trees fromboth regions received an overall topological score of 63.8% asscored by the Nye et al. algorithm [27]. Close examination of bothsubgenomic trees revealed a shifting of topological position acrossnumerous sequences in a manner suggestive of intra-subtyperecombination [28]. For example, isolates 04BR 1060 and 04BR1068 changed their topological positions over the  gag-pol   and  env  regions of their genomes. The computed topological score of theisolates in both regions was 33.3%, with a branch length mismatchof 89.5%. Similarly, isolate 05BR 1089 placed the  gag-pol   regionwith 05BR 1103 with an 100% of aLRT value, while  env   stronglygrouped (aLRT , 94%) with isolate 05BR 1081 (Figures 2a&2b).The evolutionary relationship between the representative subtypesequences was analyzed to illustratethe level of genetic diversity andto identify cross epidemic relationships that could suggest commongeographic origins of subtype B present among presumablycirculating subtype B viruses in Sa˜o Paulo. The resulting 62 puresubtype B sequences were compared with each other and with 435non-redundant B sequences (unique patient entries) available in theLos Alamos database and collected from more than 30 countriesincluding Brazil (Figure 3a). The ML tree showed that sequencesfrom Brazil appeared dispersed, even if a subcluster of Braziliansequences was observed (Figure 3a; green nodes). This subcluster Table 1.  Characteristics and main findings of the BrazilianHIV-1 recently infected individuals. Male gender 102 (90.3%)Media age (Years) 31Risk group ( n =113)homosexual men 90 (79.6%)heterosexuals 23 (20.4%)Median CD4 count (cells/mm3) 560Median viral load (log HIV RNA copies/mL) ( n =108) 15000Drug resistance mutations ( n =101) 14 (13.9%)PI 6 (5.9%)NRTI 6 (5.9%)NNRTI 5 (4.9%)Subtype distributions ( n =109)B 88 (80.7%)BF1 12 (11%)C 3 (2.8%)BC 2 (1.8%)F1 2 (1.8%)CRF31 BC 1 (0.9%)CRF2 AG 1 (0.9%)Prediction of viral tropism (n=86)X4 viral tropism (pure or dual/mixed R5/X4) 26 (30.2%)doi:10.1371/journal.pone.0025869.t001 Analysis of HIV-1 Strains in Sa˜o Paulo, BrazilPLoS ONE | 3 October 2011 | Volume 6 | Issue 10 | e25869  contains sequences from 73% (41/56) of MSM patient samples withnon-recombinant clade B HIV-1 identified in this study. Furtheranalysis of the subcluster indicated that some strains grouped withhigh aLRT (  . 98%) values with little genetic diversity (branchlength , 0.015) into various main transmission clusters composedof 2 to 3 infections per cluster within the B molecular subtype(Figure 3b). Notably, three sequences obtained from heterosexualcontact-acquired infected patients (Figure 3b; brown square) aretightly positioned within some of the MSM risk groups. As shown inFigure 3A,the sequencesidentifiedin this study and otherpublishedBrazilian clade B sequences have a close genetic relationship withthose previously found in the United States. Characterization of the NFLG recombinant sequences  Among the 13 NFLG isolates with strong evidence of recombina-tion, 10 were mosaic isolates consisting of subtype BF1, 2 of subtypeBC and 1 identified as CRF02 AG. None of the putative BF1recombinants had a recombination profile identical to the other BFmosaics described in Brazil or elsewhere (Figure 4). Moreover, therewas no obvious preferred location for breakpoints. To further test forrecombination, ML phylogenetic trees were generated for thenucleotide sequence regions on either side of the breakpoints detectedby the bootscan method (Figure 5). This analysis corroborates theresultsfromthebootscanandthusprovidesunambiguousevidenceforrecombinationeventssupportedbyhighaLRTvalues.Thenucleotidesequence of isolate 06BR 2039, characterized as CRF02 AG, wasblastedagainsttheNCBInucleotidedatabase.Asignificantnumberof similar full genome hit with a Nigerian HIV-1IbNg (GenBank:L39106) strain was indicated by the search. The distribution of HIV-1 subtypes by partial sequencing The overall proportion of subtypes and recombinants generatedby partial sequences is shown in Table 2. Consistent with theresults obtained from the NFLGs analysis, the phylogenetic tree Table 2.  HIV-1 strain subtyping by partial sequences. Sample ID Sequence fragmentSubtypeA  (546–2598)  B1  (2157–3791)  B2  (3236–5220)  C  (4890–7808)  D  (7719–9537) 03BR 1025  + +  2  + +  B03BR 1032  2 2 2  +  2  B03BR 1034  +  2 2 2 2  B03BR 1037  +  2  +  2 2  C03BR 1042  + + +  2  +  B04BR 1050  + +  2  + +  B04BR 1056  2  + + + +  B04BR 1058  2  +  2 2 2  B04BR 1059  +  2  +  2 2  B04BR 1062  + +  2  +  2  BF104BR 1063  2 2 2 2  +  B04BR 1069  + +  2 2  +  B05BR 1073  +  2  + + +  B05BR 1086  + +  2 2 2  B05BR 1087  + + +  2 2  B05BR 1088  2  + + + +  B05BR 1091  + +  2  +  2  F105BR 1096  + + +  2  +  B05BR 1099  2  +  2 2  +  B05BR 1102  + +  2 2  +  C05BR 1105  + + +  2  +  B05BR 1106  + + +  2  +  B06BR 1113  + + +  2  +  B06BR 1116  2  +  2 2 2  CRF31 BC06BR 1118  2  + +  2  +  BF106BR 1122  + + +  2 2  B06BR 1123  + +  2  + +  B03BR 2010  + + +  2  +  B03BR 2013  2  + +  2 2  B04BR 2024  2  + +  2 2  B04BR 2030  + + +  2 2  B05BR 2037  +  2  +  2 2  Bdoi:10.1371/journal.pone.0025869.t002 Analysis of HIV-1 Strains in Sa˜o Paulo, BrazilPLoS ONE | 4 October 2011 | Volume 6 | Issue 10 | e25869  analysis of the sequenced fragments showed the predominance of subtypes B strains (79%). Subtypes C and BF1 were detected infour samples (6% each). Subtype F1 and a CRF31 BC variantwere isolated in one sample (3% each). ARV drug resistance-associated mutations The HIV-1 proteaseand RT regions of viruses obtained from 101patients were scrutinized for any ARV drug resistance-associatedmutations. Primary drug resistance mutations were detected in 14patients (13.7%). Of these, 12 (85.7%) patients were infected withsubtype B HIV-1. The distributions of these mutations by drug classwere as follows: five patients had resistance to protease inhibitors(PIs), three patients to non-nucleoside RT inhibitors (NNRTI), threepatients to nucleoside RT inhibitors (NRTI), one patient toPI + NNRTI, and two patients to NRTI + NNRTI (Table 3). V3 sequence analysis and viral tropism  An evaluation of the V3 loop amino acids and predictions of  viral tropism were performed for 86 of the derived NFLG andpartial sequences. Using the computer program geno2pheno [coreceptor] for phenotype prediction, 26 patient virus strains (30.2%)were predicted to be X4 or X4/DM, and the V3 sequences of 60patient virus strains (69.8%) were predicted to be R5-tropic virus. Among the MSM group evaluated for co-receptor tropism (  n =69),19(27.5%) patient virus strains were predicted to be X4 or X4/DM and three patient samples have viruses with evidence of resistance to at least one ARV drug (Table 3). The tetrapeptideGWGR, a motif frequently detected in Brazilian subtype Bsequences, in the V3 loop apex sequence was observed in 17(19.8%) patients. Of these patients, 6 (25%) had a predicted X4 orX4/DM virus. The  N   -linked glycosylation sites (amino acids 4to 6; positions 305 to 307 in HXB2 Env), essential for viralreplication and the heptapeptide DIIGDIR (amino acids 32 to 38;positions 322 to 327 in HXB2 Env) were completely conserved in64 and 19 subjects, respectively (data not shown). Discussion In the present study, 77 NFLG and 32 partial sequences fromrecently HIV-1 infected Brazilian subjects were analyzed. Thephylogenetic analysis of the sequences presented here showed thatsubtype B is largely dominant (79.8%), followed by BF1 URF. Figure 1. Phylogenetic tree constructed using a maximum-likelihood method from the NFLG sequences of 64 patient samples and37 HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes.  Annotation of samples is asfollows: symbol-green circle (subtype B), symbol-blue square (subtype C), symbol-red triangle (subclade F1), symbol-brown square-M indicatesheterosexual male, symbol-brown square-F indicates heterosexual female, and symbol-black square indicates homosexual male. The approximatelikelihood ratio test (aLRT) values of  $ 90% are indicated. The scale bar represents 0.05 nucleotide substitutions per site.doi:10.1371/journal.pone.0025869.g001Analysis of HIV-1 Strains in Sa˜o Paulo, BrazilPLoS ONE | 5 October 2011 | Volume 6 | Issue 10 | e25869
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