JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3487–3491 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00704-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.
Low Frequency of CXCR4-Using Viruses in Patients at the Time of
Primary Non-Subtype-B HIV-1 Infection
1,21314Pierre Frange,† Marie-Laure Chaix,†* Ste?phanie Raymond,Julie Galimand,Christiane Deveau, 4513Laurence Meyer,Ce?cile Goujard,Christine Rouzioux,Jacques Izopet,
and the French ANRS CO 06 PRIMO Cohort Study Group
1Universite? Paris Descartes, EA 3620, AP-HP, Laboratoire de Virologie, Hoˆpital Necker-Enfants Malades, Paris,Unite? d’Immunologie, 2He?matologie et Rhumatologie Pe?diatriques, Hoˆpital Necker-Enfants Malades, AP-HP, Paris,INSERM U563, Laboratoire de Virologie, 3Hoˆpital Purpan, Toulouse,INSERM, U1018, Universite? Paris-Sud, Faculte? de Me?decine Paris-Sud, AP-HP, Hoˆpital Biceˆtre,
Service d’E?pide?miologie et de Sante? Publique, Le Kremlin-Biceˆtre,4 and Service de Me?decine Interne et 5Maladies Infectieuses, Hoˆpital Biceˆtre, AP-HP, Le Kremlin-Biceˆtre,France
Received 7 April 2010/Returned for modi，cation 27 June 2010/Accepted 24 July 2010
We used genotypic and phenotypic assays to estimate the frequency of X4/DM viruses in 131 patients infected
with non-subtype-B viruses at the time of primary HIV-1 infection (PHI). All patients were enrolled in the French PRIMO Cohort from 1996 to 2007. Most strains belonged to CRF02_AG (51.1%) and subtype A
(14.5%). Sixteen viruses (12.2%) were classi？ed as CXCR4 tropic (“X4 strains”) by the combined criteria of
amino acids 11 and 25 of the V3 loop (11/25) and net charge rules and/or the SVMgeno2phenoalgorithm: 10%6 strains by the combined genotypic rule, 7 by the SVMgeno2phenoalgorithm, and 3, clustering in subtype 10%D, by both algorithms. However, only one strain (0.8%), belonging to subtype A, was de？ned as a dual-tropic (DM) virus by the phenotypic assay. The 67 CRF02_AG strains included 2 classi？ed as X4 strains by the
combined genotypic rule (3%) and 2 others classi？ed as X4 strains by SVMgeno2pheno(3%), but none of 10%these 4 strains was an X4 or DM strain according to the phenotypic assay. These results suggest that the cellular virus reservoir was established with X4 strains in very few non-subtype-B-infected patients at the time
of PHI. Genotypic predictions can overestimate the proportion of non-subtype-B X4 viruses at PHI.
Human immunode，ciency virus type 1 (HIV-1) can be char- [CI], 12.3% to 19.5%) strains of X4/DM viruses in 390 HIV-1 acterized by the host chemokine coreceptor that it uses to subtype B-infected patients diagnosed at the time of primary enter CD4-expressing cells. HIV-1 variants usually bind to the HIV-1 infection (PHI) in France from 1996 to 2007 (18). CCR5 chemokine coreceptor early in the course of disease. One of the major challenges of determining tropism is to These are ―R5‖ viruses (3, 31, 48). Viruses that use another select the best method for identifying coreceptor usage. HIV chemokine coreceptor, CXCR4, are ―X4‖ viruses, and they coreceptor usage is most commonly determined with a recom- emerge later in HIV infection. They can account for up to 40 binant phenotype assay in clinical studies (28, 45). Bioinfor- to 50% of all viruses in heavily treated patients with advanced matic tools based on the virus genotype may also be able to disease (1, 32). The presence of X4 viruses has been associated predict coreceptor usage. They are faster, less expensive, and with accelerated disease progression and a precipitous loss of more suitable for studies of a large number of patients than are CD4 T cells (27, 29, 40). A recent Swiss study suggested that phenotypic recombinant assays. Each available genotypic test the presence of X4 strains and the X4-speci，c virus load is adequately speci，c but not very sensitive for detecting strongly predict clinical disease progression during combined X4/DM or X4 variants. An overall concordance of 71.2 to 92% antiretroviral therapy (cART), in addition to the CD4 T-cell between genotypic and phenotypic assays has been reported (8, count or viral load (44). This potential correlation between 15, 37, 41). However, most of these studies included HIV-1 virus tropism and disease progression has important clinical subtype B strains. Genotypic algorithms may not be suitable implications. The development of coreceptor CCR5 antago- for predicting the tropism of non-subtype-B HIV-1 strains nists for treating retroviruses and the lack of a virological (20). Two recent studies demonstrated that genotypic tests response by patients infected with X4 or dual/mixed (X4/DM) performed well for predicting the coreceptor usage of viruses have increased the need to determine HIV-1 tropism. CRF02_AG and subtype C strains (36, 38), but no study has Recent studies have found the frequency of X4/DM dual- examined the correlation between genotypic and phenotypic tropic strains in plasma samples from recently infected patients tests for predicting the tropism of non-subtype-B HIV-1 at the in the United States and Spain to be from 3.2% to 17.5% (14, time of PHI. The French PRIMO Cohort contained a large 15, 16). Similarly, we found 15.9% (95% con，dence interval proportion of patients infected with a non-subtype-B virus (25.5% in 2005 to 2006) (6). We have therefore estimated the frequency of X4/DM * Corresponding author. Mailing address: Laboratoire de Virologie, Hoˆpital Necker-Enfants Malades, 149 Rue de Se`vres, 75015 Paris, viruses in 131 patients infected with non-subtype-B viruses France. Phone: 33(1)184.108.40.206. Fax: 33(1)220.127.116.11. E-mail: marie at the time of PHI. All of them were enrolled in the French -email@example.com. PRIMO Cohort from 1996 to 2007. We also studied the † P.F. and M.-L.C. contributed equally to the work. concordance between genotypic and phenotypic assays for Published ahead of print on 4 August 2010.
3488 FRANGE ET AL. J. CLIN. MICROBIOL.
TABLE 1. Baseline characteristics of the 131 patients infected predicting the tropism of non-subtype-B viruses in these with non-subtype-B HIV-1 patients. aParameterValue
MATERIALS AND METHODS No. (%) of male patients........................................... 91 (69.5) Study population. The patients studied presented with PHI when enrolled in No. (%) of patients in risk group the multicenter ANRS PRIMO Cohort (22). The PRIMO study protocol was MSM......................................................................... 55 (41.9) approved by the Paris Cochin Ethics Committee, and all subjects gave their Heterosexual............................................................ 69 (52.7) written informed consent. Subjects diagnosed during or soon after PHI, whether i.v. drug user............................................................ 1 (0.8) symptomatic or not, were enrolled. Recent infection was con，rmed by (i) a Other ........................................................................ 6 (4.3) negative or indeterminate HIV enzyme-linked immunosorbent assay (ELISA) result together with a positive antigenemia or plasma HIV RNA result, (ii) a Median time since infection (days) (range) ............ 35.5 (18–169) Western blot (WB) pro，le compatible with ongoing seroconversion (incomplete 3Median CD4 cell count (cells/mm) (range)...........523 (128–1,393) WB with an absence of antibodies to pol proteins), or (iii) an initially negative test for HIV antibodies followed within 6 months by a positive HIV serology. The Median HIV RNA (logcopies/ml) (range) ......... 4.84 (2.65–8.33) 106date of infection was estimated as the date of symptom onset minus 15 days, the Median HIV DNA (logcopies/10PBMCs) 10bdate of the incomplete WB minus 1 month, or the midpoint between a negative (range).................................................................. 3.20 (1.84–4.55) and a positive ELISA. The interval between the estimated date of infection and a i.v., intravenous. enrollment could not exceed 6 months. Each patient underwent a physical ex- b Data not available for ，ve patients. amination at enrollment, and blood samples (plasma and peripheral blood mononuclear cells [PBMCs]) were collected for immunological and virological studies at the time of the enrollment in the cohort. No patient had received antiretroviral drugs at the time of inclusion. RESULTS V3 env sequences. Total DNA was extracted from frozen PBMCs with the QIAamp DNA minikit (Qiagen SA, Courtaboeuf, France). The env (340 bp) A total of 870 patients were enrolled in the French ANRS C2V3 regions were then ampli，ed by using ED3/ED12 as outer primers and PRIMO Cohort between 1996 and December 2007. Of these Env7/ED33 as inner primers (13). Each PCR mixture contained 1 g of template patients, 207 were infected with a non-subtype-B strain based DNA, 0.2 M each primer in Expand high-，delity buffer (5 ) with 75 mM on the phylogenetic analysis of the env gene. This substudy was MgCl2, 200 M each deoxynucleoside triphosphate (dNTP), and 2.5 U of Ex- pand high-，delity Taq polymerase (Roche Applied Science, Mannheim, Ger- carried out with 131 patients (Table 1) because samples were many) in a ，nal volume of 50 l. The ampli，ed products were separated by not available for the other 76 patients. The median time be- electrophoresis and puri，ed by using a QIAquick PCR puri，cation kit (Qiagen). tween infection and enrollment was 35.5 days (range, 18 to 169 Nucleotide sequences were obtained by directly sequencing the ampli，ed DNA days). Six strains (4.6%) were resistant to at least one class of using the inner primers of each PCR and Big Dye Terminator V1.1 (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Elec- antiretrovirals, and two viruses were resistant to two classes trophoresis and data collection were performed with an ABI 3130 genetic ana- (1.5%). The distribution of the viruses into subtypes/CRFs was lyzer sequencer (Applied Biosystems), and alignments were carried out by using as follows: 19 subtype A (14.5%), 4 subtype C (3.1%), 6 sub- Sequence Navigator software. type D (4.6%), 8 subtype F (6.1%), 9 subtype G (6.9%), 2 Phylogenetic analysis of the V3 region. The HIV-1 subtype was systematically CRF01_AE (1.5%), 67 CRF02_AG (51.1%), 2 CRF09_cpx determined after phylogenetic analysis of the V3 sequences. Phylogenetic rela- tionships between V3 env sequences were estimated by comparing sequences (1.5%), and 2 CRF27_cpx (1.5%) strains. Twelve strains with previously reported sequences representative of group M, including refer- (9.2%) remained undetermined, as they did not cluster with ence sequences of subtypes, sub-subtypes, and all the circulating recombinant any known subtype or CRF. We have previously sequenced the form (CRF) sequences available in the HIV database or GenBank (until CRF43) full lengths of four of them: three subtype B/C/U recombinant (http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). Sequences were aligned by using the Clustal X program (42). Pairwise evolutionary dis- viruses and one subtype A/K/U recombinant virus (19). tances were estimated by using Kimura’s two-parameter method. Phylogenetic We found 16 viruses (12.2%) classi，ed as X4/DM by at least trees were constructed by a neighbor-joining method (neighbor program imple- one of the two genotypic rules/algorithms. Of these, 10 strains mented in the Phylip package). The reliability of the tree topology was estimated (7.6%) were classi，ed as X4/DM with the SVMgeno2pheno 10%from 1,000 bootstrap replicates. TreeView Win16 was used to draw trees for illustration (34). algorithm: they belonged to subtype A (n 1), subtype D (n 3), Genotypic methods for determining virus tropism. We determined the HIV-1 CRF01_AE (n 2), CRF02_AG (n 2), and CRF27_cpx (n coreceptor usage of each sample by two genotypic methods. One method was the 1); the last one remained undetermined. We identi，ed 9 strains SVMgeno2pheno algorithm (available at http://coreceptor.bioinf.mpi-sb.mpg.de (6.9%) as being X4/DM strains by the combined genotypic rule: /cgi-bin/coreceptor.pl), used with a false-positive rate of 10%. The other method 1 subtype A strain, 4 subtype D strains, 2 CRF02_AG strains, 1 was a genotypic rule based on amino acid residues at positions 11 and 25 and the overall net charge of V3 (4, 10, 17). One of the following criteria was required for subtype G strain, and 1 undetermined strain. Three strains (2.3%) predicting X4 coreceptor usage: (i) R or K at position 11 of V3 and/or K at were classi，ed as X4/DM with both SVMgeno2phenoand the 10%position 25, (ii) R at position 25 of V3 and a net charge of 5, or (iii) a net combined genotypic rule: all of them belonged to subtype D. charge of 6 (36–38). The V3 net charge was calculated by subtracting the Only one strain (0.8%), which clustered into subtype A, was number of negatively charged amino acids (D and E) from the number of positively charged ones (K and R). de，ned as a dual-tropic virus by the phenotypic assay: it was Phenotypic method for determining virus tropism. A region spanning gp120 classi，ed as a X4/DM strain by the combined genotypic rule and the ectodomain of the gp41 env gene of plasma HIV-1 RNA was ampli，ed but as an R5 strain by SVMgeno2pheno. This strain was 10%by reverse transcription-PCR. The phenotype of HIV-1 coreceptor usage was isolated from a patient infected after heterosexual contact. determined by using a recombinant virus entry assay, as previously described The agreement between the genotypic classi，cation based (35). The sensitivity of the assay for detecting minor amounts of CXCR4-using viruses was 0.5% (35). on the combined criteria of the 11/25 and net charge rules and Nucleotide sequence accession numbers. Virus sequences were submitted to the SVMgeno2phenotool and the phenotype according to 10%GenBank with the following accession numbers AM851091, GQ409975 to the HIV subtype are summarized in Table 2. Two of the 67 GQ409981, GQ409985, GQ409988 to GQ409991, GQ409993 to GQ409994, CRF02_AG strains were classi，ed as X4 tropic by the com- GQ409997, GQ409999 to GQ410007, GQ410009 to GQ410010, GQ410013, GQ410015 to GQ410017, GQ410019, and GU988902 to GU989000. bined genotypic rule (3%), and two others were classi，ed as
VOL. 48, 2010 CXCR4-USING VIRUSES IN NON-SUBTYPE-B HIV-1 INFECTION 3489
TABLE 2. Genotypic prediction of HIV-1 tropism and observed phenotypes for predicting CXCR4 usage of CRF02_AG, subtype A, subtype
D, and other non-subtype-B strains
No. of strains of indicated predicted phenotype
Other subtype Prediction method CRF02_AG (n 67) Subtype A (n 19) Subtype D (n 6) (n 39)
R5 R5X4/X4 R5 R5 R5X4/X4 R5 R5X4/X4 R5X4/X4 Combined 11/25 and net charge rules 65 0 18 0 2 0 37 0 R5 X4 2 0 0 1 4 0 2 0
a Geno2pheno 10%65 0 17 1 3 0 35 0 R5 2 0 1 0 3 0 4 0 X4
a Value of the ，xed false-positive rate.
says can be useful for predicting the tropism of CRF02_AG X4 tropic by SVMgeno2pheno(3%), but none of these 10%four strains was identi，ed as an X4 strain by the phenotype recombinant subtype and subtype A strains in clinical practice,
assay. as these are the predominant non-B subtypes of HIV-1 in
We tried to evaluate the performance of the genotypic al- DISCUSSION gorithms for subtype D viruses, although these have been The phenotypic assay identi，ed very few (0.8%) X4/DM rarely found in the French PRIMO Cohort, as this subtype is viruses in the 131 patients infected with a non-subtype-B strain said to be mainly X4 tropic. The algorithms gave different and diagnosed at the time of PHI in France from 1996 to 2007. results, but the SVMgeno2phenotool was more speci，c. 10%This is one of the largest series of such patients examined to Further studies on more subtype D strains are needed to es- date. The genotypic rules identi，ed 12.2% of strains (16/131) timate the validity of such genotypic rules for predicting core- as being X4/DM viruses: 7 strains were classi，ed as X4/DM ceptor usage for this subtype. strains by the SVMgeno2phenoalgorithm, 6 were classi，ed 10%Although some studies found no difference in coreceptor as X4/DM strains by the combined genotypic rule, and 3 were tropism among the different virus subtypes (47), recent re- classi，ed as X4/DM strains by both algorithms. Because only ports suggested that syncytium-inducing (SI) or CXCR4- one strain was dual tropic with the phenotypic assay, we could using strains predominate in some subtypes (subtype D and not evaluate the sensitivity of the genotypic methods to predict CRF01_AE) (9, 23, 26, 43, 46), while non-SI/CCR5-using viral tropism. Further studies are needed to perform this eval- strains tend to predominate in some other subtypes (subtypes uation. A and C) (2, 26, 33, 43, 46). The fact that we ，nd no differences Previous studies have shown that the combined genotypic between subtypes/CRFs could be due to the small number of algorithm is better than the 11/25 and net charge rules used strains of each type (except CRF02_AG). However, 20 to 45% separately to predict HIV-1 coreceptor usage in subtype B of the strains that were classi，ed as X4-tropic viruses by the viruses (11, 37). A recent report found that genotypic tools successive genotypic algorithms belonged to subtype D, and were not very sensitive for detecting X4 variants in non-B the two CRF01_AE viruses were de，ned as X4 tropic with the subtypes. However, no details of their performance for a par- SVMgeno2phenoalgorithm. This could be due to the poor 10%ticular subtype were given (20). In another study, Raymond et sensitivity of the phenotypic assay for detecting minor CXCR4- al. found that the SVMgeno2phenotool lacked sensitivity 10%using virus variants. However, our phenotypic assay detects for predicting the CXCR4 usage of CRF02_AG strains, minor CXCR4-using variants when they account for as little as whereas the combined 11/25 and net charge rule criteria were 0.5% of the virus population, which is substantially better than equally good at predicting the CXCR4 usage of both the analytical sensitivity of direct sequencing ( 10 to 20%). In CRF02_AG and subtype B strains (36, 37). We could not addition, our study was performed at the time of PHI, when evaluate the sensitivity of these genotypic tools because none the dominant homogenous population is usually expanding of our patients was infected with an X4-tropic CRF02_AG (12, 21). These discordant results could also be due to the poor virus. However, we con，rmed their previously reported high speci，city of the genotypic algorithms for non-subtype-B speci，city for this subtype (97%; 95% CI, 89.6% to 99.6%). strains that include few X4 viruses. The combined 11/25 and net charge rule criteria seemed to We ，nd a smaller proportion of X4/DM strains in patients be more sensitive for detecting CXCR4 usage in the 19 subtype with an acute infection than the 3.2 to 17.2% reported in A strains than was the SVMgeno2phenoalgorithm, but 10%previous studies (14, 15, 16, 18). However, those earlier studies their speci，cities were similar ( 94%). included very few non-subtype-B strains: there were no non- As the phenotypic test is complex to implement in a clinical subtype-B viruses in the French and American reports and 4 setting, our results suggest that using the combined 11/25 and
(6.5%) and 10 (5%) viruses in the Spanish studies, which found net charge rule criteria or using the SVMgeno2phenoal- 10%no X4/DM non-subtype-B viruses. This rarity of X4/DM vi- gorithm can provide reliable information on HIV-1 tropism
ruses is in line with what was reported previously for large when testing CRF02_AG or subtype A strains. Genotypic as-
3490 FRANGE ET AL. J. CLIN. MICROBIOL.
cohorts of antiretroviral-naïve chronically infected individuals, derestimated the frequency of CXCR4 use in non-subtype-B
viruses. in which the proportion of non-subtype-B-infected patients
In conclusion, the frequency (0.8%) of X4/DM viruses in was very low, if it was reported (5, 25, 32).
patients infected with non-subtype-B strains of HIV-1 is very We performed our study on cell-associated viruses using
low at the time of PHI. The use of genotypic methods at this HIV DNA extracted from PBMCs, which is representative of
time could overestimate the proportion of non-subtype-B X4- the virus in the cellular reservoir. In contrast, most studies have
tropic strains. carried out tropism tests on HIV RNA extracted from plasma. A recent report by Raymond et al. described the good agree- ACKNOWLEDGMENTS ment between HIV-1 tropisms in PBMCs and plasma at the
stage of PHI (39). Our results suggest that the cellular virus This work was supported by the ANRS (Agence Nationale de Re- reservoir is established with X4/DM strains in a very small scherche sur le SIDA et les He?patites). We are indebted to the patients enrolled in the cohort. Without their proportion of patients infected with non-subtype-B viruses at participation, none of these studies would have been possible. The the time of PHI. Further studies are needed to evaluate the English text was edited by Owen Parkes. The full list of investigators in virus tropism in the tissues of patients during primary HIV-1 the French Primo Study group can be found at http://u822.kb.inserm infection, especially in cases where a patient is infected with a .fr/COHAD/participantsPRIMO.ht. X4/DM strain, as Mefford et al. recently reported that bioin- REFERENCES formatics prediction programs underestimate the frequency of 1. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptors CXCR4 usage by R5X4 strains in the brain and other tissues as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. (30). Immunol. 17:657–700. 2. Bjorndal, A., A. Sonnerborg, C. Tscherning, J. Albert, and E. M. Fenyo. X4/DM strains were found in fewer PRIMO Cohort patients 1999. Phenotypic characteristics of human immunode，ciency virus type 1 infected with non-subtype B HIV than in patients infected with subtype C isolates of Ethiopian AIDS patients. AIDS Res. Hum. Retrovi- ruses 15:647–653. subtype B, where 14.3% of X4/DM-tropic viruses were identi- 3. Bomsel, M., and V. David. 2002. Mucosal gatekeepers: selecting HIV viruses ，ed in PBMC samples using the SVMgeno2phenoalgo- 10%for early infection. Nat. Med. 8:114–116. rithm (18). These results could be explained by differences in 4. Briggs, D. R., D. L. Tuttle, J. W. Sleasman, and M. M. Goodenow. 2000. Envelope V3 amino acid sequence predicts HIV-1 phenotype (co-receptor the demographics and behaviors of the two groups of PRIMO usage and tropism for macrophages). AIDS 14:2937–2939. Cohort patients. The patients infected with subtype B strains 5. Brumme, Z. L., J. Goodrich, H. B. Mayer, C. J. Brumme, B. M. Henrick, B. Wynhoven, J. J. Asselin, P. K. Cheung, R. S. Hogg, J. S. Montaner, and P. R. were mainly native French men who had had sex with men Harrigan. 2005. Molecular and clinical epidemiology of CXCR4-using (MSM). Clusters could be frequently isolated from these men HIV-1 in a large population of antiretroviral-naive individuals. J. Infect. Dis. in phylogenetic studies. Many of the non-subtype-B-infected 192:466–474. 6. Chaix, M. L., D. Descamps, M. Wirden, L. Bocket, C. Delaugerre, C. Tama- patients were heterosexual men and women originally from let, V. Schneider, J. Izopet, B. Masquelier, C. Rouzioux, L. Meyer, and D. sub-Saharan countries. We postulate that the subtype B pa- Costagliola. 2009. Stable frequency of HIV-1 transmitted drug resistance in patients at the time of primary infection over 1996-2006 in France. AIDS tients had been infected by chronically infected, intensively 23:717–724. treated, patients whose strains were more frequently X4/DM 7. Chesebro, B., K. Wehrly, J. Nishio, and S. Perryman. 1996. Mapping of tropic. independent V3 envelope determinants of human immunode，ciency virus type 1 macrophage tropism and syncytium formation in lymphocytes. J. Vi- The difference between the two populations is also sup- rol. 70:9055–9059. ported by the fact that more subtype B group patients were 8. Chueca, N., C. Garrido, M. Alvarez, E. Poveda, J. de Dios Luna, N. Zahon- ero, J. Hernandez-Quero, V. Soriano, C. Maroto, C. de Mendoza, and F. infected with a resistant strain than were non-subtype-B pa- Garcia. 2009. Improvement in the determination of HIV-1 tropism using the tients during the ，rst decade of the PRIMO Cohort (data not V3 gene sequence and a combination of bioinformatic tools. J. Med. Virol. shown). This study, which includes the majority of non-sub- 81:763–767. 9. Church, J. D., W. Huang, A. Mwatha, J. Toma, E. Stawiski, D. Donnell, L. A. type-B-infected patients enrolled since 1996, ，nds that fewer Guay, F. Mmiro, P. Musoke, J. B. Jackson, N. Parkin, and S. H. Eshleman. non-subtype-B-infected patients were infected with resistant 2008. HIV-1 tropism and survival in vertically infected Ugandan infants. J. Infect. Dis. 197:1382–1388. viruses (4.6%) than the subtype B-infected patients (15.9%) 10. De Jong, J. J., A. De Ronde, W. Keulen, M. Tersmette, and J. Goudsmit. tested in our previous study (18), which included recently in- 1992. Minimal requirements for the human immunode，ciency virus type 1 fected patients from the PRIMO Cohort. V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution. J. Virol. 66:6777–6780. There could be another explanation for the differences in 11. Delobel, P., M. T. Nugeyre, M. Cazabat, C. Pasquier, B. Marchou, P. Mas- the coreceptor usages of the subtype B and non-subtype-B sip, F. Barre-Sinoussi, N. Israel, and J. Izopet. 2007. Population-based strains isolated from the PRIMO Cohort patients. Several sequencing of the V3 region of env for predicting the coreceptor usage of human immunode，ciency virus type 1 quasispecies. J. Clin. Microbiol. 45: studies have shown that while the V3 region is a major deter- 1572–1580. minant of coreceptor tropism, other regions outside the core- 12. Delwart, E., M. Magierowska, M. Royz, B. Foley, L. Peddada, R. Smith, C. Heldebrant, A. Conrad, and M. Busch. 2002. Homogeneous quasispecies in ceptor binding site and outside the gp120 surface subunit (in 16 out of 17 individuals during very early HIV-1 primary infection. AIDS the gp41 transmembrane subunit) can contribute to coreceptor 16:189–195. usage (7, 23, 24). These regions, which have yet to be fully 13. Delwart, E. L., E. G. Shpaer, J. Louwagie, F. E. McCutchan, M. Grez, H. Rubsamen-Waigmann, and J. I. Mullins. 1993. Genetic relationships deter- characterized, differ between HIV-1 subtypes and in(uence mined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes. the susceptibility of certain virus subtypes to use predomi- Science 262:1257–1261. 14. de Mendoza, C., C. Rodriguez, F. Garcia, J. M. Eiros, L. Ruiz, E. Caballero, nantly a CCR5 or CXCR4 coreceptor. A. Aguilera, P. Leiva, J. Colomina, F. Gutierrez, J. del Romero, J. Aguero, Finally, the lower prevalence of X4-tropic non-subtype-B and V. Soriano. 2007. Prevalence of X4 tropic viruses in patients recently strains in this report than in our previous study on subtype infected with HIV-1 and lack of association with transmission of drug resis- tance. J. Antimicrob. Chemother. 59:698–704. B-infected patients from the PRIMO Cohort could be due to 15. de Mendoza, C., K. Van Baelen, E. Poveda, E. Rondelez, N. Zahonero, L. the smaller number of subjects studied. This could have un- Stuyver, C. Garrido, J. Villacian, and V. Soriano. 2008. Performance of a
VOL. 48, 2010 CXCR4-USING VIRUSES IN NON-SUBTYPE-B HIV-1 INFECTION 3491
population-based HIV-1 tropism phenotypic assay and correlation with V3 CXCR4 usage by R5X4 HIV type 1 in brain and other tissues. AIDS Res. genotypic prediction tools in recent HIV-1 seroconverters. J. Acquir. Im- Hum. Retroviruses 24:1215–1220. mune De，c. Syndr. 48:241–244. 31. Moore, J. P., S. G. Kitchen, P. Pugach, and J. A. Zack. 2004. The CCR5 and 16. Eshleman, S. H., M. Husnik, S. Hudelson, D. Donnell, Y. Huang, W. Huang, CXCR4 coreceptors—central to understanding the transmission and patho- S. Hart, B. Jackson, T. Coates, M. Chesney, and B. Koblin. 2007. Antiret- genesis of human immunode，ciency virus type 1 infection. AIDS Res. Hum. roviral drug resistance, HIV-1 tropism, and HIV-1 subtype among men who Retroviruses 20:111–126. have sex with men with recent HIV-1 infection. AIDS. 21:1165–1174. 32. Moyle, G. J., A. Wild？re, S. Mandalia, H. Mayer, J. Goodrich, J. Whitcomb, 17. Fouchier, R. A., M. Groenink, N. A. Kootstra, M. Tersmette, H. G. Huisman, and B. G. Gazzard. 2005. Epidemiology and predictive factors for chemokine F. Miedema, and H. Schuitemaker. 1992. Phenotype-associated sequence receptor use in HIV-1 infection. J. Infect. Dis. 191:866–872. variation in the third variable domain of the human immunode，ciency virus 33. Ndung’u, T., E. Sepako, M. F. McLane, F. Chand, K. Bedi, S. Gaseitsiwe, F. Doualla-Bell, T. Peter, I. Thior, S. M. Moyo, P. B. Gilbert, V. A. Novitsky, type 1 gp120 molecule. J. Virol. 66:3183–3187. and M. Essex. 2006. HIV-1 subtype C in vitro growth and coreceptor utili- 18. Frange, P., J. Galimand, C. Goujard, C. Deveau, J. Ghosn, C. Rouzioux, L. zation. Virology 347:247–260. Meyer, and M. L. Chaix. 2009. High frequency of X4/DM-tropic viruses in PBMC samples from patients with primary HIV-1 subtype-B infection in 34. Page, R. D. 1996. TreeView: an application to display phylogenetic trees on 1996-2007: the French ANRS CO06 PRIMO Cohort Study. J. Antimicrob. personal computers. Comput. Appl. Biosci. 12:357–358. 35. Raymond, S., P. Delobel, M. Mavigner, M. Cazabat, C. Souyris, S. Encinas, Chemother. 64:135–141. 19. Frange, P., J. Galimand, N. Vidal, C. Goujard, C. Deveau, F. Souala, M. P. Bruel, K. Sandres-Saune, B. Marchou, P. Massip, and J. Izopet. 2009. Peeters, L. Meyer, C. Rouzioux, and M. L. Chaix. 2008. New and old com- Development and performance of a new recombinant virus phenotypic entry assay to determine HIV-1 coreceptor usage. J. Clin. Virol. 47:126–130. plex recombinant HIV-1 strains among patients with primary infection in 1996-2006 in France: the French ANRS CO06 Primo Cohort Study. Retro- 36. Raymond, S., P. Delobel, M. Mavigner, M. Cazabat, C. Souyris, S. Encinas, virology 5:69. K. Sandres-Saune, C. Pasquier, B. Marchou, P. Massip, and J. Izopet. 2009. Genotypic prediction of human immunode，ciency virus type 1 CRF02-AG 20. Garrido, C., V. Roulet, N. Chueca, E. Poveda, A. Aguilera, K. Skrabal, N. Zahonero, S. Carlos, F. Garcia, J. L. Faudon, V. Soriano, and C. de Men- tropism. J. Clin. Microbiol. 47:2292–2294. doza. 2008. Evaluation of eight different bioinformatics tools to predict viral 37. Raymond, S., P. Delobel, M. Mavigner, M. Cazabat, C. Souyris, K. Sandres- tropism in different human immunode，ciency virus type 1 subtypes. J. Clin. Saune, L. Cuzin, B. Marchou, P. Massip, and J. Izopet. 2008. Correlation Microbiol. 46:887–891. between genotypic predictions based on V3 sequences and phenotypic de- 21. Ghosn, J., I. Pellegrin, C. Goujard, C. Deveau, J. P. Viard, J. Galimand, M. termination of HIV-1 tropism. AIDS 22:F11–F16. Harzic, C. Tamalet, L. Meyer, C. Rouzioux, and M. L. Chaix. 2006. HIV-1 38. Raymond, S., P. Delobel, M. Mavigner, L. Ferradini, M. Cazabat, C. Souyris, resistant strains acquired at the time of primary infection massively fuel the K. Sandres-Saune, C. Pasquier, B. Marchou, P. Massip, and J. Izopet. 2010. cellular reservoir and persist for lengthy periods of time. AIDS 20:159–170. Prediction of HIV type 1 subtype C tropism by genotypic algorithms built 22. Goujard, C., M. Bonarek, L. Meyer, F. Bonnet, M. L. Chaix, C. Deveau, M. from subtype B viruses. J. Acquir. Immune De，c. Syndr. 53:167–175. Sinet, J. Galimand, J. F. Delfraissy, A. Venet, C. Rouzioux, and P. Morlat. 39. Raymond, S., P. Delobel, M. Mavigner, M. Cazabat, S. Encinas, P. Bruel, K. 2006. CD4 cell count and HIV DNA level are independent predictors of Saune, B. Marchou, P. Massip, and J. Izopet. 2010. Phenotypic character- disease progression after primary HIV type 1 infection in untreated patients. ization of HIV-1 co-receptor use in primary infection in both plasma and Clin. Infect. Dis. 42:709–715. PBMC samples, abstr. 470. Abstr. 17th Conf. Retroviruses Opportun. Infect., 23. Huang, W., S. H. Eshleman, J. Toma, S. Fransen, E. Stawiski, E. E. Paxinos, San Francisco, CA. J. M. Whitcomb, A. M. Young, D. Donnell, F. Mmiro, P. Musoke, L. A. Guay, 40. Shepherd, J. C., L. P. Jacobson, W. Qiao, B. D. Jamieson, J. P. Phair, P. J. B. Jackson, N. T. Parkin, and C. J. Petropoulos. 2007. Coreceptor tropism Piazza, T. C. Quinn, and J. B. Margolick. 2008. Emergence and persistence in human immunode，ciency virus type 1 subtype D: high prevalence of of CXCR4-tropic HIV-1 in a population of men from the multicenter AIDS CXCR4 tropism and heterogeneous composition of viral populations. J. Vi- cohort study. J. Infect. Dis. 198:1104–1112. rol. 81:7885–7893. 41. Skrabal, K., A. J. Low, W. Dong, T. Sing, P. K. Cheung, F. Mammano, and 24. Huang, W., J. Toma, S. Fransen, E. Stawiski, J. D. Reeves, J. M. Whitcomb, P. R. Harrigan. 2007. Determining human immunode，ciency virus corecep- N. Parkin, and C. J. Petropoulos. 2008. Coreceptor tropism can be in(u- tor use in a clinical setting: degree of correlation between two phenotypic enced by amino acid substitutions in the gp41 transmembrane subunit of assays and a bioinformatic model. J. Clin. Microbiol. 45:279–284. human immunode，ciency virus type 1 envelope protein. J. Virol. 82:5584– 42. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. 5593. Higgins. 1997. The CLUSTAL_X windows interface: (exible strategies for 25. Hunt, P. W., P. R. Harrigan, W. Huang, M. Bates, D. W. Williamson, J. M. multiple sequence alignment aided by quality analysis tools. Nucleic Acids McCune, R. W. Price, S. S. Spudich, H. Lampiris, R. Hoh, T. Leigler, J. N. Res. 25:4876–4882. Martin, and S. G. Deeks. 2006. Prevalence of CXCR4 tropism among anti- 43. Tscherning, C., A. Alaeus, R. Fredriksson, A. Bjorndal, H. Deng, D. R. retroviral-treated HIV-1-infected patients with detectable viremia. J. Infect. Littman, E. M. Fenyo, and J. Albert. 1998. Differences in chemokine core- Dis. 194:926–930. ceptor usage between genetic subtypes of HIV-1. Virology 241:181–188. 26. Kaleebu, P., I. L. Nankya, D. L. Yirrell, L. A. Shafer, J. Kyosiimire- 44. Weiser, B., S. Philpott, T. Klimkait, H. Burger, C. Kitchen, P. Burgisser, M. Lugemwa, D. B. Lule, D. Morgan, S. Beddows, J. Weber, and J. A. Whit- Gorgievski, L. Perrin, J. C. Piffaretti, and B. Ledergerber. 2008. HIV-1 worth. 2007. Relation between chemokine receptor use, disease stage, and coreceptor usage and CXCR4-speci，c viral load predict clinical disease HIV-1 subtypes A and D: results from a rural Ugandan cohort. J. Acquir. progression during combination antiretroviral therapy. AIDS 22:469–479. Immune De，c. Syndr. 45:28–33. 45. Whitcomb, J. M., W. Huang, S. Fransen, K. Limoli, J. Toma, T. Wrin, C. 27. Koot, M., I. P. Keet, A. H. Vos, R. E. de Goede, M. T. Roos, R. A. Coutinho, Chappey, L. D. Kiss, E. E. Paxinos, and C. J. Petropoulos. 2007. Develop- F. Miedema, P. T. Schellekens, and M. Tersmette. 1993. Prognostic value of ment and characterization of a novel single-cycle recombinant-virus assay to HIV-1 syncytium-inducing phenotype for rate of CD4 cell depletion and determine human immunode，ciency virus type 1 coreceptor tropism. Anti- progression to AIDS. Ann. Intern. Med. 118:681–688. microb. Agents Chemother. 51:566–575. 28. Low, A. J., W. Dong, D. Chan, T. Sing, R. Swanstrom, M. Jensen, S. Pillai, 46. Zhang, C., S. Xu, J. Wei, and H. Guo. 2009. Predicted co-receptor tropism B. Good, and P. R. Harrigan. 2007. Current V3 genotyping algorithms are and sequence characteristics of China HIV-1 V3 loops: implications for the inadequate for predicting X4 co-receptor usage in clinical isolates. AIDS future usage of CCR5 antagonists and AIDS vaccine development. Int. 21:F17–F24. J. Infect. Dis. 13:e212–e216. 29. Maas, J. J., S. J. Gange, H. Schuitemaker, R. A. Coutinho, R. van Leeuwen, 47. Zhang, L., C. D. Carruthers, T. He, Y. Huang, Y. Cao, G. Wang, B. Hahn, and J. B. Margolick. 2000. Strong association between failure of T cell and D. D. Ho. 1997. HIV type 1 subtypes, coreceptor usage, and CCR5 homeostasis and the syncytium-inducing phenotype among HIV-1-infected polymorphism. AIDS Res. Hum. Retroviruses 13:1357–1366. men in the Amsterdam Cohort Study. AIDS 14:1155–1161. 48. Zhu, T., H. Mo, N. Wang, D. S. Nam, Y. Cao, R. A. Koup, and D. D. Ho. 1993. 30. Mefford, M. E., P. R. Gorry, K. Kunstman, S. M. Wolinsky, and D. Gabuzda. Genotypic and phenotypic characterization of HIV-1 patients with primary 2008. Bioinformatic prediction programs underestimate the frequency of infection. Science 261:1179–1181.