1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain
2 Histocompatibilidad, Centro de Transfusión de la Comunidad de Madrid, 28032 Madrid, Spain
3 Centro Sanitario Sandoval, IMSALUD, 28010 Madrid, Spain
Correspondence
Cecilio López-Galíndez
clopez{at}isciii.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LTNPs have been associated with a low viral burden (Barker et al., 1998; Cao et al., 1995
; Hogervorst et al., 1995
; Pantaleo et al., 1995
; Rinaldo et al., 1995
) and a strong cellular (Barker et al., 1998
; Cao et al., 1995
; Harrer et al., 1996
; Hogervorst et al., 1995
; Klein et al., 1995
; Pantaleo et al., 1995
; Rinaldo et al., 1995
) or humoral (Cao et al., 1995
; Carotenuto et al., 1998
; Hogervorst et al., 1995
; Keet et al., 1994
; Lifson et al., 1991
; Montefiori et al., 1996
; Pantaleo et al., 1995
; Pilgrim et al., 1997
; Zhang et al., 1997
) specific immune response. Also, lower non-specific immune system activation (Buchbinder et al., 1994
; Lifson et al., 1991
; Sheppard et al., 1993
), specific human leukocyte antigen (HLA) haplotypes (Hogan & Hammer, 2001
) and supertypes (Trachtenberg et al., 2003
), a 32 bp deletion in the CCR5 gene (Dean et al., 1996
; Michael et al., 1997
) and infection with less virulent HIV-1 strains (Cao et al., 1995
; Keet et al., 1994
; Learmont et al., 1992
) are associated with long-term survival.
HIV-1 has a replication machinery that leads to the accumulation of mutations, due to the lack of repair systems. The correlation between viral genetic divergence and time has been used to trace the origin of the HIV-1 (Korber et al., 2000) and HIV-2 (Lemey et al., 2003
) global or country-wide epidemics (Casado et al., 2000
; Lukashov & Goudsmit, 2002
; Robbins et al., 2003
), a familial transmission case (Leitner & Albert, 1999
) and the validation of an old HIV-1 sample (Zhu et al., 1998
). Recently, we used the correlation of genetic divergence with time, derived from the Spanish epidemic, for dating different HIV-1 viral subpopulations within patient quasispecies, revealing the co-existence of ancestral and modern subpopulations (Bello et al., 2004
).
In the present study, we extended the dating of viral sequences to a group of LTNPs. This analysis permitted the identification of two subsets of patients, one showing only ancestral viral sequences and the other showing predominantly modern viral sequences. We have also compared several clinical, virological, immunological and host genetic markers between the subsets.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blood samples.
Peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained as described previously (Casado et al., 2001) and the CD4+ and CD8+ cell counts were measured with mAbs by flow cytometry (Epics-XL; Beckman Coulter).
Separation, amplification and quantification of viral nucleic acids.
PBMC-associated DNA was obtained from 1x107 cells by standard methods. Viral RNA was isolated from 200500 µl plasma according to Boom et al. (1990). Nested PCR and RT-PCR were used to amplify the C2V5 region in env as described previously (Casado et al., 2001
). DNA viral load was determined in the same region, as reported previously (Rodrigo et al., 1997
). To avoid genetic bottlenecking, the first PCR included at least 20 copies of viral DNA and primers 169ECU (5'-AATGGCAGCACAGTACAATGTACAC-3' at positions 69456969; all numbering is as for the HXB2 clone) and 96ED (5'-AGACAATAATTGTCTGGCCTGTACCGT-3', positions 78627836). The second PCR included a 1 µl aliquot of the first PCR product and primers 27EU (Casado et al., 2001
) and 167ED (5'-ATGAATTCTGGGTCCCCTCCTGAGGA-3', positions 73147339). When a low proviral load did not allow the simultaneous amplification of at least 20 copies of provirus, two to ten first-round PCR products were pooled and a 5 µl aliquot was used in the second PCR to prevent the loss of sequence heterogeneity. In this study, we analysed two viral populations: the global viral population, which is the result of PCR amplification of at least 20 copies of the DNA sample, and a quasispecies analysis, which refers to the study of 20 clones of the global population.
Plasma HIV-1 RNA was quantified with an Amplicor HIV Monitor test kit, which has a detection limit of 50 copies ml1 (Roche); in patients with undetectable viral load, a value of 50 copies was used for the estimation of mean values.
To obtain the viral RNA sequence from patient plasma, the first RT-PCR included 50100 copies of viral RNA, deduced from the viral load, and primers 169ECU and 96ED. The second PCR included a 1 µl aliquot of the first RT-PCR product and primers 27EU and 167ED.
Cloning and sequencing.
To obtain the proviral DNA and viral RNA global sequences from each patient, purified nested PCR and RT-PCR products were sequenced with primer 27EU by using an ABI PRISM Dye Terminator kit in an ABI PRISM 377 sequencer (both from Perkin-Elmer). To analyse proviral quasispecies, a 2 µl aliquot of the nested PCR product was ligated into plasmid pCR2.1, cloned according to the TOPO TA cloning kit instructions (Invitrogen) and 1820 clones per sample were sequenced.
Dating viral global nucleotide sequences.
HIV-1 subtype B V3 env nucleotide sequences from 96 Spanish samples, collected between 1993 and 2001, were used to generate a Spanish consensus nucleotide sequence, which contained the most frequent nucleotide at each position (Casado et al., 2000). Also, the most recent common ancestor (MRCA) for this nucleotide sequence set (Spanish MRCA) was inferred by the DNAML program (PHYLIP version 3.1) as explained previously (Bello et al., 2004
). The genetic distance of each patient's V3 sequence from the Spanish consensus sequence was estimated by the Kimura two-parameter model (transition/transversion ratio, 2·0) as implemented in MEGA version 2.1 (Kumar et al., 2001
) with 100 bootstrapped datasets, or to the Spanish MRCA by the F-84 method with the DNADIST program (in PHYLIP version 3.1). These values were used to estimate viral dating by using the equations derived previously (Bello et al., 2004
). Dating using the Spanish consensus or the Spanish MRCA did not result in statistically different dates and, for clarity, only the results with the Spanish consensus are shown in Table 1
.
|
Serological assays.
End-point plasma titres of antibodies to the gp160 protein were determined by an ELISA kit (Genelavia Mixt detection kit; Sanofi Diagnostics Pasteur).
Plasma 2-microglubulin (
2m) concentrations were measured by using an ELISA (DRG Diagnostics). As a control, the
2m level in 32 HIV-1-negative blood donors was determined.
Characterization of HLA alleles.
Generic exon 2 and 3 DNA amplification for the HLA-A and B genes and hybridization with sequence-specific probes coupled to fluorescent beads was performed with LifeMATCH kits (Orchid Diagnostics). Higher resolution by sequence-based typing was also used when needed in order to precisely define the HLA supertypes (Sette & Sidney, 1999; Trachtenberg et al., 2003
).
Characterization of the 32-CCR5 genotype.
Analysis of the 32-CCR5 genotype was performed on PBMC DNA by PCR as described by Michael et al. (1997)
. The primers amplified a gene fragment of 225 nt for the wild-type allele and of 193 nt for the
32-CCR5 allele, which were separated in 7 % TBE polyacrylamide gels.
Statistical analysis.
Statistical analyses were carried out with the Statgraphics Plus 5.0 program (Statistical Graphics Corporation) and SPSS 11-0 (SPSS Inc.). An unpaired Student's t-test was used to compare group means, except for DNA proviral load and CD4/CD8 ratio, where a Wilcoxon non-parametric test was used, and in the analysis of the HLA alleles, where P was calculated by a 2 test and the Bonferroni correction.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The samples analysed were taken between 1998 and 2003 and seroconversion times ranged from 1985 to 1992. Of the 16 LTNPs studied, a subset of seven LTNPs were designated ancestral LTNPs, whereas the other nine patients were identified as modern LTNPs. In the ancestral LTNPs, the global DNA and RNA viral sequences (when amplified), as well as all individual sequences in the DNA viral quasispecies, had a dating that was very distant from the sampling time (915 years earlier) and close to the patient seroconversion time (±3 years) (Table 1). This viral dating indicated very slow or arrested viral divergence in the ancestral LTNPs. In these patients, the dating of the viral global sequence was confirmed in a second sample that was separated from the first by at least 1 year, giving similar results (Table 1
). In the modern subset, the global DNA and RNA viral sequences and the majority of the individual sequences in the DNA viral quasispecies analysis had a dating close to the sampling time (±2 years). In the modern LTNPs, although the DNA viral quasispecies were dominated by modern clones, some individual sequences were ancestral. The only exception to this trend were the viral quasispecies of patient 7, where only a minority of the DNA clones were modern. However, like all the other modern LTNPs, patient 7 displayed a modern global RNA viral sequence (Table 1
). The modern dating of the viral sequences in this subset indicated a continuous process of viral evolution during HIV-1 infection.
Virological and immunological characteristics of patients
To further characterize these two subsets of patients, several virological and immunological markers were analysed. Firstly, a phylogenetic analysis of the sequences from viral quasispecies was performed. This analysis showed that all sequences from each patient formed monophyletic groups, with bootstrap values of >75 % separating the sequences between patients, except in patient 64 of the modern subset, who displayed two distinct clades that clustered in different branches of the tree (see Fig. 1). Also, the viral phenotype in all patients was deduced from the V3 sequence; all of the viruses were non-syncytial (Barker et al., 1998
; Hogervorst et al., 1995
; Keet et al., 1994
).
|
|
|
|
Clinical, epidemiological and risk behaviour characteristics of the patients
To study whether clinical, epidemiological or risk behaviour history of the patients could also be related to the differences observed between the subsets, several parameters were evaluated. No statistically significant differences were found between the groups in age, gender, infection route or time from HIV-1 infection, nor in the self-reported use of alcohol or other drugs (data not shown). The presence of serological markers for other pathogens, such hepatitis B or C viruses, Mycobacterium tuberculosis or Toxoplasma gondii, did not differ between subsets. Re-exposure to HIV after infection was evaluated by different markers: syringe sharing, sexually transmitted infections or safe-sex practices. The proportion of patients that shared syringes (67 vs 17 %) and displayed sexually transmitted infections (67 vs 29 %) after HIV infection was higher in the modern subset than in the ancestral one, respectively, but only the practice of safe sex reached statistical significance. The practice of safe sex showed 100 % compliance in the ancestral subset, versus 22 % in the modern group (P=0·0032). All patients with ancestral sequences were partners of HIV-negative individuals, who were followed thoroughly in the CSS because they were included in a cohort of discordant couples. However, as mentioned previously, we only detected a double infection in one of the patients analysed (patient 64; see Fig. 1).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA quasispecies analysis revealed a significant difference in the mean viral heterogeneity between subsets. In the modern LTNPs, the mean intrapatient quasispecies heterogeneity was >4·5 %, whereas in the ancestral LTNPs, this marker was usually <1 %. The modern dating and high heterogeneity of the viral quasispecies in the modern LTNPs indicated clearly that the virus is constantly evolving and accumulating viral divergence, although there is no clinical progression. In the ancestral LTNPs, however, after so many years of HIV-1 infection, viral evolution seems to be arrested or very slow, as suggested by the ancestral dating and low heterogeneity of the viral quasispecies. These data are compatible with viral latency. In fact, these individuals often presented viral loads of <50 copies ml1 and occasional blips (Table 2). However, these blips appear to correspond to independent events, as they did not result in the accumulation of viral genetic variation. In several studies on viral evolution, it was possible to detect some LTNPs with highly homogeneous quasispecies, similar to our ancestral patients (Mani et al., 2002
; Markham et al., 1998
; Menzo et al., 1998
; Visco-Comandini et al., 2001
). Recently, Wang et al. (2003)
described the first demonstration of lack of viral evolution in one LTNP.
The segregation of the patients could have been performed according to the RNA viral load only, and would have resulted in similar groupings. However, the RNA viral load did not identify patients with ancestral or modern viral sequences directly, as there are patients in the ancestral group with equal or higher viral loads (patients 56 and 58) than modern ones (patients 4, 7, 38 and 45) (Table 2). Moreover, the ancestral viral dating gives evidence not only of a very limited replication, but also of arrested or very limited viral evolution.
The DNA quasispecies analysis revealed that, in the ancestral subset, all clones, as well as the global DNA and RNA sequences, were ancestral. In contrast, the modern subset showed predominantly modern DNA populations, but the DNA quasispecies were heterogeneous, with the presence of ancestral sequences (up to 37 % of sequences in the quasispecies, except in patient 7). As a control on the presence of ancestral sequences in non-LTNP patients, we performed the dating in 10 individuals with high viral loads. At the global DNA level, only modern sequences were obtained, but this needs to be confirmed at the quasispecies level and in a larger number of patients.
Segregation of LTNPs into the modern and ancestral subsets was also supported by immunological markers. Whereas all ancestral LTNPs displayed almost constant CD4+ cell numbers and values of the CD8+ and 2m markers that were close to those in uninfected control patients, the modern LTNP subset displayed higher mean CD8+ cell numbers, anti-gp160 antibody titres and
2m levels, indicating a superior HIV-1-specific and non-specific immune activation than in the ancestral group. Patients with immunological characteristics similar to our ancestral subset have been reported: Hogervorst et al. (1995)
described that LTNPs could be divided into two groups according to low or high p24 antibody titres. Several studies also described a subset of LTNPs with extremely low viral loads and neutralizing antibodies (Cao et al., 1995
; Carotenuto et al., 1998
; Harrer et al., 1996
; Zhang et al., 1997
). Ferbas et al. (1995)
identified three patients, among 12 LTNPs, with a low virus burden, but without evidence of activated circulating anti-HIV CD8+ cells. Finally, Lefrère et al. (1997)
identified two LTNPs, from a group of 12, with undetectable plasma viral loads and normal concentrations of IgG, IgA,
2M and neopterin. All of these data support a correlation between HIV-1 replication, HIV antigenic stimulation in vivo and the anti-HIV immune response (Ferbas et al., 1995
). It is possible that, in the ancestral LTNPs, the number of circulating virus particles was not sufficient to elicit a high specific antibody response and a non-specific immune-system activation. At the same time, the low degree of immune activation resulted in reduced HIV-1 replication, because the massive replication of HIV-1 takes place predominantly in activated CD4+ cells (Finzi & Siliciano, 1998
). In fact, within the group of 16 LTNPs, a significantly positive correlation between RNA viral load and different markers of immune activation (CD8+ cell number,
2m concentration and anti-gp160 antibody titre) was observed (data not shown).
Within the host genetic factors analysed, the only significant difference found was a higher frequency of the sB58 supertype in the ancestral group compared to the HIV-1-negative control group (53 and 11 %, respectively) (Table 4). The sB58 and sB27 supertype alleles, alone or in combination, were associated with lower viral loads (Trachtenberg et al., 2003
). However, it seems improbable that this HLA supertype could fully explain the differences between subsets, as this supertype was also found in the modern LTNPs. These results, although derived from a small number of patients, support the notion that a particular host genotype, coupled with other viral or host factors, could contribute to explaining the difference between subsets.
Activation of the immune system has shown to be important for virus replication in patients (Fauci, 1996). This marker was quantified by the
2m marker, which was statistically significantly different between groups (1·7±0·6 vs 2·7±0·8 µg ml1) and different from the control group (1·02±0·22 µg ml1) (Table 3
). However, activation was also analysed in relation to clinical and epidemiological characteristics of the patients, such as exposure to other infectious micro-organisms or re-exposure to HIV-1. No significant difference in exposure to other infectious micro-organisms was found between subsets (data not shown). However, by the analysis of behavioural markers, the ancestral group showed a lack of risk behaviour and a lower opportunity for re-exposure to HIV than the modern subset, as measured by the safe-sex behaviour marker (100 vs 22 %, respectively) or syringe sharing (17 vs 67 %, respectively). However, it is worth highlighting that superinfection or double infection was detected by phylogenetic analysis in only one individual of the cohort examined (patient 64 of the modern subset; Fig. 1
).
Although HIV-infected individuals could be described by a continuous spectrum of disease-progression rates, the segregation of LTNPs into individuals with modern and ancestral viral sequences also corresponds to patients with very different virological and immunological characteristics. Based on the very low viral burden, limited viral evolution and almost normal immunological parameters, it could be inferred that the ancestral LTNPs could correspond to authentic non-progessors or at least to very slow progressors (Balotta et al., 1997; Ferbas et al., 1995
; Harrer et al., 1996
; Lefrère et al., 1997
), whereas modern LTNP patients with a low but detectable viral burden, high viral diversity and abnormal immunological characteristics could represent the slow progressors. Therefore, ancestral viral dating in LTNPs could have prognostic value. The existence of these differences within non-progressor patients could be particularly important and must be considered when making comparative studies between LTNPs and other progressor groups. The heterogeneity of the characteristics of LTNPs could be seen even within each subset and could be the result of differences in the relative influence of virological, immunological and host factors to disease progression within each patient.
In conclusion, by dating the viral populations that are present in HIV-1 LTNP patients, we have been able to define two subsets of individuals that are also differentiated by several virological, immunological and host characteristics. Determining the role of the viral, immunological and host factors that could contribute to the differences within these patients may provide new insights into the long-term control of HIV-1 infection.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balotta, C., Bagnarelli, P., Riva, C. & 9 other authors (1997). Comparable biological and molecular determinants in HIV type 1-infected long-term nonprogressors and recently infected individuals. AIDS Res Hum Retrovir 13, 337341.[Medline]
Barker, E., Mackewicz, C. E., Reyes-Terán, G., Sato, A., Stranford, S. A., Fujimura, S. H., Christopherson, C., Chang, S.-Y. & Levy, J. A. (1998). Virological and immunological features of long-term human immunodeficiency virus-infected individuals who have remained asymptomatic compared with those who have progressed to acquired immunodeficiency syndrome. Blood 92, 31053114.
Bello, G., Casado, C., García, S., Rodríguez, C., del Romero, J. & López-Galíndez, C. (2004). Co-existence of recent and ancestral nucleotide sequences in viral quasispecies of human immunodeficiency virus type 1 patients. J Gen Virol 85, 399407.
Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E. & van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28, 495503.[Medline]
Buchbinder, S. P., Katz, M. H., Hessol, N. A., O'Malley, P. M. & Holmberg, S. D. (1994). Long-term HIV-1 infection without immunologic progression. AIDS 8, 11231128.[Medline]
Cao, Y., Qin, L., Zhang, L., Safrit, J. & Ho, D. D. (1995). Virologic and immunologic characterization of long-term survivors of human immunodeficiency virus type 1 infection. N Engl J Med 332, 201208.
Carotenuto, P., Looij, D., Keldermans, L., de Wolf, F. & Goudsmit, J. (1998). Neutralizing antibodies are positively associated with CD4+ T-cell counts and T-cell function in long-term AIDS-free infection. AIDS 12, 15911600.[CrossRef][Medline]
Casado, C., Urtasun, I., Martin-Walther, M. V., Garcia, S., Rodriguez, C., del Romero, J. & Lopez-Galindez, C. (2000). Genetic analysis of HIV-1 samples from Spain. J Acquir Immune Defic Syndr 23, 6874.[Medline]
Casado, C., García, S., Rodríguez, C., del Romero, J., Bello, G. & López-Galíndez, C. (2001). Different evolutionary patterns are found within human immunodeficiency virus type 1-infected patients. J Gen Virol 82, 24952508.
Dean, M., Carrington, M., Winkler, C. & 14 other authors (1996). Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 273, 18561862.
Fauci, A. S. (1996). Host factors and the pathogenesis of HIV-induced disease. Nature 384, 529534.[CrossRef][Medline]
Ferbas, J., Kaplan, A. H., Hausner, M. A. & 7 other authors (1995). Virus burden in long-term survivors of human immunodeficiency virus (HIV) infection is a determinant of anti-HIV CD8+ lymphocyte activity. J Infect Dis 172, 329339.[Medline]
Finzi, D. & Siliciano, R. F. (1998). Viral dynamics in HIV-1 infection. Cell 93, 665671.[Medline]
Harrer, T., Harrer, E., Kalams, S. A. & 10 other authors (1996). Strong cytotoxic T cell and weak neutralizing antibody responses in a subset of persons with stable nonprogressing HIV type 1 infection. AIDS Res Hum Retrovir 12, 585592.[Medline]
Hendriks, J. C., Medley, G. F., van Griensven, G. J., Coutinho, R. A., Heisterkamp, S. H. & van Druten, H. A. (1993). The treatment-free incubation period of AIDS in a cohort of homosexual men. AIDS 7, 231239.[Medline]
Hogan, C. M. & Hammer, S. M. (2001). Host determinants in HIV infection and disease. Part 2: genetic factors and implications for antiretroviral therapeutics. Ann Intern Med 134, 978996.
Hogervorst, E., Jurriaans, S., de Wolf, F. & 8 other authors (1995). Predictors for non- and slow progression in human immunodeficiency virus (HIV) type 1 infection: low viral RNA copy numbers in serum and maintenance of high HIV-1 p24-specific but not V3-specific antibody levels. J Infect Dis 171, 811821.[Medline]
Keet, I. P., Krol, A., Klein, M. R. & 7 other authors (1994). Characteristics of long-term asymptomatic infection with human immunodeficiency virus type 1 in men with normal and low CD4+ cell counts. J Infect Dis 169, 12361243.[Medline]
Klein, M. R., van Baalen, C. A., Holwerda, A. M. & 7 other authors (1995). Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics. J Exp Med 181, 13651372.
Korber, B., Muldoon, M., Theiler, J., Gao, F., Gupta, R., Lapedes, A., Hahn, B. H., Wolinsky, S. & Bhattacharya, T. (2000). Timing the ancestor of the HIV-1 pandemic strains. Science 288, 17891796.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetic analysis software. Bioinformatics 17, 12441245.
Learmont, J., Tindall, B., Evans, L., Cunningham, A., Cunningham, P., Wells, J., Penny, R., Kaldor, J. & Cooper, D. A. (1992). Long-term symptomless HIV-1 infection in recipients of blood products from a single donor. Lancet 340, 863867.[Medline]
Lefrère, J.-J., Morand-Joubert, L., Mariotti, M., Bludau, H., Burghoffer, B., Petit, J.-C. & Roudot-Thoraval, F. (1997). Even individuals considered as long-term nonprogressors show biological signs of progression after 10 years of human immunodeficiency virus infection. Blood 90, 11331140.
Leitner, T. & Albert, J. (1999). The molecular clock of HIV-1 unveiled through analysis of a known transmission history. Proc Natl Acad Sci U S A 96, 1075210757.
Lemey, P., Pybus, O. G., Wang, B., Saksena, N. K., Salemi, M. & Vandamme, A.-M. (2003). Tracing the origin and history of the HIV-2 epidemic. Proc Natl Acad Sci U S A 100, 65886592.
Levy, J. A. (1993). HIV pathogenesis and long-term survival. AIDS 7, 14011410.[Medline]
Lifson, A. R., Buchbinder, S. P., Sheppard, H. W., Mawle, A. C., Wilber, J. C., Stanley, M., Hart, C. E., Hessol, N. A. & Holmberg, S. D. (1991). Long-term human immunodeficiency virus infection in asymptomatic homosexual and bisexual men with normal CD4+ lymphocyte counts: immunologic and virologic characteristics. J Infect Dis 163, 959965.[Medline]
Lukashov, V. V. & Goudsmit, J. (2002). Recent evolutionary history of human immunodeficiency virus type 1 subtype B: reconstruction of epidemic onset based on sequence distances to the common ancestor. J Mol Evol 54, 680691.[CrossRef][Medline]
Mani, I., Gilbert, P., Sankalé, J.-L., Eisen, G., Mboup, S. & Kanki, P. J. (2002). Intrapatient diversity and its correlation with viral setpoint in human immunodeficiency virus type 1 CRF02_A/G-IbNG infection. J Virol 76, 1074510755.
Markham, R. B., Wang, W.-C., Weisstein, A. E. & 8 other authors (1998). Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc Natl Acad Sci U S A 95, 1256812573.
Menzo, S., Sampaolesi, R., Vicenzi, E. & 7 other authors (1998). Rare mutations in a domain crucial for V3-loop structure prevail in replicating HIV from long-term non-progressors. AIDS 12, 985997.[CrossRef][Medline]
Michael, N. L., Chang, G., Louie, L. G., Mascola, J. R., Dondero, D., Birx, D. L. & Sheppard, H. W. (1997). The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression. Nat Med 3, 338340.[Medline]
Montefiori, D. C., Pantaleo, G., Fink, L. M., Zhou, J. T., Zhou, J. Y., Bilska, M., Miralles, G. D. & Fauci, A. S. (1996). Neutralizing and infection-enhancing antibody responses to human immunodeficiency virus type 1 in long-term nonprogressors. J Infect Dis 173, 6067.[Medline]
Muñoz, A., Wang, M.-C., Bass, S., Taylor, J. M. G., Kingsley, L. A., Chmiel, J. S. & Polk, B. F. (1989). Acquired immunodeficiency syndrome (AIDS)-free time after human immunodeficiency virus type 1 (HIV-1) seroconversion in homosexual men. Am J Epidemiol 130, 530539.[Abstract]
Pantaleo, G., Menzo, S., Vaccarezza, M. & 11 other authors (1995). Studies in subjects with long-term nonprogressive human immunodeficiency virus infection. N Engl J Med 332, 209216.
Pilgrim, A. K., Pantaleo, G., Cohen, O. J., Fink, L. M., Zhou, J. Y., Zhou, J. T., Bolognesi, D. P., Fauci, A. S. & Montefiori, D. C. (1997). Neutralizing antibody responses to human immunodeficiency virus type 1 in primary infection and long-term-nonprogressive infection. J Infect Dis 176, 924932.[Medline]
Rinaldo, C., Huang, X.-L., Fan, Z. & 8 other authors (1995). High levels of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte activity and low viral load are associated with lack of disease in HIV-1-infected long-term nonprogressors. J Virol 69, 58385842.[Abstract]
Robbins, K. E., Lemey, P., Pybus, O. G., Jaffe, H. W., Youngpairoj, A. S., Brown, T. M., Salemi, M., Vandamme, A. M. & Kalish, M. L. (2003). U.S. human immunodeficiency virus type 1 epidemic: date of origin, population history, and characterization of early strains. J Virol 77, 63596366.
Rodrigo, A. G., Goracke, P. C., Rowhanian, K. & Mullins, J. I. (1997). Quantitation of target molecules from polymerase chain reaction-based limiting dilution assays. AIDS Res Hum Retrovir 13, 737742.[Medline]
Sette, A. & Sidney, J. (1999). Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50, 201212.[CrossRef][Medline]
Sheppard, H. W., Lang, W., Ascher, M. S., Vittinghoff, E. & Winkelstein, W. (1993). The characterization of non-progressors: long-term HIV-1 infection with stable CD4+ T-cell levels. AIDS 7, 11591166.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Trachtenberg, E., Korber, B., Sollars, C. & 12 other authors (2003). Advantage of rare HLA supertype in HIV disease progression. Nat Med 9, 928935.[CrossRef][Medline]
Visco-Comandini, U., Aleman, S., Yun, Z. & Sonnerborg, A. (2001). Human immunodeficiency virus type 1 variability and long-term non-progression. J Biol Regul Homeost Agents 15, 299303.[Medline]
Wang, B., Mikhail, M., Dyer, W. B., Zaunders, J. J., Kelleher, A. D. & Saksena, N. K. (2003). First demonstration of a lack of viral sequence evolution in a nonprogressor, defining replication-incompetent HIV-1 infection. Virology 312, 135150.[CrossRef][Medline]
Zhang, Y. J., Fracasso, C., Fiore, J. R., Bjorndal, A., Angarano, G., Gringeri, A. & Fenyo, E. M. (1997). Augmented serum neutralizing activity against primary human immunodeficiency virus type 1 (HIV-1) isolates in two groups of HIV-1-infected long-term nonprogressors. J Infect Dis 176, 11801187.[CrossRef][Medline]
Zhu, T., Korber, B. T., Nahmias, A. J., Hooper, E., Sharp, P. M. & Ho, D. D. (1998). An African HIV-1 sequence from 1959 and implications for the origin of the epidemic. Nature 391, 594597.[CrossRef][Medline]
Received 30 June 2004;
accepted 15 October 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |