HIV-1 transmission between MSM and heterosexuals, and increasing proportions of circulating recombinant forms in the Nordic Countries.

Increased knowledge about HIV-1 transmission dynamics in different transmission groups and geographical regions is fundamental for assessing and designing prevention efforts against HIV-1 spread. Since the first reported cases of HIV infection during the early 1980s, the HIV-1 epidemic in the Nordic countries has been dominated by HIV-1 subtype B and MSM transmission. HIV-1 pol sequences and clinical data of 51 per cent of all newly diagnosed HIV-1 infections in Sweden, Denmark, and Finland in the period 2000-2012 (N = 3,802) were analysed together with a large reference sequence dataset (N = 4,537) by trend analysis and phylogenetics. Analysis of the eight dominating subtypes and CRFs in the Nordic countries (A, B, C, D, G, CRF01_AE, CRF02_AG, and CRF06_cpx) showed that the subtype B proportion decreased while the CRF proportion increased over the study period. A majority (57 per cent) of the Nordic sequences formed transmission clusters, with evidence of mixing both geographically and between transmission groups. Detailed analyses showed multiple occasions of transmissions from MSM to heterosexuals and that active transmission clusters more often involved single than multiple Nordic countries. The strongest geographical link was between Denmark and Sweden. Finally, Denmark had a larger proportion of heterosexual domestic spread of HIV-1 subtype B (75 per cent) compared with Sweden (49 per cent) and Finland (57 per cent). We describe different HIV-1 transmission patterns between countries and transmission groups in a large geographical region. Our results may have implications for public health interventions in targeting HIV-1 transmission networks and identifying where to introduce such interventions.

Molecular characterization of HCV in a Swedish county over 8 years (2002-2009) reveals distinct transmission patterns.

BACKGROUND: Hepatitis C virus (HCV) is a major public health concern and data on its molecular epidemiology in Sweden is scarce. We carried out an 8-year population-based study of newly diagnosed HCV cases in one of Sweden's centrally situated counties, Södermanland (D-county). The aim was to characterize the HCV strains circulating, analyze their genetic relatedness to detect networks, and in combination with demographic data learn more about transmission. METHODS: Molecular analyses of serum samples from 91% (N=557) of all newly notified cases in D-county, 2002-2009, were performed. Phylogenetic analysis (NS5B gene, 300 bp) was linked to demographic data from the national surveillance database, SmiNet, to characterize D-county transmission clusters. The linear-by-linear association test (LBL) was used to analyze trends over time. RESULTS: The most prevalent subtypes were 1a (38%) and 3a (34%). Subtype 1a was most prevalent among cases transmitted via sexual contact, via contaminated blood, or blood products, while subtype 3a was most prevalent among people who inject drugs (PWIDs). Phylogenetic analysis revealed that the subtype 3a sequences formed more and larger transmission clusters (50% of the sequences clustered), while the 1a sequences formed smaller clusters (19% of the sequences clustered), possibly suggesting different epidemics. CONCLUSION: We found different transmission patterns in D-county which may, from a public health perspective, have implications for how to control virus infections by targeted interventions.

Challenges with using primer IDs to improve accuracy of next generation sequencing.

Next generation sequencing technologies, like ultra-deep pyrosequencing (UDPS), allows detailed investigation of complex populations, like RNA viruses, but its utility is limited by errors introduced during sample preparation and sequencing. By tagging each individual cDNA molecule with barcodes, referred to as Primer IDs, before PCR and sequencing these errors could theoretically be removed. Here we evaluated the Primer ID methodology on 257,846 UDPS reads generated from a HIV-1 SG3Δenv plasmid clone and plasma samples from three HIV-infected patients. The Primer ID consisted of 11 randomized nucleotides, 4,194,304 combinations, in the primer for cDNA synthesis that introduced a unique sequence tag into each cDNA molecule. Consensus template sequences were constructed for reads with Primer IDs that were observed three or more times. Despite high numbers of input template molecules, the number of consensus template sequences was low. With 10,000 input molecules for the clone as few as 97 consensus template sequences were obtained due to highly skewed frequency of resampling. Furthermore, the number of sequenced templates was overestimated due to PCR errors in the Primer IDs. Finally, some consensus template sequences were erroneous due to hotspots for UDPS errors. The Primer ID methodology has the potential to provide highly accurate deep sequencing. However, it is important to be aware that there are remaining challenges with the methodology. In particular it is important to find ways to obtain a more even frequency of resampling of template molecules as well as to identify and remove artefactual consensus template sequences that have been generated by PCR errors in the Primer IDs.

PCR-induced transitions are the major source of error in cleaned ultra-deep pyrosequencing data.

BACKGROUND: Ultra-deep pyrosequencing (UDPS) is used to identify rare sequence variants. The sequence depth is influenced by several factors including the error frequency of PCR and UDPS. This study investigated the characteristics and source of errors in raw and cleaned UDPS data. RESULTS: UDPS of a 167-nucleotide fragment of the HIV-1 SG3Δenv plasmid was performed on the Roche/454 platform. The plasmid was diluted to one copy, PCR amplified and subjected to bidirectional UDPS on three occasions. The dataset consisted of 47,693 UDPS reads. Raw UDPS data had an average error frequency of 0.30% per nucleotide site. Most errors were insertions and deletions in homopolymeric regions. We used a cleaning strategy that removed almost all indel errors, but had little effect on substitution errors, which reduced the error frequency to 0.056% per nucleotide. In cleaned data the error frequency was similar in homopolymeric and non-homopolymeric regions, but varied considerably across sites. These site-specific error frequencies were moderately, but still significantly, correlated between runs (r=0.15-0.65) and between forward and reverse sequencing directions within runs (r=0.33-0.65). Furthermore, transition errors were 48-times more common than transversion errors (0.052% vs. 0.001%; p<0.0001). Collectively the results indicate that a considerable proportion of the sequencing errors that remained after data cleaning were generated during the PCR that preceded UDPS. CONCLUSIONS: A majority of the sequencing errors that remained after data cleaning were introduced by PCR prior to sequencing, which means that they will be independent of platform used for next-generation sequencing. The transition vs. transversion error bias in cleaned UDPS data will influence the detection limits of rare mutations and sequence variants.

Longitudinal ultradeep characterization of HIV type 1 R5 and X4 subpopulations in patients followed from primary infection to coreceptor switch.

In early infection HIV-1 generally uses the CCR5 coreceptor. During disease progression the coreceptor use switches to include CXCR4 in approximately 70% of infected individuals. The primary determinant for coreceptor use is located in the V3 loop of the viral envelope. Here, ultradeep pyrosequencing (UDPS) of the V3 loop was used to investigate if CXCR4-using (X4) virus may be present as a minority population during primary HIV infection (PHI). Three patients with HIV populations that switched coreceptor use, as determined by the MT-2 cell culture assay, were investigated. Longitudinally collected plasma samples (four to nine samples per patient) obtained from PHI until after coreceptor switch were analyzed by UDPS of the V3 loop. From each sample between 279 and 32,094 reads were generated based on template molecule availability. UDPS analysis showed that the X4 virus that emerged after switch was not present during PHI or prior to overt phenotypic switch. In addition, the phylogenetic analyses indicated that the X4 populations originated from R5 variants that had evolved after the previous R5-only sample was obtained. Finally, one to three major variants were found during PHI, supporting the idea that infection is established with one or just a few viral particles.

High intrapatient HIV-1 evolutionary rate is associated with CCR5-to-CXCR4 coreceptor switch.

In approximately 70% of individuals infected with HIV-1 subtype B, the virus switches coreceptor use from exclusively CCR5 use (R5 virus) to either inclusion of or exclusively CXCR4 use (X4 virus) during infection. This switch is associated with an accelerated loss of CD4(+) T-cells and a faster progression to AIDS. Despite intensive research, the mechanisms responsible for coreceptor switch remains elusive. In the present study, we investigated associations between viral evolutionary rate and selection pressure versus viral coreceptor use and rate of disease progression in eight patients with longitudinally sampled HIV-1 env V1-V3 sequences. By employing a Bayesian hierarchical phylogenetic model, we found that the HIV-1 evolutionary rate was more strongly associated with coreceptor switch than with rate of disease progression in terms of CD4(+)T-cell decline. Phylogenetic analyses showed that X4 variants evolved from R5 populations. In addition, coreceptor switch was associated with higher evolutionary rates on both the synonymous and non-synonymous substitution level, but not with dN/dS ratio rates. Our findings suggest that X4 viruses evolved from pre-existing R5 viral populations and that the evolution of coreceptor switch is governed by high replication rates rather than by selective pressure. Furthermore, the association of viral evolutionary rate was more strongly associated with coreceptor switch than disease progression. This adds to the understanding of the complex virus-host interplay that influences the evolutionary dynamics of HIV-1 coreceptor use.

A single early introduction of HIV-1 subtype B into Central America accounts for most current cases.

Human immunodeficiency virus type 1 (HIV-1) variants show considerable geographical separation across the world, but there is limited information from Central America. We provide the first detailed investigation of the genetic diversity and molecular epidemiology of HIV-1 in six Central American countries. Phylogenetic analysis was performed on 625 HIV-1 pol gene sequences collected between 2002 and 2010 in Honduras, El Salvador, Nicaragua, Costa Rica, Panama, and Belize. Published sequences from neighboring countries (n = 57) and the rest of the world (n = 740) were included as controls. Maximum likelihood methods were used to explore phylogenetic relationships. Bayesian coalescence-based methods were used to time HIV-1 introductions. Nearly all (98.9%) Central American sequences were of subtype B. Phylogenetic analysis revealed that 437 (70%) sequences clustered within five significantly supported monophyletic clades formed essentially by Central American sequences. One clade contained 386 (62%) sequences from all six countries; the other four clades were smaller and more country specific, suggesting discrete subepidemics. The existence of one large well-supported Central American clade provides evidence that a single introduction of HIV-1 subtype B in Central America accounts for most current cases. An introduction during the early phase of the HIV-1 pandemic may explain its epidemiological success. Moreover, the smaller clades suggest a subsequent regional spread related to specific transmission networks within each country.

Therapy failure resulting from superinfection by a drug-resistant HIV variant.

BACKGROUND: HIV-1-infected patients can be superinfected with additional HIV-1 variants. Therapy failure can be the consequence of an infection with a resistant strain. METHODS: A patient was diagnosed with a recent HIV-1 infection in April 2005 and subsequently clinically monitored. HIV-1 evolution was studied by population sequencing of the first 984 bases of the pol gene as well as 454 ultra-deep pyrosequencing (UDPS) of parts of the pol and env genes. RESULTS: The patient was diagnosed with a wild-type HIV-1 strain, but experienced rapid virological failure after initiating a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based treatment regimen 3 years later. Population sequencing and UDPS revealed the presence of a second HIV-1 strain with a Y188L NNRTI resistance mutation in a sample obtained shortly prior to initiation of therapy. Phylogenetic analyses showed that the two HIV-1 strains were genetically distinct, providing evidence for superinfection. CONCLUSIONS: The virological treatment failure in this patient was probably due to the superinfection with an NNRTI-resistant HIV-1 variant. Superinfection with drug-resistant strains can undermine HIV-1 treatment regimens selected on the basis of resistance testing at diagnosis. Patients, especially in high-risk groups, as well as their clinicians, should be aware of the risks and dangers of superinfections.

Low prevalence of transmitted drug resistance in patients newly diagnosed with HIV-1 infection in Sweden 2003-2010.

Transmitted drug resistance (TDR) is a clinical and epidemiological problem because it may contribute to failure of antiretroviral treatment. The prevalence of TDR varies geographically, and its prevalence in Sweden during the last decade has not been reported. Plasma samples from 1,463 patients newly diagnosed with HIV-1 infection between 2003 and 2010, representing 44% of all patients diagnosed in Sweden during this period, were analyzed using the WHO 2009 list of mutations for surveillance of TDR. Maximum likelihood phylogenetic analyses were used to determine genetic subtype and to investigate the relatedness of the sequences. Eighty-two patients showed evidence of TDR, representing a prevalence of 5.6% (95% CI: 4.5%-6.9%) without any significant time trends or differences between patients infected in Sweden or abroad. Multivariable logistic regression showed that TDR was positively associated with men who have sex with men (MSM) and subtype B infection and negatively associated with CD4 cell counts. Among patients with TDR, 54 (68%) had single resistance mutations, whereas five patients had multi-drug resistant HIV-1. Phylogenetic analyses identified nine significantly supported clusters involving 29 of the patients with TDR, including 23 of 42 (55%) of the patients with TDR acquired in Sweden. One cluster contained 18 viruses with a M41L resistance mutation, which had spread among MSM in Stockholm over a period of at least 16 years (1994-2010). Another cluster, which contained the five multidrug resistant viruses, also involved MSM from Stockholm. The prevalence of TDR in Sweden 2003-2010 was lower than in many other European countries. TDR was concentrated among MSM, where clustering of TDR strains was observed, which highlights the need for continued and improved measures for targeted interventions.

Phylogenetic analysis of the Latvian HIV-1 epidemic.

The Latvian HIV-1 outbreak among intravenous drug users (IDUs) in 1997-1998 involved subtype A1. To obtain a more complete picture of the Latvian HIV-1 epidemic, 315 HIV-1-infected patients diagnosed in 1990-2005 representing different transmission groups and geographic regions were phylogenetically characterized using env V3 and gag p17 sequences. Subtypes A1 and B infections were found in 76% and 22% of the patients, respectively. The subtype A1 sequences formed one large cluster, which also included sequences from other parts of the former Soviet Union (FSU), whereas most subtype B sequences formed three distinct clusters. We estimated that subtype A1 was introduced from FSU around 1997 and initially spread explosively among IDUs in Riga. A recent increase of heterosexually infected persons did not form a separate subepidemic, but had multiple interactions with the IDU epidemic. Subtype B was introduced before the collapse of the Soviet Union and primarily has spread among men who have sex with men.

Performance of ultra-deep pyrosequencing in analysis of HIV-1 pol gene variation.

INTRODUCTION: Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed. PRINCIPAL FINDING: In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858-26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11-0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log(10) of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads. CONCLUSION: This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage.

HIV-1 molecular epidemiology in Guinea-Bissau, West Africa: origin, demography and migrations.

The HIV-1 epidemic in West Africa has been dominated by subtype A and the recombinant form CRF02_AG. Little is known about the origins and the evolutionary history of HIV-1 in this region. We employed Maximum likelihood and Bayesian methods in combination with temporal and spatial information to reconstruct the HIV-1 subtype distribution, demographic history and migration patterns over time in Guinea-Bissau, West Africa. We found that CRF02_AG and subsubtype A3 were the dominant forms of HIV-1 in Guinea-Bissau and that they were introduced into the country on at least six different occasions between 1976 and 1981. These estimates also corresponded well with the first reported HIV-1 cases in Guinea-Bissau. Migration analyses suggested that (1) the HIV-1 epidemic started in the capital Bissau and then dispersed into more rural areas, and (2) the epidemic in Guinea-Bissau was connected to both Cameroon and Mali. This is the first study that describes the HIV-1 molecular epidemiology in a West African country by combining the results of subtype distribution with analyses of epidemic origin and epidemiological linkage between locations. The multiple introductions of HIV-1 into Guinea-Bissau, during a short time-period of five years, coincided with and were likely influenced by the major immigration wave into the country that followed the end of the independence war (1963-1974).

Dynamics of HIV-1 quasispecies during antiviral treatment dissected using ultra-deep pyrosequencing.

BACKGROUND: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. PRINCIPAL FINDINGS: Here, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180-220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406-35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07-0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. CONCLUSIONS: With this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.

HIV-2 genetic evolution in patients with advanced disease is faster than that in matched HIV-1 patients.

The objective of this study was to estimate and compare the evolutionary rates of HIV-2 and HIV-1. Two HIV-2 data sets from patients with advanced disease were compared to matched HIV-1 data sets. The estimated mean evolutionary rate of HIV-2 was significantly higher than the estimated rate of HIV-1, both in the gp125 and in the V3 region of the env gene. In addition, the rate of synonymous substitutions in gp125 was significantly higher for HIV-2 than for HIV-1, possibly indicating a shorter generation time or higher mutation rate of HIV-2. Thus, the lower virulence of HIV-2 does not appear to translate into a lower rate of evolution.

Towards an understanding of the migration of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo haemorrhagic fever (CCHF) is a lethal disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV). It is one of the most widespread medically significant tick-borne pathogens, with a distribution that coincides well with the geographical occurrence of its tick vector, Hyalomma marginatum marginatum. Sporadic outbreaks of CCHF have previously been recognized in Asia, Africa, the Middle East and Europe but, in the 21st century, outbreaks have become more frequent in former Yugoslavia, Turkey and Iran. It has been suggested that CCHFV is a migrating pathogen, but it is not clear to what extent. We have, for the first time, analysed the worldwide migration pattern of CCHFV. Our results showed that Turkey may be a donor in Europe, towards both the east and the west, while the United Arab Emirates acted as a donor in the Middle East, and China was found to be the origin for genotype 2. Finally, we showed that migration of CCHFV was unrestricted between Iran and Pakistan. Considering the distribution and coincidence of the tick vector with CCHFV and CCHF, and the fact that the tick vector is present in western Europe, future outbreaks may extend to include hitherto-naïve areas, suggesting that increased surveillance and geographical mapping of this lethal pathogen are needed.

Differences in molecular evolution between switch (R5 to R5X4/X4-tropic) and non-switch (R5-tropic only) HIV-1 populations during infection.

The recent introduction of entry inhibitors in the clinic as components of antiretroviral treatment has heightened the interest in coreceptor use of human immunodeficiency virus type 1 (HIV-1). Viruses using CCR5 as coreceptor (R5 viruses) are generally present over the entire course of infection whereas viruses using the CXCR4 coreceptor (R5X4/X4 viruses) emerge in about 50% of infected individuals during later stages of infection. The CCR5-to-CXCR4 switch represents a concern because CCR5 inhibitors, while suppressing R5 viruses, may allow the emergence of CXCR4-tropic viruses. In this study, HIV-1 populations that maintained CCR5 usage during infection were compared with populations that switched coreceptor usage to include CXCR4 later during infection, with the aim to find molecular properties of the virus populations associated with the CCR5-to-CXCR4 switch. We amplified and molecularly cloned the V1-V3 region of the HIV-1 envelope from 51 sequential HIV-1 isolates derived from 4 to 10 serial samples for each of the patients. Four of the patients had virus populations that switched coreceptor usage to include CXCR4 (switch populations: SP) during infection and four patients had viral populations that maintained exclusive CCR5 usage (non-switch populations: nSP). Coreceptor usage was determined experimentally on individual clones from dualtropic R5X4 isolates. In nSP we found that the number of potential N-linked glycosylation sites (PNGS) increased over time, whereas no pattern of change was observed in SP. We also found differences in V2 length and V3 charge between R5 viruses of nSP and R5 viruses of SP before the switch. The V2 region was significantly longer in R5 viruses of SP compared to viruses of nSP throughout the course of infection, and the V3 charge increased with time in R5 populations from SP, while it remained unchanged or decreased in nSP. These molecular properties could prove important for understanding the evolution of coreceptor usage in HIV-1 populations, and maybe even for predicting an upcoming coreceptor switch at early stages after primary infection.

Primary HIV-1 R5 isolates from end-stage disease display enhanced viral fitness in parallel with increased gp120 net charge.

To better understand the evolution of the viral envelope glycoproteins (Env) in HIV-1 infected individuals who progress to AIDS maintaining an exclusive CCR5-using (R5) virus population, we cloned and sequenced the env gene of longitudinally obtained primary isolates. A shift in the electrostatic potential towards an increased net positive charge was revealed in gp120 of end-stage viruses. Residues with increased positive charge were primarily localized in the gp120 variable regions, with the exception of the V3 loop. Molecular modeling indicated that the modifications clustered on the gp120 surface. Furthermore, correlations between increased Env net charge and lowered CD4(+) T cell counts, enhanced viral fitness, reduced sensitivity to entry inhibitors and augmented cell attachment were disclosed. In summary, this study suggests that R5 HIV-1 variants with increased gp120 net charge emerge in an opportunistic manner during severe immunodeficiency. Thus, we here propose a new mechanism by which HIV-1 may gain fitness.

Frequent intrapatient recombination between human immunodeficiency virus type 1 R5 and X4 envelopes: implications for coreceptor switch.

Emergence of human immunodeficiency virus type 1 (HIV-1) populations that switch or broaden coreceptor usage from CCR5 to CXCR4 is intimately coupled to CD4+ cell depletion and disease progression toward AIDS. To better understand the molecular mechanisms involved in the coreceptor switch, we determined the nucleotide sequences of 253 V1 to V3 env clones from 27 sequential HIV-1 subtype B isolates from four patients with virus populations that switch coreceptor usage. Coreceptor usage of clones from dualtropic R5X4 isolates was characterized experimentally. Sequence analysis revealed that 9% of the clones from CXCR4-using isolates had originated by recombination events between R5 and X4 viruses. The majority (73%) of the recombinants used CXCR4. Furthermore, coreceptor usage of the recombinants was determined by a small region of the envelope, including V3. This is the first report demonstrating that intrapatient recombination between viruses with distinct coreceptor usage occurs frequently. It has been proposed that X4 viruses are more easily suppressed by the immune system than R5 viruses. We hypothesize that recombination between circulating R5 viruses and X4 viruses can result in chimeric viruses with the potential to both evade the immune system and infect CXCR4-expressing cells. The broadening in cell tropism of the viral population to include CXCR4-expressing cells would gradually impair the immune system and eventually allow the X4 population to expand. In conclusion, intrapatient recombination between viruses with distinct coreceptor usage may contribute to the emergence of X4 viruses in later stages of infection.

Isolation of human immunodeficiency virus-type 1 (HIV-1) clones with biological and molecular properties of the primary isolate.

We developed a new biological cloning system for HIV-1 isolates using the U87.CD4 cell lines that express different chemokine receptors. We demonstrate that our method is sensitive and specific because the clones isolated had the same coreceptor usage and genotype as viruses of the primary isolate. We evaluated our cloning system by isolating 27 biological clones from two primary HIV-1 R3R5X4 isolates. Three HIV-1 phenotypes (R3R5X4, R3R5 and R5) were identified in isolate 29 and two (R3R5X4 or R5X4) in isolate 31. Each phenotype was distinguished by a unique genotype. Sequencing of 20 molecular clones from each isolate did not reveal additional genotypes. One of the three genotypes identified from isolate 29 was not found by molecular cloning of the original isolate, suggesting high specificity and sensitivity of the biological cloning system in isolating minor virus populations. Our results suggest that the new cloning approach can be used as an alternative to the existing method for isolating biological clones in PBMC.