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

Cerebrospinal Fluid Viral Load and Intrathecal Immune Activation in Individuals Infected with Different HIV-1 Genetic Subtypes

Background: HIV-1 exhibits a high degree of genetic diversity and is presently divided into 3 distinct HIV-1 genetic groups designated major (M), non-M/non-O (N) and outlier (O). Group M, which currently comprises 9 subtypes (A-D, F-H, J and K), at least 34 circulating recombinant forms (CRFs) and several unique recombinant forms (URFs) is responsible for most of the HIV-1 epidemic. Most of the current knowledge of HIV-1 central nervous system (CNS) infection is based on subtype B. However, subtypes other than subtype B account for the majority of global HIV-1 infections. Therefore, we investigated whether subtypes have any influence on cerebrospinal fluid (CSF) markers of HIV-1 CNS infection. Methodology/Principal Findings: CSF HIV-1 RNA, CSF neopterin and CSF white blood cell (WBC) count were measured in patients infected with different HIV-1 subtypes. Using multivariate regression analysis, no differences in the CSF WBC count, neopterin and viral load were found between various HIV-1 subtypes

Daily Sampling of an HIV-1 Patient with Slowly Progressing Disease Displays Persistence of Multiple env Subpopulations Consistent with Neutrality

The molecular evolution of HIV-1 is characterized by frequent substitutions, indels and recombination events. In addition, a HIV-1 population may adapt through frequency changes of its variants. To reveal such population dynamics we analyzed HIV-1 subpopulation frequencies in an untreated patient with stable, low plasma HIV-1 RNA levels and close to normal CD4+ T-cell levels. The patient was intensively sampled during a 32-day period as well as approximately 1.5 years before and after this period (days −664, 1, 2, 3, 11, 18, 25, 32 and 522). 77 sequences of HIV-1 env (approximately 3100 nucleotides) were obtained from plasma by limiting dilution with 7–11 sequences per time point, except day −664. Phylogenetic analysis using maximum likelihood methods showed that the sequences clustered in six distinct subpopulations. We devised a method that took into account the relatively coarse sampling of the population. Data from days 1 through 32 were consistent with constant within-patient subpopulation frequencies. However, over longer time periods, i.e. between days 1…32 and 522, there were significant changes in subpopulation frequencies, which were consistent with evolutionarily neutral fluctuations. We found no clear signal of natural selection within the subpopulations over the study period, but positive selection was evident on the long branches that connected the subpopulations, which corresponds to >3 years as the subpopulations already were established when we started the study. Thus, selective forces may have been involved when the subpopulations were established. Genetic drift within subpopulations caused by de novo substitutions could be resolved after approximately one month. Overall, we conclude that subpopulation frequencies within this patient changed significantly over a time period of 1.5 years, but that this does not imply directional or balancing selection. We show that the short-term evolution we study here is likely representative for many patients of slow and normal disease progression

High prevalence and diversity of HIV-1 non-B genetic forms due to immigration in southern Spain: A phylogeographic approach

Phylogenetic studies are a valuable tool to understand viral transmission patterns and the role of immigration in HIV-1 spread. We analyzed the spatio-temporal relationship of different HIV-1 non-B subtype variants over time using phylogenetic analysis techniques. We collected 693 pol (PR+RT) sequences that were sampled from 2005 to 2012 from naïve patients in different hospitals in southern Spain. We used REGA v3.0 to classify them into subtypes and recombinant forms, which were confirmed by phylogenetic analysis through maximum likelihood (ML) using RAxML. For the main HIV-1 non-B variants, publicly available, genetically similar sequences were sought using HIV-BLAST. The presence of HIV-1 lineages circulating in our study population was established using ML and Bayesian inference (BEAST v1.7.5) and transmission networks were identified. We detected 165 (23.4%) patients infected with HIV-1 non-B variants: 104 (63%) with recombinant viruses in pol: CRF02_AG (71, 43%), CRF14_BG (8, 4.8%), CRF06_cpx (5, 3%) and nine other recombinant forms (11, 6.7%) and unique recombinants (9, 5.5%). The rest (61, 37%) were infected with non-recombinant subtypes: A1 (30, 18.2%), C (7, [4.2%]), D (3, [1.8%]), F1 (9, 5.5%) and G (12, 7.3%). Most patients infected with HIV-1 non-B variants were men (63%, p < 0.001) aged over 35 (73.5%, p < 0.001), heterosexuals (92.2%, p < 0.001), from Africa (59.5%, p < 0.001) and living in the El Ejido area (62.4%, p<0.001). We found lineages of epidemiological relevance (mainly within Subtype A1), imported primarily through female sex workers from East Europe. We detected 11 transmission clusters of HIV-1 non-B Subtypes, which included patients born in Spain in half of them. We present the phylogenetic profiles of the HIV-1 non-B variants detected in southern Spain, and explore their putative geographical origins. Our data reveals a high HIV-1 genetic diversity likely due to the import of viral lineages that circulate in other countries. The highly immigrated El Ejido area acts as a gateway through which different subtypes are introduced into other regions, hence the importance of setting up epidemiological control measures to prevent future outbreaks

Comparative Performance of Three Viral Load Assays on Human Immunodeficiency Virus Type 1 (HIV-1) Isolates Representing Group M (Subtypes A to G) and Group O: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR Version 1.5, and Quantiplex HIV-1 RNA Version 3.0

The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of gag-encoded p24 (gag p24), pol-encoded integrase, and env-encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O