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

Significance of HIV-1 genetic subtypes [Elektronisk resurs]

The human immunodeficiency virus type 1 (HIV-1) displays high genetic variability. Phylogenetic studies have shown that a majority of HIV-1 sequences cluster into one large group called M (major), whereas a few are clearly distinct, group O (outlier) and group N (non-M-non-O). Group M sequences are further subdivided into at least nine genetic subtypes, A through H and J. Inter-subtype recombinants also exist. The geographical distribution of different subtypes varies. Subtype B dominates in Europe and North America. All subtypes have been described in Africa, but subtypes A, C and D dominates. Subtype C is probably the most prevalent subtype world-wide. This thesis was based on a prospective follow-up of all patients at Danderyd Hospital who were known or suspected to be infected with a virus of African origin. A majority of the study subjects were African immigrants who were infected before their arrival to Sweden. The subtype of the viruses was determined by phylogenetic analysis of DNA sequences corresponding to the V3 domain of the env gene. We found that almost all known subtypes (A, B, C, D, G, H and AE) were represented at Danderyd Hospital. A majority of the infections had occurred in Africa, but some transmissions of non-B-subtypes in Sweden were also documented. Subtype B, which dominates among Swedish HIV-1 infected homosexual men and i.v. drug users, was rare among the study subjects who mainly were heterosexually infected. Furthermore, we found that two patients from Zaire were infected with an earlier not described subtype. These viruses are now prototypes for a new subtype called subtype J. Plasma virus load is an important predictor of clinical prognosis. We found that two of three first generation assays for plasma HIV-1 RNA quantification failed to accurately quantify some subtypes e.g. subtype A. This finding limits the use of these assays in many parts of the world. In a later study, we found that newer versions of one of the assays more reliably quantified a majority of samples from all subtypes. Virus entry into cells requires interactions with the CD4 receptor and certain chemokine coreceptors. We investigated if coreceptor usage differs between genetic subtypes of HIV-1. We found that the ability to infect via the CXCR4 coreceptor predicted a rapid/high, SI virus phenotype for all subtypes. However, CXCR4-positive isolates were rare in subtype C, indicating subtype specific differences in biological properties. Finally, we compared the rate of disease progression in patients infected with subtypes A through D by analysis of the rate of CD4 cell decline, plasma HIV-1 RNA levels and clinical status. Disease progression among ethnic Africans and ethnic Swedes was also compared. We found that neither genetic subtype of the virus nor the ethnicity of the host was found to significantly influence disease progression. This suggests that the difference in biological phenotype of subtype C isolates does not translate into differences in disease progression. In summary, determination of the genetic subtype of HIV-1 probably has little value for routine clinical care of individual patients, but provides a powerful tool for monitoring changes in local and global transmission patterns