Ultra-deep Pyrosequencing Research Articles

Analysis of potential antiviral resistance mutation profiles within the HBV reverse transcriptase in untreated chronic hepatitis B patients using an ultra-deep pyrosequencing method

The potential antiviral resistance mutations within hepatitis B virus (HBV) reverse transcriptase (RT) region for nucleos(t)ide analogues (NA) are not well known. Especially, the effect of pre-existing antiviral drug resistance mutations in untreated patients in comparison to the resistance developed after treatment is not still clear. Sixteen naive chronic hepatitis B patients were studied. None of the patients had received NA treatment prior to the serum samples being collected. Forty-two potential NA resistance (NAr) mutation sites were screened by ultra-deep pyrosequencing (UDPS). After therapy, mutations conferring treatment resistance were detected by LiPA. Serum samples taken before treatment showed no classic primary or compensatory/secondary drug resistance mutations. However, NAr mutations found in 6 isolates (37.5%) involved 7 positions including rtL91I, rtT128I, rtQ215P, rtF221Y, rtN238D, rtC256S, and rtI266G. Substitutions at 3 NAr mutation sites (rtT128I, rtN238D, and rtC256S) were detected in 3 unresponsive patients developing drug resistance after NA treatment. One patient with rtI266G mutation also developed drug resistance after lamivudine (LAM) therapy. However, the relationship between rtI266G mutation and NA drug resistance was not previously reported. These results suggest that association of potential mutations besides the primary and secondary/compensatory resistance mutations should be investigated. Investigation of NAr mutations before treatment may be important for the success of the treatment.

Study of hepatitis B virus quasispecies by ultra-deep pyrosequencing: Resolving the nitty-gritty

Rodriguez et al.1 described the dynamics of hepatitis B virus (HBV) resistance to adefovir by ultra-deep pyrosequencing (UDPS), a validated technique to study HBV quasispecies.2 Their article reports relevant results, which, however, require further examination. HBV polymerase variants resistant to nucleos(t)ide (NUC) treatment (NUCsRV) were detected in 83% of NUC-naïve cases, in keeping with previous reports, with prevalence restricted by sensitivity: 83%-100% at 0.03% cutoff, 12%-20% at 1% cutoff (Table 1). The authors describe an interesting computational method assuming exponential outgrowth of selected variants. Nonetheless, the relatively low coverage of each sample, at best 4,853 sequences, is questionable with respect to the lowest cutoff reported (0.03%) because variants represented by a single sequence (e.g., patient AH, with viral load 2.1 log IU/mL and 0.34% of rtT184S/A/I/L) would be considered correct, a singular notion that is not backed by previous experience (Table 1). Thus, UDPS standardization (e.g., minimal viral load and sequence coverage for standardized sensitivity, computational algorithms) must still be resolved. The authors state that some previous UDPS studies assessing preexisting NUCsRV, including ours,3 have methodological flaws: lack of sensitivity, no error rate consideration, and/or no linkage studies. This is surprising, because we reported the lowest cutoff to date (0.03%), established by clonal sequence processing and Poisson filtering.3 Rodriguez et al. report different cutoffs for each variant, 0.03% being the lowest, whereas in our study 0.03% was used for all positions (0.007% average clonal error). So sensitivity was not lacking. Furthermore, we did perform a linkage study. The analysis demonstrated HBV quasispecies dynamics in a sequentially NUC-treated patient, with emergence of complex combined variants, particularly after entecavir. Lastly, although it is a secondary issue, questions also arise about patient classification in their study. The 9.57% of rtM204V/I at baseline in “naïve” patient C suggests prior lamivudine treatment (as the authors indicate). Similarly, the 11% rtA181T/V in “naïve” patient AA, 100 times greater than previously UDPS-detected at baseline (Table 1), also suggests prior NUC therapy. As the study investigates pretreatment HBV variants, inclusion of these cases, detectable by routine methods, raises the concern that some variant populations may have been overestimated. In conclusion, UDPS is consolidated for HBV quasispecies research, as supported by Rodriguez et al. However, certain factors, such as those mentioned here, should be kept in mind to avoid misinterpretation of the state-of-the-art in UDPS studies. Francisco-Rodriguez Frias, M.D.1,2 David Tabernero, M.S.1,2 Rafael Esteban, M.D.1,3 Maria Buti, M.D.1,3 1Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) Instituto de Salud Carlos IIIMadrid, Spain 2Biochemistry Department Hospital Universitari Vall d'Hebron (HUVH)Vall d'Hebron Institut de Recerca (VHIR)Universitat Autònoma de Barcelona (UAB)Barcelona, Spain 3Liver Unit Hospital Universitari Vall d'Hebron (HUVH)Vall d'Hebron Institut de Recerca (VHIR)Universitat Autònoma de Barcelona (UAB)Barcelona, Spain

Reply

We thank Rodriguez-Frias et al. for their interest in our work and their careful reading of our article. We understand that the authors mistook our criticisms of the prior literature on the topic to be specific to their work, but want to clarify this point. Our sentence “These works however suffered from important methodological flaws, including lack of sensitivity, no consideration of the error rate of the method to establish reliable cutoffs and ensure specificity, too short genomic region analyzed, and/or no linkage studies” refers to three distinct articles, including the one by Rodriguez-Frias et al.1 In this sentence, “and/or” means that the three studies did not all suffer from the same flaws. We found the analysis of ultra-deep pyrosequencing (UDPS) data by Rodriguez-Frias et al. of good quality, and their sensitivity appropriate for this type of study. However, the hepatitis B virus (HBV) reverse transcriptase fragment they analyzed, which spanned positions rt148 to rt208, was far too short to be appropriate for HBV resistance studies, especially in the context of adefovir (or even tenofovir) therapy, which selects variants with substitutions at position rt236 or neighboring positions, as shown in our study. In this context, linkage studies are of no use because mutations that play a key role in the fitness of the variants are not taken into consideration. Additionally, the authors studied only four patients, with a single baseline assessment in three of them. Several timepoints were analyzed in one patient only, who received sequential nucleos(t)ide analog-based therapies, with samples taken at treatment changes only. Overall, the study by Rodriguez-Frias et al. is a valuable pioneer study describing a new, well-handled technical approach, but it suffers from the methodological limitations of a too-short genomic region analyzed that resulted in poorly contributive linkage analyses, and provides limited novel information on HBV resistance to antiviral drugs due to limitations in the number and type of patient samples analyzed. We were a bit confused by the comments on the sensitivity of our analysis, which is in fact similar to that in the PLoS One article (0.03%). The authors question our coverage and sensitivity, and suggest that a variant present as a single viral particle (or as a single sequence?) cannot be reliably detected by our approach. They give the example of a patient with a variant representing 0.34% of 2.1 log IU/mL. First, 0.34% of 4,853 sequences means that 17 identical sequences of very good quality were generated from this single sequence, a high enough number to rely on its presence. Second, this calculation is purely theoretical if one remembers that the HBV DNA “international unit” is an arbitrary unit that in fact represents a much larger number of viral particles. That said, we completely agree with Rodriguez-Frias et al. that UDPS standardization is needed, at both the experimental and analytical levels. We did not discuss these aspects in detail in the article, as our research focus was on using the new technology and our in-house software tools to provide novel information on HBV resistance to nucleotide analogs. We agree that classification of patients as treatment-naïve or treatment-experienced is always challenging, in clinical trials and even more in real life. Primary drug resistance transmitted at the time of infection is also a possibility that would require further investigation. Nevertheless, speculations must be avoided. The patient with 9.57% of resistant variants at baseline was a treatment-naïve pediatric patient included in a clinical trial with adefovir in the United States. Prior exposure is very unlikely, but primary transmitted resistance is possible. The second patient with 11.29% of resistant variants at baseline was a French blood donor who declared no risk factor and no prior therapy for hepatitis B. Prior therapy or primary resistance are possible in this case. Finally, we would like to thank Rodriguez-Frias et al. for their comments and for giving us the opportunity for this interesting discussion. We look forward to reading more from their group on this topic, and to working with them and others to improve standardization of next-generation sequencing strategies in viral hepatitis studies. Christophe Rodriguez, Pharm.D., Ph.D. Stéphane Chevaliez, Pharm.D., Ph.D. Jean-Michel Pawlotsky, M.D., Ph.D. National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Hôpital Henri Mondor, Université Paris-Est, Créteil, France; INSERM U955, Créteil, France

Inference with viral quasispecies diversity indices: clonal and NGS approaches

josep.gregori@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.

Comparison of quasispecies diversity of HCV between chronic hepatitis c and hepatocellular carcinoma by Ultradeep pyrosequencing.

Backgrounds. Hepatitis C virus (HCV) exists as population of closely related genetic variants known as quasispecies. HCV quasispecies diversity is strongly influenced by host immune pressure on virus. Quasispecies diversity is expected to decline as host immune response to HCV decreases over natural course of progressing from chronic hepatitis C (CHC) to hepatocellular carcinoma (HCC). Methods. Ultradeep pyrosequencing (UDPS) was used to evaluate degree of quasispecies diversity in 49 patients infected with HCV including 26 with CHC and 23 with HCC. Whole structural protein of HCV genome was subjected to UDPS. Results. Shannon's indices for quasispecies diversity in HCV E1 were significantly lower in patients with HCC than in those with CHC. 14 amino acid positions differed significantly between two groups. Area under curve of ROC analysis for differentiating HCC from CHC was >0.8 for all of 14 amino acid positions. Conclusion. HCV quasispecies diversity as indicator of declining host immune functions was easily assessed by UDPS technology. Shannon's indices in 14 amino acid positions were found to differentiate between patients with CHC and those with HCC. Our data propose that degree of HCV quasispecies measured by UDPS might be useful to predict progression of HCC in chronic HCV patients.

Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%.Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.

Short duration of lamivudine for the prevention of hepatitis B virus transmission in pregnancy: lack of potency and selection of resistance mutations

This study sought to assess the antiviral efficacy of lamivudine (LMV) administered during third trimester to reduce maternal viraemia and to identify the emergence of LMV resistance. A prospective observational analysis was performed on 26 mothers with high viral load (>10⁷ IU/mL). Twenty-one women received LMV (treated group) for an average of 53 days (range 22-88 days), and the remaining five formed the untreated control group. Serum samples from two time points were used to measure HBV DNA levels and antiviral drug resistance. The LMV-treated women achieved a median HBV DNA reduction of 2.6-log10 IU/mL. Although end-of-treatment (EOT) HBV DNA in four (18%) LMV-treated women remained at >10(7) IU/mL (± 0.5 log IU/mL), no mother-to-baby transmission was observed. In contrast, a baby from the untreated mother was HBsAg positive at 9 months postpartum. Four technologies were used for drug resistance testing. Only ultra-deep pyrosequencing (UDPS) was sufficiently sensitive to detect minor viral variants down to <1%. UDPS showed that LMV therapy resulted in increased viral quasispecies diversity and positive selection of HBV variants with reverse transcriptase amino acid substitutions at sites associated with primary LMV resistance (rtM204I/V and rtA181T) in four (19%) women. These viral variants were detected mostly at low frequencies (0.63-5.92%) at EOT, but one LMV-treated mother had an rtA181T variant that increased from 2.2% pretherapy to 25.59% at EOT. This mother was also infected with the vaccine escape variant (sG145R), which was inhibited by LMV treatment. LMV therapy during late pregnancy only reduced maternal viraemia moderately, and drug-resistant viral variants emerged.

Role of the line probe assay INNO-LiPA HBV DR and ultradeep pyrosequencing in detecting resistance mutations to nucleoside/nucleotide analogues in viral samples isolated from chronic hepatitis B patients

Despite the effectiveness of nucleoside/nucleotide analogues in the treatment of chronic hepatitis B (CHB), their long-term administration is associated with the emergence of resistant hepatitis B virus (HBV) mutants. In this study, mutations resulting in antiviral resistance in HBV DNA samples isolated from 23 CHB patients (nine treatment naïve and 14 treated previously) were studied using a line probe assay (INNO-LiPA HBV DR; Innogenetics) and ultradeep pyrosequencing (UDPS) methods. Whilst the INNO-LiPA HBV DR showed no resistance mutations in HBV DNA samples from treatment-naive patients, mutations mediating lamivudine resistance were detected in three samples by UDPS. Among patients who were treated previously, 19 mutations were detected in eight samples using the INNO-LiPA HBV DR and 29 mutations were detected in 12 samples using UDPS. All mutations detected by the INNO-LiPA HBV DR were also detected by UDPS. There were no mutations that could be detected by INNO-LiPA HBV DR but not by UDPS. A total of ten mutations were detected by UDPS but not by INNO-LiPA HBV DR, and the mean frequency of these mutations was 14.7 %. It was concluded that, although INNO-LiPA HBV DR is a sensitive and practical method commonly used for the detection of resistance mutations in HBV infection, UDPS may significantly increase the detection rate of genotypic resistance in HBV at an early stage.

Ultra-Deep Pyrosequencing of Partial Surface Protein Genes from Infectious Salmon Anaemia Virus (ISAV) Suggest Novel Mechanisms Involved in Transition to Virulence

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q266→L266 substitution or insertion next to the putative cleavage site (R267) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L266 codons in screening samples and Q266 codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.

Ultradeep Pyrosequencing and Molecular Modeling Identify Key Structural Features of Hepatitis B Virus RNase H, a Putative Target for Antiviral Intervention

Last-generation nucleoside/nucleotide analogues are potent against hepatitis B virus (HBV) and have a high barrier to resistance. However, delayed responses have been observed in patients previously exposed to other drugs of the same class, long-term resistance is possible, and cure of infection cannot be achieved with these therapies, emphasizing the need for alternative therapeutic approaches. The HBV RNase H represents an interesting target because its enzyme activity is essential to the HBV life cycle. The goal of our study was to characterize the structure of the HBV RNase H by computing a 3-dimensional molecular model derived from E. coli RNase H and analyzing 2,326 sequences of all HBV genotypes available in public databases and 958,000 sequences generated by means of ultradeep pyrosequencing of sequences from a homogenous population of 73 treatment-naive patients infected with HBV genotype D. Our data revealed that (i) the putative 4th catalytic residue displays unexpected variability that could be explained by the overlap of the HBx gene and has no apparent impact on HBV replicative capacity and that (ii) the C-helix-containing basic protrusion, which is required to guide the RNA/DNA heteroduplex into the catalytic site, is highly conserved and bears unique structural properties that can be used to target HBV-specific RNase H inhibitors without cross-species activity. The model shows substantial differences from other known RNases H and paves the way for functional and structural studies as a prerequisite to the development of new inhibitors of the HBV cell cycle specifically targeting RNase H activity.

Comparison of Next-Generation Sequencing and Clone-Based Sequencing in Analysis of Hepatitis B Virus Reverse Transcriptase Quasispecies Heterogeneity

We previously reported that, based on clone-based sequencing (CBS), hepatitis B virus (HBV) heterogeneity within the reverse transcriptase (RT) region was a predictor of antiviral efficacy. Here, by comparing ultradeep pyrosequencing (UDPS), i.e., next-generation sequencing (NGS), with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV viral populations. HBV genomic DNA was extracted from serum samples from 31 antiviral treatment-naive patients with chronic hepatitis B. The RT region quasispecies were analyzed in parallel using CBS and UDPS. Characterization of quasispecies heterogeneity was conducted using bioinformatics analysis. Quasispecies complexity values were calculated with the formula Sn = -Σi(pilnpi)/lnN. The number of qualified strains obtained by UDPS was much larger than that obtained by CBS (P < 0.001). Pearson analysis showed that there was a positive correlation of quasispecies complexity values at the nucleotide level for the two methods (P < 0.05), while the complexity value derived from UDPS data was higher than that derived from CBS data (P < 0.001). Study of the prevalences of variations within the RT region showed that CBS detected an average of 9.7 ± 1.1 amino acid substitutions/sample and UDPS detected an average of 16.2 ± 1.4 amino acid substitutions/sample. The phylogenetic analysis based on UDPS data showed more genetic entities than did that based on CBS data. Viral heterogeneity determination by the UDPS technique is more sensitive and efficient in terms of low-abundance variation detection and quasispecies simulation than that by the CBS method, although imperfect, and thus sheds light on the future clinical application of NGS in HBV quasispecies studies.

Detection of Low-Frequency HIV Type 1 Reverse Transcriptase Drug Resistance Mutations by Ultradeep Sequencing in Naive HIV Type 1-Infected individuals

Genotypic resistance testing is recommended to evaluate the susceptibility of HIV to antiretroviral drugs. These tests are based on bulk population sequencing and thus consider only variants representing more than 20% of the viral population, whereas next generation sequencing methods allow detection below this threshold. We aimed to evaluate the potential use of ultradeep pyrosequencing (UDPS) for genotypic resistance testing in clinical routine at the University Hospital of Bordeaux, France. We performed UDPS on reverse transcriptase (RT) from 47 HIV-1 individuals, naive of antiretroviral treatment and for whom genotypic resistance testing was requested for clinical management in 2011-2012. In 8.5% of the patients, only low-frequency variants harboring RT drug resistance mutations were detected raising the question of their clinical significance. Rilpivirine-associated resistance mutations were detected in 19.1% of our population study. To conclude, UDPS could become a routine tool for the evaluation of HIV-infected patients in hospital laboratories.

PCR-Induced Transitions Are the Major Source of Error in Cleaned Ultra-Deep Pyrosequencing Data

BackgroundUltra-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.ResultsUDPS 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.ConclusionsA 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.

Characterization of CXCR4-using HIV-1 during primary infection by ultra-deep pyrosequencing

R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections (PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%-6.4% of subjects also harbour CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using viruses among HIV-1 quasispecies. We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population. We then used UDS of the V3 env region with the geno2pheno algorithm (false positive rate = 5.75) to identify the HIV-1 quasispecies. CXCR4-using viruses were detected in all but 1 of the 11 patients by UDS, and accounted for 0.2%-100% of the virus populations. The frequency of CXCR4-using viruses was <20% in six subjects and 100% in four subjects. Virus populations containing 100% CXCR4-using variants during PHI persisted for at least 1-2 years after the acute phase. The CCR5 Δ32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5 (15%) viruses. UDS and the phenotype were concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis.

Resistance Mutations and CTL Epitopes in Archived HIV-1 DNA of Patients on Antiviral Treatment: Toward a New Concept of Vaccine

Eleven patients responding successfully to first-line antiretroviral therapy (ART) were investigated for proviral drug resistance mutations (DRMs) in RT by ultra-deep pyrosequencing (UDPS). After molecular typing of the class I alleles A and B, the CTL epitopes in the Gag, Nef and Pol regions of the provirus were sequenced and compared to the reference HXB2 HIV-1 epitopes. They were then matched with the HLA alleles with determination of theoretical affinity (TA). For 3 patients, the results could be compared with an RNA sample of the circulating virus at initiation of therapy. Five out of 11 patients exhibited DRMs by UDPS. The issue is whether a therapeutic switch is relevant in these patients by taking into account the identity of the archived resistance mutations. When the archived CTL epitopes were determined on the basis of the HLA alleles, different patterns were observed. Some epitopes were identical to those reported for the reference with the same TA, while others were mutated with a decrease in TA. In 2 cases, an epitope was observed as a combination of subpopulations at entry and was retrieved as a single population with lower TA at success. With regard to immunological stimulation and given the variability of the archived CTL epitopes, we propose a new concept of curative vaccine based on identification of HIV-1 CTL epitopes after prior sequencing of proviral DNA and matching with HLA class I alleles.

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.

Dynamic and Rapid Changes in Viral Quasispecies by Udps in Chronic Hepatitis C Patients Receiving Telaprevir-Based Therapy

Telaprevir (TVR) is a protease inhibitor (PI) used in chronic hepatitis C treatment with pegylated interferon plus ribavirin. We analysed the prevalence and kinetic development of TVR resistance upon treatment. A total of 24 cirrhotic patients (genotype 1a, n=8; genotype 1b, n=16) previously non-responders to standard therapy were treated with TVR-based therapy. The distribution of TVR-resistant variants was assessed at every HCV-RNA-positive time point by 454 ultra-deep pyrosequencing (UDPS) during a mean follow-up period of 9.4 months. A median of 6,837 reads/specimen was studied. Based on control UDPS, we considered mutations as real when present >0.4%. TVR-resistant variants were found at baseline in 8/24 patients (33.3%). Four of the 24 patients (16.7%), all genotype 1a, did not achieve HCV RNA<100 IU/ml between week (W)2 and W12 and stopped treatment. No statistical significant difference was observed in the prevalence of resistant mutants between responders and non-responders (25% [5/20] and 75% [3/4], respectively). The proportion of genotype 1a patients with R155K/T/Q at baseline was higher in non-responders than in responders (50% versus 0%). During treatment failure, significant enrichment in V36A/M and R155K/T/Q was observed but their frequency reverted back to baseline after TVR discontinuation. TVR-resistant variants are widely present at baseline. The presence of TVR-resistant mutants at baseline, even in high abundance (>20%), did not always preclude TVR treatment success. The detection of R155K/T/Q at baseline may predict failure in genotype 1a patients. At failure, which occurred in genotype 1a patients, a significant enrichment in V36A/M and R155K/T/Q was observed.

Detection of haemagglutinin D222 polymorphisms in influenza A(H1N1)pdm09-infected patients by ultra-deep pyrosequencing

This study was aimed at establishing the genetic heterogeneity of influenza virus haemagglutinin (HA) gene quasi-species and the polymorphisms at codon 222, by application of ultra-deep pyrosequencing (UDPS) to respiratory samples from patients hospitalized for influenza A(H1N1)pdm09 infection, presenting with severe or moderate–mild disease. HA diversity was significantly higher in samples collected from patients with severe manifestations than in those from patients with moderate–mild manifestations (p 0.02). D222 polymorphism was detected in 40.7% of patients by UDPS, and in only 7.1% by Sanger sequencing. D222E, D222G, D222N and D222A were observed in 37.0%, 11.1%, 7.4% and 3.7% of patients, respectively; 10.7% of samples harboured more than two variants. The relative frequency of each single variant showed a wide range of intrapatient variation. D222G/N/A were detected, as either minor or predominant variants, only in severe cases, whereas D222E was equally represented in severe and moderate–mild infections. Other amino acid variants were observed at different positions within the analysed HA fragment. Consistent with higher heterogeneity, non-D222 variants were more frequently detected in severe cases than in moderate–mild cases. In addition, seven non-D222 mutations carried by minority variants, not previously described, were observed.

Full genome ultra‐deep pyrosequencing associates G‐to‐A hypermutation of the hepatitis B virus genome with the natural progression of hepatitis B

Human APOBEC3 (A3) cytosine deaminases are antiviral restriction factors capable of editing the genome the hepatitis B virus (HBV). Despite the importance of the human A3 protein family for the innate immune response little is known about the clinical relevance for hepatitis B. The aim of this study was to utilize ultra-deep pyrosequencing (UDPS) data to analyse the phenomenon of G-to-A hypermutation of the complete HBV genome and to relate it to fundamental characteristics of patients with chronic hepatitis B. By analysing the viral population of 80 treatment naïve patients (47 HBeAg-positive and 33 HBeAg-negative), we identified an unequal distribution of G-to-A hypermutations across the genome. Our data indicate that G-to-A hypermutation occurs predominantly in a region between nucleotide positions 600 and 1800 a region which is usually single stranded in matured HBV particles. This implies that A3 likely edits HBV in the virion. Hypermutation rates for HBeAg-negative patients were more than 10-fold higher than those of HBeAg-positive patients. For HBeAg-negative patients higher hypermutation rates were significantly associated with the degree of fibrosis. Additionally, we found that for HBeAg-positive chronic hepatitis G-to-A hypermutation rates were significantly associated with the relative prevalence of the G1764A mutation, which is related to HBeAg seroconversion. In total, our data imply an important association of hypermutation mediated by A3 deaminases with the natural progression of chronic hepatitis B infections both in terms of HBeAg seroconversion and disease progression towards cirrhosis.

Using Ultradeep Pyrosequencing to Study HIV-1 Coreceptor Usage in Primary and Dual Infection

HIV-1 dual infection (DI) and CXCR4 (X4) coreceptor usage are associated with accelerated disease progression but frequency and dynamics of coreceptor usage during DI is unknown. Ultradeep sequencing was used to interrogate for DI and infer coreceptor usage in longitudinal blood samples of 102 subjects. At baseline, X4 usage was high (23 subjects harbored X4 variants) and was not associated with infection duration or DI. Coreceptor usage changed over time in 12 of 47 participants, and X4 usage emerged in 4 of 41 monoinfections vs 2 of 5 superinfections (P = .12), suggesting a weak statistical trend toward occurrence of superinfection and acquiring X4 usage.