The ViroSeq HIV-1 Genotyping System, from Abbott GmbH, is a fully capillary- based genetic analysers (ABI PRISM , Avant, , , and Natalia M Marlowe at Abbott Laboratories The new Applied Biosystems ViroSeq HIV-1 Genotyping System (HGS) was formally released in. In this study, the Abbott RealTime HIV-1 (Abbott RealTime) assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan) and the.

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Using the protocol of Gall et al. Kellam, J Clin Microbiol Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads e.

The two amplicons cover critical regions across the HIV-1 genome including pol and envallowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]integrase strand transfer inhibitors, and virus entry inhibitors.

The two amplicons generated span 7, bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies.

The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing.

Genotypic analysis of HIV-1 drug resistance mutations

Routine antiretroviral ARV drug resistance testing is useful in choosing an optimal treatment regimen and monitoring its efficiency in clinical practice 1— While the RNA-based approach works well in antiretroviral therapy ART -naive individuals, it is viiroseq successful hvi levels of viral replication are low, such huv in individuals on ART.

The sequence length of traditional RNA-based HIV genotyping for drug resistance is relatively short and does not cover the HIV-1 region encoding viral integrase or the viral envelope, hindering analysis of drug resistance mutations associated with integrase strand transfer inhibitors or entry inhibitors. The global scale up of ARV treatment and ihv introduction of integrase strand transfer inhibitors and aabbott inhibitors into clinical trials and clinical practice necessitate modification of traditional methods of HIV genotyping.

Both genotyping kits were extensively tested and validated 36— Many experienced genotyping laboratories have ihv their own in-house amplification and sequencing protocols 1147— 56including identification of minor viral variants that are normally missed by commercial genotyping kits 57— All of these approaches generally include smaller and more restricted regions for testing HIV-1 drug resistance.

Recently, the protocol developed by Gall et al. Drug resistance mutations detected in viral RNA from plasma and proviral DNA from peripheral blood mononuclear cells PBMCs or dried blood spots DBS show substantial correlation in treated patients, suggesting that either compartment is suitable for the detection of mutations as a virological guide for clinical care 63— It abbotr known that amplified HIV sequences and sequences from proviral DNA could have substantial numbers of guanine-to-adenine transitions.

Such vidoseq inordinate number of identical G-to-A transitions is a retroviral signature known as hypermutation 66— G-to-A hypermutations produce multiple stop codons and reduce HIV replication, leading to an evolutionary dead end. It is an innate host intracellular defense mechanism. G-to-A hypermutations play an important role in the evolution of antiretroviral drug resistance 7172 and could be associated with ART failure For sequence quality control, it is important that G-to-A hypermutations are not products of PCR amplification The proposed technique is a modification of the method of Gall et al.

The key modifications include using i a proviral DNA template, ii an extra round of PCR, iii selection of robust primers, and iv modified running conditions. To illustrate the potential utility of long-range HIV genotyping, the technique was applied to a set of specimens collected in Botswana. Chan School of Public Health. All study subjects signed a consent form and donated a blood abbotr for viral genotyping. The first large fragment of the HIV-1 genome, amplicon 1, was amplified and sequenced in HIV-infected subjects a single sequence per subject firoseq from eight geographic localities agbott Botswana: The second large fragment of the HIV-1 genome, amplicon 2, was amplified and sequenced in 90 subjects the work is still in progress originating from Mochudi, Molapowabojang, Otse, and Ranaka.

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A total of specimens from the BCPP study were used for analysis of genotyping efficiency. In addition, the following combinations of the subgenomic regions included concatenated amplicon 1 plus amplicon 2 and amplicon 1 plus V1C5. The list of PI-associated mutations included 40 mutations at 18 positions across protease. The adjusted hypermutations were expressed as a number of identified hypermutations adjusted by sequence length.

The hypermutation ratio was computed as the ratio between weighted mutations matched mutations out of potential mutations and weighted controls control mutations out of potential controls and was derived as a statistical outcome of the Hypermut package An HIV cluster was defined as a aabbott lineage that gives abboty to a monophyletic subtree of the overall phylogeny with strong statistical support.

The bootstrapped maximum-likelihood ML method 84— 86 was used to determine the statistical support of clusters.

A viral lineage group or subtree with at least two viral sequences and specified statistical support was considered to be an HIV cluster. Clusters were identified using a depth-first algorithm 8788a method for traversing or searching tree or graph data structures starting from the root. This approach eliminated double counting of viral sequences in clusters when the clusters had internal structure with strong support.

The sharing of data, including generated HIV sequences, with the scientific community for the purpose of research is of key importance in ensuring continued progress in our understanding of how to contain the HIV epidemic.

The confidentiality of study subjects was protected by recoding of HIV sequences deposited in GenBank at the country level with no community or village data. The statistical support for each node was assessed by bootstrap analysis from bootstrap replicates performed with the rapid bootstrap algorithm implemented in RAxML To test whether the extent of HIV clustering is associated with any subgenomic region, the proportion of clustered sequences was compared between long amplicon 1, amplicon 2, concatenated amplicons 1 plus 2, and concatenated amplicon 1 plus V1C5 and short ViroSeq and V1C5 HIV-1C sequences.

The proportion of HIV sequences in clusters was estimated at the bootstrap thresholds for cluster definition from 0. Comparisons of continuous outcomes between two groups were performed using the Wilcoxon rank sum test.

P values of less than 0. All reported P values are 2 sided. Proportions of viral sequences in clusters between targeted loci were compared by McNemar’s test in R, and P values of less than 1. All plots were produced in R.

All figures were finalized in Adobe Illustrator CS6. The protocol of Gall et al. The modifications of the protocol of Gall et al.

The rationale for focusing on two instead of four amplicons was driven by a balance between sequencing data and cost. 3100 first amplicon corresponding to amplicon 2 in the study by Gall et al. The second amplicon corresponding to amplicon 4 in viroseqq study by Gall et al. Overview of long-range HIV genotyping. The 1st- and 2nd-round products are mapped against the HIV-1 genome structure. The 2nd-round PCR products, amplicon 1 and amplicon 2, are shown as gray bars.

Amplification of a large fragment spanning almost the entire HIV-1 genome Fig. The 1st-round product was used as the template in two separate 2nd-round PCRs with specific primers see Table S1 in the supplemental material to obtain amplicon 1 and amplicon 2 Fig.

No additional extension step was performed at the end of the run. After standard purification with USB ExoSap-It 92 Affymetrix; catalog number MLamplicon 1 was subjected to direct Sanger sequencing on both strands using a total of 12 sequencing primers see Table Viroeeq in the supplemental material.

Genotypic analysis of HIV-1 drug resistance mutations | Scientist Live

These cases were cloned and Sanger sequenced on both strands. All amplicon 2 products were cloned before Sanger sequencing on both strands, with a total of 12 sequencing primers see Table S2 in the supplemental material.

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The high diversity of HIV presents a challenge hic direct Sanger sequencing. Samples collected during the early stage of HIV infection are relatively homogeneous in the case of transmission of a single Agbott variant. In contrast, samples obtained from chronically infected individuals are likely to include a heterogeneous pool of viral quasispecies. High heterogeneity of viral quasispecies combined with numerous insertions and deletions indels could result in low quality of the directly sequenced specimens.

In this case, cloning may be considered an alternative solution to direct sequencing. If the time of HIV infection is unknown, the diversity of the targeted ihv, or subregion, could guide the initial sequencing strategy.

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Amplicon 1 spans a relatively conserved region of the HIV-1 genome. In contrast, amplicon 2 includes the most variable regions of the HIV-1 genome, with multiple indels. The goal of this study was to obtain a single HIV sequence per subject. Therefore, generation of a single amplicon 1 and a single amplicon 2 sequence was considered a success. If a study aimed to address the multiplicity of HIV infection or the diversity of viral quasispecies, multiple sequences e.

Ligation, transformation, and plating were performed according to the manufacturer’s instructions. A list of sequencing primers used with clones is presented in Table S3 in the supplemental material. Some amplification issues during long-range HIV genotyping, such as lack of or insufficient amplification, an overamplified product, or the presence of multiple bands, could be resolved by troubleshooting.

The initial amplification results could guide troubleshooting. The overamplified products could be overcome by reducing the number of cycles in the first-round PCR to 25 or in the second-round PCR to 20 to 25 or by decreasing the amount of input template. Amplicon 1 was amplified and sequenced in HIV-infected subjects a single sequence per subjectwhile amplicon 2 was amplified and sequenced in 90 subjects.

The distribution of amplified and sequenced samples from proviral DNA is presented in Table 1. Amplicon 1 was successfully amplified in Viral sequences were obtained for all amplified samples. The majority of amplified amplicon 1 sequences, of In 23 cases Cloning followed by Sanger sequencing helped to resolve the gaps in all 23 cases. The proportion of successfully amplified cases was Sequences were obtained for all amplified products.

The histogram shows the distribution of HIV-1 RNA among specimens with available viral load data both amplified and failed specimens. Amplification and sequencing of amplicon 2 were completed for 90 subjects. Given that the first-round RT PCR product is used for amplification of both amplicons 1 and 2, obtaining amplicon 1 suggests a successful amplification of amplicon 2.

This strategy has not been explored yet. A subset of 23 specimens were successfully amplified and sequenced. The nine failed cases included one sample with unknown and eight specimens with available viral loads.

The last failed sample, with a high viral load, also failed amplification from proviral DNA, apparently suggesting an intrinsic problem with mismatch of amplification primers.

Amplicon 2 covers the entire HIV-1 env gene and allows analysis of mutations associated with drug resistance to virus entry inhibitors. To illustrate the validity of long-range HIV genotyping for analysis of mutations associated with antiretroviral drug resistance, we estimated drug resistance profiles within two groups of specimens originating from the MPP and BCPP studies.

Despite relatively rare use of protease inhibitors in Botswana, mutations associated with resistance to PIs were detected at five positions in protease: Thus, it is likely that the majority of identified mutations in the protease gene were caused by G-to-A hypermutations.