Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms

Mori, Mikiko; Ode, Hirotaka; Kubota, Mai; Nakata, Yoshihiro; Kasahara, Takaaki; Shigemi, Urara; Okazaki, Reiko; Matsuda, Masakazu; Matsuoka, Katsunari; Sugimoto, Atsuko; Hachiya, Atsuko; Imahashi, Mayumi; Yokomaku, Yoshiyuki; Iwatani, Yoshinori

doi:10.1128/spectrum.01507-22

Cited by 10 publications

(15 citation statements)

References 46 publications

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“…Full genome sequencing can be used to con rm recombinants when viruses typing were unclassi ed or the results of different gene segments were inconsistent. A number of new HIV recombinants have been reported by near-full-length genome sequencing [33][34][35][36]. In this study, 12 recombinants were con rmed by performing full genome sequencing.…”

Section: Discussionmentioning

confidence: 93%

Genetic characterization of HIV-1 viruses among antiretroviral therapy failure individuals in Suzhou City, China

Dong

TIAN

et al. 2022

Preprint

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Background: This retrospective study was aimed to characterize the distribution of HIV-1 genotypes and prevalence of drug-resistance mutations in antiretroviral therapy (ART) failure individuals in Suzhou City, China. Methods: Polgene of HIV-1 viruses in EDTA anticoagulant plasma samples of 398 ART failure HIV-infected individuals were successfully amplified by using an in-house assay. Drug resistance mutations were analyzed by using the Stanford HIV Drug Resistance Database system (https://hivdb.stanford.edu/hivdb/by-mutations/). HIV-1 genotypes were determined by REGA HIV subtyping tool (version 3.46, https://www.genomedetective.com/app/typingtool/hiv). Near full-length genomes (NFLG) of HIV-1 viruses were obtained by next generation sequencing method. Results: Sequences analysis of the pol gene revealed that CRF 01_AE (57.29%, 228/398) was the dominant subtype circulating in Suzhou City, followed by CRF 07_BC (17.34%, 69/398), subtype B (7.54%, 30/398), CRF 08_BC (6.53%, 26/398), CRF 67_01B (3.02%, 12/398) and CRF55_01B (2.51%, 10/398). The overall prevalence of drug-resistant mutations in ART failure HIV-infected individuals was 64.57% (257/398), including 45.48% (181/398) for nucleotide reverse transcriptase inhibitors (NRTIs) mutations, 63.32% (252/398) for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations, and 3.02% (12/398) for protease inhibitors (PIs) mutations. Ten near full-length genomes (NFLG) of HIV-1 viruses were identified, including six recombinants of CRF 01_AE and subtype B, two recombinants of CRF 01_AE, subtype B and subtype C sequences, one recombinant of CRF 01_AE and subtype C and one recombinant of CRF 01_AE, subtype A1 and subtype C. Conclusions: The high prevalence of drug-resistant HIV-1 viruses was a serious challenge for HIV prevention and HIV-infected individuals’ treatment. Therapeutic regimens of ART failure HIV-infected individuals should be adjusted in time according to drug resistance test results. NFLG sequencing facilitates the identification of new HIV-1 recombinant strains.

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Section: Discussionmentioning

confidence: 93%

Genetic characterization of HIV-1 viruses among antiretroviral therapy failure individuals in Suzhou City, China

Dong

TIAN

et al. 2022

Preprint

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“…Moreover, other factors, like the input DNA amounts and concentrations used need to be considered in interpreting viral sequence variation. Our Illumina analysis was done using the minimum DNA requirement (around 5µg) and using 30x of coverage input, conversely, we used the maximum allowed DNA input for nanopore (around 250µg), this may have influenced the sequencing quality and under-represented sequences in Illumina as has been reported before (Illingworth et al 2017) contrasting to the higher diversity detected in nanopore, although similarly to other reports, minority reads were masked and diluted by a greater number of sequences during consensus read construction (Mori et al 2022).…”

Section: Discussionmentioning

confidence: 99%

“…Overall, the different methods were useful for different purposes with Sanger and Nanopore less reliable for iSNVs detection than Illumina, on the other hand, Nanopore was more successful for recombinant FeLV-B detection. With the growth in sequencing studies using different technologies, it will become increasingly important to consider which sequencing method was used in interpreting geographic and temporal variations in viral sequences, as well as designing and standardizing appropriate pipeline analysis to improve the quality of sequence, data interpretation and avoid sequence dilution, effects as has begun to be implemented in diagnostic HIV sequencing (Ávila-Ríos et al 2020, Lee et al 2020, Mori et al 2022). Artificial clustering and estimation of sequencing variation due to sequencing artifact may complicate the interpretation of large viral phylogenies, particularly in viruses like FeLV where there is high virus circulation and recombination.…”

Section: Discussionmentioning

confidence: 99%

Comparison Between Sanger, Illumina and Nanopore Sequencing Evidencing Intra-Host Variation of Feline Leukemia Virus That Infects Domestic Cats

Castillo-Aliaga,

Blanchard,

Castro-Seriche

et al. 2023

Preprint

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Knowledge of Feline Leukemia Virus (FeLV) sequence variation has mainly been developed using Sanger sequencing methods. However, advances in next generation sequencing methods and their broad use in laboratories has been changing our understanding of viral genetics. FeLV sequencing has specific complications with the presence of both exogenous (exFeLV) and endogenous (enFeLV) virus with frequent recombination between them, limiting sequencing approaches. Here we report an FeLV-A and FeLV-B amplicon-based comparison between Sanger, Illumina, and Oxford Nanopore sequencing methods in Chilean domestic cats. We analysed the hypervariable envelope gene, where a higher number of variants as well as recombination with endogenous strains occurs. We detected multiple variants and viral quasispecies infecting the cats. We compared these three methods to evaluate the advantages and disadvantages between them. Although the Sanger method is highly reliable, it showed a high fail rate (many amplicons did not produce useable sequence) and the sequences obtained showed artificial sequence clustering when compared with the NGS methods. Illumina sequencing showed a lower error rate but could not discriminate between exogenous and endogenous viruses. Finally, Oxford Nanopore (MinION) sequencing could successfully detect low-abundance sequences and discriminate between FeLV-A and FeLV-B sequences, although its higher error-rate requires caution in interpretation of the results. Our results indicate advantages and disadvantages for each method, with the purpose of sequencing needing to be considered in the choice of method. Results of large viral phylogenetic trees combing sequences derived from mixed sequencing methods, such as those combining historical and contemporary sequencing need to be considered with some caution as artificial clustering by sequencing method may occur.

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“…Dose escalation was performed when a cytopathic effect was observed. At the indicated time points, genomic DNA was extracted from infected cells using the DNeasy Blood and Tissue Minikit (Qiagen), and the Gag-, Pol-, Env-, and Nef-coding regions were amplified by PrimeSTAR GXL DNA polymerase (Takara Bio) and sequenced (Psomagen or Poochon Scientific) using previously described primers ( 77 , 78 ).…”

Section: Methodsmentioning

confidence: 99%

Epistatic pathways can drive HIV-1 escape from integrase strand transfer inhibitors

Hikichi,

Grover,

Schäfer

et al. 2024

Sci. Adv.

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People living with human immunodeficiency virus (HIV) receiving integrase strand transfer inhibitors (INSTIs) have been reported to experience virological failure in the absence of resistance mutations in integrase. To elucidate INSTI resistance mechanisms, we propagated HIV-1 in the presence of escalating concentrations of the INSTI dolutegravir. HIV-1 became resistant to dolutegravir by sequentially acquiring mutations in the envelope glycoprotein (Env) and the nucleocapsid protein. The selected Env mutations enhance the ability of the virus to spread via cell-cell transfer, thereby increasing the multiplicity of infection (MOI). While the selected Env mutations confer broad resistance to multiple classes of antiretrovirals, the fold resistance is ~2 logs higher for INSTIs than for other classes of drugs. We demonstrate that INSTIs are more readily overwhelmed by high MOI than other classes of antiretrovirals. Our findings advance the understanding of how HIV-1 can evolve resistance to antiretrovirals, including the potent INSTIs, in the absence of drug-target gene mutations.

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Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms

Abstract: HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases.

Cited by 10 publications

References 46 publications

Genetic characterization of HIV-1 viruses among antiretroviral therapy failure individuals in Suzhou City, China

Genetic characterization of HIV-1 viruses among antiretroviral therapy failure individuals in Suzhou City, China

Comparison Between Sanger, Illumina and Nanopore Sequencing Evidencing Intra-Host Variation of Feline Leukemia Virus That Infects Domestic Cats

Epistatic pathways can drive HIV-1 escape from integrase strand transfer inhibitors

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