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Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management.

Grossmann S, Nowak P, Neogi U - J Int AIDS Soc (2015)

Bottom Line: The N384I connection mutation was additionally detected by NFLG in two samples.Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies.Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Introduction: HIV-1 near full-length genome (HIV-NFLG) sequencing from plasma is an attractive multidimensional tool to apply in large-scale population-based molecular epidemiological studies. It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups. Thus, the main objective of this study was to develop a simplified subtype-independent, cost- and labour-efficient HIV-NFLG protocol that can be used in clinical management as well as in molecular epidemiological studies.

Methods: Plasma samples (n=30) were obtained from HIV-1B (n=10), HIV-1C (n=10), CRF01_AE (n=5) and CRF01_AG (n=5) infected individuals with minimum viral load >1120 copies/ml. The amplification was performed with two large amplicons of 5.5 kb and 3.7 kb, sequenced with 17 primers to obtain HIV-NFLG. GRT was validated against ViroSeq™ HIV-1 Genotyping System.

Results: After excluding four plasma samples with low-quality RNA, a total of 26 samples were attempted. Among them, NFLG was obtained from 24 (92%) samples with the lowest viral load being 3000 copies/ml. High (>99%) concordance was observed between HIV-NFLG and ViroSeq™ when determining the drug resistance mutations (DRMs). The N384I connection mutation was additionally detected by NFLG in two samples.

Conclusions: Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies. It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies. Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

No MeSH data available.


Amplification strategy of HIV-1 NFLG. (a) HIV-1 gene map in HXB2 [GenBank:K03455] co-ordinates Adapted from Los Alamos Database (www.hiv.lanl.gov). (b) The locations of all primers used for cDNA synthesis, nested and semi-nested PCR are shown. The 8.8 kb genome is amplified into two fragments: fragment 1, 5.5 kb (gag-vpu) and fragment 2, 3.7 kb (vif-3′-LTR) with 400 bp overlap. (c) Representative amplification of all four subtypes is presented.
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Figure 0001: Amplification strategy of HIV-1 NFLG. (a) HIV-1 gene map in HXB2 [GenBank:K03455] co-ordinates Adapted from Los Alamos Database (www.hiv.lanl.gov). (b) The locations of all primers used for cDNA synthesis, nested and semi-nested PCR are shown. The 8.8 kb genome is amplified into two fragments: fragment 1, 5.5 kb (gag-vpu) and fragment 2, 3.7 kb (vif-3′-LTR) with 400 bp overlap. (c) Representative amplification of all four subtypes is presented.

Mentions: The near full-length genome from the samples was amplified in two fragments using nested and semi-nested polymerase chain reactions (PCR) with seven sets of primers numbered as HXB2 [GenBank:K03455] co-ordinates (Figure 1a and Table 1). First-strand cDNA synthesis was performed using the SuperScript® III RT enzyme (Invitrogen, Life Technologies, MA, USA). For fragment 1 Gag-Vpu (herein F1Gag-Vpu), cDNA was synthesized with the primer 6352R (10 pmol) and for fragment 2, Tat-3LTR (herein F2Tat-3LTR), an oligo (dT)18 primer (50 pmol) (Thermo Scientific) was used. A first master mix of 5 µl extracted viral RNA was combined with 1 µl of 10 mM dNTP mix, 1 µl of 6352R or oligo(dT)18 primer and 5 µl of PCR grade water. The reaction mix was heated to 65°C for five minutes and immediately placed on ice for two minutes. A second master mix consisting of 4 µl of 5× First-Strand Buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2), 1 µl 0.1 M DTT, 1 µl of RiboLock RNase inhibitor (40 U/µl; Thermo Scientific) and 2 µl of SuperScript-III RT (200 U/µl) was subsequently added to the first mastermix. The final 20 µl reaction mix was then incubated at 25°C for five minutes followed by 55°C for one hour and finally 70°C for 10 minutes to terminate the reaction.


Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management.

Grossmann S, Nowak P, Neogi U - J Int AIDS Soc (2015)

Amplification strategy of HIV-1 NFLG. (a) HIV-1 gene map in HXB2 [GenBank:K03455] co-ordinates Adapted from Los Alamos Database (www.hiv.lanl.gov). (b) The locations of all primers used for cDNA synthesis, nested and semi-nested PCR are shown. The 8.8 kb genome is amplified into two fragments: fragment 1, 5.5 kb (gag-vpu) and fragment 2, 3.7 kb (vif-3′-LTR) with 400 bp overlap. (c) Representative amplification of all four subtypes is presented.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482814&req=5

Figure 0001: Amplification strategy of HIV-1 NFLG. (a) HIV-1 gene map in HXB2 [GenBank:K03455] co-ordinates Adapted from Los Alamos Database (www.hiv.lanl.gov). (b) The locations of all primers used for cDNA synthesis, nested and semi-nested PCR are shown. The 8.8 kb genome is amplified into two fragments: fragment 1, 5.5 kb (gag-vpu) and fragment 2, 3.7 kb (vif-3′-LTR) with 400 bp overlap. (c) Representative amplification of all four subtypes is presented.
Mentions: The near full-length genome from the samples was amplified in two fragments using nested and semi-nested polymerase chain reactions (PCR) with seven sets of primers numbered as HXB2 [GenBank:K03455] co-ordinates (Figure 1a and Table 1). First-strand cDNA synthesis was performed using the SuperScript® III RT enzyme (Invitrogen, Life Technologies, MA, USA). For fragment 1 Gag-Vpu (herein F1Gag-Vpu), cDNA was synthesized with the primer 6352R (10 pmol) and for fragment 2, Tat-3LTR (herein F2Tat-3LTR), an oligo (dT)18 primer (50 pmol) (Thermo Scientific) was used. A first master mix of 5 µl extracted viral RNA was combined with 1 µl of 10 mM dNTP mix, 1 µl of 6352R or oligo(dT)18 primer and 5 µl of PCR grade water. The reaction mix was heated to 65°C for five minutes and immediately placed on ice for two minutes. A second master mix consisting of 4 µl of 5× First-Strand Buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2), 1 µl 0.1 M DTT, 1 µl of RiboLock RNase inhibitor (40 U/µl; Thermo Scientific) and 2 µl of SuperScript-III RT (200 U/µl) was subsequently added to the first mastermix. The final 20 µl reaction mix was then incubated at 25°C for five minutes followed by 55°C for one hour and finally 70°C for 10 minutes to terminate the reaction.

Bottom Line: The N384I connection mutation was additionally detected by NFLG in two samples.Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies.Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Introduction: HIV-1 near full-length genome (HIV-NFLG) sequencing from plasma is an attractive multidimensional tool to apply in large-scale population-based molecular epidemiological studies. It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups. Thus, the main objective of this study was to develop a simplified subtype-independent, cost- and labour-efficient HIV-NFLG protocol that can be used in clinical management as well as in molecular epidemiological studies.

Methods: Plasma samples (n=30) were obtained from HIV-1B (n=10), HIV-1C (n=10), CRF01_AE (n=5) and CRF01_AG (n=5) infected individuals with minimum viral load >1120 copies/ml. The amplification was performed with two large amplicons of 5.5 kb and 3.7 kb, sequenced with 17 primers to obtain HIV-NFLG. GRT was validated against ViroSeq™ HIV-1 Genotyping System.

Results: After excluding four plasma samples with low-quality RNA, a total of 26 samples were attempted. Among them, NFLG was obtained from 24 (92%) samples with the lowest viral load being 3000 copies/ml. High (>99%) concordance was observed between HIV-NFLG and ViroSeq™ when determining the drug resistance mutations (DRMs). The N384I connection mutation was additionally detected by NFLG in two samples.

Conclusions: Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies. It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies. Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

No MeSH data available.