A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy

Weber, Jan; Rangel, Héctor R.; Chakraborty, Bikram; Tadele, Mahlet; Martı́nez, Miguel Ángel; Martínez-Picado, Javier; Marotta, Michael; Mirza, Muneer; Ruiz, Lı́dia; Clotet, Bonaventura; Wrin, Terri; Petropoulos, Christos J.; Quiñones‐Mateu, Miguel E.

doi:10.1099/vir.0.19123-0

Cited by 38 publications

(53 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Xanthi) in contrast to the other (N. clevelandii) responds with necrotic symptoms to infection with PVY N , and employing a series of selected isolates and mutants, the link between necrosis property and fitness of genotypes was tested. Thus, we measured both the number of progeny produced and the competitiveness of these viruses in mixed-infection plants for wild- (Carrasco et al, 2007a), qPCR, a method already used efficiently to determine the proportion of two animal viruses in competition experiments (Weber et al, 2003;Van Maarseveen et al, 2006), could be advantageously applied to plant viruses to estimate the fitness of selected genotypes. Thus, in addition to the previously published PVY qPCR assays (Balme-Sinibaldi et al, 2006), a new assay that specifically targeted A 2271 of the PVY sequence was developed.…”

Section: Discussionmentioning

confidence: 99%

“…The fitness of various isolates can be estimated by a measure of replication rate and competitiveness (Chao, 1990;Holland et al, 1991). Recent technologies such as quantitative PCR (qPCR) allow the accurate measurement of viral RNA concentration during in vivo studies, based on which the relative copy numbers of viral genomes can be estimated (Weber et al, 2003;Van Maarseveen et al, 2006;Carrasco et al, 2007a). In the present paper, these specific quantification tools (e.g.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants

Rolland

Kerlan

Jacquot

2009

Journal of General Virology

View full text Add to dashboard Cite

The prevalence of necrotic potato virus Y (PVY) in natural populations could reflect increased fitness of necrotic isolates. In this paper, the effects of the acquisition of molecular determinants (A/G 2213 and A/C 2271 ) involved in necrosis capacity on both the number of progeny produced and the competitiveness of PVY were characterized. The relationship between necrosis and fitness was tested using (i) Nicotiana tabacum cv. competitiveness. In contrast, nucleotides involved in necrotic properties were associated with decreased fitness. Finally, in the host that did not respond to infection with necrosis, the benefit associated with the PVY N -605 genetic background was higher than the cost associated with the acquisition of molecular determinants involved in necrosis capacity. The opposite result was obtained in the host responding to the infection with necrosis. These results indicate that the emergence of necrotic isolates from a non-necrotic population is unlikely in tobacco.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants

Rolland

Kerlan

Jacquot

2009

Journal of General Virology

View full text Add to dashboard Cite

show abstract

“…Some examples of measuring virus directly include quantitation of a viral protein, such as p24, by enzyme-linked immunosorbent assay (106,156,163); measurement of reverse transcriptase activity (164, 175); and quantitation of proviral DNA (116). In addition, in growth competition assays, the relative amounts of test and reference strains can be quantified by sequence analysis of proviral DNA or viral RNA (65,69,78,86,117,154,155,169,174), heteroduplex tracking assays (HTAs) (140), or allele-specific real-time PCR (33,142,176). It should be noted that the latter methods do not provide information on the extent of viral replication.…”

Section: Cell Culture Assaysmentioning

confidence: 99%

“…Real-time PCR has also been used to quantify the relative amounts of test and reference strains in a growth competition assay by allele-specific amplification of viral nucleic acid at a codon where the two variants differ (33,142,176). Real-time PCR has an advantage over other molecular assays used to quantify the relative proportion of the test and reference strains because of its high throughput and wide linear range of detection.…”

Section: Cell Culture Assaysmentioning

confidence: 99%

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

Dykes

Demeter

2007

Clin Microbiol Rev

View full text Add to dashboard Cite

SUMMARY The relative fitness of a variant, according to population genetics theory, is that variant's relative contribution to successive generations. Most drug-resistant human immunodeficiency virus type 1 (HIV-1) variants have reduced replication fitness, but at least some of these deficits can be compensated for by the accumulation of second-site mutations. HIV-1 replication fitness also appears to influence the likelihood of a drug-resistant mutant emerging during treatment failure and is postulated to influence clinical outcomes. A variety of assays are available to measure HIV-1 replication fitness in cell culture; however, there is no agreement regarding which assays best correlate with clinical outcomes. A major limitation is that there is no high-throughput assay that incorporates an internal reference strain as a control and utilizes intact virus isolates. Some retrospective studies have demonstrated statistically significant correlations between HIV-1 replication fitness and clinical outcomes in some patient populations. However, different studies disagree as to which clinical outcomes are most closely associated with fitness. This may be in part due to assay design, sample size limitations, and differences in patient populations. In addition, the strength of the correlations between fitness and clinical outcomes is modest, suggesting that, at present, it would be difficult to utilize these assays for clinical management.

show abstract

“…Thus, a modified drug susceptibility assay, based on RVs and a single round of replication, is one of the methods most frequently used to measure HIV-1 RC (PhenoSense TM ; Monogram Biosciences, San Francisco, CA, USA) [6,34,35]. A potential limitation of methods based on single-cycle assays is their lower sensitivity to detect differences between wt and mutated viruses carrying mutations leading to small variations in RC.Results using RVs and clinical viral isolates suggest that the major determinants of viral fitness are carried in the polymerase (pol) gene in patients treated with HAART [19,28]. Although it is widely accepted that amino acid changes conferring resistance to PR inhibitors are major determinants of replicative impairment of mutant viruses [18,21], significant replication defects have also been associated with mutations that confer resistance to RT inhibitors [17,33].…”

mentioning

confidence: 99%

A new strategy based on recombinant viruses for assessing the replication capacity of HIV‐1

et al. 2008

View full text Add to dashboard Cite

ObjectiveIn heavily pretreated patients, resistance mutations arise in both protease (PR) and reverse transcriptase (RT) sequences; however, the relative impact of PR and RT mutations on viral fitness cannot be evaluated with the majority of systems. To address this issue we have developed a model based on recombinant viruses (RVs) that allows the analysis of the replication capacity (RC) of viral populations in which PR and RT are cloned either in combination or separately. MethodsRVs were generated for full-length polymerase (pol) gene, PR or RT sequences from nine naïve and 14 heavily pretreated HIV-infected patients in therapeutic failure. The relative RC was assessed by comparing luciferase activity between mutant RV and wild-type (wt) isolates. ResultsA strong decrease (460%) in the RC of the pol RV population was observed in the 14 heavily pretreated patients as compared with the wt RVs. The analysis of PR and RT RVs from these patients showed that the decrease in RC was mainly attributable to PR sequences in three of these 14 patients and to RT sequences in seven of these patients. In the four remaining patients, PR and RT sequences independently reduced the RC of the RVs to similar extents. ConclusionsDifferent patterns of mutations in either PR or RT have a strong impact on RC in highly experienced HIV-infected patients.Keywords: drug resistance, protease, Renilla luciferase, replicative fitness, reverse transcriptase IntroductionTreatment of HIV-1-infected patients with highly active antiretroviral therapy (HAART) has significantly reduced the rate of morbidity and mortality associated with HIV-1 infections [1]. Although the main goal of HAART in the management of HIV disease is the suppression of viral replication, defined as the achievement and maintenance of undetectable viraemia in plasma [2,3], failure to achieve complete viral suppression is common [4]. However, despite virological failure, a number of patients have exhibited 'discordant CD4 cell/viral load' responses, defined as stable or increasing CD4 cell counts while viraemia persists [5][6][7][8][9]. Different immune-based mechanisms have been proposed to explain this paradoxical phenomenon: impaired viral replication in thymic tissue [10], decreased cell death by activation-induced apoptosis [11], or regeneration of HIV-specific immune responses [12,13]. However, several studies have suggested a direct decrease in replication capacity (RC) as a result of resistance mutations and a consequent reduction in cytopathic effect as the cause of the sustained immunological benefit observed in the presence of a high viral load.Viral fitness can be defined as the ability of a virus to replicate in a given environment in comparison with a reference strain. This is a complex parameter that depends on multiple viral and host factors. Alterations in any of the viral genes involved in cell entry, reverse transcription, integration, gene expression, assembly, egress or *Present address: Department of Infectious Diseases, Hospital Clinic IDIBAPS, Barcelo...

show abstract

A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy

Abstract: A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy

Cited by 38 publications

References 45 publications

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

A new strategy based on recombinant viruses for assessing the replication capacity of HIV‐1

Contact Info

Product

Resources

About