Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance

Althaus, Claudia; Gianella, Sara; Rieder, Peter; Wyl, Viktor von; Kouyos, Roger D.; Niederöst, Barbara; Schmid, A.; Metzner, Karin J.; Joos, Béda; Günthard, Huldrych F.; Fischer, Marek

doi:10.1016/j.jviromet.2010.01.012

Cited by 30 publications

(35 citation statements)

References 23 publications

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“…Concentrated RNA was then extracted using the High Pure viral RNA kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. Using extracted RNA, HIV cDNA was generated using the SuperScript III first-strand synthesis kit (Invitrogen, CA) according to the manufacturers' protocol with specific primer mf302 (2). HIV RNA in seminal plasma was quantified by real-time PCR in an ABI 7900HT thermocycler (Applied Biosystems, CA) with 0.005 M ROX dye (Invitrogen) as a passive reference.…”

Section: Methodsmentioning

confidence: 99%

Associations between Virologic and Immunologic Dynamics in Blood and in the Male Genital Tract

Gianella

Strain

Rought

et al. 2012

J Virol

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To determine the influence of asymptomatic genital viral infections on the cellular components of semen and blood, we evaluated the associations between the numbers and activation statuses of CD4 ؉ and CD8 ؉ T lymphocytes in both compartments and the seminal levels of cytomegalovirus (CMV), herpes simplex virus (HSV), and human immunodeficiency virus 1 (HIV). Paired blood and semen samples were collected from 36 HIV-infected antiretroviral-naïve individuals and from 40 HIV-uninfected participants. We performed multiparameter flow cytometry analysis (CD45, CD45RA, CD3, CD4, CD8, and CD38) of seminal and blood cellular components and measured HIV RNA and CMV and HSV DNA levels in seminal and blood plasma by real-time PCR. Compared to HIV-uninfected participants, in the seminal compartment HIV-infected participants had higher levels of CMV (P < 0.05), higher numbers of total CD3 ؉ (P < 0.01) and CD8 ؉ subset (P < 0.01) T lymphocytes, and higher CD4 ؉ and CD8 ؉ T lymphocyte activation (RA-CD38 ؉ ) (P < 0.01). Seminal CMV levels positively correlated with absolute numbers of CD4 ؉ and CD8 ؉ T cells in semen (P < 0.05) and with the activation status of CD4 ؉ T cells in semen and in blood (P < 0.01). HIV levels in semen (P < 0.05) and blood (P < 0.01) were positively associated with T-cell activation in blood. Activation of CD8 ؉ T cells in blood remained an independent predictor of HIV levels in semen in multivariate analysis. The virologic milieu in the male genital tract strongly influences the recruitment and activation of immune cells in semen and may also modulate T-cell immune activation in blood. These factors likely influence replication dynamics, sexual transmission risk, and disease outcomes for all three viruses.

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Section: Methodsmentioning

confidence: 99%

Associations between Virologic and Immunologic Dynamics in Blood and in the Male Genital Tract

Gianella

Strain

Rought

et al. 2012

J Virol

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“…HIV-1 has substantial genetic variability due to the high rate of mutation, 45,46 error-prone reverse transcriptase, 47 and frequent genetic recombination, 48 and it is therefore challenging to design universal primers and probes that detect all clades and subtypes of HIV-1. 49 It is well known that mismatches between primers, probes, and target sequences can have detrimental effects on the sensitivity of real-time PCR assays. Malnati et al performed a systematic examination of the effects of mismatches between primers and the sequences of various HIV-1 isolates.…”

Section: Fig 4 (A)mentioning

confidence: 99%

The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques

Janocko

Althouse

Brand

et al. 2015

AIDS Research and Human Retroviruses

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“…The TaqMan probe used was mf319tq (59-TGCAGCTTCCTCATTGATGGT-39), as described by Althaus et al (26). The PCR product mapped to the unspliced region of Gag.…”

Section: Reagents and Cell-culture Experimentsmentioning

confidence: 99%

TLR8 Activates HIV from Latently Infected Cells of Myeloid-Monocytic Origin Directly via the MAPK Pathway and from Latently Infected CD4+ T Cells Indirectly via TNF-α

Schlaepfer

Speck

2011

The Journal of Immunology

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We previously showed that the TLR7/8 agonist, R-848, activated HIV from cells of myeloid-monocytic origin. In this work, we show that this effect was solely due to triggering TLR8 and that NF-κB was involved in the TLR8-mediated activation of HIV from latently infected cells of myeloid-monocytic origin. Inhibition of Erk1/2 or p38α resulted in attenuation of TLR8-mediated activation of NF-κB. Western blots confirmed that TLR8 triggering activated Erk1/2 and p38α but, surprisingly, not JNK. Although the Erk1/2 inhibitors resulted in a less attenuated TLR8-mediated NF-κB response than did p38α inhibitors, they had a more pronounced effect on blocking TLR8-mediated HIV replication, indicating that other transcription factors controlled by Erk1/2 are involved in TLR8-mediated HIV activation from latently infected cells. TNF-α, which was secreted subsequent to TLR8 triggering, contributed to the activation of HIV from the latently infected cells in an autocrine manner, revealing a bimodal mechanism by which the effect of TLR8 triggering can be sustained. We also found that TNF-α secreted by myeloid dendritic cells acted in a paracrine manner in the activation of HIV from neighboring latently infected CD4+ T cells, which do not express TLR8. Notably, monocytes from highly active antiretroviral therapy-treated HIV+ patients with suppressed HIV RNA showed a robust TNF-α secretion in response to TLR8 agonists, pointing to a functional TLR8 signaling axis in HIV infection. Thus, triggering TLR8 represents a very promising strategy for attacking the silent HIV from its reservoir in HIV+ patients treated successfully with highly active antiretroviral therapy.

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Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance

Cited by 30 publications

References 23 publications

Associations between Virologic and Immunologic Dynamics in Blood and in the Male Genital Tract

Associations between Virologic and Immunologic Dynamics in Blood and in the Male Genital Tract

The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques

TLR8 Activates HIV from Latently Infected Cells of Myeloid-Monocytic Origin Directly via the MAPK Pathway and from Latently Infected CD4+ T Cells Indirectly via TNF-α

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