Detection, Quantification, and Characterisation of HIV/SIV

Berry, Neil; Herrera, Carolina; Cranage, Martin

doi:10.1007/978-1-60761-817-1_9

Cited by 9 publications

(8 citation statements)

References 19 publications

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“…Tissue samples were digested with proteinase K and phenol/chloroform following standard protocols according to [46]. After DNA extraction, qPCR was performed using Taqman Universal PCR Master mix (Applied Biosystems) and primers and complementary probes with sequences located in conserved gag regions as reported for the qRT-PCR assay.…”

Section: Methodsmentioning

confidence: 99%

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Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions

et al. 2013

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BackgroundLive attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV.ResultsUsing a novel, doxycycline dependent, replication-competent live-attenuated SIVmac239Δnef (SIV-rtTAΔnef), we show that under replication-permissive conditions SIV-rtTAΔnef is fully viable. Twelve rhesus macaques were infected with a peak plasma vRNA on average two log10 lower than in 6 macaques infected with unconditionally replication-competent SIVΔnef. Consistent with the attenuated phenotype of the viruses the majority of animals displayed low or undetectable levels of viraemia by 42-84 days after infection. Next, comparison of circulating T cells before and after chronic infection with parental SIVΔnef revealed a profound global polarisation toward CD28-CCR7- T-effector memory 2 (TEM2) cells within CD95+CD4+ and CD95+CD8+ populations. Critically, a similar effect was seen in the CD95+ CD4+ population and to somewhat lesser extent in the CD95+ CD8+ population of SIV-rtTAΔnef chronically infected macaques that were maintained on doxycycline, but was not seen in animals from which doxycycline had been withdrawn. The proportions of gut-homing T-central memory (TCM) and TEM defined by the expression of α4β7 and CD95 and differential expression of CD28 were increased in CD4 and CD8 cells under replication competent conditions and gut-homing CD4 TCM were also significantly increased under non-permissive conditions. TEM2 polarisation was seen in the small intestines of animals under replication permissive conditions but the effect was less pronounced than in the circulation. Intracellular cytokine staining of circulating SIV-specific T cells for IL-2, IFN-γ, TNF-α and IL-17 showed that the extent of polyfunctionality in CD4 and CD8 T cells was associated with replication permissivity; however, signature patterns of cytokine combinations were not distinguishable between groups of macaques.ConclusionTaken together our results show that the global T memory cell compartment is profoundly skewed towards a mature effector phenotype by attenuated SIV. Results with the replication-conditional mutant suggest that maintenance of this effect, that may be important in vaccine design, might require persistence of replicating virus.

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Section: Methodsmentioning

confidence: 99%

“…SIV proviral DNA concentrations were calculated using the Mx3005P software, expressed as copies of SIV DNA per 10 5 PBMC/mononuclear cells (MNC). A single SIV DNA copy determined the limit of assay detection, as determined by Poisson statistics and as previously reported [46]. …”

Section: Methodsmentioning

confidence: 99%

Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions

et al. 2013

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“…Several laboratory developed assays for HIV-2 RNA were developed to address the need for detection and quantification of HIV-2 nucleic acid in plasma, however, standardization, validation and regulatory approvals have been challenging [8], [10], [11], [12], [13], [14]. A significant challenge in HIV-2 assay validation is the limited resources for diverse HIV-2 isolates for nucleic acid source material.…”

Section: Introductionmentioning

confidence: 99%

Discrepant Amplification Results during the Development of an Assay Leads to Reclassification of Two AIDS Reagent Repository HIV-2 Isolates as HIV-1

et al. 2014

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The development and verification of HIV-2 assays depends in part on the availability of well-characterized samples, including those from reagent repositories. During the development of an HIV-2 RNA quantification assay, two HIV-2 viral isolates (CDC 301340 and CDC 301342) obtained from the NIAID AIDS Reagent and Reference Repository were not detected leading to an investigation. Two HIV-2 primers/probe sets of known performance in real-time viral RNA quantification assays, targeting different regions of the virus, also failed to generate RT-PCR products for these two isolates. These isolates were tested in the HIV-1 specific COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 (Roche Molecular Diagnostics) and were quantified at high copy number. Other HIV-2 isolates tested were not amplified in the COBAS HIV-1 TaqMan assay. Furthermore, the discrepant isolates were highly reactive in an HIV-1 p24 antigen test while the other HIV-2 isolates showed very weak, if any, cross-reactivity with the HIV-1 p24 assay. Phylogenetic tree analysis of sequences from the protease-reverse transcriptase regions of the discrepant HIV-2 isolates mapped with HIV-1 Group M, Subtype CRF02_AG confirming these isolates were of HIV-1 origin and had been misclassified as HIV-2. The use of misclassified isolates in the verification of molecular and immunological assays can lead to misinterpretation of test results, misdirection of efforts into assay redesign and increased development costs. The results of this study were shared with the NIAID AIDS Reagent Program, leading to the reclassification of the two discrepant isolates as HIV-1.

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“…Plasma viral load (PVL) is widely used to assess the status and progression of simian immunodeficiency virus (SIV) infections in non-human primates (NHP) [6,27,37]. Quantitative polymerase chain reaction (qPCR) is commonly used for the determination of PVL [5,12,21,28,35,40]. Plasma is a challenging specimen for the quantification of viral RNA due to the presence of PCR inhibitors and RNases, and removing such inhibitors improved analytical sensitivity for detection of viral RNA [9,10,23,26].…”

Section: Introductionmentioning

confidence: 99%

“…Commercially available aRNAs have been used in human diagnostics for quality assurance in viral load determination, including human immunodeficiency virus (HIV) . To date, however, aRNA has not been used for quality control in quantification of SIV RNA .…”

Section: Introductionmentioning

confidence: 99%

Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA

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Panganiban

et al. 2013

J of Medical Primatology

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Introduction Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay. Methods The PVL quantification procedure was optimized by inclusion of an exogenous control Hepatitis C Virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660 and the LTR region of SIVagmSAB were also optimized. Results Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV-RNA in the same samples using the “industry standard” method of branched-DNA (bDNA) signal amplification. Conclusions Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs.

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Detection, Quantification, and Characterisation of HIV/SIV

Cited by 9 publications

References 19 publications

Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions

Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions

Discrepant Amplification Results during the Development of an Assay Leads to Reclassification of Two AIDS Reagent Repository HIV-2 Isolates as HIV-1

Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA

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