Sensitive detection and quantification of particle-associated reverse transcriptase in plasma of HIV-1-infected individuals by the product-enhanced reverse transcriptase (PERT) assay

Abstract

Tests for the enzyme reverse transcriptase (RT) should permit the detection of all infectious retroviruses, provided that these are present as extracellular particles. The capability of a new procedure, named product-enhanced reverse transcriptase (PERT) assay, to detect HIV-1 in fresh human plasma was compared with that of the polymerase chain reaction (PCR) for viral RNA. Both procedures had identical dilution endpoints corresponding to 10(2) particles/ml. All 30 samples from HIV-1 positive patients at different stages contained RT activity whose level was significantly correlated with viral RNA and corresponded to 553-417,000 particles/ml. In HIV-1 low titer performance and seroconversion panels, the PERT assay detected more positives than PCR for viral RNA. Three of 160 blood donors exhibited elevated RT activity, indicating a prevalence of 1.9% (95% CI 0.4-5.3%). One positive donor, with laboratory parameters suggesting a mild chronic liver impairment, exhibited RT activity comparable to that of HIV positives, but was consistently negative by various tests for hepatitis viruses, cytomegalovirus, the HIVs and HTLVs. The results suggest that the PERT assay is more sensitive for detection of HIV-1 contamination of plasma than RNA PCR. However, it is not affected adversely by viral sequence variability, and may therefore, also detect HIV-1 subtype O, and additional retroviruses as yet undetectable by PCR.

Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma

“…Using the CRISPRi HML-2 construct or abacavir treatment to target HML-2 expression, we observed a decrease in the activity of HML-2 RT using a product-enhanced RT (PERT) assay on the media from our transfected cells. The PERT assay was developed to detect retroviral RT activity utilizing native RT enzymes from cellular supernatant ( 39 ). Using an RNA template from an MS2 bacteriophage plasmid and a standardized curve for RT activity generated from HIV-1 RT, we identified increased endogenous RT expression in our patient-derived GBM cells as compared with the A172 glioma cell line ( 39 ) ( Figure 10, C–E ).…”

“…The PERT assay was developed to detect retroviral RT activity utilizing native RT enzymes from cellular supernatant ( 39 ). Using an RNA template from an MS2 bacteriophage plasmid and a standardized curve for RT activity generated from HIV-1 RT, we identified increased endogenous RT expression in our patient-derived GBM cells as compared with the A172 glioma cell line ( 39 ) ( Figure 10, C–E ). The PERT assay demonstrated a marked decrease in HML-2 RT activity after 48-hour exposure to abacavir and after transfection with the CRISPRi-HML-2 construct ( Supplemental Figure 8E ).…”

Sensitive detection systems for infectious agents in xenotransplantation*

“…37,45 Mitogen treatment has been shown to enhance viral mRNA expression and virus release and is therefore a good tool to increase the sensitivity of these detection methods. 20,37,46 A special form of the RT assay is the product-enhanced reverse transcriptase (PERT) assay, 47 which was also used to detect PERV. 48 Production of PERV particles was shown by electron microscopy (Figure 2), 20,41,42,46 and using PERV-specific antibodies, the specificity has been demonstrated by immune gold electron microscopy.…”

Use of reverse-transcriptase-based HIV-1 viral load assessment to confirm low viral loads in newly diagnosed patients in Switzerland

“…In Switzerland, regulations on HIV confirmation testing issued by the Swiss Federal Office of Public Health (SFOPH) in 2006 therefore request that a VL ≤1’000 cp/ml be confirmed by the product-enhanced reverse transcriptase (PERT) assay, a sequence-independent test that quantifies retrovirus particles based on their content of enzymatically active reverse transcriptase (RT) [4]. This test has been available in our country since 1994, and its sensitivity is comparable to NAT [5,6]. VL based on the PERT assay, or on other RT-based tests, have been shown to correlate well with RNA-based VL over a wide dynamic range (10 2 -10 6 cp/ml) [6-8].…”

“…This test has been available in our country since 1994, and its sensitivity is comparable to NAT [5,6]. VL based on the PERT assay, or on other RT-based tests, have been shown to correlate well with RNA-based VL over a wide dynamic range (10 2 -10 6 cp/ml) [6-8]. …”