J. Simon Gronowitz

We describe a procedure (ExaVir™ Load) to carry out human immunodeficiency virus‐1 (HIV‐1) viral load testing using reverse transcriptase (RT) recovered from HIV‐1 virions in plasma. Samples from individuals infected with HIV‐1 were treated with a sulphydryl‐reactive agent to inactivate endogenous polymerases. Virions were then immobilised on a gel and washed in individual mini columns to remove RT‐inhibiting antibodies, antiviral drugs, and other RT inhibitors. Immobilised virions were lysed finally, and the viral RT eluted. The amount of RT recovered was quantified by a sensitive RT activity assay using either colorimetry or fluorimetry to detect DNA produced by RT. The “RT load” values of 390 samples from 302 HIV‐1 patients living in Sweden were compared to results obtained with an HIV‐1 RNA viral load assay. The correlation between the two tests was r = 0.90, P < 0.0001. Four of 202 samples from healthy blood donors gave low positive values in the RT test. All samples in a panel with 10 HIV‐1 subtypes were positive by the RT load. The RT load test provides a technically less demanding and cost‐effective alternative to methods based on nucleic acid amplification. Being insensitive to genetic drift occurring in HIV, the assay should be of particular use in resource‐limited settings, where different subtypes and recombinant HIV strains occur. J. Med. Virol. 71:347–359, 2003.

A new sensitive colorimetric reverse transcriptase (RT) activity assay utilizing a 96-well microtitre plate format, with solid phase-conjugated polyadenylic acid (prA), was investigated for simple analyses of the RT inhibiting capacity and mode of action of various substances. Three different technical procedures using the assay were evaluated: (i) direct lC₅₀ determinations with various substances, using four different combinations of primer and dNTP amounts; (ii) analyses of the capacity of the substances to interfere with the binding of RT to template or template-primer (BIC₅₀); (iii) analyses of the capacity of the substances to destroy the template-primer in presence or absence of RT (TDC₅₀). The assay was found to be useful for all three purposes using small amounts of recombinant RT. In the IC₅₀ analyses, the test substances gave values similar to those reported for soluble RT assays, and the values varied in accordance with their known mode of action in relation to the combination of primer and dNTP amount used. Only one of the substances, prG, in addition to DNA and RNA gave true RT binding inhibition. The template destruction assay showed that chain terminating substances gave destruction at low inhibitor concentrations. Furthermore, this destruction was RT-dependent, in contrast to the destruction obtained with substances that can base-pair with the template or primer. For optimum information on mode of action of a given substance all three assay procedures should be used. The use of the assay in relation to the screening and analyses of new RT inhibitory substances and characterization of RT in primary isolates or plasma is discussed.