Improved Mg2+-based reverse transcriptase assay for detection of primate retroviruses

Abstract

The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new 32P-based, Mg2+-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID50s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immunodeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of recombinant HIV-1 RT enzyme indicated that 10(-5) U, which is equivalent to 4.25 x 10(4) virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg2+-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans.

FIG. 1

Linear range of the RT assay. The linearity of the RT assay was determined by HIV-1_LAI and SIV_mac-mm239. Ten microliters of undiluted virus was assayed at the indicated time intervals. The RT activity is indicated. The mean ± standard deviation from one experiment is indicated. ▴, HIV-1; ■, SIV; ○, medium.

FIG. 2

Detection of recombinant HIV-1 RT enzyme. Serial dilutions of HIV-1 RT enzyme were analyzed by the RT assay. The RT activity is indicated. The mean ± standard deviation was calculated for four spots obtained from two RT assays which were done in duplicate with one dilution series.