Reverse transcriptase (RT) is an indispensable component of infectious retroviruses. We have developed an ultrasensitive RT test in which RNA of bacteriophage MS2 serves as the template for RT-mediated cDNA synthesis. A fragment of the cDNA is selectively amplified by polymerase chain reaction and the amplication product is analyzed by Southern blot hybridization or enzyme immunoassay. The procedure was 10'6 to 107 times more sensitive than a conventional RT test and detected as little as 10-9 unit of murine leukemia virus RT, which corresponded to 2.1 X 102 molecules, a number present in 3-11 virions. As a screening assay for filterable particle-associated RT, it was positive with supernatants from cell cultures producing human immunodeficiency virus (11V) type 1 or human T-cell leukemia virus (fITLV) type 1 or 2, but was negative with nonproducer cultures. It was positive with plasma samples from all tested individuals infected with HIV-1, HIV-2, or HTLV-1 and sera from cats infected with feline leukemia virus or feline immunodeficiency virus. Control samples from blood donors or uninfected cats were negative. Density banding experiments with culture supernatants showed that the RT activity was associated with virus particles. The assay should detect all replicationcompetent retroviruses or similar agents. It may be used as a screening assay for such agents, for quantitation of the viral load, drug susceptibility testing of RT, and control of virus inactivation in biological products.Infectious retroviruses are important causative agents of human and animal disease. They all possess a characteristic enzyme, reverse transcriptase (RT), and can thus be detected by assays for this activity (1,2). However, the current tests are insensitive when compared with virus-specific methods. Detection of human immunodeficiency virus type 1 (HIV-1) by RT assay, for example, is 100 times less sensitive than by antigen (Ag) assay (3). Polymerase chain reaction (PCR) is again orders of magnitude more sensitive (4).The narrow detection range of sequence-based tests such as PCR is, however, a disadvantage when detection of retroviruses in general is the aim. Attempts to detect uncharacterized retroviruses by the use of PCR with primers from conserved genomic regions have been met with some success but lack sensitivity and, therefore, require prior isolation and multiplication of the virus in a suitable host cell line usually difficult to find (5). Here, we report the development of an ultrasensitive RT assay capable of detecting retroviruses at a sensitivity hitherto reserved to virus-specific sequence amplification.tained from R. C. Gallo (National Institutes of Health, Bethesda, MD). HTLV-IIIB was also produced in C81-66-45 (hence called C81-66-45/HIV-1). The HTLV-1 producer MT-2 was from I. Miyoshi (Kochi Medical School, Kochi, Japan), the cloned HTLV-2 producer 76D9 was from J. Jendis (our laboratory), and the uninfected monocytoid cell line RC2A was from P. Stoeckbauer (Center Sample Pretreatment. One milliliter o...

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HIV-1 subtypes were determined in newly diagnosed residents of Switzerland. Blood was anonymously collected from patients with a first confirmed positive HIV-1 test result. Viral DNA from the env V3-V5 region was amplified by nested polymerase chain reaction (PCR) and screened for subtype B by heteroduplex mobility assay. All amplicons not identified as B were sequenced. From November 1996 to February 1998, 206 samples were analyzed. Main transmission risks were unprotected heterosexual (55.7%) or homosexual (27.1%) sexual contact or intravenous drug use (12.9%). Subtype B dominated in patients of Swiss, other European, American, or Asian citizenship; particularly high frequencies were found in homosexuals (97%) and drug users (94%). Non-B subtypes including A, C, D, E, F, G, H, a possible B/F recombinant, and a sequence related to J were present in 28.2% (95% confidence interval [CI], 22.9%-35.0%). Non-B were frequent in African citizens (95%), heterosexually infected individuals (44%), and women (43%). Heterosexually infected Swiss males harbored non-B strains in 18% and females in 33%. The results document a change in the epidemiology of newly diagnosed HIV-1 infections in Switzerland: predominance of heterosexual transmission and a high frequency of non-B subtypes.

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.

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.

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