Genetic variability in coding regions of the surface antigen and reverse transcriptase domain of hepatitis B virus polymerase, Colombia, 2002-2014

Introduction: Despite the availability of an effective vaccine and treatment to reduce the viral load and progressive hepatocellular injury, approximately 240 million people worldwide are chronically infected with the hepatitis B virus (HBV). In Colombia, the circulation of different viral genotypes has been confirmed. Mutations in the genome have been associated to antiviral therapy resistance, viral escape to neutralizing antibodies, occult infection and progression to hepatocellular carcinoma. Objective: To identify the genotypes and the presence of mutations in the coding region of the surface (S) antigen and the reverse transcriptase (RT) domain of the polymerase of HBV obtained from serum samples for hepatitis B diagnosis received by the Instituto Nacional de Salud during the period 2002-2014. Materials and methods: A total of 495 serum samples with previous HBsAg reactive result were used for molecular detection. A fragment of 1,591 nucleotides was sequenced, and the corresponding phylogenetic analysis was performed. Results: We detected the viral genome of HBV in 66 samples and 28 were successfully sequenced. The phylogenetic analysis allowed the identification of subgenotypes F3 and A2. The L180M and M204V resistance mutations were simultaneously identified in one sample, while the I169L resistance mutation was identified in another one. A single escape mutation, P120Q, was identified in one more. Two samples showed a deletion of 105 nucleotides in the preS1-preS2 region. Conclusions: The circulation of genotypes/subgenotypes F3 and A2 of HBV in Colombia was corroborated, as well as the presence of some resistance and escape mutations. The present study constitutes a contribution to the molecular epidemiology of HBV in Colombia.

Endogenous LINE-1 (Long Interspersed Nuclear Element-1) Reverse Transcriptase Activity in Platelets Controls Translational Events Through RNA-DNA Hybrids.

OBJECTIVE: One source of endogenous reverse transcriptase (eRT) activity in nucleated cells is the LINE-1/L1 (long interspersed nuclear element-1), a non-LTR retrotransposon that is implicated in the regulation of gene expression. Nevertheless, the presence and function of eRT activity and LINE-1 in human platelets, an anucleate cell, has not previously been determined. APPROACH AND RESULTS: We demonstrate that human and murine platelets possess robust eRT activity and identify the source as being LINE-1 ribonucleoprotein particles. Inhibition of eRT in vitro in isolated platelets from healthy individuals or in people with HIV treated with RT inhibitors enhanced global protein synthesis and platelet activation. If HIV patients were treated with reverse transcriptase inhibitor, we found that platelets from these patients had increased basal activation. We next discovered that eRT activity in platelets controlled the generation of RNA-DNA hybrids, which serve as translational repressors. Inhibition of platelet eRT lifted this RNA-DNA hybrid-induced translational block and was sufficient to increase protein expression of target RNAs identified by RNA-DNA hybrid immunoprecipitation. CONCLUSIONS: Thus, we provide the first evidence that platelets possess L1-encoded eRT activity. We also demonstrate that platelet eRT activity regulates platelet hyperreactivity and thrombosis and controls RNA-DNA hybrid formation and identify that RNA-DNA hybrids function as a novel translational control mechanism in human platelets.

Cytotoxicity of human endogenous retrovirus K-specific T cells toward autologous ovarian cancer cells.

PURPOSE: To determine whether HERV-K envelope (ENV) protein could function as a tumor-associated antigen and elicit specific T-cell responses against autologous ovarian cancer cells. EXPERIMENTAL DESIGN: The expression of HERV-K transcripts and ENV protein, the presence of serum antibodies against HERV-K, reverse transcriptase (RT) activities, and cellular immune responses in primary ovarian cancer tissues and patient blood samples were analyzed and compared with samples from patients with benign ovarian diseases and normal female donors. RESULTS: Ovarian cancer cells in primary tumors and ascites expressed markers of cancer stem cells and markers of both mesenchymal and epithelial cells. Expression of HERV transcripts and HERV-K ENV protein and reverse transcriptase activities were higher in ovarian cancer compared with adjacent normal and benign tissues. The ovarian cancer patient plasma also had high reverse transcriptase activities and the ovarian cancer patient sera contained HERV-K immunoreactive antibodies. HERV-K-specific T cells generated from autologous dendritic cells pulsed with HERV-K ENV antigens exhibited phenotypes and functions consistent with a cellular immune response including T-cell proliferation, IFNγ production, and HERV-K-specific cytotoxic T lymphocyte (CTL) activity. Significantly higher CTL lysis of autologous tumor cells than of uninvolved normal cells was demonstrated in patients with ovarian cancer than patients with benign diseases and further enhanced lysis was observed if T regulatory cells were depleted. CONCLUSION: Endogenous retroviral gene products in ovarian cancer may represent a potentially valuable new pool of tumor-associated antigens for targeting of therapeutic vaccines to ovarian cancer. Clin Cancer Res; 21(2); 471-83. ©2014 AACR.

Quantification of reverse transcriptase in ALS and elimination of a novel retroviral candidate.

BACKGROUND: Retroviral involvement in amyotrophic lateral sclerosis (ALS) has been suspected for several years since the recognition that both murine and human retroviruses can cause ALS-like syndromes. Nonquantitative studies have demonstrated the retroviral enzyme reverse transcriptase (RT) in ALS patients' sera, but the amount and source of RT activity are unknown. We therefore developed a quantitative assay to study RT levels in ALS and examined the possibility that the recently discovered human gammaretrovirus XMRV (xenotropic MuLV-related virus) might be the source of the RT activity. METHODS: A quantitative product-enhanced RT assay was used to measure RT activity levels in serum and CSF. XMRV sequences were sought by PCR analysis of DNA and RNA extracted from blood. RESULTS: Fifty percent of ALS patients' sera contained >6 x 10(-8) RT units/mL as opposed to 7% of control sera (p = 0.008). The levels of RT activity in ALS patients were comparable to the levels observed in patients infected with HIV. RT activity was detected in only 1 of 25 CSF samples tested. XMRV sequences were not found in any of 25 nucleic acid extracts obtained from ALS patients' blood. CONCLUSIONS: These findings further support the concept of retroviral involvement in amyotrophic lateral sclerosis (ALS) and demonstrate that serum is more suitable than CSF for assay of reverse transcriptase (RT) activity in this disease. The levels of serum RT activity detected are comparable to those found in HIV infection. XMRV is not detectable in the blood of ALS patients, and the agent responsible for ALS-associated RT activity therefore remains unidentified.

Reverse transcriptase viral load correlates with RNA in SIV/SHIV-infected macaques.

Monitoring of viral load in macaques has usually been carried out using in-house PCR-based methods. A novel viral load (VL) kit (ExaVir Load) based on the measurement of lentivirus reverse transcriptase (RT) activity provides a potential alternative to methods that measure plasma viral RNA. RT is a fundamental and conserved activity of all retroviruses and the method should theoretically detect RT from all lentiviruses. To test this we compared VL measured by a commercially available RT kit with an in-house QC RT-PCR in macaques infected with SIV and SHIV. Both RT and RNA levels were measured over time in both sets of macaques. Results indicated that the relationship between both tests was strong for SIV and SHIV (r = 0.95 and r = 0.92, p < 0.0001, respectively). The VL trends also followed each other, indicating that both techniques measured the same process of viral replication. Furthermore, the RT load obtained using standardized control plasma samples supplied by NIBSC gave values close to the designated VL. However, when comparing RT load with QC RT-PCR a consistently three to five time higher level was obtained with the RT assay, highlighting potential differences in assay calibration. Even so, the data suggest that the RT assay is both sensitive and robust for use in the SIV/SHIV macaque model, particularly where molecular-based assays for SIV VL determinations are not easily available. The assay is also a commercially available kit and hence has the potential to reduce the variability seen between laboratories using in-house PCR.

No indication for activation of exogenous retroviruses in patients with renal allograft rejection.

AIM: Reactivation of latent BK virus in kidney-transplanted patients results in severe graft dysfunction. The role of retroviruses infecting also latently target cells is not investigated so far in this setting. We determined the presence or induction of retroviruses in sera of immunosuppressed patients with renal allografts at the timepoint of organ rejection or ongoing polyomavirus nephropathy. PATIENTS AND METHODS: Sera of patients with acute kidney rejection or polyomavirus nephropathy (n=25) and controls (n=8) were tested for reverse transcriptase activity by the ultrasensitive product enhanced reverse transcriptase (PERT) assay. In parallel, kidney biopsies were investigated for histological signs of kidney rejection or polyomavirus nephropathy confirmed by either immunofluorescence or immunohistochemistry. RESULTS: None of the investigated sera, specifically those of patients with ongoing BK virus nephropathy, indicated reverse transcriptase activity. CONCLUSION: Our results do not support the idea of the induction of known or unknown retroviruses in patients with kidney transplantation, even under highly immunosuppressive therapies.

Detection of serum reverse transcriptase activity in patients with ALS and unaffected blood relatives.

BACKGROUND: Retroviral involvement in the etiology of sporadic ALS has been suspected for several years since the recognition that both murine and human retroviruses can cause motor neuron disease-like syndromes. In a pilot study, an increased prevalence of a retroviral marker (reverse transcriptase [RT] activity) was demonstrated in the serum of British patients with ALS. The current investigation was designed to confirm and extend these findings in a geographically distinct patient cohort under blinded testing conditions. METHODS: A highly sensitive product-enhanced RT assay was employed to test coded sera obtained from 30 American patients with sporadic ALS and from 14 of their blood relatives, 16 of their spouses, and 28 nonrelated, nonspousal control subjects. RESULTS: Serum RT activity was detected in a higher proportion of ALS patients (47%) than in non-blood-related controls (18%; p = 0.008). The prevalence of RT activity in the serum of spousal controls (13%) was similar to that in other non-blood-related controls. Unexpectedly, the prevalence of serum RT activity in blood relatives of ALS patients (43%) approached that in the ALS patients themselves. CONCLUSIONS: These results confirm that patients with ALS have a significantly higher prevalence of serum reverse transcriptase (RT) activity than that seen in unrelated control subjects. The finding of a similarly increased prevalence in blood relatives of ALS patients raises the possibility that the observed RT activity might be due to an inherited endogenous retrovirus.

High rate of resistance to antiretroviral drugs among HIV-infected prison inmates.

BACKGROUND: Resistance to antiretroviral (ARV) drugs represents a major obstacle to the success of HIV therapy. The aim of the study was to examine the prevalence of genotypic resistance to ARV drugs in a large group of HIV-infected individuals incarcerated in penal facilities. MATERIAL/METHODS: We analyzed the reverse transcriptase and protease genes on plasma samples collected from 309 HIV-infected prison inmates in Madrid. In order to compare the prevalence of resistance at different periods and detect any trend over time, half of the samples from ARV-naive and half from pre-treated subjects were randomly collected in 1999 and in 2001. RESULTS: Overall, 63.7% of specimens harbored plasma HIV-RNA above 1000 copies/ml. Genotypic data were obtained in 94.4% of them. Primary resistance mutations among 127 drug-naive subjects were recognized in 13% in 1999 vs. 15% in 2001. In contrast, drug resistance was found in 35% and 59% of 182 pre-treated subjects in 1999 and 2001. CONCLUSIONS: Drug resistance has increased over the last two years among inmates on ARV drugs and currently affects 59% of those failing treatment. A nearly 3-fold increase has been noticed for NNRTI resistance. In comparison with HIV-positive subjects outside jail on ARV drugs, prisoners are more likely to experience virological failure, but show a lower rate of drug resistance; this affects particularly drugs with a low genetic barrier (i.e. NNRTI and 3TC).

A new quantitative HIV load assay based on plasma virion reverse transcriptase activity for the different types, groups and subtypes.

BACKGROUND: Plasma viral load monitoring is an integral part of the standard of care for HIV-infected patients in industrialized countries. In developing countries, viral load assay is either unaffordable or hindered by on-site maintenance and/or technical problems. OBJECTIVES: To evaluate a new and simple quantitative assay for plasma HIV reverse transcriptase (RT) activity; and to compare RT activity-based and RNA-based quantification in plasma samples from patients infected by different subtypes of HIV-1 group-M, HIV-1 group-O and HIV-2. METHODS: The RT-based viral load assay involves separation of the virion-protected RT and quantification of its activity with an enzyme immunoassay. Plasma viraemia was quantified both by RT activity and by RNA copies in 322 samples from 236 HIV-1 group M-infected patients, including serial samples from 54 patients. Samples from 49 patients infected by HIV-1 group O or HIV-2 were also tested. RESULTS: RT activity and RNA copies were detected in 70% of plasma samples; respectively 25% and 1% of samples contained detectable RNA copies or RT activity alone. Measured RT activity corresponded to 48%, 96% and 100% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. The values of the two assays correlated independently of the HIV subtype (P < 0.0001) and group/type (P < 0.03). Patient follow-up showed a similar pattern of viraemia with the two assays. CONCLUSION: Plasma RT activity assay is a simple, cheap and reliable alternative for HIV viral load determination. As such, it could be particularly valuable for diagnosis and treatment monitoring in developing countries.

[The quantity analysis of reverse transcriptase in porcine endogenous retrovirus expressed in Banna minipig inbred].

Quantitative RT(reverse transcriptase) assay was established to detect the reverse transcriptase in plasma of thirty-four Chinese Banna minipig inbred in this work. The protocol was given in the RT kit (Roche), using HIV-1 as the positive control of the kit and supernatant of PK-15 as the PERV positive control respectively. The results show that positive reverse transcriptase reaction can be detected in the plasma of the pigs, but the levels are much lower than that of HIV-1 and lower than that of PERV in supernatant of PK-15.

A rapid phenotypic assay for detecting multiple nucleoside analogue reverse transcriptase inhibitor-resistant HIV-1 in plasma.

Zidovudine and other nucleoside analogue reverse transcriptase inhibitors (NRTIs), like zalcitabine and didanosine used for treatment of individuals infected with HIV-1, can select for viruses with Q151M and other associated mutations (for example, A62V, S68G, V751, F77L, F116Y) in the reverse transcriptase (RT) enzyme. These mutations confer resistance to multiple nucleoside analogues, and thereby compromise the efficacy of this class of drugs. Presently available phenotypic assays for detection of multiple nucleoside analogue resistant (MNR) HIV-1 require testing for each NRTI individually. Here we report an enzymatic RT assay that uses resistance to zidovudine triphosphate (zidovudine-TP) as a diagnostic biochemical marker of MNR HIV-1. This assay exploits the different biochemical mechanisms for zidovudine-resistance conferred by either Q151 M or T215Y/F mutations and the inability of conventional RT assays to detect T215Y/F-associated zidovudine resistance. The assay detects RT activity directly in plasma by using Amp-RT, an ultra-sensitive PCR-based RT assay. We show that enzymatic resistance to zidovudine-TP is specific to MNR RT and is distinguishable from both wild-type (WT) and RT containing classical zidovudine-resistant mutations (D67N, K70R, T215Y/F, K219Q). Compared to WT, MNR HIV-1 RT had 5- to 36-fold increases in the concentration of drug required to inhibit 50% (IC50) of RT activity, depending on the presence of Q151 M alone or with additional MNR mutations. A screening assay utilizing 1 microM zidovudine-TP was developed and validated on 14 reference isolates, 37 plasma specimens, and seven patient-derived viruses. Twenty-three specimens were found to have reduced susceptibility to zidovudine-TP, and all had Q151 M. In contrast, 21 specimens were sensitive to zidovudine-TP, of which 12 had WT genotypes, four had T215Y/F, and five had T69S-insertions along with T215Y/F mutations. This RT-based phenotypic assay provides a specific and rapid tool for the direct identification and monitoring of Q151M-associated MNR HIV-1 in plasma.

HIV inhibitor from Thai bitter gourd.

Thai bitter gourd protein (MRK29) was isolated from Momordica charantia ripe fruit and seed. The purification was performed by ammonium sulfate fractionation and gel filtration chromatography. MRK29 possessed one isoelectric point of (pI) > or = 9, and the time of flight mass spectrum (TOFMS) indicated its molecular weight at 28.6 kD. The twenty amino acid sequence from the N-terminus was in the following order: 1Asp Val Asn Phe Arg Leu Ser Gly Ala 10Asp Pro Arg X Tyr Gly Met Phe Ile Glu 20Asp. MRK29 inhibited the HIV-1 reverse transcriptase with 50% IR at the concentration of 18 micrograms/ml. MRK29 was concentrated in the 30-60% salt precipitated fraction, at which the concentration of 0.175 microgram/ml exerted 82% reduction of viral core protein p24 expression in HIV-infected cells. MRK29 might have modulatory role on immune cells, because it increased 3-fold TNF activity.

Detección mediante RT-PCR de células tumorales circulantes en pacientes con melanoma maligno en estadio III. Correlación con el grosor y la histología del tumor / Detection of circulating tumoral cells by RT-PCR in patients with stage III of malignant melanoma. Correlation with tumor thickness and hystologic type

El objetivo de nuestro estudio ha sido evaluar la utilidad de la Transcriptasa reversa y Reacción en cadena de la polimerasa (RT-PCR) en la detección de la enzima tirosinasa como marcador de la presencia de células derivadas de melanocitos en pacientes con melanoma maligno en estadio III y su correlación con factores pronósticos como el grosor y la forma clínico-histológica del tumor. La detección de células de melanoma circulantes mediante RT-PCR se ha realizado en un grupo de 28 pacientes con melanoma maligno en estadio III (clasificación de la AJCC). Nuestros resultados demuestran una alta positividad de la PCR en este grupo de pacientes siendo más frecuente en tumores con un grosor > 4mm (75 por ciento). No obstante, no encontramos correlación entre la positividad de la PCR y la forma clínico-histológica del tumor. Nuestros resultados sugieren la posibilidad de aplicar la detección del ARNm de la tirosinasa mediante RT-PCR como un nuevo marcador en el pronóstico del melanoma maligno (AU)

Detection and characterization of porcine endogenous retrovirus in porcine plasma and porcine factor VIII.

The pig genome contains porcine endogenous retroviruses (PERVs) capable of infecting human cells. Detection of infectious retrovirus in porcine peripheral blood mononuclear cells and endothelial cells suggested to us that pig plasma is likely to contain PERV. Both PERV env sequences and viral reverse transcriptase (RT) activity were detected in all plasma samples isolated from four NIH minipigs. To detect infectious virus from plasma, we performed a culture assay using three cell lines of feline, swine, and human origin that had previously been shown to be permissive for PERV. Infectious virus was successfully cultured from all four NIH minipig plasmas on the swine cell line ST-IOWA. Using RT-PCR with env-specific primers, we could detect expression of PERV class C envelope in the supernatant of ST-IOWA cells that had been exposed to each pig plasma. We next examined a pig plasma derivative, Hyate:C (porcine factor VIII), and found evidence of PERV particles, since all six lots examined were positive for PERV RNA and RT activity. However, infectious virus could not be detected in clinical lots of Hyate:C, suggesting that the manufacturing process might reduce the load of infectious virus to levels below detectable limits of the assay. Detection of infectious virus in porcine plasma confirms and extends the previous findings that certain porcine cells express PERV when manipulated in vitro and clearly demonstrates that there are porcine cells that express infectious PERV constitutively in vivo.

Lower HIV-2 plasma viral loads may explain differences between the natural histories of HIV-1 and HIV-2 infections.

To explain the low transmissibility and pathogenicity of HIV-2 infection's plasma viral loads in both HIV-1- and HIV-2-infected persons were compared by using the polymerase chain reaction (PCR)-based Amp-RT assay to measure levels of reverse transcriptase (RT) activity. The study comprised a total of 155 HIV-infected-people including 58 who were infected with HIV-2 with CD4+ cell counts <500 x 106/L (n = 15), CD4+ cell counts >500 x 106/L (n = 26), or with tuberculosis (TB; n = 17), and 97 HIV-1-infected people with CD4+ cell counts <500 x 106/L (n = 32), CD4+ cell counts >500 x 106/L (n = 25), or TB (n = 40). Among persons with CD4+ cell counts <500 x 106/L, 11 (73.3%) of 15 HIV-2-infected persons had detectable plasma RT activity compared with 25 (78.1%) of 32 HIV-1-infected persons (p =.725). However, the median HIV-2 plasma RT activity in this group was significantly lower (2561 x 10-10 U/ml; p =.036; detectable range, 1712-644,868 x 10-10 U/ml) than the RT activity of HIV-1-infected persons with similar CD4+ cell counts (13,241 x 10-10 U/ml; detectable range, 8482-1,478,880 x 10-10 U/ml). Among TB patients, 10 (58.8%) of 17 HIV-2-infected persons had detectable plasma RT activity compared with 30 (75%) of 40 HIV-1-infected persons (p =.342). In contrast, among patients with CD4+ cell counts >500 x 106/L, none of 26 HIV-2-infected persons had detectable RT activity compared with 13 (52%) of 25 HIV-1-infected persons (p <.001). Our data suggest that unlike HIV-1 infection, HIV-2 infections with CD4+ cell counts >500 x 106/L are associated with a low level of viral replication, which may explain the longer clinical latency and lower transmissibility seen in HIV-2 infection.

Detection of reverse transcriptase activity in the serum of patients with motor neurone disease.

The recognition that both human and murine retroviruses can cause motor neurone disease-like syndromes has raised the possibility that a retrovirus may be involved in the aetiology of motor neurone disease. This possibility was explored by looking for evidence of reverse transcriptase in the serum of motor neurone disease patients. Sera from 56 patients with motor neurone disease and 58 controls were tested by the product-enhanced reverse transcriptase assay, a technique that is approximately a million fold more sensitive than conventional reverse transcriptase assays and capable of detecting very low numbers of retroviral particles. Cell-free reverse transcriptase activity was detected in the serum of 33 of the 56 motor neurone disease patients (59%) but in only 3 of the controls (P < 0.00001). The reverse transcriptase activity was detectable in the presence of a large excess of an effective inhibitor of human cellular DNA polymerases and was therefore tentatively considered to be compatible with a retroviral origin. The reverse transcriptase activity, however, was not found to be due to the presence of known human exogenous retroviruses including HIV-1, HIV-2, HTLV-I, HTLV-II, HRV-5 or human foamy virus, as assessed by PCR-based assays. Further investigations will be required to determine the source of the reverse transcriptase activity observed in these motor neurone disease patient sera.

Effective treatment of murine leukemia with antisense poly-2'O-(2,4-dinitrophenyl)-oligoribonucleotides.

Two antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides (poly-DNP-RNA) have been synthesized and tested for the treatment of murine leukemia. Compound I was designed as a bifunctional inhibitor of either the reverse transcriptase (RT) activity or viral envelope synthesis in Moloney murine leukemia virus (MMLV). Compound II was designed as a trifunctional inhibitor of either RT activity or envelope synthesis or protease synthesis in MMLV. Administration of either I or II to MMLV-infected mice for 3 weeks decreased viremia gradually to below the level detectable by RT-PCR. Viremia did not reappear 8 weeks after termination of treatment, when most of the mice were killed for autopsy. All infected but untreated mice died within 6 months with enlarged spleens that exhibited abnormal histologic signs and were found by PCR to contain the DNA of integrated viral genome. The infected mice that had been treated subsequently with adequate dosage of compound I or II had normal spleens, continued to live on, and had no integrated MMLV genome in their spleen and bone marrow samples. The effective i.p. dosage (ED50) for compounds I and II are 0.25 and 0.1 mg/kg, respectively, which are 200-fold to 500-fold lower than that of the monofunctional RT inhibitor poly-DNP-oligo A. The estimated effective oral dosage of compound II is 1.2 mg/kg.

Increased interleukin-6 activity in the serum of ponies acutely infected with equine infectious anaemia virus.

Seven ponies were infected with the virulent wild-type Wyoming strain of equine infectious anaemia virus (EIAV). Infection status was monitored by serum reverse transcriptase activity, rectal temperature, and complete blood count. Preinfection serum and serum obtained during the initial febrile episode following infection were assayed for interleukin 6 (IL-6) activity. Postinfection IL-6 activity was significantly increased as compared to preinfection values. The magnitude of increase in IL-6 was positively correlated with reverse transcriptase activity (an indirect measure of viraemia) but was not correlated with rectal temperature. IL-6 production in response to EIAV infection may play a role in pathogenesis of disease, especially the hyperglobulinaemia and apparent polyclonal B cell activation in these horses.

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

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.