Safety and efficacy of a biodegradable implant releasing tenofovir alafenamide for vaginal protection in a macaque model.

OBJECTIVES: To advance the initiative of ending the global epidemic, long-lasting HIV protection is needed through sustained release of antiretroviral drugs for months to years. We investigated in macaques the safety and efficacy of biodegradable polycaprolactone implants releasing tenofovir alafenamide for HIV pre-exposure prophylaxis (PrEP). METHODS: Implants were administered subcutaneously in the arm using a contraceptive trocar. Efficacy against vaginal simian-HIV (SHIV) infection was investigated in six pigtailed macaques that received two tenofovir alafenamide implants (0.35 mg/day), one in each arm, for a total release rate of tenofovir alafenamide at 0.7 mg/day. Macaques were exposed to SHIV twice weekly for 6 weeks. Statistical analyses were used to compare outcome with eight untreated controls. Histological assessments were performed on skin biopsies collected near implantation sites. RESULTS: Median (range) tenofovir diphosphate level in PBMCs was 1519 (1068-1898) fmol/106 cells. All macaques with tenofovir alafenamide implants were protected against vaginal SHIV infection. In contrast, 7/8 controls were infected after a median of 4 SHIV exposures (P = 0.0047). Histological assessment of tissues near tenofovir alafenamide implant sites showed inflammation and necrosis in 5/6 animals, which were not evident by visual inspection. CONCLUSIONS: We demonstrated complete protection against vaginal SHIV infection with two implants releasing a total of 0.7 mg of tenofovir alafenamide per day. We also identified tenofovir diphosphate concentrations in PBMCs associated with complete vaginal protection. Consistent with previous findings, we observed adverse local toxicity and necrosis near the tenofovir alafenamide implant site. Improved tenofovir alafenamide implants that are safe and maintain high efficacy have the potential to provide long-lasting protection against vaginal HIV infection.

Comparative Pharmacokinetics and Local Tolerance of Tenofovir Alafenamide (TAF) From Subcutaneous Implant in Rabbits, Dogs, and Macaques.

The administration of antiretrovirals (ARVs) for HIV pre-exposure prophylaxis (PrEP) is highly efficacious and may benefit from new long-acting (LA) drug delivery approaches. This paper describes a subcutaneous, reservoir-style implant for the LA delivery of tenofovir alafenamide (TAF) and documents the preclinical assessment of implant safety and pharmacokinetics (PK) in New Zealand White (NZW) rabbits (3 groups of n = 5), beagle dogs (2 groups of n = 6), and rhesus macaques (2 groups of n = 3). Placebo implants were placed in rabbits (n = 10) and dogs (n = 12). Implant parameters, including selection of the TAF form, choice of excipient, and PCL formulation were tuned to achieve targeted concentrations of the active anabolite of TAF, tenofovir diphosphate (TFV-DP), within peripheral blood mononuclear cells (PBMCs) and mucosal tissues relevant to HIV transmission. Sustained concentrations of TFV-DP in PBMCs over 100 fmol/106 cells were achieved in all animal species indicating that the implants effectively delivered TAF for 3-6 months. Unlike placebo implants without TAF, all active implants resulted in local adverse events (AEs) proximal to the implant ranging in severity from mild to moderate and included dermal inflammation and necrosis across all species. Despite these AEs, the implant performed as designed and achieved a constant drug release profile, supporting the continued development of this drug delivery platform.

Transmission of human immunodeficiency virus and hepatitis C virus from an organ donor to four transplant recipients.

In 2007, a previously uninfected kidney transplant recipient tested positive for human immunodeficiency virus type 1 (HIV) and hepatitis C virus (HCV) infection. Clinical information of the organ donor and the recipients was collected by medical record review. Sera from recipients and donor were tested for serologic and nucleic acid-based markers of HIV and HCV infection, and isolates were compared for genetic relatedness. Routine donor serologic screening for HIV and HCV infection was negative; the donor's only known risk factor for HIV was having sex with another man. Four organs (two kidneys, liver and heart) were transplanted to four recipients. Nucleic acid testing (NAT) of donor sera and posttransplant sera from all recipients were positive for HIV and HCV. HIV nucleotide sequences were indistinguishable between the donor and four recipients, and HCV subgenomic sequences clustered closely together. Two patients subsequently died and the transplanted organs failed in the other two patients. This is the first recognized cotransmission of HIV and HCV from an organ donor to transplant recipients. Routine posttransplant HIV and HCV serological testing and NAT of recipients of organs from donors with suspected risk factors should be considered as routine practice.

Identification of a human population infected with simian foamy viruses.

Studying the transmission of simian retroviruses to humans can help define the importance of these infections to public health. We identified a substantial prevalence (4/231, 1.8%) of infection with simian foamy viruses (SFV) among humans occupationally exposed to nonhuman primates. Evidence of SFV infection included seropositivity, proviral DNA detection and isolation of foamy virus. The infecting SFV originated from an African green monkey (one person) and baboons (three people). These infections have not as yet resulted in either disease or sexual transmission, and may represent benign endpoint infections.

Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group.

Pig organs may offer a solution to the shortage of human donor organs for transplantation, but concerns remain about possible cross-species transmission of porcine endogenous retrovirus (PERV). Samples were collected from 160 patients who had been treated with various living pig tissues up to 12 years earlier. Reverse transcription-polymerase chain reaction (RT-PCR) and protein immunoblot analyses were performed on serum from all 160 patients. No viremia was detected in any patient. Peripheral blood mononuclear cells from 159 of the patients were analyzed by PCR using PERV-specific primers. No PERV infection was detected in any of the patients from whom sufficient DNA was extracted to allow complete PCR analysis (97 percent of the patients). Persistent microchimerism (presence of donor cells in the recipient) was observed in 23 patients for up to 8.5 years.

Human infection with foamy viruses.

Virtually all nonhuman primate species investigated thus far including prosimians, New World and Old World monkeys and apes all harbor distinct and species-specific clades of simian foamy virus (SFV). However, evidence supporting the existence of a human-specific foamy virus (FV) is not yet available. Early reports describing widespread infection of healthy and sick humans with FV could not be confirmed. In contrast, all FV infections documented in humans are of zoonotic origin and are identified in persons occupationally exposed to nonhuman primates. The introduction of SFV into humans raises several public health questions regarding disease outcomes and potential for human-to-human transmissibility. The available data from a very limited number of SFV-infected humans suggest that these infections are nonpathogenic and are not easily transmissible. Additional studies are needed to better define the prevalence and natural history of SFV in humans.

Viral load differences in early infection with two HIV-1 subtypes.

OBJECTIVES: Information on early HIV-1 infection has come primarily from studies of persons infected with subtype B in North America and Europe; much less is known about other subtypes. The purpose of the present study was to compare the virologic and immunologic parameters following seroconversion among recently-infected persons infected with either of two different HIV-1 subtypes. METHOD: A prospective cohort study was carried out at methadone treatment clinics administered by the Bangkok Metropolitan Administration, Thailand. A total of 130 HIV-1-infected seroconverters (103 with HIV-1 subtype E and 27 with subtype B) were included in the study. The main outcome measures were serial HIV-1 RNA viral load, natural killer cell percentage, CD4 and CD8 lymphocyte counts since seroconversion. RESULTS: The demographic and behavioral characteristics of persons with either subtype were similar. Median RNA viral levels at the earliest time within 3 months of seroconversion were more than three times higher for persons infected with subtype E than subtype B (63 100 versus 18 050 copies/ml, P = 0.001). However, this difference decreased over time such that viral loads were similar at 12, 18, and 24 months following seroconversion. The CD4 and CD8 lymphocyte counts were similar in infections with either subtype during the entire period up to 24 months post-seroconversion. CONCLUSIONS: Higher viral loads associated with subtype E may result from inter-subtype biological differences; however, the epidemiological dynamics of transmission in Bangkok may have also contributed to this phenomenon.

Indeterminate HTLV serologic results in U.S. blood donors: are they due to HTLV-I or HTLV-II?

Current screening tests to detect human T-lymphotropic virus type I (HTLV-I) in volunteer blood donors commonly yield indeterminate HTLV serologic results (mostly isolated gag reactors). To assess the significance of indeterminate HTLV serologic results in U.S. blood donors, we compared 56 persons who had such serologic patterns with 30 HTLV seropositive blood donors and with HTLV seronegative controls. Polymerase chain reaction assays showed that none of the 56 individuals with indeterminate HTLV serologic results were infected with HTLV-I or HTLV-II, while all 30 HTLV seropositive blood donors were infected with either HTLV-I (in 15) or HTLV-II (in the other 15). The seroindeterminate blood donors were also different from the HTLV seropositive blood donors and more like HTLV seronegative controls in their demographic characteristics and the presence of HTLV risk factors. These results are evidence that volunteer blood donors with isolated and persistent gag seroreactivity in the United States are unlikely to be infected with HTLV-I or HTLV-II.

The search for human infection with simian type D retroviruses.

Evidence has recently been presented for an infection with a simian type D retrovirus in a patient with AIDS and lymphoma. We tested for simian type D infection in three groups of subjects: 375 patients with lymphoproliferative diseases (255 non-Hodgkin's, 88 Hodgkin's, and 32 chronic lymphoproliferative disease), of whom 75 were human immunodeficiency virus (HIV)-1 infected; seven persons with unexplained low CD4 lymphocyte counts with clinical conditions; and 45 blood donors, of whom 37 were human T-lymphocyte virus (HTLV)-I/II seroindeterminate and eight were HTLV-I/II and HIV-1 seronegative. Serum samples were screened for antibodies against simian type D retroviruses by an enzyme immunoassay, and reactive samples were analyzed by Western blotting. None of the samples were seropositive, but eight (five from non-Hodgkin's and three from Hodgkin's lymphoma patients) were seroindeterminate. Polymerase chain reaction analysis of genomic DNA from peripheral blood lymphocytes of all eight of these patients was carried out using simian type D gag generic primers with generic internal oligoprobing. All samples were negative. We conclude that simian type D infection is rare among HIV-infected and noninfected lymphoma patients, persons with unexplained low CD4 counts, and persons with HTLV-seroindeterminate test results.

Highly sensitive and specific polymerase chain reaction assays for detection of baboon and pig cells following xenotransplantation in humans.

Pig and baboon xenotransplants in humans require assays that discriminate source from human cells to investigate engraftment and identify true infection of recipients with xenogeneic endogenous retroviruses. We developed two polymerase chain reaction assays that target a porcine-specific sequence in the beta-globin gene and a baboon-specific sequence in the mitochondrial cytochrome oxidase subunit II gene. The sensitivity and specificity of both assays were evaluated on DNA lysates from baboon, pig, and human peripheral blood lymphocytes. Both assays detected single cells in backgrounds of human DNA. Additionally, both assays were highly specific and yielded negative results in reactions containing only human DNA. The two assays reliably detected peripheral blood lymphocyte samples from 14 baboons and 16 pigs. The baboon- and pig-specific target sequences are gender independent and nonpolymorphic and allow universal applicability. The high sensitivity and specificity is of particular importance in assessing low-level engraftment in human xenotransplant chimeras.

Polymerase chain reaction assays for the diagnosis of infection with the porcine endogenous retrovirus and the detection of pig cells in human and nonhuman recipients of pig xenografts.

BACKGROUND: Pigs offer an unlimited source of xenografts for humans. However, recipients of pig xenografts are inevitably exposed to the porcine endogenous retrovirus (PERV), which is carried in the pig germline. The ability of PERV to infect human cells in vitro has heightened safety concerns regarding the transmission of PERV to pig xenograft recipients. METHODS: In response to the need to establish laboratory tests for the surveillance of PERV infection, we have developed polymerase chain reaction (PCR) assays to detect PERV pol and gag sequences by using conserved primers and probes. In addition, we have developed a PCR assay to detect pig-specific mitochondrial DNA (mtDNA) sequences as a marker of pig cells. RESULTS: Analysis of assay sensitivities using cloned target copies in a background of human DNA demonstrated a detection threshold of 1, 5, and 1 copy for the PERV gag, pol, and pig mtDNA PCR assays, respectively. All three PCR assays gave negative results on peripheral blood lymphocyte samples from 69 humans, as well as 6 baboons and 6 macaques, demonstrating 100% specificity. The PERV and pig mtDNA assays were integrated into a simple testing algorithm that allows the differentiation between pig cell microchimerism and true xenogeneic infection. To allow for monitoring of PERV expression, a reverse transcriptase-PCR assay was also developed to detect cell-free PERV RNA. CONCLUSION: The use of the diagnostic tests described here will help define the risks of PERV transmission associated with the use of pig xenografts in humans and nonhuman primates.

Development and validation of a Western immunoblot assay for detection of antibodies to porcine endogenous retrovirus.

BACKGROUND: Reports that pig endogenous retrovirus (PERV) infects human cells in vitro have heightened the importance of molecular and serologic monitoring of xenograft recipients for evidence of infection with PERV. We report the development and validation of a PERV-specific Western immunoblot assay for the diagnostic testing of porcine xenografts recipients. This assay is based upon the serological cross-reactivity observed between PERV variants capable of infecting human cells in vitro and other mammalian C type retroviruses. METHODS AND RESULTS: Strong reactivity between PERV expressing embryonic pig kidney PK-15 cells and antisera raised against whole virus preparations of murine leukemia virus, gibbon ape leukemia virus (GALV), and simian sarcoma-associated virus was demonstrated by an immunofluorescence assay, suggesting specific antigenic cross-reactivity between this group of viruses and PERV. Western immunoblot analysis demonstrated that anti-GALV antisera reacted with three proteins in PK-15 cells having molecular masses of 30, 55, and 66 kDa. Antisera specific for the Gag proteins of either GALV or simian sarcoma-associated virus reacted with the 30-kDa (major) and 55-kDa (minor) proteins present in PK-15 cells and in PERV-infected 293 human kidney cells, likely representing reactivity to the processed and precursor forms of the PERV Gag protein, respectively. No reactivity was seen in uninfected 293 cells. Analysis of plasma samples from 200 United States blood donors and from 58 human immunodeficiency virus-1, 18 human immunodeficiency virus-2, 13 human T-cell lymphotrophic virus-I, 21 human T-cell lymphotrophic virus-II, and 15 cytomegalovirus infected controls were negative. CONCLUSIONS: As this assay is based on PERV antigen derived from infected human cells, it clearly has the capacity to detect a serologic response towards PERV variants that have zoonotic potential and will allow for the accurate determination of PERV-specific seroreactivity in porcine xenograft recipients.

Lack of cross-species transmission of porcine endogenous retrovirus infection to nonhuman primate recipients of porcine cells, tissues, or organs.

BACKGROUND: Nonhuman primates (NHPs) have been widely used in different porcine xenograft procedures inevitably resulting in exposure to porcine endogenous retrovirus (PERV). Surveillance for PERV infection in these NHPs may provide information on the risks of cross-species transmission of PERV, particularly for recipients of vascularized organ xenografts for whom data from human clinical trials is unavailable. METHODS: We tested 21 Old World and 2 New World primates exposed to a variety of porcine xenografts for evidence of PERV infection. These NHPs included six baboon recipients of pig hearts, six bonnet macaque recipients of transgenic pig skin grafts, and nine rhesus macaque and two capuchin recipients of encapsulated pig islet cells. Serologic screening for PERV antibody was done by a validated Western blot assay, and molecular detection of PERV sequences in peripheral blood mononuclear cells (PBMCs) and plasma was performed using sensitive polymerase chain reaction and reverse transcriptase-polymerase chain reaction assays, respectively. Spleen and lymph node tissues available from six bonnet macaques and three rhesus macaques were also tested for PERV sequences. RESULTS: All plasma samples were negative for PERV RNA suggesting the absence of viremia in these xenografted animals. Similarly, PERV sequences were not detectable in any PBMC and tissue samples, arguing for the lack of latent infection of these compartments. In addition, all plasma samples were negative for PERV antibodies. CONCLUSION: These data suggest the absence of PERV infection in all 23 NHPs despite exposure to vascularized porcine organs or tissue xenografts and the use of immunosuppressive therapies in some animals. These findings suggest that PERV is not easily transmitted to these NHP species through these types of xenografts.

Rapid screening of open reading frames by protein synthesis with an in vitro transcription and translation assay.

The analysis of open reading frames (ORFs) to predict full-length or truncated proteins in genes is conventionally achieved by DNA sequencing. This method becomes labor-intensive when a large number of specimens or when large genes are to be examined. To circumvent this problem, we used an in vitro transcription and translation (TT) assay to identify full-length or truncated proteins in PCR-amplified genes. A total of 47 nef genes from the simian immunodeficiency virus (SIV) were cloned from 13 SIV-infected monkeys and were screened for ORFs by using the TT assay. Of these 47 genes, 20 had an intact ORF and 27 had premature stop codons at variable positions in the nef gene. All 20 nef genes with intact ORFs produced full-length proteins, while truncated proteins of different sizes were synthesized from all 27 nef genes with premature stop codons. In addition, we validated a simplified TT protocol that allows the direct screening of ORFs from transformed bacterial colonies, thus eliminating the need for plasmid preparations. By being rapid, simple and cost-effective, this technique should be widely applicable to examine the integrity of the ORF of any gene.

Endemicity and phylogeny of the human T cell lymphotropic virus type II subtype A from the Kayapo Indians of Brazil: evidence for limited regional dissemination.

Long terminal repeat (LTR)-based restriction fragment length polymorphism (RFLP) analysis of human T cell lymphotropic virus type II (HTLV-II) from 17 seropositive Kayapo Indians from Brazil showed that all 17 samples contained a unique HTLV-IIa subtype (A-II). Additional RFLP screening demonstrated the presence of this subtype in two of three Brazilian blood donors and a Mexican prostitute and her child. In contrast, 129 samples from blood donors and intravenous drug users (IDUs) from the United States, two Pueblo Indian samples, five samples from Norwegian IDUs, and two samples from blood donors from Denmark were all found to be a different HTLV-IIa subtype (A-III). Phylogenetic analysis of two Kayapo and one Mexican LTR sequences showed that they cluster with a subtype A-II sequence from a Brazilian blood donor and with sequences from two prostitutes from Ghana and Cameroon. These results demonstrate that infection with the A-II subtype is endemic among the Kayapo Amerindians, has disseminated to non-Indian populations in Brazil, and is also present in Mexico. Furthermore, the A-II subtype does not appear to represent an origin for the HTLV-IIa infection in urban areas of the United States and Europe. This study provides evidence that HTLV-IIa may be a Paleo-Indian subtype as previously suggested for HTLV-IIb.

Investigation of proviral load in individuals infected with human T-lymphotropic virus type II.

Human T-lymphotrophic virus type II (HTLV-II) has not yet been associated with any disease. Little is known about the proviral loads of HTLV-II in vivo and its relationship, if any, to lack of pathogenicity. We determined the HTLV-II proviral copy number in peripheral blood lymphocyte (PBL) samples from 49 HTLV-II-infected individuals, of whom 25 were coinfected with human immunodeficiency virus type 1 (HIV-1). The HTLV-II copy numbers were determined by polymerase chain reaction (PCR) amplification of end-point dilutions of PBL lysates, followed by hybridization to a 32P-labeled HTLV-II-specific probe. The proviral copy number for the 49 samples ranged from < 0.02 to 200 per 1000 PBLs; 6% had < 0.02, 16% had 0.02, 20% had 0.2, 18% had 2, 31% had 20, and 8% had 200 copies per 1000 PBLs. The distributions of HTLV-II copy numbers in the coinfected and singly infected subgroups were not significantly different (Wilcoxon rank sum, p = 0.24). In the coinfected subgroup, there was no significant correlation between the HTLV-II proviral load and the counts of CD4-positive lymphocytes or CD8-positive lymphocytes (Spearman Coefficient = 0.26, p = 0.20; = 0.091, p = 0.67, respectively). Our data demonstrate the presence of a wide range of viral loads in HTLV-II-infected individuals. The high viral loads (> or = 20 copies/1000 lymphocytes) detected in 39% of our samples suggest that the low pathogenicity of HTLV-II is not related to the presence of low viral loads in the infected subjects. Our data from the HIV-1 coinfected individuals show no apparent effect of HIV-1 on HTLV-II proviral loads.

Rapid screening of phenotypic resistance to nevirapine by direct analysis of HIV type 1 reverse transcriptase activity in plasma.

Drug susceptibility testing for the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected persons is often curtailed because such testing is expensive and time consuming. We describe a non-culture-based phenotypic assay for the rapid analysis of HIV-1 resistance to nevirapine. The assay measures the susceptibility of plasma reverse transcriptase (RT) activity to inhibition by nevirapine by using the PCR-based Amp-RT assay. Assay validation was made using two reference wild-type (WT) and six other nevirapine-resistant (>100-fold) HIV-1 isolates. Amp-RT IC50 values were found to correlate with those obtained by a conventional replication-based assay. The results also indicated that 50 microM nevirapine can be used in a single screening test to detect nevirapine resistance. Analysis of virus mixtures showed a detection threshold of 10% of nevirapine-resistant HIV-1 in a background of WT virus. To evaluate the assay on clinical samples, 30 plasma specimens collected longitudinally from 4 patients before and after treatment with nevirapine were analyzed, and results were compared with codon 181 genotypes. Preteatment samples and those obtained during the first 6 days of therapy (n = 21) were sensitive to nevirapine, and none had detectable Y181C mutation. Phenotypic resistance was seen in eight samples obtained after 1 week of treatment and was correlated with detection of the Y181C mutation. An increase in the level of phenotypic resistance was seen over time. These data validate this rapid and simple assay for monitoring phenotypic resistance to nevirapine.

Amplification of simian retroviral sequences from human recipients of baboon liver transplants.

Investigations into the use of baboons as organ donors for human transplant recipients, a procedure called xenotransplantation, have raised the specter of transmitting baboon viruses to humans and possibly establishing new human infectious diseases. Retrospective analysis of tissues from two human transplant recipients with end-stage hepatic disease who died 70 and 27 days after the transplantation of baboon livers revealed the presence of two simian retroviruses of baboon origin, simian foamy virus (SFV) and baboon endogenous virus (BaEV), in multiple tissue compartments. The presence of baboon mitochondrial DNA was also detected in these same tissues, suggesting that xenogeneic "passenger leukocytes" harboring latent or active viral infections had migrated from the xenografts to distant sites within the human recipients. The persistence of SFV and BaEV in human recipients throughout the posttransplant period underscores the potential infectious risks associated with xenotransplantation.

Resistance of human immunodeficiency virus type 1 to reverse transcriptase and protease inhibitors: genotypic and phenotypic testing.

Treatment of HIV-1-infected persons with antiretroviral drugs including reverse transcriptase (RT) and protease inhibitors has significantly reduced the rate of HIV and AIDS-related morbidity and mortality. However, these treatments can select for drug-resistant viruses which are associated with poor virologic responses to the antiretroviral therapies and loss of clinical benefit. Drug resistance is conferred by single or several amino acid changes in the pol gene. These mutations can be classified as primary when they directly confer reduced drug susceptibility, or secondary when their influence is primarily on replication capabilities of resistant viruses. Both genotypic and phenotypic methods are used for drug resistance testing. Genotypic assays detect resistance-related mutations by sequence analysis or point mutations assays. Phenotypic testing measures drug susceptibility of patient-derived viruses in culture assays. Viruses can be conventionally isolated from peripheral blood lymphocytes, or generated more rapidly through recombination of plasma-derived RT/protease sequences and modified HIV-1 vectors. Phenotypic testing provides direct evidence of resistance, is easy to interpret, but is laborious and expensive. In contrast, genotypic testing provides indirect evidence of resistance, is relatively faster and cheaper, but some complex mutation patterns may be difficult to interpret. Non-culture based phenotypic assays that measure susceptibility of RT activity in plasma to RT inhibitors have been described recently, and provide new tools for rapid phenotypic testing. Resistance testing is currently recommended to help guide the choice of new regimens after treatment failure and for guiding therapy in pregnant women.

Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: optimization, validation, and comparative analysis with other detection systems.

An ultra-sensitive assay for reverse transcriptase (RT) activity called Amp-RT has been developed. An in vitro transcribed heteropolymeric RNA sequence was used as a template, and polymerase chain reaction (PCR) amplification with Southern-blot hybridization served as a detection system for the cDNA product of the reaction. Titration of Mg2+ and Mn2+ concentrations using the human immunodeficiency virus type 1 (HIV-1) and the human T lymphotropic virus type 1 (HTLV-I), respectively, showed optimal assay reactivity for both viruses at 2-20 mM of Mg2+. Analysis of density banded HIV-1 showed that the peak RT activity of the assay was associated with the fractions consistent with retrovirus particles. The sensitivity of Amp-RT was also compared with that of three conventional RT assays by using seven different retroviruses including HIV-1, simian immunodeficiency virus (SIV), caprine arthritis-encephalitis virus (CAEV), HTLV-I and HTLV-II, simian retrovirus type 2 (SRV-2), and gibbon ape leukemia virus (GALV). HTLV-I, HTLV-II, and GALV could not be detected by the three conventional RT assays. Amp-RT was able to detect all these viruses in 10(1)-10(3)-fold dilutions. Similarly, Amp-RT was found to be 10(3)-10(6)-fold more sensitive than the other RT assays in detecting HIV-1, SIV< or CAEV. Culture supernatants from uninfected cell lines were all Amp-RT negative. A quantitative Amp-RT assay was also developed by using recombinant HIV-1 RT and signal quantitation. The assay was found to have a 5 log linear range, and therefore, provides a useful tool for quantitating RT and retroviruses. Amp-RT offers a sensitive generic tool for the qualitative and quantitative detection of known and unknown retroviruses.