Evaluation of HIV type 1 western blot-indeterminate blood donors for the presence of human or bovine retroviruses.

From 1985 through 1990, 1100 of 500,000 human blood donations in Syracuse, New York were repeatedly reactive by ELISA for antibodies to the human immunodeficiency virus type 1 (HIV-1). Nine hundred of the ELISA-reactive samples were confirmed as negative by Western blot (WB), 40 were confirmed as positive, and the remaining 160 sera were indeterminate, reacting mainly with HIV-1 gag gene products. Twenty donors with the most reactive indeterminate WB were selected for follow-up studies. Four of these 20 donors admitted to retroviral risk factors and, interestingly, 12 (60%) had exposure to dairy cattle and drank unpasteurized milk. These 20 donors were analyzed over a 3-year period for the presence of the pathogenic human retroviruses HIV-1, HIV-2, human T cell lymphoma/leukemia virus types I and II (HTLV-I and HTLV-II), as well as bovine immunodeficiency virus (BIV) and leukemia virus (BLV). Retroviral analyses included serology, plasma antigen capture, virus culture, and the polymerase chain reaction. Only one donor seroconverted and was clearly infected with HIV-1. None of the other 19 donor serological reactivities to HIV-1 changed, nor were they positive for any of the above-mentioned retroviruses. Although we cannot ascertain whether these latter 19 HIV-1 WB-indeterminate donors were exposed to human or bovine retroviral proteins, it is unlikely that their HIV-1 seroreactivity was caused by infection with HIV-1, HIV-2, HTLV-I, HTLV-II, BLV, or BIV.

The application of quantitative polymerase chain reaction to therapeutic monitoring.

UNLABELLED: Use of polymerase chain reaction in HIV monitoring: The polymerase chain reaction can be used not only to detect the presence of viral sequences but also to provide a semiquantitative or a precise evaluation of the number of copies of genome present. Integral to the development of accurate and reproducible assays have been critical advances in polymerase chain reaction technology. In addition, standardization of protocols and reagents has proved invaluable. The HIV life cycle permits both DNA and RNA to be targeted. Although early cross-sectional studies provided little insight into disease progression, recent longitudinal studies have provided valuable information on HIV infection. CONCLUSION: A rapid and simple quantitative assay for HIV RNA in plasma or sera has been developed that should prove valuable in determining the natural history of infection, dissecting viral pathogenesis and monitoring the efficacy of therapeutic intervention.

Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma.

Pancreatic adenocarcinomas are known to have a high incidence of K-ras gene mutations. Differential diagnosis of pancreatic cancer and chronic pancreatitis sometimes presents a clinical dilemma. We recently developed a highly sensitive and specific polymerase chain reaction capable of detecting 3-30 copies of mutant K-ras genes harboring codon 12 single base changes in the presence of 300,000 normal copies. Mutant ras genes were detected in DNA purified from pancreatic juice from all 6 cases of pancreatic adenocarcinoma and 1 case of intraductal papillary neoplasms of the pancreas. In 2 of 6 other cases with pancreatic adenocarcinoma, circulating metastatic cells were detected in DNA purified from peripheral blood. Activated ras genes were not found in pancreatic juice of three control cases (chronic pancreatitis and choledocholithiasis) or in the peripheral blood of two patients with insulinomas. Notable conclusions of this study are that there can be significant levels of shed tumor cells in peripheral blood and an even higher number in pancreatic juice. In addition, two different K-ras mutations were found in some patients.

Amplification and analysis of specific DNA and RNA sequences of bovine leukemia virus from infected cows by polymerase chain reaction.

Bovine leukemia virus (BLV) is the etiologic agent of leukemia in cattle and is believed to cause decreases in milk productivity, fertility, and life span in infected cows. BLV is a type C retrovirus in the Oncovirinae subfamily. It is most closely related to human T-cell lymphoma/leukemia virus type I (HTLV-I) and type II (HTLV-II). Since the polymerase chain reaction (PCR) provides rapid and efficient amplification of DNA sequences, primers were designed to amplify regions of the polymerase (pol) and pX genes specific for BLV targets. These sets of primers consistently amplified as few as 10 copies of BLV DNA contained in a plasmid in the background of 1 microgram of either human or bovine chromosomal DNA. In addition, no amplification products were detected from cell lines infected with HTLV-I, HTLV-II, or human immunodeficiency virus type 1 or 2 by the BLV PCR systems. Samples of peripheral blood mononuclear cells from 18 cows, previously determined to be serologically positive or negative, were correctly identified in a blind study as containing proviral DNA by use of the BLV primers and probes. Cloning and sequencing of amplified products revealed finite sequence variations among a previously cloned BLV isolate, the wild-type virus, and the published genome. Reverse transcriptase-directed PCR with the primers for both BLV pol and BLV pX was performed on plasma from a BLV-infected cow and detected in vivo BLV RNA expression. In summary, we have developed a specific and sensitive assay using PCR for the detection and identification of BLV infections; this assay can now be applied to clinical and basic research questions in veterinary medicine.

Modified Western blot assay for confirmation and differentiation of human T cell lymphotropic virus types I and II.

Disease association studies of human T cell lymphotropic virus (HTLV) types I and II are hindered by the need for multiple assays to confirm and differentiate between the viruses. A modified Western blot assay has been developed using HTLV-I viral lysate and unique (MTA-4) and shared (p21E) HTLV recombinant proteins. By defining confirmation of infection as the presence of antibodies to p24 gag protein and to p21E, all 56 HTLV-I and 49 HTLV-II antisera were confirmed by this modified Western blot alone. Differentiation was determined by reactivity to MTA-4. All HTLV-I antisera reacted with MTA-4 and all HTLV-II antisera did not react with MTA-4. These findings indicate the utility of selected HTLV-I recombinant proteins in a single assay format to confirm and differentiate infections with HTLV-I and HTLV-II.

Significance of human T-lymphotropic virus type I indeterminant serological findings among healthy individuals.

Follow-up studies on 67 blood donors with indeterminant serological findings for human T-lymphotropic virus (HTLV) type I by standard immunoassays showed no evidence of infection by polymerase chain reaction analysis for HTLV-I or HTLV-II nucleic acids or by antibody reactivity to a unique HTLV-I recombinant envelope protein, MTA-4. Among HTLV-I- or -II-infected individuals, a history of blood transfusion, past residence in established HTLV-I endemic areas or some association with intravenous drug use were common. In contrast, 85% of indeterminant cases had none of these risk factors. These observations suggest that healthy individuals with indeterminant serology for HTLV-I should not require additional studies.

Determination of a unique and immunodominant epitope of human T cell lymphotropic virus type I.

The identification and isolation of unique and immunogenic recombinant epitopes for human T cell lymphotrophic virus (HTLV) type I might allow the development of an antibody-based assay to differentiate between HTLV-I and HTLV-II infections. To test the feasibility of this approach, an HTLV-I envelope epitope was isolated by immunoscreening of a lambda gt11 recombinant HTLV-I DNA library with a human monoclonal antibody to HTLV-I. This recombinant epitope. MTA-4, when tested with sera from HTLV-I- or HTLV-II-infected individuals, was reactive with all HTLV-I and nonreactive with all HTLV-II antisera. These results indicate that MTA-4 is a unique and immunodominant epitope on HTLV-I and confirm the usefulness of human-derived monoclonal antibodies in an experimental approach to dissect the human humoral response to a viral pathogen.

The polymerase chain reaction (PCR): a valuable method for retroviral detection.

Although the detection of antibodies to a specific pathogen is used initially as the assay of choice, direct detection of human retroviruses is difficult. First, only a small fraction of cells are infected in the peripheral blood and lymphatic tissue may serve as a reservoir for infection. Second, infected cells may harbor only a small number of copies of the viral sequences. Third, a latent infection marked by transcriptional dormancy is often established thereby obviating the use of proteins or RNA to detect the viruses. Fourth, closely related but distinct members of the onco-and lenti-virus families may complicate specific detection of a particular virus. An additional hurdle is viral heterogeneity. HIV variants, for example, have been identified within and among individuals harboring this virus. Accordingly, sensitive and specific detection of the human retroviruses seemingly requires specific amplification of viral DNA sequences prior to detection. In this regard, an in vitro DNA amplification procedure using DNA polymerase and termed the polymerase chain reaction (PCR) initially applied to human genetic diseases has been successfully applied to human retroviruses. A PCR-based assay has demonstrated utility for detecting infection: (1) prior to the generation of detectable antibodies, (2) in individuals with ambiguous or indeterminate serological status, (3) for neonatal screening, (4) by a specific type or multiple viruses, and (5) in therapeutic trials to allow the monitoring of infected cell load and viremia. It is also unlikely that the viruses identified to date represent all of the retroviruses responsible for human disease. Lymphatic disorders, in general, and immunodeficiencies, in particular, merit closer scrutiny for a retroviral etiologic agent.(ABSTRACT TRUNCATED AT 250 WORDS)

High prevalence of HTLV-II among intravenous drug abusers: PCR confirmation and typing.

The polymerase chain reaction (PCR) was used to confirm the presence of human T-cell lymphotropic viruses (HTLV) in intravenous drug users (IVDU) whose sera were reactive by immunofluorescence assay (IFA) for HTLV-1/-II antibody. Peripheral blood mononuclear cells from 41 IFA-positive and 19 IFA-negative individuals were analyzed. HTLV sequences were detected in 39/41 IFA-positive samples; 36 were HTLV-II positive and 3 were HTLV-I positive. Two IFA antibody-positives were negative by both PCR and by enzyme immunoassay (EIA). One IFA and EIA antibody-negative sample was positive for HTLV-II by PCR. This study indicates a high prevalence of HTLV-II among IVDUs and further demonstrates the feasibility of using PCR to differentiate between HTLV-I and -II.

Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.

We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3'-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches were efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3' terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3'-terminus, however, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.

Human immunodeficiency virus type 1 (HIV-1) infection a median of 18 months before a diagnostic western blot. Evidence from a cohort of homosexual men.

STUDY OBJECTIVE: To study the natural history of human immunodeficiency virus type 1 (HIV-1) infection, we used an in-vitro amplification technique to detect HIV-1 nucleic acid sequences in sequential aliquots of peripheral blood mononuclear cells from homosexual men enrolled in the Multicenter AIDS Cohort Study. DESIGN AND PATIENTS: Blinded, longitudinal study of 24 homosexual men who were positive for HIV-1 antibodies at a recent follow-up visit. MEASUREMENTS AND MAIN RESULTS: Coded clinical samples were evaluated using two enzyme-linked immunosorbent assays (whole virus and gp120-gp41 fragment), Western blot, a p24 antigen capture assay, virus cocultivation, and in-vitro amplification of conserved regions from the HIV-1 gag and env open-reading frames. In 20 of the 24 men an HIV-1 enzymatically amplified product was detected before HIV-1 antibody seroconversion: at 42 months before seroconversion in two cases; at 36 months in one case; at 30 months in one case; at 24 months in four cases; at 18 months in eight cases; at 12 months in one case; and at 6 months in three cases (median, 18 months). In the four other men, detection of an HIV-1 enzymatically amplified product was concurrent with confirmation of antibody seroconversion by Western blot. CONCLUSIONS: There is a long and variable interval between virus acquisition and a diagnostic serum antibody response, perhaps due to the prolonged, persistent infection characteristic of the lentiviruses (family Retroviridae).

Enzymatic amplification of the human immunodeficiency virus in peripheral blood mononuclear cells from pediatric patients.

The presence of the human immunodeficiency virus type 1 (HIV) provirus was assessed in peripheral blood mononuclear cells (PBMCs) from 27 offspring of HIV-infected mothers, 12 of these mothers, and 4 HIV-uninfected mother-infant control pairs. Enzymatic amplification of specific conserved regions of the gag and env genes was performed directly in PBMC lysates using the polymerase chain reaction (PCR) technique. The enzymatically amplified gene products were evaluated using the oligomer hybridization detection procedure. PBMCs from infected infants (as determined by Centers for Disease Control clinical criteria) and from HIV-infected mothers manifested a characteristic HIV oligomer hybridization product. Clinically uninfected seropositive infants with declining HIV antibody titers and infants who became seronegative lacked an enzymatically amplified HIV gene product. These preliminary data indicate that PCR is a valuable diagnostic technique to detect or exclude HIV infection in young infants and children.

Prevalence of human T-cell leukemia/lymphoma virus (HTLV) type II infection among high-risk individuals: type-specific identification of HTLVs by polymerase chain reaction.

The extent of human T-cell leukemia/lymphoma virus type II (HTLV-II) infection and its rate of spread have been difficult to determine owing to the serological cross-reactivity between HTLV-I and HTLV-II. The present study overcame this problem by directly detecting type-specific proviral sequences by means of the polymerase chain reaction (PCR) and liquid hybridization. Screening was performed on a cohort of primarily white intravenous drug abusers (IVDAs), and individuals of other behaviorally defined risk groups from the New York City area. Eleven percent (19 of 169) of the individuals in these high-risk groups were determined by PCR to have HTLV-II proviral infections. One of these patients displayed an exfoliative erythrodermatitis. Thirteen of the 19 subjects were positive in an HTLV-II enzyme-linked immunosorbent assay (ELISA). The remaining six individuals, although negative in the HTLV-II ELISA, were confirmed as HTLV-II positive by analyzing their DNA with a second HTLV-II-specific primer detector system. Four additional individuals were reactive in the HTLV-II ELISA but were PCR-negative for HTLV-II. PCR analysis for HTLV-I revealed that all four were positive for that virus. Thirty-seven percent (seven of 19) of the HTLV-II PCR-positive subjects were also PCR-positive for HTLV-I, and 84% (16 of 19) of the HTLV-II positive individuals were infected with human immunodeficiency virus (HIV-1). Six individuals were triply infected with HTLV-I, HTLV-II, and HIV-1.

A polyclonal CD4+ and CD8+ lymphocytosis in a patient doubly infected with HTLV-I and HIV-1: a clinical and molecular analysis.

HTLV-I is associated with adult T-cell leukemia/lymphoma (ATL) characterized by monoclonal expansions of CD4+ T-lymphocytes. In this report we describe a histologically benign, polyclonal HTLV-I infection in a patient exhibiting both an absolute CD4+ and CD8+ lymphocytosis. Three T-cell lines containing integrated HTLV-I proviral copies established from this patient were initially polyclonal, but with time all grew out the same two clones as determined by analysis of their T-cell antigen receptor beta chain gene rearrangements. The patient subsequently developed pulmonary and nasopharyngeal nodules containing HTLV-I infected cells. Restriction analysis of the patient's HTLV-I provirus revealed no differences from prototype HTLV-I and the tax gene was normally expressed in vivo and in vitro. The patient's T-lymphocytosis and HTLV-I+ pulmonary tract nodules were put into a complete clinical remission by treatment with alkylating agents and steroids. Subsequently, the patient developed a severe immunodeficiency state and expired. Retrospective serologic and gene amplification assays for HIV-1 demonstrated that he had been doubly infected from the time of presentation. Postmortem analysis by polymerase chain reaction revealed the presence of both HTLV-I and HIV-1 in lymphatic tissues and the testes; HIV-1 was also detected in brain tissue.

Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro.

We evaluated various detection methods to identify amplified human retroviral sequences after Thermus aquaticus-directed polymerase chain reaction (PCR). A combination of hybridization formats and direct incorporation assays provided the most information. This multiphasic approach enabled us to detect specific human T cell leukemia virus type I (HTLV-I)-homologous regions in several HTLV-I-seronegative patients with T cell lymphoma, as well as variants of HTLV-I and human immunodeficiency virus type 1 in patients with prototype disease. In all diagnostic assays designed to detect a particular retrovirus, it was necessary to include a hybridization step, because sequences (endogenous or exogenous) homologous to certain primers were present in most human DNA preparations and yielded discrete products, sometimes of the predicted molecular weight, after amplification. These products could be discriminated by hybridization from amplified prototype proviral sequences. The intensity of the signal generated after hybridization was proportional to input target DNA, an observation making it feasible to quantitatively measure the proviral load in a DNA sample.

Characterization of a sequence of human T cell leukemia virus type I from a patient with chronic progressive myelopathy.

DNA from peripheral blood mononuclear cells of individuals with chronic progressive myelopathy (CPM) were extensively analyzed for the presence of human T cell leukemia virus (HTLV) type I-like sequences by using the polymerase chain reaction. The DNA samples were amplified with oligonucleotides from three separate regions of HTLV viral genomes. A portion of the amplified viral genome from a representative patient was sequenced after molecular cloning into bacteriophage M13. Sequence data indicate that HTLV type I and not a related virus is associated with CPM.

Hepatitis B virus particles contain a polypeptide encoded by the largest open reading frame: a putative reverse transcriptase.

A segment of the largest open reading frame of hepatitis B virus (HBV) was inserted into an open reading frame vector directing the expression in Escherichia coli of a fusion molecule containing 143 HBV-encoded amino acids. The fusion protein was used to generate antiserum which served in immunoblots to identify a polypeptide with a molecular mass of 65 kilodaltons in HBV particles. Because of the small number of molecules in virus particles, unambiguous detection required the development of a highly sensitive immunoblot procedure.

Enzymatic amplification of HTLV-I viral sequences from peripheral blood mononuclear cells and infected tissues.

Human T-cell lymphotropic virus type I (HTLV-I) and human T-cell lymphotropic virus type II (HTLV-II) have been associated with adult T-cell leukemia/lymphoma (ATL) and a rare T-cell variant of hairy cell leukemia, respectively. Direct detection of viral nucleic acid in peripheral blood lymphocytes (PBLs) and infected tissues in carrier patients and those with chronic disease has proven refractory due to viral transcriptional dormancy and the small number of infected cells present. The investigators report here the successful application of the DNA amplification procedure, termed PCR, to the detection of these human oncoviruses. Judicious selection of specific oligonucleotides for primers and probes provides type-specific and simultaneous detection of these two retroviruses. The ability to amplify and detect highly conserved regions of these medically relevant viruses may facilitate the identification of, as yet, uncharacterized retroviruses.

A sensitive method for the identification of uncharacterized viruses related to known virus groups: hepadnavirus model system.

Amino acid sequence similarity of the reverse transcriptases encoded by retroviruses and hepadnaviruses was first reported by Toh, H., Hayashida, H. & Miyata, T. (1983) Nature (London) 305, 827-829. The regions of similarity extend over a small number of amino acids and require the introduction of gaps through the open reading frame. By using an octapeptide region as the sole criterion for "taxonomic" classification, we have grouped the oncoviruses into two distinct categories and the lentiviruses and hepadnaviruses into two additional groupings. This classification suggests that murine and feline leukemia viruses may be more closely related to the viruses that are associated with leukemia in primates and cattle than had been appreciated. We have exploited a portion of this region because of the minimal translational codon degeneracy of the conserved residues. Unique oligonucleotides from this region have been designed and used in the primer-directed in vitro DNA amplification of the hepadnaviruses as a model system. In addition, mixtures of oligonucleotides with various sequences but of the same length were demonstrated to be efficient primers. The amplification procedure enabled dramatic increases in sensitivity and coincident detection of mammalian and avian genomes. This approach will be a valuable tool to detect and characterize members of viral groups. In addition, since short stretches of similarity have been frequently identified in related but distinct genes, such an approach could prove a valuable asset to molecular studies in general.

Loss of human immunodeficiency virus type 1 (HIV-1) antibodies with evidence of viral infection in asymptomatic homosexual men. A report from the Multicenter AIDS Cohort Study.

Four asymptomatic homosexual men reverted from positive to negative serologic results for the human immunodeficiency virus, type 1 (HIV-1) over 2.5 years, as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot. Antibody bands in the Western blot from three men were undetectable 6 to 12 months after being positive; gradual fading of the number and intensity of bands was seen in the other man. No HIV-1-p24 antigenemia was detected; cryopreserved peripheral blood mononuclear cells were negative for HIV-1 by standard culture assay. Polymerase chain reaction (gene amplification) assays were done on peripheral blood mononuclear cells and showed the HIV-1 provirus in all subjects 6 to 18 months after the last positive antibody test. Serum specimens from each participant were genetically identical. Polymerase chain reaction showed that peripheral blood mononuclear cells from one subject at different times matched by HLA DNA typing. Clinical and laboratory features of these four men were similar to those of other seronegative subjects. Rare, asymptomatic persons seropositive for HIV-1 may not remain seropositive, but may remain latently infected with HIV-1.