End-point Dilution Research Articles

Quantitation of human immunodeficiency virus type 1 during pregnancy: relationship of viral titer to mother-to-child transmission and stability of viral load.

To develop strategies to prevent mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1), it is important to define the factors determining it. We examined the relationship between maternal HIV-1 titer and the occurrence of mother-to-child transmission. In addition, we quantitated HIV-1 longitudinally in mothers during pregnancy, at delivery, and up to 1 year postpartum. To examine transmission, we prospectively studied 19 mother-child pairs; in 5 pairs, HIV-1 transmission occurred. We used endpoint dilution culture of peripheral blood mononuclear cells to determine maternal viral titer and found that although 4 of 6 (67%) women with viral titers of > or = 125 HIV-1 infectious units per 10(6) cells transmitted HIV-1 to their infants, only 1 of 13 (7.6%) women with lower viral titers transmitted (P = 0.01). Twelve of the 19 mothers had HIV-1 loads determined serially 3-8 times over periods ranging from 18 to 65 weeks. Viral titers varied greatly between the 12 women, but the viral load in each woman remained stable over time. In this cohort, HIV-1 viral load remained stable during pregnancy and the greater the maternal viral burden, the more likely that transmission occurred. These two related findings suggest that determination of HIV-1 titers early in pregnancy may predict which women are at high risk of transmitting to their infants and may be used to counsel HIV-1-infected women of childbearing age. These data identify maternal viral titer as a major determinant of mother-to-child HIV-1 transmission and thereby provide the scientific rationale for therapeutic strategies designed to interrupt transmission by lowering viral load.

HIV type 2 proviral load measured by quantitative polymerase chain reaction correlates with CD4+ lymphopenia in HIV type 2-infected individuals.

The efficiency of detection of 2 sets of primer pairs from putatively conserved regions of the human immunodeficiency virus type 2 (HIV-2) genome were assessed in 86 seropositive individuals from The Gambia by nested polymerase chain reaction (PCR). HIV-2 long terminal repeat (LTR) target sequences were detected in DNA extracted from peripheral blood mononuclear cells (PBMCs) in 84 of 86 (97%) individuals whereas HIV-2 integrase (pol) gene sequences were detected in 39 of 41 (95%) individuals. The use of LTR target sequences and recombinant Pfu DNA polymerase, rather than Taq polymerase, in a modified secondary amplification reaction mediated the incorporation of 35S-labeled nucleotides in a quantitative radiometric assay. This sensitive assay was used to quantify HIV-2 proviral DNA in clinical samples and compared well with estimations by limiting end-point dilution of target molecules. A linear response between counts and the number of copies amplified from serial dilutions of pROD10 plasmid DNA (3-2000 copies) yielded a standard curve to allow extrapolation to clinical data. Increased levels of HIV-2 proviral DNA, expressed as copies per 10(5) CD4-positive lymphocytes, were associated with declining CD4 count in 63 adult patients (Spearman rank correlation, r = -0.71, n = 63, p < 0.001) and with the occurrence of HIV-related clinical disease. Kruskall-Wallis analysis of variance analysis showed the mean proviral copy number (log10) to be significantly different between groups (p < 0.001) where CD4 counts were grouped as < 200/mm3 (3.4 +/- 1.05 copies), 200-500/mm3 (2.84 +/- 0.93 copies), and > 500/mm3 (1.88 +/- 0.43 copies).(ABSTRACT TRUNCATED AT 250 WORDS)

The duration of fixation influences the yield of HCV cDNA-PCR products from formalin-fixed, paraffin-embedded liver tissue

The extent of hepatitis C virus (HCV) RNA loss during increasing periods of fixation of liver tissue in formalin was examined. For this purpose human liver tissue, known to be HCV RNA positive and stored at − 70°C until use, was cut into small slices ( n = 9), which were fixed in phosphate-buffered formalin for increasing periods of time before embedding in paraffin. Nucleic acids were extracted from each slice of formalin-fixed, paraffin-embedded liver tissue and HCV RNA loss during fixation was semi-quantified by testing 10-fold dilutions of each extract in an HCV cDNA-PCR assay. The endpoint dilution for HCV RNA detection by cDNA-PCR in liver slices fixed in buffered formalin for 8–24 h was comparable to the endpoint dilutions found for ‘fresh’, non-fixed liver slices. After fixation for 2–3 days the endpoint dilution for HCV RNA detection was 10 2 to 10 3-fold less. After 2–4 weeks of formalin-fixation, HCV RNA was detectable from undiluted nucleic acid extracts only. It is concluded that formalin-fixed, paraffin-embedded liver biopsies can be used for HCV RNA detection by cDNA-PCR, on condition that the liver tissue has been embedded in paraffin within 24 h of formalin-fixation.

Quantitative analysis of wild‐type and HBeAg minus hepatitis B viruses by a sequence‐dependent primer extension assay

The ratio between wild-type hepatitis B virus (HBV) and HBV mutant, unable to secrete "e" antigen (HBeAg minus HBV) appears to be an important determinant of the outcome of chronic hepatitis B. Quantitative analysis of wild-type and HBeAg minus HBVs in the blood could be useful to monitor chronic hepatitis B patients. We developed a solid-phase minisequencing assay for both viruses using a primer-guided incorporation of a single labeled nucleotide on an affinity captured biotinylated amplified HBV-DNA template. A standard curve was constructed by mixing increasing quantities of wild type and mutant virus DNAs. The detection of wild-type and HBeAg minus sequences, ranging from 10% to 90% of overall viremia, was linear and reproducible till 0.1 pg/microliter of serum HBV-DNA. The assay yields numerical values and the ratio of incorporated nucleotides defines the relative proportions (%) of the two viral sequences with accuracy. We tested the sensitivity and accuracy of the minisequencing on mixed end point dilutions of wild-type and HBeAg minus reference sera and amplified products. The feasibility and reproducibility of the assay were tested in 35 sera from 21 HBsAg positive patients with chronic hepatitis B using both minisequencing and oligo-hybridization assays. A high correlation was found between the two assays (r = 0.957 P < 0.0001). In conclusion, the minisequencing assay provides a precise and reproducible quantitative analysis of wild-type and HBeAg minus HBVs in clinical specimens. It is proposed to study the relations between HBV heterogeneity and the course of hepatitis B and its response to therapy.

Quantitation of HIV-1 in whole blood of infected children

To validate the technique of HIV-1 culture from whole blood for the quantitation of viral load in infected children. Forty-three HIV-1-infected children were followed in two paediatric centres. Quantitative HIV-1 cultures from unfractionated whole blood using an end-point dilution technique were compared with simultaneous quantitative cultures of peripheral blood mononuclear cells (PBMC) and plasma. Good sensitivity (93%) of the methods used was confirmed. A close correlation (r = 0.80) was observed between HIV-1 titres measured directly from whole blood and those expected from PBMC and plasma titres. The mean whole blood viral load was higher in patients with more severe signs of disease, but the difference did not reach statistical significance. The whole blood viral titres measured sequentially at monthly intervals remained within one dilution of each other in 16 of the 22 patients studied. In this study, the quantitation of HIV-1 in unfractionated blood allowed for a reliable and sensitive measurement of the whole blood viral load in infected children.

Autoantigenic Properties of Native and Denatured Glutamic Acid Decarboxylase: Evidence for a Conformational Epitope

The frequency of antibodies to GAD (anti-GAD) in insulin-dependent diabetes mellitus (IDDM) varies greatly according to the type of assay employed. We therefore examined the immunoassay characteristics of diabetic sera using GAD purified from porcine brain and shown to contain both isoforms. Sera from 38 patients with IDDM, including 1 patient with both stiff-man syndrome (SMS) and IDDM, were studied for anti-GAD by radioimmunoprecipitation (RIP), Western blotting, and dot-blotting. The sera were selected according to reactivity in the RIP assay. There was a good correlation between potency of the RIP reaction at the screening dilution of 1:2 and the endpoint dilution in the assay which ranged from 1:2 to 1:30,000 for IDDM sera, and 1:300,000 for the SMS serum. Of the 38 sera positive for anti-GAD by RIP, only 6 had antibodies detectable by Western blotting, and all gave an RIP titer of at least 1:250. The low frequency of antibodies by Western blotting was explicable by denaturation of the antigen. Thus, using a dotblotting assay in which reactivity to untreated "native" GAD was compared with reactivity to GAD after denaturation by reduction with 2-mercaptoethanol and boiling, 20 of the 38 IDDM sera reacted unequivocally with the native GAD compared with only 2 that reacted with denatured GAD after reduction and boiling. The sera were tested for their capacity to inhibit the catalytic activity of GAD, but only the high-titer serum from the patient with SMS did so. Our study further validates the RIP assay for anti-GAD and establishes that anti-GAD exists in IDDM over a wide range of titers that correlate with other assay characteristics, and also indicates that the conformational autoantibody epitope on GAD is susceptible to alteration under denaturing conditions.

Mucosal infection of neonatal rhesus monkeys with cell-free SIV.

Although the mechanisms for maternal transmission are unknown, approximately half of the infants congenitally infected with the human immunodeficiency virus type 1 (HIV-1) seem to become infected late in gestation or during delivery. Previously, we have developed a rhesus monkey model for congenital infection by injecting cell-free simian immunodeficiency virus (SIV) directly into amniotic fluid. Our results suggested that fetal infection may have occurred via skin or mucous membrane exposure. Mucosal surfaces have also been implicated as a portal of virus entry by a study in which the presence of serosanguinous fluid in neonatal gastric aspirates correlated with an increased rate of HIV-1 transmission. To test whether cell-free virus could transverse intact neonatal mucosal surfaces, we administered SIVmac251 orally to four rhesus monkey neonates within 1 hr following cesarean section delivery. All four neonates developed viremia and were positive by cocultivation and PCR. Seroconversion occurred in three of the four neonates. The SIV dose given was within physiological range as shown by end-point dilution of virus stock and viremic plasma samples of juvenile rhesus monkeys. This primate model for mucosal transmission of cell-free virus features a high infection rate, thus making studies of mucosal immunity and the development of strategies to prevent intrapartum virus transmission possible.

Quantitative assessment of human immunodeficiency virus type 1 replication in human xenografts of acutely infected Hu-PBL-SCID mice.

Replication of the IIIB strain of human immunodeficiency virus type 1 (HIV-1IIIB) in CB.17 scid/scid mice reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID) was investigated. Hu-PBL-SCID mice could be infected, in a dose-dependent manner, when HIV-1IIIB was injected intraperitoneally. Replication-competent HIV-1 could be recovered from spleen, peripheral blood, bone marrow, thymus, lymph node, and peritoneal cavity, indicating the ability of the infection to spread to human tissue throughout the chimeric animals. HIV-1 infection was quantitated using p24 determination, end-point dilution culture, and polymerase chain reaction amplification. The level of HIV-1 replication in Hu-PBL-SCID mice was found to approximate the level reported in human infection. No HIV-1-specific immune response was generated to this infection, but nonspecific immune activation did occur, as also reported in human infection. Similarities therefore exist between HIV-1 infection of humans and Hu-PBL-SCID mice, which makes the latter an in vivo model in which to study HIV-1 replication.

Hepatitis C virus replication and antibodies to structural and nonstructural viral proteins in chronic hepatitis C

The correlation between hepatitis C virus replication and antibodies to both structural (core) and nonstructural (C100-3) hepatitis C virus proteins (anti-HCVcore and anti-C100-3, respectively) was assessed. The concentration of serum hepatitis C virus RNA was determined by a competitive reverse transcription-polymerase chain reaction assay, and antibody titers were determined by endpoint dilution. No correlation was found between viremic levels and antibody titers in 42 chronic hepatitis C patients. At the end of a 6-month course of interferon-alpha therapy, 18 patients became negative for hepatitis C virus RNA. In the other 24 patients, post-treatment viremic levels ranged from 10(-6.5)-10(0.5) of pretreatment levels. Both anti-C100-3 and anti-HCVcore frequently decreased in patients whose viremic levels dropped to the negative range or to < 10(-2) of pretreatment levels. Anti-C100-3 decreased in all such cases (25/25), while anti-HCVcore decreased in 18/25 (72%) (p < 0.01), indicating that anti-C100-3 is more likely to decrease following suppression of viral replication than anti-HCVcore. These data suggest that hepatitis C virus antibodies may serve as a marker of suppression of viremia following interferon therapy even in patients who do not clear the virus.

Detection of hepatitis C virus RNA by reverse-transcriptase and polymerase chain reaction: clinical applications of quantitative analysis

Polymerase chain reaction (PCR) applications to diagnostics allowed the detection of viral nucleic acids in expected and unexpected clinical circumstances. This has raised some scepticism on the practical usefulness of PCR in the routine laboratory and emphasized the need for quatitative analysis. We addressed this question detecting HCV-RNA by a single step RT-PCR in serum samples from 50 patients with chronic non-A, non-B hepatitis included in clinical trials for recombinant alpha-interferon therapy. We obtained at least 5 serum specimens from each patient (baseline, during and after therapy samples) during an 18-month mean follow-up (range 12–45 months). RT-PCR was performed on total RNA extracted from 100 μl serum aliquots using primers for the highly conserved 5′NCR of HCV-RNA and 35 amplification cycles. PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridization against a P 32-oligonucleotide probe. Sensitivity was evaluated in separate experiments on tenfold dilutions of a reference Chimp serum containing 10 6 CID 50/ml. The overall sensitivity of our assay ranged between 10 2 and 10 3 genome Eq./ml. We establish a semiquantitative score system to evaluate viremia levels: 2 = HCV-RNA levels > 10 4 genome Eq./ml; 1 = levels between 10 3 and 10 4 g.Eq./ml; 0 = levels less than 10 2 g.Eq./ml. The reproducibility of this scoring system was confirmed testing repeatedly in duplicate end-point dilutions of positive serum samples. A statistically significant relation was observed between elevated HCV-RNA and ALT values (83.8%, chi-square 159.963 P < 0.0001). Response to IFN therapy was significantly better in patients with lower baseline HCV-RNA levels. A time relation was found between flare-ups of serum HCV-RNA levels and ALT elevations higher than 3 × normal values viremia elevations coincident or occuring about 1 month earlier than ALT elevations. This finding suggests that immuno pathogenesis might be responsible of HCV-induced liver damage as in chronic hepatitis B where identical relations were observed between viremia and ALT serum levels. In conclusion, single-step HCV-RNA RT-PCR can be a specific and reproducible semiquantitative assay and provides useful diagnostic informations for therapeutic decision making and monitoring of HCV-infected patients.

Colorimetric diagnosis of prolonged bluetongue viremia in sheep, using an enzyme-linked oligonucleotide sorbent assay of amplified viral nucleic acids

Summary Each of 5 US-origin serotypes of bluetongue virus (btv) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a btv serogroup-specific nested polymerase chain reaction (pcr) method and an embryonating chicken egg (ece) inoculation procedure. Mean duration of viremia was 100 and 38 days for the pcr and ece methods, respectively. This difference was significant (P &lt; 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of btv-specific pcr products. This enzyme-linked oligonucleotide sorbent assay (elosa) relied on annealing of separate biotinylated and fluores-ceinated probes to the amplified btv nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of pcr products indicated that the elosa was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The btv pcr-elosa system represents a more sensitive and expeditious means of diagnosing btv-induced viremia than does the ece procedure currently used. The combination of elosa with pcr should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.

Replication and tropism of human immunodeficiency virus type 1 as predictors of disease outcome in infants with vertically acquired infection

In a series of 97 infants born to mothers who were seropositive for human immunodeficiency virus type 1 (HIV-1), 18 were identified as infected within the first 60 days of life on the basis of viral culture and polymerase chain reaction findings. We studied viral burden in vivo by quantitative polymerase chain reaction and the in vitro replication pattern of the HIV-1 infecting strain by culturing patient cells with normal phytohemagglutinin-stimulated peripheral blood mononuclear cells. According to the lag phase before p24 antigen detection and the level of p24 production on peripheral blood mononuclear cells, HIV-1 isolates from these patients were classified as rapid/high (R/H), slow/high (S/H), and slow/low (S/L). The pattern of HIV-1 replication in vitro was significantly associated with the viral burden in vivo; the range of HIV-1 copies per 10(5) peripheral blood mononuclear cells was 10 to 38, 44 to 314, and 360 to 947 in children with isolates of the S/L, S/H, and R/H types, respectively. Viral tropism was assessed by culturing patient cells under end-point dilution conditions with either CD4+ T-lymphocytes or monocyte-derived macrophages. We found that children with S/L isolates harbored mainly monocytotropic variants; all infants with S/H or R/H isolates had T-lymphotropic variants and, in 7 of 11 cases, monocytotropic or amphitropic variants. All children with R/H isolates had HIV-related symptoms by the age of 4 months, and five had acquired immunodeficiency syndrome by the age of 1 year. At 1 year of age, four and no infants with S/H or S/L isolates, respectively, had HIV-1-related symptoms (p < 0.001), and none had acquired immunodeficiency syndrome (p = 0.006).

Analysis of the role of the bel and bet open reading frames of human foamy virus by using a new quantitative assay

We have constructed a BHK-21-derived indicator cell line containing a single integrated copy of the beta-galactosidase (beta-Gal) gene under control of the human foamy virus (HFV) long terminal repeat promoter (from -533 to +20). These foamy virus-activated beta-Gal expression (FAB) cells can be used in a quantitative assay to measure the infectious titer of HFV. Our results show that the FAB assay is 50 times more sensitive than determination of the virus titer by the end-point dilution method. Using the FAB assay, we have found that HFV can productively replicate in several erythroblastoid cell lines as well as in the Jurkat T-cell line. We have also examined the roles of bel2, bet, and bel3 in viral replication by constructing proviral HFV clones in which the reading frame of Bel2, Bet, or Bel3 is disrupted by placement of translation stop codons. Analysis of these mutants reveals that while the bel3 gene is not required for viral replication in vitro, mutations in the bel2 or bet gene decrease cell-free viral transmission approximately 10-fold.

Determination of plasma viral load in HIV-1 infection by quantitative competitive polymerase chain reaction

To better characterize viral load profiles through the course of HIV-1 disease and in response to treatment, and to further evaluate quantitative competitive polymerase chain reaction for measurement of viral load, we extended our comparative evaluation of this and other viral load measurements to a total of 118 patients, representing all stages of HIV-1 disease. For cross-sectional analysis across the spectrum of HIV-1 disease, plasma viral load was evaluated in 112 HIV-1-infected patients by quantitative competitive polymerase chain reaction analysis, plasma p24 antigen assay, plasma immune complex-dissociated p24 antigen assay and an endpoint dilution viral culture. Longitudinal specimens from six additional patients were analyzed, extending from the time of presentation with symptomatic acute HIV-1 infection through up to more than 2 years of follow-up. Longitudinal specimens were also studied for three patients over the period of initiation of zidovudine treatment, for 6 weeks of treatment and following temporary withdrawal of the treatment. All measurement techniques were assessed in replicate aliquots of plasma. Quantitative competitive polymerase chain reaction was the most sensitive measure of viral load, and was best correlated with CD4+ T-cell counts. In longitudinally studied patients, this technique also allowed measurement of plasma virus levels throughout the period of follow-up, even when culture and p24 assays became negative following resolution of acute HIV-1 infection. The quantitative competitive polymerase chain reaction was also able to detect rapid and substantial changes in viral load associated with initiation and temporary withdrawal of antiviral treatment. The quantitative competitive polymerase chain reaction is promising as a sensitive and accurate method for measuring plasma viral load in HIV-1-infected patients, and is useful for following changes in viral load over the natural history of infection and following treatment intervention. The technique is particularly useful for patients with > 200 x 10(6) CD4+ T cells/l, in whom other viral markers are typically negative.

Productive expression state confers resistance of human immunodeficiency virus (HIV)-2-infected lymphoma cells against superinfection by HIV-1.

In the past, positive as well as negative results pertaining to HIV-1/HIV2 interference have been obtained. Therefore, in the present study attention was paid to the viral expression state of preinfected cells at the time of exposure to secondary virus. A clonal HIV-2 infected HUT-78 cell line was derived by endpoint dilution and subsequently inoculated with cell-free HIV-1. Superinfection with HIV-1 was ruled out by Western blot and PCR analysis. The chronically HIV-2 infected cells used for these studies showed a highly productive expression state, as evidenced by immunoperoxidase staining (IPS), Western blot profile and levels of reverse transcriptase (RT) activity. We discuss several mechanisms of interference in productively infected cells, which may confer resistance to superinfection with secondary virus.

Enhanced susceptibility of blood monocytes from patients with pulmonary tuberculosis to productive infection with human immunodeficiency virus type 1.

Blood monocytes from patients with active pulmonary tuberculosis and age-matched healthy purified protein derivative-reactive donors were infected with human immunodeficiency virus type 1 (HIV-1)JR-FL in vitro to assess their susceptibility to productive infection by HIV-1. HIV-1 p24 levels (enzyme-linked immunosorbent assay) in supernatants of infected cells from patients with tuberculosis, albeit variable, were significantly higher at days 10-20 of culture; the maximum levels of p24 antigen were greater in supernatants of HIV-1-infected monocytes from patients than maximum levels for controls (p < 0.05). The maximum increment in p24 levels for patients also exceeded that for controls (p < 0.05). Entry of HIV-1 and/or initiation of reverse transcription, measured by polymerase chain reaction using HIV-1 R/U5 primer pairs, was variable and low in infected monocytes from both patients and controls, and did not correlate with HIV-1 p24 levels. The frequency of infected cells as assessed by endpoint dilution viral cultures was similar for both groups. Therefore, blood monocytes from patients with active tuberculosis can develop a highly productive infection with HIV-1 that does not appear to be due to enhanced HIV entry or higher frequency of infected cells. The enhanced susceptibility may result directly from activation of monocytes by exposure to Mycobacterium tuberculosis and its products in situ.

Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals.

The rate of clinical progression is variable among individuals infected with human immunodeficiency virus type 1 (HIV-1). Changes in viral burden which correlate with disease status have been demonstrated in cross-sectional studies; however, a detailed longitudinal study of the temporal relationship between viral burden, CD4+ T-cell numbers, and clinical status throughout the course of infection has not been reported. Multiple longitudinal blood samples were obtained from four HIV-1-infected individuals with clinically divergent profiles. Levels of HIV-1 were measured in sequential samples of peripheral blood mononuclear cells, using both end-point dilution cultures and quantitative polymerase chain reaction methods. Serial HIV-1 isolates from each case were also evaluated to determine their biological properties in vitro. For the three patients with clinical progression, a dramatic increase in the level of HIV-1 was observed concurrent with or prior to a marked drop in CD4+ T lymphocytes. This increase in viral burden was temporally associated with the emergence of a more cytopathic viral phenotype. In contrast, consistently low levels of HIV-1 were observed in the one patient who was clinically and immunologically stable for more than a decade. Moreover, viral isolates from this patient were less cytopathic in vitro compared with HIV-1 isolates from those patients with disease progression. The temporal association between increased viral burden and CD4+ T-cell decline suggests a direct role for HIV-1 in the cytopathology of CD4+ T cells in vivo. Our results indicate that the pathogenic mechanisms responsible for CD4+ T-cell depletion may be related to both quantitative and qualitative changes in HIV-1.

High Levels of HIV-1 in Plasma During All Stages of Infection Determined By Competitive PCR

Quantitative competitive polymerase chain reaction (QC-PCR) methods were used to quantify virion-associated human immunodeficiency virus type-1 (HIV-1) RNA in plasma from 66 patients with Centers for Disease Control stage I to IVC1 infection. HIV-1 RNA, ranging from 100 to nearly 22,000,000 copies per milliliter of plasma (corresponding to 50 to 11,000,000 virions per milliliter), was readily quantified in all subjects, was significantly associated with disease stage and CD4+ T cell counts, and decreased by as much as 235-fold with resolution of primary infection or institution of antiretroviral therapy. Plasma virus levels determined by QC-PCR correlated with, but exceeded by an average of 60,000-fold, virus titers measured by endpoint dilution culture. Quantitation of HIV-1 in plasma by QC-PCR may be useful in assessing the efficacy of antiretroviral agents, especially in early stage disease when conventional viral markers are often negative.

Isolation and quantitation of HIV in peripheral blood.

Quantitation of replication-competent human immunodeficiency virus (HIV) in peripheral blood of infected individuals is critical for investigations of HIV pathogenesis and therapy. In this unit, the basic protocol determines the HIV titer in seropositive blood by measuring the tissue culture infectious dose (TCID) by an end-point dilution method. A second basic protocol utilizes the PHA-stimulated T cell blasts (activated T cells) in co-culture with PBMC as described in the first basic protocol for the short-term growth of HIV in vitro. An Alternate Protocol describes the accumulative method of determining 50% tissue culture infectious dose (TCID(50)) of HIV using the Reed-Muench equation when multiple replicates of a given sample are employed in the assay. A consequence of HIV infection is the depletion of CD4(+) target cells, evidenced by syncytia formation or single-cell death; two support protocols detail the evaluation of these cytopathic effects.

Tumor necrosis factor-alpha induces circular forms of human immunodeficiency virus type-1 DNA in the persistently infected low-level expressing cell line, ACH-2

The low human immunodeficiency virus type-1 (HIV-1) expressing T-cell line, ACH-2, was used to investigate accumulation of the circular, extrachromosomal form of HIV DNA (HD) after tumor necrosis factor-alpha (TNF-α) induction. We chose the 2 long terminal repeat (LTR) circular form to analyze unintegrated HD by polymerase chain reaction (PCR), using primer pairs which flank the 2 LTR HD. Approximately a 10-fold increase in 2 LTR HD was detected intracellularly in the TNF-α-induced ACH-2 cells using an end point-dilution assay. To examine the cellular compartment location of the 2 LTR HD accumulation, ACH-2 cells were fractionated into cytoplasmic and nuclear components and further subjected to PCR. A 4- to 5-fold increase in the 2 LTR HD signal was observed in the nuclear fraction. These results indicate that unintegrated HD increases in a chronically infected cell line after TNF-α induction. This phenomenon, which previously had been observed only with acute infections, may offer insight into basic pathogenic mechanisms.