Development of a sensitive, quantitative assay with broad subtype specificity for detection of total HIV-1 nucleic acids in plasma and PBMC.

An LTR-based quantitative PCR (qPCR) assay was modified and optimized for the quantification of total HIV-1 nucleic acids in plasma and PBMC. TaqMan qPCR primers and probes were designed against the NCBI/LANL HIV-1 compendium database by analyzing sequences used in assays for sensitive cross-clade detection of HIV-1 as reported in the literature and elucidating regions of improved cross-subtype specificity. Inosine and mixed nucleotide bases were included at polymorphic sites. Real-time RT-qPCR and qPCR were performed on plasma viral RNA and cellular lysates. A step-up amplification approach to allow binding of primers across polymorphic regions showed improved sensitivity compared to universal cycling. Unlike a lead competing laboratory-developed assay, all major HIV-1 subtypes, and a wide range of recombinants from a 127-member diversity panel were detected and accurately quantified in spiked plasmas. Semi-nested PCR increased detection sensitivity even further. The assay was able to detect down to 88 copies/mL of HIV-1 in plasma with 95% efficiency or the equivalent of a single infected cell. The PCR assay will be valuable in studies that monitor very low viral levels including residual or break through HIV-1 in patients receiving antiretroviral therapy, in HIV-1 cure, and in other research studies.

Pressure cycling technology: a novel approach to virus inactivation in plasma.

BACKGROUND: Hydrostatic-pressure virus inactivation is a novel approach to the inactivation of pathogens in plasma and blood-derived components, that retains the therapeutic properties of these products. STUDY DESIGN AND METHODS: A custom-built apparatus was used to pressurize human plasma samples spiked with lambda phage. Phage titer and plasma protein activities were monitored after pressure treatment. RESULTS: Pressure-mediated inactivation of lambda phage was found to be an effective means for virus inactivation, particularly when performed at near-zero (0 degrees C) temperatures, rather than at temperatures above 20 degrees C and below -40 degrees C. The efficiency of inactivation was improved by an increase in applied pressure and repeated cycling from atmospheric to high pressure. In contrast, activities of plasma proteins alkaline phosphatase and total amylase did not vary with temperature and remained within 29 percent and 6 percent, respectively, of starting values after the same pressure treatments. By combining cycling, near-zero temperatures, and high pressure, phage titers in serum were reduced approximately 6 log after 10 to 20 minutes of treatment. Activities of plasma proteins IgG, IgM, and factor X were at 104 percent, 89 percent, and 80 percent, respectively, of starting values after 20 minutes of the same temperature and pressure treatment. CONCLUSION: High-pressure procedures may be useful for the inactivation of viruses in blood and other protein-containing components.

The western immunoblot for Lyme disease: determination of sensitivity, specificity, and interpretive criteria with use of commercially available performance panels.

Recent recommendations for the serological diagnosis of Lyme disease include statements on quality assurance and the use of performance panels to assess laboratory competency. We used two performance panels--one from the Centers for Disease Control and Prevention (CDC) and one from Boston Biomedica Inc. (West Bridgewater, MA)-to evaluate the sensitivity and specificity of four western blot kits. We used the same panels to compare the interpretive criteria for western blots as proposed by participants in the Centers for Disease Control and Prevention, Association of State and Territorial Public Health Laboratory Directors Conference and those proposed by BBI Clinical Laboratories (BBICL; New Britain, CT). Our results indicated that the BBICL western blots were more sensitive than those of the CDC, MarDx (Carlsbad, CA), or Cambridge Biotech (Rockville, MD). However, use of the CDC criteria with the BBICL western blots increased specificity to 100% but reduced sensitivity to 74.3%. A sample table is provided as an example of the test results obtained with the BBI performance panel. Obviously, this work should be confirmed by other investigators.

In situ hybridization detection of human herpesvirus 6 in biopsy specimens from Chinese patients with non-Hodgkin's lymphoma.

An in situ hybridization assay was developed for the detection of human herpesvirus 6 in formaldehyde-fixed, paraffin-embedded tissue. This test was applied to specimens obtained from 45 patients with non-Hodgkin's lymphoma seen in Fujian, People's Republic of China, who had been classified by the working formulation and immunohistologically characterized. Human herpesvirus 6 sequences were detected in eight of 45 (mean incidence +/- SD, 18% +/- 6%) non-Hodgkin's lymphoma tumor samples tested. The significance of human herpesvirus 6-infected cells in lymphoma tissue remains to be determined.

Detection of human herpesvirus 6 in tissues involved by sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease).

After preliminary serologic data demonstrated elevated antibody titers to human herpesvirus (HHV) 6 in patients with sinus histiocytosis with massive lymphadenopathy (SHML) or Rosai-Dorfman disease, tissues were examined from 9 patients with classical SHML to search for evidence of HHV-6 infection. Involved tissues from 7 of the 9 patients had detectable HHV-6 by in situ hybridization: Tissue from 1 had detectable Epstein-Barr virus genome but no HHV-6 and tissue from another had no detectable HHV-6 or Epstein-Barr virus. These studies suggest that HHV-6 and, to a lesser extent, Epstein-Barr virus may be involved in the etiology of SHML.

Curdlan sulfate and HIV-1: II. In vitro long-term treatment of HIV-1 infection with curdlan sulfate.

Short-term (1 h) treatment with a newly synthesized sulfated polysaccharide, curdlan sulfate (CRDS), showed relatively weak blocking effects on the binding of human immunodeficiency virus type 1 (HIV-1) to the surface of H9 cells. To investigate whether long-term treatment with CRDS could strengthen this effect, CRDS in various doses (0.1, 1, 10, and 100 micrograms/ml) was used in 2-week treatment periods in four separate protocols or "Procedures." SF titers and p24 antigen levels were partially suppressed during long-term CRDS treatment but returned to control levels after the treatment was terminated. In addition, no direct cytotoxicity of CRDS to H9 cells or H9/HIV-1 cells was observed in vitro in the course of continuous exposure to 100 micrograms/ml CRDS for 2 weeks. These results demonstrate the effectiveness of long-term treatment of cells infected with HIV-1 in inhibiting virus expression. The most dramatic inhibition results were obtained when the compound was present both at the time of exposure of cells to virus and during a long-term follow-up treatment. These results show that CRDS inhibits both the cell-free and cell-associated transmission of HIV-1 to host cells and interferes with early events in virus infection. In contrast, CRDS exhibits no significant virucidal activity and has little effect on already infected cells.

Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells.

We developed an improved microtiter-based assay for the detection of polymerase chain reaction (PCR)-amplified DNA sequences. The synthetic DNA sequences used to prime the PCR were labeled with biotin at their 5' ends so that the specific PCR product was labeled with biotin. Following amplification, an aliquot of the PCR product was denatured and hybridized to a capture DNA sequence immobilized in a microtiter well. The capture sequence was complementary to a portion of the sequence between the primers, so that only extended primers were captured. The captured PCR product was detected colorimetrically by using a streptavidin-peroxidase conjugate and tetramethylbenzidine substrate.

Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization.

We have developed a microtiter sandwich hybridization assay for the detection of polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences. This assay utilizes an enzyme-linked immunosorbent assay-like format in which cloned DNA containing a sequence complementary to half of one PCR product strand is immobilized in microtiter wells. A biotin-labeled DNA sequence complementary to the other portion of the same PCR product strand is used as the probe. The DNAs from 69 hepatitis B surface antigen-positive serum samples and 16 antigen-negative control samples were amplified by the PCR procedure, and the product was detected by Southern and sandwich hybridization. Both detection procedures were capable of detecting as few as five copies of HBV DNA. Compared with Southern hybridization, the sandwich hybridization assay exhibited a sensitivity of 100% and a specificity of 95% for the detection of amplified HBV sequences. Unlike Southern hybridization, however, the sandwich hybridization assay employs a nonradioactive probe and allows easy handling of large numbers of samples. DNA was detected in 74% of the antigen-positive samples. All of the antigen-negative samples (healthy blood donors) were negative for HBV DNA by both procedures.

Persistent active herpes virus infection associated with atypical polyclonal lymphoproliferation (APL) and malignant lymphoma.

This study focuses on lymphoproliferative diseases associated with persistent infection by Epstein-Barr virus (EBV) and human herpes virus 6 (HHV-6). A suggestive premalignant lymphoproliferative state is distinguished from malignant lymphoma and identified as "atypical polyclonal lymphoproliferation" (APL). Sixteen cases of herpes virus (HHV)-associated APL are compared with 21 cases of HHV-associated malignant lymphomas (ML), with 108 cases of EBV or HHV-6 related acute infections mononucleosis, with 14 cases of seronegative non-specific lymphoid hyperplasia and with 304 cases of HHV-unrelated ML. Six cases of APL and two ML occurred in AIDS patients, two cases in Sjogren's syndrome, one in a kidney allograft recipient, and the remaining cases had no identified underlying disease. APL was histologically reminiscient of excessive infectious mononucleosis, while other cases of Castleman's disease or even of malignant lymphoma. Seropositive APL and ML contained significantly more virus genome than is found in latent background infection. There was no histologic difference between HHV-6 or EBV-positive APL or ML, although both viruses infect different lymphocyte populations. From histology alone, seropositive ML cases were not distinguished from seronegative ones, yet persistent active EBV and HHV-6 appear to predominate in follicular center cell- and immunoblastic lymphoma and in HOdgkin's disease. Although no a direct oncogenic activity of these viruses could be observed in our cases, they may contribute to lymphomagenesis by inducing progressive lymphoproliferation.

Labeling of DNA probes with a photoactivatable hapten.

A photoactivatable reagent for introducing haptens onto DNA probes has been prepared using a commercially available bifunctional linker arm reagent and amino-derivatized 2,4-dinitrophenyl (DNP). The resulting compound (photo-DNP) couples efficiently to DNA using an ordinary sunlamp. Under optimum conditions, about 7-23 DNP molecules per 1000 bases are incorporated into the DNA. Hybridization experiments demonstrate that as little as 1.5 x 10(5) copies of target DNA can be detected by filter hybridization with a photo-DNP-labeled probe and immunochemical detection.

A sensitive nonisotopic hybridization assay for HIV-1 DNA.

We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10(5) cells or about 30 molecules of HIV-1 DNA.

Polymerase chain reaction amplification and in situ hybridization for the detection of human B-lymphotropic virus.

Polymerase chain reaction amplification (PCR) is a recently described technique that allows for the amplification of a given sequence of DNA. It can be used to reliably amplify sequences of up to 3 kb within hours. The amplified sequence can then be recognized by hybridization with a specific probe after transfer onto nitrocellulose or nylon paper. We used PCR to recognize human B-lymphotropic virus (HBLV or HHV-6) specific sequences in various tumors as well as in the blood of patients with AIDS. Sixty-three specimens of DNA extracted from peripheral blood of patients with AIDS as well as DNA extracted from various lymphoproliferative disorders were analysed; 52 out of 63 (83%) patients with AIDS were found to have amplification of the HHV-6 specific sequence; 2 out of the 63 (3%) had equivocal amplification and 9 (14%) were found to be negative. Twenty out of 23 tumors were found to have amplified HBLV-specific sequences. Only one of these tumors was positive by Southern hybridization on restriction enzyme digested genomic DNA. In situ hybridization of clinical specimens using radiolabelled RNA probes or hapten-labelled DNA probes was used to detect the presence of HBLV in tumors. Three tumors of B cell origin were found to be positive for HBLV.

A chemical method for introducing haptens onto DNA probes.

We have developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. We have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 X 10(-17) mol (1 X 10(6) copies) of HBV DNA in total DNA from human serum.

Prevention of rotavirus-induced diarrhea in neonatal mice born to dams immunized with empty capsids of simian rotavirus SA-11.

Female CF-1 mice were immunized subcutaneously with a rotavirus vaccine consisting of noninfectious, purified empty capsids from simian rotavirus SA-11 and were given a booster dose at 14-17 days of gestation. The vaccinated animals developed high titers of rotavirus-specific IgG in colostrum and milk. Virus-specific IgA titers were significantly (150-fold) lower, and virus-specific IgM was not detected. These results contrast with those obtained in natural and experimental infections of the gastrointestinal tract with epizootic-diarrhea infant-mouse ( EDIM ) virus; in such cases, intestinal and lacteal virus-specific antibody is primarily of the IgA isotype. Suckling mice born to vaccinated dams were challenged with infectious EDIM virus and monitored for the development of diarrheal disease; these neonates acquired maternal antibody and did not develop diarrhea.

Focus formation and neoplastic transformation by herpes simplex virus type 2 inactivated intracellularly by 5-bromo-2'-deoxyuridine and near UV light.

The induction of focus formation in low serum and of neoplastic transformation of Syrian hamster embryo cells was examined after the expression of herpes simplex virus type 2 functions. Syrian hamster embryo cells infected at a high multiplicity (5 PFU/cell) with 5-bromo-2'-deoxyuridine-labeled herpes simplex virus type 2 (11% substitution of thymidine residues) were exposed to near UV light irradiation at various times postinfection. This procedure specifically inactivated the viral genome, while having little, if any, effect on the unlabeled cellular DNA. Focus formation in 1% serum and neoplastic transformation were observed in cells exposed to virus inactivated before infection, but the frequency was enhanced (15- to 27-fold) in cells in which the virus was inactivated at 4 to 8 h postinfection. Only 2 to 45 independently isolated foci were capable of establishing tumorigenic lines. The established lines exhibited phenotypic alterations characteristic of a transformed state, including reduced serum requirement, anchorage-independent growth, and tumorigenicity. They retained viral DNA sequences and, even at relatively late passage, expressed viral antigens, including ICP 10.