Amount Of Viral DNA Research Articles

Identification of herpes simplex virus DNA in lesions of erythema multiforme by the polymerase chain reaction

An association between erythema multiforme and herpes simplex virus infection has been supported by clinical studies and by the detection by immunofluorescence of herpes viral antigen in sera and skin biopsy specimens of patients with erythema multiforme. In rare cases, the virus has also been isolated in cultures of skin biopsy specimens of erythema multiforme. To investigate further the association between erythema multiforme and herpes simplex virus, we used the polymerase chain reaction for herpes simplex virus to examine skin lesions from patients with erythema multiforme. In this study herpes simplex virus DNA was detected in 11 of 31 biopsy specimens of erythema multiforme; six additional cases showed equivocal amplification results, which is suggestive of low amounts of viral DNA. Seven skin and mucosal biopsy specimens with the histologic changes of herpes virus infection served as positive controls; all were positive for herpes simplex virus DNA. Viral DNA was not detected in control biopsy specimens from skin excised for unrelated conditions. These studies support the association of herpes simplex virus in the pathogenesis of some cases of erythema multiforme. The polymerase chain reaction provides a quick and effective method of detecting herpes simplex virus in lesions of herpes-associated erythema multiforme. Furthermore, the polymerase chain reaction may delineate those cases of erythema multiforme that are etic-logically related to herpes virus infection and therefore might be treated with acyclovir to prevent recurrence.

Detection of Epstein-Barr virus and human herpes virus type 6 in saliva from patients with lymphoproliferative diseases by the polymerase chain reaction

Direct detection of these viruses was made by using the PCR for amplifying viral DNA. In virtually all adults low levels of the herpesvirus can be detected. Therefore it is necessary to quantitate the amount of viral DNA, and a method to compare the plasmid containing the cloned target gene was used here. After 35 cycles of amplification, 10 copies of the viral DNA per 100 μl saliva were detected. The PCR was used to detect increased levels of EBV and HHV-6 in saliva from patients with lymphoproliferative diseases, suggesting that these viruses may play a part in their pathogenesis. Viral detection by such highly sensitive methods as PCR may allow better monitoring of medication, as well as early detection of EBV- and HHV-6 related diseases that may arise in these patients. The great sensitivity of PCR and its ability to analyse very small samples make this technique most suitable for clinical diagnosis.

Quantitation of cytomegalovirus DNA by blot hybridization in blood leukocytes of viremic patients

We describe a technique for quantitation of viral DNA in blood leukocytes during viremic infection with human cytomegalovirus (CMV). Using a cloned subgenomic DNA probe and a blot hybridization assay, small amounts of viral DNA within samples of leukocyte DNA could be quantitated reproducibly using a videodensitometer. Critical components of the assay were: (1) a direct relationship between optical density and known amounts of viral DNA diluted in cellular DNA as positive standard samples and, (2) determination of the proper duration of autoradiographic exposure. The technique was sufficiently sensitive to detect 10 picograms of CMV DNA in the presence of microgram quantities of host cell DNA. In addition, samples containing minute amounts of CMV DNA could reliably be distinguished from samples that were negative. We have used the technique to characterize the pathogenesis of CMV viremia and to monitor the effects of antiviral chemotherapy. This procedure could easily be applied to pathogenetic studies of a variety of other infectious agents.

Enteric adenoviruses

Human adenoviruses are classified into 47 serotypes and six subgenera (A–F) with different tropisms. In recent years adenovirus type 40 (Ad40) and 41 (Ad41) of subgenus F have been shown to be causative agents in enteric infections, which is second in importance only to rotaviruses as a cause of infantile gastroenteritis. Infection with EAds occurs worldwide and has been associated with 4–17% of cases of diarrhoea in children. Ad40 and Ad41 primarily affect young children less than 2 years of age and occur throughout the year. The clinical characteristics include watery diarrhoea accompanied by vomiting, low grade fever and mild dehydration. A distinct feature of EAds infection is the protracted diarrhoea (mean 8.6 and 12.2 days for Ad40 and Ad41, respectively). Respiratory symptoms are infrequent. Serotypes Ad40 and Ad41 differ from all other (established) adenoviruses by being unable to replicate in conventional cell cultures. These fastidious viruses only grow in selected cell lines, 293 cells being the most commonly used. In spite of the difficulty of isolating Ad40 and Ad41, they can be directly identified and typed by ELISA and solid-phase immune electron microscopy. The amount of viral DNA in stool specimens is sufficient for identification by DNA restriction and dot-blot assays. The recent development of highly sensitive and specific monoclonal antibody-based ELISAs enable accurate diagnosis of adenovirus gastroenteritis in routine work and make possible the evaluation of the role of the enteric adenoviruses in diarrhoeal disease in the developing countries.

Extrahepatic Hepatitis B Virus Dna Sequences in Patients With Acute Hepatitis B Infection

Recent studies have demonstrated the presence of hepadnavirus-related nucleic acids in extrahepatic tissues in various animal models. The prevalence and biological significance of extrahepatic infection in humans remains undetermined. To characterize the tissue distribution and physical state of extrahepatic hepatitis B virus nucleic acids in acute hepatitis infection, we examined serum, liver and multiple extrahepatic tissues obtained at autopsy from two patients with fulminant hepatitis and one patient with resolving hepatitis who died of an unrelated cause. Southern-blot hybridization analysis was used to analyze the physical state of hepatitis B virus-related DNA. Hepatitis B virus-related RNA sequences were examined by slot-blotting total RNA extracted from corresponding tissues. Hepatitis B virus nucleic acids were demonstrated in lymph nodes, spleen, gonads, thyroid gland, kidneys, pancreas and adrenal glands. The most intense signal of hybridization was obtained with DNA extracted from lymph nodes. In general, the levels of hepatitis B virus RNA correlated with the amount of viral DNA. Fast-migrating DNA sequences resembling replicative intermediates and ranging in size from 1 to 3.2 kb were detected in EcoRI digests. Faint high-molecular-weight smears suggesting random integration also were observed. Remarkably, little or no hepatitis B virus nucleic acid was detected in the serum or liver. In control specimens obtained from hepatitis B virus carriers, most hybridizable hepatitis B virus nucleic acid was present in liver, but hepatitis B virus DNA was also detected in extrahepatic tissues. Finally, no specific histological changes were observed in extrahepatic tissues harboring hepatitis B virus.(ABSTRACT TRUNCATED AT 250 WORDS)

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.

Rate of increase of Autographa californica nuclear polyhedrosis virus in Trichoplusia ni larvae determined by DNA:DNA hybridization

The rate of increase and doubling time of the HOB clone of Autographa californica nuclear polyhedrosis virus (AcMNPV-HOB) in neonate Trichoplusia ni larvae was determined by measuring the increase in viral DNA through time following inoculation with average doses of 50 or 17,400 occulusion bodies per larva. Changes in total DNA and viral DNA through time were followed by fluorescence spectroscopy and quantitative slot-blot DNA:DNA hybridization, respectively. Total DNA content (i.e., larval DNA and viral DNA) of larvae infected with the intermediate dose lagged behind that of noninfected larvae 30 hr post-inoculation (p.i.), reached a maximum at 51 hr p.i., and stayed constant thereafter. The total DNA content of larvae inoculated with the high dose lagged behind that of the control group from 18 hr p.i. and increased slowly untildeath of the larvae (ca. 48 hr p.i.). The amount of viral DNA in larvae inoculated with the intermediate dose increased exponentially between 15 and 42 hr p.i., reached a maximum at 48 hr p.i., and stayed constant until 68 hr p.i., by which time most larvae had died. The amount of viral DNA in larvae inoculated with the high dose did not increase exponentially; initially the rate of increase was the same as that for larvae inoculated with the intermediate dose but became progressively lower after 13 hr p.i. Calculations of the rate of increase for AcMNPV-HOB in neonate T. ni larvae inoculated with the intermediate dose and incubated at 29°C resulted in a value of 0.264 hr −1 (doubling time: 2.63 hr). These values agree very well with 0.274 hr −1 (doubling time: 2.53 hr) previously calculated from dose-survival time assays using a birth-death model and lend support to the applicability of this model to insect-baculovirus interactions.

Latent infections in spinal ganglia with thymidine kinase-deficient herpes simplex virus

A herpes simplex virus type 1 variant [C239(TK-)] harboring a deletion in the thymidine kinase (TK) gene was assessed for capacity to establish latent infections. Outbred Swiss Webster mice were inoculated on both hind footpads, and numbers of neurons expressing latency-associated transcript and amounts of viral DNA in latently infected lumbosacral spinal ganglia were scored. C239(TK-) established levels of latent infection that were only slightly lower than those found for either a TK rescued variant of this agent or the parent wild-type KOS. However, in contrast to the TK+ viruses, C239(TK-) could not be reactivated when spinal ganglia were cultured in vitro. The results presented show that expression of the viral TK gene plays no major role in establishment of the latent state but that it functions during reactivation of latent virus from explanted ganglia maintained in vitro.

Hybridization techniques provide improved sensitivity for HCMV detection and allow quantitation of the virus in clinical samples

Hybridization techniques (slot-blot and in-situ hybridization assays) and immunostaining using murine monoclonal antibodies directed against different proteins of the human cytomegalovirus (HCMV) were compared for their sensitivity and specificity for detection of HCMV. A model system with HCMV infected human embryonic lung fibroblasts and lung biopsy specimens obtained from patients with culture positive HCMV interstitial pneumonia were used for evaluation of these techniques. The hybridization techniques were found to provide an improved sensitivity compared to immunostaining. Additionally a good correlation was found between the virus dose determined by TCID 50 and the amount of viral DNA detected by slot-blot hybridization and by the number of autoradiographic silver grains per 100 cells per 2 weeks exposure time detected in the infected fibroblasts by in-situ hybridization. Thus, at least in the model system quantification of the virus was achieved by hybridization assays.

Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression.

The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes.

Limitations to the expression of parvoviral nonstructural proteins may determine the extent of sensitization of EJ- ras-transformed rat cells to minute virus of mice

The FR3T3 and NRK rat cell lines and their human EJ Ha- ras-1 oncogene-transformed derivatives, termed FREJ and NREJ, were compared for their susceptibility to the parvovirus MVMp. For a similar production of p21 ras protein, FREJ clones are markedly sensitized to killing by MVMp, whereas the NREJ cells are not. Such a contrasting effect of ras transformation on the sensitivity of cells of different origins to MVMp can be traced back to their respective abilities to support the parvoviral life cycle. The FR3T3 line produces a substantial amount of viral DNA whose expression in the form of the nonstructural protein NS-1 is stimulated in its transformed derivatives. Conversely, NRK cells offer an early block to parvoviral DNA replication and expression that appears to persist in the ras-transformed clones. Thus, at least two intracellular restrictions can protect normal rat cells against MVMp infection, and transformation by ras relieves one of them at the level of parvoviral gene expression. A fair correlation was also found between the degree of sensitivity of the various lines to MVMp-induced killing and their capacity to synthesize the nonstructural viral proteins, suggesting a possible role of parvoviral nonstructural proteins in cytotoxicity.

Regulation by dimethylsulfoxide, insulin, and corticosteroids of hepatitis b virus replication in a transfected human hepatoma cell line

A human hepatoblastoma cell clone E4 was obtained by transfection of HepG2 cells with a plasmid DNA containing four tandem copies of hepatitis B virus (HBV) genome. Analysis of both intracellular and extracellular viral DNA revealed that this clone exhibited the main steps of the replication process previously found in normal hepatocyte primary cultures experimentally infected in vitro. Indeed, relaxed-circular, covalently closed circular, and single-stranded forms of viral DNA were identified in the cells together with complete virions and immature cores in the medium. Furthermore, the ability of these secreted particles to infect normal human hepatocyte cultures was established. These E4 cells were used to evaluate the effect of various soluble factors on HBV replication. Corticosteroids and, to a greater extent, dimethylsulfoxide (DMSO) increased intracellular viral DNA, whereas insulin reduced it dramatically. Parallel changes in the amounts of viral DNA secreted in the medium were observed. Measurement of the albumin secretion rate indicated that cellular and viral activities could be regulated, at least in part, in a coordinated manner.

Human immunodeficiency virus type 1 infection in homosexual men who remain seronegative for prolonged periods.

Infection with the human immunodeficiency virus type 1 (HIV-1), as demonstrated by viral cultures, has been described in some patients before antibodies to HIV-1 can be detected, but the duration and frequency of such latent infections are uncertain. We selected prospectively a cohort of 133 seronegative homosexual men who continued to be involved in high-risk sexual activity, and we cultured 225 samples of their peripheral-blood lymphocytes, using mitogen stimulation to activate the integrated HIV-1 genome. HIV-1 was isolated in blood samples from 31 of the 133 men (23 percent), 27 of whom have remained seronegative for up to 36 months after the positive culture. The other four men seroconverted 11 to 17 months after the isolation of HIV-1. In three of them, we studied cryopreserved lymphocytes obtained earlier, using the polymerase chain reaction to amplify small amounts of viral DNA, and we demonstrated that HIV-1 provirus had been present 23, 35, and 35 months before seroconversion. We conclude that HIV-1 infection in homosexual men at high risk may occur at least 35 months before antibodies to HIV-1 can be detected. A prolonged period of latency in such infections may be more common than previously recognized; the degree of infectiousness during such periods is unknown.

Intramolecular homologous recombination in cells infected with temperature-sensitive mutants of vaccinia virus

I have used a plasmid containing two copies of the Saccharmyces cerevisiae his3 gene to study intramolecular homologous recombination in vaccina virus-infected cells. Recombination of the plasmid was monitored by restriction enzyme digestion and Southern blot hybridization in cells infected with representatives from each of 32 complementation groups of temperature-sensitive mutants ts42 and ts17 did not replicate nor detectably recombine the input plasmid. All except one of the mutants that synthesized normal amounts of viral DNA and protein replicated and recombined the plasmid in a manner indistinguishable from wild-type virus. The remaining mutant, ts13, only poorly replicated and recombined the input plasmid. Thus, the processes of replication and recombination could not be separated by using this battery of mutants. Viral mutants defective in late protein synthesis were unable to resolve the vaccinia virus concatemer junction in plasmids but carried out intramolecular homologous recombination with plasmids as efficiently as did wild-type virus at the conditionally lethal temperature. This result distinguishes homologous recombination, which requires early gene products, from resolution of concatemer junctions, which requires additional late gene products.

Inhibition of CMV replication in human fibroblasts by human monocyte-derived macrophages: implication for CMV persistent infection

The effect of monocytes (M) and monocyte-derived macrophages (MdM) on cytomegalovirus (CMV) replication in human fibroblasts (HF) was studied by one step growth experiments, plaque formation and dot hybridization with a labeled CMV DNA probe. HF were infected with CMV at multiplicities of infection (mol) of 0.001 to 1. After virus adsorption, M or MdM were added at an effector target ratio of 2:1. MdM reduced both infectious viral yield and the quantity of viral DNA and inhibited viral plaque formation. M, however, affected these parameters to a lesser extent. Electron microscopic studies showed that MdM treated CMF-infected HF, 4 days pi, contained only a few capsids in their nuclei and many vacuoles in their cytoplasm as compared to CMV infected HF (control). A reduction of CMV DNA inhibition was observed upon incubation of the infected HF cells with MdM separated from the infected cells by a membrane. Addition of tumor necrosis factor (TNF) antibody to CMV-infected HF incubated with MdM either in direct contact or separated by a membrane from the infected cells reduced the inhibition of CMV-DNA production. The results of this study suggest that MdM may modulate CMV replication in vivo and may also have a role in CMV persistence or chronic infection.

Transcriptional activity of human papillomavirus type 6 in respiratory tract papillomata.

We have investigated the molecular basis for differences that we observed in the biological activities of genetically related but distinguishable human papillomavirus type 6 (HPV-6) subtypes. To analyse tissue-specific differences in replication and transcription, and to identify viral gene products important in the benign transformation of epithelial cells, we have modified procedures utilizing guanidinium isothiocyanate and density gradient centrifugation to facilitate the extraction of relatively undegraded DNA and RNA from 21 biopsy specimens of respiratory tract papillomata. Southern transfer analysis was used to characterize the viral genome, and to demonstrate that relative quantities of viral DNA in lesions varied but that the range was similar in lesions induced by HPV-6c, -6d, -6e and -6f. Dot blot analysis of the amount of viral RNA in comparison with the amount of 28S ribosomal RNA indicated that the relative level of viral RNA in each lesion varied considerably and that on average there was approximately twice as much viral RNA in HPV-6c-induced lesions as in HPV-6d-, -6e- or -6f-induced lesions. In dot blot and Northern analyses, hybridization of RNA from HPV-6c-induced lesions with HPV-6c DNA gave a stronger signal than hybridization of the same RNA with an HPV-6e probe, and vice versa. These differences in hybridization intensities with subtype-specific probes indicate that the most abundant RNA species are transcribed from parts of the genome that show sequence divergence between these two subtypes. Northern analysis demonstrated the predominant viral transcript to be about 1200 nucleotides in length in lesions induced by each of the four subtypes.

Porcine parvovirus: replication in and inhibition of selected cellular functions of swine alveolar macrophages and peripheral blood lymphocytes.

The ability of four isolates of Porcine Parvovirus (NADL-8, NADL-2, KBSH, and Kresse) to replicate in and affect the functions of swine peripheral blood lymphocytes and alveolar macrophages was studied in vitro. V-strand and C-strand viral DNA was present in both concanavalin A- and non-treated lymphocytes as well as alveolar macrophages following infection with all four isolates. Indirect fluorescent antibody assays on swine testis cells, inoculated with cell lysates of NADL-8-infected peripheral blood lymphocytes (both concanavalin A- and non-treated) and alveolar macrophages, indicated that these immune cells supported the production of progeny virus. The quantity of viral DNA and progeny virus was dependent upon the multiplicity of infection and length of time following infection. Infection of lymphocytes and alveolar macrophages with PPV was associated with a decrease in cell viability. Peripheral blood mononuclear cells and alveolar macrophages infected with any of the four isolates demonstrated reduced lymphocyte blastogenesis and non-Fc-mediated alveolar macrophage phagocytosis, respectively.

Histological and molecular analysis of cottontail rabbit papillomavirus-induced papillomas treated with hematoporphyrin derivative photodynamic therapy.

The clinical regression of cottontail rabbit papillomavirus-induced cutaneous papillomas on Dutch-belted rabbits following treatment with hematoporphyrin derivative photodynamic therapy was recently reported. Those papillomas that underwent total regression failed to recur at 18 months. Tissues from totally regressed papillomas and adjacent sites failed to reveal the presence of papilloma on histological examination. These findings were confirmed by the absence of viral DNA following analysis by Southern blots for cottontail rabbit papillomavirus viral copy number and DNA restriction pattern. Partially regressed papillomas and untreated papillomas revealed histologically typical papillary features with approximately equal amounts of viral DNA.

Tumour-associated antigens in primary mouse fibroblasts induced by transformation with bovine papillomavirus type 1

Bovine papillomavirus type 1 (BPV 1) DNA was used to transform primary fibroblasts of C57BL/6J mice. Transformation frequency in these cells was much lower than in C127 cells and not associated with the appearance of morphologically distinct foci. However, continuous lines of transformed C57BL/6J cells were developed by serial subculturing of transfected cells. These transformed cell lines showed phenotypic properties associated with transformation including abnormal karyotypes. They contained variable amounts of viral DNA, but the copy number was in the same range as in six C127 transformants tested for comparison. In two cell lines monomeric viral DNA in an episomal form was detected. Slowly migrating viral sequences in these and in the third line were probably episomal concatamers, but the possibility of integration could not be excluded. There was some variation in immunogenicity, but all cell lines induced a cell-mediated immune response in syngeneic mice detected by the chromium release assay. In addition to BPV 1-transformed cell lines, the effector cells also reacted against an unidentified antigen shared by 2 cell lines transformed by SV40 and UV irradiation, respectively.

Further characterization of the interaction of polyoma virus and simian virus 40 with embryonal carcinoma cells

The host restriction imposed on polyoma virus by embryonal carcinoma cells can be circumvented by mutations in the enhancer region of the viral genome. In addition, expression of the early viral genes can be induced by differentiating cells transfected with purified viral DNA. Although no host-range mutants of SV40 have been isolated, expression of T antigen from episomal genomes can be induced by differentiating the embryonal carcinoma cells transfected with microgram quantities of DNA. Further, transient expression of T antigen can be observed in the embryonal carcinoma cells following transfection with large amounts of viral DNA. In addition, replication of the A2 strain of polyoma in F9 cells is enhancer dependent but the SV40 enhancer can functionally substitute for the polyoma enhancer. The F9 host-range mutant TT340 contains five tandem repeats of the region surrounding the origin of replication, and it requires T antigen for replication. The parental strain (Toronto) of the mutant is able to replicate at low levels in a T antigen-dependent manner in F9 cells. This strain also has an unselected host range for PCC4 cells and the mutation in TT340 required for growth on F9 cells does not alter this inherent host range.