Favourable effect of chemotherapy on clinical symptoms and human herpesvirus-8 DNA load in a patient with Kaposi's sarcoma presenting with fever and anemia.

The case of a patient infected with human immunodeficiency virus type 1 (HIV-1) with Kaposi's sarcoma who presented with fever of unknown origin, severe anemia, thrombocytopenia and hypoalbuminemia but only limited involvement of the skin is presented. Chemotherapy directed at Kaposi's sarcoma resulted in resolution of these clinical signs and symptoms and was associated with a significant reduction in human herpesvirus-8 DNA load in serum, despite continued HIV-1 replication. Such a decreasing human herpesvirus-8 load following Kaposi's sarcoma-directed chemotherapy has not been reported previously. These findings suggest that Kaposi's sarcoma was indeed responsible for the clinical syndrome and that this neoplasm is a source of human herpesvirus-8 virus particle production, which can be inhibited by chemotherapy-induced reduction in tumor burden.

Human immunodeficiency virus type 1 in faeces and serum: evidence against independently evolving subpopulations.

It is not known whether independent tissue-specific evolution accounts for the differences between human immunodeficiency virus type 1 (HIV-1) subpopulations in intestinal tissue and blood. To study this, sequential serum samples from three persons were analysed for the presence of HIV-1 V3 genotypes which were detected exclusively in faeces at a specific time-point. For two persons the faeces genotype was found in serum samples collected before the time of faeces collection: 7 months for one person and 32 months for the other person. In the third person, serum collected 1 month after faeces collection contained the faeces genotype in abundance. These data indicate that a difference between intestinal tissue and blood HIV-1 subpopulations is not the result of complete compartmentalization and independent HIV-1 evolution in intestinal tissue, but that it reflects an unequal distribution of HIV-1 in different tissues.

Genetic differences between human immunodeficiency virus type 1 subpopulations in faeces and serum.

To study human immunodeficiency virus type 1 (HIV-1) compartmentalization between intestine and blood, paired faecal and serum samples were collected from 204 HIV-1-infected persons. Direct sequencing of the gp120 V3 region obtained from 33 persons showed that faecal and serum sequences could be nearly homologous (0.3% different) or very dissimilar (11.3% different). Individual clones were obtained and sequenced from the faecal and serum samples of 13 persons. In 6 persons the HIV-1 subpopulations in faeces and serum were similar, whereas in 7 persons, distribution of V3 genotypes showed a marked difference. Genetic characterization of the HIV-1 subpopulations showed less heterogeneity in faecal subpopulations than in serum subpopulations in 5 of the 7 subjects. Furthermore, faecal and serum subpopulations differed predominantly by nonsynonymous nucleotide substitutions (in 6 of 7 persons). Comparison of the HIV-1 subpopulations in faeces and serum of these 7 persons, using resampling techniques, revealed a significant difference between faecal and serum subpopulations at an N-linked glycosylation site, C-terminal of the V3 loop (amino acids 331-333). Sequences from faecal subpopulations of all 7 persons contained a glycosylation site at amino acid position 331-333. Four of these 7 harboured serum variants lacking a glycosylation site at this position. The faecal subpopulations in these 4 persons showed limited nonsynonymous substitutions compared to synonymous substitutions, indicating that purifying selection is operational on these subpopulations.

Diverse patterns of recognition of hepatitis C virus core and nonstructural antigens by antibodies present in Egyptian cancer patients and blood donors.

Serum samples from 429 cancer patients, 82 unpaid blood donors, and 74 paid blood donors were tested for hepatitis C virus (HCV) markers in two commercially available enzyme immunoassays (EIAs). A total of 229 of 429 (53.4%) cancer patients were positive by the two EIAs. A total of 34 of 156 (21.8%) of the blood donors were positive by the EIAs, with a higher prevalence among paid blood donors (20/74; 27%) compared with that among the unpaid blood donors (14 of 82; 17%). EIA-positive sera were tested for confirmation of the results in an immunoblot assay (LiaTek) in which reactivities to four synthetic peptides representing the HCV core protein and two synthetic peptides representing nonstructural proteins 4 and 5 were measured. Of 243 first and/or second EIA-positive samples from cancer patients, 188 (77.2%) were confirmed to be positive in the synthetic peptide immunoblot. A total of 33 of 35 (94.3%) blood donor samples were confirmed to be positive. A great diversity in reactivity patterns was seen. However, all sera from the group of paid blood donors were exclusively reactive to core peptides 1 and 2. A subset of LiaTek assay-positive samples were tested by the four-antigen RIBA-2 assay. The sera from the paid blood donors were all nonreactive. A subset of the LiaTek-positive sera was analyzed for the presence of the HCV genome by reverse transcriptase-PCR. Eleven of the 20 serum samples with reactivity to LiaTek core peptides 1 and 2 only were HCV reverse transcriptase-PCR positive, as were the majority of the sera with other reactivity patterns by the LiaTek assay. The results confirm the very high prevalence of HCV infection in Egypt. Furthermore, the results indicate that there is circulating in Egypt, particularly in the group of blood donors paid for their donation, an HCV variant which elicits an immune response that is not detected by the RIBA-2 assay.

Human immunodeficiency virus type 1 RNA populations in faeces with higher homology to intestinal populations than to blood populations.

To determine whether human immunodeficiency virus type 1 (HIV-1) in faeces is representative of the HIV-1 population in intestinal tissue, we studied HIV-1 V3 variation in faeces, intestinal biopsies and serum from two individuals. Phylogenic analysis of HIV-1 V3-coding RNA in faeces from one individual showed three distinct genotypes. Viruses belonging to all three genotypes were also present in sigmoidal tissue and in serum. Jejunal tissue contained two of these three genotypes. Analysis of the V3-coding RNA in faeces of the other individual showed five distinct genotypes. One of these genotypes was present in all specimens from this individual. Besides this shared genotype, jejunal tissue and serum contained sequences belonging to one other genotype. In addition, one of the other three V3 variants was detected in sigmoidal tissue. For both persons the shared HIV-1 RNA genotypes in faeces and serum displayed a distinctly different frequency distribution. In one individual, the genotype which was detected in a majority of the clones in faeces (59%) and as a minority in serum (11%), was the most abundant genotype in jejunal and sigmoidal tissue (61% and 80%, respectively). For the other individual the genotype that was present in faeces in a significant number of clones (43%) was detected in serum as a minority (8%), whereas this genotype composed 47% of the clones isolated from jejunal tissue. Taken together these data suggest that faeces contain HIV-1 sequences that are derived from local HIV-1 replication in intestinal tissue.

Isolation of human immunodeficiency virus type 1 (HIV-1) RNA from feces by a simple method and difference between HIV-1 subpopulations in feces and serum.

A simple method for the isolation and subsequent detection of human immunodeficiency virus type 1 (HIV-1) RNA from feces is described. Viral RNA was isolated by the method developed by Boom et al. (R. Boom, C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990), which was adapted for feces. HIV-1 RNA was detected by reverse transcription (RT) followed by a nested PCR encompassing the V3 region. Reconstruction experiments revealed that the efficiencies of the extraction technique and the subsequent RT-PCR were not considerably affected by the varied composition of feces. The method was applied on fecal specimens from 18 HIV-1-infected individuals, among which were samples that had been stored for 9 years. It appeared that HIV-1 RNA was detectable in the feces of 12 persons (67%). Viral RNA was present in the feces of persons who fulfilled the criteria for CDC class II and CDC class III HIV infection as well as in patients who were diagnosed with AIDS (CDC class IV). Direct sequencing of amplimers obtained from paired fecal and serum specimens showed that differences in sequence heterogeneity existed. In one patient a remarkable difference in the HIV-1 sequences between isolates from feces and serum was observed. In conclusion, HIV-1 RNA is frequently present in the feces of HIV-1-infected individuals, and in some cases the HIV-1 subpopulation in feces differs from the HIV-1 subpopulation in serum.

Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences.

The technique of nonradioactive in situ hybridization has been used to visualize the DNA and mRNA expression of human cytomegalovirus (HCMV) immediate early antigen (IEA) in a transfected rat fibroblast cell line. Expression of the transfected HCMV immediate early DNA can be induced by a cycloheximide treatment and is S-phase-dependent. In addition to cytoplasmic mRNA localization, a nuclear RNA hybridization signal was found. In a substantial part of the cells the nuclear signal was in the form of a "track," possibly showing transport of IEA mRNA from the site of transcription to the cytoplasm. The use of PCR-generated intron- and exon-specific probes in a double hybridization revealed that intron and exon mRNA sequences coexist in the nuclear RNA signal. This shows the applicability of multiple-color fluorescence hybridization to obtain information about the site of pre-mRNA splicing in the nucleus. In addition, by combining the technique of in situ hybridization with an immunocytochemical procedure we illustrate the possibility of visualizing transcribed mRNAs simultaneously with their translation products.

Rapid purification of hepatitis B virus DNA from serum.

We describe two rapid, simple, and reliable procedures for routine purification of hepatitis B virus (HBV) DNA from serum. HBV DNA could be purified from 24 serum samples in 1.5 to 2 h and was recovered in the initial reaction vessel. Both procedures have in common that HBV DNA is complexed with silica particles in the chaotropic agent guanidinium thiocyanate (GuSCN) but differ in lysis conditions and in the conditions used to elute HBV DNA from the silica particles after purification of the silica-DNA complexes. In one procedure (protocol H), serum HBV lysis was mediated by sodium dodecyl sulfate-proteinase treatment and HBV DNA was subsequently complexed with silica particles in the presence of GuSCN. After washing and drying of the silica-DNA complexes, HBV DNA was eluted from the silica particles in a low-salt buffer. In the other procedure (protocol Y*), serum HBV was directly lysed in GuSCN and HBV DNA was simultaneously complexed with silica particles. After washing and drying of the complexes, HBV DNA was eluted by proteinase treatment in low-salt buffer. Omission of proteinase treatment prevented efficient elution, presumably because of copurification of the protein which is covalently bound to the HBV DNA genome. We show, by Southern blot analysis, that HBV DNA could be reproducibly purified from human serum with the same yields by either procedure (30 to 50% relative to a classic procedure) and apparently independent of serum composition. HBV DNA purified by either method was a good substrate in the polymerase chain reaction compared with DNA purified by the classic procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and simple method for purification of nucleic acids.

We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

The SV40 small t antigen is essential for the morphological transformation of human fibroblasts.

The morphological transformation of human fibroblasts as measured in an assay for dense focus formation required, besides the SV40 large T antigen, an intact SV40 small t antigen. Using a G418-resistant colony formation assay it also was found that expression of the SV40 large T antigen only is not sufficient for the morphological transformation of human fibroblasts. Therefore it is concluded that the SV40 small t antigen is essential for the morphological transformation of human fibroblasts.

Induction of gene expression under human cytomegalovirus immediate early enhancer-promoter control by inhibition of protein synthesis is cell cycle-dependent.

In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.

Morphological transformation by early region human polyomavirus BK DNA of human fibroblasts with deletions in the short arm of one chromosome 11.

Human fibroblasts derived from four individuals with various deletions in the short arm of one chromosome 11 were susceptible to morphological transformation by early region BK virus DNA, whereas diploid human fibroblasts were not. This difference in susceptibility to transformation by early region BK virus DNA might be explained by a putative 'transformation suppressor' locus situated within the deleted region on the short arm of chromosome 11.

Resistance to methylation de novo of the human cytomegalovirus immediate early enhancer in a model for virus latency and reactivation in vitro.

Rat-9G cells carry several stably integrated copies of the major immediate early (IE) transcription unit of the human cytomegalovirus (HCMV). In these cells IE expression is repressed but inducible. In this report we describe the DNA methylation status of HpaII, HhaI and AhaII sites within the IE gene, determined at different passage levels. Most, if not all, of the resident IE genes were progressively methylated in a similar fashion. This resulted in DNA methylation patterns in which sites surrounding the IE upstream region were preferentially methylated to a high degree. In contrast, sites within the 19 bp IE enhancer elements were markedly under-methylated. This particular DNA methylation pattern probably resulted from differences in DNA methylation rates, sites within the IE enhancer being methylated at only a very low rate. Methylation of the IE genes did not affect their inducibility, which might be related to the very low methylation level of the IE enhancer.

Transcriptional activation of the major immediate early transcription unit of human cytomegalovirus by heat-shock, arsenite and protein synthesis inhibitors.

In Rat-9G cells several copies of the major immediate early (IE) transcription unit (regions 1 and 2) of the human cytomegalovirus (HCMV) are stably integrated. The cells show a repressed phenotype for IE expression but can be induced by inhibition of protein synthesis. In this report we present evidence that the repressed phenotype is due to the absence of IE transcription and that heat-shock and sodium arsenite treatments each result in the transcriptional activation of the repressed IE transcription unit. Either treatment resulted in the induction of HCMV IE transcripts and IE nuclear antigen expression. An octameric DNA sequence present in three of the 18 bp IE enhancer elements (GGACTTTC) resembles the cellular heat-shock element core consensus sequence and may therefore be involved in the heat-shock response.

Two domains within the early coding region of SV40 involved in the transformation of human fibroblasts.

The early gene products of simian virus 40 (SV40) transform human fibroblasts, whereas those of the human papovavirus BK (BKV) do not. The early gene products of both SV40 and BKV transform baby rat kidney cells. SV40-BKV chimerics were constructed which were able to transform baby rat kidney cells. Using the SV40-BKV chimerics, two domains within the SV40 large T protein were identified which are involved in the transformation of human fibroblasts.

Persistence of BK virus in human foetal pancreas cells.

High multiplicity BK virus (BKV) infection of primary cells derived from human foetal pancreas resulted in massive cytopathology and subsequent outgrowth of cells. Intranuclear BKV T-antigen was present in all cells and viral antigen was detected in 10 to 30% of these cells. The subcultured cells yielded BKV in the supernatant (approx. 10(5) TCID50/ml) and in the cells free viral DNA was present (approx. 10% of total cellular DNA content). Analysis of the viral DNA indicated the presence of deleted and rearranged BKV DNA molecules. Although all cells continuously expressed BKV T-antigen they did not exhibit the transformed phenotype. This persistent infection of human foetal pancreas cells represents a novel type of in vitro interaction between BKV and human cells which may correspond to the in vivo findings on BKV tropism for pancreatic cells.

Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate-early gene products by protein synthesis inhibition.

Upon transfection of Rat-2-TK- cells with plasmid pES, containing the cloned 7.0-kilobase (kb) EcoRI-SalI fragment (0.063 to 0.089 map units) of the human cytomegalovirus genome, major immediate-early antigen expression was obtained in 1 to 2% of the nuclei of the transfected cells, as determined by immunofluorescence with the E3 monoclonal antibody. Cotransfection of pES with the cloned herpes simplex virus type 1 thymidine kinase gene resulted in the establishment of a hypoxanthine-aminopterin-thymidine-resistant cell line which expressed a major immediate-early antigen in approximately 1% of the cells at early passages, with expression gradually declining to less than 0.1% upon subculturing. Southern blot analysis of DNA extracted from this cell line revealed the presence of multiple integration events of pES DNA sequences into cellular DNA, including a head-to-tail tandem array of approximately 10 copies of pES. The integration pattern was stable for at least 80 passages. Metaphase chromosomes prepared from this cell line showed, upon in situ hybridization, a strong hybridization signal in both sister chromatids of a large submetacentric chromosome which is considered to have harbored the tandemly integrated pES molecules. Whereas in most cells of the population, immediate-early expression seemed to be repressed, this repression could be overcome by protein synthesis inhibition, resulting in a massive induction of human-cytomegalovirus-specific transcripts of 2.1 and 1.9 kb and a minor species of 2.9 kb. After release from protein synthesis inhibition, approximately 20% of the cells showed nuclear fluorescence when the E3 monoclonal antibody was used.

Naturally occurring BK virus variants (JL and Dik) with deletions in the putative early enhancer-promoter sequences.

The genomes of two independently isolated BK virus (BKV) variants (JL and Dik) were compared with prototype BKV DNA by restriction endonuclease mapping and sequence analysis. Differences were mainly detected in two regions: the BKV (JL) and BKV (Dik) putative early enhancer-promoter regions and the middle of the T-antigen-coding regions. Base sequence analysis of these two regions showed the following. (i) The putative enhancer-promoter regions of BKV (Dik) and BKV (JL) contained only one 68-base-pair (bp) unit of the 68-bp triplication (the central copy of which is missing 18 bp) present in prototype BKV. (ii) In the same region, BKV (JL) and BKV (Dik) contained unique stretches of DNA 33 and 63 bp long, respectively. In these 63 bp, a sequence which was very similar to the proposed simian virus 40 enhancer core sequence (GGAGTGGAAAG) was present. (iii) The altered restriction endonuclease recognition sites in the sequenced part of the T-antigen-coding region of BKV (JL) and BKV (Dik) were due to base sequence changes, leaving the amino acid sequence unchanged.