Detection of cytomegalovirus DNA in short term cultures.

Detection of human cytomegalovirus (CMV) by in situ DNA hybridisation six days after incubation of human diploid fibroblasts (ISDH-6) was evaluated prospectively in 205 urine samples, obtained from 57 kidney transplant and 17 bone marrow transplant recipients. The results were compared to those of conventional virus isolation (CVI) and the detection of CMV early antigens after one day of cultivation (EA-1). Of 42 samples positive for CMV by at least one of these methods, 40 (95%) were detected with ISDH-6.

Prevalence of Chlamydia trachomatis infection in a population of asymptomatic women in a screening program for cervical cancer.

The prevalence of Chlamydia trachomatis infection in a population of women with no symptoms of sexually transmitted disease was investigated. These women, aged 35-55 years, participated in a screening program for cervical cancer. With the use of a direct immunofluorescence method, 109 out of 2,470 smears tested were positive for Chlamydia trachomatis, indicating an overall prevalence of 4.

Optimization of human papillomavirus genotype detection in cervical scrapes by a modified filter in situ hybridization test.

Optimal conditions for the screening of cervical scrapes for human papillomavirus (HPV) were investigated by using filter in situ hybridization. Since integrated and episomal HPV can be found, cell lines containing viral DNA in an integrated form (HPV in CaSki) or in an episomal state (BK virus-induced hamster tumor cells) were used for optimization experiments. An increase in sensitivity was achieved by alkaline denaturation and neutralization before the specimens were spotted onto the membrane.

Detection of Chlamydia trachomatis in culture and urogenital smears by in situ DNA hybridization using a biotinylated DNA probe.

The detection of Chlamydia trachomatis by in situ DNA hybridization in urogenital smears was investigated using a commercially available biotinylated DNA probe. Intracellular staining of inclusion bodies was used as the criterion for positivity. Of 35 patients with a culture proven chlamydial infection 19 had smears in which C.

Immunological detection of cytomegalovirus early antigen on monolayers inoculated with urine specimens by centrifugation and cultured for 6 days as alternative to conventional virus isolation.

Detection methods for human cytomegalovirus were evaluated with 431 urine samples from 30 bone marrow and 88 kidney transplant recipients. Low-speed centrifugal inoculation was followed by early antigen (EA) detection by means of indirect immunofluorescence with a monoclonal antibody after 1 (EA-1) and 6 (EA-6) days of cultivation. The results were compared with those of conventional virus isolation (CVI).

Sensitivity of in situ detection with biotinylated probes of human papilloma virus type 16 DNA in frozen tissue sections of squamous cell carcinomas of the cervix.

The sensitivity of human papilloma virus type 16 (HPV-16) DNA detection by DNA in situ hybridization using biotinylated probes (bio-DISH) was estimated by performing this technique on snap-frozen tissue sections of 10 cervical squamous cell carcinomas containing increasing amounts of HPV-16 as determined by Southern blot hybridization. A protocol using serial sections for bio-DISH and DNA extraction was used. The number of positively stained cells and the detection limit were strongly dependent on the treatment of the sections with proteinase K prior to hybridization.

Regeneration of herpesviruses from molecularly cloned subgenomic fragments.

The ability to manipulate the genomes of herpesviruses is of eminent importance for obtaining insight into gene function and regulation of gene expression of these complex viruses. Here we report the use of in vivo overlap recombination to generate pseudorabies virus mutants. Cotransfection of up to five overlapping cloned subgenomic fragments, which together constitute the entire genomic information of pseudorabies virus, results in the efficient reconstitution of virus.

Prevalence of genital HPV infections in a regularly screened population in The Netherlands in relation to cervical cytology.

To determine the prevalence of human papilloma virus (HPV) genotypes in relation to cervical cytology, 1,290 cervical samples from a regularly screened population of 30-55-year-old women were investigated. Gynaecological specimens, obtained from the cervix, were cytologically classified and screened for the presence of HPVs 6/11 and 16/18 using dot-spot DNA hybridisation. Of the cervical samples containing unequivocally normal cells, 21 of 1,271 (1.

Comparison of in situ DNA hybridization and immunological staining with conventional virus isolation for the detection of human cytomegalovirus infection in cell cultures.

The sensitivity of immunochemical staining and in situ DNA hybridization for the detection of human cytomegalovirus (HCMV) was compared with that of virus isolation. Human diploid fibroblasts were infected with serial, 10-fold dilutions of HCMV strain AD169 and examined at various intervals between 1 and 42 days after inoculation, using the three methods being compared. HCMV-DNA was detected by in situ hybridization using a biotin-labeled HCMV probe and CMV early antigen (EA) by immunochemistry using a specific monoclonal antibody.

Construction and characterization of deletion mutants of pseudorabies virus: a new generation of 'live' vaccines.

Various deletions were introduced into a cloned subgenomic fragment (BamHI-7), located in the unique short (US) region of the DNA from the virulent Northern Ireland Aujeszky-3 (NIA-3) strain of pseudorabies virus (PRV). In the cloned HindIII-B fragment, the MluI-BglII fragment was replaced by different MluI-BglII fragments of the deleted BamHI-7 clones. Transfection of the deleted HindIII-B fragments together with the HindIII-A fragment of either the NIA-3 or the non-virulent NIA-4 strain yielded replication-competent deletion mutants.

Ecotropic and mink cell focus-forming murine leukemia viruses integrate in mouse T, B, and non-T/non-B cell lymphoma DNA.

Structures of somatically acquired murine leukemia virus (MuLV) genomes present in the DNA of a large panel of MuLV-induced C57BL and BALB/c B and non-T/non-B cell lymphomas were compared with those present in MuLV-induced T-cell lymphomas induced in the same low-"spontaneous"-lymphoma-incidence mice. Analyses were performed with probes specific for the gp70, p15E, and U3-long terminal repeat (LTR) regions of ecotropic AKV MuLV and a mink cell focus-forming virus (MCF)-LTR probe annealing with U3-LTR sequences of a unique endogenous xenotropic MuLV, which also hybridizes with U3-LTR sequences of a substantial portion of somatically acquired MCF genomes in spontaneous AKR thymomas. The DNAs of both T- and B-cell tumors induced by neonatal inoculation with the highly oncogenic C57BL-derived MCF 1233 virus predominantly contain integrated MCF proviruses.

Immunologic induction of malignant lymphoma: graft-vs-host reaction-induced B cell lymphomas contain integrations of predominantly ecotropic murine leukemia proviruses.

The induction of a graft-vs-host reaction in (BALB/c X A)F1 mice by i.v. injection with BALB/c lymphoid cells leads to a lymphoid hyperplasia that may progress to malignant lymphoma.

Presence of markers for virulence in the unique short region or repeat region or both of pseudorabies hybrid viruses.

The unique short region and part of the repeat region of virulent pseudorabies virus strain NIA-3 was replaced by the corresponding region of the avirulent NIA-4 strain by transfection with subgenomic DNA fragments. The resulting hybrid virus showed a reduced virulence in both mice and pigs. Therefore, important markers for virulence are located in the unique short or repeat region or both of pseudorabies virus.

Generation of AKR mink cell focus-forming virus: nucleotide sequence of the 3' end of a somatically acquired AKR-MCF.

The 3' end of an AKR-MCF provirus (MCFr35) was cloned and found to be biologically active. Comparison of the nucleotide sequence of MCFr35 with the sequence of other MuLVs revealed that the MCFr35 was most likely derived from the same xenotropic and ecotropic parents, which were involved in the generation of AKR-MCF247. Ecotropic sequences are present around the XbaI site at position 7.

Murine leukemia virus-induced T-cell lymphomagenesis: integration of proviruses in a distinct chromosomal region.

A number of mink cell focus-forming (MCF) proviruses was molecularly cloned from mouse lymphoma DNA. From each clone, flanking probes were prepared to detect common integration regions in other MuLV-induced lymphomas. One clone frequently revealed variations in the molecular structure of the corresponding region (Pim-1) in other lymphomas.

Generation of AKR mink cell focus-forming viruses: a conserved single-copy xenotrope-like provirus provides recombinant long terminal repeat sequences.

AKV and AKR mink cell focus-forming virus-specific probes from the envelope and long terminal repeat (LTR) regions were prepared for study of the structure of recombinant proviruses in tumor tissues of AKR mice. The results showed that (i) all somatically acquired proviruses possessed, besides a recombinant gp70 gene, an altered U3 LTR; (ii) in a substantial portion of the somatically acquired AKR mink cell focus-forming proviruses, the LTR comprised sequences derived from the same xenotropic-like provirus; (iii) this U3 LTR donating parental provirus (Xeno-dL) was present only once per genome equivalent in several mouse strains; (iv) in the strains containing the Xeno-dL provirus, the provirus was present in the same chromosomal site; (v) restriction analysis of the Xeno-dL revealed that the mink cell focus-forming gp70 sequences were derived from a parental provirus, different from Xeno-dL. Therefore, at least two non-ecotropic parents participate in the generation of leukemogenic AKR mink cell focus-forming viruses: a xenotropic-like virus, Xeno-dL, donating U3 LTR sequences, and another xenotropic-like virus or viruses providing gp70 sequences.

Mobility of endogenous ecotropic murine leukemia viral genomes within mouse chromosomal DNA and integration of a mink cell focus-forming virus-type recombinant provirus in the germ line.

Characterization of endogenous ecotropic Akv proviruses in DNA of low and high leukemic mouse strains revealed the presence of one to six copies of the Akv genome per haploid genome equivalent integrated in the germ line. Low leukemic strains analyzed so far contained only one complete copy of the Akv proviral DNA. The site of integration varied among strains, although genetically related strains often carried the Akv proviral gene in the same chromosomal site.

Moloney murine leukemia virus-induced tumors: recombinant proviruses in active chromatin regions.

The DNase I sensitivity of chromosomal DNA regions carrying integrated proviral genomes of Moloney (M-MuLV) and AKR Murine Leukemia Virus (AKR-MuLV), and the cellular homologue of the mos-gene (c-mos) of Moloney Sarcoma Virus (MSV) were studied in tumor tissues of leukemic mice. The genetically transmitted sequences of M-MuLV, AKR-MuLV, and the c-mos gene are all in DNase I resistant chromatin conformations in M-MuLV-induced tumors. Each M-MuLV-induced tumor contained at least one somatically acquired integrated recombinant MuLV genome that displayed two main characteristic features of active chromatin: a) a configuration hypersensitive to DNase I, and b) extensive hypomethylation.

Characterization of AKR murine leukemia virus sequences in AKR mouse substrains and structure of integrated recombinant genomes in tumor tissues.

A specific cDNA probe of AKR murine leukemia virus (AKR-MLV) was prepared to detect AKR-MLV sequences in normal and tumor tissues in a variety of AKR mouse substrains. AKR strains contained up to six endogenous AKR-MLV genomes. All substrains tested had one AKR-MLV locus in common, and closely related substrains had several proviruses integrated in an identical site.

M-MuLV-induced leukemogenesis: integration and structure of recombinant proviruses in tumors.

M-MuLV-specific DNA probes were used to establish the state of integration and amplification of recombinant proviral sequences in Moloney virus-induced tumors of Balb/Mo, Balb/c and 129 mice. The somatically acquired viral sequences contain both authentic M-MuLV genomes and recombinants of M-MuLV with endogenous viral sequences. All reintegrated genomes carry long terminal repeat (LTR) sequences at both termini of their genome.

Expression of bacteriophage M13 DNA in vivo. Localization of the transcription initiation and termination signal of the mRNA coding for the major capsid protein.

During the infection cycle of the filamentous bacteriophage M13 a phage specific RNA species is made which selectively directs in vitro the synthesis of the precursor of the major capsid protein encoded by gene VIII. This RNA is unstable (its mean half-life is 11 min) and is made in amounts representing at least 2% of the newly synthesized RN. Nucleotide sequence analysis have indicated that the synthesis of this RNA species is initiated and terminated at the same promoter (G0.