Biomedical subjects

G Darai

Publications and source records attributed to G Darai.

At least 127 records · Page 7Linked to original sources

PubMed1986

Replacement of the deletion in the genome (0.762-0.789 mu) of avirulent HSV-1 HFEM using cloned MluI DNA fragment (0.7615-0.796 mu) of virulent HSV-1 F leads to generation of virulent intratypic recombinant.

The HFEM strain of HSV-1 is apathogenic for the tree shrew by the intraperitoneal (i.p.) route because of a deletion in the genome coordinates 0.762-0.789. Insertion of the MluI DNA fragment (coordinates 0.7615-0.789) cloned from HSV-1 strain F, which is pathogenic for the tree shrew, restored the i.p. pathogenicity to strain HFEM. The recombinant designated R-M1-C1 was highly pathogenic for the tree shrew, but slightly virulent for inbred mouse strain A. It thus appears that the viral DNA sequence involved in the i.p. pathogenicity of HSV-1 is located within the genome coordinates 0.761-0.796. This sequence is recognized differently by the cellular elements involved in HSV-1 infection in the tree shrew and the mouse.

Animals↗

PubMed1986

Isolation and characterization of a tupaia rhabdovirus.

Liver tumor biopsies of a 9-year-old moribund Tupaia (tree shrew) were explanted and cultured in vitro. The cell cultures degenerated spontaneously. A virus was isolated from the cell-free supernatant of these cultures and subsequent electron microscopy revealed rhabdovirus-like particles. Negative staining showed typical bullet-shaped particles 125-220 nm in length with a diameter of 68 nm studded with a dense layer of surface projections 9-11 nm in length. One end of the virion was flat, the other end was open; a distinct ribonucleoprotein (RNP) core was visible. The pitch of the RNP was 4.5 nm. Virus was assembled and matured by budding primarily into regions of dilated endoplasmic reticulum. The dimensions of the virion also were determined in ultrathin sections: the diameter and length of the virion were 52 and 125-255 nm, respectively, those of the RNP core were 39 and 120-240 nm. Only tupaia embryonic fibroblasts and kidney cells were susceptible to the rhabdovirus. The virus, when plaque-assayed on tupaia embryonic fibroblasts, grew to a titer of 1 X 10(6) PFU.

Animals↗

PubMed1985

Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus.

A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.

Animals↗

PubMed1985

Investigation of the virulence genes of herpes simplex virus 2 by experimental infection in vivo with defined intertypic recombinants of a virulent HSV-2 X an avirulent HSV-1.

For determination of the gene loci for virulence and tropism of herpes simplex virus 2 (HSV-2) intertypic recombinants of HSV-1 strain HFEM and HSV-2 strain 3345 were screened for their pathogenicity in the tree shrew. HSV-1 strain HFEM is not pathogenic in the tree shrew and can be recovered as a latent virus only from the spleen of the animals; but HSV-2 strain 3345, the other parental virus of the recombinants, was found to be virulent for the tree shrew and colonized the ganglia of latently infected animals. No clinical pictures were observed when the animals were inoculated with HSV recombinants RS1(3), RB2(3), RB2(8), RB2(9), RB2(10), RB5(2), RB7(3), and RB7(7). It was found, however, that HSV recombinant RB5(0) is pathogenic for the tree show. Studies of the state of viral latency in the animals infected with these recombinants revealed that only HSV recombinant RB5(0) was able to persist as latent virus in the spleen of latently infected animals. The genome of recombinant HSV-RB5(0) contains DNA sequences of HSV-2 strain 3345 with two interruptions at 0.22-0.33 and 0.54-0.59 viral map units. In these regions of HSV-RB5(0), DNA sequences of HSV-strain HFEM at 0.26-0.33 and about 0.57-0.58 viral map units, respectively, had been recombined. The results obtained in this study indicate that the recombinant HSV-RB5(0) acquired the virulent phenotype (v+) from parental virus HSV-2 strain 3345 but lost the phenotype for colonizing the ganglia (g+). Furthermore, it acquired the phenotype of parental virus HSV-1 strain HFEM for persisting in a latent state only in the spleen (s+ and g-) of the latently infected animals.

Animals↗

PubMed1985

Transduction of virulence in herpes simplex virus type 1 from a pathogenic to an apathogenic strain by a cloned viral DNA fragment.

Herpes simplex virus (HSV) type 1 strain HFEM, the genome of which contains a deletion of about 4 kilobasepairs (kbp) between 0.7 and 0.8 viral map units, is apathogenic in the tree shrew when the animals are inoculated intravenously, intraperitoneally and/or subcutaneously. Similar results were obtained using Balb/c mice. Studies of the state of viral latency in animals infected with HSV-1 strain HFEM revealed that this strain was unable to colonize the ganglia of tree shrews. Infectious virus could be recovered only from the spleens of latently infected tree shrews. Thus, this system offers new opportunities for investigating the gene functions responsible for the virulence of HSV-1. Marker rescue experiments were performed by the contransfection technique using native DNA of HSV-1 strain HFEM and Bam H I DNA fragment B derived from the pathogenic HSV-1 strain F and cloned in a bacterial vector. A number of different intratypic recombinants were established in which the deleted region of HSV-1 strain HFEM had been repaired. The pathogenicity of these recombinants was examined in vivo. One of the recombinants (HSV-R-HFehx-C19) caused generalized and lethal herpes virus infection in juvenile and adult tupaias, indicating that the virulence of the pathogenic HSV-1 strain F can be transduced by the cloned Bam H I DNA fragment B to the apathogenic HSV-1 strain HFEM.

Animals↗

PubMed1985

Mapping of the deletion in the genome of HSV-1 strain HFEM responsible for its avirulent phenotype.

To determine the exact map position of the deletion in the genome of HSV-1 strain HFEM, the Bam HI DNA fragment B of the HSV-1 strains HFEM, F, and of HSV-R-HFehx-C19 were cloned in the Bam HI site of the bacterial plasmid vector pAT153 and analysed in detail. Analysis of the insert of the three recombinant plasmids harbouring the Bam HI DNA fragment B of HSV-1 strain F (pHSF-B-B), HSV-1 strain HFEM (pHSHF-B-B), and recombinant HSV-R-HFehx-C19 (pHSR19-B-B) was performed using a variety of restriction enzymes and Southern blot hybridization. These comparative studies revealed that the HpaI DNA fragment P of the intact viral genome of HSV-1 strain F is not present in the genome of HSV-1 strain HFEM, indicating that the deletion in HSV-1 strain HFEM corresponds to this fragment, which spans the coordinates 0.762 to 0.790 of the HSV-1 DNA. The size of the deletion was found to be 4.1 kbp, corresponding to 2.7 Md.

Animals↗

PubMed1985

The nucleotide sequence of the early region of the Tupaia adenovirus DNA corresponding to the oncogenic region E1b of human adenovirus 7.

The nucleotide sequence of the early region E1b of the tree shrew (Tupaia) adenovirus (TAV) DNA has been determined. The sequenced region includes the genes for polypeptides of Mr 15 000, 44 000 and 13 400, which are analogous to the small and large E1b proteins and protein IX, respectively, of the three human adenovirus serotypes 5, 7, and 12. The hexanucleotide consensus signal AATAAA occurs only at the 3' terminus of the gene for protein IX suggesting that the E1 region of TAV encompasses one transcription unit. The amino acid sequences of the TAV polypeptides have a higher degree of homology to those of Ad7 and Ad5 than to those of Ad12.

Adenoviridae↗

PubMed1985

Infectious hepatitis B virus from cloned DNA of known nucleotide sequence.

The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA.

Animals↗

PubMed1985

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.

Animals↗

PubMed1985

Analysis of the genomic termini of tupaia herpesvirus DNA by restriction mapping and nucleotide sequencing.

A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging.

Animals↗

PubMed1985

Characterization of the DNA of canine adenovirus by restriction enzyme analysis.

The DNA of canine adenovirus (canAV 1, strain Behring H.c.c. 269) was characterized in detail. The molecular weight of canAV DNA was (20.1 +/- 0.1) x 10(6) daltons as determined by contour length measurements, and 19.9 x 10(6) daltons as determined by restriction enzyme analysis. A terminal protein was found to be covalently linked to the 5' ends of canAV DNA. Physical maps of viral DNA were constructed for BamHI, ClaI, SacI, SalI, and SmaI enzymes. Three international strains of canAV [American Type Culture Collection (ATCC) catalogue No. VR-800, VR-293, and VR-133] were analyzed under the same conditions. CanAV 1 (ATCC VR-133 and VR-293) had DNA cleavage patterns identical to the H.c.c. 269 strain, whereas the DNA cleavage pattern of canAV 2 (ATCC VR-800) was clearly different.

Adenoviridae↗

PubMed1985

Amplification and stability of recombinant plasmid molecules with a very large insert of foreign genetic material.

The capacity of bacterial plasmid vector pAT 153 for harbouring and for amplifying in a stable way a very large insert of foreign genetic material was screened. The DNA of a tree shrew herpesvirus (strain 2) with a molecular weight of about 130 megadalton (200 kilobasepairs) consisting of a long unique DNA sequence without direct and inverted repeats (greater than 40 basepairs), was used for molecular cloning. A successful insertion and stable amplification of a DNA fragment with 49 megadalton (74 kilobasepairs) cloned in Eco RI site of pAT 153 was performed, which resulted in the construction of recombinant DNA plasmid pTH2-E-C1. The stability of the recombinant plasmid pTH2-E-C1 with a super insert of 74 kilobasepairs (49 MD) indicates that the bacterial plasmid vector pAT 153 has really a high capacity for harbouring foreign genetic material.

Bacteria↗

PubMed1985

Molecular cloning and a stable amplification of the DNA molecules heavily methylated at CpG sequences using a new E. coli cell system (GC3).

Fish lymphocystis disease virus (FLDV) DNA is heavily methylated in CpG sequences and therefore the amplification of recombinant DNA of FLDV inserted into a bacterial plasmid vector and/or a bacteriophage system poses severe problems. The problem was solved using a newly constructed and developed bacterial system (E. coli GC-3 system), which allowed the amplification of foreign DNA material heavily methylated at CpG sequences. E. coli GC-3 (Gc-1, rifd) is derived from E. coli GC-1 (K 12, hsd-, MDU) after transformation with phage lambda drifd 18 Hind III-B-fragment. A defined and complete gene library of the FLDV DNA sequences was established by insertion of FLDV DNA fragments (Eco RI, Eco RI/Bam HI, Eco RI/Hind III) into the corresponding restriction sites of bacterial plasmid vector pAT 153. Bacterial colonies harbouring recombinant plasmids were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridizing plasmid DNA to viral DNA. The analyses revealed that sequences representative for the complete viral genome were cloned.

Animals↗

PubMed1985

Vaccination against hepatitis B in patients with renal insufficiency.

To define the degree of renal insufficiency at which the immune response to vaccination against hepatitis B is impaired, anti-HB concentrations after vaccination with 20 micrograms HB-Vax at 0, 1 and 6 months were examined in 76 dialysis patients, 24 patients with incipient renal failure (S-creatinine 1.4-3.5mg/dl) and in 43 controls. Compared with controls, seroconversion rate and anti-HB concentrations were significantly (p less than 0.02) lower in patients with incipient renal failure. The time course of anti-HBs in dialysis patients with successful vaccination, either with three doses (0, 1, 6 months) or with five doses (0, 1, 2, 4, 6 months) of HB-Vax was compared with healthy controls. The proportion of patients losing anti-HBs and the decrease of antibody concentration was significantly greater in dialysis patients immunised with three doses of the vaccine. In dialysis patients vaccinated with five doses, the percentage losing HB antibodies was slightly higher than in controls, but final titres after 24 months were comparable.

Adult↗

PubMed1984

Morphogenesis and fine structure of eel virus (Berlin), a member of the proposed Birnavirus group.

Eel Virus (Berlin) is associated with the occurrence of skin tumors in European eels. The genome of the virus consists of two segments of double-stranded (ds) RNA. The agent is assembled exclusively in the cytoplasm. Isometric particles with a diameter of 61 nm and in addition tubular structures and smaller particles were observed. The virion has a single shell: its capsid is composed of 132 interconnected morphological units with T = 13 dextro symmetry. According to particle size and bipartite nature of the genome, this virus has to be assigned to the tentatively proposed group of bisegmented ds RNA animal viruses.

Anguilla↗

PubMed1984

DNA analysis of insect iridescent virus 6: evidence for circular permutation and terminal redundancy.

DNA analysis of small insect iridovirus 6 was performed. Combined exonuclease-restriction endonuclease digestions revealed that all resulting fragments were degraded without preference for any one DNA fragment. Upon denaturation and reannealing of native linear Chilo iridescent virus DNA (158 x 10(6) daltons), duplex DNA circles of a smaller size (140 x 10(6) daltons) with protruding tails were formed.

Journal Article↗

PubMed1983

SSA (Ro)-antibodies in Wegener's granulomatosis.

In 4 consecutive cases of Wegener's granulomatosis with glomerulonephritis, antibodies against the extractable nuclear antigen SSA (Ro) could be demonstrated with counterimmunoelectrophoresis. Antibodies were demonstrable on admission and paralleled disease activity while patients were under therapy. This potential serological marker may be of value for diagnosis and follow-up of patients with Wegener's granulomatosis.

Adult↗

PubMed1983

Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumours of pleuronectes.

Virions of fish lymphocystis disease virus (FLDV), a member of the iridovirus family, were isolated directly from lymphocystis disease lesions of individual flatfishes and purified by sucrose and subsequent cesium chloride gradient centrifugation to homogeneity as judged by electron microscopy. The isolated FLDV DNAs appear to be heterogeneous in size. Contour length measurements of 43 DNA molecules gave an average length of 49 +/- 23 microns, corresponding to 93 +/- 44 X 10(6) D. Molecular weight estimations of FLDV DNA by restriction enzyme analysis resulted in only 64.8 X 10(6) D indicating an excess length of the DNA of about 50%. FLDV DNA was sensitive to lambda 5'-exonuclease and to E. coli 3'-exonuclease III without preference of any one terminal DNA restriction fragment. Denaturation and reannealing experiments of FLDV DNA resulted in the formation of circular DNA molecules of 34.25 microns contour length (= 65.22 X 10(6) D). This result suggests that FLDV DNA contains directly repeated sequences at both ends and that it is terminally redundant. FLDV DNA is methylated in cytosine. FLDV DNA did not hybridize with frog virus DNA indicating that the two iridoviruses are not closely related to each other. Restriction enzyme analysis and Southern blot hybridizations revealed that FLDV isolates can be classified into two different strains: FLDV strain 1 occurs in flounders and plaice, whereas strain 2 is usually found in lesions of dabs.

Animals↗