- Volume 73, Issue 6, 1992
Volume 73, Issue 6, 1992
- Animal
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Cloning and sequencing of the structural region and expression of putative core gene of hepatitis C virus from a British case of chronic sporadic hepatitis
More LessWe report the cloning and sequencing of the putative structural region of the hepatitis C virus (HCV) genome (2229 nucleotides) from an isolate derived from a British case of chronic sporadic non-A, non-B hepatitis. The overall sequence shows a higher similarity with one type of HCV, HCV1 (92%), than with HCV2 (80%), is very highly conserved at the 5′ end (99%) preceding the long open reading frame, is well conserved also in the putative core region (90 to 97%), but shows marked variation in the putative envelope region, particularly in the envelope 2/non-structural 1 region (70%). The putative core gene was cloned in pJ3Θ under the early simian virus 40 promoter and expressed in human hepatoma cells. A predominantly cytoplasmic 22K polypeptide was expressed which was antigenically reactive with serum from chronically infected HCV patients.
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Bovine papillomavirus type 1-transformed primary mouse fibroblasts show no correlation between tumorigenicity and viral gene expression, but c-myc gene expression is elevated in tumorigenic cell lines
More LessBovine papillomavirus type 1 (BPV-1)-transformed primary mouse fibroblasts containing episomal or integrated BPV-1 sequences were analysed for virus-specific transcripts and c-myc gene expression. Total BPV-1-specific expression was high in cell lines containing episomal BPV-1 DNA in comparison to lines containing integrated BPV-1 sequences, mainly due to higher expression of the E6/E7 sequences. No correlation was found between the viral transcription and tumorigenicity, although BPV-1 gene expression occurred in all cell lines. High levels of c-myc expression were found in all cell lines exhibiting a tumorigenic phenotype as compared to the non-tumorigenic lines. These data suggest that expression of BPV-1 genes may be essential for transformation but not tumorigenicity, whereas high levels of expression of cellular oncogenes like c-myc may be associated with tumorigenicity.
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Efficient in vivo encapsidation of a shuttle vector into pseudo-simian virus 40 virions using a shuttle virus as helper
C. Madzak, A. Margot and A. SarasinWe have designed shuttle vectors containing the late region of simian virus 40 (SV40) DNA (coding for the capsid proteins) which could be encapsidated into pseudo-SV40 virions during passage in monkey cells. We describe here the use of these shuttle viruses as helpers for the encapsidation of another shuttle vector into viral particles. Following cotransfection into monkey cells, the efficiency of encapsidation was similar for the shuttle virus and the other plasmid. The amounts of pseudo-SV40 virions recovered from the two vectors reflected the amounts of their DNAs present in monkey cells. Thus, the presence of the SV40 late region did not confer any significant advantage for encapsidation. The encapsidation of any shuttle vector into pseudo-SV40 virions is therefore possible and efficient, shuttle viruses constituting an interesting alternative to the use of SV40 as helper in this process.
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Analysis of human immunodeficiency virus type 1 LTR-LTR junctions in peripheral blood mononuclear cells of infected individuals
More LessCircularized DNA species containing two long terminal repeat circle junctions were analysed in peripheral blood mononuclear cells of human immunodeficiency virus type 1 (HIV-1)-infected individuals. The circle junction fragments found could be classified into four groups: fragments containing a normal circle junction, fragments with deletions at the circle junction, fragments containing the primer binding site inserted at the circle junction, and fragments containing insertions at the circle junction derived from other regions of the HIV-1 genome.
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Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone
An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) non-lymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAGM LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.
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Cell growth effects of Epstein—Barr virus leader protein
More LessB lymphoblastoid cell lines immortalized with P3HR1/633 Epstein—Barr virus (EBV), which has a deletion in the EBV nuclear antigen leader protein (EBNA-LP) gene, were transfected with a vector expressing wild-type EBNA-LP. The EBNA-LP trans-fectants grew out faster under G418 selection than control cells but expression of EBNA-LP made no significant difference to growth rate or saturation density of the resulting established cell lines. When the cells expressing EBNA-LP were allowed to grow to saturation and then diluted in fresh medium they underwent DNA synthesis more rapidly than control cultures.
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Recombination between a herpes simplex virus type 1 vector deleted for immediate early gene 3 and the infected cell genome
More LessWe have used a vector derived from a herpes simplex virus type 1 (HSV-1) mutant deleted for 3.6 kbp of the essential immediate early gene 3 to transduce the Tn5 neomycin phosphotransferase (neo r) gene into rodent and primate cells in culture. The transgene was flanked by genomic sequences from the human hprt gene. We demonstrate in this study that sequences introduced by infection with the replication-defective HSV vector can be stably inserted into the cell genome by recombination. Both the efficiency of stable transduction, measured by the number of neo r-positive colonies, and the number and length of the transgene sequences inserted into the cell genome were found to be a function of cell type. The transduction efficiency appeared to be influenced, at least in part, by the cytopathic potential of the replication-defective HSV-1 vector, which was more pronounced in primate than in rodent cells.
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Non-permissiveness of synovial membrane cells to human parvovirus B19 in vitro
More LessThe ability of cultured human synovial cells derived from synovial membrane and cartilage to support the replication of human parvovirus B19 was assessed. No viral DNA synthesis nor viral antigens were detected suggesting that B19 virus is not capable of replicating in synovial cells. The significance of this finding in relationship to the pathogenesis of parvovirus arthritis is discussed.
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Bovine respiratory syncytial virus fusion protein gene: sequence analysis of cDNA and expression using a baculovirus vector
More LessThe nucleotide sequence of bovine respiratory syncytial virus (RSV), ATCC strain A51908 fusion (F) glycoprotein gene cDNA was determined. The amino acid sequence deduced was then compared to those of two different isolates of bovine RSV, strains RB 94 and 391–2, and the A and B subtypes of human RSV, strains 18537 and A2. The bovine RSV F protein is highly conserved between the three isolates, A51908 has 97% amino acid identity to RB 94, and 99% identity to 391–2. The F proteins of both the A and B types of human RSV are 81% identical to that of A51908. The cDNA clone was expressed using a baculovirus vector and the expressed recombinant F protein produced in SF9 cells was characterized by Western blot analysis. The recombinant F protein was post-translationally cleaved into the active form and reacted with serum from bovine RSV-infected calves.
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Diversity of the antibody responses produced in ponies and mice against the equine influenza A virus H7 haemagglutinin
More LessA large panel of mouse monoclonal antibodies was produced and tested against field isolates of the equine H7N7 influenza A virus subtype. Only a limited degree of H7 haemagglutinin variation was detected. At least four antigenic sites were identified by selecting variant viruses in eggs. The limited variation in the field did not correlate with the frequency of variant viruses detected in eggs; this frequency was similar to those reported for other influenza viruses. We sought to determine whether the limited amount of variation could be correlated with an epitope-restricted antibody response in vaccinated horses. To this end, limiting dilution cultures were established with peripheral blood leukocytes from vaccinated ponies and the antibodies released into culture supernatants were assayed for binding to variant H7 viruses in ELISA. Three neutralizable antigenic sites mapped by mouse anti-bodies were also recognized by antibodies in pony limiting dilution culture supernatants, indicating that the equine antibody response against the influenza virus H7 haemagglutinin is diverse, and should be effective in selecting variant viruses.
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Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice
More LessSendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.
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A measles virus isolate from a child with Kawasaki disease: sequence comparison with contemporaneous isolates from ‘classical’ cases
We examined the relationship between a measles virus isolate from a child with Kawasaki disease and two contemporaneous wild-type isolates from children with ‘classical’ measles and the Schwarz vaccine strain. Sequence analysis of 3118 bp from the nucleoprotein, matrix, fusion and haemagglutinin genes of each virus revealed that the isolate from the child with Kawasaki disease was not related to measles vaccine strains and did not contain any of the marked abnormalities previously found in subacute sclerosing panencephalitis isolates, but was more akin to wild-type isolates currently circulating in the U.K. A comparison of our sequences with those obtained from earlier wild-type U.K. isolates suggests significant evolution of measles virus in the U.K. over the last decade.
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The genes encoding the phospho- and matrix proteins of phocine distemper virus
More LessThe nucleotide sequences of the phosphoprotein (P)/V/C and matrix (M) protein genes of phocine distemper virus (PDV) have been determined and the deduced amino acid sequences of the proteins derived from these genes compared with those of the other morbilliviruses. The 1655 nucleotides of the P gene encode a phosphoprotein of 507 amino acid residues (from nucleotide numbers 60 to 1583) which is 75% identical to that of canine distemper virus (CDV). The C proteins of the two viruses are 73% identical. The C protein has the same length, 174 amino acid residues, as C of CDV. The nucleotide sequences of the P genes are 78% identical. The editing site in the P gene is present as a stretch of 18 nucleotides, conserved in all other morbilliviruses. A V-like protein can be accessed by insertion of one G residue at this site. The P and M genes are separated from adjacent genes and each other by the CTT trinucleotide sequence which is totally conserved in morbilliviruses. The M gene is 1447 nucleotides long and encodes a typical paramyxovirus M protein of 335 amino acids (identical in length to those of CDV, measles virus and rinderpest virus). The gene is 78% identical to CDV in the coding region (nucleotides 32 to 1039) but only 34% identical to CDV in the 407 nucleotides of the 3′ untranslated sequence of the M mRNA. The M protein of PDV is 91% identical to the M protein of CDV. The data demonstrate interesting variations in the level of conservation of various genome areas and prove the distinct nature of PDV.
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- Plant
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Comparisons of the genomic sequences of erysimum latent virus and other tymoviruses: a search for the molecular basis of their host specificities
More LessThe nucleotide sequence of the genome of erysimum latent tymovirus (ELV) has been determined. It closely resembles those of the other four sequenced tymoviral genomes in its gene organization and composition, but is the smallest (6034 nucleotides) and most distinct of them. Furthermore the 78 non-coding nucleotides at the 3′ terminus of the ELV genome are unable to form a complete tRNA-like structure like that reported for other tymoviruses. Comparisons of the five tymovirus genomes and their encoded proteins indicate that they have probably evolved from the progenitor tymovirus by independent progressive mutational change without genetic recombination. Comparisons of the sequences of the two non-virion proteins of five tymoviruses, and virion proteins of 17 tymoviruses, revealed no specific similarities between those of ELV and turnip yellow mosaic virus that could explain why their host ranges and symptoms are so similar, yet differ, in this respect, from ononis yellow mosaic, kennedya yellow mosaic and eggplant mosaic tymoviruses.
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The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains
More LessWe inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.
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Rice hoja blanca virus genome characterization and expression in vitro
More LessNo information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nucleocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro.
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Relationships among the viroids derived from grapevines
More LessThere have been numerous reports of grapevine viroids, describing physical and biological properties suggestive of similar or identical molecular forms. With consideration of these properties and the application of random-primed and specific cDNA probes, four major groups of grapevine viroids have been defined. Designations which can be used to describe distinct viroids within the four groups include (i) CEVd-g, a grapevine isolate of citrus exocortis viroid, (ii) GVd-c, a grapevine viroid recovered from cucumber, and AGVd, Australian grapevine viroid, (iii) GYSVd-1 and GYSVd-2, two viroids inducing yellow speckle disease and (iv) HSVd-g, a grapevine isolate of hop stunt viroid.
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Conserved terminal nucleotide sequences in the genome of rice black streaked dwarf virus
More LessThe terminal regions of the dsRNA genome segments of rice black streaked dwarf virus (RBSDV) were sequenced. The individual dsRNAs, which were 32P-labelled at their 3′ termini by incubation with [32P]pCp and T4 RNA ligase, were separated by 5% PAGE, and the 10 dsRNA segments were sequenced by two-dimensional electrophoresis. The common 3′-terminal sequences ---GUC 3′ and ---AAAAACUU 3′ were found in the plus and minus strands, respectively. The strictly conserved terminal sequences (5′ AAGUUUUU….GUC 3′) of the genome segments of RBSDV differ from those of the phytoreoviruses and rice ragged stunt virus.
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Complete replication of a satellite RNA in vitro by a purified RNA-dependent RNA polymerase
More LessThe 334 nucleotide R satellite RNA was used as a template for purified RNA-dependent RNA polymerase (RdRp) from cucumber mosaic virus-infected tobacco plants. The products of the reaction were dsRNA and positive-strand RNA of the same size as the R satellite RNA. Similar products were obtained when T7 RNA polymerase positive-strand transcripts of a cDNA clone of the satellite RNA, designed to have the same 5′ and 3′ ends as the satellite RNA, were used as templates. The formation of the positive strands demonstrates complete replication of the satellite RNA. A positive-strand transcript with 65 and 255 additional nucleotides at the 5′ and 3′ ends of the satellite RNA respectively was also utilized as a template by the RdRp, but only dsRNA was formed. However, no products could be detected when the RdRp was programmed with transcripts corresponding to the negative-strand satellite RNA, either with no additional terminal nucleotides or with 24 and 310 additional nucleotides at the 5′ and 3′ ends respectively.
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- Corrigendum
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Volumes and issues
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Volume 105 (2024)
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Volume 73 (1992 - 2024)
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