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At least 73 records · Page 4Linked to original sources
PubMed1997

Improvement of the specific infectivity of the rubella virus (RUB) infectious clone: determinants of cytopathogenicity induced by RUB map to the nonstructural proteins.

A plasmid, Robo102, which contains a cDNA copy of the rubella virus (RUB) genomic RNA from which infectious transcripts can be synthesized in vitro, was recently developed (C. Y. Wang, G. Dominguez, and T. K. Frey, J. Virol. 68:3550-3557, 1994). To increase the specific infectivity of Robo102 transcripts (approximately 5 plaques/10 microg of transcripts), a modified reverse transcription-PCR method was used to amplify nearly 90% of the RUB genome in three fragments, which were then used to replace the corresponding fragments in Robo102. Replacement of a fragment covering nucleotides (nt) 5352 to 9759 of the RUB genome yielded a construct, Robo202, which produced highly infectious transcripts (10(4) plaques/microg), indicating the presence of an unrecognized deleterious mutation (or mutations) in this region of the Robo102 cDNA. Robo102 was based on the w-Therien strain of RUB, which forms opaque plaques in Vero cells, while the PCR replacement fragments were generated from a variant, f-Therien, which produces clear plaques in Vero cells. Although Robo202 contains over 4,000 nt from f-Therien, Robo202 virus produces opaque plaques. However, when the other two PCR fragments amplified from f-Therien (nt 1 to 1723 and nt 2800 to 5352) were introduced into Robo202, the resulting construct, Robo302, yielded transcripts that produced a virus that formed clear plaques. This indicates that the determinants of plaque morphology map to the regions of the genome covered by these two fragments, both of which are in the nonstructural open reading frame. Generation of Robo202/302 chimeras indicated that the most 5' terminal fragment (nt. 1 to 1723) had the greatest effect on plaque morphology. The plaque morphology was correlated with the ability of the viruses to kill infected cells. The only difference at the molecular level detected among the viruses was that the more cytopathic viruses produced more nonstructural proteins than did the less cytopathic viruses. This finding, as well as the mapping of the genetic determinants to the region of the genome encoding these proteins, indicates that the nonstructural proteins can mediate cell killing.

Animals↗
PubMedDate not supplied

Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus.

Postweaning multisystemic wasting syndrome (PMWS) is a recently emerged disease affecting pigs. Type 2 porcine circovirus (PCV2) has been associated with this syndrome although other factors are required in association with this virus for PMWS expression. The aim of this study was to investigate whether general immunostimulation (injections of keyhole limpet hemocyanin emulsified in incomplete Freund adjuvant and of thioglycollate medium) could strengthen the severity of PMWS in six-week-old specific-pathogen-free (SPF) piglets transfected with pure tandem-cloned PCV2 DNA by the intramuscular route. Non-immunostimulated piglets transfected with the viral clone did not present clinical signs but only mild pathological microlesions characteristic of PMWS. These piglets seroconverted and high viral genome loads and infectious titers were detected in the lymphoid organs at the end of the trial. Mild-to-moderate forms of PMWS were generally observed in the immunostimulated transfected piglets, as well as one severe form for a piglet (8003) which died. These piglets with mild-to-moderate forms had higher DNA loads than the transfected-only animals. Thus, viral replication was enhanced by immunostimulation. This is the first time that clinical PMWS has been reported in an SPF immunostimulated piglet infected with a pure inoculum consisting of tandem-cloned PCV2 DNA. This result confirms that PCV2 is the agent of PMWS and that immunostimulation could enhance PMWS in SPF piglets transfected with a PCV2 DNA clone.

Animals↗
PubMed2004

Sequence analysis, viral rescue from infectious clones and generation of recombinant virions of the avian adeno-associated virus.

Aiming at the generation of a viral-vectored system for gene delivery and vaccination in poultry, the entire genomes of the VR-865 and DA-1 strains of the avian adeno-associated virus have been cloned and sequenced. Sequence analysis of the clones showed that the genomic distribution of the structural and non-structural protein-coding genes of these viruses is conserved and in agreement with what has been previously described for the primate adeno-associated viruses. Amino acid differences between the avian adeno-associated viruses and the primate adeno-associated viruses are more evident in the genes that code for the non-structural (Rep) proteins of the virus, while the Cap region amino acid sequence was found to be more conserved. Since all the regulatory and coding sequences of the virus were present in the plasmids obtained, complete infectious viral particles were rescued from these clones, and these rescued viral populations were amplified by co-infecting primary embryo liver cells with the rescued virus and the CELO strain of the avian adenovirus type 1. As a proof of concept of the validity of this system for the purpose of gene delivery, recombinant viruses encoding for the LacZ gene as a reporter system were also generated. These recombinant viruses were used to express beta galactosidase activity in primary chicken embryo cell cultures.

Animals↗
PubMed1984

Cloned infectious complementary DNA of the poliovirus Sabin 1 genome: biochemical and biological properties of the recovered virus.

A complete cDNA copy of the genome of the attenuated type 1 poliovirus vaccine (Sabin 1) strain was constructed and inserted into the EcoRI site of the plasmid pBR325. When cultured mammalian cells were transfected with this recombinant plasmid, 20 to 50 poliovirus plaques per 10 micrograms plasmid DNA were observed. Fingerprints of the RNA of the recovered virus showed no changes when compared with those of the parental virus genome, an observation indicating that the primary structure of the cloned cDNA is a reflection of authentic poliovirus RNA. The recovered virus had the same properties as those of the Sabin 1 strain in regard to antigenicity, sensitivity to temperature, and dependency on bicarbonate concentration. These results suggest that the virus obtained by DNA transfection is indistinguishable from the Sabin 1 strain. The recombinant plasmid could therefore be used as a stable repository of the virus and as inoculum for the oral polio live vaccine.

Animals↗
PubMed1992

Complete nucleotide sequence of the cloned infectious genome of Junonia coenia densovirus reveals an organization unique among parvoviruses.

We previously constructed a recombinant plasmid, pBRJ, encompassing an infectious Junonia coenia densovirus (JcDNV) genome (M. Jourdan et al. (1990). Virology 179, 403-409). We report here the complete viral sequence of pBRJ. The genome, 5908 nucleotides (nt) long, consists of an internal unique sequence flanked by long (517 nt) inverted terminal repeats. The first 96 bases of one extremity can fold into a typical Y-shaped hairpin structure. The opposite extremity is incomplete, lacking 88 nt. These terminal structures, similar to those of dependoviruses, human parvovirus B19 and Bombyx mori densovirus (BmDNV), strongly suggest a common mechanism of DNA replication for these parvoviruses. JcDNV genomic organization is unique among parvoviruses in that coding sequences are evenly distributed in the 5' half of both strands. On one strand, the major open reading frame (ORF1) encodes the four structural proteins. On the complementary strand, ORF2, ORF3 (included in ORF2), and ORF4 probably encode nonstructural proteins. JcDNV genome has little DNA homology with vertebrate parvoviruses and surprisingly even less with the two densoviruses presently sequenced. ORF1 contains the highly conserved PGY and G-rich regions and ORF2 the NTP-binding domain common to most structural and to all nonstructural vertebrate parvoviral ORFs, respectively. The single homology between JcDNV and BmDNV is unexpectedly located in JcDNV NTP-binding domain and BmDNV ORF2 assumed to encode structural polypeptides. Only a weak homology exists between JcDNV and Aedes DNV in their NTP-binding domain.

Amino Acid Sequence↗
PubMed1994

High similarity among the tomato yellow leaf curl virus isolates from the west Mediterranean basin: the nucleotide sequence of an infectious clone from Spain.

An isolate of tomato yellow leaf curl geminivirus, from the first epidemic outbreaks that occurred in Murcia, Spain (TYLCV-M) in 1992, was cloned and its nucleotide sequence was determined. The circular single stranded DNA consisted of 2777 nucleotides. The genome organization resembled that of other TYLCV sequenced so far; regulatory signal sequences for bidirectional transcription and for polyadenylation of the transcripts were localized in the sequence. Infectivity of the cloned DNA was demonstrated by subcloning a 1.8 mer of TYLCV-M in pBin19 and agroinoculating it into tomato and Nicotiana benthamiana plants. Symptoms and viral DNA forms in agroinfected plants did not differ from those of field infected ones. Sequence comparisons with other TYLCV isolates show a high homogeneity between isolates from the West Mediterranean Basin, suggesting the presence of a geographical cluster.

Amino Acid Sequence↗
PubMed1988

Low-cytopathic infectious clone of human immunodeficiency virus type I (HIV-I).

Single genotypic variants of HIV-I, contained in a parental cytopathic HIV-I isolate, were isolated by molecular cloning and propagated in susceptible cells. Two such HIV-I clones, designated N1T-E and N1T-A, exhibited similar restriction endonuclease maps but strikingly different biological activities. Infection of T lymphocytes or monocytes by clone N1T-E was characterized by slow kinetics and lack of significant cytopathic effects, but high reverse transcriptase activity levels in culture supernatants of chronically-infected cells. Clone N1T-A, like the parental HIV-I isolate, exhibited fast kinetics of infection in T cells and monocytes and strong cytopathicity in these cells. Full characterization of the low-cytopathic virus in comparison to the structurally similar cytopathic clone may facilitate the elucidation of the molecular basis of HIV cytopathogenicity.

Cloning, Molecular↗
PubMed2006

The pathogenesis of Newcastle disease: a comparison of selected Newcastle disease virus wild-type strains and their infectious clones.

The effect of mutations of Newcastle disease virus (NDV) fusion (F) gene, hemagglutinin-neuraminidase (HN) gene, and phosphoprotein (P) gene and HN chimeras between the virulent Beaudette C and low virulence LaSota strains on pathogenesis and pathogenicity was examined in fully susceptible chickens. A virulent F cleavage site motif within a LaSota backbone increased pathogenicity and severity of clinical disease. A LaSota HN within a Beaudette C backbone decreased pathogenicity indices and disease severity. A Beaudette C HN within a LaSota backbone did not change either pathogenicity indices or severity of disease in chickens. Loss of glycosylation at site 4 of the HN or modified P gene of Beaudette C decreased pathogenicity indices and caused no overt clinicopathologic disease in chickens. Both pathogenicity indices and clinicopathologic examination demonstrated that the F, HN, and P genes of NDV collectively or individually can contribute to viral virulence.

Animals↗
PubMed2004

Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone.

We have established a T7-based model system for hepatitis C virus (HCV) 1a strain, which involves the use of a replication-defective adenovirus that carries the gene for T7 RNA polymerase and a transcription plasmid containing full-length HCV cDNA clone. To facilitate high-level expression of HCV, sub-confluent Huh7 cells were first infected with adenovirus containing the gene for the T7 RNA polymerase and then transfected with the transcription plasmid. As a negative control, part of NS5B gene of this clone was deleted which abolishes the HCV RNA-dependent RNA polymerase and prevents replication of viral RNA. This model produces high levels of structural (core, E1, E2) and nonstructural proteins (NS5), which were detected by Western blot analysis and immunofluorescence assay. Negative-strand HCV RNA was detected only in the wild-type clone in the presence of actinomycin D, and no RNA was detected with the NS5B deleted mutant control. As a practical validation of this model, we showed that IFN alpha-2b selectively inhibits negative-strand RNA synthesis by blocking at the level of protein translation. The inhibitory effect of IFN alpha-2b is not due reduction of transcription by T7 polymerase or due to intracellular degradation of HCV RNA. This in vitro model provides an efficient and reliable means of assaying negative-strand RNA, protein processing, and testing the antiviral properties of interferon.

Animals↗
PubMed2000

Identification of a novel plant virus promoter using a potyvirus infectious clone.

A putative promoter from the strawberry vein banding caulimovirus (SVBV) genome was identified by its ability to drive infection with full-length cDNA of the zucchini yellow mosaic RNA potyvirus (ZYMV). A high rate of infection was obtained with the cDNA under control of the SVBV promoter using particle bombardment technology. The SVBV promoter shows 60% homology to the cauliflower mosaic virus 35S promoter in the domain spanning the conserved motifs of CCACT (at -83) and the TATA box (at -31), to the transcription start. The 3'-end one-third of the putative promoter (328 bp) was sufficient to invoke full infectivity with the ZYMV clone, and drove transient reporter gene expression in Solanaceae and Cucurbitaceae transformed with a binary plant transformation vector. Stable expression of a reporter gene (GUS) under control of the truncated SVBV promoter was shown in transformed tobacco shoots in roots, leaves and stems.

Base Sequence↗
PubMed1983

Complete nucleotide sequence of an infectious clone of Friend spleen focus-forming provirus: gp55 is an envelope fusion glycoprotein.

The Friend spleen focus-forming provirus is 6,296 base pairs (bp) in length. Compared to Moloney murine leukemia virus, it has undergone five major deletions, three substitutions, and a number of minor alterations. Otherwise, these viruses are about 90% homologous. A 16-bp palindrome is found in the region thought to be involved in packaging and dimerization of the RNA genome. Premature termination of translation of the gag polyprotein is attributed to a 13-bp deletion in the p12 region. A substitution of xenotropic env sequences was identified in the 5' region of the env gene; 150 nucleotides 3' to this substitution, a deletion of 585 bp removes the site where the normal env precursor protein is cleaved to form gp70 and p15(E), resulting in a fusion protein of Mr 44,725. Due to these changes, the env product gp55 is expected to have a substantially different conformation on the cell surface compared to either a xenotropic or ecotropic gp70 protein, and may be responsible for the rapid erythroleukemic potential of spleen focus-forming virus.

Amino Acid Sequence↗
PubMed1991

The nucleotide sequence of the infectious cloned DNA components of potato yellow mosaic virus.

The complete nucleotide sequence of a Venezuelan isolate of potato yellow mosaic virus (PYMV) has been determined, showing it to be typical of subgroup I geminiviruses in that it is whitefly-transmitted, has a circular, bipartite ssDNA genome and possesses bidirectionally orientated open reading frames (ORFs). The two genomic components have little sequence similarity apart from a common region of 268 nucleotides (nt) which is almost identical. Analysis of ORFs revealed six potential coding regions encoding proteins of Mr greater than 10K, four in PYMV A (2593 nt) and two in PYMV B (2547 nt), which are preceded by regulatory transcription elements and have polyadenylation signals present at the ends. Amino acid sequence alignments of PYMV DNA ORF-encoded proteins with those encoded by other previously sequenced geminivirus ORFs show that PYMV is closely related to those geminiviruses isolated from the New World, especially in the putative coat protein gene regions.

Amino Acid Sequence↗