SEARCH · PubMed Health

Results for “Infectious clone”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,135 records · Page 63Linked to original sources

PubMed2001

Rescue of infectious bursal disease virus from mosaic full-length clones composed of serotype I and II cDNA.

Infectious Bursal Disease Virus (IBDV) is the causative agent of one of the most important and wide-spread infectious diseases among commercial chicken flocks. IBDV causes a depletion of B-lymphoid cells in the bursa of Fabricius, inducing immunosuppression, morbidity, or even acute mortality. Because currently used live IBDV vaccines are derivatives from field isolates no serologic discrimination between field isolates and live vaccines can be made. The recently developed reverse genetics techniques for IBDV allows one to generate genetically modified IBDVs which might have altered biological and antigenic properties. Here, we describe the rescue of mosaic serotype I IBDVs, of which the polyprotein encoding region was partly replaced by the corresponding region of a serotype II strain. A mosaic virus, containing the C-terminal part of serotype II VP3 showed only a slightly delayed release of progeny virus compared to unmodified serotype I virus, while maximum viral titers at 25 h post infection were equal. Since serotype specific epitope(s) are present in the C-terminal part of VP3, we were able to discriminate this rescued virus from serotype I and II IBDV strains. These findings make the use of a chimeric VP3 a promising approach to develop an IBDV marker vaccine.

Amino Acid Sequence↗

PubMed1999

Activation of promoter P4 of the autonomous parvovirus minute virus of mice at early S phase is required for productive infection.

Autonomous parvoviruses are tightly dependent on host cell factors for various steps of their life cycle. In particular, DNA replication and gene expression of the prototype strain of the minute virus of mice (MVMp) are closely linked to the onset of host cell DNA replication, pointing to the involvement of an S-phase-specific cellular factor(s) in parvovirus multiplication. The viral nonstructural protein NS-1 is absolutely required for parvovirus DNA replication and is able to transcriptionally regulate parvoviral and heterologous promoters. We previously showed that the promoter P4, which directs the transcription unit encoding the NS proteins, is activated at the onset of S phase. This activation is dependent on an E2F motif in the proximal region of promoter P4. An infectious MVM DNA clone was mutated in the E2F motif of P4. The wild type and the E2F mutant derivative were tested for their ability to produce progeny viruses after transfection of permissive cells. In the context of the whole MVMp genome, the E2F mutation abolished P4 induction in S phase and inactivated the infectious molecular clone, which failed to become amplified and generate progeny particles. The virus could be rescued when NS proteins were supplied in trans, showing that P4 hyperactivity in S is needed to reach a level of NS-1 expression that is sufficient to drive the viral replication cycle. These data show that E2F-mediated P4 activation at the early S phase is a limiting factor for parvovirus production. The primary barrier to parvovirus gene expression in G1 is thought to be promoter formation rather than activation, due to the poor conversion of the parental single-strand genome to a duplex form. The S dependence of P4 activation may therefore be a sign of the virus adaptation to life in the S-phase host cell. If the conversion block in G1 were to be leaky, the S induction of promoter P4 could be envisioned as a safeguard against the production of toxic NS proteins until cells reach the S phase and provide the full machinery for parvovirus replication.

Animals↗

PubMed1984

DNAs of two molecularly cloned endogenous ecotropic proviruses are poorly infectious in DNA transfection assays.

Endogenous ecotropic murine leukemia virus expression varies with inbred mouse strain and age. The mechanism(s) regulating virus expression is unknown, but expression is thought to be controlled at the transcriptional level by linkage to cis-acting cellular DNA sequences or DNA methylation or both. To begin to differentiate between these different control mechanisms, we molecularly cloned two endogenous ecotropic proviruses, Emv-3 and Emv-13, complete with flanking cellular DNA sequences. Both proviruses are poorly expressed in vivo and in vitro, although they appear to be structurally nondefective by restriction enzyme analysis. Cloned DNAs of both proviruses were poorly infectious in DNA transfection experiments, suggesting that methylation may not regulate the expression of these genes in vivo. Removal of their flanking cellular sequences did not increase their infectivity. However, these DNAs were highly infectious when mixed together, indicating that both proviruses carry mutations, that inhibit their expression and belong to different complementation groups. Marker rescue experiments suggested that Emv-3 is defective in the gag region and Emv-13 is defective in p15E-U3. The infectivity of Emv-3, but not of Emv-13, DNA was increased by the addition of AKR xenotropic murine leukemia virus DNA, consistent with known regions of homology between ecotropic and xenotropic proviruses. Recombination between defective endogenous viruses also appears to occur in vivo, suggesting that this may be a common mechanism controlling endogenous murine leukemia virus expression.

Animals↗

PubMed2003

The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences.

The role of the hepatitis C virus (HCV) p7 protein in the virus life cycle is not known. Previous in vitro data indicated that this 63-aa polypeptide is located in the endoplasmic reticulum and has two transmembrane domains (TMDs) connected by a cytoplasmic loop; the amino- and carboxyl-terminal tails are oriented toward the endoplasmic reticulum lumen. Furthermore, recent in vitro studies suggested that HCV p7 could function as a virus-encoded ion channel. It might therefore be a relevant target for future drug development. We studied the role of HCV p7 in vivo. Because HCV does not replicate efficiently in cell culture, we mutagenized p7 of an infectious genotype 1a cDNA clone and tested RNA transcripts of each mutant for infectivity in chimpanzees by intrahepatic transfection. Appropriate processing of mutant polypeptides was confirmed by studies in transfected mammalian cells. Mutants with deletions of all or part of p7 and a mutant with substitutions of two conserved residues in the cytoplasmic loop were not viable. Thus, p7 is essential for infectivity of HCV. A chimera in which the p7 of the 1a clone was replaced with p7 from an infectious genotype 2a clone also was not viable. This finding suggests a genotype-specific interaction between p7 and other genomic regions. To define which portions of p7 played the most significant role for this interaction, we tested three chimeras with the 1a backbone in which only specific domains of p7 had the 2a sequence. A p7 chimera with 2a tails and TMDs and the 1a cytoplasmic loop was not viable. A mutant with 2a tails and cytoplasmic loop and 1a TMDs also was not viable. However, a p7 chimera with 2a TMDs and cytoplasmic loop and 1a tails was viable. The transfected chimpanzee became viremic at week 2, and recovered viruses had the chimeric sequence. These data indicate that the amino- and/or carboxyl-terminal intraluminal tails of p7 contain sequences with genotype-specific function.

Animals↗

PubMed2004

Influence of long terminal repeat and env on the virulence phenotype of equine infectious anemia virus.

The molecular clones pSPeiav19 and p19/wenv17 of equine infectious anemia virus (EIAV) differ in env and long terminal repeats (LTRs) and produce viruses (EIAV(19) and EIAV(17), respectively) of dramatically different virulence phenotypes. These constructs were used to generate a series of chimeric clones to test the individual contributions of LTR, surface (SU), and transmembrane (TM)/Rev regions to the disease potential of the highly virulent EIAV(17). The LTRs of EIAV(19) and EIAV(17) differ by 16 nucleotides in the transcriptional enhancer region. The two viruses differ by 30 amino acids in SU, by 17 amino acids in TM, and by 8 amino acids in Rev. Results from in vivo infections with chimeric clones indicate that both LTR and env of EIAV(17) are required for the development of severe acute disease. In the context of the EIAV(17) LTR, SU appears to have a greater impact on virulence than does TM. EIAV(17SU), containing only the TM/Rev region from the avirulent parent, induced acute disease in two animals, while a similar infectious dose of EIAV(17TM) (which derives SU from the avirulent parent) did not. Neither EIAV(17SU) nor EIAV(17TM) produced lethal disease when administered at infectious doses that were 6- to 30-fold higher than a lethal dose of the parental EIAV(17). All chimeric clones replicated in primary equine monocyte-derived macrophages, and there was no apparent correlation between macrophage tropism and virulence phenotype.

Amino Acid Sequence↗

PubMed1990

Production of infectious swine vesicular disease virus from cloned cDNA in mammalian cells.

Full-length cDNA clones of the swine vesicular disease virus (SVDV) were constructed from subgenomic cDNA clones in the expression vector pSVL (pSVLS00). The direct transfection of mammalian cells with plasmid pSVLS00 results in the production of infectious virus. The recovered virus was neutralized completely by anti-SVDV guinea-pig serum, but did show a difference in plaque morphology from the parental virus.

Animals↗

PubMed1986

Molecular cloning and physical characterization of integrated equine infectious anemia virus: molecular and immunologic evidence of its close relationship to ovine and caprine lentiviruses.

Molecular clones of the integrated form of the genome of equine infectious anemia virus (EIAV), the etiologic agent of a naturally occurring, worldwide disease of horses, were obtained. The restriction map of a full-length genome was determined. Additional evidence for the close evolutionary relationship between EIAV and a prototype lentivirus (caprine arthritis encephalitis virus) was acquired by Southern blotting and immunological analyses. An interspecies radioimmunoassay was developed in which EIAV and ovine and caprine lentiviruses could be detected equally well. These studies make available precisely defined reagents to pursue the study of the mechanisms of pathogenesis of lentiviral induced diseases.

Animals↗

PubMed1995

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Base Sequence↗

PubMed1990

Infectious transcripts and nucleotide sequence of cloned cDNA of the potexvirus white clover mosaic virus.

The complete nucleotide sequence of white clover mosaic virus (WCIMV) strain O has been determined and compared to the WCIMV strain M sequence. The two virus strains show 12% divergence at the nucleotide level and 3% divergence at the amino acid level (open reading frames 1 to 5). Two open reading frames, 10 and 21 kDa, identified in the strain M sequence were not found in the strain O sequence. High levels of infectious RNA transcripts were produced from a full-length cDNA clone of strain O constructed downstream of the SP6 RNA polymerase promoter (greater than 2 micrograms RNA/micrograms template DNA). Symptoms characteristic of WCIMV developed when host plants were inoculated with SP6 RNA transcripts. The specific infectivity of transcript RNA produced in the presence of m7GpppG was similar to that of virion RNA. The SP6 transcripts lacked 5' nonviral nucleotides, but contained 6, 14, or 198 3' nonviral nucleotides (derived from the pUC19 vector). Transcripts were infectious without m7GpppG, or with long nonviral 3' nucleotide stretches. Progeny virus RNA from infections initiated with transcripts containing 198 nonviral 3' nucleotides did not maintain these sequences.

Base Sequence↗

PubMed1982

Biological and biochemical studies of African green monkey lymphotropic papovavirus.

The growth of African green monkey lymphotropic papovavirus (LPV) in human lymphoblastoid cell line BJA-B was found to be slow and inefficient due to the accumulation of defective particles. An analysis of molecularly cloned LPV DNAs showed that 3 of 19 clones had DNAs that were longer (5.1 kilobases) than the DNAs of the other clones. The 5.1-kilobase DNA was infectious for BJA-B cells, whereas the shorter (4.8-kilobase) molecules were defective. Unlike the wild-type virus, stocks of LPV made from cloned, infectious DNAs were homogeneous and had higher titers. Using stocks of nondefective LPV, we investigated other biological properties. LPV replication in another human B-lymphoblastoid cell line was observed. The virus did not cause tumors when it was inoculated into newborn hamsters. Serological surveys of human and nonhuman primate sera indicated that virtually all primates, including humans, show evidence of infection by viruses antigenically related to LPV.

Animals↗

PubMed1991

Nonmutant forms of the avirulent satellite D of turnip crinkle virus are produced following inoculation of plants with mutant forms synthesized in vitro.

The avirulent satellite RNA D (sat D) of turnip crinkle virus (TCV) has been cloned as a copy DNA. RNA transcripts synthesized in vitro from the cloned cDNA are infectious when coinoculated with RNA transcripts of the cloned TCV genome. Sat D is the smallest and most likely the progenitor of the other TCV satellites and, in contrast to the virulent satellite RNA C (sat C), does not intensify viral symptoms. Mutant forms of sat D including internal deletions up to 50 bases yielded sat D in infected plants. However, sat D mutants were not recovered in mutant form, but reverted to normal size and sequence in infected turnip plants. Mutations at a site with homology to the catalytic strand of self-cleaving sequences in certain viroids and satellites appeared to confer virulence on sat D in that test plants showed severe crinkling and stunting normally associated with sat C. However, sat C appeared along with a restored form of sat D in the progeny RNAs of these severely infected plants. Sat C was presumably generated by recombination between sat D and the TCV genome. In contrast, when plants were inoculated with transcripts containing the equivalent mutations in sat C, sat C was recovered from infected plants in mutant form. These findings demonstrate the tendency for mutant forms of the avirulent satellite, sat D, to revert, but raise questions about the source of information used in the reversion process.

Base Sequence↗

PubMed2001

Genetics and genomics in infectious disease susceptibility.

The past decade has witnessed a rapid transition from the first positional cloning of an infectious disease susceptibility gene (Slc11a1, also called Nramp1) in the mouse to genome-wide scans in human multicase families and the identification of potential disease-causing genes by simple inspection of the public human genome databases. Pathogen genome projects have facilitated multilocus sequence typing of pathogen isolates and studies of ecological fitness and virulence patterns in disease-causing isolates. Comparative sequence analysis of pathogen strains and functional genomics studies are now underway, hopefully providing new insight into infectious disease susceptibility.

Cloning, Molecular↗

PubMed1986

Molecular studies of the differential replication at pyrexial temperatures of two influenza viruses differing in virulence for ferrets.

Replication of a virulent clone (7a) of the reassortant influenza virus A/Puerto Rico/8/34-A/England/939/69 (H3N2) in ferret nasal turbinate tissue is less affected than that of an attenuated clone (64d) by temperatures which occur during pyrexia in ferrets. This is a factor which contributes to the difference in virulence of the two clones. The differential replication of the two clones at pyrexial temperatures has been reproduced in allantois-on-shell (egg-bit) cultures, and the synthesis of viral polypeptides and RNA species examined. This virus-host system was chosen because it was more convenient to use than organ cultures but, like the latter, might provide information relevant to the in vivo situation. With this system it was not possible to achieve single cycle replication: the observed effects are cumulative over several (2 to 3) cycles of replication (24 h) and therefore conclusions from them may not be as definitive as those from single cycle conditions. However, in cells infected with clone 64d both A(+) cRNA and polypeptide synthesis were little affected at 40 degrees C but levels were decreased by about 70-80% at 41 degrees C; A(+) cRNA and polypeptide levels were unaffected even at 41 degrees C with clone 7a. These reductions seem insufficient to account for the 10-fold reduction in infectious yields of clone 64d at 40 degrees C or the 100-fold and 10-fold reductions in yields of clones 64d and 7a respectively at 41 degrees C. There was no evidence of increased production of non-infectious virus at elevated temperatures by either clone. Levels of vRNA were considerably reduced at 40 and 41 degrees C for both clones, but the levels were considerably greater at all temperatures in clone 7a-infected cells than in those infected with clone 64d; vRNA levels were higher for clone 7a at 41 degrees C than for clone 64d at 37 degrees C. The different levels of vRNA do not reflect differences in the availability of template A(-) cRNAs since levels of these were similar for both clones at 37 and 40 degrees C and only reduced for clone 64d at 41 degrees C. Although the interpretation of these data is complicated by multiple cycles of replication it appears that limited availability of vRNA could be an important constraint on the ability of clone 64d to replicate at pyrexial temperatures.

Animals↗

PubMed2005

Development of mouse hepatitis virus and SARS-CoV infectious cDNA constructs.

The genomes of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) have been generated with a novel construction strategy that allows for the assembly of very large RNA and DNA genomes from a panel of contiguous cDNA subclones. Recombinant viruses generated from these methods contained the appropriate marker mutations and replicated as efficiently as wild-type virus. The MHV cloning strategy can also be used to generate recombinant viruses that contain foreign genes or mutations at virtually any given nucleotide. MHV molecular viruses were engineered to express green fluorescent protein (GFP), demonstrating the feasibility of the systematic assembly approach to create recombinant viruses expressing foreign genes. The systematic assembly approach was used to develop an infectious clone of the newly identified human coronavirus, the serve acute respiratory syndrome virus (SARS-CoV). Our cloning and assembly strategy generated an infectious clone within 2 months of identification of the causative agent of SARS, providing a critical tool to study coronavirus pathogenesis and replication. The availability of coronavirus infectious cDNAs heralds a new era in coronavirus genetics and genomic applications, especially within the replicase proteins whose functions in replication and pathogenesis are virtually unknown.

Animals↗

PubMed1997

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts.

Bovine viral diarrhea virus (BVDV) is the most insidious and devastating viral pathogen of cattle in the United States. Disease control approaches must be based on detailed knowledge of virus biology. To develop reverse-genetic systems to study the molecular biology of the virus, we first constructed a plasmid containing the entire genome of BVDV cloned as cDNA. Subsequently, we showed that infectious BVDV was produced by cells transfected with uncapped RNA transcribed in vitro from the cDNA clone. This result defined functional 5' and 3' termini in viral genomic RNA and established the biological importance of the proposed internal ribosome entry site element in the 5' untranslated region of the viral genome. BVDV rescued from the infectious cDNA clone has an in vitro phenotype similar to that of the wild-type parent, the National Animal Disease Laboratory strain of BVDV. A deletion of a single codon in the full-length genomic BVDV cDNA clone, encoding glutamic acid at position 1600, gave rise to sequence-tagged virus easily identified by restriction fragment length polymorphism analysis of reverse transcription-PCR amplicons. Suitability of the molecular clone of BVDV for genomic manipulations was shown by substitution of the major envelope glycoprotein E2/gp53 with that of the Singer strain, giving rise to a chimeric virus. The predicted change in antigenic structure of the chimeric virus could be readily identified with strain-specific monoclonal antibodies by neutralization and immunofluorescence assays. Immediate applications of this system include development of safe and effective live vaccine strains possessing predetermined defined attenuating mutations.

Animals↗

PubMed1996

Continuous production of minute virus of mice by an untransformed variant of Fisher rat fibroblast (FR3T3).

Many tumour cells are killed by the lytic replication of the autonomous parvoviruses H-1 and minute virus of mice (MVMp), whereas most untransformed cells (although they take up these viruses efficiently) are resistant, i.e. they do not produce infectious virus and are not lysed. Therefore, cells able to continuously produce large quantities of infectious virus have not yet been described. We have isolated such cells from the resistant cell line FR3T3 (Fisher rat fibroblast). These cells (called FR3T3C) produce infectious MVMp virions without being detectably lysed. Furthermore, a persistently infected population (R100FR3T3C) was generated by repetitive infection of FR3T3C cells with MVMp. Indeed, R100FR3T3C cells were successfully cultivated for two years and continuously produced infectious virus. Seventeen clones of R100FR3T3C cells isolated by limiting dilution produced infectious virions, indicating that in the R100FR3T3C cell population, virus production was not limited to a few cells. These cell lines may be useful for the production of MVMp and for the generation of a cell line for the packaging of recombinant viral genomes.

Animals↗

PubMed1997

Promonocytic U937 subclones expressing CD4 and CXCR4 are resistant to infection with and cell-to-cell fusion by T-cell-tropic human immunodeficiency virus type 1.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary markedly in the ability to infect cells of the monocyte/macrophage (M/M) lineage. M/M are generally resistant to infection with T-cell-tropic (T-tropic) strains of HIV-1. Recently, the chemokine receptors CCR5 and CXCR4 were identified as cofactors for fusion/entry of macrophage- and T-tropic strains of HIV-1, respectively. To investigate the mechanisms of resistance of M/M to T-tropic HIV-1 infection, we examined a number of subclones of the U937 promonocytic cell line. We found that certain subclones of U937 (plus clones) could, while others (minus clones) could not, support replication of T-tropic strains of HIV-1. We demonstrate that (i) both minus and plus clones support HIV-1 replication when transfected with an infectious molecular cDNA clone of a T-tropic HIV-1; (ii) minus clones do not, but plus clones do, efficiently support fusion with cells expressing HIV-1 IIIB Env; (iii) both plus and minus clones (with the exception of one clone) express physiologically functional CXCR4 protein as well as CD4 on the cell surface; (iv) introduction of CXCR4 into the CXCR4-negative clone does not restore fusogenicity with or susceptibility to T-tropic HIV-1; and (v) a ligand (stromal cell-derived factor 1) for or a monoclonal antibody (12G5) to CXCR4 does not effectively inhibit HIV-mediated cell-to-cell fusion of U937 cells. These data indicate that resistance to T-tropic HIV-1 infection of U937 minus clones occurs at fusion/ entry events and that expression of functional CXCR4 and CD4 is not a sole determinant for susceptibility to T-tropic HIV-1 infection; furthermore, they suggest that other factors are positively or negatively involved in HIV-mediated cell-to-cell fusion in U937 promonocytic cells.

Antibodies↗

PubMed1987

Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.

We constructed full-length cDNA clones of Sindbis virus that can be transcribed in vitro by SP6 RNA polymerase to produce infectious genome-length transcripts. Viruses produced from in vitro transcripts are identical to Sindbis virus and show strain-specific phenotypes reflecting the source of RNA used for cDNA synthesis. The cDNA clones were used to confirm the mapping of the causal mutation of ts2 to the capsid protein. A general strategy for mapping Sindbis virus mutations is described and was used to identify two lethal mutations in an original full-length construct which did not produce infectious transcripts. An XbaI linker was inserted in the cDNA clone near the transcriptional start of the subgenomic mRNA; the resulting virus retains the XbaI recognition sequence, thus providing formal evidence that viruses are derived from in vitro transcripts of cDNA clones. The potential applications of the cDNA clones are discussed.

Animals↗