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Results for “Infectious clone”

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At least 631 records · Page 35Linked to original sources

PubMed1997

Construction of a genome-length cDNA clone for human astrovirus serotype 1 and synthesis of infectious RNA transcripts.

We have constructed a genome-length cDNA clone for human astrovirus serotype 1. When a human colon cancer-derived cell line, CaCo-2, is transfected with RNA transcribed in vitro from this cDNA clone, infectious virus is produced at titers close to those observed after infection with intact astrovirus. A rodent cell line, BHK, which is largely refractory to astrovirus infection, was found to support efficient growth of the virus if transfected with viral RNA. The high transfection efficiency seen in the BHK cells allows studies of the viral replication in the transfected cells and thus should prove useful for the characterization of noninfectious astroviral mutants.

Cloning, Molecular↗

PubMed1998

Nucleotide sequence and infectivity of a full-length cDNA clone of panicum mosaic virus.

The sequence of an infectious cDNA clone of panicum mosaic virus (PMV) showed that the single-stranded RNA genome is 4326 nucleotides (nt) and a single highly abundant subgenomic (sg) RNA of 1475 nt was synthesized during PMV infection of pearl millet plants and protoplasts. Computer comparisons revealed strong similarities between the predicted amino acid sequences of the p48 and p112 open reading frames (ORFs) and replicase proteins of members of the Tombusviridae. The sgRNA has the potential to encode five proteins. Three small ORFs, p8, p8-FS, and/or p6.6 have similarity to ORFs of carmo-, necro-, and machlomoviruses thought to be involved in virus spread in plants. The sgRNA also has the potential to encode a 26-kDa capsid protein and a 15-kDa nested gene (p15) of unknown function. PMV transcripts also supported replication and movement of SPMV, the satellite virus. Genome organization, physicochemical properties, and biological features indicate that PMV is a member of the Tombusviridae family. However, PMV differs sufficiently from previously described members to warrant its placement in a new genus provisionally designated Panicovirus.

Amino Acid Sequence↗

PubMed2001

Recovery of infectious pariacoto virus from cDNA clones and identification of susceptible cell lines.

Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.

Animals↗

PubMed1995

Insertion of primary syncytium-inducing (SI) and non-SI envelope V3 loops in human immunodeficiency virus type 1 (HIV-1) LAI reduces neutralization sensitivity to autologous, but not heterologous, HIV-1 antibodies.

The aim of the study was to investigate the influence of V3 loops from naturally occurring viruses on the neutralization sensitivity of a molecularly cloned virus. A selection of well-defined syncytium-inducing (SI) and non-SI V3 loops of a single human immunodeficiency virus type 1-infected individual (H594) and the V3 regions of two SI laboratory strains were inserted in an infectious molecular clone of human immunodeficiency type 1 LAI. Neutralization was performed with a heterologous serum pool and autologous patient serum, using the virus reduction neutralization assay and peripheral blood lymphocytes as target cells. High sensitivity of the chimeric viruses containing the laboratory strain V3 regions to neutralization by H594 sequential sera as well as the heterologous serum pool was found. A statistically significant correlation between the sensitivities of these viruses was seen. In contrast, insertion of the primary isolate NSI and SI envelope V3 loops significantly reduced the neutralization by autologous serum but not by the heterologous serum pool. No correlation was found between the neutralization of the viruses with laboratory strain-derived V3 regions and the viruses with primary isolate V3 domains. We conclude that heterologous antibodies are able to neutralize infectious molecular clones with V3 loops of both SI and NSI viruses, regardless of whether they originated from laboratory strains or primary isolates. However, serum of patient H594 discriminated between the two types of viruses and showed reduced neutralization of the viruses with the autologous NSI and SI primary isolate V3 loops. These results indicated that the neutralization sensitivity of the viruses depended on the capacity of the V3 region to influence the conformation of the virus envelope. These V3-dependent conformational changes partially explain the neutralization sensitivity of laboratory strains and the relative neutralization resistance of primary isolates.

Acquired Immunodeficiency Syndrome↗

PubMed1986

Infectious RNA transcripts from cloned cDNAs of cucumber mosaic viral satellites.

Complete cDNA copies of two variants of CARNA 5, the satellite of cucumber mosaic virus, have been cloned in the transcription vector pPM1. These two naturally-occurring CARNA 5s are capable and incapable, respectively, of inducing a lethal necrotic disease of tomato upon coinoculation with the genomic RNAs 1, 2, and 3 of cucumber mosaic virus. Uncapped transcripts synthesized in vitro from the two linearized, recombinant plasmids are infectious and each induces the appropriate symptomatology upon coinfection with cucumber mosaic viral RNAs on tomato plants. Progeny CARNA 5s isolated from such infected plants correspond to their natural CARNA 5 counterparts.

Cloning, Molecular↗

PubMed2000

Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique.

Bovine papillomavirus type 1 (BPV-1) induces fibropapillomas in its natural host and can transform fibroblasts in culture. The viral genome is maintained as an episome within fibroblasts, which has allowed extensive genetic analyses of the viral functions required for DNA replication, gene expression, and transformation. Much less is known about BPV-1 gene expression and replication in bovine epithelial cells because the study of the complete viral life cycle requires an experimental system capable of generating a fully differentiated stratified bovine epithelium. Using a combination of organotypic raft cultures and xenografts on nude mice, we have developed a system in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures were established with bovine keratinocytes plated on a collagen raft containing BPV-1-transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after the rafts were lifted to the air interface, they were grafted on nude mice. After 6-8 weeks, large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.

Animals↗

PubMed2001

Development of new vaccines against dengue fever and Japanese encephalitis.

Mosquito-borne dengue (DEN) and Japanese encephalitis (JE) viruses are the leading causes of arthropod-transmitted viral disease in humans. A licensed tetravalent vaccine that provides effective, long-term immunity against all four serotypes of DEN virus is needed, but is currently unavailable. Improvements to currently available JE vaccines are also needed. Past and recent strategies for the development of new DEN and JE vaccines include inactivated and live attenuated viruses, engineered viruses and chimeric viruses derived from infectious cDNA clones of DEN or JE virus, recombinant poxviruses, recombinant baculoviruses, protein expression in Escherichia coli, and naked DNA vaccines. This report summarizes some of the recent developments in DEN and JE vaccinology, particularly vaccine strategies that involve live attenuated viruses, engineered viruses derived from infectious cDNA clones, and naked DNA vaccines.

Amino Acid Sequence↗

PubMed1998

Production of infectious RNA transcripts from full-length cDNA clones representing two subgroups of peanut stunt virus strains: mapping satellite RNA support to RNA1.

Full-length cDNA clones from which infectious transcripts could be generated were constructed from the genomic RNAs of two distinct strains of peanut stunt cucumovirus (PSV), PSV-ER and PSV-W. PSV-ER, a subgroup I strain, is known to support efficient replication of satellite RNA (satRNA) in infected plants, whereas PSV-W, a subgroup II strain, does not support satRNA replication. Although artificial reassortants (pseudorecombinants) of all possible combinations of infectious transcripts representing RNA1, RNA2 and RNA3 were infectious, only those having RNA1 from PSV-ER supported the replication of satRNA. These results demonstrate conclusively that support of PSV satRNA replication maps to RNA1. Comparisons of secondary structure predictions of the C-terminal helicase-like domain of the 1a proteins of four PSV strains belonging to two subgroups did not reveal any obvious differences between strains that differ in satRNA support. The complete nucleotide sequence of RNA1 from strains PSV-ER and PSV-W were determined and found to be 79% identical. Sequence comparison analysis of RNA1 sequences of cucumoviruses confirmed the placement of the PSV strains into two distinct subgroups.

Amino Acid Sequence↗

PubMed2000

Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry.

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp(390), which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp(390) on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly(390), Ala(390), and His(390)) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

Animals↗

PubMed1992

Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells.

Animals↗

PubMed1997

Proviral organization and sequence analysis of feline immunodeficiency virus isolated from a Pallas' cat.

The nucleotide sequence and genomic organization have been determined for a highly cytopathic feline immunodeficiency virus (FIV) isolated from a Pallas' cat. The 9747-bp provirus of this virus, FIV-Oma, has typical lentivirus organization with LTRs, gag, pol, and env open reading frames (ORFs), putative vif and rev ORFs, and an ORF similar to ORF2/ORFA of domestic cat FIV isolates. Although the FIV-Oma provirus is 300 to 600 bp longer than other FIV proviruses, these additional bases are distributed throughout the genome. Phylogenetic analysis of a conserved region of the pol gene suggests that FIV-Oma is more closely related to some of the puma and lion lentiviruses than it is to domestic cat FIV isolates; however, many regions of the genome exhibit extensive nucleotide sequence divergence. None of the eight molecular proviral clones isolated from a genomic library are infectious, but we have constructed an infectious, cytopathic clone of FIV-Oma from subcloned and PCR-amplified fragments of these proviral clones. This clone will be useful for identifying the genetic determinants of FIV-Oma's biological activities.

Amino Acid Sequence↗

PubMed1998

Growth properties of HSIVnef: HIV-1 containing the nef gene from pathogenic molecular clone SIVmac239.

To elucidate the function of nef, we constructed infectious chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) of macaques, by deleting a part of the nef sequence from the HIV-1 genome and inserting the entire nef gene from the pathogenic molecular clone, SIVmac239. We have named this construct HSIVnef. While there was no difference in the replication kinetics of HSIVnef+ and HSIVnef- in transformed cell lines, there was a profound difference in the replication of these virus in primary cells. These data indicated that the SIV nef gene could complement the functions of the HIV-1 nef gene in the context of the HIV-1 genome, providing further evidence that results observed in the macaque model are relevant to HIV infections in man. HSIVnef is a useful model for the study of the nef gene.

Animals↗

PubMed1999

cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses.

The replication of viral genomes and the production of recombinant viral vectors from infectious molecular clones of parvoviruses MVMp and H1 were greatly improved by the introduction of a consensus NS-1 nick site at the junction between the left-hand viral terminus and the plasmid DNA. Progressive deletions of up to 1600 bp in the region encoding the structural genes as well as insertions of foreign DNA in replacement of those sequences did not appreciably affect the replication ability of the recombinant H1 virus genomes. In contrast, the incorporation of these genomes into recombinant particles appeared to depend on in cis-provided structural gene sequences. Indeed, the production of H1 viral vectors by cotransfection of recombinant clones and helper plasmids providing the structural proteins (VPs) in trans, drastically decreased when more than 800 bp was removed from the VP transcription unit. Furthermore, titers of viral vectors, in which most of the VP-coding region was replaced by an equivalent-length sequence consisting of reporter cDNA and stuffer DNA, were reduced more than 50 times in comparison with recombinant vectors in which stuffer DNA was not substituted for the residual VP sequence. In addition, viral vector production was restricted by the overall size of the genome, with a mere 6% increase in DNA length leading to an approximately 10 times lower encapsidation yield. Under conditions fulfilling the above-mentioned requirements for efficient packaging, titers of virus vectors from improved recombinant molecular DNA clones amounted to 5 x 10(7) infectious units per milliliter of crude extract. These titers should allow the assessment of the therapeutic effect of recombinant parvoviruses expressing small transgenes in laboratory animals.

Capsid↗

PubMed1989

Molecular cloning of SIV from sooty mangabey monkeys.

We derived two infectious molecular clones of SIV from sooty mangabey monkeys (Cercocebus atys) and compared them by restriction enzyme mapping and limited DNA sequencing to other known primate lentiviruses. These analyses show that SIVsmm is closely related to, but distinct from, SIVmac and HIV-2. Our data suggest that SIVmac may have been derived from SIVsmm by cross-species transmission in captivity.

Amino Acid Sequence↗

PubMed1993

Localization of the viral antigen of feline immunodeficiency virus in the lymph nodes of cats at the early stage of infection.

Immunohistochemical examinations of localization of feline immunodeficiency virus (FIV) Gag protein were performed on lymph nodes of cats experimentally inoculated with three different strains of FIV (infectious molecular clone of TM 1, Petaluma, and KYO-1 strains), using rabbit anti-FIV Gag serum. The FIV Gag antigens were observed in many follicular dendritic cells (FDCs) and sparsely in small lymphocytes of paracortical area in the lymph nodes of cats inoculated with Petaluma and KYO-1 strains. However, the antigens were present only in small lymphocytes, and not in FDCs of a cat inoculated with infectious molecular clone of the TM1 strain. The cell type differences in expression of the viral antigen in vivo might reflect on the cell tropisms of the FIV strains in vitro. By double immunohistochemical staining with rabbit anti-FIV Gag serum and monoclonal antibodies which recognize feline CD4, feline CD8 or feline pan-T molecules, the FIV Gag-positive lymphocytes were characterized as feline CD4-positive T cells. Since the distributions of FIV Gag antigens were mainly in the FDCs, the FDCs may play an important role as a major reservoir and may be a primary target of FIV at early stages of infection.

Animals↗

PubMed1997

A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543-3.

An infectious molecular clone of simian immunodeficiency virus SIVsm was derived from a biological isolate obtained late in disease from an immunodeficient rhesus macaque (E543) with SIV-induced encephalitis. The molecularly cloned virus, SIVsmE543-3, replicated well in macaque peripheral blood mononuclear cells and monocyte-derived macrophages and resisted neutralization by heterologous sera which broadly neutralized genetically diverse SIV variants in vitro. SIVsmE543-3 was infectious and induced AIDS when inoculated intravenously into pig-tailed macaques (Macaca nemestrina). Two of four infected macaques developed no measurable SIV-specific antibody and succumbed to a wasting syndrome and SIV-induced meningoencephalitis by 14 and 33 weeks postinfection. The other two macaques developed antibodies reactive in Western blot and virus neutralization assays. One macaque was sacrificed at 1 year postinoculation, and the survivor has evidence of immunodeficiency, characterized by persistently low CD4 lymphocyte subsets in the peripheral blood. Plasma samples from these latter animals neutralized SIVsmE543-3 but with much lower efficiency than neutralization of other related SIV strains, confirming the difficulty by which this molecularly cloned virus is neutralized in vitro. SIVsmE543-3 will provide a valuable reagent for studying SIV-induced encephalitis, mapping determinants of neutralization, and determining the in vivo significance of resistance to neutralization in vitro.

Amino Acid Sequence↗

PubMed1983

Comparison of infectious JC virus DNAs cloned from human brain.

We cloned JC virus DNA obtained directly from brain tissue of 10 cases of progressive multifocal leukoencephalopathy and compared DNAs by restriction endonuclease mapping. Before cloning, each DNA preparation was homogeneous with respect to restriction patterns, but with the cloned DNAs we found variability in three regions of the genome among DNAs from different cases. There was a region of hypervariability between 0.67 and 0.725 map units; no two DNAs were exactly alike in this region. We determined that the origin of DNA replication also was in this region at 0.69 +/- 0.02 map units. In 4 of the 10 DNAs examined there was a deletion of approximately 75 base pairs between 0.14 and 0.235 map units, the region presumed to contain the codons for the C-terminal ends of the structural protein Vpl and for T antigen. JC virus DNA from these same four cases had an additional HincII-HpaI site at 0.895 map units in the presumptive Vp3 and Vp2 coding regions. Overall, no two JC virus genomes were identical although all were from fatal central nervous system infections and were infectious in vitro. Our restriction patterns suggest that there are two subtypes of JC virus circulating in the population.

Base Sequence↗

PubMed1998

A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene.

A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter. Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA. ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2. A coat protein deficient mutant of the clone, pO delta CP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished. Transcripts from pO delta CP1 were infective, able to move in N. benthamiana but produced no coat protein. This demonstrates that the coat protein was dispensable for RNA replication and for movement. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.

Blotting, Northern↗