PubMed Health⌕ Search

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 253 records · Page 14Linked to original sources
PubMed1989

Identification of the initiation codons for translation of cowpea mosaic virus middle component RNA using site-directed mutagenesis of an infectious cDNA clone.

A full-length cDNA copy of CPMV M RNA has been cloned downstream of a phage lambda promoter in the plasmid pPMI. Transcripts obtained from this clone can be translated in vitro and replicated in cowpea mesophyll protoplasts in the presence of viral B RNA. We have constructed a series of site-directed mutants of this clone to investigate the mechanism of translation of CPMV M RNA. The results obtained confirm that the AUG at position 161 is used to direct the synthesis of a 105K protein in vitro and the detection of a 58K protein in infected cowpea protoplasts suggests that it is also used in vivo. The synthesis of the 95K protein can be initiated from either of the AUGs at positions 512 and 524, though synthesis of this protein does not appear to be essential for CPMV replication in protoplasts.

Blotting, Northern↗
PubMed1991

Biological characterization of infectious molecular clones derived from a human immunodeficiency virus type-1 isolate with rapid/high replicative capacity.

In order to molecularly characterize rapidly and slowly replicating HIV-1 variants, molecular clones were obtained from a rapid/high virus isolate. This isolate, 4803, had only been passaged in peripheral blood mononuclear cells (PBMC) prior to cloning. Molecular cloning was done in bacteriophage lambda-dash using high molecular weight DNA of isolate 4803 infected PBMC. Seven recombinant phages were identified. The clones were found to be related to each other and differed only at 1 or 2 restriction sites (out of 28). The molecular clones were transfected into various cell types by electroporation. The phenotype of progeny viruses was found to be dependent on the cell type used for transfection. Progeny viruses produced by PBMC cultures differed from the parental isolate in that they did not form syncytia and lacked the capacity to replicate in cell lines. Since transfection of PBMC yielded progeny viruses within 1 week, this phenotype is considered to be the true phenotype of the clones. Transfection of the T-lymphoid HUT-78 cell line and of the monocytoid U937-2 cell line yielded progeny viruses after considerable delay (more than 1 month). Progeny viruses from HUT-78 cells were similar to the parental isolate in that they formed syncytia in PBMC and replicated in all cell lines tested. Progeny viruses from U937-2 cells showed an intermediate phenotype in that they replicated in U937-2 but not in T-lymphoid cell lines. These results indicate that molecular clones of a rapid/high virus may have a restricted replicative capacity compared to the parental, genetically heterogenous virus isolate.

Cell Line↗
PubMed1992

Isolation and characterization of a highly divergent HIV-2[GH-2]: generation of an infectious molecular clone and functional analysis of its rev-responsive element in response to primate retrovirus transactivators (Rev and Rex).

A highly divergent HIV-2 designated as HIV-2[GH-2] was obtained from an AIDS-related complex (ARC) patient in Ghana. A full-length molecular clone of this isolate was obtained and a biologically active clone was constructed. Its restriction pattern differed from that of prototype HIV-2[GH-1] in 25 of 35 restriction sites, but was strikingly similar to a previously characterized HIV-2 isolate from a Ghanaian (HIV-2ALT). The conserved integrase region (288-bp fragment) previously displayed 95% identity with that of ALT but 17-20% divergence from the HIV-2 prototype member, and a new distinct subgroup (HIV-2b) of HIV-2 consisting of GH-2 and ALT was postulated (Miura et al. 1991.) These isolates, however, were biologically distinguishable from each other by its replication capacity in a monocyte line, U937, in which GH-2 could not grow but ALT grew well. In addition, the nucleotide sequence of the LTR of this new isolate displays 21% divergence from that of prototype HIV-2[GH-1], but the core enhancer, Sp1 binding sites and TATA box were conserved. Although the 3' half of the env gene sequence which is deleted in HIV-2ALT clone showed 27% diversity from the prototype, functional differences in the rev-responsive element were not observed.

Cloning, Molecular↗
PubMed2005

HIV-1 envelope pseudotyped viral vectors and infectious molecular clones expressing the same envelope glycoprotein have a similar neutralization phenotype, but culture in peripheral blood mononuclear cells is associated with decreased neutralization sensitivity.

Recombinant lentiviral vectors pseudotyped with heterologous HIV-1 envelope glycoproteins allow rapid and accurate measurement of antibody-mediated HIV-1 neutralization. However, the neutralization phenotypes of envelope pseudoviruses have not been directly compared to isogenic replication competent HIV-1. We produced pseudoviruses expressing three different HIV-1 envelope glycoproteins and subcloned the same three env genes into a replication competent NL4-3 molecular clone. For each of the antibodies tested, the neutralization dose-response curves of pseudoviruses and corresponding replication competent viruses were similar. Thus, envelope pseudoviruses can be used to study the anti-HIV-1 neutralizing antibody response. A single passage of replication competent virus derived from 293T cells through peripheral blood mononuclear cells (PBMC) caused a substantial decrease in sensitivity to neutralizing antibodies. This was associated with an increase in average virion envelope glycoprotein content of the PBMC-derived virus. Replication competent HIV-1 and isogenic envelope pseudoviruses have similar neutralization characteristics, but passage into PBMC is associated with decreased sensitivity to neutralization.

HIV Antibodies↗
PubMed2000

Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2.

The nucleotide sequence of the RNA1 of broad bean wilt virus 2 (BBWV2) isolate B935 has been determined from overlapping cDNA clones. It contains 5956 nucleotides in length excluding the 3' terminal poly(A) tail and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 234 to 5846. A repeated motif has been found in the 5' non-coding region. The predicted polyprotein encoded by the long ORF is 1870 amino acid in length with a molecular weight of 210 K. Amino acid sequence comparisons between portions of the BBWV2 RNA1-encoded polyprotein and proteins encoded by several species in Comoviridae revealed the putative functions of BBWV2 RNA1-encoded proteins and the same general genetic organization as that of comoviruses and nepoviruses. Based on the determined sequence, full-length cDNA clone of RNA1 designated as pU1FL was constructed. Together with transcripts from full-length cDNA clone of RNA2 (pU2FL), transcripts from pU1FL infected Chenopodium quinoa successfully.

Amino Acid Motifs↗
PubMed2000

Construction and biological characterization of an infectious molecular clone of HIV type 1GB8.

Here we report the construction, sequencing, and repair of a molecular clone of HIV-1GB8, a virus representative of HIV-1 subtype B strains circulating in the UK. The phenotype of virus produced by the clone matches that of the parental virus. The molecular clone will be used in the production of attenuated virus stocks for chemical inactivation to allow development of faccines based on killed whole virus preparations.

Base Sequence↗
PubMed2002

Construction and characterization of a full-length HIV-1(92UG001) subtype D infectious molecular clone.

Here we report the construction, sequencing, and biological characterization of a molecular clone of HIV-1(92UG001), a virus representative of subtype D strains circulating in Uganda. The virus produced by the clone has an aggressive syncytium-inducing phenotype, which matches that of the parental virus. This phenotype may be related to duplication of a binding site for a transcription factor, T cell factor 1alpha (TCF-1alpha), in the long terminal repeat of the virus.

Adult↗
PubMed1995

Acquired immune dysfunction in rabbits experimentally infected with an infectious molecular clone of the bovine immunodeficiency virus (BIV127).

To investigate the effect of bovine immunodeficiency virus (BIV) infection on the rabbit immune system, we studied the proliferative responses of peripheral blood lymphocytes (PBLs) of rabbits experimentally inoculated with BIV. All BIV127-inoculated rabbits seroconverted after 6 weeks and remained seropositive over a prolonged period of time. Assays for specific lymphocyte reactivity to concanavalin A (Con A) were performed monthly for over 1 year. One-hundred percent of infected rabbits developed abnormally low T cell responses, as measured by Con A stimulation. By 3 months postinoculation, the PBL response to Con A was diminished and remained depressed for 6 months. All animals were clinically asymptomatic within 14 months of BIV inoculation. By 15 and 16 months postinoculation, two of three infected rabbits exhibited recurrent lowering of the T cell responsiveness including a decrease in absolute PBL counts. One of these animals died unexpectedly. Our results further confirmed that a functional impairment of lymphocytes was induced early in the course of BIV infection, prior to clinical disease. These findings suggested that BIV infection may mimic asymptomatic infection of human immunodeficiency virus (HIV) and provided further evidence of the importance of BIV-induced disease in rabbits as a relevant model for the study of AIDS.

Animals↗
PubMed2005

Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone.

A stable plasmid DNA, pMWJEAT, was constructed by using full-length Japanese encephalitis virus (JEV) cDNA isolated from the wild-type strain JEV AT31. Recombinant JEV was obtained by synthetic RNA transfection into Vero cells and designated rAT virus. JEV rAT exhibited similar large-plaque morphology and antigenicity to the parental AT31 strain. Mutant clone pMWJEAT-E138K, containing a single Glu-to-Lys mutation at aa 138 of the envelope (E) protein, was also constructed to analyse the mechanisms of viral attenuation arising from this mutation. Recombinant JEV rAT-E138K was also recovered and displayed a smaller-plaque morphology and lower neurovirulence and neuroinvasiveness than either AT31 virus or rAT virus. JEV rAT-E138K exhibited greater plaque formation than rAT virus in virus-cell interactions under acidic conditions. Heparin or heparinase III treatment inhibited binding to Vero cells more efficiently for JEV rAT-E138K than for rAT virus. Inhibition of virus-cell interactions by using wheatgerm agglutinin was more effective for JEV rAT than for rAT-E138K on Vero cells. About 20 % of macropinoendocytosis of JEV rAT for Vero cells was inhibited by cytochalasin D treatment, but no such inhibition occurred for rAT-E138K virus. Furthermore, JEV rAT was predominantly secreted from infected cells, whereas rAT-E138K was more likely to be retained in infected cells. This study demonstrates clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV.

Animals↗
PubMed1990

Generation and characterization of infectious chimeric clones between human immunodeficiency virus type 1 and simian immunodeficiency virus from an African green monkey.

A series of chimeric clones of human immunodeficiency virus type 1 and simian immunodeficiency virus isolated from an African green monkey was constructed in vitro. In transient transfection experiments, all clones produced virion-associated reverse transcriptase, gag proteins, and env proteins. Eight out of 10 chimeric viruses clearly grew in the human CD4+ cell line C8166. Susceptibility of other CD4+ cell lines, MT-4, A3.01, and Molt4 clone 8, to infection with these viruses was also demonstrated.

Animals↗
PubMed1998

Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection in vitro.

Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), and DIS (TG), when introduced into deletion mutants of HIV-1 recombined efficiently, and the progeny virions replicated with comparable kinetics. A fourth DIS loop form, containing an artificial AAAAAA sequence disrupting the putative DIS loop-loop interactions [DIS (A6)], supported efficient recombination with DIS loop variants; however, DIS (A6) progeny virions exhibited a modest replication disadvantage in mixed cultures. Our studies indicate that the nonhomologous DIS sequences found in different HIV-1 subtypes are not a primary obstacle to intersubtype recombination.

Cloning, Molecular↗
PubMed1998

Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus.

An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3' long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV(PV3.3), and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV(PV3.3#3) (redesignated EIAV(UK)), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6 degrees C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV(PV) strain into the infectious nonpathogenic molecular clone 19-2-6A leads to the production of progeny virus particles with the ability to induce clinical signs of EIA. Therefore, EIAV(UK), which is the first pathogenic, cell culture-adapted molecular clone of EIAV to be described, should be of value in identifying viral determinants of pathogenicity.

Amino Acid Sequence↗
PubMedDate not supplied

Development and evaluation of a non-isotopically labeled DNA probe for the diagnosis of infectious laryngotracheitis.

A digoxigenin-labeled cloned infectious laryngotracheitis virus (ILTV) DNA fragment was evaluated as a nonradioactive alternative probe in the diagnosis of infectious laryngotracheitis. The dot-blot hybridization protocol was optimized and was capable of detecting 40 pg of purified ILTV DNA and as few as 50 ILTV-infected chicken embryo liver cells. The utility of this approach for diagnostic use was evaluated through four ILTV inoculation trials using a mild field isolate, a virulent challenge strain, a tissue-culture-origin vaccine, and an egg-origin vaccine. Birds were examined for clinical signs of ILT, and conjunctival and pharyngeal swabs from inoculated and sentinel birds were tested for ILTV by the digoxigenin-labeled probe and by virus isolation. In general, higher numbers of ILTV-positive samples were detected by both assays from conjunctival swabs. For the non-vaccine strains, detection by dot-blot hybridization was equivalent to that for virus isolation. However, for the two vaccine strains, there was some lack of correlation between the dot-blot results and the virus-isolation results. The kappa values between virus-isolation results and dot-blot results for the tissue-culture-origin vaccine, egg-origin vaccine, Ont 1598 field isolate, and virulent strain were 0.00, 0.16, 0.39, and 0.24, respectively, for pharyngeal samples and 0.19, 0.29, 0.58, and 0.48, respectively, for conjunctival samples.

Animals↗
PubMed1990

Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4.

Minus strand RNA synthesis by the positive strand alphaviruses, Sindbis and Semliki Forest viruses, normally occurs early in infection, is coupled to synthesis of viral nonstructural proteins and to formation of viral replication complexes, and terminates and does not occur late in infection. Previously, ts24 of the A complementation group of Sindbis virus RNA-negative mutants was found to possess, among its other temperature sensitive defects, a temperature sensitivity in the normal cessation of minus strand synthesis which enabled minus strands to be synthesized late in infection at 40 degrees in the absence of protein synthesis. Revertants of ts24 (ts24R1, ts24R2) retained the defect in the shutoff of minus strand synthesis, indicating the lesion was not conditionally lethal and could map outside the A cistron. The studies reported here used an infectious clone of Sindbis virus to identify the mutation responsible for this phenotype. Hybrid viruses were prepared from constructs containing restriction fragments of the cDNA of ts24R1 in place of the corresponding fragments in the infectious SIN HR clone and screened for their ability to synthesize minus strands at 40 degrees in the presence of cycloheximide. A unique base change of an A for a C residue at nt 6339, predicting a change from glutamine to lysine at amino acid 195 in nsP4, was found in genomes of ts24, ts24R1, and ts24R2. Other nucleotide changes present at the 5' and 3' termini did not affect minus strand synthesis. The substitution of the parental Sindbis virus sequence that encompassed nt 6339 in an infectious clone of the ts24R1 revertant eliminated the mutant phenotype. We conclude that the ability to continue minus strand synthesis at 40 degrees exhibited by ts24 and its revertants is caused by an alteration in nsP4, which is the alphavirus replicase or an essential component of the replicase. We hypothesize that this domain of nsP4 functions to fix the minus strand as the stable template of alphavirus replication complexes.

Amino Acid Sequence↗
PubMed1999

Generation of transmissible hepatitis C virions from a molecular clone in chimpanzees.

Multiple alignments of hepatitis C virus (HCV) polyproteins from six different genotypes identified a total of 22 nonconsensus mutations in a clone derived from the Hutchinson (H77) isolate. These mutations, collectively, may have contributed to the failure in generating a "functionally correct" or "infectious" clone in earlier attempts. A consensus clone was constructed after systematic repair of these mutations, which yielded infectious virions in a chimpanzee after direct intrahepatic inoculation of in vitro transcribed RNAs. This RNA-infected chimpanzee has developed hepatitis and remained HCV positive for more than 11 months. To further verify this RNA-derived infectivity, a second naive chimpanzee was injected intravenously with serum collected from the first chimpanzee. Infectivity analysis of the second chimpanzee demonstrated that the HCV infection was successfully transmitted, which validated unequivocally the infectivity of our repaired molecular clone. Amino acid sequence comparisons revealed that our repaired infectious clone had 4 mismatches with the isogenic clone reported by Kolykhalov et al. (1997, Science 277, 570-574) and 8 mismatches with that reported by Yanagi et al. (1997, Proc. Natl. Acad. Sci. USA 94, 8738-8743). At the RNA level, more mismatches (43 and 67, respectively) were identified; most of them were synonymous substitutions. Further comparisons with 16 isolates from different genotypes demonstrated that our repaired clone shares greater consensus than the reported isogenic clones. This approach of generating infectious HCV RNA validates the importance of amino acid sequence consensus in relation to the biology of HCV.

Amino Acid Sequence↗
PubMed1993

Genomic organization and mapping of transcription and translation products of the NADL-2 strain of porcine parvovirus.

The NADL-2 strain of PPV was cloned into pUC19 and independent infectious clones were sequenced. This permitted a correction of published sequences and to predict a cruciform structure as an alternative to the 5'-hairpin of the "-" strand. This 5'-end structural covariance is shared with other parvoviruses of the same group and two alternative sequences ("flip" and "flop") were present in the region of the cruciform. Transcript and translation product mapping allowed the prediction of the location of the different expression signals. The 5'-startpoints of the transcripts were located at nucleotides 225 and 2035, respectively, and the polyadenylation site at nucleotides 4829-4833. This indicated that the TATA boxes at 196-TATA and 2004-AATA and the 4813-AATAAA polyadenylation sequence would be functional. Alternative splicing of capsid gene (VP) transcripts (either 2280-AG/GT or 2313-AG/GT spliced with 2386-AG/GA), to maintain or remove the first AUG (at 2287) in the ORF, yielded two 2.9-kb mRNAs containing a nested set of protein-coding sequences (VP-1 and VP-2 with predicted molecular mass 80.9 and 64.3 kDa, respectively). Three nonstructural (NS) protein gene transcripts were identified. The 4.7-kb transcript was not spliced in the NS gene and was predicted to code for a 75.5-kDa protein (NS-1; published value of phosphorylated form 84 kDa). The splicing sites of two different 3.3-kb NS transcripts were analyzed. These transcripts were predicted to code for the NS-2 protein (18.1 kDa). Of the two NS-2 transcripts, one had also the VP-intron removed downstream of the NS-2 coding sequences. A 2.9-kb transcript would code for an NS-3 protein (12.4 kDa) although such a protein has not been described before. A flow chart of the information from the viral DNA to the viral proteins is presented and several differences, both for the NS and the VP genes, with closely related parvoviruses are noted.

Amino Acid Sequence↗