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PubMedDate not supplied

Isolation and characterization of infectious full-length DNA clones of chimpanzee foamy viruses SFV6 and SFV7: evidence for a Taf-dependent internal promoter.

We have cloned complete viral genomes directly from Hirt supernatant DNAs of simian foamy virus types 6 and 7 (SFV6 and SFV7) -infected cells. These clones were shown to be infectious by transfection into cells and subsequent infection of susceptible cells either by cocultivation or by passage of cell-free supernatants. The presence of virus particles, suggested by a typical cytopathic effect, was confirmed by electron microscopy. These viruses were characterized at different levels of the replication cycle. The proviral genomes revealed a taf deletion comparable to that previously described in the human foamy virus (HFV) bel1 gene. Analysis of viral RNAs revealed similar patterns of transcripts for SFV6- and SFV7-infected cells, with predominant expression of accessory genes. Characteristic major viral polypeptides were identified by radioimmunoprecipitation for both isolates. Sequences homologous to the gene encoding Taf and to a potential internal promoter were identified in the infectious clones and subcloned into expression vectors. Their functional properties were tested by transfection assays, which provided evidence for the presence of a Taf-dependent internal promoter in both SFV6 and SFV7 isolates.

Animals↗
PubMed1991

Isolation of replication-competent molecular clones of visna virus.

Visna virus is the prototypic member of a subfamily of retroviruses responsible for slow infections of animals and humans. As a part of our investigation of the functions of viral gene products in virus replication, we have isolated three infectious molecular clones and determined the complete nucleotide sequences of two of the clones. We have also characterized the progeny of the biologically cloned viral stocks and of the infectious clones and document considerable heterogeneity in plaque size and antigenic phenotype of the former that is reduced to near homogeneity in the progeny of the infectious clones. It thus should now be possible to trace the emergence of antigenic variants of visna virus as well as ascribe defined functions to structural and regulatory genes of the virus in determining neurovirulence and the slow tempo of infection.

Amino Acid Sequence↗
PubMed1991

Gallid herpesvirus 1 (infectious laryngotracheitis virus): cloning and physical maps of the SA-2 strain.

Clones representing 90% of the genome of Gallid herpesvirus 1 (infectious laryngotracheitis virus; ILTV) were obtained and used in hybridization experiments to construct EcoRI, KpnI amd SmaI physical maps. The genome was 155 kilobase pairs (kbp) and comprised of a long unique sequence (120 kbp) and a short unique sequence (17 kbp) bounded by repeat sequences each of 9 kbp. An unrelated second pair of repeat sequences was located at 0.67 and 0.88 map untis. A terminal repeat of the unique long region (UL) was also detected, but no isomerization of UL was detected.

Animals↗
PubMed1994

Infectious rabies viruses from cloned cDNA.

The generation of infectious rabies virus (RV), a non-segmented negative-stranded RNA virus of the Rhabdoviridae family, entirely from cloned cDNA is described. Simultaneous intracellular expression of genetically marked full-length RV antigenome-like T7 RNA polymerase transcripts and RV N, P and L proteins from transfected plasmids resulted in formation of transcriptionally active nucleocapsids and subsequent assembly and budding of infectious rabies virions. In addition to authentic RV, two novel infectious RVs characterized by predicted transcription patterns were recovered from modified cDNA. Deletion of the entire non-translated pseudogene region, which is conserved in all naturally occurring RVs, did not impair propagation of the resulting virus in cell culture. This indicates that non-essential genetic material might be present in the genomes of non-segmented RNA viruses. The introduction of a functional extra cistron border into the genome of another virus resulted in the transcription of an additional polyadenylated mRNA containing pseudogene sequences. The possibility of manipulating the RV genome by recombinant DNA techniques using the described procedure--potentially applicable also for other negative-stranded viruses--greatly facilitates the investigation of RV genetics, virus-host interactions and rabies pathogenesis and provides a tool for the design of new generations of live vaccines.

Animals↗
PubMed1996

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Animals↗
PubMed2003

Mutagenesis of a bovine herpesvirus type 1 genome cloned as an infectious bacterial artificial chromosome: analysis of glycoprotein E and G double deletion mutants.

The genome of bovine herpesvirus type 1 Schönböken was cloned as a bacterial artificial chromosome (BAC) by inserting mini F plasmid sequences into the glycoprotein (g) E gene. The resulting BAC clone, pBHV-1DeltagE, was transfected into bovine kidney cells and viable gE-negative BHV-1 (BHV-1DeltagE) was recovered. By RecE/T mutagenesis in Escherichia coli, the gG open reading frame was deleted from pBHV-1DeltagE. From the mutated BAC, double negative BHV-1DeltagE-gG was reconstituted and its growth properties were compared to those of rescuant viruses in which the gE gene was restored (BHV-1rev, BHV-1DeltagG). The mutant viruses did not exhibit markedly lowered virus titres. Plaque sizes of BHV-1DeltagE, BHV-1DeltagE-gG and BHV-1DeltagG, however, were reduced by 19 to 55 % compared to parental strain Schönböken or BHV-1rev. Our results suggested that gE and gG function independently from each other in cell-to-cell spread, because an additive effect on plaque formation was observed in the gE/gG double deletion mutant.

Animals↗
PubMed1984

Production of infectious poliovirus from cloned cDNA is dramatically increased by SV40 transcription and replication signals.

Sub-genomic cDNA clones representing the entire genomic RNA of poliovirus Type 1 (Mahoney) have been isolated in E. coli. Construction of a complete cDNA copy of the poliovirus genome in the EcoRI site of plasmid vector pBR325 from these clones is described. Introduction of plasmid DNA containing the complete cDNA copy of polio RNA into cultured primate cells by transfection produces infectious poliovirus. The virus produced by such a transfection appears to be identical to wild type poliovirus. Isolation of a polio recombinant plasmid containing SV40 transcription and replication signals is also described. Transfection of COS-1 cells with this plasmid yields greater than 1,600 plaque-forming units (PFU) per microgram of input DNA.

Base Sequence↗
PubMed1997

Recovery of infectious SV5 from cloned DNA and expression of a foreign gene.

A complete cDNA clone of the genome (15,246 nucleotides) of the paramyxovirus SV5 was constructed from cDNAs such that an anti-genome RNA could be transcribed by T7 RNA polymerase and the correct 3' end generated by cleavage using hepatitis delta virus ribozyme. The plasmid encoding the antigenome sequence was transfected into cells previously infected with recombinant vaccinia virus that expressed T7 RNA polymerase, together with helper plasmids that expressed the viral replication proteins, NP, P, and L, under the control of the T7 polymerase promoter. Rescue of the RNA genome from DNA was demonstrated by recovering SV5 with the tag restriction sites introduced into the DNA clone, using RT-PCR of the genome RNA and nucleotide sequencing. Rescue of SV5 from DNA did not require expression of the viral V protein as a helper plasmid, suggesting that V protein is not essential for initial replication. The infectious cDNA of SV5 was also manipulated to express green fluorescent protein (GFP) under the control of SV5 transcriptional start and stop signals introduced between the HN and L genes. The amount of GFP that was expressed varied depending on the nature of the newly introduced transcription signals.

DNA, Complementary↗
PubMed1997

An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription.

Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.

Animals↗
PubMedDate not supplied

Antigenicity, pathogenicity, and immunogenicity of small and large plaque infectious bursal disease virus clones.

A serotype 1 variant strain of infectious bursal disease virus designated IN was passaged 40 times in BGM-70 cell line. A small plaque (SP) clone and a large plaque (LP) clone were then isolated and plaque purified four times. The SP and LP viruses formed circular plaques about 0.5 mm and 6.0 mm in diameter, respectively. Both clones lost their pathogenicity for specific-pathogen-free (SPF) chickens and did not elicit significant levels of virus-neutralizing antibody titers. However, the SP and LP clones maintained their immunogenicity when used as inactivated vaccines in SPF chickens. The restriction enzyme profiles of both clones were similar. Back passage of the SP and LP clones in SPF chickens resulted in loss of their phenotypic characteristics.

Animals↗
PubMed2000

Nucleotide sequence and synthesis of infectious RNA from cloned cDNA of broad bean wilt virus 2 RNA 2*.

The complete nucleotide sequence of broad bean wilt virus -2 (BBWV-2) RNA 2 was determined. The sequence is 3593 nucleotides in length, and contains a single open reading frame which has the potential to encode a 119 kDa polyprotein. Direct amino acid sequencing of the virus coat proteins suggests that the polyprotein is processed to produce three proteins, 52 kDa, 44 kDa and 22 kDa in size. A comparison of several fabaviruses in terms of nucleotide sequence and amino acid sequences showed that BBWV-2 isolates display high sequence identity. A full-length cDNA constructed under the control of the T7 promoter could be successfully transcribed to produce biologically active RNA. This is the first report on generating infectious transcripts of BBWV-2 RNA 2.

Base Sequence↗
PubMed2003

Shuttle PCR-based cloning of the infectious adeno-associated virus type 5 genome.

Adeno-associated virus type 5 (AAV5), which is distinct from the other serotypes of AAV, has attracted considerable interest as a premier gene delivery vector. As do the other serotypes, AAV5 contains its 4.7 kb-sized, single-stranded genome flanked with inverted terminal repeats (ITRs) in a hairpin conformation, which serves frequently as pause and arrest sites for DNA polymerases during PCR. To amplify the full-length of the AAV5 genome in single step, we established a shuttled, long and accurate PCR (LA-PCR) procedure in the present study. Furthermore, helper oligonucleotides, which hybridize with the palindromic sequence elements in ITR, were designed and employed in PCR to prevent the formation of hairpin structures by highly GC-rich ITRs. Consequently, a 4.7 kb-sized PCR product was amplified successfully, and cloned into a pBluescript II KS(+) plasmid. Six plasmids, harboring the full-length AAV5 genome, rescued wild type AAV5 viruses on transfection to HeLa and HEK 293 cells, which were co-infected with helper adenoviruses. Western and Southern blot analyses supported further the fact that the pAAV5 plasmids harbored the full-length AAV5 genome. The PCR method described in this study is applicable for the cloning of genomes containing variable palindromic structures, in addition to AAV genomes of other serotypes.

Cell Line↗
PubMed2001

Palindromic sequence plays a critical role in human foamy virus dimerization.

The retroviral RNA genome is dimeric, consisting of two identical strands of RNA linked near their 5' ends by a dimer linkage structure. Previously it was shown that human foamy virus (HFV) RNA transcribed in vitro contained three sites, designated SI, SII, and SIII, which contributed to the dimerization process (O. Erlwein, D. Cain, N. Fischer, A. Rethwilm, and M. O. McClure, Virology 229:251-258, 1997). To characterize these sites further, a series of mutants were designed and tested for their ability to dimerize in vitro. The primer binding site and a G tetrad in SI were dispensable for dimerization. However, a mutant that changed the 3' end of SI migrated slower on nondenaturing gels than wild-type RNA dimers. The sequence composition of the SII palindrome, consisting of 10 nucleotides, proved to be critical for in vitro dimerization, since mutations within this sequence or replacement of the sequence with a different palindrome of equal length impaired in vitro dimerization. The length of the palindrome also seems to play an important role. A moderate extension to 12 nucleotides was tolerated, whereas an extension to 16 nucleotides or more impaired dimerization. When nucleotides flanking the palindrome were mutated in a random fashion, dimerization was unaffected. Changing the SIII sequence also led to decreased dimer formation, confirming its contribution to the dimerization process. Interesting mutants were cloned into the infectious molecular clone of HFV, HSRV-2, and were transfected into BHK-21 cells. Mutations in SII that reduced dimerization in vitro also abolished virus replication. In contrast, constructs containing mutations in SI and SIII replicated to some extent in cell culture after an initial drop in viral replication. Analysis of the SIM1 mutant revealed reversion to the wild type but with the insertion of an additional two nucleotides. Analysis of cell-free virions demonstrated that both replication-competent and replication-defective mutants packaged nucleic acid. Thus, efficient dimerization is a critical step for HFV to generate infectious virus, but HFV RNA dimerization is not a prerequisite for packaging.

Animals↗
PubMed2001

Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences.

We studied the in vivo biological properties of viruses reconstituted from the genome of murine gammaherpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus RgammaHV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus RgammaHV68A98.02, with biological properties comparable to those of wild-type MHV-68. On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAC-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.

Animals↗
PubMed1996

Infectious in vitro transcripts from cloned cDNA of the potato A potyvirus.

A full-length cDNA copy of potato virus A RNA was constructed downstream from the bacteriophage T7 RNA polymerase promoter. A single extra guanosine not present in PVA RNA was added to the 5'-end of the cDNA. The capped in vitro transcripts from cloned cDNA were infectious in the mesophyll protoplasts and intact plants of Nicotiana tabacum. PVA coat protein accumulated in transcript-inoculated tobacco protoplasts and infected systematically intact plants producing high PVA titres according to the ELISA and immunoblot assay. The infected tobacco plants produced similar mild mosaic symptoms in the systematically infected leaves, irrespective of whether the RNA transcripts or parental virus were used as inoculum; the virus particles were also morphologically similar according to immunosorbent electron microscopy.

Base Sequence↗
PubMed2025

Infectious Subgenomic Amplicon Strategies for Japanese Encephalitis and West Nile Viruses.

Classical methods for constructing infectious cDNA clones of flaviviruses are often hindered by instability and toxicity. The Infectious-Subgenomic-Amplicons (ISA) method is an advancement which utilizes overlapping DNA fragments representing viral genomic sequence and in-cell recombination to bypass bacterial plasmid assembly. However, the ISA method has limitations due to the toxicity of some ISA DNA fragments in bacteria during synthetic production. We validated modified ISA strategies for producing toxic ISA Japanese encephalitis virus (JEV) and West Nile virus (WNV) DNA fragments. Three approaches were explored, including subdividing toxic DNA fragments into two sub-fragments for synthetic clonal production, using a low-copy bacterial plasmid, and subdividing the toxic DNA fragments into four short overlapping sub-fragments, each up to 1.8 kb. The latter novel approach in ISA applications enabled the synthesis of entirely bacteria-free ISA DNA fragments. Our results demonstrate that subdividing toxic fragments into sub-fragments smaller than 1.8 kb for synthesis is the efficient strategy, circumventing the need for bacterial plasmids and ensuring rapid production of synthetic flaviviruses. This method also shortens the production timeline. We also compared the efficacy of JEV and WNV ISA in zinc finger antiviral protein 1 (ZAP) wild-type and knockout cells and found that knockout cells may be more effective for ISA rescue of flaviviruses, including CpG-enriched strains for live attenuated vaccines. The validated modified ISA strategies provide an efficient approach for producing synthetic JEV and WNV. This will enable rapid research during outbreaks of emerging flaviviruses by facilitating the quick generation of new virus variants.

West Nile virus↗
PubMed2005

Point mutations in an infectious bovine viral diarrhoea virus type 2 cDNA transcript that yields an attenuated and protective viral progeny.

An infectious cDNA clone of the hypervirulent bovine viral diarrhoea virus (BVDV) strain 890 (isolate 256) was produced by a streamlined PCR procedure. As compared to the published sequence of strain 890, the nucleotide sequencing of cloned cDNA corresponding to isolate 256 revealed several mutations seven of which were attributed to the cloning procedure. The infectious transcript was transfected into permissive cells and led to viral multiplication (AvrII+ strain). In vitro, viral titres reached by the parental strain exceed those of the AvrII+ strain by more than one order of magnitude. The latter was clearly less virulent to young calves as indicated by clinical, haematological and virological parameters. Thirty-four days after inoculation with AvrII+ strain, calves were challenged with the virulent parental strain. The animals were protected as compared to unvaccinated controls. Therefore, our approach led to the production of an attenuated strain with potential use as a vaccine strain and will be useful for studies of virulence determinants in BVDV-2.

Animals↗