Molecular studies of brome mosaic virus using infectious transcripts from cloned cDNA.
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Acetyltransferases↗
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The long terminal repeat is a determinant of cell tropism of maedi-visna virus.
Maedi-visna virus (MVV) is a lentivirus of sheep, mainly affecting the lungs and the central nervous system. Long terminal repeat (LTR) sequence variability is common in tissue culture-derived isolates of MVV as well as those of other lentiviruses. The role of this sequence variation in MVV replication has not been explored. PCR amplification of the LTRs of an MVV isolate revealed two product sizes, the larger containing a 53 bp duplication. PCR products containing the two size variants of the LTRs were cloned into an infectious molecular clone of MVV and the resulting chimeric viruses were tested for growth in various cell types. The chimeric virus containing only one copy of the 53 bp sequence was found to grow more slowly in sheep choroid plexus cells, sheep fibroblasts and sheep synovial cells than the virus with the 53 bp duplication. Both viruses grew equally well in macrophages. These results indicate that the LTRs determined the extended cell tropism of MVV.
Animals↗
[Construction of infectious Japanese encephalitis virus clone based on the cDNA template of the attenuated live vaccine production strain SA14-14-2].
OBJECTIVE: To construct infectious Japanese encephalitis virus (JEV) based on the in vitro-ligated cDNA template of the vaccine strain SA14-14-2, and identify the virus. METHODS: Full-length genomic cDNA of JEV SA14-14-2 strain was ligated and then RNA was transcribed in vitro, the infective virus was obtained by transfecting the RNA into Vero cells and identified. RESULTS: The infective clone of JEV was constructed, the virulence was weaker than the wild virus. CONCLUSION: It was possible to construct infectious clone from the production strain of live attenuated Japanese B encephalitis vaccine.
Animals↗
Both SU and TM env proteins are responsible for monkey cell tropism of simian immunodeficiency virus SIV mac.
While simian immunodeficiency virus (SIV) derived from an infectious molecular clone pMA239 is tropic and pathogenic for monkeys, the virus derived from another infectious clone pMA142 does not replicate in monkey cells. To determine genetic sequences responsible for this tropism, a series of recombinant clones were constructed from pMA142 and pMA239. The determinant in pMA239 was mapped within regions encompassing the env gene. Viruses, which carry the 239 env gene encoding surface and/or transmembrane proteins, were tropic for monkey cells.
Animals↗
West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy.
A candidate live attenuated vaccine strain was constructed for West Nile virus (WN), a neurotropic flavivirus that has recently emerged in the U.S. Considerable attenuation for mice was achieved by chimerization with dengue virus type 4 (DEN4). The genes for the structural premembrane and envelope proteins of DEN4 present in an infectious cDNA clone were replaced by the corresponding genes of WN strain NY99. Two of 18 cDNA clones of a WN/DEN4 chimera yielded full-length RNA transcripts that were infectious when transfected into susceptible cells. The two infectious clones shared a motif in the transmembrane signal domain located immediately downstream of the NS2B-NS3 protease cleavage site that separates the DEN4 capsid protein and the WN premembrane protein of the chimera. This motif, Asp and Thr at a position 3 and 6 amino acids downstream of the cleavage site, respectively, was not present in the 16 noninfectious cDNA clones. The WN/DEN4 chimera was highly attenuated in mice compared with its WN parent; the chimera was at least 28,500 times less neurovirulent in suckling mice inoculated intracerebrally and at least 10,000 times less virulent in adult mice inoculated intraperitoneally. Nonetheless, the WN/DEN4 chimera and a deletion mutant derived from it were immunogenic and provided complete protection against lethal WN challenge. These observations provide the basis for pursuing the development of a live attenuated WN vaccine.
Aedes↗
Infectious hepatitis B virus from cloned DNA of known nucleotide sequence.
The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA.
Animals↗
Cauliflower mosaic virus 35S promoter-controlled DNA copies of cowpea mosaic virus RNAs are infectious on plants.
Clones have been constructed that contain full-length cDNA copies of cowpea mosaic virus RNA1 and RNA2, downstream of the cauliflower mosaic virus 35S promoter. The clones, when linearized downstream of the viral sequences, give rise to cowpea mosaic virus-like symptoms when inoculated onto cowpea plants. Viral RNA and virions can be detected in the inoculated plants, demonstrating that the clones are directly infectious.
Base Sequence↗
The nucleotide sequence of an infectious insect-transmissible clone of the geminivirus Panicum streak virus.
The infectious genome of a Kenyan isolate of Panicum streak virus (PSV) has been cloned and sequenced. Infection of host plants was done using an Agrobacterium binary vector containing a partial repeat of the genome. Progeny virus from resultant infections proved to be transmissible by the leafhopper Cicadulina mbila (Naude). Comparisons of the amino acid sequences of PSV DNA-encoded proteins with those of previously characterized geminiviruses infecting monocotyledonous plants, including maize streak virus, revealed high levels of identity. The evolutionary relationship between PSV and other geminiviruses infecting monocotyledons is discussed.
Amino Acid Sequence↗
Construction of a "mutagenesis cartridge" for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants.
By following a strategy of genetic analysis of poliovirus, we have constructed a synthetic "mutagenesis cartridge" spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type 1 (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed us to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. We suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).
Cloning, Molecular↗
Construction and analysis of infectious transcripts synthesized from full-length cDNA clones of both genomic RNAs of pea early browning virus.
Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.
Base Sequence↗
Analysis of the infectivity of monomeric clones of pepper huasteco virus.
The infectivity of several monomeric clones of pepper huasteco virus was investigated. All clones were infectious when inoculated excised from the plasmid DNA. However, only certain clones were infectious when inoculated in the non-excised form. Constructs in which the cloning site lies inside regions or genes involved in replication (e.g. Rep-binding site, rep and AC2-AC3 genes) were not infectious, whereas constructs in which the site was located inside the CP or BC1 genes were infectious. A clone that interrupts the BV1 gene was not infectious suggesting an early of BV1 during the establishment of the infection. Linear viral clones containing different DNA fragments at both extremes were also infectious although with a lower efficiency. Analysis of the progeny suggested a precise excision mechanism since in most cases only wild type virus was recovered. The results suggest that excision could be linked to replication through a very specific recombination process.
Geminiviridae↗
Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone.
A yeast artificial chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the YAC. The application of powerful yeast genetic techniques to an infectious adenovirus clone promises to significantly enhance the genetic analysis of adenovirus and to simplify the construction of adenovirus-based vectors for vaccines or for gene transfer to mammalian cells or whole animals. The adenovirus YAC was produced by homologous recombination in vivo between adenovirus 2 virion DNA and YAC vector plasmids carrying segments of the viral left and right genomic termini. This recombinational cloning strategy is generally applicable to the construction of YACs containing other DNA segments, such as the genomes of other viruses. Further, it is very efficient and may permit the targeted cloning of segments of the genomes of higher organisms directly from genomic DNA.
Adenoviruses, Human↗
Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.
Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.
Animals↗
A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication.
Yellow fever virus (YF) is the prototype member of the Flavivirus genus. Here, we report the successful construction of a full-length infectious cDNA clone of the vaccine strain YF-17D. YF cDNA was cloned into a low-copy-number plasmid backbone and stably maintained in several E. coli strains. Transcribed RNAs had a specific infectivity of 10(5)-10(6) p.f.u. ( micro g RNA)(-1), and the resulting virus exhibited growth kinetics, plaque morphology and proteolytic processing similar to the parental virus in cell culture. This clone was used to analyse the importance of conserved flavivirus RNA sequences and the 3' stem-loop structure in virus replication. The conserved sequences 5'CS and CS1, as well as the 3' stem-loop structure, were found to be essential for virus replication in cell culture, whereas the conserved sequence CS2 and the region containing YF-specific repeated sequences were dispensable. This infectious clone will aid future studies on YF replication and pathogenesis, as well as facilitate the development of YF-17D-based recombinant vaccines.
3' Untranslated Regions↗
Development of a rapid antiviral screening assay based on GFP reporter virus of bovine enterovirus.
In recent years, bovine enterovirus (BEV) has been increasingly associated with diarrhea in cattle in China, posing new challenges for disease control in the cattle industry. However, the mechanisms underlying BEV pathogenesis and virulence remain poorly understood. Infectious cDNA clones provide a powerful tool for dissecting viral replication and pathogenic mechanisms. In this study, we generated a full-length infectious cDNA clone of the BEV-F isolate HB19-1. Three overlapping fragments spanning the complete viral genome were amplified by RT-PCR and assembled downstream of a cytomegalovirus (CMV) promoter placed immediately upstream of the 5' untranslated region (5'UTR). To establish a reporter virus system, the green fluorescent protein (GFP) gene was inserted between the 5'UTR and the N terminus of VP4, followed by a 2A cleavage sequence (IKTAG) at the C terminus of GFP. The recombinant rHB19-GFP virus was successfully rescued. Growth curve analysis demonstrated that rHB19-GFP exhibited slower replication kinetics at early time points relative to the parental HB19-1 virus, with no significant difference in their peak viral titers. This GFP-expressing reporter virus enables convenient monitoring of BEV replication and provides a useful platform for antiviral screening. Using this system, we found that 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) inhibited BEV replication, suggesting its potential as an antiviral candidate. Overall, the rHB19-GFP infectious clone developed here offers a practical tool for studying BEV biology and for identifying antiviral compounds against BEV.
Animals↗
A cDNA clone to clover yellow vein potyvirus genome is highly infectious.
We obtained a highly infectious cDNA clone of clover yellow vein virus (CIYVV). The cDNA fragments, from which a full-length cDNA clone was constructed, were sequenced, and the complete nucleotide sequence of C1YVV RNA was determined. The viral genome is 9584 nucleotides (nt) in length excluding the poly(A) tail and contains one open reading frame (ORF) encoding a large polyprotein of 3072 amino acids. The non-coding region preceding the ORF is 190 nt long. The termination codon is followed by a 175-nt sequence. Seven potential protease NIa, one HC-pro and one P1 protease recognition sites were found in the C1YVV polyprotein by searching for cleavage consensus sequences among the potyvirus group. The cleavage dipeptides of C1YVV NIa protease are Q(E)/S(A,G). The F is conserved at the -2 position from the cleavage site except for at the P3/6K1 junction, and the V conserved at the -4 position among many potyviruses is not present at all. The genome organization of C1YVV was determined, and the amino acid sequence was compared with that of other potyviruses. The full-length cDNA clone of C1YVV was constructed by combining cDNA fragments and placed it under the control of the cauliflower mosaic virus 35S promoter. The full-length cDNA was constructed so that no extra nucleotide was present at the transcription initiation site and only 10 adenine residues were present at the 3' end of the C1YVV cDNA clone. Mechanical inoculation of a circular-formed plasmid DNA onto broad bean seedlings led to systemic infection, and the symptoms were similar to those caused by the wild-type virus but rather mild. Plasmid diluted as low as 500 pg/microl was able to induce symptoms, demonstrating that this full-length C1YVV cDNA is more infectious than any other infectious cDNAs so far reported. Filamentous particles reacting with the antiserum to C1YVV were observed in the crude sap of infected plants by immunoelectron microscopy, and genome replication was demonstrated by RT-PCR of 3' non-coding regions of C1YVV genome in total plant RNAs.
Amino Acid Sequence↗
Human immunodeficiency virus type 1 cloning vectors for antiretroviral resistance testing.
Better detection of minority human immunodeficiency virus type 1 (HIV-1) populations containing gene mutations may improve the usefulness of antiretroviral resistance testing for clinical management. Molecular cloning of HIV-1 PCR products which might improve minority detection can be slow and difficult, and commercially available recombinant virus assays test drug susceptibility of virus pools. We describe novel plasmids and simple methods for rapid cloning of HIV-1 PCR products from patient specimens and their application to generate infectious recombinant virus clones for virus phenotyping and genotyping. Eight plasmids with differing deletions of sequences encoding HIV-1 protease, reverse transcriptase, or Gag p7/p1 and Gag p1/p6 cleavage sites were constructed for cloning HIV-1 PCR products. A simple HIV-1 sequence-specific uracil deglycosylase-mediated cloning method with the vectors and primers designed here was more rapid than standard ligase-mediated cloning. Pooled and molecularly cloned infectious recombinant viruses were generated with these vectors. Replicative viral fitness and drug susceptibility phenotypes of cloned infectious viruses containing patient specimen-derived sequences were measured. Clonal resistance genotyping analyses were also performed from virus isolates, plasma HIV-1 RNA, and infected cell DNA. Sequencing of a limited number of molecular clones detected minorities of resistant virus not identified in the pooled population PCR product sequence and linkage of minority mutations.
Anti-HIV Agents↗
In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14.
Development of a novel infectious cDNA assay is described for human rhinovirus type 14. A full-length cDNA clone of the human rhinovirus type 14 genome RNA was assembled and transcribed in vitro by using the SP6 transcription system. Transfection of HeLa cells with the nascent RNA resulted in the production of rhinovirus indistinguishable from the parental virus by both immunological and polyacrylamide gel analysis.
Cloning, Molecular↗