Genetic Heterogeneity ; Hepacivirus ; genetics ; Hepatitis C ; virology ; Humans ; Replicon ; genetics

Plasmid-mediated quinolone resistance (PMQR) genes: qnr, aac(6')-Ib-cr, and qepA were investigated among 153 armA and 51 rmtB-positive transconjugants and their 204 clinical isolates of Enterobacteriaceae. Overall, qnrB4 and aac(6')-Ib-cr genes were identified in 52.3% (63 K. pneumoniae, 10 E. coli, 4 E. cloacae, and 3 E. aerogenes) and 24.8% (16 K. pneumoniae, 8 E. coli, 6 S. marcescens, 4 E. cloacae, 3 C. freundii and 1 K. oxytoca) of 153 armA-positive isolates, respectively. Four isolates of K. pneumoniae and two isolates of E. coli positive for armA co-harbored both qnrB4 and aac(6')-Ib-cr. The qepA gene was detected in 11.8% (5 E. coli and 1 K. pneumoniae) of 51 rmtB-positive clinical isolates and their transconjugants. Southern hybridization confirmed the co-localization of qepA and rmtB on a large conjugative plasmid of size between 90 to 170 kb. Inc replicon typing showed that qnrB4/6, aac(6')-Ib-cr, and qepA genes were principally disseminated by IncFIIAs, IncL/M, and IncF plasmids, respectively. This study constitutes the first report of the three known PMQR genes among the 16S rRNA methylase producing Enterobacteriaceae isolates of human origin from Korea.
Chimera ; Cloaca ; Enterobacteriaceae ; Humans ; Korea ; Methyltransferases ; Plasmids ; Pneumonia ; Replicon

The resolution recognization sites of transposon Tn4430 of Bacillus thuringiensis was inserted into cloning vector pRSET B and pUC19, resulting recombinant plasmids pBMB1201 and pBMB1202. Both of the mini res fragments, BamHI/HindIII fragment in pBMB1201 and EcoRI/HindIII fragment in pBMB1202, were ligated to the 3.3 kb EcoRI/HindIII fragment of shuttle vector pHT3101, which contained the ori. Ec, ampr and emr antibiotic resistant genes, resulting recombinant plasmid pBMB1203. After deleted the BamHI and EcoRI sites which located ouside the two res sites, resolution vector pBMB1204 was resulted. There are multiple cloning sites between two copies of resolution sites which have the same direction. The plasmid replication origin ori44, which come from B. thuringiensis sub sp. kurstaki strain YBT-1520, was inserted into the multiple cloning sites of pBMB1204 and then resolution shuttle vector pBMB1205 was obtained. With spectinomycin resistant gene as target, it was found that the resolution rate is 100% and the stability of the resolved plasmid is 93%. Using this shuttle vector, antibiotic resistance markers and other non-B. thuringiensis DNA can be selectively eliminated after the selection of transformants by antibiotic resistance marker. This vector is very useful to solve the gene safety problem while has no effect on target gene expression.
Bacillus thuringiensis ; genetics ; DNA Transposable Elements ; Genetic Vectors ; Plasmids ; Replicon

Genome, Viral ; Hepacivirus ; genetics ; physiology ; Models, Biological ; RNA, Viral ; biosynthesis ; genetics ; Replicon ; Transformation, Genetic ; Virus Replication

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.
Animals ; Swine ; Circovirus/genetics* ; Vaccines, DNA/genetics* ; Replicon/genetics* ; Genetic Vectors/genetics* ; Plasmids/genetics*

Severe fever with thrombocytopenia syndrome bunyavirus is a newly emerging virus in China, enveloped with a tripartite, single-stranded RNA genome of negative polarity. The regulatory elements for viral transcription and replication, as well as encapsidation and packaging signals, are thought to be located within these noncoding regions (NCRs). The terminal nucleotides are genus specific and highly conserved. The function of the remaining nucleotides of the NCRs is still not well understood. In this study, we developed the plasmid-driven RNA polymerase I minireplicon system for SFTSV firstly, using reporter genes GFP and luciferase. The function of the noncoding regions of the three Bunyaviridae RNA segments (L, M, S) in transcription was analyzed. Reporter genes are successfully expressed in SFTSV minireplicon system. Our results suggest that the NCRs of SFTSV from all three segments contain the necessary signals to initiate transcription. Quantitative detection of the luciferase expression level shows that promoter activity in the three segments is different.
Bunyaviridae Infections ; virology ; Cloning, Molecular ; Genome, Viral ; Humans ; Phlebovirus ; genetics ; physiology ; Replicon ; Viral Proteins ; genetics ; metabolism ; Virus Replication

Viral replicon is a kind of self-replicating viral RNA sourced from viral genome, which contains viral non-structural genes that are critical for viral genome replication with structural proteins deleted or replaced by foreign genes. Kunjin virus is a member of the Flavivirida family, Flavivirus genus, and Kunjin virus replicon is the first and the clearly defined flavivirus replicon. Kunjun virus replicon has been regarded as an excellent viral vector on account of its high expression, lower cytotoxicity and genetic stability. These unique characteristics of kunjin virus replicons make them suitable for the study of viral genome replication, recombinant proteins production, vaccine development and gene therapy. In this article, recent progress in the development, properties and applications of kunjin virus replicon system was briefly reviewed.
Genetic Vectors ; genetics ; Genome, Viral ; Recombination, Genetic ; Replicon ; genetics ; Virus Replication ; physiology ; West Nile virus ; genetics ; metabolism

The ability to introduce recombinant DNA molecules back into mycobacteria would greatly increase the potential of molecular genetic approaches for the study of mycobacteria as well as for the use in clinical purposes. We have initiated the construction of vectors that facilitates the introduction of recombinant DNA into mycobacteria. The vector was designed to contain replicons for multiplication in mycobacteria and Escherichia coli, a promoter for gene expression, a drug resistant gene for selecting transformants, and a few restriction enzyme sites for convenient cloning. Constructed Mycobacterium-E. coli shuttle vector named p YMC (hsp60) was shown to transform M. smegmatis at high efficiency and maintain plasmid at stable level. The ability of the vector to express cloned foreign gene was also monitored by measuring the expressed level of luciferase gene which was used as a reporter. High level of luciferase activity in M. smegmatis with pYMC (hsp60:luc) was detected confirming successful construction of Mycobacterium-E. coli shuttle vector.
Clone Cells ; Cloning, Organism ; DNA, Recombinant ; Escherichia coli* ; Escherichia* ; Gene Expression ; Genetic Vectors* ; Luciferases ; Molecular Biology ; Mycobacterium* ; Plasmids ; Replicon

Japanese encephalitis virus (JEV) is a member of mosquito-borne flaviviruses. To investigate whether there is a cis-acting genetic element in the coding region of the JEV C protein, which is required for viral replication, we generated four mutants by introducing a various size of deletions in each structural protein-coding region, designated as pJEV/Rep/deltaCC/LUC, pJEV/Rep/deltaC/LUC, pJEV/Rep/deltaprM/LUC, and pJEV/Rep/deltaE/LUC, of these, all replicons except for pJEV/Rep/deltaCC/LUC were competent in replication. Since pJEV/Rep/deltaCC/LUC is the same as pJEV/Rep/ deltaC/LUC except for an additional 5' deletion (nt 132~201) in the coding region of the C protein, this region appeared to be essential for RNA replication. This is consistent with the proposed cyclization sequence motif in the 5' region of the C gene, which has been recently shown to be required for replication in other mosquito-borne flaviviruses such as DV, YFV, KUN, and WNV. Thus, our results suggest that a cis-acting genetic element in the coding region of the JEV C protein may play an important role in RNA replication. This study will facilitate the current understanding of JEV RNA replication.
Asian Continental Ancestry Group* ; Capsid Proteins ; Clinical Coding* ; Cyclization ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Flavivirus ; Humans ; Replicon ; RNA

OBJECTIVETo investigate the impact of interferon-stimulated exonuclease 20 kDa (ISG20) on replication of genotype 2a hepatitis C virus (HCV) subgenomic replicon RNA and infectivity of the cell culture-derived HCV strain JFH1 to determine the potential of exogenously expressed ISG20 as an anti-viral therapy of chronic hepatitis C.

METHODSPlasma vectors containing wild-type (WT) ISG20 or a catalytically-inactive mutant ISG20m were transiently transfected into Huh7, Huh7.5 and HEK293 cells, and the replication of a monocistronic subgenomic JFH1 RNA replicon, SGRm-JFH1BlaRL, was measured. Huh7.5 cells stably expressing ISG20, ISG20m, or the control vector were established by transducing replication incompetent pCX4-Bsr-myc retroviruses encoding WT ISG20, D94G mutant ISG20, or the empty vector, respectively, and selecting with 5 mug/mL of blasticidin for approximately three weeks. The stable Huh7.5 cells were then transfected with HCV replicon RNA and infected with cell culture-derived HCV to investigate inhibition capacity of ISG20 against HCV.

RESULTSHuh7.5-ISG20, Huh7.5-ISG20m, and Huh7.5-Bsr controls cells stably expressing ISG20, ISG20m, or the control vector, respectively, were constructed successfully; the ectopically expressed ISG20 and ISG20m were distributed in both nucleus and cytoplasm, as detected by immuno uorescence. SGRm-JFH1BlaRL replicated efficiently and with similar kinetics in the Huh7.5-Bsr and Huh7.5-ISG20m cells, with expression levels plateauing at 48-96 h post-transfection. In contrast, at all time points examined, SGRm-JFH1BlaRL replication was 9.1% to 16.7% in the Huh7.5-ISG20 cells. The Huh7, Huh7.5 and HEK293 cells transiently expressing ISG20 also showed 16.7% to 25.0% of HCV replication that the respective controls. In addition, the amount of infectious progeny JFH1 virus released in culture supernatants was 9.1% to 12.5% from the Huh7.5-ISG20 cells than from the Huh7.5-Bsr and Huh7.5-ISG20m cells at 48-72 h post-infection, and the latter two cultures produced similar JFH1 virus yields. Finally, the expression of HCV core protein was also lower in the Huh7.5-ISG20 cells, as detected by immunoblot analysis.

CONCLUSIONExogenous expression of ISG20, either in a transient or stable manner, suppresses not only replication of genotype 2a HCV RNA replicons but also JFH1 virus propagation in cultured hepatocytes. The exonuclease activity of ISG20 is required for its antiviral activities against HCV.

Antiviral Agents ; pharmacology ; Cell Line ; Genome, Viral ; HEK293 Cells ; Hepacivirus ; genetics ; Humans ; RNA, Viral ; genetics ; Replicon ; Virus Replication ; drug effects