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*

PURPOSE: Hepatitis C virus (HCV) infection is a major cause of liver disease. Several miRNAs have been found to be associated with HCV infection. This study aimed to investigate the functional roles and possible molecular mechanisms of miR-215 in HCV replication. MATERIALS AND METHODS: The expression levels of miR-215 and TRIM22 were detected by quantitative real-time PCR (qRT-PCR) and western blot analysis in Con1b subgenomic genotype 1b HCV replicon cells (Con1b cells) and JFH1 full genome infecting Huh7.5.1 cells (Huh7.5.1 cells). HCV RNA levels were measured by qRT-PCR. The protein levels of NS3, NS5A, p65 subunit of NF-κB (p65), and phosphorylated p65 (p-p65) were determined by western blot analysis. The relationship between miR-215 and TRIM22 were explored by target prediction and luciferase reporter analysis. RESULTS: miR-215 overexpression enhanced HCV replication in Con1b cells, while miR-215 knockdown suppressed HCV replication in Huh7.5.1 cells. TRIM22 was confirmed to be a direct target of miR-215. TRIM22 upregulation resulted in a decline in HCV replication, while TRIM22 inhibition led to enhancement of HCV replication. Additionally, exogenous expression of TRIM22 reversed the facilitating effect of miR-215 on HCV replication, while TRIM22 downregulation counteracted the inhibitory effect of miR-215 knockdown on HCV replication. Furthermore, miR-215 targeted TRIM22 to block the NF-κB pathway, and exerted a positively regulatory role on HCV replication. CONCLUSION: miR-215 facilitated HCV replication via inactivation of the NF-κB pathway by inhibiting TRIM22, providing a novel potential target for HCV infection.
Blotting, Western ; Down-Regulation ; Genome ; Genotype ; Hepacivirus ; Liver Diseases ; Luciferases ; MicroRNAs ; Real-Time Polymerase Chain Reaction ; Replicon ; RNA ; Up-Regulation

Hepatitis B virus (HBV) infection remains a major global health problem; indeed, there are 250 million carriers worldwide. The host range of HBV is narrow; therefore, few primates are susceptible to HBV infection. However, ethical constraints, high cost, and large size limit the use of primates as suitable animal models. Thus, in vivo testing of therapies that target HBV has been hampered by the lack of an appropriate in vivo research model. To address this, mouse model systems of HBV are being developed and several are used for studying HBV in vivo. In this review, we summarize the currently available mouse models, including HBV transgenic mice, hydrodynamic injection-mediated HBV replicon delivery systems, adeno-associated virus-mediated HBV replicon delivery systems, and human liver chimeric mouse models. These developed (or being developed) mouse model systems are promising and should be useful tools for studying HBV.
Animals ; Global Health ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Host Specificity ; Humans ; Hydrodynamics ; Liver ; Mice* ; Mice, Transgenic ; Models, Animal ; Primates ; Replicon

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is considered a serious global threat. However, little is known regarding the multidrug resistance (MDR) mechanisms of CRKP. This study investigated the phenotypes and MDR mechanisms of CRKP and identified their clonal characteristics. METHODS: PCR and sequencing were utilized to identify antibiotic resistance determinants. Integron gene cassette arrays were determined by restriction fragment length polymorphism (RFLP) analysis. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis. Plasmids were typed by using a PCR-based replicon typing and analyzed by conjugation and transformation assays. RESULTS: Seventy-eight strains were identified as resistant to at least one carbapenem; these CRKP strains had a high prevalence rate (38.5%, 30/78) of carbapenemase producers. Additionally, most isolates harbored MDR genes, including Extended spectrum β-lactamases (ESBLs), AmpC, and quinolone and aminoglycoside resistance genes. Loss of porin genes was observed, and Class 1 integron was detected in 66.7% of the investigated isolates. PFGE and MLST results excluded the occurrence of clonal dissemination among these isolates. CONCLUSIONS: A high prevalence of NDM-1 genes encoding carbapenem resistance determinants was demonstrated among the K. pneumoniae isolates. Importantly, this is the first report of bla(NDM-1) carriage in a K. pneumoniae ST1383 clone in China and of a MDR CRKP isolate co-harboring bla(NDM-1), bla(KPC-2), bla(CTX-M), bla(SHV), acc(6′)-Ib, rmtB, qnrB, and acc(6′)-Ib-cr.
China* ; Clone Cells ; Drug Resistance, Bacterial ; Drug Resistance, Microbial ; Drug Resistance, Multiple* ; Electrophoresis, Gel, Pulsed-Field ; Genes, MDR ; Integrons ; Klebsiella pneumoniae* ; Klebsiella* ; Molecular Epidemiology ; Phenotype ; Plasmids ; Pneumonia ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Prevalence ; Replicon

BACKGROUND: The emergence of fosfomycin resistance and extended-spectrum β-lactamase (ESBL) genes is a serious threat to public health and a new challenge in shigellosis treatment. The purpose of this study was to identify fosfomycin resistance and characterize β-lactamase genes in fos-carrying isolates of Shigella flexneri from patients in China. METHODS: A total of 263 S. flexneri isolates were collected from 34 hospitals in the Anhui Province of China during September 2012-September 2015 and screened for fosA3, fosA, and fosC2 by PCR amplification and sequencing. The fos-carrying isolates were then screened for β-lactamase genes. The clonal relationships between fosA3-carrying isolates, the transmissibility of fosfomycin resistance, replicon types of plasmids carrying fosfomycin resistance genes and other associated resistance genes were investigated. RESULTS: Twenty-five of the 263 isolates (9.5%) showed resistance to fosfomycin, and 18 (6.8%) were positive for fosA3. None of the isolates was positive for fosA or fosC2. Seventeen of the isolates carrying fosA3 (94%) were CTX-M producers (seven CTX-M-55, five CTX-M-14, and five CTX-M-123), while three (16.7%) were TEM producers (TEM-1).Sixteen (88.9%) fosA3-carrying isolates exhibited multi-drug resistance. The replicon types of the 13 fosA3-carrying plasmids were IncF (n=13), IncHI2 (n=3), IncIl-Ir (n=2), and IncN (n=1). CONCLUSIONS: Our results indicated that fosA3 could spread through plasmids in S. flexneri isolates, along with the bla(CTX-M) and bla(TEM), which facilitate its quick dispersal. To the best of our knowledge, this is the first report of CTX-M-123-type ESBLs in S. flexneri isolates from patients in China.
China* ; Drug Resistance, Multiple ; Dysentery, Bacillary ; Fosfomycin* ; Humans ; Plasmids ; Polymerase Chain Reaction ; Public Health ; Replicon ; Shigella flexneri* ; Shigella*

In this study, 78 isolates of Escherichia coli isolated from Korean beef cattle farms were investigated for the production of extended-spectrum beta-lactamase (ESBL) and/or AmpC beta-lactamase. In the disc diffusion test with ampicillin, amoxicillin, cephalothin, ceftiofur, cefotaxime, ceftazidime, and cefoxitin, 38.5% of the isolates showed resistance to all of ampicillin, amoxicillin, and cephalothin. The double disc synergy method revealed that none of the isolates produced ESBL or AmpC beta-lactamases. DNA sequencing showed that all isolates encoded genes for TEM-1-type beta-lactamase. Moreover, 78.2% of the isolates transferred the TEM-1-type beta-lactamase gene via conjugation. In plasmid replicon typing of all donors, IncFIB and IncFIA were identified in 71.4% and 41.0% of plasmids, respectively. In transconjugants, IncFIB and IncFIA were the most frequent types detected (61.5% and 41.0%, respectively). Overall, the present study indicates that selection pressures of antimicrobials on beta-lactamases in beef cattle may be low relative to other livestock animals in Korea. Moreover, to reduce selection pressure and dissemination of beta-lactamase, the long-term surveillance of antimicrobial use in domestic beef cattle should be established.
Amoxicillin ; Ampicillin ; Animals ; beta-Lactamases* ; Cattle* ; Cefotaxime ; Cefoxitin ; Ceftazidime ; Cephalothin ; Diffusion ; Escherichia coli* ; Escherichia* ; Humans ; Korea ; Livestock ; Plasmids* ; Replicon* ; Sequence Analysis, DNA ; Tissue Donors

PURPOSE: Foot-and-mouth disease (FMD) is an economically important global animal disease. To control FMD virus (FMDV) outbreaks, a lot of different novel approaches have been attempted. In this study, we proposed a novel porcine reproductive and respiratory syndrome virus (PRRSV) as a replicon vector to express FMDV structural protein. MATERIALS AND METHODS: PRRSV infectious clone (PRRSVK418DM) was used to develop an expression vector through the reverse genetic manipulation of PRRSV; FMDVP12A3C gene of serotype O was synthesized and used for an antigen. MARC-145 cells (African green monkey kidney epithelial cell line) were used for electroporation mediated transfection. The transfection or the expression of P12A3C and N protein of PRRSV was analyzed by either replicon containing PRRSV alone or by co-infection of helper PRRSV. RESULTS: We constructed PRRSVK418DM replicon vector containing FMDVP12A3C, and genome sequences were confirmed by subsequent sequence analysis. In vitro expression of P12A3C and PRRSV N protein was confirmed by immunofluorescence antibody assay using antibodies specific for PRRSV N protein (anti-PRRSV N MAb), FMDV-VP1 (anti-VP1 MAb). CONCLUSION: The results indicate that PRRSV replicon vector can be a promising novel vector system to control FMDV and useful for vaccine development in the future.
Animals ; Antibodies ; Cercopithecus aethiops ; Clone Cells ; Coinfection ; Disease Outbreaks ; Electroporation ; Epithelial Cells ; Fluorescent Antibody Technique ; Foot-and-Mouth Disease* ; Genetic Vectors ; Genome ; Kidney ; Porcine respiratory and reproductive syndrome virus* ; Replicon* ; Sequence Analysis ; Transfection ; Viruses

OBJECTIVETo construct a full-genome hepatitis C virus (HCV) replicon that will allow for direct initiation of replication and generation of infectious viral particles in an in vitro and in vivo cell system.

METHODSSelf-cleaving ribozyme sequences were added to each side of the HCV cDNA clone JFH1 and the replication-deficient clone JFH1/GND, then inserted into the pcDNA3.1 vector downstream of the CMV promoter. The resultant recombinant plasmids, pcDNA3.1-RZ-JFH1 and pcDNA3.1-RZ-JFH1/GND, were tested for activity in vitro and in vivo by transiently transfecting into Huh7.5 cells (5 mug/100 mm culture dish) and injecting by high-pressure tail vein injection into Kunming mice (10 - 30 mug/mouse). Quantitative reverse transcription-PCR, immunofluorescence, immunohistochemistry, and serological testing were performed to determine the replication ability and assess the properties of the recombinant plasmids in the two systems.

RESULTSHCV RNA (1 - 3 * 10(6) copies/ml) was detected in the supernatant of transfected Huh7.5 cells up to 16 weeks after transfection. In addition, the viral particles from the supernatant were able to infect nave Huh7.5 cells. However, only transient viremia was achieved upon tail vein injection of the plasmid, and no HCV antigen-positive cells were detected by immunohistochemistry nor HCV-specific antibodies by serological testing.

CONCLUSIONThe constructed HCV replicon was capable of stable expression in cultured cells and of efficiently generating infectious viral particles in the in vitro system over a long period. However, the HCV replicon did not show infective characteristics in an in vivo mouse system. The full-length HCV replicon may represent a useful tool for in vitro study of HCV pathological mechanisms, possibly including anti-HCV drug screening.

Animals ; Base Sequence ; Cell Line ; Genetic Vectors ; Genome, Viral ; Hepacivirus ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred Strains ; RNA, Catalytic ; genetics ; Recombination, Genetic ; Replicon ; Virus Replication ; genetics

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

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