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*

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

OBJECTIVETo construct the recombinant HCV-1b replicon by replacing NS5A region using serum samples from patients with chronic hepatitis C (CHC) in South China and explore the biological characteristics of NS5A protein in response to antiviral therapy.

METHODSThe on-off plasmid containing the cutting sites of the restriction endonucleases MIu I and Bcl I was designed based on the backbone of robust HCV 1b replicon. The full-length fragments of HCV NS5A were amplified from different CHC patients by RT-PCR and cloned into pMD-18 vector, followed by sequence analysis of amino acid mutation of ISDR, PKRBD, V3 and IRRDR within the NS5A region. If the amplicon obtained contained no MIu I or Bcl I cutting sites, the NS5A fragment was re-amplified using primers containing the cutting sites and inserted into the replicon for replacement.

RESULTSThe full-length fragments of NS5A were obtained successfully from CHC patients. The core region of ISDR-V3 of NS5A was replaced in the HCV replicon plasmid and showed correct sequences. The amino acid mutations of ISDR and PKRBD within NS5A were more frequent in patients with sustained viral response (SVR) than those without SVR. A high variability in the amino acid sequence was observed in both IRRDR and V3 regions.

CONCLUSIONThe plug-in type recombinant HCV replicon for replacement of NS5A region in the virus from CHC patients has been successfully constructed, which provides a basis for further investigation of the biological characteristics of NS5A protein, the mechanisms of interferon-resistance, and antiviral therapy of difficult-to-treat CHC.

Amino Acid Sequence ; Antiviral Agents ; administration & dosage ; pharmacology ; Drug Resistance ; genetics ; Genetic Variation ; Genotype ; Hepacivirus ; genetics ; Hepatitis C, Chronic ; drug therapy ; virology ; Humans ; Interferons ; administration & dosage ; pharmacology ; Molecular Sequence Data ; Recombination, Genetic ; Replicon ; genetics ; Sequence Analysis ; Viral Nonstructural Proteins ; 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

To optimize a self-replicate Japanese enciphalitis virus (JEV) replicon, and to make it as an efficient vector to express the heterologous protein, we constructed three JEV replicons by PCR-based shortening the length of capsid genes. The vectors remained full or part of C gene, based on the JEV replicon pCTCJEV. Lac Z was selected as the reporter gene to verify the self-replicate ability of these DNA-based replicons. While transfected into the cell lines CME-4, which continuously expressing the JEV structure proteins C-prM-E, the JEV replicons pCMW-2M-1LACZ, pCMW-2M-3LACZ, which remained the first 23aa and 68aa of C protein, can express the reporter protein as the same level as pCMW-2M-LACZ with the full-length C protein. These results illustrated that the JEV replicon vector with 69-nt of the C gene can retain the self-replicate ability, and provide valuable tools to construct a possible vector for a long-lasting JEV RNA virus expression system.
Capsid Proteins ; genetics ; physiology ; Cell Line ; Encephalitis Virus, Japanese ; genetics ; physiology ; Genetic Vectors ; genetics ; Humans ; Replicon ; genetics ; physiology ; Transfection ; Virus Replication ; genetics ; physiology

To investigate the effects of site-directed mutagenesis at nsP2-726Pro on the characteristics of replicon vector derived from XJ-160 virus, a Sindbis virus (SINV) isolated in China. The mutant vector pBRep-726L, pBRep-726S, pBRep-726V or pBRep-726A was constructed by introducing nsP2-726Pro --> Leu, nsP2-726Pro --> Ser, nsP2-726Pro --> Val or nsP2-726Pro --> Ala into XJ-160 viral replicon vector pBRepXJ respectively. To quantitatively and qualitatively determine the site-directed mutagenesis on the replicon, the recombinant plasmids expressing Neomycinr (Neo(r)), enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) were constructed by cloning report genes into pBRepXJ or mutant XJ-160 vector respectively. And in vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. Compared with the wild-type replicon, the mutation nsP2-726Pro --> Val or nsP2-726Pro --> Ala accelerated the processing of CPE on BHK-21 cells and simultaneously enhanced its self-replicating capacity. The mutant vector pBRep-726L with Leu substitution exhibited similar packaging capacity to that of pBRepXJ. In contrast, pBRep-726S exhibited a medium phenotype, including the process of CPE and the activity of R. luc expression in BHK-21 cells. The site-directed mutagenesis at nsP2-726Pro not only regulates directly XJ-160 virus vector-host cell interactions, but also plays an important role in its packaging capacity. All of these results lay a basis for researching the relation between the structure and function of alphavirus genome and developing alphavirus vector system with Chinese intellectual property.
Amino Acid Substitution ; Animals ; Cell Line ; China ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Microscopy, Fluorescence ; Mutagenesis, Site-Directed ; methods ; Mutation ; Plasmids ; genetics ; Proline ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Replicon ; genetics ; Sindbis Virus ; genetics ; isolation & purification ; Transfection

To construct vector system of XJ-160 virus, a Sindbis virus isolated in China, recombinant vector pBRepXJ together with its helper plasmid pBR-H were derived from XJ-160 viral infectious clone pBR-XJ160 by overlap-PCR. To quantitatively and qualitatively verify the function of the replicon system, recombinant plasmids pSinRep-EGFP, pBRepXJ-EGFP, pSinRep-R and pBRepXJ-R were constructed by cloning report genes of enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) into pBRepXJ or pSinRep5, a commercial Sindbis vector. And in Vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. The results indicated that the replicon vector system was capable of self-replicating in host cell, and the expression efficiency of heterologous genes corresponded with that of the commercial Sindbis vector (pSinRep5). Our study laid the basis for developing alphavirus vector system with Chinese intellectual property.
Alphavirus Infections ; genetics ; Cloning, Molecular ; DNA, Complementary ; analysis ; genetics ; Genetic Vectors ; Genome, Viral ; Replicon ; genetics ; Sindbis Virus ; genetics ; Virus Replication ; physiology

Animals ; Antiviral Agents ; pharmacology ; Disease Models, Animal ; Hepacivirus ; drug effects ; genetics ; metabolism ; Hepatitis C ; virology ; Interferons ; pharmacology ; Mice ; MicroRNAs ; Replicon ; Signal Transduction ; Viral Proteins ; genetics ; Virus Replication