OBJECTIVETo construct and characterize EGFP reporter gene labeled Sindbis virus (SINV).

METHODSThe reporter gene EGFP was inserted into the genome of infectious clone pBR-XJ160 by using multi-fusion long fragment PCR method. Then apply reverse genetic manipulation technique to rescue and obtain EGFP labeled SINV.

RESULTSWe successively obtained labeled SINV, which has good fluorescent expression characteristics and genetic stability.

CONCLUSIONThe labeled virus can be seen in living cells and living body, and this serves as a good tool for cell and tissue tropism and biological function study of viruses. This study laid a foundation for further studying the cell tropism, biological functions and infection mechanism of SINV.

Base Sequence ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; Molecular Sequence Data ; Sindbis Virus ; genetics

OBJECTIVETo construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector.

METHODSThe gene encoding HCV envelope glycoprotein E1E2 was cloned into sindbis virus vector to construct recombinant plasmids pBR-XJE1E2 and pVA-XJE1E2, and transfect them into BHK-21 cells to obtain recombinant virus-like particles. The expression of E1 and E2 protein were verified by Western Blot and indirect immunofluorescent assay (IFA).

RESULTSThe results of restriction enzyme digestion, PCR and sequencing analysis showed that the recombinant plasmids were constructed successfully. And the results of RT-PCR, Western blotting and IFA detection showed that the transfect cells could package HCV-like particles of expressing structural proteins E1E2.

CONCLUSIONThe recombinant expression plasmids pBR-XJE1E2 and pVA-XJE1E2 based on sindbis virus vector could package HCV-like particles in eukaryotic cell, which provides a foundation for further study of its in vivo animal immune response.

Animals ; Cells, Cultured ; Cricetinae ; Genetic Vectors ; Hepacivirus ; genetics ; Plasmids ; Recombination, Genetic ; Sindbis Virus ; genetics ; Viral Envelope Proteins ; genetics

To investigate the feasibility of using Real-Time PCR to evaluate the effectiveness of Sindbis virus inactivation by Methylene Blue with visible light. Sindbis virus was treated by Methylene Blue with different intensity of visible light and the transcribed cDNA was quantified by Real-Time PCR. Residual infectivity of treated virus was tested by cell infection method as parallel control at the same time. The residual infectivity of virus decreased from 6.50 lgTCID50/mL to under the limit of detection as light intensity increased. Meanwhile, the quantity of virus cDNA decreased significantly (P < 0.05), which correlated to the decline of virus infectivity (R2 > 0.98). Methylene Blue with visible light could cause lesion to nucleic acid of Sindbis virus, the extent of which was light intensity-dependent and correlated to the decrease of virus infectivity. The results demonstrated that Real-Time PCR can be a useful tool for evaluating effect of virus inactivation after Methylene Blue treatment with light.
Light ; Methylene Blue ; pharmacology ; Polymerase Chain Reaction ; methods ; Sindbis Virus ; drug effects ; genetics ; physiology ; radiation effects ; Virus Inactivation ; drug effects ; radiation effects

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

Japanese encephalitis virus (JEV) is one of the most important human pathogens, which causes the permanent neuropsychiatric sequelae and even fatal diseases with high mortality and morbidity, especially among children. In this study, we expressed the structural proteins (C, prM, and E) of JEV using a Sindbis virus-based heterologous gene expression vector, the pSinRep5. We designed two expression vectors (pSinRep5/JEV C-E and pSinRep5/JEV C-NS1), which encode the precise coding sequence of JEV C-E and JEV C-NS1 proteins, respectively. These cloned JEV structural protein genes were designed to express under the Sindbis virus subgenomic promoter. Upon the transfection and expression of the pSinRep5/JEV C-E or pSinRep5/JEV C-NS1 plasmid, the transfected cells expressed approximately 55 kDa JEV E prtiens. As designed, the JEV NS1 proteins were expressed only in the SinRep5/JEV C-NS1 RNAtransfected cells. In addition, we found in the pSinRep5/JEV C-NS1-transfected cells that the viral proteins were predominantly localized around the perinuclear membranes. On the other hand, cytoplasmic staining was mainly observed in the pSinRep5/JEV C-E RNA-transfected cells in the absence of NS1 protein. Thus, our system will provide a useful tool to dissect intracellular membrane localization signals located in the JEV structural proteins without handling the infectious JEV viral particles and to characterize viral morphogenesis of this pathogen.
Asian Continental Ancestry Group* ; Child ; Clinical Coding ; Clone Cells ; Cytoplasm ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Flavivirus ; Gene Expression ; Hand ; Humans ; Intracellular Membranes ; Membranes ; Morphogenesis ; Mortality ; Plasmids ; Sindbis Virus ; Transfection ; Viral Proteins ; Virion

Porcine reproductive and respiratory syndrome virus (PRRSV) is a small enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. It causes the porcine reproductive and respiratory syndrome in swine. The virus has 7 structural proteins: Of the seven, the N protein is the nucleocapsid that comprises a core of the virus particle. We have expressed the N protein of PRRSV PL97-1/LP1 strain using a heterologous gene expression vector derived from Sindbis virus, called pSinrep5. Immunofluorescence analysis showed that the N proteins were mainly found in the cytoplasm as well as in the nucleus of BHK-21 cells transfected with pSinrep5-N-derived RNA. Moreover, expression of the N protein did not change the incompetence of RNA replication of Mutant/nt14900 that lacks a 3' cis-acting replication element and the efficiency of RNA replication of Mutant/nt14800 that has a low level of RNA replication. Overall, our findings are consistent with previous results and help to understand a role of the N protein in PRRSV biology.
Arteriviridae ; Cytoplasm ; Fluorescent Antibody Technique ; Gene Expression ; Humans ; Nucleocapsid ; Porcine Reproductive and Respiratory Syndrome ; Porcine respiratory and reproductive syndrome virus ; Proteins ; RNA ; RNA Viruses ; Sindbis Virus ; Sprains and Strains ; Swine ; Virion ; Viruses

BACKGROUNDTo develop a rapid, specific and sensitive method for detecting Sindbis virus (SINV) with SYBR GREEN I real time PCR.

METHODSTotal RNA of strains of Sindbis virus and a related virus were extracted and reverse transcribed to cDNAs. With the cDNAs as template, the SYBR GREEN I real time PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Sindbis virus, and the sensitivity, specificity and reproducibility were evaluated.

RESULTSFor the PCR, 55 degrees C was chosen as the optimal anneal temperature and 0.5 micromol/L as the optimal primer concentration. Using this method, all the selected SINV were detected as positive, while the results of control arboviruses such as Geta virus, Japanese encephalitis virus (JEV), Batai virus, Seadornavirus Orbiviruses and synthesized WEEV cDNA were negative. With this system, 0.1 PFU/ml SINV cDNA could be detected; The sensitivity of this assay was about 100 times higher than standard RT-PCR. All the results were reproducible within two compatible tests, and the stability of the detection system was very good. The test results of simulated infection human serum samples showed that human serum had no obvious interference with this system. With this system, 6 of 151 clinical samples with unknown fever or encephalitis were determined as positive.

CONCLUSIONThe developed SYBR GREEN I real time PCR assay for detecting Sindbis virus was highly sensitive, specific and showed a good reproducibility and stability. It is our belief that the present method can be further used in clinic sample to verify its stringency.

Alphavirus Infections ; diagnosis ; virology ; DNA, Complementary ; chemistry ; genetics ; Organic Chemicals ; chemistry ; RNA, Viral ; genetics ; isolation & purification ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Sindbis Virus ; genetics

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.
Alphavirus ; genetics ; immunology ; physiology ; Amino Acid Sequence ; Animals ; Cell Line ; DNA, Recombinant ; genetics ; Female ; Genetic Engineering ; Glycoproteins ; chemistry ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neutralization Tests ; Sindbis Virus ; immunology ; Viral Load ; Viral Proteins ; chemistry ; metabolism

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