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Construction of mammalian cell lines continuously expressing JEV structural proteins.

Shan LIU ; Ying HUANG ; Peng YANG ; Zhi-Wei SUN ; Wei-Yuan YU

Chinese Journal of Virology.2009;25(4):279-285.

Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vector. The recombinant plasmid pCJE-ME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJE-CME, the cell lines continuously expressing structural proteins could be selected with G418. And the expression products of pCJE-CME vector could be detected by ELISA, Western Blot and IFA assay. It showed that the JEV capsid protein could enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.
Animals ; Apoptosis ; CHO Cells ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; cytology ; virology ; Cricetinae ; Cricetulus ; Encephalitis Virus, Japanese ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Viral Structural Proteins ; genetics ; metabolism

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The cloning, expression and structural analysis of putative unknown protein Orf 9b in SARS-CoV.

Wen LI ; Yang LIU ; Lei LAN ; Ming-Nan LIU

Chinese Journal of Virology.2009;25(4):274-278.

Orf 9b was amplified by PCR from SARS-CoV genome and cloned into the Nco I and Bam HI sites of the pET32c expression vector, and then recombinant plasmid pET32c-Orf 9b was constructed. The recombinant fusion protein Orf 9b was expressed by IPTG induction and purifed. After being cleaved by rEK, Orf 9b protein with MW 11 kD was separated and collected. It was demonstrated by ELISA that the purified Orf 9b protein could react with sera of SARS rehabilitaion patients but not with sera from healthy donors. CD and Infrared spectroscopy were used to predict the secondary structure of the purified Orf 9b protein. The distribution percentages for the the secondary structures of alpha-helix, beta-sheet, and random coil in the Orf 9b protein estimated by CD were 12.5%, 40%, and 47.5%, respectively, while the same parameters estimated by Infrared spectroscopy were 13.7%, 47.5%, and 37.9%, respectively. The results obtained by the two methods were substantially identical and showed that the structure of the Orf 9b protein consisted mostly of beta-sheet, and comprised only few alpha-helix. The acquisition of purified protein and the structural information presented herein may provide foundation for further functional study.
Antibodies, Viral ; immunology ; Cloning, Molecular ; Gene Expression ; Humans ; Open Reading Frames ; Protein Structure, Secondary ; SARS Virus ; chemistry ; genetics ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; virology ; Viral Proteins ; chemistry ; genetics ; immunology

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The preparation of monoclonal antibodies against AAV2 and study on their characterizations.

Shu-Ping TAN ; Zhen-Hua YUAN ; Wen-Hong TIAN ; Xiao-Yan DONG ; Xiao-Bing WU

Chinese Journal of Virology.2009;25(4):267-273.

7 strains of stable cell lines secreting monoclonal antibodies against AAV2 capsids were obtained by immunizing BALB/C mice with highly purified recombinant adeno-associated virus. Among them, the monoclonal antibodies B10 and G4 had neutralizing activity, and their subtypes were IgG1 and IgG2a, respectively. The binding characterizations of the two neutralizing antibodies were studied. Both B10 and G4 showed serotype specific binding activities to rAAV2 virus particles other than AAV1, AAV5, and AAV8, and the binding could not be blocked by heparin. After incubating with the two antibodies separately, rAAV2 viruses could still bind to sensitive cell line BHK-21, suggesting that the binding sites of the two antibodies to rAAV2 located at different positions on viral particle surface from the primary receptor binding sites of AAV2. Western blotting assay showed that B10 could bind to VP1, VP2 and VP3 of rAAV2. However, G4 bound none of them. The results suggested that B10 recognized a linear epitope of AAV2 capsid, whereas G4 probably recognized a conformational epitope on the surface of AAV2 virus particle. The two antibodies with different characteristics provided valuable tools for AAV2 virus particles detection and infection processes.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Capsid ; immunology ; Cell Line ; Dependovirus ; genetics ; immunology ; Epitopes ; immunology ; Female ; Humans ; Immunoglobulin G ; immunology ; Mice ; Mice, Inbred BALB C

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Two HCV vaccines based on adeno-associated virus vectors elicited durable and effective immune responses in mice.

Yao DENG ; Ke ZHANG ; Hong CHEN ; Hong-Mei LIU ; Xiao-Bing WU ; Li RUAN ; Wen-Jie TAN

Chinese Journal of Virology.2009;25(4):261-266.

Recombinant adeno-associated viruses (rAAV) vectors have been shown to mediate long-term transgene expression in mice and nonhuman primates. We have adapted viral vector system based on two rAAV vectors, namely rAAV1 and rAAV2. We have generated rAAV vectors expressing the envelope glycoprotein (E1 and E2) derived from Chinese HCV patient (genotype 1b) and used these to immunize BALB/c mice. We detected the total antibody titer by IFA and neutralizing antibody (nAb) using in vitro HCV neutralizing assays based on HCV pseudotyped particles. Furthermore, IFN-gamma ELISpot assay was used to assess the T cellular response against HCV at 12 weeks after rAAV1-E1E2 immunization. We also analyzed HCV envelope glycoprotein expression in muscle of rAAV1-E1E2 immunized mice. Our data showed: (i) rAAV1 directed long-term expression of HCV genes in mice; (ii) immunized intramuscularly with a single dose of rAAV elicited durable and effective immune responses in mice; and (iii) Moreover, rAAV1-E1E2 induced higher total antibody and nAb titers than rAAV2-E1E2 did. These data suggest that rAAV1 vectors could stimulate robust, durable, and effective immune responses against HCV.
Animals ; Antibodies, Viral ; blood ; Dependovirus ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; metabolism ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; virology ; Humans ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Envelope Proteins ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

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The impact of the HIV-1 envelope (Env) mutation on its assembly of functional pseudovirus.

Su-Ting WANG ; Jian-Hui NIE ; Hui-Hui CHONG ; Chun-Tao ZHANG ; Xue-Ling WU ; You-Chun WANG

Chinese Journal of Virology.2009;25(4):257-260.

To find out whether the mutations of HIV-1 Env have influence on the assembly of pseudovirus and their abilities to infect cells, site-directed mutation (A457D)was performed using cycling mutagenesis and selection of mutants with DpnI. Transformation and plasmid purification technologies were used to obtain mutated env clone. Then both the prototype and the mutant were co-transfected with pSG3(delta(env)) to 293FT cells, respectively. Single-cycle infection assay was employed to analyze the effect of the prototype and the mutant on the ability of functional pseudovirus assembly. The transient expression of both the prototype S12-42-1 and mutant S12-42M were confirmed by Western blot essay. The S/CO value was less than 1 for S12-42-1 and 6.65 for S12-42M, demonstrating the functional pseudovirus was generated only for S12-42M. So mutation on HIV-1 Env has influence on the assembly of pseudovirus and their abilities to infect cells.
Amino Acid Sequence ; Base Sequence ; Cell Line ; HIV Infections ; virology ; HIV-1 ; chemistry ; genetics ; physiology ; Humans ; Molecular Sequence Data ; Mutation ; Sequence Alignment ; Virion ; genetics ; physiology ; Virus Assembly ; env Gene Products, Human Immunodeficiency Virus ; chemistry ; genetics ; metabolism

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Outbreak of acute hemorrhagic conjunctivitis in Beijing City in 2007 caused by coxsackievirus A24 variant: molecular identification and phylogenetic analysis.

Li-Xin DING ; Yong ZHANG ; Jie LI ; Xiang-Feng DOU ; Dong-Mei YAN ; Shuang-Li ZHU ; Hong-Qiu AN ; Wen-Bo XU

Chinese Journal of Virology.2009;25(4):251-256.

In 2007, an outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Beijing. In order to identify the etiology of this outbreak, 57 eye conjunctival swabs were collected from 57 outpatient patients, and detected for adenovirus, human enterovirus 70 (HEV70) and Coxsackievirus A24 variant (CVA24v) genes by using RT-PCR or PCR methods. The results showed that 38 were positive for CVA24v, the positive rate was 66.7%, but none was positive for HEV70 and adenovirus, showing that this outbreak was caused by CVA24v. 9 viral isolates were obtained from 57 clinical specimens by using viral isolation method, and all were identified as CVA24v by molecular typing method. All 9 CVA24v isolates were performed by VP1 sequencing, the results showed that except for strain 0744/BJ/CHN/2007, the variability at nucleotide acid level and amino acid level among other 8 CVA24v were relatively low, and the homologies were more than 99.6% and 100.0%, respectively; the homologies of nucleotide acid and amino acid between strain 0744/BJ/CHN/2007 and other 8 CVA24v were 96.8%-97.2% and 99.7%, respectively. Phylogenetic analysis of 9 CVA24v revealed that they represented the Clade 4 and Clade 5 in Group I, showed that this outbreak was caused by at least 2 viral transmission chains. Comparing to 3C region of CVA24v frequently used before, VP1 region was considered as the most rigorous target for molecular epidemiology study of CVA24v. To enhance the research of sero-epidemiology and molecular epidemiology of CVA24v and to know the genetic characterizations and molecular evolution of CVA24v are most important to prevent and control the outbreaks of AHC in China.
China ; epidemiology ; Conjunctivitis, Acute Hemorrhagic ; epidemiology ; virology ; Disease Outbreaks ; Enterovirus C, Human ; classification ; genetics ; isolation & purification ; Humans ; Molecular Epidemiology ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

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Production of human papillomavirus type 16 virus-like particles and its immunogenicity.

Min-Xi WEI ; Shao-Wei LI ; Bo HUANG ; Wen-Tong SHEN ; Yong-Zai SU ; Chun-Hua ZHANG ; Ying GU ; Hai-Lian DU ; Jun ZHANG ; Ning-Shao XIA

Chinese Journal of Virology.2009;25(4):245-250.

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.
Animals ; Antibodies, Viral ; immunology ; ultrastructure ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; Goats ; Human papillomavirus 16 ; genetics ; immunology ; ultrastructure ; Humans ; Male ; Oncogene Proteins, Viral ; genetics ; immunology ; isolation & purification ; Papillomavirus Infections ; immunology ; virology ; Rabbits ; Recombinant Proteins ; genetics ; immunology ; Vaccination ; Virion ; genetics ; immunology

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Marek's disease virus encoded miRNAs--an update review.

Bo-Xue NIU ; Li-Qiong TENG ; Ping WEI

Chinese Journal of Virology.2009;25(2):154-158.

Animals ; Forecasting ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; genetics ; metabolism ; virology ; MicroRNAs ; metabolism

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Research advance of TRIM5alpha on structure and restriction mechanism of HIV-1 replication.

Xia TANG ; Yi-Qun KUANG ; Yong-Tang ZHENG

Chinese Journal of Virology.2009;25(2):148-153.

HIV Infections ; virology ; HIV-1 ; physiology ; Virus Replication ; physiology

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Construction and identification of replicon vector derived from an infectious full-length cDNA clone of a Sindbis virus.

Wu-Yang ZHU ; Shi-Hong FU ; Li-Hua WANG ; Ying HE ; Qing TANG ; Guo-Dong LIANG

Chinese Journal of Virology.2009;25(2):143-147.

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

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