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2000 Vol.15(4)

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Review

Expression of Hepatitis C Virus RNA Polym erase in Insect Cells

LAN Shui-Yun, ZHENG Ling-Ji, HU Yun-Wen, CHEN Chong, QIU Hai-Jun, YUAN Zheng-Hong

2000, 15(4): 313

The genes of C terminal of HCV NS3 region of two chronic patients in two different time were sequenced to analyse the conservation of a helper T cell epitope in HCV NS3 protein (amino acids 1248 to 1261). The epitope was synthesized and the immune response to it in a self limited patient and two chronic patients was detected using T lymphocyte proliferation assay and inhibition experiment. The epitope specific T cell line was established by several cycles of stimulation relax stimulation and analysed with a FACScan. The results indicated that the epitope didn't change in two chronic patients, all three patients had strong CD4 + T cell reponse to the epitope, and epitope specific CD4 + T cell line was established. These data suggest that the eptiope is a conserved strong helper T cell epitope and could become a promising candidate for designing a CD4 + T cell vaccine.

The Study of a Helper T-cell Epitope in Hepatitis C Virus Non-structural 3 Protein

XU Dun-Jie, HONG Chao, JI Lian-Quan, WANG Hai-Chao

2000, 15(4): 318

The genes of C terminal of HCV NS3 region of two chronic patients in two different time were sequenced to analyse the conservation of a helper T cell epitope in HCV NS3 protein (amino acids 1248 to 1261). The epitope was synthesized and the immune response to it in a self limited patient and two chronic patients was detected using T lymphocyte proliferation assay and inhibition experiment. The epitope specific T cell line was established by several cycles of stimulation relax stimulation and analysed with a FACScan. The results indicated that the epitope didn't change in two chronic patients, all three patients had strong CD4 + T cell reponse to the epitope, and epitope specific CD4 + T cell line was established. These data suggest that the eptiope is a conserved strong helper T cell epitope and could become a promising candidate for designing a CD4 + T cell vaccine.

Studies on Growth Characteristics of Human Herpesvirus 7 in vitro

BANG Guang-Yong, TAO Kun, LIN Jiang, JI Xiao-Hui, LE Mei-Zhao

2000, 15(4): 323

The growth characteristics of Human herpesvirus 7 local strain YY5 were investigated in cord blood mononuclear cells (CBMCs) and CD4 + lymphoblastic cell line SUPT1. Virus growth was demonstrated by cytopathic effect (CPE), percentage of dead cells count, indirect immunofluorescence test with viral antigen expression at different days post infection. At the same time, ultrastructural appearance of infected cells and of HHV 7 in various period were investigated by transmission electron microscopy. The results are as follows: (1) The CPE peak time of HHV 7 was latter than that of HHV 6, and CPE of HHV 7 was less pronounced. (2) Although the CPE was obvious and the viral antigen was found in SUPT1 cells, mature virus particles were hardly observed intra and extra these cells. (3) The virions mainly laid in the giant syncytia. HHV 7 infected cells exhibited heterochromatin clumping and margination or pyknosis, vacuolized cytoplasm, discontinuation of the plasma membrane and necrosis. (4) Mature virus parti

Amplification and Cloning of Full-length JEV cDNA and Preparation of Infectious RNA by in vitro Transcription

HUANG Qiang-Sheng, MA Wen-Yu, JIANG Chao-Zhun, FAN Yang-Ru, ZHANG Fu-Quan, XIAO Yi, DING Tian-Bing

2000, 15(4): 330

Preparation of infectious RNA by in vitro transcription from full length cDNA has led to advances in the study of many positive strand RNA viruses. There are two strategies of preparing full length cDNA of RNA viruses: (1)construction of infectious clone. (2)amplification of full length cDNA by long template RT PCR with the RNA promoter incorporated to 5 primer. In this study both ways were established to generate Japanese Encephalitis Virus (JEV) infectious RNA. Firstly JEV (SA14 strain) full length cDNA was amplified by long template RT PCR and then cloned into the cosmid vector. One clone in which JEV cDNA was downstream of T7 promoter was selected and the RNA derived from this clone showed infectious after transfection of BHK cell. In another way of preparing JEV full length cDNA by long template RT PCR, T7 promoter was introduced to 5 primer. RNA derived from this cDNA also showed infectious after transfection of BHK cell. The recovered viruses were further confirmed by intracerebral inocu

The Long-term Culture of hum an bone marrow stromai cells in vitro and their susceptibiiltty to viruses

YANG Ji-Cheng, CHENG Wei-Hua, LI Li-E, DONG Ning-Zheng, YANG Zong-Hua, WANG Xiao-Dong

2000, 15(4): 336

To culture human bone marrow stromal cells long term in vitro , and investigate their susceptibility to virus, the fetus, child and adult bone marrow stromal cell were cultured for long time in vitro by the adherent assay. The myoid bone marrow stromal cells were obtained after being generated more than five times, then their susceptibility to five kinds of virus was assayed by microdose CPE. All myoid stromal cells were susceptible to VSV, PolioV, HSV I and Ⅱ. Except CoxB 3, producing obvious CPE with TCID 50 up to 10 -3 10 -4 . Fetal myoid stromal cells were two times higher than child, and four times than adult in terms of virus susceptibility. Our research supplies not only a simple. practicable assay for ex vivo long term culture of human bone marrow stromal cell, but also experimental reference for estimating the virus susceptibility of these cells, at the same time, contributes to a more extensive prospect for applying these cells.

A Study on the Relation between Changes of lnterleukin and Receptor in Serum in Patients with Chronic Hepatitis B and Action of Treatment with IFN.a

HE Sheng-Song, LI Chu-Chi, XIONG Chi-Juan, LIU Wei, CENG Lian-Lan

2000, 15(4): 342

Objective\ To study the relationship between the changes of Interleukin and Receptor in serum of patients with chronic hepatitis B (CHB) before and after Interferon (IFN ) treatment and expression of HBV marker. Methods: Enzyme liked immunosorbent assay (ELISA) were performed in 25 patients with CHB to determine levels of IL 6\, IL 2\,TNF \, IL 8\, sIL 2R in serum. Results: Our date showed that the IL 6 level in response group after IFN treatment was significantly lower than that befor (P0.05). The level of IL 2 in unresponse group after IFN treatment was significantly decreased, but the level in response group was significantly increased (P0.01), but the serum sIL 2R level in patients with response was significantly decreased (P0.01). The TNF level in both response and unresponse group after IFN treatment was significantly decreased (P0.01), but the serum IL 8 level was increased. Conclusion\ The results suggest that HBV replication in patients with CHB could be also affected

Construction of Helicoverpa armigera Library During Latent Periods by Nuclear Polyhedrovirus cDNA Polym erase Chain Reaction

BAI Zhi-Jiang, ZHANG You-Qing

2000, 15(4): 346

A general cDNA library which was from infected larvae dueing the latent periods has been constructed. The first cDNA strand was synthesized from total RNA by M MLV transcriptase. After oligo(dG) tailing the cDNA was amplified by polymerase chain reaction. The double strands cDNA were ligated to pGEM T easy vectors. Screeing of cDNA library showed that the length of inserts was from 0.3 to 1.1 kilobases. The capacity of cDNA library was 1.6610 5 clones. Moreover, the number of cDNA clones of HaNPV was more than 50%.

Studieson Association of the Replication of AcMNPV with the Nuclear Matrix

LIU Xiao-Qiong, TU Ze-Hua, WANG Jia-Kun, TAO Han-Chao, GUO Hua-Shan, CHEN Qu-Hou-

2000, 15(4): 350

The structure of nuclear matrix in Tn5B1 cell infected with AcMNPV was observed by means of sequential cell fractionation and whole mount technique of electron microscopy. Whereas early infection of AcMNPV resulted in no dramatic change in the nuclear matrix morphologically, polyhedra formed in the network of the nuclear matrix in the late stage of infection. Results of Dot boltting showed that both ie 1 gene and polyhedrin gene tightly binded on the nuclear matrix in early stage of infection.

Cloning of Grapevine Leafroll Associated Virus 3 Coat Protein Gene

DIAO Dong-Lan, SHU Shui-Fang, HUANG Wen-Qing, ZHANG Man-Liang

2000, 15(4): 357

A pair of primers were designed and synthesized based on the nucleotide sequence of coat protein gene of GLRaV 3 from America. The expected size of CP gene of GLRaV 3 was amplified by IC (Immuno capture) RT PCR method. The CP gene of GLRaV 3 was cloned into pGEM 3zf(+) vector and sequenced. The nucleotide sequence was compared with American isolate. Result showed that their homology was 94.1%.

Com parative Analysis of Serological and M olecular M ethods for the detection of Rice Grassy Stunt Virus

ZHANG Chun-Mei, LIN Ai-Yang, XIE Lian-Hui

2000, 15(4): 361

Methods of ELISA, nonradioactive molecular hybridization and RT PCR were applied in the detection of rice grassy stunt virus (RGSV). The detection sensitivity of indirect ELISA using antiserum against fusion protein GST NC was 1 mg of infected leaves or 84 ng of purified virus. The method of dot hybridization using NC, a DIG labelled DNA probe was 50 g diseased leaves, or 6 ng purified preparations. The detection endpoint of RT PCR was 10 g diseased leaves, or 2 ng purified virus preparation. Comparisons of sensitivity and maneuverability were made among these methods.

Detection of Papaya Ringspot Virus by Imm uno-capture PCR,Nested-PCR and ELISA-PCR Assays

XIAO Huo-Gen, HU Jin-Sheng, FAN Fu-Zhong

2000, 15(4): 367

Sensitive, rapid, and reliable immuno capture PCR (IC PCR), ELISA PCR and Nested PCR assays were developed for the first time to detect PRV. It was detected from dilutions of 10 -4 , 10 -7 and 10 -14 (equivalent to 0.5 ug, 0.5 ng and 510 -8 ng of papaya leaf tissues) by IC PCR, ELISA PCR and Nested PCR, respectively.

Study on the Feline Caticivirus Infected Tiger

FAN Quan-Shui, JIA Xian-Zhu, QIU Wei, HUANG Geng, HE Hong-Ban, ZHANG Jian-Bin, JIAO Jun, TU Chun, JU Chi-Huan, LI Jin-Zhong, WU Yin-Lian, YAN Shen-

2000, 15(4): 373

A virus strain like calicivirus was isolated from organs of tiger with feline kidney (FK) cell. The viruses were nonenveloped, 35~39 nm in diameter, multiplied in cytoplasm and put in crystal order. The viruses multipied easily in feline kidney cell cultures and produced cytopathic effects (CPE). Virus cross neutralization (VN) showed that the virus is closely relation with standard vaccine virus strain. Felines infected with FK cell cultures viruses clinically appeared FCV signs. The RT PCR amplified product around 540 bp which was the same as result of Designed. The PCR products were directly sequenced. The gene sequences were compared with FCV strains extracted from the GenBank database and showed 87% identity. Systematical identification has proved that the viruse isolated is feline calicivirus of tiger. This is the first report of feline calicivirus infected tiger in China.

Molecular Cloning and Sequencing of VP2 Gene of Canine Parvovirus Inner Mongolia Isolate(CPV-IM)

DONG Jiang-Li, LI Chu-Fen, ZHANG He-Ling

2000, 15(4): 379

The DNA of Canine Parvovirus Inner Mongolia isolate (CPV IM) was isolated from the enteric lysate of virus infected dog showing enteritis symptoms. The VP2 gene of CPV IM was amplified by PCR using designed and synthesized two primers and cloned into plasmid pUC19 at Bam H I/ Sac I sites. Recombinant plasmid pUCVP2 was identified by PCR and restriction enzymes analysis and nucleotide sequence analysis. Results show that the full length VP2 gene clone of CPV IM was obtained. VP2 gene of CPV IM isolate consists of 1 755 nt, and there is a high homology in nucleotide sequence and amino acid sequence in comparison with CPV N, CPV B and Feline Panleukopenia Virus (FPLV). The rate of nucleotide sequence homology is 99.32%, 98.29% , 98.52%, and the rate of amino acid homology is 98.97%, 97.09%, 97.77%, respectively.

Construction of Physical M apping of Goose-derived Adenovirus Genomic DNA

XU Fu-Zhou, CUI Chi-Zhong, LIU Yue-Long, DUAN Yu-You, SUN Fu-Chang

2000, 15(4): 388

The genomic DNA library of goose derived adenovirus (strain Y 81 G 4) was constructed after ten fragments of viral genomic DNA digested with Hin dⅢ were cloned into plasmid pUC18 respectively. The inserted fragments of the ten recombinant plasmids were labelled with digoxigenin. Viral genomic DNA digested with Bam HI\, Eco RI\, Pst I or Eco RV were transferred to NC membranes after electropheresis and probed with DIG labelled cloned Hin dⅢ fragments respectively. The physical mapping of adenovirus Y 81 G 4 genomic DNA was constructed successfully by Southern blot. To compare strain Y 81 G 4 genomic DNA mapping orientation with EDSV strain AA 2, the cloned right terminal fragment of EDSV strain AA 2 was labelled with digoxigenin and probed with the two cloned terminal fragments of Y 81 G 4 genomic DNA by Southern blot. By comparison of homology the orientation of Y 81 G 4 genomic DNA were determined. These results will be helpful for construction of avian adenovir .

Study on Reovirus-like Virus of Eriocheir sinensis

GONG Cheng-Liang, XUE Ren-Yu, CAO An-Li, WEI Yo-Gong, SHU Huo-Xiong, CHEN Hui, TUN Xiang-Fu-

2000, 15(4): 395

In order to find out the reason for causing shiver disease of Chinese mitten crab ( Eriocheir sinensis ) a virus was isolated from diseased Chinese mitten crab. Symptom of normal crab was positive after it had been infected artificially with preliminary pure virion from the shiver crab. It was decided that this virus could be detected in the blood, intestinal canal and germinative gland. The virus was also infective to Sesarna dehanni . The virion was globose, no envelope and about 55 nm in size by electron microscope observation. The electrophoretype of the viral nucleic acid in 1% agarose was 3/3/4/2 in four different size classes, and total molecular weight was about 20 kb. The nucleic acid should be a dsRNA by the eletrophoretype and the character of sensitivity to RNase and not sensitivity to DNase I. The classification of the virus was proposed to reoviridae family by genome and morphology. The research is significant in the discussing the cause of shiver disease and preventing and tr

Cloning,Sequence Analysis and Expression of Partial HCV Nonstructural Protein 2,3 Gene

FAN Chao, GAO Jian-En, DAO Ji-Min

2000, 15(4): 400

Complementary DNA of partial HCV NS2 NS3 protein gene which encodes viral Zn 2+ dependent metalloprotease and serine protease was amplified by RT nested PCR from serum of a Chinese patient with chronic hepatitis C.The product was digested with Eco R Ⅰ/ Xba Ⅰ and cloned into pcDNA3.The nucleotide sequence was determined.Comparison of NS2 NS3 with the reported typical isolates HCV 1,HCV J,HC C2,HCV J6,HCV J8 showed as 73.72%,90.20%,91.02%,64.56%,63.37% identity in nucleotide sequence and 83.24%,92.09%,93.13%, 70.88% ,67.86% identity in amino acid sequence respectively, so the isolate could be classified as HCV genotype Ⅱ. This gene fragment was cloned into pBV220 to construct recombinant expression vector pBV220 NS2 3.It was expressed efficiently in E.coli . The cloning and expression of HCV NS2 NS3 may contribute to the investigation of the relation between structure and function of viral encoded protease.

Preliminary Study on Chinese Tobacco Leaf Curl Gem nivirus-Guangxi Specie

ZHANG Ying, YAN Qi-Yan, LIU Yu-Le, CHEN Gang, CA Jian-He, TIAN Bei

2000, 15(4): 405

Using DNA of Chinese Tobacco Leaf Curl Virus Guangxi(TbLCV Guangxi)as the template,a 0.4 kb virus specific fragment induding the total common region of the virus and DNA sequence of 52 aa of N terminal of AV1 was obtained by PCR with primers specific to whitefly transmittal gemniviruses,cloned and sequenced.This fragment can basically represent the characteristics of the virus full length genome.The sequence of this fragment is the same as that of chinese tomato yellow leaf curl virus (TYLCV Chi), which indicates that TbLCV Guangxi and TYLCV Chi are the same gemniviruses. According to the sequence of the common region of the 0.4 kb fragment, the virus full length genome DNA was amplified from the infected tobacco plant by PCR with primers back to back.

Study on the Phage Display Expressing Anti-H antavirus Capsid Protein scFv

LI Guang-Fu, TANG Jia-Qi, CHEN Yu-Ping, WANG Yan, CAO Min, LI Xian-Fu, BO Xiu-Zhen

2000, 15(4): 409

The expression vector of anti Hantavirus(HV) capsid protein scFv was constructed by DNA recombination and PCR technique,and expressed in E.coli . The expressed scFv was fused with the phage GeneⅢ protein, and located on the surface of phage.The sequencing of scFv demonstrated that the sequence of scFv is consistent with that of cloned antibody variable region from F3 strain hybridom against HV capsid protein.The expressed scFv was proved to be able to bind HV antigen by ELISA.