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2001 Vol.16(2)


Study on Biological Characters of Sindbis Virus Yunnan Strain

NG Wen---, ZHANG Hal, WANG Jing, ZHANG Yu, ZHOU Guo-linz, M I, SHI Hua, ZI Deng, LIAN Guo

2001, 16(2): 97

The biological characters of Sindbis virus strain of Yunnan(YN87448 strain)were studied by the test of the filtration, acid—resistant, ether—resistant, CPE, susceptibility of animal, HA, plague,determination of virus titres,and the cross—HI,cross-lFAT and PRNT as welI The results in— dicated that YN87448 strain belongs to Sindbis virus pha、rirus,Toga~ indae.YN87448 strain virus was plaque purified(PYN87448).The biological character of PYN87448 strain virus was studied too PYN87448 strain virus will be used in the molecule biological test
Research Article

Enhancing Effect of Thl Cytokine on Imm une Responses of M ice to Recom binant Plasmid Encoding Hepatitis C Virus Core Gene

FENG Zhi—hua, ZHOU Yong—xing, JIAO Cheng—song, et al

2001, 16(2): 101

BALB/c mice were immunized with a recombinant plasmid containing HCV—C gene(pcD NAHCV—C)alone or together with a constructs encoding IL一2 or IL12(pIL一2 or pIL一12).Anti— HCV—C specific antib0dy in the zer8 was measured with ELISA Spleen cells from experimental and control mice were extracted The mouse myloma SP2/0 cells expressing HCV C antigen served as tar get cells CTL activity was measured by~andard 4h Cr release ossays The results showed that anti— HCV—C antibody A value increased from 1.21 of pcDNAHCV C alone to 2 O9 and 1 73 of pHCV—C coimmunized with PIL一2 or PIL一12 respectively CTL activity to HCV core prote~n was substantially enhanced after coimmunization with the IL 12 expressing plasmid In contrast.the improvement of HCV core—specific CTL activity with an IL一2一pmdudng construct coimmunization was not obvious. whieh could related to the lOW—leve1 expression of IL 2 vector-pDOR DNA vaccine of HCV combined with IL一12 expresing plasmid co uld get better immunizing effects,and【c might represent a gene ther appeudc strategy against the HCV infection of human

Identification and Expression of Human ScFv Against Surface Antigen of Hepatitis B Virus in E .coil

ZHONG Yan wei, CHENG Jun.SHI Shuang-shuang, el al

2001, 16(2): 105

Abstract:Using HBsAg antigen purified from }ⅢsAg positive$ar~Ii1 as the coating antigen,the sin— gle—chain variable fragment(ScFv)antibodv to HBsAg has been screened and idemifed from a semi synthetic phage library by phage display technique The SfiI/NotI scFv DNA fragment was harvested from PHENI—ScFv and inserted into pCANTAB5E.and the recombinant plasmid DNA was used to transf0rm competent host XL1 Blue.After induction with IP,rG fnr 20 hrs,the expressed Ⅲ sAg ScFv in the supernantant of XL1一B1He was precipitated with 50% ammonium sulfated and demonstrat ed the molecular weight is about 28kDa bv SD PAGE The reactivity and spedficity have be n con— firmed by enzyme—linked irnmuno-sorbent assay f ELISA).The identification and expression of HB— sAg ScFv from E coli were successfully achieyed

High Expression,Purification and Imm uno-indentification of Recombinant HIV Surface Antigen gpl20

CHEN Chen, LIU Kun, WANG Jin zhong, IVIA Ming, CENG Yun-qi CHEN Qi min .ZHU Xiao-ke .XIA Jian-hui

2001, 16(2): 109

Different—length fragments of the HIV trans-membrane antigen (gp120)gene were ex pressed in Escherichia coti,using T7 expression system .SDS-PAGE stained by Coomassic Brilliant Blue showed no expression of full—length gpl20 and poor expresion 0f half—length gpl20 fragments from the N—terminal,but high expression of 1/3一lgenth gpl20 gene fragments from the N term inal (including V1/V2 epitopes).more than 18% of total bacterial protein Western blot showed fairly good reactivity to serum from HIV-infected individua1.On this basis,we expresed the corresponding tp 120 fragments of the HIV strains epidemic in China This study laid a solid foundation for expIo— ration of high expression of gP 120 in E coli and development of HIV serological diagnostic system for Chinese people.

Expression in E .Coti and Antigenicity of HGV NS5 Protein

GONG Rui, MA Hul—wen, TONG Li—heng

2001, 16(2): 114

880 bp cD A localized to the putative NS5 region of HGV genome was expressed in E. colf BL2I(DE3) The eDNA fragment was inserted into a plasmid pGEX-5X一1,at the downstream of the DNA sequence encoding Schistosoma japonicum glutathioue S_transferase(GST),in the S~TIe reading frame with the gene of GST.A 60KD GS丁_ S53 fusion protein was expressed at 37℃ in a forill of nclusion bodies amounting to 30 percent of total host protein whereas at 20℃ mainly in a form of soluble pmtein The fusion protein was extracted and purified to homologue,The purified GST— NS53 fusion protein could be specifically recognized with either the sera from the patient infected by HGV or the antisera directed against GST

Establishment of Fluorescent Real Time Quantitative PCR for Detecting HIV-1 and Its Application

XU Huang—bin, BEN Kun—long, ZENG Tao, LI Jin-guang

2001, 16(2): 119

Accurate determination of HIV一1 pmviral burden and viralload is very useful in prognosis of HIV一1 infected patients and in assessment of drug for therapy of AIDS patients.In order to establish a quantitative method in detecting HIV一1 proviral burden and viralload.8E5 cell line and a recombinant RNA constructs were used as the HIV一1 proviral DNA and viral RNA external references,respective— ly The PCR products were Labeled with the fluorescent D A dye SYBR green.The amount of burden or load was measured by GeneAmp 5700 Sequence Detection System Using this method.the HIV 1 pmvlral burdens in PBMC of patient and in cell suspension treated with the compounds AZT.GL and W T were measured.HIv_1 viraIloads in supernatant of the cell culture treated with the above com— pounds were also determined.The therapeutic indices(TIs)of the compounds calculated based on the inhibition of virus induced syncytlal formation.and inhibitionn of proviral burdens and viralloads were compared,and their TIs successively increased.The fluorescent real time quantitative PCR poseses very good specificity.sensitivity and duplication.TI value of a drug based on inhibition of proviral burden in cell culture,and the proviral burden in PBMC of patient may be useful in evaluating a drug on eradicating provirus from resting and memory CD4 T cells

Studies on Hantaviras Aerosol Transmission in Laboratory and the Field Sampling

LI Jin—song, LU Jian chun, CHE Feng—xiang, et al

2001, 16(2): 124

Aerosol transmission of Hanlavirus was studled by animal experiments in laboratory and air sampling in the field A agrarlus were subcutaneously inoculated hantavirus,breed in the chamber of the upwind and saved as the sources of virus aerosol Sucking mice,weanling mice and A .agrarius were put in the chamber of the downwind after the different days of infected A agrarius were breed Two,chambers were parted by the stainless steel net The results suggested that the seventh day could be the time sign that infected A .agrarius delivered infectious virus aeroso1.The epldemlolng ical in— vestigation of the field suggested that there may have an infectious virus unit at least,in 96L a by threshing machine and 350L air by the nest of A agrarius,respectively.W e believe this study may provide evidence that the aerosol transmission of hantavirus is probably a main route of transmision for hantvlruses in autumn and winter

Immunoreaetivity Studies of Various HEV Antigen with Anti-HEV IgM of the Sear from Patients Infected with Hepatitis E Virus

RONG Ouang-ya, ZhOU ji—wen, LEI Li, ZHAO Gui—lan, SUN Jie

2001, 16(2): 128

Objective To compare the Iramunoreactivity of various HEV Antigen with An ti—HEV IgM Methods Solid-phase erazyrne immunoassay(EIA )vcas developed for detecting anti HEV IgM by using synthetic peptides E30,E42,E33,and recombinant antigen from HEV ORF一2 Results Of 60 anti-HEV positive sera by using E30,E42,E33 and recombina nt antigen as coating antigen,Anti— HEV IgM positive rates were 76.7% .26,6% .18 3% and 66 7% respectively InAcute-phase and convalescence—phase sera of the patients with Hepa titis E.An ti—HEV IgM positive rate was 90% and 3 3% respectively.Condusions The HEV E30一based EIA will be very useful in the early diagnosis of Hepatitis E.

DNA Immunization of Hepatitis E Virus(HEV)

LU ng—mei, CHEN Er-jla, XIE Tian-hor~, ZHUANG Jun—ying.LIU Yong LI Chun—hong, SUN Mao-sheng”, DAI Chang—bai

2001, 16(2): 131

Two plasmid constructs,pcE2 and peE3,containing 3’fragment of open reading frame 2 (ORF2,1163 bp)of hepatitis E virus(HEV)and full—length ORF3(369 bp).were injected into bi— lateral tibiaLs of Swiss mice respectively.for three times(0,2 and 4 weeks)and observed the HEv IgG by ELIsA HEV IgG was induced after the injection of pcE2 or pcE3 or both,and the percentage of seraconversion was i00% after two weeks of the third iniection.Compared with injection of either construct,the antibo dy titers were higher in the group with combined injection of two constructs.

Morphology and M orphogenesis of the Isolated Varicelia.Zoster Virus in Rabbit Brain Nerve Cell

SONG Yan—yan, WANG Z —yu, W ANG Gui-ting, et al.

2001, 16(2): 135

To Investigate the morphology and morphogenesis of the isolated varicella—zoster virus (VZV)in rabbit brain nerve cell(RNC),ultrathin section electron microscopy was used for study A small number of nucleocapsids were observed in the nuclei of tLNC at 6 h post infection.A 1arge hum— her of necleocapsids were seen in the nuclei and cytoplasm at 12 h post infection.and the peak was at 24 h,but end,elope d virions were rarely seen in the nuclei and cytoplasm.The virions were spherical Cores,necleocapsids and enveloped virions are 30~50 nm,74~96肌and 110-180 nm in diameter respectively.There were three kinds of nucleocapsids with electron dense,particle and empty cores Electron dense body—like oDre and plate structure were observed both in nuclei and cytoplasm “Repli— cation complexes”composed of vacuoles enveloped by membrane was seen in the cytoplasm .These re— suits indicated that VZVJ1 showed diferent morphogenesis in RNC and other cells.

Complete Nucleotide Sequennce of the S Genome Segment of Hantavirus HTN261 Strain Isolated in the Northeast China

SUN Cheng, CHEN Lu, ZHANG Bao, et a

2001, 16(2): 140

To determine the complete nuelcotide sequence of the S genome segment of HTN261 virus strain which was isolated from the striped field mouse(Apodemus agraius)in Heilongjiang province of the Northeast China.the complete nucleotide sequence of the S genome segment of HTN261 virus strain was determined with two clones,which were obtained using RT-PCR,and by linking two over lapping fragments into PUC19 vector respectively.The complete nueleotide sequence of the S genome segment of HTN261 virus strain WaS 1697 nucleotides in length and contained one ORF encoding the N protein starts at position 37 and ends at position 1326 The length of predicted protein,429 amino acids,is identieal tO the N protein of HTN,DOB,SEO types of hantaviruses Comparison of S seg— ment sequence of HTN261 strain with the corresponding sequences of other hantaviruses showed that the dentities of HTN261 strain with HTN76—118 strain were 89% (complete nueleotide sequence) andd 98% (amino acid sequence).Phylogenetie ana lysis showed that HTN261 strain with HTN76— 1 18 strain form one lineage within the clade containing hantaviruses of HTN type.But the identities of HTN261 strain with HTN76—1 18 strain differ by 1 1% in complete nucleotide sequence of S segment and 2% in amino acid sequence of N protein.HTN261 strain belongs to hantavirus HTN type and may be a new subtype

The Reproduction of Nuclear Polyhedrosis Virus in the M idgut Epithelial Cell and the Other Sensitive Cell of Helicoverpa armigera

ZHANG Yan-jun, TAN Jun, LIN Yu-qin

2001, 16(2): 151

The reproduction of Helicoverpa armigera nueleopolyhedmvirus in the midgut epithelia cells and the other sensitive tissues-vvas observed by electron microscopy.The reproducing viruses in the midgut epithelia cells were mostly without envelopes,and thte polyhedrons were seldom formed .The reprodueiing viruses in the other sensitive cells were with envelopes,and packed in polyhedrons

Comparison of Producing Dendrolimus punctatus Cytoplasm ic Polyhedrosis Virus in Substitutive Host Insect

MA Yong-ping, MENG Xiao—lin .HU Rong, XU Jing—ping

2001, 16(2): 155

Argyrogramma agnata has been selected as a substitutive host insect for producing Den drolimus punctatus Cytoplasmic Polyhedmsis Virus(DpCPV),In our is very suscepti— ble to DpCPV.The DpCPV produced in A.agnata is designated Aa-DpCPV The cytoplasmic poly— hedra body(CPB).the virion size and the shape of Aa—DpCPV are same as that of its original DpCPV (DpCPV-W 1984) The RNA bands of Aa-DpCPV and DpCPV—W 1984 al1 have 10 RNA segments re— spectively in 3% PAGE.which molecular weights ranged in size from 2 98×10。to 0 66× 10。Dal— ton.Aa—DpCPV has the saro.e strong toxicity as that of DpCPV-W 1984(from D punctatus)tO D. punctatus(Walker)larva So it cai2 he applied tO the pine caterpillar contro1.The DpCPV yield in A.agnata iS 2.5 x 10 CPB/larva

Maize Dwarf Mosaic Disease Occurred in Hangzhou Isolate Caused by Sugarcane M osaic Virus

CHENG Ye, CHEN Jiong‘一, ZHENG Tao .YANG ]Jan—ping, CHEN ]ianpins

2001, 16(2): 170

Recently maize dwarf mosaic disease WaS occurred on maize crop seriously in large scale in Hangzhou district.Purified preparations from the infected maize leaves~ontained numerous filamen— t0us virus particles of e 750 nm in leng th.Cells of infected plants contained typical pinwheels and lam— inated aggregates.The coat protein of the virus was 33.6 kD A 1.8 kb fragment of 3’一terminus of the viral RNA was amplified by RT—PCR,cloned and its sequence was determined。Sequence compar— isons showed that it shared 71 5% -99 1% homology with isolates of sugarcane mosaic virus.67。8% ~ 68.5% with sorghum mosaic virus and 38。4% ~48.4% with ma ize dwarf mosaic virus,indicating that the pathogen of this disease on maize in Hangzhou was sugarcane mosaic virus.In addition.the relationships of sugarcane mosaic virus isolates from different origins all OVer world were discussed based 0n coat protein sequences

Studies on Apoptosis of Host Cells Induced by Foot-and-M outh Disease Virus

WU Hai, ZHENG Cong, QU San, WANG Yan

2001, 16(2): 175

Apoptosis of PK—t5 ceils induced by Foot—and-Mouth Disease Virus(FMDV)in vitro was reported in this paper.Typical cell apoptosis was detected hy of Hoechst 33258 fluorescence probe. agarose gel electrophoresis and in situ end-labeling(TUNEL).After PK一15 cells were infected by titration of 4.8 lg TCIDsD/mL FMDV for 32 h,apoptosis characteristics of nuclear condensation, fragmentation,accompanied by apoptotic bodies formation(Hoeehst 33258 staining).180—200 inte— ger-fold sized pieces DNA Ladders(agarose gel e1.ectrophoresis)and strong green fluorescence clots (TUNEL)were alI exhibited,and cell apoptosis was approximately 20% In addition.the quantira— tire analysis of apoptosis in PK一15 ceils induced by FMDV showed that apoptosis was correlated with infection of virus.and it was also tim~dependent.Results indicare that FMDV can induce apoptosis of host ceils and apoptosis plays an important role in the cytopathogencity effect of FMDV

Detection of SFV Gene with Nested·PCR in Rhesus M onkey Blood

REN Li—hong.BAI W ei, LIN Ling, SUN Ming, LI Hua, YI Hong—kun, HOU Zhong—liu

2001, 16(2): 179

Two pairs of primers were used for amplifying SFV gene with nested—PCR in Rhesus Mon— key blood The neady 500 bp PCR products were cloned into vector pUC-19.The fragment was de tected as 465 bp SFV pol gene by sequence analysis.The homology of the fragment to 425 bp pol gene of the SFV-1 was the was 92 00% 158 samples of Rhesus Monkey blood were used to de teet SFV gene with nested—PCR Among them,54 were positive.the poskive rate was 34 2% h ap— peared a h/gh present of SFV infection in Rhesus M onkey.

Cloning,Sequence Analysis and Expression in E .coli of the EP0 Gene of Pseudorabies Virus Ea Strain

FANG Liu—rang, CHEN Huan—chun, XIAO Shao—bo, MA Xiang—ru, WANG fei

2001, 16(2): 183

The 1 23 kb DNA fragment encoding the early protein EP0 of pseudombie8 virus(PRV)Ea strain was amplified by PCR technique and doned inm pBluescriptlI sk+.Thiee sequencing plasmids containing the partial fragment of the EPO gene were constructed and the sequences were obtained by ganger’S sequencing technique Cc~npared wi山PRV InFh strain.there were multipile site-muratiens and a deleted-mutation in the EP0 gene of PRV strain Ea,and the diversity-of anlino acid residu also existed,Then,the EP0 gene was in— serted into an expression vector,pET-28a,fused into the downstream of the 6xHis-Tag in fram e,to yidd the expression plasmid pETEP0 After induction by 球TG.a high expression of fusion protein was obtained. PAGE analysis and Western blotting showed that the fusion protein WaS 62kD an d the protein WaS specific toantisera against PRVEa strain si~dicated thatthe EP0 germ be expressedin l( )andthe ex— pression products have immtmo-genicity
Brief Reports

The Synchronous Detection on HCV·RNA and HGV·RNA in Plasm a and in Peripheral Blood M ononuclear Cells of Patients with Hepatitis C

LI Shu—li.ZENG Ling—lan.LUO Duan—de, YANG Xiao—ming, et al

2001, 16(2): 188

To understand the incidence of hepatitis C and hepatitis G cointection,and positive rate of HCV RNA or HGV—RNA in plasida and PBMC.HCV—RNA and HGV—RNA in phsma and in periph— eral blood mononuclear ceILs(PBMC)of the 40 patients were amplified with R r-PCR.There were 6 an d 8 HGV—RNA positive cases in plasma and PBM C.respectively.And there were 5 HGV—RNA po s— itive cam both in plasma an d PBM C At the same time.there were 5,6,3 both HCV—RNA and HGV— RNA positive cases in PBMC and in both plasma and PBMC.respectively.It accounted for 13% ,15% and 8% .rasp~tivdy.The HCV—RNA and HGV—RNA positive incidence of PBM C Was higher than that of plasma The incidence of HCV—RNA and HGV.RNA coinfection was similar to European,American and Japanese.The synchronous detection has an important signficanee in avoid— ing leaked diagnosis

Expression of Hepatitis C Virus NS5A Gene In E .coli and Its Application in HCV Antibody Detection

RUAN Hua, GAO Jin—rang, YE Lin hai, XU Jing—ping, W ANG Xiao-ling.ZHAO Yue-e.W U Zheng—hui

2001, 16(2): 190

Full-length NS5A gene of the hepatitis C virus was amplified by PCR using plasmid pBAC25 containing HCV nonstructural gene as template.The am plified fragment(about 1.34 kb)was cloned into plaS d pQE32.and the recombinant plasmid pQENSSA was expressed in JM109 strain The NS5A protein WaS purified by NiS04 metal chelating resin,and characterized by W estern—blot Its antigenecity was de~ermined by ELISA The positive detection rate of anti—NS5A was 75% (69/92) in ninety—two clinic sera The positive rate of anti—NS5A was 82.5% (33/40)in fourty positive stan— dand sera,and the negative rate of anti—NS5A was 10O% (40/40)in fourty negative standand sera The results showed that the Full—length NSSA proteinn had the higher sensitivity and specificity in the detection of HCV antibody in sera,we suggested that NS5A protein was a useful antigen for blood screening.