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2001 Vol.16(3)

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Contents

Molecular Mechanism of Synergism of Plant Virus

LU Ru fang, Li Wei—min, PENG Xue一xian

2001, 16(3): 195

The widespread synergism of plant virus is olle of main factors leading to crop loss.The discover of synergism in virus—resistant transgenic plants severely constrains the of transgenic plants.The types,characters, interactions of virus and virus,x4rus and environment,and molecular mechanism of s~mergism are re~iewed in this paper.
Research Article

Study Oil S Gene Nucleotide Sequence of Hantan SEO Type Virus in China

2001, 16(3): 202

Epidemiologic feature of Hemorrhagic fever with renal syndrome(HFRS)is endemic outbrak by SEO type nowadays.This article is to study SEO viral genes and the cause of endemic outbreak of HFRS.Virus isolation,McAbs typing and TR-PCR were used to determine a SEO strain isolated from Hebei province in 1996.Nucleotide sequence of S gene was detected.Compared with 75-118.Seoul and SRll strains,the homologies of nucleotide sequence were 64.2%,91.8%,96.8%,respectively,amino acid sequence were 80.5%,95.4%,97.3%,repectively.The high epidemics of HFRS wasnt because of recombinant of viral genes.This study provides important material for the cause of HFRS outbreak.

Detection of Trv DNA in Various Populations by Polymerase Chain Reaction-microplate Hybridization

GUO Nai—zhou.MA Da, WANG Wan-xlang

2001, 16(3): 208

TTV DNA were detected by polymerase chain reaction-microplate bybridization.The results showed that the positive rate of TTV DNA in 81 normal samples,92 blood donation samples,123 A to G hepatitis,32non-A to G hepatitis samples and 48 proimitive hepatic-carcinoma samples was 3.7%?4.3%?21.1%? 28.1% ?52.0%,respectively.There were significant difference between the normal?the hepatitis group and the primitive hepatic-carcinoma group.Among multiinfection,the infection rate of TTV with HBV was as high as 54.0%.The data showed that TTV infection was very popular in different groups.Some of the normal and blood donation group infected with TTV could become virus carrier.And the hepatits patients were high risk group.In addition,TTV may be transmitted by other means besides blood.TTV infection has close relationship with the increase of ALT and TBIL.

Isolation and Adaptation of HAV on M EK Cells and Vero Cells

JING Shen—rong, JianG Shu—de, YU feng, WANG Yan

2001, 16(3): 212

HAV strains W and X were isolated and adapted to Rhesus monkey embryo kidney(MEK) cells from feces of hepatitis A patients,and identified to be HAV with blocking experiment,neutralizing experiment,indirect immunofluorescence (IF),immune electron microscopy(IEM) and RT-PCR.Antigenic titers of strains W and X P7 on MEK cells were 1∶512,1∶1024,respectively and infective titers(logTCID 50 /mL)of that were 8.17 ,8.50 respectively.Only the isolate W was adapted to Vero cells.Antigenic titer of adapted strain W P6 on Vero cells on 21th day was 1∶256 and infective titer(logTCID 50 /mL)was 8.00.

Preliminary Study of Hepatitis C Viral Quasispecies in NS2 Region in Patients wiUl Hepatitis C Virus Infection

LENG Yan, CHEN Xiuzhu, DU Yong, MAO Chun-sheng, Du Guixin, WANG Haitao

2001, 16(3): 215

To explore the genetic variation of nonstmetural(NS)2 region and its gene structural characteristics in hepatitis C vims quasispecies,NS2 genes ofHCV from a HCV as)~nptomatie~[Tier and apatien twith ab. nomud ALT level tteile amphfied by RT-nested-PCR respectively and cloned into T Yector.5 positive clones form each of them were selected~ domly for sequencing.Eve~-clone WaS unique in nuclectide sequence and amino acid sequence.A 0Bt all the NS2 gene clones from the asymptomatic carrier had complete ORF ex。ept OIle done had a stop codon at 835aain HCV polypmtein.Butwhenit ean3~tothepatientwith abnorm al ALT level,3 clones of them had stop cadom.at 835aa and 1 clone had a stop ced on at 887aa in polypretein. 0nly 1 clone had complete ORF.Comparing this ORF with the s~ ence of the as)~lptomatic carrier,most of the~aino acid changes were confined to the N-termlnai.Simulating the so。ondarv strucRu~s of Ns2 proteins with intact openreadingframe demomUated thatthe structure ofthe HCV patientwith abnormalA levelwas similart0 the dominant structure ofthe asymptomatic carrier.It could beseenfrom thetwopatientswith HCV infection thatHepatitis C viral quasispeciesin NS2 r 0nhadan obvious diversity between HCv as)~lptoraatic p~riier and HCV patient with abnomaal A leve1.The diversity suggested that the stale of HCV qua~species in NS2 region WSSlikelyto correlatewith the progression ofliver diseas

Expression of Soluble Human SeFv Against Envelope Protein E2 of Hepatitis C Virus Antigen in E.coli

CHENG Jun, Shi Shuang—shuang.ZHONG Yan—wei, XIA Xiao—bingWANG Gang, WANG Lin, LIU Yan, CHEN Ju-mei

2001, 16(3): 220

To construct expressive vector for human ScFv against E2 protein of hepatitis C virus (HCV-E2-ScFv),and express soluble HCV-E2-ScFv in E.coli JM109,using phage display technique,the recombinant phages were panned by recombinant E2 antigen which was coated in a microtiter plate,after five rounds of biopanning,46 clones were identified specific to E2 antigen.750 bp fragment could be released from the plasmid of positive phage colones,and the sequenle analysis indicated that we have got the ScFv BNA fragment.And then DNA fragment was inserted into expressive vector pCANTAB5E and E.coli host JM109 was transformed and induced by IPTG.The specificity of ScFv in the culture medium was evaluated by enzyme-linked immunosorbent assay(ELISA).The molecular weight of expressed HCV-E2-ScFv is 28kD by SDS-polyacrymide gel electrophoresis(PAGE).The expressed HCV-E2-ScFv will be useful in the immunohistochemical study of liver tissue and gene therapy against HCV infectiion.

Cloning and Sequence Analysis of Human Hepatitis B Virus preS Gene Variants from Hangzhou an d Lanzhou in China

WANG Miao, JIN Yongfeng, Du Guang, ZHANG Yao-zhou

2001, 16(3): 224

PreS gene fragments were inserted into pBluescript SK after being amplified by PCR from HBV DNA,which was extracted from HBsAg-positive sera(Hangzhou and Lanzhou samples)and the cloned genes were sequenced.The complate nucleotide sequences of the cloned preS genes(preS-HZ and preS-LZ,GenBank accession numbers:Af325674 and AF325675 respectively) were 522bp long,encoding 174aa.The identity of preS-HZ with published preS gene Shagnhai isolate(adr subtype:Gan,et al.,1986),Beijing isolate (adr subtype:Qi,et al.,1989),Japan isolate(adr subtype:Ono,et al.,1983),adw subtype(Valenzuela,et al.,1980),ayw subtype (Galibert,et al.,1979)was respectively 96.7%,96.2%,97.3%,88.7%,84.1% for nucleotide sequence and 96.0%,94.9%,97.1%,85.1%,85.4% for amino acid sequence;correspondingly,preS-LZ was respectively 96.4%,96.2%,96.9%,88.7%,83.7 for nucleotide sequence and 94.9%,94.9%,96.0%,85.1%,84.1% for amino acid sequence.Only two nucleotide substitutions(resulting in two amino acid changes) were observed between preS-HZ and preS-LZ

Sequence Analysis of the Putative-cathepsin Gene of Heliothis Armigera Single-nudeocapsid Baculovirus

U Lan, CHEN Xin-wen, SUN Xiulian, Hilary Lallzon, Just M, Vlak Basil, HU Zhi-hong

2001, 16(3): 229

A putative -cathespin(cath)gene was identified from the single(S) nucleocapsid nucleopolyhedrovirus of Helicoverpa armigera(HaSNPY).This open reading frame is 1098 nucleotides long,encoding a putative protein of 366 amino acids.The amino acids sequence alignment of eleven baculoviral v-CATHs showed that the primary structure and the catalytic sited of the proteins were conserved.A pholygenetic tree of these v-CATHs was constructed by using maximun parsimony analysis,the result indicated that the -cath gene of HaSNPV had a different evolutionary history from that of other NPVs.

Nucleotide Sequence of Spodoptera litura Nucleopolyhedrovirus DNA Xba I 4.0 kb Containing the Zinc Finger Protein Gene and Repetitive Sequence Regions

HU Guo-dong, WANG Li—hua, HU Xiao-hui, YANG Kaia, PANG Yi

2001, 16(3): 236

The complete nucleotide sequence of Spodoptera litura nueleopolyhedrovims(SpltNPV)DNA Xba I 4.0 kb has been determined.It contains a zinc finger protein gene and three repetitive sequence regions·The open madil1g frame of this zinc finger gene is 2 196 bp long,enc0d_mg a putative protein of 731 amino acids withmo lecular wei出0f 83.09 kD andisodeeⅡic point of4.61.The 5 noncoding region ofthis gene has olle baculovims early promotermotif(GAGT)and olle TATA box.Five polyadenylafion sitwats,AATAAA,arefound inthe do~ stream ofthetranslation stop codon.A ringfingermotifwhichis a C3HG4type of zincfinger pm— telnlnotifand alqUOlearlocalization si 丑l present atthe am/no acid residues 232~241 and 323~340 — spectively.This proteinis probablyhi y curled.Th repetitive SG~XleneA2:regions(SR1,SR2,SR3)are present in this fragment.SRI and SR3 contain nlllTl~rous repetitive SOqlfleDCe$,,and one of these in SRI is 41bp long. SR1 and SR3 possibly act且s the transcfiption aI enha兀ceI音or or;~s of replication.

Sequence Analysis of the Helicoverpa armigera Nuclear Polyhedrosis Virusp40 Gene

WU Jia-cai, ZHANG Chuan-xi

2001, 16(3): 242

The Hind Ⅲ-H?J fragments of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) qenome were cloned. By sequencing the terminus of the cloned fragments, an ORF encoding the HaSNPV p40 was identified. The coding region was 966 bp long and potential to encode a 36.6kD pepetide. The HaSNPV p40 exhibits 98% identity with HzSNPV p40 at the nucleotide sequence. The two genes diverged significantly from BmNPV p40 and AcMNPV gp41.The predicted amino acid sequences have less than 45% homology between HaSNPV p40?HzSNPV p40 and BmNPV p40 or AcMNPV gp41, but four genes have a conserved hydrophilic domain. In this region they have 62% homology. Further more, we determined that Hind Ⅲ-H fragment links with J fragment in the genome and the two fragments were analasysed with four restriction endonucleases (BamH Ⅰ?EcoR Ⅰ?Hind Ⅲ?Pst Ⅰ) .

A Dwarf Disease on Rice,Wheat and Maize from Zhejiang and Hebei is Caused by Rice Black-streaked Dwarf Virus

ZHAJNG Heng, LEI Juan-l-i-, CHEN Jian, LU Yong-ping, CHEN Sheng-xiang, XUE Qirrg-zhong

2001, 16(3): 246

Two isolates of plant reoviruses causing severe stunting and dark leaf symptoms on wheat and maize from Hebei province and on rice from Zhejiang province have been characterized.The virus particles of both isolates were about 60~80nm in diameter and closely related serologically.Their dsRNA electrophoretic profiles in agarose gel were similar.They were both transmitted efficiently by the planthopper Laodelphax striatellus to maize and rice from their original hosts but maize was not a very suitable host for the vector and was a poor source of virus.Two fragments corresponding to dsRNA parts of rice black-streaked dwarf virus(RBSDV)S7 and S8 were amplified by RT-PCR from both isolates and sequenced.There were 97.0~98.9% identical nucleotides between the two Chinese isolates,which appear to be RBSDV(92.2%~95.5% identical nucleotides to Japanese isolate)rather than maize rough dwarf virus(79.6%~88.4% identical nucleotides to Italian isolate).

Construction of cDNA Clone and Nucleotde Sequence of RNA3 of Cucumber Mosaic Virus Isolate Xb from Banana

WANG Hai-he.XIE Iian—hui .LIN Qi-ying

2001, 16(3): 252

The nucleotide sequence of RNA3 of a cucumber mosaic virus strain from banana of subgroup Ⅱ(CMV-Xb) was cloned and analyzed at nucleotide and protein level after RT-PCR amplificaion with two pairs of primers.The results showed that CMV-Xb is composed of 2205 nucleotides(nt) with two open reading frames.The one near 5'termini(97~936nt) encodes a 3a protein with 279 amino acids(aa),the other near 3'termini (1225~1871nt)codes for coat protein with 218aa,The 5'non-coding region(NR),intercistron region(IR) and 3'NR are 96nt,288nt and 324nt, respectively.Comparison at the nucleotide and putative amino acid level with other strains in subgroup Ⅱ showed that the nucleotide sequence of Xb is very conservative in the encoding regions as well as non-coding regions.The evolution of RNA3s of subgroup Ⅱ members of CMV is consecutive.

Expression of Envelope Gene gp85 of Avian Leukodls Virus in E.coli

LIU Gong-ping, ZHAO zhen-fen, LIU Fu—an

2001, 16(3): 257

A part of the cloned fragment was excised from the recombinant with Bgl II and Sal I and subcloned into the expression vector pET-21d (+) to yield another recombinant named pET-21d-RAV-1 env (Bgl II/Sal I). The recombinant was sequenced and the result showed that the inserted fragment was identical to the counterpart in RAV-1 env gene both in nucleotide sequence and open reading frame. The E.coli BL2 (DE3) transformed with recombinant plasmid were induced with 1 mmol/L IPTG and the expression product found to be 20 kD in size on SDS-PAGE. The size of the expression product was the same as that theoretically calculated. In addition, the effects of start time and duration for IPTG induction on expression efficiency were analyzed and the result indicated that the start time for IPTG induction was more important to high expression efficiency than its duration.

L6565 M ouse Leukemia Induced by L6565 Murine Leukemi a Virus

LI Yong, YANG ZI, LIU Jian-jun, GUO Guang-shong, XIAO Hong, WEN Li

2001, 16(3): 261

Suckling mice were inoculated with L6565 blLV for researehlng the pathogeny of L6565 moL!se Leukemia Afterinoeulatica~,叽emol3y,eⅧ sacrificed r3r week.Theperipheral blood,thymus,[ymp畸node, spleen and liver weTe removed from l’lflOIl~.|I11e tissues were used to observe tIl0 pathological changes and to detect1.6565 MLV RNA by RT-PC~ .At 3~5 weeks postinoeulation.routine thynms.1ymphy node began to swell,And IIIOU~ leukemia was indueed at 10~12 weeks postinoeulatlon.L6565 MLV RNA Ⅵ∞ detected first inⅡl0use thymus and s een at the se~nd week曲er inoculadon,and the viral RNA *,vaS detected in rnanv /131iillle oigalls 8s time goes on.The results sh㈣d that L6565 l/lOUSe leukemia COIlld be jnduced by L6565 naalne leukemia、TirllS,and it is related to the transformation of lvmph0cyte by 1.6565 MLV.

Detection of Ilfeetious Laryngotracheitis Virus by Nested PCR

HUANG Geng—ming, Xin Chao-an

2001, 16(3): 265

Two pairs of oligonucleotides flanking 33aymidine Kinase(TK)seonent of infectious laryngotracheifs virus(Ⅲ )were chosen as primers for polymerase chaln reaction(nested PCRj. e method of nested PCR W~LS.used to detect of virus DNA in ILTV infected diferent chorlollantoic membrane,specimens from laboratoO, and 7 clinical specimens from various species, m ILTV gene TK seglllent p1 d DNA as positive contro1. The vlms DNA was extracted by acid guanidinium thlocyanate-phenol—chloroform slngle-step method .Th e re— suhs sho,a-ed that allⅡ V infected diferent chorloUantoic memb rane were positive.The highest detection rate ofⅡ Ⅳ from tracheal swabs of Iqon—hnmmtized chicken was 7/10 on the tenth day P.i..and that from tracheal swal3~of SPF chickens was 8/10 on the tenth day P.i..The best time for唧detection in tracheal swabs fmm both rloii—inmmnized chickens and SPF chickens wasthefifth day P.i. detectionfrom clinical 蚋m— ples by nested PCR W~LS.at a rate of7/7.To~,erify the nucleotide hybridization test th TKc probe .it suggests that nested PCR is sensitive,specific,rapid mad simple,and can be印 ed for study of pathogenesis on moiec— ular level and for early clinical diagnosis.

Study On the Inhibition of Specific 01igonucleotides On Classical Swine fever Virus in vitro

ZHOU Peng-cheng, NIE Yu—chun, CAO Qi, CHEN Jian·guo, DING Ming—xiao

2001, 16(3): 270

The effect of oligodeoxynuclcotldes(ODN)OI1 replication of CSFV(Shimen strain)WSIS investigated in this papor.Ph~phorthimte 01igedeox.vnuel·eoddes(PS-ODN)targeted to the~gion of the 3’,5._nonceding re,on(NCR)as well SIS PS-ODNtargetedto NS3 gone region㈨maIke叭inhibitedthereplication of CSFV in PK-15 cells.Antiviral activity of PS·ODN Writs evaluated bY fluorescent plaque fomaation assav.The results presented here indicated that ODN 175 complementary to 5’temalnal conserved sequence mad it’s sense ODN P5S。as wel as1’8 complementartoNS3 region exhibited strongly antiviral activity,whereasthe anfiviral acfivi一 押ofP3 andit’s enese PS-ODN W86 muchlower.Atthe sametime,antMral activity of sense ODN is sli higher than corresponding antlsense ODN The inhibition ofODN is in a dose·and sequence—dependant]xlanller. Inhibition of CSFV’s replication in PK一15 ceils Call be signllicanfly increased in the presense of lipofecfin by either sense or anfisense 0DN.These data suggested both 5’·NCR and 3I_NCR may play important roles in CSFV replication.

Construction of the Transgenic Plasmid Containing GFP Gene and Four Ribozyme Genes Targeting the Prawn Baclll0virus Gene

ZOU Jian, SHEN Xuc-ten, WU Jhn·hua, ZHOU Guo·ying, GONG Zu·xllnl“

2001, 16(3): 275

TheⅡ lsg c plasⅡl|d of haⅡⅡI1e r}Iead ribozymes Rzl and Rz2 targetirg one of the Prat,m bac· ulovims genes has been eoustmctae.The transgenic plasmld eontains double series o0pjes of Rzl and Rz2 as Rzl·Rz2·Rzl·Rz2.The Green Fluorescent Protein(GFP)gene was selected a8 the report gene of the transgenic plasmid.The GFP gene was cloned into plasmld peDNA3 at site 2093bp with Sma I.cont~lled by SV40 pro· illlOter.mb0 Ⅱ】e gene Seile8wclonedintotheMCS ofplasmld peDNA3with proper rest6cfion endonuclease, conmdled byT7 andCMV promoter.The constructedtransgenlc plasmidw∞ named andwill be usedfor obtainingta’ansgenic Prawn.

Construction of a Chimeric Gene encoding Core,NS3 and NS4 Region Antigens of Hepatitis C Virus and Its Expression

LI Ming, SHEN Xiao-chuan, LIN Jiao-jiao, NI Zhen-ya, YUAN Chun-xiu, LIU Jin-ming, YANG Guan-zhen, WU Xiang-fu

2001, 16(3): 283

DNA fragments of Core(300bp)and NS4(400bp),encoding the nucleocapsid region(Core)protein CS and nonstructural region(NS4)protein NS4a of HCV,have been obtained from HCV genome by PCR,both of the two fragments were liked with C33c(700bp)and formed a chineric gene C33c-Core-NS4(HCV-CCN).The chimeric gene was recombined with expression vector pET24a(+)and was expressed in Escherichia coli.The expressed protein was purified by Ni(+)NTB affinity chromatography.Its molecular weight was about 58kD.Western blotting analysis showed that the chimerical antigen had good antigenicity,which could play an important role in anti-HCV assay.

Construction of Gene Library and Localization of Chitinase Gene of Parocneria orienta Nuclear polyhedrovirus

ZHAO Xiao-dong, ZHOU Jian-hua, DING Qing-quan

2001, 16(3): 286

The gene library of Parocneria oriema nuclear polyhedlovims(PaorNPV)was panly constructed by doing$oiriefragnen~ ofBamH I and EcoR I ofP~rNPV genomic DNA into pGEM3Z(f)as a vector.Using the chifinase gene of Hear SNPV probed wlth[a-32p]dCTP,the chifinase gene ofPaorNPV localized OI3_the fragraents of BamH I-D,EcoR[-A,‰ dⅢ一A,Pst LD and Xho tG/H respectively.Atthe time,the fragment ofBamH I-D eontniningthe chiA has been cloned.