2001 Vol.16(4)


Studies Oil Interferon-inducible Mx Proteins in Fish

ZHANG Yi-bing, GUI Jian-fang

2001, 16(4): 291

Mx proteins are members of a family of interferon inducible genes expressed in organisms or cells which are treated with all sorts of inducers or virus infection.These proteins together with other interferon inducible proteins form the antiviral state in host cells and the first line of the body’s defense against virus infection.Apart from the inhibtion of a specific virus,Mx proteins may be related with other basic cellular functions such as development/differentiation,protein sorting and growth.Mx proteins in several fishes are found and a comparsion of their sequences with that of avian and mammalian species reveals striking conservation of domains.They all maintain a tripartite ATP/GTP binding motif and a signature of the dynamin family in the amino terminal of the protein.In addition,the C terminal region of the Mx proteins contains the localization signals and the leucine zipper motif which account for the trimerization of Mx in the cell.So far,the antiviral function of the fish Mx proteins has not b

The Development of The Hantan Virus Vaccine

XIAO Chun-hui, YANG Zhan—qiu

2001, 16(4): 299

We outlinethe development ofthe hantaanvims vaccine in ree~t years∞ brain dsaneinavtivated vaccine,cultural cell bivalent and univalent inactivated vaccine,recu~ inadou vaccine,DNA vaccine,attenuat— ed live vaccine,adjuvarIt,inacfivatima methods and lmnlulle strategy.
Research Article

A Possible M echanism of Apoptosis Induced by Simian Immunoddideny Virus in PolymorphonudearNeutrophils

LI Gang, YANG Fei—yi, LIU Xin—hua, WANG Dan, LI Ping-feng

2001, 16(4): 304

The aim ofthis stud),istoinvestigatethemechanis~s of apoptosisin polymorphonudear neutrophils (PMNs)induced by simian immunodeficiency v~ (siv).In this experiment.gag gene was amplified by the polymerase chaill reactio~andthe expression ofp53 and bc/一2 gC~12e were determined byWestern blot assay. The results showedtllattl1e viability ofPMNs aftertreated with SIV was declined with the cLlk time pro 1~ged. gene could be amplified from PMNs at 24 h postinfection. expression of p53 gene in PMNs W83 enhaneed at 24 h postinfee6on.At tlle s8me time。the expression of bcI一2 gene were elevated in both of control and SIV infected group.but the production of 一2 protein in SIV infected group was obviously lower than contro1.0tilt"dataindicatethat PMN$could beinfected witll SIV andthe ch~ged expression ofp53 and bcI一2 gene~,66t be one oftlle meehanlsms of apoptosis occurred in PMN infected witll sⅣ.

Structure Characterstics of M S Segment of Hantavirus,K24,strain

YAO Zhi—hui, DONG Guan-mu, YU Yong-xin

2001, 16(4): 309

Abstract:Our pmvious studv showedthatK24 strain懈arI antigenicity broadHantavims SE0 type strain.In this paper we studied the molecular characteristics of K24 strain.The total ceUular RNA Wilts extracted from in. fected cell culture andtheM and S segment genewere ampficated byRT-PCR andthen cloned and sequenced. The resultsindlcated thattheK24 strain completeM genome segment w丑s 3 652 nucleotidein length encoding 1 133amino acid,andthe complete S gmaon~segmoatwas1 772 nucleotideeneoa~ 429mnino acid.C0Ⅱ . ingwith other hmatavirus rIs。the homology. of M genmne segment with that of H] ,Arms is 70.7% 一 71.2% in nucleotide leveland75.9% 一76.9% in amino acid level respectively.wh wit}l that ofSEO virus strains it WaS 95.2% 一99.2% at nucleotide level and 95.9—99.4% at amino acidleve1.The homology ofS gen ome segment with H] 76.118 and SEO SR一1l showed 74.O% and 95.6% at nucleotide leve1.and 83. 3% and 97.9% at amino acidlevel_Thetesul:s a similartothat ofM gt~qome segment.Wh印c哪I with 10 SEO virus stu ns,therewere 4 amino acid diferencesintheM gencnae segment,whcihmi t be 叩m1sible an tigeniclty characteristics broad.Phylogenictrees We constructed depending OI1the es ofril1. cleotide and mnino acids respectively.

Molecular Characterristics of the Seed Virus 0f Hl S Candidate Vaccine A16 Strain

YAO Zhi, DONC Cuan-mu, YU Yong-xin, JU Ke, YAN Yu-ehe

2001, 16(4): 315

For further studying on the molecular characteristics of A16 strain,which is the seed virus of HFRS candidate vaccine,the M and S segments of A16 strain were amplified by RT PCR and then sequenced respectively.Sequence assay showed that Msegment of A16 strain contained 3615 nucleotides,encoding 1133 amino acids,and S segment of A16 strain contained 1 770 nucleotides,encoding 429 amino acids.The homology of M segment of A16 strain’s nucleotide sequences and induced amino acid sequences with that of other HTN type virus varied from 75.7% to 90.3% and 85.9% to 97.1%respectively.Namely,the mutation of M segment of A16 strain’s nucleotide sequence and amino acid sequence with that of other HTN type virus varied from 9.7% to 24.3% and 2.9% to 14.1% respectivedly.Like M segment,the homology of S segment of A16 strain’s nucleotide sequences and induced amino acid sequences with that of other HTN type virus varied from 76.0% to 90.0% and 92.1% to 97.7%,and 67.0% to 67.7% and 81.7% to 82.6% with that of SE

The Sequence Analysts of I-IB~ S Gene Isolated from Both m 3sAgand Anti-l-IBs Antibody Positive Blood Donors

CU Wen-jie.LIN Jing-xiang, WANC You-chun“, ZHANC.I~a-yuan, LAN Hai.yun, LI He-rai

2001, 16(4): 321

In order to investigate HBV genotype and variants from the blood donors positive for both HbsAg and anti HBs antibody,389 sera from HBsAg positive blood donors were tested for anti HBs antibody with RIA assay.The sera positive for both HbsAg and anti HBs were tested for HBV DNA with polymerase chain reaction(PCR).The positive PCR products were cloned and sequenced.The sequences were compared with the representative sequences of different HBV genotypes.The results showed that 10/389 sera positive for HBsAg were positive for anti HBs.Five of these ten sera were positive for HBV DNA.The sequence analysis showed that all five sequences belonged to HBV genotype B,four sequences were adw serotype and one was adr serotype.Amino acids of antigen determinant "a" of two sequences have been changed.

Distribution and Significance of HCV and HBV Genes and Their Related Antigens in Hepatocellular Carcinoma

WANG Wen—liang, WNAG Cbun-jie, SHI Jian-guo, WANG Bengfung

2001, 16(4): 325

HCV RNA and HBV X gene were detected by in situ hybridization and HCV core?C33c antigens and HBxAg were localized by immunohistochemical method.Results showed(1) The positive rate of HCV RNA and HBV X gene were 40%(55/136) and 82% (112/136) in HCC respectively,both of them were positive simultaneously,34%(46/136).HCV RNA was localized in the cytoplasm of cancerous cells and hepatocytes,the positive cells distributed in scatter,local or diffusion pattens.The distribution of HBV X gene in HCC cancerous cells showed cytoplasm,nuclear and nuclear cytoplasm,the positive cells distributed just as that of HCV RNA.(2) The positive rates of HCV C33c and core antigens in HCC were 81%(133/164) and 86%(141/164) respectively,and C33c antigen was localized in the cancerous cells intracytoplasmically,either in the form of focal or diffuse in distribution,some were access to the nuclear membrane.Not only was HCV core antigen positive stain distributed in the nuclei of the cancerous cell but it was also found in the cytoplas

Sequence Analysis of Helicoverpa armigera Single-nucleocapsid Nucleopolyhedrovirus odv-e66 Gene

WU Dong, HU Zhihong, Just M, CHEN Xinwen

2001, 16(4): 330

ODV E66 is the major protein of occlusion derived virions envelope of baculovirus.An odv e66 gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus was identified and sequenced.The HaSNPV odv e66 gene had a size of 2 109 bp and potentially coding a protein of 672 amino acids,with a molecular weight of 74.5 kD.A late transcriptional motif,ATAAG,was found at 20~ 24nt uppstream of the translational start codon ATG.There was a TATA box,a GATA box and a CAGT motif at 13nt, 92nt, 141nt upstream of ATG,respectively.Amino acid sequences alignment with so far identified baculovirus’ODV E66 had revealed a conserved hydrophobic region at the N terminal of ODV E66 proteins.A potential nuclear locating signal RR(K)IW was found conserved in all ODV E66 proteins.Two leu zipper motifs were found in the middle of ODV E66 proteins.Six potential transmembrane regions were found in HaSNPV ODV E66 protein.Two of them were conserved in the other baculoviruses’.Analysis with computer shown

Cloning and Sequence Analysis of the iap2 Gene of Helicoverpa armigera Single-nudeocapsid Nudeopolyhedrovinm

ZHANG Xiao-xia.ZHANG Zhong-xin.DING Qing-quan

2001, 16(4): 338

An iap 2 gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus was identified.It was localized Bam H I F fragment of the genome.Nucleotide sequencing showed the open reading frame was 753nt,encoded 250 amino acids with a predicted size of 29.2kD.A"TATA"box was found-30~-33nt upstream of the translational start codon ATG.There is an early consensus translational motif CAAT,-50~-53nt upstream of the translational start codon ATG.A typical poly(A)signal was identified 46~51nt downstream of the translational stop codon TAA.HaSNPV iap 2 gene encode one typical RING and two BIRs.The alignment of AcMNPV?OpMNPV?LdMNPV?LdMNPV?SeMNPV?DIAP and XIAP indicated that they had consensus BIR motifs and RING zinc finger motif.It was suggested HaNPV IAP2 had a potential anti apoptotic function.

Sequence Analysis of Helicoverpa armigera Single—nucleocapsid Nucleopolyhedrovirus heficase Gene

FAN Lingyun, HU Zhihong, Just.M.Vlak, CHEN Xinwen

2001, 16(4): 344

In order to investigate the genomic organization of the single nucleocapid nucleopolyhedrovirus of Helicoverpa armigera ,the Eco R I N fragment located at 54.8~59.3 kbp of the viral genome was sequenced.The fragment contained 3 762 bp helicase gene potentially encoding a protein with a molecular mass of 146kDa.A late transcriptional motif,ATAAG,was found at 50nt upstream of the translational start codon ATG,while two TATA box was located at 112nt and 189nt upstream of ATG.A typical poly(A) signal was found at 12nt downstream of the translational stop codon.Compared HaSNPV helicase with the helicases of Autographa colifornica MNPV(AcMNPV),( Bombyx bori NPV (BmNPV), Lymantria dispar MNPV(LdMNPV) Spodoptera exigue MNPV (SeMNPV), Orgyia pseudotsugata MNPV (OpMNPV),and Xestia c nigrum granuloviurs(XcGV),only 5motifs(I,Ia,II,III,IV)were found conserved in baculovirus.The homology of the other two notifs (V and VI),which are also quite conserved in other helicases,were lowe

Molecular Cloning and Sequencing of Dendt~limuspunctatus Cytoplasmic Polyhedrosis Virus(DpCPV)oolyhedrin gene

Du Jian-yu, IANG Jia-rnin, GUO Hai-tao, ZHANG xiao-cto

2001, 16(4): 350

CPVs belong to the genus Cypovirus in the family Reoviridae and infect midgut epithelial cells of the wide range of insects.For the synthesis of cDNA of Dendrolimus punctatus CPV (DpCPV) polyhedrin gene,the primers were constructed on the basis of the terminal RNA sequence of BmCPV segment 10,After RT PCR,the amplified cDNA was cloned into the pGEM T.The S10 cDNA of DpCPV was found to be 763 nucleotides in length with a single open reading frame in one strand capable of coding a predicted protein of 248 residues (Mr of 28411).Comparison of the nucleotide sequence of the polyhedrin gene of DpCPV with that of BmCPV showed that nucleotides homology is 89.3%,but there are only 6 amino acid differences between the two CPVs.

Sequence Analysis of the H/rid m -L Region of the Helicoverpa armigera Single-nudeocapsid Nucleopolyhedrovirus(HaSNPV)

FANG Ming-gang, CHEN Xin, WANG IJuan-zhong, Just M, HU Zhi-hong

2001, 16(4): 355

In order to investigate the genetic organization of the single nucleocapsid nucleopolyhedrovirus (SNPV) of Helicoverpa armigera (HaSNPV),the Hin d III L fragment of the viral genome was sequenced.The fragment contained five complete open reading frames(ORFs) representing ORF227 (a unique gene),a late expression factor 10 gene ( lef 10),a baculovirus structural protein gene( vp 1 054), Ac 55(a homologue of the AcMNPV ORF55) and Ac 56 (a homologue of the AcMNPV ORF56),respectively.These five genes were identified for the first time in HaSNPV.Alignment of baculovirus LEF10 s indicates that they have conserved leucine zipper regions,and aligament of baculovirus VP1054s indicates that they are highly conserved.

Isolation,Identification,and Determination of Virulence of Spodoptera exigua Nuclear Polyhedrosis Virus Chinese Strain

2001, 16(4): 361

A strain of Spodoptera exigua Nuclear Polyhedrosis Virus(SeNPV)isolated from China was purified and identified.The results of bioassay indicated that the LC 50 of 2nd and 3rd instar of Spodoptera exigua was 6.6 ×10 4 and 2.6×10 5 PIB/ml

Expression of 2.6kb Enhancin Gene Fragment from Helicoverpa atmigera Granulosis Virus

HU Rong, MENG Xiao-Lin, XU Jin, WANG Jian, LIJ Wei

2001, 16(4): 364

The 2.6kb fragment of enhancing gene from Helicoverpa armigera granulosis virus was inserted into vector pQE32 and expressed successfully in E.coli M15.The synergy of expression product(P 102 ) on Helicoverpa armigera nuclear polyhedrosis virus(HaNPV)and Bt against the 2nd instar larvae of H.armigera was studied.The results indicated that the accumulated mortality of the larvae increased 6.25%~27.09% on 168h post infection and the median lethal time decrease at least 12.3h in HaNPV+En treated compared with those in HaNPV treated.The accumulated mortality of the larvae increase 28.18% on 72h postinfection and the median lethal time decreased 12.33h in Bt+En treated compared with that in Bt treated.

Cloning and Sequencing of Coat Protein Geng of Malformed Leaf Strain of Papaya Ringspot Virus

HE Guo-an, XL~.O Huo—gen一, ZHANG Shu—guang, LI Hua—p吨, FAN Huai-zhong

2001, 16(4): 369

The coat protein(CP)gene of an isolate of papaya malformed leaf virus(PMaLV),preserved in Plant Virology Laboratory of South China Agricultural University by Luo Xuehai,was synthesized by reverse transcription and polymerase chain reaction(RT PCR) and cloned into pGEM T and pGEM T Easy Vector System(T vector).The full length of the CP gene was sequenced,and it shown that the CP gene was 861 nucleotides(nt) in length,with 3nt deletion beginning from 66nt as compared with that of Hawaii strain(HA) and Australian strain W of papaya ringspot virus(PRSV).Compared with Ys(yellow ringspot strain)?Sm(severe mosaic strain)?G(Hainan isolate)?HA and W strains of PRSV,the CP gene sequence of PMaLV shared 96%?98%?95%?89% and 89% nucleotide sequence identity respectively.The coat protein encoded by CP gene of PMaLV consisted of 287 amino acids residues,and shared 98%?97%?97%?96% and 95% amino acid identity with those of the five strains respectively.This results of sequence analysis shown conclusively that PMaLV is a s

李馨 ,秦启联卜 ,丁翠 ,刘海虹

LI Xin, QIN Qilian, DING Cui, LIU Haihong

2001, 16(4): 373

A polydnavirus(PDV)was purified by sucrose density gradient centrifugation from the calyx region of female parasitoid wasp Microplitis mediator (Hymenoptera:Braconidae).Negative stain technique showed that the virus was tadpole like and measured 130×35nm.A diverse protein profile of the virus’capsid proteins was displayed by SDS PAGE.Its genome was composed of over 14 DNA molecules,which were different in molecular size and abundance.Molecular size of the genome was approximate 108 kbp calculated by patterns from six restrict endonucleases digestion.Micro injection experiment with calyx fluid of the female wasps showed that the virus suppressed growth and development of host of the wash.

Sequencing and Sequence Analysis of Sl Spike Protein Gene from Avian Infectious Bronchitis Virus Proventriculus Strain Isolated in Qingdao(SD/97/02)

PAN Jie—yah, CHEN De—sheng, DAI Ya-bin, CHEN Pu-yan”

2001, 16(4): 377

A pair of primers were designed and synthesized according to the reported S1 spike protein gene sequence of IBV.The viral RNA of SD/97/02 strain was amplified by reverse transcription polymerase chain reaction(RT PCR).The amplified product was analyzed by agarose gel electrophoresis,all appeared a fragment of 1657 bp as expected.The RT PCR product was cloned into pMD18 T vector,and then the recombinant plasmid was sequenced.The result suggested that it was the S1 spike gene of IBV.The recombinant plasmid was designated pMDQXS1.The sequence has been published in the Genbank,and the accession number is AF193423.Sequence analysis showed that the S1 gene has low G+C content at about 37.0%.There existed several RE cleavage sites such as Hin d III, Bam H I, Bgl III, Sac I and Sal I,but have no Eco R I site,which is different from that of other strains.Its homology compared with other IBV strains was between 87.02% and 94.21%.In the site between 154 and 429 of this S1 gene is a highly variation r

Morphology and Morphogenesis of Manderin fish Virus

FANG Qinl, AI Tao-shan, WANG Ya-pingl, ZHU Zuo-yan

2001, 16(4): 382

The structure and morphogensis of Mandarin fish( Siniperca chuats ) virus were studied.The matured virons were about 135nm±10 in diameter with envelope outside.The intact virus is composed of three parts,its nucleocapsid was about 90nm±5,and the envelope was around 18nm±3,the air space between core and envelope was about 27nm±2?The morphogenesis of the virus was observed in the ultra thin section of spleen tissue cells with outbreak lethal disease,which were collected in different stages of infected mandarin fish.At early stage infected fish section,it showed typical virus entry into cell cytoplasm via viropexis.There were many viromatrix,unmatured,matured and viron released phases,which were found in middle and late phase of diseased fish.In addtion,the same virions were also detected in diseased kidney,liver,heart and gill tissue.Artificial infected fish also caused acute lethal disease with same virus found in its tissue section.

Epitope Mapping of HTNV NP Using mAb 5H5 from a Phage-displayedRalldom Nonapeptide Ia’brary

LI Gueng-yu, B.M Xue—fan, PAN Lei, YANG Wei—song

2001, 16(4): 386

To screen the antigenic epitope with monoclonal antibody (mAb)5H5 against Hantaan virus nucleocapsid protein from phage-displayed constrained nonapeptide library,we have completed the work as follows.(1) Purification and biotinylation of mAb 5H5.(2)Biopanning of the phage peptide library by using the mAb 5H5 as selective moleculars.(3)Identification of the epitope.The positive clones were confirmed by sandwich ELISA,competition ELISA and DNA sequencing.Amino acid sequences of 10 positive clones were compared with the nucleocapsid protein of Hantaan virus strain 76 118.The ten positive clones were selected by mAb 5H5 and the binding between the short peptide and the mAb can be inhabited by natural Hantaan virus antigen.Amino acid sequences of 10 positive clones were consensus:VRDAEEQYE,that is roughly identical with the sequence of amino acid of N terminal of the nucleocapsid protein.These results demonstrate that the linear epitope is the antigenic epitope of Hantaan virus nucleocapsid protein and the rando
Brief Reports

Location and Clone of the Polyhedrin Gene of Ecproctis pseudoconspera Nuclear Polyedrosis Virus

CHANG Guo-hui.CHEN Sheng—hang“, LUO Ban-jun, u Tian-xia

2001, 16(4): 390

The polyhedrin gene of EpNPV was located on Hin d III E, Xba I B, Eco RI J, Kpn I A, Pst I F, Bam H I G fragments under stringent condition(68℃,in water),by using the DIG labeled Bam H I F fragment of AcMNPV DNA as the probe.In order to sequence the EpNPV polyhedrin gene,the Bam H I G fragment was cloned into pTZ19R vector.Then the fragment was digested by Xba I? Hin d III,and subcloned into pTZ19R.

Development of RT-PCR Assay for the De tection of Bluetongue Virus

ZHANG Peng-wei, DONG Charlg-yuan“, TU Pan, GUO Shu-fang, CHENG Xian, YAN Yin-fang

2001, 16(4): 393

Using primers complementary to the conserved sequence previously published of BTV genomic dsRNA segment 7,a part of the 5′end of segment 7 was synthesized and amplified by RT PCR method.We adapted this method to test on 12 blood samples suspected to be with BTV and the blood sample of a health sheep as a negative control.The results suggested that eleven of the twelve samples were positive,one was negative.In order to compare the sensitivity and reliability of RT PCR technique,Vero cells were used to isolate BTV from these suspected samples and the results will be a control.The study proved that RT PCR is a rapid,sensitive and precise method for detection and identification of bluetongue virus in clinical samples,animal quarantine and scientific research.