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2001 Vol.16(1)

Research Article

M olecular Evolution of Guangdong Influenza Virus Isolates in Influenza Outbreak in 1996

HUANG Ping, SHEN Gui—zhang, NI Han—zhong, ZHOU Hui-qiong

2001, 16(1): 1

It was demonstrated that there vcas significantly antigenic drift in the influenza virus strains from Guangdong in 1996 I r was the mokcular basis of the variation oll A、B、C、D and E domain en— coded by HA gene,especially on A、C and E domain while the change of receptor binding domain played the slight roles in the 1996 influenza outbreak 1n the other hand.the variation of No 145 and No 193 encoded by klA gene resuked in biological—feature changes of epidemical influenzu iso~.ates. which could be iso[ated and cultured by MDCK cell—lines。but difficultly by embryonated.eggs

Preliminary Study on the Activation of TRAFs M ediated by Epstein-Barr Virus Encoded LM P1 in Nasopharyngeal Carcinoma

WANG Cheng—xing, LI Xiao—yan, GU Huan—hui, DENG Xu-yun, CAO Ya

2001, 16(1): 6

The present is aimed at identifying the activation of TRAFs in nasopharyngeal carcinoma (NPC)ill"oitro The differentia1 expression of TRAF2、TRAF3 was not detected in RNA and protein 1evel,whereas the expression of TRAF1 in HNE2一LM P1 eel1 1ines was much more abundant than that in HNE2 cell lines, suggesting that TRAF1 niay be an inducible molecule: More importantly. TRAF1,TRAF2 or TRAF3 were activated in the HNE2一LMP1 cells,whereas TRAF1,TRAF2 or TRAF3 were not activated in HNE2 cells as detected by the immunoprecipitation-W estern blotting as— say These data provide an experimental basis for our study beginning from the signal transducation pathway for the eluccidation of the mechanism of LM P1 carcinogenesis in NPC

HSP72.1ike Immunoreactivities in the Brains of M ice Experimentally Infected with Hantavirus

ZHAO Jun, YANG Shou-jing“.LIU Yan-fang

2001, 16(1): 11

Sueking mice.2~3 day after birth,were intraperitoneally inoculated with 0 O5mL F1M lIl 0f Chen Strain Hantaan virus At different time point after inoculation,the brains were taken,rou— tinely fixed and embedded in paraffin for preparing 5 um serial sections Traditional and confocal im— munochemicaI detection of viral antigens and heat shock protein 70 were performed to explore the cere— hral stress response after viral infection.The results showed that HSP70 imm ur~reactivities could be stably detected in the viral antigen positive neurons,hut not in the viral antigen negative or uninfected control tissues By confocaI microscopic examination.the H趼,70 and viral antigens were colocolized in the neuronal cytoplasm .Our result,comparable to our previous findings in human tissues and culture cells。indicated that Hantavirus infection can induce the expression of H趼,70 in the infected cells。and HSP70 expresion might he necessary hut not sufficient to keep the cell surviva1.

Molecular Cloning and Nucleotide Sequence Analysis of the M Genome Segment of Hantavirus Z37,a Chinese HFRS Vaccine Strain

ZHU Han-ping, LIU He—bin, YAO Ping·ping, ZHU Zhi—yong

2001, 16(1): 15

0ne of the strain of bivalent HFRS vaccine.Z37 strain was isolated from Rattus norvegicus and identified aS SEO virus by serological test The M segment cDNA of Hantavirus Z37 strain WaS obtained by reverse transcription and polymerase chain reaction,subsequently cloned into pGEM—T vector.The sequence of positive recombinants was determined by the method of dideoxy chain term[一 nation,which revea led that the M genomic segment is 3651 nucleotide in length with a predicated bng open reading frame encoding a protein of 1133 amino acids Comparison with HNT type(76—118.A9, HV一114 strains)indicatedthattherewere 71 8% ~72.1% homolng y atthe nucleotideleve1.76.2~ 76.7% homology at the amino acid leve1.Co mparison with SEO type (R22,L99.80-39 strains) showed 95.3~ 96 l homology at the nucleotide leve1.95 3~99.2% homology at the amino acid 1ev d The results of nucleotide and amino acid comparison indicated that Z37 strain is SEO viruses in molecular Ieve 1.

Sequencing and Homology Analysis for the Nucleocapsid Gene of JS/95/03 Strain of Avian Infectious Bronchitis Virus Isolated from Jiangsu,China

CHEN De, PAN Jie-yan, JIA Liqun, CAI Bao-xiang, CHEN Puyan

2001, 16(1): 22

Field avian infectious bronchitis virus(IBV)designated as JS/95/o3。which was isolated from Jiangsu province of china,was cultivated in chicken embryo.It’S single strain RNA was extract— ed from purified virus and worked as template of reverse transcription polymexase chain reaction f RT— PCR),a pair of primer designed according to megalign results of published IBV sequences in Genbank was used to amplify the neucleocapsid gene,the RT·PCR product was sequenced directly Sequence analysis revealed that the sequence of JS/95/03 is most homologized with that of M41 strain

Sequence Analysis of ORF3 and Partial ORF1 Region from Two Patients Infected with New Genotype of Hepatitis E Virus(HEV)

W ANG Youchun。, ZHANG Hua—yuan, GU Wen—jie .Roger Ling .LI He-min。.Tim J Harrison

2001, 16(1): 28

ORF3 and partial ORF1 regions were amplified with RT-PCR from ta~vo patients(T1 and TII)infected with new genotype of hepatitis E Virus.The PCR products were cloned and sequenced The results showed that G-C rich region in ORF3 was deleted when amplified with norrflal PCR reac— tion However.PCR reaction containing G-C melt solution can overcome this problem The sequence analysis showed that T1 and Tll belong tO a new genotype of HEV which differs from genotype I,II and III reported.T1 and T11 have 79% -82% .80% -81% and 83% ~85% identical tO genotype I. II and Ill respectively

Analysis of HIV-1 Sequence W orked Abroad and Subtype in Returned Laborers in Shandong Province

SU Sheng—li.XING Hui, LIU Chuan—xin, PAN Pin—liang, HUANG Tao FU Ji-hua, ZHAO Shi-li, SHAO Yi—ming

2001, 16(1): 34

To understand the molecular epidemiologica[characteristic of HIV-1 virus strain in laborers worked abroad in Shandong province since 2992,11 specimens of peripheral blood of identified HIV infected eases were sampled and PBM C was separated by Ficoll density gradient centrifuge. The pre— virus DNA was extracted and the membrane protein gene enzt DNA was multiplied by nested PCR, and then the sequence of env C2一V3 region*as measured and analyzed.The results indicated that 10 labo rers worked abroad and 1 female—spouse of a labo rer worked abroad weAiTe found catching HIV一1 A , B ,C,E subtype by gene sequencing and analyzing,with subtype C 7,subtype B 2,subtype A and E 1 respectively Thus it can be seen that the infection of HIV virus strain in the 1aborers worked abroad was complex The gene variation rate of these virus strains was greatly different.It is suggest— ed that we strengthen the detection and control of the 1aborers worked abroad to prevent the spread of the virus strains from other countries


ZHANG Dongwei, LIAN G, QI Zi, LING Shi

2001, 16(1): 40

HCV RNA positive serum was first selected by RT PCR test kit from several anti HCV positive sera obtained from Xi’an.HCV RNA extracted from the elected sera was converted to cDNA by reverse transcription with random primer.Half nested PCR was performed.The amplified product was 852 bp.The purified PCR product was digested by restriction endonucleases and then ligated to epressio vector pET 22b\++.Its nucleotide sequence was determined by dideoxy chain termination method.A comparison of the sequence with several isolates reported previously showed that the sequence belonged to HCV type Ⅱ.

Cloning of 3’-terminal Part of Sugarcane M osaic Virus RNA and Sequence Comparison of CP Gene Between Chinese Isolate and Other Strains of SCMV

LI Li.jun.ZHOU Zhong.ju.XIE Lian-hui

2001, 16(1): 45

ScMV.CA,an isolate of the doraJnant A strain in China continent was propagated on sweet maize and purified.Cloned cDNAs representing more than 0.8kb of the 3’termini were obtained after a reverse transcription and ligation to pUC19 in Sma1 site using an ollgo(dT)primer One clone na med SCMV.CA54 was sequenced and the 1ength of the SCMV—CA54 was 1296 bp.The sequenee covered the 3’untranslated region(3’UTR).coat protein(cP)mad part of the nuclear inclusion b (NIb)genes of the virus.The nucleotide and deduced amino acid~equenee in the CP gene hatweell Chinese isolate SCMV.CA and other isolates or strains of SCMV subgroup were compa red.Results showed that the homology of nucleotide sequence in CP gene between SCMV—CA and other isolates or strains of SCMV subgroup ranged from 63.7% to 77.6% .and amino acid sequence from 64% to 89% .This homology degree lies betwen the range for distinct viruses and tha t betwen related strains of potyviruses,arguing tha t the Chinese isolate might represent a distinct virus.

Cloning and Sequencing of M ovement Protein Gene of Cymbidium M osaic Virus

LIU Zhi-xin, WU Hao.PAN Jun-song, ZHENG Xue-qin

2001, 16(1): 51

The viral RNA was extracted from purified cymbidium mosaic virus(CyMV)isolated from Oendrobium orchid cultivated in Hainan island.The gene of the movement protein(MP)was amplifled by mealls of reverse transcription—polymerase chain reaction(RT.PCR),and cloned into pGEM T easy vector. Sequence analysis showed that the gene fragment contained 3 open reading frames (ORbs)which may be encoding 14 kD、12 kD and 10 kD peptides The nucleotide sequence of the doned gene~agment shared 97.8% homology with the MP genes of CyMV isolated from orchids cultivated in Hawaii and Sing apore

Block of Viral M ovement in Papaya M utant Plants Resistant to Papaya Ringspot Virus

ZHoU Guohui, LI Hua—ping ZHANG Shu—guang FAN Hwai—zhong

2001, 16(1): 55

One—step RT—PCR was used to detect papaya ringspot virus(PRSV)movement in susceptible and resistant(mutant)papaya(Carla papaya L )plants.The time of post—inoculation at which PRSV Wc-lS first detected in susceptible plants was 48 hours in the other area of inoculated leaves OUt Of inoculated sites.4 days in the petioles of inoculated leaf and 6 days in all parts of plant.Compa rative— ly,in resistant plants,the inoculated sites were the only area at which PRSV can be detected any was proposed that the mutated resistance to PRSV protects the virus from moving out of inoculated sites or/and into non- noculated areas.

Comparison of Dynamics in Viremia Levels in Chickens Inoculated with M arek’s Disease Virus Strains of Different Pathotypes

YIN Jun, CUI Zhi-zhong

2001, 16(1): 59

Abstract:One day-old unvaccinated chicks were inoculated with serotype I virulent strain GA and vac— cine strain CVI988.semtype III vaccine strain HVT of Marek’s disease viruses(MDV).By using an indirect immunofluorescence assay of suspensions of peripheral blood mononuclear 0e11s(PBMC)with monoclonal antibodies(MAb)BA4 and B specific to glycoprotein B of MDV,The dynamic courses of viremia levels for each of 3 strains from 4 days to 45 days postinfection(PI)were determind.Viremia f0r virulent strain GA—infected chiekens were detected from 4 days PI untiI days before death of infect— ed got its peak at about 14 days PI.Viremm in serotype 1 vaccine CVI988一inoculated chickensweremeasured between 4t022 days couldbe detectedfrom 4 days PIan d ended at18 days PI,it got its peak at 8 days PI.For serotype 3 vaccine HVT-inoculated chickens,viremia could be detected beween 4 to 14 days PI,its peak happened at about 6 days PI.Such IFA with MAbs BA4 and B could be used r。evaluate quality of vaccination process in chicken flocks.Tbe differentiM dy— namie courses of viremia for different strains may be used t0 diagnose virulent V infection in vacci— hated flocks.The viremia levels measured by IFA with the MAbs an d co-cultivation assay for plague forming unit(PFU)were compared.The results indicated that IFA assay of PBMC suspension was more sensitive and specific than co—cultivation assay.i.e.viremia levels measured by IFA were 30 t0 100一fold bigher than that by plague—form ing assay in CEF for the same sampie

Study on Purification and Immunogenicity of Rabies Virus Nucleoprotein

SU Yan, W ANG Ji—lln, YANG Xiang-ping, XUE Hong-gang.ZHU Jia-hong

2001, 16(1): 64

Abstract:In order tO establish the optimum condition for purification of the nucleopmtein(NP)of rabies virus by immuno0finity chromatography,the efficient and non-danatulative eluants(Mg-d)Ⅵ忸s obtained by using ELISA elution model; didn’t damage the activity of NP.Two kind of NPs,expressed by Te— combimnt vacclnia virus(rVac—N)and~,o3mbinant haculovirus(BRN),were purified by a Sepharose CL 413 column and a 2C12一Sepham~e 4B oolurrm ByWestern-blot and SDS-PAGE.highpurityandgood antigenie,~ intact NPs were identified. purified ribonucleopmtein(RNP) rabies virus 5aG strain was also 0brained. Aftel"immunized with NP and RNP.mice developed a strong anti-nudeoproteln response and were protected against a lethal chaHenge of rabies virus CVS strain There were not diference been observed araong the mice immunized with different purified protein These data indicate that the NPs are antigenical and immunagenical oomparabletothe authentm rabies RNP aridtherefore present a potantial sour~ ofan dk t ,safeand e∞ nomical subunit vaccine

Cloning and Sequencing of Envelope Gene of Subgroup J Avian Lenkosis Virus

QIN Ai, CUI Zhi, Lucy Lee, Aly Fadly

2001, 16(1): 68

The envelope gene of ADOL-4817 strain of avian leukosis v[ros subgroup J(ALV—J)was amplified by polymerase chain reaction(PCR)and cloned into TA vector.The sequence analysis re— suits showed that tbe envelope gene is composed of 1 746 bp.1 554 bp of which could be translated in— to 517 amino acids for gp85 and gp37.The molecular weight of envelope protein is 57.7kD There are I5 potential glycosy[ation sites in the envelope protein,13 of which is located in gp85,Analysis of se— quences of envelope gene indicate that ADOL-48 17 showed high degree of sequence identity to other ALV—J strains,and most closely related to the like—envelope gene of endogenous virus EAV—HP but di— vergent from these of other ALV subgroup A-E .These data support the hypothesis that envelope gene of avian leukosis virus suhgroup J maybe acquired hy l:ecombination with expressed sequences

Isolation of Japanese Encephalitis Virus from Brain Tissues of Bat in Yunnan Province

ZHANG Hai, ZHANG Yun-zhi, HUANG W, MI Zhu, GONG He-qin, WANG Jing

2001, 16(1): 74

In July 1997, a strain (GB30) of virus was isolated from 60 samples of brain tissues of Murina aurata (Chiroptera: Vespertilionidae) collected in Gengma county, Yunnan province, China. Isolation of virus was negative from 4 samples of brain tissues of Rousettus leschenaulti (Chiroptera: Pteropodidae) collected in Gengma. GB30 virus strain could regularly cause illness and death in suckling mice, produced evident CPE in BHK21 cells. It agglutinated red blood cells of dove at pH5.75~7.4. This virus has been identified serologically by hemagglutination inhibition and immunofluorescent tests using Japanese encephalitis (JE), dengue (DEN) type 1,2,3,4, and chikungunya (CHIK) viruses monoclonal antibodies, and JE and sindbis (SIN) viruses immune sera. It showed specific reaction to JE virus only and no reaction with DEN 1~4, CHIK and SIN viruses. Therefore it can be identified as JE virus. This is the first report on the isolation of JE virus from Murina aurata . The results showed that bats are consider

Expression of P 19 Gene of Avian Leukosis Virus in Escherichia coli

LIU Gong, ZHAO Zhen-fen, LIU Fu-an

2001, 16(1): 78

Abstract:Based on avian 1eukosis virus(ALV)p19 gene termina1 nucleotide sequence,a 82 bp dou— ble.stranded DNA fragment was chemically synthesized and cloned into the expression eect~r pGE— M EX.1 The sequencing result indicated that the cloned fragmentⅥw a correct version of the one de— signed both in nucleotide sequence and in its open reading frame.The recombinant was used to transfo/ Tn E.∞“ BL21(DE3) The cloned fragment was expressed as a fused protein with T7 gene 10 leader peptide and was shown to be 34 kD in size on SDS—PAGE ge1 when induced with l mmol/L IPTG.The expression product was able to bind immunologically to rabbit anti—ALV serum in W est— ern-blot assay and is being tested to differentiate exogenous from endogenous ALV .

Homology Comparison of White Spot Syndrome Baculovirus(WSSV) from Penaeid shrimp

HUANG Can-hua, SHI Zheng, ZHANG Lu, XIE Yun-li, ZHANG Li, CHEN Di, wU Qing-jiang

2001, 16(1): 81

Homology of three WSSV isolates.which were sampled from representative maritime space of China:Tanghai isolate(13o Bay of China),Ningbo isolate(East China Sea).Shenzhen isolate (South China Sea)was compared.Both of the genome RFLP patterns and the characteristic structural proteins SDS-PAGE electropherograms showed that they were quite$a/’ae.It suggested that they were the same kind of WSSV virus that caused explosive epidemic diseases of shrimps(EEDs)throughout southern and northern China The$drne large PCR products achieved when using the PCR primers from RV—PJ:PRDV (P japonicus.Japan)and WSBV=PmNOBIII(P.morR~don Taiwan,Chi— na)respectively to amplify the genome from P.chinensis(Tanghai,China)with high fidelity Taq Polymerase.The sequence identities of WSSV from P.chinens~ with those from RV—PJ=PRDV (P 血ponicus,Japan)and WSBV=PmNOBill(P.monodon Taiwan.China)are 97% and 100% respectively,the results provided additiona l evidence that W SSV reported in different parts of the Asian and Pacific regions maybe quite the same or jLISt different variants of the same virus
Brief Reports

Study of Rheum officinale Against Coxsackievirus B3 in vivo

SHEN Yuan—ying, YANG Zhanqiu, LIU Jian.iun . al

2001, 16(1): 85

In order to search a effective therapy for viral myocardifis,antivial activity of Rheum offici— hale on Coxsackieivirus B3(CVB3)was evaluated in BALB/C mice.After being determined optium scheme with orthogonal design,mice infected with CVB3 were treated with Rheum officinale.rib— avirin and 0.9% NaC1 The survival rates of the mice were 35% ,5% and 5% ;M ed Jan survival time were 23.11 and 10.5 days;Infectious viml titers repecfively,The lesions of cardiac tissues lightened were 10一 ,10一 ’and 10一 ’TCID50/0.1 mL inmicetreated withRheum offinicale.These re— suits showed that Rheum officinale was effective in treatment of CVB3 infected mice The study pro— vided scientific basis for further research and widen antiviral spectrum of Rheum officinale

Observation of the Expression of HCV NS 5 Antigen in vitro by the SABC Immunological Techniques and Gold·labeled Colloid Electron M icroscopy Method

YE Jin, ZENG Linglan, YANG Mu, LUO Duan, GUO Jinsong

2001, 16(1): 88

To study the expression of HCV non.structure 5 antigen in vitro,a human HepG2 cell Iine was incubated with a HCV RNA positive serum.The SABC immunologieaI techniques and gold.1abeled colloid electron microscopy method were employed tO examine for the viral proteins in those calls.The HCV non.structure 5 antigen was first detected in the HepG2 cells at 72 hours poSt ineubation.The antigen WaS continuously observed in the cytoplasm or on the membrane O.S we1I on the eell wall of the HepG2 ceils even after 1.2,3 and 4 weeks post incubation.The observation of HCV non.structure 5 antigen continuously expressed in the HepG2 cells strongly indicates that the cells may have been in. fected bv HCV virus and the virus ma y have replicated in the ce lls.Therefore.the HepG2 eell line may be served as a potentiM host for establishment of HCV infection and propagation in vitro

Dynamic Distribution of Shrimp White spot syndrome virus(WSSV)Qingdao Strain in Crawfishes by Dot Hybridization

ZHU Jian—zhong, XIA Xiao-qin。.LU Cheng—ping .GUO Fu.shengz

2001, 16(1): 92

A fragment sized 40Obp ofWhite spot syndrome virus(WSSV,formerly designatedNOSV), recovered from recombinant plasmid pAFD.was labeled with Digoxigenin as a probe to detect dynamic distribution of WSSV within 120h and 72h in crawfishes(Cambarus procla ii)inoculated WSSV by oral taking and injection respectively.Stomach epithelium, intestine epithelium, heart, gill, haemolymph,muscle,hepatopancreas-hypoderm ,connective tNsue and ovary of infected crawfishes were examined for W SSV In both groups.W SSV was first detected in heamolymph at 12h P.i.and then disappeared Again it was detected at 96h P.i.only in oral infection group and maintained till 120h P.i.,but it didn’t appear at 72h injection group.WSSV in heart.muscle was detected at 36h P i in oral infection group and 24h P.i in injection group respectively.and then increased generally.In addition,W SSV in intestine epithelium,con ective tissue,ovary of oral infection group and intestine epithelium.hypoderm,ovary of injection group could also be detected.In dead crawfish— es after l20h and 72h P i in two groups.WSSV could be detected in all the examined tissues and it demonstrated that systemic infection occurred in the animales.The tissue containing more amounts of W SSV was hypoderm in oral infection group,while intestine epithelium.gil1.hypoderm,ovary in in· jection infection group It deduced that W SSV first appears in haemolymph and then goes into hean, muscle and other tissues and proliferates in them.Once again,W SSV is released into heamolymph re— sulting in systemic infection till crawfishes’death