2003 Vol.18(4)
Abstract：In this Paper we repoaed that Cytomegalovirus infection induced high levels of Cyclin E， increases of Cyclin E／cdk2 activity，leading to cell cycle arrest．By 72h postinfection(at a multiplicity of infection of 1 0 PFU／per cel1)，29％ of the cells was S phase，69％ of the cells in Gz／M phase，only 2％ of the cells in Go／G1 phase by flow cytometry an alysis．By 20h postinfection，the levels of cyclinE in infected M RC．5 cells were eightfold greater than those in mock-infectcd cells by ELISA an alysis．The cyclin E／cdk2 kinase activities in HCM V-infected M RC一5 cells were relative to the levels of cyclinE essentially．
Abstract：To study the apoptosis of malignant carcinoma cells induced by Hantaan virus．SP2／0 cells cultured in vitro were infected with Hantaan virus．Their apoptosis profiles were detected using Giem sa staining，flow cytometry and immunohistochemistry staining．Giemsa staining showed typical apoptotic changes in infected cells；Apoptosis peaks were detected by flow cytometry； Immunohistochemistry staining showed apparent increase of Fas and FasL expression in infected cells compared with norm al cells．Th e results suggest that Hantaan virus could induce SP2／0 cells in vitro to undergo apoptosis which might be associated with Fas an d FasL．
Abstract：A human endothelial cell line(ECV304)derived from umbilical vein was used to study the mechanism of Dengue virus type 2 fDEN2)infection on endothelial cells．VimS growth curve on the ECV304 cells showed that the cells were sensitive to DEN2 infection．M embrane preparations from ECV304 cells were collected either by mechanical scraping or by trypsin digestion．SDS-PAGE an alysis showed that there lacks of a protein with a molecular size 43 kDa in the membran e preparation treated by trypsin．Virus overlay protein binding assays(VOPBA)w． ere performed by binding∞S-M et labeled DEN2 with separated membrane proteins of ECV304 cells．Three proteins each of 29．34 an d 43 kDa located on the surface of ECV304 were found to bind with the labeled viru s．The virus．binding efects of the 34 and 43 kDa proteins could be inhibited when the ECV304 cells were treated with trypsin． Preincubation of ECV304 cells with rEgp could inhibit DEN2．binding an d block viru s infection． Preincubation of the protein electro—transferred nitrocellulose membran e with the rEgP could alSO block DEN2 binding with the three identified proteins on the surface of ECV304 cells in VOPBA．Th e result suggests that three proteins each of 29．34 and 43 kDa on the surface of ECV304 cell might associate with the receptor complex for DEN2 binding，and envelope E glycoprotein could mediate initial binding of DEN2 to human vascular endothelial cells．
Abstract：To assign the ck．borne encephalitis virus(TBEV)Senzhang strain isolated in China to one of three TBEV subtypes and understand the relation between the polyprotein structure an d biological function an d aid in the development of new TBEV vaccines．the nucleotide sequence of the polyprotein of Senzhan g strain was determined．Nine pairs of primers were designed according to the nucleotide sequence of Sofjin．HO。Oshima 5·10 strain．By RT·PCR，cDNA fragments of Senzhang strain were acquired from infected BHK．21 cells．Then the cDNA fragments was cloned into the vector pGEM ％T and then tran sforrned to competent DH5 Q cells．Positive clones were screened an d identified by EcoR I cleavage an d PCR．Th e sequences of inserted fragment were determined bv 3 1o0 Genetic Analyzer． Sequence analysis showed that Senzhang strain contains a sigle open reading frame(ORF)of 10245 nucleotides(nt)which encodes a polyprotein of 34 1 4 amino acids(aa)．Senzhang strain is more similar to Oshima5·10，So1]in·HO，So1]in，and assigned to the Far Eastern Subtype．
Abstract：Recombinant plasmid pCDNA3一Core was constructed by inserting Hepatitis C virus(HCV一1 1 core gene into the site between EcoR I and Xba I of eukaryotic expression vector pCDNA3．The recombinan t plasmid pCDNA3一Core was tran sfected to COS一7 cellline by LipoVec ．Expression of core protein was detected by immunology Dot blot 24h after transfection．Th e nuclear cracks in tran sfected cell was observed by Hoechst 3325 8 staining and the 1 80—200bp DNA ladders alSO was detected 72h after transfection．Th ese results reveal that HCV Core protein Can induce apoptosis of COS一7 cell and its function nlay play an importan t role in HCV chronic and persistan t infection．
Abstract：The genome sequences of SARS．associated coronavirus (SARS．COV)were compared witIlother Coronaviruses．Similar sequences were found by searching biomolecular databases．Sequenceanalysis showed that SARS．CoV had similar genomi c organization and structural proteins whilecompared with other Coronaviruses．SARS．CoV is related to other known Coronaviruses．But therewere some specific sequences in SARS．CoV genome．Th e virulence of SARS may deftve fromvaxi~ions of ORF l a an d S protein an d the specificity of some nonstructural proteins．Th e result of wordfrequency an alysis indicated that SARS．CoV does not closely resemble an y of kn own Coronaviruses．
Abstract：Not only the output of insecticide DpCPV is always afected by climate and circumstan ces ， but also af ected by the technology of production highly．Studies have been done on DpCPVHn indoors and outdoors for the Dendrolimus punctatus quanlity of every collection an d the inoculation concentration an d the inoculation frequency an d the harvest time． The optimal conditions of multiplication have been gained．Th e optimal inoculation concentration is 1× 1 07CPB／mL ．Th e optimal Dendrolimus punctatus larvae for multiplication are 4 or 5 instar larvae．Th e optimal CPV ingathering time iS 13th or 14th day after inoculation．Th e OUtdoor yield of each larva can be 7．5× 10 CPB ．It iS higher than other reports in China．It is very important to guide high output of DpCPV multiplication．
Abstract：In order to make bases for construction of animal tumor model which expresses EGFP— HPV l 6E7，recombinant expression plasmid was constructed by techniques of gene recombination after screening an d identifing by restriction and PCR．Then the recombinant was tran sfected into mouse liver cancer cell by techniques of gene transfection and detected expression by fluoroscopy．pEGFP—HPV 1 6 E7 was identified by enzyme digestion an d PCR．Th e resent showed that the length ．inserted location and direction of the target gene was correct an d the expression of EGFP in tran sfected cell was observed Because of it’S pEGFP—HPV 1 6E7 expression plasmid that mak es it easy to assess the expression of EGFP-HPV 1 6E7 fusion protein and to sift the transfected cells．
Abstract：Korean isolate was studied on the shape and structure，structural polypeptide，restriction pattern．Diameter of polyhedron is 0．36~1．3Um， size of virion is 326 am × 69nm．Assayed by SDS—PAGE，polyhedron of Korean isolates contained a single polypeptide whose molecular is 28．7kDa． genome is 1 30．1 8kb．Genome digested by BamH I，EcoR I，Hind III and Pst I，map attained is very similar to the other isolates reported．
Abstract：DendrolimUS punctatus polyhedra were purified from infected Dendrolimus punctatus Larvae using density centrifugation．Dendrolimus punctatus cytoplasmic polyhedrosis viruses(DpCPV)were subsequently extracted from polyhedra by SDS treatment，followed by l％ agrose gel electrophoresis， and the segement dsRNA 5 was purified after being excised from the ge1．On the basis of nucleotides homology，five pairs of primers were designed．After the RT—PCR，five sub—clone segments were obtained，following sequencing and splicing，the complete nucleotide seq uence of the genome segment 5(S5)of DpCPV was determined．The 285 1-nucleotide sequence contains a single long open reading frame which span s nucleotides 7 1 to 27 1 4 an d was predicted to encode a protein with a molecular mass of about 100．3kDa．Sequence an alysis an d deduced amino acid seq uence of DpCPV S5 revealed high homology with proteins from LdCPV一1 an d BmCPV．From the phylogenetic tree，we can an alyse the classification an d evolutionary relationship in viruses．
Abstract：As the close consan guinity of human race，the common cotton—eared marmoset(Callithrbc Jacchus)has been used in a wide range of research as a primate mode1．Acute respiratory infection(ARI) broke out in a colony reared in the experimental animal center of Tianjin Medical University in 1 999．A virus strain with high hemagglutinin titer was isolated out from embryonated eggs an d M DCK cells after inoculating with the lung tissues suspension of the dead marmosets． The data of the hemagglutination inhibition (HI)test with paried serum samples and the animal inoculation test confirmed that the isolated virus strain is the pathogen of the ARI．Th e isolated virus strain was then identified by the cross HI test。electron microscope observation。RT—PCR an d BLAST analyses．The virus was verified to be Sendai virus，which belongs to Parainfluenza virus type 1．virus was verified to be Sendai virus，which belongs to Parainfluenza virus type 1．
One ORF of White spot syndrome virus(WSSV)genome has homology with the cytokine receptor．This ORF contains 2022 nucleotides and codes for 674 amino acids with a theoretical molecular weight of 76 kDa．Th is putative gene contains one conserved motif of eukaryotic cytokine receptor gpl30．To investigate the function of this protein，the gene was amplified from the genome of WSSV by polymerase chain reaction(PCR)，then cloned into pET28b and expressed in E．coli．Th e expressed fusion protein tagged with six consecutive histidines was 76kDa after SDS—PAGE．Th e purified fusion protein was used as antigen to immunize rabbit for further gene function study．
According to the published OI 2 gene sequence of PCV一2．two primers were designed to amplify the ORF2 gene from PK一1 5 cells inoculated by PCV一2．It was cloned into the pSecTag2 vector,and the recombinant plasmid named pSecORF2 was obtained．Another primer was designed to amplify OI 2 gene including signal peptide sequence from pSecORF2．Then it was cloned into pIREShyg vector．Th e recombinan t expression vector plRESiORF2 was constructed．CHO cells were tran sfected in vitro with them． Th e expression of ORF2 gene was detected by indirect immunofluorescence assay(If1A、．Further research iS underway to study the biological activity of OI protein an d to establish the diagnostic kit of PCV
The complete genome of type 2 Porcine circovirus(pcv一2)was amplified by polymerase chain reaction(PCR and cloned directly into me EcoRI sites of plasmid pcDNA3，and recominant plasmid carrying the complete genome was constructed，designated pcDNApcv2．The entire genome of PCV一2 was purified and recycled from pcDNApcv2 with the digestion of EcoRI enzyme an d then circular genomic DNA was generated by self-ligating with T4 DNA ligase in vitro．Th e non—infected PK一1 5 cells were transfected with the PCV一2 circular genome using Lipofectin Reagent．After four continuous passages，PCV一2 virus and specific antigens were visualized by electron microscopy an d IFA，respectively．Th us，the cloned circular PC V一2 genomi c DNA generated in this study was infectious in vitro．
Abstract：The complete nucleotide sequences of the RNA2 of two Chinese isolates of Rice stripe virus are determined．One is isolated from endemic sites at Chuxiong(cx)，Yunnan Province。the other is isolated from outbreak sites at Hongze(HZ)，Jiangsu Province．Th e total nucleotide sequences of the RNA2 Of RSV CX and RSV HZ are 3506 bp an d 35 14 bp long．respectively．W hen compared with RNA2 Of T and O isolates of Japan ．we find that these four isolates could be divided into two groups． HZ．T and O isolates share 97．2％ ～98．O％ an d 96．8％ ～97．1％ identities in vORF2 an d VCORF2 at the nucleotide leve1．respectively an d form one group．The sequences in 5 an d 3 terminal non-encoding region are completely identical among these three isolates．In this group．HZ isolate is more closely related to T isolate than to O isolate．Th e length of intergenic region fIR)of HZ isolate iS the same as those of T and O isolates．CX isolate belongs to an other group．which shares only 95．0％ ～95．7％ an d 93．9％ ～94．4％ sequences identities in vORF2 an d VCORF2 between two groups at the nucleotide leve1． respectively．Th ere is a deletion of 8 nt in length in the IR Of CX isolate compared with HZ isolate．Even though no base variation occu~ed in 5 terminal non．coding region，there is one base substitution in 3’terminal non—coding region between CX and HZ isolates．These results show that the isolates aregrouped according to their geographical location． Additionally， highly consensus in 5 and 3non—encod ing region suggests that these regions play a very important role in tran scription andreplication of viral genome．Finally，the molecular epidemiology and gene functions of RSV arediscussed in this paper．
Abstrat：Plaques counting is a routine method in the theory research and practiced application of microbiology．Because of weak contrast between plaque an d the background of the plate，an d the plaques linking together,a serious error was produced using automatic counting by computer．So that plaques have been counted by artificial method．In the present study，a new method was established for automatic counting plaques by computer．At first，The plaques were formed by infecting Escherichia coli with 入bacteriophage，and prepared as the method of soft—agar layer plate．Th e electrical images counted the plaques were obtained by screening the plaques plates．A representative area，in which the plaques were linked each other,was picked out from each image，an d han ded with watershed algorithm． Th e plaques linking together were separated into individual plaque alone in the images．And then，the images were counted with the method of growth algorithm based on area by computer．Th e exact effect of counting plaques was in agreement with artificial method ．It indicates the new method of counting plaques can be used in automatic counting by computer．
Abstract：The viral RNAs were isolated from the infected Vero—E6 cells．then the templates were produced by reverse tran scription reaction．Two pairs of primers were used to amplify the 5 一ends of four strains of SAR． ssociated coronavirus(SARS—CoV)by RACE．cDNA fragments with the length of about 420 bp were amplified．The fragments were then purified an d sequenced．The sequences of 5 一UTRs of SARS —CoV isolated in China were the same as those isolated in other countries an d regions． such as Tor2 strain，Urbani strain，HKU—Su l 0 strain，CUHK—W l strain，S 2500 strain an d SIN2677 strain．Th e secondary structures formed by 5 一UTRs of all known SARS—CoV were almost identica1．but significan tly diferent from other kn own non—SARS coronaviruses．Sequence an alysis indicated that the conserved core sequence(5 一CUAAAC一3 1 of transcription regulating sequence in some coronaviruses was about—l 97 nt upstream from the start codon．
With the specific primers YP1 and YP2 which were designed based on the reported Potato virus Y(PVY)pl gene sequence，the target gene(0．83kb)was amplified by RT—PCR using the total I A extracted from PVY infected tobacco plant as templates．The gene was cloned into plasmid an d tran sformed then E coli．DH5a sequenced．The result of sequence analysis shows it iS the PVY pJ gene． The comparison shows that there are significant differences on the P1 amino acid sequence am ong the known PVY isolates。the identity ran ges from 64％ tO 94％ 。Based Oil P1 amino acid sequence，the PVY phylogenic tree was established an d the isolates of PVY were clustered into man y bran ches．
Abstrat：Plaques counting is a routine method in the theory research and practiced application of microbiology．Because of weak contrast between plaque an d the background of the plate，an d the plaques linking together,a serious error was produced using automatic counting by computer．So that plaques have been counted by artificial method．In the present study，a new method was established for automatic counting plaques by computer．At first，The plaques were formed by infecting Escherichia coli with 入bacteriophage，and prepared as the method of soft—agar layer plate．Th e electrical images counted the plaques were obtained by screening the plaques plates．A representative area，in which the plaques were linked each other,was picked out from each image，an d han ded with watershed algorithm． Th e plaques linking together were separated into individual plaque alone in the images．And then，the images were counted with the method of growth algorithm based on area by computer．Th e exact effect of counting plaques was in agreement with artificial method ．It indicates the new method of counting plaques can be used in automatic counting by computer．
Abstract：The envelope proteins of White spot syndrome virus(WSSV)are very fragile and easy to be destroyed during purification．It was difi cult to obtain a large quan tity of intact virions by routine sucrose gradient centrifugation．After modifying the sucrose gradient by adding citrate sodium ，we can obtain a large quantity of intact virions and nucleocapsids．Th is purified virions and nucleocapsids were subsequently used for an alyzing viral structural proteins an d DNA extraction．Th e result showed that this modified technique is very efi cient for virus purification．
Virion RNA was abstracted from purified type I Avian paramyxovirus strain YN—PA0 1 (isolated from parrot)and used as a template．The fragment containing the fusion gene(F)and hemagglutinin-·neuraminidase gene(HN)of the isolate was amplified by RT-·PCR and cloned to the pMD 1 8一T Vector．Using primer walking method the complete sequence of F-HN genes was obtained finally． And the respective amino acid sequence was deduced．Through relative software the phylogenetic trees on F gene an d HN gene were constructed between strain YN—PA0 1 an d reference strains．The results showed that strain YN—PA01 comparing with reference strains displays 98．7％ -83．2％ nucleotide homology an d 98．1％-86．2％ amino acid homology on F gene；97．4％-79．1％ nucleotide homology an d 97．2％ -83．2％ amino acid homology on HN gene．Additional 6 amino acids are encoded by the HN gene ORF of strain YN—PA01 comparing with national reference strains．Th e studied strain YN—PA01 exhibits highest identities with strain JS／5／0 1／Go either an alyzed on F gene or HN gene．
Abastract：Most of the cyanophages were adsorbed onto the ultrafilter membrance when pre—filtrated water sample passed through the tangential flow ultrafiltration system．In order to recover much more cyanophages，we used a technique named‘‘backflushing ultrafiltration”．The results indicated that when the volume of the backflush liquid was raised step by step，the concentration multiple of the cyanophage would gradually decrease，while the recovery rate of the cyanophage would gradually increase．In our performance，the maximal concentration multiple an d the recovery rate of cyan ophage could reach up to 30 times an d more than 90％ severally．Th is method was tested to be efective for concentrating cyan ophage from large volume liquid sample．M oreover，the concertration multiple of the cyan ophage could be elevated to nearly one—hundred times when two—step backflushing ultrafiltration was adopted．
Two pairs of PCR primers were designed according to the sequances of the vaccine strain andvirulent strain of CPV．Heminested PCR method was established．Result of the first PCR amplificationshowed the same am plified products of 574bp length ，after the second PCR am plification，the viru lentstrain produced the length 364bp fragment，but the vaccine strain couldn’t produce that．The products ofPCR were examined by electrophoresis an d restriction enzyme digestion．Th e result showed the lengthof the fragment an d enzyme sites were as the same as those designed．Th e PCR assay of CPV wasproved to be spec ific an d sensitive．It shows that this method may be used in discriminating the vaccinestrain an d viru lent strain of CPV or monitoring the vaccinated can ine in order to aviod disease andfinan ciallosing．
Morphological characterization of purified SARS．associated virus(SARS-CoV)from Hubei patient was carried out by negative stain and ultrathin section electronmicroscopy．The spike of isolated SARS．CoV virus is shorter an d smaller than Human coronavirus．A large quan tity of SARS-CoV particles could be observed in the infected Vero cells．Th e process of infection，assembly an d morphogenesis was observed．
