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2002 Vol.17(2)

Contents

Cloning and Analyzing a New PEBP Like Protein Gene in Human Fibroblast Cell Infected by HSV I

W ANG Jlong.LIU Long—Ding, SUN M ing, DONG Cheng-Hong, WANG Li—Chun, WANG Jing-Jing.LI Qi—han

2002, 17(2): 97

In our study on the gene response to virus infection,a colony of 705bp—HSP一714 was se— lected from the cell specific cDNA library induced by HSVI infection.The gene sequence analysis re· vealed that it was a new complete gene sequence(Gene Bank Accession number~AF372624)with a complete ORF encoding 126 amino acids.The biological informatics aiualysis demonstrated that this pmtein contained a phosphatidylethanolamine—binding protein(PEBP)like domain,which had a high homology to the CEN—like family of plant and mam malian ete.genome.Furthermore,the evolution tree demonstrates that this gene shows more homologous to CEN—like family Of plant Therefore.it can be predicted that this gene might be a new member of human PEBP—like family with special significance

Research of Antiviral Activity of Pcnciclovir to Herpesvirus in vitro

XIAO Chun—hui, YANG Zhan—qiu, XIAO Hong, XIONG Zeng-hui, W EN Li, HOU W ei, LIU Hong

2002, 17(2): 102

In order to evaluate the cytotoxicity and antiherpesvlral activity of penciclovir,the antiher— pesvlral effect of PCV was investigated by observing eytopathie efect(CPE)and the change of viral infectious titre jn cells infected with herpesvirus,antiviral index and treatment index,The result shows the 50% toxicity dose(TD50)。f PCV to HEL ceil and Hep-2 ceil are 105.2txg/mL and 85.1~g/mL respectively;the average 50% inhibitory coneertration(IC50)of PCV to HSV一1,HSV一2,VZV.HSV一1 wU strain are 21 78fxg/mL,20 15~tg/mL,23.19~g/ml and 17,875g/mL respectively:teat index are 5 84,6 31.5,49 and 7 1 1 respectively.It is indicated that penciclovir is an effective anti—herpesvirus compound in vitro,
Research Article

The Analysis of HEV Genotypes Isolated from Sporadic Acute Hepatitis E in Shanghai

LAN Hai, W ANG, u chun, ZHANG Hua, PENG Hui, LIN Jing, GU Wen, WU Xing, LI He

2002, 17(2): 106

In order to analyze HEV genotypes isolated from sporadic acute hepatitis E in Shanghai, HEV RNAs were detected with RT—nPCR and the positive PCR products were cloned and sequenced The genotypes of the isolated sequences were analyzed with the molecular biological software.The re— suits showed that 9 of 35 sera from patients diagnosed as acute sporadic hepatitis E were positive for RT-nPCR.8 of 9 PCR positive products were confirmed to be related to HEV sequence by sequencing. 1 of 8 sequences belongs to HEV genotype l and 7 of 8 sequences belongs to HEV genotype 4 This in— dicates that the infection rate of HEV genotype 4 is higher than that of HEV genotype 1 among the pa— tients with the diagnosis of acute spo radic hepatitis in Shanghai.

The Studies on the Host Cell Apoptosis of Yunnan Sindbis Virus

HUANG Wen— ZHANG Yu-zeng.WANG Jing—lin.ZHANG Hal lin

2002, 17(2): 110

The one step growth curve of YN87448 strain virus in BHK21 cell showed that a lot of progeny viruses could be observed at 16 hr.after infection The tiler of virus reached the highest about 10一 0TCDS0 at 48 hour post infection.The titer of virus is not reduced by Co1.The apo ptosis was observed by the electron m icroscope and the DNA ladder was detected by agarose gel eletrophoresis. YN87448 strain virus can cause apo tosis in BHK21 cell and mice

Constraction of Replication—defective Hum an Foamy Virus Vector Directing Expression of Foreign Genes

LI Zhi, YANG Pin, LIU Hui, LI Wen—xin

2002, 17(2): 114

A replication—defective vector(pGPSN1一GFP)base on an infections molecular clone of HFV (pHSRV13)was constructed by deletion and replacement of the en’u and bet genes with exprssion cas— setts for neomycin—resistance and EGFP.Vector stocks weir,prodaced by cationic lipid reagents co — transfection of BHK一21 cells with pGPSNI—GFP and helper plasmid P△ GP These vectors are able to tra~sfer foreign genes into several vertebrate cells and steadily express them .

Genetic Organization of the EcoR I-G Region of the Spodoptera litura Nucleopolyhedrovirus

LI Zhen, LONG Qing-xing, YUAN Mei-jing, HE Xiong—lei, PANG Yi, WANG Xun—zhang

2002, 17(2): 119

In order to investigate the genomic organzation of the S/,o’ptera litura multinucleoeapsid nucleopolyhedrovirus(SphNPV),the EcoR 1-G[ragment located at map units(mu)49 2-54.1 ol the viral genome was sequenced.The fragment contains 2 partial and 5 complete open reading frames (ORF)representing the viral capsid protein gene p 39 and P 91,[ate expression factor 4(z 4), cg30 gene,tlp20 gene,including homologous region(hr)+Sequence analysis further revealed that these ORFs have the same conserved motifs and gene structure as those observed in 【heir homologues from other baculovirus do.The genomic arrangement of the ORFs in the SpltNPV is similar in aI1 the bac— oloviirus genome,it maybe reveals that this fragment is the conserved part in the baculovirus

Sequence Analysis of the Xba I-I Region of the Single Nucleocapsid Nllcle0polyhedr0Virus of Helicoverpa armigera

FENG Zong—di, CHEN Xin—wen, Just M .Vlak, HU Zhi—hong

2002, 17(2): 125

The Xba I-I fragment located at 18.4~22.8 m.u. of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus genome was sequenced. The fragment contains eight complete ORFs, including putative ubiquitin, 39K/pp31, lef-11, Lsel25, orf507, orf1080, Ac34,Ac38 and a partial orf which potentially encodes C-terminal of P47. orf507 and orf1080 are two putative unique genes of HaSNPV, both contain an early-expression gene motif CAGT and a TATA box in upstream sequences. The ubiquitin gene of HaSNPV shows high identity with baculoviruses and human or yeast ubiquitins. The 39K/pp31 promotor of HaSNPV does not contain late promotor motifs, which exists in other so far identified baculoviral homologues, suggesting it probably has different transcriptional pattern.

Affection of Infection of Autographa californica nucleopolyhedroviruson Sf9 Cell Cycle

WANG Ying, YU Ze, YAO Han, TAO De, CHEN Qu

2002, 17(2): 132

Baculovirus infection resulted in many changes in host cells.The progress of the cell cycle of Spodoptera frgiperda IPLB-Sf21-AE clonal isolate 9 (Sf9) cells was directed by Flow cytometry analysis(FACS).The results showed that the whole time of the cell cycle was about 18 hours and the gapes between every two phases were about 6 hours.At 12-18 hours post AcNPV infection,Sf9 cells were arrested in G\-2/M phase.When the cells in G\-1/S phase synchronized by druge were infected by AcNPV,2/3 of the cells was in G\-2/M phase and 1/3 of the cells in S phase.

Amplification of Dendrolimus puctatus whenshanesis Cytovirus (DpCPV-W)in vitro in Sf21 Cell

MA Yong ping, MENG Xiao—lin, XU Jin—ping

2002, 17(2): 137

We have studied amphificatlon of DpCPV W in vitro in Sf21 cell ln this paper.The results showed that DpCPV W could amplify is vitro in Sf2I ce t[and form normal potyhedra Acce~ional TeV eombinant enhancln in medium coutd synergist the infective rate of DpCPV—W in Sf21 cel1 in vitro. Bioassay indicated that the toxici’ty of DpCPV—W polyhedra amplified in Sf2 I cell was as violence a5 the polyhedra amplified in pine caterpillar By using the plaque purfieation technique in Sf21 cell,DpCPV— W could be purified n a single virion clone Ievd

cDNA Clone and Sequence Analysis of Segment 10 of Rice Black.Streaked Dwarf Fijivirus in Jiangsu

WANG Zhanghui, ZHOU Yi-jun, FAN Yong jian.CHENG Zhao-bang, ZHANG Wen—hui

2002, 17(2): 142

An isolate of rice black-streaked dwarf Fijivirus originally from Lianyuangng,Jiangsu,China was used tO inoculated maize tO propagate the virus Improved single primer amplification technique was adopted tO clone the vira1 genomic dsRNA SIO for sequence determination The result showed:the full length of S10 is 1 801 bp and has the same organization with M RDV in Italy and RBSDV in Japan The similarity of nucleotide and predicted protein weDe 87 5% and 92.6% with MRDV. 93.3% and 96.4% with RBSDV .This study provides a new methodforthe sequence determination of viral dsRNA

Cloning,Construction of gE Gene and gI Gene Partial Deletion Mutant of Pseud0rabies Virus SH Strain

JIANG Yan .HOU Yu—feng, CHEN De—sheng, CHEN Pu yan “

2002, 17(2): 149

According to the Genbank open sequence,two pairs of primer were designed in order to am — plify gE gene and partial gI gene of pseudorabies virus Shanghai strain,respectively.The g£gene and partial gI gene were obtained by po]ymerase chain reaction(PCR),subsequently cloned into pGEM T vector.Then both of them were inserted into pUCI8 vector.The recombinant plasmid pgEI deleted part of gI gene was constructed.

Nucleotide Sequence of Non-structural Protein P3 Coding Region of Foot-and-mouth Disease Virus

IAU Zai—xin, ZHAO Q._ZU, ZHANG Xian—sheng, JIANG Peng—fei, CHANG Hui—yong, XIE Qing-ge

2002, 17(2): 153

Abstract:Two i.5kb DNA were amplified by reverse transcription and PCR(RT—PCR)from the ep— ithelial tongue tissue of cattle infected with FMDV akesu/58 strain after 53 passages,and then cloned A continuous 2,724 nuclco tlde sequence spanning the P3 codiiag region was obtained,including a end— ing codon TAA 907 amino acids were encoded.3A protein gene was composted of 459 nuclcotides. encoding 153 amino acids Three 3B(VPg)genes had 69.72 and 72 nuclcotides and encoded 23,24 and 24 amino acids, respectively 639 nuclcotides and 213 amino acids were attributed to 3C and 1,4 13 nucleotides and 471 amino acids to 3D.All proteins were linked by cleavage sites G1u/Gly (Set).In addition,the varlateCterminal part of 3A protein hasbeenidentified by sequence alignment with other FMDV strains.

Sequence Comparison and Analysis of L Gene from A Foot—and·month Disease Virus Strain China/99

ZHANG Xian, ZHAO Qi, IAU Zai, LIU Xiang, CHANG Hui, JIANG Peng, fei CHENG, LI Dong, WEI Huai, XIE Qing

2002, 17(2): 158

Foot and mouth disease virus strain China/99 RNA5 were used as templet for RT—PCR.The amplifled eDNA products were cloned into pGEM—T Easy Vectors and transformed into JM109.The re— combinant plasroids were identified by electrophoresis.E∞R l cleavage,and PCR The nucleotide and amino acid sequences were compared with the I,genes of the other four reference strains.The resuIts shown that the differential ratio of L gene of China/99 with A10.OIK,OlCaropos and TW45 strains was l5 27% ,15.56% .15 56% and 15 49% respectively;and that of amio acid se0uences was 8.29%.8.76%。9 22% and 10 14% respectively.All the conjunctions of L/el for five fot—and— mouth viruses are Gly/lie.The higher conversions in T.C.A-G and A-c mostly affect the stabgle of amino acid residuses. The regions of 43.53th. 95.105th, 108—11lth, 146—153th 。161—173th. 183— 188th and 182·187[h in I protease roost probably are the active sites that play an important role 1n keeping configuration andfunction of proteins,andH 8.C5l,E65,H95.H109,Hl38,Hl48 andEI65may be active amino acids

Study on the HN Gene Cloning of NDV Isolates in South China and their Phylogenesis

HE Dong—sheng, QIN Zhi—Ieng, LIU Fu an

2002, 17(2): 163

Three HN genes of NDV isolates from South China were am plified by one-step RT-PCR . They where subsequently cloned and sequenced. The Phylogenesis of these NDV isolates was also studied.The gene types of So uth China NDV strains were found to be separated from those of Eum — Ameriean NDV strains exhibiting farther genetic distance NDV isolates from Taiwan Province be, longed to the same gene type as those from Mainland China.However,one of the Chinese NDV strain, FS1.belonged to none of these gene types.Significantly,a paramyxovirus strain.named GPM V that was isolated ftom Guangdong Province,had a rather close genetic relationship with FS2 stain that was isolated from chicken in Guangdong

Analysis of the Structure and Prokaryotic Expressing of the Glycoprotein G Gene of Pseudorabies Virus

MIN Ping, ZHANG Chu—yu, PAN Zi—shu

2002, 17(2): 166

The glycoprotein G (gG)gene of pseudorahies virus Hubei strain(PRV HB)was amplified by PCR,sequenced and analyzed.The results revealed that the fragment cloned and sequenced was 1804 base pairs(bp)with a G+C content of 68.78% and an ORF containing 1500bpto encode a pro— rein of 500 amino acids Comparison of the gG gene of PRV HB with PRV Rice strain showed the se— quence homologies of the nucteotide and deduced amino acid welne 98% and 84.1% . respectively. Most of thes,e diferences were located within amino acid residues 320 to 380 According to the nu— cleotide sequence analysis,two different length segments of gG gene were cloned into the prokaryotic vector pET28a(+)and expre~ed.The specific polypeptides of gG.whose molecular weights were about 55kD.63kD respectlve]y,x,ere identified by SDS—PAGE and Dot—El ISA.This results might be contribute to the study of the structure and function of G gene and gG—EI ISA diagnosis kit of PRV.

Am plification,Cloning and Sequence Analysis of S1 Genes from Infectious Bronchitis Virus Isolates JS/95/03 and SD/97/01

DAI Ya, CHEN De, PAN Jie, JIANG Yan, LIU Xing, LU Yin, LIU Mei, DING Chan, CHEN Pu, CAI Bao-xiang

2002, 17(2): 172

Abstract:The entire S1 genes from nephropathognic strain JS/95/03 and respiratory strain SD/97/01 of infectious bronchitis virus were amplified,cloned and sequenced The nucleotide and deduced amino acid sequences of S1 genes from two isolates and ten reference strains were compared. The results showed that JS/95/03 and SD/97/01 were geneticalty closely related to M41 and H120.respectively. So jt speculated that they were possible variants of raceine strains Two isolates were different in pathogenicity.but they genetically related.Only 24 amino acid differences were found between S1 glycoproteins of two isolates, and 15 of them occurred in the region of the first 130 amino acid residues A amino acid differece Rt the position 1 I6 might he involved in pathDgenicity The corctpa~一 son of the predicted secondary structures of S1 glycoproteins from two isolates revealed that only one or a few amino acid differences were r~ponsible for different antigenicity and structure.

四株草鱼呼肠孤病毒毒株的细胞感染特性比较研究

2002, 17(2): 178

Some characterizations of four Grass Carp Reovirus (GCRV) strains were comparatively studied at the first time. It showed that the stored GCRV\-\{873\},GCRV\-\{875\} and GCRV\-\{876\} at minus 30℃ for ten years still remained infectious to CIK cells, and the 3 virus titers could reach above 10\+2 TCID\-\{50\}/mL, which was a little lower than the newly isolated GCRV\-\{991\} strain′s. After inoculation 3-4 generations, their titers rised gradually, and the titer of GCRV\-\{873\} could be up to the highest, which was almost 6.4×10\+\{11\} TCID\-\{50\}/mL with 0.05PFU/cell multiplicity of infection (MOI) at 28℃. The infective ability of the four strains at different temperatures (28℃,31℃,34℃,37℃ and 41℃) was also detected, which revealed that CPE appeared in CIK cells under above conditions, but titers depressed as the temperature rised. In addition, 4 GCRV-ds RNAs were also compared in a same electronic condition

Infection Characterizations of four Grass Carp Reovirus(GCRV)Strain

FANG Qin, XIAO Tiao, IA I, ZOU Gui, ZHANG Huai, WANG Ya

2002, 17(2): 182

Abstract:Some characterizations of four Grass Carp Reovirus(GCRV)strains were comparatively studied at the first time.It showed that the stored GCRV873,GCRVs75 and GCRv876 at minus 3012 for ten years still remained infectious to CIK cells,and the 3 virus titers could reach above 10 TCIDs0/ mI ,which was a little lower than the newly isolated GCRV99l strain s.After inoculation 3 4 genera— tions,their titers rised gradually,and the titer of GCRV873 could be up to the highest,which was al— most 6 4x10“TCIDso/mI with 0.05PFu/cellmultiplicity ofinfection(MOI)at 2812 .Theinfec— tive ability of the four strains at different temperatures(28℃ ,3l℃ 。34℃ ,37℃ and 41℃ )was also detected,which revealed that CPE appeared in CIK cells under above conditions,but titers depressed as the temperature rised In addition,4 GCRV—ds RNAs were also compared in a same electronic con— dition。

Isolation and Purification of the Eriocheir sinensis “Tremble Disease”Virus and Its Pathongenity on Crabs

SUN Xue-qiang, LU Cheng-ping

2002, 17(2): 185

Abstract:The Eriocheir sinensis(Es)that suffered“tremble diease”by artificial infection were ho— mogenated The supernatant was concentrated with p。】yethyIeneg1ycol(PEG)6000 and purified by gel filtration chromatogmph with Sepia,arose 4B.The eluates collected were concentrated at 4℃ .The virus particles in the first part of the eluates could be seen under electron microscopy.The diameter of virions varified jn 50~ 100nm Sinopotamon denticu缸£“?II artilicia】inlected with the purified virus became j1].Bv double antibody sandwich EI ISA detection.the results showed that there was signifi— cai]t diflerence between the jnfected and the centre crabs
Brief Reports

Sequencing and Expression of PP71 Gene of Human Cytomegalor Virus

ZHANG Yu—ping, CHEN Sheng—liang, CHEN Li—qu, Tian xian

2002, 17(2): 189

Abstract:PP7I gene Of the Hut~tan cytomegalor virur(HCMV AD一169)strain W,qS amplified by PCR. After digestion by Barn H I and Hin dⅢ.the fragment was cloned into the high level expre~ion vec— tot pET28a Recombinant plasmid pET28a—PP71 was transfonned into E.coli BI 2l(DE3)and ln— duced by IPTG,high 1evel protein V,’as produced,and the target protein WaS 40% of all proteins.Purifl— eatlo~ recovery rate was high and up to 92 4% of the target protein before purification.which provides scientific basis on research of HCM V pathogenesis and diagnosis.

The Cooperative Anti-influenza Virus Activities of Amantadine, Ribavirin and Herby Houttuynia

YAN Yin—fang, CHEN Xiao .YANG Xiao-qind, wu Shao-hua, FANG Dong—feng .DONG Chang yuan

2002, 17(2): 192

The research Of the cooperative antivirus activities was carried OUt using anaantadine. rib— avirin and herhy houttuynia.The inhibitory effect 0f CPE caused by influenza virus A3 in MDCK cd and the therapeutic effect on BALB/c mice pneumonia caused by influenza virus FM1 were examined and analyzed when amantadine,ribavirin and herby houttuynia were used alone or assoeiatively The results show that the concentrations Of amantadine,ribavirin and herby houttuynia with sanle effeet were 256,512 and 1024 times cooperatively as many as alone in vitro,respectively.The ablities of treating pneumonia of mice when three kinds of drugs were used coperatively were far higher than those alone in BAI B/c mice