2002 Vol.17(3)

Research Article

Study on the Endonuelease Site of G1 Region of Hantavirus Genome

XIONG Li—juan, LUO Duan—de, ZENG Ling—lan, CAI Shu—qing

2002, 17(3): 195

Typing RT—PCR was used to amplify hantaviruses genome from peripheral blood samples of 34 patients with hemorrhagic fever with renal syndrom e hospitalized in W uhan.The products contained within G 1 gene of M segm ent, w ere digested with H in C II and Sac I respectively, thereby, han— tavirus could be typed into HTN and SEO.The results of RFLP—PCR were highly consistent with typ— ing RT—PCR,and furthermore,RFI P—PCR could quickly screen the variant strain with gene mutation at the restriction site of HiI"l C II or SacI.

Secreted Expression of D2V Envelope Glycoprotein in Pichia pastoris

WEI Hui—yong, JIANG Li—fang, XUE Yao—hua, GUO Hui—yu

2002, 17(3): 198

Abstract:The E gene of dengue一2 virus was amplified using RT—PCR mothod from the C6/36 cells in— fected by D2V NGC strain and inserted into pPICZ a B vector.The recom binant plasm id was integret— ed into Pichia pastoris X一33 by electroporation and the expressed products were analyzed w ith SDSPAGE and W estern blotting.High level secreted expression was performed by determining the M ut phenotype and screening m ulti—copy integrants in the recombinant Pichia strains.The molecular m ass of recombinant E glycoprotein was approx. 69kD and secreted into supernatants when induced with methano1.The expression product was able tO reacted with D2V polyclone antibody and D2V E specif— ic M cAb.M CAC purified E—hisidine—tagged protein from the expressed product w ith HisTrap Kit can bind im m unologically tO H is antibody and D2V E specific M cAb in W estern—blot as ay. This study suggests the entire E gene coding D2V envelope glycoprotein can be expressed efficiently in Pichia pastoris and the expressed products of E fusion protein could amount to 0.1 g/L.

Epitope M apping of Hepatitis C Virus NS3 Antigen from a Phage Peptide Library by Using Imm obilized Specific M onocionai Antibody

ZHONG Yan—wei, CHENG Jun, CHEN Xin—hua, WANG Gang, HONG Yuan, W ANG Lin, LI Li, ZHANG Ling—xia, CHEN Ju~mei

2002, 17(3): 202

Using HCV NS3 m onoclonal antibody as selective molecule, a 12 m ar phage peptide library was biopanned and positive clones were selected by ELISA . com petition assay and DN A sequencing. Eleven positive clones were chosen for DN A sequencing.From the experim ent and sequencing com par— ison results,one epitope was confirm ed as m imotope of HCV N S3.HCV N S3 m im otope was obtained by phage peptide library screening.The result provides a new approach for HCV therapy and vaccine developm ent.

Com plete Sequence Analysis of the Full-length Genom e of the Japanese Encephalitis Virus Attenuated Strain SA14-12-1-7

ZENG Ming, YU Yong—xin, DONG Guan—mu, FAN Xing—liang, YAO Zhi—hui, LI De—fu

2002, 17(3): 206

To sequence the full length genome of Japanese encephalitis virus attenuated strain SA14—12— 1—7.direct information about the genomic structure and the possible relationship to the attenuated and stability m echanism has been provided in this study.Six pairs of prim ers were designed according tO the published sequences of SA14—14—2 and SA14 strains,covering full—length genome of the JEV virus.Using RT—PCR,cDN A fragm ents Of SA14—12—1—7 strain were got and cloned into pGEM —T vector,then trans— formed into com petent TG1 hosts.The positive colones were screened and then the inserted fragm ents were sequenced.The results of sequence analysis show ed that genom e of SA14—12—1—7 strain consistes of 10976 nucleotides and containes a single open reading frame of 10299 nt which encodes a polyprotein of 3432 am ino acids.Com pared with the published sequence of SA14—14—2 and SA 14 strain,the hom ology of the nucleotide sequence and deduced am ino acid sequence were all more than 99% .the mutation sites were located in different regions.In E region,5 sites are the same as SA14—14—2 and other 3 sites are the same as SA14 strain.Some sites in NS3,NS5 and 3’NTR m ay relate w ith the attenuated stability.So the sequence of SA14—12—1—7 strain genome is sim ilar tO the published inform ation,several mutation sites are responsibe for the attenuation Of the virus and the virulence stability.The sequence analysis would aid in understanding attenuated mechanism of the vaccine.

Cloning,Sequencing and Com paring Analysis of Hum an Papillom avirus Type 16 E 7 Gene Isolated from Different Region

XIONG Jin—hu, W U Xing—xin“, TAN Yun

2002, 17(3): 211

In order tO study the structure specificity of HPV 1 6 E 7 gene of cervical carcinoma in W uhan city and W ufeng county of Hubei the tissue DNA was abstracted from cervical carcinom a biopsies from W uhan and high incidence region of cervical carcinoma in W uhan county. HPV 16 E 7 gene was am pli— fied by PCR from the cervical carcinoma tissus DNA with the infection of HPV1 6.and then was cloned into pGEM —T vector.After sequencing the double strands,the gene was com pared with the prototype E 7 gene of HPV 16,HPV 16 Hubei strain and com pared with each other.Sequencing results showed one point mutation in the viral neocleotide sequence of high incidence region that the 229 th neocleotide “C’’was changed into “T”,which caused the Arg cordon TGT tO convert into a Cys cordon CGT . This may be the cause of high incidence of HPV infection and high oncogenisis.The results also show that there is only a synonymous mutation in the viral neocleotide sequence of W uhan which is different from the HPV 16 HB E 7 gene,suggesting that HPV 16 HB and the HPV16 prototype are CO—exist in Hubei, but the HPV 16 prototype are the m ain type.

Comparison of Nucleotide and Deduced Amino Acid Sequences of E Protein Gene of the W ild-Type Japanese Encephalitis Viurs Strain SA14 and Its Different Attenuated Derivatives

FAN Xing—liang, YU Yong—xin, LI De—fu, YAO Zhi—hui

2002, 17(3): 216

For understanding the mechanism Of neurovirulence attenuation Of the Japanese encephalitis virus(JEV),nucleotides of the E coding region of five JE strains,one wild and four attenuated viruses, were am plified by PCR and sequenced.A comparison of the five sequences indicated that there were tO— tally twelve nucleotides and eight amino acid differences between the vaccine strain SA14—14—2(PHK一8) and its parent SA14,in which three amino acids(E一107,E一176,E一439)were different between SA14 and SA14-12—1—7,and other three amino acids(E一138,E一279,E一315)were different between SAx4-12— 1—7 and SA14-9—7 as well as SA14-5—3.The SA14-12—1—7 strain showed low neurovirulence but unstable, whereas al1 the other attenuated strains showed high stability of attenuation.Our results suggest that the mutations of E一176(I1e Va1),E一439(Lys Arg)and E一107(Leu-+Phe)may contribute for the attenuation of neuroinvasion and neurovirulence and the m utations of E一1 38,E一279,E一3 1 5 may con— tribute tO the attenuation of the neuroviru1ence and itS stability

Sequence Analysis of Com plete L Segm ent of 84FLi, a Hantann Virus Isolated from Xi’an

Li Xin, YANG Wei, HANG Chang, BAI Xue, MA Ben, H UANG, LI Guang, ZHA NG

2002, 17(3): 221

In order tO study the nucleotide sequence of L segment of Hantaan virus strain 84FI i,the cDNA of L segment was amplified fragment by fragment by RT—PCR .The purified PCR products were sequenced directly or cloned into pM D18一T vector and then sequenced.The I genom e segm ent is 6533 nucleotides in length with a predicated region(from 38 to 6493)encoding 2151 amino acids.The entire sequence com position was A 33.39% ,C16.43% ,G20.74% ,T29.44% ,and the GC and AT contents were 37.17 and 62.83% .Homology analysis showed that the homologies of 84FLi L segment nu— cleotide sequence with HTN isolates especially Chinese isolates were higher than those of other Han— tavirus.It showed 83.7% homology with HTN foreign standard strain 76—118。while the amino acid homology between the two strains was 97.5% .The result suggested that 84FLi strain was one of the Hantann virus,and highly related tO other Chinese Hantaan Virus iso lates.

Comparison of Expression Results of Different Connection Manners of Hantaan Virus M and S Gene Segments in Insect Cells

LUO Wen, XU Zhi—kai”, ZHANG Fang—lin, YAN Yan, W U Xing—an, LIU Yong, BA I W en—tao, W ANG Hai—tao

2002, 17(3): 226

H antaan virus glycoprotein G1 and nucleoprotein partial fragment w ere connected in differ— ent ways.The constructed chim eric gene G 1 SO .7 or SO.7G 1 was inserted into baculovirus expression vector pFBD.The recombinant shuttle plasmids(Bacmids)containing chimeric genes were obtained in E .coli DH 10Bac.Sf9 cells were transfected by the recom binant Bacm ids,and then recom binant bac— uloviruses were selected and fusion proteins were expressed in insect cells.The expression w as identified by ELISA,im m unofluorescence and W estern blot.It show es that,the recom binant baculovirus contain— ing the chim eric gene G1SO .7 could express the fusion protein in insect cells.This protein,whose molecular weight was about 97kD,could be recognized by the hantaan virus nucleoprotein specific mAb and glycoprotein G1 specific m Ab.The fusion protein,which was expres ed by the recom binant bac— uloviurs containing the chim eric gene SO .7G1 in insect cells,could only be recognized by the hantaan virus nucleoprotein specific mA b.Its molecular weight was about 43kD .It suggests that the chimeric gene G 1SO .7 can express biologically active integrated fusion protein in insect cells,while the product of SO.7G 1 isn’t integrated and its biological activity isn’t as good as the product of G1SO .7.

Interaction of Adenovirus E 1 B 55kD Oncoproteins with hDaxx

WAN Yan—ping, W U Yi—mou, TANG Li—zhi, LIAO Duan—fang

2002, 17(3): 230

Abstract:To question the reason of CO—localization of hDaxx and PM L in PODs disrupted by Aden— ovirus(Ad)E1 B 55一kilodalton oncoproteins(Ad E1 B 55kD),the interaction of Ad E1 B 55kD with hDaxx was analyzed by the yeast two-hybrid assay, and the direct binding of Ad E1 B 55kD with hDaxx was studied by coimm unoprecipitation reaction in vivo or in vitro.The results show that Ad E1B 55kD oncoproteins interact with hDaxx by binding the C-terminus 58 am ino acids of A d2 E1B 55kD or the full length of Adl2 E1B 55kD.Ad2/5 or Adl2 E1B 55kD directly binds hDaxx in vivo or in vitro.

Sequence Analysis of the 1ef-3 Gene of Helicoverpa armigera Single-nucleocapsid Nucle0pOlyhedr0virus

ZHANG Xiao—xia, LIANG Zhen—pu, NIU Guo—dong, ZHANG Zhong—xin .DING Qing—quan

2002, 17(3): 234 doi: 1003—5125(2002)03—0234—05

An lef一3 gene of HaSNPV was identified.It was 1ocalized in Bam H 工一F fragment of the genome.Nucleotide sequencing showed the open reading frame was 1 1 40 nt,encoded 379 amino acids with a predicted size of 44kD.A ”TATA”box was found in 40~ 43nt upstream of the transcriptiona1 start codon ATG.A typical poly(A)signal,AATAAA,was found in the downstream of the tran— scriptional stop codon.HaSPV LEF一3 protein also contains the conserved motif。which exists in nearly 20 kinds of SSBs,SO HaSNPV LEF一3 protein iS a probable SSB protein

Enhancement to Helicoverpa armigera NucleOpOlyhedrOVirus by Chitinase of W HS3 Strain

XU Hong—ge, PENG Hui—yin, LIU Jia—xin, BAI Zhi—qiang, XIE Wei

2002, 17(3): 239

WHs3 Serratia marcesscens is a high yield bacterium of Chitinase.The yield of chtinase in induce medium was 84.4t~g/mI .The chitinase(A3) can enhance the toxicity of Helicoverpa arm igera nucleopolyhedrovirus(HaSNPV)up tO 20% ~70% .The LT50 and LT90 were shortened 1.1 and 1.3 days respectively com paring with contro1

Cucum ber M osaic Virus and Tobacco M osaic Virus Proliferation in Transgenic Chili Peppers and its Protoplast

XU Bing—liang, SHANG Hong—sheng, WANG Xu

2002, 17(3): 243

Transgenic chili pepper plant expressing CM V and TM V coat protein gene were infected with CM V and TM V to com paring the characteristic of CP—M R in transgenic chili pepper and virus content in inoculated plants.The results showed that the transgenic chili pepper exhibited high level of coat protein—mediated resistance to both CM V and TM V .The developm ent of systemic sym ptom was 7 ~ 15 days delayed,percentages of plants showing sym ptoms and severity of disease sym ptom in inocu— lated plants were strongly reduced,and virus accumulat and spread were obviously repressed in inocu— lated and lewly—developed leaves in CP(+)plants challenged with CMV and TMV.The protoplasts from transgenic chili pepper expressing CM V—CP and TMV—CP were infected with CMV virion.The results showed that protoplasts from transgenic chili pepper can also inhibit the virus proliferation.The protoplasts were infected with 40btg/mL virus concentration,48 hours later,the amount of virus in con— trol plant protoplasts was 4.2 times than that in CP(+)plant protoplasts.This results demonstrated that the resistance characteristic of transgenic chili pepper was tO reduce virus proliferation in plants.

The Clone and Expression of the Coat Protein Gene of Potato Virus S in E .coli

WU Xing—quan, W U Zu—j ian, XIE Lian—hui“, I IN Qi—ying

2002, 17(3): 248

Based on the PVS coat protein gene sequence(885bp)a pair of specific primers were de— signed and the coat protein(CP)gene of potato virus S(PVS)was amplified by RT—PCR.The gene was cloned into pGEX一2T vector and then transformed into E .coli DH5a.The gene has high homolo— gy(95% )comparing with other isolate genes.The expression vector p2TSCP was constructed.The coat protein gene was expressed in E .coli DH5a and a 58kD protein was analysed by SDS—PAGE.

Isolation and IdentificatiOn Of Very Virulent Infectious Bursal Disease Virus(vvIBDV)Strain NH99

SUN Jian—he, I U Ping, LI Hui, ZHAO Yu

2002, 17(3): 252

An infectious bursal disease virus(IBDV)field strain,named NH99,was isolated and iden— tifed from some flocks with imm une failure in Shanghai according tO the clinical symptom ,pathology, serological test,animal challenge and virus morphology,The virus was characterized as a very virulent infectious bursal disease virus(vvIBDV)through nucleic acid isolation and related DNA sequence analy— sis which have the same molecular characteristics as vvIBDV .This is the first report of outbreak of a— cute infectious bursal disease caused by vvIBDV in Shanghai.

Identification and Characterization of the RNase lit Gene of the Tiger Frog Virus

LU Ling, HE Jian—guo, HE Hau—hong, DENG Min, WENG Shao-ping, WANG Xiao-hong

2002, 17(3): 257

From the tiger frog virus(TFV)genome,an open reading frame(ORF),whose deduced amino acid sequence(aa)shows identity with RNase 111。was identified.The ORF has 1 113bp long and codes a putative protein of 371 aa with a predicted molecular mass of 40.47kD and a G+ C content of 56.63% . Stem loops and short dyad sequences appear in the downstream of the translation stop codon.Compared with the RNase 111 S found in TFV and other species.the higher homology among TFV and the tWO iridovirus is present,and TFV has lower identity with other species, especially yeast and rhabditida.

Preliminary Studies on Two Strains of Reovirus from Crab Eriocheir sinensis

ZHANG Shu—yong。, ZHANG Jian—hong。, HUANG Can-hua。, Bonami Jean—Robert, SHI Zheng—li

2002, 17(3): 263

Two different reoviruses were isolated during investigation on pathogens of diseased crab E— riocheir sinensis(designated as Es RV816 and Es RV905). RV816 was isolated from crabs collected from Jiangsu province.Es RV905 was purchased from a local farm in W uhan, Hubei province.The spherical viral particles of Es RV8 1 6 and Es RV905 are about 65 and 55 nm in diameter.The tWO viral particles contain two different genom es of 10 and 12 RNA segm ents, respectively. Based on their hosts,number of segments and electrophoresis type of the genomes,they may represent tWO new gen— era of the reoviridae.
Brief Reports

Cloning and Determ ination of S 1 Gene of IBV GD05 Strain and Its Expression in E .coli.

HUANG Ya, ZHEN Qing, WANG Lin, LI Xiao-kun, HUANG Zi

2002, 17(3): 266

Acording tO Avian Infectious Brochitis virus(IBV)Beaudette strain S 1 gene sequence,a pair of primers were designed and synthesized.W ith the primers, IBV Guangdong isolation strain GD05 S 1 gene was successively amplified by RT — PCR .PCR product was digested w ith BstY I,Hae III and Pst I respectively,the result showed RFLP pattern of was the same as that of M41 S 1 gene. IBV GD05 strain was thought as M ass serotype primaryly.IBV GD05 S 1 gene was cloned into pGEM — T vector and sequenced, its sequence was consisted of 1611 base pairs.By comparison , the nu— cleotide sequence was 97.14% identical tO that of IBV M 41. IBV GD05 S 1 gene was subcloned into expression vector pET21 d.SDS—PAGE experiment showed that it expresed in E .coli.

Research Progress in Som e Genes of Insect Baculovirus

HONG Jingjun, DUAN Jialong, PENG Hui

2002, 17(3): 270

昆虫杆状病毒(Insect baculo irus)对鳞翅目、双 翅目和膜翅目等昆虫具有病原性,是一种开发应用 较广、高效的生物杀虫剂,具有杀虫专一、效果好、有 流行传播作用等优点。为了更好地利用昆虫杆状病 毒为防治农林害虫服务,加快昆虫杆状病毒杀虫剂 研究的步伐,本文归纳了近几年来有关昆虫杆状病 毒基因研究进展供从事研究的人员参考。

Application of Tupaia on Studying of Virus Diseases

HU Xi—liu, LI ji—qiang

2002, 17(3): 276

树匡鞫(Tupaia)是一种在生物医学研究中很有应 用价值的新型实验动物,其分类尚有争议,有人认为 是食虫类,有人则将之列为低等灵长类,还有人认为 树鳓是一个独立目的,称为攀鼢目(Scandentia)⋯ , 但目前基本定为低等灵长类。由于其具有体积小, 类似松鼠,比较容易饲养和操作,管理方便,繁殖力 高,廉价经济等优点,树鼢存在多种自发性疾病,对 多种病毒易感,而且其进化程度高,新陈陈谢和大体 解剖与人较为接近,因此其作为较理想的实验动物, 已经被广泛地应用于医学与生物学的研究中。在病 毒学方面,树匡鞫不但被用作疱诊病毒、腺病毒、EB病 毒、甲型和乙型肝炎病毒、轮状病毒的研究L2 J,而且 还用于流感病毒、丙型和丁型肝炎病毒、基孔肯雅病 毒等研究。本文仅就树匡鞫在病毒学方面的应用概况 作一介绍。

Advance in Aquatic Crustacean Viruses-I.Bacilliform Viruses

SHI Zheng—li .ZHU Hai—yan

2002, 17(3): 279

水生甲壳类动物病毒的研究始于一种地中海螃 蟹(Portunus depurator或Liocarcinus depurator)体 内发现的类呼肠孤病毒⋯ 。随后,人们相继在这类 动物体内发现多种病毒,其中约17种为杆形病毒。 这些杆形病毒的多数往往只是被它们的宿主携带 (尤其是螃蟹和淡水甲壳类动物)而不引发疾病,其 研究仅限于组织病理和形态的描述。而在对虾体内 发现的杆形病毒却对对虾养殖业造成巨大的经济损 失,成为研究的热点。其中,有两种形成核多角体的 对虾病毒在感染部位、形态和血清学特征等方面非 常接近杆状病毒科的核多角体病毒属,被称为对虾 杆状病毒。其它不形成核多角体的杆形病毒没有确 定的分类地位。为了避免概念上的混淆,本综述将 所有不形成核多角体或核包埋体的 J、具有杆状形 态的病毒通称为杆形病毒。对形成核多角体或核包 埋体的、具有杆状形态的病毒称为杆状病毒。本文 将着重介绍水生甲壳类动物体内发现的杆状病毒和 杆形病毒的动态研究。表1列出了在水生甲壳类动 物体内发现的杆形病毒的基本特性。

The RelatiOnship Between the Geonome and Its Variation of Coxasckievirus B3 and Myocardium Inj ury

LI Xiang—jun, REN Li—qun “, LI Guang—sheng, WAN Min

2002, 17(3): 286

肠道病毒感染是人类急性和慢性心肌炎的常见 病因之一,而且也与人类扩张型心肌病(DCM)的发 生发展有密切关系⋯ 。病毒分离,血清学研究,免 疫组化技术、原位核酸杂交技术以及聚合酶链反应 技术(PCR)均提示肠道病毒与心肌炎关系密切。最 近,LI等[ ]用肠道病毒特异性单克隆抗体,采用改 良的免疫组化技术对心肌炎或DCM 病人心肌切片 中肠道病毒抗原进行检测,此法直接证明了心肌炎 或DCM 病人体内确实有子代病毒产生。但是,肠 道病毒究竟通过怎样的机制引起心肌损伤还未阐 明,推测可能有多种机制参与。本文仅就肠道病毒 (柯萨奇病毒B3,CVB3)基因组结构及其基因变异 与心肌损伤的关系方面加以综述。

Research Advances on the M echanism of Viruses Affecting Apoptosis

ZHANG Xiao-xia, ZHANG Zhong—xin

2002, 17(3): 289

自Kerr等1972年提出细胞凋亡(apoptosis)概 念以来,这一生物学现象的研究已受到广泛重视,细 胞凋亡是细胞在一定的生理或病理条件下,遵循自 身程序进行自杀的过程。但它与程序化细胞死亡 (PCD)不是同一现象⋯1。程序化细胞死亡具有严格 的基因时控性,一定时间内使某些细胞死亡而让另 外一些细胞新生,其调控是非常精确的。细胞凋亡 虽也涉及基因调控,但它只是瞬时发生的形态学变 化过程,它以细胞皱缩。胞浆致密,染色质浓缩成大 小不等的块状,DNA 梯级性降解,细胞膜内陷形成 多个凋亡小体等生化和形态学变化为其特征。