1996 Vol.11(3)



Molecular biology of parvovirus


1996, 11(3): 193

Research progress on hog cholera virus and its vaccine

LU Yu, CHEN Jian-Guo, DING Ming-Xiao

1996, 11(3): 201

Structure and Characterization of M Genome Segment of HFRS Virus

DONG Guan-Mu, YU Yong-Xin

1996, 11(3): 208

Study on morphogenesis and development process of MCV by silver - DNA staining and electron microscopy

LI De-Zhong, XIAO Tong-Hao, WU Ning

1996, 11(3): 214

After using the methenamine silver-DNA staining,15 cases of eariy molluscum contagiosumtissues were examined by electron microscopy.The results showed that there was a silver-stainedDNA area(inclusion body,IB)in every cell infected with MCV. The morphogenesis and develop-ment of MCV tcok place in the IB,not in the cytoplasm area. The process of MCV morphogenesiswas firstly to synthesize virus materials(DNA) which secondly accumulated into a filament-granu-lar-like area(previrus inclusion body,PIB),and next to form some dense thin-granular-chain-likesturctures(previrus matrix,PM)in PIB,and then the bilayer membranes which cut and surroundPM to form primary MCV. The development process was from primary to intermediate,then tomatural and finally to degenerate MCV. And in the process the outer membrane,matrix,nucleusand the nuclear outermembrane of MCV had many changes.The lateral bodies were the temporalstructures and the viroplasm containing DNA in the intermediate MCV.The results suggested thatthe MCV gene may

The isolating, cloning and sequencing of HPV 16 E7 gene from cervical carcinoma biopsy DNA of Hubei Province

WU Xin-Xing, ZHAO Wen-Xian, DING Xiao-Hua, SU Ying-Bin, DING Hong-Zhen

1996, 11(3): 220

The technique of add-on PCR was used for isolating HPV16 E7 gene from cervical carcinomabiopsy DNA in Hubei province.The E7 gene was inserted into vector pUC18.By restriction en-zyme cleavage and nucleotide sequence detection,a new recombinant plasmid was constructed andnamed it pHPV16 E7-HB. The result of nucleotide sequencing showed that the overall length ofHPV16 E7-HB gene is 294 bp,it is the same as the standard strain.But there were two C→Tmutation in HPV16 E7-HB.Anonsense mutation was identifed at codon 43.a Gln codon(CAA)was converted into ochre termination codon (TAA).The mutations occurring in 294 bp ofDNA PCR production seemed to be a structure difference between HB strain and standard strainrather than a mismatch of PCR itself.

Study on Immunogenecity and Immunoprotection of Hantaan Virus Nucleocapsid Protein Expressed in E .coli

HUANG Chang-Xing, LI Guang-Yu, YANG Wei-Song, HANG Chang-Shou, BAI Xue-Fan, SUN Yong-Tao, ZHANG Wen-Bin, LI Xin-Hong

1996, 11(3): 225

Immunogenecity and immunoprotection of recombinant Hantaan virus nucleocapsid protrein(NP)expressed in E.coli were studied.First,mice were immunized with the recombinant NP,the titer of antibody against Hantaan virus was determineted using indirect immunofluorescenceassay,it was1:12800 in 4 weeks after immunized.Then,the spleen cells and sera of immunizedmice were transferred to 3 ̄4 day-old mice in 24 hours after the mice infected with lethal dose ofHantaan virus.The protection rates of spleen cells were 10~38%,but the sera could not protectmice from death,at the same time, it also did not change the living time of mice with lethal Han-taan virus infection. The results indicate that NP ean induce the cell-mediated immune protectionbut cannot induce the antibody-mediated immune protection and the early death in Hantaan virusinfection mice.

Transovarial transmission of dengue viruses in Aedes albopictus and Aedes aegypti mosquitoes

ZHANG Hai-Lin, MI Zhu-Qing, ZHANG Yun-Zhi

1996, 11(3): 230

edes albopictus and Ae. aegypti mosquitoes from maialand China were tested for their abil-ity to transovarial transmission of dengue(DEN) viruses. DEN1~4 viruses for experiment wereprototype dengue strains(Hawaii,New Guinea,H87 and H241)and Ban18 strain of DEN4 isolat-ed from Yunnan,China.Parental females of Ae. albopictus and Ae aegypti were orally infectedwith DEN1~4 viruses,and 4559 larvae and adult of F1 and F2 generations from infected motherswere divided into 10 pools for the detection of the viruses. The positive rates of pools from Ae.albopictus of F1 were 10%(1/10) for DEN1,22.22%(2/9) for DEN2,33.33%(4/12)forDEN3 and 28.95%(11/38)for DEN4.The minimum filial infection rates(MFIR) of Ae.al-bopictus of F1 were successively 0.20%,0.71%,0.70%and 0.63%for DEN1~4.The positiverates of pools and MFIR from Ae.albopictus of F2 were 35.29%(6/17)and 0.93% for DEN 4.The positive rates of pcols from Ae.aegypti of F1 and F2 were 60%(3/5)and 33.33%(3/9),and their MFIR were 0.63 % and 0.60% for DEN4,respectively.

Construction of HPV 16 genomes containing mutated and deleted long control regions from cevical cancers

LIU Gong, DONG Xiao-Beng, LIU Yan-Na, CHU Yong-Lie

1996, 11(3): 237

Y1 is an imporiant cellular transcriptional regulator which is ubiquitously expressed in vari-ous kinds of cells.The specific binding sites of YY1 protein are found in the flanking sequences ofnlany cellular aiid viral promoters.In the "high risk"huinan papilloniaviruses(HPVs)YY1 works as a traxiscriptional repressor for the viral early gene promoter.In the episomal HPV 16 genomesextracted from cervieal cancers,removal of YY1 binding sites in LCR is a repeated event, resultingin increasing the activity of viral oncogene promoter P97.In order to study the effect of revomal ofYY1 binding sites in LCR on the viral tumorigenicity,HPV16 wild-tny plasmid p1203 was usedas basic sequences,through mltiple-step-clone to construct the new HPV16 plasnuds containingdeleted and mutated LCRs. houence analysis coofirmed that pDV390 had a G to A exchiinge inthe second YY1 site,whereas PDV 1326 and pDV401 contained 115 bp aiid 143 bp deletions in therespective LCRs that revomed 2 and 4 YY1 binding sites.

Screening,Cloning and Genetic Heterogeneity of the Hepatitis Delta Vires Antigen——Coding Regions Isolated from Different Areas an d Population in China

TAN Wen-Jie, DAN Mei-Yun, YI Yan-Jie, BI Sheng-Li, MIAO Ji

1996, 11(3): 244

More than ten Chinese hepatitis,delta virus isolates were coml,ared by sequencing the antigen-ceding regions after cloned to PGEM-3Zf(-)or PGEM-Tvector.The viruses compared in-cluded the Henan-1,Henan-2,Henan-3,Xinjiang ,Neimeng,Sichuan,Xizang,Liaoning,Beijing,Guangxi and Shanghai strains derived from asymptomatic carriers and chronic or fulminant hepatitis patients.It was found that all HDV isolates prevalente in China were genotype I but there wereat least two subgroups. The genet ic heterogeneity of HDAg-coding regions existed among theHDV isolates from different areas or population.These strains from Henan and Xinjiang areasshared over 92.1%nucleotide and 86.9%amino acid homology with Taiwan strain that repre-sents genotype IA.But the strains from Sichuan,Xizang-1,Neimeng-1,Liaoning,Beijing andGuangxi are closely related to US-1strain representing genotype IB(over 94.3%and 88.8%i-dentity in the nucleotide and amino acid sequences respectively).The Shanghai strain is closely re-lated to Italy strain and ov

Effect of cytokines on the infection of monocyte - macrophages with HSV- 1

JI Xiao-Hui, YAO Kun, LI Huan-Di, ZHOU Yao-Xi

1996, 11(3): 251

In vitro hunian monocyte-line U937 cells and mouse peritoneal macrophages were inoculatedwith HSV-1,which were persistently treated with difterent cytokines since several days beforeor after inoculation. The effect of these cytokines on the infection of monocyte-niacrophages withHSV -1 was studied by virus titration,indirect immunofluorescent test for viral antigen and poly-merase chaln reaction technique for viral DNA in infected cells.The results showed that TNF - α50 u/mL,M-CSF 200 u/mL,IFN -γ1000 u/mL and IFN - α 1000 u/mL all enhanced the re-sistance of monocyte - macrophages to HSV - 1 infection,accelerated the clearance of virus DNAand inhibited the expression of viral antigen in the cells,moreover,they were antergic to the en-hancement activity of LPS for the virus replication in monocyte macrophages.

Initial analysis of the process of poliovirus entry into cells


1996, 11(3): 257

This study analyzed the configurational changes of poliovirus as it crossed the membrane of cells,and investigated the relationship between the configuration changes of this virus and its entryinto cells,The function of VP4 in the process of poliovirus entry into cells was studied also.Amodel about poliovirus entry into cells was suggested

Preliminary study on the method for detecting IgM antibody to hepatitis C virus

HE Hong-Xia, HONG Shi-Wen, MAO Fan-Yong, BAI Yan-Ping

1996, 11(3): 264

Indirect ELISA for detecting IgM antibody to HCV was established based on recombinantantigens of HCV structural and nonstructural regions.The result showed it is easy, rapid,repro-ducible, specific and sensitive.Asti HCV IgM was positive only in the sera of patients with hepatitis C,and all negative in healthy donors.There were no cross reactions with IgM antihady toHAV and HBV.The false positive and negative caused by RF and specific IgG were removed.Itis suitable for the clinical detection of IgM antibody to HCV.The sera of 24 patients with hepatitisC were assayed for anti HCV lgM.The data indicated that the positive rate is 75i)fj in thepatients with acute hepatitis C.As ALT being nornial,anti HCV IgM became undetectable or itslevel dropped. Aiiti HCV IgM was detected in 9 of 16(56.3%) patients with chronic hepatitis.Among these 9 patients with anti HCV IgM,7 were with abnormal ALT and 2 with norinalAlT. puiti HCV IgM may persist in chronic active patients.The results suggested that AntiHCV lgM is a useful

Isolation and characterization of tomato mosaic virus from tomato

ZHOU Xue-Ping, QIAN Xiu-Gong, LIU Yong, XUE Chao-Yang, LI De-Bao

1996, 11(3): 268

virus isolate(To-Sl)from tomato was collected in Ha11gzhou,Zhejiang Province.To-Slcould infect 24 plant species of three families.To-Sl produced mosaic symptoms on tomato,andlocal leision on N.tabocum White Burley. Its thern1al inactivation point was 85~90℃,dilutionend point was 10 ̄(-6)~10 ̄(-7).The virus particles were rod-shaped with the average length of288nm.The molecular weight cf To-Sl coat protein estimated was 2 1 000 Da.dsRNA analysisshowed that To-Sl corLqisted of one single st rand RNA with the length of 6.4 kb.In double-diffusion tests,spur forn1ation was observed between To-Sl and TMV,and the resuIts of in-tragel cross-absorption tests agreed with the double-diffusion tests.In gel electrophoresis,thepatterns of To-Sl virions were different from TMV.To-Sl shared one bands with TMV,butTMV had another band with slow migration.Cross protect ion tests showed To-Sl had no prrvtection eHect to TMV,and vice versa.On the basis of these characteristics theTo-Sl was identi-fied as tomato mosaic virus(ToMV).

Inhibition of replication of rice dwarf virus in transgenic rice plant expressing antisense ribozyme gene

YANG Wen-Ding, WU Zu-Jian, WANG Su-Yan, LIU Wei-Ping, XIE Yin, XIE Lian-Hui, TIAN Bo

1996, 11(3): 277

he plant expression vector pROK II containing antisense ribozyme gene of rice dwarf virus(RDV)S5 was transferred to rice immature embryo by microprojectile bombardrnent.The resis-tant calli were obtained in the presence of G418. Two or three months later these calli could differ-entiate to shoot after transferred to differential medium. The transformation of the regeneratedrice plant was confirmed by Southern blot.Transgenic plants were inocuiated with viruliferous leafhopper(Nephotettix cincticeps)to determine their RDV resistance.The results showed thatthe infection of RDV was not prevented but the symptoms were milder than control.Most of thetransgenic rice plants were fertile while the control was not.In order to assay the RDV replica-tion,virus concentration was detected by ELISA. The results indicated that the repliea.tion of RDVin transgenic plants was obviously inhibited.

Detection of pseudorabies virus by biotinylated DNA probe

WANG Qin, GUO Wan-Zhu, LIN Wen-Qi

1996, 11(3): 284

dot blot hybridization method was developed to detect pseudorbies virus DNA in infectedcultured cells.Seven strains of pseudorabies virus were detected by hiotinylated pseudorabies virusFa DNA probe,and recomhinant plasmid pPB11DNA(BamHI-11 fragment of PRV Fa was clonedbetween the BamHI sites of plasmid pBR322)probe.PRV Fa DNA probe could detect 10 pg nu-cleic acid in the samples. The nucleic acid hybridization method is sensitive and specific for diag-nosing pseudorabies virus.

Amplification of the Intact Gene Coding Epstein-Barr Virus-Specific tIIym idine Kinase

CHEN Shang-Wu, CHEN Rui-Jun, HUANG Di, SHU Zhen-Yu, MA Jian-Quan

1996, 11(3): 287

Based o1"1 Epstein—Barr virus(EBV)DNA (B95—8)genome sequenc3e data and the strut—ture BXLFlopen readingframe codingforEBV ~hymidinekinase(TK),a pairofPCR pr/mers was designed and svnthesized Template DNA was extracted rapidly from B95—8 calIs harboring EBV bv using guanidinium isothioeyanate(GuSCN)and silica∞urse(cs).A 1843bp A frag—ment containing intact EBV TK gene was amplified by polymerase chain reaction.The PCR prod—uct was verified by cleaving with NcoI