SONG Hong, FU Shi—hong, W ANG Huan—Yu and LIANG Guo—dong. The Relationships of Serotypes and Nucleotide Sequences 0f The Dengue viruses Genome[J]. Virologica Sinica, 2002, 17(4): 297-303.
The aim of this study was to prove the relationships of serotypes and nucleotide sequences of the Dengue viruses genome．To this end，the nucleotide sequences of 5 noncoding region，3 一noncod— ing region，envelope E glycoprotein，nonstructural protein NS1 were aligned correspondingly．W ith four Dengue viruses phylogenetic groups which were correspo nding to four serotypes each other being identified，Our results demonstrate that there were no intergenomic recombination observed．The po s— sibility of intragenomic recombination and the similarity of nucleotide sequences of different Dengue viruses were analysed．
DING Yong, W U, ZHANG W, ZHAO M and TAN Yun. Study on Recombinant HPV DNA Vaccine Inducing Specific Lymphoproliferation[J]. Virologica Sinica, 2002, 17(4): 304-307.
Using molecular cloning technique，HPV16 E7 gene was cloned into eukarytic expression vector．HPV16 E7 DNA vaccine was constructed and Balb／c mice were immunized by intradermal ad— ministration with HPV16 E7 vaccine．After immunization，mice spleen lymphocytes were prepared and restimulated by E7 protein in vitro．Specific lympproliferation were detected by MTT colorimetric as— say．Because detection of specific lymphoproliferation is a simple and efficient way，which can reflect cell—mediated immunity．The study showed that the construction of HPV16 E7 vaccine was correct．It can induce a specific cell—mediated immunization
LUAN Yi, YU Xiu—ping, SONG Chang—qin, BIAN Ji—feng, ZHAO Wa—ming, JIA Ji—hui, ZHOU Ya—bin and QI Mei. Comparison of Detection Antibodies to Human papillomaviruses 16 L1 in the Cervical Cancer People with Different Recombinant Antigen[J]. Virologica Sinica, 2002, 17(4): 308-311.
In order to use major capsid protein L1 of Human papillomaviruses(HPV)16 produced in a fused fom in E ．coli and HPV16 L1 VLP produced in recombinant adenovirus in 293 cells as antigen to detecton antibodies of HPV 1 6 L1 in the cervical cancer people．and compare the serological differ— enee of the two antigen in the diagnosis of cervical cancer，we used PCR to amplify HPV1 6L1 gene from the cervical cancer， then cloned into pUC18一T． After DNA sequencing， HPV16L1 gene was cloned into pGEX一2T expressing vector，and induced by IPTG to express in E ．coli as glutathione—S— transferase—L1(GST—LI)fusions and purified to near homogeneity as antigen，together with HPV16 L1 VLP produced in recombinant adenovirus in 293 cells，to test antibo dies of human—papillomavirus (HPV)16 L1 of 12 cervical cancer and 53 blood donors．The gene derived from the cervical cancer HPV16 genome was 1535 bp in length，and expressed by E ．coli to the full—length 83 kD polypep— tide，which recognized 1)y HPV16L1 monocloned antibody．In the 12 cervical cancer sera，there were 7 positive in HPV16L1 antibody(58．3％ )；while 8 positive in HPV16L1 VLP antibody(66．7％)．A— mong 7 positive in anti—HPV16 L1using HPV16L1 protein from the E ．coli as antigen，all are positive when using HPV 16L1一V LP as antigen．W hile among 5 negative in anti—HPV16 Llusing HPV 16L1 protein from the E ．coli as antigen， there is 1 positive when using HPV1 6L1一VI P as antigen．There are no difference between two group as antigens in ELISA detection．(P0．05)Our research sug． gested that HPV 1 6 is highly associated with cervical cancer．The sensitivity of the test is the same whether using HPV 16I 1 protein from the E ． coli or HPV16L1一VLP as antigens． To detect HPV16I 1 antibody is helpful to diagnosis cervical cancer．
CHEN Hongmei BAI Xue—fan, PAN Lei, LI Guang—yu, WEI San—hua, HUANG Chang—xing and Construction and Expression of Eukaryotic Expression Vector Bearing Fusion Gene Of HBV PreS2+S Gene and IFN— Gene[J]. Virologica Sinica, 2002, 17(4): 312-314.
To construct a recombinant eukaryotic expression vector bearing fusion gene Of Hepatitis B viruS(HBV)S2+S gene and IFN-a gene，technique of splicing by overlapping extension and two times PCR were used．Fusion gene fragment was obtained and directly cloned into pcDNA3．1 V5／His TOPO TA cloning vactor to get recombinant eukaryotic expression vector pcDNA3．1$2S／IFN—a． Then the recombinant vector was transferred into Vero E6 cells using LipofectAM INE．The recombi— nant vector was constructed and correctly checked by digestion with restriction enzymes and poly— merase chain reaction．The vector bearing fusion gene could be expresed in eukaryotic cells detected by indirect immunofluotescence technique．The relative efficient expression of the fusion gene in Vero E6 cells might provide an experimental basis for specific immunotherapy for HBV infection．
ZHAN Fa—xian, PAN Nan—sheng, YE Guo-jun, CHEN Si—li, GONG Zhen—kui, LIU Chuan—nan, ZHU Hong—hao and CHEN Fen. Influenza Epidem ic Situation Year of 2001 in Analysis in the Epidemic Hubei Area[J]. Virologica Sinica, 2002, 17(4): 315-318.
The influenza epidemic situation in the epidemic year of M arch 200 1 to March 2002 in Hubei area was analyzed according to the results of isolations of Infzuenza viruses，detection of human antibodies against Infzuenza viruses and epidemiological survey．It was concluded that there were two epidemics during the epidemic year．One in August and September of 200 1 was mainly caused by A1 type of Infzuenza viruses．Another was caused by the dominant strains of A3 type．It was also shown that the isolated strains of A3 type during this epidemic year likely underwent some antigenic draft， compared with the A3 type of Infzuenza viruses isolated in 1 999．
W ANG Han—zhong, HUANG Yi, SI Yan—hong, FANG Ming．gang ． CHEN Xinwen and Just M ．Vlak ．HU Zhi．hong. A Novel Expression System of Helicoverpa armigera single-nucleocapsid nucleopolydrovirus[J]. Virologica Sinica, 2002, 17(4): 319-325.
A 8．5kb fragment containing an E ．coli low—copy number mini F replicon，a selectable kanamycin resistance marker and lacZ gene with attTn7(the target site for bacterial transposon Tn7) replaced polyhedrin gene in HaSNPV genome using homologous recombination．HaSNPV genome was cloned and maintained as 1 32 kb bacterial artificial chromosome(Bacmid)in E．coli，transfection of the Bacmid(HaBacmid．HZ8)into HzAml cells led to a productive virus infection．In this paper．the donor plasmid HapFastPhP10 was constructed using the Ha—polh gene and the Ha．P10 promoter tO re． place the original Ac P1 0 promoter and Ac—Polh promoter of the pFastBacDual donor plasmid respec． tively．The eGFP gene was inserted into multiple cloning site(MCS)downsream of the P10 promoter in the pFastBacHaPhpP10 donor plasmid．Recombinant HaBacmid HZ8 was constructed by transpo s． ing a mini—Tn7 element from a HaDFastPhP1 0 donor plasmid tO the mini．attTn7 attachment site on the HaBacmid—HZ8 when the Tn7 transposition functions are provided in trans by a helper plasmid．Re． combinant HaBacmid．HZ8 DNA was transfected HzAml cells．occlusion bodies and green flusecent were found within HzAml cells 5 days after transfection．The results indicated the Habac to Bac ex． pression system based on HaSNPV can effectively express foreign gene．
LI Zhi—guang, YIN Jun and ZHONG Jiang. Expression of Full Length and N Termial Truncated Enhancin from Trochoplusia ni granulovirus in AcM NPV and Analysis of Their Activit[J]. Virologica Sinica, 2002, 17(4): 326-330.
Enhancin is a group of baculovirus proteins capable of increasing the infectivity of viruses in insect larvae，as well as the insecticidal effect of other biology control agents． In order to study the structure and function of enhancin from Trichoplusia i gran ulovirus， recombinant baculoviruses were constrcuted to express truncated forms of enhancin with 150， 186 and 250 am ino acids deleted from N terminal of native enhancin，respectively．The truncated enhancins were expressed successfully in Tn一5B1—4 cells． In vitro peritrophic membrane assay showed that all three truncated enhancin lost their mucin degrading ability，while a full length recombinant enhancin was active in the assay．It is concluded that the N terminal amino acids is essential for the mucin degrading activity of enhancin．
WU Dong, W ANG Hua—lin, DENG Fei, CHEN Xin—wen, PENG Hui—yin。HU Zhi—hong and Molecular Cloning and Expression of the Chitinase Gene from Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in E.coli[J]. Virologica Sinica, 2002, 17(4): 331-335.
The PCR product of the HaSNPV chitinase gene，which was without the N—terminal signal peptide and the C-terminal endoplasmic reticulum location signal，was cloned into a prokaryotic expres— sion vector pProEXHTb．After the induction of IPTG，the chitinase gene was successfully expressed in Eschechia coli DH5a．The expression product shown a molecular weight of 60 kDa，and it covered about 40％ of the total protein in E ．coli．W estern blot analysis using an antibody derived from AcM — NPV ehitinase confirm ed the expression product was a homologue of baculoviral chitinase．
LIANG Jun, GONG Min, YUAN Zhi—ming’’ and LIANG Bu—feng. Expression of Helicobacter pylori cagA gene in Insect Cells by Baculovirus[J]. Virologica Sinica, 2002, 17(4): 336-339.
A recombination transposing vector pBlueBacHis2一CagA was constructed by inserting Heli— cobacter pylori f口gA gene into pBlueBacHis2A vector of baculovirus expession system ．Co—transfecting Sf9 cells with Bac—N—blue DNA and pBlueBacHis2一CagA，recombinant viral DNA was obtained by pu— rification of recombinant plaques．PCR result showed ca gene intergrating in correct position of bac— ulovirus gemone．The expression of Helicobacter pylori ca gene was confirmed by SDS-PAGE and W esternbolt．Using indirect ELISA assay，it was found that the protein exhibited good specific reac— tivity with sera of HP—infected individuals
xu Jin—ping, MENG Xiao-lin“, WANG Jian and LU Wei. Expression，Purification and Bioactivity Detection of Recombinant Hum an IL-12 in Argyogramma agnata[J]. Virologica Sinica, 2002, 17(4): 340-343.
Two cDNA fragments encoding human interleukin一12(hII一12)P35 and P40 subunits were isolated by RT．PCR from KB cells stimulated with PDBu，and then cloned into pCR ．1 respcetively and down the double promoters of pAcUw 5 1．The recombinant baculovirus Ac—hiL2 was obtained by cotransfecting with pAeUW 5 1一ILl2 and baeuloGold M Linearized baculovirus DNA．Argyrogramma agnata larvae were infected with recombinant baculovirus Ac—hiLl2 by hemoeoel injection．The rhII， 1 2 was purified from the supernatant with affinity chromatography．The blood lymph supernatant har— vested and rhII，12 purified were SUbjected to SDS-PAGE(silver stain)and Western blot．The level of rhIL-12 was detected by EU SA．Bioactivity of purified rhII，12 samples were detected by M 1vr method．It is indicated that Ae-hlL12 can replicate in fat body and midgut cells of Argyrogramma Agnata larvae．The MW of rhlL一12 expressed was 75kD．The expresion levels of rhIL-12 were 17．8 ／~g／lO cells and 200—300mg／L in Sf9 cells and Argyrogramma agnata Staudinger larvae blod lymph respectively．The purified rhII，12 has significant activities which enhanced NK eytotoxieity and increased the proliferation of human PBMC PHA-P activated significantly．
CHEN Jiong．CHEN Jian—ping and Phylogenetic Tree Analysis of UTR and Transmembrane Structure Prediction of Proteins of Genus Bymovirus[J]. Virologica Sinica, 2002, 17(4): 344-349.
Homology and phylogenetic tree analysis of genus Bymovirus suggested that the 5 一UTR of RNA1 and RNA2 of the same virus were more similar than those of the same RNAs of different virus— es．but the instance of 3 UTR was opposite．Secondary structure analysis on polypoteins showed that P3 and 14K encoded by RNA1 contained transmembrane(TM)stuctures．TM region in P3 protein of BaM MV ALS1 isolates was toxic to the E ．coli．By analogy to other members of family Potyviridae． these structures were predicted to have the function as attachment to the membranes．P2 protein en— coded by RNA2 also contained two TM regions．Some BaMMV isolates which were maintained me— chanically for a long period of times occurred deletions in these regions and were not transmitted by Polymyxa graminis．It was suggested that the TM structures were closely related to vector transmis— sion．
FU Ming—jia, WU Zu—jian, LIN Qi—ying’’ and XIE Lian—hui. Purification of a Antiviral Protein in Plearotus citrinopileatus and Its Activities against Tobacco mosaic virus and Hepatitis B virus[J]. Virologica Sinica, 2002, 17(4): 350-353.
A antiviral protein。YP46—46，was isolated and purified to homogeneity from the mushroom， Plearotus citrinopileatus．by precipitation of 40％ 一60％ saturation of ammonium sulfate followed by DEAE-sepharose FF ion—exchange column chromatography。 Sephacryl M S一200 High Resolution molecular sieve chromatography．The protein has a molecular weight of about 27．4kD by SDS-PAGE． The concentration of the protein was 0．24／~g／mL when the inhibition rate against Tobacco mosaic irus was 50％ ．Using an assay system based on HepG2．2．2．1 5 cell line，YP46—46 was studied for its inhibitory ability against Hepatitis B virus．The result shows that the concentration of 50％ inhibition of HBsAg was 0．08ttg／mL，but low effect on HBeAg．
WANG Shu—hui 。XIONG Kun, YANG Yi—shu, ZHU Yi—xin, XIA Qiu—yu, CHEN Guo-min, WANG Jin—zhong, CHEN Qi—min, GENG Yun—qi 一 and ZENG Yi. The Study on BIV Activity in Human Cells M T -4[J]. Virologica Sinica, 2002, 17(4): 354-357.
Bovine immunodeficiency virus(BIV)is a kind of Lentivirus belonging tO Retrovirus，and up tO now．there is no report about itS infection tO human．In order to check BIV S infection to human cells，we transfected BIVl27 cDAN into human cells MT一4．By RT～PCR，we checked the transcription of BIV S gag gene，and determined the expression of gag or gag-pol gene in M T一4 cell by IFA．Re— verse Transcriptase(RT)assay showed the reverse transcriptase of BIVl27 had been expressed，while cell infection experiment indicated that BIV couldn t replicate in MT一4 cells．
SUN Jian—he, JIANG Jing, LU Ping and ZHAO Yu. Amplification and Clone of VP2-4-3 Gene of Very Virulent Infectious Bursal disease virus[J]. Virologica Sinica, 2002, 17(4): 358-361.
The methods of reverse transcription，polymerase chain reaction(PCR)amplification，and cloning of full—length VP2—4—3 gene of a very virulent infectious Bursal disease virus(vvlBDV)strain SH95 were developed．The use of random primer and a reverse transcriptase lacking RNase—H activity produced full—length coding region and non—coding region eDNA copies of the viral genomic segments． The 3060 base—pairs(bp)of VP2—4—3 were amplified by long and accurate PCR in a single step，SUC— cessfully cloned and sequenced revealing their identity of IBDV．
TANG Xing—san, DONG Chang—yuan’’, GUO Shu—fang, CHEN Xiao, CHEN Dong—e, GUI Yi—rui, LU Li—li and LUO Xiang. Effect of Interaction Between BTV-Hbc and Different Species Cell and Charateristics of Group Specific Antigen[J]. Virologica Sinica, 2002, 17(4): 362-366.
Bluetongue virus(BTV)HbC strain and BTV 10(the international standard strain)were respectively inoculated onto the different species monolayer cells such as Vero cell(monkey renal cel1)， C6 cell(mouse neuroglioma cel1)and Hela cell(human cervical cancer cel1)．We studied comparatively the replicated characteristics of BTV—HbC in different species cells，with BTV 10 in the same cells， morphologic characteristics under microscopy and electron microscopy after interaction between BTV— HbC and different cells．the serologic relation between BTV—HbC and BTV一10．It is further proved that the BTV—HbC strain is probably a new type of Bluetongue virus when connecting our previous re— search about BTV—HbC genome map and RT—PCR analyses of BTV—HbC gene coding group speci fic antigen．
FAN Cheng—peng, FANG Cheng—xiang, ZHANG Luo-zhen and DAI Shu—xiang. New Methods for Determining Induction Rate of Prophage in Lysogenic Bacteria[J]. Virologica Sinica, 2002, 17(4): 367-370.
Two new methods，colony counting method and plate inducing method，were established for determining ultraviolet induction rate of prophage in the present study．Culture of lysogenic bacteria (λ)which was induced hy ultraviolet irradiation and cultivated in dark was directly spread on the plate，and induction rate of prophage was calculated based on the CFUs(colony form ing units)．The plate with mixture of lysogenic bacteria and indicator strain was induced by ultraviolet irradiation．The induction rate of prophage was calculated based on the PFUs(plaque form ing units)．These two methods not only can determ ine precisely induction frequency of prophage。but also are easy tO ma— nipulate ，and they can save efforts and apparatus，they are also easy to repeat．Furthermore，the m ix— ture of Cordyceps sinensis extract and lysogenic bacteria was irradiated with U V ．The results demon— strate that the new methods are practical and easy tO conduct，and that Cordyceps sinensis has a strong protection effect against U V—irradiation．
CHENG Zi—qiang, ZHAO Zhen—hua, HAO Yong—qing, ZHAO Xin—li and H ASI Agual. Serology Investigatio and PCR Diagnosis of Avian M yelocytomatosis[J]. Virologica Sinica, 2002, 17(4): 371-373.
We investigated the infection of Avian leukosis virus subgroup J myelocytomatosis usingEI ISA method in Inner Mongolia Region．The result indicated that the meat—type flocks of three dif—ferent farms infection rate'．of ALV—J were 28．12％(9／32)、9．38％(3／32)and 8．33％(10／12)，respec—tively，and all laying chickens infection rate were 0％(0／35，0／10)．We chose 6 chickens of ALV—J an—tibody positive and 13 chickens of ALV—J antibody negative to do PCR test．The result indicated that6 chickens of ALV—J antibody positive were all PCR positive，and 4 chickens were PCR positive among1 3 chickens of AI V—J antibody negative．The result indicated that there were ALV—J virus not only inAI V—J antibody positive chickens but also in some AI V—J antibody negative chickens．
CHENG Kai, WANG Chun—yan, GUO Ya—xin, SHI Zheng—li 。ZHAO Yi—jun and Measurement of Lysing Cycle and Burst Size of Cyanophage Infecting Filamentous Cyan0bacteria(blue-green algae)[J]. Virologica Sinica, 2002, 17(4): 374-376.
Several methods were t~ted for measurement of lysing cycle and burst size of the cyanophage infecting the ilia— mentous cyanobacterium—Plectonema boryanum IU594．The rm~ts indicated that the lysing curve of host cells was con— sistent with the one-step growth curve of cyanophage，thus it was more convenient tO substitute measuring the lysing curve of host cells for the one—step growth curve measurement of cyanophage．Besides，the burst size increased with the re— ducing cyanophage inoculation．reaching the maxium of 206PFU／Cel by 1 PFU infection．It was suggested that more ex— act burst size of cyanophage could be obtained under 1PFU cyanophage inoculation．