2002 Vol.17(1)


Apoptosis of Vaginal Epithelial Cells Induced by Infection 0f HSV-2 in Mice

CHEN Bao-ping, CHEN Dao-ping, IAN Yu, ZHOU Feng, ZHANG W, LIU jun, CHEN Min

2002, 17(1): 1

Abstract:Recently 8ome reports have shown that some viruses could induce apoptosis of infected cells in uitro to limit their spread.To clarify the effect of HSV-2 strain 333 on induction of apoptosis and its morphological characters in vaginal epithelia the mice were infected with the virus intravagina lly,an d killed at the intervals between day 1-11 post infection(P.I.).The vaginas weTe taken and fixed in 10% neutral formalin for paraffinembeding section and TUNEL staining to demonstrate apoptosis. The results were as follows.Numerous apoptotic cells appeared in the epithelia on the first day of in— fection .the number of the apoptotic cells reached to a high level on day 2—11 P.I..At early stage of apoptosis,the cell nuclei displayed norma1.but their chrornatin were stained by TUNEL staining. W hen the chromatin condensed and migrated to the periphery.then left a blank space in the center of the nuc .The apoptotic area spread widely.occupying 1/4·2/4 of the vaginal epithelia At the S;~L,’TLe time,the necrotic areas and herpetic vesicles in the epithelia could be found to restrict only in Solve areas not as widely as the apoptotic areas and were surrounded by the apoptotic areas The apoptotic cells fell off into the vaginal hanen without form ing apoptotic bodies an d phagocyosis.The results showed that the HSV一2 strain 333 could induce apoptosis and necrosis of vaginal mucosal epithelia at the 8an~e time in mice.The apoptos~ might play an impomant role in limiting both the range of viral spreading an dthe production of progenyvirus

Cloning and Sequence Analysis of Chinese CXCR4

FAN Zhao, I I, LUO gao-jun

2002, 17(1): 6

Abstrach In order to acquire the CXCR4 gene and develop the method to diagnose and treat AIDS pa— tient,the cDNA of CXCR4 was doned by RT-nPCR from peripheral blood mononuclear cells of a Chi— nese.An expected target fragment was identified by restriction enzyme digestion and DNA sequence data.The sequence data showed that CXCR4 gene is 1 059 nt encoding 352 amino acids.Compared with the sequence from GeneBank,there were only two deferent sites.Searched in GenBank,it was found that the segment had high homologenicity with M99293、XM 051223、XM051224、XM05 1225. The comparison result showed that the cloned segment included intact cod ing sequence and two base. pair deference
Research Article

The Immunization Study of Hepatitis C Virus E2 Gene DNA in Experimental Animal

2002, 17(1): 11

Abstract:HCV E2 DNA fragment encoding E2 pmtein(amino acid 417—750)was cloned into the eu— caryotic expressing plasmid pCDNA3 to get the recombinant plasmid pcE2.The pcD2 DNA was used as gene vaccine to immunize rabbi~ and mice respectively.The specific antibody against HCV E2 was detected by ELISA at 20 day after immunization,and WaS raising to highest level at 40 day,then main· tained at the titer of 1:1 600.The CD4 and CD8 T-lymphocyte in immune mice increased compared with the control group.the CD8 T_ 1 increased 35 46% aS measured by FACS.The immuuo-histo— chemicaI assay of the tissue from immune mice demostrated that the E2 gene had expressed inside the cells.this results indicated that E2 DNA has a good immunity

The Preliminary Study on Hepatitis B Virus Quasispecies X Gene and its Mutants

2002, 17(1): 22

The HBV genetic heterogeneity in the patients with chroni c HBV infection was reported in this article. A set of spectific primers was syn thesized according to HBV DNA sequence of Chinese strain, the whole X region was amplified by PCR method from the serum of 9 patients with chronic HBV infection , and then the PCR products were subcloned into pGEM Teasy vectors. Clones were randomly selected to be sequenced. Comparison of the cloned sequence was made to find the difference. After being compared, each sequence of selected clones is o f difference. The point mutation scattered through X region. Deletion mutations were detected in 19 clones of 37(51.4%), which caused different carboxyl endings of X protein. There is a hot region (after 123 aa code) where deletion mutation frequently happens. There were HBV quasispecies groups in sera from patients wi t h chronic HBV infection. There was a hot deletion region near the 3’ end of X ge ne, resulting in losing its transactivating activity

Study on M Genomic Segment of Hantaviros Strains Isolated from Different Areas in Hubei Province

HOU Wei‘, YANG Zhan-qiu‘”, Qu Hong .ZHAO Ming, XIAO Hong, CAI H 一lan

2002, 17(1): 26

Abstract:According tO Genebank sofeware of HV.the type-specific primers were desisgned cori-~s— pording tO the conserved sequences in the GI region of M genomic segment of HV.A reverse—transcrip— tion heminested polymerase chain reaction for HV was developed.Then.RT-heminested PCR was ap— plied tO type 3O strains of HV isolated from different areas in Hube i province.The results are shown that:(1)The 76/118、A9、H-114(HTNV)and R22(SEOV)Strains of HV were amplified and typed using the RT-heminested PCR.And HV RNA could only be detected by this method.but other vira1 RNA couldn’t be detected .(2)30 strains isolated from different areas in Hubei province were typed as HTNV amd SEOV。respectively.Those results suggested that:(1)The RT-heminested PCR was lea— sible for detectiong and typing HV in Hubei province.(2)Both HINV and SEOV were found in eoid— mic aro-.~.S of HFRS in Hubei province.Distinct hantaviruses could coexist in either different or the sam e geographic or ecological zones in Hubei province.

Immune Responses in Rhesus M onkeys Vaccinated with M ulti·epit0pe Antigen of HCV and Challenged by HCV Virus

HE Shao-qing, YANG Fang, DAI Jie, HU Fang, HUANG Jian-sheng, H Qjhan

2002, 17(1): 30

It is a new approach to study and produce HCV vaccine by using multi-epitope antigen of HCV. In this study, multi-epitope antigen gene, PCX, which consists of 5 epitopes of HCV and an epitope of T cell stimulation o f tetanus toxoid, was expressed in E.coli. And rhesus monkeys were vaccinate d by expressed antigen and elicited the high humoral response, the titer of anti bodies to PCX was high to 1∶1000. In 60th week after vaccination, the titer of Abs was still to 1∶40. Meanwhile, positive human sera of HCV challenged vaccina ted monkeys after 6 weeks immunized, ALT value rose transiently in monkeys vacci nated by PCX. It was positive in monitoring RNA of HCV using RT-PCR in monkey s era. The result demonstrated that Rhesus monkey immunized by multi-epitope PCX could elicit high-level immune responses

A Epidemiological Survey on CM V Infection of Child·bearing Age W omen and Congenital Infection in Wuhan

YAN Ying, fang CHEN, YANG Xiao, KUANG Er-sheng, ZHU Bing, DONG Chang-yuan

2002, 17(1): 34

Abstract:A survey of CMV seroepidemiology of child—bearing age women and new-born babies and his (her)mother was carried out with EI ISA and PCR technique.The CMV-IgG and IgM antibody posi— tire ratios of 1246 an~yzed women were 82.2% and 3.6% .respectively.There were 20 mothers and 3 babies with CMV.I)NA ;n their urine among 195 mo thers.There were 98 women who had abnormal borne history in assayed women and their CMV IgG and IgM antibody were ohviously higher than that of normal women(p0.01).

Primary Studies on DNA Imm unization of Hum an Rotavirus

PENG Shi—yong, WANG Jian-wei”, WEN Le-ying, HE Jin—sheng, HONG Tao

2002, 17(1): 38

Abstract:The probability of DNA vaccine against human rotavirus was studied,by constructing three recombinant plasmid clones,pCI/vp7,pCI/vp4,and pCI/vp6,and transferring into TA muscle (quadriceps)of BALW C mous~.with intramuscular inoculation.The sera were collected and antibody activity was evaluated with E1 ISA and neutralizing test pCI/vp7,pCI/vp4,and pCI/vp6 have been successfully Constructed,moreover,pCl/vp7 has transfected 293 cells and the vp7 gene could expres thoroughly.Intramuscular inoculation of these plasmids induced efficient immuniation,and the anti— body induced by pCl/vp7 presents some definite protective of a strain of rotavirus Wa.As the results showed,DNA vaccine wil1 be a helpful means to protect children from severe acute diarrhea caused by Gro upA ro taviruse s.

Construction of HV S and 5’Terminal of S Gene Segment Eukarytic Expression Vectors and Transfection into Vero Cells

PAN Lei, BAI Xue—fan, HUANG Chang—xing, LI Guang-yu.CHEN Hong—mei, W EI San—hua.YANG W ei—song

2002, 17(1): 47

Abstract:In order tO provide some data for study of T cell epitope.HV S and 5’termirmI of S gene segment were inserted into an eukarytic expression vector pcDNA 3.1/V5一His TOPO.The recombi— nant expression plasmids peDNA3 1-S and pcDNA3.1-S—N were constructed.Vero cells were trans— fected in vitro with them.The transient expresion of HV S and 5’terminal of S gene segment in veto cells w∞ detected by;ndirect immunofluoreseence assay(IFA).

The Immune Response Induced by Recombinant Adenovirus with Administration of Interaperitonea Injection

wu Juan。LI Jia—qi, SUN Mao-sheng, DAI Chang-bai

2002, 17(1): 51

Abstract:In order to investigate the immunity of the recombinant adenoviruses inoculated to the ani— mals,structural gene from RNA of HAV was cloned by RT-PCR and inserted into the shuttle plasmid (pXCX2Not I).A replication-defective adenovirus was rescued in 293 packing cells via homologous re— combination of both ptasmid pXCX2·CMV—HAV and plMI7 containing AdS gertome with de[eforts of E1 region by calcium phosphate technique.A series of methods were employed to identify the generat— ed recombinant ademovirus(rAdI-IAV) The titer of rAdHAV stocks was up to I x 10 TCIDso/mL. The Kunming mice were administrated with rAdHAV by intraperitonea injeetion(i.P.).Anti—HAV IgG and HAV neutralizing antibodies were induced.In Conclusion,a recombinant replication—defective adenoviros is an efficient delivery system to develop recombinant virus vaccine.

Experimental Studies and Comparison on Clinic Diagnosis of TORCH IgM Antibody

KUANG Er, YANG Fu-hua, HE Xiang-Wan, LIU Jian, YAN Yin

2002, 17(1): 56

Abstract:To research ToRCH IgM clinic diagnosis,four kinds for methods for non—specific interfer— enee removed have been compared in IgM detection;then ToRCH IgM in sera of 256 female donors. 143 normal pregnant women and 61 abnormal pregnant women were detected with the best method . anti—human IgG( chain specific),resolving non-specific interference The results show that the posi— tive late of T。RCH IgM in normal population was very high,it was up to 23 1% in health pregnant women(Tox 5.6%.CMV9.8%.RuV8.4%,HSV4 9% respectively),while it increased dramatically in abnorm_al pregnantwomen(p0.01).SO didTox,CMV.RuVIgM(9.7%,26.2%,24.6% respec— tively).Then T0RCH pathogen active infection during pregnancy can Cause abnorma l pregnancy and should be inspe cted carefully.esp.Tox,CMV,RuV.

Study on Expression and Location of Hepatitis C Virus NS5A Protein in Insect Cells

FENG Lei.YE IAn-bai“, RUAN Hua, GAO Jin·rong, XU Jin·ping, LIU Yuan.jie, WU Zhenghui

2002, 17(1): 60

Full—length NS5A gene of Hepatitis C Virus was amplified by PCR.using plasmid pBAC25 containing HCV nonstructural protein genes as template.The amplified fragment(about 1.34kb)was cloned into transfer plasmid pBuleBacHisA.The recombinant plasmid DNA was cotransfected with genome DNA of wild type AcNPV to SF-9 il~ect cells.The recombinant baculovirus AcNS5A was ob— rained by plaque selection.Restriction analysis and PCR identification indicated that the NS5A gene was integrated into the genome of AcNSSA.and HCV NSSA gene was expre~sad as a 64kD protein in SF·9 cells infected with AcNS5A.This protein can he recognized by anti.NS5A antib0dy when idend— fied bv Western—blot.The Immune fluorescence assay and immune histo-chemistry assay were used tO study the distribution of NS5A protein inside the cel1.Distribution of NS5A protein are changed with the time in the recombinant virus infected SF一9 cells.Ns5A protein are changed with the time in the recombinant virus infected SF-9 cells.NS5A protein iS 1ocated at the cel1 membrance at 24 hours post infection and located at both the membrance and the nucleus after 48 hours.and ful1 of the cells after 72 hours.The result suggests that NSSA protein Was located on plasma membrance and in nucleus.it may play an important role in RNA replication and in controlling the expression of the cell gene~during the replication of the virus.

Identification of the Receptor for PDV of Spodoptera Iitura M ultinucleoeapsid Nucleopolyhedrovirus

YIN Chong.YU Jian—xiu.PANG Yi*

2002, 17(1): 66

Abstract:This research provides the evidence about the identification of haculovirus receptors present in midgut cell lystes.Rabbit antiserum against purified PDV of SpltM NPV was produced Total midgut eel1 proteins were prepared with SDS lysis buffer with or wi出OUt 2-mere~,ptoethanal(2-ME).After SDS—Polyacrylarnid gel eleetmphoresis(SDS—PAGE),the midgut eel1 proteins were tranfered to nitro cellulose membrance.Transferred proteinS were renaturalized overnight with 4% bovine serum albumin (BSA).Then with PDV—specific antiserum.a virus overlay protein blot as~y(VOPBA)was performed to identify the protein that binds PDV of SpltMNPV.h was shown that PDV of SpltMNPV bound c0 three molecules of approximately 40kD,73kDand 85kD in midgut cel1 lysates.

The Isolation and Identification of A New M ember of Tetraviridae Family from China

HU Yuan—yank, OUYANG Guo-jie, ZHANG ]ia-min, LI Li, wU Kai—lang YI Fu—ming, YIN Yi—nong

2002, 17(1): 73

A New member of Tetraviridae family was found and is olate d from Dendrolimus punetatus larvae in China.Purified virion is icosahedral spheroid with the diameter 44nm and 240 identical subunits in the particle with T=4 symmetry.The nucleic acid in the virion was determined to be single-strande d (ss) RNA by means of diphenylamine reaction,orcinol reaction and formaldehyde reaction.The viral RNA showed one band in agarose gel eletrophoresis (5.2kb).Usi n g SDS-PAGE,there were a major polypeptide band of molecular weight 52 kD and a small polypeptide at 39kD. According to the viral taxonomic principle,the virus belongs to Nudaurelia capensis β-like viruses in Tetraviridae.

M odel Establishment of the Cell Persistently Infected by a Field—isolated Virulent Strain of CSFV

YING Song-cheng, ZHENG Cong, QU San, ZHANG Chu, WANG Zai

2002, 17(1): 77

Abstract:A porcine kidney cell line(PK一15)was infected by a field—isolated virulent strain of classical swine fever virus(CSFV39) After the infected cells had subeultured 79 passages for near eight months.a steady modeI of the eell persistently infected by virus has been establisheel,hence the CS— FV39.PK15 cell line was procured.In this experiment immunofluorescenee,RT-PCR assay and elec— tron microscopy observation were used to identify the basic viral fearums in the CSFV39一PK15 cel1. The results showed the 9th,29th.79th CSFV39一PK15 cell pasages still harhored CSFV and they ha d the radical characteristics of viral persistent infectlon.This endeavor should be the fotmdation for the further research of CSFV persistent infection.

Comparison and Analysis 0f The CAV Fiber Protein Gene

FAN Quan, XIA Xian-zhu, QIU Wei, HUANG Geng, HE Hong, Yu Chun, QIAO Jun, wu Yin, ZHOU Xiao, YlN Zhen

2002, 17(1): 82

Abstract:Accordilag to CAV Fip genes sequence,4 paris of primer were designed tO amplify the Fip genes with PCR method.Complete Fip gene sequences of al1 CAV strains were shown tO be consisted of 1 629nt and 1 63lnt and encoded 542 and 543 amino acids from ATG start cod en.The deduced amino acid sequences of the Fip of the CAV一2 strains were compared one another and with standardard CAV strain extracted from the GenBank database.There are point mutation(transversion)occurring in at— tenuated SY—V6O and SY—V5 The Fip genes of CAV of virulent strain of different area were 8e— quenced There are less differences among domestic CAV一1 strains but more differences with foreign CAV.1 strains.Honology of Fip between CAV—l and CAV一2 iS 80.48% .

Sequence Analysis of ORF2 Gene of Type 2 Porcine Circovirus and Construction of Eukaryotic Expressing Vector of ORF2


2002, 17(1): 87

Abstract:According to the published ORF2 gene sequence of PCV一2,two primers were designed.and ORF2 gene was amplified from suspected PMW S sample using PCR.Then the PCR product was cloned into pGEM .T easy vector,and recombinant plasmid nam ed pTORF2 was obtained The cloned ORF2 gene in pTORF2 was sequenced and compared with other PCV isolates in GenBank,the results demonstrated that the ORF2 gene is closely related with a USA isolate AF264039.the homology of their nucleotide and amino acid sequence both were 100% . In addition. there existed 92.3% to 98.6% nucleotide sequence identity and 92.3% to 96 6% amino acid sequence identity with other PCV-2 isolates.Recombinant plasmid pTORF2 was digested with Bam H I/EcoR V.ORF2 gene Was purified.and introduced into eukaryotic expressing veCtor pSecTag2/HygroB between BarnH I and E∞R V enzyme site.then pSecTagORF2 recom binant plasmid was constructed .Further research is underway to study the biological activity of ORF2 protein and establish the diagnostic kit of PCV.

Cloning and Sequence Analysis of Buchnera GroEL Genefrom Endosybiotic Bacterium of M yzus persicae

2002, 17(1): 69

Buchnera groEL gene of endosymbiotic bacterium (Buc hnera sp.) from Myzus persicae Yangling boitype (P.R.China) was isloated,a n d the complete nucleotides sequence was determined.It is indicated that Buchne ra groEL gene of M.persicae includes 1647 base pairs,has 99% and 91% sequ ce similarity with Buchnera groEL gene of M.persicae The Nether land bi otype (AF003957) and Acyrthosiphon pisum Japanese biotype (X61150),respectiv ely.Buchnera GroEL-YL contains 548 amino acide,and has 3 different amino ac ids comparing with Buchnera GroEL-NT,that is AA111MetLys、AA222ValMet and AA348GlnHis.
Brief Reports

The Studies on Expression of 1 197 Gene of Prawn Baculovirus and on Purifications of the Expressed Product

YING Zhi-wei, ZHOU Guo-ying.GONG Zu-Xun

2002, 17(1): 92

In previous report we have cloned a putative early and late transcriptional gene 1 197 of prawn baculovirus.In order to detect the biological function of this gene ,1197 gene was inserted into expressing vector pGEX-3X.The fusion protein expressed with high yield gua$obtained,but it~1)as foundthattheAsion proteinlocatesininclusion bodies ofE.coli andit causod troubleinitspurfica— tion.Wefoundthatthe expressed productwithM .of 66kD could bepurifiedfrom inclusion hod— ies by using a serious of treatments of inclusion bodies r~,ith ug’ea and guanidine hydrochloride and then by sequential FPLC chromatography.