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2006 Vol.21(3)

Research Article

Specificity and Epitope Mapping of Four Monoclonal Antibodies against SARS-CoV Nucleocapsid Protein

WANG Jian-wei, WANG Yan-bin, CHANG Zhao-rui, YAN Ke-xia, TAN Wen-jie, QU Jian-guo, XIA Ning-shao, RUAN Li, HUNG Tao

2006, 21(3): 207

In order to characterize the specificity of the monoclonal antibodies (McAbs) against SARS- associated coronavirus (SARS-CoV) nucleocapsid protein and to identify the epitopes recognized by the McAbs,the nucleocapsid proteins of human coronavirus OC43 (HCoV-OC43) and 229E (HCoV-229E) was expressed in E.coli. The specificities of four McAbs (1-1C2, 2-2E5, 1-1D6, and 2-8F11) were examined by Western blotting as well as indirect fluorescence assay. Twelve different recombinant truncated N proteins were then used to identify the epitopes recognized by the McAbs by Western blotting. The results showed that: (1) McAbs 1-1C2, 2-2E5, and 1-1D6 recognized neither human coronviruses HCoV-OC43 and HCoV-229E nor their nucleocapsid proteins, implying a possible specificity of these 3 McAbs to SARS-CoV; (2)The epitopes recognized by McAbs 2-8F11, 2-2E5, and 1-1D6 were located between the 30th and 60th amino acid (a.a.) residues of the SARS-CoV N protein, while the epitopes recognized by the McAb1-1C2 were located between 170th and 184 th a.a. residues. The identification of the specific epitopes of SARS-CoV N protein is paramount for the immunological characterization, the development of accurate immunological assay as well as for exploring the pathogenesis of SARS-CoV.

Cloning and Expression of N and P of Human Respiratory Syncytial Virus RNA Polymerasa Complex

YU Jie-mei, He Jin-sheng*, LI Dong-liang, YANG Bing, YUAN Yuan

2006, 21(3): 213

The n and p genes from subgroup A human respiratory syncytial virus (HRSV), were amplified by RT-PCR. The n product was cloned into pGEM-T easy vector and that of p into pcDNAII vector and the two genes were subcloned into the eukaryotic expression vector pcDNA3.1(+). The resulting recombinant plasmids pcDNA3.1(+)/N and pcDNA3.1(+)/P were checked by restriction enzymes and DNA sequencing and were tranfected into COS-7 cells with the aid of Lipofectamine 2000. Expression of N and P were detected by western blots at 72 hours post transfections. Expressed N and P in COS-7 cells was confirmed by western blot. The constructed expression vectors will be used for the further reverse genetics experimentations on HRSV.

Cloning of HCV ns2 Gene and its Expression in Prokaryotic and Mammalian cells

LIU Chao-hong, JIAO Chen-song, CHEN Xin-wen

2006, 21(3): 217

The full-length and truncated ns2 gene (2881-3078 bp) were gained through PCR using p90-HCV as template. The truncated ns2 gene was cloned into the prokaryotic expression vector PET32a. The fusion protein Trx-His-NS2C expressed in a soluble form in E.coli, and then was purified by His resin. Highly specific and efficient antibody was produced by immunizing the rabbit with purified fusion protein. In addition to this, recombinant adenovirus containing the full-length ns2 gene was constructed. The NS2 protein was expressed successfully in a size of 23.2kDa by infecting 293 and HepG2 cells with recombinant adenovirus. These results had set a base for the further stulies on the structure and function of the NS2 protein.

Localization of HSV-I ICP22 Protein in Cells by Antibody Against a predicted Antigenic Peptide

CUN Wei, MA Shao-hui, ZHAO Hong-ling, LIU Long-ding, WANG Li-chun, DONG Cheng-Hong, ZHANG Ying, WANG Xiang, LI Qi-han*

2006, 21(3): 221

As one of the viral proteins expressed in the immediate-early phase of HSVI infection, ICP22 functions in the regulation of viral replication. However, it is difficult to produce specific antibody against ICP22 protein with homology of antigenicity in this protein. Based upon prediction of antigenicity of ICP22, a peptide of 1-36 amino acids was thought to possess a specific antigenic epitope, which was expressed as a fusion protein with GST. The antibody, raised in mice immunized with a purified fusion protein, was able to recognize ICP22 expressed in eukaryotic cells. Analysis with this antibody suggested a nuclear locatization of ICP22 and specific punctuative structure in cells.

Localization of HSV-I ICP22 Protein in Cells by Antibody Against a predicted Antigenic Peptide

CUN Wei, MA Shao-hui, ZHAO Hong-ling, LIU Long-ding, WANG Li-chun, DONG Cheng-Hong, ZHANG Ying, WANG Xiang, LI Qi-han*

2006, 21(3): 226

As one of the viral proteins expressed in the immediate-early phase of HSVI infection, ICP22 functions in the regulation of viral replication. However, it is difficult to produce specific antibody against ICP22 protein with homology of antigenicity in this protein. Based upon prediction of antigenicity of ICP22, a peptide of 1-36 amino acids was thought to possess a specific antigenic epitope, which was expressed as a fusion protein with GST. The antibody, raised in mice immunized with a purified fusion protein, was able to recognize ICP22 expressed in eukaryotic cells. Analysis with this antibody suggested a nuclear locatization of ICP22 and specific punctuative structure in cells.

Construction of Recombinant Adenovirus Expressing Protective Antigen of Bacillus Anthracis and Analysis of Immuno-responses

XU Lin, GUO Yan, GUAN Jie, YU Hong, ZHOU Yu-sen, LI Jin-song*

2006, 21(3): 231

In this study, we constructed recombinant adenovirus containing protective antigen gene of Bacillus anthracis. The PA gene was obtained from pcDNA3.1-PA vector by PCR , and cloned into plasmid pAdTrack-CMV. The positive clone was linearized with PmeI restriction enzyme and transformed into E. coli BJ5183 cells with the adenoviral plasmid pAdEasy-1. Then vAd-PA was generated by homologous recombination. After linearized with PacI restriction enzyme, vAd-PA was transfected into HEK-293 cell line by lipofection. Expression of PA in 293 cells was confirmed by SDS-PAGE and Western-blot. The BALB/c mice were intramuscularly inoculated with the recombinant adenovirus and the specific antibody was detected by ELISA . The induced antibody titer was 2.8×103 (Geometric Mean).

Molecular Characterization of Hantavirus Zhejiang Isolate ZT10 Strain

WENG Jing-qing, XIE Rong-hui, LU Yi-yu, YAO Ping-ping, LI Min-hong, LI Chan, XU Fang, MAO Hai-yan, ZHU Han-ping, CUI Qing-rong, DONG Ying, ZHU Zhi-yong

2006, 21(3): 235

The effects of the M gene on the molecular epidemiological characteristics of the Hantavirus ZT10 strain isolated from M. arvalis was investigated. The gene was amplified by RT-PCR and yielded the expected product of 3651 bp. The amplified fragment was then cloned into the T vector and confirmed by PCR and sequencing. Analysis of the sequence revealed that the ZT10 M segment belongs to seoul virus.The nucleotide sequence identity of this M gene with eight virus strains was 84.0%~96.3%. The identity with Hantanvirus(Prospect Hill virus, Tula virus, Khabarovsk virus, Isla vista virus) isolated from arvalis was 57.5%~60.9%. Also, the identiry with the SEO types and with the Guo3 isoated from ZheJiang province was 84.0%. Seoul virus ZT10 strain is a new specific seoul subtype virus in ZheJiang province.

Inhibition of Rabies Virus Replication and Infection by Rabies Virus Nucleoprotein Gene siRNA Cocktail

WANG Ji-lin, ZHU Jia-hong*, XU Ge-lin

2006, 21(3): 238

Small interference RNA mixtures (siRNA Cocktails ) of Rabies virus (RV) N gene, P gene and G gene were prepared by digesting long double-stranded RNA (dsRNA) with RNase Ⅲ. These siRNA cocktails were transfecteing into BSR or MNA cell monolayers and the direct immunofluores- cence assay (FA) was used to measure the replication and infection of RV on the cell monolayers. We found that the siRNA Cocktails of N gene can obviously and stably inhibit the replication and infection of RV. A descent of mRNA of N gene was also detected by RT-PCR. Under the same situation, the siRNA Cocktails of P gene or G gene showed no or weak inhibition to the replication and infection of RV. All of these results provide a basis for selecting target gene of RV in RNA itreatment and its further application.

Characterization of a LTR Chimeric Infectious Clone of Chinese Equine Infectious Anemia Virus

WEI Li-li, WANG Xiao-jun, GENG Qing-hua, TONG Xiao, LIANG Hua, SHEN Tao, ZHANG Xiao-yan, XIANG Wen-hua, SHAO Yi-ming, SHEN Rong-xian

2006, 21(3): 244

Equine infectious anemia virus full-length-gene chimeric clone derived virus, vLGFD9-12, was inoculated into healthy horses. Over an observation period of 150 days, all inoculated groups showed no overt abnormal symptoms. The results from routine blood assays showed that the counts of WBC and HGB had no obvious changes between vLGFD9-12 group and its parental virus pLGFD3-8. Low levels of viral RNA in the plasma of vLGFD9-12- and pLGFD3-8-infected horses was detected, The results from cytolytic T lymphocyte (CTL) and lymphocyte proliferation assay (LPA) showed that all inoculated animals had similar changes on virus-specific CTL responses and virus-specific lymphocyte proliferation activity. These results provide an important foundation for further study on mechanism of attenuation of the EIAV and the protective immunity against EIAV.

Expression of CSFV E2 Envelope Protein Gene in Eukaryotic Cells and Preparation of Monoclonal Antibodies against CSFV E2 Envelope Protein

LIU Jian-ling, ZHANG Yan-ming, SU Zheng-yuan, XU Xin-gang

2006, 21(3): 249

The recombinant retroviral vector pBABE-puro-E2 was constructed by inserting full-length cDNA of CSFV Shimen strain E2 gene into pBABE-puro. Both the recombinant retroviral vector and pVSVg plasmid were transfected into eukaryotic cells 293GP by calcium phosphate transfection method, and the pseudovirus were produced. The pseudovirus-infected eukaryotic cells PK-15 and expression of E2 protein were determined by puromycin-resistant and FACS analysis. Balb/c mice were intraperitoneal injected with PK-15 cells expressing the CSFV E2 protein. Anti-CSFV E2 antibody was screened by ELISA. The results showed that CSFV E2 protein was expressed in PK-15 cells’ envelope protein successfully. ELISA could detect specific anti-E2 antibody in mouse serum immuned by PK-15 cells.

Detection of Apoptosis of the Human Hepatic Carcinoma Cells Induced by Bluetongus Virus by PI and Annexin V/PI Staining Methods

WANG Yan, LIU Chun-xin, YI You-rong, CUI Ye-jian, ZHU Jun, DONG Chang-yuan

2006, 21(3): 253

Flow cytometry was used to quantify the percentage of apoptotic hepatic Caracimoma cells infected with bluetongue virus HbC3 and stained with propidium iodide (PI) and Annexin V/PI. The percentages of the apoptotic cells measured by PI at 24h, 36h and 48h were 10.8 ±3.05, 21.7±6.28, 28.3±10.6, respectively, and those by Annexin V-PI were 20.42±3.70, 49.3±8.11, 79.6±11.5, respectively. The two staining methods and the rate of apoptotic cells infected by bluetengue virus hours were significantly different (P0.01). The early apoptotic cells and secondary necrosis could be detected by AnnexinV/PI assay with good sensitivity and specificity. The apoptotic cells induced by bluetengue virus was one of the important forms in the pathological changes and cell necrosis.

Construction of Recombinant Marek’s Disease Virus Vaccine Strain CVI988/Rispens Expressing the VP2 Protein of IBDV

LIU Hong-mei, QIN Ai-jian*, YE Jian-qiang, CHEN Hong-jun, SHAO Hong-xia, JIN Wen-jie, LIU Yue-long

2006, 21(3): 257

An expression cassette containing the VP2 gene of infectious bursal disease virus (IBDV) strain JS under the control of the CMV promoter and an enhancer in pcDNA3.1-VP2 was amplified by PCR and cloned into pUC18-US10 plasmid to yield the transferring vector pUC18-US10-VP2. The recombinant virus, designated rCVI988-VP2, was generated by co-transfection of CEF with pUC18-US10-VP2 and the Marek’s disease virus (MDV) vaccine CVI988/Rispens virus. PCR analysis and immunofluorenscence assays indicated that the recombinant virus was stable over 8 passages and that VP2 was expressed in CEF infected with the rCVI988-VP2. The data showed that the recombinant MDV expressing the VP2 gene of IBDV was successfully constructed and could prove to be a good strategy for IBD control.

Sequencing and Analysis of the Genomes of Three Newcastle Disease Viruses Isolated from Guangxi

XIE Zhi-xun, TANG Xiao-fei, DONG Jian-bao, LIU Jia-bo, PANG Yao-shan, DENG Xian-wen, XIE Zhi-qin

2006, 21(3): 261

Eight pairs of specific primers were designed and synthesized based on the entire genomic sequence data of Newcastle Disease Virus (NDV) present in GenBank. Complete nucleotide sequences of GX7/02, GX9/03 and GX11/03 strains were acquired by reverse transcription-polymerase reaction. The entire genomic sequences of three NDV strains consisted of 15 192 nt, which was the same in length to 7 NDV ZJ1, U.S/Largo/71, Italy/2736/00 strains, but was 6 nt longer than LaSota, Clone-30 and B1 strains in GenBank. The additional 6 nt motif was located in the non-coding region of nucleoprotein (NP) gene between nucleotides 1647 and 1648 of the entire genomic sequences of NDV LaSota, B1 and Clone-30 strains. The results of identity analysis of nucleotide and amino acid sequences showed that the identities between GX7/02, GX9/03, GX11/03 and ZJ1 strains were higher than that of the three strains of LaSota, B1 and Clone-30.

Genome Sequencing and Structure Prediction of ZHZC3 Strain of Taura Syndrome Virus Isolated in China

HUANG Xin-xin, LU Cheng-ping*

2006, 21(3): 267

Eight overlapping clones covering the whole viral genome of TSV ZHZC3 isolate were amplified by RT-PCR and the 5′ and 3′ ends were amplified with RACE. The PCR products were cloned into pMD-18T vector and sequenced. The result showed that the genome of ZHZC3 consisted of 10202nt excluding the poly (A) tail, and contained two ORFS (ORFS1-2) which encoded polyproteins of 2107aa and 1011aa. No insertions or deletions were detected in the coding regions while 3nt of A were deleted at 5′ UTR when compared with HI94. The overall nucleotide sequence identity between ZHZC3 and HI94 was 97.6%. Compared with HI94 and BLZ01, the ORF1 of ZHZC3 shared 97.6% and 97.7% nucleotide sequence identity. The ORF2 shared 98.3 and 97% sequence identity at the nucleotide level. Compared with partial sequences of ORF2 from foreign isolates, both ZHZC3 and the Taiwan isolates shared higher nucleotide identity with HI94. Amino acid sequence analysis of six TSV isolates from different regions of mainland China revealed unique aa changes at 312 (S), 449 (A), 451 (Q), and 468 (H) in the highly variable region CP2 of ORF2, indicating that the isolates spread through the South-easten China most closely related to the other isolates with it’s own evolutionary character.

Study on a Synergistic Effect of Tinopal LPW on Parocneria orienta NPV

ZHOU Jian-hua, HU Ying-zhi, XIAO Yu-gui, XIAO Yin-bo, GUO Heng-xiao

2006, 21(3): 273

Bioassay of PoNPV+1%TinopalLPW was conducted on second instar larvae of a laboratory colony of Parocneria orienta. The results showed that 1%TinopalLPW had a marked synergistic effect. Various doses of PoNPV along with 1%TinopalLPW were sprayed on second and third instar larvae of an overwintering population in the field. The combinations were 3.6×1011PIB/hm2+1%TinopalLPW, 1.8×1011PIB/hm2+1%TinopalLPW, 9.0×1010PIB/hm2+1%TinopalLPW and 3.6×1011PIB/hm2, 1.8×1011PIB/hm2, 9.0×1010PIB/hm2. The most marked synergistic effect was obtained with 9.0×1010PIB/hm2+1%TinopalLPW. Other combinations did not have significant synergistic effects.

Method for Detecting Tobacco Ringspot Virus by IC-RT-Realtime PCR

YANG Wei-dong, ZHENG Yun, ZHANG Gui-ming, CHEN Zhi-nan, LI Ming-fu, YU Dao-jian, ZHANG Yong-jiang

2006, 21(3): 277

The technique of immune capture reverse transcription real-time PCR (IC-RT-Realtime PCR) was applied for the first time to detect Tobacco ringspot virus. When detecting virus, it was shown to be advantageous in terms of specificity and sensitivity to integrate several techniques, such as specific bond of antigen and antibody, nucleic acid hybridization and real-time PCR over the traditional technique of DAS-ELISA. Application of the integrated techniques common problems such as masking of symptom, low concentration of virus and disturbing-materials.

Anti-CVB3 Polypeptides Selected from A Phage Display Random Peptide Library

LIU Nian, LI Fan, YUAN Hang

2006, 21(3): 281

CVB3 was propagated in Hep-2 cells and purified by sucrose gradient centrifugation. The purified CVB3 reacted with random peptides library displaying 9 amino acids. After 3 rounds of screening, the capability of phage peptides in the inhibition of CVB3 replication was determined. DNA was extracted from the positive phage clones, sequenced and the amion acid sequence was deduced. The results showed that 3 phage positive peptides were identified and bound to CVB3 with high affinity. The binding of the peptide to CVB3 was shown to inhibit replication substantially. The TCID50 of progeny virus was reduced from 10-7.5SFU/mL to 10-5.25, 10-6, 10-5.5SFU/mL by the three peptides, respectively. The data demonstrate that peptides against CVB3 can be selected from phage peptide library and may prove useful as antiviral peptides agents.

Inhibition of Hepatitis B Virus Replication by Homoharringtonine, IFN-α1b, Hydroxycamptothecin and Norfloxacin

RAO Min, ZHANG Shu-ling, DONG Ji-hua, LI Shu-li

2006, 21(3): 284

The inhibitory effects of homoharringtonine、IFN-α1b 、hydroxycamptothecin and norfloxacin on Hepatitis B virus(HBV)replication in vitro was investigated. Different concentrations of each drug were added to the culture medium of HepG2.2.15 cell line transfected with HBV. The Cytopathic effects(CPE)and cell toxicity of each drug was observed. Under the maximum non-toxic conentration(TC0),ELISA and fluorescent quantified PCR were used for determining HBsAg、HBeAg and HBVDNA in the culture medium. The results showed that homoharringtonine and IFN-α1b had an apparent inhibitory effect to HBV while Hydroxycamptothecinhad had only a weak effect to HBV replication. Norfloxacin had no inhibition effect to HBV at any concentration. This data t provide part of the experimental evidence needed to initiate clinical trials to evaluate homoharringtonine as an anti-HBV drub.

Animal Model of Respiratory Syncytial Virus Infection in Nude Mice

ZHOU Juan, WANG Li-jia, CUI Yu-xia, LIU Xu-mei, YANG Xi-qiang*

2006, 21(3): 288

Respiratory syncytial virus ( RSV) is a worldwide leading viral cause of lower respiratory tract infection in children. Immunodeficient individuals are vulnerable to severe RSV infection. No ideal animal model is available at the present time. To establish an ideal murine model, RSV was dripped in tranasally into cellular immunodeficient nude mice, then pulmonary virus was isolated and viral antigen in bronchi-alveolar lavage fluid was identified by direct immunofluorescent assay. Pulmonary viral titers detected by plaque forming assay, peaked on the 3rd day post infection, and were still detectable by the 9th day post infection. RSV antigen was mainly localized in cytoplasm of alveolus and bronchioles by immunohistochemistry assay. RSV infected nude mice developed interstitial pneumonia with prominent lymphocyte infiltration; meanwhile the virus destroyed the air-blood barrier and viral inclusions were found by electron microscopy. The numbers of leukocyte in bronchi-alveolar lavage fluid increased significantly and peaked at the 9th day post infection. All these results indicated that RSV infected nude mice showed similar patterns of viral replication and pulmonary histopathology as with infected humans, and had a high level, durative viral replication. Therefore, RSV infected nude mice appears to be a workable murine model for assessing the effect of antiviral agents against RSV.

A Visual Gene Array for the Detection of HSV-2 Based on Nanogold Probes

LIU Ming-gui, WANG Ye-fu, SUI Fang, WAN Zhi-xiang, GAO Wen-jun

2006, 21(3): 294

A visual HSV-2 detection gene array was developed based on nanogold probes。The specific primer and probe of HSV-2 were designed based on the highly conservative region of HSV-2 DNA polymerase gene and the HSV-2 PCR amplicon were labeled by biotin through PCR reaction.Amino-modified probes were spotted on an activated glass surface on which hybridized with the PCR amplicon.Subsequently, the streptavidin-nanogold was added to form the amplification system of the biotin-streptavidin-nanogold complex.At last, the step of silver staining amplified the signal of hybridization, Therefore it was easy to detect the HSV-2 by naked eyes.As little as 100fmol/L of HSV-2 amplicon could be detected on the array, The HSV-2 detection gene array can detect the HSV-2 virus infection with high sensitivity and low cost.
Brief Reports

Clinical Application of Flow Cytometry for Quantification of Hanta Virus Antigenemia in Patients with Hemorrhagic Fever with Renal Syndrome

WANG Wei-hua, WANG Feng, CHEN Jie, WANG Li, ZHOU Xun-liang, GU Xin, ZHOU Bin-Lei, YU Hong-shen

2006, 21(3): 298

The clinical value of the detection Hanta virus antigenemia in Peripheral blood mononuclear cells (PBMC) in patients with Hemorrhagic fever with renal syndrome was evaluated. Samples from 32 cases with varying degrees of HFRS were subjected to quantitative detection for HV membrane antigen in PBMC by flow cytometry. The correlation between clinical severity and positive rates was analyzed. On the first day of onset of illness, positive PBMC rates were highly different from group to group (P0.01), which suggested a close relationship between clinical severity and the positive rate. Dynamic observations showed that in the light and moderate groups, positive rates differed significantly from day to day. This was not the case in the severe group with no statistical significance between the 7th and 10th day(P0.05), which implied a relatively slow rate of virus elimination. Quantitative analysis of HV antigen in PBMC by FCM is a promising method in early diagnosis, individualized therapy and in evaluating the prognosis of HFRS.

Cloning and Phylogenetic Analyses of the Nucleoprotein Gene from Coronavirus of Pigeon Origin

QIAN HUA-dong, ZHU JIAN-guo, WU ZHU-li, HUA XIU-guo*

2006, 21(3): 301

The nucleoprotein (N) gene of the coronavirus of pigeon origin (PSH) was cloned and sequenced A pair of primers based on the nucleoprotein gene sequences of group 3 coronaviruses was designed and used to amplify the N gene of PSH The PCR product was purified and cloned into the pMD18-T vector. The positive recombinant pTN was used for sequencing. The cloned 1275bp fragment covered all the open reading frame of N gene and potentially encoded 409 amino acids. The results of comparative sequence analysis indicated that the nucleotide sequence of N gene of this strain shared 67.4%~96.6% identity with group 3 coronaviruses, and the predicted amino acid sequence shared 67.4%~97.8% identity, while only limited identity (41.5%) was observed with group 1 and group 2 coronaviruses. Based on these results, PCoV PSH strain was identified as a member of group 3 coronaviruses.
Review

Function of RNA-dependent RNA Polymerase of Influenza A Virus on the Regulation of Viral Genome Replication and Transcription

TAO Pan, PAN Zi-shu*, WU Jian-guo

2006, 21(3): 304