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2006 Vol.21(1)

Contents

Genetic and Immunogenic Characteristics of Measles Virus Isolated in Zhejiang Province

YAN Ju-ying, LU Yi-yu, ZHANG Jian-hua, FEN Yan, SHI Wen, MAO Hai-yan, GONG Li-ming, GE Qiong, ZHOU Min, LI Min-hong

2006, 21(1): 1

In order to elucidate the relationship between genetic characteristics and measles outbreak,and the influence of antigenic variation on measles vaccine effect,we investigated the genetic characteristics and antigenic variation of measles viruses isolated in Zhejiang province during 1999~ 2003.Rats were immunized with the measles virus isolated from patients and the titers of the antibody were measured by cross neutralization test using the antigen of the isolated measles virus,vaccine strains of Shanghai191...

Prokaryotic Expression and Purification of Hsp70-NP and Its Immunity

LI Jing, YE Zheng-xu, YANG Shou-jing, GAO Juan, LIU Yan-fang

2006, 21(1): 11

Recombinant fusion prokaryotic expression vector pGEX-4T-1/hsp70-S was constructed by cloning genes Hsp70 and Hantaan virus S gene coding region into pGEX-4T and was expressed in E. coli. The Hsp70-NP fusion protein was separated and purified with GSTrapFF purification system. We also constructed the prokaryotic expression vectors pGEX-4T-1/hsp70 and pET28a/S to prepare the purified protein Hsp70 and NP. Then BANB/c mice were vaccinated with the protein Hsp70-NP, Hsp70 and NP separately. ELISA results showed that both Hsp70-Npand NP could induce specific antibodies against NP. The specific antibody titers stimulated by Hsp70-NP were obviously higher than that of NP. The lymphoproliferative responses of spleen cells clearly showed that the spleen cells from both groups of mice were able to proliferate in the presence of NP protein. The stimulation indexes of splenocytes to NP induced by Hsp70-NP were significantly higher than that induced by NP. It indicated that linkage of Hsp70 to NP enhanced the immune response to Hantaan virus nucleocapsid protein.
Research Article

Expression of Herpes Simplex Virus 1 Glycoprotein D in Pichia pastoris

LI Zhi-hua, XU Wei-ming, JI Qiu-yan, LONG Hai-ting, PEN Zheng-hua, LI Jian-feng

2006, 21(1): 6

The extracellular portion of glycoprotein D gene(gDt),encoding 1-314 amino acids,was amplified from extracted HSV-1 DNA and cloned into expression vector pPIC9K.After electroporation transformation of Pichia pastoris GS115 and selection with G418,a strain of P.pastoris with high yield of secreted recombinant gD was obtained.The yield of recombinant gD was 250mg/liter in shake flask cultures.The recombinant protein could be identified with specific monoclonal antibody 1-I-9 using ELISA and Western blotting assays. Recombinant gD was purified to almost homogeneity by Q-Sepharose ion exchange, Chelating Sepharose immobilized metal ion affinity and Sephacryl S-200 gel filtration chromatographies. Purified gD expressed in P. pastoris elicited high level of specific antibodies in BALB/c mice, indicating recombinant gD were characteristic high immunogenicity and capable of inducing significant titers of specific antibodies.

Action and Mechanism of Epstein Barr virus Latent Membrane Protein1 induced Immortalization of Mouse Embryonic Fibroblasts

HE Zhi-min*, ZHANG Zhi-wei, YANG Fang, YU Yan-Hui, OUYANG Yong-mei, CHEN Zhu-Chu

2006, 21(1): 16

To investigate the action and mechanisms of Epstein Barr-virus(EBV) latent membrane protein1(LMP1) in stimulating the proliferation of primary mouse embryonic fibroblast (MEF) cells the retrovirus (RV) containing wild type LMP1(wt-LMP1) and mutant type LMP1(mt-LMP1);which replaced YYD with ID in tumor necrosis factor receptor associated death domain(TRADD)-binding site;were constructed and used to infect the MEF cells;respectively. Then cytokinetic and morphologic changes from infected cells at the course of passage were observed and found that control cells (MEF-LNSX) showed an apparent growth arrest from passages 8 (P8). In contrast;mt-LMP1 prolonged the cell (MEF-LMP1TRADD) doubling time since P10 and arrested growth at P19;while the MEF-LMP1 cell with wt-LMP1 had an accurate doubling time and displayed a immortalization. Meantime;the expression of cyclinA had a decrease in MEF-LNSX and MEF-LMP1TRADD after P4and P10 respectively. while in MEF-LMP1;cyclinA increased from P10 and kept at a high level. These results suggested that LMP1 can stimulate proliferation of MEF cells and induce their immortalization;while LMP1TRADD only induces an early proliferation of MEF cells. It is implied that TRADD domain might involve in LMP1-induced MEF cells immortalization.

Screening and Cloning of the Target Genes Down-regulated by β-interferon plasmid Using Suppression Subtractive Hybridization Technique

ZHONG Yan-Wei, CHENG Jun, QU Jian-Hui, ZHANG Li-Ying, GUO Jiang, LI Xiao-Dong

2006, 21(1): 21

The suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes down-regulated byβ-interferon in HepG2. The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)IFNβand pcDNA3.1(-) empty vector, respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA from pcDNA3.1(-) empty vector transfected cell was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the products were subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search. The subtractive library of genes down-regulated byβ-interferon was constructed successfully. The amplified library contained 50 clones, of which 37 clones had 200-1000 bp inserts. Sequence analysis indicated that 22 clones containing the coding sequences , of which 19 had homology in the GenBank and 3 were unknown. The obtained sequences may be target genes regulated by β-interferon, and some genes encode proteins involved in cell cycle regulation, metabolism, and cell apoptosis.

Immunogenicity of a Hepatitis C Virus Multiple-mimotope Proteinof Hypervariable Region

GONG Yu-ping, GAO Jun, ZHAO Ping, QIN Zhao-ling, YANG Miao, QI Zhong-tian

2006, 21(1): 24

A synthesized multiple-mimotope gene of Hepatitis C virus was cloned into a prokaryotic expression vector pGEX-4T-1 to generate a fusion protein GST-HCVME. The reactivity of the GST- HCVME with the sera of HCV patients was measured by Western-blot and ELISA. BALB/c mice were injected with GST-HCVME, and an ELISA approach was applied to determine the specific antibody titers in the mouse serum. The cross-reactivity of the antibodies was also checked with 2 synthesized HCV hyper-variable region 1 (HVR1) peptides. The results showed that the purified GST-HCVME fusion protein was able to react with 25 out of 35 HCV patients’ serum samples. The serum antibody response was effectively elicited in BALB/c mice injected with GST-HCVME. The highest titer of antibody against HCV (GST-HCVME) was about 1:104 at the eighth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the 2 synthesized HCV HVR1 peptides. These findings suggest that the multiple-mimotope protein of hepatitis C virus can efficiently induce HCV-specific immune responses in mice, and may be of potential use in the development of HCV vaccines.

Construction and Identification of Recombinant Adenoviruses Containingthe Cap Protein of Porcine Circovirus 2

Cap protein

2006, 21(1): 29

Porcine circovirus 2 (PCV2) has been implicated as the etiological agent of a new disease, named postweaning multisystemic wasting syndrome (PMWS). PCV2 contains two major open reading frames (ORFs): ORF1 and ORF2, encoding replication-associated (Rep) protein and capsid (Cap) protein, respectively. The cap protein is a candidate 2 for recombinant PCV vaccine, because it possesses several potential antigen epitopes. In the study, the cap gene was amplified by PCR, and cloned into the transfer vector pShuttle-CMV subsequentially. The recombinant plasmid and bone vector pAdEasy-1 were cotransformed into BJ5183 to generate the recombinant plasmid rAd-ORF2 pAd-Cap. The recombinant adenovirus (rAd-Cap) was generated by transfection and plaque purification in in 293 cells. The transcription and expression of Cap protein in rAd-Cap infected 293 cells were confirmed by RT-PCR、indirect-ELISA, Western blot and IPMA. The study laid the foundation for development of the recombinant vaccine against PCV2.

Immuno-Suppression of Reticuloendotheliosis Virus Infection on Antibody Responses to Different Vaccines in SPF chickens

SUN Shu-hong, CUI Zhi-zhong, QIU Yu-Yu

2006, 21(1): 34

Avian reticuloendotheliosis virus (REV) infection was reported to be very common in chicken flocks in China, but its economic impact on the poultry industry was not clear. The results in this study indicated that REV infection in 1-day-old SPF chickens could severely suppressimmune reactions to inactivated vaccines against Newcastle disease virus (NDV) and Avian influenza virus (AIV). Hemagglutination inhibition (HI) antibody titers to NDV, AIV-H9 and AIV-H5 in REV-infected birds were significantly lower than that in the control group 3, 4, and 5 weeks after vaccination at the age of 7 days. REV infection of high doses caused more severely immuno-suppression than that with low doses, but the difference between high and low doses was not significant. REV infection also caused severe atrophy of central immune organs, the ratios of thymus and the Bursa to body weight in REV-infected birds were significantly lower than that in thecontrol birds. This study demonstrated that the early REV-infection interfered vaccinations to NDV and AIV.

Roles of ICAM-1 and it’s Receptors in HPAIV Pneumonia

ZHAO Hong-xia, XIA Xian-zhu, LU Ji-rong, YANG Song-tao, ZHU Ping

2006, 21(1): 38

The intercellular adhension molecular-1(ICAM-1)and it’s its receptors in the mice’s lungs of HPAIV and control mice pneumonia were detected by using immunohistochemistry staining after infected mice with Highly pathogenic avian influenza virus (HPAIV). The expressive level of ICAM–1, and its receptors of CD11b and CD18 were first detected after 24 hours infection in bronchial alveolar epithelial cells, bronchopulmonary capillary endothelial cells, and iinfiltrativing lymphomonocyte and monocytes, and reached were the peaks 4 days after infection. The highest expression of ICAM-1 and its receptors were detected when infected with 100LD50/50 μL HPAIV. The change was confirmed by the method of ELASA. Our results indicate that ICAM-1 and its receptors have the high expression level in HPAIVP pneumonia, and may play an important roles in the pathogenesis.

Cloning and Expression of Neuraminidase Gene of H5N1Subtype Avian Influenza Virus

SONG Jian-ling, ZHANG Fu-qiang*, WANG Jun-ping, HU Yuan-yuan

2006, 21(1): 43

A pair of clone primers were designed and synthesized based on neuraminidase(NA) gene sequences of known Avian influenza virus H5N1 subtypes. NA gene were amplified from total RNA, which had been extracted from allantoic fluid of H5N1 subtype virus inoculated embryo, by reverse transcriptase-polymerase chain reaction using high proofreading polymerase (PyobestTM DNA Polymerase), and expressed using Invitrogen championTM pET directional TOPO expression system. 53.8kDa recombinant NA fusion with polyhistidine (6xHis) tag in N-terminal was expressed and purified. Its immunoreactivity and immunogenicity had been analyzed. The results showed the recombinant NA could not only bind to antiserum against H5N1 subtype virus with specificity but also elicit specific antibody in immunizing animal, and possessed good antigenicity.

The Transcription Profiles of Nine HaSNPV Genes

DAI Wen-tao, DENG Fei, WANG Hua-lin, YUAN Li, HU Zhi-hong

2006, 21(1): 47

The transcription profiles of nine selected HaSNPV genes, e.g. early phase genes, early and late phase genes, late phase genes and very late phase genes were analyzed using reverse transcription and real-time quantitative PCR. The results indicated that for most of these genes the transcriptional start point was coincident with the prediction of their promoter. But the predicted early phase gene pkip started to be transcribed at late phase, and the predicted early and late phase gene ha107 was only transcribed at late phase. At 72hpi, the transcription level of these genes was generally the highest. The transcription level of polyhedrin was obviously higher than the other genes. The iap2 was the second highly transcribed gene, indicating that it might be functional. The HaSNPV p10, unlike its homologue in AcMNPV, was not highly transcribed.

Expression and Localization of the Dendrolimus punctatus CypovirusPolyhedrin gene

LI Dou-lin, LI Yan-qiu, ZHANG Jia-min, YANG Bo, CHENG Wu-guo, ZHOU Wei-dong, HU Yuan-yang*

2006, 21(1): 52

To express the polyhedrin (the tenth segment, S10) of Dendrolimus punctatus Cypovirus (DpCPV) and investigate the localization of the polyhedrin in eukaryote, The polyhedrin gene was amplified from DpCPV by RT-PCR and cloned into pET-28a vector. The polyhedrin was then expressed in bacteria. The purified protein was used to immunized rabbit to produce the antibody. The S10 ORF was also cloned into the baculovirus vector pFASTBAC-HTb. The recombinantplasmid pFASTBACS10 was transformed to E. coli containing AcMNPV bacmid to generate the recombinant Bacmid AcS10. AcS10 bacmid DNA transefect Sf9 cell and the recombinant baculoviruse vAcS10 was produced. After S10 was expressed, the products were detected and analyzed by the SDS-PAGE, Western-blot and Immunofluorescence technique. The polyhedrin expressed in SF-9 cells was mainly located in cytoplasm.

Cloning and Expression of the gp41 Gene from SpltMNPV Japan Strain and Analysis of Phylogeny Based on gp41 and ph gene

HU Zhao-li, ZHU Jiang, SHENG Ye, WANG Wen-bing, LI Xin-ping, SHEN Song-dong

2006, 21(1): 57

The gp41 gene was cloned from Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) Japan-C3 strain. This gene is 993bp long and encodes a polypeptide with a predicted molecular mass of 36.9 kDa. Analysis showed the gp41of SpltMNPV Japan-C3 strain was most closely related to that of SpltMNPV Chinese-G2 strain with 99.9% identity in nucleotide sequence and 99.9% in amino acid sequence. The phylogenetic trees based on gp4l and polyhedrin had the similar topologies. The combined tree of gp41 and polyhedrin had an identical topology as the gp41 gene. Mutation rate analysis showed the gp41 gene had a higher nucleotide substitution rate than that of the polyhedrin gene, implying that the polyhedrin gene may have a different selection constraint than the gp41 gene.

Identification and Serological Study of Odontoglossum ringspot virus

MING Yan-lin*, ZHENG Guo-hua, LI Mei

2006, 21(1): 64

A virus isolated from cattleya in Xiamen, China was identified as an isolate of Odontoglossum ringspot virus, based on its biological properties, particle morphology, serological characterization and RT-PCR. Mechanical inoculation test with the isolate was carried out to 27 species of 6 families of indicative plants, of which 8 species of 4 families showed symptoms. The result is different from that of Tobacco mosaic virus. The thermal inactivation point of the isolate was about 92℃~94℃, and the longevity in vitro was beyond 3 months. Electron microscopic examination of the purified virus particle preparations showed straight particles of about 300nm long and 18nm wide. The pure viral coat protein was estimated to be about 17 kDa. The serological test showed that the virus isolation reacted positively with the antiserum against ORSV while weakly with TMV antiserum. Antiserum was prepared by immunizing rabbit, and used to detect ORSV in Xiamen orchid’s plant by Dot-ELISA.
Brief Reports

Characterization of Spike Gene of Infectious Bronchitis Coronavirus CK/CH/ LDL97Ⅰ/97 Isolated in China

LIU Xin, LIU Sheng-wang, KONG Xian-gang, ZHANG Qing-xia, RONG Jun-gong, HAN Zong-xi, SHAO Yu-hao

2006, 21(1): 68

The spike gene (S) of Infectious bronchitis virus (IBV) CK/CH/LDL97Ⅰ/97 isolate was amplified,cloned, sequenced and analyzed. Results showed that the S gene was 3501 bp encoding a polypeptide of 1167 amino acids. The precursor of the S protein was cleaved into S1 and S2 fragments, which comprised 541 and 626 amino acid residues, respectively. The cleavage site sequence was RRTGR. The homologies of nucleotide and amino acid sequences of S1 gene between CK/CH/LDL97Ⅰ/97 and 27 reference IBV strains ranged from 60.6% to 99.6%, and 50.5% to 99.1%, respectively. The CK/CH/LDL97Ⅰ/97 S1 was most similar to that of three proventriculus IBV isolates, Q1(99.1%), J2(98.9%), T3 (98.9%), which were isolated in China. Phylogenetic analysis of S1 proteins indicated that CK/CH/LDL97Ⅰ/97, Q1, J2 and T3 formed a separated cluster, which might belong to a novel genotype of IBV.

Construction and Characterization of Recombinant Marek’s Disease Virus CVI988 Expressing the E. coli LacZ Gene

QIU Ya-feng, GE Fei-fei, ZhANG Xue-lian, XU Xue-qing, CHEN Pu-yan*

2006, 21(1): 71

The LacZ gene controlled under the Marek’s disease virus (MDV) glycoprotein B (gB) promoter was excised from plasmid pSK-gB-LacZ and inserted into US10 gene. Two plasmids were constructed, pUS-gB-LacZ(L)and pUS-gB-LacZ(U)),which differ with regard to the orientation of the expression cassette. Then pUS-gB-LacZ(L)was transfected into secondary Chicken embryo fibroblasts (secondary CEF) cells and super-infected with the MDV CVI988 strain. The recombinant virus (rCVILacZ) expressing the lacZ gene was isolated and purified in secondary CEF. The growth curve of rCVILacZ was similar to that of the parent virus in CEF.

Expression and Purification of Erns Gene of Classical Swine Fever Virus in E. coli

YIN Shuang-hui, LIU Xiang-tao*, HAN Xue-qing, SHANG You-jun, SUN Shi-qi

2006, 21(1): 75

The Erns gene was amplified by RT-nPCR from C-strain of Classical swine fever virus (CSFV). Then the Erns gene was cloned into pGEX6P-1 vector. The expression products were analysed by SDS-PAGE and Western blotting methods. The inclusion bodies were recovered from bacterial lysate by centrifugation and washed with buffer, and then dissolved in denaturing agents. The protein was refolded by dilution dialysis. Refold protein reacted strongly with the positive serum of CSFV, which indicated that the protein has immunogenic.

Expression of Avian Infectious Bronchitis Virus E Protein in E.coli

ZHOU Yu-chang, LIU Sheng-wang*, KONG Xian-gang

2006, 21(1): 78

Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the Small Envelope (E) gene of avian Infectious bronchitis virus (IBV) LX4 strain and the gene was cloned into the pMD18-T vector. By digestion with restriction enzymes SalⅠand BamHⅠ, the E gene was subcloned into pET-30a vector to construct recombinant plasmid pET-30a-E. The recombinant plasmid was transformed into E. coli BL21(DE3) and induced with IPTG. It was demonstrated by SDS-PAGE that a protein of 14kDa, which was comparable in size to the native E protein, was expressed in E. coli. The 14KDa protein was purified and used to prepare the rabbit antiserum against IBV E protein. The result showed that the antibody could react with purified E protein in Western blotting.

The Study on the Differentiation between the EIAV American prevalent strain and Attenuated Vaccine strain

GENG Qing-hua, WANG Xiao-lun, ZHAO Li-ping, LV Xiao-ling, SHEN Rong-xian, XIANG Wen-hua*

2006, 21(1): 81

In order to develop an assay to distinguish the infection of Equine infectious anemia virus (EIAV) American strains from Donkey-leukocyte attenuated virus (DLV) strain, eight primers were designed based on the comparison of complete sequences of four EIAV American strains and DLV. Corresponding viral genome fragments were amplified and analyzed from donkey leucocytes by PCR using these eight primers. Four primers from gag gene were selected to detect the differences between EIAV American strains and DLV. Six horses were inoculated with DLV and two with EIAV American strains, the Wyoming strain and the Argentina strain, respectively. Two fragments, 675bp and 400bp, were amplified from DLV-infected cells, while only one segment of 675bp was obtained from cells inoculated with American strains. Our results indicate that this nest multiplex PCR can be used to distinguish EIAV American strains from the donkey leucocyte-attenuated vaccine strain of EIAV.
Review

The Universal System of Virus Taxonomy in the 8th ICTV Report

HONG Jian, ZHOU Xue-ping

2006, 21(1): 84