2007 Vol.22(1)


The Functional Motif of SARS-CoV S Protein Involved in the Interaction with ACE2

Yi ZHANG, Wei WANG, Jin-rong GAO, Li YE, Xiao-nan FANG, Ying-chun ZENG, Zheng-hui WU, Ying-long SHE, Lin-bai YE

2007, 22(1): 1

Received: 17 July 2006 Accepted: 31 July 2006
SARS-CoV is a newly discovery pathogen causing severe acute respiratory syndrome, S protein play an important rule in the adsorption and penetration of SARS-CoV into the cell by interaction with ACE2 receptor. To determinant the functional motif of S protein involved in the interaction with ACE2, seven truncated S proteins deleted from N or C terminal were obtained by E.Coli expression system and purified by column chromatography to homogeneity. Each truncated S protein was fixed on to the well of ELISA plate and interacted with ACE2 protein. The adsorption were quantified by ELISA, the results demonstrated that the amino acid from 388 to 496 of S protein was responsible for interaction with ACE2 receptor, and the interaction can be disrupted completely by the antibody specific against amino acid 388 to 496 of S protein. The deletion besides this domain did not show significant effects on the interaction with ACE2, suggested that S protein of SARS-CoV could be developed as vaccines to prevent the spread of SARS-CoV.

Construction and Characterization of a Hepatitis B Virus Replicon

Yin-ping LU, Bao-ju WANG, Ji-hua DONG, Zhao LIU, Shi-he GUAN, Meng-ji LU, Dong-liang YANG

2007, 22(1): 8

Received: 04 July 2006 Accepted: 24 October 2006
To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was transient transfected into Huh7 cells, HBsAg,HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.

Inhibition of Hepatitis B Virus Replication by Rheum palmatum L. Ethanol Extract in a Stable HBV-producing Cell Line

Yan SUN, Li-jun LI, Jing LI, Zhi LI

2007, 22(1): 14

Received: 31 May 2006 Accepted: 06 July 2006
Hepatitis B virus(HBV) infection is a severe health problem in the world. However,there is still not a satisfactory therapeutic strategy for the HBV infection. To search for new anti-HBV agents with higher efficacy and less side-effects,the inhibitory activities of traditional Chinese medicine Rheum palmatum L. ethanol extract(RPE) against HBV replication were investigated in this study. Quantitative real-time polymerase chain reaction(PCR) was employed to analyze the inhibitory activity of RPE against HBV-DNA replication in a stable HBV-producing cell line HepAD38; the expression levels of HBV surface antigen(HBsAg) and e antigen(HBeAg) were also determined by enzyme linked immunosorbent assay(ELISA) after RPE treatment. RPE could dose-dependently inhibit the production of HBV-DNA and HBsAg. The concentration of 50% inhibition(IC50) was calculated at 209.63,252.53μg /mL,respectively. However,its inhibitory activity against HBeAg expression was slight even at high concentrations. RPE had a weak cytotoxic effect on HepAD38 cells(CC50= 1 640μg /mL) and the selectivity index(SI) was calculated at 7.82. Compared with two anthraquinone derivatives emodin and rhein,RPE showed higher ability of anti-HBV and weaker cytotoxicity. So Rheum palmatum L. might possess other functional agents which could effectively inhibit HBV-DNA replication and HBsAg expression. Further purification of the active agents,identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

Wan-gang GU, Jun-fa YUAN, Ge-lin XU, Li-juan LI, Ni LIU, Cong ZHANG, Jian-hong ZHANG, Zheng-li SHI

2007, 22(1): 21

Received: 24 April 2006 Accepted: 07 September 2006
The BALB/c mice were immunized with purified White Spot Syndrome Virus (WSSV). Six monoclonal antibody were selected, by ELISA, with VP28 protein expressed in E. Coli. The in vitro neutralization experiments showed that 4 of them could inhibit the virus infection to crayfish. Western-blot revealed that all these monoclonal antibodies were against the nature structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labelling. These monoclonal antibodies could be used to develop immunological diagnosis methods of WSSV infection.

Molecular Epidemiology and Sequencing of the G-L Intergenic Region of RabiesViruses Isolated in China

Sheng-Li MENG, Ge-Lin XU, Jia-Xin YAN, Ping-Gang MING, Jie WU, Xiao-Ming YANG, He-Tian MING, Feng-Cai ZHU, Dun-Jin ZHOU, QI-You XIAO, Guan-Mu DONG

2007, 22(1): 26

Received: 31 July 2006 Accepted: 31 August 2006
A group of 25 rabies viruses (RABVs), recovered from 24 dogs and one human case, were collected from various areas in China between 2004 and 2006. Genetic and phylogenetic analyses of the G-L intergenic region were carried out in 25 street RABV isolates and CTN vaccine strains of 7 generations. The study was based on the comparison of a 519 bp nucleotide sequence, encompassing the G-L intergenic region. The nucleotide sequence homologies of Chinese street strains were from 95.5% to 100%. The phylogenetic analysis showed that all Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and they were distributed according to their geographical origins. All of the Chinese strains were closely related but they could still be divided into two groups: group of street strains and group of CTN strains. This study presents details about the molecular epidemiology of rabies viruses based on the sequences of the G-L Intergenic region.

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

Shun-lin HU, Qin SUN, Qu-zhi WANG, Yu-liang LIU, Yan-tao WU, Xiu-fan LIU

2007, 22(1): 34

Received: 14 August 2006 Accepted: 17 October 2006
Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF)cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.

Complete Genomic Sequence Analysis of a New SV40 Isolate

Xue-mei ZHANG, Yan-chun CHE, Jing-jing WANG, Long-ding LIU, Ming-xue XIE, Qi-han LI

2007, 22(1): 41

Received: 14 August 2006 Accepted: 20 November 2006
The genome of a new SV40 strain(SV-IMB)isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the average identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region consists of a complete enhancer and a defective enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. Our findings demonstrate that the SV-IMB is a new SV40 isolate.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

Hong-bo FAN, Jun-wei LI, Zhi-lin LI, Wei ZHENG, Po Tien, De-yin GUO

2007, 22(1): 46

Received: 30 August 2006 Accepted: 23 October 2006
The M and NP gene of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA, and cloned into pMD18-T vector respectively. Then the expression plasmid containing M gene (pHM6-m) or NP gene (pHM6-np) was constructed by inserting M or NP gene into the pHM6 eukaryote expression vector, and sequenced. 32 BALB/c mice (6-week-old) were divided into four groups at random. Three groups of BALB/c mice were inoculated with 30 μg of plasmid pHM6-m, 30 μg of plasmid pHM6-np or the mixture of plasmid pHM6-m (15 μg ) and pHM6-np(15 μg) once via the intramuscular route respectively. A group of mice were injected with 100 μl PBS as controls. Two weeks later, all mice were challenged with homologous H5N1 avian influenza virus, and observed in the following 12 days. The survival rates of mice in pHM6-m group, pHM6-np group and mixed plasmids group were 62.5%, 25.0% and 50.0%, respectively. Results showed that effective protection could be provided by either pHM6-m or pHM6-np, but pHM6-m provided a better protective effect than pHM6-np.

Pathogenicity of diatraea saccharalis Densovirus to Host Insets and Characterization of its Viral Genome

Nazaire Kouassi, Jian-xin PENG, Yi LI, Cristina Cavallaro, Jean-Claude Veyrunes, Max Bergoin

2007, 22(1): 53

Received: 04 December 2006 Accepted: 30 December 2006
Pathogenicity of the Diatraea Saccharalis densovirus (DsDNV) was tested on its host larvae. The results showed that up to 4 days after inoculation, no larvae mortality was observed and the infected larvae started to exhibit the infection symptoms from the forth day. After 5 days of infection, the cumulative mortality of infected larvae increased significantly and reached 60% after 12 days and 100% after 21 days of infection, whereas that of the control group was only 10% and 20%, respectively, after same periods of infection, suggesting that the high mortality of infected larvae groups was due to high pathogenicity of DsDNV. The size of the double stranded DNA (dsDNA) was determined by Electron microscopy visualization of viral DNA molecules and gel electrophoresis of both native and endonuclease digested DNA fragments. The total length of the native dsDNA was about 5.95 kb. The DsDNV DNA was digested with 16 restriction enzymes and a restriction map of those enzymes was constructed with 41 restriction sites. Comparison of the restriction map of the DsDNV genome with those of the genomes of Junonia Coenia densovirus (JcDNV) and Galleria mellonella densovirus (GmDNV) indicated that the three densovirus genomes were found to share many identical restriction sites. Thus, most of the restriction sites of the following endonucleases Bam HI, Hha I, Xba I, Cla I, Asp 700, Spe I, Nco I and Bcl I, were found to be conserved among the three densovirus genomes. Symmetrical cleavage sites mapped at the both ends of the genome suggested the presence of inverted terminal repeats (ITRs) whose size was estimated to be about 500 bp. The similar genome size, most identical restriction sites and presence of an ITR of about 500 bp of these three densoviruses suggested that they belong to the same group of ambisense densoviruses.

Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP (19+28) Fusion Protein

Qing-yu CHENG, Xiao-lin MENG, Jin-ping XU, Wei LU, Jian WANG

2007, 22(1): 61

Received: 20 June 2006 Accepted: 18 July 2006
We developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV, using a colloidal gold as an indicator. The fusion protein, VP (19+28), was expressed in E. coli, purified and used to prepare polyclonal antibodies. The purified anti-VP (19+28) IgG were conjugated with colloidal gold. Unconjugated anti-VP (19+28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes. After assembly, three groups (5 individual animals in each group)of shrimp samples were tested which included healthy, moribund and dead shrimps. For each group, three different tissues (body juices, gills and hepatopancreas) were tested at the same time. In parallel, all the samples were also analyzed using an existing PCR for comparison. Out of 45 samples tested, 30 were detected as positive while 15 negative The results of LFIA correlate with those obtained by the PCR analysis, indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.

Using the SELDI ProteinChip System to Detect Changes in Protein Expression in Vero Cells after Infection

Zhi-jun LIU, Bin WANG, Zhi-yong YAN, Xu-xia SONG, Dong-meng QIAN, Zhi-qiang BAI

2007, 22(1): 68

Received: 30 October 2006 Accepted: 15 December 2006
Human herpes simplex virus 1 (HSV-1) causes facial, ocular, and encephalitic disease and is associated with latent infection and cancer. Here, we developed a means of studying the pathogenesis of HSV-1 infection at the protein level by using the SELDI Protein Chip to detect changes of protein expression in Vero cells cultured in vitro. After infection with HSV-1 and culture for 12, 24 or 48 h, cells were harvested and lysed. IMAC3 arrays were applied to SELDI-TOF-MS to detect proteomic differences before and after infection. The chip detected a series of differentially expressed protein peaks. Interestingly, both peaks at 16 912 Da and 17 581 Da corresponded precisely with the molecular mass of ISG15, which may participate in antiviral activity during the process of infection.Thus, the results we obtained can serve as a basis to study the pathogenesis of HSV-1 and the interaction between the virus and its host. In addition, they can help in the discovery of new therapeutic targets for treatment of HSV-1 infection.

Comparison of Three ELISA Kits for the Differentiation of Foot-and-mouth Disease Virus-infected from Vaccinated Animals

Yi-mei CAO, Zeng-jun LU, Zai-xin LIU, Qing-ge XIE

2007, 22(1): 74

Received: 14 April 2006 Accepted: 22 May 2006
A study was performed in order to validate FMDV 3ABC-I-ELISA kit developed in China for the differentiation of FMDV-infected animals from those had merely been vaccinated. Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits, Ceditest? FMDV-NS (Ceditest? kit), UBI? FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI? kit) and FMDV 3ABC-I-ELISA kit developed at the LanZhou Veterinary Research Institute. The test parameters (sensitivity and specificity) of the three kits were primarily determined, and the result obtained from domestic FMD 3ABC-I-ELISA kit was compared with that of obtained from two foreign kits respectively. The results indicate that the coincidence rate between FMD 3ABC-I-ELISA and Ceditest? kit is 98.05%, and the coincidence rate between FMD 3ABC-I-ELISA and UBI? kit is 94.4%; the sensitivity of both Ceditest? and FMD 3ABC-I-ELISA kit is 100%. However, the sensitivity of UBI? kit is only 81.8%. With sera from naive or vaccinated, but non-infected animals, the specificity of all tests exceeds 90%.