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2008 Vol.23(3)

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Proteomic Analyses of the Shrimp White Spot Syndrome Virus

Yan-wei TAN, Zheng-li SHI

2008, 23(3): 157 doi: 10.1007/s12250-008-2924-0

Received: 30 November 2007 Accepted: 28 December 2007
White spot syndrome virus (WSSV), a unique member within the virus family Nimaviridae, is the most notorious aquatic virus infecting shrimp and other crustaceans and has caused enormous economic losses in the shrimp farming industry worldwide. Therefore, a comprehensive understanding of WSSV morphogenesis, structural proteins, and replication is essential for developing prevention measures of this serious parasite. The viral genome is approximately 300kb and contains more than 180 open reading frames (ORF). However, most of proteins encoded by these ORF have not been characterized. Due to the importance of WSSV structural proteins in the composition of the virion structure, infection process and interaction with host cells, knowledge of structural proteins is essential to understanding WSSV entry and infection as well as for exploring effective prevention measures. This review article summarizes mainly current investigations on WSSV structural proteins including the relative quantities, localization, function and protein-protein interactions. Traditional proteomic studies of 1D or 2D gel electrophoresis separations and mass spectrometry (MS) followed by database searches have identified a total of 39 structural proteins. Shotgun proteomics and iTRAQ were initiated to identify more structural proteins. To date, it is estimated that WSSV is assembled by at least 59 structural proteins, among them 35 are defined as the envelope fraction (including tegument proteins) and 9 as nucleocapsid proteins. Furthermore, the interaction within several major structural proteins has also been investigated. This identitification and characterization of WSSV protein components should help in the understanding of the viral assembly process and elucidate the roles of several major structural proteins.

One New Method of Nucleic Acid Amplification —Loop-mediated Isothermal Amplification of DNA

Xue-en FANG, Jian LI, Qin CHEN

2008, 23(3): 167 doi: 10.1007/s12250-008-2929-8

Received: 17 December 2007 Accepted: 31 January 2008
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

Lan LI, Yi-shu YANG, Ze-lin LI, Yi ZENG

2008, 23(3): 173 doi: 10.1007/s12250-008-2909-z

Received: 10 September 2007 Accepted: 19 February 2008
To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from plasmid of HIV-1 NL4.3 cDNA, and APOBEC3G gene was achieved by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by two enzymes digestion technology, SDS-PAGE, and Western blotting. Rabbits were immuzied by Vif or APOBEC3G protein. Serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assay were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of anti-APOBEC3G antibodies was 1:102400. The antibidies could detect the antigen expressing in the cells. These fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved. These results ensure the immunogenicity and antigenicity of the purified recombinant proteins.

The Function of SeMNPV IAP3 in Mammalian Cells

Li-na ZHU, Chun-lei MEI, Zhong-xin ZHANG

2008, 23(3): 183 doi: 10.1007/s12250-008-2883-5

Received: 25 May 2007 Accepted: 04 January 2008
The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I /Bam H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in HEK293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in HEK293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

Jing LU, Li QIN, Guang-jie LIU, Si-ting ZHAO, Xiao-ping CHEN

2008, 23(3): 189 doi: 10.1007/s12250-008-2896-0

Received: 17 August 2007 Accepted: 12 December 2007
Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.

Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65

Bi-shi FU, Bao-lin LI, Lin-ding WANG

2008, 23(3): 196 doi: 10.1007/s12250-008-2925-z

Received: 06 December 2007 Accepted: 06 January 2008
To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA

Isolation and Identification of Canine Parvovirus Serotype 2a and Its VP2 Protein Expression in Transgenic Tobacco

Ning XIONG, Yong ZHANG, Yao WANG, Bao-yu YANG, Shi-yun CHEN

2008, 23(3): 203 doi: 10.1007/s12250-008-2928-9

Received: 13 December 2007 Accepted: 10 April 2008
A strain of canine parvovirus(CPV)was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5’ region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.

Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

Lei GUO, Ying ZHANG, Yan-chun CHE, Wen-juan WU, Wei-zhong LI, Li-chun WANG, Yun LIAO, Long-ding LIU, Qi-han LI

2008, 23(3): 211 doi: 10.1007/s12250-008-2936-9

Received: 18 January 2008 Accepted: 22 February 2008
An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β–D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

YVDD Mutation of Hepatitis B Virus, a Dominant Lamivudine-Resistant Type in Guangzhou, South China

Zhi-ying OU, Rong ZHOU, Yun-shao HE

2008, 23(3): 218 doi: 10.1007/s12250-008-2963-6

Received: 27 March 2008 Accepted: 02 April 2008
The epidemiological effects of native and mutated YMDD motif in the HBV genome under the selective pressure of lamivudine were investigated. YMDD wild and mutation motif in HBV genome were detected by flow through reverse dot blots (FT-RDB) with KaiPuTM DNA HybriMax Rapid Hybridization Machine based on the principle of “Flow-through hybridization” and by the traditional Reverse Dot Blot assay. Sera from 1 021 suspected lamivudine-resistant chronic HBV carriers after more than 8 months of lamivudine therapy and the corresponding archived sera were collected and assayed. We found 35.94% were single type infections with 8.03% YMDD, 7.93% YIDD and 19.98% YVDD. It was also found that 64.06% were mixed infections including 1.96% YMDD and YIDD, 51.62% YMDD and YVDD, 1.96% YIDD and YVDD, 8.52% YMDD, YIDD and YVDD. The levels of infections containing YVDD motif reached 82.08%. The pretreatment infectious status were: YMDD single infection was 36.93%; YIDD single infection was 6.07%; YVDD single infection was 17.04%; YMDD and YIDD mixed infection was 0.97%; YMDD and YVDD mixed infection was 33.99%; YIDD and YVDD mixed infection was 0.98%; YMDD, YIDD and YVDD mixed infection was 4.02%. Infections containing YVDD motif were only 56.03%. The 34.32% mutation rate of YMDD motif to YVDD was significantly higher than the 10.97% of YMDD to YIDD (U=10.98, P<0.05), as estimated by Mann-Whitney U-test for non-parametric data. HBV containing YVDD motif might have an evolutionary ascendancy and become the dominant type under the selective pressure of lamivudine.

Expression of Human Papillomavirus in Mammary Carcinoma and its Possible Mechanism in Carcinogenesis

Chun-ling FAN, Jing-hua ZHOU, Cheng-yi HU

2008, 23(3): 226 doi: 10.1007/s12250-008-2914-2

Received: 13 September 2007 Accepted: 04 May 2008
To explore the role of Human papillomavirus (HPV) in mammary carcinogenesis, the expression of the HPV-16, iNOS , P53 and hTERT proteins in breast carcinomas and their relationships were investigated. 52 samples of breast cancer and 16 samples of benign breast tumors were assayed using the immunohistochemical SP method for detection of protein expression levels. The expression of HPV-16, iNOS, P53 and hTERT proteins in a mammary carcinoma was 44.2%, 57.7%, 63.5% and 59.6% respectively, which was significantly greater than the corresponding levels in the benign group. The expression of iNOS, P53 and hTERT was correlated with the presence of an HPV-16 infection in a mammary carcinoma(P<0.05). The connection between these events might also involve the iNOS, mutated type P53 and the hTERT protein.