2007 Vol.22(3)


Research Article

Expression of Prothrombinase/fibroleukin Gene fg12 in Lung Impairment in a Murine Severe Acute Respiratory Syndrome Model

Wei-ming YAN, Jia-quan HUANG, Xiao-ping LUO, Qin NING

2007, 22(3): 181

Received: 31 October 2006 Accepted: 25 December 2006
To evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in Severe acute respiratory syndrome (SARS), a murine SARS model induced by Murine hepatitis virus strain 3 (MHV-3) through trachea was established. Impressively, all the animals developed interstitial pneumonia with extensive hyaline membranes formation within alveoli, and presence of micro-vascular thrombosis in the pulmonary vessels. MHV-3 nucleocapsid gene transcripts were identified in multiple organs including lungs, spleen etc. As a representative proinflammatory gene, mfgl2 prothrombinase expression was evident in terminal and respiratory bronchioles, alveolar epithelia and infiltrated cells in the lungs associated with fibrin deposition and micro-vascular thrombosis. In summary, the established murine SARS model could mimic the pathologic characteristics of lungs in patients with SARS. Besides the physical damages due to virus replication in organs, the up-regulation of novel gene mfgl2 in lungs may play a vital role in the development of SARS associated lung damage.

Antiviral Effect of Interferon-Induced Guanylate Binding Protein-1 against Coxsackie Virus and Hepatitis B Virus B3 in Vitro

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

2007, 22(3): 193

Received: 25 June 2006 Accepted: 24 December 2006
Guanylate binding protein-1(GBP-1) is an interferon-induced protein. To observe its antiviral effect against Hepatitis B virus (HBV) and Coxsackie virus B3 (CVB3), we constructed an eukaryotic expression vector of human GBP-1(hGBP-1). Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector, then subcloned into a pCDNA3.1(-) vector. Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells, and inhibition effect of hGBP-1 against HBV replication was observed. Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3, and viral yield in cultures were detected. The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells. hGBP-1 inhibit CVB3 but not HBV replication in vitro. These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.

P19 of Tomato Bushy Stunt Virus Suppresses RNA Silencing Induced by Short Hairpin RNA in Mammal Cells

Wei-xian CHEN, Juan CHEN, Zhen-zhen ZHANG, Ai-long HUANG

2007, 22(3): 199

Received: 01 September 2006 Accepted: 24 January 2007
To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

Phylogenetic Analysis of Homologous Proteins Encoded by UL2 and UL23 genes of Herpesviridae

Long-ding LIU, Wen-juan WU, Min HONG, Hai-jing SHI, Jing-jing WANG, Hong-ling ZHAO, Yun LIAO, Qi-han LI

2007, 22(3): 207

Received: 26 October 2006 Accepted: 20 February 2007
The proteins encoded by the Herpesviridae -gene play a critical role in the replication stage of the virus. In this paper, phylogenetic analyses provided evidence that some -gene products, such as UL2 and UL23 from HSV1, have their homologous genes in its family, and also exist in prokaryotic organisms, indicating that these viruses appear to have been assembled over evolutionary time by numerous independent events of horizontal gene transfer.

Differences in Variation of Human Immunodeficiency Virus Type 1 Sequences from Henan and Shanghai Regions of China

Chun-yu YIN, Hong-zhou LU, Wei-ming JIANG, Maria Pia De Pasquale, Yue-kai HU, Xiao-zhang PAN, Xin-hua WENG, Richard T. D'Aquila, Yi-Wei TANG

2007, 22(3): 212

Received: 03 November 2006 Accepted: 27 December 2006
Illegally paid blood donation was a risk factor for HIV acquisition exclusively in Henan and Hubei Provinces of China, and not in Shanghai. Nucleotide sequences in the gag and env genes of HIV-1 were compared between isolates from Henan and Shanghai regions of China to test whether an expected higher degree of a common source of infections from this unique blood donation transmission risk would be evident as decreased variation among Henan isolates in an exploratory cross-sectional analysis. Among 38 isolates studied, 23 of 23 (100%) from Henan and 8 of 15 (54%) from Shanghai were subtype B. In addition, fewer sequence differences were found in gp41 of subtype B isolates from Henan than from Shanghai isolates. Further studies with additional controls are therefore warranted to confirm the role of the degree of a common source of infections in differences in HIV variation across populations.

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

Jin-shan HUANG, Bi-fang HAO, Xiu-lian SUN, Fei DENG, Hua-lin WANG, Zhi-hong HU

2007, 22(3): 218

Received: 13 November 2006 Accepted: 30 January 2007
To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.

Synonymous Codon Usage Bias and Overexpression of a Synthetic Gene Encoding Interferon α2b in Yeast

Bin FANG, Bu-feng LIANG, Guang-yuan HE

2007, 22(3): 226

Received: 22 November 2006 Accepted: 17 January 2007
To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut+ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×105 IU/mL.

Study of HIV-1 Drug Resistance in Patients Receiving Free Antiretroviral Therapy in China

Xin-ping LI, Hui XING, Zhe WANG, Xue-feng SI, Lian-en WANG, Hua CHENG, Wei-guo CUI, Shu-lin JIANG, Ling-jie LIAO, Hai-wei ZHOU, Jiang-hong HUANG, Hong PENG, Peng-fei MA, Yi-ming SHAO

2007, 22(3): 233

Received: 21 December 2006 Accepted: 06 March 2007
To investigate the prevalence of drug-resistance mutations, resistance to antiretroviral drugs, and the subsequent virological response to therapy in treatment-naive and antiretroviral-treated patients infected with HIV/AIDS in Henan, China, a total of 431 plasma samples were collected in Queshan county between 2003 and 2004, from patients undergoing the antiretroviral regimen Zidovudine + Didanosine + Nevirapine (Azt+Ddi+Nvp). Personal information was collected by face to face interview. Viral load and genotypic drug resistance were tested. Drug resistance mutation data were obtained by analyzing patient-derived sequences through the HIVdb Program (http://hivdb.stanford.edu). Overall, 38.5% of treatment-naive patients had undetectable plasma viral load (VL), the rate significantly increased to 61.9% in 0 to 6 months treatment patients (mean 3 months) (P<0.005) but again significantly decrease to 38.6% in 6 to 12 months treatment patients (mean 9 months) (P<0.001) and 40.0% in patients receiving more than 12 months treatment (mean 16 months) (P<0.005). The prevalence of drug resistance in patients who had a detectable VL and available sequences were 7.0%, 48.6%, 70.8%, 72.3% in treatment-na?ve, 0 to 6 months treatment, 6 to 12 months treatment, and treatment for greater than 12 months patients, respectively. No mutation associated with resistance to Protease inhibitor (PI) was detected in this study. Nucleoside RT inhibitor (NRTI) mutations always emerged after non-nucleoside RT inhibitor (NNRTI) mutations, and were only found in patients treated for more than 6 months, with a frequency less than 5%, with the exception of mutation T215Y (12.8%, 6/47) which occurred in patients treated for more than 12 months. NNRTI mutations emerged quickly after therapy begun, and increased significantly in patients treated for more than 6 months (P<0.005), and the most frequent mutations were K103N, V106A, Y181C, G190A. There had been optimal viral suppression in patients undergoing treatment for less than 6 months in Queshan,Henan. The drug resistance strains were highly prevalent in antiretroviral-treated patients, and increased with the continuation of therapy, with many patients encountering virological failure after 6 months therapy.

The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

Yi-qin WANG, Yu-cheng YANG, Wen-lu ZHANG, Su-ling HONG

2007, 22(3): 241

Received: 05 July 2006 Accepted: 24 December 2006
To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.

Pathogenesis and Associated Diseases of Kaposi's Sarcoma-associated Herpesvirus

Lin-ding WANG

2007, 22(3): 248

Received: 21 November 2006 Accepted: 02 February 2007
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the primary etiological agent of Kaposi’s sarcoma, primary effusion lymphoma and muticentric Castleman’s disease. In common with the other herpesviruses, KSHV exhibits both latent and lytic life cycles, both of which are characterized by distinct gene expression profiles and programs. KSHV encodes proteins which play essential roles in the inhibition of host adaptive and innate immunity, the inhibition of apoptosis, and the regulation of the cell cycle. KSHV also encodes several proteins which have transforming and intrcellular signalling activity.