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2009 Vol.24(6)

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Review

Driving Forces of AIDS Pathogenesis: Massive CD4+ T Lymphocyte Depletion and Abnormal Immune Activation

Chang LI, Qin-xue HU

2009, 24(6): 501 doi: 10.1007/s12250-009-3063-y

Received: 21 April 2009 Accepted: 09 June 2009
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The occurrence of massive CD4+ T cell depletion is one of the most prominent characteristics of human immunodeficiency virus type 1 (HIV-1) infection during acute phase, resulting in unrestorable destruction to the immune system. The infected host undergoes an asymptomatic period lasting several years with low viral load and ostensibly healthy status, which is presumably due to virus-specific adaptive immune responses. In the absence of therapy, an overwhelming majority of cases develop to AIDS within 8-10 years of latent infection. In this review, we discuss the roles in AIDS pathogenesis played by massive CD4+ T lymphocytes depletion in gut-associated lymphoid tissue (GALT) during acute infection and abnormal immune activation emerging in the later part of chronic phase.

Structural Characteristics and Molecular Mechanism of Hepatitis B Virus Reverse Transcriptase

Hui FENG, Kang-hong HU

2009, 24(6): 509 doi: 10.1007/s12250-009-3076-6

Received: 19 May 2009 Accepted: 24 June 2009
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Hepatitis B virus (HBV), a typical member of the Hepadnaviridae family, is responsible for infections that cause B-type hepatitis which leads to severe public health problems around the world. The small enveloped DNA-containing virus replicates via reverse transcription, and this unique process is accomplished by the virally encoded reverse transcriptase (RT). This multi-functional protein plays a vital role in the viral life cycle. Here, we provide a summary of current knowledge regarding the structural characteristics and molecular mechanisms of HBV RT. Improved understanding of these processes is of both theoretical and practical significance for fundamental studies of HBV and drug discovery.
Research Article

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Shi-meng ZHANG, Rong FAN, Tian-yi YANG, Yi1 SUN, Jing-yun LI, Qin-zhi XU, Ping-kun ZHOU

2009, 24(6): 518 doi: 10.1007/s12250-009-3068-6

Received: 05 May 2009 Accepted: 16 August 2009
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Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

A Comparison of Complete Genome Sequences of a Rabies Virus Chinese Isolate SH06 with the Vaccine Strains

Sheng-li MENG, Ge-Lin XU, Jie WU, Xiao-Ming YANG, Jia-Xin YAN

2009, 24(6): 529 doi: 10.1007/s12250-009-3030-7

Received: 09 February 2009 Accepted: 24 July 2009
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In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus (SH06) obtained from the brain of a rabid dog. The overall length of the genome was 11 924 nucleotides. Comparison of the genomic sequence showed the homology of SH06 at nucleotide level with full-length genomes of reference vaccine strains ranged from 82.2% with the PV strain to 86.9% with the CTN strain. A full-length genome-based phylogenetic analysis was performed with sequences available from GenBank. Phylogenetic analysis of the complete genome sequences indicated that the SH06 exhibited the highest homology with rabies street virus BD06 and CTN vaccine strain originated from China.

Involvement of CXCR3-associated Chemokines in MHV-3 Induced Fulminant Hepatic Failure

Yong ZOU, Ge SONG, Lin DING, Tao CHEN, Wei-ming YAN, Xiao-jing WANG, Xiao-ping LUO, Qin NING

2009, 24(6): 537 doi: 10.1007/s12250-009-3071-y

Received: 11 May 2009 Accepted: 01 September 2009
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The role of chemokines in murine hepatitis virus strain 3 (MHV-3) induced fulminant hepatic failure (FHF) is not well defined. In this study, we investigated the role of the CXC chemokine receptor 3 (CXCR3)- associated chemokine [monokine induced by IFN-gamma (Mig/CXCL9) and interferon-gamma-inducible protein 10 (IP-10/CXCL10)] in the recruitment of intrahepatic lymphocytes and subsequent fulminant hepatic failure induced by MHV-3. Balb/cJ mice (6-8 weeks, female) were intraperitioneally injected with 100 PFU MHV-3.The proportions and numbers of T cells and NK cells as well as the expression of CXCR3 on T cells and NK cells in the liver, spleen and blood were analyzed by flow cytometry. The hepatic mRNA level of the CXCR3-associated chemokines (CXCL9 and CXCL10) was detected by realtime PCR. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes on the splenic lymphocytes. Following MHV-3 infection, the number of hepatic NK cells and T cells and the frequencies of hepatic NK cells and T cells expressing CXCR3 increased markedly; however, in the spleen and peripheral blood, they both decreased significantly. Moreover, the hepatic mRNAs levels of CXCL9 and CXCL10 were significantly elevated post infection. The transwell migration assay demonstrated that MHV-3-infected hepatocytes have the capacity to attract and recruit the splenic NK cells and T cells, and CXCL10 plays a key role in lymphocyte mobilization from the spleen. These results suggest that the CXCR3- associated chemokines (CXCL9 and CXCL10) may play an important role in the recruitment of intrahepatic lymphocytes and subsequent necroinflammation and hepatic failure in MHV-3 infection.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Lan-lan ZHANG, Jin-yu SHEN, Cheng-feng LEI, Chao FAN, Gui-jie HAO, Qin FANG

2009, 24(6): 545 doi: 10.1007/s12250-009-3038-z

Received: 12 March 2009 Accepted: 12 May 2009
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Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Human Cytomegalovirus Infection Inhibits the Differentiation of Human Hippocampus Neural Precursor Cells into Astrocytes

Ling LI, Bin WANG, Peng LI, Zhi-qiang BAI, Hai-tao WANG, Hai-yan LIU, Dong-meng QIAN, Xiu-xia SONG, Shou-yi DING, Zhi-yong YAN, Guang-yu JIANG

2009, 24(6): 552 doi: 10.1007/s12250-009-3019-2

Received: 23 December 2008 Accepted: 26 May 2009
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HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.

Construction and Identification of the Helper Plasmids for Reverse Genetic System of Rabies Virus Street Strain

Ping-gang MING, Ying HUANG, Qing TANG, Jia-liang DU, Xiao-yan TAO, Jia-xin YAN, Rong-liang HU

2009, 24(6): 559 doi: 10.1007/s12250-009-3039-y

Received: 17 March 2009 Accepted: 08 June 2009
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To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.

Preparation and Characterization of Monoclonal Antibodies against VP1 Protein of Foot-and-mouth Disease Virus O/China99

Shuai SONG, Tong LIN, Jun-jun SHAO, Shan-dian GAO, Guo-zheng CONG, Jun-zheng DU, Hui-yun CHANG

2009, 24(6): 566 doi: 10.1007/s12250-009-3061-0

Received: 08 April 2009 Accepted: 15 September 2009
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Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.

Confirmed Diagnosis by RT-PCR and Phylogenetic Analysis of Peste des Petits Ruminants Viruses in Tibet, China

Wen-hua ZHAO, Shi-biao YANG, Jian-qiang HAN, Mei JIANG, Hua-chun LI, Nian-zu ZHANG, Qi-han LI

2009, 24(6): 573 doi: 10.1007/s12250-009-3064-x

Received: 21 April 2009 Accepted: 06 July 2009
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This paper reports the confirmed diagnosis by nested RT-PCR of PPR cases in Tibet, China in 2007, and results of phylogenetic analysis. Results showed that the 11 tested samples were PPRV positive by nested RT-PCR, of which 2 samples were genetically close to the X7443 strain (Nigeria 75/1) of lineage I, and 3 samples close to the strain AY560591 (Sungri96) of linage IV with 96.6%、97.3%、97.6% and 98% nucleotide sequence homogeneity respectively, based on partial sequencing of the F gene from 5 samples and complete sequencing of the N/M/F/H genes from one sample. This study suggested that there are at least 2 origins of PPRV in China.