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2008 Vol.23(1)

Review

Integrin Activation and Viral Infection

Shan-dian GAO, Jun-zheng DU, Jian-hua ZHOU, Hui-yun CHANG, Qing-ge XIE

2008, 23(1): 1 doi: 10.1007/s12250-008-2886-2

Received: 13 June 2007 Accepted: 12 September 2007
Integrins are members of a ubiquitous membrane receptor family which includes 18 different α subunits and 8 β subunits forming more than 20 α/β heterodimers. Integrins play key functions in vascular endothelial cell and tumour cell adhesion, lymphocyte trafficking, tumor growth and viral infection. Current understanding of the molecular basis of integrins as viral receptors has been achieved through many decades of study into the biology of transmembrane glycoproteins and their interactions with several viruses. This review provides a summary of the current knowledge on the molecular bases of interactions between viruses and integrins, which are of potential practical significance. Inhibition of virus-integrin interactions at the points of virus attachment or entry will provide a novel approach for the therapeutic treatment of viral diseases.
Research Article

Sequence Variation in the Gp120 region of SHIV-CN97001 during in vivo Passage

Qiang LIU, Gui-bo YANG, Yue MA, Chen-li QIU, Jie-jie DAI, Hui XING, Yi-ming SHAO

2008, 23(1): 8 doi: 10.1007/s12250-008-2892-4

Received: 23 July 2007 Accepted: 10 November 2007
SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances (divergence, diversity) were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif (GPGQ) and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses (enJSRV)

Yu WANG, Shu-ying LIU, Jian-yun LI, Min HAN, Zhen-ling WANG

2008, 23(1): 15 doi: 10.1007/s12250-008-2876-4

Received: 18 April 2007 Accepted: 28 November 2007
In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19- T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame (orf-x) that overlaps the 3’ end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain (Accession No. AF153615) and USA JSRV21 strain (Accession No. AF105220). The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Ting JIANG, Xiang LI, Jian-hua SONG, Chang-yong LIANG, Xin-wen CHEN

2008, 23(1): 25 doi: 10.1007/s12250-008-2888-0

Received: 29 June 2007 Accepted: 23 October 2007
Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV–HzAM1 system can be used to analyze the mechanism of ODV entry.

Isolation and Complete Genomic Sequence Analysis of a New Sindbis-like Virus

Jing-jing WANG, Hai-lin ZHANG, Yan-chun CHE, Li-chun WANG, Shang-hui MA, Long-ding LIU, Yun LIAO, Qi-han LI

2008, 23(1): 31 doi: 10.1007/s12250-008-2891-5

Received: 23 July 2007 Accepted: 16 October 2007
The Sindbis-like virus was first discovered in China in 1986. Its complete genomic sequence consists of more than 11 000 bp encoding more than 3 700 amino acids. It contains a 5’ non-transcriptional region (5’-NTR) in a non-structural region, four non-structural proteins (nsP1, nsP2, nsP3, nsP4) regions, capsids in conserved and non-conserved regions and structural E1, E2, E3, 6K regions and a 3’ non-transcriptional region (3’-NTR). The Sindbis-IMB was isolated from the blood of a patient suspected to have encephalitis, and was followed by identification and passage. The virus RNA was extracted from virus supernatant in infected cells and the whole genome was divided into 12 fragments; RT-PCR was then performed to amplify the 12 fragments for complete sequencing. The results showed that the whole genomic sequence of Sindbis-IMB consists of 11 717 bp encoding 3 773 amino acids. Homology comparison with other Sindbis-like isolates demonstrated the highest similarity was the YN87448 with a variation of 1% strain isolated in Yunnan Province and the second highest to the SAAR86 strain with a variation of ~1.2%. The nucleotide sequence variations were present in non-structural regions, resulting in amino acids K, E, N, R, H, and L in protein sequences in positions 230, 231, 443,781, 1 582, and 1746 in the new isolation respectively. Furthermore, three additional amino acids--glutamic acid, serine and alanine--were noted in nsp4 terminus as compared to the YN87448 isolate.

Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein

Juan TAN, Kai WU, Rui CHANG, Qi-min CHEN, Yun-qi GENG, Wen-tao QIAO

2008, 23(1): 37 doi: 10.1007/s12250-008-2893-3

Received: 23 July 2007 Accepted: 20 December 2007
The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.

Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

Min HONG, Yan-chun CHE, Gui-zhen TANG, Wei CUN, Xue-mei ZHANG, Long-ding LIU, Qi-han LI

2008, 23(1): 43 doi: 10.1007/s12250-008-2903-5

Received: 06 September 2007 Accepted: 20 December 2007
HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Lan-lan ZHANG, Jin-yu SHEN, Cheng-feng LEI, Xiao-ming LI, Qin FANG

2008, 23(1): 51 doi: 10.1007/s12250-008-2921-3

Received: 07 November 2007 Accepted: 25 December 2007
Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

Research on Hepatitis B virus Genotypes and Subgenotypes among Bai Nationality in Dali, Yunnan Province

Wei LI, Yuan-ying SHEN, Xuan-rong ZHANG, Lai-feng REN, Qiang LI, Ru SHEN, Hai-ping ZHAO

2008, 23(1): 57 doi: 10.1007/s12250-008-2923-1

Received: 22 November 2007 Accepted: 26 December 2007
To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B+C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B+C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B+C) (P=0.077/P=0.663). In Dali, genotypes B, B+C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B+C) in patients.
Brief Reports

The Syndrome of Sea Cucumber (Apostichopus japonicus) Infected by Virus and Bacteria

Huan DENG, Zun-chun ZHOU, Nian-bin WANG, Chang LIU

2008, 23(1): 63 doi: 10.1007/s12250-008-2863-9

Received: 27 February 2007 Accepted: 18 July 2007
A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.

Expression and Assembly Mechanism of the Capsid Proteins of a Satellite Virus (XSV) Associated with Macrobrachium rosenbergii Nodavirus

Jian-min WANG, Hua-jun ZHANG, Zheng-li SHI

2008, 23(1): 73 doi: 10.1007/s12250-008-2926-y

Received: 06 December 2007 Accepted: 20 December 2007
The extra small virus (XSV) is a satellite virus associated with Macrobrachium rosenbergii nodavirus (MrNV) and its genome consists of two overlapping ORFs, CP17 and CP16. Here we demonstrate that CP16 is expressed from the second AUG of the CP17 gene and is not a proteinase cleavage result of CP17. We further expressed CP17 and several truncated CP17s (in which the N- or C-terminus or both was deleted), respectively, in Escherichia coli. Except for the recombinant plasmid CP17ΔC10, all recombinant plasmids expressed soluble protein which assembled into virus-like particles (VLPs), suggesting that the C-terminus is important for VLP formation.

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Hui-qiong YIN, Gai-ping ZHANG, Hong ZHANG, Jin-gang ZHANG

2008, 23(1): 68 doi: 10.1007/s12250-008-2901-7

Received: 29 August 2007 Accepted: 14 November 2007
Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5’ end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.