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2009 Vol.24(3)



A Multiple Functional Protein: the Herpes Simplex Virus Type 1 Tegument Protein VP22*

Mei-li LI, Hong GUO, Qiong DING, Chun-fu ZHENG

2009, 24(3): 153 doi: 10.1007/s12250-009-3035-2

Received: 19 February 2009 Accepted: 08 April 2009
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The herpes simplex virus type 1 (HSV-1) VP22, is one of the most abundant HSV-1 tegument proteins with an average stoichiometry of 2 400 copies per virion and conserved among alphaherpesvirinae. Many functions are attributed to VP22, including nuclear localization, chromatin binding, microtubule binding, induction of microtubule reorganization, intercellular transport, interaction with cellular proteins, such as template activating factor I (TAF-I) and nonmuscle myosin II A (NMIIA), and viral proteins including tegument protein VP16, pUS9 and pUL46, glycoprotein E (gE) and gD. Recently, many novel functions performed by the HSV-1 VP22 protein have been shown, including promotion of protein synthesis at late times in infection, accumulation of a subset of viral mRNAs at early times in infection and possible transcriptional regulation function.
Research Article

Genetic Analysis of the VP1 Region of Human Enterovirus 71 Strains Isolated in Fuyang, China, During 2008*

Shao-hui MA, Jian-sheng LIU, Jing-jing WANG, Hai-jing SHI, Hui-juan YANG, Jun-ying CHEN, Long-ding LIU, Qi-han LI

2009, 24(3): 162 doi: 10.1007/s12250-009-3033-4

Received: 16 February 2009 Accepted: 27 March 2009
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Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.

Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Hua-qi PAN, Nan WANG, Li LIU, Lei LIU, Jiang-chun HU, Pu-yan CHEN, Shu-jin WANG, Rui-bing CAO

2009, 24(3): 171 doi: 10.1007/s12250-009-3002-y

Received: 16 September 2008 Accepted: 16 January 2009
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Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.

Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX*

Jian-qiang LI, Ji-xing LIU, Xi LAN, Jie CHENG, Run WU, Zhong-Zi LOU, Xiang-ping YIN, Xue-rui LI, Bao-yu LI, Bin YANG, Zhi-yong LI

2009, 24(3): 179 doi: 10.1007/s12250-009-2982-y

Received: 31 July 2008 Accepted: 08 January 2009
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The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM,M and N genes open reading frame (ORF) of DX were 4 152, 231,681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.

Evaluation of Sensitivities and Specificities of SARS-CoV Detection by Real-time Quantitative Reverse Transcription-PCR Assays*

Li-li XU, Zhi-hong HU, Hua-lin WANG, Xiao HAN, Fei DENG

2009, 24(3): 187 doi: 10.1007/s12250-009-3021-8

Received: 30 December 2008 Accepted: 17 February 2009
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The etiological agent of severe acute respiratory syndrome (SARS) was identified as a new coronavirus, termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study, we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreenTM dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities, 13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection, but because of its sequence variability in the different viral strains, primers and a probe based on the N gene were suitable substitutions. Meanwhile, we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus*

Chao FAN, Lan-lan ZHANG, Cheng-feng LEI, Qin FANG

2009, 24(3): 194 doi: 10.1007/s12250-009-3028-1

Received: 13 January 2009 Accepted: 17 March 2009
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Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335-742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335-742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Expression of Kaposi's Sarcoma-associated Herpesvirus ORFK8.1 and Its Preliminary Diagnostic Application*

Bi-shi FU, Bao-lin LI, Xin-xing OUYANG, Yan ZENG, Fan-hong XU, Lin-ding WANG

2009, 24(3): 202 doi: 10.1007/s12250-009-3029-0

Received: 14 January 2009 Accepted: 14 April 2009
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The ORFK8.1 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate–polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant ORFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 μg/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein’s specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.

HCMV Infection Depress NGF Expression in Human Glioma Cells

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

2009, 24(3): 209 doi: 10.1007/s12250-009-3018-3

Received: 11 December 2008 Accepted: 08 April 2009
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Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

Application of VP1 Protein to Develop Monoclonal Antibody against Foot-and-mouth Disease Virus Asia1 Type*

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

2009, 24(3): 215 doi: 10.1007/s12250-009-2986-z

Received: 05 August 2008 Accepted: 10 March 2009
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In order to develop an anti-FMDV Asia1 type monoclonal antibody (mAb), BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5E1 and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asia1 mAbs had no cross-reactivity with Swine vesicular disease (SVD) and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1:5×106, 1:2×106 and 1:5×106, respectively. 1B8 was found to be of IgG1 subtype, 5E1 and 5E2 belonged to IgG2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis.

Genetic Variation Analyses of nsp2 Gene of PRRSV in Ningxia Hui Autonomous Region of China

Hong TIAN, Jing-yan WU, Shuang-hui YIN, You-jun SHANG, Zi-ping MAN, Na ZHAO, Ye JIN, Xiang-tao LIU

2009, 24(3): 221 doi: 10.1007/s12250-009-3017-4

Received: 10 December 2008 Accepted: 12 April 2009
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To gain a better understanding of the genetic diversity and evolution of PRRSV in the Ningxia Hui Nationality Autonomous Region (Ningxia) of China, the nsp2 genes from a series of PRRSV strains collected from the region in 2007 were partially sequenced. These sequences were then analyzed along with the classical strain (ch-1a) and two other epidemic strains SD (3) and SD2006. Comparison of the nucleotide sequence with ch-1a indicated that nsp2 genes of seventeen Ningxia isolates (NX strain) have deletions of 87 nucleotides. Sequence analysis indicated that homology between the Ningxia strain and ch-1a was 60.3%-79.9% in the nucleotide sequence, and homology between the NX strains and SD strains was 80.3%-98.8% in the nucleotide sequence. The nsp2 genes of the seventeen isolates had 74.9%-100% nucleotide sequence identities with each other. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.