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Investigation of the immunological properties of the bovine viral diarrhea virus protein NS3 expressed by an adenovirus vector in mice

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ARCHIVES OF VIROLOGY 1999年第6期144卷 1057-1070页

作者： Elahi, SM Shen, SH Harpin, S Talbot, BG Elazhary, Y Univ Montreal Fac Vet Med Virol Sect St Hyacinthe PQ J25 7C6 Canada Natl Res Council Canada Biotechnol Res Inst Montreal PQ H4P 2R2 Canada Univ Sherbrooke Fac Sci Dept Biol Quebec City PQ Canada

Two replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-gamma in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2.

关键词：腺病毒科抗原病毒/遗传学抗原病毒/免疫学细胞系 DNA 重组腹泻病毒组牛病毒性/免疫学基因表达遗传载体免疫细胞肽水解酶类 RNA解螺旋酶类重组蛋白质类/遗传学重组蛋白质类/免疫学病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/免疫学动物牛人类小鼠

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Subcellular localisation of NS3 in HCV-infected hepatocytes

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JOURNAL OF MEDICAL VIROLOGY 1999年第4期59卷 456-462页

作者： Errington, W Wardell, AD McDonald, S Goldin, RD McGarvey, MJ Univ London Imperial Coll Sci Technol & Med Sch Med Dept Med London W2 1NY England Univ London Imperial Coll Sci Technol & Med Sch Med Dept Histopathol London W2 1NY England

Hepatitis C virus (HCV) NS3 is a multifunctional protein with both protease and helicase activities and has been shown to interact with host cell proteins. It is shown that NS3 is present in the hepatocytes from patients with chronic HCV infection by using anti-NS3 antisera. NS3 is detectable in approximately 4% of the hepatocytes from these patients. In most infected cells, NS3 is present in the cytoplasm;however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own are positive for NS3. The presence of NS3 in the nuclei of hepatocytes in chronically infected patients indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection. 1999. (C) 1999 Wiley-Liss, Inc.

关键词：印迹法蛋白质细胞系细胞核/病毒学大肠杆菌荧光抗体技术肝炎病毒属/代谢丙型肝炎抗体/免疫学丙型肝炎慢性/代谢丙型肝炎慢性/病毒学肝/细胞学肝/病毒学重组蛋白质类/代谢染色与标记亚细胞部分病毒非结构蛋白质类/分析病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/免疫学人类

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Improvement of a serodiagnostic strategy for foot-and-mouth disease virus surveillance in cattle under systematic vaccination:: a combined system of an indirect ELISA-3ABC with an enzyme-linked immunoelectrotransfer blot assay

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ARCHIVES OF VIROLOGY 2000年第3期145卷 473-489页

作者： Bergmann, IE Malirat, V Neitzert, E Beck, E Panizzutti, N Sánchez, C Falczuk, A WHO PAHO Pan Amer Foot & Mouth Dis Ctr BR-20001 Rio De Janeiro Brazil Univ Giessen Klinikum Inst Biochem Giessen Germany LARA Lab Reg Apoio Anim Porto Alegre RS Brazil ICA Inst Colombiano Agropecuaria Bogota Colombia SENASA Serv Nacl Sanidad Anim Gelab Genercia Labs Buenos Aires DF Argentina

Foot-and-mouth disease (FMD) recombinant non-capsideal viral antigens 3A, 3B, 2C, 3D and 3ABC were assessed individually in an indirect enzyme-linked immunosorbent assay (T-ELISA) for their ability to screen for persistent infection-specific antibodies in cattle, regardless of vaccination condition. Results of sequential serum samples from non-vaccinated animals with experimentally induced persistent infection, and their correlation with virus isolation, indicated that the polypeptides 3A, 3B and 3ABC showed the most adequate characteristics for further field studies. Reliable performance of the I-ELISA with the selected antigen 3ABC was indicated by the distinct patterns observed for the frequency distribution values of naive and true positive samples. For regularly vaccinated livestock, a clear negative profile was proved in samples from regions without recent history of FMD. In contrast, at 90 and 900 days post-outbreak, coexistence of a positive and a negative population was established. These findings indicated that, irrespective of vaccination, the test allowed a classification of the herd-disease status. A high degree of agreement was observed between I-ELISA-3ABC and EITB results for clearly reactive and non-reactive sera. For samples with reactivity values close to that of the cut-off, the EITB profiles upheld the definition of the infection condition. On this basis, screening by I-ELISA-3ABC, together with confirmation of suspect or positive samples by EITB is proposed as an adequate and accurate approach for large-scale epidemiological surveillance.

关键词：抗体病毒/血液口蹄疫病毒属/免疫学牛疾病/诊断牛疾病/流行病学牛疾病/病毒学酶联免疫吸附测定/方法口蹄疫/诊断口蹄疫/流行病学口蹄疫/病毒学免疫印迹法/方法人群监测重组蛋白质类/免疫学敏感性与特异性血清学试验接种/兽医学病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/免疫学病毒疫苗/免疫学动物牛

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ISDR pattern and evolution in patients with chronic hepatitis C treated with standard or Peg-IFN plus ribavirin

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ANTIVIRAL THERAPY 2003年第2期8卷 105-110页

作者： Cappiello, G Abbate, I Lo Iacono, O Longo, R Solmone, M Ferraro, D Antonucci, G Di Marco, V Di Stefano, R Croxi, A Ippolito, G Capobianchi, MR Natl Inst Infect Dis L Spallanzani Rome Italy S Pertini Hosp Rome Italy Univ Palermo Palermo Italy

The aim of the study was to characterize the interferon sensitivity determining region (ISDR) mutation pattern and its changes at 4 weeks of treatment in a population of patients infected with hepatitis C virus (HCV) genotype 1b receiving standard or PEG-IFN plus ribavirin (RBV), to find possible early correlates of therapy outcome. Forty-five patients with chronic hepatitis due to HCV 1b were treated by PEG-IFN-alpha 2b (n=23) or IFN-alpha 2b (n=22) plus RBV 1000-1200 mg/day. They were classified 24 weeks after stopping therapy as sustained responders (SR), relapsers (REL) or non-responders (NR). Sixteen patients were SIR, 12 REL and 17 *** mutations were evaluated by direct sequencing at baseline in all and after 4 weeks in patients with detectable viraemia (n=30).

关键词：氨基酸序列抗病毒药/治疗应用队列研究药物疗法联合基因型肝炎病毒属/药物作用肝炎病毒属/遗传学丙型肝炎慢性/药物疗法干扰素α/治疗应用分子序列数据突变聚乙烯二醇类/治疗应用重组蛋白质类利巴韦林/治疗应用序列比对治疗结果病毒非结构蛋白质类/遗传学女(雌)性人类男(雄)性中年人

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The Rift Valley fever virus nonstructural protein NSs is phosphorylated at serine residues located in casein kinase II consensus motifs in the carboxy-terminus

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VIROLOGY 1999年第2期263卷 517-525页

作者： Kohl, A di Bartolo, V Bouloy, M Inst Pasteur Unite Arbovirus & Virus Fievres Hemorrag Grp Bunyavirides F-75724 Paris France Inst Pasteur Unite Immunol Mol F-75724 Paris France

The S segment of Rift Valley fever Virus (Bunyaviridae, Phlebovirus) codes for two proteins, the nucleoprotein N and the nonstructural protein NSs. The NSs protein is a phosphoprotein of unknown function that is localized in the cytoplasm and the nuclei of infected cells where it forms filamentous structures. To characterize further the protein expressed in VC10 cells infected with the MP12 strain, we analyzed its phosphorylation states and showed that phosphorylated forms were found in both compartments. Cytoplasmic and nuclear NSs were phosphorylated only at serine residues. Phosphopeptide mapping and molecular analysis of mutants obtained by site-directed mutagenesis allowed us to map the major phosphorylation sites of nuclear and cytoplasmic forms of NSs to serine residues 252 and 256, located at the carboxy-terminus in consensus sequences for casein kinase II. A similar map was obtained when the protein was purified from mosquito cells infected with MP12. In addition, we showed that the purified unphosphorylated NSs protein expressed from pET-NSs plasmid in a coupled transcription-translation reaction containing Escherichia coli S30 extracts did not possess autophosphorylation activity but was phosphorylated in vitro after incubation with recombinant casein kinase II. (C) 1999 Academic Press.

关键词：氨基酸特征结构氨基酸序列酪蛋白激酶Ⅱ 细胞系细胞核/代谢共有序列/遗传学蚊科/细胞学蚊科/病毒学溴化氰/代谢细胞质/代谢分子序列数据诱变定点突变肽谱磷酰化磷酸丝氨酸/分析磷酸丝氨酸/代谢蛋白质丝氨酸苏氨酸激酶/分离和提纯蛋白质丝氨酸苏氨酸激酶/代谢重组融合蛋白质类/化学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢立夫特山谷热病毒/遗传学立夫特山谷热病毒/代谢丝氨酸/遗传学丝氨酸/代谢病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学动物

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Infectious hypodermal and hematopoietic necrosis virus of shrimp is related to mosquito Brevidensoviruses

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VIROLOGY 2000年第1期277卷 167-177页

作者： Shike, H Dhar, AK Burns, JC Shimizu, C Jousset, FX Klimpel, KR Bergoin, M Univ Calif San Diego Sch Med Dept Pediat La Jolla CA 92093 USA Super Shrimp Grp Inc National City CA 91950 USA Univ Montpellier 2 Mol Virol Unit UMR 5087 F-34095 Montpellier France

We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small DNA virus of shrimp, from wild Penaeus stylirostris. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The left, mid, and right ORFs on the complementary (plus) strand have potential coding capacities of 666 amino acids (aa) (75.77 kDa), 363 aa (42.11 kDa), and 329 aa (37.48 kDa), respectively. The overall genomic organization is similar to that of the mosquito brevidensoviruses. The left ORF most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest aa sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. A search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae, and we therefore propose to rename this virus Penaeus stylirostris densovirus(PstDNV). (C) 2000 Academic Press.

关键词：氨基酸序列碱基序列蚊科/病毒学 DNA病毒/分类 DNA病毒/遗传学 DNA病毒/生理学十足目(甲壳纲)/病毒学基因组病毒分子序列数据开放读码框架种系发生聚合酶链反应序列比对序列同源性氨基酸病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学病毒粒子/分类病毒粒子/遗传学病毒粒子/分离和提纯病毒复制动物

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Subcellular localization of hepatitis C viral proteins in mammalian cells

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ARCHIVES OF VIROLOGY 1999年第2期144卷 329-343页

作者： Kim, JE Song, WK Chung, KM Back, SH Jang, SK Pohang Univ Sci & Technol Dept Life Sci Pohang 790784 South Korea Kwangju Inst Sci Technol Dept Life Sci Kwangju South Korea

We determined the subcellular localization of hepatitis C viral (HCV) proteins as a first step towards the understanding of the functions of these proteins in the mammalian cell (CHO-KI). We used fluorescence emitted from green fluorescent protein (GFP)-fused to the viral proteins to determine the subcellular localization of the viral proteins. We found that most of the viral proteins were excluded from the nucleus. Core exhibited a globular pattern near the nucleus. NS2 was concentrated in the perinuclear space. NS4A accumulated in the ER and the Golgi regions. NS3 was detected in the nucleus as well as the cytoplasm, when it was expressed by itself. However, NS3 became restricted to the cytoplasm, when it was produced together with NS4A. NS4B showed a spot-like pattern throughout the cytoplasm. NS5A and NS5B were distributed throughout the cytoplasm in a mesh-like pattern. These results can provide a basis for further investigations into the functions of the HCV proteins.

关键词： CHO细胞 COS细胞细胞核/代谢仓鼠亚科细胞质/代谢基因表达绿色荧光蛋白质类肝炎病毒属/遗传学肝炎病毒属/代谢发光蛋白质类/分析发光蛋白质类/遗传学显微镜检查荧光重组融合蛋白质类/分析重组融合蛋白质类/遗传学病毒非结构蛋白质类/分析病毒非结构蛋白质类/遗传学病毒蛋白质类/分析病毒蛋白质类/遗传学动物

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GB virus C/Hepatitis G virus infection in Saudi Arabian blood donors and patients with cryptogenic hepatitis

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ARCHIVES OF VIROLOGY 2000年第1期145卷 73-84页

作者： Al-Ahdal, MN Rezeig, MA Kessie, G Chaudhry, F Al-Shammary, FJ King Faisal Specialist Hosp & Res Ctr Dept Pathol & Lab Med Riyadh Saudi Arabia King Faisal Specialist Hosp & Res Ctr Dept Med Riyadh 11211 Saudi Arabia King Saud Univ Coll Appl Med Sci Dept Clin Lab Sci Riyadh Saudi Arabia

Viral hepatitis is a common infection in the developing countries. Aside from Hepatitis A-E viruses, a novel hepatitis virus termed GBV-C, or HGV, was recently described. We have studied the prevalence of this virus among Saudi Arabian healthy blood donors (n = 200) and patients with cryptogenic (non-A-E) hepatitis (n = 71). After serum extraction and RNA reverse transcription, amplification was carried out by the polymerase chain reaction (PCR), using primers for the 5' noncoding region (NCR), NS5A region and NS3 helicase region. Among the patients with cryptogenic hepatitis, PCR-positivity was 18/71 (25.4%) for the 5' NCR, 14/71 (19.7%) for the NS5A region, and 15/71 (21.1%) for the NS3 helicase region. Among the healthy blood donors, PCR-positivity was 4/200 (2%) for the 5' NCR, 0/200 (0%) for the NS5A region, and 1/200 (0.5%) for the NS3 helicase region. Since the 5' NCR is considered the most conserved segment of the virus genome, it is not unusual to find higher positivity rate when that region is used for amplification. It is noted that the positivity rate is not far different among the three amplified regions, indicating that the heterogeneity of GBV-C/HGV is not as extensive as in hepatitis C virus. Phylogenetic analysis of 5' NCR DNA sequences showed that all isolates in this study belong to genotype 2. We conclude that the prevalence of GBV-C/HGV is similar to what is reported worldwide among the general Saudi population but relatively higher among Saudi patients with cryptogenic hepatitis.

关键词： 5'非翻译区/遗传学氨基酸序列碱基序列供血者克隆分子 DNA引物 DNA探针黄病毒科/化学黄病毒科/分类黄病毒科/遗传学肝炎病毒性人/流行病学肝炎病毒性人/病毒学分子序列数据患病率 RNA解螺旋酶类/化学 RNA解螺旋酶类/遗传学 RNA 病毒/分离和提纯逆转录聚合酶链反应沙特阿拉伯/流行病学序列分析 DNA 丝氨酸内肽酶类病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学人类

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Prevalence of, and antigenic variation in, serotype G10 rotaviruses and detection of serotype G3 strains in diarrheic calves: Implications for the origin of G10P11 or P11 type reassortant asymptomatic strains in newborn children in India

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ARCHIVES OF VIROLOGY 2002年第1期147卷 143-165页

作者： Varshney, B Jagannath, MR Vethanayagam, RR Kodhandharaman, S Jagannath, HV Gowda, K Singh, DK Rao, CD Indian Inst Sci Dept Microbiol & Cell Biol Bangalore 560012 Karnataka India Natl Dairy Dev Board Div Biotechnol Anand Gujarat India

Previous studies have shown predominant association of G10P11 type bovine rotavirus-derived reassortant strains with asymptomatic infections in newborn children in India. To understand the epidemiological and genetic basis for the origin of these strains in humans, the relative frequencies of different serotypes among bovine rotaviruses (BRVs) isolated from southern, western and central regions of the country were determined by subgroup and serotype analysis as well as nucleotide (nt) sequence analysis of the genes encoding the outer capsid proteins VP4 and VP7. Since the human G10P11 asymptomatic neonatal strain I321 possessed NSP1 from a human rotavirus, to determine its genetic origin in the bovine strains, comparative analysis of partial gene sequences from representative G10P11 strains was also carried out. The following observations were of great epidemiological significance, (i) G10P11 strains predominated in all the three regions with frequencies ranging between 55.6% and 85.2%. In contrast to the high prevalence of G6 strains in other countries, only one G6 strain was detected in this study and G8 strains represented 5.8% of the isolates, (ii) among the G10 strains, in serotyping ELISA, four patterns of reactivity were observed that appeared to correlate with the differences in electropherotypic patterns and amino acid (aa) sequence of the VP7, (iii) surprisingly, strains belonging to serotype G3 were detected more frequently (10.7%) than those of serotypes G6 and G8 combined, while strains representing the new serotype (G15) were observed in a single farm in Bangalore, and (iv) about 3.9% of the isolates were nontypeable as they exhibited high cross-reactivity to the serotyping MAbs used in the study. Comparative analysis of the VP7 gene sequence from the prototype G3 MAb-reactive bovine strain J63 revealed greatest sequence relatedness (87.6% nt and 96.0% aa) with that of serotype G3 rhesus-monkey strain RRV. It also exhibited high sequence homology with

关键词：氨基酸序列抗原变异抗原病毒碱基序列病毒壳体/遗传学病毒壳体/免疫学衣壳蛋白质类牛疾病/流行病学牛疾病/病毒学电泳聚丙烯酰氨凝胶印度/流行病学分子序列数据患病率再装配病毒/遗传学轮状病毒属/遗传学轮状病毒属/免疫学轮状病毒感染/流行病学轮状病毒感染/兽医学轮状病毒感染/病毒学序列比对序列分析 DNA 血清分型病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/免疫学动物猫牛狗人类婴儿新生小鼠

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Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1

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VIROLOGY 1999年第1期264卷 66-75页

作者： Hall, RA Khromykh, AA Mackenzie, JM Scherret, JH Khromykh, TI Mackenzie, JS Univ Queensland Dept Microbiol Brisbane Qld 4072 Australia Royal Childrens Hosp Sir Albert Sakzewski Virus Res Ctr Brisbane Qld 4029 Australia

The flavivirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native slate NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline(250) to leucine) when compared with the parental KUN virus. When site-directed mutagenesis was used to substitute leucine(250) with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious virus early (12-24 h) in infection. This correlated with our observations that FLSD required approximately IO-fold more infectious virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows virus replication, and reduces virulence in mice. (C) 1999 Academic Press.

关键词：氨基酸序列氨基酸取代长尾猴黑保守序列二聚作用脑炎病毒亚组日本/致病力脑炎病毒亚组日本/生理学脑炎虫媒病毒/病理生理学动力学小鼠近交BALBC 分子序列数据诱变定点点突变重组蛋白质类/化学重组蛋白质类/代谢序列比对序列同源性氨基酸 Vero细胞病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢毒力动物小鼠

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