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Functional analysis of the Autographa californica nucleopolyhedrovirus IAP1 and IAP2
XianDong Zeng,Fang Nan,ChangYong Liang,JianHua Song,Qian Wang,Just M. Vlak,XinWen Chen
Science China Life Sciences , 2009, DOI: 10.1007/s11427-009-0105-5
Abstract: The Autographa californica nucleopolyhedrovirus (AcMNPV) contains three apoptosis suppressor genes: p35, iap1 and iap2. AcMNPV P35 functions as a pancaspase inhibitor, but the function of IAP1 and IAP2 has not been entirely resolved. In this paper, we analyze the function of IAP1 and IAP2 in detail. AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4 (Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV (HearNPV) infection and rescued the replication of HearNPV and BV production in these cells. Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection. Recombinant HearNPVs expressing AcMNPV iap1, iap2 and p35, respectively, not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells. However, the iap1, iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production. These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors.
曾宪东,南方,梁昌镛,宋建华,王倩,Just M. Vlak,陈新文
中国科学 生命科学 , 2009,
Abstract: 苜蓿银纹夜蛾核多角体病毒(Autographa californica nucleopolyhedrovirus,AcMNPV)基因组含有3个细胞凋亡抑制基因,即p35,iap1和iap2.其中,p35作为一个有效的依赖于天冬氨酸的半胱氨酸蛋白酶(caspase)抑制因子,能够抑制多种因素诱发细胞凋亡,而iap1和iap2的功能仍未完全明晰,本研究对IAP1和IAP2的功能进行了详细分析.缺失了p35的AcMNPV仍可抑制棉铃虫核多角体病毒(Helicoverpa armigera single nucleocapsid NPV,HearNPV)诱导的BTI-Tn-5B1-4(Tn-Hi5)细胞凋亡并挽救HearNPV在Tn-Hi5细胞中复制及HearNPV出芽型病毒粒子的产生.进一步构建了瞬时表达质粒以及分别表达AcMNPV的p35,iap1和iap2基因的重组HearNPV,转染瞬时表达的IAP1和IAP2对HearNPV感染诱导的Tn-Hi5细胞凋亡有抑制效果,而重组病毒感染Tn-Hi5细胞也可抑制其凋亡并在其中复制,然而重组HearNPV表达的p35,iap1和iap2并未能挽救出芽型病毒粒子的产生....
Effects of Early or Overexpression of the Autographa californica Multiple Nucleopolyhedrovirus orf94 (ODV-e25) on Virus Replication  [PDF]
Xiao-Chun Luo, Shan-Shan Wang, Jie Zhang, Duo-Duo Qian, Si-Min Wang, Lu-Lin Li
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0065635
Abstract: odv-e25(e25) is one of the core genes of baculoviruses. To investigate how it functions in the replication cycle of a baculovirus, a number of Autographa californica multiple nucleopolyhedrovirus recombinants with e25 under control of the promoter of immediate early gene ie1, or the promoter of the very late hyperexpressed gene p10, were constructed using a bacmid system, and the effects of early expression or overexpression of e25 on replication of the virus were evaluated. Microscopy and titration assays demonstrated that bacmids with e25 under control of ie1 promoter were unable to produce budded viruses; and that the recombinant viruses with e25 under control of p10 promoter generated budded virus normally, but formation of occlusion bodies were dramatically reduced and delayed in the infected cells. Electron microscopy showed that there were no mature virions or intact nucleocapsids present in the cells transfected with a recombinant bacmid with e25 under control of ie1 promoter. Quantitative real-time PCR analysis demonstrated that alteration of the e25 promoter did not affect viral DNA synthesis. The reporter gene expression from the promoter of the major capsid protein gene vp39 was reduced 63% by early expression of e25. Confocal microscopy revealed that E25 was predominantly localized in nuclei by 24 hours post infection with wild-type virus, but it remained in the cytoplasm in the cells transfected with a recombinant bacmid with e25 under control of the ie1 promoter, suggesting that the transport of E25 into nuclei was regulated in a specific and strict time dependent manner.
苜蓿银纹夜蛾核多角体病毒iap1和iap2基因功能分析  [PDF]
中国科学 生命科学 , 2009,
Abstract: 苜蓿银纹夜蛾核多角体病毒(Autographacalifornicanucleopolyhedrovirus,AcMNPV)基因组含有3个细胞凋亡抑制基因,即p35,iap1和iap2.其中,p35作为一个有效的依赖于天冬氨酸的半胱氨酸蛋白酶(caspase)抑制因子,能够抑制多种因素诱发细胞凋亡,而iap1和iap2的功能仍未完全明晰,本研究对IAP1和IAP2的功能进行了详细分析.缺失了p35的AcMNPV仍可抑制棉铃虫核多角体病毒(HelicoverpaarmigerasinglenucleocapsidNPV,HearNPV)诱导的BTI-Tn-5B1-4(Tn-Hi5)细胞凋亡并挽救HearNPV在Tn-Hi5细胞中复制及HearNPV出芽型病毒粒子的产生.进一步构建了瞬时表达质粒以及分别表达AcMNPV的p35,iap1和iap2基因的重组HearNPV,转染瞬时表达的IAP1和IAP2对HearNPV感染诱导的Tn-Hi5细胞凋亡有抑制效果,而重组病毒感染Tn-Hi5细胞也可抑制其凋亡并在其中复制,然而重组HearNPV表达的p35,iap1和iap2并未能挽救出芽型病毒粒子的产生.结果表明,AcMNPV的iap1和iap2基因表达产物作为细胞凋亡抑制因子是有功能的.
Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus  [cached]
Neil?M. J. O’Flynn,Avnish Patel,Jan Kadlec,Ian?M. Jones
Bioscience Reports , 2012, DOI: 10.1042/bsr20120093
Abstract: The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268–281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell-to-cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies, suggesting that additional factors also govern entry.
Isolation and Characterization of a Nucleopolyhedrovirus from Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae)
International Journal of Virology and Molecular Biology , 2012, DOI: 10.5923/j.ijvmb.20120103.02
Abstract: Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae) is an important pest of vegetable crops widely distributed in South America. At present, its control is based on the use of broad spectrum chemical insecticides. In order to develop a more selective alternative to be applied in integrated or organic pest management programs, we isolated and characterized a baculovirus that infects R. nu, denominated RanuMNPV (Rachiplusia nu multiple nucleopolyhedrovirus) in view of its morphology and pattern of infection. According to optical microscope images of in vitro infected cell cultures, data from bioassays carried out with various species of larvae, and DNA sequences from p74, polyhedrin, v-cathepsin, v-chitinase, lef8 and lef9 genes, this isolate could be considered as a variant of Autographa californica MNPV with a different host range.
Characterization of an Egyptian Spodoptera littoralis Nucleopolyhedrovirus with Cloning and Sequencing of a Highly Conserved Region From Polyhedrin Gene  [cached]
AlaaEddeen Seufi
Erciyes Medical Journal , 2007,
Abstract: Purpose: An Egyptian isolate of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was tested for its potential as biocontrol agent in comparison to Autographa californica multiple nucleopolyhedrovirus (AcMNPV).Material and Methods: Comparative assays of SpliNPV and AcMNPV against 2nd instar larvae of Spodoptera littoralis revealed 4-fold greater susceptibility of S. littoralis to AcMNPV than to SpliNPV based on LC50 values for the two viruses. The LT50s determined for SpliNPV and AcMNPV using LC50 of the virus against 2nd instar larvae were 4.2 and 5.8 days, respectively. A DNA segment of 405 bp containing highly conserved region from polyhedrin gene of SpliNPV (Polh-cr) was successfully amplified by PCR. Subsequently, this DNA segment was cloned and sequenced. Nucleotide sequence and its deduced amino acid sequence were compared to all available sequences in GenBank. Results: Sequence alignment results revealed that Polh-cr showed significant similarities with 91 different baculovirus isolates. The percentage of homology ranged from 78% for Plusia orichalcea NPV to 99 % for SpliNPV. Conclusion: This highly conserved region provides a candidate that could be used in easy, fast and economic prospective systems for virus detection as well as in biological control strategies.
Reduction of liver macrophage transduction by pseudotyping lentiviral vectors with a fusion envelope from Autographa californica GP64 and Sendai virus F2 domain
David M Markusic, Niek P van Til, Johan K Hiralall, Ronald Elferink, Jurgen Seppen
BMC Biotechnology , 2009, DOI: 10.1186/1472-6750-9-85
Abstract: Reduction of macrophage sequestration is therefore essential for efficient lentiviral liver gene therapy.Fusions were made of Autographa californica GP64 and the hepatocyte specific Sendai Virus envelope proteins. Lentiviral vectors were produced with either wild type GP64, Sendai-GP64, or both wild type GP64 and Sendai-GP64 and tested in vitro and in vivo for hepatocyte and macrophage gene transfer.Sendai-GP64 pseudotyped vectors showed specific gene transfer to HepG2 hepatoma cells, with no detectable transduction of HeLa cervical carcinoma cells, and a decreased affinity for RAW mouse macrophages. Co-expression of wild type GP64 and Sendai-GP64 resulted in improved viral titers while retaining increased affinity for HepG2 cells.In vivo, the Sendai-GP64 vectors also showed decreased transduction of murine liver macrophages.We demonstrate reduced macrophage transduction in vitro and in vivo with GP64/Sendai chimeric envelope proteins.HIV-1 derived lentiviral vectors efficiently transfer genes and mediate long-term gene expression in non-dividing cells [1-3]. These properties make lentiviral vectors attractive candidates for the correction of inherited liver disorders. Lentiviral vectors pseudotyped with the envelope glycoprotein from Vesicular Stomatitis Virus (VSVg) can efficiently transduce primary hepatocytes in vitro [4,5]. In contrast, in vivo lentiviral vector delivery in rodents results in relatively poor gene transfer to hepatocytes because nonparenchymal liver cells are preferentially transduced [6-8]. We have previously shown that the main target of VSVg pseudotyped lentiviral vectors are liver macrophages, the Kupffer cells [9]. Although depletion of Kupffer cells leads to a significant increase in the gene transfer to hepatocytes [9] it would be preferable to develop lentiviral vectors that are less efficiently sequestered by macrophages. This would make more virus available for hepatocyte transduction and could also reduce the immune response to the vi
Characterization of an Egyptian Spodoptera littoralis nucleopolyhedrovirus and a possible use of a highly conserved region from polyhedrin gene for nucleopolyhedrovirus detection
AlaaEddeen M Seufi
Virology Journal , 2008, DOI: 10.1186/1743-422x-5-13
Abstract: Baculoviruses are considered to be the largest and most broadly studied insect viruses. Although they infect over 600 species of insects [1], individual isolates normally show a limited host range and infect only closely related species. It is believed that baculoviruses are potentially useful as safe biological control agent and in some cases they were used successfully to control different insect pests [2-4]. However the overall use of baculoviruses for biological control is limited compared to other pest control means [3,5]. This is due to the virulence and speed of action, as related to dose, host range, cost of production and patent registration are important effectiveness-determining properties of insect-pathogenic biocontrol agents.Baculoviruses were not only isolated from the insect orders Hymenoptera, Diptera and Trichoptera, but also were isolated from the crustacean order Decapoda (shrimps) [6]. Furthermore, some NPVs (Penaeus monodon-NPV) are considered as serious pests for marine crustaceans [7]. The infection of marine crustaceans (shrimps, prawns, crabs, eustacosa...etc.) with NPV will reduce their economic value and thus negatively affecting the national income. This problem will arise in countries that depend on marine wealth as a source of national income and/or where marine crustaceans are a major in most of the peoples' food (Australia and Far East nations like Japan, China, etc...). Although, the shrimp viruses have now been classified as Nimaviridae in the genus Whispovirus and are no longer baculoviruses [8], their diagnosis by PCR using primers of the polyhedrin and DNA polymerase genes of Autographa californica nucleopolyhedrovirus (AcMNPV) and Lymantria dispar nucleopolyhedrovirus (LdMNPV) was confirmed by Hsu et al. (2000). For the above mentioned reasons, many authors were interested in developing an accurate and easy diagnostic method to detect baculoviral infection in larval shrimps as well as insects [9,7-19].Because baculoviruses are
Differential expression of early viral gene BmORF51 in Bombyx mori nucleopolyhedrovirus infection of resistant and susceptible silkworms
F Lin, Q Yao, H Chen, Y Zhou, K Chen
African Journal of Biotechnology , 2010,
Abstract: Open reading frame 51 of Bombyx mori nucleopolyhedrovirus (Bm51) is a homologue of autographa californica multiple NPV ORF63. In this study, the expression profiles of Bm51 in the resistant strain NB and the susceptible strain 306 were characterized, and Bm51 gene was amplified from BmNPV genomic DNA by polymerase chain reaction (PCR) and cloned into Escherichia coli expression vector pET-30a (+). The recombinant His-tagged Bm51 protein was expressed in E. coli BL21 (DE3) and purified by metal chelating affinity chromatography to produce antibodies against Bm51 protein. The amino acid sequence of recombinant protein was confirmed by mass spectroscopic analysis. The transcription and protein product of early viral gene, Bm51, was detected at 6 h post-infection (p.i.) in resistant strain NB by quantitative real-time (qRT)-PCR and western blotting, and the expression of Bm51 in NB reached the maximal level at 36 h p.i. in NB, and then gradually decreased to undetectable level at 72 h p.i. In contrast, the Bm51 protein was undetectable until 12 h p.i. in susceptible strain 306 and the expression of Bm51 progressively increased during the 72 h post-infection.
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