The lowered drop method for the preparation of specimens of partially purified virus lysates for quantitative electron micrographic analysis

Pinteric, L.; Taylor, Joyce

doi:10.1016/0042-6822(62)90027-2

Cited by 39 publications

(21 citation statements)

References 6 publications

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“…Viral and subviral particles were counted by transmission electron microscopy (TEM) of negatively stained DLPs, using an adaptation of a previously described technique (47). Droplets containing purified DLPs mixed with known quantities of 85-nm-diameter latex bead standards (Polysciences, Inc.) were deposited on Formvar grids.…”

Section: Methodsmentioning

confidence: 99%

Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

Trask

Dormitzer

2006

J Virol

109

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Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic doublelayered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10 7 , yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.To initiate rotavirus infection, the nonenveloped, icosahedral, triple-layered particle (TLP or virion) must translocate a large transcriptionally active subviral particle, the double-layered particle (DLP), across a membrane and into the cytoplasm of a target cell. The DLP consists of concentric VP2 and VP6 icosahedral protein shells, which encapsidate 11 doublestranded RNA genome segments, the viral polymerase (VP1), and a capping enzyme (VP3). Biochemical and structural studies indicate that conformational rearrangements in VP4 and VP7, the two proteins that make up the outermost shell of the TLP, deliver the DLP into the cytoplasm. The dissociation of trimers of the plate-like protein VP7 in low-calcium environments mediates uncoating in vitro (17, 51). The spike protein VP4 is anchored in the DLP and protrudes through the VP7 layer (53,58). This protein undergoes a fold-back rearrangement that resembles the fusogenic rearrangements of enveloped virus fusion proteins (18), although the function of the VP4 conformational change has yet to be demonstrated experimentally.Because VP4 and VP7 are both targets of neutralizing antibodies, understanding the mechanism of cell entry is linked to understanding protection against rotavirus gastroenteritis, which kills approximately 500,000 children each year (43). The development of efficient techniques to incorporate recombinant VP4 and VP7 into infectious rotavirus virions would provide powerful tools to investigate how these ...

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Section: Methodsmentioning

confidence: 99%

Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

Trask

Dormitzer

2006

J Virol

109

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“…aPinteric and Taylor, 1962;Watson, 1962;Watson et al, 1963;Horne, 1967;Oliver, 1973; Adrian et al, 1984. b No wick/wick. EM, electron microscopy; FESEM, field emission scanning electron microscopy; rAd/p53, p53 transgene-expressing recombinant adenovirus.…”

mentioning

confidence: 98%

The Use of Field Emission Scanning Electron Microscopy to Assess Recombinant Adenovirus Stability

Obenauer-Kutner¹,

Ihnát²,

Yang³

et al. 2002

Human Gene Therapy

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A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

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“…The former rely on the infection of cells in culture followed by a quantitative determination of the biological functionality of the vector (e.g., plaque assay) 50 or histochemical or immunostaining of products of the reporter or effector gene delivered by Ad vector in targeted cells. 54 The latter methods involve physical measurements of viral particle or DNA quantity (e.g., by direct visualization by electron microscopy 55 or by measurement of the optical absorbance of the virion DNA). 56 Different concentrations or dose units of Ad vectors have been used, such as PFU, 50% tissue culture infective dose, infectious units, transducing units, particle units, etc.…”

Section: Methods For Ad Vector Quantitationmentioning

confidence: 99%

“…All relevant assays associated with the development of the Ad vector for gene therapy of cystic fibrosis have provided a good history of how a systematic Ad vector assay methodology for the purpose of preclinical study and clinical trial support was performed. 5,50,[55][56][57] Development of the assays for the Ad vectors carrying the p53 tumor-suppressor expression cassette is another good example. These assays have been developed not only to analyze and verify the virological, biochemical, and biological features of the vectors, but also to fulfill the collection of supporting data for the approval of the clinical trial protocol, to support the conduct of the clinical trial, and to develop the manufacturing process.…”

Section: Development Of Assays For Ad Vectorsmentioning

confidence: 99%

Development and application of adenoviral vectors for gene therapy of cancer

Zhang¹

1999

Cancer Gene Ther

164

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For successful gene vaccination and therapy of cancer, it is essential to develop gene delivery vectors that can meet clinical and social requirements. The need for improved vectors was clearly manifested during the peak of the first wave of gene therapy. Adenoviral (Ad) vectors have recently drawn the attention of many of those involved in the field of gene therapy for cancer because of their practical advantages and application potential. Many experiments, innovations, preclinical studies, and clinical trials have generated an overwhelming amount of data and literature concerning this vector system. It is hoped that the comprehensive review presented here, which includes the principles, potential, capacity, and limitations of the current Ad systems, will help to further the rational development of the system. The literature in this article is organized in an attempt to emphasize Ad vector development in the aspects of technical approaches, practical hurdles and strategies to overcome them, significant experimental results, recent advances, future directions, etc. The technical range of this review covers details that are intended to serve as a reference for advanced technical readers and provide a foundation for initiates in the field of Ad vector gene therapy. The material presented here is also intended for nontechnical persons who want to have an overview of the significance and potential of the technology.

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The lowered drop method for the preparation of specimens of partially purified virus lysates for quantitative electron micrographic analysis

Cited by 39 publications

References 6 publications

Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

The Use of Field Emission Scanning Electron Microscopy to Assess Recombinant Adenovirus Stability

Development and application of adenoviral vectors for gene therapy of cancer

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