High‐pressure freezing in the study of animal pathogens

Monaghan, Paul; Cook, H.; Hawes, Philippa C.; Simpson, Jennifer; Tomley, Fiona M.

doi:10.1046/j.1365-2818.2003.01245.x

Cited by 32 publications

(17 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…BHK-21 cells infected with FMDV quickly "round up" in this way, with the majority of the cells floating into the medium in which they are cultured within 3 hours post infection (Monaghan et al, 2003). The exact time until cytopathic effect is likely to vary with strain virulence: Baranowski et al (1998) observed complete cytopathic effects 4-6h with MARLS FMDV (a monoclonal antibody escape mutant) compared to 16-20h with C-58c1 FMDV.…”

Section: Parameter Estimatesmentioning

confidence: 99%

Modelling Foot-and-Mouth Disease Virus Dynamics in Oral Epithelium to Help Identify the Determinants of Lysis

2010

View full text Add to dashboard Cite

Section: Parameter Estimatesmentioning

confidence: 99%

Modelling Foot-and-Mouth Disease Virus Dynamics in Oral Epithelium to Help Identify the Determinants of Lysis

2010

View full text Add to dashboard Cite

“…The problem becomes even more complicated, when the sample is planned to be used for subsequent protein detection by immunocytochemistry at the ultrastructural level, which also requires preservation of the antigen to be recognized by a specific antibody. A range of protocols are available for the freeze-substitution method and they have to be selected empirically since comparative studies are scarce and our mechanistic understanding of the substitution process barely exists (Buser and Walther 2008;Giddings 2003;Lonsdale et al 1999;Monaghan et al 2003;Takahashi et al 1997;Takizawa et al 1999;Wild et al 2001). These procedures are usually optimized to bring solution for a specific problem, and they vary in solvent polarity, substitution schedule, fixative additives, and resin embedment.…”

Section: Introductionmentioning

confidence: 99%

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

Sobol

Philimonenko

Hozák

2010

Histochem Cell Biol

View full text Add to dashboard Cite

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.

show abstract

“…Importantly, because routine preparations for electron microscopy provide poor preservation of the nucleocapsid, we used high-pressure freezing followed by freeze-substitution to visualize the nucleocapsid. High-pressure freezing and freeze-substitution techniques ensure better preservation of fine ultrastructure than conventional fixation methods (26,27). We then reinvestigated the morphogenesis of an inducible mutant in L4 that has been characterized previously (12).…”

mentioning

confidence: 99%

Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

Jesus

Moussatché

Condit

2014

J Virol

View full text Add to dashboard Cite

Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCEThe vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid.

show abstract

High‐pressure freezing in the study of animal pathogens

Cited by 32 publications

References 17 publications

Modelling Foot-and-Mouth Disease Virus Dynamics in Oral Epithelium to Help Identify the Determinants of Lysis

Modelling Foot-and-Mouth Disease Virus Dynamics in Oral Epithelium to Help Identify the Determinants of Lysis

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

Contact Info

Product

Resources

About