Enumeration of Vaccinia Virus Particles in Crude Extracts of Infected Tissues by Electron Microscopy.

Sharp, D. G.; Overman, John R.

doi:10.3181/00379727-99-24366

Cited by 15 publications

(8 citation statements)

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“…The surface film was then floated off in water, mounted on an electron microscope grid, shadowed lightly with chromium, and examined. Direct comparison of the spray gun and the sedimentation methods showed the results with vaccinia virus to be in excellent agreement (11). Two important modifications have been used recently.…”

Section: Virus Particlementioning

confidence: 93%

“…Recent experiments have demonstrated the feasibility of virus particle counts on crude extracts of vaccinia virus infected tissues (10,11), thus obviating virus loss and inactivation during purification. In these studies tissue was homogenized to make a 10 per cent extract by weight.…”

Section: Many Virus Particle Count Experiments Have Utilized Vacciniamentioning

confidence: 99%

“…Egg membrane material was diluted 1:300 in 0.005 M phosphate buffer and skin extract usually diluted 1:666; however, in some instances various dilutions were used. The procedure has been described in detail recently (11). In brief, the Servan type SU particle-counting rotor with 8 sector-shaped cells was used.…”

Section: Virus Particlementioning

confidence: 99%

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Ratios of Vaccinia Virus Particles to Virus Infectious Units

Overman

Sharp

1959

The Journal of Experimental Medicine

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Total virus particle counts, infectivity titrations and the ratios between particles and infective units have been determined for vaccinia virus infected tissues. Growth curves of vaccinia in the chorioallantoic membrane are characterized by relatively low ratios from 1 to 4 days after inoculation and a marked rise in the ratio at more prolonged intervals. Ratio determinations of vaccinia virus passages in the egg, rabbit skin, and guinea pig skin have been made to study the phenomenon of adaptation in different hosts. The embryonated egg chorioallantoic membrane shows no variation in the ratio of particles to infectious units during passage and it is concluded that this host is completely susceptible to vaccinia. During adaptive passages on the skin of rabbits and guinea pigs relatively large amounts of non-infective virus appear as indicated by a rise in the particle-infectivity ratios. The extent of ratio increase appears related to the general resistance of the host to the virus. Finally, treatment of crude tissue extracts with sonic vibration is described as an aid in dispersing the virus particles for quantitative particle counts.

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Section: Virus Particlementioning

confidence: 93%

Section: Many Virus Particle Count Experiments Have Utilized Vacciniamentioning

confidence: 99%

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Ratios of Vaccinia Virus Particles to Virus Infectious Units

Overman

Sharp

1959

The Journal of Experimental Medicine

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“…However, we suspect that their method of estimating the total count may well have given too low a figure because some virus was not picked up on the supports and some was obscured by debris. In view of such criticisms, an attempt was recently made to demonstrate the accuracy of the sedimentation technique (Sharp & Overman, 1958). It appears that the method does give a reliable estimate of the number of vaccinia particles in a sample provided this has been adequately diluted.…”

Section: Discussionmentioning

confidence: 99%

The Infectivity of Purified and Partially Purified Preparations of Vaccinia and Cowpox Viruses

Kaplan

Valentine

1959

Journal of General Microbiology

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SUMMARY: Twenty-three preparations of vaccinia virus and 5 of cowpox virus were examined by electron microscopy of spray droplets and by infectivity titrations at various stages of purification. Extraction of virus-containing tissues with a fluorocarbon compound was a useful early step in purification, but preparations of the highest purity were obtained by flocculation of the virus with M-NaCl. The average ratio of total particle count to infectivity for the vaccinia virus preparations was 12. The lowest observed ratio based on reliable particle counts was 3.4.During inactivation of vaccinia virus, a very small, but constant, fraction of infectivity was observed t o survive extremely high doses of gamma-radiation .In order to assess the likelihood of multiple reactivation being responsible for this persistence, it was necessary to know the ratio of infective units to total particle count in the type of virus preparation being irradiated. The two groups of published figures for vaccinia virus are not in close agreement, so several preparations of vaccinia virus, and a few of cowpox virus, were counted by electron microscopy and titrated for infectivity. The influence of various methods of purification on the preparations was also studied. METHODS Viruses.The vaccinia virus strains were all sublines of the Lister Institute vaccine strain. The rabbit dermal virus (RV) was a homogeneous line (Amies, 1938) in its 80th skin passage. Chick embryo adapted virus (LC) had been passed from 13 to 16 times in the chick embryo chorioallantois. Tissue culture virus (T) was taken from the first five batches of virus grown for smallpox vaccine preparation. This virus was a first passage of bacteriologically sterile RV in trypsinized chick embryo cells. The cozupoe virus was a strain lyophilized in 1939 and recently cultivated. Its history is unknown, but it produced typical haemorrhagic lesions in the chick embryo chorioallantois after 3 days of incubation.Diluent. All extractions and dilutions were made in McIlvaine's phosphate + citrate buffer (pH 7.2, 0.004 M-phosphate). Treatments with trypsin were done with Armour's crystalline, salt-free product.Extractions were made by mechanical disruption of the virus-infected tissues in a Servall 'Omnimix' homogenizer a t 14,000 rev./min. for 5 min., with an ice-bath round the stainless steel vessel. Trifluorotrichloroethane (Gessler,

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“…Electron microscopy. Purified samples of rickettsiae were prepared for electron microscopy by centrifugation onto agar by using the Sorvall SU rotor and the method of Sharp and Overman (16). Rickettsiae were shadowed lightly with chromium.…”

Section: Bumeti Materials and Methodsmentioning

confidence: 99%

Changes in buoyant density relationships of two cell types of Coxiella burneti phase I

Wachter¹,

Briggs²,

Gangemi³

et al. 1975

Infect Immun

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Coxiella burneti phase I, purified from a formalin-inactivated yolk-sac vaccine, was separated into two bands of morphologically distinct cell types when subjected to sucrose gradient centrifugation. Recycling of the less dense, rod-shaped cells in unbuffered sucrose gradients (pH 5.5 to 6.0) resulted in the formation of bands having the location and appearance of the original two bands. Recycling of the denser band of larger ovoid-shaped cells yielded a single band, suggesting that the larger cell type arose from the smaller cell. In contrast to vaccine-derived rickettsiae, live, cell culture-propagated phase I organisms formed a single band in unbuffered sucrose gradients, at the same density as the upper band of the vaccine preparation. Centrifugation of cell culture-derived rickettsiae for 26 to 48 h in sucrose gradients of pH 5.5 resulted in the formation of a second band, at the same density as the lower band of the vaccine preparation. This did not occur in gradients of pH 7.0. Treatment of cell culture-propagated rickettsiae with formalin or germicidal ultraviolet radiation induced a total shift of the less dense cell population to a zone of higher density when centrifuged isopycnically in CsCl gradients. This density change did not occur in sucrose gradients, suggesting a difference in the effect of these treatments on the permeability of the cell membrane to sucrose and CsCl.

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Enumeration of Vaccinia Virus Particles in Crude Extracts of Infected Tissues by Electron Microscopy.

Cited by 15 publications

References 0 publications

Ratios of Vaccinia Virus Particles to Virus Infectious Units

Ratios of Vaccinia Virus Particles to Virus Infectious Units

The Infectivity of Purified and Partially Purified Preparations of Vaccinia and Cowpox Viruses

Changes in buoyant density relationships of two cell types of Coxiella burneti phase I

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