Reproducible Plaquing System for Rabies, Lymphocytic Choriomeningitis, and Other Ribonucleic Acid Viruses in BHK-21 / 13S Agarose Suspensions

Sedwick, W. David; Wiktor, Tadeusz J.

doi:10.1128/jvi.1.6.1224-1226.1967

Cited by 119 publications

(32 citation statements)

References 10 publications

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“…The various preparations indicated in Table 1 were then added to the cultures. After adsorption for 25 min at 29 C, the cultures received 5 ml of Eagle's medium containing 20%c, fetal calf serum and were placed in 5% CO2 at 37 C. After a single cycle of virus replication (24 hr), the cover slips were removed and strained for AAV antigen by the indirect immunofluorescence technique (3). At this time, early adenovirus cytopathic changes were evident.…”

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confidence: 99%

Purification of Rabies Virus Grown in Tissue Culture

Sokol

Kuwert

Wiktor

et al. 1968

J Virol

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Extracellular rabies virus, grown in monolayer cultures of BHK21 cells in the presence of medium supplemented with bovine serum albumin, was purified by the following procedure. Virus was precipitated from infectious tissue culture fluid by zinc acetate and was resuspended in a solution of ethylenediaminetetraacetate. The suspension was filtered through a Sephadex column and was treated with ribonuclease and deoxyribonuclease. The virions were then pelleted by centrifugation at high speed and were resuspended in buffer solution. Banding of the virus by centrifugation in a sucrose density gradient was the final step in the purification procedure. Purified preparations contained bullet-shaped virus particles of variable length and little (up to 5%) contaminating host-cell material. Most of the virions were “complete”, i.e., 180 nm long, but some virus particles were shorter. The length distribution of the virions was nonrandom. Shorter virions seemed to be noninfectious and showed markedly decreased hemagglutinating activity. The complement-fixing activity and the ribonucleic acid to protein ratio of the virions were not related to the length of the virus particles. Although the properties of extracellular and intracellular viruses were similar, the procedure was not suitable for purification of intracellular rabies virus.

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Section: Downloaded Frommentioning

confidence: 99%

Purification of Rabies Virus Grown in Tissue Culture

Sokol

Kuwert

Wiktor

et al. 1968

J Virol

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“…Inoculum was removed, the cell cultures were washed three times with phosphate-buffered saline (Dulbecco), and the cultures were fed with Eagle minimal essential medium supplemented with 0.2% bovine serum albumin. After 4 to 7 days of further incubation at 35°C, culture fluids were harvested for assay of released virus by the agarosesuspended BHK-21 13S cell technique (33).…”

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Rabies serogroup viruses in neuroblastoma cells: propagation, "autointerference," and apparently random back-mutation of attenuated viruses to the virulent state

Clark¹

1980

Infect Immun

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Each of several strains of fixed rabies virus was found to replicate to high titers in C1300 mouse neuroblastoma (clone NA) cells, without adaptation. Rabies serogroup Lagos bat, Mokola, and Duvenhage viruses also replicated efficiently in NA cells. Kotonkan and Obodhiang viruses replicated efficiently after adaptation, to titers not previously obtained in vitro. Infection in NA cells was frequently more cytopathic than in BHK-21 cells, allowing titration of Kotonkan and Obodhiang viruses by plaque assay. Duvenhage virus caused syncytium formation. Serial propagation of rabies viruses at a high multiplicity of infection in NA cells led to a rapid decline in virus yields; similar "autointerference" has not previously been demonstrated with rabies virus in other cell systems. Rabies virus infection in NA cells exhibited extreme sensitivity to interference by experimentally added defective interfering virions. Although several strains of attenuated rabies virus consistently reverted rapidly to virulence after propagation in NA cells, other strains of attenuated rabies and rabies serogroup viruses acquired increased virulence at a more gradual rate or not at all, suggesting that diverse characters may control virulence. When attenuated Flury HEP rabies virus was serially propagated at a low multiplicity of infection in either NA cells or suckling mouse brain, virulence appeared at a very variable rate, indicating that these systems may selectively enhance replication of randomly occurring virulent virus mutants.

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“…The fact that rabies virus requires static cells for its maximal growth may not be a new discovery, since an insight into previous data pointing to an enhanced growth in ultraviolet-irradiated cells [15] and to an efficient plaguing in agarose-suspended BHK-21 cells . [22] would naturally lead to this conclusion.…”

Section: Discussionmentioning

confidence: 77%

“…Still, however, there are many examples for a peculiar behavior of this virus that cells from a highly susceptible host do not necessarily support its multiplication in vitro. Also, plaque assay of this virus has been achieved only with agarose-suspended BHK21 cells [22] and monolayered primary chick embryo (CE) cells [31]. In the latter case, only the egg-adapted HEP Flury strain formed countable plaques under an agar overlay.…”

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Enhanced Growth and Plaquing of Rabies Virus in Static Chick Embryo Cell Culture

Sekine

Yoshino

1976

Japanese Journal of Microbiology

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The 7-day egg passage line of HEP Flury strain of rabies virus was inoculated to primary chick embryo (CE) cells prepared in different ways to compare efficiencies of viral growth and plaguing. Special care to minimize cellular damage due to trypsin at the step of monodispersion and sowing a comparatively large number of cells for monolayer preparation were required for rabies plaguing, whereas such cares were not necessary for plaguing of vesicular stomatitis virus. Plaque number and size were increased by incorporation of a high concentration of thymidine into cell growth medium. Various other means to produce a static state of CE cells were tested, and a maximal plaguing efficiency was obtained when dishes receiving a massive number of dispersed cells in MEM plus 1% calf serum were incubated at 37 C for 1 day without any buffering for monolayer preparation and postinfection incubation was done at 32 C in a CO2-incubator.Bottle cultures of CE cells prepared in a similar manner, when infected with HEP Flury virus, yielded a markedly higher titer of virus than CE cells prepared by our previous standard method.

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Reproducible Plaquing System for Rabies, Lymphocytic Choriomeningitis, and Other Ribonucleic Acid Viruses in BHK-21 / 13S Agarose Suspensions

Cited by 119 publications

References 10 publications

Purification of Rabies Virus Grown in Tissue Culture

Purification of Rabies Virus Grown in Tissue Culture

Rabies serogroup viruses in neuroblastoma cells: propagation, "autointerference," and apparently random back-mutation of attenuated viruses to the virulent state

Enhanced Growth and Plaquing of Rabies Virus in Static Chick Embryo Cell Culture

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