Einar Everitt

Purification of infectious pancreatic necrosis virus by anion exchange chromatography increases the specific infectivity

An improved method for the isolation and purification of infectious pancreatic necrosis virus (IPNV) is described. Virions released into the clarified growth medium are adsorbed to an anion exchange resin of diethylaminoethyl cellulose at pH 8.1. IPNV together with the likewise released and accumulated excess pool of the precursor to the major capsid protein, ICP62, are eluted at a salt concentration between 100 and 125 mM NaCl. The bovine serum albumin content of the growth medium supplement also elutes close to this position. Upon one step of combined sucrose- and CsCl-gradient centrifugation the recovered viruses display lower levels of aggregation, higher specific nucleic acid contents and an approximately 350% higher specific infectivity as compared with pools of viruses processed in parallel and isolated according to the established method relying on precipitation with poly(ethylene glycol).

Spectrophotometric quantitation of anchorage-dependent cell numbers using extraction of naphthol blue-black-stained cellular protein

A convenient method is described by which the actual or relative number of cells in anchorage culture is determined. After removal of the growth medium, cells are subjected to a double-fixation procedure. The cellular protein content is subsequently quantitatively stained with naphthol blue-black. After a period of removal of unbound stain, dye-protein complexes are hydrolytically released and measured spectrophotometrically at 620 nm. A linear correlation exists (r = 0.994) between cell concentration, in the range 3 X 10(4) to 8 X 10(5) cells/ml of final assay volume, and absorbance up to reading values of 3.8. The technical reproducibility of the assay, as judged from assessments of cell numbers in suspension culture, displays a coefficient of variation of 5%. The method was developed for 9.6-cm2 culture dishes, but it should be possible to transform it for the use of microtiter plates.

Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins.

Summary. The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm.

Preparative centrifugation - a new method for preparing pollen concentrates suitable for radiocarbon dating by AMS

Methods of preparative centrifugation eliminate many of the difficulties involved in preparing pollen concentrates from deposits rich in resistant organic material. Density centrifugation for the separation of pollen from a gyttja sample rich in resistant organic matter was tested. Combining centrifugations in two CsCl solutions, one of higher density and one of lower density than pollen, a pure pollen fraction was successfully prepared. Data on the isodensity and sedimentation rate of ‘fossilized’ recent pollen from twelve tree taxa are also presented, and the potential for separating a single taxon from pollen assemblages is demonstrated.

TCID50 determination by an immuno dot blot assay as exemplified in a study of storage conditions of infectious pancreatic necrosis virus

Some cell lines are difficult to grow in confluent monolayers and, therefore, the plaque assay cannot be applied since plaques may be hard to distinguish from other blank areas of the cell monolayers. To avoid this problem a rapid and sensitive immuno dot blot TCID50 method was developed using antibodies against virus antigens to detect infection and virus production. An alternative statistical method was developed to treat the scoring data and thereby obtained a coefficient of variation of 10%. To speed up the total procedure and to increase the proliferation rate of cells grown in 96-well cell culture plates, the plates were pretreated for 4 h at 4 degrees C with growth medium obtained from cell culturing flasks containing confluent cell monolayers. This immuno dot blot TCID50 method was applied for a study of the infectivity maintenance upon storage of infectious pancreatic necrosis virus (IPNV). Storage of IPNV at -70 degrees C with a cryoprotective agent (10% glycerol) preserved the TCID50 level even after as many as ten cycles of freezings and thawings, whereas the infectivity decreased by four orders of magnitude after storage at 4-8 degrees C for 2 months in the salt buffer used commonly.

Identification of IPNV-specified components released from productively infected RTG-2 cells following massive cytopathic effect

SummaryRainbow trout gonad cells (RTG-2) display a dramatic cytopathic effect and lysis following productive infection by infectious pancreatic necrosis virus (IPNV). In this study viruses were efficiently released into the growth medium together with low amounts of the monomeric free form of the structural protein VP3, heterodimers of VP2-VP3, aggregates of pVP2 and viral RNA associated with VP3. Ribonucleoprotein complexes of RNA-VP3 contained RNA equivalent to at the most 25% of full length viral genomes. Infectivity of material released into the growth medium late in infection was only associated with fully assembled viruses and isolated subviral RNA-VP3 complexes were not infectious. Upon purification of IPNV, viral hexa- and pentagonal particles of approx. 15 nm diameter were occasionally co-purified with the virus and then appeared in large quantities. Similar particle-like structures were seen as substructures of purified viruses that were treated with and partially disintegrated by CsCl of virus isodensity concentration.

Enhancement of intracellular uncoating of adenovirus in HeLa cells in the presence of benzyl alcohol as a membrane fluidizer.

SummaryThe early extra- and intra-cellular interaction between adenovirus type 2 and HeLa cells was studied in the presence of benzyl alcohol as a fluidizing agent. The processes of virus attachment and internalization were not affected at 5–15 mM of benzyl alcohol at 25°C. Under the same conditions an enhancement by 45% at the most was demonstrated for the cell-mediated virion uncoating. By completely blocking virion internalization with 50 mM azide the uncoating was reduced to 20% of the normal level. The remaining surfacelocated uncoating was not affected by benzyl alcohol. It was demonstrated that an enhancement of the intracellular virion uncoating was followed by a raised production of the hexon antigen, which was interpreted as an increase in the specific infectivity of the virus.

Adenovirus cellular receptor site recirculation of HeLa cells upon receptor-mediated endocytosis is not low pH-dependent.

SummaryHeLa cells were depleted of 75% of the available adenovirus type 2 specific cellular receptor sites (CRSs) by controlled attachment of viruses at a high multiplicity of infection. Upon removal of unattached viruses and further incubation, the cells were able to recycle the CRSs to 75% of the level of uninfected control cells within 5 h. The rate of virus receptor recycling was approx. 1 CRS × min−1 × cell−1. The existense of receptor recycling further strengthens the involvement of a total process of receptor-mediated endocytosis in adenovirus internalization. The recirculation process was neither affected by the lysosomotropic agents ammonium chloride and amantadine-HCl, nor by the ionophore monensin or the multifunctional weak-base amine chloroquine.

Cultivation of HeLa cells with fetal bovine serum or ultroser G: Effects on the plasma membrane constitution

SummaryPlasma membranes isolated from HeLa cells cultivated in suspension cultures supplemented with 3.5% fetal bovine serum or 2% of the commercially available serum substitute Ultroser G contained the same amounts of protein, cholesterol, and phosphate on a cellular basis. Minor differences in the plasma membrane fatty acid composition were seen, with the most pronounced alteration observed for palmitic acid, which amounted to 27 and 20% in fetal bovine serum- and Ultroser G-supplemented cells, respectively. Plasma membranes from cells growth with Ultroser G contained almost twice as much phosphatidylethanolamine and displayed two thirds of the phosphatidylcholine content, compared to plasma membranes obtained from fetal bovine serum supplemented cells. The former membranes also showed a 3 times higher specific [3H]acetate labeling of cholesterol, indicating a higherde novo synthesis of cholesterol. Both quantitative and qualitative alterations were revealed among the plasma membrane polypeptides when these were subjected to immuno- and lectin blottings. Fluorescence anisotropy measurements at different temperatures produced similar results irrespective of the growth medium supplement when the plasma membrane specific probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene was used on intact cells. However, the average cellular rigidity was higher for Ultroser G supplemented cells, determined with 1,6-diphenyl-1,3,5-hexatriene as a probe.

Human adenovirus 2 as immunogen in rabbits yields antisera with high titers of antibodies against the nonstructural 72K DNA-binding protein.

Immunization in rabbits with intact, highly purified adenovirus type 2 (Ad2) virions, yielded antisera with high titers of antibodies against the 72 000 dalton DNA-binding protein (DBP). This was established by rocket immunoelectrophoresis when an anti-intact Ad2-antiserum was analyzed against fractions from an ion-exchange chromatogram of soluble antigens remaining after virus isolation from virus-infected HeLa cells. The high anti-DBP titer did not reflect the composition of the immunogen, since no DBP was detectable within virions. An antiserum raised in response to mildly disrupted virions showed no specificity against the DBP, but contained antibodies against the same structural proteins as the anti-intact Ad2-antiserum, when analyzed by the immunoblotting technique. These findings indicate that the nonpermissive rabbit as an experimental host permits early gene expression of a human adenovirus.