Antibody-mediated uncoating of adenovirus in vitro.

In a virus destabilization assay in vitro it was demonstrated that exposure of adenovirus to proteins will non-specifically protect the virus from being uncoated following transfer to low pH and hypotonic conditions. Such uncoating was also fully inhibited upon pretreatment of virus with 0.05% of the non-ionic detergent polyoxyethylenesorbitan monolaurate (Tween 20). However, in the presence of low concentrations of Tween 20 it was shown that monospecific immunoglobulins, directed against the fiber antigen and polyspecific antibodies produced in response to intact virions, were able to overcome the detergent-protecting effect of uncoating. Immunoglobulins directed towards the remaining outer-capsid components, the hexon, the penton base and the protein IIIa, revealed no such effects. The antifiber-mediated uncoating was paralleled by an aggregation of the virions. The data suggest that the virion-stabilizing effect of salt is enhanced by the hydrophobic action of a non-ionic detergent. Under these conditions the interaction between antifiber antibodies and fibers of the virion will trigger a destabilization of the virion upon transfer to low pH and hypotonic conditions.

Using an electronic nose for determining the spoilage of vacuum-packaged beef.

The use of an electronic nose in the quantitative determination of the degree of spoilage of vacuum-packaged beef was evaluated. Beef from four different slaughterhouses was sliced, vacuum-packaged and stored at 4 degrees C for 8 weeks. Samples were withdrawn for bacterial (aerobic bacteria, lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas and Enterobacteriaceae) and sensorial analyses and analysis of the volatile compounds during the storage period. A trained panel was used for the sensorial evaluations. The volatile compounds were analysed using an electronic nose containing a sensory array composed of 10 metal oxide semiconductor field-effect transistors, four Tagushi type sensors and one CO2-sensitive sensor. Four of the 15 sensors were excluded due to lack of response or overloading. Partial least-squares regression was used to define the mathematical relationships between the degree of spoilage of vacuum-packaged beef, as determined by the sensory panel, and the signal magnitudes of the sensors of the electronic nose. The mathematical models were validated after 6 months using a new set of samples. The stability of the sensors during this period was examined and it was shown that the sensitivity of five of the 11 sensors used had changed. Using the six remaining sensors, the signal patterns obtained from the meat from the different slaughterhouses did not change over a period of 6 months. It was shown that the degree of spoilage, as calculated using a model based on two Tagushi sensors, correlated well with the degree of spoilage determined by the sensory panel (r2 = 0.94).

Bacterial spoilage of meat and cured meat products.

The influence of environmental factors (product composition and storage conditions) on the selection, growth rate and metabolic activity of the bacterial flora is presented for meat (pork and beef) and cooked, cured meat products. The predominant bacteria associated with spoilage of refrigerated beef and pork, are Brochothrix thermosphacta, Carnobacterium spp., Enterobacteriaceae, Lactobacillus spp., Leuconostoc spp., Pseudomonas spp. and Shewanella putrefaciens. The main defects in meat are off-odours and off-flavours, but discolouration and gas production also occur. Bacteria associated with the spoilage of refrigerated meat products, causing defects such as sour off-flavours, discolouration, gas production, slime production and decrease in pH, consist of B. thermosphacta, Carnobacterium spp. Luctobacillus spp. Leuconostoc spp. and Weissella spp. Analysis of spoilage as measured by bacterial and chemical indicators is discussed. It is concluded that a multivariate approach based on spectra of chemical compounds, may be helpful in order to analyse spoilage, at least for spoilage caused by lactic acid bacteria. The consequences of bacteria bacteria interactions should be evaluated more.

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components.

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0 x 10(2) CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0 x 10(1) CFU per ml of Y. enterocolitica and incubated at 25 degrees C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.

Exchange of the cellular growth medium supplement from fetal bovine serum to Ultroser G increases the affinity of adenovirus for HeLa cells.

A comparative investigation was performed on the process of attachment of adenovirus type 2 to HeLa cells cultivated in the presence of 3.5% fetal bovine serum (FBS-cells) or 2% Ultroser G (USG-cells). The initial rates of virus attachment were markedly higher at temperatures between 10 and 35 degrees C for the virus binding to USG-cells than to FBS-cells. This was not caused by a higher amount of available virus-recognizing cellular receptor sites or cellular receptor units recognizing the viral fiber, but could be explained by a higher affinity of virions for USG-cells as compared to FBS-cells. Studies of virus attachment to cells, pretreated with neuraminidase and/or wheat germ agglutinin, suggested that the cellular receptor sites of FBS-cells were masked to a higher extent by sialic acid than the cellular receptor sites of USG-cells.

Cultivation of HeLa cells with fetal bovine serum or Ultroser G: effects on the plasma membrane constitution.

Plasma 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 grown 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 higher de 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.

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

The 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 process of virus attachment and internalization were not affected at 5-15 mM of benzyl alcohol at 25 degrees 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 surface-located 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.