Comparison of two methods of virus detection by immunosorbent electron microscopy (ISEM) using protein A

The efficiency of two methods of immunosorbent electron microscopy has been compared. The first method consists in trapping virus particles by means of Staphylococcus aureus cells coated with a layer of viral antibodies; the second method consists in trapping virus particles on electron microscope grids coated with specific antibody. A suspension containing 10⁷ antibody-coated bacteria trapped the total number of virions present in 1 ml of a 500 ng/ml virus preparation; the cells were then fully saturated with virions, and approximately 100 virions (of 30 nm diameter) were visible at the periphery of each cell. When 10 cells/ml were used the minimum virus concentration needed to see one virion at the cell periphery was 5 ng/ml. Antibody-coated grids allowed for the detection of approximately the same quantity of virus, but the data obtained with this method were more reproducible and suitable for quantitation.     

Some factors affecting infectivity assays of wound-tumor virus on cell monolayers from an insect vector

Measurements of wound-tumor virus (WTV)-infectivity were made by counting infection foci in cell monolayers of Agallia constricta. The optimum seeding density was about 2.9 × 10⁵ cells per 0.1 ml per coverslip. The foci were revealed by staining with fluorescent antibody and examination with the fluorescence microscope. The focus count method employed in these studies, is shown to be an accurate method for determining WTV infectivities. The number of infected cells per focus was related to the concentration of WTV in the inoculum and is probably an expression of the rate of virus multiplication and spread in the cell monolayer. The effects of glycine, histidine, phosphate, and MgCl₂ in inocula were studied; a histidine-MgCl₂ (His-MgCl₂) solution was best for experiments with WTV on cell monolayers. Tumor extracts diluted to 10⁻² in His-MgCl₂ solution were quick-frozen with no detectable loss in infectivity and maintained a constant infectivity at −80° for at least 9 months. The optimal temperature for thawing was about 37°. Optimal inoculation and incubation periods at 30° were dependent upon the WTV-concentration in His-MgCl₂ solution. Optimal periods of inoculation with 8.3 × (10^7.5, 10^7.0, and 10^6.5) virions per milliliter were 1.5, 2, and 3 hr, respectively; optimal incubation periods for the same concentrations were 36, 42, and 54 hr, respectively. A satisfactory assay procedure for inocula containing about 8.3 × 10^7.0 to 8.3 × 10⁶ virions per milliliter consisted of applying 0.01 ml of inoculum per coverslip for 1 hr at 30°; after this an incubation period of 40–60 hr was allowed before focus counts were made.     

A sensitive bioassay system for detecting defective interfering particles of rabies virus

Rabies virus has been known to produce CPE after several days incubation. By increasing the incubation temperature to 39–41° after an initial period at 34–36°, we found a rapid CPE accompanied by a cessation of viral replication. When this procedure was utilized with a mixed infection of infectious and DI rabies virions, the surviving cells infected with DI particles stood out against a background of lysed cells which had been infected with infectious virions alone. With this finding, an interference focus-forming assay of rabies virus DI particles was devised. The critical m.o.i. of helper infectious virus was determined to be 0.2 PFU/cell. When a sample had many infectious virions which interfered with this narrow limit of the optimal m.o.i., that problem was overcome by UV irradiation, which inactivated infectious virions at a much higher rate than DI particles. Numbers of interference focus-forming units (IFU) were in linear proportion to the input dose of DI particles. Under optimal conditions, this temperature increase procedure was sensitive enough to measure as few as 20 IFU/ml, almost equal in sensitivity to the plaque assay.     

Solid-phase immune electron microscopy (SPIEM) by use of protein A and its application for characterization of selected adenovirus serotypes

Staphylococcal protein A was used for the anchoring of specific antibodies for a solid-phase immune electron microscope method (SPIEM). More IgG adsorbed to grids in the absence of protein A than in its presence. However, the virus trapping efficiency was markedly improved in the presence of protein A. The specificity of the test was evaluated by use of different adenovirus preparations and matching rabbit hyperimmune sera. The degree of serological reaction was evaluated by counting the number of particles attached to the grid. Type specific reactions and inter- and intrasubgroup reactions were identified.     

Immune electron microscopy of avian infectious bronchitis virus serotypes.

An immune electron microscopy agglutination technique in which emphasis is placed upon the importance of antigen-antibody equivalence has been developed as a possible method for the serotyping of avian infectious bronchitis viruses. The Connecticut and Massachusetts 41 serotypes were used as a model system. Stock virus concentrations were standardized by physical particle counts of virions sedimented directly onto electron microscope specimen grids. Suspensions containing approximately 150 virions per grid square were allowed to react with dilutions of homologous and heterologous antisera. Virions in these constant virus-variable serum mixtures were sedimented directly onto electron microscope specimen grids, and the relative degree of aggregation per grid was determined from the mean percent aggregation of five randomly selected grid squares. In homologous assays, regions of relative antibody excess, of equivalence, and of relative antigen excess were clearly evident. At equivalence, the mean percent aggregation was significantly higher than in the regions of relative antibody or antigen excess. In the heterologous systems, the degree of aggregation differed little from that of the virus controls containing no antiserum.     

A simplified microwell pseudoreplica for the detection of viruses by electron microscopy and immunoelectron microscopy

Simplified procedures for immunoelectron microscopy (IEM) and electron microscopy (EM) are described. The procedures employ the principle of agar filtration and pseudoreplication. The modification consisted of the use of microwells for storage of gels with or without antiserum (for IEM or EM, respectively) and an array of containers in which pseudoreplication and negative staining were performed. The containers were prepared from 5 ml syringes from which the needle holding parts were cut. This device enabled simultaneous and rapid handling of specimens. With Sindbis virus as a model, our microwell pseudoreplica IEM (MW-PR-IEM) was compared to six other IEM techniques and was found to be the most rapid and sensitive technique. With the MW-PR-IEM technique, the specific minimal detection limit (detection of clumps) was 1.5 x 10(7) virus particles per ml, and the non-specific detection limit (detection of single virions) was 1.8 x 10(6) virus particles per ml.     

One more probable structural transition in potato virus X virions and a revised model of the virus coat protein structure

We found that a 2-h incubation of potato virus X (PVX) virions in 10 mM Tris-HCl buffer pH 7.5 at -20 degrees C results in a strong but reversible drop in virion stability. Under these conditions, the PVX virions are completely disrupted by low (starting from 50 mM) concentrations of LiCl and CaCl(2) but not of NaCl. Incubation of PVX samples with 0.05-2 M LiCl at +4 degrees C did not result in virion disassembly and the virions were not disrupted upon incubation at -20 degrees C in 10 mM Tris-HCl buffer pH 7.5 without LiCl. We suggest that a 2-h incubation of the PVX virions at -20 degrees C in 10 mM Tris-HCl pH 7.5 results in a structural transition in the virus particles. A revised model of the three-dimensional organization of coat protein subunits in the PVX virions is proposed. This two-domain model explains better the high plasticity of the PVX CP structure.     

Factors influencing immune electron microscopy of flexuous potato viruses.

Effects of pH of extraction buffers, pH and titer of trapping antisera and their combinations, virus acquisition time and virus host on the trapping efficiency of flexuous potato viruses X, S and Y (PVX, PVS and PVY) in immune electron microscopy were evaluated. Addition of ethylene-diamine-tetraacetic acid to the extraction buffer improved trapping of PVY, adversely affected PVS but not PVX. Combinations of antisera had differential adverse effect on trapping which was maximum with the mixture of three antisera. Mixture of antisera to PVX and PVY had the least adverse effect on trapping of PVX and PVY as compared to the mixture with PVS antiserum. Trapping of PVX and PVY was good and almost at par at all the dilutions of the antisera while that of PVS was good up to 1000-fold only. Prolonged virus acquisition time significantly increased the number of virions trapped. Trapping was affected both by the pH of the antiserum and the extraction buffer, while in the case of PVY it was also affected by the host species.     

Assessment of specific virus infectivity and virus neutralization by a progeny virus immunotitration method as exemplified in an adenovirus system

An alternative method for the determination of specific virus infectivity and quantitative measurement of inhibitory activity of antibodies was developed using an adenovirus system. HeLa cells in 37 ml suspension cultures of 1.5 X 10(7) cells were infected with purified adenovirus 2 (Ad2) at different multiplicities of infection. After appropriate incubations, total progeny virus was isolated by a one-step CsCl gradient sedimentation procedure. Recovered virions were disrupted in the presence of 5 M urea and directly quantitated by rocket immunoelectrophoresis against an anti-hexon-antiserum. One infectious unit (IU) was defined as the lowest amount of virions capable of producing maximum yield of progeny virus in the standardized system, and corresponded to 32 physical particles, which also equalled one plaque-forming unit (pfu). The coefficient of the inter-experimental variation of the total virus yield determination was 13%. Reduction in progeny virus synthesis was taken as a measurement of the degree of the inhibitory effect of neutralizing antibodies. A linear relationship was obtained between dilution of a neutralizing antiserum and reduction in synthesis of progeny virus. Separate determinations of such neutralization revealed a coefficient of variation of 5.5%.     

Detection of rotavirus by serological trapping on antibody-coated electron microscope grids.

A serological trapping technique for detecting rotaviruses is described which involves coating electron microscope grids with protein A and specific rotavirus antiserum. The presence of a layer of antibodies on the grid increases the number of rotavirus particles that can be visualized. Thirty-five crude fecal extracts from infants suffering from diarrhea were examined by the serological trapping technique and by standard electron-microscopy. When the specimens were deposited on antibody-coated girds, 71% of them were found to contain virus particles, compared with 20% on standard uncoated grids. The method is simple and rapid and does away with the need to concentrate the specimens.     

An evaluation of some factors important for maximising sensitivity of plant virus detection by immuno-electron microscopy

We report the results of a comparison of the variations to the basic technique developed by Derrick and called 'serologically specific electron microscopy' by him. We have found that the reaction time of the antibody-coated grid with the virus preparation (virus acquisition time) is one of the key factors in determining the efficiency of the method at trapping virus particles. The lower the virus concentration, the longer the reaction time should be. The use of protein A to pretreat the grids before coating with antiserum did not appear to be of benefit under our conditions.     

Improved ELISA for the detection of HBsAg

A sandwich ELISA for hepatitis B virus surface antigen (HBsAg) was developed using monoclonal anti-HBs for the solid phase and horse-radish peroxidase labelled sheep anti-HBs.

The sensitivity was approx. 0.3 U/ml HBsAg, in the standard test procedure,comprising two incubation steps of 1 h at 37°C, or in a shortened procedure comprising two incubation steps of 30 min at 50°C. A slightly reduced sensitivity (approx. 0.5 U/ml) was obtained when the two incubations were combined in a one-step incubation for 1 h at 37°C. All three procedures were completed by an incubation for 30 min at room temperature with peroxide and tetra-methylbenzidine.

The number of false positives obtained when donor sera were screened was below 0.5%. False positive reactions occurred more frequently, but still to a limited extent, when previously selected sera containing rheumatoid factor or other possibly interfering factors were tested with the standard procedure. Most sera containing factors that interfere with a commercial ELISA for HBsAg using sheep anti-HBs coated plates, were negative. Rheumatoid factor positive sera seldom gave false positive results.

The lower detection limit became approx. 0.1 U/ml when the cut-off was reduced, while the number of false positives in a donor population only increased to 1.5%. The results obtained with reagents from four different batches indicate a stability of up to 4 wk at 37°C and for at least 26 wk at 4°C.     

Decline in infectivity of simian virus 40 by sodium deoxycholate and its restoration with the extract of monkey kidney cells

Plaque-forming activity and T-antigen-synthesizing activity in the crude preparation of simian virus 40 (SV40) decreased to 1/20-27 after treatment with 0.5% sodium deoxycholate (DOC) for 30 min at 37 degrees C. A full restoration of the activity occurred after incubation of DOC-treated virions with the extract of monkey CV-1 cells, host cells for productive infection with SV40. Analysis by sedimentation through 15% sucrose to CsCl cushion (rho = 1.327 g/cm3) revealed that virions in the [35S]methionine-labeled crude virus preparation sedimented to the interface between CsCl and sucrose, and that treatment with DOC resulted in the loss of infectivity and the appearance of virions sedimentable into CsCl cushion. The [35S]methionine-labeled purified virions (prepared after treatment with DOC and sedimentable into CsCl cushion) sedimented to the CsCl-sucrose interface after incubation with the cell extract, with restoration of infectivity. The infectivity-restoring activity of the cell extract was sensitive to ethyl ether, partially sensitive to heating at 75 degrees-97 degrees for 30 min, but resistant to treatment with DNase (50 micrograms/ml), RNase (40 micrograms/ml), or trypsin (0.05%) for 30 min at 37 degrees. These results suggest that lipid-related cellular components bind stably to virions of SV40 and facilitate an efficient infection.     

Determination of Optimal Conditions for the Immobilization of Cells in a Cell Capture Enzyme Immunoassay (CC-EIA) by a Simple Giemsa Assay

To determine the optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA), the most suitable diluent, and the optimal pH, temperature and period of incubation were examined using WI-38, a human embryonic lung fibroblast cell line. For the evaluation, we devised a simple Giemsa assay method, in which immobilized cells on a microplate were stained with Giemsa solution, the stained dye was eluted with ethanol after washing the plate, and the optimal density (O.D.) was measured at wavelength 620 nm. the optimal conditions for the immobilization were determined to be treatment with 5% formalin in phosphate-buffered saline (PBS) (pH 7.2) for 15 minutes at room temperature, which were confirmed to be suitable for the measurement of cell associated collagen by CC-EIA. Additionally, we found that the simple Giemsa staining method was also useful for evaluating the number of immobilized cells on the microplate after CC-EIA.     

Estimate of absolute specific infectivity of wound tumor virus purified with polyethylene glycol

Purification of wound tumor virus (WTV) was made simpler and quicker and a much greater proportion of active virions were obtained in the purified preparations of this labile virus by precipitating it with polyethylene glycol (PEG) 6000 before using density gradient centrifugation. Adjustment of clarified extracts to pH 5.7 gave the best yield of total (active plus inactive) virus and the highest specific infectivity. An optimal NaCl concentration (0.3 m) was important in obtaining good separation of the virus from host components during subsequent zonal density gradient centrifugation and maximal yields. NaCl at 0.2 m yielded purified virus of somewhat greater specific infectivity. Maximal yields of total virus were obtained with the use of 4% PEG for 2 hr although 3% PEG gave virus of about 1.5 times greater specific infeetivity. A histidine-MgCl₂ buffer solution for resuspending virus precipitated by PEG resulted in at least 50% greater total virus yields than did a glycine-MgCl₂ solution. WTV purified under the optimal conditions determined in this investigation yielded 3–5 times more total virus and at least 50 times more infective virus than did the standard method used previously. The purified virus had an absolute specific infectivity of about 25–30%, that is, it contained about 2 or 3 inactivated virions to each infective one. Purified preparations containing about 3 × 10¹¹ total virions per ml could be quick-frozen and stored at ?80 ° for at least a year without detectable loss of infectivity.     

Effect of pH, ions, bovine serum albumin and heterologous antisera on the stability of immunosorbed flexuous potato viruses

Electron microscopic studies on the stability of immunosorbed (trapped) virions of potato viruses X, S and Y0 (PVX, PVS and PVY0) revealed disintegration and dislodging of PVY0 virions upon incubation with (1) antisera to PVX, PVS, or both diluted in saline, (2) 0.86% NaCl (saline) or 0.1 mol/l CaCl2 but not with 0.1 mol/l CaSO4 or 0.1 mol/l MgSO4. PVX virions, on the other hand, showed partial dislodging upon incubation with an antiserum to PVS diluted in saline, but complete disintegration and dislodging with saline. 0.1 mol/l CaCl2 caused partial dislodging while MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) had no apparent adverse effect. PVS virions were not affected by saline, CaCl2, MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) and were only partially dislodged by antisera to PVX or PVY0. Disintegration and/or dislodging of the PVX and PVY0 virions was prevented when (1) they were fixed with glutaraldehyde prior to incubation or (2) the virus extract contained bovine serum albumin (BSA) or (3) heterologous antisera were diluted in 0.1 mol/l phosphate buffer (PB) before use except the PVS antiserum which still caused disintegration and dislodging of PVY0 virions. Prior fixation of virions prevented their disruption and dislodging by saline only in the case of PVY0 but not PVX. On the other hand, BSA reverted the adverse effect of saline but not that of the PVS antiserum on PVY0 virions. The results presented here suggest (1) a disruptive effect of Cl' on PVX and PVY0 virions particularly when it was associated with Na+ and (2) an interaction between the immunosorbed virions of PVX or PVY0 and the antiserum to PVS.     

On the study of Sendai virus hemolysis. II. Morphological study of envelop fusion and hemolysis.

Interaction of human erythrocytes was studied morphologically with intact nonhemolytic Sendai virus and an artificially induced hemolytic one which was obtained by freezing and thawing of the former virus. Fusion of the viral envelope with the erythrocyte membrane was observed with both viruses. In the nonhemolytic preparation, virions were uniformly spherical in shape and completely devoid of uranyl acetate in negative staining. During the whole period of the envelope-membrane fusion, the integrated viral envelope appeared to be intact, and the release of hemoglobin into the extracellular space was never observed even after the incubation period of 1 hr at 36°. On the contrary, in the hemolytic preparation, virions had damages on their envelopes so that they were permeable to uranyl acetate stain. The release of hemoglobin occurred immediately after formation of the envelope-membrane fusion and was complete by the incubation period of 10 min at 36°. The intact nonhemolytic virion also could become hemolytic after treatment with the antiviral antibody and complement. The results indicate that Sendai virus hemolysis is caused exclusively by virions having injured envelopes but not by virions having intact envelopes. The study also revealed that the envelope-membrane fusion was followed by dispersal of the viral envelope antigens on the erythrocyte membrane, migration of the nucleocapsids into cytoplasm, and cell-cell fusion of the erythrocytes.     

Preparation and selection of monoclonal antibodies for detecting hepatitis B surface antigen in blood sera

HV monoclonal antibodies (MAb) were produced in order to improve the quality of HBsAg detection and their specific characteristics were compared with those of other MAbs. MAbs were characterized by asymmetric interactions with the antigen when used as first or second antibodies. The reactivity of a panel of HV and X MAb to ad and ay subtypes was studied by enzyme immunoassay. Mutual blocking (epitope mapping) of MAb helped select antibody couples for the creation of highly effective test system for the diagnosis of the major HBsAg subtypes. The sensitivity and specificity of MAbs were evaluated on reference and control panels of HBsAg sera and on serum specimens from a random sampling of 300 blood donors. The sensitivity of the most specific MAb pairs was 0.1 ng/ml for HBsAg subtype ay and 0.25 ng/ml for subtype ad. The specificity of attested MAb was 98.5% in incubation with stirring and 97% in static incubation. The optimal combinations of attested MAbs were used in the manufacture of Recomnathep B test system in the sandwich format.     

Evidence for the secretion of soluble peptidoglycans by clinical isolates of Staphylococcus aureus.

Four isolates of Staphylococcus aureus from patients with endocarditis and bacteremia were capable of secreting high-molecular-weight soluble peptidoglycans when grown in a minimal cell wall medium containing penicillin G. Vancomycin was not able to substitute for penicillin G in triggering this secretion. Secretion reflected de novo synthesis of soluble peptidoglycan and was strongly dependent on time of incubation (30 to 60 min), and number of bacteria (2 X 10(8) to 5 X 10(8) colony-forming units per ml), but not on penicillin G concentration (10 to 250 micrograms/ml). The incorporation of alanine into the peptidoglycans secreted in vitro by these isolates incubated in the presence of penicillin G under optimal conditions was variable. The least incorporation of alanine into peptidoglycan occurred with an isolate from a patient treated with nafcillin who had no detectable antipeptidoglycan titer.     

A possible baculovirus in the insect-parasitic fungus, Strongwellsea magna

Particles similar in appearance to the virions of the invertebrate baculoviruses have been found in large numbers in hyphae of the insect-parasitic fungus, Strongwellsea magna, growing in the fly, Fannia canicularis. The particles (100 X 390 nm) consisted to a densely staining core (50 X 340 nm) within a poorly staining envelope. The envelope of some of the particles was contiguous with vesicles derived from unit membranes originating at the hyphal plasma membrane. The structure and aspects of the apparent morphogenesis of these particles suggest they may belong to a virus of the genus Baculovirus.