Solid-phase immune electron microscopy (SPIEM) for rapid viral diagnosis.

Immune electron microscopy (IEM) is one of the fastest and most sensitive methods for the detection and diagnosis of viruses. This technique is based on formation of immune complexes of the virus with its corresponding antibody. In IEM optimal precipitation depends on a correct ratio, and there is a prozone effect. These problems can be overcome by using the solid-phase immune electron microscopic (SPIEM) technique. In this technique the antibody is attached to a particle which is used for 'fishing' the virus to be examined out of the suspension. After low speed centrifugation the preparation is treated either for observation in the transmission electron microscope or in the scanning electron microscope. In 'positive' samples the virus is seen attached to the surface of the particle. We report here results with S. aureus as the solid phase for the detection of Sindbis virus. The anti-Sindbis gamma globulins are attached to the bacteria by means of protein A present on their surface.

Evaluation of immunoadsorbent electron microscopic techniques for detection of Sindbis virus.

Two immunosorbent electron microscopic techniques (ISEM), the protein A coated grid technique (PA-CGT) and the antibody coated grid technique (AB-CGT) were applied and evaluated for the detection of Sindbis virus from infected tissue culture fluids. At optimal conditions, the efficiency of trapping the virions was only about 1.5 higher with the PA-CGT as compared to the AB-CGT, but the PA-CGT was less dependent on the antiserum dilution used in the test. Both methods were suitable for quantitation experiments, since the number of virions trapped was proportional to the virus concentration. The influence of virus incubation time and temperatures, staining solutions, buffers and washing procedures on the trapping efficiency and specificity was further studied with the PA-CGT. Maximal trapping on coated grids was obtained after 3 h incubation of the virus. At room temperature, less debris was found on the grids, as compared to 37 degrees C, and the numbers of virions counted were only slightly lower. The optimal staining solution was alcohol uranyl acetate. The specificity of the PA-CGT was dependent on washing steps with phosphate buffered saline containing bovine serum albumin. With the standard procedure, at room temperature around 3 X 10(7) virions/ml (1 X 10(6) PFU/ml) were specifically detected in about 1.5 h.

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.

Differentiation and myelination of transplanted mouse dorsal root ganglia.

Dorsal root ganglia from newborn mice develop and myelinate following transplantation under the kidney capsule of adult syngeneic mice. The transplanted dorsal root ganglia in the subcapsular environment developed more rapidly and formed more abundant myelin than comparable ganglia in an in vitro culture.

Silicates: non-specific adsorbents in purification of water from viruses.

Amorphous silicates having small particles with large surface area, were found to have high non-specific adsorption capacity. Escherichia coli T4 bacteriophage was used as a model in this research. Investigations have shown an adsorption capacity of up to 99% on different tested commercially available silicates. Optimal conditions for the adsorption of T4 bacteriophage on various silicates were determined. Amorphous silicates may be used in purification of water from viral contaminants. A silicate-containing model unit for filtration by adsorption of viruses is described.

Silicates as nonspecific adsorbents of bacteriophage: a model for purification of water from viruses.

Amorphous silicates having small particles with a large surface area were found to have high nonspecific adsorption capacity. Investigations with Escherichia coli T4 bacteriophage have shown adsorption capacity of up to 99.9% on different tested silicates. Optimal conditions for the adsorption of T4 bacteriophage on various commercially available silicates were determined. A continuous-flow adsorption of T4 bacteriophage on silicates appears to be a model system for the removal of viruses from potable water and sewage.

Etiology of acute gastroenteritis in children in Israel: role of human reoviruslike agent and bacterial pathogens.

A study of acute gastroenteritis in children was carried out with the aim of establishing the prevalence of human reoviruslike agent (HRLA) and its relation to other enteric pathogens in Israel. The stools of 384 children with acute diarrhea referred to a pediatric emergency service were screened for HRLA by counterimmunoelectroosmorphoresis (CIEOP) and for pathogenic bacteria. Evidence of HRLA infection was found in 65 patients (17%). The highest infection rate prevailed during the cool season (25%), with a peak prevalence (41%) in November, when both the temperature and humidity were low. A very high proportion of HRLA was found in children younger than 36 months and no HRLA infection was observed in those older than nine years. The highest prevalence occurred in infants younger than six months, a situation rarely encountered in other countries. The main clinical features of HRLA infection were fever, vomiting, dehydration, signs of upper respiratory infection and carbohydrate intolerance. Bacterial pathogens accounted for 45% of enteric infections. Shigella species predominated (28%) during the summer season, especially in older children. In 38% of the study group, no etiologic agent could be detected. None of the 50 control subjects showed evidence of viral or bacterial pathogens in stools.