Method for detecting viruses in aerosols

A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 micron; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AlCl3 to 0.0005 M, collecting the virus on a 0.25-micron-pore Filerite disk (diameter, 25 mm) and and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 X 10(8) PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 X 10(7) to 4.5 X 10(7) PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools.     

Improved methods for detecting enteric viruses in oysters.

New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of less than or equal to 2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.     

Improved methods for detecting enteric viruses in oysters.

New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of less than or equal to 2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.     

Multilaboratory evaluation of methods for detecting enteric viruses in soils.

Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated "Berg" and "Goyal," was based on results of an initial screening which indicated that both met basic criteria considered essential by the task group. Both methods utilized beef extract solutions to achieve desorption and recovery of viruses from representative soils: a fine sand soil, an organic muck soil, a sandy loam soil, and a clay loam soil. One of the two methods, Goyal, also used a secondary concentration of resulting soil eluants via low-pH organic flocculation to achieve a smaller final assay volume. Evaluation of the two methods was simultaneously performed in replicate by nine different laboratories. Each of the produced samples was divided into portions, and these were respectively subjected to quantitative viral plaque assay by both the individual, termed independent, laboratory which had done the soil processing and a single common reference laboratory, using a single cell line and passage level. The Berg method seemed to produce slightly higher virus recovery values; however, the differences in virus assay titers for samples produced by the two methods were not statistically significant (P less than or equal to 0.05) for any one of the four soils. Despite this lack of a method effect, there was a statistically significant laboratory effect exhibited by assay titers from the independent versus reference laboratories for two of the soils, sandy loam and clay loam.     

Multilaboratory evaluation of methods for detecting enteric viruses in soils.

Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated "Berg" and "Goyal," was based on results of an initial screening which indicated that both met basic criteria considered essential by the task group. Both methods utilized beef extract solutions to achieve desorption and recovery of viruses from representative soils: a fine sand soil, an organic muck soil, a sandy loam soil, and a clay loam soil. One of the two methods, Goyal, also used a secondary concentration of resulting soil eluants via low-pH organic flocculation to achieve a smaller final assay volume. Evaluation of the two methods was simultaneously performed in replicate by nine different laboratories. Each of the produced samples was divided into portions, and these were respectively subjected to quantitative viral plaque assay by both the individual, termed independent, laboratory which had done the soil processing and a single common reference laboratory, using a single cell line and passage level. The Berg method seemed to produce slightly higher virus recovery values; however, the differences in virus assay titers for samples produced by the two methods were not statistically significant (P less than or equal to 0.05) for any one of the four soils. Despite this lack of a method effect, there was a statistically significant laboratory effect exhibited by assay titers from the independent versus reference laboratories for two of the soils, sandy loam and clay loam.     

Sampling strategy for detecting viruses in a sewage treatment plant.

A study of pollutant flows was carried out at a wastewater treatment plant in Nancy, France, which used activated-sludge treatment. To carry out observation of hourly flow variation, a sampling strategy needs to be defined. A comparison between two methods of sampling was conducted: dip samples every 2 h over a period of 24 h and one 24-h composite sample were taken from raw and treated wastewater and then analyzed for enteroviruses, fecal coliforms, chemical oxygen demand, biochemical oxygen demand, and suspended solids. The results showed that the hourly variations of these pollutants in the effluents are in good agreement with expectations based upon the customers' usage and the characteristics of the wastewater network. Significant correlations were found between all tested parameters and enteroviruses in raw wastewater. After biological treatment, no correlation remained in treated wastewater between viruses and other parameters. As for the two sampling methods, a rather good representation of the daily load was given by the composite mode of sampling as concerns physicochemical and microbiological parameters. Biological treatment removed an average of 83% of viruses.     

Improved methods for detecting enteric viruses in oysters

[This corrects the article on p. 127 in vol. 36.].     

Sampling strategy for detecting viruses in a sewage treatment plant

[This corrects the article on p. 1769 in vol. 45.].     

Mechanisms by which ambient humidity may affect viruses in aerosols

Many airborne viruses have been shown to be sensitive to ambient humidity, yet the mechanisms responsible for this phenomenon remain elusive. We review multiple hypotheses, including water activity, surface inactivation, and salt toxicity, that may account for the association between humidity and viability of viruses in aerosols. We assess the evidence and limitations for each hypothesis based on findings from virology, aerosol science, chemistry, and physics. In addition, we hypothesize that changes in pH within the aerosol that are induced by evaporation may trigger conformational changes of the surface glycoproteins of enveloped viruses and subsequently compromise their infectivity. This hypothesis may explain the differing responses of enveloped viruses to humidity. The precise mechanisms underlying the relationship remain largely unverified, and attaining a complete understanding of them will require an interdisciplinary approach.     

Method for recovering viruses from river water solids.

Small numbers of virions (poliovirus 1) that had been adsorbed to river water solids were eluted by mixing the solids for 30 min with a 10% solution of beef extract that contained sufficient Na2HPO4 to bring the molarity of the salt to 0.05 and sufficient citric acid to bring the pH to 7. The virions were recovered by inoculating the beef extract onto cell cultures. With this method, 39 to 63% of the poliovirions that had been adsorbed onto the river water solids were recovered.     

Method for improving the detection of viruses in fecal samples.

A method is described that improved the detection of viruses in fecal samples by electron microscopy. The virus particles were concentrated, and much of the background debris was removed by adsorption of viruses on meat protein added to the fecal sample at a low pH and a low salt concentration. Viruses were eluted by raising the pH and the salt concentration. Further concentration was achieved by acid precipitation and vacuum dialysis.     

A comparison of serological techniques for detecting and identifying honeybee viruses

The sensitivity and specificity of conventional Ouchterlony gel-diffusion, immuno-osmoelectrophoresis (IO), immune serum electron microscopy (ISEM), “decoration,” radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) tests for detecting black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), and sacbrood virus (SBV) particles in extracts of diseased honeybees were compared. A “slow” ISEM method detected virus particles in extracts of individuals or groups of individuals diluted to 10^?3 and 10^?4, respectively, whereas the IO method and a “fast” ISEM method using protein A were one-tenth as sensitive, and Ouchterlony gel-diffusion tests were only one-thousandth as sensitive. Using the antibody “decoration” technique, mixtures of serologically unrelated virus particles could be resolved. RIA and ELISA were found to be one thousand times more sensitive than ISEM in detecting the particles of BQCV, CBPV, KBV, and SBV; however, nonspecific reactions occurred when using RIA with very dilute particle suspensions, and this made dilution endpoints difficult to assess, but this did not occur when using the ELISA method. There was little difference in the effectiveness of rabbit or hen antisera in the tests, except when protein A was used as it does not combine with hen antibodies.     

Animal viruses, coliphages, and bacteria in aerosols and wastewater at a spray irrigation site.

Aerosol samples collected at the Muskegon County Wastewater Management System Number 1 spray irrigation site in Michigan by using the Army prototype XM2 Biological Sampler/Collector were examined for the presence of animal viruses, coliphages, and bacteria. Air samples, collected in Earle lactalbumen hydrolysate, and wastewater samples were filtered through a 0.45- and 1.2-micron membrane filter sandwich, pretreated with 10% beef extract (pH 7.0), and assayed for animal viruses by the plaque method on Buffalo green monkey kidney cells. Untreated air and wastewater samples were assayed for coliphages by the soft agar overlay method with three Escherichia coli hosts (ATCC 13706, 15597, and 11303) and for bacteria by the heterotrophic plate count method. Filtered air samples were assayed for coliphages by the most-probable-number method with the same three hosts. Although no animal viruses were detected in the aerosol samples, coliphages and bacteria were recovered. E. coli ATCC 13706 coliphage were recovered more often and in greater numbers than either of the other two types of coliphages. Concentrations of animal viruses, coliphages, and bacteria detected in the raw influent decreased as the wastewater was aerated and stored in the lagoons. No animal viruses were detected in the wastewater at the pump station just before distribution to the spray irrigation rigs. The most-probable-number method was more sensitive and consistent than the overlay procedure in detecting low levels of coliphages in air samples.     

Method for detecting small numbers of Vibrio cholerae in very polluted substrates.

A method is presented for the indirect detection of Vibrio cholerae by the multiplication of two specific bacteriophages: phiH74/64 for El-Tor vibrios, and phage group IV (Mukerjee) for classical vibrios. The product to be examined is seeded in alkaline tryptone water for enrichment, as in the classical method, and is then incubated for 6 h at 37 C. Thereafter, a loopful is transferred to each of two nutrient broth (pH 9) tubes. One of these receives a drop of phage phiH74/64; the other receives a drop of phage group IV. The stock phages are diluted so as to contain about 3,800 plaque-forming units in one drop; this is the maximum amount which, when added to 10 ml of broth, will not be detected in a loopful of 1 mm diameter. The tubes containing phage phiH74/64 are incubated at 42 C; those with phage group IV are incubated at 37 C. After 18 h the cultures are killed by agitation with chloroform, and a 1-mm loopful is deposited on a layer seeded with the detector strains: Makassar 757 for El-Tor phage and V. cholerae 154 for classical cholera phage. After 4 to 5 h at 37 C, lysis appears on the spot areas if there has been phage multiplication in the respective broth tubes. With experimentally contaminated sewage water, vegetables, or stools, 1 to 10 cholera vibrios were detected in every sample. In rare cases, false-positive results were obtained by multiplication of the phage on non-cholera vibrios.     

Therapeutic aerosols in children.

The use and variety of drugs administered to children as inhaled aerosols is increasing, but little is known about how much drug reaches the lung and how it is distributed there in different age groups. In this article the reasons for measuring aerosol deposition in children are discussed and the potential methods for doing this described. Of the methods available, only the use of radiolabelled aerosols gives accurate information on total lung deposition and distribution. The potential risk of the radiation exposure required for these measurements varies with the age of the child but seems to be small. Properly designed studies are expected to clarify the factors affecting lung deposition in children and identify methods of inhalation associated with efficient and predictable delivery of the drug. Measurements of radioaerosol deposition may therefore be justified in children when this information is expected to lead to improvements in the effectiveness or safety of their treatment.