Isolation of Ether-Resistant Enteroviruses from Sewage: Methodology

Experiments were conducted to determine whether polio type 1 (Mahoney and coxsackie A8 viruses adsorb onto cotton fibers of sewer swabs. Negative results were obtained. It has been shown that viruses may exist in sewage as free virus particles or as bound (adsorbed) virus particles. The sewer-swab method of sampling is superior because it filters out the bound virus over several days; when collected, it represents a catch (grab) sample at that particular time which may or may not contain free virus. A simple method for the preparation of sewage inocula for virus isolations is described which samples the bound virus fraction. Only ether-resistant viruses can be isolated, and an ultracentrifuge is not required. By this method, an isolation rate between 60 and 80% of positive sewer swabs can be achieved. Corresponding figures of 84 and 96% were achieved by concentration of sewer-swab eluates with an ultracentrifuge. Quantitative studies showed that the virus concentration in raw sewage can be as high as one infectious particle per 0.5 ml.     

Concentration of Enteroviruses on Membrane Filters

Enteroviruses can be made to adsorb or to pass through membrane filters by manipulation of the suspending medium. Salts facilitate virus adsorption, but membrane-coating components (MCC) interfere. Because cells release MCC into the culture medium during viral growth, MCC must be removed before virus can be adsorbed to membranes. Adsorbed virus can be eluted with diluents containing MCC (cell extracts or serum) or agents that reduce surface tension (sodium lauryl sulfate). By membrane adsorption and elution, enteroviruses can be readily concentrated and quantitatively recovered from crude virus harvests.     

Evaluation of Filters for Removal of Bacteriophages from Air

Glass wool, nonabsorbent cotton, fiberglass filter medium, and a commercial absolute filter were tested for effectiveness in removing aerosolized bacterial viruses under low flow rate (1 ft(3)/min) and high flow rate (10 to 25 ft(3)/min) air-flow conditions. Special equipment was designed for measurement of filter efficiencies under the two air-flow conditions. Under low air-flow rate test conditions, glass wool was only 98.543 to 99.83% efficient, whereas cotton (five layers), fiberglass medium (three layers), and the commercial absolute filter were at least 99.900, 99.999, and 99.999 efficient, respectively. Glass wool and cotton were not used under higher air-flow conditions because they were difficult to assemble in leak-tight filters. The commercial absolute filter and fiberglass medium (three layers) were at least 99.990 and 99.999% efficient, respectively, under the higher air flow conditions. A stainless-steel filter of simple design and fitted with three layers of fiberglass medium was found to be greater than 99.999% efficient in removing high concentrations (20,000 to 70,000 plaque-forming units per cubic foot) of aerosolized bacteriophages from air moving at a low flow rate (1 ft(3)/min). Use of this filter on pressure-vacuum tanks in the fermentation industry is suggested. Several other uses of such a filter are proposed.     

Prevalence of Nonpolio Enteroviruses in the Sewage of Guangzhou City,China, from 2009 to 2012

The human-pathogenic viruses in urban sewage have been extensively monitored to obtain information on circulating viruses in human communities. Enteroviruses (EVs) excreted by patients who present with diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 4-year (2009 to 2012) surveillance study was conducted to detect nonpolio enteroviruses (NPEVs) in the urban sewage of Guangzhou city, China. After the viruses in the sewage samples were concentrated and isolated, molecular identification was used to detect and type the NPEVs. During the 4-year study, 17 different NPEV serotypes were identified in the sewage of Guangzhou city. The most common serotypes were echovirus 11 (ECHO11), ECHO6, ECHO7, and ECHO12 and coxsackie group B viruses 5 (CVB5) and CVB3. The predominant serotypes were influenced by spatial and temporal factors and differed each year. CVB5 was commonly detected in 2009 and 2010 but was rarely isolated in 2011 and 2012. In contrast, CVB3 was not observed in 2009 and 2010 but was increasingly detected in 2011 and 2012. Our study provides an overview of the serotype distribution and circulation patterns of NPEVs in the sewage of Guangzhou, China. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.     

Removal of Nonferrous Heavy Metals from Industrial Sewage Using Biosorbents

Producers of the antibiotics neomycin and lincomycin were most potent in sorbing nonferrous metals; they removed 99% Zn and Cd and 95% Ni. The degree of metal sorption increased with an increase in solution pH and calcium content in the biomass. Dynamic studies of biosorbents prepared from a microbial biomass showed that neomycin industrial waste products are highly efficient in treating galvanic washing water. The total dynamic exchange capacities by nickel and cadmium were 6.85 and 7.16 mg/ml, respectively.     

Filtration Methods for Recovering Enteroviruses from Foods

A set of simple, rapid filtration methods has been developed for recovering enteroviruses from foods. The methods employ quite inexpensive equipment, and the resulting extract might be concentrated in any of several ways. Three model enteroviruses were apparently recovered with at least 80% efficiency when inoculated, at levels ranging from thousands to one plaque-forming unit per 20-g sample, into eleven foods representing six major food groups.     

毛蚶中肠道病毒检测方法的建立

本文建立了检测毛蚶中肠道病毒的方法。病毒在pH4.5时吸附于毛蚶组织,离心后用pH9.5甘氨酸盐水将病毒从毛蚶组织中洗脱出来,并用0.0005mol/L三氯化铝代替Johnson法中的Cat—Floc,达到去除细胞毒性物质的目的。经过再一次离心,利用牛肉浸膏在pH3.5—4.0时凝聚并吸附病毒的特性使洗脱液中的病毒得到浓缩。离心后沉淀溶解于0.1mol/L磷酸氢二钠溶液。用本法提取毛蚶中脊髓灰质炎病毒,当病毒含量为每克组织3.5×10~5至35PFU时,平均回收率为71.2％。     

Development of a Simple Method for Concentrating Enteroviruses from Oysters

The development of a simple method for concentrating enteroviruses from oysters is described. In this method viruses in homogenized oyster tissues are efficiently absorbed to oyster solids at pH 5.5 and low salt concentration. After low-speed centrifugation, the supernatant is discarded and viruses are eluted from the sedimented oyster solids by resuspending them in pH 3.5 glycine-buffered saline. The solids are then removed by low-speed centrifugation, and the virus-containing supernatant is filtered through a 0.2-micronm porosity filter to remove bacteria and other small particulates without removing viruses. The virus-containing filtrate is then concentrated to a volume of a few milliliters by ultrafiltration, and the concentrate obtained is inoculated directly into cell cultures for virus assay. When tested with pools of oysters experimentally contaminated with small amounts of different enteroviruses, virus recovery efficiency averaged 63%.     

Environmental Factors Influencing Isolation of Enteroviruses from Polluted Surface Waters

The influence of water quality upon the concentration of virus on location was assessed in field studies conducted in the Houston ship channel, Galveston Bay, and Houston waste treatment plants. Clarification of polluted surface waters was accomplished with minimal loss of virus. Virus from clarified sewage effluents and saline waters was then adsorbed and concentrated on textile and membrane filter surfaces. Direct measurements of virus from large volumes of polluted surface waters under existing field conditions were then made using the virus concentrator equipment.     

Degradation of Linseed Oil Vapors by Soil Bacteria in Trickling Biofilters

Oxidation products of linseed oil were produced by impinging a stream of air onto the surface of pure linseed oil and injecting the vapor-laden air into soil percolation columns to enrich the population of bacteria capable of degrading linseed oil vapors. As the populations of bacteria increased, the linseed oil vapors were consumed by these organisms, and the air that emerged from the columns was free of linseed oil contaminants. Five different kinds of bacteria capable of growing on the linseed oil oxidation products as sole source of carbon and energy were found and isolated in pure culture. Chromatographic analyses showed that individual organisms removed specific components of the vapor at specific rates, but none was able to remove them all within a 30-day period of time. When the five were grown together and presented the linseed oil vapor, all vapor constituents were utilized, and the rate of utilization was greater than that seen when the isolates were tested in pure culture. This indicated that the five organisms operated as a bacterial consortium in the degradation of linseed oil vapors. Trickling biofilters prepared from pregrown populations of the five organisms challenged with linseed oil vapors were able to remove all volatile constituents found in linseed oil vapor. Bioremediation of the air was complete and it was accomplished in a single pass of the air through the filter.

This work shows that bacteria found in the soil are capable of degrading linseed oil vapors and that they can be grown in the laboratory and used successfully in bench scale trickling biofilters.     

Estuaries as Filters: The Role of Tidal Marshes in Trace Metal Removal

Flux calculations demonstrate that many estuaries are natural filters for trace metals. Yet, the underlying processes are poorly investigated. In the present study, it was hypothesized that intertidal marshes contribute significantly to the contaminant filter function of estuaries. Trace metal concentrations and sediment characteristics were measured along a transect from the subtidal, over an intertidal flat and marsh to a restored marsh with controlled reduced tide. Metal concentrations in the intertidal and restored marsh were found to be a factor two to five higher than values in the subtidal and intertidal flat sediments. High metal concentrations and high accretion rates indicate a high metal accumulation capacity of the intertidal marshes. Overbank sedimentation in the tidal marshes of the entire estuary was calculated to remove 25% to 50% of the riverine metal influx, even though marshes comprise less than 8% of the total surface of the estuary. In addition, the large-scale implementation of planned tidal marsh restoration projects was estimated to almost double the trace metal storage capacity of the present natural tidal marshes in the estuary.     

Ecology of Enteroviruses in Tropics

A continuing surveillance on enterovirus infection in healthy infants was conducted from October 1971 through February 1973 in urban areas in Ghana, West Africa. About 40 infants were visited in every two months for collection of faecal specimens and examined for infection. Enteroviruses were recovered in tissue culture. The overall isolation rate of enteroviruses was approximately 44%, and there was no seasonal difference between rainy and dry seasons. The rate of virus isolation in urban areas was significantly higher than in rural areas. Within the urban areas, however, no difference in the rate of virus isolation was detected between densely populated and sparsely populated areas. The results of virus identification revealed that all three types of Poliovirus, many types of Echovirus and a few Coxsackieviruses were isolated during the course of the study. It was observed that improvement of sanitary facilities decreased the frequency of virus infection among infants, but the condition of water supply did not influence the virus infection rate. Neither the age of infants nor the size of siblings showed any relation to the virus isolation rate. It was suggested from the results that many types of enteroviruses have been circulating continuously in the tropical urban community throughout the year.     

Comparative Inactivation of Enteroviruses and Adenovirus 2 by UV Light

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm², respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.     

Polyelectrolyte Flocculation as an Aid to Recovery of Enteroviruses from Oysters

A simple and rapid method for recovering enteroviruses from oysters is described. A polycation sewage flocculant promoted cohesion of oyster solids and thereby aided separation of these from the viruses. Recovery of 80 to 100% of experimentally inoculated virus was achieved, and the suspension or extract obtained could be inoculated directly into tissue cultures or concentrated first for greater sensitivity.     

Bovine Enteroviruses as Indicators of Fecal Contamination

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.