William H. Benton

Detection of viruses in water: A review of methods and application☆

Abstract One of the major problems facing environmental health officials in regard to water quality is related principally to the unavailability of reliable and standard methods to concentrate, detect, and isolate low-multiplicities of virus from very large volumes of water. The critical examination of all water supplies for the presence of viruses (including waters used for drinking, recreation, and food production) requires a quantitative approach. In order to be quantitative, measurable quantities of water must be examined. This is the only way in which a definitive assessment can be made as to the distribution and extent of virus contamination of our water resources. The challenge to the virologist is related to the need for developing new and/or improved techniques in the laboratory that have a high likelihood for adaptation to the real world situation. In this regard, a number of techniques have been shown experimentally to be good candidates for assessing the occurrence of viruses in various types of water. The most promising methods are: (i) membrane-adsorption technique; (ii) adsorption to precipitable salts, iron oxide, and polyelectrolytes; (iii) aqueous polymer two-phase separation technique; and (iv) soluble alginate filter technique. Most of these methods have shown good-to-excellent virus recovery efficiencies as well as a reasonable efficacy for concentrating viruses from water in controlled laboratory experiments. Other methods such as (i) continuous-flow ultracentrifugation; (ii) forced-flow electrophoresis and electro-osmosis, and (iii) hydroextraction have also shown favorable virus recovery efficiencies under laboratory-controlled conditions but fall short as candidate techniques for real world virus-in-water problems. From the data, it would appear that the most promising methods for detecting and isolating low-multiplicities of virus in clean and finished waters are those that rely on virus adsorption and/or retention coupled with a flow-through sampling system. For waters that are moderately or grossly turbid, it would appear that aqueous polymer two-phase separation may be the better approach. In this review paper, the above methods are briefly described in terms of mechanisms, procedure and efficiency. The methods are evaluated in terms of speed, simplicity, and economy of application.

Comparison of cytopathogenicity, immunofluorescence and in situ DNA hybridization as methods for the detection of adenoviruses

Abstract Three different methods were compared for their efficiency at detection of adenoviruses. The samples examined for viral analysis consisted of concentrates prepared from raw sewage, chosen as providing a representation of the spectrum of viruses being intestinally shed from a large population at any given time. When using one single cell line, HEp-2, the overall numbers of adenoviruses detected using cytopathogenicity and immunofluorescence were roughly equal. In situ hybridization was approx. 40% more sensitive than either of these other methods as determined by average virus titers for the different samples, and also proved to be better by means of a nonparametric comparison. The 293 cell line was approx. 5 times more sensitive for detecting adenoviruses by cytopathogenicity as compared with the HEp-2 cell line, but proved unsuitable in our hands for quantitatively detecting indigenous adenoviruses by immunofluorescence. The relative number of indigenous adenoviruses present in the sewage concentrates we examined was, on average, 94-fold greater than that of enteroviruses. Assay of enteroviruses was performed by plaque assay in the BGM cell line.

The loss of poliovirus 1 infectivity in marine waters

Abstract The loss of infectivity (LOI) of poliovirus 1 in marine water from the Gulf of Mexico was studied. Typically, three logs of infectivity were lost in 5–6 days at 24°C. Experiments described in this report suggested that this LOI was not a result of container adsorption or virion aggregation: nor was a resistant component within the stock virus found that would have explained the two-component curves often observed with the virus loss. Viral infectivity loss occurred in raw, filter-sterilized, and autoclaved marine water. Loss was also observed when the virus was suspended in artificial seawater of 1, 10 and 20 g kg −1 salinity. No explanation for the LOI other than true inactivation of the virion was found. The specific component(s) of marine water responsible for virion inactivation remains to be ascertained.

Detecting Viruses in Water

Various and divergent approaches that have been used to concentrate and assay viruses from tap water and environmental freshwaters are summarized and briefly explained. The basic principles behind the different methodologies and descriptions of the most recent developments are emphasized. Comparisons help demonstrate the relative sensitivities of different concentration and assay techniques.

Suppression of viral replication by guanidine: a comparison of human adenoviruses and enteroviruses

A comparison was made between the relative sensitivities of laboratory strain human adenoviruses and enteroviruses, and recently isolated human enteroviruses, to the presence of guanidine hydrochloride in cell culture media. The concentration of guanidine hydrochloride used was 100 micrograms per ml. Representatives of all six human Adenovirus subgenera were unaffected in their replication at this concentration of guanidine. The different human Enterovirus types examined varied in their sensitivity, with suppression ranging from less than 1 to 3 log10 units for laboratory strains, and from 2 to 7 log10 units for recently isolated viruses. The findings suggest a novel role for antiviral drugs; serving as an adjunct in facilitating selective isolation of specific virus groups which may be present as part of mixed viral populations.

Sequential inoculation as an adjunct in enteric virus plaque enumeration

Abstract The potential utility of sequentially inoculating a virus sample onto two different cultures of similar vs dissimilar cell lines was evaluated in conjunction with IDU (5-iodo-2′-deoxyuridine) treatment of the cells as a potential adjunct in viral plaque formation assays. This evaluation was done using laboratory grown human echovirus 7, human enterovirus 69 and human poliovirus 1, plus an environmental concentrate derived from sewage that contained indigenous untyped enteroviruses. The cell lines employed were BGM, RD, L-132 and HEL-299. Sequential inoculation generally yielded higher viral assay titers when compared with the more traditional method of simply introducing viral inoculum onto a culture of the first (initial) cell line and then completing the assay without removing that inoculum. When a permissive cell line (BGM or RD) was used for both the initial and final cultures in a sequential inoculation technique, the total plaque count titer from both the initial plus final cultures represented an average 35% improvement over the traditional method.