Effect of sludge type on poliovirus association with and recovery from sludge solids

Sludge type was found to affect the degree of association between seeded poliovirus type 1 (LSc) and sludge solids. The mean percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested sludges was 57.2, 70.4, and 94.7, respectively. The degree of association between poliovirus and sludge solids was significantly greater for aerobically digested sludges than for the other two sludge types. Sludge solids associated viruses were eluted using 0.05 M glycine buffer, pH 10.5-11.0, and subsequently concentrated by organic flocculation. The effectiveness of the glycine method in the recovery of solids-associated viruses was also found to be affected by sludge type. Significantly lower mean poliovirus recovery was found for aerobically digested sludges (14.5%) than for mixed liquors or anaerobically digested sludges (72.3 and 60.2%, respectively). The eluent used in the method was not as effective in dissociating the virus from aerobic sludge solids as it was for the other two sludge types. All other virus adsorption-elution steps of the method (i.e., virus concentration steps) were equally effective in poliovirus recovery for all three sludge types. It is suggested that future methods developed for the recovery of viruses from sludges be evaluated for the various sludge types likely to be tested.     

Association of Enteroviruses with Natural and Artificially Introduced Colloidal Solids in Water and Infectivity of Solids-Associated Virions

Encephalomyocarditis viruses adsorb to introducted organic and inorganic solids in water over a wide range of pH and with various concentrations and species of metal cations. Visible flocculation of solids was not a prerequisite for significant virus association. Virus adsorption to natural solids in various types of natural waters was significant but variable. Clay-adsorbed virus retained its infectivity in tissue culture monolayers. These solids-associated viruses also retained infectivity in mice.     

Demonstration of solids-associated virus in wastewater and sludge.

Data presented demonstrate the relatively high multiplicity of solids-associated virus in field samples, i.e., wastewater, sludge, and soils. Influent, effluent, and chlorinated effluent samples showed 16.1 to 100% of the total virus demonstrated in samples to be solids associated. Three techniques for freeing solids-associated virus are described and compared. Using sonication of solids and polyethylene glycol concentration, virus was demonstrated in fully digested sludge (60 days at 34 C), sand at the site of a sewer leak, and dried sludge cake and mud 900 m downstream from a sewage disposal site. These data emphasize the inadequacy of virus concentration techniques that do not include the processing of solids. In situ elution failed to free solids-associated virus.     

Demonstration of solids-associated virus in wastewater and sludge.

Data presented demonstrate the relatively high multiplicity of solids-associated virus in field samples, i.e., wastewater, sludge, and soils. Influent, effluent, and chlorinated effluent samples showed 16.1 to 100% of the total virus demonstrated in samples to be solids associated. Three techniques for freeing solids-associated virus are described and compared. Using sonication of solids and polyethylene glycol concentration, virus was demonstrated in fully digested sludge (60 days at 34 C), sand at the site of a sewer leak, and dried sludge cake and mud 900 m downstream from a sewage disposal site. These data emphasize the inadequacy of virus concentration techniques that do not include the processing of solids. In situ elution failed to free solids-associated virus.     

Virus attachment to transparent exopolymeric particles along trophic gradients in the southwestern lagoon of New Caledonia

Viruses on organic aggregates such as transparent exopolymeric particles (TEP) are not well investigated. The number of TEP-attached viruses was assessed along trophic gradients in the southwestern lagoon of New Caledonia by determining the fraction of viruses removed after magnetic isolation of TEP. In order to isolate TEP magnetically, TEP were formed in the presence of magnetic beads from submicrometer precursors collected along the trophic gradients. The mixed aggregates of TEP-beads-viruses were removed from solution with a magnetic field. The percentage of viruses associated with newly formed TEP averaged 8% (range, 3 to 13%) for most of the stations but was higher (ca. 30%) in one bay characterized by the low renewal rate of its water mass. The number of viruses (N) attached to TEP varied as a function of TEP size (d [in micrometers]) according to the formulas N = 100d(1.60) and N = 230d(1.75), respectively, for TEP occurring in water masses with short (i.e., <40 days) and long (i.e., >40 days) residence times. These two relationships imply that viral abundance decreases with TEP size, and they indicate that water residence time influences viral density and virus-bacterium interactions within aggregates. Our data suggest that the fraction of viruses attached to TEP is highest in areas characterized by a low renewal rate of the water mass and can constitute at times a significant fraction of total virus abundance. Due to the small distance between viruses and hosts on TEP, these particles may be hot spots for viral infection.     

Simple apparatus for collecting estuarine sediments and suspended solids to detect solids-associated virus

Laboratory trials of a new sampler for collection of estuarine sediment-associated virus resulted in a recovery effectiveness averaging 30% for two enteroviruses and rotavirus SA11. A minimal recovery potential of 54% was calculated when losses caused by virus concentration procedure inadequacies were excluded. Both sediment-associated and suspended solids-associated viruses were collected with the sampler. Recoveries of 61 and 60% poliovirus and rotavirus, respectively, were obtained from salt water-suspended, solids-associated virus. The unique advantage of the sampler for selective collection of virus-associated top layers of sediment, plus collection over extensive areas, resulted in recovery of more virus than was obtained with a commonly used dredge-type sampler.     

Simple apparatus for collecting estuarine sediments and suspended solids to detect solids-associated virus.

Laboratory trials of a new sampler for collection of estuarine sediment-associated virus resulted in a recovery effectiveness averaging 30% for two enteroviruses and rotavirus SA11. A minimal recovery potential of 54% was calculated when losses caused by virus concentration procedure inadequacies were excluded. Both sediment-associated and suspended solids-associated viruses were collected with the sampler. Recoveries of 61 and 60% poliovirus and rotavirus, respectively, were obtained from salt water-suspended, solids-associated virus. The unique advantage of the sampler for selective collection of virus-associated top layers of sediment, plus collection over extensive areas, resulted in recovery of more virus than was obtained with a commonly used dredge-type sampler.     

Effects of bentonite clay solids on poliovirus concentration from water by microporous filter methods.

To determine whether suspended solids interfere with enteric virus recovery from water by microporous filter methods, the effects of bentonite clay solids at a concentration of 10 nephelometric turbidity units on the recovery of poliovirus type 1 from seeded, activated carbon-treated, filtered tap water were studied. Volumes (500 ml) of virus-laden water at pH 5.5 or 7.5, with and without 50 mM MgCl2, were filtered through 47-mm-diameter, electropositive (Virosorb 1MDS) and electronegative (Filterite) filters that had been pretreated with Tween 80 to minimize direct virus adsorption to filter surfaces. Bentonite solids enhanced virus retention on both types of filters, even under conditions in which viruses were not solids associated. However, bentonite solids also interfered with elution of retained viruses when eluting with 0.3% beef extract-50 mM glycine (pH 9.5). Under some conditions, overall virus recoveries were lower from water with bentonite solids than from solids-free control water. The results of this study indicate that clay turbidity can interfere somewhat with virus recovery by current microporous filter methods.     

Inactivation of poliovirus and other enteroviruses by solutions of sodium fluoride at low pH

Solutions of sodium fluoride at pH 3 to 4 inactivated enteroviruses, whereas other sodium salts had little or no effect on virus infectivity. Solutions of potassium fluoride also inactivated viruses under similar conditions. Light, temperature, and the presence of organic compounds such as detergents and fecal matter did not affect inactivation of virus by 0.4 M solutions of sodium fluoride at pH 3. to 4. Decreasing the sodium fluoride concentration below 0.04 M or raising the pH above 4 reduced the viricidal properties of the solutions. Virus adsorbed to membrane filters and sludge flocs could not be recovered after treatment of solids-associated virus with solutions of sodium fluoride.     

Inactivation of poliovirus and other enteroviruses by solutions of sodium fluoride at low pH.

Solutions of sodium fluoride at pH 3 to 4 inactivated enteroviruses, whereas other sodium salts had little or no effect on virus infectivity. Solutions of potassium fluoride also inactivated viruses under similar conditions. Light, temperature, and the presence of organic compounds such as detergents and fecal matter did not affect inactivation of virus by 0.4 M solutions of sodium fluoride at pH 3. to 4. Decreasing the sodium fluoride concentration below 0.04 M or raising the pH above 4 reduced the viricidal properties of the solutions. Virus adsorbed to membrane filters and sludge flocs could not be recovered after treatment of solids-associated virus with solutions of sodium fluoride.     

Effects of bentonite clay solids on poliovirus concentration from water by microporous filter methods

To determine whether suspended solids interfere with enteric virus recovery from water by microporous filter methods, the effects of bentonite clay solids at a concentration of 10 nephelometric turbidity units on the recovery of poliovirus type 1 from seeded, activated carbon-treated, filtered tap water were studied. Volumes (500 ml) of virus-laden water at pH 5.5 or 7.5, with and without 50 mM MgCl2, were filtered through 47-mm-diameter, electropositive (Virosorb 1MDS) and electronegative (Filterite) filters that had been pretreated with Tween 80 to minimize direct virus adsorption to filter surfaces. Bentonite solids enhanced virus retention on both types of filters, even under conditions in which viruses were not solids associated. However, bentonite solids also interfered with elution of retained viruses when eluting with 0.3% beef extract-50 mM glycine (pH 9.5). Under some conditions, overall virus recoveries were lower from water with bentonite solids than from solids-free control water. The results of this study indicate that clay turbidity can interfere somewhat with virus recovery by current microporous filter methods.     

Isolation of enteroviruses from water, suspended solids, and sediments from Galveston Bay: survival of poliovirus and rotavirus adsorbed to sediments.

The distribution and quantitation of enteroviruses among water, suspended solids, and compact sediments in a polluted estuary are described. Samples were collected sequentially from water, suspended solids, fluffy sediments (uppermost layer of bottom sediments), and compact sediment. A total of 103 samples were examined of which 27 (26%) were positive for virus. Polioviruses were recovered most often, followed by coxsackie B viruses and echoviruses 7 and 29. Virus was found most often attached to suspended solids: 72% of these samples were positive, whereas only 14% of water samples without solids yielded virus. Fluffy sediments yielded virus in 47% of the samples, whereas only 5% of compact bottom-sediment samples were positive. When associated with solids, poliovirus and rotavirus retained their infectious quality for 19 days. The same viruses remained infectious for only 9 days when freely suspended in seawater. Collection of suspended solids at ambient water pH appears to be very useful for the detection of virus; it has advantages over collecting and processing large volumes of water, with accompanying pH adjustment and salt addition for processing.     

Isolation of enteroviruses from water, suspended solids, and sediments from Galveston Bay: survival of poliovirus and rotavirus adsorbed to sediments

The distribution and quantitation of enteroviruses among water, suspended solids, and compact sediments in a polluted estuary are described. Samples were collected sequentially from water, suspended solids, fluffy sediments (uppermost layer of bottom sediments), and compact sediment. A total of 103 samples were examined of which 27 (26%) were positive for virus. Polioviruses were recovered most often, followed by coxsackie B viruses and echoviruses 7 and 29. Virus was found most often attached to suspended solids: 72% of these samples were positive, whereas only 14% of water samples without solids yielded virus. Fluffy sediments yielded virus in 47% of the samples, whereas only 5% of compact bottom-sediment samples were positive. When associated with solids, poliovirus and rotavirus retained their infectious quality for 19 days. The same viruses remained infectious for only 9 days when freely suspended in seawater. Collection of suspended solids at ambient water pH appears to be very useful for the detection of virus; it has advantages over collecting and processing large volumes of water, with accompanying pH adjustment and salt addition for processing.     

Impact of hydrology on free-living and particle-associated microorganisms in a river floodplain system (Danube, Austria)

1. A floodplain segment of the Danube River downstream of Vienna was studied during the hydrologically most dynamic phase (spring–summer) to evaluate the significance of connection between the main channel and the floodplain segment for particle abundance and quality as well as for bacterial and viral parameters, both free‐living and attached to particles. 2. Hydrological connectivity between the main channel and its floodplain segment (expressed as water age) influenced particle abundance and quality. Polysaccharide‐containing particles [Alcian positive‐stained particles (ASP)] and protein‐containing particles [Coomassie positive‐stained particles (CSP)] each contributed a substantial fraction to total suspended solids and were both positively related to water age. ASP were about twice as abundant as CSP. 3. Water age influenced bacterial and viral abundance and the bacterioplankton productivity in the surrounding water. Free‐living bacterial abundance and their bacterial secondary production (BSP) increased continuously with water age, best described by a linear regression. Water age also significantly impacted BSP and per cell BSP of bacteria attached to particles. The abundance of attached bacteria and viruses was not influenced by water age. 4. Bacteria and viruses on particles were influenced by particle quality. Their abundance on particles was closely related to particle size. Particle‐attached bacteria accounted on average for 30.34% (± 3.09) of the total bacterial abundance. A variable and occasionally significant proportion of viruses, between 0.43% and 35.06%, were associated with particulate material. 5. Bacteria attached to particles were significantly more productive than their free‐living counterparts. Their per‐cell activity was on average 8.6 times higher than that of the free‐living fraction. 6. Hydrological connectivity between the Danube River and its floodplain is crucial not only for the exchange of water, sediment and nutrients, but also for microbiota, thus influencing microbial life, distribution and activity.     

Virus Attachment to Transparent Exopolymeric Particles along Trophic Gradients in the Southwestern Lagoon of New Caledonia

Viruses on organic aggregates such as transparent exopolymeric particles (TEP) are not well investigated. The number of TEP-attached viruses was assessed along trophic gradients in the southwestern lagoon of New Caledonia by determining the fraction of viruses removed after magnetic isolation of TEP. In order to isolate TEP magnetically, TEP were formed in the presence of magnetic beads from submicrometer precursors collected along the trophic gradients. The mixed aggregates of TEP-beads-viruses were removed from solution with a magnetic field. The percentage of viruses associated with newly formed TEP averaged 8% (range, 3 to 13%) for most of the stations but was higher (ca. 30%) in one bay characterized by the low renewal rate of its water mass. The number of viruses (N) attached to TEP varied as a function of TEP size (d [in micrometers]) according to the formulas N = 100d^1.60 and N = 230d^1.75, respectively, for TEP occurring in water masses with short (i.e., <40 days) and long (i.e., >40 days) residence times. These two relationships imply that viral abundance decreases with TEP size, and they indicate that water residence time influences viral density and virus-bacterium interactions within aggregates. Our data suggest that the fraction of viruses attached to TEP is highest in areas characterized by a low renewal rate of the water mass and can constitute at times a significant fraction of total virus abundance. Due to the small distance between viruses and hosts on TEP, these particles may be hot spots for viral infection.     

Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost.     

Persistent infection of cultured mammalian cells by Japanese encephalitis virus.

Persistent infections were established by serial undiluted passage of flavivirus Japanese encephalitis virus in a line of rabbit kidney cells (MA-111). The persistently infected cells resembled uninfected cells in most respects. Low levels of infectious virions were released from a small percentage of cells, and a larger and more variable percentage was shown to possess viral antigen by fluorescent-antibody staining. Released viruses were shown to interfere with replication of wild-type Japanese encephalitis virus. Persistently infected MA-111 cells could not be superinfected with homologous wild-type Japanese encephalitis virus but could be superinfected with two heterologous viruses. Transfer of cell culture medium from persistently infected MA-111 cells to a line of African green monkey kidney cells (Vero) resulted in similar persistent infections in the latter cells. Temperature sensitivity and host-cell interferon production were not involved in establishment or maintenance of persistence. Determination of ratios of physical particles to infectious particles revealed that many defective, noninfectious viruses were present, suggesting that defective interfering particles may be responsible for persistency.     

Infantile enteritis viruses: morphogenesis and morphology.

A new virus was found to be associated with acute gastroenteritis in children. In duodenal biopsies, it was observed infecting only intestinal epithelial cells, and it resembled orbiviruses in its morphogenesis. For diagnsotic purposes the virus was readily demonstrated by negative staining of fecal extracts. Two forms of particles were seen: double-sheeled particles (70 to 75 nm in diameter) resembling those of reovirus with a sharper outline, and single-shelled particles (60 nm in diameter) with obvious capsomer structure and resembling those of orbiviruses. The morphological resemblance of this human virus to the viruses of "Nebraska" calf scours and epizootic diarrhoea of infant mice is emphasized.     

Removal of Waterborne Human Enteric Viruses and Coliphages with Oxidized Coal

Human enteric viruses and coliphages have been detected in water that has undergone what is generally considered adequate treatment, including chlorination. Because small numbers of virus particles are needed for the initiation of a productive virus infection, the presence of any number of virus particles in water resources will always be of concern. In this investigation the ability of oxidized coal to remove viruses from water was investigated. The oxicoal product was found to be able to remove not only coliphages, but also various pathogenic human viruses from seeded water sources. Removal was dependent upon the type of virus, the period of exposure, and the concentration of oxidized coal. Received: 20 August 1997 / Accepted: 22 January 1998     

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.