Morphometric analysis demonstrates that metabolically active cardiac triglycerides are 1H NMR visible.

The relationship between myocardial triglyceride content and 1H NMR visible fatty acid resonance intensity was investigated. Hearts from rats fed a 20% rapeseed oil diet contained markedly increased levels of triglycerides as judged by thin layer chromatographic analysis. This elevation in cardiac triglycerides was associated with sharp increases in the cell volume occupied by lipid droplets and in 1H NMR visible fatty acid resonances. Spin-lattice and spin-spin relaxation times of the 1H NMR visible fatty acid resonances from myocardium of rapeseed oil-fed rats were similar in value to those measured for neat triolein. Additionally, the fatty acids constituting these enhanced 1H NMR visible resonances were metabolically active. Perfusion of triglyceride enriched hearts in the presence or absence of glucose caused a time-dependent decrease in the intensity of their 1H NMR visible fatty acid resonances. In contrast, perfusion with glucose+acetate essentially prevented this time-dependent decrease in 1H NMR visible fatty acid resonances. Morphometric analysis of these hearts demonstrated that the decrease in 1H NMR resonance intensity correlated with changes in the cell volume of triglyceride-enriched lipid droplets. These results demonstrate that metabolically active stores of cardiac fatty acids, presumably triglycerides, are 1H NMR visible. Further, they indicate the possible utility of 1H NMR spectroscopy in the future study of myocardial triglyceride metabolism.

Intracellular calcium during photodynamic permeabilization of cardiomyocytes.

The purpose of this study was to determine whether an early increase in [Ca2+]i preceding generalized lysis of cardiomyocytes occurred during photodynamic permeabilization. A method was developed which facilitated the simultaneous measurement, in real time, of permeabilization of the sarcolemma to Ca2+ and Mn2+ during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to measure the rate and extent of change in [Ca2+]i. The same system was used in the presence of extracellular Mn2+ to determine how quickly the cardiomyocytes became permeable to either Mn2+ or quin-2. Calcium ions were observed to enter the myocytes prior to permeabilization of the sarcolemma to either Mn2+ or quin-2, and thus before membrane lysis. Lysis of cardiomyocytes did not appear to be dependent upon increases in [Ca2+]i. Controls were performed to rule out fluorescent artifacts. Reperfusion injury and photodynamic therapy involve both the production of free radicals and an early increase in [Ca2+]i. This study demonstrates a direct correlation between the production of reactive oxygen species and prelytic increases in [Ca2+]i in neonatal cardiomyocytes and demonstrates that this phenomenon may be common to many cell types.

Effects of ozone treatment on the infectivity of hepatitis A virus.

The inactivation of a large-focus-forming variant of hepatitis A virus (HM-175) by ozone was investigated. Experiments using mainly single-particle virus preparations suspended in phosphate-carbonate buffer were conducted over a range of pH levels (6-8) at 4 degrees C. Viral enumerations involved the use of a radioimmunofocus assay. While some tolerance to lower (i.e., 0.1-0.5 mg/L) ozone residuals was noted, the exposure of virus particles to ozone concentrations of 1 mg/L or greater at all pH levels resulted in their complete (5 log) inactivation within 60 s. The pH-related effects that were observed were not considered to be significant.

Inactivation of human and simian rotaviruses by chlorine dioxide.

The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.

Inactivation of human and simian rotaviruses by ozone.

The inactivation of simian rotavirus SA-11 and human rotavirus type 2 (Wa) by ozone was compared at 4 degrees C by using single-particle virus stocks. Although the human strain was clearly more sensitive, both virus types were rapidly inactivated by ozone concentrations of 0.25 mg/liter or greater at all pH levels tested. Comparison of the virucidal activity of ozone with that of chlorine in identical experiments indicated little significant difference in rotavirus-inactivating efficiencies when the disinfectants were used at concentrations of 0.25 mg/liter or greater.

Inactivation of human and simian rotaviruses by chlorine.

The inactivation of simian rotavirus SA-11 and human rotavirus type 2 (Wa) by chlorine was compared at 4 degrees C by using single-particle virus stocks. Both virus types were usually more readily inactivated at pH 6.0 than at pH 8.0 when low chlorine concentrations (0.05 to 0.2 mg/liter) were used. A complete (5 log) reduction of both was obtained within 20 s at all pH levels when chlorine concentrations were increased to 0.3 mg/liter. Slight differences in the chlorine sensitivities of SA-11 and human rotavirus type 2 were noted but were not considered to be significant.

Entrainment of viruses from septic tank leach fields through a shallow, sandy soil aquifer.

A study was conducted which focused on movement of naturally occurring human enteroviruses from a subsurface wastewater disposal system through a shallow aquifer. The potential for significant entrainment of virus particles was evidenced by their recovery at down-gradient distances of 67.05 m and from aquifer depths of 18 m. A significant negative correlation was observed between virus occurrence and the distance from the "septage" (leaching pool) source. Virus occurrence could not be statistically correlated with either total or fecal coliforms, indicating the limitations of current microbial water quality indicators for predicting the virological quality of groundwater.

Accumulation of sediment-associated viruses in shellfish.

The present study focused on the importance of contaminated sediments in shellfish accumulation of human viruses. Epifaunal (Crassostrea virginica) and infaunal (Mercenaria mercenaria) shellfish, placed on or in cores, were exposed to either resuspended or undisturbed sediments containing bound poliovirus type 1 (LSc 2ab). Consistent bioaccumulation by oysters (four of five trials) was only noted when sediment-bound viruses occurred in the water column. Virus accumulation was observed in a single instance where sediments remained in an undisturbed state. While the incidence of bioaccumulation was higher with resuspended rather than undisturbed contaminated sediment, the actual concentration of accumulated viruses was not significantly different. The accumulation of viruses from oysters residing on uninoculated sediments. When clams were exposed to undisturbed, virus-contaminated sediments, two of five shellfish pools yielded viral isolates. Bioaccumulation of undisturbed sediments by these bivalves was considered marginal when related to the concentration of virus in contaminated sediments; they would only represent a significant threat when suspended in the water column. Arguments were advanced for water-column sampling in the region of the water-sediment interface to provide an accurate determination of the virological quality of shellfish harvesting waters.

Inefficient accumulation of low levels of monodispersed and feces-associated poliovirus in oysters.

The accumulation of low levels (0.002 to 0.18 PFU/ml) of both feces-associated and monodispersed poliovirus by oysters (Crassostrea virginica or C. gigas) and clams (Mercenaria mercenaria) was investigated. These levels were chosen to duplicate the conditions present in light to moderately polluted waters. Experiments were performed in both small- and large-scale flowing seawater systems, developed to mimic the natural marine habitats of shellfish. Under these experimental conditions, viral accumulation by physiologically active shellfish was only noted when water column concentrations exceeded approximately 0.01 PFU/ml. Bioaccumulation increased with increasing concentrations of both monodispersed and feces-associated viruses. At virus concentrations below this level, viruses were seldom detected in either clams or oysters. Evidence indicated that the lack of accumulation was not the result of inefficient extraction or detection methods. The modified Cat-Floc-beef extract procedure used in the experiment was found to be capable of detecting as few as 1.5 to 2.0 PFU per shellfish. Evidence is presented to indicate that an uptake-depuration equilibrium was present at virus exposure levels of 0.10 PFU/ml, but not at 0.01 PFU/ml. The results suggested that viral accumulation by shellfish may not be efficient at water column concentrations below congruent to 0.01 PFU/ml.

Virus removal during groundwater recharge: effects of infiltration rate on adsorption of poliovirus to soil.

Studies were conducted to determine the influence of infiltration rate on poliovirus removal during groundwater recharge with tertiary-treated wastewater effluents. Experiments were conducted at a uniquely designed, field-situated test recharge basin facility through which some 62,000 m3 of sewage had been previously applied. Recharge at high infiltration rates (75 to 100 cm/h) resulted in the movement of considerable numbers of seeded poliovirus to the groundwater. Moderately reduced infiltration rates (6 cm/h) affected significantly improved virus removal. Very low infiltration rates (0.5 to 1.0 cm/h), achieved by partial clogging of the test basin, yielded the greatest virus removal efficiencies.

Poliovirus retention in 75-cm soil cores after sewage and rainwater application.

The adsorption rate of a guanidine-resistant strain of poliovirus LSc 2ab was measured in Long Island soils with in situ field cores (10.1 by 75 cm). The test virus was chosen because it exhibited soil adsorption and elution characteristics of a number of non-polioviruses. After the inoculation of cores with seeded sewage effluent at a 1-cm/h infiltration rate, cores were extracted, fractionated, and analyzed for total plaque-forming units per each 5-cm fraction. The results showed that 77% of the viruses were adsorbed in the first 5 cm of soil. An additional 11% were found in the 5- to 10-cm fraction, and a total of 96% of the viruses were adsorbed by 25 cm. The remaining 4% were uniformly distributed over the next 50 cm of soil, with a minimum of 0.23% in each soil section. Few viruses (< 0.22%) were observed in core filtrates. Analysis of the viral distribution pattern in seeded cores, after an application of a single rinse of either sewage effluent or rainwater, indicated that large-scale viral mobilization was absent. However, localized areas of viral movement were noted in both of the rinsed cores, with the rainwater-rinsed cores exhibiting more expensive movement. All mobilized viruses were resorbed at lower core depths.

Modified procedure for the recovery of naturally accumulated poliovirus from oysters.

Methods were compared for their ability to recover poliovirus from oysters (Crassostrea gigas) which had been allowed to accumulate virus via normal filtration activities. Clarification procedures included glycine-NaCl and polyelectrolyte extraction methods followed by a variety of acid precipitation concentration methods. Polyelectrolyte flocculation followed by a beef extract-supplemented acid precipitation carried out at pH 3.5 yielded the most efficient recoveries. Direct assay of homogenates was found to be an unreliable method for determining the initial virus concentration in "naturally infected" oysters.

Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater.

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.

Survey of human enterovirus occurrence in fresh and marine surface waters on Long Island.

A variety of surface water systems, including a lake, a creek, and two marine embayments, were analyzed on a monthly basis for indigenous human enteroviruses and coliform bacteria. Findings are discussed in terms of the probable pollution sources to each system and their relationship to data from previous studies.

Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.

Survey of human virus occurrence in wastewater-recharged groundwater on Long Island.

Treated wastewater effluents and groundwater observation wells from three sewage recharge installations located on Long Island were assayed on a monthly basis for indigenous human enteroviruses and coliform bacteria for a period of 1 year. Viruses were detected in groundwater at sites where recharge basins were located less than 35 feet (ca. 10.6 m) above the aquifer. Results from one of the sites indicated the horizontal transfer of viable viruses through the groundwater aquifer.