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.     

Concentration of Viruses from Sewage and Excreta on Insoluble Polyelectrolytes

The concentration of viruses from sewage by adsorption on and elution from an insoluble cross-linked copolymer of maleic anhydride is described. Viruses either added to sewage or naturally contained in sewage were preferentially adsorbed to this polyelectrolyte at a pH range of 5.0 to 6.0 and were eluted at pH 8.0 to 9.0. In a 2-month survey of viruses in sewage in the spring (April to May 1968), when viruses are at low levels, efficient and economical detection of these agents was accomplished with the polyelectrolyte-concentration method. This method lends itself to the detection of viruses present in minute amounts in fecal samples, urine, sewage, and other natural waters. Large volumes of these fluids can be treated with the polymer described, and virus can be concentrated sufficiently for detection.     

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%.     

Survival of enteric viruses under natural conditions in a subarctic river.

The survival of enteric viruses was studied in the vicinity of Fairbanks, Alaska at selected stations along a 317-km section of the Tanana River. This section was located downstream from all known domestic wastewater sources and was effectively sealed by a total ice cover. The mean flow time through the region was 7.1 days, during which initial viral population showed a relative survival rate of 34%. The tracing of native viruses at such great distances in the complete absence of other point and nonpoint viral sources has not been previously reported. Of the two methods of virus concentration used, viral recoveries from the disk adsorption virus elution procedure were far greater than those achieved with the Aquella system employed at that time. The fact the ratio of enteric viruses to fecal indicator bacteria was not constant clearly inferred that these bacteria were not an effectual measure of virus concentration. The persistence of fecal coliforms and fecal streptococci, however, attested to the microbiological health risk involved.     

Survival of enteric viruses under natural conditions in a subarctic river.

The survival of enteric viruses was studied in the vicinity of Fairbanks, Alaska at selected stations along a 317-km section of the Tanana River. This section was located downstream from all known domestic wastewater sources and was effectively sealed by a total ice cover. The mean flow time through the region was 7.1 days, during which initial viral population showed a relative survival rate of 34%. The tracing of native viruses at such great distances in the complete absence of other point and nonpoint viral sources has not been previously reported. Of the two methods of virus concentration used, viral recoveries from the disk adsorption virus elution procedure were far greater than those achieved with the Aquella system employed at that time. The fact the ratio of enteric viruses to fecal indicator bacteria was not constant clearly inferred that these bacteria were not an effectual measure of virus concentration. The persistence of fecal coliforms and fecal streptococci, however, attested to the microbiological health risk involved.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10(4) cells were 4.66 at 30 degrees C, 4.36 at 10 degrees C and 4.19 at 5 degrees C. These limits were salt dependent, low (4-6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4.66 at 30 degrees C but this was increased to 4.83 at 10 degrees C. At 5 degrees C growth occurred at pH 7.0 but not at pH 5.13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10 degrees C and 30 degrees C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

混合盐碱胁迫对芹菜种子萌发的影响

NaCl、Na2SO4、NaHCO3和Na2CO3按不同比例混合,模拟盐度和pH变化规律与天然盐碱地相似的15种盐碱条件,探讨混合盐碱胁迫对芹菜(Apium graveolens)种子萌发的影响.结果表明:芹菜种子的发芽率、发芽势、发芽指数和活力指数均随盐浓度的升高,pH的增大呈下降趋势.芹菜种子的萌发主要受盐浓度的影响,不同盐浓度间的影响差异大;当盐浓度为200 mmol/L时,基本不萌发.     

Impact of Bifidobacterium longum on human fecal microflora.

The effects of Bifidobacterium longum feedings for five weeks on the fecal microflora, water contents, pH values, ammonia concentration, and beta-glucuronidase activity were investigated in five healthy human volunteers. Although numbers of major bacterial groups of the fecal microflora were not changed by the bifidobacteria feedings, a remarkably decreasing number of lecithinase-negative clostridia was observed. The percentage of lecithinase-negative clostridia and bacteroides to the total bacterial numbers isolated were decreased during the feedings and numbers of C. paraputrificum and C. innocuum were reduced. A significant reduction of fecal pH values for the last week of the feeding was observed. Ammonia concentration and beta-glucuronidase activity in the feces during the feedings were significantly lower than those before or after the feedings. The oral supplement of B. longum may be introduced to improve the fecal properties such as fecal ammonia concentration and beta-glucuronidase activity, but not the composition of fecal flora.     

Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII

Virus inactivation by solvent/detergent treatment using 0.3% tri-n-butyl phosphate and 1% Triton X-100 in the high purity factor VIII concentrate Replenate((R)) has been investigated. A wide range of model enveloped viruses were confirmed to be inactivated by >4 to >6log after 30min at 22 degrees C under standard conditions. Using Sindbis as a representative enveloped virus, the effect of various parameters on the inactivation process was tested. Virus inactivation was confirmed to be effective in different batches of product and was not influenced by changing the process conditions with regard to protein and salt concentration or pH. Virus inactivation was effective even at a temperature as low as 4-5 degrees C. Although solvent/detergent concentration was the most critical parameter, a concentration as low as 0.15% TnBP/0.5% Triton X-100 was still completely effective. At a lower concentration an extended incubation period was required. These studies demonstrate the robustness of this solvent/detergent procedure based on Triton X-100 and allow suitable process limits to be set for this manufacturing step.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10⁴ cells were 4·66 at 30†C, 4·36 at 10†C and 4·19 at 5†C. These limits were salt dependent, low (4–6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4·66 at 30†C but this was increased to 4·83 at 10†C. At 5†C growth occurred at pH 7·0 but not at pH 5·13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10†C and 30†C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

Role of pH on dimeric interactions for DENV envelope protein: an insight from molecular dynamics study

The entry of dengue viruses is mediated by pH triggering in the host cells. Here we have studied the DENV E protein stability and binding of its units at low and normal pH using MD and MM-PB/SA method for the first time. To investigate the role of pH in dissociation of dimeric protein, we have performed a concise study of hydrogen bonding and other interactions between units of dimer at low and normal pH. The Generalized Born calculation connotes that dimeric unit was relatively less stable and less proned for dimerisation at low pH. Our results provide a theoretical verification for previous assumptions of pH triggering mechanism of dengue envelope protein. During the pH alteration, we found a large decrement in salt bridges which were observed at normal pH. We also compared the flexibility of each unit and found that they exhibit different fluctuations during molecular dynamics simulations.     

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.     

Kinetics of salt-dependent unfolding of [2Fe–2S] ferredoxin of <Emphasis Type="Italic">Halobacterium salinarum</Emphasis>

The [2Fe–2S] ferredoxin from the extreme haloarchaeon Halobacterium salinarum is stable in high (>1.5 M) salt concentration. At low salt concentration the protein exhibits partial unfolding. The kinetics of unfolding was studied in low salt and in presence of urea in order to investigate the role of salt ions on the stability of the protein. The urea dependent unfolding, monitored by fluorescence of the tryptophan residues and circular dichroism, suggests that the native protein is stable at neutral pH, is destabilized in both acidic and alkaline environment, and involves the formation of kinetic intermediate(s). In contrast, the unfolding kinetics in low salt exhibits enhanced rate of unfolding with increase in pH value and is a two state process without the formation of intermediate. The unfolding at neutral pH is salt concentration dependent and occurs in two stages. The first stage, involves an initial fast phase (indicative of the formation of a hydrophobic collapsed state) followed by a relatively slow phase, and is dependent on the type of cation and anion. The second stage is considerably slower, proceeds with an increase in fluorescence intensity and is largely independent of the nature of salt. Our results thus show that the native form of the haloarchaeal ferredoxin (in high salt concentration) unfolds in low salt concentration through an apparently hydrophobic collapsed form, which leads to a kinetic intermediate. This intermediate then unfolds further to the low salt form of the protein.     

Effect of distal enterectomy on cholesterol and bile salt levels in the rat

The effect of 50% or 80% distal enteroctomy on cholesterol and bile salt levels in male Wistar rats have been investigated. Short time measurements showed that serum cholesterol levels were maximal after 20 days from 50% intestinal resection and after 10 days from 80% intestinal resection. This increase was maintained in 50% resected rats 1 and 5 months after operation, whilts in 80% resected group the values became normal. Portal blood and bile cholesterol levels remain almost normal except 5 months after 50% intestinal resection. Bile salt concentration and bile salt output in the bile decrease after 1 and 5 months from 50% intestinal resection and after 1 month from 80% intestinal resection. These results together with data of fecal loss of bile salts indicate that in 50% resected rats new steady states have been reached, with low levels of bile salts in the bile. One month after 80% resection the fecal loss of bile salts was so high that the conversion of cholesterol into bile salts was increased. After 5 months from 80% resection values in serum and bile were almost normal suggesting either an increase in extrahepatic cholesterol synthesis or a partial prevention of fecal loss that can be explained by the observed caecal enlargement.     

Salinity Regulation of the Interaction of Halovirus SNJ1 with Its Host and Alteration of the Halovirus Replication Strategy to Adapt to the Variable Ecosystem

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.     

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.     

Distribution of viruses associated with particles in waste water.

The distribution of solids-associated viruses in wastewater was studied to determine the effect of treatment processes on viruses associated with solids. Solids less than 0.3 micrometers in diameter were separated from the liquid phase of each sample by using a continuous-flow centrifuge. The percentage of virus associated with solids larger than 0.3 micrometers decreased from 28% in the influent to 3.4% in unchlorinated effluent, and this was accompanied by a 92% decrease in the total concentration of virus. These results indicate that the original solids-associated virus as well as that is secondarily adsorbed to mixed liquor-suspended solids is lost during clarification. The total concentration of virus was reduced by 82% by chlorination, and the percentage of virus associated with solids increased to 7.7% upon chlorination, indicating some protection due to association with particles larger than 0.3 micrometers. When a suspension of fecal particles and a 0.22-micrometers filtrate of a fecal homogenate were sonicated, a threefold increase in virus titer was observed in each. This demonstrated that viruses may be attached to particles smaller than 0.22 micrometers. Thus, small viral aggregates or viruses attached to submicron particles represented the major portion of solids-associated virus in treated sewage.