Survival of poliovirus within organic solids during chlorination.

Poliovirus in fecal homogenates was used to determine the protection against inactivation by chlorination afforded virus that was occluded within particulates. Virus that was closely associated with or occluded within small fecal particulates was protected. A fourfold increase in combined residual chlorine was required to achieve the same degree of inactivation for occluded virus as for free or secondarily adsorbed virus. A combined chlorine residual of 6.6 mg/liter was necessary to achieve 50% inactivation in 15 min at pH 8.0 and 22 degrees C in a particulate suspension containing occluded virus compared to 1.4 mg/liter for free virus. These differences were found to be relatively small compared to differences due to the presence of dissolved organics or between free and combined chlorine residuals. The results suggest different mechanisms of protection due to adsorption and occlusion.     

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.     

Optimal replication of poliovirus within cells

We construct a mathematical model of the within-cell replication of poliovirus, a prototypic RNA virus, and use realistic parameter estimates to describe the increase of copy number of the viral genome. Our initial model is essentially an exponential growth model; we also consider modifications of this model to account for resource utilization. The saturation of viral replication dynamics observed in experimental systems can be explained in terms of heavy resource use by the virus. We then use our models to consider the conditions under which the growth of poliovirus is optimized. Intriguingly, if poliovirus has optimized its replication within cells, the predicted ratio of positive to negative strands is close to what is actually observed. We interpret our findings in terms of the evolution of life-history traits.     

Cleavage sites within the poliovirus capsid protein precursors.

Partial amino-terminal sequence analysis was performed on radiolabeled polio-virus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.     

Orbitally magnetized organic solids

The preparation is discussed of polymeric organic solids in which large ring-like delocalized electronic orbitals can be made to enclose or trap magnetic flux. The situation is analogous to snapping shut a loop of superconductor while it is in a magnetic field. The operation of the Meissner effect acts to retain the trapped flux and create remant magnetization.     

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.     

Enhanced intracellular calcium concentration during poliovirus infection.

The infection of human fibroblasts by poliovirus leads to a notable increase in the intracellular calcium concentration, [Ca2+]i, measured by microfluorimetry or by flow cytometry. [Ca2+]i increases from 2 to 3 h postinfection, and by the fifth hour there is a 5- to 10-fold increase in [Ca2+]i. At this time postinfection there is active viral protein synthesis. The modifications in [Ca2+]i are not observed in the presence of cycloheximide, guanidine, or Ro 09-0179, indicating that virus gene expression is required for the increase in [Ca2+]i. Attempts to identify the source of the intracellular Ca2+ by using different inhibitors of calcium fluxes suggest that calcium enters from the culture medium through voltage-sensitive calcium channels.     

Stable hairpin structure within the 5''-terminal 85 nucleotides of poliovirus RNA.

The primary sequence of a 5'-terminal fragment of poliovirus type 1 RNA, generated by digestion with RNase III, has been determined. This sequence reveals the presence of a stable hairpin structure beginning nine nucleotides from the terminally linked protein VPg. The sequence does not contain (i) the initiation codons AUG or GUG or (ii) the putative ribosome-binding sequence complementary to the 3' end of eucaryotic ribosomal 18S RNA. The stem-and-loop structure identified can be drawn in either plus or minus RNA strands. It is unclear to which strand functional significance (if any) can be assigned. It is possible that the hairpin structure is involved in ribosomal recognition and translation or in RNA synthesis by interacting with replicase molecules.     

Chloroquine induces empty capsid formation during poliovirus eclipse.

The poliovirus capsid (160S) is modified during eclipse in HeLa cells, which results in at least three types of particles having sedimentation coefficients of 135, 110, and 80S. The lysosomotropic agent chloroquine redirected the production of eclipse products from 135 and 110S particles (containing RNA) to 80S particles (without RNA). The effect started at 5 microM and was fully developed with 20 microM chloroquine. Viral protein synthesis and virion production remained unaffected. The results show that chloroquine can redirect the processing of input virions without interfering with productive uncoating.     

Survival of bacteria during aerosolization.

One form of commercial application of microorganisms, including genetically engineered microorganisms is as an aerosol. To study the effect of aerosol-induced stress on bacterial survival, nonrecombinant spontaneous antibiotic-resistant mutants of four organisms, Enterobacter cloacae, Erwinia herbicola, Klebsiella planticola, and Pseudomonas syringae, were sprayed in separate experiments in a greenhouse. Samples were collected over a distance of 15 m from the spray site for enumeration. Spores of Bacillus subtilis were used as tracers to estimate the effects of dilution on changes in population over distance. Viable counts of P. syringae, Enterobacter cloacae, and K. planticola decreased significantly over a distance of 15 m. Erwinia herbicola showed no significant decline in counts over the same distance. The degree of survival of P. syringae during aerosolization was dependent on ambient environmental conditions (i.e., temperature, relative humidity), droplet size of the aerosol, and prior preparative conditions. Survival was greatest at high relative humidities (70 to 80%) and low temperatures (12 degrees C). Survival was reduced when small droplet sizes were used. The process of washing the cells prior to aerosolization also caused a reduction in their survival. Results from these experiments will be useful in developing sound methodologies to optimize enumeration and for predicting the downwind dispersal of airborne microorganisms, including genetically engineered microorganisms.     

Anomalous behavior of certain poliovirus polypeptides during SDS-gel electrophoresis.

Urea is shown to be a useful additive to sodium dodecyl sulfate gels prior to electrophoresis and offers an alternative means for the resolution of some SDS-protein complexes. Two types of effect are described, one of which causes increases in the relative mobilities of certain polypeptides found in poliovirus-infected cells. It is postulated that urea plus SDS is able to achieve a more complete denaturation of some polypeptides, which leads to faster electrophoretic mobilities. The molecular weight estimations for such proteins in these conditions are therefore lower than those determined in the presence of SDS alone. A second effect of some urea solutions is to cause the multiple banding of the structural polypeptide VP3 when included in gels at high concentrations. This effect is variable and appears to be unrelated to the presence of cyanate ions.     

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis.

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.     

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.     

Survival of poliovirus in flowing turbid seawater treated with ultraviolet light

[This corrects the article on p. 533 in vol. 15.].     

Survival of intracellular pathogens within macrophages

Summary The threat caused by intracellular pathogens increases as conventional drag treatments are less and less effective against a wide range of microorganisms. Understanding the molecular mechanisms used by intracellular pathogens to avoid killing and degradation in their host cells is likely to point at new ways to threat infectious diseases. We discuss some of the strategies used by various microorganisms to avoid killing and degradation in phagolysosomes. Interestingly, it appears that microbes have a lot to teach us about the cell biology and molecular mechanisms of organelle sorting in macrophages.     

Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water.

The potential for human immunodeficiency virus (HIV) to enter domestic sewers via contaminated body fluids such as blood has spurred interest in the survival of this virus in water and wastewater. This study focused on establishing the inactivation of HIV and productively infected lymphocytes in dechlorinated tap water. In addition, HIV survival was compared with that of poliovirus. Results indicated that either free HIV or cell-associated HIV was rapidly inactivated, with a 90% loss of infectivity within 1 to 2 h at 25 degrees C and a 99.9% loss by 8 h. In comparison, poliovirus showed no loss of infectivity over 24 h. The presence of human serum in tap water slowed the rate of HIV inactivation through 8 h but did not stabilize the virus through 24 h. In addition, blood from stage IV AIDS patients was introduced into tap water, and the recovery of HIV was monitored by using both an infectivity assay and polymerase chain reaction amplification of viral sequences. Virally infected cells were no longer detectable after 5 min in dechlorinated tap water, while little diminution in amplifiable sequences was observed over 2 h. Thus, detection of viral sequences by polymerase chain reaction technology should not be equated with risk of exposure to infectious HIV.     

Cellular origin and ultrastructure of membranes induced during poliovirus infection.

Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes.     

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.