Infectivity and antigenicity reduction rates of human rotavirus strain Wa in fresh waters

The rates of inactivation of human rotavirus type 2 (strain Wa) (HRV-Wa) and poliovirus type 1 (strain CHAT) were compared in polluted waters (creek water and secondary effluent before chlorination) and nonpolluted waters (lake water, groundwater, and chlorinated tap water). Viral infectivity titers were determined by plaque assays, while HRV-Wa antigenicity also was monitored by an enzyme-linked immunosorbent assay. Both viruses persisted longest in lake water and shortest in tap water. The actual inactivation times (i.e., times required for two-log10 reductions of initial viral titers) for the two viruses were significantly different in all waters except tap water. With the exception of the groundwater and secondary effluent results, the HRV-Wa inactivation times in the fresh waters tested were significantly different. Owing perhaps to aggregation, HRV-Wa appeared less susceptible to the effects of chlorine than previously reported for this virus and for the simian rotavirus SA11. HRV-Wa displayed prolonged survival in lake water and groundwater exceeding that previously reported for the SA11 virus. The HRV-Wa infectivity reduction rate (ki) was significantly correlated with the water pH (i.e., as pH increased, ki increased). The water pH may have influenced viral aggregation and thereby HRV-Wa susceptibility to other virucidal factors in the water. Enzyme-linked immunosorbent assay results showed similar inactivation patterns with the most significant reduction in HRV-Wa antigenicity occurring in polluted waters and tap water. In all waters, particularly tap water, infectivity declined at a faster rate than antigenicity. It is proposed that HRV-Wa can be used as a model for future studies of rotaviral persistence in the aquatic environment.     

Stability of simian rotavirus in fresh and estuarine water.

The rates of inactivation of poliovirus 1, echovirus 7, coxsackievirus B3, and simian rotavirus SA11 were compared in polluted and nonpolluted fresh and estaurine water samples. The study was done in two parts, comparing virus survival in samples taken 1 year apart from the same sites. The survival studies were performed at 20 degrees C and at the natural pH of the water samples. In the first part of the study, the time required for a 3-log10 reduction in the initial virus titers was 2 to 3 days in the estaurine water samples and varied from 3 to greater than 14 days in the freshwater samples. In the second part of the study, no clear distinction was found between survival of viruses in freshwater samples and survival in estaurine water samples. The time required for a 3-log10 reduction in the initial virus titers in the second part of the study varied from 12 to greater than 14 days. This indicates that there is a nonseasonal change in factors in the water which affect virus survival. In this study SA11 survival time (used as a model for human virus) was well within the range exhibited by the enteroviruses, indicating that it also is environmentally stable in natural waters.     

Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water.

The survival in mineral water of hepatitis A virus (HAV) and poliovirus type 1 was compared, under controlled experimental conditions, at 4 degrees C and room temperature. Viral infectivity titers were determined by cell culture titration, while HAV antigenicity was monitored by radioimmunoassay-endpoint titration. Both viruses persisted longest at 4 degrees C. At this temperature, after 1 year of exposure, the inactivation of either HAV or poliovirus type 1 was not important. At room temperature, poliovirus type 1 was not detected after 300 days, whereas HAV was still infectious. For both temperatures, the computed regression coefficients of best-fit lines for inactivation rates for the two viruses were significantly different. The survival of HAV was also studied at 4 degrees C and room temperature in mineral water with 5- and 50-micrograms/ml protein concentrations (i.e., purity of the virus suspension) for 120 days. As shown by a comparison of the regression coefficients for the inactivation rates, the stability of HAV in mineral water depends on protein concentration and temperature. Radioimmunoassay-endpoint titration results showed inactivation patterns similar to those of cell culture titration, with the most significant reduction in HAV antigenicity at room temperature. At the two temperatures, the infectivity of HAV declined at a faster rate than the antigenicity.     

Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters

Aims: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters. Methods and Results: Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity. Conclusions: Solar UV can rapidly inactivate C. parvum in environmental waters. Significance and Impact of the Study: This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.     

Physicochemical stability and inactivation of human and simian rotaviruses

The effects of various physical and chemical treatments on the stability of a human serotype 1 rotavirus and simian agent 11 (SA11) were compared by using a fluorescence focus assay. The infectivity of both strains was retained after storage at room temperature for 14 days, 4 degree C for 22 days, and -20 degree C for 32 days; lyophilization; and treatment at pH 3 to 11. Both viruses were inactivated at pH 12, as was the human virus at pH 2, although this pH resulted in only partial inactivation of SA11. The human virus also appeared to be more sensitive than SA11 to the action of ether and chloroform. The infectivity of both viruses was lost after UV irradiation for 15 min and after treatment with 8% formaldehyde for 5 min, 70% (vol/vol) ethanol for 30 min, and 2% lysol, 2% phenol, and 1% H2O2 for 1 h each.     

Physicochemical stability and inactivation of human and simian rotaviruses.

The effects of various physical and chemical treatments on the stability of a human serotype 1 rotavirus and simian agent 11 (SA11) were compared by using a fluorescence focus assay. The infectivity of both strains was retained after storage at room temperature for 14 days, 4 degree C for 22 days, and -20 degree C for 32 days; lyophilization; and treatment at pH 3 to 11. Both viruses were inactivated at pH 12, as was the human virus at pH 2, although this pH resulted in only partial inactivation of SA11. The human virus also appeared to be more sensitive than SA11 to the action of ether and chloroform. The infectivity of both viruses was lost after UV irradiation for 15 min and after treatment with 8% formaldehyde for 5 min, 70% (vol/vol) ethanol for 30 min, and 2% lysol, 2% phenol, and 1% H2O2 for 1 h each.     

RNA of rotavirus: comparison of RNAs of human and animal rotaviruses.

Single-stranded RNA (ssRNA) was transcribed in vitro from inner-shell particles of human rotavirus strain Wa (HRV-Wa) and a bovine rotavirus (neonatal calf diarrhea virus [NCDV]) by virion-associated RNA polymerase activity. The ssRNA product consisted of 11 RNA segments which were separated by polyacrylamide gel electrophoresis. In vitro-transcribed 32P-labeled ssRNA was used to study the genetic relatedness between rotaviruses by annealing with genomic double-stranded RNA (dsRNA) of homologous or heterologous rotavirus. All segments of HRV-Wa ssRNA were hybridized with dsRNA of HRV TK80, collected from the feces of a gastroenteritis patient, at the level of 88 to 100% of the homologous reaction. On the other hand, no segments of ssRNA from HRV-Wa hybridized with dsRNA of NCDV or simian rotavirus (simian agent 11). Similarly, ssRNA from NCDV did not hybridize with dsRNA of HRV-Wa, but hybridized with dsRNA of simian agent 11 at the level of 30% of the homologous value.     

In situ inactivation of animal viruses and a coliphage in nonaerated liquid and semiliquid animal wastes.

The persistence of five animal viruses, representing picorna-, rota-, parvo-, adeno-, and herpesviruses, and the coliphage f2 was determined in the field by exposing the viruses to different animal wastes and by adopting an established filter sandwich technique. This technique allows us to copy the natural state of viruses in the environment, where adsorption onto or incorporation into suspended solids may prolong virus survival. Using filter sandwiches either equipped with porous (15 nm in diameter) or poreless polycarbonate (PC) membranes, it was possible to differentiate between overall virus inactivation and the effect of virucidal agents that act through poreless PC membranes. Depending on ambient temperature, pH, and type of animal waste, values for time, in days, required for a 90% reduction of virus titer varied widely, ranging from less than 1 week for herpesvirus to more than 6 months for rotavirus. Virus inactivation progressed substantially faster in liquid cattle manure, a mixture of urine and water (pH > 8.0), than in semiliquid wastes that consisted of mixtures of feces, urine, water, and bedding materials (pH < 8.0). Hitherto unidentified virucidal agents that permeate poreless PC membranes contributed substantially to the overall inactivation. On the other hand, substances that protect rotavirus and possibly other viruses from inactivation may be present in animal wastes. Together, the study showed that viruses contained in manure may persist for prolonged periods of time if stored under nonaerated conditions. At times of land application, this may lead to environmental contamination with pathogens.     

Simian rotavirus SA11 replication in cell cultures.

Understanding the basic virology of rotavirus infections has been hampered by the fastidiousness of most isolates and by the lack of a rapid quantitative assay method. The growth characteristics of the simian rotavirus SA11 were studied because it grows to high titers in tissue culture and infectivity can be quantitated by plaque assay. SA11 replication was analyzed in a variety of primary cell cultures or continuous cell lines derived from both homologous and heterologous hosts. Viral replication was observed in each of the cell cultured examined. The individual cell cultures demonstrated marked variability in their susceptibility to rotavirus infection. The highest titers were obtained with MA104, BSC-1, CV-1, and BGM cells. Observable cytopathic effect was found to correlate with the percentage of infected cells in the culture. This study presents growth curves of the simian rotavirus in a variety of cell cultures.     

Development of a method for detection of human rotavirus in water and sewage.

The simian rotavirus SA11 was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA11 adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter-bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SA11 was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SA11 serum which would detect the simian, human bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SA11. The buoyant density of the sewage isolates in CsCl gradients was 1.36 g/cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 +/- 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.     

Development of a method for detection of human rotavirus in water and sewage

The simian rotavirus SA11 was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA11 adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter-bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SA11 was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SA11 serum which would detect the simian, human bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SA11. The buoyant density of the sewage isolates in CsCl gradients was 1.36 g/cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 +/- 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.     

Penetration of different human pathogenic viruses into sand columns percolated with distilled water, groundwater, or wastewater

The adsorption of several enteroviruses and rotavirus SA11 to sand from an aquifer in the Federal Republic of Germany was estimated in sand-filled columns loaded with ca. 10(7) PFU and run at a velocity of 2.5 m/day for 12 h. After either distilled water, groundwater, secondary effluent, or tertiary effluent was percolated, the sand core was slowly extruded out of the column and cut in 1-cm slices. The slices were eluted with nutrient broth, and the amount of viruses in the broth was estimated. The best adsorption was promoted by groundwater and tertiary effluent, followed by distilled water and secondary effluent. Similar experiments, carried out at different percolation rates, indicated that a 50-day underground stay of recharged water probably suffices to eliminate viruses in the groundwater-recharged tertiary effluent. However, when viruses and sand were incubated in the presence of the surfactants sodium dodecyl sulfate, nonyl phenol, dodigen 226, or alkylbenzylsulfonate, the adsorption of the viruses was substantially diminished. Experiments in the presence of nonyl phenol seem to indicate that hydrophobic interactions are involved in the adsorption of viruses to sand.     

Penetration of different human pathogenic viruses into sand columns percolated with distilled water, groundwater, or wastewater.

The adsorption of several enteroviruses and rotavirus SA11 to sand from an aquifer in the Federal Republic of Germany was estimated in sand-filled columns loaded with ca. 10(7) PFU and run at a velocity of 2.5 m/day for 12 h. After either distilled water, groundwater, secondary effluent, or tertiary effluent was percolated, the sand core was slowly extruded out of the column and cut in 1-cm slices. The slices were eluted with nutrient broth, and the amount of viruses in the broth was estimated. The best adsorption was promoted by groundwater and tertiary effluent, followed by distilled water and secondary effluent. Similar experiments, carried out at different percolation rates, indicated that a 50-day underground stay of recharged water probably suffices to eliminate viruses in the groundwater-recharged tertiary effluent. However, when viruses and sand were incubated in the presence of the surfactants sodium dodecyl sulfate, nonyl phenol, dodigen 226, or alkylbenzylsulfonate, the adsorption of the viruses was substantially diminished. Experiments in the presence of nonyl phenol seem to indicate that hydrophobic interactions are involved in the adsorption of viruses to sand.     

Solar Disinfection of Viruses in Polyethylene Terephthalate Bottles

Solar disinfection (SODIS) of drinking water in polyethylene terephthalate (PET) bottles is a simple, efficient point-of-use technique for the inactivation of many bacterial pathogens. In contrast, the efficiency of SODIS against viruses is not well known. In this work, we studied the inactivation of bacteriophages (MS2 and ϕX174) and human viruses (echovirus 11 and adenovirus type 2) by SODIS. We conducted experiments in PET bottles exposed to (simulated) sunlight at different temperatures (15, 22, 26, and 40°C) and in water sources of diverse compositions and origins (India and Switzerland). Good inactivation of MS2 (>6-log inactivation after exposure to a total fluence of 1.34 kJ/cm²) was achieved in Swiss tap water at 22°C, while less-efficient inactivation was observed in Indian waters and for echovirus (1.5-log inactivation at the same fluence). The DNA viruses studied, ϕX174 and adenovirus, were resistant to SODIS, and the inactivation observed was equivalent to that occurring in the dark. High temperatures enhanced MS2 inactivation substantially; at 40°C, 3-log inactivation was achieved in Swiss tap water after exposure to a fluence of only 0.18 kJ/cm². Overall, our findings demonstrate that SODIS may reduce the load of single-stranded RNA (ssRNA) viruses, such as echoviruses, particularly at high temperatures and in photoreactive matrices. In contrast, complementary measures may be needed to ensure efficient inactivation during SODIS of DNA viruses resistant to oxidation.     

Initial interaction of rotavirus strains with N-acetylneuraminic (sialic) acid residues on the cell surface correlates with VP4 genotype, not species of origin

We examined 41 human and animal rotavirus strains representative of all known P genotypes for their dependency on cellular N-acetylneuraminic (sialic) acid (SA) residues for infectivity. Our results showed that all rotaviruses studied, whether of animal or human origin, belonging to P genotypes [1], [2], [3], and [7] depended on SA residues on the cell surface for efficient infectivity but that all human and animal rotavirus strains representative of the remaining known P genotypes were SA independent. The SA residue requirement for efficient infectivity did not change for reassortant rotavirus strains with altered VP4-VP7 combinations. The initial interaction of rotavirus strains with SA residues on the cell surface correlated with VP4 genotype specificity, not with species of origin or VP7 G serotype specificity (P = 0.001; r2 = 1.00, Pearson's correlation coefficient). In addition to being a requirement for infectivity, the presence of SA residues on the cell surface is a requirement for efficient growth in cell culture; recognition of the association of specific P genotypes with the binding of rotavirus to SA residues will facilitate our understanding of the molecular basis of the early events of rotavirus-cell interactions in cell culture models and of pathogenicity in vivo.     

Viral replication, persistence in water and genetic characterization of two influenza A viruses isolated from surface lake water

Water-borne transmission has been suggested as an important transmission mechanism for Influenza A (IA) viruses in wild duck populations; however, relatively few studies have attempted to detect IA viruses from aquatic habitats. Water-isolated viruses have rarely been genetically characterized and evaluation for persistence in water and infectivity in natural hosts has never been documented. In this study, we focused on two IA viruses (H3N8 and H4N6 subtypes) isolated from surface lake water in Minnesota, USA. We investigated the relative prevalence of the two virus subtypes in wild duck populations at the sampling site and their genetic relatedness to IA viruses isolated in wild waterbirds in North America. Viral persistence under different laboratory conditions (temperature and pH) and replication in experimentally infected Mallards (Anas platyrhynchos) were also characterized. Both viruses were the most prevalent subtype one year following their isolation in lake water. The viruses persisted in water for an extended time period at constant temperature (several weeks) but infectivity rapidly reduced under multiple freeze-thaw cycles. Furthermore, the two isolates efficiently replicated in Mallards. The complete genome characterization supported that these isolates originated from genetic reassortments with other IA viruses circulating in wild duck populations during the year of sampling. Based on phylogenetic analyses, we couldn't identify genetically similar viruses in duck populations in the years following their isolation from lake water. Our study supports the role for water-borne transmission for IA viruses but also highlights that additional field and experimental studies are required to support inter-annual persistence in aquatic habitats.     

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.     

An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.     

Effects of environmental variables and soil characteristics on virus survival in soil

Because of the increasing emphasis placed upon land application as a means of wastewater disposal, it is important to evaluate the influences of different factors upon virus survival in soil. The objective of this study was to measure the effects of various environmental variables on virus persistence. Test samples of soil were placed in vials, and the soil was wetted with suspensions of virus in either distilled water, unchlorinated secondary sewage effluent, or mixtures of effluent and water. The viruses used were coxsackieviruses A9 and B3, echovirus 1, poliovirus 2, rotavirus SA11, and bacteriophages T2 and MS2. The rate of virus inactivation was evaluated statistically with regard to conditions under which the vials were incubated and to the soil characteristics. The factors that were found to influence virus survival were temperature, soil moisture content, presence of aerobic microorganisms, degree of virus adsorption to the soil, soil levels of resin-extractable phosphorus, exchangeable aluminium, and soil pH. Overall, temperature and virus adsorption to soil appeared to be the most important factors affecting virus survival.