Isolation of enterovirus and reovirus from sewage and treated effluents in selected Puerto Rican communities

Sewage treatment plant effluents were surveyed for viral contributions to gastroenteritis outbreaks in Puerto Rico. Of the 15 sewage treatment plants studied, all discharged their effluents upstream from water treatment plant intakes. No base-line data on the degree of viral challenge to these sewage treatment plants or the subsequent reduction of viruses before discharge existed. Enterovirus counts were generally much higher than those found in the continental United States. At four plants, viruses in the incoming sewage exceeded 100,000 PFU/liter, and one of these, a trickling filter plant, was discharging 24,000 PFU/liter to receiving waters. Virus identification showed that more than 80% of the enterovirus isolates were coxsackievirus B5. These overwhelming viral numbers pointed to defects in the sewage treatment processes. Without reasonable barriers to protect receiving waters, several of the downstream communities were using raw waters that posed extraordinary demands on the ability of their water treatment plants to supply virologically safe drinking water.     

Isolation of enterovirus and reovirus from sewage and treated effluents in selected Puerto Rican communities.

Sewage treatment plant effluents were surveyed for viral contributions to gastroenteritis outbreaks in Puerto Rico. Of the 15 sewage treatment plants studied, all discharged their effluents upstream from water treatment plant intakes. No base-line data on the degree of viral challenge to these sewage treatment plants or the subsequent reduction of viruses before discharge existed. Enterovirus counts were generally much higher than those found in the continental United States. At four plants, viruses in the incoming sewage exceeded 100,000 PFU/liter, and one of these, a trickling filter plant, was discharging 24,000 PFU/liter to receiving waters. Virus identification showed that more than 80% of the enterovirus isolates were coxsackievirus B5. These overwhelming viral numbers pointed to defects in the sewage treatment processes. Without reasonable barriers to protect receiving waters, several of the downstream communities were using raw waters that posed extraordinary demands on the ability of their water treatment plants to supply virologically safe drinking water.     

Environmental surveillance and molecular characterization of human enteric viruses in tropical urban wastewaters

Aims: To study the prevalence and genotypes of waterborne pathogenic viruses in urban wastewaters in the tropical region. Methods and Results: Viruses in wastewaters collected at three water reclamation plants in Singapore were studied by molecular methods. Over a 6‐month sampling period, adenoviruses, astroviruses and both norovirus genogroups I (GI) and II (GII) were detected in 100% of the sewage and secondary effluent. Enteroviruses and hepatitis A viruses (HAV) were found in 94 and 78% of sewage, and 89 and 28% of secondary effluent, respectively. By using quantitative real‐time PCR, estimated concentrations of astrovirus in the sewage were 1–2 orders of magnitude higher than those for adenovirus, noroviruses GI and GII. Genotyping of environmental isolates revealed multiple genotypes of GI and GII noroviruses. Coxsackieviruses A, astrovirus type 1 and adenovirus type 41 were prevalent. Norovirus GII/4 and coxsackievirus A24 isolates in wastewaters were closely related to respective outbreak strains isolated previously in Singapore. Conclusions: This study showed the widespread occurrence of all tested enteric virus groups in urban wastewaters. Genetic diversity of astroviruses, enteroviruses and noroviruses in the tropical region was observed. Significance and Impact of the Study: The high prevalence and great genetic diversity of human enteric viruses in urban wastewaters strongly supports the need of further comprehensive studies for evaluating the public health risk associated with viral pathogens in water environments.     

Human norovirus occurrence and diversity in the Llobregat river catchment, Spain

Human noroviruses (NoV) were quantified and characterized in an 18 month survey conducted along the Llobregat river catchment in Spain. Sample types included freshwater, untreated and treated wastewater and drinking water. High NoV genome copy numbers were reported, reaching up to 10(6) l(-1) and 10(9) l(-1) in freshwater and raw sewage respectively. In both types of samples, GII NoV genome copies outnumbered those of GI, although without significance. All samples of semi-treated and treated drinking water were negative for NoV. A clear seasonality of NoV occurrence was observed both in river water and sewage samples, with significantly higher genome copy numbers in the cold than in the warm months period. Mean NoV log reduction rates after biological treatment of sewage were 2.2 and 3.1 for GI and GII respectively. A total of 77 NoV strains isolated in the Llobregat river catchment could be phylogenetically characterized, 44 belonging to GI and 33 to GII. The most prevalent genotype was GI.4, followed by GII.4 and GII.21. Several variants of the pandemic GII.4 strain were detected in the environment, corroborating their circulation among the population.     

Long-Term Inactivation Study of Three Enteroviruses in Artificial Surface and Groundwaters, Using PCR and Cell Culture

Since the transmission of pathogenic viruses via water is indistinguishable from the transmission via other routes and since the levels in drinking water, although significant for health, may be too low for detection, quantitative viral risk assessment is a useful tool for assessing disease risk due to consumption of drinking water. Quantitative viral risk assessment requires information concerning the ability of viruses detected in drinking water to infect their host. To obtain insight into the infectivity of viruses in relation to the presence of virus genomes, inactivation of three different enteroviruses in artificial ground and surface waters under different controlled pH, temperature, and salt conditions was studied by using both PCR and cell culture over time. In salt-peptone medium, the estimated ratio of RNA genomes to infectious poliovirus 1 in freshly prepared suspensions was about 10⁰. At 4°C this ratio was 10³ after 600 days, and at 22°C it was 10⁴ after 200 days. For poliovirus 1 and 2 the RNA/infectious virus ratio was higher in artificial groundwater than in artificial surface water, but this was not the case for coxsackievirus B4. When molecular detection is used for virus enumeration, it is important that the fraction of infectious virus (based on all virus genomes detected) decays with time, especially at temperatures near 22°C.     

Environmental Transmission of Human Noroviruses in Shellfish Waters

Human noroviruses (NoV) are the most common cause of epidemic gastroenteritis following consumption of bivalve shellfish contaminated with fecal matter. NoV levels can be effectively reduced by some sewage treatment processes such as activated sludge and membrane bioreactors. However, tertiary sewage treatment and substantial sewage dilution are usually required to achieve low concentrations of virus in shellfish. Most outbreaks have been associated with shellfish harvested from waters affected by untreated sewage from, for example, storm overflows or overboard disposal of feces from boats. In coastal waters, NoV can remain in suspension or associate with organic and inorganic matter and be accumulated by shellfish. Shellfish take considerably longer to purge NoV than fecal indicator bacteria when transferred from sewage-polluted estuarine waters to uncontaminated waters. The abundance and distribution of NoV in shellfish waters are influenced by the levels of sewage treatment, proximity of shellfish beds to sewage sources, rainfall, river flows, salinity, and water temperature. Detailed site-specific information on these factors is required to design measures to control the viral risk.     

F-specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water.

Culturable enteroviruses were detected by applying concentration techniques and by inoculating the concentrates on the BGM cell line. Samples were obtained from a wide variety of environments, including raw sewage, secondary effluent, coagulated effluent, chlorinated and UV-irradiated effluents, river water, coagulated river water, and lake water. The virus concentrations varied widely between 0.001 and 570/liter. The same cell line also supported growth of reoviruses, which were abundant in winter (up to 95% of the viruses detected) and scarce in summer (less than 15%). The concentrations of three groups of model organisms in relation to virus concentrations were also studied. The concentrations of bacteria (thermotolerant coliforms and fecal streptococci) were significantly correlated with virus concentrations in river water and coagulated secondary effluent, but were relatively low in disinfected effluents and relatively high in surface water open to nonhuman fecal pollution. The concentrations of F-specific RNA bacteriophages (FRNA phages) were highly correlated with virus concentrations in all environments studied except raw and biologically treated sewage. Numerical relationships were consistent over the whole range of environments; the regression equations for FRNA phages on viruses in river water and lake water were statistically equivalent. These relationships support the possibility that enteric virus concentrations can be predicted from FRNA phage data.     

Absence of therapeutic blood concentrations of tetracycline in rats after administration in drinking water

Because of ease of administration and broad antibacterial spectrum, tetracycline often is administered in drinking water to control infectious diseases of rats. Assay of serum after a gavage bolus of tetracycline (300 mg/kg body weight) revealed little absorption of tetracycline by this route. Rats were given water containing tetracycline at several concentrations (400 mg/liter, 4g/liter, and 4 g tetracycline plus 50 g sucrose/liter) ad libitum and serum concentrations of tetracycline were monitored. Bioassay of serum samples from these animals, taken during 72 hours of water medication, revealed no detectable tetracycline concentrations (greater than 0.2 mcg/ml) in the 400 mg and 4 g/liter groups. Two of eighteen serum samples from the group given 4 g tetracycline with 50 g sucrose/liter had minimal therapeutic tetracycline concentrations (0.3 mcg/ml) effective for Mycoplasma pulmonis. Some of the animals given tetracycline ad libitum in drinking water drank very little and lost weight compared to control animals. These findings indicate that the practice of adding tetracycline to drinking water of rats may be ineffective in controlling systemic diseases, and also be detrimental to the treated animals.     

Inactivation Kinetics and Mechanism of a Human Norovirus Surrogate on Stainless Steel Coupons via Chlorine Dioxide Gas

Acute gastroenteritis caused by human norovirus is a significant public health issue. Fresh produce and seafood are examples of high-risk foods associated with norovirus outbreaks. Food contact surfaces also have the potential to harbor noroviruses if exposed to fecal contamination, aerosolized vomitus, or infected food handlers. Currently, there is no effective measure to decontaminate norovirus on food contact surfaces. Chlorine dioxide (ClO₂) gas is a strong oxidizer and is used as a decontaminating agent in food processing plants. The objective of this study was to determine the kinetics and mechanism of ClO₂ gas inactivation of a norovirus surrogate, murine norovirus 1 (MNV-1), on stainless steel (SS) coupons. MNV-1 was inoculated on SS coupons at the concentration of 10⁷ PFU/coupon. The samples were treated with ClO₂ gas at 1, 1.5, 2, 2.5, and 4 mg/liter for up to 5 min at 25°C and a relative humidity of 85%, and virus survival was determined by plaque assay. Treatment of the SS coupons with ClO₂ gas at 2 mg/liter for 5 min and 2.5 mg/liter for 2 min resulted in at least a 3-log reduction in MNV-1, while no infectious virus was recovered at a concentration of 4 mg/liter even within 1 min of treatment. Furthermore, it was found that the mechanism of ClO₂ gas inactivation included degradation of viral protein, disruption of viral structure, and degradation of viral genomic RNA. In conclusion, treatment with ClO₂ gas can serve as an effective method to inactivate a human norovirus surrogate on SS contact surfaces.     

Prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments

Aims: To study the virological quality of surface water from highly urbanized tropical water catchment areas and to determine predominant enteric viral genotypes in surface water. Methods and Results: A wide range of human pathogenic viruses in urban surface waters was screened by nested PCR assays after concentration by ultrafiltration. Among the 84 water samples collected, at least one virus was detected in 70 (83·3%) of these samples. Noroviruses were determined to be the most prevalent enteric viruses detected in urban surface water samples, followed by astroviruses, enteroviruses, adenoviruses and hepatitis A viruses. The molecular characterization of environmental viral isolates suggested co‐circulation of multiple genotypes of both noroviruses GI and GII, astroviruses and enteroviruses in urban surface waters. Conclusions: Human enteric viruses with great genetic diversity were detected in surface waters, indicating the presence of human origin of faecal contamination in highly urbanized water catchment areas. Significance and Impact of the Study: The present study identifies and characterizes potential viral hazards of source waters for drinking water supply and recreational activities. This will enable scientific decisions to be made regarding the selection and prioritization of human pathogenic viruses to be included in the future risk assessment and treatment evaluation for water and wastewater.     

Chlorination of indicator bacteria and viruses in primary sewage effluent

Wastewater disinfection is used in many countries for reducing fecal coliform levels in effluents. Disinfection is therefore frequently used to improve recreational bathing waters which do not comply with microbiological standards. It is unknown whether human enteric viruses (which are responsible for waterborne disease) are simultaneously inactivated alongside fecal coliforms. This laboratory study focused on the chlorination of primary treated effluent with three doses (8, 16, and 30 mg/liter) of free chlorine as sodium hypochlorite. Seeding experiments showed that inactivation (>5 log(10) units) of Escherichia coli and Enterococcus faecalis was rapid and complete but that there was poor inactivation (0.2 to 1.0 log(10) unit) of F(+)-specific RNA (FRNA) bacteriophage (MS2) (a potential virus indicator) at all three doses. However, seeded poliovirus was significantly more susceptible (2.8 log(10) units) to inactivation by chlorine than was the FRNA bacteriophage. To ensure that these results were not artifacts of the seeding process, comparisons were made between inactivation rates of laboratory-seeded organisms in sterilized sewage and inactivation rates of organisms occurring naturally in sewage. Multifactorial analysis of variance showed that there was no significant difference (P > 0.05) between the inactivation rates for seeded and naturally occurring FRNA bacteriophage. However, laboratory-grown poliovirus was inactivated much more rapidly than were naturally occurring, indigenous enteroviruses (P < 0.001). This may reflect differences in the way indigenous virus is presented to the disinfectant. Inactivation rates for indigenous enteroviruses were quite similar to those seen for FRNA bacteriophage at lower doses of chlorine. These results have significance for the effectiveness of chlorination as a sewage treatment process, particularly where virus contamination is of concern, and suggest that FRNA bacteriophage would be an appropriate indicator of such viral inactivation under field conditions.     

Removal of trace metal ions from industrial waters, nuclear effluents and drinking water, with the aid of cross-linked N-carboxymethyl chitosan

Chitoplex (cross-linked N-carboxymethyl chitosan) is particularly effective in removing trace metal ions (0·01 mg/liter) from brines (50 g/liter), carrier-free radioisotopes such as cobalt-60 from nuclear effluents, and toxic contaminants such as lead and cadmium (0·01 mg/liter) from drinking waters. Chitoplex is indifferent to large concentrations of magnesium.     

Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006

Noroviruses are the causative agents of the majority of viral gastroenteritis outbreaks in humans. During the past 15 years, noroviruses of genotype GGII.4 have caused four epidemic seasons of viral gastroenteritis, during which four novel variants (termed epidemic variants) emerged and displaced the resident viruses. In order to understand the mechanisms and biological advantages of these epidemic variants, we studied the genetic changes in the capsid proteins of GGII.4 strains over this period. A representative sample was drawn from 574 GGII.4 outbreak strains collected over 15 years of systematic surveillance in The Netherlands, and capsid genes were sequenced for a total of 26 strains. The three-dimensional structure was predicted by homology modeling, using the Norwalk virus (Hu/NoV/GGI.1/Norwalk/1968/US) capsid as a reference. The highly significant preferential accumulation and fixation of mutations (nucleotide and amino acid) in the protruding part of the capsid protein provided strong evidence for the occurrence of genetic drift and selection. Although subsequent new epidemic variants differed by up to 25 amino acid mutations, consistent changes were observed in only five positions. Phylogenetic analyses showed that each variant descended from its chronologic predecessor, with the exception of the 2006b variant, which is more closely related to the 2002 variant than to the 2004 variant. The consistent association between the observed genetic findings and changes in epidemiology leads to the conclusion that population immunity plays a role in the epochal evolution of GGII.4 norovirus strains.