Human enteric viruses in a wastewater treatment plant: evaluation of activated sludge combined with UV disinfection process reveals different removal performances for viruses with different features.

This study assess the quality of wastewater through the detection and quantification of important viruses causing gastroenteritis at different stages of the wastewater treatment process in an activated-sludge wastewater treatment plant with ultraviolet disinfection. Ten sampling events were carried out in a campaign along a period of 18 months collecting wastewater samples from the influent, after the activated-sludge treatment, and after the final disinfection with UV radiation. Samples were concentrated through ultracentrifugation and analysed using retro-transcription, PCR and real time quantitative PCR protocols, for detection and quantification of Group A Rotavirus (RVA), Human Astrovirus (HAstV), Norovirus Genogroup II (NoV GII) and Human Adenovirus (HAdV). HAdV (100%), NoV GII (90%), RVA (70%) and HAstV (60%) were detected in influent samples with concentration from 1·4 (NoV GII) to 8·0 (RVA) log10  gc l-1 . Activated-sludge treatment reached well quality effluents with low organic material concentration, although nonstatistical significant differences were registered among influent and postactivated sludge treatment samples, regarding the presence and concentration for most viruses. All post-UV samples were negative for NoV GII and HAstV, although RVA and HAdV were detected in 38% and 63% of those samples respectively, with concentration ranging from 2·2 to 5·5 and 3·1 to 3·4 log10  gc l-1 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that an activated-sludge wastewater treatment plant with UV disinfection reduces to levels below the detection limit those single-stranded RNA viruses as noroviruses and astroviruses and reach significant lower levels of rotaviruses and adenoviruses after the complete treatment process.

The efficacy of simulated solar disinfection (SODIS) against coxsackievirus, poliovirus and hepatitis A virus.

The antimicrobial activity of simulated solar disinfection (SODIS) against enteric waterborne viruses including coxsackievirus-B5, poliovirus-2 and hepatitis A virus was investigated in this study. Assays were conducted in transparent 12-well polystyrene microtitre plates containing the appropriate viral test suspension. Plates were exposed to simulated sunlight at an optical irradiance of 550 Wm(-2) (watts per square metre) delivered from a SUNTEST™ CPS+ solar simulator for 6 hours. Aliquots of the viral test suspensions were taken at set time points and the level of inactivation of the viruses was determined by either culture on a HeLa cell monolayer for coxsackievirus-B5 and poliovirus-2 or by utilising a chromogenic antibody-based approach for hepatitis A virus. With coxsackievirus-B5, poliovirus-2 and hepatitis A virus, exposure to SODIS at an optical irradiance of 550 Wm(-2) for 1-2 hours resulted in complete inactivation of each virus. The findings from this study suggest that under appropriate conditions SODIS may be an effective technique for the inactivation of enteric viruses in drinking water. However, further verification studies need to be performed using natural sunlight in the region where the SODIS technology is to be employed to validate our results.

Evaluation of resistance development and viability recovery by a non-enveloped virus after repeated cycles of aPDT.

Nowadays, the emergence of drug resistant microorganisms is a public health concern. The antimicrobial photodynamic therapy (aPDT) has an efficient action against a wide range of microorganisms and can be viewed as an alternative approach for treating microbial infections. The aim of this study was to determine if a model target virus (T4-like bacteriophage), in the presence of the tricationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py(+)-Me-PF), can develop resistance to aPDT and recover its viability after photodynamic treatments. To assess the development of aPDT resistance after repeated treatments, a suspension of T4-like bacteriophage was irradiated with white light (40 Wm(-2)) for 120 min in the presence of 5.0 µM of Tri-Py(+)-Me-PF (99.99% of inactivation) and new phage suspensions were produced from the surviving phages, after each cycle of light exposure. The procedure was repeated ten times. To evaluate the recovery of viral viability after photoinactivation, a suspension of T4-like bacteriophage was irradiated with white light for 120 min in the presence of 5.0 µM of Tri-Py(+)-Me-PF on five consecutive days. In each day, an aliquot of the irradiated suspension was plated and the number of lysis plaques was counted after 24, 48, 72, 96 and 120 h of dark incubation at 37 °C. The profile of bacteriophage photoinactivation did not change after ten consecutive cycles and no recovery of viability was detected after five accumulated cycles of photodynamic treatment. The results suggest that aPDT represents a valuable and promising alternative therapy to treat viral infections, overcoming the problem of microbial resistance.

Comparative reductions of bacterial indicators, bacteriophage-infecting enteric bacteria and enteroviruses in wastewater tertiary treatments by lagooning and UV-radiation.

A two-year monitoring program of microbiological and physical-chemical parameters at 2 waste water treatment plants (WWTPs) in Mallorca (Spain) was performed in order to (1) evaluate the efficiency of lagooning and UV radiation as tertiary treatment processes; (2) determine the characteristics of wastewater effluent for its potential agricultural reuse; and (3) establish correlations between bacteriological and virological parameters. The presence of currently established bacterial indicators (total coliforms, faecal coliforms, Escherichia coli, enterococci, and spores of sulphite-reducing clostridia), virological (enteroviruses, somatic coliphages, F-specific coliphages, and phages infecting Bacteroides fragilis and Bacteroides thetaiotaomicron), and helminth eggs were tested during this study. Bacterial and viral indicators were removed at least with one log reduction in the lagooning system, and to a lesser extent with UV-radiation treatment. The lagooning system was less efficient in removing phages and viruses than were bacterial indicators, with the exception of F-specific phages. Phages of B. fragilis and B. thetaiotaomicron were less removed than all of the other microbiological parameters. In the UV-radiation treatment, however, the faecal coliforms proved the most sensitive, while clostridial spores, somatic coliphages, Bacteroides phages, and enteric viruses were the more resistant. Helminth eggs were not detected in any samples from effluents of either the secondary or tertiary treatments.Indicator levels in both treatments met the established regulations of both local and national authorities for the disposal or reuse of wastewater in irrigation for non-human crop. We demonstrate that somatic coliphages are effective indicators of enteric viruses in both of the WWTPs studied.

Phosphorylation of PI3K/Akt and MAPK/ERK in an early entry step of enterovirus 71.

Viruses have been known to subvert the anti-apoptotic pathways of the host cell in order to delay apoptosis. However, the mechanisms utilized by enterovirus 71 (EV71) to mediate anti-apoptotic activity remained undetermined. We observed that EV71 infection induced an early activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK/ERK signaling pathways. The activity of GSK3beta, a downstream target of these pathways, was negatively regulated by the activation of both MAPK/ERK and PI3K/Akt. The phosphorylation of GSK3 could be inhibited by treatment with the specific inhibitors of MAPK/ERK and PI3K/Akt. Other Akt downstream targets, BAD, caspase-9 and the Forkhead transcription factor (FKHR), were not phosphorylated during the course of infection by EV71. We further demonstrated that infection by UV-irradiated, inactivated virus triggered early Akt activation but was insufficient to trigger late Akt activation. These data suggest that with the phosphorylation of MAPK/ERK and PI3K/Akt the subsequent inactivation of GSK3beta is utilized by EV71 as a potential mechanism to delay host cell apoptosis.

Human endothelial cell activation and apoptosis induced by enterovirus 71 infection.

Enterovirus 71 (EV71), a neurotropic virus, its infection is transmitted mainly by the oral-fecal route. However, it is unclear how EV71 is disseminated/spread from initial replication sites to the central nervous system. Since endothelial cells form the interface between blood and tissues throughout the body, it is likely that EV71 can infect and then exit endothelial cells to establish infection. In this study, human endothelial cells were examined for susceptibility to EV71 infection using human microvascular endothelial cell line (HMEC-1 cell). Immunofluorescence assay confirmed EV71 infection of HMEC-1. Viable viruses were cultured from both the culture supernatant and the cell lysate. Live but not UV-inactivated EV71 induced HMEC-1 to secrete IL-6, macrophage migration inhibition factor, and macrophage chemo-attractant protein 1, and to express toll-like receptor 4. In addition, EV71 decreased the viability and increased the apoptosis of HMEC-1 cells after 36-48 hr of infection. These results demonstrate that EV71 is able to infect, activate, and induce apoptosis of endothelial cells, which may play a role in the pathogenesis of EV71 infection.

Comparative inactivation of enteroviruses and adenovirus 2 by UV light.

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm(2), respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.

Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21(ras) GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.

Ultraviolet sterilisation of model viruses important to finfish aquaculture in Australia.

Mortalities approaching 100% have been recorded in larval barramundi and cultured rainbow trout. These have been attributed to a picorna-like virus and an iridovirus, respectively. Two Australian-made ultraviolet sterilising units were tested for their effectiveness in inactivating water-suspended model viruses. These were an iridovirus isolated from frogs and a picornavirus, bovine enterovirus, both of similar structure and size to the pathogenic fish viruses. Both viruses were inactivated by both ultraviolet units at a flow rate comparable to that used in nurseries in aquaculture (5000 L/h). With transmittance reduced to 27.7%, the minimum effective dose was 2.6 x 10(4) uW.sec/cm2 for both viruses.

Survival and disinfection of adenovirus type 19 and enterovirus 70 in ophthalmic practice.

In an experimental model designed to simulate actual clinical situations, the risk period of adenovirus type 19 (AdV-19) and enterovirus 70 (EV-70) transmission by contaminated instruments has been investigated, as well as the effective methods of disinfection against these viruses. No EV-70 could be detected 6 hours after inoculation on applanation tips contaminated with viruses and naturally dried, indicating that drying the instruments and keeping them in a moisture-free environment is a convenient disinfection procedure against EV-70. In contrast, AdV-19 maintained its initial infectious level for 11 days on contaminated and subsequently dried tips. Both viruses when placed in eyedrop solutions at room temperature sustained their infectivity for more than 20 days. It was found that heating at 90 degrees C for 5 seconds is the most simple and effective disinfection against AdV-19 and EV-70. AdV-19 was about 60 times more resistant to ultraviolet irradiation than EV-70.

The inactivation of a bovine enterovirus and a bovine parvovirus in cattle manure by anaerobic digestion, heat treatment, gamma irradiation, ensilage and composting.

A bovine enterovirus and a bovine parvovirus seeded into liquid cattle manure were rapidly inactivated by anaerobic digestion under thermophilic conditions (55 degrees C), but the same viruses survived for up to 13 and 8 days respectively under mesophilic conditions (35 degrees C). The enterovirus was inactivated in digested liquid manure heated to 70 degrees C for 30 min, but the parvovirus was not inactivated by this treatment. The enterovirus, seeded into single cell protein (the solids recovered by centrifugation of digested liquid manure), was inactivated by a gamma irradiation dose of 1.0 Mrad, but the parvovirus survived this dose. When single cell protein seeded with bovine enterovirus or bovine parvovirus was ensiled with cracked corn, the enterovirus was inactivated after a period of 30 days, while the parvovirus survived for 30 days in one of two experiments. Neither the enterovirus nor the parvovirus survived composting for 28 days in a thermophilic aerobic environment when seeded into the solid fraction of cattle manure. It was concluded that, of the procedures tested, only anaerobic digestion under thermophilic conditions appeared to be reliable method of viral inactivation to ensure the safety of single cell protein for refeeding to livestock. Composting appeared to be a suitable method for the disinfection of manure for use as a soil conditioner.

[Virological analyses of irradiated sewer sludge (author's transl)]. / Virologische Untersuchungen an bestrahlten Klärschlämmen.

The virus-inactivating effect of Co60 irradiation was verified by a field test in the sewer sludge irradiation plant at Geiselbullach near Munich. The following samples were tested: 16 waste water inlets 15 waste water outlets 32 fresh sludge samples 62 sewer sludge samples prior to irradiation 52 sewer sludge samples and 9 fresh sludge samples after irradiation. We supplemented these analyses by inoculating sewer sludge samples with polio inoculation virus type 1 before irradiation and by the admixing of virus-pure suspensions in MEM + 2 per cent fetal calf serum and virus sludge mixtures packed in plastic capsules with the material to be irradiated. After irradiation these capsules were picked up again and analyzed for their viral content. As test viruses we selected polio wild virus type 1, echovirus type 6, coxsackie virus B5, coxsackie virus A9 and adeno virus type 1. After the sludge samples had been sufficiently homogenized by an adequately long decaying period and provided they contained 3-per cent solid matter, we managed to obtain the same irradiation results in field tests, as with tests under laboratory conditions. For entero viruses the D-value (decimal reduction) was 300 to 400 krad, for adeno viruses it amounted to 700 krad.

Gamma radiation inactivation of coxsackievirus B-2.

The radioresistance of coxsackievirus B-2 was studied when the virus was suspended in Eagle minimal essential medium, distilled water, cooked ground beef, and raw ground beef and irradiated at various temperatures in a cobalt-60 gamma radiation source. The number of surviving viruses at given doses of radiation was determined by a plaque assay system. All destruction curves indicated a first-order reaction. When the virus was irradiated in minimal essential medium at temperatures of -30, -60, and -90 C, D values (in Mrad) were 0.69, 0.59, and 0.64, respectively. When the virus was suspended in water and irradiated at -90 C, the D value was 0.53. Cooked ground beef containing the virus was irradiated at temperatures ranging from 16 to -90 C. The D values were 0.70 (16 C), 0.76 (0.5 C), 0.68 (-30 C), 0.78 (-60 C), and 0.81 (-90 C). Raw ground beef containing the virus was irradiated at -30, -60, and -90 C, and the D values were respectively 0.75, 0.71, and 0.68. The D values indicate that the rate of viral inactivation was dependent on the suspending menstrum.

Ultraviolet devitalization of eight selected enteric viruses in estuarine water.

The effect of ultraviolet (UV) radiation on the devitalization of eight selected enteric viruses suspended in estuarine water was determined. The surviving fractions of each virus were calculated and then plotted against the UV exposure time for purposes of comparison. Analytical assessment of the survival data for each virus consisted of least squares regression analysis for determination of intercepts and slope functions. All data were examined for statistical significance. When the slope function of each virus was compared against the slope function of poliovirus type 1, the analytical findings indicated that poliovirus types 2 and 3, echovirus types 1 and 11, and coxsackievirus A-9 exhibited similar devitalization characteristics in that no statistically significant difference was found (P > 0.05). Conversely, the devitalization characteristics of coxsackievirus B-1 and reovirus type 1 were dissimilar from those of poliovirus type 1 in that a statistically significant difference was found between the slope functions (P < 0.05). This observed difference in devitalization of coxsackievirus B-1 and reovirus type 1 was attributed primarily to the frequency distribution of single and aggregate virions, the geometric configuration, the size of the aggregates, and the severity of aggregation. The devitalization curve of coxsackievirus B-1 was characteristic of a retardant die-away curve. The devitalization curve of reovirus type 1 was characteristic of a multihittype curve. The calculated devitalization half-life values for poliovirus types 1, 2, and 3; echovirus types 1 and 11; coxsackievirus types A-9 and B-1; and reovirus type 1 were 2.8, 3.1, 2.7, 2.8, 3.2, 3.1, 4.0, 4.0 sec, respectively. These basic data should facilitate an operative extrapolation of the findings to the applied situation. It was concluded that UV can be highly effective and provide a reliable safety factor in treating estuarine water.

Lasting biological effects of early environmental influences. IV. Notes on the physicochemical and immunological characteristics of an enterovirus that depresses the growth of mice.

Physicochemical and immunological techniques have been used in an attempt to characterize a filterable agent, separated from the intestines of mice raised under ordinary conditions of husbandry, which produces a lasting depression of weight in specific pathogen-free (SPF) mice when administered to them orally shortly after birth. Although this agent has not yet been identified, it will be tentatively designated here as enterovirus. The mouse enterovirus can be readily sedimented by ultracentrifugation and by precipitation at pH 4.3; it does not pass through cellophane membranes. Its infective power is completely destroyed by ultraviolet radiation, but is resistant to heating at 56 degrees C, exposure to ether, treatment with trypsin, ribonuclease, and deoxyribonuclease. Dialysis and treatment with ether and nucleases greatly increase the infective activity of the intestinal filtrates containing the enterovirus, a finding which suggests that these procedures eliminate or destroy some inhibitory substance(s). The mouse enterovirus causes hemagglutination of mouse red blood cells. When injected into rabbits, it elicits in them an immune response that renders their serum capable of neutralizing its weight-depressing activity. As measured by inhibition of hemagglutination or complement fixation, the sera of infected mice do not exhibit any significant activity against usual mouse viruses. Centrifugation of the mouse enterovirus in 50%-20% sucrose gradient gave almost complete recovery of the infectivity and of hemagglutinating activity in the same fraction. In contrast, the protein content of the material was distributed through the various fractions. Consequently, this procedure resulted in a marked increase of specific activity.