Adhesion-Aggregation and Inactivation of Poliovirus 1 in Groundwater Stored in a Hydrophobic Container

Viral inactivation and adhesion-aggregation in water are often studied as separate phenomena. When the focus is placed on viral adhesion-aggregation, inactivation is neglected because the phenomena under investigation occur over a short period measured in days. When viral inactivation is studied, adhesion-aggregation phenomena are considered to be negligible because viral survival is traced over several days or months. In the present work, we took a global approach, examining the relative contributions of each of these processes in a complex system composed of groundwater, Poliovirus 1, and a hydrophobic container (polypropylene) maintained in a dark environment at 20°C. We demonstrated that infectious viral load fell off 2.8 log₁₀ during the first 20 days. During this time, adhesion was far from negligible because it accounted for most of the decline, 1.5 log₁₀. Adhesion was undoubtedly favored by the presence of divalent ions in the groundwater. After 20 days, aggregation may also have been the cause of 0.66 to 0.92 log₁₀ of viral loss. Finally, viral inactivation was quantitatively the lowest phenomena because it only explained 0.38 to 0.64 log₁₀ of the viral loss. This study thus clearly demonstrated that estimates of viral survival in a given system must always take into account adhesion-aggregation phenomena which may be responsible for the majority of viral loss in the aqueous phase. Adhesion and aggregation are reversible processes which may lead to an underestimation of viral load in certain studies.     

Thermal and water source effects upon the stability of enteroviruses in surface freshwaters

The long-term survival of three human enterovirus serotypes, Coxsackievirus B3, echovirus 7, and poliovirus 1 was examined in samples of surface freshwater collected from five sites of physically different character. These were an artificial lake created by damming a creek, a small groundwater outlet pond, both a large- and a medium-sized river, and a small suburban creek. Survival was studied at temperatures of -20, 1, and 22 degrees C. The average amount of viral inactivation was 6.5-7.0 log10 units over 8 weeks at 22 degrees C, 4-5 log10 units over 12 weeks at 1 degree C, and 0.4-0.8 log10 units over 12 weeks at -20 degrees C. The effect of incubation temperature upon viral inactivation rate was statistically significant (p less than 0.00001). As determined by pairing tests, survival was also significantly related to both viral serotype and water source at each of the three incubation temperatures (p less than or equal to 0.05). Efforts were made to determine whether the rate of viral inactivation observed at the different incubation temperatures was related to characteristics inherent to the water that was collected from the different locations. The characteristics examined included physical and chemical parameters, indigenous bacterial counts, and the amount of bacterial growth that the waters would support (measured as the maximum number of generations which seeded bacteria could undergo after being placed into either pasteurized or sterile-filtered water samples). Analysis of viral inactivation rate versus these characteristics revealed three apparent effectors of viral persistence.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Quantitative viral load measurement for BKV infection in renal transplant recipients as a predictive tool for BKVAN

Infection by polyomavirus BK (BKV) is an emerging problem in the clinical management of renal transplant patients because it is responsible for nephropathy and consequently can cause loss of the transplanted organ (BKV associated nephropathy, BKVAN). Aim of this study was to evaluate the use of blood viral load measurement as a screening tool for diagnosis of BKV infection and to identify a threshold value for the management of patients. A total of 75 kidney transplant patients, corresponding to 338 consecutive plasma samples, were analyzed by an automatic system for nucleic acid extraction and quantitative real-time polymerase chain reaction (PCR) for detection of BKV. BKV was detected in 170 samples (26 patients) with a median viral load of 4.1 log10 copies/mL; among these 26 patients, seven (34.7%) were found to have BKVAN on allograft biopsy together with a median viral load of 5 log10 copies/mL. The ROC curve analysis identified a viral load equal to 4.1 log10 copies/mL as the best discriminant cut-off value to predict the disease and to identify patients at risk of developing BKVAN.     

Use of cell culture to assess Cryptosporidium parvum survival rates in natural groundwaters and surface waters

Bench-scale survival studies with Cryptosporidium parvum were conducted with representative aquifer and reservoir waters of Florida. C. parvum inactivation rates ranged from 0.0088 log(10)/day at 5 degrees C to -0.20 log(10)/day at 30 degrees C. Temperature, surface water or groundwater type, and the interaction of these factors had statistically significant effects on the survival of C. parvum.     

Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.     

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation.

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation.

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Inactivation of MS-2 phage and poliovirus in groundwater

Since temperature affects the inactivation rate of viruses in natural water systems, the aim of this study was to determine if a temperature shift could influence the structural integrity of model viruses. When crude lysates of MS-2 phage were seeded into groundwater microcosms and incubated at 27 degrees C, complete virus inactivation took place in eight days. The temperature was then shifted to 4 degrees C. Three days after the temperature shift, a two-log increase in virus titer (reactivation) occurred. However, when purified MS-2 lysates were added to groundwater microcosms, no reactivation was obtained. No reactivation of poliovirus took place when similar microcosm experiments were done. The sedimentation coefficients of MS-2 shifted from 80S to 58S, 48S, 37S, 32S, and 18S as inactivation proceeded in groundwater and distilled water controls. Similarly, the sedimentation coefficients of polioviruses changed from 156S to 142S, 135S, 117S, 105S, 95S, and 80 S as inactivation took place. There was no correlation between % virus inactivation and % decrease in virions with intact sedimentation coefficients, as reported earlier for poliovirus inactivated by chlorine. The results presented support our hypothesis that virus inactivation proceeds gradually, involving the rearrangement and (or) loss of capsomere components that may eventually lead to the ejection of nucleic acids. The intermediate particles generated as inactivation proceeds may be in a reversibly inactivated state, and may revert back to a fully infectious state when chemical components stabilize the virus particle.     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater.

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins

Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves showed that mobility of haemagglutinin measured at pH 7.3 increased considerably with temperature with the greatest change occurring over the range 20-30 degrees C. However, at pH 5.2 no mobility was detectable over the time range of the experiment. The activity of the virus was determined by assaying haemolysis of human erythrocytes. The haemolytic activity showed an optimum at pH 5.2 and increased markedly with temperature, being negligible below 20 degrees C. In addition, inactivation of the virus by incubation at pH 5.2 was also strongly temperature dependent. A 15 min incubation at pH 5.2 inactivated the virus above 30 degrees C but had no effect below 20 degrees C. On the basis of these results, it is proposed that mobility of haemagglutinin is significant for its functional properties. When the pH is reduced from 7.3 to 5.2, the mobility observed at higher temperatures is required for the molecular rearrangements which accompany the fusion event. In the absence of an apposing membrane, these rearrangements result in irreversible aggregation of haemagglutinin in the viral membrane, and hence loss of mobility and activity.     

Effects of pH on plaque forming unit counts and aggregation of MS2 bacteriophage

AIM: The aim of this study was to determine whether aggregation processes in aqueous phase may explain the decrease in plaque forming unit (PFU) counts for pH close to the isoelectric point (pI) of viral particles (MS2 phages). METHODS AND RESULTS: Loss in PFU was observed for pH < or = pI (pI(MS2) = 3.9): for example, at pH 2.5, loss was approx. 3 log(10) PFU. Particle size analysis combining results of dynamic light scattering and flow particle image analysis was then applied to determine the aggregate state of viral suspensions by recording size distributions. The size of major population significantly changed to 30 nm at neutral pH to more than several micrometres when passing below the isoelectric point. CONCLUSIONS: Our study shows that MS2 phages exhibit significant aggregation processes for pH < or = pI leading to aggregate with sizes of few micrometres. This aggregation process can largely explain the decline in PFU counts. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clear that viral aggregation can be a source of significant bias for PFU assays because in the presence of an aggregate the PFU count can be less than the sum of its constituent particles. Therefore, cautions should be taken in terms of conditions of storage (pH far from pI) to avoid such aggregation artefact.     

Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation.

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.     

Quantifying the impact of human immunodeficiency virus-1 escape from cytotoxic T-lymphocytes

HIV-1 escape from the cytotoxic T-lymphocyte (CTL) response leads to a weakening of viral control and is likely to be detrimental to the patient. To date, the impact of escape on viral load and CD4(+) T cell count has not been quantified, primarily because of sparse longitudinal data and the difficulty of separating cause and effect in cross-sectional studies. We use two independent methods to quantify the impact of HIV-1 escape from CTLs in chronic infection: mathematical modelling of escape and statistical analysis of a cross-sectional cohort. Mathematical modelling revealed a modest increase in log viral load of 0.051 copies ml(-1) per escape event. Analysis of the cross-sectional cohort revealed a significant positive association between viral load and the number of "escape events", after correcting for length of infection and rate of replication. We estimate that a single CTL escape event leads to a viral load increase of 0.11 log copies ml(-1) (95% confidence interval: 0.040-0.18), consistent with the predictions from the mathematical modelling. Overall, the number of escape events could only account for approximately 6% of the viral load variation in the cohort. Our findings indicate that although the loss of the CTL response for a single epitope results in a highly statistically significant increase in viral load, the biological impact is modest. We suggest that this small increase in viral load is explained by the small growth advantage of the variant relative to the wildtype virus. Escape from CTLs had a measurable, but unexpectedly low, impact on viral load in chronic infection.     

Death of enterohemorrhagic Escherichia coli O157:H7 in real mayonnaise and reduced-calorie mayonnaise dressing as influenced by initial population and storage temperature.

This study was undertaken to determine the survivability of low-density populations (10(0) and 10(2) CFU/g) of enterohemorrhagic Escherichia coli O157:H7 inoculated into real mayonnaise and reduced-calorie mayonnaise dressing and stored at 20 and 30 degrees C, temperatures within the range used for normal commercial mayonnaise distribution and storage. Inactivation patterns at 5 degrees C and inactivation of high-inoculum populations (10(6) CFU/g) were also determined. The pathogen did not grow in either mayonnaise formulation, regardless of the inoculum level or storage temperature. Increases in storage temperature from 5 to 20 degrees C and from 20 to 30 degrees C resulted in dramatic increases in the rate of inactivation. Populations of E. coli O157:H7 in the reduced-calorie and real formulations inoculated with a population of 0.23 to 0.29 log10 CFU/g and held at 30 degrees C were reduced to undetectable levels within 1 and 2 days, respectively; viable cells were not detected after 1 day at 20 degrees C. In mayonnaise containing an initial population of 2.23 log10 CFU/g, viable cells were not detected after 4 days at 30 degrees C or 7 days at 20 degrees C; tolerance was greater in real mayonnaise than in reduced-calorie mayonnaise dressing stored at 5 degrees C. The tolerance of E. coli O157:H7 inoculated at the highest population density (6.23 log 10 CFU/g) was less in reduced-calorie mayonnaise dressing than in real mayonnaise at all storage temperatures. In reduced-calorie mayonnaise dressing and real mayonnaise initially containing 2.23 log10 CFU/g, levels were undetectable after 28 and 58 days at 5 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature, relative humidity, absolute humidity, and evaporation potential on survival of airborne Gumboro vaccine virus

Survival of airborne virus influences the extent of disease transmission via air. How environmental factors affect viral survival is not fully understood. We investigated the survival of a vaccine strain of Gumboro virus which was aerosolized at three temperatures (10°C, 20°C, and 30°C) and two relative humidities (RHs) (40% and 70%). The response of viral survival to four metrics (temperature, RH, absolute humidity [AH], and evaporation potential [EP]) was examined. The results show a biphasic viral survival at 10°C and 20°C, i.e., a rapid initial inactivation in a short period (2.3 min) during and after aerosolization, followed by a slow secondary inactivation during a 20-min period after aerosolization. The initial decays of aerosolized virus at 10°C (1.68 to 3.03 ln % min(-1)) and 20°C (3.05 to 3.62 ln % min(-1)) were significantly lower than those at 30°C (5.67 to 5.96 ln % min(-1)). The secondary decays at 10°C (0.03 to 0.09 ln % min(-1)) tended to be higher than those at 20°C (-0.01 to 0.01 ln % min(-1)). The initial viral survival responded to temperature and RH and potentially to EP; the secondary viral survival responded to temperature and potentially to RH. In both phases, survival of the virus was not significantly affected by AH. These findings suggest that long-distance transmission of airborne virus is more likely to occur at 20°C than at 10°C or 30°C and that current Gumboro vaccination by wet aerosolization in poultry industry is not very effective due to the fast initial decay.