F-specific RNA Research Articles

A Longitudinal Study of Bacteriophages as Indicators of Norovirus Contamination of Mussels (Mytilus edulis) and Their Overlying Waters

Sewage pollution leads to the contamination of bivalve shellfish by pathogenic microorganisms. Bacterial indicators support the management of risks associated with the consumption of shellfish; however, they often fail to indicate adequately the potential hazard to human health posed by certain human enteric viruses. Bacteriophages have been proposed as alternative indicators that may more effectively predict the presence of enteric viral pathogens. This study explored the relationships between bacterial indicators (Escherichia coli (E. coli), faecal coliforms (FC) and intestinal enterococci (IE)), phages (somatic (SOMPH), F-specific RNA (F + PH) and human-specific Bacteroides GB-124 phages (GB124PH)) and Norovirus (NoV) (GI/GII) in mussels (Mytilus edulis) and their overlying waters. The bioaccumulation of these indicators and Norovirus in shellfish matrices (e.g., flesh, digestive gland) was investigated bimonthly over a 12-month period in an English estuary. The findings revealed a marked seasonality in the distribution of all organisms, with the highest levels occurring during the autumn/winter months. The levels of all phages in shellfish and their overlying waters correlated better with the levels of Norovirus than with those of bacterial indicators. Somatic coliphages were the indicator that exhibited the strongest correlations with NoV (rho = 0.929). This study suggests that relatively low-cost culture-based phage enumeration appears to offer a more accurate indication of the likely presence of Norovirus in mussels than traditional bacterial indicators.

Whole-Genome Sequence of Salmonella enterica subsp. enterica Serovar Typhimurium Strain WG49 and Escherichia coli Strain WG5 Used in South Africa for Phage Detection in Water Samples.

Salmonella enterica subsp. enterica serovar Typhimurium WG49 is widely used for enumeration of F-specific RNA (F-RNA) coliphages in water. Escherichia coli WG5 is broadly used for the detection and enumeration of somatic coliphages in water samples. We report here the genome sequences of these bacterial strains used in South Africa under ISO methods 10705-1 and 10705-2.

Waterborne Viruses and F-Specific Coliphages in Mixed-Use Watersheds: Microbial Associations, Host Specificities, and Affinities with Environmental/Land Use Factors.

This study is one of the first, to our knowledge, to evaluate relationships among F-specific coliphages and a large suite of enteric viruses in mixed-use but agriculturally dominated surface waters in Canada. This study suggested that relationships between viable F-specific coliphages and molecularly detected viruses do exist, but they are not always positive. Caution should be employed if viable F-specific coliphages are to be used as indicators of virus presence in surface waters. This study elucidates relative effects of agriculture, wildlife, and human activity on virus and F-specific coliphage detection. Seasonal and meteorological attributes play a strong role in the detection of most virus and F-specific coliphage targets.

Detection, fate and inactivation of pathogenic norovirus employing settlement and UV treatment in wastewater treatment facilities

It is accepted that discharged wastewaters can be a significant source of pathogenic viruses in receiving water bodies contributing to pollution and may in turn enter the human food chain and pose a risk to human health, thus norovirus (NoV) is often a predominant cause of gastroenteritis globally. Working with NoV poses particular challenges as it cannot be readily identified and detection by molecular methods does not assess infectivity. It has been proposed that the infectivity of NoV may be modelled through the use of an alternative virus; F-specific RNA (FRNA) bacteriophages; GA genotype and other FRNA bacteriophages have been used as a surrogate in studies of NoV inactivation.This study investigated the efficiency of novel pulsed ultraviolet irradiation and low pressure ultraviolet irradiation as a potential pathogen inactivation system for NoV and FRNA bacteriophage (GA) in secondary treated wastewaters. The role of UV dose and the impact of suspended solids concentration on removal efficiency were also examined. The study also investigated the role of settlement processes in wastewater treatment plants in removing NoV. While NoV inactivation could not be determined it was found that at a maximum UV dose of 6.9J/cm2 (6900mJ/cm2) an average 2.4 log removal of FRNA bacteriophage (GA) was observed; indicating the potential need for high UV doses to remove NoV if FRNA bacteriophage prove a suitable indicator for NoV. The study found that increasing concentrations of suspended solids impacted on PUV efficiency however, it appears the extent of the impact may be site specific. Furthermore, the study found that settlement processes can play a significant role in the removal of FRNA bacteriophage, thus potentially NoV.

Effectiveness of cooking to reduce norovirus and infectious F-specific RNA bacteriophage concentrations in Mytilus edulis.

The aim of this study was to determine if domestic cooking practices can reduce concentrations of norovirus (NoV) and F-specific RNA (FRNA) bacteriophage in experimentally contaminated mussels. Mussels (n=600) contaminated with NoV and FRNA bacteriophage underwent four different cooking experiments performed in triplicate at ~70°C and >90°C. Concentrations of infectious FRNA bacteriophage (using a plaque assay) were compared with concentrations of FRNA bacteriophage and NoV determined using a standardised RT-qPCR. Initial concentrations of infectious FRNA bacteriophage (7·05log10 PFUg(-1) ) in mussels were not significantly reduced in simmering water (~70°C); however, cooking at higher temperatures (>90°C) reduced infectious FRNA bacteriophage to undetected levels within 3min. Further investigation determined the time required for a 1-log reduction of infectious FRNA bacteriophage at 90°C to be 42s therefore a >3-log reduction in infectious virus can be obtained by heating mussel digestive tissue to 90°C for 126s. Domestic cooking practices based on shell opening alone do not inactivate infectious virus in mussels, however, cooking mussels at high temperatures is effective to reduce infectious virus concentrations and the risk of illness in consumers. The data will contribute towards evidence-based cooking recommendations for shellfish to provide a safe product for human consumption.

Simulated sunlight inactivation of norovirus and FRNA bacteriophage in seawater

To investigate norovirus (NoV) and F-specific RNA (FRNA) bacteriophage inactivation in seawater under simulated sunlight and temperature conditions representative of summer (235Wm(-2) ; 17°C) and winter (56Wm(-2) ; 10°C) conditions in Ireland. Inactivation experiments were carried out using a collimated beam of simulated sunlight and 100ml of filtered seawater seeded with virus under controlled temperature conditions. NoV concentrations were determined using RT-qPCR, and FRNA bacteriophage concentrations were determined using RT-qPCR and by plaque assay. For all virus types, the fluence required to achieve a 90% reduction in detectable viruses (S90 value) using RT-qPCR was not significantly different between summer and winter conditions. S90 values for FRNA bacteriophage determined by plaque assay were significantly less than those determined by RT-qPCR. Unlike S90 values determined by RT-qPCR, a significant difference existed between summer and winter S90 values for infectious FRNA bacteriophage. This study demonstrated that RT-qPCR significantly overestimates the survival of infectious virus and is therefore unsuitable for determining the inactivation rates of viruses in seawater. Results from this study provide initial data on the inactivation of NoV and FRNA bacteriophage in seawater under representative summer and winter conditions and will be of interest to shellfish and water management agencies alike.

Antiviral effect of cationic compounds on bacteriophages

The antiviral activity of several cationic compounds – cetyltrimethylammonium bromide (CTAB), chitosan, nisin, and lysozyme – was investigated on the bacteriophage c2 (DNA head and non-contractile tail) infecting Lactococcus strains and the bacteriophage MS2 (F-specific RNA) infecting E. coli. Firstly, these activities were evaluated in a phosphate buffer pH 7 – 10 mM. The CTAB had a virucidal effect on the Lactococcus bacteriophages, but not on the MS2. After 1 min of contact with 0.125 mM CTAB, the c2 population was reduced from 6 to 1.5 log(pfu)/mL and completely deactivated at 1 mM. On the contrary, chitosan inhibited the MS2 more than it did the bacteriophages c2. No antiviral effect was observed for the nisin or the lysozyme on bacteriophages after 1 min of treatment. A 1 and 2.5 log reduction was respectively observed for nisin and lysozyme when the treatment time increased (5 or 10 min). These results showed that the antiviral effect depended both on the virus and structure of the antimicrobial compounds. The antiviral activity of these compounds was also evaluated in different physico-chemical conditions and in complex matrices. The antiviral activity of CTAB was impaired in acid pH and with an increase of the ionic strength. These results might be explained by the electrostatic interactions between cationic compounds and negatively charged particles such as bacteriophages or other compounds in a matrix. Milk proved to be protective suggesting the components of food could interfere with antimicrobial compounds.

Difference in behaviors of F-specific DNA and RNA bacteriophages during coagulation–rapid sand filtration and coagulation–microfiltration processes

Difference in behaviors of F-specific DNA and RNA bacteriophages during coagulation–rapid sand filtration and coagulation–microfiltration (MF) processes were investigated by using river water spiked with F-specific DNA bacteriophage f1 and RNA bacteriophage f2. Because the particle characteristics of f1 (filamentous) and f2 (spherical) are quite different and the surface charge of f1 in the river water was slightly more negative than that of f2, the removal ratios of f1 were approximately 1-log lower than the removal ratio of f2 after any treatment process used in the present study. This result indicates that the behaviors of the two bacteriophages during the treatment processes were different, and that the removal of f1 by the combination of coagulation and filtration processes was more difficult than that of f2. The removal ratios for f1 and f2 were approximately 3-log and 4-log, respectively, in the coagulation–rapid sand filtration process, and 6-log and 7-log, respectively, in the coagulation–MF filtration process. Therefore, as expected, the coagulation–MF process appears to be more effective than the coagulation–rapid sand filtration process for the removal of not only spherical viruses but also filamentous viruses.

Genogroup distribution of F-specific coliphages in wastewater and river water in the Kofu basin in Japan

To determine the genogroup distribution of F-specific coliphages in aquatic environments using the plaque isolation procedure combined with genogroup-specific real-time PCR. Thirty water samples were collected from a wastewater treatment plant and a river in the Kofu basin in Japan on fine weather days. F-specific coliphages were detected in all tested samples, 187 (82%) of 227 phage plaques isolated were classified into one of the 4 F-specific RNA (F-RNA) coliphage genogroups and 24 (11%) plaques were F-specific DNA coliphages. Human genogroups II and III F-RNA coliphages were more abundant in raw sewage than animal genogroups I and IV, excluding one sample that was suspected to be heavily contaminated with sporadic heavy animal faeces. The secondary-treated sewage samples were highly contaminated with genogroup I F-RNA coliphages, probably because of different behaviours among the coliphage genogroups during wastewater treatment. The river water samples were expected to be mainly contaminated with human faeces, independent of rainfall effects. A wide range of F-specific coliphage genogroups were successfully identified in wastewater and river water samples. Our results clearly show the usefulness of the genogroup-specific real-time PCR for determining the genogroups of F-specific coliphages present in aquatic environments.

Sequence Variation among Group III F-Specific RNA Coliphages from Water Samples and Swine Lagoons

Typing of F-specific RNA (FRNA) coliphages has been proposed as a useful method for distinguishing human from animal fecal contamination in environmental samples. Group II and III FRNA coliphages are generally associated with human wastes, but several exceptions have been noted. In the present study, we have genotyped and partially sequenced group III FRNA coliphage field isolates from swine lagoons in North Carolina (NC) and South Carolina (SC), along with isolates from surface waters and municipal wastewaters. Phylogenetic analysis of a region of the 5' end of the maturation protein gene revealed two genetically different group III FRNA subclusters with 36.6% sequence variation. The SC swine lagoon isolates were more closely related to group III prototype virus M11, whereas the isolates from a swine lagoon in NC, surface waters, and wastewaters grouped with prototype virus Q-beta. These results suggest that refining phage genotyping systems to discriminate M11-like phages from Q-beta-like phages would not necessarily provide greater discriminatory power in distinguishing human from animal sources of pollution. Within the group III subclusters, nucleotide sequence diversity ranged from 0% to 6.9% for M11-like strains and from 0% to 8.7% for Q-beta-like strains. It is demonstrated here that nucleotide sequencing of closely related FRNA strains can be used to help track sources of contamination in surface waters. A similar use of phage genomic sequence information to track fecal pollution promises more reliable results than phage typing by nucleic acid hybridization and may hold more potential for field applications.

Elimination of Fecal Coliforms and F-Specific RNA Coliphage from Oysters (Crassostrea virginica) Relaid in Floating Containers

Declining oyster (Crassostrea virginica) production in the Chesapeake Bay has stimulated aquaculture based on floats for off-bottom culture. While advantages of off-bottom culture are significant, the increased use of floating containers raises public health and microbiological concerns, because oysters in floats may be more susceptible to fecal contamination from storm runoff compared to those cultured on-bottom. We conducted four commercial-scale studies with market-size oysters naturally contaminated with fecal coliforms (FC) and a candidate viral indicator, F-specific RNA (FRNA) coliphage. To facilitate sampling and to test for location effects, 12 replicate subsamples, each consisting of 15 to 20 randomly selected oysters in plastic mesh bags, were placed at four characteristic locations within a 0.6- by 3.0-m “Taylor” float, and the remaining oysters were added to a depth not exceeding 15.2 cm. The float containing approximately 3,000 oysters was relaid in the York River, Virginia, for 14 days. During relay, increases in shellfish FC densities followed rain events such that final mean levels exceeded initial levels or did not meet an arbitrary product end point of 50 FC/100 ml. FRNA coliphage densities decreased to undetectable levels within 14 days (16 to 28°C) in all but the last experiment, when temperatures fell between 12 and 16°C. Friedman (nonparametric analysis of variance) tests performed on FC/Escherichia coli and FRNA densities indicated no differences in counts as a function of location within the float. The public health consequences of these observations are discussed, and future research and educational needs are identified.

The Fate and Transport of Viruses through Surface Water Constructed Wetlands

AbstractColiphage removal efficiency and the effects of wetland hydrology on virus transport were determined for constructed wetlands in San Jacinto, CA. Mathematical models were used to further characterize virus transport. MS2, an F‐specific RNA (FRNA) coliphage was used as a model for human enteric viral behavior. Two wetland types were studied, a one‐phase cell and three‐phase cell. These wetlands received unchlorinated secondary effluent at a constant rate. The mean residence time in the wetlands was 9 ± 3 d as determined using bromide as a conservative tracer. Assuming 100% porosity, a plug flow model predicts this mean residence time within the experimental standard deviation (8 d). This suggests that a negligible volume was occupied by vegetation and settled solids. The convection‐dispersion equation adequately simulated the residence time distribution of the conservative tracer. MS2 removal in the wetlands was experimentally determined to be 97 ± 3%. There was no distinction between the two wetland types in terms of removal efficiency. The average coliphage decay rate was calculated to be 0.44 per day. However, the error involved with using the first order decay rate was high, 83 ± 12%. Therefore, first order decay does not adequately describe removal processes within the wetland. Most virus removal occurred within the first 3 m (k = 4.0 ± 1.8 d−1) with a removal efficiency of 85.3 ± 0.6%. The remainder had a decay rate of 0.20 ± 0.17 d−1 with a removal efficiency of 56 ± 33%.

Replication of coliphage Q beta as affected by host cell number, nutrition, competition from insusceptible cells and non-FRNA coliphages.

F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, might serve as indicators of human enteric viruses in groundwater, provided these phages do not replicate in groundwater and replicate only to a limited extent in wastewater. Several factors that could influence phage replication in either of these environments were examined. Q beta did not replicate when host cells were fewer than 10(4) cfu ml-1. Replication selected for insusceptible cells when Q beta was incubated with its E. coli host. Loss of Q beta, presumably by inactivation, occurred in autoclaved on-site and urban wastewater, autoclaved groundwater, and in filter-sterilized spent LB broth. Replication did not occur in LB broth diluted with sterile saline to 1% of its original strength, which indicates that replication of FRNA coliphages cannot occur in such nutrient-poor environments as wastewater and groundwater. Competition from non-FRNA coliphages and insusceptible cells tended to reduce Q beta replication, as predicted, but phage yields unexpectedly increased significantly when Enterococcus faecalis was added to cultures.

Effects of temperature and host cell growth phase on replication of F-specific RNA coliphage Q beta.

Human enteric viruses have been found in groundwater in the absence of fecal coliforms. Because detection of human enteric viruses is costly, time-consuming, and lacking in sensitivity, F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, are being examined for suitability as indicators of human enteric viruses in groundwater. Temperatures and host cell growth conditions that constrain F-pilus expression will limit FRNA coliphage replication in groundwater and wastewater, as is desirable in an indicator. Below 25 degrees C F-pilus synthesis ceases; FRNA coliphage Qbeta did not replicate below this temperature in batch cultures. One-step replication studies indicated that the replicative cycle is prolonged and that fewer progeny are released as the temperature decreases. The decreases in phage replication observed in the one-step replication studies were a consequence of fewer cells infected as the temperature was lowered or as host cells entered stationary phase. The numbers of phage particles released from infected cells did not change. The minimum temperature for replication of Qbeta, 25 degrees C, is not maintained in wastewater and does not occur in Wisconsin groundwater. On the basis of temperature and host cell growth phase, we have concluded that extensive replication of FRNA coliphages does not occur in wastewater and groundwater in Wisconsin and areas with similar cool climates.

AIDS, death, and the drug approval process: Can we deny patients the right to determine the level of risk they are willing to take?

The present study aimed to determine the differences in the behaviors of four F-specific RNA (F-RNA) coliphage genogroups (GI–GIV) during wastewater treatment. Raw sewage, aeration tank effluent, secondary-treated sewage, and return activated sludge were collected from a wastewater treatment plant in Japan at monthly intervals between March and December 2011 (n = 10 each). F-specific coliphages were detected by plaque assay in all tested samples, with a concentration ranging from − 0.10 to 3.66 log10 plaque-forming units/ml. Subsequently, eight plaques were isolated from each sample, followed by genogroup-specific reverse-transcription quantitative PCR (qPCR) for F-RNA coliphages and qPCR for F-specific DNA (F-DNA) coliphages. GI F-RNA coliphages were the most abundant in the secondary-treated sewage samples (73% of the plaque isolates), while GII F-RNA coliphages were the most abundant in the other three sample types (41–81%, depending on sample type). Based on the results of the quantification and genotyping, the annual mean concentrations of each F-specific coliphage type were calculated, and their reduction ratios during wastewater treatment were compared with those of indicator bacteria (total coliforms and Escherichia coli) and enteric viruses (human adenoviruses and GI and GII noroviruses). The mean reduction ratio of GI F-RNA coliphages was the lowest (0.93 log10), followed by those of the indicator bacteria and enteric viruses (1.59–2.43 log10), GII–GIV F-RNA coliphages (> 2.60–3.21 log10), and F-DNA coliphages (> 3.41 log10). These results suggest that GI F-RNA coliphages may be used as an appropriate indicator of virus reduction during wastewater treatment.

F-Specific RNA Bacteriophages as Model Viruses in UV Disinfection of Wastewater

F-specific RNA (FRNA) bacteriophages can be assayed in wastewater by simple and rapid methods. Their inactivation by UV radiation follows first-order kinetics and relatively simple formulas can describe the effect of UV absorbance by wastewater and the wavelength-dependent killing efficiency of polychromatic lamps. Because the organisms can be grown in high concentrations, biological calibration of full-scale reactors requires only relatively small volumes of phage culture. Naturally occurring FRNA-phages were more resistant to UV than somatic coliphages, Escherichiacoli and faecal streptococci. The inactivation rate constant was almost equal to that of reoviruses, which underlines the suitability of FRNA-phages as a process indicator for UV inactivation of viruses. A pure culture of MS2 was inactivated at a rate which was almost twice that of naturally occurring FRNA-phages, indicating the necessity of designing reactors for practical applications on field-data rather than laboratory experiments.

Virus, bacteriophages and water purification.

Water can be a vector of viral disease, but direct virological analysis of water has logistic and practical limitations. Viruses of major importance for water hygiene (e.g. hepatitis and gastro-enteritis viruses) cannot yet be grown in tissue culture. Therefore, as in bacteriological quality procedures, model organisms are required for the evaluation of virological quality of water and the effectiveness of virus removal by water treatment processes. On the basis of published information, the F specific RNA (FRNA) phages have been chosen for this purpose. For the enumeration of the phages a particular Salmonella typhimurium strain with an artificially introduced F plasmid was developed as a host strain and was found to give accurate and reliable results. FRNA phages were found in very high numbers (10(2)-10(5) pfu/ml) in all types of waste water investigated. FRNA phages are seldom found in non-faecally contaminated waste water. Surprisingly low numbers are found in faeces. FRNA phages in waste water effluent were found to be highly resistant to chloramines and relatively resistant to UV inactivation. The FRNA phages can thus effectively be used as indicator organisms for human pathogenic viruses in the evaluation of disinfection processes for water treatment plants.

F Specific RNA Bacteriophages in Sewage: Methodology and Occurrence

F specific RNA (FRNA) bacteriophages are attractive models for the study of the fate of animal viruses in water treatment processes and pure cultures (MS2, f2) have frequently been used in model experiments. The use of indigenous FRNA phages has been hampered by the lack of selective enumeration methods. Usually, bacteriophages in sewage are enumerated by counting the number of plaques produced in a lawn of an Escherichia coli host strain. Both somatic and F specific phages will contribute to the count. We report the development of a simple and highly selective method, based on a specially constructed host strain of Salmonella typhimurium (WG 49, phage type 3 Nalr (F'42 lac :: Tn5)). Counts on this host strain normally originate exclusively from F specific phages, because there are only few somatic Salmonella phages in most sewage samples. FRNA phage counts from natural sewage samples using this host strain are more reliable and higher than those obtained on E. coli hosts. F specific bacteriophages were detected in all sewage samples investigated in numbers ranging from 102 − 5 × 104 pfu/ml. Data are presented on the number and types of F specific phages in mixed domestic/industrial sewage; in poultry processing wastewater and in hospital wastewater. The potential of F specific phages as water quality indicators is discussed.