Isolation and grouping of RNA phages by Itaru Watanabe et al. (1967).

I. Watanabe et al. isolated approximately 30 strains of RNA phages from various parts of Japan. To isolate RNA phages, they assessed the infection specificity of male Escherichia coli and RNase sensitivity. They found that the isolated strains of RNA phages could be serologically separated into three groups. Furthermore, most of them were serologically related, and the antiphage rabbit serum prepared by one of these phages neutralized most of the other phages. The only serologically unrelated phage was the RNA phage Qß, which was isolated at the Institute for Virus Research, Kyoto University, in 1961.

RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses.

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

Recent Advances in Structural Studies of Single-Stranded RNA Bacteriophages.

Positive-sense single-stranded RNA (ssRNA) bacteriophages (phages) were first isolated six decades ago. Since then, extensive research has been conducted on these ssRNA phages, particularly those infecting E. coli. With small genomes of typically 3-4 kb that usually encode four essential proteins, ssRNA phages employ a straightforward infectious cycle involving host adsorption, genome entry, genome replication, phage assembly, and host lysis. Recent advancements in metagenomics and transcriptomics have led to the identification of ~65,000 sequences from ssRNA phages, expanding our understanding of their prevalence and potential hosts. This review article illuminates significant investigations into ssRNA phages, with a focal point on their structural aspects, providing insights into the various stages of their infectious cycle.

Activity- and gene-based quantification of enteric viruses, F- specific RNA phage genogroups, pepper mild mottle virus, and Escherichia coli in surface water.

Polymerase chain reaction (PCR) is widely applied for the monitoring of pathogenic viruses in water environments. To date, several pretreatments to selectively detect genes from infectious viruses via PCR have been developed. This study was aimed to characterize and validate methods for quantifying active viruses and indicators and to evaluate the proportion of their active fractions in surface water (n = 42). Active E. coli and F-specific RNA phage (FRNAPH) genogroups were quantified using culture assays. In addition to these microbes, norovirus genogroups I (GI) and II, Aichi virus 1, and pepper mild mottle virus (PMMoV) were quantified by (reverse transcription)-quantitative PCR (RT-qPCR) with and without cis-dichlorodiammineplatinum (CDDP) treatment to exclude genes in inactive viruses. CDDP-RT-qPCR showed concentrations and detection frequencies comparable to or higher than culture assays. Consequently, although CDDP-RT-qPCR can suggest the presence of an inactive virus, it can also overestimate the activity of the virus in the environment. Differences between culture and CDDP-RT-qPCR and between CDDP-RT-qPCR and RT-qPCR varied among the viruses. CDDP-RT-qPCR showed a concentration comparable to the culture assay (within 1 log10 difference) in 93 % of positive samples for GI-FRNAPH but in <63 % of positive samples for GII- and GIII-FRNAPHs. GII-NoV was detected from 5 and 30 out of 42 samples via CDDP-RT-qPCR and RT-qPCR, respectively, and was suggested as inactivated by 2.0 log10 or higher in most of the samples. By contrast, concentrations of PMMoV determined by these two assays were not notably different. It is suggested that the operational conditions of wastewater treatment plants around the sites, rather than environmental stresses, affected the microbial inactivation. To better understand the infectivity of viruses in the environment, it is important to investigate them using sensitive detection methods at various sites, including the source of contamination.

Toward better monitoring of human noroviruses and F-specific RNA bacteriophages in aquatic environments using bivalve mollusks and passive samplers: A case study.

Monitoring pathogenic enteric viruses in continental and marine water bodies is essential to control the viral contamination of human populations. Human Noroviruses (NoV) are the main enteric viruses present in surface waters and foodstuff. In a context of global change, it is currently a challenge to improve the management of viral pollutions in aquatic environments and thereby limit the contamination of vulnerable water bodies or foodstuffs. The aim of this study is to evaluate the potential of specific accumulation systems for improving the detection of NoV in water bodies, compared to direct water analyses. Passive samplers (Zetapor filters) and three species of bivalve molluscan shellfish (BMS) (Dreissena polymorpha, Mytilus edulis and Crassostreas gigas) were used as accumulation systems to determine their performance in monitoring continental and marine waters for viruses. F-specific RNA bacteriophages (FRNAPH) were also analyzed since they are described as indicators of NoV hazard in many studies. During a one-year study in a specific area frequently affected by fecal pollution, twelve campaigns of exposure of passive samplers and BMS in continental and coastal waters were conducted. Using suitable methods, NoV (genome) and FRNAPH (infectious and genome) were detected in these accumulation systems and in water at the same time points to determine the frequency of detection but also to gain a better understanding of viral pollution in this area. The reliability of FRNAPH as a NoV indicator was also investigated. Our results clearly showed that BMS were significantly better than passive samplers and direct water analyses for monitoring NoV and FRNAPH contamination in water bodies. A dilution of viral pollution between the continental and the coastal area was observed and can be explained by the distance from the source of the pollution. Viral pollution is clearly greater during the winter period, and stakeholders should take this into consideration in their attempts to limit the contamination of food and water. A significant correlation was once again shown between NoV and FRNAPH genomes in BMS, confirming the reliability of FRNAPH as a NoV indicator. Moreover, a strong correlation was observed between NoV genomes and infectious FRNAPH, suggesting recent viral pollution since infectious particles had not been inactivated at sufficient levels in the environment. More generally, this study shows the value of using BMS as an active method for improving knowledge on the behavior of viral contamination in water bodies, the ranking of the contamination sources, and the vulnerability of downstream water bodies.

Effects of surface hydrophobicity on the removal of F-specific RNA phages from reclaimed water by coagulation and ceramic membrane microfiltration.

Microfiltration (MF) has been widely adopted as an advanced treatment process to reduce suspended solids and turbidity in treated wastewater effluents designated for potable reuse. Although microfilter pores are much larger than viruses, the addition of a coagulant upstream of a microfilter system can achieve stable virus removal. Ceramic membranes have a narrow pore size distribution to achieve the high removal of contaminants. This study aims to evaluate virus log reduction using bench-scale coagulation and ceramic membrane MF. To investigate the effects of differences in net surface hydrophobicity, 18 sewage-derived F-specific RNA phages (FRNAPHs) were used for batch hydrophobicity and coagulation-MF tests. The capability of bench-scale coagulation and ceramic membrane MF under continuous automated long-term operation was tested to remove the lab reference strain MS2 and three selected FRNAPH isolates which varied by surface property. Median virus log reduction values (LRVs) exceeding 6.2 were obtained for all three isolates and MS2. Although coagulation and hydrophobicity were positively correlated, the virus isolate demonstrating the lowest level of hydrophobicity and coagulation (genogroup I) still exhibited a high LRV. Thus, coagulation and ceramic membrane MF systems may serve as viable options for virus removal during water reclamation and advanced treatment.

Quantification and fate of plasmid-specific bacteriophages in wastewater: Beyond the F-coliphages.

Plasmid-specific bacteriophages specifically infect bacteria carrying conjugal plasmids. While wastewater has been used as isolation source for such phages, to date, only the distribution and ecology of RNA phages specific to the F plasmid have been described, because they serve as a water quality indicator. Yet, several other plasmid classes have higher clinical and ecological relevance, and the distribution, fate, and ecology of the phages that target them remain uncharacterized. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages applicable to several different conjugal plasmid classes, (ii) describe the distribution of such phages in wastewater systems, and (iii) relate their abundance to plasmid abundance and to municipal wastewater treatment processes. We introduced four model conjugal plasmids, belonging to incompatibility groups IncP-1, IncN, IncHI1, or IncF into an avirulent Salmonella enterica strain, for which somatic phages are at low abundance in wastewater. These strains were used in double layer agar assays with waters from contrasting sources. Plasmid-specific phages were common in wastewater but rare in river water. Hospital wastewater contained significantly more IncP-1-, but fewer IncF- and IncN- specific phages than domestic wastewater. This pattern did not match that of plasmid abundance estimated by Inc group targeting high-throughput quantitative PCR. The comparison between influent and effluent of wastewater treatment plants revealed a reduction in phage concentration by ca. 2 log, without significant contribution of primary settling. Overall, the ubiquity of these phages hints at their importance for plasmid ecology, and can provide opportunities in water quality monitoring and in ecological management of mobile resistance genes.

Engineering an Antibody V Gene-Selective Vaccine.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information.

Intra-Population Competition during Adaptation to Increased Temperature in an RNA Bacteriophage.

Evolution of RNA bacteriophages of the family Leviviridae is governed by the high error rates of their RNA-dependent RNA polymerases. This fact, together with their large population sizes, leads to the generation of highly heterogeneous populations that adapt rapidly to most changes in the environment. Throughout adaptation, the different mutants that make up a viral population compete with each other in a non-trivial process in which their selective values change over time due to the generation of new mutations. In this work we have characterised the intra-population dynamics of a well-studied levivirus, Qß, when it is propagated at a higher-than-optimal temperature. Our results show that adapting populations experienced rapid changes that involved the ascent of particular genotypes and the loss of some beneficial mutations of early generation. Artificially reconstructed populations, containing a fraction of the diversity present in actual populations, fixed mutations more rapidly, illustrating how population bottlenecks may guide the adaptive pathways. The conclusion is that, when the availability of beneficial mutations under a particular selective condition is elevated, the final outcome of adaptation depends more on the occasional occurrence of population bottlenecks and how mutations combine in genomes than on the selective value of particular mutations.

Characterization of a novel group I F-specific RNA bacteriophage isolated from human stool.

A novel F-specific RNA bacteriophage (FRNAPH) YM1, affiliating to genogroup I (GI) of Levivirus, is isolated for the first time from human stool samples using double-layer agar plates with the Escherichia coli ATCC700891 as the host. The complete genomic sequence of YM1 is 3551 nt in length, obtained through next-generation sequencing, and contains four genes encoding for maturation protein, coat protein, lysis protein, and RNA-dependent RNA polymerase (RdRp). The genomic sequence of YM1 shares the highest similarity of 95.3% with that of a GI FRNAPH DL16 isolated from surface water of Great Bay. The YM1 possesses a non-enveloped, icosahedral virion of 23 ± 0.45 nm in diameter. One-step growth curve analysis shows that the burst time of YM1 is 30 min post-infection (p.i.) with the average burst size of 264 PFU/cell. The YM1 lyses only E. coli strains tested, revealing high host specificity. This newly discovered phage may serve as a candidate for viral indicator to monitor human enteric virus, especially norovirus, contamination in the environments.

Assembly of the virome in newborn human infants.

Healthy human infants are typically born without high concentrations of viral particles in their intestines, but after a few weeks of life particle counts typically reach a billion per gram of stool. Where do these vast populations come from? Recent studies support the idea that colonization is stepwise. First pioneer bacteria seed the infant gut. Bacteria commonly harbor prophage sequences integrated in their genomes, which periodically induce to make particles, providing a first wave of viral particles. Later more viruses infecting human cells are detected. Analysis showed that lower accumulation of viruses that grow in human cells is associated with breastfeeding. Thus these studies emphasize the environmental influences on formation of the early life virome, and begin to point the way toward modulating viral colonization to optimize health.

Application of crAssphage, F-RNA phage and pepper mild mottle virus as indicators of human faecal and norovirus contamination in shellfish.

Shellfish growing waters contaminated with inadequately treated human wastewater is a major source of norovirus in shellfish and poses a significant human health risk to consumers. Microbial source tracking (MST) markers have been widely used to identify the source (s) of faecal contamination in water but data are limited on their use for shellfish safety. This study evaluated the source specificity, sensitivity, occurrence and concentration of three viral MST markers i.e. cross-assembly phage (crAssphage), F-specific RNA bacteriophage genogroup II (F-RNA phage GII) and pepper mild mottle virus (PMMoV) using animal faeces (n = 119; 16 animal groups), influent wastewater (n = 12), effluent wastewater (n = 16) and shellfish (n = 33). CrAssphage, F-RNA phage GII and PMMoV had source specific values of 0.97, 0.99 and 0.91, respectively. The sensitivity of MST markers was confirmed by their 100% detection frequency in influent wastewaters. The frequency of detection in effluent wastewater ranged from 81.3% (F-RNA phage GII) to 100% (PMMoV). Concentration of F-RNA phage GII was one log10 (influent wastewater) and 2-3 log10 (effluent wastewater) lower than crAssphage and PMMoV, respectively. Despite lower prevalence of F-RNA phage GII in oysters and mussels compared to crAssphage and PMMoV, concentrations of the three MST markers were similar in mussels. As an indicator of norovirus contamination in shellfish, crAssphage and PMMoV had greater predictive sensitivity (100%; [95% CI; 81.5%-100%)]) and F-RNA phage GII had greater predictive specificity (93.3%; [95% CI; 68.1%-99.8%]). In contrast, crAssphage and F-RNA phage GII have similar accuracy for predicting norovirus in shellfish, however, PMMoV significantly overestimated its presence. Therefore, a combination of crAssphage and F-RNA phage GII analysis of shellfish could provide a robust estimation of the presence of human faecal and norovirus contamination.

Investigation of F-RNA Bacteriophage as a Tool in Re-Opening Australian Oyster Growing Areas Following Sewage Spills.

Oysters contaminated with human enteric viruses from sewage are implicated in foodborne outbreaks globally. Bacteriophages have been identified as potential indicators for these viruses, but have not been used in shellfish management outside of the USA. This study aimed to determine the background levels of F-RNA phage in five Australian oyster growing areas with a history of sewage spills and closures, over an 18-month period. In addition, oysters from five growing areas impacted by adverse sewage events were investigated for F-RNA phage, Escherichia coli, norovirus (NoV) and hepatitis A virus (HAV). F-RNA phage ≤ 60 pfu/100 gm shellfish flesh were found to represent a conservative background level in the surveyed areas. Following two of the five sewage spills, elevated phage levels were observed in most sample sites less than 4 days post spill. By 7 days, most sites from all events had phage < 30 pfu/100 gm. NoV was detected in day 1 and day 6 samples from one event when all phage were ≤ 30 pfu/100 gm. NoV was also detected in a day 3 sample from another event with < 30 phage pfu/100 gm, however, multiple replicate samples had elevated phage levels. The results of this study add evidence on the potential use of F-RNA phage as a tool in early re-opening of oyster harvest areas post sewage spills. However, it also highlights the need to better understand situations where phage testing may be ineffectual, and the importance of sampling at multiple sites and over multiple time points, to effectively capture evidence of contamination.

The Single-Stranded RNA Bacteriophage Qß Adapts Rapidly to High Temperatures: An Evolution Experiment.

Single-stranded (ss)RNA viruses are thought to evolve rapidly due to an inherently high mutation rate. However, it remains unclear how ssRNA viruses adapt to novel environments and/or how many and what types of substitutions are needed to facilitate this evolution. In this study, we followed the adaptation of the ssRNA bacteriophage Qß using thermally adapted Escherichia coli as a host, which can efficiently grow at temperatures between 37.2 and 45.3 °C. This made it possible to evaluate Qß adaptation to the highest known temperature that supports growth, 45.3 °C. We found that Qß was capable of replication at this temperature; within 114 days (~1260 generations), we detected more than 34 novel point mutations in the genome of the thermally adapted Qß population, representing 0.8% of the total Qß genome. In addition, we returned the 45.3 °C-adapted Qß populations to 37.2 °C and passaged them for 8 days (~124 generations). We found that the reverse-adapted Qß population showed little to no decrease in fitness. These results indicate that Qß can evolve in response to increasing temperatures in a short period of time with the accumulation of point mutations.

F-Specific RNA Bacteriophages Model the Behavior of Human Noroviruses during Purification of Oysters: the Main Mechanism Is Probably Inactivation Rather than Release.

Noroviruses (NoV) are responsible for many shellfish outbreaks. Purification processes may be applied to oysters before marketing to decrease potential fecal pollution. This step is rapidly highly effective in reducing Escherichia coli; nevertheless, the elimination of virus genomes has been described to be much slower. It is therefore important to identify (i) the purification conditions that optimize virus removal and (ii) the mechanism involved. To this end, the effects of oyster stress, nutrients, and the presence of a potential competitor to NoV adhesion during purification were investigated using naturally contaminated oysters. Concentrations of NoV (genomes) and of the viral indicator F-specific RNA bacteriophage (FRNAPH; genomes and infectious particles) were regularly monitored. No significant differences were observed under the test conditions. The decrease kinetics of both virus genomes were similar, again showing the potential of FRNAPH as an indicator of NoV behavior during purification. The T90 (time to reduce 90% of the initial titer) values were 47.8 days for the genogroup I NoV genome, 26.7 days for the genogroup II NoV genome, and 43.9 days for the FRNAPH-II genome. Conversely, monitoring of the viral genomes could not be used to determine the behavior of infectious viruses because the T90 values were more than two times lower for infectious FRNAPH (20.6 days) compared to their genomes (43.9 days). Finally, this study highlighted that viruses are primarily inactivated in oysters rather than released in the water during purification processes.IMPORTANCE This study provides new data about the behavior of viruses in oysters under purification processes and about their elimination mechanism. First, a high correlation has been observed between F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and norovirus (NoV) in oysters impacted by fecal contamination when both are detected using molecular approaches. Second, when using reverse transcription-quantitative PCR and culture to detect FRNAPH-II genomes and infectious FRNAPH in oysters, respectively, it appears that genome detection provides limited information about the presence of infectious particles. The comparison of both genomes and infectious particles highlights that the main mechanism of virus elimination in oysters is inactivation. Finally, this study shows that none of the conditions tested modify virus removal.

Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.

Occurrence and reduction of F-specific RNA bacteriophage genotypes as indicators of human norovirus at a wastewater treatment plant.

F-specific RNA bacteriophages (FRNAPHs) have been suggested as good indicators of the presence of human enteric viruses in water treatment facilities. The occurrence and reduction of norovirus (NoV) and FRNAPH genotypes in wastewater treatment plants (WWTPs) have been well studied; however, the relationship between these genotypes in WWTPs has not been fully elucidated. Thus, we aimed to investigate the occurrence and reduction of FRNAPH genotypes in an attempt to identify NoV indicators in a WWTP via a 1-year survey. All FRNAPH and NoV genotypes were detected in WWTP influents at high rates (71-100%), including the infectious FRNAPH genotype IV (GIV), which has been rarely detected in previous studies. The reductions of FRNAPH GII and NoV GII during wastewater treatment indicated a relationship between the two (r = 0.69, P < 0.01), and the mean values were not significantly different. These results suggested that FRNAPH GII could be used as an appropriate indicator of NoV GII during wastewater treatment. FRNAPH GI was also found to be an appropriate indicator of viral reduction because of its high resistance to wastewater treatment compared with the other FRNAPH and NoV genotypes; therefore, it can be considered as a worst-case scenario organism.

Influence of physico-chemical characteristics of sediment on the in situ spatial distribution of F-specific RNA phages in the riverbed.

Riverbed sediment is commonly described as an enteric virus reservoir and thought to play an important role in water column contamination, especially during rainfall events. Although the occurrence and fate of faecal-derived viruses are fairly well characterized in water, little information is available on their presence as their interactions with sediment. This study aimed at determining the main environmental factors responsible for the presence of enteric viruses in riverbed sediment. Using a combination of microbiological and physico-chemical analyses of freshly field-sampled sediments, we demonstrated their contamination by faecal phages. The in situ spatial distribution of phages in sediment was mainly driven by sediment composition. A preferential phage accumulation occurred along one bank of the river, where the quantity of fine sands and clay particles smaller than 0.2 mm was the highest. Additionally, a mineralogical analysis revealed the influence of the heterogeneous presence of virus sorbents such as quartz, calcite, carbonates and iron-bearing phases (goethite) on the phage spatial pattern. A more precise knowledge of the composition of riverbed sediment is therefore useful for predicting preferential areas of enteric virus accumulation and should allow more accurate microbial risk assessment when using surface water for drinking water production or recreational activities.

New approach for the simultaneous detection of somatic coliphages and F-specific RNA coliphages as indicators of fecal pollution.

Two groups of coliphages have been recently included in different water management policies as indicators of viral fecal pollution in water and food: somatic coliphages, which infect E. coli through cell wall receptors, and F-specific RNA coliphages, which infect through the F-pili. Somatic coliphages are more abundant in fecally contaminated waters, except reclaimed waters, those disinfected by UV irradiation, and some groundwater samples that show a higher level of F-specific coliphages. Somatic coliphages are morphologically similar to DNA enteric viruses while F-specific coliphages are similar to RNA viruses such as norovirus and hepatitis A viruses, which are the viral pathogens of concern in sewage. The use of strains sensitive to both types of phages has been proposed for total coliphage enumeration, thereby avoiding double analysis. The standardized methods available for coliphage detection are robust and cost-effective, but the introduction of ready-to-use methods would facilitate routine implementation in laboratories. The fastest available tool for somatic coliphage enumeration is the recently developed Bluephage, which uses a modified ß-glucuronide-overexpressing E. coli strain unable to take up the glucuronide substrate. The overexpressed enzyme accumulates inside the bacterial cells until released by phage-induced cell lysis, whereupon it encounters its substrate and the medium changes from yellow to blue. The present method uses E. coli strain CB12, sensitive to somatic coliphages and F-specific coliphages due to the expression of the F-pili. The Bluephage approach incorporating CB12 detects both types of coliphages in a time range of 1:30 to 4:00 h, as assayed with coliphages from raw sewage, river water, sludge and mussels. This strategy can be applied to obtain qualitative and quantitative results and is applicable to microplates as well as to large sample volumes (100 ml). Moreover it can provide monitoring of water bodies at real time, as for example for ambient recreational beach monitoring.

Existing Host Range Mutations Constrain Further Emergence of RNA Viruses.

RNA viruses are capable of rapid host shifting, typically due to a point mutation that confers expanded host range. As additional point mutations are necessary for further expansions, epistasis among host range mutations can potentially affect the mutational neighborhood and frequency of niche expansion. We mapped the mutational neighborhood of host range expansion using three genotypes of the double-stranded RNA (dsRNA) bacteriophage φ6 (wild type and two isogenic host range mutants) on the novel host Pseudomonas syringae pv. atrofaciens. Both Sanger sequencing of 50 P. syringae pv. atrofaciens mutant clones for each genotype and population Illumina sequencing revealed the same high-frequency mutations allowing infection of P. syringae pv. atrofaciens. Wild-type φ6 had at least nine different ways of mutating to enter the novel host, eight of which are in p3 (host attachment protein gene), and 13/50 clones had unchanged p3 genes. However, the two isogenic mutants had dramatically restricted neighborhoods: only one or two mutations, all in p3. Deep sequencing revealed that wild-type clones without mutations in p3 likely had changes in p12 (morphogenic protein), a region that was not polymorphic for the two isogenic host range mutants. Sanger sequencing confirmed that 10/13 of the wild-type φ6 clones had nonsynonymous mutations in p12, and 2 others had point mutations in p9 and p5. None of these genes had previously been associated with host range expansion in φ6. We demonstrate, for the first time, epistatic constraint in an RNA virus due to host range mutations themselves, which has implications for models of serial host range expansion.IMPORTANCE RNA viruses mutate rapidly and frequently expand their host ranges to infect novel hosts, leading to serial host shifts. Using an RNA bacteriophage model system (Pseudomonas phage φ6), we studied the impact of preexisting host range mutations on another host range expansion. Results from both clonal Sanger and Illumina sequencing show that extant host range mutations dramatically narrow the neighborhood of potential host range mutations compared to that of wild-type φ6. This research suggests that serial host-shifting viruses may follow a small number of molecular paths to enter additional novel hosts. We also identified new genes involved in φ6 host range expansion, expanding our knowledge of this important model system in experimental evolution.