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.

Evolutionary Adaptation of an RNA Bacteriophage to Repeated Freezing and Thawing Cycles.

Bacteriophage fitness is determined by factors influencing both their replication within bacteria and their ability to maintain infectivity between infections. The latter becomes particularly crucial under adverse environmental conditions or when host density is low. In such scenarios, the damage experienced by viral particles could lead to the loss of infectivity, which might be mitigated if the virus undergoes evolutionary optimization through replication. In this study, we conducted an evolution experiment involving bacteriophage Qß, wherein it underwent 30 serial transfers, each involving a cycle of freezing and thawing followed by replication of the surviving viruses. Our findings show that Qß was capable of enhancing its resistance to this selective pressure through various adaptive pathways that did not impair the virus replicative capacity. Notably, these adaptations predominantly involved mutations located within genes encoding capsid proteins. The adapted populations exhibited higher resistance levels than individual viruses isolated from them, and the latter surpassed those observed in single mutants generated via site-directed mutagenesis. This suggests potential interactions among mutants and mutations. In conclusion, our study highlights the significant role of extracellular selective pressures in driving the evolution of phages, influencing both the genetic composition of their populations and their phenotypic properties.

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.

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.

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.

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.

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.

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.

Hyperexpansion of RNA Bacteriophage Diversity.

Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.

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.

Protein-RNA Interactions in the Single-Stranded RNA Bacteriophages.

Bacteriophages of the Leviviridae family are small viruses with short single-stranded RNA (ssRNA) genomes. Protein-RNA interactions play a key role throughout the phage life cycle, and all of the conserved phage proteins - the maturation protein, the coat protein and the replicase - are able to recognize specific structures in the RNA genome. The phage-coded replicase subunit associates with several host proteins to form a catalytically active complex. Recognition of the genomic RNA by the replicase complex is achieved in a remarkably complex manner that exploits the RNA-binding properties of host proteins and the particular three-dimensional structure of the phage genome. The coat protein recognizes a hairpin structure at the beginning of the replicase gene. The binding interaction serves to regulate the expression of the replicase gene and can be remarkably different in various ssRNA phages. The maturation protein is a minor structural component of the virion that binds to the genome, mediates attachment to the host and guides the genome into the cell. The maturation protein has two distinct RNA-binding surfaces that are in contact with different regions of the genome. The maturation and coat proteins also work together to ensure the encapsidation of the phage genome in new virus particles. In this chapter, the different ssRNA phage protein-RNA interactions, as well as some of their practical applications, are discussed in detail.

F-Specific RNA Bacteriophages, Especially Members of Subgroup II, Should Be Reconsidered as Good Indicators of Viral Pollution of Oysters.

Norovirus (NoV) is the leading cause of gastroenteritis outbreaks linked to oyster consumption. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as indicators of viral contamination in oysters by focusing especially on FRNAPH subgroup II (FRNAPH-II). These viral indicators have been neglected because their behavior is sometimes different from that of NoV in shellfish, especially during the depuration processes usually performed before marketing. However, a significant bias needs to be taken into account. This bias is that, in the absence of routine culture methods, NoV is targeted by genome detection, while the presence of FRNAPH is usually investigated by isolation of infectious particles. In this study, by targeting both viruses using genome detection, a significant correlation between the presence of FRNAPH-II and that of NoV in shellfish collected from various European harvesting areas impacted by fecal pollution was observed. Moreover, during their depuration, while the long period of persistence of NoV was confirmed, a similar or even longer period of persistence of the FRNAPH-II genome, which was over 30 days, was observed. Such a striking genome persistence calls into question the relevance of molecular methods for assessing viral hazards. Targeting the same virus (i.e., FRNAPH-II) by culture and genome detection in specimens from harvesting areas as well as during depuration, we concluded that the presence of genomes in shellfish does not provide any information on the presence of the corresponding infectious particles. In view of these results, infectious FRNAPH detection should be reconsidered as a valuable indicator in oysters, and its potential for use in assessing viral hazard needs to be investigated.IMPORTANCE This work brings new data about the behavior of viruses in shellfish, as well as about the relevance of molecular methods for their detection and evaluation of the viral hazard. First, a strong correlation between the presence of F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and that of norovirus (NoV) in shellfish impacted by fecal contamination has been observed when both viruses are detected using molecular approaches. Second, when reverse transcription-PCR and culture are used to detect FRNAPH-II in shellfish, it appears that the genomes of the viruses present a longer period of persistence than infectious virus, and thus, virus genome detection fails to give information about the concomitant presence of infectious viruses. Finally, this study shows that FRNAPH persist at least as long as NoV does. These data are major arguments to reconsider the potential of FRNAPH as indicators of shellfish viral quality.

Influence of adaptive mutations, from thermal adaptation experiments, on the infection cycle of RNA bacteriophage Qß.

A population's growth rate is determined by multiple 'life history traits'. To quantitatively determine which life history traits should be improved to allow a living organism to adapt to an inhibitory environment is an important issue. Previously, we conducted thermal adaptation experiments on the RNA bacteriophage Qß using three independent replicates and reported that all three end-point populations could grow at a temperature (43.6°C) that inhibited the growth of the ancestral strain. Even though the fitness values of the endpoint populations were almost the same, their genome sequence was not, indicating that the three thermally adapted populations may have different life history traits. In this study, we introduced each mutation observed in these three end-point populations into the cDNA of the Qß genome and prepared three different mutants. Quantitative analysis showed that they tended to increase their fitness by increasing the adsorption rate to their host, shortening their latent period (i.e., the duration between phage infection and progeny release), and increasing the burst size (i.e., the number of progeny phages per infected cell), but all three mutants decreased their thermal stability. However, the degree to which these traits changed differed. The mutant with the least mutations showed a smaller decrease in thermal stability, the largest adsorption rate to the host, and the shortest latent period. These results indicated that several different adaptive routes exist by which Qß can adapt to higher temperatures, even though Qß is a simple RNA bacteriophage with a small genome size, encoding only four genes.

Rapid and sensitive method to assess human viral pollution in shellfish using infectious F-specific RNA bacteriophages: Application to marketed products.

F-specific RNA bacteriophages (FRNAPH) have been used as indicators of environmental fecal pollution for many years. While FRNAPH subgroup I (FRNAPH-I) are not host specific, some FRNAPH-II and -III strains appear specific to human pollution. Because a close relationship has been observed between FRNAPH-II genome and human norovirus (NoV) in shellfish, and because FRNAPH infectivity can easily be investigated unlike that of NoV, the detection of human infectious FRNAPH could therefore provide a valuable tool for assessing viral risk. In this study, an integrated cell culture real-time RT-PCR method has been developed to investigate infectious FRNAPH subgroup prevalence in oysters. This rapid screening method appears more sensitive than E. coli or NoV genome detection, and allows an FRNAPH subgroup present in low concentrations (0.05 PFU/g of oyster) to be detected in the presence of another 1000 times more concentrated, without any dissection step. Its application to marketed oysters (n = 135) over a 1-year period has allowed to identify the winter peak classically described for NoV or FRNAPH accumulation. Infectious FRNAPH were detected in 34% of batches, and 7% were suspected of having a human origin. This approach may be helpful to evaluate oyster's depuration processes, based on an infectious viral parameter.

Quantitative Distribution of Infectious F-Specific RNA Phage Genotypes in Surface Waters.

UNLABELLED: F-specific RNA phages (FRNAPHs) are considered potential viral indicators of water pollution due to their occurrence and stability in water environments. However, their suitability as viral indicators is not fully elucidated because the characteristics of FRNAPHs are variable depending on the genotype. In this study, for the characterization of infectious FRNAPH genotypes, integrated culture reverse transcription-PCR coupled with the most probable number approach was applied to surface water samples. Further, to recover low concentrations of FRNAPH genotypes, an FRNAPH recovery method was developed. The novel FRNAPH recovery method using a noncharged microfiltration membrane could effectively recover FRNAPH strains without inactivation, while a method using an electronegative microfiltration membrane resulted in the inactivation of some strains. Infectious FRNAPH genotypes in surface water samples were successfully quantified with an efficiency comparable to that of the conventional plaque assay. Genotype I (GI) and GII FRNAPHs tended to be predominant at locations impacted by treated and untreated municipal wastewater, respectively. The numbers and proportions of infectious FRNAPHs tended to be higher during the winter season when water temperature decreased. IMPORTANCE: Properties of FRNAPHs are highly variable depending on their genotypes. Previous typing methods for FRNAPHs are not quantitative and/or are based on molecular assays, which cannot differentiate infective strains from inactive strains. Due to the reasons mentioned above, the utility of FRNAPHs as viral indicators of water pollution has not been fully validated. In this study, a quantitative genotyping method for infectious FRNAPHs was developed and applied to surface water samples. The method enabled characterization of infectious FRNAPH genotypes in terms of their occurrence and seasonality. Moreover, comparison of the method to a conventional molecular assay (reverse transcription-quantitative PCR) enabled characterization of their stability. Our approach can provide novel findings for further validation of FRNAPHs as viral indicators of water pollution.

Relevance of F-Specific RNA Bacteriophages in Assessing Human Norovirus Risk in Shellfish and Environmental Waters.

UNLABELLED: Human noroviruses (HuNoVs) are the main cause of shellfish-borne gastroenteritis outbreaks. In the absence of routine technical approaches allowing infectious particles to be detected, this viral pathogen is currently targeted by genome research, leading to difficult interpretations. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as fecal and viral contamination indicators in shellfish and water from a local harvesting area. FRNAPH were also used as microbial source tracking tools. Constraints imposed by detection limits are illustrated here by the detection of infectious FRNAPH in several samples in the absence of FRNAPH genomes. The opposite situation was also observed, likely explained by the persistence of the genomes being greater than infectivity. Similar considerations may be applied to HuNoVs, suggesting that HuNoV genome targeting is of limited relevance in assessing infectious risks. While FRNAPH did not provide any benefits compared to Escherichia coli as fecal pollution indicators in water, novel observations were made in shellfish: contrary to E. coli, a seasonal trend of infectious FRNAPH concentrations was observed. These concentrations were higher than those found in water, confirming bioaccumulation in shellfish. This study also underlines a relationship between the presence of HuNoV genomes and those of human-specific FRNAPH subgroup II (FRNAPH-II) in shellfish collected throughout Europe. Further research should be undertaken to evaluate FRNAPH potential as an indicator of the presence of infectious HuNoVs. To this end, shellfish involved in HuNoV-caused gastroenteritis outbreaks should be analyzed for the presence of infectious FRNAPH-II. IMPORTANCE: This work provides new data about the use of F-specific RNA phages (FRNAPH) as a tool for evaluating fecal or viral contamination, especially in shellfish. In our case study, FRNAPH did not provide any benefits compared to E. coli as fecal pollution indicators in water but were found to be very useful in shellfish. Their concentrations in shellfish were higher than those found in the surrounding water, confirming bioaccumulation. This study also underlines a relationship between the presence of human norovirus genomes (HuNoVs) and those of FRNAPH subgroup II (FRNAPH-II). Considering that the two virus types have similar behaviors and since FRNAPH infectivity can be investigated, the specific detection of infectious FRNAPH-II could be regarded as an indication of the presence of infectious HuNoVs. The contribution of infectious human FRNAPH targeting for assessing the viral risk associated with HuNoVs in shellfish should thus be investigated.

The True Story and Advantages of RNA Phage Capsids as Nanotools.

RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools.

Contribution of silent mutations to thermal adaptation of RNA bacteriophage Qß.

UNLABELLED: Changes in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qß in which the culture temperature was increased from 37.2°C to 41.2°C and finally to an inhibitory temperature of 43.6°C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qß strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2°C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature. IMPORTANCE: Recent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qß based on experimental evolution during which Qß showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and one mutation in the untranslated region that spread widely in the Qß population during the adaptation process at moderately high temperature provided a suitable genetic background to alter the fitness contribution of subsequent mutations from deleterious to beneficial at a higher temperature.

Occurrence of and Sequence Variation among F-Specific RNA Bacteriophage Subgroups in Feces and Wastewater of Urban and Animal Origins.

F-specific RNA bacteriophages (FRNAPH) have been widely studied as tools for evaluating fecal or viral pollution in water. It has also been proposed that they can be used to differentiate human from animal fecal contamination. While FRNAPH subgroup I (FRNAPH-I) and FRNAPH-IV are often associated with animal pollution, FRNAPH-II and -III prevail in human wastewater. However, this distribution is not absolute, and variable survival rates in these subgroups lead to misinterpretation of the original distribution. In this context, we studied FRNAPH distribution in urban wastewater and animal feces/wastewater. To increase the specificity, we partially sequenced the genomes of phages of urban and animal origins. The persistence of the genomes and infectivity were also studied, over time in wastewater and during treatment, for each subgroup. FRNAPH-I genome sequences did not show any specific urban or animal clusters to allow development of molecular tools for differentiation. They were the most resistant and as such may be used as fecal or viral indicators. FRNAPH-II's low prevalence and low sequence variability in animal stools, combined with specific clusters formed by urban strains, allowed differentiation between urban and animal pollution by using a specific reverse transcription-PCR (RT-PCR) method. The subgroup's resistance over time was comparable to that of FRNAPH-I, but its surface properties allowed higher elimination rates during activated-sludge treatment. FRNAPH-III's low sequence variability in animal wastewater and specific cluster formation by urban strains also allowed differentiation by using a specific RT-PCR method. Nevertheless, its low resistance restricted it to being used only for recent urban pollution detection. FRNAPH-IV was too rare to be used.

Enzyme treatment reverse transcription-PCR to differentiate infectious and inactivated F-specific RNA phages.

F-specific (F+) RNA phages are recommended as indicators of fecal contamination and the presence of enteric viruses and as viral surrogates to elucidate the resistance of viruses to adverse conditions or to assess the effectiveness of inactivating processes. Reverse transcription (RT)-PCR methods have been used to detect, quantify, or identify subgroups of F+ RNA phages. However, these methods may overestimate the infectivity of F+ RNA phages in test samples, since the presence of both infectious and inactivated phages (or naked RNA) can lead to positive RT-PCR signals. In this study, we evaluated the ability of an enzyme treatment (ET) with proteinase K and RNase A prior to RNA extraction, followed by RT-PCR, to differentiate infectious and inactivated F+ RNA phages. The results indicated that ET RT-PCR reduced, but did not completely eliminate, false-positive signals encountered with RT-PCR alone. The two-step ET RT-PCR, in which the enzymes were added sequentially, was more effective at reducing false-positive signals than the one-step ET RT-PCR, which involved addition of both enzymes together. Despite its inability to completely eliminate false-positive signals, ET RT-PCR gave more reliable information on the infectivity of F+ RNA phages. Thus, the method is better than RT-PCR alone for detecting F+ RNA phages as indicators to assess the risk of fecal contamination by enteric pathogens or to evaluate the effectiveness of virus-inactivating processes.