EXPERIMENTAL EVOLUTION OF RNA VERSUS DNA VIRUSES

Based on their extremely high mutation rates, RNA viruses have been traditionally considered as the fastest evolving entities in nature. However, recent work has revealed that, despite their greater replication fidelity, single-stranded (ss) DNA viruses can evolve fast in a similar way. To further investigate this issue, we have compared the rates of adaptation and molecular evolution of ssRNA and ssDNA viruses under highly controlled laboratory conditions using the bacteriophages ΦX174, G4, f1, Qβ, SP, and MS2 as model systems. Our results indicate that ssRNA phages evolve faster than ssDNA phages under strong selective pressure, and that their extremely high mutation rates appear to be op…

Unequal distribution of RT-PCR artifacts along the E1-E2 region of Hepatitis C virus.

Although viral variability studies have focused traditionally on consensus sequences, the relevance of molecular clone sequences for studying viral evolution at the intra-host level is being increasingly recognized. However, for this approach to be reliable, RT-PCR artifacts do not have to contribute excessively to the observed variability. Molecular clone sequences were obtained from an in vitro transcript to estimate the maximum error rate associated to RT-PCR for the Hepatitis C virus (HCV) E1-E2 region. On average, the frequency of RT-PCR errors was one order of magnitude lower than the level of intra-host genetic variability observed in samples from an HCV outbreak. However, RT-PCR err…

Phage Therapy in Livestock and Companion Animals

The irrational use of antibiotics has led to a high emergence of multi-drug resistant (MDR) bacteria. The traditional overuse of antibiotics in the animal feed industry plays a crucial role in the emergence of these pathogens that pose both economic and health problems. In addition, antibiotics have also recently experienced an increase to treat companion animal infections, promoting the emergence of MDR bacteria in pets, which can reach humans. Phages have been proposed as an alternative for antibiotics for the treatment of livestock and companion animal infections due to their multiple advantages as adaptative drugs, such as their ability to evolve, to multiply at the site of infections, …

Experimental Evolution in Viruses

Experiments in which evolution takes place in real time can help us establish cause–effect relationships that are difficult to infer from the analysis of natural populations. The simplicity, rapid evolution and biomedical relevance of viruses make them a particularly interesting model system for experimental evolution. Bacterial, animal and plant viruses can be passaged under a variety of conditions, either in simple cell culture systems or in vivo to test population biology hypotheses, study the genetic basis of evolution, or predict evolutionary change in nature. Experimental evolution is a conceptually simple and flexible tool which allows us to address issues ranging from the molecular …

Isolation of Four Lytic Phages Infecting Klebsiella pneumoniae K22 Clinical Isolates from Spain

This article belongs to the Special Issue Bacteriophage—Molecular Studies.

Inference of the Life Cycle of Environmental Phages from Genomic Signature Distances to Their Hosts

The environmental impact of uncultured phages is shaped by their preferred life cycle (lytic or lysogenic). However, our ability to predict it is very limited. We aimed to discriminate between lytic and lysogenic phages by comparing the similarity of their genomic signatures to those of their hosts, reflecting their co-evolution. We tested two approaches: (1) similarities of tetramer relative frequencies, (2) alignment-free comparisons based on exact k = 14 oligonucleotide matches. First, we explored 5126 reference bacterial host strains and 284 associated phages and found an approximate threshold for distinguishing lysogenic and lytic phages using both oligonucleotide-based methods. The an…

Bacteriophages: Protagonists of a Post-Antibiotic Era

This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond.

Correlation between mutation rate and genome size in riboviruses: mutation rate of bacteriophage Qβ.

Abstract Genome sizes and mutation rates covary across all domains of life. In unicellular organisms and DNA viruses, they show an inverse relationship known as Drake’s rule. However, it is still unclear whether a similar relationship exists between genome sizes and mutation rates in RNA genomes. Coronaviruses, the RNA viruses with the largest genomes (∼30 kb), encode a proofreading 3′ exonuclease that allows them to increase replication fidelity. However, it is unknown whether, conversely, the RNA viruses with the smallest genomes tend to show particularly high mutation rates. To test this, we measured the mutation rate of bacteriophage Qβ, a 4.2-kb levivirus. Amber reversion-based Luria–D…

A Method for Isolation of the Virome from Plasma Samples

Virome studies are of special interest nowadays. Understanding viral communities in different body compartments will help guide future personalized treatments and to discern between homeostasis and disease. High-throughput sequencing technologies allow us to detect all the nucleic acids present in a sample, including viral ones, by random sequencing. One of the major challenges in virome studies is the correct isolation of the viral nucleic acids from a specific sample. This can be done during the extraction steps (e.g., enrichment of viral capsids), or during the bioinformatic analysis (e.g., removing all human and bacterial sequences). Furthermore, it is an important remark that the treat…

Phage Therapy in Gastrointestinal Diseases

Gastrointestinal tract microbiota plays a key role in the regulation of the pathogenesis of several gastrointestinal diseases. In particular, the viral fraction, composed essentially of bacteriophages, influences homeostasis by exerting a selective pressure on the bacterial communities living in the tract. Gastrointestinal inflammatory diseases are mainly induced by bacteria, and have risen due to the emergence of antibiotic resistant strains. In the lack of effective treatments, phage therapy has been proposed as a clinical alternative to restore intestinal eubiosis, thanks to its immunomodulatory and bactericidal effect against bacterial pathogens, such as Clostridioides difficile in ulce…

Selection for thermostability can lead to the emergence of mutational robustness in an RNA virus

Mutational robustness has important evolutionary implications, yet the mechanisms leading to its emergence remain poorly understood. One possibility is selection acting on a correlated trait, as for instance thermostability (plastogenetic congruence). Here, we examine the correlation between mutational robustness and thermostability in experimental populations of the RNA bacteriophage Qβ. Thermostable viruses evolved after only six serial passages in the presence of heat shocks, and genome sequencing suggested that thermostability can be conferred by several alternative mutations. To test whether thermostable viruses have increased mutational robustness, we performed additional passages in …

The external domains of the HIV-1 envelope are a mutational cold spot

In RNA viruses, mutations occur fast and have large fitness effects. While this affords remarkable adaptability, it can also endanger viral survival due to the accumulation of deleterious mutations. How RNA viruses reconcile these two opposed facets of mutation is still unknown. Here we show that, in human immunodeficiency virus (HIV-1), spontaneous mutations are not randomly located along the viral genome. We find that the viral mutation rate experiences a threefold reduction in the region encoding the most external domains of the viral envelope, which are strongly targeted by neutralizing antibodies. This contrasts with the hypermutation mechanisms deployed by other, more slowly mutating …

Learning From Mistakes: The Role of Phages in Pandemics

The misuse of antibiotics is leading to the emergence of multidrug-resistant (MDR) bacteria, and in the absence of available treatments, this has become a major global threat. In the middle of the recent severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic, which has challenged the whole world, the emergence of MDR bacteria is increasing due to prophylactic administration of antibiotics to intensive care unit patients to prevent secondary bacterial infections. This is just an example underscoring the need to seek alternative treatments against MDR bacteria. To this end, phage therapy has been proposed as a promising tool. However, further research in the field is mandatory to assure…

Reliability of wastewater analysis for monitoring COVID-19 incidence revealed by a long-term follow-up study

Background Wastewater-based epidemiology has been used for monitoring human activities and waterborne pathogens. Although wastewaters can also be used for tracking SARS-CoV-2 at the population level, the reliability of this approach remains to be established, especially for early warning of outbreaks. Methods We collected 377 samples from different treatment plants processing wastewaters of >1 million inhabitants in Valencia, Spain, between April 2020 and March 2021. Samples were cleaned, concentrated, and subjected to RT-qPCR to determine SARS-CoV-2 concentrations. These data were compared with cumulative disease notification rates over 7 and 14 day periods. Results We amplified SARS-CoV-2…

Enhanced Antibacterial Activity of Repurposed Mitomycin C and Imipenem in Combination with the Lytic Phage vB_KpnM-VAC13 against Clinical Isolates of Klebsiella pneumoniae.

Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) on behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC).

Metropolitan wastewater analysis for COVID-19 epidemiological surveillance

The COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a rapidly emerging pandemic which has enforced extreme containment measures worldwide. In the absence of a vaccine or efficient treatment, cost-effective epidemiological surveillance strategies are urgently needed. Here, we have used RT-qPCR for SARS-CoV-2 detection in a series of longitudinal metropolitan wastewaters samples collected from February to April 2020, during the earliest stages of the epidemic in the Region of Valencia, Spain. We were able to consistently detect SARS-CoV-2 RNA in samples taken in late February, when communicated cases in that region were only incipient. We also find…

Social Bacteriophages

This article belongs to the Section Medical Microbiology.

Isolation and Characterization of Two Klebsiella pneumoniae Phages Encoding Divergent Depolymerases

The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics is Klebsiella pneumoniae, whose hypervariable extracellular capsule makes treatment particularly difficult. Here, we describe two new K. pneumoniae phages, &pi

Nucleoside Analogue Mutagenesis of a Single-Stranded DNA Virus: Evolution and Resistance

ABSTRACT It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of conce…

CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was t…

Lack of evidence for infectious SARS-CoV-2 in feces and sewage

Purpose: The SARS-CoV-2 coronavirus is a respiratory virus whose primary route of transmission is airborne. However, it has been shown that the virus can replicate in gastrointestinal cells, can be excreted in feces, and can reach sewage systems. Although viral RNA is known to be found in patient feces and sewage, little is known about the possible fecal-oral transmission of the coronavirus. Determining the presence of infective viral particles in feces and sewage is necessary to take adequate control measures and to discover new routes of coronavirus transmission. Here, we analyzed feces and urine of COVID-19 patients and wastewater samples at the time of high prevalence in the region unde…

The fitness effects of synonymous mutations in DNA and RNA viruses.

Despite being silent with respect to protein sequence, synonymous nucleotide substitutions can be targeted by natural selection directly at the DNA or RNA level. However, there has been no systematic assessment of how frequent this type of selection is. Here, we have constructed 53 single random synonymous substitution mutants of the bacteriophages Qb and UX174 by site-directed mutagenesis and assayed their fitness. Analysis of this mutant collection and of previous studies undertaken with a variety of single-stranded (ss) viruses demonstrates that selection at synonymous sites is stronger in RNA viruses than in DNA viruses. We estimate that this type of selection contributes approximately …

Phages as Promising Biomedical Tools

Social Interactions Among Bacteriophages

Although viruses lack many of the social adaptations shown by more complex organisms, different types of social interactions have been unraveled in viruses. Phage research has contributed significantly to the development of this field, called sociovirology, with the discovery of processes such as intracellular and extracellular public good production, prudent host exploitation, cheating, and inter-phage communication. We here review and discuss these processes from a social evolution approach. Similar to other organisms, the origin and maintenance of phage-phage interactions can be explained using kin selection, group selection and game theory approaches. Key determinants of phage social ev…

An unusually high substitution rate in transplant-associated BK polyomavirus in vivo is further concentrated in HLA-C-bound viral peptides

Infection with human BK polyomavirus, a small double-stranded DNA virus, potentially results in severe complications in immunocompromised patients. Here, we describe the in vivo variability and evolution of the BK polyomavirus by deep sequencing. Our data reveal the highest genomic evolutionary rate described in double-stranded DNA viruses, i.e., 10−3–10−5 substitutions per nucleotide site per year. High mutation rates in viruses allow their escape from immune surveillance and adaptation to new hosts. By combining mutational landscapes across viral genomes with in silico prediction of viral peptides, we demonstrate the presence of significantly more coding substitutions within predicted cog…

Experimental Evolution and Population Genetics of RNA Viruses

Viral studies have contributed substantially to the field of experimental evolution during the last two decades. The rapid evolution of RNA viruses makes them especially suitable for investigating real-time evolution, while their small genomes facilitate the analysis of the genetic basis of evolutionary change. We review recent advances in RNA virus experimental evolution, focusing on genetic properties that differentiate them from DNA-based organisms, such as their high mutation rates, small genome sizes, low genetic robustness, and the predominance of antagonistic epistasis. We argue that these properties can explain many aspects of RNA virus evolution, including rapid evolution, marked f…

Viral evolution and Immune responses

Antiviral responses are activated rapidly after viral infection in order to control and prevent dissemination of the virus. Different pathways are activated in the immune system, including innate and adaptive responses. On the other hand, viruses have evolved specifi c strategies to evade these responses. Due to the high viral evolutionary rates, escape variants can emerge and spread fast in the population. The co-evolution between viruses and their host is a constant arms race, and is of special interest to understand the viral escape mechanisms that may guide the future development of antiviral treatments and vaccines.

Directed evolution of a Mycobacteriophage

This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond.

Virus-Host Coevolution as a Tool for Controlling Bacterial Resistance to Phage Therapy

Bacterial resistance to antibiotics is a global public health concern. New treatments are needed to combat resistant strains, among which phage therapy is a promising option. Probably the main advantages of phage therapy are its high specificity as well as rapid viral adaptability, which in principle allows using phage evolution to overcome resistance. Here, we have performed serial coevolution passages between Escherichia coli and its phage T7 to investigate the ability of coevolved phages to reduce the emergence of resistances. We find that the initial bacterial population is less likely to undergo resistance when challenged with experimentally coevolved phages than when challenged with t…

The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages

Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar…

Genetic Diversity and Evolution of Viral Populations

Abstract Population genetic diversity plays a prominent role in viral evolution, pathogenesis, immune escape, and drug resistance. Different mechanisms are responsible for creating and maintaining genetic diversity in viruses, including error-prone replication, repair avoidance, and genome editing, among others. This diversity is subsequently modulated by natural selection and random genetic drift, whose action in turn depends on multiple factors including viral genetic architecture, viral demography, and ecology. Understanding these processes should contribute to the development of more efficient control and treatment strategies against viral pathogens.

Experimental virus evolution in cancer cell monolayers, spheroids, and tissue explants

Viral laboratory evolution has been used for different applications, such as modeling viral emergence, drug-resistance prediction, and therapeutic virus optimization. However, these studies have been mainly performed in cell monolayers, a highly simplified environment, raising concerns about their applicability and relevance. To address this, we compared the evolution of a model virus in monolayers, spheroids, and tissue explants. We performed this analysis in the context of cancer virotherapy by performing serial transfers of an oncolytic vesicular stomatitis virus (VSV-Δ51) in 4T1 mouse mammary tumor cells. We found that VSV-Δ51 gained fitness in each of these three culture systems, and t…

Social evolution of innate immunity evasion in a virus

Antiviral immunity has been studied extensively from the perspective of virus−cell interactions, yet the role of virus−virus interactions remains poorly addressed. Here, we demonstrate that viral escape from interferon (IFN)-based innate immunity is a social process in which IFN-stimulating viruses determine the fitness of neighbouring viruses. We propose a general and simple social evolution framework to analyse how natural selection acts on IFN shutdown and validate it in cell cultures and mice infected with vesicular stomatitis virus. Furthermore, we find that IFN shutdown is costly because it reduces short-term viral progeny production, thus fulfilling the definition of an altruistic tr…

Phages for biofilm removal

This article belongs to the Special Issue Phage Therapy, Lysin Therapy, and Antibiotics, a Trio Due to Come.

Mechanisms of viral mutation

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be intro…