Lysogeny destabilizes computationally simulated microbiomes.

Microbiomes are ecosystems, and their stability can impact the health of their hosts. Theory predicts that predators influence ecosystem stability. Phages are key predators of bacteria in microbiomes, but phages are unusual predators because many have lysogenic life cycles. It has been hypothesized that lysogeny can destabilize microbiomes, but lysogeny has no direct analog in classical ecological theory, and no formal theory exists. We studied the stability of computationally simulated microbiomes with different numbers of temperate (lysogenic) and virulent (obligate lytic) phage species. Bacterial populations were more likely to fluctuate over time when there were more temperate phages species. After disturbances, bacterial populations returned to their pre-disturbance densities more slowly when there were more temperate phage species, but cycles engendered by disturbances dampened more slowly when there were more virulent phage species. Our work offers the first formal theory linking lysogeny to microbiome stability.

The virome in allergy and asthma: A nascent, ineffable player.

Allergic diseases can be affected by virus-host interactions and are increasingly linked with the tissue-specific microbiome. High-throughput metagenomic sequencing has offered the opportunity to study the presence of viruses as an ecologic system, namely, the virome. Even though virome studies are technically challenging conceptually and analytically, they are already producing novel data expanding our understanding of the pathophysiologic mechanisms related to chronic inflammation and allergy. The importance of interspecies and intraspecies interactions is becoming apparent, as they can significantly, directly or indirectly, affect the host's response and antigenic state. Here, we emphasize the challenges and potential insights related to study of the virome in the context of allergy and asthma. We review the limited number of studies that have investigated the virome in these conditions, underlining the need for prospective, repeated sampling designs to unravel the virome's impact on disease development and its interplay with microbiota and immunity. The potential therapeutic use of bacteriophages, which are highly complex components of the virome, is discussed. There is clearly a need for further in-depth investigation of the virome as a system in allergic diseases.

Respiratory virome profiles reflect antiviral immune responses.

BACKGROUND: From early life, respiratory viruses are implicated in the development, exacerbation and persistence of respiratory conditions such as asthma. Complex dynamics between microbial communities and host immune responses shape immune maturation and homeostasis, influencing health outcomes. We evaluated the hypothesis that the respiratory virome is linked to systemic immune responses, using peripheral blood and nasopharyngeal swab samples from preschool-age children in the PreDicta cohort. METHODS: Peripheral blood mononuclear cells from 51 children (32 asthmatics and 19 healthy controls) participating in the 2-year multinational PreDicta cohort were cultured with bacterial (Bacterial-DNA, LPS) or viral (R848, Poly:IC, RV) stimuli. Supernatants were analysed by Luminex for the presence of 22 relevant cytokines. Virome composition was obtained using untargeted high throughput sequencing of nasopharyngeal samples. The metagenomic data were used for the characterization of virome profiles and the presence of key viral families (Picornaviridae, Anelloviridae, Siphoviridae). These were correlated to cytokine secretion patterns, identified through hierarchical clustering and principal component analysis. RESULTS: High spontaneous cytokine release was associated with increased presence of Prokaryotic virome profiles and reduced presence of Eukaryotic and Anellovirus profiles. Antibacterial responses did not correlate with specific viral families or virome profile; however, low antiviral responders had more Prokaryotic and less Eukaryotic virome profiles. Anelloviruses and Anellovirus-dominated profiles were equally distributed among immune response clusters. The presence of Picornaviridae and Siphoviridae was associated with low interferon-λ responses. Asthma or allergy did not modify these correlations. CONCLUSION: Antiviral cytokine responses at a systemic level reflect the upper airway virome composition. Individuals with low innate interferon responses have higher abundance of Picornaviruses (mostly Rhinoviruses) and bacteriophages. Bacteriophages, particularly Siphoviridae, appear to be sensitive sensors of host antimicrobial capacity, while Anelloviruses are not correlated with TLR-induced immune responses.

Development of an in vitro homeostasis model between airway epithelial cells, bacteria and bacteriophages: a time-lapsed observation of cell viability and inflammatory response.

Bacteriophages represent the most extensive group of viruses within the human virome and have a significant impact on general health and well-being by regulating bacterial population dynamics. Staphylococcus aureus, found in the anterior nostrils, throat and skin, is an opportunistic pathobiont that can cause a wide range of diseases, from chronic inflammation to severe and acute infections. In this study, we developed a human cell-based homeostasis model between a clinically isolated strain of S. aureus 141 and active phages for this strain (PYOSa141) isolated from the commercial Pyophage cocktail (PYO). The cocktail is produced by Eliava BioPreparations Ltd. (Tbilisi, Georgia) and is used as an add-on therapy for bacterial infections, mainly in Georgia. The triptych interaction model was evaluated by time-dependent analysis of cell death and inflammatory response of the nasal and bronchial epithelial cells. Inflammatory mediators (IL-8, CCL5/RANTES, IL-6 and IL-1ß) in the culture supernatants were measured by enzyme-linked immunosorbent assay and cell viability was determined by crystal violet staining. By measuring trans-epithelial electrical resistance, we assessed the epithelial integrity of nasal cells that had differentiated under air-liquid interface conditions. PYOSa141 was found to have a prophylactic effect on airway epithelial cells exposed to S. aureus 141 by effectively down-regulating bacterial-induced inflammation, cell death and epithelial barrier disruption in a time-dependent manner. Overall, the proposed model represents an advance in the way multi-component biological systems can be simulated in vitro.

Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses.

BACKGROUND: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. OBJECTIVE: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. METHODS: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue. RESULTS: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. CONCLUSIONS: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials.

Rhinovirus Species-Specific Antibodies Differentially Reflect Clinical Outcomes in Health and Asthma.

Rationale: Rhinoviruses (RVs) are major triggers of common cold and acute asthma exacerbations. RV species A, B, and C may have distinct clinical impact; however, little is known regarding RV species-specific antibody responses in health and asthma.Objectives: To describe and compare total and RV species-specific antibody levels in healthy children and children with asthma, away from an acute event.Methods: Serum samples from 163 preschool children with mild to moderate asthma and 72 healthy control subjects from the multinational Predicta cohort were analyzed using the recently developed PreDicta RV antibody chip.Measurements and Main Results: RV antibody levels varied, with RV-C and RV-A being higher than RV-B in both groups. Compared with control subjects, asthma was characterized by significantly higher levels of antibodies to RV-A and RV-C, but not RV-B. RV antibody levels positively correlated with the number of common colds over the previous year in healthy children, and wheeze episodes in children with asthma. Antibody levels also positively correlated with asthma severity but not with current asthma control.Conclusions: The variable humoral response to RV species in both groups suggests a differential infectivity pattern between RV species. In healthy preschoolers, RV antibodies accumulate with colds. In asthma, RV-A and RV-C antibodies are much higher and further increase with disease severity and wheeze episodes. Higher antibody levels in asthma may be caused by a compromised innate immune response, leading to increased exposure of the adaptive immune response to the virus. Importantly, there is no apparent protection with increasing levels of antibodies.

Promising approaches for the treatment and prevention of viral respiratory illnesses.

Viral respiratory tract infections are the most common human ailments, leading to enormous health and economic burden. Hundreds of viral species and subtypes have been associated with these conditions, with influenza viruses, respiratory syncytial virus, and rhinoviruses being the most frequent and with the highest burden. When considering prevention or treatment of viral respiratory tract infections, potential targets include the causative pathogens themselves but also the immune response, disease transmission, or even just the symptoms. Strategies targeting all these aspects are developing concurrently, and several novel and promising approaches are emerging. In this perspective we overview the entire range of options and highlight some of the most promising approaches, including new antiviral agents, symptomatic or immunomodulatory drugs, the re-emergence of natural remedies, and vaccines and public health policies toward prevention. Wide-scale prevention through immunization appears to be within reach for respiratory syncytial virus and promising for influenza virus, whereas additional effort is needed in regard to rhinovirus, as well as other respiratory tract viruses.

Virus Type and Genomic Load in Acute Bronchiolitis: Severity and Treatment Response With Inhaled Adrenaline.

BACKGROUND: Acute bronchiolitis frequently causes infant hospitalization. Studies on different viruses or viral genomic load and disease severity or treatment effect have had conflicting results. We aimed to investigate whether the presence or concentration of individual or multiple viruses were associated with disease severity in acute bronchiolitis and to evaluate whether detected viruses modified the response to inhaled racemic adrenaline. METHODS: Nasopharyngeal aspirates were collected from 363 infants with acute bronchiolitis in a randomized, controlled trial that compared inhaled racemic adrenaline versus saline. Virus genome was identified and quantified by polymerase chain reaction analyses. Severity was assessed on the basis of the length of stay and the use of supportive care. RESULTS: Respiratory syncytial virus (83%) and human rhinovirus (34%) were most commonly detected. Seven other viruses were present in 8%-15% of the patients. Two or more viruses (maximum, 7) were detected in 61% of the infants. Virus type or coinfection was not associated with disease severity. A high genomic load of respiratory syncytial virus was associated with a longer length of stay and with an increased frequency of oxygen and ventilatory support use. Treatment effect of inhaled adrenaline was not modified by virus type, load or coinfection. DISCUSSION: In infants hospitalized with acute bronchiolitis, disease severity was not associated with specific viruses or the total number of viruses detected. A high RSV genomic load was associated with more-severe disease. CLINICAL TRIALS REGISTRATION: NCT00817466 and EudraCT 2009-012667-34.

The common cold: potential for future prevention or cure.

The common cold is the most frequent, although generally mild, human disease. Human Rhinoviruses are the prevalent causative agents, but other viruses are also implicated. Being so common, viral colds, have significant implications on public health and quality of life, but may also be life-threatening for vulnerable groups of patients. Specific diagnosis and treatment of the common cold still remain unmet needs. Molecular diagnostic techniques allow specific detection of known pathogens as well as the identification of newly emerging viruses. Although a number of medications or natural treatments have been shown to have some effect, either on the number or on the severity of common colds, no single agent is considerably effective. Virus-specific management remains in most cases a challenging potential as many factors have to be taken into account, including the diversity of the viral genomes, the heterogeneity of affected individuals, as well as the complexity of this long standing host-virus relationship.

Exploring the Impact of Airway Microbiome on Asthma Morbidity: A Focus on the "Constructing a 'Eubiosis Reinstatement Therapy' for Asthma-CURE" Project.

The asthma pandemic imposes a huge burden on patients and health systems in both developed and developing countries. Despite available treatments, symptom control is generally suboptimal, and hospitalizations and deaths remain at unacceptably high levels. A pivotal aspect of asthma that warrants further exploration is the influence of the respiratory microbiome and virome in modulating disease activity. A plethora of studies report that the respiratory microbiome is characteristically dysbiotic in asthma. In addition, our data suggest that dysbiosis is also observed on the respiratory virome, partly characterized by the reduced abundance of bacteriophages (phages). Even though phages can naturally infect and control their bacterial prey, phage therapy has been grossly neglected in the Western world, although more recently it is more widely used as a novel tool against bacterial infections. However, it has never been used for tackling microbiome dysbiosis in human non-communicable diseases. This review provides an up-to-date understanding of the microbiome and virome's role within the airways in relation to asthma morbidity. It also advances the rationale and hypothesis for the CURE project. Specifically, the CURE project suggests that managing the respiratory microbiome through phage therapy is viable and may result in restoring eubiosis within the asthmatic airway. This entails controlling immune dysregulation and the clinical manifestation of the disease. To accomplish this goal, it is crucial to predict the effects of introducing specific phage mixtures into the intricate ecology of the airways and devise suitable interventions.

Relevance of the bacteriophage adherence to mucus model for Pseudomonas aeruginosa phages.

Pseudomonas aeruginosa infections are getting increasingly serious as antimicrobial resistance spreads. Phage therapy may be a solution to the problem, especially if improved by current advances on phage-host studies. As a mucosal pathogen, we hypothesize that P. aeruginosa and its phages are linked to the bacteriophage adherence to mucus (BAM) model. This means that phage-host interactions could be influenced by mucin presence, impacting the success of phage infections on the P. aeruginosa host and consequently leading to the protection of the metazoan host. By using a group of four different phages, we tested three important phenotypes associated with the BAM model: phage binding to mucin, phage growth in mucin-exposed hosts, and the influence of mucin on CRISPR immunity of the bacterium. Three of the tested phages significantly bound to mucin, while two had improved growth rates in mucin-exposed hosts. Improved phage growth was likely the result of phage exploitation of mucin-induced physiological changes in the host. We could not detect CRISPR activity in our system but identified two putative anti-CRISPR proteins coded by the phage. Overall, the differential responses seen for the phages tested show that the same bacterial species can be targeted by mucosal-associated phages or by phages not affected by mucus presence. In conclusion, the BAM model is relevant for phage-bacterium interactions in P. aeruginosa, opening new possibilities to improve phage therapy against this important pathogen by considering mucosal interaction dynamics.IMPORTANCESome bacteriophages are involved in a symbiotic relationship with animals, in which phages held in mucosal surfaces protect them from invading bacteria. Pseudomonas aeruginosa is one of the many bacterial pathogens threatening humankind during the current antimicrobial resistance crisis. Here, we have tested whether P. aeruginosa and its phages are affected by mucosal conditions. We discovered by using a collection of four phages that, indeed, mucosal interaction dynamics can be seen in this model. Three of the tested phages significantly bound to mucin, while two had improved growth rates in mucin-exposed hosts. These results link P. aeruginosa and its phages to the bacteriophage adherence to the mucus model and open opportunities to explore this to improve phage therapy, be it by exploiting the phenotypes detected or by actively selecting mucosal-adapted phages for treatment.

Respiratory eukaryotic virome expansion and bacteriophage deficiency characterize childhood asthma.

Asthma development and exacerbation is linked to respiratory virus infections. There is limited information regarding the presence of viruses during non-exacerbation/infection periods. We investigated the nasopharyngeal/nasal virome during a period of asymptomatic state, in a subset of 21 healthy and 35 asthmatic preschool children from the Predicta cohort. Using metagenomics, we described the virome ecology and the cross-species interactions within the microbiome. The virome was dominated by eukaryotic viruses, while prokaryotic viruses (bacteriophages) were independently observed with low abundance. Rhinovirus B species consistently dominated the virome in asthma. Anelloviridae were the most abundant and rich family in both health and asthma. However, their richness and alpha diversity were increased in asthma, along with the co-occurrence of different Anellovirus genera. Bacteriophages were richer and more diverse in healthy individuals. Unsupervised clustering identified three virome profiles that were correlated to asthma severity and control and were independent of treatment, suggesting a link between the respiratory virome and asthma. Finally, we observed different cross-species ecological associations in the healthy versus the asthmatic virus-bacterial interactome, and an expanded interactome of eukaryotic viruses in asthma. Upper respiratory virome "dysbiosis" appears to be a novel feature of pre-school asthma during asymptomatic/non-infectious states and merits further investigation.

Corrigendum: Interactions of Bacteriophages and Bacteria at the Airway Mucosa: New Insights Into the Pathophysiology of Asthma.

[This corrects the article DOI: 10.3389/falgy.2020.617240.].

Broad and unexpected phenotypic expression in Greek children with steroid-resistant nephrotic syndrome due to mutations in the Wilms' tumor 1 (WT1) gene.

Mutations in the Wilms' tumor suppressor gene 1 (WT1), most commonly within exons 8 or 9 or intron 9, are found in cases with the overlapping conditions of Denys-Drash and Frasier syndromes, as well as in patients with steroid-resistant nephrotic syndrome (SRNS). This study investigated the presence of WT1 gene mutations in cases with childhood SRNS, along with an evaluation of their clinical outcome. Twenty-seven Greek children with sporadic (19 cases) and familial (8 cases) SRNS were tested. Four phenotypically female patients with sporadic SRNS were found to carry de novo WT1 mutations, including two cases with p.R394W, and one case each with p.R366H, or n.1228+5G>A. Karyotype analysis found 46XX in three cases, but 46XY in one. No phenotype-genotype correlations were apparent in the WT1 gene positive cases since their clinical presentation varied broadly. Interestingly, one patient with a pathological WT1 nucleotide variation responded fully to combined therapy with cyclosporine A and corticosteroids. This study further illustrates that investigation of WT1 gene mutations is clinically useful to support definitive diagnosis in children presenting with SRNS in order to direct the most appropriate clinical management.

Investigation of Salmonella Phage-Bacteria Infection Profiles: Network Structure Reveals a Gradient of Target-Range from Generalist to Specialist Phage Clones in Nested Subsets.

Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus-host infection networks have been used to characterize the "predator-prey" interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage-bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

Recurrent Wheeze Exacerbations Following Acute Bronchiolitis-A Machine Learning Approach.

Introduction: Acute bronchiolitis is one of the most common respiratory infections in infancy. Although most infants with bronchiolitis do not get hospitalized, infants with hospitalized bronchiolitis are more likely to develop wheeze exacerbations during the first years of life. The objective of this prospective cohort study was to develop machine learning models to predict incidence and persistence of wheeze exacerbations following the first hospitalized episode of acute bronchiolitis. Methods: One hundred thirty-one otherwise healthy term infants hospitalized with the first episode of bronchiolitis at a tertiary pediatric hospital in Athens, Greece, and 73 age-matched controls were recruited. All patients/controls were followed up for 3 years with 6-monthly telephone reviews. Through principal component analysis (PCA), a cluster model was used to describe main outcomes. Associations between virus type and the clusters and between virus type and other clinical characteristics and demographic data were identified. Through random forest classification, a prediction model with smallest classification error was identified. Primary outcomes included the incidence and the number of caregiver-reported wheeze exacerbations. Results: PCA identified 2 clusters of the outcome measures (Cluster 1 and Cluster 2) that were significantly associated with the number of recurrent wheeze episodes over 3-years of follow-up (Chi-Squared, p < 0.001). Cluster 1 included infants who presented higher number of wheeze exacerbations over follow-up time. Rhinovirus (RV) detection was more common in Cluster 1 and was more strongly associated with clinical severity on admission (p < 0.01). A prediction model based on virus type and clinical severity could predict Cluster 1 with an overall error 0.1145 (sensitivity 75.56% and specificity 91.86%). Conclusion: A prediction model based on virus type and clinical severity of first hospitalized episode of bronchiolitis could predict sensitively the incidence and persistence of wheeze exacerbations during a 3-year follow-up. Virus type (RV) was the strongest predictor.

Analysis of human total antibody repertoires in TIF1γ autoantibody positive dermatomyositis.

We investigate the accumulated microbial and autoantigen antibody repertoire in adult-onset dermatomyositis patients sero-positive for TIF1γ (TRIM33) autoantibodies. We use an untargeted high-throughput approach which combines immunoglobulin disease-specific epitope-enrichment and identification of microbial and human antigens. We observe antibodies recognizing a wider repertoire of microbial antigens in dermatomyositis. Antibodies recognizing viruses and Poxviridae family species are significantly enriched. The identified autoantibodies recognise a large portion of the human proteome, including interferon regulated proteins; these proteins cluster in specific biological processes. In addition to TRIM33, we identify autoantibodies against eleven further TRIM proteins, including TRIM21. Some of these TRIM proteins share epitope homology with specific viral species including poxviruses. Our data suggest antibody accumulation in dermatomyositis against an expanded diversity of microbial and human proteins and evidence of non-random targeting of specific signalling pathways. Our findings indicate that molecular mimicry and epitope spreading events may play a role in dermatomyositis pathogenesis.