Mucosal Microbiome Disruption in Acute Laryngeal Injury Following Intubation.

The objective of this study was to characterize mucosal microbial shifts in patients with acute laryngeal injury (ALgI) after intubation. This cross-sectional study included 20 patients with ALgI who underwent early endoscopic intervention with tissue culture, 20 patients with idiopathic subglottic stenosis (iSGS) who underwent tissue culture during the routine endoscopic intervention, and 3 control patients who underwent mucosal swab culture. 70% of the ALgI patients had a positive culture compared to 5% of the iSGS patients and none of the controls. The most identified microbes isolated from ALgI patients included Staphylococcus species in 30% and Streptococcus species in 25%. The high rate of pathologic bacterial infiltration into postintubation laryngeal wounds supports efforts to reduce bacterial colonization of endotracheal tubes and highlights the role of culture-directed antibiotic therapy as a part of early intervention to improve outcomes for patients with ALgI.

Proton Pump Inhibitors Modulate Gene Expression Profile in Esophageal Mucosa and Microbiome.

OBJECTIVE: Proton pump inhibitors (PPIs) are commonly used to manage children with upper gastrointestinal symptoms and without a formal diagnosis. We investigated the effect of PPIs on esophageal mucosal transcriptome and active microbiota in children with normal esophagi. Furthermore, we examined whether the differences in host esophageal mucosal gene expression were driven by an underlying esophageal epithelial cell type composition. METHODS: Using metatranscriptomics, the host transcriptional and active microbial profiles were captured from 17 esophageal biopsy samples (PPI naïve [PPI-], n = 7; PPI exposed [PPI+], n = 10) collected from children without any endoscopic and histologic abnormalities in their esophagus (normal esophagus). Deconvolution computational analysis was performed with xCell to assess if the observed epithelial gene expression changes were related to the cell type composition in the esophageal samples. RESULTS: The median (IQR) age of our cohort was 14 years (12-16) with female (63%) preponderance. Both groups were similar in terms of their demographics and clinical features. Compared with PPI-, the PPI+ had upregulation of 27 genes including the MUC genes. The cell type composition was similar between the PPI- and PPI+ groups. Prevotella sp and Streptococcus sp were abundant in PPI+ group. CONCLUSIONS: In children with normal esophagus, PPI exposure can be associated with upregulation of esophageal mucosal homeostasis and epithelial cell function genes in a cell-type independent manner, and an altered esophageal microbiome. Additional studies are warranted to validate our findings and to investigate the causal effect of PPIs on the normal esophageal epithelium and microbial communities.

The relationship between the intestinal microbiome and body mass index in children with cystic fibrosis.

BACKGROUND: The nutritional status of children with cystic fibrosis (CF), as assessed by their body mass index percentile (BMIp), is a critical determinant of long-term health outcomes. While the intestinal microbiome plays an important role in nutrition, little is known regarding the relationship of the microbiome and BMIp in children with CF. METHODS: Pediatric patients (< 18 years old) with CF and healthy comparison patients (HCs) were enrolled in the study and stool samples obtained. BMIp was categorized as Green Zone (BMIp > 50th), Yellow Zone (BMIp 25th-49th) and Red Zone (BMIp < 25th). Intestinal microbiome assessment was performed via 16S rRNA gene sequencing; microbial richness, diversity, and differential species abundance were assessed. RESULTS: Stool samples were collected from 107 children with CF and 50 age-matched HCs. Compared to HCs, children with CF were found to have lower bacterial richness, alpha-diversity, and a different microbial composition. When evaluating them by their BMIp color zone, richness and alpha-diversity were lowest in those in the Red Zone. In addition, an unclassified amplicon sequence variant (ASV) of Blautia, a known butyrate-producing anaerobe, was of lowest abundance in children in the Red Zone. CONCLUSION: Children with CF have a dysbiotic intestinal microbiome with specific changes that accompany changes in BMIp. Longitudinal assessments of the microbiome and its metabolic activities over time are needed to better understand how improvements in the microbiome may improve nutrition and enhance long-term survival in children with CF.

Idiopathic subglottic stenosis arises at the epithelial interface of host and pathogen.

Background: Idiopathic subglottic stenosis (iSGS) is a rare fibrotic disease of the proximal airway affecting adult Caucasian women nearly exclusively. Life-threatening ventilatory obstruction occurs secondary to pernicious subglottic mucosal scar. Disease rarity and wide geographic patient distribution has previously limited substantive mechanistic investigation into iSGS pathogenesis. Result: By harnessing pathogenic mucosa from an international iSGS patient cohort and single-cell RNA sequencing, we unbiasedly characterize the cell subsets in the proximal airway scar and detail their molecular phenotypes. Results show that the airway epithelium in iSGS patients is depleted of basal progenitor cells, and the residual epithelial cells acquire a mesenchymal phenotype. Observed displacement of bacteria beneath the lamina propria provides functional support for the molecular evidence of epithelial dysfunction. Matched tissue microbiomes support displacement of the native microbiome into the lamina propria of iSGS patients rather than disrupted bacterial community structure. However, animal models confirm that bacteria are necessary for pathologic proximal airway fibrosis and suggest an equally essential role for host adaptive immunity. Human samples from iSGS airway scar demonstrate adaptive immune activation in response to the proximal airway microbiome of both matched iSGS patients and healthy controls. Clinical outcome data from iSGS patients suggests surgical extirpation of airway scar and reconstitution with unaffected tracheal mucosa halts the progressive fibrosis. Conclusion: Our data support an iSGS disease model where epithelial alterations facilitate microbiome displacement, dysregulated immune activation, and localized fibrosis. These results refine our understanding of iSGS and implicate shared pathogenic mechanisms with distal airway fibrotic diseases.

Evolutionary dynamics of respiratory syncytial virus in Buenos Aires: Viral diversity, migration, and subgroup replacement.

Globally, the human respiratory syncytial virus (RSV) is one of the major causes of lower respiratory tract infections (LRTIs) in children. The scarcity of complete genome data limits our understanding of RSV spatiotemporal distribution, evolution, and viral variant emergence. Nasopharyngeal samples collected from hospitalized pediatric patients from Buenos Aires tested positive for RSV LRTI during four consecutive outbreaks (2014-2017) were randomly subsampled for RSV complete genome sequencing. Phylodynamic studies and viral population characterization of genomic variability, diversity, and migration of viruses to and from Argentina during the study period were performed. Our sequencing effort resulted in one of the largest collections of RSV genomes from a given location (141 RSV-A and 135 RSV-B) published so far. RSV-B was dominant during the 2014-2016 outbreaks (60 per cent of cases) but was abruptly replaced by RSV-A in 2017, with RSV-A accounting for 90 per cent of sequenced samples. A significant decrease in RSV genomic diversity-represented by both a reduction in genetic lineages detected and the predominance of viral variants defined by signature amino acids-was observed in Buenos Aires in 2016, the year prior to the RSV subgroup predominance replacement. Multiple introductions to Buenos Aires were detected, some with persistent detection over seasons, and also, RSV was observed to migrate from Buenos Aires to other countries. Our results suggest that the decrease in viral diversity may have allowed the dramatic predominance switch from RSV-B to RSV-A in 2017. The immune pressure generated against circulating viruses with limited diversity during a given outbreak may have created a fertile ground for an antigenically divergent RSV variant to be introduced and successfully spread in the subsequent outbreak. Overall, our RSV genomic analysis of intra- and inter-outbreak diversity provides an opportunity to better understand the epochal evolutionary dynamics of RSV.

Influence of Sex on Respiratory Syncytial Virus Genotype Infection Frequency and Nasopharyngeal Microbiome.

Respiratory syncytial virus (RSV) has a significant health burden in children, older adults, and the immunocompromised. However, limited effort has been made to identify emergence of new RSV genotypes' frequency of infection and how the combination of nasopharyngeal microbiome and viral genotypes impact RSV disease outcomes. In an observational cohort designed to capture the first infant RSV infection, we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes, during which the 2012/2013 season was dominated by RSV-A, whereas 2013 and 2014 was dominated by RSV-B. We found non-G-72nt-duplicated RSV-A strains were more frequent in male infants (P = 0.02), whereas G-72nt-duplicated genotypes (which is ON1 lineage) were seen equally in both males and females. DESeq2 testing of the nasal microbiome showed Haemophilus was significantly more abundant in infants with RSV-A infection compared to infants with RSV-B infection (adjusted P = 0.002). In addition, the broad microbial clustering of the abundant genera was significantly associated with infant sex (P = 0.03). Overall, we show sex differences in infection by RSV genotype and host nasopharyngeal microbiome, suggesting an interaction between host genetics, virus genotype, and associated nasopharyngeal microbiome. IMPORTANCE Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infections in young children and is responsible for high hospitalization rates and morbidity in infants and the elderly. To understand how the emergence of RSV viral genotypes and viral-respiratory microbiome interactions contribute to infection frequency and severity, we utilized an observational cohort designed to capture the first infant RSV infection we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes. We found non-G-72nt-duplicated RSV-A genotypes were more frequent in male infants, whereas G-72nt-duplicated RSV-A strains (ON1 lineage) were seen equally in both males and females. Microbiome analysis show Haemophilus was significantly more abundant in infants with RSV-A compared to infants with RSV-B infection and the microbial clustering of the abundant genera was associated with infant sex. Overall, we show sex differences in RSV genotype-nasopharyngeal microbiome, suggesting an interaction host genetics-virus-microbiome interaction.

Upper respiratory tract microbiota dynamics following COVID-19 in adults.

To date, little is known about the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, on the upper respiratory tract (URT) microbiota over time. To fill this knowledge gap, we used 16S ribosomal RNA gene sequencing to characterize the URT microbiota in 48 adults, including (1) 24 participants with mild-to-moderate COVID-19 who had serial mid-turbinate swabs collected up to 21 days after enrolment and (2) 24 asymptomatic, uninfected controls who had mid-turbinate swabs collected at enrolment only. To compare the URT microbiota between groups in a comprehensive manner, different types of statistical analyses that are frequently employed in microbial ecology were used, including ⍺-diversity, ß-diversity and differential abundance analyses. Final statistical models included age, sex and the presence of at least one comorbidity as covariates. The median age of all participants was 34.00 (interquartile range=28.75-46.50) years. In comparison to samples from controls, those from participants with COVID-19 had a lower observed species index at day 21 (linear regression coefficient=-13.30; 95 % CI=-21.72 to -4.88; q=0.02). In addition, the Jaccard index was significantly different between samples from participants with COVID-19 and those from controls at all study time points (PERMANOVA q<0.05 for all comparisons). The abundance of three amplicon sequence variants (ASVs) (one Corynebacterium ASV, Frederiksenia canicola, and one Lactobacillus ASV) were decreased in samples from participants with COVID-19 at all seven study time points, whereas the abundance of one ASV (from the family Neisseriaceae) was increased in samples from participants with COVID-19 at five (71.43 %) of the seven study time points. Our results suggest that mild-to-moderate COVID-19 can lead to alterations of the URT microbiota that persist for several weeks after the initial infection.

Mucosal Gene Expression in Response to SARS-CoV-2 Is Associated with Viral Load.

Little is known about the relationships between symptomatic early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and upper airway mucosal gene expression and immune response. To examine the association of symptomatic SARS-CoV-2 early viral load with upper airway mucosal gene expression, we profiled the host mucosal transcriptome from nasopharyngeal swab samples from 68 adults with symptomatic, mild-to-moderate coronavirus disease 19 (COVID-19). We measured SARS-CoV-2 viral load using reverse transcription-quantitative PCR (RT-qPCR). We then examined the association of SARS-CoV-2 viral load with upper airway mucosal immune response. We detected SARS-CoV-2 in all samples and recovered >80% of the genome from 95% of the samples from symptomatic COVID-19 adults. The respiratory virome was dominated by SARS-CoV-2, with limited codetection of other respiratory viruses, with the human Rhinovirus C being identified in 4 (6%) samples. This limited codetection of other respiratory viral pathogens may be due to the implementation of public health measures, like social distancing and masking practices. We observed a significant positive correlation between SARS-CoV-2 viral load and interferon signaling (OAS2, OAS3, IFIT1, UPS18, ISG15, ISG20, IFITM1, and OASL), chemokine signaling (CXCL10 and CXCL11), and adaptive immune system (IFITM1, CD300E, and SIGLEC1) genes in symptomatic, mild-to-moderate COVID-19 adults, when adjusting for age, sex, and race. Interestingly, the expression levels of most of these genes plateaued at a cycle threshold (CT) value of ~25. Overall, our data show that the early nasal mucosal immune response to SARS-CoV-2 infection is viral load dependent, potentially modifying COVID-19 outcomes. IMPORTANCE Several prior studies have shown that SARS-CoV-2 viral load can predict the likelihood of disease spread and severity. A higher detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity. However, the relationship between SARS-CoV-2 viral load, airway mucosal gene expression, and immune response remains elusive. We profiled the nasal mucosal transcriptome from nasal samples collected from adults infected with SARS-CoV-2 during spring 2020 with mild-to-moderate symptoms using a comprehensive metatranscriptomics method. We observed a positive correlation between SARS-CoV-2 viral load, interferon signaling, chemokine signaling, and adaptive immune system in adults with COVID-19. Our data suggest that early nasal mucosal immune response to SARS-CoV-2 infection was viral load dependent and may modify COVID-19 outcomes.

Ligation-based assay for variant typing without sequencing: Application to SARS-CoV-2 variants of concern.

BACKGROUND: COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. METHODS: A ligation-based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS-CoV-2) by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time polymerase chain reaction (PCR) instrumentation for assay performance. RESULTS: The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 104 genome copies/mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. CONCLUSIONS: The assay demonstrates the potential for robust variant typing with performance comparable with next-generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

Viral Genetic Determinants of Prolonged Respiratory Syncytial Virus Infection Among Infants in a Healthy Term Birth Cohort.

BACKGROUND: Respiratory syncytial virus (RSV) is associated with acute respiratory infection. We sought to identify RSV variants associated with prolonged infection. METHODS: Among healthy term infants we identified those with prolonged RSV infection and conducted (1) a human genome-wide association study (GWAS) to test the dependence of infection risk on host genotype, (2) a viral GWAS for association with prolonged RSV infection using RSV whole-genome sequencing, (3) an analysis of all viral public sequences, (4) an assessment of immunological responses, and (5) a summary of all major functional data. Analyses were adjusted for viral/human population structure and host factors associated with infection risk. RESULTS: We identified p.E123K/D and p.P218T/S/L in G protein that were associated with prolonged infection (Padj = .01). We found no evidence of host genetic risk for infection. The RSV variant positions approximate sequences that could bind a putative viral receptor, heparan sulfate. CONCLUSIONS: Using analysis of both viral and host genetics we identified a novel RSV variant associated with prolonged infection in otherwise healthy infants and no evidence supporting host genetic susceptibility to infection. As the capacity of RSV for chronicity and its viral reservoir are not defined, these findings are important for understanding the impact of RSV on chronic disease and endemicity.

Species-specific transcriptomic changes upon respiratory syncytial virus infection in cotton rats.

The cotton rat (Sigmodon) is the gold standard pre-clinical small animal model for respiratory viral pathogens, especially for respiratory syncytial virus (RSV). However, without a reference genome or a published transcriptome, studies requiring gene expression analysis in cotton rats are severely limited. The aims of this study were to generate a comprehensive transcriptome from multiple tissues of two species of cotton rats that are commonly used as animal models (Sigmodon fulviventer and Sigmodon hispidus), and to compare and contrast gene expression changes and immune responses to RSV infection between the two species. Transcriptomes were assembled from lung, spleen, kidney, heart, and intestines for each species with a contig N50 > 1600. Annotation of contigs generated nearly 120,000 gene annotations for each species. The transcriptomes of S. fulviventer and S. hispidus were then used to assess immune response to RSV infection. We identified 238 unique genes that are significantly differentially expressed, including several genes implicated in RSV infection (e.g., Mx2, I27L2, LY6E, Viperin, Keratin 6A, ISG15, CXCL10, CXCL11, IRF9) as well as novel genes that have not previously described in RSV research (LG3BP, SYWC, ABEC1, IIGP1, CREB1). This study presents two comprehensive transcriptome references as resources for future gene expression analysis studies in the cotton rat model, as well as provides gene sequences for mechanistic characterization of molecular pathways. Overall, our results provide generalizable insights into the effect of host genetics on host-virus interactions, as well as identify new host therapeutic targets for RSV treatment and prevention.

Mucosal gene expression in response to SARS-CoV-2 is associated with early viral load.

Little is known about the relationships between symptomatic early-time SARS-CoV-2 viral load and upper airway mucosal gene expression and immune response. To examine the association of symptomatic SARS-CoV-2 early viral load with upper airway mucosal gene expression, we profiled the host mucosal transcriptome from nasopharyngeal swab samples from 68 adults with symptomatic, mild-to-moderate COVID-19. We measured SARS-CoV-2 viral load using qRT-PCR. We then examined the association of SARS-CoV-2 viral load with upper airway mucosal immune response. We detected SARS-CoV-2 in all samples and recovered >80% of the genome from 85% of the samples from symptomatic COVID-19 adults. The respiratory virome was dominated by SARS-CoV-2, with limited co-detection of common respiratory viruses i.e., only the human Rhinovirus (HRV) being identified in 6% of the samples. We observed a significant positive correlation between SARS-CoV-2 viral load and interferon signaling (OAS2, OAS3, IFIT1, UPS18, ISG15, ISG20, IFITM1, and OASL), chemokine signaling (CXCL10 and CXCL11), and adaptive immune system (IFITM1, CD300E, and SIGLEC1) genes in symptomatic, mild-to-moderate COVID-19 adults, when adjusted for age, sex and race. Interestingly, the expression levels of most of these genes plateaued at a CT value of ~25. Overall, our data shows that early nasal mucosal immune response to SARS-CoV-2 infection is viral load dependent, which potentially could modify COVID-19 outcomes. AUTHOR SUMMARY: Several prior studies have shown that SARS-CoV-2 viral load can predict the likelihood of disease spread and severity. A higher detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity. However, the relationship between SARS-CoV-2 viral load and airway mucosal gene expression and immune response remains elusive. We profiled the nasal mucosal transcriptome from nasal samples collected from adults infected with SARS-CoV-2 during Spring 2020 with mild-to-moderate symptoms using a comprehensive metatranscriptomics method. We observed a positive correlation between SARS-CoV-2 viral load with interferon signaling, chemokine signaling, and adaptive immune system in adults with COVID-19. Our data suggest that early nasal mucosal immune response to SARS-CoV-2 infection was viral load-dependent and may modify COVID-19 outcomes.

COVID-19 severity from Omicron and Delta SARS-CoV-2 variants.

The Omicron variant of SARS-CoV-2 achieved worldwide dominance in late 2021. Early work suggests that infections caused by the Omicron variant may be less severe than those caused by the Delta variant. We sought to compare clinical outcomes of infections caused by these two strains, confirmed by whole genome sequencing, over a short period of time, from respiratory samples collected from SARS-CoV-2 positive patients at a large medical center. We found that infections caused by the Omicron variant caused significantly less morbidity, including admission to the hospital and requirement for oxygen supplementation, and significantly less mortality than those caused by the Delta variant.

Exclusive breast-feeding, the early-life microbiome and immune response, and common childhood respiratory illnesses.

BACKGROUND: The impact of breast-feeding on certain childhood respiratory illnesses remains controversial. OBJECTIVE: We sought to examine the effect of exclusive breast-feeding on the early-life upper respiratory tract (URT) and gut microbiome, the URT immune response in infancy, and the risk of common pediatric respiratory diseases. METHODS: We analyzed data from a birth cohort of healthy infants with prospective ascertainment of breast-feeding patterns and common pediatric pulmonary and atopic outcomes. In a subset of infants, we also characterized the URT and gut microbiome using 16S ribosomal RNA sequencing and measured 9 URT cytokines using magnetic bead-based assays. RESULTS: Of the 1949 infants enrolled, 1495 (76.71%) had 4-year data. In adjusted analyses, exclusive breast-feeding (1) had an inverse dose-response on the ⍺-diversity of the early-life URT and gut microbiome, (2) was positively associated with the URT levels of IFN-α, IFN-γ, and IL-17A in infancy, and (3) had a protective dose-response on the development of a lower respiratory tract infection in infancy, 4-year current asthma, and 4-year ever allergic rhinitis (odds ratio [95% CI] for each 4 weeks of exclusive breast-feeding, 0.95 [0.91-0.99], 0.95 [0.90-0.99], and 0.95 [0.92-0.99], respectively). In exploratory analyses, we also found that the protective association of exclusive breast-feeding on 4-year current asthma was mediated through its impact on the gut microbiome (P = .03). CONCLUSIONS: Our results support a protective causal role of exclusive breast-feeding in the risk of developing a lower respiratory tract infection in infancy and asthma and allergic rhinitis in childhood. They also shed light on potential mechanisms of these associations, including the effect of exclusive breast-feeding on the gut microbiome.

Upper respiratory tract bacterial-immune interactions during respiratory syncytial virus infection in infancy.

BACKGROUND: The risk factors determining short- and long-term morbidity following acute respiratory infection (ARI) due to respiratory syncytial virus (RSV) in infancy remain poorly understood. OBJECTIVES: Our aim was to examine the associations of the upper respiratory tract (URT) microbiome during RSV ARI in infancy with the acute local immune response and short- and long-term clinical outcomes. METHODS: We characterized the URT microbiome by 16S ribosomal RNA sequencing and assessed the acute local immune response by measuring 53 immune mediators with high-throughput immunoassays in 357 RSV-infected infants. Our short- and long-term clinical outcomes included several markers of disease severity and the number of wheezing episodes in the fourth year of life, respectively. RESULTS: We found several specific URT bacterial-immune mediator associations. In addition, the Shannon ⍺-diversity index of the URT microbiome was associated with a higher respiratory severity score (ß =.50 [95% CI = 0.13-0.86]), greater odds of a lower ARI (odds ratio = 1.63 [95% CI = 1.10-2.43]), and higher number of wheezing episodes in the fourth year of life (ß = 0.89 [95% CI = 0.37-1.40]). The Jaccard ß-diversity index of the URT microbiome differed by level of care required (P = .04). Furthermore, we found an interaction between the Shannon ⍺-diversity index of the URT microbiome and the first principal component of the acute local immune response on the respiratory severity score (P = .048). CONCLUSIONS: The URT microbiome during RSV ARI in infancy is associated with the acute local immune response, disease severity, and number of wheezing episodes in the fourth year of life. Our results also suggest complex URT bacterial-immune interactions that can affect the severity of the RSV ARI.

Metatranscriptomics to characterize respiratory virome, microbiome, and host response directly from clinical samples.

We developed a metatranscriptomics method that can simultaneously capture the respiratory virome, microbiome, and host response directly from low biomass samples. Using nasal swab samples, we capture RNA virome with sufficient sequencing depth required to assemble complete genomes. We find a surprisingly high frequency of respiratory syncytial virus (RSV) and coronavirus (CoV) in healthy children, and a high frequency of RSV-A and RSV-B co-detections in children with symptomatic RSV. In addition, we have identified commensal and pathogenic bacteria and fungi at the species level. Functional analysis revealed that H. influenzae was highly active in symptomatic RSV subjects. The host nasal transcriptome reveled upregulation of the innate immune system, anti-viral response and inflammasome pathway, and downregulation of fatty acid pathways in children with symptomatic RSV. Overall, we demonstrate that our method is broadly applicable to infer the transcriptome landscape of an infected system, surveil respiratory infections, and to sequence RNA viruses directly from clinical samples.

Microbial community structure and composition is associated with host species and sex in Sigmodon cotton rats.

BACKGROUND: The cotton rat (genus Sigmodon) is an essential small animal model for the study of human infectious disease and viral therapeutic development. However, the impact of the host microbiome on infection outcomes has not been explored in this model, partly due to the lack of a comprehensive characterization of microbial communities across different cotton rat species. Understanding the dynamics of their microbiome could significantly help to better understand its role when modeling viral infections in this animal model. RESULTS: We examined the bacterial communities of the gut and three external sites (skin, ear, and nose) of two inbred species of cotton rats commonly used in research (S. hispidus and S. fulviventer) by using 16S rRNA gene sequencing, constituting the first comprehensive characterization of the cotton rat microbiome. We showed that S. fulviventer maintained higher alpha diversity and richness than S. hispidus at external sites (skin, ear, nose), but there were no differentially abundant genera. However, S. fulviventer and S. hispidus had distinct fecal microbiomes composed of several significantly differentially abundant genera. Whole metagenomic shotgun sequencing of fecal samples identified species-level differences between S. hispidus and S. fulviventer, as well as different metabolic pathway functions as a result of differential host microbiome contributions. Furthermore, the microbiome composition of the external sites showed significant sex-based differences while fecal communities were not largely different. CONCLUSIONS: Our study shows that host genetic background potentially exerts homeostatic pressures, resulting in distinct microbiomes for two different inbred cotton rat species. Because of the numerous studies that have uncovered strong relationships between host microbiome, viral infection outcomes, and immune responses, our findings represent a strong contribution for understanding the impact of different microbial communities on viral pathogenesis. Furthermore, we provide novel cotton rat microbiome data as a springboard to uncover the full therapeutic potential of the microbiome against viral infections.

The respiratory microbiome after lung transplantation: Reflection or driver of respiratory disease?

With the introduction of high-throughput sequencing methods, our understanding of the human lower respiratory tract's inhabitants has expanded significantly in recent years. What is now termed the "lung microbiome" has been described for healthy patients, as well as people with chronic lung diseases and lung transplants. The lung microbiome of lung transplant recipients (LTRs) has proven to be unique compared with nontransplant patients, with characteristic findings associated with disease states, such as pneumonia, acute rejection, and graft failure. In this review, we summarize the current understanding of the lung microbiome in LTRs, not only focusing on bacteria but also highlighting key findings of the viral and the fungal community. Based on our knowledge of the lung microbiome in LTRs, we propose multiple opportunities for clinical use of the microbiome to improve outcomes in this population.

SARS-CoV-2 infection and viral load are associated with the upper respiratory tract microbiome.

BACKGROUND: Little is known about the relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the respiratory virus responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, and the upper respiratory tract (URT) microbiome. OBJECTIVE: We sought to compare the URT microbiome between SARS-CoV-2-infected and -uninfected adults and to examine the association of SARS-CoV-2 viral load with the URT microbiome during COVID-19. METHODS: We characterized the URT microbiome using 16S ribosomal RNA sequencing in 59 adults (38 with confirmed, symptomatic, mild to moderate COVID-19 and 21 asymptomatic, uninfected controls). In those with COVID-19, we measured SARS-CoV-2 viral load using quantitative reverse transcription PCR. We then examined the association of SARS-CoV-2 infection status and its viral load with the ⍺-diversity, ß-diversity, and abundance of bacterial taxa of the URT microbiome. Our main models were all adjusted for age and sex. RESULTS: The observed species index was significantly higher in SARS-CoV-2-infected than in -uninfected adults (ß linear regression coefficient = 7.53; 95% CI, 0.17-14.89; P = .045). In differential abundance testing, 9 amplicon sequence variants were significantly different in both of our comparisons, with Peptoniphilus lacrimalis, Campylobacter hominis, Prevotella 9 copri, and an Anaerococcus unclassified amplicon sequence variant being more abundant in those with SARS-CoV-2 infection and in those with high viral load during COVID-19, whereas Corynebacterium unclassified, Staphylococcus haemolyticus, Prevotella disiens, and 2 Corynebacterium_1 unclassified amplicon sequence variants were more abundant in those without SARS-CoV-2 infection and in those with low viral load during COVID-19. CONCLUSIONS: Our findings suggest complex associations between SARS-CoV-2 and the URT microbiome in adults. Future studies are needed to examine how these viral-bacterial interactions can impact the clinical progression, severity, and recovery of COVID-19.

Molecular characterization of respiratory syncytial viruses circulating in a paediatric cohort in Amman, Jordan.

Respiratory syncytial viruses (RSVs) are an important cause of mortality worldwide and a major cause of respiratory tract infections in children, driving development of vaccine candidates. However, there are large gaps in our knowledge of the local evolutionary and transmission dynamics of RSVs, particularly in understudied regions such as the Middle East. To address this gap, we sequenced the complete genomes of 58 RSVA and 27 RSVB samples collected in a paediatric cohort in Amman, Jordan, between 2010 and 2013. RSVA and RSVB co-circulated during each winter epidemic of RSV in Amman, and each epidemic comprised multiple independent viral introductions of RSVA and RSVB. However, RSVA and RSVB alternated in dominance across years, potential evidence of immunological interactions. Children infected with RSVA tended to be older than RSVB-infected children [30 months versus 22.4 months, respectively (P value = 0.02)], and tended to developed bronchopneumonia less frequently than those with RSVB, although the difference was not statistically significant (P value = 0.06). Differences in spatial patterns were investigated, and RSVA lineages were often identified in multiple regions in Amman, whereas RSVB introductions did not spread beyond a single region of the city, although these findings were based on small sample sizes. Multiple RSVA genotypes were identified in Amman, including GA2 viruses as well as three viruses from the ON1 sub-genotype that emerged in 2009 and are now the dominant genotype circulating worldwide. As vaccine development advances, further sequencing of RSV is needed to understand viral ecology and transmission, particularly in under-studied locations.