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1AbstractBackgroundRespiratory syncytial virus (RSV) is associated with acute respiratory infection. We sought to identify RSV variants associated with prolonged infection.MethodsAmong healthy term infants we identified those with prolonged RSV infection and conducted 1) a human 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.ResultsWe identified p.E123K/D and p.P218T/S/L in G protein that were associated with prolonged infection (Padj= 0.01). 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.ConclusionsUsing analysis of both viral and host genetics we identified a novel RSV variant associated with prolonged infection in 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.Key pointsUsing a comprehensive computational analysis of viral and host genetics we identified a novel RSV variant associated with prolonged infection and no evidence supporting host genetic infection susceptibility, findings important to understanding RSV contribution to chronic disease and viral endemicity.SummaryA comprehensive computational statistical analysis of both host and viral genetics provided compelling evidence for RSV viral persistence in healthy human infants, a finding of significant importance to understanding the impact of RSV on chronic disease and viral endemicity.

The impact of breast-feeding on certain childhood respiratory illnesses remains controversial.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.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.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).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.

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.

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.

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.

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.

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.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.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 × 10The 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.

Background: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. Methods: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. Conclusions: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

Background: Esophageal conditions result in significant morbidity and mortality worldwide. There is growing enthusiasm for discerning the role of microbiome in esophageal diseases. Conceivably, the focus has been on examining the role of local microbiome in esophageal diseases although this is somewhat limited by the invasive approach required to sample the esophageal tissue. Given the ease of sampling the oral cavity combined with the advances in genomic techniques, there is immense interest in discovering the role of the oral microbiome in esophageal conditions. Summary: In this review, we aim to discuss the current evidence highlighting the association between the oral microbiome and esophageal diseases. In particular, we have focused on summarizing the alterations in oral microbiome associated with malignant, premalignant, and benign esophageal cancers, inflammatory and infectious conditions, and esophageal dysmotility diseases. Identifying alterations in the oral microbiome is a key to advancing our understanding of the etiopathogenesis and progression of esophageal diseases, promoting novel diagnostics, and laying the foundation for personalized treatment approaches. Key Messages: Further studies are needed to unravel the mechanisms by which the oral microbiome influences the development and progression of esophageal diseases, as well as to investigate whether alterations in the oral microbiome can impact the natural history of various esophageal diseases.

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 has proven to be unique compared to non-transplant 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 lung transplant recipients, focusing on bacteria, but also highlighting key findings of the viral and the fungal community. Based on our knowledge of the lung microbiome in lung transplant recipients, we propose multiple opportunities for clinical use of the microbiome to improve outcomes in this population.

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 SUMMARYSeveral 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.

Background: African ancestry is associated with a higher prevalence and greater severity of asthma than European ancestries, yet genetic studies of the most common locus associated with childhood-onset asthma, 17q12–21, in African Americans have been inconclusive. The aim of this study was to leverage both the phenotyping of the Children's Respiratory and Environmental Workgroup (CREW) birth cohort consortium, and the reduced linkage disequilibrium in African Americans, to fine map the 17q12–21 locus. Methods: We first did a genetic association study and meta-analysis using 17q12–21 tag single-nucleotide polymorphisms (SNPs) for childhood-onset asthma in 1613 European American and 870 African American children from the CREW consortium. Nine tag SNPs were selected based on linkage disequilibrium patterns at 17q12–21 and their association with asthma, considering the effect allele under an additive model (0, 1, or 2 effect alleles). Results were meta-analysed with publicly available summary data from the EVE consortium (on 4303 European American and 3034 African American individuals) for seven of the nine SNPs of interest. Subsequently, we tested for expression quantitative trait loci (eQTLs) among the SNPs associated with childhood-onset asthma and the expression of 17q12–21 genes in resting peripheral blood mononuclear cells (PBMCs) from 85 African American CREW children and in upper airway epithelial cells from 246 African American CREW children; and in lower airway epithelial cells from 44 European American and 72 African American adults from a case-control study of asthma genetic risk in Chicago (IL, USA). Findings: 17q12–21 SNPs were broadly associated with asthma in European Americans. Only two SNPs (rs2305480 in gasdermin-B [GSDMB] and rs8076131 in ORMDL sphingolipid biosynthesis regulator 3 [ORMDL3]) were associated with asthma in African Americans, at a Bonferroni-corrected threshold of p

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. Diverse diseases in divergent organ systems are associated with fibrosis, suggesting common biologic mechanisms. One well characterized pathway is chronic inflammation secondary to pathogen. In the present study, we explored the role of the proximal airway microbiome in iSGS pathogenesis. In human samples, abundant bacteria are detectable in iSGS scar as well as in health subglottic controls or patients that developed subglottic stenosis following endotracheal intubation. Interestingly, the community structure of the iSGS proximal airway microbiome does not appear disrupted. Rather, in iSGS defects in the airway epithelial barrier allow displacement of the native microbiome into the immunoprivileged lamina propria and are associated with adaptive immune activation. Animal models of iSGS confirm both bacteria and an adaptive immune response are necessary for pathologic proximal airway fibrosis. Single cell RNA sequencing of the affected airway in iSGS offers an unbiased characterization of the observed epithelial barrier dysfunction. The airway scar in iSGS patients demonstrates basal cell depletion and epithelial acquisition of a mesenchymal phenotype. The epithelial alterations are associated with the observed 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.

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.

Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT., In polyploid cells, copy number is not uniform throughout the genome. Underreplication causes pericentric heterochromatin and defined regions of euchromatin to have reduced copy number relative to overall ploidy. SUUR and Rif1 are key regulators of underreplication, and Rif1 is a known regulator of replication timing (RT). Das et al. take a computational approach to measure replication timing in polyploid cells. Their results demonstrate that, while Rif1 and SUUR both promote underreplication, they differentially affect RT.

The nose is the portal for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, suggesting the nose as a target for topical antiviral therapies. The purpose of this study was to assess both the in vivo and in vitro efficacy of a detergent-based virucidal agent, Johnson and Johnson's Baby Shampoo (JJ), in SARS-CoV-2-infected subjects.Subjects were randomized into three treatment groups: (1) twice daily nasal irrigation with JJ in hypertonic saline, (2) hypertonic saline alone, and (3) no intervention. Complementary in vitro experiments were performed in cultured human nasal epithelia. The primary outcome measure in the clinical trial was change in SARS-CoV-2 viral load over 21 days. Secondary outcomes included symptom scores and change in daily temperature. Outcome measures for in vitro studies included change in viral titers.Seventy-two subjects completed the clinical study (n = 24 per group). Despite demonstrated safety and robust efficacy in in vitro virucidal assays, JJ irrigations had no impact on viral titers or symptom scores in treated subjects relative to controls. Similar findings were observed administering JJ to infected cultured human airway epithelia using protocols mimicking the clinical trial regimen. Additional studies of cultured human nasal epithelia demonstrated that lack of efficacy reflected pharmacokinetic failure, with the most virucidal JJ detergent components rapidly absorbed from nasal surfaces.In this randomized clinical trial of subjects with SARS-CoV-2 infection, a topical detergent-based virucidal agent had no effect on viral load or symptom scores. Complementary in vitro studies confirmed a lack of efficacy, reflective of pharmacokinetic failure and rapid absorption from nasal surfaces.

Despite being commonly used to collect upper airway epithelial lining fluid, nasal washes are poorly reproducible, not suitable for serial sampling, and limited by a dilution effect. In contrast, nasal filters lack these limitations and are an attractive alternative. To examine whether nasal filters are superior to nasal washes as a sampling method for the characterization of the upper airway microbiome and immune response, we collected paired nasal filters and washes from a group of 40 healthy children and adults. To characterize the upper airway microbiome, we used 16S ribosomal RNA and shotgun metagenomic sequencing. To characterize the immune response, we measured total protein using a BCA assay and 53 immune mediators using multiplex magnetic bead-based assays. We conducted statistical analyses to compare common microbial ecology indices and immune-mediator median fluorescence intensities (MFIs) between sample types. In general, nasal filters were more likely to pass quality control in both children and adults. There were no significant differences in microbiome community richness, α-diversity, or structure between pediatric samples types; however, these were all highly dissimilar between adult sample types. In addition, there were significant differences in the abundance of amplicon sequence variants between sample types in children and adults. In adults, total proteins were significantly higher in nasal filters than nasal washes; consequently, the immune-mediator MFIs were not well detected in nasal washes. Based on better quality control sequencing metrics and higher immunoassay sensitivity, our results suggest that nasal filters are a superior sampling method to characterize the upper airway microbiome and immune response in both children and adults.

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.

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 (β =.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 (β = 0.89 [95% CI = 0.37-1.40]). The Jaccard β-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.

Background Little is known about the relationships between SARS-CoV-2, the respiratory virus responsible for the ongoing COVID-19 pandemic, and the upper respiratory tract (URT) microbiome. Objective Our objectives were 1) to compare the URT microbiome between SARS-CoV-2-infected and -uninfected adults, and 2) 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, β-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 (β linear regression coefficient=7.53, 95%CI=0.17-14.89, p=0.045). In differential abundance testing, 9 amplicon sequence variants (ASVs) were significantly different in both of our comparisons, with Peptoniphilus lacrimalis, Campylobacter hominis, Prevotella 9 copri, and an Anaerococcus unclassified ASV 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 ASVs were more abundant in those without SARS-CoV-2 infection and in those with low viral load during COVID-19. Conclusion 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., Capsule Summary: In our study, we found substantial differences in the upper respiratory tract microbiome between SARS-CoV-2-infected and -uninfected adults. Furthermore, we show that the SARS-CoV-2 viral load is associated with the upper respiratory tract microbiome during COVID-19.

Surveillance for the emerging infectious disease Eastern equine encephalitis, and its causative virus in mosquitoes, continued within New York State from 2013 to 2019. There were increases in geographic area and number of consecutive years, with cases in four mammalian species, and virus in 11 mosquito species. The first cases in a goat and in an emu were reported. The first detection of virus in Aedes cinereus was reported. Virus in phylogenetic group NY4 was isolated from a horse and from mosquitoes 6 kilometers and 13 days apart in 2013. Phylogenetic groups NY4 and NY5 were found 15 days apart in two towns 280 kilometers distant in 2013. Within four adjacent counties there was a pattern of overlap, where four had NY5, two adjacent counties had NY6, two adjacent counties had NY7, and one county had NY5, NY6, and NY7, reducible to a Euler diagram. Virus in phylogenetic group NY5, found within an 11-kilometer wide area in New York State, was related to FL4 found in Florida 1,398 kilometers distant. This was consistent with a phylogenetic group originating in Florida, then being moved to a specific location in New York State, by migratory birds in consecutive years 2013 and 2014.

Infection with influenza can be aggravated by bacterial co-infections, which often results in disease exacerbation. The effects of influenza infection on the upper respiratory tract (URT) microbiome are largely unknown. Here, we report a longitudinal study to assess the temporal dynamics of the URT microbiomes of uninfected and influenza virus-infected humans and ferrets. Uninfected human patients and ferret URT microbiomes have stable healthy ecostate communities both within and between individuals. In contrast, infected patients and ferrets exhibit large changes in bacterial community composition over time and between individuals. The unhealthy ecostates of infected individuals progress towards the healthy ecostate, coinciding with viral clearance and recovery. Pseudomonadales associate statistically with the disturbed microbiomes of infected individuals. The dynamic and resilient microbiome during influenza virus infection in multiple hosts provides a compelling rationale for the maintenance of the microbiome homeostasis as a potential therapeutic target to prevent IAV associated bacterial co-infections.

Immune checkpoint inhibitors (ICIs) are used for the treatment of numerous cancers, but risks associated with ICI-therapy during the COVID-19 pandemic are poorly understood. We report a case of acute lung injury in a lung cancer patient initially treated for ICI-pneumonitis and later found to have concurrent SARS-CoV-2 infection. Post-mortem analyses revealed diffuse alveolar damage in both the acute and organizing phases, with a predominantly CD68+ inflammatory infiltrate. Serum was positive for anti-SARS-CoV-2 IgG, suggesting that viral infection predated administration of ICI-therapy and may have contributed to a more fulminant clinical presentation. These data suggest the need for routine SARS-CoV-2 testing in cancer patients, where clinical and radiographic evaluations may be non-specific.

The adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 arose on the background of the spike D614G change and strains harboring these changes have become dominant circulating strains globally. Sequence analysis suggests that the three adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence (CS) of the leader transcription-regulating sequence (TRS) as opposed to a step-wise mutation model. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain. Deep sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. The K203/R204 variant results in a novel sub-genomic RNA. The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans resulting in both coding changes and novel sub-genomic RNA transcripts suggests this as a mechanism for diversification and adaptation within its new host. IMPORTANCE: A major variant of the SARS-CoV-2 virus (R203K/G204R in the nucleocapsid) results in changes to the nucleocapsid at both the protein and RNA level. We show this variant likely arose by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) elsewhere in the viral genome. This recombination event introduced a new TRS between the RNA binding and dimerization domains of nucleocapsid that generates a novel sub-genomic RNA transcript. Presence of the K203/R204 variant was significantly associated with increased expression of nucleocapsid and sub-genomic RNA from other open reading frames. The mechanism by which the virus generates diversity, specifically how this KR variant arose and how it generated a novel major sub-genomic transcript is relevant to how future major shifts may arise and their potential functional implications.

BACKGROUND Olfactory dysfunction is a common symptom of chronic rhinosinusitis (CRS). We previously identified several cytokines potentially linked to smell loss, potentially supporting an inflammatory etiology for CRS-associated olfactory dysfunction. In the current study we sought to validate patterns of olfactory dysfunction in CRS using hierarchical cluster analysis, machine learning algorithms, and multivariate regression. METHODS CRS patients undergoing functional endoscopic sinus surgery were administered the Smell Identification Test (SIT) preoperatively. Mucus was collected from the middle meatus using an absorbent polyurethane sponge and 17 inflammatory mediators were assessed using a multiplexed flow-cytometric bead assay. Hierarchical cluster analysis was performed to characterize inflammatory patterns and their association with SIT scores. The random forest approach was used to identify cytokines predictive of olfactory function. RESULTS One hundred ten patients were enrolled in the study. Hierarchical cluster analysis identified 5 distinct CRS clusters with statistically significant differences in SIT scores observed between individual clusters (p < 0.001). A majority of anosmic patients were found in a single cluster, which was additionally characterized by nasal polyposis (100%) and a high incidence of allergic fungal rhinosinusitis (50%) and aspirin-exacerbated respiratory disease (AERD) (33%). A random forest approach identified a strong association between olfaction and the cytokines interleukin (IL)-5 and IL-13. Multivariate modeling identified AERD, computed tomography (CT) score, and IL-2 as the variables most predictive of olfactory function. CONCLUSION Olfactory dysfunction is associated with specific CRS endotypes characterized by severe nasal polyposis, tissue eosinophilia, and AERD. Mucus IL-2 levels, CT score, and AERD were independently associated with smell loss.

Rationale: Recurrent wheeze and asthma are thought to result from alterations in early life immune development following respiratory syncytial virus (RSV) infection. However, prior studies of the n...

Background Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine’s elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012–2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3). Methods We investigated the basis of low VE in 2012–2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination. Results We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ≥4-fold increase in titer. Conclusions In contrast to analyses based on ferret studies, low H3N2 VE in 2012–2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population., Low H3N2 vaccine effectiveness in 2012–2013 appears attributable to low vaccine immunogenicity in a subset of adults, and not the induction of a narrow, specific response to the egg-adapted strain suggested by previous experiments in immunologically naive ferrets.

Introduction: The gut microbiome is a potentially modifiable factor in treatment related outcomes in allogeneic hematopoietic cell transplant (HCT). Prior studies have linked pre- or mid-treatment gut microbiome diversity with risk for treatment related morbidity and mortality. However, these studies have been limited by the inclusion of one or only a few institutions and the lack of longitudinal sampling with high quality metadata. These limitations complicate the interpretation of microbiome alterations over the course of HCT. Methods: To overcome these, we devised and implemented a large-scale biospecimen collection protocol in conjunction with BMT CTN 1703, a randomized, multicenter, Phase III trial of tacrolimus/methotrexate vs. post-transplant cyclophosphamide/tacrolimus/mycophenolate mofetil in reduced intensity conditioning (RIC) allogeneic HCT (NCT03959241). Patients enrolled on 1703 were optionally co-enrolled in the companion immune and microbiome profiling study, 1801 (MI-IMMUNE). This involved blood, urine and stool sampling before conditioning (PCON), then weekly starting on day 0 through day 77 (through day 84 for blood), and on days 98, 180, 270, 365 and 730. For all enrolled participants where the donor consented, residual donor cells were saved from the empty hematopoietic stem cell product bag for later analysis. Additionally, the protocol included one-time blood, urine and stool sample collection from consented matched related donors (MRDs) prior to stem cell collection. Starting with protocol version 4.0 on February 1, 2021, participation in 1801 stool collection was required for the first six out of eighteen sample collection timepoints. Participation in later stool and urine timepoint collections remained optional. Here we review the feasibility of creating a multi-institutional biobank. Additionally, we assess the success of our strategies by calculating sample collection compliance and standard deviation in compliance across centers for each timepoint. Results: On June 18, 2021, BMT CTN 1703/1801 closed to accrual with 431 patients enrolled on 1703; 323 patients from 36 centers were co-enrolled on 1801. 304 (94%) provided study samples, making this the largest prospective microbiome and immune profiling study in HCT patients to date. As of July 6, 2021, 3,683 blood, 2,668 urine, and 2,098 stool samples had been collected. Across the first 6 timepoints for all participating centers, blood, urine and stool sample collection averaged 93%, 82%, and 74% compliance, respectively. Of the 99 (30%) patients enrolled on 1801 with a MRD, 34 (34%) donors consented to sample collection. Sample collection compliance was lower for MRDs than for patients on the study with 76%, 74%, and 62% of expected blood, urine and stool samples collected, respectively, from this group. For stool collection exclusively, a median of 5 samples were collected per patient across the first 6 timepoints (median of 6 across all timepoints) with 93 (31%) of patients completing a full sample set through Day 28. 139 (46%) patients provided at least one sample after day 28; these represented 37% of the total samples collected to date. The PCON sample, which provides an important measure of pre-treatment gut microbiome diversity, had the third highest compliance with 74% of patients providing a sample. Surprisingly, Day 28 had the lowest compliance (66%) and highest standard deviation (37%) possibly because this timepoint often falls around the time of hospital discharge. Between PCON and day 28, the standard deviation between sites in the average collection compliance (24%) and number of samples collected per patient (1.1) was small indicating the successful adoption of stool collection across institutions. Table 1 summarizes sample collection statistics. Conclusion: Overall this study has resulted in a large, novel biobank of blood, urine and stool samples from patients undergoing RIC allogeneic HCT at 36 centers across the US. This will serve as a valuable resource for investigating the role of the gut microbiome in long term health outcomes following HCT. Although the results of 1801 are forthcoming given ongoing sample collection, the size and composition of the biobank to date clearly demonstrate the feasibility of implementing multi-institutional stool collection. This study represents a critical step towards the large-scale adoption of microbiome sampling as a diagnostic tool. Figure 1 Figure 1. Disclosures Chhabra: GSK: Honoraria. Clark: Kadmon: Consultancy. Horowitz: Mesoblast: Research Funding; Shire: Research Funding; Vertex: Research Funding; Stemcyte: Research Funding; Vor Biopharma: Research Funding; Janssen: Research Funding; Miltenyi Biotech: Research Funding; Kiadis: Research Funding; Sobi: Research Funding; Kite/Gilead: Research Funding; Pfizer, Inc: Research Funding; Jazz Pharmaceuticals: Research Funding; Magenta: Consultancy, Research Funding; Medac: Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Daiicho Sankyo: Research Funding; Xenikos: Research Funding; Omeros: Research Funding; Orca Biosystems: Research Funding; Pharmacyclics: Research Funding; Regeneron: Research Funding; Tscan: Research Funding; Takeda: Research Funding; CSL Behring: Research Funding; Genentech: Research Funding; Gamida Cell: Research Funding; Chimerix: Research Funding; Bristol-Myers Squibb: Research Funding; bluebird bio: Research Funding; Astellas: Research Funding; Amgen: Research Funding; Allovir: Consultancy; Actinium: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Jenq: Microbiome DX: Consultancy; Merck: Consultancy; Prolacta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kaleido: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seres: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; LisCure: Consultancy, Membership on an entity's Board of Directors or advisory committees; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karius: Consultancy. Levine: Equillium Bio: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Research Funding; Kamada: Research Funding; Biogen: Research Funding; Omeros: Membership on an entity's Board of Directors or advisory committees; Symbio: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Talaris Therapeutics: Membership on an entity's Board of Directors or advisory committees; Viracor: Patents & Royalties: GVHD biomarker patent with royalties from Viracor; Mesoblast: Consultancy, Research Funding; X4 Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Murthy: CRISPR Therapeutics: Research Funding. Riches: ATARA Biotherapeutics: Other: Payment; BioIntelect: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Other: Payment. Sung: Merck: Research Funding; Novartis: Research Funding; Enterome: Research Funding; Seres: Research Funding; AVROBIO: Consultancy; Abbott Nutrition: Honoraria; Clasado: Other: Research Product; DSM: Other: Research Product. Al Malki: Neximmune: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy. Rezvani: Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding; US Department of Justice: Consultancy. Bolaños-Meade: Incyte Corp: Consultancy. Holtan: Incyte: Consultancy, Research Funding; Generon: Consultancy. Saber: Govt. COI: Other. Hamadani: Sanofi, Genzyme, AstraZeneca, BeiGene: Speakers Bureau; Janssen, Incyte, ADC Therapeutics, Omeros, Morphosys, Kite: Consultancy; Takeda, Spectrum Pharmaceuticals and Astellas Pharma: Research Funding. Kean: Bluebird Bio: Research Funding; Bristol Myers Squibb: Patents & Royalties: From clinical trial data, Research Funding; Vertex: Consultancy; Novartis: Consultancy; Gilead: Research Funding; Regeneron: Research Funding; EMD Serono: Consultancy. Perales: Cidara: Honoraria; Servier: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Takeda: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; NexImmune: Honoraria; MorphoSys: Honoraria; Omeros: Honoraria; Karyopharm: Honoraria; Sellas Life Sciences: Honoraria; Merck: Honoraria; Miltenyi Biotec: Honoraria, Other; Medigene: Honoraria; Bristol-Myers Squibb: Honoraria; Kite/Gilead: Honoraria, Other; Celgene: Honoraria.

Sampling of mallards in Alaska during September 2014-April 2015 identified low pathogenic avian influenza A virus (subtypes H5N2 and H1N1) that shared ancestry with highly pathogenic reassortant H5N2 and H5N1 viruses. Molecular dating indicated reassortment soon after interhemispheric movement of H5N8 clade 2.3.4.4, suggesting genetic exchange in Alaska or surrounds before outbreaks.

Immunodominance defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody immunodominance despite its importance to immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible immunodominance hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changes as the immune response progresses and depending on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrate “original antigenic suppression”, where antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody immunodominance to viruses and other immunogens.

Here, we report 17 nearly complete genome sequences of enterovirus D68 (EV-D68) isolated from Kansas City, MO, in 2018. Phylogenetic analysis suggests that these strains belong to subclade B3, similar to the ones that caused the 2016 epidemics in the United States but different from the 2014 outbreak B1 strains.

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.

Following its introduction into New York State (NYS) in 1999, West Nile virus (WNV; Flavivirus, Flaviviridae) underwent a rapid expansion throughout the USA and into Canada and Latin America. WNV has been characterized as being evolutionarily stable, with weak geographic structure, a dominance of purifying selection and limited adaptive change. We analyzed all available full-genome WNV sequences, focusing on the 543 available sequences from NYS, which included 495 newly sequenced 2000–15 isolates. In addition, we analyzed deep-sequencing data from 317 of these isolates. While our data are generally in agreement with the limited pace of evolutionary change and broad geographic and temporal mixing identified in other studies, we have identified some important exceptions. Most notably, there are 14 codons which demonstrated evidence of positive selection as determined by multiple models, including some positions with evidence of selection in NYS exclusively. Coincident with increased WNV activity, genotypes possessing one or more of these mutations, designated NY01, NY07, and NY10, have increased in prevalence in recent years and displaced historic strains. In addition, we have found a geographical bias with many of these mutations, which suggests selective pressures and adaptations could be regional. Lastly, our deep-sequencing data suggest both increased overall diversity in avian tissue isolates relative to mosquito isolates and multiple non-synonymous minority variants that are both host-specific and retained over time and space. Together, these data provide novel insight into the evolutionary pressures on WNV and the need for continued genetic surveillance and characterization of emergent strains.