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.

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.

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.

Severe COVID-19 Is Associated With an Altered Upper Respiratory Tract Microbiome.

Background: The upper respiratory tract (URT) is the portal of entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and SARS-CoV-2 likely interacts with the URT microbiome. However, understanding of the associations between the URT microbiome and the severity of coronavirus disease 2019 (COVID-19) is still limited. Objective: Our primary objective was to identify URT microbiome signature/s that consistently changed over a spectrum of COVID-19 severity. Methods: Using data from 103 adult participants from two cities in the United States, we compared the bacterial load and the URT microbiome between five groups: 20 asymptomatic SARS-CoV-2-negative participants, 27 participants with mild COVID-19, 28 participants with moderate COVID-19, 15 hospitalized patients with severe COVID-19, and 13 hospitalized patients in the ICU with very severe COVID-19. Results: URT bacterial load, bacterial richness, and within-group microbiome composition dissimilarity consistently increased as COVID-19 severity increased, while the relative abundance of an amplicon sequence variant (ASV), Corynebacterium_unclassified.ASV0002, consistently decreased as COVID-19 severity increased. Conclusions: We observed that the URT microbiome composition significantly changed as COVID-19 severity increased. The URT microbiome could potentially predict which patients may be more likely to progress to severe disease or be modified to decrease severity. However, further research in additional longitudinal cohorts is needed to better understand how the microbiome affects COVID-19 severity.

Pharmacokinetic-based failure of a detergent virucidal for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) nasal infections: A preclinical study and randomized controlled trial.

BACKGROUND: 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 (J&J), in SARS-CoV-2-infected subjects. METHODS: Subjects were randomized into three treatment groups: (1) twice daily nasal irrigation with J&J 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. RESULTS: Seventy-two subjects completed the clinical study (n = 24 per group). Despite demonstrated safety and robust efficacy in in vitro virucidal assays, J&J irrigations had no impact on viral titers or symptom scores in treated subjects relative to controls. Similar findings were observed administering J&J 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 J&J detergent components rapidly absorbed from nasal surfaces. CONCLUSION: 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.

Generation of a Novel SARS-CoV-2 Sub-genomic RNA Due to the R203K/G204R Variant in Nucleocapsid: Homologous Recombination has Potential to Change SARS-CoV-2 at Both Protein and RNA Level.

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.

Pharmacokinetic-based failure of a detergent virucidal for SARS-COV-2 nasal infections.

The nose is the portal for SARS-CoV-2 infection, suggesting the nose as a target for topical antiviral therapies. Because detergents are virucidal, Johnson and Johnson's Baby Shampoo (J&J) was tested as a topical virucidal agent in SARS-CoV-2 infected subjects. Twice daily irrigation of J&J in hypertonic saline, hypertonic saline alone, or no intervention were compared (n = 24/group). Despite demonstrated safety and robust efficacy in in vitro virucidal assays, J&J irrigations had no impact on viral titers or symptom scores in treated subjects relative to controls. Similar findings were observed administering J&J 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 J&J detergent components rapidly absorbed from nasal surfaces. This study emphasizes the need to assess the pharmacokinetic characteristics of virucidal agents on airway surfaces to guide clinical trials.

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.