Biology subjects

Piedra, P. A.

Publications and source records attributed to Piedra, P. A..

17 recordsLinked to original sources

bioRxiv2026

RSV & rhinovirus coinfection order dictates outcome via type III interferon-mediated interference in infant respiratory epithelium

Coinfection with respiratory syncytial virus (RSV) and rhinovirus (RV) is the most frequently detected coinfection in infants, yet mechanisms governing viral interference and disease outcome remain poorly understood. Although infection order is a key determinant of coinfection outcome involving RSV, influenza, rhinovirus, and SARS-CoV-2, its role in RSV/RV coinfections has not been defined. Using infant-derived human nasal organoids at air-liquid interface, we demonstrate that coinfection order fundamentally determines the outcome of RSV/RV interactions. An initial RSV infection almost completely inhibits secondary RV coinfection, whereas primary RV infection only partially impairs a subsequent RSV coinfection. These distinct outcomes are accompanied by divergent temporal antiviral and pro-inflammatory responses. Mechanistically, we identify type III interferons as a sufficient mediator of RSV-induced restriction of RV, establishing a host-driven mechanism of viral interference. In contrast, partial impairment of RSV following RV infection occurs through an interferon-independent mechanism. Despite marked differences, RSV/RV coinfections do not exacerbate epithelial injury or innate immune responses beyond that induced by RSV alone, indicating RSV is the dominant driver of pathology in coinfections. Collectively, our data establishes coinfection order as a critical determinant of RSV/RV outcome and reveals novel and mechanistic insight into critical viral-host interactions in the infant nasal epithelium.

microbiology↗

bioRxiv2026

RSV Infects the Human Nasal Epithelium via the Basolateral Route with Distinct Subgroup Infectivity and Basal Cell Tropism

Respiratory syncytial virus (RSV) causes millions of lower respiratory tract infections (LRTIs) in young children, older adults, and immunocompromised populations every year. RSV infection initiates in the upper respiratory tract and can progress to the lower airways resulting in bronchiolitis, pneumonia, and even death. RSV primarily infects epithelial cells apically, but we hypothesized that basolateral exposure of the respiratory epithelium could provide an alternative mechanism of infection that contributes to LRTI development. Using a human nose organoid-air liquid interface (HNO-ALI) model, we performed apical and basolateral inoculations with contemporaneous RSV strains (RSV/A/ON and RSV/B/BA) representing the two RSV subgroups (A and B) in both adult and infant derived HNO-ALIs. Basolateral RSV exposure resulted in delayed viral replication and apical release compared to apical infection. A statistically significant difference in basolateral infection frequency was observed between RSV/B/BA and RSV/A/ON (81.3% versus 25%). Basolateral infection selectively targeted a rare basal cell population, while preserving epithelial integrity. Using undifferentiated HNO-ALIs, we determined for the first time that Krt23+ activated basal cells (ABCs) are uniquely susceptible to RSV infection, a finding we confirmed in fully differentiated HNO-ALIs. Together, our findings show that RSV can infect the respiratory epithelium from the basolateral side by initially targeting a rare subset of basal cells before spreading apically to ciliated cells. Moreover, RSV/B/BA may have an advantage over RSV/A/ON in utilizing the basolateral infection route. These findings highlight an alternative RSV infection pathway and could be a potential mechanism for RSV spread to the lower airways. ImportanceUnderstanding the pathogenesis of RSV is essential to understanding and preventing acute and long-term sequelae from infection. The canonical understanding of RSV infection is that the virus infects and is restricted to the apical ciliated cells upon inhalation or fomite exposure. We demonstrate that an alternative route of infection - the basolateral route, can be utilized by RSV to infect the apical ciliated cells of the respiratory epithelium. We also show for the first time a novel difference in infectivity between the two contemporaneous RSV strains (RSV/A/ON and RSV/B/BA). In addition, we describe a rare basal subset-the Krt23+ activated basal cells that are uniquely susceptible to RSV thus expanding the known cellular tropism of RSV. Infection of basal cells can impact airway differentiation, homeostasis, and remodeling. Overall, our findings expand on the pathogenesis of RSV and indicate there are alternative mechanisms of infection and cell populations that are susceptible to RSV.

microbiology↗

bioRxiv2026

Streptococcus pneumoniae colonization modulates human nasal epithelial responses to respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality in infants globally. Specific nasal bacterial genera are differentially associated with RSV severity in infants: Haemophilus and Streptococcus with more severe disease and Dolosigranulum with healthy controls or milder outcomes. We hypothesized these differential bacterial effects begin at the epithelial level. Therefore, we established human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) as a model system to assess effects of individual nasal microbionts on the epithelial response to subsequent RSV infection and of RSV on those microbionts. Infant-derived HNO-ALI were monocolonized with either Streptococcus pneumoniae, nontypeable Haemophilus influenzae, or Dolosigranulum pigrum one day before viral infection. RSV reduced colonizing S. pneumoniae and D. pigrum levels without affecting H. influenzae. S. pneumoniae precolonization uniquely reduced RSV levels during infection. S. pneumoniae precolonization also modulated the epithelial transcriptional response to RSV infection more so than H. influenzae or D. pigrum, with a pronounced effect on genes involved in immune response, cell cycle, stress, and growth signaling. Gene set enrichment analysis showed S. pneumoniae precolonization blunted RSV-induced increase in inflammatory and immune responses, consistent with S. pneumoniae also modulating RSV-induced cytokine production. Furthermore, S. pneumoniae precolonization blocked RSV-mediated dysregulation of cell-cycle genes, consistent with preventing arrest. Bacterial rescue of cell-cycle progression is a potential mechanism for reduced infectious virion production, since cell-cycle arrest enhances RSV replication. HNO-ALI facilitated elucidation of bacterial-viral-epithelial interplay at a frequent site of viral infection, directly linking nasal bacterial colonization to RSV infection dynamics. IMPORTANCEMost RSV-related hospitalizations occur in healthy young children, yet predictors of RSV severity in this population are limited. The nasal passages are a major microbiota site exposed to frequent respiratory viral infections. Specific nasal bacterial genera/species correlate with RSV clinical outcomes; however, the biological basis of these associations is poorly defined. Here, we established human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) as a tractable experimental system for defining the interplay between bacteria, virus, and epithelial cells in the human nasal passages, enabling mechanistic insight into how the nasal microbiota contribute to RSV disease severity. Using this approach, we found that precolonization with each of the common nasal bacteria S. pneumoniae, H. influenzae, or D. pigrum differentially affected the nasal epithelial response to RSV infection. This work highlights that the nasal epithelium actively integrates microbial and viral signals and that variation in bacterial colonization can shape viral infection trajectories.

microbiology↗

bioRxiv2025

Evolutionary dynamics of Respiratory Syncytial Virus in pre-pandemic, pandemic, and post-pandemic periods in Houston, Texas, USA

BackgroundRespiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants and poses significant risks to immuno-compromised individuals and the elderly. The COVID-19 pandemic disrupted typical RSV seasonality, leading to unusual patterns of viral circulation and resurgence. However, the impact of these disruptions on RSV evolutionary dynamics remains incompletely understood. This study aimed to investigate the evolutionary dynamics of RSV in Houston, Texas, across pre-pandemic (November 2015 - March 2020), pandemic (April 2021 - June 2023), and post-pandemic periods (July 2023 - December 2024), focusing on genetic diversity, lineage dynamics, and selective pressures. MethodsIn one of the largest comprehensive RSV genomic evolutionary analyses, we analyzed 1,344 RSV-positive respiratory samples collected from children seeking outpatient care or hospitalized with acute respiratory infections between November 2015 to February 2024, which is one of the largest comprehensive RSV genomic evolutionary analyses. We successfully sequenced the whole genomes for 87.5% of the isolates (606 RSV/A and 570 RSV/B). To assess genetic diversity, lineage dynamics, and selective pressures, we conducted phylogenetic analysis, variant calling, and dN/dS ratio calculations. We clustered variant prevalence patterns using k-means and used Shannon entropy to quantify genetic variability. We performed statistical regression analyses to evaluate the accumulation of variants and their impact on gene expression. ResultsRSV circulation in Houston was markedly disrupted during the pandemic, with an absence of cases in 2020-2021 followed by off-season surges from 2021 to 2023. Phylogenetic analysis revealed distinct lineage dynamics, with RSV/A showing remarkable persistence of specific lineages such as A.D.1 and emergence of new lineages including A.D.3. In contrast, RSV/B underwent a dramatic restructuring, dominated by the B.D.E.1 lineage in the pandemic and post-pandemic period. Bayesian molecular clock analysis estimated nucleotide substitution rates of 9.37 x 10-4 (95% HPD: 7.99 x 10-4 - 1.07 x 10-3) substitutions/site/year for RSV/A and 9.79 x 10-4 (95% HPD: 8.96 x 10-4 - 1.06 x 10-3) for RSV/B. The time to the most recent common ancestor (tMRCA) of the dominant RSV/A clade, A.D.1, is dated to approximately 2010 (95% HPD: 2008-2011), while for RSV/B the dominant clade- B.D.E.1 traces back to 2002 (95% HPD: 1997-2007). Despite reduced lineage diversity, RSV/B accumulated non-synonymous variants at nearly twice the rate of RSV/A, notably in the M2-2 gene, reflecting high within-lineage variability. The significant increase in non-synonymous mutations in M2-2 in RSV/B during the pandemic and post-pandemic time phases strongly correlated with increased transcriptional activity. Additionally, antigenic site mutations in the F protein, particularly in RSV/B, were also observed, with implications for immune evasion and effectiveness of the current monoclonal antibody immunoprophylaxis intervention. ConclusionsThe COVID-19 pandemic significantly impacted RSV evolution, leading to reduced genetic diversity during the pandemic and the emergence of novel lineages post-pandemic. RSV/B exhibited more dynamic evolutionary changes, particularly in the M2-2 gene, suggesting potential adaptive advantages. Ongoing genomic surveillance coupled with functional studies is crucial for monitoring variants and assessing the clinical impact of these mutations on transmission, disease severity, fitness, and long-term effectiveness of RSV prevention and treatment strategies.

evolutionary biology↗

bioRxiv2025

Oral dosing of the nucleoside analog obeldesivir is efficacious against RSV infection in African green monkeys

Respiratory syncytial virus (RSV) is a significant cause of morbidity and mortality in high-risk populations. Although prophylactic options are available, there are no effective oral therapeutics for RSV infection. Obeldesivir (ODV) is an orally bioavailable prodrug of the nucleoside analog GS-441524, which is converted intracellularly to its active nucleoside triphosphate and inhibits the RSV RNA polymerase. Here we report the potent antiviral activity of ODV against geographically and temporally diverse RSV A and B clinical isolates (EC50: 0.20-0.66 M). Resistance selection studies with ODV and GS-441524 against RSV identified a single amino acid substitution, I777L, in the L polymerase with reduced susceptibility (3.3-3.8-fold) to ODV and GS-441524, indicating a high barrier for resistance development. In an African green monkey RSV infection model, once-daily oral ODV doses of 30 or 90 mg/kg initiated [~]24 hours post-infection significantly reduced log10 viral RNA copies/mLxday area under the curve by 69-92% in the upper and lower respiratory tracts. Together, these preclinical data support the clinical evaluation of ODV for the treatment of RSV infection.

microbiology↗

bioRxiv2024

Nasal microbionts differentially colonize and elicit cytokines in human nasal epithelial organoids

Nasal colonization by Staphylococcus aureus or Streptococcus pneumoniae is associated with an increased risk of infection by these pathobionts, whereas nasal colonization by Dolosigranulum species is associated with health. Human nasal epithelial organoids (HNOs) physiologically recapitulate human nasal respiratory epithelium with a robust mucociliary blanket. We reproducibly monocolonized HNOs with these three bacteria for up to 48 hours with varying kinetics across species. HNOs tolerated bacterial monocolonization with localization of bacteria to the mucus layer and with minimal cytotoxicity compared to uncolonized HNOs. Human nasal epithelium exhibited both species-specific and general cytokine responses, without induction of type I interferons, consistent with colonization rather than infection. Only live S. aureus colonization robustly induced IL-1 family cytokines, suggestive of inflammasome signaling. D. pigrum and live S. aureus decreased CXCL10, whereas S. pneumoniae increased CXCL11, chemokines involved in antimicrobial responses to both viruses and bacteria. Overall, HNOs are a compelling model system to reveal host-microbe dynamics at the human nasal mucosa. IMPORTANCEHuman nasal microbiota often includes highly pathogenic members, many of which are antimicrobial resistance threats, e.g., methicillin-resistant Staphylococcus aureus and antibiotic-resistant Streptococcus pneumoniae. Preventing colonization by nasal pathobionts decreases infections and transmission. In contrast, nasal microbiome studies identify candidate beneficial bacteria that might resist pathobiont colonization, e.g., Dolosigranulum pigrum. Discovering how these microbionts colonize the human nasal passages and means to reduce pathobiont colonization is limited by previous models. This creates an urgent need for human-based models that exemplify bacterial nasal colonization. We addressed this need by developing human nasal epithelial organoids (HNOs) as a new model system of bacterial nasal colonization. HNOs accurately represent the mucosal surface of the human nasal passages enabling exploration of bacterial-epithelial interactions, which is crucial since the epithelium instigates the initial innate immune response to bacteria. Here, we identified differential epithelial cytokine responses to these three bacteria setting the stage for future research.

microbiology↗

bioRxiv2024

Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples--a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants. IMPORTANCERespiratory syncytial virus (RSV) and human noroviruses (HuNoV) are NIAID category C and category B priority pathogens, respectively, that inflict significant health consequences on children, adults, immunocompromised patients, and the elderly. Due to the high strain diversity of RSV and HuNoV genomes, obtaining complete genomes to monitor viral evolution and pathogenesis is challenging. In this paper, we present the design, optimization, and benchmarking of a comprehensive oligonucleotide target capture method for these pathogens. All 85 RSV samples and 49/55 HuNoV samples were patient-derived with six human intestinal enteroids. The methodology described here results has a higher success rate in obtaining full-length RSV and HuNoV genomes, enhancing the efficiency of studying these viruses and mutations directly from patient-derived samples.

molecular biology↗

bioRxiv2024

Pediatric human nose organoids demonstrate greater susceptibility, epithelial responses, and cytotoxicity than adults during RSV infection.

Respiratory syncytial virus (RSV) is a common cause of respiratory infections, causing significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe as compared to healthy adults is not fully understood. In the present study, we infect both pediatric and adult human nose organoid-air liquid interface (HNO-ALIs) cell lines with two contemporary RSV isolates and demonstrate how they differ in virus replication, induction of the epithelial cytokine response, cell injury, and remodeling. Pediatric HNO-ALIs were more susceptible to early RSV replication, elicited a greater overall cytokine response, demonstrated enhanced mucous production, and manifested greater cellular damage compared to their adult counterparts. Adult HNO-ALIs displayed enhanced mucus production and robust cytokine response that was well controlled by superior regulatory cytokine response and possibly resulted in lower cellular damage than in pediatric lines. Taken together, our data suggest substantial differences in how pediatric and adult upper respiratory tract epithelium responds to RSV infection. These differences in epithelial cellular response can lead to poor mucociliary clearance and predispose infants to a worse respiratory outcome of RSV infection.

microbiology↗

bioRxiv2023

Longitudinal host transcriptional responses to SARS-CoV-2 infection in adults with extremely high viral load

Current understanding of viral dynamics of SARS-CoV-2 and host responses driving the pathogenic mechanisms in COVID-19 is rapidly evolving. Here, we conducted a longitudinal study to investigate gene expression patterns during acute SARS-CoV-2 illness. Cases included SARS-CoV-2 infected individuals with extremely high viral loads early in their illness, individuals having low SARS-CoV-2 viral loads early in their infection, and individuals testing negative for SARS-CoV-2. We could identify widespread transcriptional host responses to SARS-CoV-2 infection that were initially most strongly manifested in patients with extremely high initial viral loads, then attenuating within the patient over time as viral loads decreased. Genes correlated with SARS-CoV-2 viral load over time were similarly differentially expressed across independent datasets of SARS-CoV-2 infected lung and upper airway cells, from both in vitro systems and patient samples. We also generated expression data on the human nose organoid model during SARS-CoV-2 infection. The human nose organoid-generated host transcriptional response captured many aspects of responses observed in the above patient samples, while suggesting the existence of distinct host responses to SARS-CoV-2 depending on the cellular context, involving both epithelial and cellular immune responses. Our findings provide a catalog of SARS-CoV-2 host response genes changing over time.

microbiology↗

bioRxiv2022

The efficiency of p27 peptide cleavage during in vitro respiratory syncytial virus (RSV) infection is cell line and RSV subtype dependent

Respiratory Syncytial Virus (RSV) Fusion protein (F) is highly conserved between RSV/A and RSV/B subtypes. To become fully active, F precursor undergoes enzymatic cleavage to yield F1 and F2 subunits and releases a 27 amino acid peptide (p27). Virus-cell fusion occurs when RSV F undergoes a conformational change from pre-F to post-F. Previous immunological and cell-surface expression data show that p27 is detected on RSV F, but questions remain on how p27 effects the conformation of mature RSV F. Monoclonal antibodies against p27, Site O (pre-Fusion specific), and Site II were used to monitor RSV F conformation by ELISA and Imaging Flow Cytometry. Pre-F to post-F conformational change was induced by temperature-stress test. We found that p27 cleavage efficiency was lower on sucrose purified (sp) RSV/A than on spRSV/B. In addition, in vitro cleavage of RSV F was cell-line dependent, higher levels of p27 expression were observed on surface of RSV infected HEp-2 cells than A549 cells. Higher levels of p27 were also found on RSV/A infected cells compared to RSV/B. We observed that RSV/A F with higher levels of p27 could better sustain the pre-F conformation during the temperature-stress challenge in both spRSV and as well RSV-infected cell lines. Our findings suggest that despite F sequence similarity, the p27 of RSV subtypes is cleaved with different efficiencies, which were also dependent on the cell lines used for infection. We therefore speculate that partially cleaved p27 may confer higher stability to the pre-F and provides a fitness advantage. IMPORTANCEThe RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F protein undergoes proteolytic cleavages by furin protease resulting in the release of a 27 amino acid peptide (p27) to become fully functional. During this process, the F protein also undergoes a conformational change from metastable pre-F to highly stable post-F. For decades, the consensus in the RSV field was that p27 was not present in the fully mature RSV F protein. Therefore, the role of the p27 peptide in viral entry and the function of the partially cleaved F protein containing p27 has been overlooked. However, recent developments have shown that p27 elicits an immune response during natural infection and that p27 can be found in vitro and in animal models infected with the prototypical RSV/A strain. In this study, we were able to detect p27 on purified RSV virions and on the surface of virus-infected cells of two widely used cell lines, HEp-2 and A549 cells for both prototypical and contemporary circulating RSV strains of both subtypes. Also, higher levels of partially cleaved F protein containing p27 could better sustain the pre-F conformation during the temperature-stress challenge. Our findings highlight that the cleavage efficiency of p27 is different between RSV subtypes and among cell lines and that the presence of p27 in partially cleaved F protein likely contributes to the stability of the pre-F conformation.

microbiology↗

bioRxiv2022

Diversity and Evolution of Computationally Predicted T Cell Epitopes against Human Respiratory Syncytial Virus

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory infection. Despite more than 60 years of research, there is no licensed vaccine. While B cell response is a major focus for vaccine design, the T cell epitope profile of RSV is also important for vaccine development. Here, we computationally predicted putative T cell epitopes in the Fusion protein (F) and Glycoprotein (G) of RSV wild circulating strains by predicting Major Histocompatibility Complex (MHC) class I and class II binding affinity. We limited our inferences to conserved epitopes in both F and G proteins that have been experimentally validated. We applied multidimensional scaling (MDS) to construct T cell epitope landscapes to investigate the diversity and evolution of T cell profiles across different RSV strains. We find the RSV strains are clustered into three RSV-A groups and two RSV-B groups on this T epitope landscape. These clusters represent divergent RSV strains with potentially different immunogenic profiles. In addition, our results show a greater proportion of F protein T cell epitope content conservation among recent epidemic strains, whereas the G protein T cell epitope content was decreased. Importantly, our results suggest that RSV-A and RSV-B have different patterns of epitope drift and replacement and that RSV-B vaccines may need more frequent updates. Our study provides a novel framework to study RSV T cell epitope evolution. Understanding the patterns of T cell epitope conservation and change may be valuable for vaccine design and assessment. Author SummaryLower respiratory infections caused by human respiratory syncytial virus (RSV) is a global health challenge. B cell epitope immune response has been the major focus of RSV vaccine and therapeutic development. However, T cell epitope induced immunity plays an important role in the resolution of RSV infection. While RSV genetic diversity has been widely reported, few studies focus on RSV T epitope diversity, which can influence vaccine effectiveness. Here, we use computationally predicted T cell epitope profiles of circulating strains to characterize the diversity and evolution of the T cell epitope of RSV A and B. We systematically evaluate the T epitope profile of RSV F and G proteins. We provide a T cell epitope landscape visualization that shows co-circulation of three RSV-A groups and two RSV-B groups, suggesting potentially distinct T cell immunity. Furthermore, our study shows different levels of F and G protein T cell epitope content conservation, which may be important to correlate with duration of vaccine protection. This study provides a novel framework to study RSV T cell epitope evolution, infer RSV T cell immunity at population levels and monitor RSV vaccine effectiveness.

evolutionary biology↗

bioRxiv2021

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

microbiology↗

bioRxiv2021

Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host. IMPORTANCEInfection with respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. In vitro data from two historic RSV/A strains and two cell lines, HEp-2 and A549, constitute most of our knowledge; but RSV contains ample variation from two evolving subgroups (A and B) showing recent convergent evolution. Here we measure viral action and host response in HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporaneous strains. We discover a subgroup-dependent difference in viral gene expression and find A549 cells are more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings reveal important differences between RSV subgroups and two widely used cell lines and provide baseline data for experiments with model systems better representative of natural RSV infection.

microbiology↗

bioRxiv2020

Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

genomics↗

bioRxiv2020

NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge

There is an urgent need for a safe and protective vaccine to control the global spread of SARS-CoV-2 and prevent COVID-19. Here, we report the immunogenicity and protective efficacy of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length SARS-CoV-2 spike (S) glycoprotein stabilized in the prefusion conformation. Cynomolgus macaques (Macaca fascicularis) immunized with NVX-CoV2373 and the saponin-based Matrix-M adjuvant induced anti-S antibody that was neutralizing and blocked binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. Following intranasal and intratracheal challenge with SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support ongoing phase 1/2 clinical studies of the safety and immunogenicity of NVX-CoV2327 vaccine (NCT04368988). HighlightsO_LIFull-length SARS-CoV-2 prefusion spike with Matrix-M1 (NVX-CoV2373) vaccine. C_LIO_LIInduced hACE2 receptor blocking and neutralizing antibodies in macaques. C_LIO_LIVaccine protected against SARS-CoV-2 replication in the nose and lungs. C_LIO_LIAbsence of pulmonary pathology in NVX-CoV2373 vaccinated macaques. C_LI

immunology↗

bioRxiv2020

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

microbiology↗

bioRxiv2020

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).

microbiology↗