Characterization of bacterial and viral pathogens in the respiratory tract of children with HIV-associated chronic lung disease: a case-control study.

INTRODUCTION: Chronic lung disease is a major cause of morbidity in African children with HIV infection; however, the microbial determinants of HIV-associated chronic lung disease (HCLD) remain poorly understood. We conducted a case-control study to investigate the prevalence and densities of respiratory microbes among pneumococcal conjugate vaccine (PCV)-naive children with (HCLD +) and without HCLD (HCLD-) established on antiretroviral treatment (ART). METHODS: Nasopharyngeal swabs collected from HCLD + (defined as forced-expiratory-volume/second < -1.0 without reversibility postbronchodilation) and age-, site-, and duration-of-ART-matched HCLD- participants aged between 6-19 years enrolled in Zimbabwe and Malawi (BREATHE trial-NCT02426112) were tested for 94 pneumococcal serotypes together with twelve bacteria, including Streptococcus pneumoniae (SP), Staphylococcus aureus (SA), Haemophilus influenzae (HI), Moraxella catarrhalis (MC), and eight viruses, including human rhinovirus (HRV), respiratory syncytial virus A or B, and human metapneumovirus, using nanofluidic qPCR (Standard BioTools formerly known as Fluidigm). Fisher's exact test and logistic regression analysis were used for between-group comparisons and risk factors associated with common respiratory microbes, respectively. RESULTS: A total of 345 participants (287 HCLD + , 58 HCLD-; median age, 15.5 years [IQR = 12.8-18], females, 52%) were included in the final analysis. The prevalence of SP (40%[116/287] vs. 21%[12/58], p = 0.005) and HRV (7%[21/287] vs. 0%[0/58], p = 0.032) were higher in HCLD + participants compared to HCLD- participants. Of the participants positive for SP (116 HCLD + & 12 HCLD-), 66% [85/128] had non-PCV-13 serotypes detected. Overall, PCV-13 serotypes (4, 19A, 19F: 16% [7/43] each) and NVT 13 and 21 (9% [8/85] each) predominated. The densities of HI (2 × 104 genomic equivalents [GE/ml] vs. 3 × 102 GE/ml, p = 0.006) and MC (1 × 104 GE/ml vs. 1 × 103 GE/ml, p = 0.031) were higher in HCLD + compared to HCLD-. Bacterial codetection (≥ any 2 bacteria) was higher in the HCLD + group (36% [114/287] vs. (19% [11/58]), (p = 0.014), with SP and HI codetection (HCLD + : 30% [86/287] vs. HCLD-: 12% [7/58], p = 0.005) predominating. Viruses (predominantly HRV) were detected only in HCLD + participants. Lastly, participants with a history of previous tuberculosis treatment were more likely to carry SP (adjusted odds ratio (aOR): 1.9 [1.1 -3.2], p = 0.021) or HI (aOR: 2.0 [1.2 - 3.3], p = 0.011), while those who used ART for ≥ 2 years were less likely to carry HI (aOR: 0.3 [0.1 - 0.8], p = 0.005) and MC (aOR: 0.4 [0.1 - 0.9], p = 0.039). CONCLUSION: Children with HCLD + were more likely to be colonized by SP and HRV and had higher HI and MC bacterial loads in their nasopharynx. The role of SP, HI, and HRV in the pathogenesis of CLD, including how they influence the risk of acute exacerbations, should be studied further. TRIAL REGISTRATION: The BREATHE trial (ClinicalTrials.gov Identifier: NCT02426112 , registered date: 24 April 2015).

Performance evaluation of the Alinity m system for quantifying cytomegalovirus DNA in samples of the respiratory, gastrointestinal, and urinary tract.

Quantitation of cytomegalovirus (CMV) DNA load in specimens other than blood such as bronchoalveolar lavages, intestinal biopsies, or urine has become a common practice as an ancillary tool for the diagnosis of CMV pneumonitis, intestinal disease, or congenital infection, respectively. Nevertheless, most commercially available CMV PCR platforms have not been validated for CMV DNA detection in these specimen types. In this study, a laboratory-developed test based on Alinity m CMV ("Alinity LDT") was evaluated. Reproducibility assessment using spiked bronchial aspirate (BAS) or urine samples showed low standard deviations of 0.08 and 0.27 Log IU/mL, respectively. Evaluating the clinical performance of Alinity LDT in comparison to a laboratory-developed test based on RealTime CMV ("RealTime LDT") showed good concordance across 200 clinical specimens including respiratory specimens, intestinal biopsies, urine, and stool. A high Pearson's correlation coefficient of r = 0.92, a low mean bias of -0.12 Log IU/mL, a good qualitative agreement of 90%, and a Cohen's kappa value of 0.76 (substantial agreement) were observed. In separate analyses of the sample types BAS, tracheal aspirates, bronchoalveolar lavage, biopsies, and urine, the assay results correlated well between the two platforms with r values between 0.88 and 0.99 and a bias <0.5 Log IU/mL. Overall, the fully automated, continuous, random access Alinity LDT yielded good reproducibility, high concordance, and good correlation to RealTime LDT in respiratory, gastrointestinal, and urine samples and may enhance patient management with rapid result reporting.IMPORTANCEIn transplant recipients, a major cause for morbidity and mortality is end-organ disease by primary or secondary CMV infection of the respiratory or gastrointestinal tract. In addition, sensorineural hearing loss and neurodevelopmental abnormalities are frequent sequelae of congenital CMV infections in newborns. Standard of care for highly sensitive detection and quantitation of the CMV DNA load in plasma and whole blood specimens is real-time PCR testing. Beyond that, there is a need for quantitative determination of CMV DNA levels in respiratory, gastrointestinal, and urinary tract specimens using a highly automated, random access CMV PCR assay with a short turnaround time to enable early diagnosis and treatment. In the present study, clinical performance of the fully automated Alinity m analyzer in comparison to the current RealTime LDT assay was evaluated in eight different off-label sample types.

Neuroimmune recognition and regulation in the respiratory system.

Neuroimmune recognition and regulation in the respiratory system is a complex and highly coordinated process involving interactions between the nervous and immune systems to detect and respond to pathogens, pollutants and other potential hazards in the respiratory tract. This interaction helps maintain the health and integrity of the respiratory system. Therefore, understanding the complex interactions between the respiratory nervous system and immune system is critical to maintaining lung health and developing treatments for respiratory diseases. In this review, we summarise the projection distribution of different types of neurons (trigeminal nerve, glossopharyngeal nerve, vagus nerve, spinal dorsal root nerve, sympathetic nerve) in the respiratory tract. We also introduce several types of cells in the respiratory epithelium that closely interact with nerves (pulmonary neuroendocrine cells, brush cells, solitary chemosensory cells and tastebuds). These cells are primarily located at key positions in the respiratory tract, where nerves project to them, forming neuroepithelial recognition units, thus enhancing the ability of neural recognition. Furthermore, we summarise the roles played by these different neurons in sensing or responding to specific pathogens (influenza, severe acute respiratory syndrome coronavirus 2, respiratory syncytial virus, human metapneumovirus, herpes viruses, Sendai parainfluenza virus, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, amoebae), allergens, atmospheric pollutants (smoking, exhaust pollution), and their potential roles in regulating interactions among different pathogens. We also summarise the prospects of bioelectronic medicine as a third therapeutic approach following drugs and surgery, as well as the potential mechanisms of meditation breathing as an adjunct therapy.

A Statovirus-like virus from respiratory tracts of patients, China.

The emerging evidence of human infections with emerging viruses suggests their potential public health importance. A novel taxon of viruses named Statoviruses (for stool-associated Tombus-like viruses) was recently identified in the gastrointestinal tracts of multiple mammals. Here we report the discovery of respiratory Statovirus-like viruses (provisionally named Restviruses) from the respiratory tracts of five patients experiencing acute respiratory disease with Human coronavirus OC43 infection through the retrospective analysis of meta-transcriptomic data. Restviruses shared 53.1%-98.8% identities of genomic sequences with each other and 39.9%-44.3% identities with Statoviruses. The phylogenetic analysis revealed that Restviruses together with a Stato-like virus from nasal-throat swabs of Vietnamese patients with acute respiratory disease, formed a well-supported clade distinct from the taxon of Statoviruses. However, the consistent genome characteristics of Restviruses and Statoviruses suggested that they might share similar evolutionary trajectories. These findings warrant further studies to elucidate the etiological and epidemiological significance of the emerging Restviruses.

Metaproteomics for Coinfections in the Upper Respiratory Tract: The Case of COVID-19.

The upper respiratory tract (URT) is home to a diverse range of microbial species. Respiratory infections disturb the microbial flora in the URT, putting people at risk of secondary infections. The potential dangers and clinical effects of bacterial and fungal coinfections with SARS-CoV-2 support the need to investigate the microbiome of the URT using clinical samples. Mass spectrometry (MS)-based metaproteomics analysis of microbial proteins is a novel approach to comprehensively assess the clinical specimens with complex microbial makeup. The coronavirus that causes severe acute respiratory syndrome (SARS-CoV-2) is responsible for the COVID-19 pandemic resulting in a plethora of microbial coinfections impeding therapy, prognosis, and overall disease management. In this chapter, the corresponding workflows for MS-based shotgun proteomics and metaproteomic analysis are illustrated.

Exploring the Complexity of the Human Respiratory Virome through an In Silico Analysis of Shotgun Metagenomic Data Retrieved from Public Repositories.

BACKGROUND: Respiratory viruses significantly impact global morbidity and mortality, causing more disease in humans than any other infectious agent. Beyond pathogens, various viruses and bacteria colonize the respiratory tract without causing disease, potentially influencing respiratory diseases' pathogenesis. Nevertheless, our understanding of respiratory microbiota is limited by technical constraints, predominantly focusing on bacteria and neglecting crucial populations like viruses. Despite recent efforts to improve our understanding of viral diversity in the human body, our knowledge of viral diversity associated with the human respiratory tract remains limited. METHODS: Following a comprehensive search in bibliographic and sequencing data repositories using keyword terms, we retrieved shotgun metagenomic data from public repositories (n = 85). After manual curation, sequencing data files from 43 studies were analyzed using EVEREST (pipEline for Viral assEmbly and chaRactEriSaTion). Complete and high-quality contigs were further assessed for genomic and taxonomic characterization. RESULTS: Viral contigs were obtained from 194 out of the 868 FASTQ files processed through EVEREST. Of the 1842 contigs that were quality assessed, 8% (n = 146) were classified as complete/high-quality genomes. Most of the identified viral contigs were taxonomically classified as bacteriophages, with taxonomic resolution ranging from the superkingdom level down to the species level. Captured contigs were spread across 25 putative families and varied between RNA and DNA viruses, including previously uncharacterized viral genomes. Of note, airway samples also contained virus(es) characteristic of the human gastrointestinal tract, which have not been previously described as part of the lung virome. Additionally, by performing a meta-analysis of the integrated datasets, ecological trends within viral populations linked to human disease states and their biogeographical distribution along the respiratory tract were observed. CONCLUSION: By leveraging publicly available repositories of shotgun metagenomic data, the present study provides new insights into viral genomes associated with specimens from the human respiratory tract across different disease spectra. Further studies are required to validate our findings and evaluate the potential impact of these viral communities on respiratory tract physiology.

Gut and respiratory tract microbiota in children younger than 12 months hospitalized for bronchiolitis compared with healthy children: can we predict the severity and medium-term respiratory outcome?

Growing evidence indicates that gut and respiratory microbiota have a potential key effect on bronchiolitis, mainly caused by respiratory syncytial virus (RSV). This was a prospective study of 96 infants comparing infants with bronchiolitis (n = 57, both RSV and non-RSV associated) to a control group (n = 39). Gut (feces) and respiratory [nasopharyngeal aspirate (NPA)] microbial profiles were analyzed by 16S rRNA amplicon sequencing, and respiratory viruses were identified by PCR. Clinical data of the acute episode and follow-up during the first year after infection were recorded. Pairwise comparisons showed significant differences in the gut (R2 = 0.0639, P = 0.006) and NPA (R2 = 0.0803, P = 0.006) microbiota between cases and controls. A significantly lower gut microbial richness and an increase in the NPA microbial diversity (mainly due to an increase in Haemophilus, Streptococcus, and Neisseria) were observed in the infants with bronchiolitis, in those with the most severe symptoms, and in those who subsequently developed recurrent wheezing episodes after discharge. In NPA, the higher microbial richness differed significantly between the control group and the non-RSV bronchiolitis group (P = 0.01) and between the control group and the RSV bronchiolitis group (P = 0.001). In the gut, the richness differed significantly between the control group and the non-RSV group (P = 0.01) and between the control group and the RSV bronchiolitis group (P = 0.001), with higher diversity in the RSV group. A distinct respiratory and intestinal microbial pattern was observed in infants with bronchiolitis compared with controls. The presence of RSV was a main factor for dysbiosis. Lower gut microbial richness and increased respiratory microbial diversity were associated with respiratory morbidity during follow-up. IMPORTANCE: Both the intestinal and respiratory microbiota of children with bronchiolitis, especially those with respiratory syncytial virus infection, are altered and differ from that of healthy children. The microbiota pattern in the acute episode could identify those children who will later have other respiratory episodes in the first year of life. Preventive measures could be adopted for this group of infants.

Yeast species in the respiratory samples of COVID-19 patients; molecular tracking of Candida auris.

Introduction: Although the existence of Candida species in the respiratory tract is often considered commensal, it is crucial to recognize the significance of Candida colonization in immunocompromised or COVID-19 patients. The emergence of Candida auris as an emerging pathogen further emphasizes the importance of monitoring yeast infection/colonization, particularly in COVID-19 patients. Methods: In this study, respiratory samples mainly from COVID-19 patients, primarily those suspected of having a fungal infection, were cultured on Sabouraud dextrose agar plates and the yeast colonies were identified using a two-step multiplex PCR method. The samples suspected of C. auris underwent specific nested PCR followed by sequence analysis. Results: A total of 199 respiratory samples were collected from 73 women and 126 men, ranging in age from 1.6 to 88 years. Among the patients, 141 had COVID-19, 32 had cancer, 5 were hospitalized in ICU, 2 had chronic obstructive pulmonary disease)COPD(, and others were patients with combination diseases. From these samples, a total of 334 yeast strains were identified. C. albicans (n=132, 39.52%) was the most common species, followed by C. tropicalis (n=67, 20%), C. glabrata (n=56, 16.76%), C. krusei (n=18, 5.4%), C. parapsilosis (n=17, 5.08%), Saccharomyces cerevisiae (n=10, 3%), C. kefyr (n=9, 2.6%), C. dubliniensis (n=7, 2.1%), C. lusitaniae (n=5, 1.5%), C. auris (n=3, 0.9%), C. guilliermondii (n=2, 0.6%), C. rugosa (n=1, 0.3%), C. intermedia (n=1, 0.3%), and Trichosporon spp. (n=1, 0.3%). C. auris was detected in a patient in ICU and two COVID-19 patients. While its presence was confirmed through sequence analysis, our extensive efforts to isolate C. auris were unsuccessful. Conclusion: While C. albicans colonization remains prevalent, our study found no evidence of Candida lung infection. Since the role of Candida colonization in airway secretions remains ambiguous due to limited research, further studies are imperative to shed light on this matter.

The characteristics of microbiome in the upper respiratory tract of COVID-19 patients.

BACKGROUND: Co-infection with other pathogens in coronavirus disease 2019 (COVID-19) patients exacerbates disease severity and impacts patient prognosis. Clarifying the exact pathogens co-infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is premise of the precise treatment for COVID-19 patients. METHODS: Sputum samples were collected from 17 patients in the COVID-19 positive group and 18 patients in the COVID-19 negative group. DNA extraction was performed to obtain the total DNA. Sequencing analysis using 16S and ITS rRNA gene was carried out to analyze the composition of bacterial and fungal communities. Meanwhile, all the samples were inoculated for culture. RESULTS: We did not observe significant differences in bacterial composition between the COVID-19 positive and negative groups. However, a significantly higher abundance of Candida albicans was observed in the upper respiratory tract samples from the COVID-19 positive group compared to the COVID-19 negative group. Moreover, the Candida albicans strains isolated from COVID-19 positive group exhibited impaired secretion of aspartyl proteinases. CONCLUSION: COVID-19 positive patients demonstrate a notable increase in the abundance of Candida albicans, along with a decrease in the levels of aspartyl proteinases, indicating the alteration of microbiota composition of upper respiratory tract.

Molecular characteristics and biofilm formation capacity of nontypeable Haemophilus influenza strains isolated from lower respiratory tract in children.

With the widespread introduction of the Hib conjugate vaccine, Nontypeable Haemophilus influenzae (NTHi) has emerged as the predominant strain globally. NTHi presents a significant challenge as a causative agent of chronic clinical infections due to its high rates of drug resistance and biofilm formation. While current research on NTHi biofilms in children has primarily focused on upper respiratory diseases, investigations into lower respiratory sources remain limited. In this study, we collected 54 clinical strains of lower respiratory tract origin from children. Molecular information and drug resistance features were obtained through whole gene sequencing and the disk diffusion method, respectively. Additionally, an in vitro biofilm model was established. All clinical strains were identified as NTHi and demonstrated the ability to form biofilms in vitro. Based on scanning electron microscopy and crystal violet staining, the strains were categorized into weak and strong biofilm-forming groups. We explored the correlation between biofilm formation ability and drug resistance patterns, as well as clinical characteristics. Stronger biofilm formation was associated with a longer cough duration and a higher proportion of abnormal lung imaging findings. Frequent intake of ß-lactam antibiotics might be associated with strong biofilm formation. While a complementary relationship between biofilm-forming capacity and drug resistance may exist, further comprehensive studies are warranted. This study confirms the in vitro biofilm formation of clinical NTHi strains and establishes correlations with clinical characteristics, offering valuable insights for combating NTHi infections.

General dynamics of the URT microbiome and microbial signs of recovery in COVID-19 patients.

COVID-19 is caused by an airborne virus, SARS-CoV-2. The upper respiratory tract (URT) is, therefore, the first system to endure the attack. Inhabited by an assemblage of microbial communities, a healthy URT wards off the invasion. However, once invaded, it becomes destabilised, which could be crucial to the establishment and progression of the infection. We examined 696 URT samples collected from 285 COVID-19 patients at three time-points throughout their hospital stay and 100 URT samples from 100 healthy controls. We used 16S ribosomal RNA sequencing to evaluate the abundance of various bacterial taxa, α-diversity, and ß-diversity of the URT microbiome. Ordinary least squares regression was used to establish associations between the variables, with age, sex, and antibiotics as covariates. The URT microbiome in the COVID-19 patients was distinctively different from that of healthy controls. In COVID-19 patients, the abundance of 16 genera was significantly reduced. A total of 47 genera were specific to patients, whereas only 2 were unique to controls. The URT samples collected at admission differed more from the control than from the samples collected at later stages of treatment. The following four genera originally depleted in the patients grew significantly by the end of treatment: Fusobacterium, Haemophilus, Neisseria, and Stenotrophomonas. Our findings strongly suggest that SARS-CoV-2 caused significant changes in the URT microbiome, including the emergence of numerous atypical taxa. These findings may indicate increased instability of the URT microbiome in COVID-19 patients. In the course of the treatment, the microbial composition of the URT of COVID-19 patients tended toward that of controls. These microbial changes may be interpreted as markers of recovery.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.