Immunological Landscapes in Lung Transplantation: Insights from T Cell Profiling in BAL and PBMC.

Lung transplant recipients frequently encounter immune-related complications, including chronic lung allograft dysfunction (CLAD). Monitoring immune cells within the lung microenvironment is pivotal for optimizing post-transplant outcomes. This study examined the proportion of T cell subsets in paired bronchoalveolar lavage (BAL) and peripheral PBMC comparing healthy (n = 4) and lung transplantation patients (n = 6, no CLAD and n = 14 CLAD) using 14-color flow cytometry. CD4+ T cell proportions were reduced in CD3 cells in both PBMC and BAL, and positive correlations were discerned between T cell populations in peripheral PBMC and BAL, suggesting the prospect of employing less invasive PBMC sampling as a means of monitoring lung T cells. Furthermore, regulatory T cells (Tregs) were enriched in BAL when compared to peripheral PBMC for transplant recipients. A parallel positive correlation emerged between Treg proportions in BAL and peripheral PBMC, underscoring potential avenues for monitoring lung Tregs. Finally, the most promising biomarker was the Teff (CD8+Granzyme B+)-Treg ratio, which was higher in both the PBMC and BAL of transplant recipients compared to healthy individuals, and increased in the patients with CLAD compared to no CLAD and healthy patients. Conclusions: Distinct T cell profiles in BAL and peripheral PBMC underscore the significance of localized immune monitoring in lung transplantation. The Teff (CD8+granzyme B+)-Treg ratio, particularly within the context of CLAD, emerges as a promising blood and BAL biomarker reflective of inflammation and transplant-related complications. These findings emphasize the imperative need for personalized immune monitoring strategies that tailored to address the unique immunological milieu in post-transplant lungs.

Toward Standardized Aerovirology: A Critical Review of Existing Results and Methodologies.

Understanding the airborne survival of viruses is important for public health and epidemiological modeling and potentially to develop mitigation strategies to minimize the transmission of airborne pathogens. Laboratory experiments typically involve investigating the effects of environmental parameters on the viability or infectivity of a target airborne virus. However, conflicting results among studies are common. Herein, the results of 34 aerovirology studies were compared to identify links between environmental and compositional effects on the viability of airborne viruses. While the specific experimental apparatus was not a factor in variability between reported results, it was determined that the experimental procedure was a major factor that contributed to discrepancies in results. The most significant contributor to variability between studies was poorly defined initial viable virus concentration in the aerosol phase, causing many studies to not measure the rapid inactivation, which occurs quickly after particle generation, leading to conflicting results. Consistently, studies that measured their reference airborne viability minutes after aerosolization reported higher viability at subsequent times, which indicates that there is an initial loss of viability which is not captured in these studies. The composition of the particles which carry the viruses was also found to be important in the viability of airborne viruses; however, the mechanisms for this effect are unknown. Temperature was found to be important for aerosol-phase viability, but there is a lack of experiments that directly compare the effects of temperature in the aerosol phase and the bulk phase. There is a need for repeated measurements between different research groups under identical conditions both to assess the degree of variability between studies and also to attempt to better understand already published data. Lack of experimental standardization has hindered the ability to quantify the differences between studies, for which we provide recommendations for future studies. These recommendations are as follows: measuring the reference airborne viability using the "direct method"; use equipment which maximizes time resolution; quantify all losses appropriately; perform, at least, a 5- and 10-min sample, if possible; report clearly the composition of the virus suspension; measure the composition of the gas throughout the experiment. Implementing these recommendations will address the most significant oversights in the existing literature and produce data which can more easily be quantitatively compared.

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Dynamics and Viability of Airborne Respiratory Syncytial Virus under Various Indoor Air Conditions.

The factors governing the viability of airborne viruses embedded within respiratory particles are not well understood. This study aimed to investigate the relative humidity (RH)-dependent viability of airborne respiratory syncytial virus (RSV) in simulated respiratory particles suspended in various indoor air conditions. We tested airborne RSV viability in three static indoor air conditions, including sub-hysteresis (RH < 39%), hysteresis (39% < RH < 65%), and super-hysteresis (RH > 65%) air as well as in three dynamic indoor air conditions, including the transitions between the static conditions. The dynamic conditions were hysteresis → super-hysteresis → hysteresis, sub-hysteresis → hysteresis, and super-hysteresis → hysteresis. We found that after 45 min of particle aging in static conditions, the viability of RSV in sub-hysteresis, hysteresis, and super-hysteresis air was 0.72% ± 0.06%, 0.03% ± 0.006%, and 0.27% ± 0.008%, respectively. After 45 min of aging in dynamic conditions, the RSV viability decreased for particles that remained in a liquid (deliquesced) state during aging when compared with particles in a solid (effloresced) state. The decreased viability of airborne RSV for deliquesced particles is consistent with prolonged exposure to elevated aqueous solutes. These results represent the first measurements of the survival of airborne RSV over particle aging time, with equal viability in low, intermediate, and high RHs at 5 and 15 min and a V-shaped curve after 45 min.

Single-Cell Profiling of Cells in the Lung of a Patient with Chronic Hypersensitivity Pneumonitis Reveals Inflammatory Niche with Abundant CD39+ T Cells with Functional ATPase Phenotype: A Case Study.

This study investigated immune cell characteristics in chronic hypersensitivity pneumonitis (HP), focusing on CD39-expressing cells' impact on inflammation and tissue remodelling. Lung tissue from an HP patient was analysed using single-cell transcriptomics, flow cytometry, and gene expression profiling. The tissue revealed diverse cell types like macrophages, T cells, fibroblasts, and regulatory T cells (Tregs). CD39-expressing Tregs exhibited heightened ATP hydrolysis capacity and regulatory gene expression. CD39hi cells displayed markers of both Tregs and proinflammatory Th17 cells, suggesting transitional properties. Communication networks involving molecules like SPP1, collagen, CSF1, and IL-1ß were identified, hinting at interactions between cell types in HP pathogenesis. This research provides insights into the immune response and cell interactions in chronic HP. CD39-expressing cells dual nature as Tregs and Th17 cells suggests a role in modulating lung inflammation, potentially affecting disease progression. These findings lay the groundwork for further research, underscoring CD39-expressing cells as potential therapeutic targets in HP.

International Pediatric COVID-19 Severity Over the Course of the Pandemic.

Importance: Multiple SARS-CoV-2 variants have emerged over the COVID-19 pandemic. The implications for COVID-19 severity in children worldwide are unclear. Objective: To determine whether the dominant circulating SARS-CoV-2 variants of concern (VOCs) were associated with differences in COVID-19 severity among hospitalized children. Design, Setting, and Participants: Clinical data from hospitalized children and adolescents (younger than 18 years) who were SARS-CoV-2 positive were obtained from 9 countries (Australia, Brazil, Italy, Portugal, South Africa, Switzerland, Thailand, UK, and the US) during 3 different time frames. Time frames 1 (T1), 2 (T2), and 3 (T3) were defined to represent periods of dominance by the ancestral virus, pre-Omicron VOCs, and Omicron, respectively. Age groups for analysis were younger than 6 months, 6 months to younger than 5 years, and 5 to younger than 18 years. Children with an incidental positive test result for SARS-CoV-2 were excluded. Exposures: SARS-CoV-2 hospitalization during the stipulated time frame. Main Outcomes and Measures: The severity of disease was assessed by admission to intensive care unit (ICU), the need for ventilatory support, or oxygen therapy. Results: Among 31 785 hospitalized children and adolescents, the median age was 4 (IQR 1-12) years and 16 639 were male (52.3%). In children younger than 5 years, across successive SARS-CoV-2 waves, there was a reduction in ICU admission (T3 vs T1: risk ratio [RR], 0.56; 95% CI, 0.42-0.75 [younger than 6 months]; RR, 0.61, 95% CI; 0.47-0.79 [6 months to younger than 5 years]), but not ventilatory support or oxygen therapy. In contrast, ICU admission (T3 vs T1: RR, 0.39, 95% CI, 0.32-0.48), ventilatory support (T3 vs T1: RR, 0.37; 95% CI, 0.27-0.51), and oxygen therapy (T3 vs T1: RR, 0.47; 95% CI, 0.32-0.70) decreased across SARS-CoV-2 waves in children 5 years to younger than 18 years old. The results were consistent when data were restricted to unvaccinated children. Conclusions and Relevance: This study provides valuable insights into the impact of SARS-CoV-2 VOCs on the severity of COVID-19 in hospitalized children across different age groups and countries, suggesting that while ICU admissions decreased across the pandemic in all age groups, ventilatory and oxygen support generally did not decrease over time in children aged younger than 5 years. These findings highlight the importance of considering different pediatric age groups when assessing disease severity in COVID-19.

Physicochemical characterization of porcine respiratory aerosol and considerations for future aerovirology.

Understanding the mechanisms which inactivate airborne viruses is a current challenge. The composition of human respiratory aerosol is poorly understood and needs to be adequately investigated for use in aerovirology studies. Here, the physicochemical properties of porcine respiratory fluid (PRF) from the trachea and lungs were investigated both in bulk solutions and in aerosols. The mass ratio of Na:K in PRF compared with cell culture media (Dulbecco's Modified Eagle Medium, DMEM), which is frequently used in aerovirology studies, was significantly lower (∼2:1 vs ∼16:1). PRF contained significantly more potassium and protein than DMEM. PRF aerosols of all samples were similarly hygroscopic to human respiratory aerosol. PRF particles could nucleate with spatially separated crystals, indicating that the protein matrix was sufficiently viscous to prevent the complete coalescence of aqueous salts prior to efflorescence. The effects of these differences in compositions on the viability of viruses are currently not well understood. The virus suspensions in aerovirology studies need to be reconsidered to adequately reflect a real-world expiration scenario.

Cytotoxic and Bactericidal Effects of Inhalable Ciprofloxacin-Loaded Poly(2-ethyl-2-oxazoline) Nanoparticles with Traces of Zinc Oxide.

The bactericidal effects of inhalable ciprofloxacin (CIP) loaded-poly(2-ethyl-2-oxazoline) (PEtOx) nanoparticles (NPs) with traces of zinc oxide (ZnO) were investigated against clinical strains of the respiratory pathogens Staphylococcus aureus and Pseudomonas aeruginosa. CIP-loaded PEtOx NPs retained their bactericidal activity within the formulations compared to free CIP drugs against these two pathogens, and bactericidal effects were enhanced with the inclusion of ZnO. PEtOx polymer and ZnO NPs did not show bactericidal activity alone or in combination against these pathogens. The formulations were tested to determine the cytotoxic and proinflammatory effects on airway epithelial cells derived from healthy donors (NHBE), donors with chronic obstructive pulmonary disease (COPD, DHBE), and a cell line derived from adults with cystic fibrosis (CFBE41o-) and macrophages from healthy adult controls (HCs), and those with either COPD or CF. NHBE cells demonstrated maximum cell viability (66%) against CIP-loaded PEtOx NPs with the half maximal inhibitory concentration (IC50) value of 50.7 mg/mL. CIP-loaded PEtOx NPs were more toxic to epithelial cells from donors with respiratory diseases than NHBEs, with respective IC50 values of 0.103 mg/mL for DHBEs and 0.514 mg/mL for CFBE41o- cells. However, high concentrations of CIP-loaded PEtOx NPs were toxic to macrophages, with respective IC50 values of 0.002 mg/mL for HC macrophages and 0.021 mg/mL for CF-like macrophages. PEtOx NPs, ZnO NPs, and ZnO-PEtOx NPs with no drug were not cytotoxic to any cells investigated. The in vitro digestibility of PEtOx and its NPs was investigated in simulated lung fluid (SLF) (pH 7.4). The analysed samples were characterized using Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), and UV-Vis spectroscopy. Digestion of PEtOx NPs commenced one week following incubation and was completely digested after four weeks; however, the original PEtOx was not digested after six weeks of incubation. The outcome of this study revealed that PEtOx polymer could be considered an efficient drug delivery carrier in respiratory linings, and CIP-loaded PEtOx NPs with traces of ZnO could be a promising addition to inhalable treatments against resistant bacteria with reduced toxicity.

Titanium dioxide nanostructures that reduce the infectivity of respiratory syncytial virus.

The spread of respiratory diseases has gained significant attention since the detection and rapid global spread of COVID-19. Respiratory viruses are commonly transmitted when an infected person coughs or sneezes onto a surface, infecting persons who subsequently contact this surface. For this reason, developing surfaces with inherent antipathogenic properties is crucially needed for controlling the spread of deadly pathogens. Recent studies have established the antipathogenic potential of hydrothermally synthesised titanium dioxide (TiO2) nanostructured surfaces against bacteria strains (Gram-positive and negative) and several respiratory viruses, including SARS-CoV-2, HRV-16 and HCoV-NL63. This study investigates the antiviral behaviour of TiO2 nanostructured surfaces against Respiratory Syncytial Virus (RSV), a respiratory virus commonly contracted by children, to reduce viral transmission in high-traffic environments such as hospitals and childcare centers. Mimicking droplets produced when a person coughs or sneezes, RSV droplets were exposed to nanostructured surfaces to investigate their antiviral potential. Results show that nanostructured TiO2 reduced the RSV infectious viral load at all timepoints compared to control surfaces, showing 1.7, 2.6 and 3.2 log reductions after 2-, 5- and 7-hours exposure, respectively. Interestingly, virus exposed to nanostructured surfaces showed little to no infectivity after 5 h exposure while viable virus was still detected on control surfaces after 7 h exposure. These encouraging results establish TiO2 nanostructured surfaces as a potential method for reducing transmission and spread of respiratory viruses and bacterial strains.

Bioengineered textiles with peptide binders that capture SARS-CoV-2 viral particles.

The use of personal protective equipment (PPE), face masks and ventilation are key strategies to control the transmission of respiratory viruses. However, most PPE provides physical protection that only partially prevents the transmission of viral particles. Here, we develop textiles with integrated peptide binders that capture viral particles. We fuse peptides capable of binding the receptor domain of the spike protein on the SARS-CoV-2 capsid to the cellulose-binding domain from the Trichoderma reesei cellobiohydrolase II protein. The hybrid peptides can be attached to the cellulose fibres in cotton and capture SARS-CoV-2 viral particles with high affinity. The resulting bioengineered cotton captures 114,000 infective virus particles per cm2 and reduces onwards SARS-CoV-2 infection of cells by 500-fold. The hybrid peptides could be easily modified to capture and control the spread of other infectious pathogens or for attachment to different materials. We anticipate the use of bioengineered protective textiles in PPE, facemasks, ventilation, and furnishings will provide additional protection to the airborne or fomite transmission of viruses.

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells.

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

TiO2 Nanostructures That Reduce the Infectivity of Human Respiratory Viruses Including SARS-CoV-2.

The rapid emergence and global spread of the COVID-19 causing Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its subsequent mutated strains has caused unprecedented health, economic, and social devastation. Respiratory viruses such as SARS-CoV-2 can be transmitted through both direct and indirect channels, including aerosol respiratory droplets, contamination of inanimate surfaces (fomites), and direct person-to-person contact. Current methods of virus inactivation on surfaces include chemicals and biocides, and while effective, continuous and repetitive cleaning of all surfaces is not always viable. Recent work in the field of biomaterials engineering has established the antibacterial effects of hydrothermally synthesized TiO2 nanostructured surfaces against both Gram-negative and -positive bacteria. The current study investigates the effectiveness of said TiO2 nanostructured surfaces against two enveloped human coronaviruses, SARS-CoV-2 and HCoV-NL63, and nonenveloped HRV-16 for surface-based inactivation. Results show that structured surfaces reduced infectious viral loads of SARS-CoV-2 (5 log), HCoV-NL63 (3 log), and HRV-16 (4 log) after 5 h, compared to nonstructured and tissue culture plastic control surfaces. Interestingly, infectious virus remained present on control tissue culture plastic after 7 h exposure. These encouraging results establish the potential use of nanostructured surfaces to reduce the transmission and spread of both enveloped and nonenveloped respiratory viruses, by reducing their infectious period on a surface. The dual antiviral and antibacterial properties of these surfaces support their potential application in a wide variety of settings such as hospitals and healthcare environments, public transport and community hubs.

MLKL Regulates Rapid Cell Death-independent HMGB1 Release in RSV Infected Airway Epithelial Cells.

Respiratory syncytial virus (RSV)-induced bronchiolitis is a significant contributor to infant morbidity and mortality. Previously, we identified that necroptosis, a pro-inflammatory form of cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3, and mixed lineage kinase domain like protein (MLKL), occurs in RSV-infected human airway epithelial cells (hAECs), mediating the release of the alarmin high mobility group box 1 (HMGB1). Here, we show that RSV infection of hAECs induces the biphasic release of HMGB1 at 6 ("early") and 24 ("late") hours post infection (hpi). The early phase of HMGB1 release at 6 hpi is cell death-independent, however, this release is nonetheless attenuated by inhibition of MLKL (primarily associated with necroptosis). The early release of HMGB1 promotes the late phase of HMGB1 release via the activation of RAGE (receptor for advanced glycation endproducts) and occurs with cell death. Treatment of hAECS with exogenous HMGB1 combined with a pan-caspase inhibitor induces hAEC necroptosis, and is attenuated by the RAGE antagonist, FPS-ZM1. Together, these findings demonstrate that RSV infection of hAECs leads to the early release of HMGB1, followed by a paracrine feed-forward amplification loop that further increases HMGB1 levels and promotes cell death. As the inhibition of MLKL or targeting of HMGB1/RAGE pathway attenuates the release of pro-inflammatory HMGB1 and decreases viral load, this suggests that the pharmacological targeting of these pathways may be of benefit for the treatment of severe RSV bronchiolitis.

Targeting the P2Y13 Receptor Suppresses IL-33 and HMGB1 Release and Ameliorates Experimental Asthma.

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.

IFI27 transcription is an early predictor for COVID-19 outcomes, a multi-cohort observational study.

Purpose: Robust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness. Methods: We conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients. Results: We show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients. Conclusion: These data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus.

Innate Immunity in the Middle Ear Mucosa.

Otitis media (OM) encompasses a spectrum of clinical presentations ranging from the readily identifiable Acute OM (AOM), which is characterised by otalgia and fever, to chronic otitis media with effusion (COME) where impaired hearing due to middle ear effusion may be the only clinical symptom. Chronic suppurative OM (CSOM) presents as a more severe form of OM, involving perforation of the tympanic membrane. The pathogenesis of OM in these varied clinical presentations is unclear but activation of the innate inflammatory responses to viral and/or bacterial infection of the upper respiratory tract performs an integral role. This localised inflammatory response can persist even after pathogens are cleared from the middle ear, eustachian tubes and, in the case of respiratory viruses, even the nasal compartment. Children prone to OM may experience an over exuberant inflammatory response that underlies the development of chronic forms of OM and their sequelae, including hearing impairment. Treatments for chronic effusive forms of OM are limited, with current therapeutic guidelines recommending a "watch and wait" strategy rather than active treatment with antibiotics, corticosteroids or other anti-inflammatory drugs. Overall, there is a clear need for more targeted and effective treatments that either prevent or reduce the hyper-inflammatory response associated with chronic forms of OM. Improved treatment options rely upon an in-depth understanding of OM pathogenesis, particularly the role of the host innate immune response during acute OM. In this paper, we review the current literature regarding the innate immune response within the middle ear to bacterial and viral otopathogens alone, and as co-infections. This is an important consideration, as the role of respiratory viruses as primary pathogens in OM is not yet fully understood. Furthermore, increased reporting from PCR-based diagnostics, indicates that viral/bacterial co-infections in the middle ear are more common than bacterial infections alone. Increasingly, the mechanisms by which viral/bacterial co-infections may drive or maintain complex innate immune responses and inflammation during OM as a chronic response require investigation. Improved understanding of the pathogenesis of chronic OM, including host innate immune response within the middle ear is vital for development of improved diagnostic and treatment options for our children.

Prevalence of Neutralising Antibodies to HCoV-NL63 in Healthy Adults in Australia.

The COVID-19 pandemic has highlighted the importance of understanding the immune response to seasonal human coronavirus (HCoV) infections such as HCoV-NL63, how existing neutralising antibodies to HCoV may modulate responses to SARS-CoV-2 infection, and the utility of seasonal HCoV as human challenge models. Therefore, in this study we quantified HCoV-NL63 neutralising antibody titres in a healthy adult population using plasma from 100 blood donors in Australia. A microneutralisation assay was performed with plasma diluted from 1:10 to 1:160 and tested with the HCoV-NL63 Amsterdam-1 strain. Neutralising antibodies were detected in 71% of the plasma samples, with a median geometric mean titre of 14. This titre was similar to those reported in convalescent sera taken from individuals 3-7 months following asymptomatic SARS-CoV-2 infection, and 2-3 years post-infection from symptomatic SARS-CoV-1 patients. HCoV-NL63 neutralising antibody titres decreased with increasing age (R2 = 0.042, p = 0.038), but did not differ by sex. Overall, this study demonstrates that neutralising antibody to HCoV-NL63 is detectable in approximately 71% of the healthy adult population of Australia. Similar titres did not impede the use of another seasonal human coronavirus (HCoV-229E) in a human challenge model, thus, HCoV-NL63 may be useful as a human challenge model for more pathogenic coronaviruses.

Low Genetic Diversity of Hepatitis B Virus Surface Gene amongst Australian Blood Donors.

Variants in the small surface gene of hepatitis B virus (HBV), which codes for viral surface antigen (HBsAg), can affect the efficacy of HBsAg screening assays and can be associated with occult HBV infection (OBI). This study aimed to characterise the molecular diversity of the HBV small surface gene from HBV-reactive Australian blood donors. HBV isolates from 16 HBsAg-positive Australian blood donors' plasma were sequenced and genotyped by phylogenies of viral coding genes and/or whole genomes. An analysis of the genetic diversity of eight HBV small surface genes from our 16 samples was conducted and compared with HBV sequences from NCBI of 164 international (non-Australian) blood donors. Genotypes A-D were identified in our samples. The region of HBV small surface gene that contained the sequence encoding the 'a' determinant had a greater genetic diversity than the remaining part of the gene. No escape mutants or OBI-related variants were observed in our samples. Variant call analysis revealed two samples with a nucleotide deletion leading to truncation of polymerase and/or large/middle surface amino acid sequences. Overall, we found that HBV small surface gene sequences from Australian donors demonstrated a lower level of genetic diversity than those from non-Australian donor population included in the study.

Utility of Three Nebulizers in Investigating the Infectivity of Airborne Viruses.

Laboratory-generated bioaerosols are widely used in aerobiology studies of viruses; however, few comparisons of alternative nebulizers exist. We compared aerosol production and virus survival for a Collison nebulizer, vibrating mesh nebulizer (VMN), and hydraulic spray atomizer (HSA). We also measured the dry size distribution of the aerosols produced and calculated the droplet sizes before evaporation and the dry size distribution from normal saline solution. Dry count median diameters of 0.11, 0.22, and 0.30 µm were found for normal saline from the Collison nebulizer, VMN, and HSA, respectively. The volume median diameters were 0.323, 1.70, and 1.30 µm, respectively. The effect of nebulization on the viability of two influenza A viruses (IAVs) (H1N1 and H3N2) and human rhinovirus 16 (HRV-16) was assessed by nebulization into an SKC BioSampler. The HSA had the least impact on surviving fractions (SFs) of H1N1 and H3N2 (89% ± 3% and 94% ± 2%, respectively), followed by the Collison nebulizer (83% ± 1% and 82% ± 2%, respectively). The VMN yielded SFs of 78% ± 2% and 76% ± 2%, respectively. Conversely, for HRV-16, the VMN produced higher SFs (87% ± 8%). Our findings indicate that there were no statistical differences between SFs of the viruses nebulized by these nebulizers. However, VMN produced higher aerosol concentrations within the airborne size range, making it more suitable where high aerosol mass production is required. IMPORTANCE Viral respiratory tract infections cause millions of lost days of work and physician visits globally, accounting for significant morbidity and mortality. Respiratory droplets and droplet nuclei from infected hosts are the potential carriers of such viruses within indoor environments. Laboratory-generated bioaerosols are applied in understanding the transmission and infection of viruses, modeling the physiological aspects of bioaerosol generation in a controlled environment. However, little comparative characterization exists for nebulizers used in infectious disease aerobiology, including Collison nebulizer, vibrating mesh nebulizer, and hydraulic spray atomizer. This study characterized the physical features of aerosols generated by laboratory nebulizers and their performance in producing aerosols at a size relevant to airborne transmission used in infectious disease aerobiology. We also determined the impact of nebulization mechanisms of these nebulizers on the viability of human respiratory viruses, including IAV H1N1, IAV H3N2, and HRV-16.