Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

Cruciferous vegetable-derived indole-3-carbinol prevents coronavirus cell egression mechanisms in tracheal and intestinal 3D in vitro models.

The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected. These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung.

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3-/- in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

SARS-CoV-2 N-antigenemia in critically ill adult COVID-19 patients: Frequency and association with inflammatory and tissue-damage biomarkers.

The current study aimed at characterizing the dynamics of SARS-CoV-2 nucleocapsid (N) antigenemia in a cohort of critically ill adult COVID-19 patients and assessing its potential association with plasma levels of biomarkers of clinical severity and mortality. Seventy-three consecutive critically ill COVID-19 patients (median age, 65 years) were recruited. Serial plasma (n = 340) specimens were collected. A lateral flow immunochromatography assay and reverse-transcription polymerase chain reaction (RT-PCR) were used for SARS-CoV-2 N protein detection and RNA quantitation and in plasma, respectively. Serum levels of inflammatory and tissue-damage biomarkers in paired specimens were measured. SARS-CoV-RNA N-antigenemia and viral RNAemia were documented in 40.1% and 35.6% of patients, respectively at a median of 9 days since symptoms onset. The level of agreement between the qualitative results returned by the N-antigenemia assay and plasma RT-PCR was moderate (k = 0.57; p < 0.0001). A trend towards higher SARS-CoV-2 RNA loads was seen in plasma specimens testing positive for N-antigenemia assay than in those yielding negative results (p = 0.083). SARS-CoV-2 RNA load in tracheal aspirates was significantly higher (p < 0.001) in the presence of concomitant N-antigenemia than in its absence. Significantly higher serum levels of ferritin, lactose dehydrogenase, C-reactive protein, and D-dimer were quantified in paired plasma SARS-CoV-2 N-positive specimens than in those testing negative. Occurrence of SARS-CoV-2 N-antigenemia was not associated with increased mortality in univariate logistic regression analysis (odds ratio, 1.29; 95% confidence interval, 0.49-3.34; p = 0.59). In conclusion, SARS-CoV-2 N-antigenemia detection is relatively common in ICU patients and appears to associate with increased serum levels of inflammation and tissue-damage markers. Whether this virological parameter may behave as a biomarker of poor clinical outcome awaits further investigations.

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

The Upper Respiratory Tract of Felids Is Highly Susceptible to SARS-CoV-2 Infection.

Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS-CoV-2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS-CoV-2. Strong viral replication was observed for nasal mucosa explants and tracheal air-liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS-CoV-2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS-CoV-2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS-CoV-2 spillover from humans to felids.

Visible blue light inhibits infection and replication of SARS-CoV-2 at doses that are well-tolerated by human respiratory tissue.

The delivery of safe, visible wavelengths of light can be an effective, pathogen-agnostic, countermeasure that would expand the current portfolio of SARS-CoV-2 intervention strategies beyond the conventional approaches of vaccine, antibody, and antiviral therapeutics. Employing custom biological light units, that incorporate optically engineered light-emitting diode (LED) arrays, we harnessed monochromatic wavelengths of light for uniform delivery across biological surfaces. We demonstrated that primary 3D human tracheal/bronchial-derived epithelial tissues tolerated high doses of a narrow spectral band of visible light centered at a peak wavelength of 425 nm. We extended these studies to Vero E6 cells to understand how light may influence the viability of a mammalian cell line conventionally used for assaying SARS-CoV-2. The exposure of single-cell monolayers of Vero E6 cells to similar doses of 425 nm blue light resulted in viabilities that were dependent on dose and cell density. Doses of 425 nm blue light that are well-tolerated by Vero E6 cells also inhibited infection and replication of cell-associated SARS-CoV-2 by > 99% 24 h post-infection after a single five-minute light exposure. Moreover, the 425 nm blue light inactivated cell-free betacoronaviruses including SARS-CoV-1, MERS-CoV, and SARS-CoV-2 up to 99.99% in a dose-dependent manner. Importantly, clinically applicable doses of 425 nm blue light dramatically inhibited SARS-CoV-2 infection and replication in primary human 3D tracheal/bronchial tissue. Safe doses of visible light should be considered part of the strategic portfolio for the development of SARS-CoV-2 therapeutic countermeasures to mitigate coronavirus disease 2019 (COVID-19).

Ultra- and micro-structural changes of respiratory tracts in SARS-CoV-2 infected Syrian hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.

IAPV-Induced Paralytic Symptoms Associated with Tachypnea via Impaired Tracheal System Function.

Although it had been reported that Israeli acute paralysis virus (IAPV) can cause systemic infection in honey bees, little is known about how it establishes this infection and results in the typical symptoms, paralysis and trembling. Here, we used our previously constructed IAPV infectious clone to investigate viral loads in different tissues of honey bees and further identify the relation between tissue tropism and paralytic symptoms. Our results showed that tracheae showed a greater concentration of viral abundance than other tissues. The abundance of viral protein in the tracheae was positively associated with viral titers, and was further confirmed by immunological and ultrastructural evidence. Furthermore, higher viral loads in tracheae induced remarkable down-regulation of succinate dehydrogenase and cytochrome c oxidase genes, and progressed to causing respiratory failure of honey bees, resulting in the appearance of typical symptoms, paralysis and body trembling. Our results showed that paralysis symptoms or trembling was actually to mitigate tachypnea induced by IAPV infection due to the impairment of honey bee tracheae, and revealed a direct causal link between paralysis symptoms and tissue tropism. These findings provide new insights into the understanding of the underlying mechanism of paralysis symptoms of honey bees after viral infection and have implications for viral disease prevention and specific therapeutics in practice.

The Molecular Mechanism of Multiple Organ Dysfunction and Targeted Intervention of COVID-19 Based on Time-Order Transcriptomic Analysis.

Coronavirus disease 2019 (COVID-19) pandemic is caused by the novel coronavirus that has spread rapidly around the world, leading to high mortality because of multiple organ dysfunction; however, its underlying molecular mechanism is unknown. To determine the molecular mechanism of multiple organ dysfunction, a bioinformatics analysis method based on a time-order gene co-expression network (TO-GCN) was performed. First, gene expression profiles were downloaded from the gene expression omnibus database (GSE161200), and a TO-GCN was constructed using the breadth-first search (BFS) algorithm to infer the pattern of changes in the different organs over time. Second, Gene Ontology enrichment analysis was used to analyze the main biological processes related to COVID-19. The initial gene modules for the immune response of different organs were defined as the research object. The STRING database was used to construct a protein-protein interaction network of immune genes in different organs. The PageRank algorithm was used to identify five hub genes in each organ. Finally, the Comparative Toxicogenomics Database played an important role in exploring the potential compounds that target the hub genes. The results showed that there were two types of biological processes: the body's stress response and cell-mediated immune response involving the lung, trachea, and olfactory bulb (olf) after being infected by COVID-19. However, a unique biological process related to the stress response is the regulation of neuronal signals in the brain. The stress response was heterogeneous among different organs. In the lung, the regulation of DNA morphology, angiogenesis, and mitochondrial-related energy metabolism are specific biological processes related to the stress response. In particular, an effect on tracheal stress response was made by the regulation of protein metabolism and rRNA metabolism-related biological processes, as biological processes. In the olf, the distinctive stress responses consist of neural signal transmission and brain behavior. In addition, myeloid leukocyte activation and myeloid leukocyte-mediated immunity in response to COVID-19 can lead to a cytokine storm. Immune genes such as SRC, RHOA, CD40LG, CSF1, TNFRSF1A, FCER1G, ICAM1, LAT, LCN2, PLAU, CXCL10, ICAM1, CD40, IRF7, and B2M were predicted to be the hub genes in the cytokine storm. Furthermore, we inferred that resveratrol, acetaminophen, dexamethasone, estradiol, statins, curcumin, and other compounds are potential target drugs in the treatment of COVID-19.

Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor.

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.

Protección incrementada por el virus recombinante de la enfermedad de Newcastle que expresa el ectodominio de la espícula del virus de la bronquitis infecciosa y el factor estimulante de colonias de granulocitos y macrófagos del pollo. Anteriormente se reportó que la cepa LaSota recombinante del virus de la enfermedad de Newcastle (rLS) que expresa el ectodominio de la espícula trimérica (S) de tipo Arkansas (Ark) del virus de la bronquitis infecciosa (IBV) (Se; rLS/ArkSe) proporciona una protección subóptima contra la exposición al virus de la bronquitis infecciosa. Ahora se ha desarrollado hemos desarrollado una cepa LaSota recombinante (rLS) que expresa el factor estimulante de colonias de granulocitos y macrófagos de pollo (GMCSF) y la espícula del virus de bronquitis Arkansas en un intento para mejorar la efectividad de la vacuna. En el estudio actual, primero se comparó la protección conferida por la vacunación con los virus rLS/ArkSe y rLS/ArkSe.GMCSF. Los pollos vacunados se desafiaron con un virus Arkansas virulento y la protección se determinó mediante los signos clínicos, la carga viral y la histomorfometría de la tráquea. Los resultados mostraron que la coexpresión del factor estimulante de colonias de granulocitos y macrófagos de pollo y la espícula de la cepa recombinante LaSota redujo significativamente la carga viral traqueal y las lesiones traqueales en comparación con los pollos vacunados con el virus rLS/ArkSe. En un segundo experimento, se evaluó el incremento en la protección cruzada por una vacuna atenuada de Massachusetts (Mass) mediante la primovacunación o la vacunación de refuerzo con rLS/ArkSe.GMCSF. Los pollos vacunados fueron desafiados con el virus Arkansas y se evaluó la protección. Los resultados mostraron que la primovacunación o la vacunación de refuerzo con el virus recombinante aumentó significativamente la protección cruzada conferida por el virus Massachusetts contra el desafío virulento con el virus Arkansas. Se observaron mayores reducciones de las cargas virales en los fluidos traqueales y lagrimales en pollos primovacunadoss con rLS/ArkSe.GMCSF y con vacunación de refuerzo con Massachusetts. De manera consistente, los niveles de anticuerpos Ark Se medidos con placas de ELISA recubiertas con proteína Ark Se recombinante a los 14 días después del refuerzo fueron significativamente más altos en estos pollos. De manera inesperada, el esquema de vacunación inverso, es decir, la primovacunación con Massachusetts y el refuerzo con la vacuna recombinante, resultó menos efectivo. Se concluye que una estrategia de primovacunación y refuerzo mediante el uso de rLS/ArkSe.GMCSF y la vacuna atenuada con Massachusetts usada en todo el mundo proporciona una protección cruzada aumentada. Por tanto, el virus rLS/GMCSF que coexpresa la proteína de la espícula de los serotipos regionales relevantes de bronquitis infecciosa podría usarse en combinación con una vacuna viva Massachusetts para proteger contra cepas variantes del virus de la bronquitis infecciosa que circulan regionalmente.

Intravenous, Intratracheal, and Intranasal Inoculation of Swine with SARS-CoV-2.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the susceptibility of animals and their potential to act as reservoirs or intermediate hosts for the virus has been of significant interest. Pigs are susceptible to multiple coronaviruses and have been used as an animal model for other human infectious diseases. Research groups have experimentally challenged swine with human SARS-CoV-2 isolates with results suggesting limited to no viral replication. For this study, a SARS-CoV-2 isolate obtained from a tiger which is identical to human SARS-CoV-2 isolates detected in New York City and contains the D614G S mutation was utilized for inoculation. Pigs were challenged via intravenous, intratracheal, or intranasal routes of inoculation (n = 4/route). No pigs developed clinical signs, but at least one pig in each group had one or more PCR positive nasal/oral swabs or rectal swabs after inoculation. All pigs in the intravenous group developed a transient neutralizing antibody titer, but only three other challenged pigs developed titers greater than 1:8. No gross or histologic changes were observed in tissue samples collected at necropsy. In addition, no PCR positive samples were positive by virus isolation. Inoculated animals were unable to transmit virus to naïve contact animals. The data from this experiment as well as from other laboratories supports that swine are not likely to play a role in the epidemiology and spread of SARS-CoV-2.

Clinical and Histopathologic Features of a Feline SARS-CoV-2 Infection Model Are Analogous to Acute COVID-19 in Humans.

The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics for and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. In this study, n = 12 specific-pathogen-free domestic cats were infected intratracheally with SARS-CoV-2 to evaluate clinical disease, histopathologic lesions, and viral infection kinetics at 4 and 8 days post-inoculation; n = 6 sham-inoculated cats served as controls. Intratracheal inoculation of SARS-CoV-2 produced a significant degree of clinical disease (lethargy, fever, dyspnea, and dry cough) consistent with that observed in the early exudative phase of COVID-19. Pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were also observed with SARS-CoV-2 infection, replicating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation was observed between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were also quantified in nasal turbinates, distal trachea, lungs, and other organs. Results of this study validate a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with acute COVID-19 in humans, thus encouraging its use for future translational studies.

Histopathologic Lesion Scoring and Histomorphometric Methods for Measuring Vaccine Reactions in the Trachea of Broiler Chickens.

Severity of the tracheal histologic inflammatory response induced in broilers by ocular inoculation of two infectious bronchitis (IBV) and three Newcastle disease virus (NDV) commercial vaccines were evaluated. The vaccine was delivered by eye drop with a coarse spray to day-old chicks. The vaccines were given individually or in various combinations and were evaluated relative to nonvaccinated controls. Evaluations were performed on postvaccination (PV) days 7 and 14. Histologic endpoints included semiquantitative severity scoring of inflammatory components and quantitative morphometric determinations of inflammatory cell concentration, mucosal thickness, and percentage of ciliated mucosal surface. Strong positive correlations were observed between routine severity scoring and morphometric inflammatory parameters, whereas a negative correlation was present between inflammation severity and the percentage of mucosal ciliation. Variable, sometimes extensive, and often statistically significant differences in inflammatory responses were observed between the various vaccines. One IBV Massachusetts strain vaccine (IBV-A) produced the greatest overall inflammatory response when given alone or in combination with the NDV vaccines. Enhancement of tracheitis was seen on PV day 14 by covaccination of IBV-A with the NDV vaccines, but not by covaccination of another IBV Massachusetts strain vaccine (IBV-B) with NDV. Reduction in cilia percentage was observed for all vaccine groups relative to controls on PV day 7. However, although reactive cilia regeneration occurred on PV day 14 for most vaccine groups, a cilia regenerative response was not apparent for individual or NDV combination vaccination for IBV-A. The study also demonstrates that substantial microscopic trachea pathology may be present in vaccinated birds not exhibiting apparent clinical respiratory signs.

Artículo regular­Métodos de calificación de lesiones histológicas e histomorfométricos para medir las reacciones a las vacunas en la tráquea de pollos de engorde. Se evaluó la gravedad de la respuesta inflamatoria histológica traqueal en pollos de engorde inducida mediante la inoculación ocular de dos vacunas comerciales contra la bronquitis infecciosa (IBV) y tres vacunas del virus de la enfermedad de Newcastle (NDV). Las vacunas se administraron mediante aplicación ocular a pollitos de un día de edad. Las vacunas se administraron individualmente o en varias combinaciones y se evaluaron en relación con los controles no vacunados. Las evaluaciones se realizaron en los días 7 y 14 después de la vacunación (PV). Los criterios de valoración histológicos incluyeron puntuación semicuantitativa de la severidad de los componentes inflamatorios y determinaciones morfométricas cuantitativas de la concentración de células inflamatorias, el grosor de la mucosa y el porcentaje de superficie de la mucosa con cilios. Se observaron fuertes correlaciones positivas entre la puntuación rutinaria de severidad y los parámetros morfométricos inflamatorios, mientras que se observó una correlación negativa entre la severidad de la inflamación y el porcentaje de la superficie con cilios en la mucosa. Se observaron diferencias variables, a veces extensas y a menudo estadísticamente significativas en las respuestas inflamatorias entre las diversas vacunas. Una vacuna de la cepa de Massachusetts del virus de la bronquitis infecciosa (IBV-A) produjo la mayor respuesta inflamatoria general cuando se administró sola o en combinación con las vacunas de Newcastle. Se observó un aumento de la traqueítis en el día 14 después de la vacunación mediante la vacunación simultánea de la vacuna de bronquitis infecciosa A con las vacunas de Newcastle, pero no mediante la vacunación simultánea de la otra vacuna de la cepa Massachusetts (IBV-B) con Newcastle. Se observó una reducción en el porcentaje de los cilios para todos los grupos vacunados en comparación con los controles en el día siete después de la vacunación. Sin embargo, aunque la regeneración de cilios reactivos ocurrió en el día 14 después de la vacunación para la mayoría de los grupos vacunados, no fue evidente una respuesta de regeneración de cilios para la vacunación individual o combinada de Newcastle con la vacuna de bronquitis infecciosa Massachusetts A. El estudio también demuestra que puede estar presente una patología microscópica sustancial de la tráquea en aves vacunadas que no presentan signos respiratorios clínicos aparentes.

Laryngotracheal aspiration test reduce the false negative rate in patients with suspected SARS-COV-2 pneumonia despite a negative nasopharyngeal swab.

BACKGROUND: In the emergency department (ED) definitive diagnosis of SARS-COV-2 pneumonia is challenging as nasopharyngeal swab (NPS) can give false negative results. Strategies to reduce false negative rate of NPS have limitations. Serial NPSs (24-48 h from one another) are time-consuming, sputum can not be collected in the majority of patients, and bronchoalveolar lavage (BAL), the most sensitive test, requires specific expertise. Laryngotracheal aspiration (LTA) is easy to perform and showed a similar accuracy to BAL for diagnosis of other pulmonary diseases, however it was not studied to diagnose SARS-COV-2 pneumonia. OBJECTIVE: An observational cross-sectional study was performed to evaluate the negative predictive value of LTA in patients with suspected SARS-COV-2 pneumonia despite a negative NPS. METHODS: In the EDs of two university hospitals, consecutive patients with suspected SARS-COV-2 pneumonia despite a negative NPS underwent LTA performed with a nasotracheal tube connected to a vacuum system. Final diagnosis based on all respiratory specimen tests (NPS, LTA and BAL) and hospital data was established by two reviewers and in case of discordance by a third reviewer. RESULTS: 117 patients were enrolled. LTA was feasible in all patients and no patients experienced adverse events. Fifteen (12.7%) patients were diagnosed with community-acquired SARS-COV-2 pneumonia: 13 LTA positive and only 2 (1.7%) LTA negative. The negative predictive value of NPS and LTA was 87.3% (79.9% - 92.7%) and 98.1% (93.3%99.8%) respectively. CONCLUSIONS: LTA resulted feasible, safe and reduced false negative rate in patients with suspected SARS-COV-2 pneumonia despite a negative NPS.

Effects of immunosuppression on the efficacy of vaccination against Mycoplasma gallisepticum infection in chickens.

Immunosuppression can increase the susceptibility of chickens to other disease-causing pathogens and interfere with the efficacy of vaccination against those pathogens. Chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) are common causes of immunosuppression in chickens. Immunosuppression was induced by experimental infection with either CAV or IBDV to assess the effect of immunosuppression on the efficacy of vaccination with Mycoplasma gallisepticum strain ts-304 against infection with virulent M. gallisepticum, a common bacterial pathogen of chickens worldwide. Birds were experimentally infected with either CAV or IBDV at 1 week of age, before vaccination and challenge with M. gallisepticum to examine the effect of immunosuppression at the time of vaccination, or at 6 weeks of age, after vaccination against M. gallisepticum but before challenge with virulent M. gallisepticum, to investigate the effect of immunosuppression at the time of challenge. All birds were vaccinated with a single dose of the ts-304 vaccine at 3 weeks of age and experimentally challenged with the virulent M. gallisepticum strain Ap3AS at 8 weeks of age. In immunosuppressed chickens there was a reduction in protection offered by the ts-304 vaccine at two weeks after challenge, as measured by tracheal mucosal thicknesses, serum antibody levels against M. gallisepticum, air sac lesion scores and virulent M. gallisepticum load in the trachea. Immunosuppressed birds with detectable serum antibodies against M. gallisepticum were less likely to have tracheal lesions. This study has shown that immunosuppression caused by infection with CAV or IBDV can interfere with vaccination against mycoplasmosis in chickens.

A cohort autopsy study defines COVID-19 systemic pathogenesis.

Severe COVID-19 disease caused by SARS-CoV-2 is frequently accompanied by dysfunction of the lungs and extrapulmonary organs. However, the organotropism of SARS-CoV-2 and the port of virus entry for systemic dissemination remain largely unknown. We profiled 26 COVID-19 autopsy cases from four cohorts in Wuhan, China, and determined the systemic distribution of SARS-CoV-2. SARS-CoV-2 was detected in the lungs and multiple extrapulmonary organs of critically ill COVID-19 patients up to 67 days after symptom onset. Based on organotropism and pathological features of the patients, COVID-19 was divided into viral intrapulmonary and systemic subtypes. In patients with systemic viral distribution, SARS-CoV-2 was detected in monocytes, macrophages, and vascular endothelia at blood-air barrier, blood-testis barrier, and filtration barrier. Critically ill patients with long disease duration showed decreased pulmonary cell proliferation, reduced viral RNA, and marked fibrosis in the lungs. Permanent SARS-CoV-2 presence and tissue injuries in the lungs and extrapulmonary organs suggest direct viral invasion as a mechanism of pathogenicity in critically ill patients. SARS-CoV-2 may hijack monocytes, macrophages, and vascular endothelia at physiological barriers as the ports of entry for systemic dissemination. Our study thus delineates systemic pathological features of SARS-CoV-2 infection, which sheds light on the development of novel COVID-19 treatment.

Discordant SARS-CoV-2 Detection in the Nasopharynx Versus Trachea for Patients With Tracheostomies.

OBJECTIVES/HYPOTHESIS: Patients with tracheostomies have an anatomically altered connection between their upper and lower airways that could impact SARS-CoV-2 testing. Our goal was to evaluate for discordance in SARS-CoV-2 detection in hospitalized patients with COVID-19 and tracheostomies based on the site analyzed. STUDY DESIGN: Retrospective chart review. METHODS: This single-institution study evaluated hospitalized patients with COVID-19 who had tracheostomies placed during their treatment. We analyzed SARS-CoV-2 RNA nucleic acid amplification test (NAAT) results after tracheostomy. All included patients had nasopharyngeal (NP) and tracheal (TR) samples taken within a 48-hour period, allowing us to characterize rate of test concordance. RESULTS: Forty-five patients met our inclusion criteria. Thirty-two (71.1%) patients had entirely concordant results after tracheostomy. However, 13 (28.9%) patients had at least one set of discordant results, the majority of which were NP negative and TR positive. There were no statistically significant differences in demographic or clinical variables, including time to tracheostomy and time to testing, among patients with concordant versus discordant SARS-CoV-2 results. CONCLUSION: This represents the first study to examine SARS-CoV-2 RNA NAAT concordance between NP and TR sites in hospitalized patients with COVID-19 and tracheostomies. One-third of patients demonstrated discordant testing when NP and TR specimens were collected within a 48-hour time period. Thus, patients with tracheostomies may have a higher false-negative rate if only one site is assessed for SARS-CoV-2. We recommend analyzing samples from both the nasopharynx and trachea for these patients until more prospective data exist. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E2634-E2638, 2021.

Isolation and development of bovine primary respiratory cells as model to study influenza D virus infection.

Influenza D virus (IDV) is a novel type of influenza virus that infects and causes respiratory illness in bovines. Lack of host-specific in vitro model that can recapitulate morphology and physiology of in vivo airway epithelial cells has impeded the study of IDV infection. Here, we established and characterized bovine primary respiratory epithelial cells from nasal turbinate, soft palate, and trachea of the same calf. All three cell types showed characteristics peculiar of epithelial cells, polarized into apical-basolateral membrane, and formed tight junctions. Furthermore, these cells expressed both α-2,3- and α-2,6-linked sialic acids with α-2,3 linkage being more abundant. IDV strains replicated to high titers in these cells, while influenza A and B viruses exhibited moderate to low titers, with influenza C virus replication not detected. These findings suggest that bovine primary airway epithelial cells can be utilized to model infection biology and pathophysiology of IDV and other respiratory pathogens.