Identification of Host Proteins Interacting with IBV S1 Based on Tracheal Organ Culture.

Infectious bronchitis virus (IBV) belongs to the gamma-coronavirus genus of Coronaviridae and causes serious infectious diseases in the poultry industry. However, only a few IBV strains can infect avian passage cell lines, seriously hindering the progress of basic research on IBV pathogenesis. Whereas IBV field strains can replicate in tracheal ring organ culture (TOC) without any previous adaptation in chicken embryos or primary cells. In this study, to investigate the potential use of TOC as an in vitro infection model for the study of IBV-host interaction, we first established a chicken embryo TOC culture system and carried out an investigation on the IBV replication kinetics in the system. We found that the selected strains of the IBV GI-1, GI-7, GI-13, GI-19, and GI-22 genotypes could successfully replicate in TOC and bring about damage to the infected trachea. Next, we identified host proteins of the chicken embryo trachea that interact with the IBV S1 protein by immunoprecipitation and protein mass spectrometry. A total of 127 candidate proteins were initially identified with major involvement in cell adhesion pathways and apoptosis- and autophagy-related pathways. The heat shock protein 70 (HSP70) was selected for further investigation in the interaction with IBV viral proteins. Our results showed that HSP70 interacted with IBV S1 in both TOC and CEK cells, whereas HSP70 overexpression inhibited viral replication. This study indicates that TOC is a good system for the elucidation of IBV-host interactions and HSP70 is a potential host antiviral factor.

Bronchoscopy in the post-acute phase of COVID-19: an observational study.

BACKGROUND: Bronchoscopy is a useful technique adopted in the management of patients with COVID-19. 10-40% of COVID-19 survivors experience persistent symptoms. A comprehensive description of the utility and safety of bronchoscopy in the management of patients with COVID-19 sequelae is lacking. The aim of the study was to evaluate the role of bronchoscopy in patients with suspected post-acute sequelae of COVID-19. METHODS: An observational, retrospective study was carried out in Italy. Patients requiring bronchoscopy for suspected COVID-19 sequelae were enrolled. RESULTS: 45 (21, 46.7%, female) patients were recruited. Bronchoscopy was more frequently indicated for patients with a previous critical disease. The most frequent indications were tracheal complications, mostly performed in patients who were hospitalized during the acute phase than treated at home (14, 48.3% VS. 1, 6.3%; p-value: 0.007) and persistent parenchymal infiltrates, more frequent in those treated at home (9, 56.3% VS. 5, 17.2%; p-value: 0.008). 3 (6.6%) patients after the first bronchoscopy required higher oxygen flow. Four patients were diagnosed with lung cancer. CONCLUSION: Bronchoscopy is a useful and safe technique in patients with suspected post-acute sequelae of COVID-19. The severity of acute disease plays a role in the rate and indications of bronchoscopy. Endoscopic procedures were mostly performed for tracheal complications in critical, hospitalized patients and for persistent lung parenchymal infiltrates in mild-moderate infections treated at home.

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.

A rabbit model of tracheal collapse for optimal self-expanding metal stents.

Investigating the characteristics of tracheas can help the understanding of diseases related to the trachea, particularly tracheal collapse (TC) in dogs. This study aimed to compare the mechanical properties of tracheas from New Zealand White (NZW) rabbits and dogs and to introduce a method for inducing a model of TC in the normal trachea. Tracheal samples were obtained from NZW rabbit cadavers (n=5) weighing 3.62-3.92 kg and from dog cadavers (n=5) weighing 2.97-3.28 kg. Three live NZW rabbits weighing 3.5-4.0 kg were used to establish the model. The radial forces of both sample sets were measured using a digital force gauge and statistically compared. Subsequently, TC was surgically induced in three female NZW rabbits by physically weakening their tracheal cartilage under general anesthesia. Their clinical signs were monitored for 3 months, and radiographic examinations were performed monthly for 3 months. The mean radial forces of the two sample sets were comparable (P>0.05). The clinical signs, radiographic examinations, and macroscopic examinations were all comparable to those of dogs with TC. The cadaveric study between the rabbits and dogs demonstrated that the surgically induced rabbit model of TC is an excellent candidate for the experimental study of dogs with TC. This study also provides a reference of tracheal radial force values to enable selection of appropriate mesh types and wire diameters of self-expanding metal stents.

Multiplex RT-qPCR Application in Early Detection of Bovine Respiratory Disease in Healthy Calves.

Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.

Evaluation of the Trachea and Bronchi in COVID-19 Patients Using the 3-dimensional Reconstruction Method.

OBJECTIVE: To evaluate changes in the trachea and bronchi of COVID-19 patients using the 3-dimensional reconstruction images obtained from chest CT (computed tomography) scans. STUDY DESIGN: An observational study. Place and Duration of the Study: Departments of Anatomy and Radiology, Faculty of Medicine, Lokman Hekim University, Ankara, Turkey, between March 2021 and January 2022. METHODOLOGY: There were 150 COVID-19 patients in the acute period and 150 individuals as the control group. The CT images were transferred to Mimics software, and a 3-dimensional reconstruction was performed. COVID-19 patients were grouped separately by gender, and their total lung severity score was classified as absent (Grade 0), mild (Grade 1), moderate (Grade 2), and severe (Grade 3). RESULTS: The cross-sectional area and diameter of the right upper lobar bronchus decreased as the grade increased (p<0.05 and p<0.001, respectively). The circumference of the right upper lobar bronchus and the cross-sectional area and circumference of the left lower lobar bronchus were found to be narrower in Grade 1-2-3 COVID-19 patients compared to those of the control group (p<0.01, p<0.05, and p<0.05, respectively). The cross-sectional area, circumference, and diameter of the middle lobar bronchus were found to be narrower in Grade 3 COVID-19 patients (p<0.05, p<0.05, and p<0.05, respectively). CONCLUSION: Although mostly independent of the grade increase, narrowing of the trachea and bronchi was observed in COVID-19 patients in the acute period. Further research is required with to reveal whether the narrowings are permanent. KEY WORDS: COVID-19, Trachea, Bronchus, 3-dimensional reconstruction.

High positive rate after consecutive negative tests of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a global public health event since 2019. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays of upper respiratory tract specimens were used as the most common method for confirmation of SARS-CoV-2. Patients diagnosed with coronavirus disease 2019 and hospitalized in Cancer Center of Wuhan Union Hospital were retrospectively enrolled. Epidemiological, clinical, and laboratory records were analyzed with highlights on the pattern of results of repeated RT-PCR tests. Nine hundred eighty-four patients admitted to hospital between February 13, 2020 to March 10, 2020 were enrolled. The median age was 62.0 years (interquartile range 49.0-68.0) and 44.5% was male. Three thousand-three hundred eleven specimens were collected for RT-PCR tests with a median of 3 tests (interquartile range 2.0-4.0) per patient. Three hundred sixty-two (36.8%) patients showed positive records from repeated RT-PCR tests. For the 362 confirmed patients, 147 cases received further RT-PCR tests after 2 consecutive negative records of SARS-CoV-2 and 38 (26%) of them obtained a positive result. Ten (23%) of 43 patients showed positive results after 3 consecutive negative tests and 4 (24%) of 17 patients were positive after 4 negative tests. Consecutive negative RT-PCR tests with respiratory specimens could not guarantee a viral clearance.

[Treatment of post-intensive care tracheal stenosis after previous covid-19 pneumonia]. / Lechenie postreanimatsionnykh rubtsovykh stenozov trakhei posle perenesennoi koronavirusnoi pnevmonii Covid-19.

OBJECTIVE: To analyze postoperative outcomes and perioperative management of patients with post-intensive care tracheal stenosis and previous COVID-19 pneumonia. MATERIAL AND METHODS: There were 8 patients with post-intensive care tracheal stenosis and previous COVID-19 pneumonia aged 34-61 years between January 2021 and April 2021. Lung damage CT-3 was observed in 2 (25%) patients, CT-4 - in 5 (62.5%) patients. In one case, COVID-19 pneumonia with lung damage CT-2 joined to acute cerebrovascular accident. Post-tracheostomy stenosis was detected in 7 (87.5%) cases, post-intubation stenosis - in 1 patient. Duration of invasive mechanical ventilation ranged from 5 to 130 days. In 75% of cases, tracheal stenosis was localized in the larynx and cervical trachea. Two patients admitted with tracheostomy. In one case, an extended tracheal stenosis was combined with atresia of infraglottic part of the larynx. One patient had tracheal stenosis combined with tracheoesophageal fistula (TEF). Length of tracheal stenosis was 15-45 mm. Tracheomalacia was observed in 4 (50%) patients. All patients had severe concomitant diseases. RESULTS: To restore airway patency, we used circular tracheal resection with anastomosis, laryngotracheoplasty and endoscopic methods. Tracheal resection combined with TEF required circular tracheal resection with disconnection of fistula. Adequate breathing through the natural airways was restored in all patients. There was no postoperative mortality. Three patients with baseline tracheal stenosis had favorable postoperative outcomes after circular tracheal resection. Four patients are at the final stage of treatment after laryngotracheoplasty and tracheal stenting. CONCLUSION: Patients after invasive mechanical ventilation for COVID-19 pneumonia are at high risk of cicatricial tracheal stenosis and require follow-up. Circular tracheal resection ensures early rehabilitation and favorable functional results. Laryngotracheoplasty is preferred if circular tracheal resection is impossible. This procedure ensures adequate debridement of tracheobronchial tree and respiratory support. Endoscopic measures are an alternative for open surgery, especially for intrathoracic tracheal stenosis and intractable tracheobronchitis.

[Treatment of cicatricial tracheal stenosis and tracheoesophageal fistula in patients with COVID-19 pneumonia]. / Lechenie rubtsovogo stenoza trakhei i trakheopishchevodnogo svishcha u bol'nykh, perenesshikh COVID-19-pnevmoniyu.

OBJECTIVE: To describe treatment of cicatricial tracheal stenosis and tracheoesophageal fistula in patients with COVID-19 pneumonia. MATERIAL AND METHODS: There were 91 patients with cicatricial tracheal stenosis for the period from August 2020 to April 2022 (21 months). Of these, 32 (35.2%) patients had cicatricial tracheal stenosis, tracheoesophageal fistula and previous coronavirus infection with severe acute respiratory syndrome. Incidence of iatrogenic tracheal injury following ventilation for viral pneumonia in the pandemic increased by 5 times compared to pneumonia of other genesis. Majority of patients had pneumonia CT grade 4 (12 patients) and grade 3 (8 patients). Other ones had pulmonary parenchyma lesion grade 2-3 or mixed viral-bacterial pneumonia. Isolated tracheoesophageal fistula without severe cicatricial stenosis of trachea or esophagus was diagnosed in 4 patients. In other 2 patients, tracheal stenosis was combined with tracheoesophageal fistula. Eight (25%) patients had tracheostomy at the first admission. This rate was almost half that of patients treated for cicatricial tracheal stenosis in pre-pandemic period. RESULTS: Respiratory distress syndrome occurred in 1-7 months after discharge from COVID hospital. All patients underwent surgery. In 7 patients, we preferred palliative treatment with dilation and stenting until complete rehabilitation. In 5 patients, stent was removed after 6-9 months and these ones underwent surgery. There were 3 tracheal resections with anastomosis, and 2 patients underwent tracheoplasty. Resection was performed in 3 patients due to impossible stenting. Postoperative course in these patients was standard and did not differ from that in patients without viral pneumonia. In case of tracheoesophageal fistula, palliative interventions rarely allowed isolation of trachea. Four patients underwent surgery through cervical approach. There were difficult surgeries in 2 patients with tracheoesophageal fistula and cicatricial tracheal stenosis. One of them underwent separation of fistula and tracheal resection via cervical approach at primary admission. In another patient with thoracic fistula, we initially attempted to insert occluder. However, open surgery was required later due to dislocation of device. CONCLUSION: Absolute number of patients with tracheal stenosis, tracheoesophageal fistula and previous COVID-19 has increased by several times compared to pre-pandemic period. This is due to greater number of patients requiring ventilation with risk of tracheal injury, non-compliance with preventive protocol for tracheal injury including anti-ischemic measures during mechanical ventilation. The last fact was exacerbated by involvement of allied physicians with insufficient experience of safe ventilation in the «red zone¼, immunodeficiency in these patients aggravating purulent-inflammatory process in tracheal wall. The number of patients with tracheostomy was 2 times less that was associated with peculiarity of mechanical ventilation in SARS-CoV-2. Indeed, tracheostomy was a poor prognostic sign and physicians tried to avoid this procedure. Incidence of tracheoesophageal fistula in these patients increased by 2 times compared to pre-pandemic period. In subacute period of COVID-associated pneumonia, palliative measures for cicatricial tracheal stenosis and tracheoesophageal fistula should be preferred. Radical treatment should be performed after 3-6 months. Absolute indication for circular tracheal resection with anastomosis is impossible tracheal stenting and ensuring safe breathing by endoscopic methods, as well as combination of cicatricial tracheal stenosis with tracheoesophageal fistula and resistant aspiration syndrome. Incidence of postoperative complications in patients with cicatricial tracheal stenosis and previous mechanical ventilation for COVID-19 pneumonia and patients in pre-pandemic period is similar.

Alpha and Omicron SARS-CoV-2 Adaptation in an Upper Respiratory Tract Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing an unprecedented pandemic. Although vaccines and antivirals are limiting the spread, SARS-CoV-2 is still under selective pressure in human and animal populations, as demonstrated by the emergence of variants of concern. To better understand the driving forces leading to new subtypes of SARS-CoV-2, we infected an ex vivo cell model of the human upper respiratory tract with Alpha and Omicron BA.1 variants for one month. Although viral RNA was detected during the entire course of the infection, infectious virus production decreased over time. Sequencing analysis did not show any adaptation in the spike protein, suggesting a key role for the adaptive immune response or adaptation to other anatomical sites for the evolution of SARS-CoV-2.

Characterisation of the Upper Respiratory Tract Virome of Feedlot Cattle and Its Association with Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is a major health problem within the global cattle industry. This disease has a complex aetiology, with viruses playing an integral role. In this study, metagenomics was used to sequence viral nucleic acids in the nasal swabs of BRD-affected cattle. The viruses detected included those that are well known for their association with BRD in Australia (bovine viral diarrhoea virus 1), as well as viruses known to be present but not fully characterised (bovine coronavirus) and viruses that have not been reported in BRD-affected cattle in Australia (bovine rhinitis, bovine influenza D, and bovine nidovirus). The nasal swabs from a case-control study were subsequently tested for 10 viruses, and the presence of at least one virus was found to be significantly associated with BRD. Some of the more recently detected viruses had inconsistent associations with BRD. Full genome sequences for bovine coronavirus, a virus increasingly associated with BRD, and bovine nidovirus were completed. Both viruses belong to the Coronaviridae family, which are frequently associated with disease in mammals. This study has provided greater insights into the viral pathogens associated with BRD and highlighted the need for further studies to more precisely elucidate the roles viruses play in BRD.

Infectious bronchitis virus infection in chicken: viral load and immune responses in Harderian gland, choanal cleft and turbinate tissues compared to trachea.

1. The role of the Harderian gland (HG), choanal cleft (CC) and turbinate in terms of IBV M41 viral load compared to the trachea, and immune (innate, cellular and mucosal) responses were studied in 21-day-old commercial broiler chickens.2. After virulent IBV M41 challenge, the antigen concentration detected either by quantitative RT-PCR or immunohistochemistry peaked at 2-3 days post challenge (dpc) in all tissues. Significant increases of lachrymal IBV-specific IgA and IgY levels were found at 4-5 dpc.3. Gene transcription showed a significant up-regulation of TLR3, MDA5, IL-6, IFN-α and IFN-ß, where patterns and magnitude fold-change of mRNA transcription were dependent on the gene and tissue type.4. The results demonstrated active IBV M41 replication in the HG, CC and turbinate, comparable to levels of replication found in the trachea. Data on immune-related genes in head-associated tissues provide further understanding on the immunobiology of IBV and offer opportunities to identify their use as quantitative biomarkers in pathogenicity and vaccination-challenge studies.

CFD simulation of porous microsphere particles in the airways of pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Pulmonary fibrosis (PF) is a chronic progressive disease with an extremely high mortality rate and is a complication of COVID-19. Inhalable microspheres have been increasingly used in the treatment of lung diseases such as PF in recent years. Compared to the direct inhalation of drugs, a larger particle size is required to ensure the sustained release of microspheres. However, the clinical symptoms of PF may lead to the easier deposition of microspheres in the upper respiratory tract. Therefore, it is necessary to understand the effects of PF on the deposition of microspheres in the respiratory tract. METHODS: In this study, airway models with different degrees of PF in humans and mice were established, and the transport and deposition of microspheres in the airway were simulated using computational fluid dynamics. RESULTS: The simulation results showed that PF increases microsphere deposition in the upper respiratory tract and decreases bronchial deposition in both humans and mice. Porous microspheres with low density can ensure deposition in the lower respiratory tract and larger particle size. In healthy and PF humans, porous microspheres of 10 µm with densities of 700 and 400 kg/m³ were deposited most in the bronchi. Unlike in humans, microspheres larger than 4 µm are completely deposited in the upper respiratory tract of mice owing to their high inhalation velocity. For healthy and PF mice, microspheres of 6 µm with densities of and 100 kg/m³ are recommended. CONCLUSIONS: The results showed that with the exacerbation of PF, it is more difficult for microsphere particles to deposit in the subsequent airway. In addition, there were significant differences in the deposition patterns among the different species. Therefore, it is necessary to process specific microspheres from different individuals. Our study can guide the processing of microspheres and achieve differentiated drug delivery in different subjects to maximize therapeutic effects.

Interference between avian corona and influenza viruses: The role of the epithelial architecture of the chicken trachea.

Respiratory viral infections are among the major causes of disease in poultry. While viral dual infections are known to occur, viral interference in chicken airways is mechanistically hardly understood. The effects of infectious bronchitis virus (IBV) infection on tissue morphology, sialic acid (sia) expression and susceptibility of the chicken trachea for superinfection with IBV or avian influenza virus (AIV) were studied. In vivo, tracheal epithelium of chickens infected with IBV QX showed marked inflammatory cell infiltration and loss of cilia and goblet cells five days post inoculation. Plant lectin staining indicated that sialic acids redistributed from the apical membrane of the ciliated epithelium and the goblet cell cytoplasm to the basement membrane region of the epithelium. After administration of recombinant viral attachment proteins to slides of infected tissue, retained binding of AIV hemagglutinin, absence of binding of the receptor binding domain (RBD) of IBV M41 and partial reduction of IBV QX RBD were observed. Adult chicken trachea rings were used as ex vivo model to study the effects of IBV QX-induced pathological changes and receptor redistribution on secondary viral infection. AIV H9N2 infection after primary IBV infection was delayed; however, final viral loads reached similar levels as in previously uninfected trachea rings. In contrast, IBV M41 superinfection resulted in 1000-fold lower viral titers over the course of 48 h. In conclusion, epithelial changes in the chicken trachea after viral infection coincide with redistribution and likely specific downregulation of viral receptors, with the extend of subsequent viral interference dependent on viral species.

A defined road to tracheal reconstruction: laser structuring and cell support for rapid clinic translation.

One of the severe complications occurring because of the patient's intubation is tracheal stenosis. Its incidence has significantly risen because of the COVID-19 pandemic and tends only to increase. Here, we propose an alternative to the donor trachea and synthetic prostheses-the tracheal equivalent. To form it, we applied the donor trachea samples, which were decellularized, cross-linked, and treated with laser to make wells on their surface, and inoculated them with human gingiva-derived mesenchymal stromal cells. The fabricated construct was assessed in vivo using nude (immunodeficient), immunosuppressed, and normal mice and rabbits. In comparison with the matrix ones, the tracheal equivalent samples demonstrated the thinning of the capsule, the significant vessel ingrowth into surrounding tissues, and the increase in the submucosa resorption. The developed construct was shown to be highly biocompatible and efficient in trachea restoration. These results can facilitate its clinical translation and be a base to design clinical trials.

COVID-19 symptoms are reduced by targeted hydration of the nose, larynx and trachea.

Dehydration of the upper airways increases risks of respiratory diseases from COVID-19 to asthma and COPD. We find in human volunteer studies involving 464 human subjects in Germany, the US, and India that respiratory droplet generation increases by up to 4 orders of magnitude in dehydration-associated states of advanced age (n = 357), elevated BMI-age (n = 148), strenuous exercise (n = 20) and SARS-CoV-2 infection (n = 87), and falls with hydration of the nose, larynx and trachea by calcium-rich hypertonic salts. We also find in a protocol of exercise-induced airway dehydration that hydration of the airways by calcium-rich salts increases oxygenation relative to a non-treatment control (P < 0.05). In a random control study of COVID-19 positive subjects (n = 40), thrice-a-day delivery of the calcium-rich hypertonic salts (active) suppressed respiratory droplet generation by 51% ± 11% and increased oxygen saturation over three days of treatment by 48.08% ± 9.61% (P < 0.001), while no changes were observed in the nasal-saline control group. Self-reported symptoms significantly declined in the active group and did not decline in the control group. Hydration of the upper airways appears promising as a non-drug approach for reducing risks of respiratory diseases such as COVID-19.

Comparative trachea transcriptome analysis in SPF broiler chickens infected with avian infectious bronchitis and avian influenza viruses.

Infectious bronchitis virus (IBV) and avian influenza virus (AIV) are two major respiratory infections in chickens. The coinfection of these viruses can cause significant financial losses and severe complications in the poultry industry across the world. To examine transcriptome profile changes during the early stages of infection, differential transcriptional profiles in tracheal tissue of three infected groups (i.e., IBV, AIV, and coinfected) were compared with the control group. Specific-pathogen-free chickens were challenged with Iranian variant-2-like IBV (IS/1494), UT-Barin isolates of H9N2 (A/chicken/Mashhad/UT-Barin/2017), and IBV-AIV coinfection; then, RNA was extracted from tracheal tissue. The Illumina RNA-sequencing (RNA-seq) technique was employed to investigate changes in the Transcriptome. Up- and downregulated differentially expressed genes (DEGs) were detected in the trachea transcriptome of all groups. The Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology databases were examined to identify possible relationships between DEGs. In the experimental groups, upregulated genes were higher compared to downregulated genes. A more severe immune response was observed in the coinfected group; further, cytokine-cytokine receptor interaction, RIG-I-like receptor signaling, Toll-like receptor signaling, NOD-like receptor signaling, Janus kinase/signal transducer, and activator of transcription, and apoptotic pathways were important upregulated genes in this group. The findings of this paper may give a better understanding of transcriptome changes in the trachea during the early stages of infection with these viruses.