Early Stages Of Infection Research Articles

Proteomic analysis of global protein acetylation in response to WSSV infection in a crustacean Cherax quadricarinatus

Protein acetylation, a crucial post-translational modification, plays a significant role in antiviral immunity of host and viral replication processes after infection. Our previous research showed that an acetyltransferase KAT2A significantly enhanced white spot syndrome virus (WSSV) replication in hematopoietic tissue (Hpt) cells from Cherax quadricarinatus, suggesting that alterations in intracellular acetylation states affect viral infection. Nevertheless, the mechanisms by which WSSV invasion regulates intracellular protein acetylation remain unexplored. To explore the connection between protein acetylation and WSSV infection, differentially acetylated proteins (DAcPs) were identified in Hpt cells during WSSV infection by proteomic analysis. The results revealed that 25 DAcPs were identified with primarily involving in transporter activity and proton ATPase activity complex during the early stage of WSSV infection, i.e., the intracellular transport of the virions. Furthermore, that 36 DAcPs associated with chromatin assembly and acetyltransferase complex were identified in the replication stage of WSSV infection, i.e., the process of extensive viral replication. Additionally, acetylation at the K27 and K36 sites of histone H3 was strongly activated during WSSV replication; similarly, the inhibition of histone acetylation by acetyltransferase inhibiter C646 significantly suppressed WSSV replication, implying that WSSV replication requires high levels of histone acetylation, while the acetylation at the K27 and K36 sites of histone H3 probably serves as critical viral regulatory targets. This study deepens the understanding of WSSV-host interaction, and lays the groundwork for future research into the relationship between protein acetylation and viral infection in crustaceans.

A Narrative Review on the Potential of Black Seeds (Nigella Sativa) in the Management of West Nile Virus Infection

Abstract: West Nile virus (WNV) infection is a mosquito-borne illness that is endemic to Africa, Asia, the Middle East, Australia, and Europe. WNV-infected individuals can only be managed with symptomatic therapy and supportive care at this point of time. Thus, we have analyzed the possible beneficial effects of black seeds (N. sativa) in this literature review. To find relevant articles, various online databases, such as the Web of Science, Medline, PMC, PubMed, Embase, EBSCO, Google Scholar, Science Direct, and reference lists were searched. The antiviral, analgesic, anti-inflammatory, antioxidant, and immunomodulatory effects of black seeds (N. sativa) have been established by numerous clinical, animal, in vivo, in vitro, and in silico studies. It has been found that black seeds (N. sativa) have the potential to be included as adjuvant therapy in addition to symptomatic treatment and supportive care in the early stages of WNV infection once randomized controlled clinical trials (RCTs) affirm the safety and efficacy of black seeds (N. sativa) in WNV infection management as they have been considered the gold standard of treatment in clinical studies.

Lithospermic acid inhibits dengue virus infection through binding with envelope proteins

The dengue virus has emerged as a global pandemic, highlighting the urgent need for the immediate development of antiviral therapeutics. Lithospermum erythrorhizon, a medicinal plant commonly used in China for various ailments including viral infections, inflammation, rheumatism, and cancer, showed promising antiviral properties in our research. Specifically, both the ethanol extract of Lithospermum erythrorhizon and its active component, lithospermic acid, demonstrated significant inhibitory effects on Dengue virus (DENV) replication in Vero cells, with EC50 values of 6.50 μg/mL(95% CI: 2.25 to 18.98)and 15.00 μM(95% CI: 12.13 to 18.07), respectively. Notably, lithospermic acid exhibited potent antiviral activity across multiple cell lines against DENV, impeding virus replication and specifically impeding the expression of viral E and NS3 proteins during the early stages of DENV infection. Experimental assays involving RNase digestion and sucrose density gradient analysis confirmed that lithospermic acid did not directly inactivate DENV but rather interfered with viral processes. Furthermore, the compound was found to bind to the E protein of DENV, effectively inhibiting viral infection and mitigating the cytopathic effects induced by DENV. Collectively, these findings underscore the potential of lithospermic acid as a promising candidate for the development of therapeutic strategies targeting DENV infection.

A virulent milRNA inhibits host immunity by silencing a host receptor-like kinase MaLYK3 and facilitates infection by Fusarium oxysporum f. sp. cubense.

MicroRNA-like RNAs (milRNAs) play a significant role in the infection process by plant-pathogenic fungi. However, the specific functions and regulatory mechanisms of fungal milRNAs remain insufficiently elucidated. This study investigated the function of Foc-milR138, an infection-induced milRNA secreted by Fusarium oxysporum f. sp. cubense (Foc), which is the causal agent of Fusarium wilt of banana. Initially, through precursor gene knockout and phenotypic assessments, we confirmed that Foc-milR138 acts as a virulent milRNA prominently upregulated during the early stages of Foc infection. Subsequent bioinformatic analyses and transient expression assays in Nicotiana benthamiana leaves identified a host receptor-like kinase gene, MaLYK3, as the direct target of Foc-milR138. Functional investigations of MaLYK3 revealed its pivotal role in triggering immune responses of N. benthamiana by upregulating a suite of resistance genes, bolstering reactive oxygen species (ROS) accumulation and callose deposition, thereby fortifying disease resistance. This response was markedly subdued upon co-expression with Foc-milR138. Expression pattern analysis further verified the specific suppression of MaLYK3 by Foc-milR138 during the early root infection by Foc. In conclusion, Foc secretes a virulent milRNA (Foc-milR138) to enter the host banana cells and inhibit the expression of the plant surface receptor-like kinase MaLYK3, subverting the disease resistance activated by MaLYK3, and ultimately facilitating pathogen invasion. These findings shed light on the roles of fungal milRNAs and their targets in resistance and pathogenicity, offering promising avenues for the development of disease-resistant banana cultivars.

Redox Status and Protein Glutathionylation in Binase-Treated HPV16-Positive SiHa Carcinoma Cells

Human papillomavirus type 16 (HPV16) belongs to viruses of the high-risk type and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus, such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of the cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the level of reactive oxygen species (ROS) and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis in HPV-16-positive SiHa cells of cervical squamous cell carcinoma,. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.e., decreases ROS and reduced glutathione (GSH) levels, suppresses the expression of the E6 oncoprotein, activates the expression of the p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the integrity of the mitochondrial membrane is a signal for activation of the mitochondrial apoptosis pathway.

Single-cell transcriptional profiling reveals cell type-specific responses to duck reovirus infection in the Bursa of Fabricius of Cairna moschata

Duck reovirus (DRV) is a universal waterfowl virus that causes significant economic losses in the duck industry. However, the role of the host innate immune response of the Bursa of Fabricius to DRV infection is largely unknown. In the present study, we constructed a single-cell resolution transcriptomic atlas of the Bursa of Fabricius of Cairna moschata after infection with HN10 (a novel DRV). Ten cell-type marker genes were used to annotate the cell type, indicating a high degree of cell heterogeneity in the Bursa of Fabricius. Most of the innate and adaptive immune system-related genes were highly expressed in T cells, B cells, neutrophils, macrophages, and DCs. In the Bursa of Fabricius, the proportions of DCs and macrophages were largely increased by HN10 infection at 14 d, suggesting that DCs and macrophages play important roles in the long-term viral response. Notably, a number of innate and adaptive immune system-related genes were highly expressed at 24 h after HN10 infection, indicating that the Bursa of Fabricius has a very strong immune function even in the early developmental stage. In the immune system, the NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway were significantly activated at the early stage of HN10 infection, while the Toll-like receptor signaling pathway was significantly activated at the late stage. Enrichment analysis suggested that different immune signaling pathways play roles in specific developmental stages. Our data provide an opportunity to reveal the immune response to DRV infection at the single-cell level.

Development of a One-Step Multiplex qPCR Assay for Detection of Methicillin and Vancomycin Drug Resistance Genes in Antibiotic-Resistant Bacteria.

The most common antibiotic-resistant bacteria in Korea are methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Pathogen identification in clinical laboratories can be divided into traditional phenotype- and genotype-based methods, both of which are complementary to each other. The genotype-based method using multiplex real-time polymerase chain reaction (PCR) is a rapid and accurate technique that analyzes material at the genetic level by targeting genes simultaneously. Accordingly, we aimed to develop a rapid method for studying the genetic characteristics of antibiotic-resistant bacteria and to provide an experimental guide for the efficient antibiotic resistance gene analysis of mecA detection for MRSA and vanA or vanB detection for VRE using a one-step multiplex qPCR assay at an early stage of infection. As a result, the sensitivity and specificity of the mecA gene for clinical S. aureus isolates, including MRSA and methicillin-susceptible S. aureus, were 97.44% (95% CI, 86.82-99.87%) and 96.15% (95% CI, 87.02-99.32%), respectively. The receiver operating characteristic area under the curve for the diagnosis of MRSA was 0.9798 (*** p < 0.0001). Therefore, the molecular diagnostic method using this newly developed one-step multiplex qPCR assay can provide accurate and rapid results for the treatment of patients with MRSA and VRE infections.

Increased viperin expression induced by avian infectious bronchitis virus inhibits viral replication by restricting cholesterol synthesis: an in vitro study

With the emergence of new variant strains resulting from high mutation rates and genome recombination, avian infectious bronchitis virus (IBV) has caused significant economic losses to the poultry industry worldwide. Little is known about the underlying mechanisms of IBV-host interactions, particularly how IBV utilizes host metabolic pathways for efficient viral replication and transmission. In the present study, the effects of the cell membrane, viral envelope membrane, and viperin-mediated cholesterol synthesis on IBV replication were explored. Our results revealed significant increase in cholesterol levels and the expression of viperin after IBV infection. Acute cholesterol depletion in the cell membrane and viral envelope membrane by treating cells with methyl-β-cyclodextrin (MβCD) obviously inhibited IBV replication; thereafter, replenishment of the cell membrane with cholesterol successfully restored viral replication, and direct addition of exogenous cholesterol to the cell membrane significantly promoted IBV infection during the early stages of infection. In addition, overexpression of viperin effectively suppressed cholesterol synthesis, as well as IBV replication, whereas knockdown of viperin (gene silencing with siRNA targeting viperin, siViperin) significantly increased IBV replication and cholesterol levels, whereas supplementation with exogenous cholesterol to viperin-transfected cells markedly restored viral replication. In conclusion, the increase in viperin induced by IBV infection plays an important role in IBV replication by affecting cholesterol production, providing a theoretical basis for understanding the pathogenesis of IBV and discovering new potential antiviral targets.

A novel BH3DNet method for identifying pine wilt disease in Masson pine fusing UAS hyperspectral imagery and LiDAR data

Pine Wilt Disease (PWD) is a forest infectious disease that inflicts substantial economic losses to China’s forestry. Its rapid spread and the significant challenges associated with its control make early detection of infected trees crucial for disaster prevention. Unmanned aerial systems (UASs) hyperspectral imaging (HSI) and light detection and ranging (LiDAR) technologies provide high-resolution spectral diagnostic information coupled with intricate three-dimensional structural data, which has potential for fine grained monitoring of PWD. However, how to fuse HSI and LiDAR data to identify the early infected individual trees is still a challenge. This study presents a novel instance segmentation network, BH3DNet, to identify individual trees at different PWD-infected stages by extracting high-level abstract features based on the fusion of drone HSI and LiDAR data. BH3DNet introduces the PointNet++ model as the base network, and incorporates a shared encoder and twin parallel decoders to align semantic category prediction and instance segmentation of individual trees in an end-to-end approach. By applying an enhanced point cloud dataset that fuses drone HSI and LiDAR point cloud data, this model facilitates the identification of PWD infection stages at the individual tree scale. We evaluated the proposed model in a Masson pine forest stand sparsely mixed with broadleaf trees in a variety of infection states ranging from healthy to severely infected by PWD, and compared the performance of the model using the RGB bands, full HSI bands and screened bands as inputs, respectively. BH3DNet achieves an overall accuracy of 89.65 % with a Kappa × 100 of 87.29 for identifying individual trees using screened HSI bands and LiDAR point cloud, significantly outperforming the Mask R-CNN using only HSI data (overall accuracy: 70.81 %, Kappa × 100: 64.16). Moreover, BH3DNet’s accuracy at the early infection stage reaches 83.75 %. It proves that fusing HSI and point cloud data reflects the information of individual trees distribution and infection status, and the BH3DNet is suitable for high-precision monitoring of PWD.

Time-course whole blood transcriptome profiling provides new insights into Microtus fortis natural resistance mechanism to Schistosoma japonicum

Microtus fortis is known as a non-susceptible animal host of S. japonicum. A better understanding of this animal immune defense mechanism during the early stage of infection may offer an alternative route for vaccine development or therapy. Here, we analyzed the whole blood transcriptome of M. fortis using next-generation sequencing (NGS) to identify immune genes of biological relevance that might be involved in the mechanism of its resistance. The blood samples were collected from uninfected animals (control group) and infected animals at different time points (3, 7, 10 and 14 days post-infection). We identified 5310 sequences as unigenes and successfully annotated 4636 of them. The immune response was more intense at 10 dpi. The upregulated genes at this time point were mainly activated in the TNF and NF-kappa B signaling pathways, Th1, Th2and Th17 cell differentiation as well as cytokine-cytokine receptor interaction. Based on the differentially expressed genes analysis, we report that the IF27L2B, RETN, PGRP, IFI35, TYROBP, S100A8, S100A11, CD162, CD88, CYBA, and LBP could play important roles in the mechanism of M. fortis resistance.

Synergistic blockade of TIGIT and PD-L1 increases type-1 inflammation and improves parasite control during murine blood-stage Plasmodium yoelii non-lethal infection.

Pro-inflammatory immune responses are rapidly suppressed during blood-stage malaria but the molecular mechanisms driving this regulation are still incompletely understood. In this study, we show that the co-inhibitory receptors TIGIT and PD-1 are upregulated and co-expressed by antigen-specific CD4+ T cells (ovalbumin-specific OT-II cells) during non-lethal Plasmodium yoelii expressing ovalbumin (PyNL-OVA) blood-stage infection. Synergistic blockade of TIGIT and PD-L1, but not individual blockade of each receptor, during the early stages of infection significantly improved parasite control during the peak stages (days 10-15) of infection. Mechanistically, this protection was correlated with significantly increased plasma levels of IFN-γ, TNF, and IL-2, and an increase in the frequencies of IFN-γ-producing antigen-specific T-bet+ CD4+ T cells (OT-II cells), but not antigen-specific CD8+ T cells (OT-I cells), along with expansion of the splenic red pulp and monocyte-derived macrophage populations. Collectively, our study identifies a novel role for TIGIT in combination with the PD1-PD-L1 axis in regulating specific components of the pro-inflammatory immune response and restricting parasite control during the acute stages of blood-stage PyNL infection.

An autophagy-related gene ATG14 promotes WSSV infection by facilitating autophagy in Cherax quadricarinatus

The activation of autophagy was found to be positively correlated with white spot syndrome virus (WSSV) entry into host cells; however, the molecular regulation between autophagic initiation and WSSV entry is still poorly known. Herein, ATG14, a major member of autophagy initiation complex class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), playing a vital role in the formation of autophagy precursors, was studied on WSSV infection in a crustacean Cherax quadricarinatus. The CqATG14 gene was cloned with 1476 bp encoding 491 amino acids, which was widely expressed in all examined tissues of crayfish with the highest expression in hemocytes. Moreover, the gene expression of CqATG14 was significantly up-regulated at 6 and 12 hpi in crayfish hematopoietic tissue (Hpt) after WSSV infection. Importantly, the internalized WSSV virions was clearly decreased at an early infection stage of 2 hpi in CqATG14-silenced Hpt cells, causing the decrease of viral replication as the expression of both WSSV genes and viral protein VP28 were significantly down-regulated at a late infection stage after CqATG14 knocking down. Furthermore, the binding of CqATG14 to CqBeclin1 was determined by co-immunoprecipitation assay; meanwhile, the presence of CqGABARAP-II protein (a marker of autophagy activation) was significantly decreased in CqATG14-silenced Hpt cells. These data suggest that CqATG14 might promote the early infection of WSSV, probably resulting from the promotion of viral endocytosis via facilitating autophagy, which benefits the further exploration of molecular mechanism around WSSV-autophagy in crustaceans.

E-nose detection of changes in volatile profile associated with early decay of ‘Golden Delicious’ apple by Penicillium expansum

The ability of an electronic nose system to differentiate alterations in the volatile organic compound (VOCs) profile caused by early spoilage of ‘Golden Delicious’ apples due to Penicillium expansum was thoroughly evaluated. Three strains of this mould species (M639, pe2280 and pe2278) were individually used to inoculate ‘Golden Delicious’ apples, resulting in four different batches: (i) M639, (ii) pe2280, (iii) pe2278 and (iv) a control group (inoculated with sterile water). Volatile compounds were identified and quantified by gas chromatography/mass spectrometry (GC/MS), and signals from an electronic nose system (E-nose) were recorded for each batch with three different growth diameters: early stage (10 mm), middle stage (20 mm) and late stage (40 mm). A strong correlation was observed between the responses from multiple E-nose biosensors and the aromatic profile associated with fungal alterations in apples. Using linear discriminant analysis (LDA) models based on E-nose sensor data from the specified batches, we successfully differentiated healthy and infected samples, specifically three distinct groups: control, M639+pe2280 and pe2278. Remarkably, the recognition rates for total external samples exceeded 87% and reached 97% for samples in the early stage of fungal infection. These results validate the online capability of E-nose technology for the non-destructive evaluation of apple storage processes.

MAPK and phenylpropanoid metabolism pathways involved in regulating the resistance of upland cotton plants to Verticillium dahliae.

Verticillium dahliae causes a serious decline in cotton yield and quality, posing a serious threat to the cotton industry. However, the mechanism of resistance to V. dahliae in cotton is still unclear, which limits the breeding of resistant cultivars. To analyze the defense mechanisms of cotton in response to V. dahliae infection, we compared the defense responses of two upland cotton cultivars from Xinjiang (JK1775, resistant; Z8,susceptible) using transcriptome sequencing at different infection stages. The results revealed a significant differential expression of genes in the two cotton cultivars post V. dahliae infection, with the number of DEGs in JK1775 being higher than that in Z8 at different infection stages of V. dahliae. Interestingly, the DEGs of both JK1775 and Z8 were enriched in the MAPK signaling pathway in the early and late stages of infection. Importantly, the upregulated DEGs in both cultivars were significantly enriched in all stages of the phenylpropanoid metabolic pathway. Some of these DEGs were involved in the regulation of lignin and coumarin biosynthesis, which may be one of the key factors contributing to the resistance of upland cotton cultivars to V. dahliae in Xinjiang. Lignin staining experiments further showed that the lignin content increased in both resistant and susceptible varieties after inoculation with V. dahliae. This study not only provides insights into the molecular mechanisms of resistance to Verticillium wilt in Xinjiang upland cotton but also offers important candidate gene resources for molecular breeding of resistance to Verticillium wilt in cotton.

Innovative biomarkers TCN2 and LY6E can significantly inhibit respiratory syncytial virus infection

BackgroundRespiratory syncytial virus (RSV) is a prominent etiological agent of lower respiratory tract infections in children, responsible for approximately 80% of cases of pediatric bronchiolitis and 50% of cases of infant pneumonia. Despite notable progress in the diagnosis and management of pediatric RSV infection, the current biomarkers for early-stage detection remain insufficient to meet clinical needs. Therefore, the development of more effective biomarkers for early-stage pediatric respiratory syncytial virus infection (EPR) is imperative.MethodsThe datasets used in this study were derived from the Gene Expression Omnibus (GEO) database. We used GSE188427 dataset as the training set to screen for biomarkers. Biomarkers of EPR were screened by Weighted Gene Co-expression Network Analysis (WGCNA), three machine-learning algorithms (LASSO regression, Random Forest, XGBoost), and other comprehensive bioinformatics analysis techniques. To evaluate the diagnostic value of these biomarkers, multiple external and internal datasets were employed as validation sets. Next, an examination was performed to investigate the relationship between the screened biomarkers and the infiltration of immune cells. Furthermore, an investigation was carried out to identify potential small molecule compounds that interact with selected diagnostic markers. Finally, we confirmed that the expression levels of the selected biomarkers exhibited a significant increase following RSV infection, and they were further identified as having antiviral properties.ResultsThe study found that lymphocyte antigen 6E (LY6E) and Transcobalamin-2 (TCN2) are two biomarkers with diagnostic significance in EPR. Analysis of immune cell infiltration showed that they were associated with activation of multiple immune cells. Furthermore, our analysis demonstrated that small molecules, 3ʹ-azido-3ʹ-deoxythymine, methotrexate, and theophylline, have the potential to bind to TCN2 and exhibit antiviral properties. These compounds may serve as promising therapeutic agents for the management of pediatric RSV infections. Additionally, our data revealed an upregulation of LY6E and TCN2 expression in PBMCs from patients with RSV infection. ROC analysis indicated that LY6E and TCN2 possessed diagnostic value for RSV infection. Finally, we confirmed that LY6E and TCN2 expression increased after RSV infection and further inhibited RSV infection in A549 and BEAS-2B cell lines. Importantly, based on TCN2, our findings revealed the antiviral properties of a potentially efficacious compound, vitamin B12.ConclusionLY6E and TCN2 are potential peripheral blood diagnostic biomarkers for pediatric RSV infection. LY6E and TCN2 inhibit RSV infection, indicating that LY6E and TCN2 are potential therapeutic target for RSV infection.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of Hirame novirhabdovirus

Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/μl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections.

Characterization of Neutrophil Functional Responses to SARS-CoV-2 Infection in a Translational Feline Model for COVID-19.

There is a complex interplay between viral infection and host innate immune response regarding disease severity and outcomes. Neutrophil hyperactivation, including excessive release of neutrophil extracellular traps (NETs), is linked to exacerbated disease in acute COVID-19, notably in hospitalized patients. Delineating protective versus detrimental neutrophil responses is essential to developing targeted COVID-19 therapies and relies on high-quality translational animal models. In this study, we utilize a previously established feline model for COVID-19 to investigate neutrophil dysfunction in which experimentally infected cats develop clinical disease that mimics acute COVID-19. Specific pathogen-free cats were inoculated with SARS-CoV-2 (B.1.617.2; Delta variant) (n = 24) or vehicle (n = 6). Plasma, bronchoalveolar lavage fluid, and lung tissues were collected at various time points over 12 days post-inoculation. Systematic and temporal evaluation of the kinetics of neutrophil activation was conducted by measuring markers of activation including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (citH3) in SARS-CoV-2-infected cats at 4 and 12 days post-inoculation (dpi) and compared to vehicle-inoculated controls. Cytokine profiling supported elevated innate inflammatory responses with specific upregulation of neutrophil activation and NET formation-related markers, namely IL-8, IL-18, CXCL1, and SDF-1, in infected cats. An increase in MPO-DNA complexes and cell-free dsDNA in infected cats compared to vehicle-inoculated was noted and supported by histopathologic severity in respiratory tissues. Immunofluorescence analyses further supported correlation of NET markers with tissue damage, especially 4 dpi. Differential gene expression analyses indicated an upregulation of genes associated with innate immune and neutrophil activation pathways. Transcripts involved in activation and NETosis pathways were upregulated by 4 dpi and downregulated by 12 dpi, suggesting peak activation of neutrophils and NET-associated markers in the early acute stages of infection. Correlation analyses conducted between NET-specific markers and clinical scores as well as histopathologic scores support association between neutrophil activation and disease severity during SARS-CoV-2 infection in this model. Overall, this study emphasizes the effect of neutrophil activation and NET release in SARS-CoV-2 infection in a feline model, prompting further investigation into therapeutic strategies aimed at mitigating excessive innate inflammatory responses in COVID-19.

Early and late responses to Fusarium Head blight in durum wheat: Focus on phenylpropanoid biosynthetic pathway

Durum wheat is among the cereal crops most susceptible to Fusarium Head Blight (FHB), a fungal disease that can lead to significant yield losses. Despite this, only limited research efforts have been directed towards understanding FHB resistance in durum wheat. Wheat grains naturally contain phenolic compounds, and anthocyanins are particularly present in the so-called pigmented wheat genotypes, such as purple pericarp ones. In this study the effects of the biotic stress caused by Fusarium graminearum infection on phenylpropanoid biosynthetic pathway in durum wheat spikes were explored, considering three genotypes with different susceptibility (including a purple pericarp genotype), and two time points (an early stage time point: 2 days post infection, and a late stage time point: 21 days post infection). At early infection stage, the F. graminearum infection triggered upregulation of all the considered genes involved in the phenylpropanoid pathway in the resistant genotype, while, in the purple pericarp genotype, the infection caused an increase in quercetin accumulation in the soluble fraction of spike extract. At late infection stage, the infection caused (in all the genotypes) a degradation of secondary cell wall and the release of the hydroxycinnamic acids esterified with arabinoxylans (ferulic acid and p-coumaric acid) and lignin-derived monomers (vanillic acid). Furthermore, chalcone synthase gene (CHS) and the transcription factor Ppm1 (Purple pericarp MYB 1) were boosted in the pigmented genotype due to infection at late infection stage. These findings contribute to the understanding of host-pathogen interactions for future breeding programs focused on improving FHB resistance in durum wheat varieties, with a particular focus on pigmented genotypes.

Coxsackievirus group B3 regulates ASS1-mediated metabolic reprogramming and promotes macrophage inflammatory polarization in viral myocarditis.

Viral myocarditis (VMC) is a common and potentially life-threatening myocardial inflammatory disease, most commonly caused by CVB3 infection. So far, the pathogenesis of VMC caused by CVB3 is mainly focused on two aspects: one is the direct myocardial injury caused by a large number of viral replication in the early stage of infection, and the other is the local immune cell infiltration and inflammatory damage of the myocardium in the adaptive immune response stage. There are few studies on the early innate immunity of CVB3 infection in myocardial tissue, but the appearance of macrophages in the early stage of CVB3 infection suggests that they can play a regulatory role as early innate immune response cells in myocardial tissue. Here, we discovered a possible new mechanism of VMC caused by CVB3, revealed new drug targets for anti-CVB3, and discovered the therapeutic potential of citrulline for VMC.

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.