Feruloylated Oligosaccharides Prevented Influenza-induced Lung Inflammation via RIG-I/MAVS/TRAF3 Pathway (preprint)

Background: and Purpose Uncontrollable inflammation has been the leading cause of mortality in many acute respiratory infections, including influenza. In light of the current COVID-19 pandemic, it is vital to develop valid therapeutics or supplements that are effective in suppressing lung inflammation. Feruloylated oligosaccharides (FOs) is a phytochemical constituent that exerts anti-inflammatory activities. Experimental Approach We established the influenza-induced lung inflammation model by infecting C57BL/6J mice or MAVS knockout (Mavstm1Zjc/J) mice with influenza A virus (H1N1). Lung index, histology analysis, hemagglutination inhibition assay as well as neuraminidase inhibition assay were performed to evaluate the therapeutic effect of FA and FOs. PCR, western blot, docking simulation and metabolomics were done to elucidate the mechanism of FOs anti-inflammatory function in lung. Key Results Herein, we found that oral administration of FOs moderately inhibited H1N1 virus infection and reduced lung inflammation in influenza-infected mice by decreasing a wide spectrum of cytokines in the lungs. FOs also suppressed transduction of the RIG-I/MAVS/TRAF3 signaling pathway and lowered the expression of NF-κB. Moreover, we found that the anti-inflammatory function of FOs against influenza depends on MAVS, which is closely associated with activation of the downstream signaling cascades and the eventual production of pro-inflammatory cytokines and type I interferons. Conclusions and Implications In conclusion, we demonstrated that FOs is an effective anti-inflammatory agent for treating the lung inflammation caused by influenza. Such therapeutic effect was likely mediated by RIG-I/MAVS/TRAF signaling.

Elucidating the enhanced binding affinity of a double mutant SP-D with trimannose on the influenza A virus using molecular dynamics.

Surfactant protein D (SP-D) is an essential component of the human pulmonary surfactant system, which is crucial in the innate immune response against glycan-containing pathogens, including Influenza A viruses (IAV) and SARS-CoV-2. Previous studies have shown that wild-type (WT) SP-D can bind IAV but exhibits poor antiviral activities. However, a double mutant (DM) SP-D consisting of two point mutations (Asp325Ala and Arg343Val) inhibits IAV more potently. Presently, the structural mechanisms behind the point mutations' effects on SP-D's binding affinity with viral surface glycans are not fully understood. Here we use microsecond-scale, full-atomistic molecular dynamics (MD) simulations to understand the molecular mechanism of mutation-induced SP-D's higher antiviral activity. We find that the Asp325Ala mutation promotes a trimannose conformational change to a more stable state. Arg343Val increases the binding with trimannose by increasing the hydrogen bonding interaction with Glu333. Free energy perturbation (FEP) binding free energy calculations indicate that the Arg343Val mutation contributes more to the increase of SP-D's binding affinity with trimannose than Asp325Ala. This study provides a molecular-level exploration of how the two mutations increase SP-D binding affinity with trimannose, which is vital for further developing preventative strategies for related diseases.

A Multicentre Study on the Prevalence of Respiratory Viruses in Children with Community Acquired Pneumonia Requiring Hospitalization in the Setting of the Zero-COVID Policy in China (preprint)

Stringent nonpharmaceutical interventions (NPIs) have been implemented worldwide to combat the COVID-19 pandemic, and the circulation and seasonality of common respiratory viruses have subsequently changed. Multicentre studies and comparisons of the prevalence of respiratory viruses accounting for community-acquired pneumonia (CAP) in hospitalized children between the pre-COVID period and the period after community and school reopening in the setting of the zero-COVID policy are rare. In this study, we included 1543 children with CAP who required hospitalization from November 1st, 2020 to April 30th, 2021 (Period 1) and 629 children with the same conditions from November 1st, 2018 to April 30th, 2019 (Period 2) in our study. All respiratory samples from the included patients were screened for six respiratory viruses (respiratory syncytial virus [RSV], adenovirus [ADV], influenza A virus [Flu A], influenza B virus [Flu B], parainfluenza virus type 1 [PIV1], and parainfluenza virus type 3 [PIV3]) using a multiplex real-time PCR assay. The median ages of enrolled patients at the time of diagnosis were 1.5 years and 1.0 years for period 1 and period 2, respectively. In period 1, viral pathogens were detected in 50.3% (776/1543) of enrolled patients. The most frequently identified viral pathogen was RSV (35.9%, 554/1543), followed by PIV3 (9.6%, 148/1543), PIV1 (3.6%, 56/1543), ADV (3.4%, 52/1543), Flu A (1.0%, 16/1543) and Flu B (0.8%, 13/1543). The total detection rates of these six viruses in the peak season of CAP were at the pre-COVID level. The prevalence of Flu A decreased dramatically and circulation activity was low compared to pre-COVID levels, while the incidence of PIV3 increased significantly. There were no significant differences in the detection rates of RSV, ADV, Flu B and PIV1 between the two periods. Our results showed that respiratory viruses accounted for CAP in hospitalized children at pre-COVID levels as communities and schools reopened within the zero-COVID policy, although the prevalence aetiology spectrum varied.

Proteome reveals antiviral host response and NETosis during acute COVID-19 in high-risk patients (preprint)

SARS-CoV-2 remains an acute threat to human health, endangering hospital capacities worldwide. Many studies have aimed at informing pathophysiologic understanding and identification of disease indicators for risk assessment, monitoring, and therapeutic guidance. While findings start to emerge in the general population, observations in high-risk patients with complex pre-existing conditions are limited. To this end, we biomedically characterized quantitative proteomics in a hospitalized cohort of COVID-19 patients with mild to severe symptoms suffering from different (co)-morbidities in comparison to both healthy individuals and patients with non-COVID related inflammation. Deep clinical phenotyping enabled the identification of individual disease trajectories in COVID-19 patients. By the use of this specific disease phase assignment, proteome analysis revealed a severity dependent general type-2 centered host response side-by-side with a disease specific antiviral immune reaction in early disease. The identification of phenomena such as neutrophil extracellular trap (NET) formation and a pro-coagulatory response together with the regulation of proteins related to SARS-CoV-2-specific symptoms by unbiased proteome screening both confirms results from targeted approaches and provides novel information for biomarker and therapy development. Graphical AbstractSars-CoV-2 remains a challenging threat to our health care system with many pathophysiological mechanisms not fully understood, especially in high-risk patients. Therefore, we characterized a cohort of hospitalized COVID-19 patients with multiple comorbidities by quantitative plasma proteomics and deep clinical phenotyping. The individual patients disease progression was determined and the subsequently assigned proteome profiles compared with a healthy and a chronically inflamed control cohort. The identified disease phase and severity specific protein profiles revealed an antiviral immune response together with coagulation activation indicating the formation of NETosis side-by-side with tissue remodeling related to the inflammatory signature. O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/22271106v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@bab525org.highwire.dtl.DTLVardef@1cac7e7org.highwire.dtl.DTLVardef@a3ab1org.highwire.dtl.DTLVardef@19375fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

An acceptable methodological evaluation method of real-time fluorescent RT-PCR targeting SARS-CoV-2 (preprint)

Background: it is necessary to evaluate real-time fluorescent reverse-transcription PCR (RT-PCR) methods to detect the nucleic acids of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: considering lack of positive specimens in some particular locations in China, the specimens from healthy individuals were used to perform the methodology evaluations, in which the indexes were the differences of quantification cycle values (ΔCq) between human-derived internal reference control (IRC) genes of a specimen and quality control gene (QC). A series of experiments was conducted to evaluate various factors that might affect the results, such as types of virus transport media, methods of specimen pretreatment and template preparation, specimen vortex strength, specimen storage temperature, and duration. Results: it was better to store specimens in normal saline (NS) transport medium, release more virus particles from swabs by vortex mixing, extract nucleic acids with centrifugation methods, and perform amplification assays timely. The above-mentioned options and optimum conditions were further confirmed using SARS-CoV-2 pseudoviruses and positive clinical specimens. Conclusions: this study provides a solution for the accurate detection of SARS-CoV-2. Specifically, this study also indicates that the routine specimens from healthy individuals could be used to methodological evaluation of real-time fluorescent RT-PCR targeting SARS-CoV-2, of which the indexes were the ΔCq values.

Gastrointestinal symptoms and fecal nucleic acid testing of children with 2019 coronavirus disease: a systematic review and meta-analysis (preprint)

In order to understand the clinical manifestations and incidence of gastrointestinal symptoms of coronavirus disease (COVID-19) in children and discuss the importance of fecal nucleic acid testing.We retrospectively analyzed studies on gastrointestinal symptoms and fecal nucleic acid detection in pediatric COVID-19 patients from January 1, 2020 to August 10, 2020, including prospective clinical studies and case reports. The results of fecal nucleic acid detection were analyzed systematically. Stata12.0 software was used for meta-analysis.The results showed that the most common gastrointestinal symptoms in children with COVID-19 were vomiting and diarrhea, with a total incidence of 17.7% (95% Cl: 13.9%-21.5%). However, the prevalence of gastrointestinal symptoms in other countries (21.1%, 95% CI: 16.5%-25.7%) was higher compared to China (12.9%, 95% CI: 8%-17.7%). In Wuhan, the pooled prevalence was much higher (41.3%, 95 % CI: 3.2%-79.4%) compared to areas outside Wuhan in China (7.1%, 95 % CI: 4.0%-10.3%).The positive rate of fecal nucleic acid testing in COVID-19 children was relatively high at 85.8% (91/106). Additionally, 71.2% (52/73) were still positive for fecal nucleic acid after respiratory tract specimens turned negative. One and two weeks after the respiratory tract specimens turned nucleic acid-negative, 45.2% (33/73) and 34.2% (25/73) patients, respectively, remained fecal nucleic acid-positive. The longest interval between the respiratory tract specimens turning negative and fecal specimens turning negative exceeded 70 days.Conclusions and Relevance:Gastrointestinal symptoms in pediatric COVID-19 are relatively common. Attention should be paid to the detection of fecal nucleic acids in children. Fecal nucleic acid-negative status should be considered as one of the desegregation standards.

An acceptable method to evaluate the analytical performance of real-time fluorescent RT-PCR targeting SARS-CoV-2 (preprint)

It was necessary to carry out methodologies evaluations of real-time fluorescent reverse-transcription PCR (RT-PCR) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Considering biosafety issues and lack of positive specimens in some special locations in China, the routine specimens from healthy individuals were used to perform methodologies evaluations, in which the indexes were the differences of quantification cycle values ({Delta}Cq) between human derived internal reference control (IRC) genes of a specimen and quality control (QC). Serial experiments were carried out to evaluate various factors that might affect aforementioned methodologies, such as types of virus transport mediums, methods of specimen pretreatment and template preparation, specimen vortex strength, specimen storage temperature and duration. The results showed that using {Delta}Cq values as indexes, among various factors that might affect analytical performance, it was better to store specimens in the normal saline transport mediums, inactivate pathogens using water or metal bath, release more virus particles from swabs by vortex mixing, extract nucleic acids with centrifuge methods, and perform amplification assays timely. Aforementioned opinions and optimum conditions were further confirmed by SRAS-CoV-2 pseudovirus and clinical positive specimens. Altogether, the results of this study indicated that the routine specimens from healthy individuals could be used to evaluate the analytical performance of real-time fluorescent RT-PCR targeting SRAS-CoV-2, of which the indexes were the {Delta}Cq values between IRC genes of a specimen and QC. This acceptable method was extremely valuable in both theoretical and practical significance under current pandemic of coronavirus disease 2019 (COVID-19).