A novel method to titrate Herpes simplex virus-1 (HSV-1) using laser-based scanning of near-infrared fluorophores conjugated antibodies

Among several strategies used for Herpes simplex virus (HSV) detection in biological specimens, standard plaque assay (SPA) remains the most reliable method to evaluate virus infectivity and quantify viral replication. However, it is a manual procedure, thereby affected by operator subjectivity, and it may be particularly laborious for multiple sample analysis. Here we describe an innovative method to perform the titration of HSV type 1 (HSV-1) in different samples, using the “In-Cell WesternTM” Assay (ICW) from LI-COR, a quantitative immunofluorescence assay that exploits laser-based scanning of near infrared (NIR). In particular, we employed NIR-immunodetection of viral proteins to monitor foci of HSV-1 infection in cell monolayers, and exploited an automated detection of their fluorescence intensity to evaluate virus titre. This innovative method produced similar and superimposable values compared to SPA, but it is faster and can be performed in 96 well plate, thus allowing to easily and quickly analyze and quantify many samples in parallel. These features make our method particularly suitable for the screening and characterization of antiviral compounds, as we demonstrated by testing acyclovir (ACV), the main anti-HSV-1 drug. Moreover, we developed a new data analysis system that allowed to overcome potential bias due to unspecific florescence signals, thus improving data reproducibility. Overall, our method may represents a useful tool for both clinical and research purposes

Antiviral and antioxidant activity of a hydroalcoholic extract from Humulus lupulus L.

A hydroalcoholic extract from female inflorescences of Humulus lupulus L. (HOP extract) was evaluated for its anti-influenza activity. The ability of the extract to interfere with different phases of viral replication was assessed, as well as its effect on the intracellular redox state, being unbalanced versus the oxidative state in infected cells. The radical scavenging power, inhibition of lipoperoxidation, and ferric reducing activity were assayed as antioxidant mechanisms. A phytochemical characterization of the extract was also performed. We found that HOP extract significantly inhibited replication of various viral strains, at different time from infection. Viral replication was partly inhibited when virus was incubated with extract before infection, suggesting a direct effect on the virions. Since HOP extract was able to restore the reducing conditions of infected cells, by increasing glutathione content, its antiviral activity might be also due to an interference with redox-sensitive pathways required for viral replication. Accordingly, the extract exerted radical scavenging and reducing effects and inhibited lipoperoxidation and the tBOOH-induced cytotoxicity. At phytochemical analysis, different phenolics were identified, which altogether might contribute to HOP antiviral effect. In conclusion, our results highlighted anti-influenza and antioxidant properties of HOP extract, which encourage further in vivo studies to evaluate its possible application

COVID-19 as a Potential Cause of Muscle Injuries in Professional Italian Serie A Soccer Players: A Retrospective Observational Study

the COVID-19 pandemic has shocked the entire planet. the soccer world has also suffered major upheavals, and many professional soccer players have been infected with the virus. The aim of this study was to evaluate the incidence of injuries in Italian Serie A professional soccer players before and during the COVID-19 pandemic. methods: we evaluated the incidence of muscle injuries between four competitive seasons of the Italian Serie A (2016-2017, 2017-2018, and 2018-2019 pre-COVID-19 vs. 2020/2021 post-COVID-19) in professional soccer players. results: significant differences were found in muscular injuries between the post-COVID-19 season and the previous seasons (p < 0.001). The median split of the players' positivity duration was of 15 days. The players' long positivity (PLP) group showed a significant number of muscular injuries compared to the players' short positivity (PSP) group (p < 0.0014, ES = 0.81, large). the total teams' days of positivity were significantly related to the total team number of muscular injuries (r = 0.86; CI 95% 0.66 to 0.94; p < 0.0001). In conclusion, this data showed that the competitive season post-COVID-19 lockdown has a higher incidence of muscle injuries in Italian serie a soccer players compared to the pre-pandemic competitive season

Sex Differences in the Response to Viral Infections: TLR8 and TLR9 Ligand Stimulation Induce Higher IL10 Production in Males

BACKGROUND: Susceptibility to viral infections as well as their severity are higher in men than in women. Heightened antiviral responses typical of women are effective for rapid virus clearance, but if excessively high or prolonged, can result in chronic/inflammatory pathologies. We investigated whether this variability could be in part attributable to differences in the response to the Toll-Like Receptors (TLR) more involved in the virus recognition. METHODS: Cytokine production by peripheral blood mononuclear cells (PBMCs) from male and female healthy donors after stimulation with Toll-like receptors (TLR) 3, 7, 8, 9 ligands or with viruses (influenza and Herpes-simplex-1) was evaluated. RESULTS: Compared to females, PBMCs from males produced not only lower amounts of IFN-α in response to TLR7 ligands but also higher amounts of the immunosuppressive cytokine IL10 after stimulation with TLR8 and TLR9 ligands or viruses. IL10 production after TLR9 ligands or HSV-1 stimulation was significantly related with plasma levels of sex hormones in both groups, whereas no correlation was found in cytokines produced following TLR7 and TLR8 stimulation. CONCLUSIONS: Given the role of an early production of IL10 by cells of innate immunity in modulating innate and adaptive immune response to viruses, we suggest that sex-related difference in its production following viral nucleic acid stimulation of TLRs may be involved in the sex-related variability in response to viral infections

Role of miR-9 in Modulating NF-κB Signaling and Cytokine Expression in COVID-19 Patients

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a significant impact on global health, with severe cases often characterized by a worsening cytokine storm. Since it has been described that the NF-kappa B signaling pathway, regulated by microRNAs, could play a pivotal role in the inflammatory response, in this study, the role of miR-9 in modulating NF-kappa B signaling and inflammatory cytokine expression in COVID-19 patients was investigated. This observational retrospective single-center study included 41 COVID-19 patients and 20 healthy controls. Serum samples were analyzed for miR-9, NF-kappa B, and I kappa B alpha expression levels using RT-PCR. The expression levels and production of pro-inflammatory cytokines IL-6, IL-1 beta, and TNF-alpha were measured using RT-PCR and ELISA. Statistical analyses, including correlation and regression, were conducted to explore relationships between these variables. COVID-19 patients, particularly non-survivors, exhibited significantly higher miR-9 and NF-kappa B levels compared to controls. A strong positive correlation was found between miR-9 and NF-kappa B expression (r = 0.813, p < 0.001). NF-kappa B levels were significantly correlated with IL-6 (r = 0.971, p < 0.001), IL-1 beta (r = 0.968, p < 0.001), and TNF-alpha (r = 0.968, p < 0.001). Our findings indicate that miR-9 regulates NF-kappa B signaling and inflammation in COVID-19. Elevated miR-9 levels in non-survivors suggest its potential as a severity biomarker. While COVID-19 cases have decreased, targeting miR-9 and NF-kappa B could improve outcomes for other inflammatory conditions, including autoimmune diseases, highlighting the need for continued research in this area

SARS-CoV-2. Comparative analysis of different RNA extraction methods

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection

Correlation between Chest Computed Tomography Score and Laboratory Biomarkers in the Risk Stratification of COVID-19 Patients Admitted to the Emergency Department

background: it has been reported that mid-regional proadrenomedullin (MR-proADM) could be considered a useful tool to stratify the mortality risk in COVID-19 patients upon admission to the emergency department (ED). during the COVID-19 outbreak, computed tomography (CT) scans were widely used for their excellent sensitivity in diagnosing pneumonia associated with SARS-CoV-2 infection. however, the possible role of CT score in the risk stratification of COVID-19 patients upon admission to the ED is still unclear. aim: the main objective of this study was to assess if the association of the CT findings alone or together with MR-proADM results could ameliorate the prediction of in-hospital mortality of COVID-19 patients at the triage. moreover, the hypothesis that CT score and MR-proADM levels together could play a key role in predicting the correct clinical setting for these patients was also evaluated. methods: epidemiological, demographic, clinical, laboratory, and outcome data were assessed and analyzed from 265 consecutive patients admitted to the triage of the ED with a SARS-CoV-2 infection. results and conclusions: the accuracy results by AUROC analysis and statistical analysis demonstrated that CT score is particularly effective, when utilized together with the MR-proADM level, in the risk stratification of COVID-19 patients admitted to the ED, thus helping the decision-making process of emergency physicians and optimizing the hospital resources

Influenza virus replication is affected by glutaredoxin1-mediated protein deglutathionylation

Several redox modifications have been described during viral infection, including influenza virus infection, but little is known about glutathionylation and this respiratory virus. Glutathionylation is a reversible, post-translational modification, in which protein cysteine forms transient disulfides with glutathione (GSH), catalyzed by cellular oxidoreductases and in particular by glutaredoxin (Grx). We show here that (i) influenza virus infection induces protein glutathionylation, including that of viral proteins such as hemagglutinin (HA); (ii) Grx1-mediated deglutathionylation is important for the viral life cycle, as its inhibition, either with an inhibitor of its enzymatic activity or by siRNA, decreases viral replication. Overall these data contribute to the characterization of the complex picture of redox regulation of the influenza virus replication cycle and could help to identify new targets to control respiratory viral infection

GSH-C4 Acts as Anti-inflammatory Drug in Different Models of Canonical and Cell Autonomous Inflammation Through NFκB Inhibition

An imbalance in GSH/GSSG ratio represents a triggering event in pro-inflammatory cytokine production and inflammatory response. However, the molecular mechanism(s) through which GSH regulates macrophage and cell autonomous inflammation remains not deeply understood. Here, we investigated the effects of a derivative of GSH, the N-butanoyl glutathione (GSH-C4), a cell permeable compound, on lipopolisaccharide (LPS)-stimulated murine RAW 264.7 macrophages, and human macrophages. LPS alone induces a significant production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α and a significant decrement of GSH content. Such events were significantly abrogated by treatment with GSH-C4. Moreover, GSH-C4 was highly efficient in buffering cell autonomous inflammatory status of aged C2C12 myotubes and 3T3-L1 adipocytes by suppressing the production of pro-inflammatory cytokines. We found that inflammation was paralleled by a strong induction of the phosphorylated form of NFκB, which translocates into the nucleus; a process that was also efficiently inhibited by the treatment with GSH-C4. Overall, the evidence suggests that GSH decrement is required for efficient activation of an inflammatory condition and, at the same time, GSH-C4 can be envisaged as a good candidate to abrogate such process, expanding the anti-inflammatory role of this molecule in chronic inflammatory states