In Vitro Virucidal Effects of Ultraviolet Light Prototypes on RNA viruses.

Ultraviolet-C (UVC) has been used to cause virus inactivation. The virucidal activity of three UV light lamps [UVC high frequencies (HF), UVC+B LED and UVC+A LED] was evaluated against the enveloped feline coronavirus (FCoVII), a surrogate model of SARS-CoV-2, the enveloped vesicular stomatitis virus (VSV), and the naked encephalomyocarditis virus (EMCV). Virucidal assays were performed at different time points of UV-light exposure (i.e., 5, 30 minutes and 1, 6, and 8 hours), placing each virus 180 cm below the perpendicular irradiation of the lamp and 1 and 2 meters from the perpendicular axis. We found that the UVC HF lamp had virucidal effects (≥96.8% of virus inactivation) against FCoVII, VSV and EMCV after 5 minutes of irradiation at each distance analyzed. Moreover, the UVC+B LED lamp had the highest inhibitory effects on FCoVII and VSV infectivity (≥99% of virus inactivation) when these viruses were settled below the perpendicular axis of the lamp for 5 minutes. Conversely, the UVC+A LED lamp was the least effective, achieving ≥85.9% inactivation of enveloped RNA viruses after 8 hours of UV exposure. Overall, UV light lamps, and in particular UVC HF and UVC+B LED ones, had a rapid and strong virucidal activity against distinct RNA viruses, including coronaviruses.

The non-functional ACE2 isoform, but not the SARS-CoV-2 receptor, is induced as an interferon-stimulated gene, in SARS-CoV-2 infected adults.

The recently discovered truncated, non-functional, ACE2 transcript (dACE2), but not the full-length ACE2 (f-lACE2), is induced by IFNs in differentiated airway cells. We measured expression of both ACE2 isoforms in SARS-CoV-2 positive and negative subjects, in relation to Interferon-stimulated genes. A significant activation of dACE2 transcript was found, in SARS-CoV-2 positive adults either hospitalized or not, showing a positive correlation with ISG15; f-lACE2 expression was weakly activated and not ISG-related. We confirmed a specific activation of dACE2 transcript in nasopharyngeal cells, related to the mucosal IFN response.

Potential Use of Tea Tree Oil as a Disinfectant Agent against Coronaviruses: A Combined Experimental and Simulation Study.

The COVID-19 pandemic has highlighted the relevance of proper disinfection procedures and renewed interest in developing novel disinfectant materials as a preventive strategy to limit SARS-CoV-2 contamination. Given its widely known antibacterial, antifungal, and antiviral properties, Melaleuca alternifolia essential oil, also named Tea tree oil (TTO), is recognized as a potential effective and safe natural disinfectant agent. In particular, the proposed antiviral activity of TTO involves the inhibition of viral entry and fusion, interfering with the structural dynamics of the membrane and with the protein envelope components. In this study, for the first time, we demonstrated the virucidal effects of TTO against the feline coronavirus (FCoVII) and the human coronavirus OC43 (HCoV-OC43), both used as surrogate models for SARS-CoV-2. Then, to atomistically uncover the possible effects exerted by TTO compounds on the outer surface of the SARS-CoV-2 virion, we performed Gaussian accelerated Molecular Dynamics simulations of a SARS-CoV-2 envelope portion, including a complete model of the Spike glycoprotein in the absence or presence of the three main TTO compounds (terpinen-4-ol, γ-terpinene, and 1,8-cineole). The obtained results allowed us to hypothesize the mechanism of action of TTO and its possible use as an anti-coronavirus disinfectant agent.

High frequency of neutralizing antibodies to type I Interferon in HIV-1 patients hospitalized for COVID-19.

The presence of anti-IFN neutralizing antibodies (NAB) has been reported in critically ill COVID-19 patients. We found that 87.5% (7/8) of HIV-1 patients co-infected with SARS-CoV-2 had serum anti-IFN-I NAB against IFN-α subtypes, IFN-ß and/or IFN-ω. Anti-IFN-I NAB were also detected in oropharyngeal samples. Patients with NAB were males, and those with high serum anti-IFN-α/ω NAB titer had severe illness and exhibited reduction in the expression of IFN-stimulated genes. Thus, high titer of anti-IFN-α/ω NAB may contribute to the greater severity of COVID-19 in HIV-1 infected patients.

High prevalence of Merkel cell polyomavirus is associated with dysregulation in transcript levels of TLR9 and type I IFNs in a large cohort of CF patients from the Italian (Lazio) reference center for cystic fibrosis.

Merkel cell polyomavirus (MCPyV) has been detected in respiratory specimens including those from Cystic Fibrosis (CF) patients, raising questions about its immunological and clinical relevance in the respiratory tract. MCPyV might promote an inappropriate antiviral response contributing to a chronic inflammatory response and resulting in detrimental effects in CF. Respiratory samples (n = 1138) were randomly collected from respiratory tract of CF patients (n = 539) during July 2018-October 2019. MCPyV-DNA detection was performed by real time PCR and positive samples were characterized by sequencing of the NCCR genomic region. The transcript levels of Toll-like receptor 9 (TLR9) and type I interferon (IFN-I) genes (IFNα, IFNß and IFNε) were examined by real-time RT-PCR assays. MCPyV-DNA was detected in 268 out of 1138 respiratory specimens (23.5%) without any difference in the prevalence of MCPyV-DNA according to age, gender or bacteriological status of CF individuals. Thirteen out of 137 CF patients remained positive for MCPyV-DNA over the time (a median follow-up period of 8.8 months). Detection of MCPyV-DNA in respiratory specimens was not associated with the occurrence of exacerbation events. Both MCPyV positive adolescents (11-24 years) and adults (≥25 years) had lower mRNA levels of TLR9, IFNß, IFNε and IFNα than the negative patients of the same age group, while MCPyV positive children produced increased levels of TLR9 and IFN-I genes (p < 0.05 for TLR9, IFNß, IFNε) with respect to the negative ones. There were significant differences in TLR9 levels (p < 0.01), but not in those of IFNs, between MCPyV-DNA positive and negative patients with S. aureus, P. aeruginosa or both. Overall, these results indicate that MCPyV-DNA is frequently detected in the respiratory samples of CF patients and might influence the expression levels of IFN-related genes in an age dependent manner. The concomitant detection of MCPyV together with S. aureus and/or P. aeruginosa correlated with alterations in TLR9 levels suggesting that virus-bacteria coinfections might contribute to affect antiviral immunity in CF patients.

Anti-IFN-α/-ω neutralizing antibodies from COVID-19 patients correlate with downregulation of IFN response and laboratory biomarkers of disease severity.

A significant number of COVID-19 patients were shown to have neutralizing antibodies (NAB) against IFN; however, NAB specificity, fluctuation over time, associations with biochemical and hematological parameters, and IFN gene expression are not well characterized. Binding antibodies (BAB) to IFN-α/-ß were screened in COVID-19 patients' serum. All BAB positive sera, and a subset of respiratory samples, were tested for NAB against IFN-α/-ß/-ω, using an antiviral bioassay. Transcript levels of IFN-α/-ß/-ω and IFN-stimulated genes (ISGs) were quantified. Anti-IFN-I BAB were found in 61 out of 360 (17%) of patients. Among BAB positive sera, 21.3% had a high NAB titer against IFN-α. A total of 69.2% of anti-IFN-α NAB sera displayed cross-reactivity to IFN-ω. Anti-IFN-I NAB persisted in all patients. NAB to IFN-α were also detected in 3 out of 17 (17.6%) of respiratory samples. Anti-IFN-I NAB were higher in males (p = 0.0017), patients admitted to the ICU (p < 0.0001), and patients with a fatal outcome (p < 0.0001). NAB were associated with higher levels of CRP, LDH, d-Dimer, and higher counts of hematological parameters. ISG-mRNAs were reduced in patients with persistently NAB titer. NAB are detected in a significant proportion of severe COVID-19. NAB positive patients presented a defective IFN response and increased levels of laboratory biomarkers of disease severity.

Analysis of serum microRNAs and rs2910164 GC single-nucleotide polymorphism of miRNA-146a in COVID-19 patients.

Alteration of micro-RNAs (miRNAs) expression, including miRNA-122a, -146a and -205 family members, can have profound effects on inflammatory and IFN pathways (miRNA-146a), known as hallmarks of COVID-19. SARS-CoV-2-infected patients were recruited at Policlinico Umberto I Hospital of Sapienza University of Rome (Italy). MiRNA-122a, -146a, -205 and IFI27 (Interferon Alpha Inducible Protein 27) levels were screened in SARS-CoV-2 patients (n = 14) and healthy controls (n = 10) by real-time RT-PCR assays. Then, miRNA-146a rs2910164 GC single-nucleotide polymorphism (SNP) was genotyped in a larger group of COVID-19 patients (n = 129), and its relationship with severe disease [Intensive Care Unit (ICU) support or survival/death] was assessed. SARS-CoV-2-positive patients had increased PCR, D-Dimer and Fibrinogen levels compared to healthy controls (p < .05 for all measurements). MiRNA-122a and -146a serum levels were upregulated in COVID-19 patients (miRNA-122a: p = .002; miRNA-146a: p < .001). Decreased IFI27 levels were observed in COVID-19 patients with higher miRNA-146a levels (p = .047). Moreover, miRNA-146a rs2910164 C/G genotypes distributions were similar in COVID-19 patients and in validated European healthy subjects (n = 37,214). MiRNA-146a SNP was not associated with severe COVID-19 outcome (ICU or death). MiRNA-122a and -146a levels were elevated in SARS-CoV-2 infected patients, with miRNA-146a upregulation possibly contributing to IFN pathways dysregulation (e.g., reduced IFI27 levels) observed in severe COVID-19, although there is no evidence for the involvement of rs2910164 SNP.

Distribution of Interferon Lambda 4 Single Nucleotide Polymorphism rs11322783 Genotypes in Patients with COVID-19.

Type III interferons (IFN-III), also known as IFN-Lambda, have a pivotal role during SARS-CoV-2 infection. IFN-Lambda response among individuals is heterogeneous and its association with COVID-19 symptoms severity needs to be further clarified. We analyzed the genotype frequencies of IFNL4 single nucleotide polymorphism (SNP) rs11322783 in patients with COVID-19 (n = 128), in comparison with a validated data set of European healthy controls (n = 14152). The IFNL4 SNP was also analyzed according to the haematological and clinical parameters of patients with COVID-19. The distributions of IFNL4 genotypes among SARS-CoV-2 positive patients [TT/TT 41.4% (n = 53), TT/ΔG 47.7% (n = 61) and ΔG/ΔG 10.9% (n = 14)] and healthy controls were comparable. Different levels of white blood cells (p = 0.036) and neutrophils (p = 0.042) were found in the IFNL4 different genotypes in patients with COVID-19; the ΔG/ΔG genotype was more represented in the groups with low white blood cells and neutrophils. There were no differences in major inflammation parameters (C-reactive protein, D-dimer, Albumin, and Lactate-dehydrogenase (LDH)] and survival rate according to the IFNL4 genotypes. In conclusion, although patients with COVID-19 did not exhibit a different distribution of the IFNL4 SNP, the ΔG/ΔG genotype was associated with a lower count of immune cell populations. These findings need to be confirmed in larger groups of patients with COVID-19 and the role of IFNL4 SNP needs to be also investigated in other respiratory viral infections.

Potential IFNγ Modulation of Inflammasome Pathway in Chlamydia trachomatis Infected Synovial Cells.

Following a Chlamydia trachomatis infection, the host immune response is characterized by its recognition via Toll-like and Nod-like Receptors, and the subsequent activation of interferon (IFN)-γ-mediated signaling pathways. Recently, the inflammasome-mediated host cell response has emerged to play a role in the physiopathology of C. trachomatis infection. Here we investigated, for the first time, the interaction of IFN-γ and inflammasome in an in vitro model of C. trachomatis-infected primary human synovial cells. Chlamydial replication as well as the expression of caspase-1, IL-1ß, as well as IL-18 and IL-6, were assayed. Our results demonstrated the inhibitory activity of IFN-γ by interfering with the inflammasome network through the downregulation of caspase-1 mRNA expression. In addition, the ability of C. trachomatis to hinder the inflammasome pathway favoring its intracellular survival within synovial cells, was observed. Overall, our data suggest a potential mechanism of immune evasion by C. trachomatis in synovial cells, that may be contested by IFN-γ.

KI and WU Polyomavirus in Respiratory Samples of SARS-CoV-2 Infected Patients.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been declared a global pandemic. Our goal was to determine whether co-infections with respiratory polyomaviruses, such as Karolinska Institutet polyomavirus (KIPyV) and Washington University polyomavirus (WUPyV) occur in SARS-CoV-2 infected patients. Oropharyngeal swabs from 150 individuals, 112 symptomatic COVID-19 patients and 38 healthcare workers not infected by SARS-CoV-2, were collected from March 2020 through May 2020 and tested for KIPyV and WUPyV DNA presence. Of the 112 SARS-CoV-2 positive patients, 27 (24.1%) were co-infected with KIPyV, 5 (4.5%) were positive for WUPyV, and 3 (2.7%) were infected simultaneously by KIPyV and WUPyV. Neither KIPyV nor WUPyV DNA was detected in samples of healthcare workers. Significant correlations were found in patients co-infected with SARS-CoV-2 and KIPyV (p < 0.05) and between SARS-CoV-2 cycle threshold values and KIPyV, WUPyV and KIPyV and WUPyV concurrently detected (p < 0.05). These results suggest that KIPyV and WUPyV may behave as opportunistic respiratory pathogens. Additional investigations are needed to understand the epidemiology and the prevalence of respiratory polyomavirus in COVID-19 patients and whether KIPyV and WUPyV could potentially drive viral interference or influence disease outcomes by upregulating SARS-CoV-2 replicative potential.

Alteration of type I interferon response is associated with subclinical atherosclerosis in virologically suppressed HIV-1-infected male patients.

Given human immunodeficiency virus-1 (HIV-1)-infected patients have alterations in the type I interferon (IFN-I) pathway and are also at elevated risk of atherosclerosis, we evaluated IFN-I response and subclinical cardiovascular disease (CVD) association in HIV-1-infected patients. Transcript levels of IFN-α/ß and IFN-stimulated gene 56 (ISG56) were evaluated by RT/real-time PCR in peripheral blood mononuclear cells collected from asymptomatic HIV-1-positive male patients at high risk of developing CVD (n = 34) and healthy subjects (n = 21). Stenosis degree (≥ or <50%), calcium volume score, calcium Agatston score, and myocardial extracellular volume were examined by coronary computerized tomography scan. Carotid intima-media thickness (cIMT), Framingham risk score, atherosclerotic cardiovascular disease (ASCVD) score, and risk score developed by data collection on adverse effects of anti-HIV drugs (D:A:D) were also measured. Increased IFN-α, IFN-ß, and ISG56 levels were observed in all HIV-1-infected males compared to healthy controls (p < .001 for all genes analyzed). HIV-1-infected patients with a stenosis degree ≥50% showed a higher Framingham risk score (p = .019), which was correlated with IFN-ß and ISG56 levels. HIV-1-infected males with enhanced IFN-I levels and stenosis displayed a higher ASCVD calculated risk (p = .011) and D:A:D score (p = .004). Also, there was a trend toward higher IFN-α and ISG56 mRNA levels in HIV-1-positive patients with an increased cIMT (p > .05). Dysregulation of IFN-I response might participate in the pathogenesis of HIV-1-associated CVD.

ACE2 expression is related to the interferon response in airway epithelial cells but is that functional for SARS-CoV-2 entry?

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS-CoV-2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs' activation, ISG15, in children (n = 59) and adults (n = 48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 expression, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs' activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS-CoV-2 infection in the respiratory tract.

SARS-CoV-2 Entry Genes Expression in Relation with Interferon Response in Cystic Fibrosis Patients.

The expression rate of SARS-CoV-2 entry genes, angiotensin-converting enzyme 2 (ACE2), the main viral receptor and the proteases, furin and transmembrane serine protease 2 (TMPRSS2) in cystic fibrosis (CF) individuals is poorly known. Hence, we examined their levels in upper respiratory samples of CF patients (n = 46) and healthy controls (n = 45). Moreover, we sought to understand the interplay of type I interferon (IFN-I) with ACE2, furin and TMPRSS2 by evaluating their gene expression with respect to ISG15, a well-known marker of IFN activation, in upper respiratory samples and after ex vivo IFNß exposure. Lower ACE2 levels and trends toward the reduction of furin and TMPRSS2 were found in CF patients compared with the healthy controls; decreased ACE2 amounts were also detected in CF individuals with pancreatic insufficiency and in those receiving inhaled antibiotics. Moreover, there was a strong positive correlation between ISG15 and ACE2 levels. However, after ex vivo IFNß stimulation of nasopharyngeal cells, the truncated isoform (dACE2), recently demonstrated as the IFN stimulated one with respect to the full-length isoform (flACE2), slightly augmented in cells from CF patients whereas in those from healthy donors, dACE2 levels showed variable levels of upregulation. An altered expression of SARS-COV-2 entry genes and a poor responsiveness of dACE2 to IFN-I stimulation might be crucial in the diffusion of SARS-CoV-2 infection in CF.

Differential induction of type I and III interferon genes in the upper respiratory tract of patients with coronavirus disease 2019 (COVID-19).

The natural course of type I and III interferon (IFN) response in the respiratory tract of COVID-19 patients needs to be better defined. We showed that type I/III IFNs, IFN-regulatory factor 7 (IRF7), and IFN stimulated genes (ISGs), are highly expressed in the oropharyngeal cells of SARS-CoV-2 positive patients compared to healthy controls. Notably, the subgroup of critically-ill patients that required invasive mechanical ventilation had a general decrease in expression of IFN/ISG genes. Heterogeneous patterns of IFN-I/III response in the respiratory tract of COVID-19 patients may be associated to COVID-19 severity.

High abundance of genus Prevotella is associated with dysregulation of IFN-I and T cell response in HIV-1-infected patients.

OBJECTIVE: HIV-1-associated dysbiosis is most commonly characterized by overall decreased diversity, with abundance of the genus Prevotella, recently related to inflammatory responses. DESIGN: A pilot study including 10 antiretroviral therapy-treated HIV-1-infected men and 50 uninfected controls was performed to identify the main gut dysbiosis determinants (e.g. Prevotella enrichment), that may affect mucosal antiviral defenses and T cell immunity in HIV-1-infected individuals. METHODS: 16rRNA gene sequencing was applied to the HIV-1-infected individuals' fecal microbiota and compared with controls. Measurements of CD4 and CD8 T cell activation [CD38, human leukocyte antigen (HLA)-DR, CD38 HLA-DR] and frequencies of Th17, obtained from lamina propria lymphocytes isolated from five different intestinal sites, were performed by flow cytometry. IFNß, IFNAR1 and MxA gene expression level was evaluated by real-time PCR in lamina propria lymphocytes. Nonparametric t tests were used for statistical analysis. RESULTS: HIV-1-infected men had a significant fecal microbial communities' imbalance, including different levels of genera Faecalibacterium, Prevotella, Alistipes and Bacteroides, compared with controls. Notably, Prevotella abundance positively correlated with frequencies of CD4 T cells expressing CD38 or HLA-DR and coexpressing CD38 and HLA-DR (P < 0.05 for all these measures). The same trend was observed for the activated CD8 T cells. Moreover, Prevotella levels were inversely correlated with IFN-I genes (P < 0.05 for IFNß, IFNAR1 and MxA genes) and the frequencies of Th17 cells (P < 0.05). By contrast, no statistically significant correlations were observed for the remaining bacterial genera. CONCLUSION: Our findings suggest that Prevotella enrichment might affect gut mucosal IFN-I pathways and T cell response in HIV-1-infected patients, thus contributing to immune dysfunction.