Bronchiolitis before and after the SARS-CoV-2 pandemic: Twelve years of experience in a Spanish paediatric hospital.

INTRODUCTION: Acute bronchiolitis is the main cause of hospitalization in children under 2 years of age, with a regular seasonality, mostly due to the respiratory syncytial virus. OBJECTIVES: To describe the epidemiology of bronchiolitis hospitalizations in our center in the last 12 years, and analyze the changes in clinical characteristics, microbiology, and adverse outcomes during the SARS-CoV-2 pandemic. METHODS: Observational study including patients admitted for bronchiolitis between April 2010 and December 2021 in a Spanish tertiary paediatric hospital. Relevant demographic, clinical, microbiological, and adverse outcome variables were collected in an anonymized database. The pandemic period (April 2020 to December 2021) was compared to 2010-2015 seasons using appropriate statistical tests. RESULTS: There were 2138 bronchiolitis admissions, with a mean of 195.6 per year between 2010 and 2019 and a 2-4-month peak between November and March. In the expected season of 2020, there was a 94.4% reduction of bronchiolitis hospitalizations, with only 11 cases admitted in the first year of the pandemic. Bronchiolitis cases increased from the summer of 2021 during a 6-month long peak, reaching a total of 171 cases. Length of stay was significantly shorter during the pandemic, but no differences were found in clinical and microbiological characteristics or other adverse outcomes. CONCLUSIONS: The SARS-CoV-2 pandemic has modified the seasonality of bronchiolitis hospitalizations, with a dramatic decrease in cases during the expected season of 2020-2021, and an extemporaneous summer-autumn peak in 2021 with longer duration but similar patient characteristics and risk factors.

Discovery and intranasal administration of a SARS-CoV-2 broadly acting neutralizing antibody with activity against multiple Omicron subvariants.

BACKGROUND: The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. METHODS: Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. FINDINGS: STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. CONCLUSIONS: With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials. FUNDING: Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).

Differences in saliva ACE2 activity among infected and non-infected adult and pediatric population exposed to SARS-CoV-2.

BACKGROUND: Variations in the ACE2 activity in saliva could explain the striking differences of susceptibility to infection and risk of severe disease. METHODS: We analyze the activity of ACE2 in saliva in different population groups across a wide age range and disease status during April to June 2020, before SARS-CoV-2 vaccine implementation, and we establish differences between infected people and participants considered resistant (highly exposed healthcare workers and children who cohabited with parents with COVID-19 without isolation and remain IgG negative). RESULTS: We included 74 adults, of which 47 (64%) were susceptible and 27 (36%) were resistant, and 79 children, of which 41 (52%) were susceptible and 38 (48%) were resistant. Resistant adults have significantly lower ACE2 activity in saliva than susceptible adults and non-significant higher values than susceptible and resistant children. ACE2 activity is similar in the susceptible and resistant pediatric population (p = 0.527). In contrast, we observe an increase in activity as the disease's severity increases among the adult population (mild disease vs. severe disease, 39 vs. 105 FU, p = 0.039; severe disease vs. resistant, 105 vs. 31 FU, p < 0.001). CONCLUSIONS: using an enzymatic test, we show that ACE2 activity in saliva correlates with the susceptibility to SARS-Cov-2 infection and disease severity. Children and adults with low-susceptibility to SARS-Cov-2 infection showed the lowest ACE2 activity. These findings could inform future strategies to identify at-risk individuals.

Metabolic Snapshot of Plasma Samples Reveals New Pathways Implicated in SARS-CoV-2 Pathogenesis.

Despite the scientific and human efforts to understand COVID-19, there are questions still unanswered. Variations in the metabolic reaction to SARS-CoV-2 infection could explain the striking differences in the susceptibility to infection and the risk of severe disease. Here, we used untargeted metabolomics to examine novel metabolic pathways related to SARS-CoV-2 susceptibility and COVID-19 clinical severity using capillary electrophoresis coupled to a time-of-flight mass spectrometer (CE-TOF-MS) in plasma samples. We included 27 patients with confirmed COVID-19 and 29 healthcare workers heavily exposed to SARS-CoV-2 but with low susceptibility to infection ("nonsusceptible"). We found a total of 42 metabolites of SARS-CoV-2 susceptibility or COVID-19 clinical severity. We report the discovery of new plasma biomarkers for COVID-19 that provide mechanistic explanations for the clinical consequences of SARS-CoV-2, including mitochondrial and liver dysfunction as a consequence of hypoxemia (citrulline, citric acid, and 3-aminoisobutyric acid (BAIBA)), energy production and amino acid catabolism (phenylalanine and histidine), and endothelial dysfunction and thrombosis (citrulline, asymmetric dimethylarginine (ADMA), and 2-aminobutyric acid (2-AB)), and we found interconnections between these pathways. In summary, in this first report several metabolic pathways implicated in SARS-CoV-2 susceptibility and COVID-19 clinical progression were found by CE-MS based metabolomics that could be developed as biomarkers of COVID-19.

Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus.

Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium-revealed by the changing composition of the mutant spectrum-may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed.

Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture.

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir's antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir.

COVID-19: Famotidine, Histamine, Mast Cells, and Mechanisms.

SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. SARS-CoV-2 infection is necessary but not sufficient for development of clinical COVID-19 disease. Currently, there are no approved pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We have investigated several plausible hypotheses for famotidine activity including antiviral and host-mediated mechanisms of action. We propose that the principal mechanism of action of famotidine for relieving COVID-19 symptoms involves on-target histamine receptor H2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release. Based on these findings and associated hypothesis, new COVID-19 multi-drug treatment strategies based on repurposing well-characterized drugs are being developed and clinically tested, and many of these drugs are available worldwide in inexpensive generic oral forms suitable for both outpatient and inpatient treatment of COVID-19 disease.

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.