African Swine Fever Virus Ubiquitin-Conjugating Enzyme Is an Immunomodulator Targeting NF-kappa B Activation

African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKβ. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme

Expression of the Ebola Virus VP24 Protein Compromises the Integrity of the Nuclear Envelope and Induces a Laminopathy- Like Cellular Phenotype

ABSTRACT Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein that inhibits interferon (IFN) gene expression and counteracts the IFN-mediated antiviral response, preventing nuclear import of signal transducer and activator of transcription 1 (STAT1). Proteomic studies to identify additional EBOV VP24 partners have pointed to the nuclear membrane component emerin as a potential element of the VP24 cellular interactome. Here, we have further studied this interaction and its impact on cell biology. We demonstrate that VP24 interacts with emerin but also with other components of the inner nuclear membrane, such as lamin A/C and lamin B. We also show that VP24 diminishes the interaction between emerin and lamin A/C and compromises the integrity of the nuclear membrane. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, expression of VP24 also promoted the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), a common interactor of lamin A/C and emerin, leading to repression of the BAF-regulated CSF1 gene. Importantly, we found that EBOV infection results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. In summary, here we demonstrate that VP24 acts at the nuclear membrane, causing morphological and functional changes in cells that recapitulate several of the hallmarks of laminopathy diseases. IMPORTANCE The Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein with multiple functions. Proteomic studies have identified the cellular nuclear membrane component emerin as a potential VP24 interactor. Here, we demonstrate that VP24 not only interacts with emerin but also with lamin A/C and lamin B, prompting nuclear membrane disruption. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, VP24 also promotes the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), leading to repression of the BAF-regulated CSF1 gene. Finally, we show that EBOV infection also results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. These results reveal novel activities of EBOV VP24 protein, resulting in a cell phenotype similar to that of most laminopathies, with potential impact on EBOV replication

Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target

Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol traffic. This protein has been shown to play an important role for several viruses. It has been reported that SARS-CoV-2 enters the cell through plasma membrane fusion and/or endosomal entry upon availability of proteases. However, the whole process is not fully understood yet and additional viral/host factors might be required for viral fusion and subsequent viral replication. Here, we report a novel interaction between the SARS-CoV-2 nucleoprotein (N) and the cholesterol transporter NPC1. Furthermore, we have found that some compounds reported to interact with NPC1, carbazole SC816 and sulfides SC198 and SC073, were able to reduce SARS-CoV-2 viral infection with a good selectivity index in human cell infection models. These findings suggest the importance of NPC1 for SARS-CoV-2 viral infection and a new possible potential therapeutic target to fight against COVID-19

Quantification of Ebola virus replication kinetics in vitro

Mathematical modelling has successfully been used to provide quantitative descriptions of many viral infections, but for the Ebola virus, which requires biosafety level 4 facilities for experimentation, modelling can play a crucial role. Ebola virus modelling efforts have primarily focused on in vivo virus kinetics, e.g., in animal models, to aid the development of antivirals and vaccines. But, thus far, these studies have not yielded a detailed specification of the infection cycle, which could provide a foundational description of the virus kinetics and thus a deeper understanding of their clinical manifestation. Here, we obtain a diverse experimental data set of the Ebola virus infection in vitro, and then make use of Bayesian inference methods to fully identify parameters in a mathematical model of the infection. Our results provide insights into the distribution of time an infected cell spends in the eclipse phase (the period between infection and the start of virus production), as well as the rate at which infectious virions lose infectivity. We suggest how these results can be used in future models to describe co-infection with defective interfering particles, which are an emerging alternative therapeutic

Deep Sequencing of RNA from Blood and Oral Swab Samples Reveals the Presence of Nucleic Acid from a Number of Pathogens in Patients with Acute Ebola Virus Disease and Is Consistent with Bacterial Translocation across the Gut.

In this study, samples from the 2013-2016 West African Ebola virus outbreak from patients in Guinea with Ebola virus disease (EVD) were analyzed to discover and classify what other pathogens were present. Throat swabs were taken from deceased EVD patients, and peripheral blood samples were analyzed that had been taken from patients when they presented at the treatment center with acute illness. High-throughput RNA sequencing (RNA-seq) and bioinformatics were used to identify the potential microorganisms. This approach confirmed Ebola virus (EBOV) in all samples from patients diagnosed as acute positive for the virus by quantitative reverse transcription-PCR in deployed field laboratories. Nucleic acid mapping to Plasmodium was also used on the patient samples, confirming results obtained with an antigen-based rapid diagnostic test (RDT) conducted in the field laboratories. The data suggested that a high Plasmodium load, as determined by sequence read depth, was associated with mortality and influenced the host response, whereas a lower parasite load did not appear to affect outcome. The identifications of selected bacteria from throat swabs via RNA-seq were confirmed by culture. The data indicated that the potential pathogens identified in the blood samples were associated with translocation from the gut, suggesting the presence of bacteremia, which transcriptome data suggested may induce or aggravate the acute-phase response observed during EVD. Transcripts mapping to different viruses were also identified, including those indicative of lytic infections. The development of high-resolution analysis of samples from patients with EVD will help inform care pathways and the most appropriate general antimicrobial therapy to be used in a resource-poor setting. IMPORTANCE Our results highlight the identification of an array of pathogens in the blood of patients with Ebola virus disease (EVD). This has not been done before, and the data have important implications for the treatment of patients with EVD, particularly considering antibiotic stewardship. We show that EVD patients who were also infected with Plasmodium, particularly at higher loads, had more adverse outcomes than patients with lower levels of Plasmodium. However, the presence of Plasmodium did not influence the innate immune response, and it is likely that the presence of EBOV dominated this response. Several viruses other than EBOV were identified, and bacteria associated with sepsis were also identified. These findings were indicative of bacterial translocation across the gut during the acute phase of EVD

Elucidating variations in the nucleotide sequence of Ebola virus associated with increasing pathogenicity

Background Ebolaviruses cause a severe and often fatal haemorrhagic fever in humans, with some species such as Ebola virus having case fatality rates approaching 90%. Currently, the worst Ebola virus outbreak since the disease was discovered is occurring in West Africa. Although thought to be a zoonotic infection, a concern is that with increasing numbers of humans being infected, Ebola virus variants could be selected which are better adapted for human-to-human transmission. Results To investigate whether genetic changes in Ebola virus become established in response to adaptation in a different host, a guinea pig model of infection was used. In this experimental system, guinea pigs were infected with Ebola virus (EBOV), which initially did not cause disease. To simulate transmission to uninfected individuals, the virus was serially passaged five times in naïve animals. As the virus was passaged, virulence increased and clinical effects were observed in the guinea pig. An RNAseq and consensus mapping approach was then used to evaluate potential nucleotide changes in the Ebola virus genome at each passage. Conclusions Upon passage in the guinea pig model, EBOV become more virulent, RNA editing and also coding changes in key proteins become established. The data suggest that the initial evolutionary trajectory of EBOV in a new host can lead to a gain in virulence. Given the circumstances of the sustained transmission of EBOV in the current outbreak in West Africa, increases in virulence may be associated with prolonged and uncontrolled epidemics of EBOV

Procalcitonin Is Not a Reliable Biomarker of Bacterial Coinfection in People With Coronavirus Disease 2019 Undergoing Microbiological Investigation at the Time of Hospital Admission

Abstract Admission procalcitonin measurements and microbiology results were available for 1040 hospitalized adults with coronavirus disease 2019 (from 48 902 included in the International Severe Acute Respiratory and Emerging Infections Consortium World Health Organization Clinical Characterisation Protocol UK study). Although procalcitonin was higher in bacterial coinfection, this was neither clinically significant (median [IQR], 0.33 [0.11–1.70] ng/mL vs 0.24 [0.10–0.90] ng/mL) nor diagnostically useful (area under the receiver operating characteristic curve, 0.56 [95% confidence interval, .51–.60]).</jats:p

Implementation of corticosteroids in treating COVID-19 in the ISARIC WHO Clinical Characterisation Protocol UK:prospective observational cohort study

BACKGROUND: Dexamethasone was the first intervention proven to reduce mortality in patients with COVID-19 being treated in hospital. We aimed to evaluate the adoption of corticosteroids in the treatment of COVID-19 in the UK after the RECOVERY trial publication on June 16, 2020, and to identify discrepancies in care. METHODS: We did an audit of clinical implementation of corticosteroids in a prospective, observational, cohort study in 237 UK acute care hospitals between March 16, 2020, and April 14, 2021, restricted to patients aged 18 years or older with proven or high likelihood of COVID-19, who received supplementary oxygen. The primary outcome was administration of dexamethasone, prednisolone, hydrocortisone, or methylprednisolone. This study is registered with ISRCTN, ISRCTN66726260. FINDINGS: Between June 17, 2020, and April 14, 2021, 47 795 (75·2%) of 63 525 of patients on supplementary oxygen received corticosteroids, higher among patients requiring critical care than in those who received ward care (11 185 [86·6%] of 12 909 vs 36 415 [72·4%] of 50 278). Patients 50 years or older were significantly less likely to receive corticosteroids than those younger than 50 years (adjusted odds ratio 0·79 [95% CI 0·70–0·89], p=0·0001, for 70–79 years; 0·52 [0·46–0·58], p80 years), independent of patient demographics and illness severity. 84 (54·2%) of 155 pregnant women received corticosteroids. Rates of corticosteroid administration increased from 27·5% in the week before June 16, 2020, to 75–80% in January, 2021. INTERPRETATION: Implementation of corticosteroids into clinical practice in the UK for patients with COVID-19 has been successful, but not universal. Patients older than 70 years, independent of illness severity, chronic neurological disease, and dementia, were less likely to receive corticosteroids than those who were younger, as were pregnant women. This could reflect appropriate clinical decision making, but the possibility of inequitable access to life-saving care should be considered. FUNDING: UK National Institute for Health Research and UK Medical Research Council