Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets.

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen.

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

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.

Alcoholic versus aqueous chlorhexidine for skin antisepsis: the AVALANCHE trial.

BACKGROUND: Preoperative skin antisepsis is routine practice. We compared alcoholic chlorhexidine with aqueous chlorhexidine for skin antisepsis to prevent surgical site infection after minor skin excisions in general practice. METHODS: We conducted this prospective, multicentre, randomized controlled trial in 4 private general practices in North Queensland, Australia, from October 2015 to August 2016. Consecutive adult patients presenting for minor skin excisions were randomly assigned to undergo preoperative skin antisepsis with 0.5% chlorhexidine in 70% ethanol (intervention) or 0.5% chlorhexidine aqueous solution (control). Our primary outcome was surgical site infection within 30 days of excision. We also measured the incidence of adverse reactions. RESULTS: A total of 916 patients were included in the study: 454 underwent antisepsis with alcoholic chlorhexidine and 462 with aqueous chlorhexidine. Of these, 909 completed follow-up. In the intention-to-treat analysis of cases available at follow-up, there was no significant difference in the incidence of surgical site infection between the alcoholic chlorhexidine arm (5.8%, 95% confidence interval [CI] 3.6% to 7.9%) and the aqueous chlorhexidine arm (6.8%, 95% CI 4.5% to 9.1%). The attributable risk reduction was 0.010 (95% CI -0.021 to 0.042), the relative risk was 0.85 (95% CI 0.51 to 1.41), and the number needed to treat to benefit was 100. Per protocol and sensitivity analyses produced similar results. The incidence of adverse reactions was low, with no difference between groups (p = 0.6). INTERPRETATION: There was no significant difference in efficacy between alcoholic and aqueous chlorhexidine for the prevention of surgical site infection after minor skin excisions in general practice. Trial registration: https://www.anzctr.org.au, no. ACTRN12615001045505.

Characterization of a second open reading frame in genome segment 10 of bluetongue virus.

Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50-59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.