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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. Using a multi-OMICS approach, Haas et al identify 54 human genes and 16 host-targeting chemical compounds that regulate influenza A virus infection in lung epithelial cells, including AHNAK and COBP1 which are also essential for SARS-CoV-2 infection.

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. Here, Mac Kain and Maarifi et al. perform a functional CRISPR/Cas9 screen to identify SARS-CoV-2 restriction factors in A549 cells. They identify DAXX, a scaffold protein of nuclear bodies with diverse functions, that has anti-viral activity post SARS-CoV-2 entry, while SARS-CoV-2 has evolved a mechanism to counteract its action via PLpro-mediated proteasomal degradation.

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.

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. 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. 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). 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

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. 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.

Background Increased renal phosphate loss can be Fibroblast Growth Factor-23 (FGF-23) dependent or independent. FGF-23 reduces expression of Sodium-Phosphate co-transporters, responsible for phosphate reabsorption; and by inhibition of 1-alpha hydroxylation of 25-OH-D3. Reduced serum concentration of 1, 25-OH2-D3 results in secondary hyperparathyroidism. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mutation is one of the rarest causes of hypophosphatemia, which causes FGF-23 dependent hypophosphatemia. Case description 31-year-old female referred to endocrinology for evaluation of recurrent nephrolithiasis despite normalization of PTH and serum Calcium (Ca) after right inferior parathyroid adenoma resection. Repeated renal stone analysis revealed calcium phosphate as the major stone composition. Patient achieved mid parental height, denied history of calcium or phosphate metabolism disorders/ rickets in the family. Physical exam was not suggestive of rickets and skeletal survey showed normal bone mineralization. Initial labs following parathyroid adenoma resection showed normal Ca 9.7 mg/dl, PTH 47 pg/ml, Phosphorus (Ph) 2.3 mg/dl, 25-OH-D3 51 ng/ml, albumin 3.9 g/dl, Mg 1.8 mg/dl. Repeat fasting labs six months later revealed Ca 9 mg/dl, Ph 1.7 mg/dl, 25-OH-D3 52 ng/ml, PTH 67.5 pg/ml and albumin 3.7 g/dl. Persistent low serum phosphate was evaluated further by a 24-hour urine collection which revealed high urine phosphate of 828 mg/day, Ca 181 mg/day, Mg 23 mg/day and creatinine was 1485 mg/day. Tubular reabsorption of phosphorus was 77% (normal: >80%), indicating renal phosphate wasting. Serum FGF-23 on two different days was >180 RU/ml. Next-generation sequencing tested positive for heterozygous ENPP-1 gene mutation variant c1441C>T (p. Arg481Trp). Patient was started on oral Phosphorus supplementation & calcitriol. Repeat labs showed normalization of Ph 2.4 mg/dl. Audiology evaluation suggested mild sensorineural hearing loss. Heart calcium score was zero. Discussion ENPP1 gene mutation's clinical presentations can vary from no symptoms, hypophosphatemic rickets to death in the first six months of life from generalized arterial calcification. ENPP1 is an enzyme, which degrades ATP into adenosine monophosphate (AMP) and pyrophosphate (PPi). PPI is an inhibitor of hydroxyapatite crystallization in vascular tissue and organs, while Ph is a pro-mineralization factor. ENPP1 gene mutation results in a low concentration of PPi, predisposing the patient to pathological calcification. It is speculated that ENPP-1 modulates FGF-23 secretion. FGF-23 elevation in these patients is a protective response, as hypophosphatemia prevents pathological calcification. Treatment target is to keep phosphate at low end of normal. In our patient, ENPP- 1 mutation likely contributed to recurrent renal stones and possible parathyroid adenoma. Evaluation of genetic mutations is necessary to decide for or against FGF-23 antibody/Burosumab treatment. In ENPP-1 mutation. Burosumab should be avoided as it can bind to FGF-23 resulting in pathological calcification. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

BackgroundAlthough NSAIDs and COX2 inhibitors show clinical promise, their toxicities limit their cancer therapeutic use. PGE2 is a metabolite from the COX2 pathway, well known to mediate immunosuppressive functions through EP2 and EP4 receptors and downstream cAMP signaling in immune cells, which suppresses their anti-tumoral activities. Similarly, PGD2, a metabolite of HPGDS, known to bind DP1, has recently been proposed to play pro-tumoral functions. OKN4395 is a novel small molecule, highly selective and potent against EP2, EP4, and DP1, intended to block the pro-tumor activities of PGE2 and PGD2, while avoiding other prostanoid receptors to reduce side effects.MethodsOKN4395’s efficacy and selectivity were rigorously characterized in a series of vitro and vivo preclinical assays. Given that the biology of DP1 is not fully understood, we confirmed using primary immune cell that its signaling was similar to the one of EP2/EP4 and further evaluated the redundancy of PGD2 with PGE2 in vitro. In addition, combination potential with anti-PD1 was evaluated.ResultsA comprehensive selectivity screen confirmed that OKN4395 selectively inhibits EP2, EP4, and DP1 receptors, with no off-target effects on other prostanoid receptors. In vitro, OKN4395 exhibited robust potency in counteracting the immunosuppressive effects of PGE2 and PGD2 in human T cells and NK cells. Our data demonstrated that PGE2 and PGD2 can fully compensate for each other’s effects; notably, the blockade of EP2, EP4, and DP1 receptors was able to effectively restore NK and CD8+ T cell anti-tumor functions when both PGE2 and PGD2 were present at high concentrations. In a mixed lymphocyte reaction assay, we showed that the well-characterized activity of anti-PD1 on IFN-γ secretion was fully blocked by prostaglandins and could only be rescued by the activity of OKN4395. Furthermore, the potent anti-tumor effect of OKN4395 in combination with anti-PD1 was demonstrated using an in vitro killing assay. In vivo, OKN4395 significantly reduced tumor growth as a monotherapy and demonstrated instances of complete tumor regression when combined with anti-PD1. Finally, pharmacokinetic and tolerability profiles from non-clinical studies supported further clinical development.ConclusionsOur studies demonstrate that triple inhibition of EP2, EP4, and DP1 with OKN4395 is more effective than dual EP2/EP4 inhibition in restoring anti-tumor immune responses in presence of both PGE2 and PGD2. These compelling results strongly endorse the clinical development of OKN4395, currently in a Phase 1 trial (NCT06789172), as a novel immunotherapy for solid tumors, both as a standalone treatment and in combination with anti-PD1.Ethics ApprovalAll human derived material used in the study were obtained under ethics and regulatory approvals.

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

Bluetongue is a major disease of ruminants and is caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges. BTV infects essentially all domestic and wild ruminants, however, the clinical outcome of infection differs substantially between host species. Clinical disease induced by BTV, including haemorrhagic fever in severe cases, is normally evident only in sheep, which are therefore susceptible hosts. Conversely, cattle are more resilient to BTV infection, as they rarely develop clinical signs despite showing high levels of viraemia and being considered potential reservoirs of the virus. The variability in the clinical outcome of BTV infection is determined by both virus and host factors. However, the exact mechanisms involved in bluetongue pathogenesis, in particular the differences between host species is not yet fully understood. In this study, we aimed to investigate whether susceptibility or resilience to BTV could correlate with features of viral replication and cellular antiviral responses that could be assessed in vitro. We concentrated on studying virus-host cell interactions using primary cells derived from sheep and cattle, the two animal species exhibiting a different clinical outcome to BTV infection. We established that BTV reaches higher titres in ovine cells compared to cells derived from cattle. This phenotype was consistent using two types of primary cells (endothelial cells and fibroblasts) and two different BTV serotypes. An interferon (IFN) production assay validated that antiviral cytokines were produced in all cell types. Importantly, the species-specific differences in the replication kinetics of BTV were abolished by inhibiting the JAK/STAT signalling pathway. In addition, pre-treatment with type-I IFN severely hampered BTV replication in bovine, but not in ovine primary cells. These data suggest that bovine IFN-stimulated genes (ISGs), unlike their ovine orthologues, are effective in controlling BTV replication and could be important host restriction factors. Using a high-throughput flow cytometry approach, we subsequently screened a library of over 1200 genes, including 300 bovine ISGs, to identify proteins with antiviral properties against BTV. We successfully identified a subset of bovine ISGs such as IFIT1, RSAD2 and OAS2 which negatively impacted BTV replication. The restricting potential of the sheep orthologues to the bovine ISGs of interest was also investigated using similar over-expression assays. This approach has identified species-specific differences, with the ovine genes showing either similar or decreased levels of restriction compared to their bovine counterparts. Finally, RNA sequencing was performed in ovine and bovine primary cells to fully characterise and compare the host anti-viral responses upon BTV infection. We specifically observed that genes responding to IFN were more highly up- (or down)-regulated in bovine compared to ovine cells. We also demonstrated the expression of inflammatory mediators and highlighted the up-regulation of genes associated with an activation of the endothelium. Our study offers novel insights on how bovine cells might control BTV replication and provides an intellectual framework to understand how virus-host interactions may influence host resilience and susceptibility to virus infections.