Dysregulated pulmonary inflammatory responses exacerbate the outcome of secondary aspergillosis following influenza.

IMPORTANCE: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by Aspergillus fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus, influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model of IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

Dysregulated Pulmonary Inflammatory Responses Exacerbate the Outcome of Secondary Aspergillosis Following Influenza.

Inhalation of airborne conidia of the ubiquitous fungus Aspergillus fumigatus commonly occurs but invasive aspergillosis is rare except in profoundly immunocompromised persons. Severe influenza predisposes patients to invasive pulmonary aspergillosis by mechanisms that are poorly defined. Using a post-influenza aspergillosis model, we found that superinfected mice had 100% mortality when challenged with A. fumigatus conidia on days 2 and 5 (early stages) of influenza A virus infection but 100% survival when challenged on days 8 and 14 (late stages). Influenza-infected mice superinfected with A. fumigatus had increased levels of the pro-inflammatory cytokines and chemokines IL-6, TNFα, IFNß, IL-12p70, IL-1α, IL-1ß, CXCL1, G-CSF, MIP-1α, MIP-1ß, RANTES and MCP-1. Surprisingly, on histopathological analysis, superinfected mice did not have greater lung inflammation compared with mice infected with influenza alone. Mice infected with influenza had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus , but only if the fungal challenge was executed during the early stages of influenza infection. However, influenza infection did not have a major effect on neutrophil phagocytosis and killing of A. fumigatus conidia. Moreover, minimal germination of conidia was seen on histopathology even in the superinfected mice. Taken together, our data suggest that the high mortality rate seen in mice during the early stages of influenza-associated pulmonary aspergillosis is multifactorial, with a greater contribution from dysregulated inflammation than microbial growth. Importance: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by A. fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus , influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

Abatacept, Cenicriviroc, or Infliximab for Treatment of Adults Hospitalized With COVID-19 Pneumonia: A Randomized Clinical Trial.

Importance: Immune dysregulation contributes to poorer outcomes in COVID-19. Objective: To investigate whether abatacept, cenicriviroc, or infliximab provides benefit when added to standard care for COVID-19 pneumonia. Design, Setting, and Participants: Randomized, double-masked, placebo-controlled clinical trial using a master protocol to investigate immunomodulators added to standard care for treatment of participants hospitalized with COVID-19 pneumonia. The results of 3 substudies are reported from 95 hospitals at 85 clinical research sites in the US and Latin America. Hospitalized patients 18 years or older with confirmed SARS-CoV-2 infection within 14 days and evidence of pulmonary involvement underwent randomization between October 2020 and December 2021. Interventions: Single infusion of abatacept (10 mg/kg; maximum dose, 1000 mg) or infliximab (5 mg/kg) or a 28-day oral course of cenicriviroc (300-mg loading dose followed by 150 mg twice per day). Main Outcomes and Measures: The primary outcome was time to recovery by day 28 evaluated using an 8-point ordinal scale (higher scores indicate better health). Recovery was defined as the first day the participant scored at least 6 on the ordinal scale. Results: Of the 1971 participants randomized across the 3 substudies, the mean (SD) age was 54.8 (14.6) years and 1218 (61.8%) were men. The primary end point of time to recovery from COVID-19 pneumonia was not significantly different for abatacept (recovery rate ratio [RRR], 1.12 [95% CI, 0.98-1.28]; P = .09), cenicriviroc (RRR, 1.01 [95% CI, 0.86-1.18]; P = .94), or infliximab (RRR, 1.12 [95% CI, 0.99-1.28]; P = .08) compared with placebo. All-cause 28-day mortality was 11.0% for abatacept vs 15.1% for placebo (odds ratio [OR], 0.62 [95% CI, 0.41-0.94]), 13.8% for cenicriviroc vs 11.9% for placebo (OR, 1.18 [95% CI 0.72-1.94]), and 10.1% for infliximab vs 14.5% for placebo (OR, 0.59 [95% CI, 0.39-0.90]). Safety outcomes were comparable between active treatment and placebo, including secondary infections, in all 3 substudies. Conclusions and Relevance: Time to recovery from COVID-19 pneumonia among hospitalized participants was not significantly different for abatacept, cenicriviroc, or infliximab vs placebo. Trial Registration: ClinicalTrials.gov Identifier: NCT04593940.

Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

An Early Islet Transcriptional Signature Is Associated With Local Inflammation in Autoimmune Diabetes.

Identifying the early islet cellular processes of autoimmune type 1 diabetes (T1D) in humans is challenging given the absence of symptoms during this period and the inaccessibility of the pancreas for sampling. In this article, we study temporal events in pancreatic islets in LEW.1WR1 rats, in which autoimmune diabetes can be induced with virus infection, by performing transcriptional analysis of islets harvested during the prediabetic period. Single-cell RNA-sequencing and differential expression analyses of islets from prediabetic rats reveal subsets of ß- and α-cells under stress as evidenced by heightened expression, over time, of a transcriptional signature characterized by interferon-stimulated genes, chemokines including Cxcl10, major histocompatibility class I, and genes for the ubiquitin-proteasome system. Mononuclear phagocytes show increased expression of inflammatory markers. RNA-in situ hybridization of rat pancreatic tissue defines the spatial distribution of Cxcl10+ ß- and α-cells and their association with CD8+ T cell infiltration, a hallmark of insulitis and islet destruction. Our studies define early islet transcriptional events during immune cell recruitment to islets and reveal spatial associations between stressed ß- and α-cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.

Abatacept for Treatment of Adults Hospitalized with Moderate or Severe Covid-19.

Background: We investigated whether abatacept, a selective costimulation modulator, provides additional benefit when added to standard-of-care for patients hospitalized with Covid-19. Methods: We conducted a master protocol to investigate immunomodulators for potential benefit treating patients hospitalized with Covid-19 and report results for abatacept. Intravenous abatacept (one-time dose 10 mg/kg, maximum dose 1000 mg) plus standard of care (SOC) was compared with shared placebo plus SOC. Primary outcome was time-to-recovery by day 28. Key secondary endpoints included 28-day mortality. Results: Between October 16, 2020 and December 31, 2021, a total of 1019 participants received study treatment (509 abatacept; 510 shared placebo), constituting the modified intention-to-treat cohort. Participants had a mean age 54.8 (SD 14.6) years, 60.5% were male, 44.2% Hispanic/Latino and 13.7% Black. No statistically significant difference for the primary endpoint of time-to-recovery was found with a recovery-rate-ratio of 1.14 (95% CI 1.00-1.29; p=0.057) compared with placebo. We observed a substantial improvement in 28-day all-cause mortality with abatacept versus placebo (11.0% vs. 15.1%; odds ratio [OR] 0.62 [95% CI 0.41- 0.94]), leading to 38% lower odds of dying. Improvement in mortality occurred for participants requiring oxygen/noninvasive ventilation at randomization. Subgroup analysis identified the strongest effect in those with baseline C-reactive protein >75mg/L. We found no statistically significant differences in adverse events, with safety composite index slightly favoring abatacept. Rates of secondary infections were similar (16.1% for abatacept; 14.3% for placebo). Conclusions: Addition of single-dose intravenous abatacept to standard-of-care demonstrated no statistically significant change in time-to-recovery, but improved 28-day mortality. Trial registration: ClinicalTrials.gov ( NCT04593940 ).

Quantitative structural analysis of influenza virus by cryo-electron tomography and convolutional neural networks.

Influenza viruses pose severe public health threats globally. Influenza viruses are extensively pleomorphic, in shape, size, and organization of viral proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships and aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we leveraged convolutional neural networks and cryoET to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza morphology, glycoprotein density, and conducted subtomogram averaging of influenza glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.

Identification of a Permissive Secondary Mutation That Restores the Enzymatic Activity of Oseltamivir Resistance Mutation H275Y.

Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. IMPORTANCE The potential for influenza A virus (IAV) to cause pandemics makes understanding evolutionary mechanisms that impact drug resistance critical for developing surveillance and treatment strategies. Oseltamivir is the most widely used therapeutic strategy to treat IAV infections, but mutations in IAV can lead to drug resistance. The main oseltamivir resistance mutation, H275Y, occurs in the neuraminidase (NA) protein of IAV and reduces drug binding as well as NA function. Here, we identified a new helper mutation, Y276F, that can rescue the functional defects of H275Y and contribute to the evolution of drug resistance in IAV.

Anti-SARS-CoV-2 Activity of Adamantanes In Vitro and in Animal Models of Infection.

Coronavirus disease 2019 (COVID-19) has had devastating effects worldwide, with particularly high morbidity and mortality in outbreaks on residential care facilities. Amantadine, originally licensed as an antiviral agent for therapy and prophylaxis against influenza A virus, has beneficial effects on patients with Parkinson's disease and is used for treatment of Parkinson's disease, multiple sclerosis, acquired brain injury, and various other neurological disorders. Recent observational data suggest an inverse relationship between the use of amantadine and COVID-19. Adamantanes, including amantadine and rimantadine, are reported to have in vitro activity against severe acute respiratory syndrome coronavirus (SARS-CoV) and, more recently, SARS-CoV-2. We hypothesized that adamantanes have antiviral activity against SARS-CoV-2, including variant strains. To assess the activity of adamantanes against SARS-CoV-2, we used in vitro and in vivo models of infection. We established that amantadine, rimantadine, and tromantadine inhibit the growth of SARS-CoV-2 in vitro in cultured human epithelial cells. While neither rimantadine nor amantadine reduces lung viral titers in mice infected with mouse-adapted SARS-CoV-2, rimantadine significantly reduces viral titers in the lungs in golden Syrian hamsters infected with SARS-CoV-2. In summary, rimantadine has antiviral activity against SARS-CoV-2 in human alveolar epithelial cells and in the hamster model of SARS-CoV-2 lung infection. The evaluation of amantadine or rimantadine in human randomized controlled trials can definitively address applications for the treatment or prevention of COVID-19.

US201 Study: A Phase 2, Randomized Proof-of-Concept Trial of Favipiravir for the Treatment of COVID-19.

BACKGROUND: Favipiravir is used to treat influenza, and studies demonstrate that it has antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We performed a randomized, open-label, multicenter, phase 2 proof-of-concept trial of favipiravir in hospitalized adult patients with polymerase chain reaction (PCR)-positive coronavirus disease 2019 (COVID-19). Patients were randomized to standard of care (SOC) or favipiravir treatment (1800mg per os twice a day [b.i.d.] on day 1, followed by 1000mg b.i.d. for 13 days). The primary end point was time to viral clearance on day 29. RESULTS: Fifty patients were enrolled and stratified by disease severity (critical disease, severe disease, or mild to moderate disease). Nineteen patients were censored from the event of viral clearance based on being SARS-CoV-2 PCR-negative at the study outset, being PCR-positive at day 29, or because of loss to follow-up. Data from the 31 remaining patients who achieved viral clearance show enhanced viral clearance in the favipiravir group compared with the SOC group by day 29, with 72% of the favipiravir group and 52% of the SOC group being evaluable for viral clearance through day 29. The median time to viral clearance was 16.0 days (90% CI, 12.0 to 29.0) in the favipiravir group and 30.0 days (90% CI, 12.0 to 31.0) in the SOC group. A post hoc analysis revealed an effect in the subgroup of patients who were neutralizing antibody-negative at randomization. Treatment-emergent adverse events were equally distributed between the groups. CONCLUSIONS: We demonstrate that favipiravir can be safely administered to hospitalized adults with COVID-19 and believe that further studies are warranted. CLINICALTRIALSGOV REGISTRATION: NCT04358549.

Type I IFN-Driven Immune Cell Dysregulation in Rat Autoimmune Diabetes.

Type 1 diabetes is a chronic autoimmune disease, characterized by the immune-mediated destruction of insulin-producing ß cells of pancreatic islets. Essential components of the innate immune antiviral response, including type I IFN and IFN receptor (IFNAR)-mediated signaling pathways, likely contribute to human type 1 diabetes susceptibility. We previously showed that LEW.1WR1 Ifnar1 -/- rats have a significant reduction in diabetes frequency following Kilham rat virus (KRV) infection. To delineate the impact of IFNAR loss on immune cell populations in KRV-induced diabetes, we performed flow cytometric analysis in spleens from LEW.1WR1 wild-type (WT) and Ifnar1 -/- rats after viral infection but before the onset of insulitis and diabetes. We found a relative decrease in CD8+ T cells and NK cells in KRV-infected LEW.1WR1 Ifnar1 -/- rats compared with KRV-infected WT rats; splenic regulatory T cells were diminished in WT but not Ifnar1 -/- rats. In contrast, splenic neutrophils were increased in KRV-infected Ifnar1 -/- rats compared with KRV-infected WT rats. Transcriptional analysis of splenic cells from KRV-infected rats confirmed a reduction in IFN-stimulated genes in Ifnar1 -/- compared with WT rats and revealed an increase in transcripts related to neutrophil chemotaxis and MHC class II. Single-cell RNA sequencing confirmed that MHC class II transcripts are increased in monocytes and macrophages and that numerous types of splenic cells harbor KRV. Collectively, these findings identify dynamic shifts in innate and adaptive immune cells following IFNAR disruption in a rat model of autoimmune diabetes, providing insights toward the role of type I IFNs in autoimmunity.

Human nasal wash RNA-Seq reveals distinct cell-specific innate immune responses in influenza versus SARS-CoV-2.

BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) "COVID-19 Expansion for AIRe Program."

SARS-CoV-2 Initiates Programmed Cell Death in Platelets.

[Figure: see text].

Unique structural solution from a VH3-30 antibody targeting the hemagglutinin stem of influenza A viruses.

Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.

Coxsackievirus B Type 4 Infection in ß Cells Downregulates the Chaperone Prefoldin URI to Induce a MODY4-like Diabetes via Pdx1 Silencing.

Enteroviruses are suspected to contribute to insulin-producing ß cell loss and hyperglycemia-induced diabetes. However, mechanisms are not fully defined. Here, we show that coxsackievirus B type 4 (CVB4) infection in human islet-engrafted mice and in rat insulinoma cells displays loss of unconventional prefoldin RPB5 interactor (URI) and PDX1, affecting ß cell function and identity. Genetic URI ablation in the mouse pancreas causes PDX1 depletion in ß cells. Importantly, diabetic PDX1 heterozygous mice overexpressing URI in ß cells are more glucose tolerant. Mechanistically, URI loss triggers estrogen receptor nuclear translocation leading to DNA methyltransferase 1 (DNMT1) expression, which induces Pdx1 promoter hypermethylation and silencing. Consequently, demethylating agent procainamide-mediated DNMT1 inhibition reinstates PDX1 expression and protects against diabetes in pancreatic URI-depleted mice . Finally, the ß cells of human diabetes patients show correlations between viral protein 1 and URI, PDX1, and DNMT1 levels. URI and DNMT1 expression and PDX1 silencing provide a causal link between enterovirus infection and diabetes.

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

Proteomic and Transcriptional Profiles of Human Stem Cell-Derived ß Cells Following Enteroviral Challenge.

Enteroviral infections are implicated in islet autoimmunity and type 1 diabetes (T1D) pathogenesis. Significant ß-cell stress and damage occur with viral infection, leading to cells that are dysfunctional and vulnerable to destruction. Human stem cell-derived ß (SC-ß) cells are insulin-producing cell clusters that closely resemble native ß cells. To better understand the events precipitated by enteroviral infection of ß cells, we investigated transcriptional and proteomic changes in SC-ß cells challenged with coxsackie B virus (CVB). We confirmed infection by demonstrating that viral protein colocalized with insulin-positive SC-ß cells by immunostaining. Transcriptome analysis showed a decrease in insulin gene expression following infection, and combined transcriptional and proteomic analysis revealed activation of innate immune pathways, including type I interferon (IFN), IFN-stimulated genes, nuclear factor-kappa B (NF-κB) and downstream inflammatory cytokines, and major histocompatibility complex (MHC) class I. Finally, insulin release by CVB4-infected SC-ß cells was impaired. These transcriptional, proteomic, and functional findings are in agreement with responses in primary human islets infected with CVB ex vivo. Human SC-ß cells may serve as a surrogate for primary human islets in virus-induced diabetes models. Because human SC-ß cells are more genetically tractable and accessible than primary islets, they may provide a preferred platform for investigating T1D pathogenesis and developing new treatments.

Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

The role of platelets in mediating a response to human influenza infection.

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.

A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection.

Encephalomyocarditis virus (EMCV) is a picornavirus that produces lytic infections in murine and human cells. Employing a genome-wide CRISPR-Cas9 knockout screen to find host factors required for EMCV infection, we identified a role for ADAM9 in EMCV infection. CRISPR-mediated deletion of ADAM9 in multiple human cell lines rendered the cells highly resistant to EMCV infection and cell death. Primary fibroblasts from ADAM9 KO mice were also strongly resistant to EMCV infection and cell death. In contrast, ADAM9 KO and WT cells were equally susceptible to infection with other viruses, including the picornavirus Coxsackie virus B. ADAM9 KO cells failed to produce viral progeny when incubated with EMCV. However, bypassing EMCV entry into cells through delivery of viral RNA directly to the cytosol yielded infectious EMCV virions from ADAM9 KO cells, suggesting that ADAM9 is not required for EMCV replication post-entry. These findings establish that ADAM9 is required for the early stage of EMCV infection, likely for virus entry or viral genome delivery to the cytosol.IMPORTANCE Viral myocarditis is a leading cause of death in the United States, contributing to numerous unexplained deaths in people ≤35 years old. Enteroviruses contribute to many cases of human myocarditis. Encephalomyocarditis virus (EMCV) infection causes viral myocarditis in rodent models, but its receptor requirements have not been fully identified. CRISPR-Cas9 screens can identify host dependency factors essential for EMCV infection and enhance our understanding of key events that follow viral infection, potentially leading to new strategies for preventing viral myocarditis. Using a CRISPR-Cas9 screen, we identified adisintegrin and metalloproteinase 9 domain (ADAM9) as a major factor required for the early stages of EMCV infection in both human and murine infection.