Human neonates infected with herpes simplex virus 1 (HSV-1) develop one of three distinct patterns of infection: (i) infection limited to the skin, eye or mouth; (ii) infection of the CNS; or (iii) disseminated infection. The disseminated form usually involves the liver, adrenal gland, and lung, and resembles the clinical picture of bacterial sepsis. This spectrum of symptoms in HSV-1-infected neonates suggests that inflammatory cytokines play a significant role in the pathogenesis of the disease. Recent studies suggest that the Toll-like receptors (TLRs) may play an important role in the induction of inflammatory cytokines in response to viruses. TLRs are mammalian homologues of Toll, aDrosophilaprotein that is essential for host defense against infection. Engagement of TLRs by bacterial, viral, or fungal components leads to the production and release of cytokines and other antimicrobial products. Here, we demonstrate that TLR2 mediates the inflammatory cytokine response to HSV-1 by using both transfected cell lines and knockout mice. Studies of infected mice revealed that HSV-1 induced a blunted cytokine response in TLR2^–/–mice. Brain levels of monocyte chemoattractant protein 1 chemokine were significantly lower in TLR2^–/–mice than in either wild-type or TLR4^–/–mice. TLR2^–/–mice had reduced mortality compared with wild-type mice. The differences between TLR2^–/–mice and both wild-type and TLR4^–/–mice in the induction of monocyte chemoattractant protein 1, brain inflammation, or mortality could not be accounted for on the basis of virus levels. Thus, these studies suggest the TLR2-mediated cytokine response to HSV-1 is detrimental to the host.

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.

Rationale: Coronavirus disease 2019 (COVID-19) is characterized by increased incidence of microthrombosis with hyperactive platelets sporadically containing viral RNA. It is unclear if SARS-CoV-2 (severe acute respiratory syndrome, corona virus-2) directly alters platelet activation or if these changes are a reaction to infection-mediated global inflammatory alterations. Importantly, the direct effect of SARS-CoV-2 on platelets has yet to be studied. Objective: To characterize the direct SARS-CoV-2–platelet interactions using in vitro studies with purified infectious virions and samples from infected patients. Methods and Results: Platelet RNA analyzed by ARTIC v3 sequencing for SARS-CoV-2 showed presence of fragmented viral genome in all patients with COVID-19. Immunofluorescent imaging of platelets from patients with COVID-19 confirmed presence of SARS-CoV-2 proteins, whereas there was no detection of viral RNA by real-time quantitative polymerase chain reaction. Transmission electron microscopy of platelets incubated with purified SARS-CoV-2 virions demonstrated rapid internalization and digestion leading to distinct morphological changes and resulted in a release of extracellular vesicles. Interactions between SARS-CoV-2 and platelets occurred with or without ACE (angiotensin-converting enzyme) 2 presence as measured by immunofluorescence. Transmission electron microscopy showed that SARS-CoV-2 virions became internalized when they were attached to microparticles, bypassing the need for ACE2. Enrichment analysis of platelet transcriptome from patients with acute COVID-19, compared with those with clinical thrombosis, suggested upregulation of pathways related to virally mediated cell death, specifically necroptosis and apoptosis. Platelets incubated with infectious virus appeared to undergo cell death in 30 minutes postincubation as assessed by transmission electron microscopy and platelets from patients with COVID-19 showed evidence of increased markers of apoptosis and necroptosis by Western blot. Immunofluorescence confirmed colocalization of SARS-CoV-2 with phospho-MLKL (mixed lineage kinase domain-like pseudokinase) and caspase 3 on nonpermeabilized platelets in vitro and in COVID-19 platelets. Conclusions: Platelets internalize SARS-CoV-2 virions, directly or attached to microparticles, and viral internalization leads to rapid digestion, programmed cell death, and extracellular vesicle release. During COVID-19, platelets mediate a rapid response to SARS-CoV-2 and this response can contribute to dysregulated immunity and thrombosis.

TLR7 senses RNA in endosomal compartments. TLR7 expression and signaling have been demonstrated in plasmacytoid and myeloid dendritic cells, B cells, and T cells. The regulation of TLR7 signaling can play a crucial role in shaping the immune response to RNA viruses with different cellular tropisms, and in developing adjuvants capable of promoting balanced humoral and cell-mediated immunity. We used unique characteristics of two ssRNA viruses, dengue virus and influenza virus, to delineate factors that regulate viral RNA-human TLR7 signaling beyond recognition in endosomal compartments. Our data show that TLR7 recognition of enveloped RNA virus genomes is linked to virus fusion or uncoating from the endosome. The signaling threshold required to activate TLR7-type I IFN production is greater than that required to activate TLR7-NF-κB-IL-8 production. The higher order structure of viral RNA appears to be an important determinant of TLR7-signaling potency. A greater understanding of viral RNA-TLR7 activity relationships will promote rational approaches to interventional and vaccine strategies for important human viral pathogens.

The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures. In recent years, considerable attention has been given to the evolution of drug resistance in the influenza A H1N1 strain. As a major annual cause of morbidity and mortality, combined with the rapid global spread of drug resistance, influenza remains as one of the most important global health concerns. Our work here focuses on a novel multi-faceted population-genetic approach utilizing unique whole-genome multi-time point experimental datasets in both the presence and absence of drug treatment. In addition, we present novel theoretical results and two newly developed and widely applicable statistical methodologies for utilizing time-sampled data – with a focus on distinguishing the relative contribution of genetic drift from that of positive and purifying selection. Results illustrate the available mutational paths to drug resistance, and offer important insights in to the mode and tempo of adaptation in a viral population.

Plasmacytoid dendritic cells (pDCs) were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs) containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs). The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation. While plasmacytoid dendritic cells (pDCs) are known to be important immune cells involved in protection from viruses and tumors, their role in protection against fungal infections is less clear. Our laboratory has been studying the interplay between pDCs and the fungal pathogen, Aspergillus fumigatus. Our previous work demonstrated that human pDCs bind to and inhibit the growth of A. fumigatus hyphae. Moreover, depletion of pDCs rendered mice very susceptible to experimental infection with A. fumigatus. Here, we show that Dectin-2, a receptor on pDCs, recognizes A. fumigatus hyphae and contributes to cytokine release and antifungal activity. In addition, using confocal and electron microscopy, we demonstrate that upon contact with hyphae, some human pDCs die and form antimicrobial structures called extracellular traps. Finally, using microarrays, we analyzed human pDC gene expression upon A. fumigatus infection and found distinct patterns including the activation of genes previously associated with viral infections and apoptosis. These results provide new insights into the mechanisms by which pDCs might help the immune system when confronted with a fungal invader.