Abstract
Human noroviruses are a major cause of diarrheal illness, but pathogenesis is poorly understood. Here, we investigate the cellular tropism of norovirus in specimens from four ...immunocompromised patients. Abundant norovirus antigen and RNA are detected throughout the small intestinal tract in jejunal and ileal tissue from one pediatric intestinal transplant recipient with severe gastroenteritis. Negative-sense viral RNA, a marker of active viral replication, is found predominantly in intestinal epithelial cells, with chromogranin A-positive enteroendocrine cells (EECs) identified as a permissive cell type in this patient. These findings are consistent with the detection of norovirus-positive EECs in the other three immunocompromised patients. Investigation of the signaling pathways induced in EECs that mediate communication between the gut and brain may clarify mechanisms of pathogenesis and lead to the development of in vitro model systems in which to evaluate norovirus vaccines and treatment.
The outstanding elastomeric properties of natural resilin, an insect protein, have motivated the engineering of resilin‐like polypeptides (RLPs) as a potential material for cardiovascular tissue ...engineering. The RLPs, which incorporate biofunctional domains for cell‐matrix interactions, are cross‐linked into RLP–PEG hybrid hydrogels via a Michael‐type addition of cysteine residues on the RLP with vinyl sulfones of an end‐functionalized multi‐arm star PEG. Oscillatory rheology indicated the useful mechanical properties of these materials. Assessments of cell viability via confocal microscopy clearly show the successful encapsulation of human aortic adventitial fibroblasts in the three‐dimensional matrices and the adoption of a spread morphology following 7 days of culture.
Engineered polypeptide–PEG hydrogels based upon the elastomeric structural protein, resilin, are cross‐linked via Michael‐type addition reaction between cysteine residues of the protein polymer and the end‐functionalized vinyl sulfones of a multi‐arm PEG macromer. The precursors quickly form elastic hydrogels and successfully support the adhesion and spreading of human aortic adventitial fibroblasts.
Type I interferons (IFN-I) were first discovered as an antiviral factor by Isaacs and Lindenmann in 1957, but they are now known to also modulate innate and adaptive immunity and suppress ...proliferation of cancer cells. While much has been revealed about IFN-I, it remains a mystery as to why there are 16 different IFN-I gene products, including IFNβ, IFNω, and 12 subtypes of IFNα. Here, we discuss shared and unique aspects of these IFN-I in the context of their evolution, expression patterns, and signaling through their shared heterodimeric receptor. We propose that rather than investigating responses to individual IFN-I, these contexts can serve as an alternative approach toward investigating roles for IFNα subtypes. Finally, we review uses of IFNα and IFNβ as therapeutic agents to suppress chronic viral infections or to treat multiple sclerosis.
Noroviruses are a major cause of acute gastroenteritis worldwide and can establish chronic infection in immunocompromised individuals. To investigate the mechanisms of norovirus evolution during ...chronic infection, we selected seven representative patients from a National Institutes of Health study cohort who sustained norovirus infection for periods ranging from 73 to 1,492 days. Six patients shed viruses belonging to a single genotype (GII.2PNA, GII.4 New OrleansP4, GII.4 Den HaagP4, GII.3P21, GII.6P7, or GII.14P7) over the period examined, while one patient sequentially shed two genotypes (GII.6P7 followed by GII.4 SydneyP31). Norovirus genomes from consecutive stool samples were sequenced at high resolution (>3,300 reads/nucleotide position) using the Illumina platform and subjected to bioinformatics analysis. Norovirus sequences could be resolved into one or more discrete clonal RNA genomes that persisted within these patients over time. Phylogenetic analyses inferred that clonal populations originated from a single founder virus and not by reinfection with community strains. Estimated evolutionary rates of clonal populations during persistent infection were similar to those of noroviruses from acute infection in the global database, suggesting that inherently higher RNA-dependent polymerase error rates were not associated with the ability to persist. The high-resolution analysis of norovirus diversity and evolution at the population level described here should allow a better understanding of adaptive mutations sustained during chronic infection. IMPORTANCE Noroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations.
Murine norovirus (NoV) is genetically similar to human NoV and offers both an efficient in vitro cell culture system and an animal model by which to investigate the molecular basis of replication. In ...this study, we present a detailed global view of host alterations to cellular pathways that occur during the progression of a NoV infection. This was accomplished for both
BALB/c-derived RAW264.7 (RAW) cells, an immortalized cell line widely used in in vitro replication studies, and primary bone marrow-derived macrophages (BMDM), representing a permissive in vivo target cell in the host. Murine NoV replicated in both cell types, although detected genome copies were approximately one log lower in BMDM compared with RAW cells. RAW and BMDM cells shared an IRF3/7-based IFN response that occurred early in infection. In RAW cells, transcriptional upregulation and INF-β expression were not coupled in that a significant delay in the detection of secreted INF-β was observed. In contrast, primary BMDM showed an early upregulation of transcripts and immediate release of INF-β that might account for lower virus yield. Differences in the transcriptional pathway responses included a marked decrease in expression of key genes in the cell cycle and lipid pathways in RAW cells compared with that of BMDM. Our comparative analysis indicates the existence of varying host responses to virus infection in populations of permissive cells. Awareness of these differences at the gene level will be important in the application of a given permissive culture system to the study of NoV immunity, pathogenesis, and drug development.
The outstanding elasticity, excellent resilience at high-frequency, and hydrophilic capacity of natural resilin have motivated investigations of recombinant resilin-based biomaterials as a new class ...of bio-elastomers in the engineering of mechanically active tissues. Accordingly, here the comprehensive characterization of modular resilin-like polypeptide (RLP) hydrogels is presented and their suitability as a novel biomaterial for in vivo applications is introduced. Oscillatory rheology confirmed that a full suite of the RLPs can be rapidly cross-linked upon addition of the tris(hydroxymethyl phosphine) cross-linker, achieving similar in situ shear storage moduli (20 k ± 3.5 Pa) across various material compositions. Uniaxial stress relaxation tensile testing of hydrated RLP hydrogels under cyclic loading and unloading showed negligible stress reduction and hysteresis, superior reversible extensibility, and high resilience with Young's moduli of 30 ± 7.4 kPa. RLP hydrogels containing MMP-sensitive domains are susceptible to enzymatic degradation by matrix metalloproteinase-1 (MMP-1). Cell culture studies revealed that RLP-based hydrogels supported the attachment and spreading (2D) of human mesenchymal stem cells and did not activate cultured macrophages. Subcutaneous transplantation of RLP hydrogels in a rat model, which to our knowledge is the first such reported in vivo analysis of RLP-based hydrogels, illustrated that these materials do not elicit a significant inflammatory response, suggesting their potential as materials for tissue engineering applications with targets of mechanically demanding tissues such as vocal fold and cardiovascular tissues.
Polysaccharide vaccines such as the Vi polysaccharide (ViPS) of
serovar Typhi induce efficient Ab responses in adults but not in young children. The reasons for this difference are not understood. ...IL-7 dependency in B cell development increases progressively with age. IL-7Rα-mediated signals are required for the expression of many V
gene segments that are distal to D
-J
in the IgH locus and for the complete diversification of the BCR repertoire. Therefore, we hypothesized that B cells generated in the absence of IL-7 do not recognize a wide range of Ags because of a restricted BCR repertoire. Compared with adult wildtype mice, young wildtype mice and IL-7-deficient adult mice generated a significantly reduced Ab response to ViPS. Additionally, ViPS-binding B cells in adult wildtype mice predominantly used distal V
gene segments. Transgenic expression of either IL-7 or a BCR encoded by a distal V
gene segment permitted young mice to respond efficiently to bacterial polysaccharides. These results indicate that restricted V
gene usage early in life results in a paucity of Ag-specific B cell precursors, thus limiting antipolysaccharide responses.
Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide motifs are agonists of Toll-like receptor 9 and are currently being investigated for use as vaccine adjuvants through the ...promotion of type I immunity. Several classes of ODN have been developed which differ in their propensity to aggregate, which in turn alters cytokine profiles and cellular subsets activated. Although aggregation state is correlated with the change in cytokine response, it is unknown if this results from a change in the number of ODNs available for binding and/or the possible engagement of multiple TLR9 molecules. Here, we examined the role of ligand valency on the activation of TLR9 through the synthesis of ODN–poly(acrylic acid) (PAA) conjugates. The compositions and size of the conjugates were characterized by UV-vis spectroscopy, proton nuclear magnetic resonance, gel permeation chromatography and dynamic light scattering. Enzyme-linked immunosorbent assays of cytokine secretion by murine-like macrophages indicate that these ODN–PAA polymer conjugates show enhanced immunostimulation at 100-fold lower concentrations than those required for ODN alone, for both TNF-α and IL-6 release, and are more potent than any other previously reported multivalent ODN constructs. Increasing valency was shown to significantly enhance cytokine expression, particularly for IL-6. Knockdown by siRNA demonstrates that these polymer conjugates are specific to TLR9. Our results define valency as a critical design parameter and polymer conjugation as an advantageous strategy for producing ODN immunomodulatory agents.
In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that ...rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles. In addition, stools of infected hosts contain norovirus and rotavirus within vesicles of exosomal or plasma membrane origin. These vesicles remain intact during fecal-oral transmission and thereby transport multiple viral particles collectively to the next host, enhancing both the MOI and disease severity. Vesicle-cloaked viruses are non-negligible populations in stool and have a disproportionately larger contribution to infectivity than free viruses. Our findings indicate that vesicle-cloaked viruses are highly virulent units of fecal-oral transmission and highlight a need for antivirals targeting vesicles and virus clustering.
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•Rotaviruses and noroviruses are shed in stool as viral clusters inside vesicles•Vesicles containing virus clusters remain intact during fecal-oral transmission•Vesicles achieve a high MOI and induce severe disease•Vesicle-cloaked viral clusters are more virulent units than free viral particles
Freely disseminating standalone viral particles are considered the optimal agents for spreading infection. Santiana et al. discover that enteric viruses are also shed in feces as viral clusters cloaked in vesicles. These virus-containing vesicles are a more potent infectious form that enhances the MOI and disease severity.
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