Abstract
Background
Norovirus is a leading cause of acute gastroenteritis worldwide. Improved diagnostic capability has been instrumental in the characterization of archival norovirus strains ...associated with gastroenteritis outbreaks that were investigated decades ago. One such investigation was that of 2 sequential gastroenteritis outbreaks that occurred in 1971 at the former Henryton State Hospital in Maryland. Approximately 40% of the resident population experienced clinical symptoms in both outbreaks, which occurred 11 months apart.
Methods
Stored stools and paired sera were re-analyzed to investigate the etiology of the 2 outbreaks.
Results
Different norovirus genotypes were identified as the etiological agents responsible for the illnesses, with GII.2 associated with the first outbreak and GII.6 with the second. The viruses were antigenically distinct as determined by analyses of hyperimmune sera raised against the corresponding virus-like particles in animals, as well as paired sera from infected individuals.
Conclusions
The observed antigenic differences were consistent with the failure of the GII.2 strain to provide cross-protective immunity to the GII.6 strain a few months later. An understanding of antigenic diversity among norovirus genotypes will be important in the design of norovirus vaccines.
Abstract Insertion of nucleotide sequences encoding “tags” that can be expressed in specific viral proteins during an infection is a useful strategy for purifying viral proteins and their functional ...complexes from infected cells and/or for visualizing the dynamics of their subcellular location over time. To identify regions in the poliovirus polyprotein that could potentially accommodate insertion of tags, transposon-mediated insertion mutagenesis was applied to the entire nonstructural protein-coding region of the poliovirus genome, followed by selection of genomes capable of generating infectious, viable viruses. This procedure allowed us to identify at least one site in each viral nonstructural protein, except protein 2C, in which a minimum of five amino acids could be inserted. The distribution of these sites is analyzed from the perspective of their protein structural context and from the perspective of virus evolution.
Multivalent, glycopolymer inhibitors designed for the treatment of disease and pathogen infection have shown improvements in binding correlated with general changes in glycopolymer architecture and ...composition. We have previously demonstrated that control of glycopolypeptide backbone extension and ligand spacing significantly impacts the inhibition of the cholera toxin B subunit pentamer (CT B5) by these polymers. In the studies reported here, we elucidate the role of backbone charge and linker length in modulating the inhibition event. Peptides of the sequence AXPXG (where X is a positive, neutral or negative amino acid), equipped with the alkyne functionality of propargyl glycine, were designed and synthesized via solid‐phase peptide synthetic methods and glycosylated via Cu(I)‐catalyzed alkyne‐azide cycloaddition reactions. The capacity of the glycopeptides to inhibit the binding of the B5 subunit of cholera toxin was evaluated. These studies indicated that glycopeptides with a negatively charged backbone show improved inhibition of the binding event relative to the other glycopeptides. In addition, variations in the length of the linker between the peptide and the saccharide ligand also affected the inhibition of CT by the glycopeptides. Our findings suggest that, apart from appropriate saccharide spacing and polypeptide chain extension, saccharide linker conformation and the systematic placement of charges on the polypeptide backbone are also significant variables that can be tuned to improve the inhibitory potencies of glycopolypeptide‐based multivalent inhibitors.
The presence of negative or positive charges on glycopeptides significantly affects the inhibition of the cholera toxin B‐subunit exhibited by the glycopeptides. The positioning, in the linker between peptide and saccharide, of introduced triazole groups also impacts inhibitory potency. Negatively charged glycopeptides equipped with the shortest triazole‐containing linkers showed the greatest inhibition, followed by neutral and positively charged glycopeptides. These results emphasize the roles that charge and linker composition play in modulating the binding of polypeptide‐based inhibitors to the CT B5 target.
Noroviruses are a major cause of infectious gastroenteritis worldwide, and viruses can establish persistent infection in immunocompromised individuals. Risk factors and transmission in this ...population are not fully understood.
From 2010 through 2013, we conducted a retrospective review among immunocompromised patients (n = 268) enrolled in research studies at the National Institutes of Health Clinical Center and identified a subset of norovirus-positive patients (n = 18) who provided stool specimens for norovirus genotyping analysis.
Norovirus genome was identified by reverse-transcription quantitative polymerase chain reaction in stools of 35 (13%) of the 268 immunocompromised patients tested, and infection prevalence was 21% (11 of 53) in persons with primary immune deficiencies and 12% (20 of 166) among persons with solid tumors or hematologic malignancies. Among 18 patients with norovirus genotyping information, norovirus GII.4 was the most prevalent genotype (14 of 18, 78%). Persistent norovirus infection (≥6 months) was documented in 8 of 18 (44%) individuals. Phylogenetic analysis of the GII.4 capsid protein sequences identified at least 5 now-displaced GII.4 variant lineages, with no evidence of their nosocomial transmission in the Clinical Center.
Norovirus was a leading enteric pathogen identified in this immunocompromised population. Both acute and chronic norovirus infections were observed, and these were likely community-acquired. Continued investigation will further define the role of noroviruses in these patients and inform efforts toward prevention and treatment.
Polysaccharide vaccines such as the Vi polysaccharide of
Salmonella enterica
serovar Typhi induce efficient antibody responses in adults, but not in young children. The reasons for this difference ...are not understood. Interleukin-7 (IL-7) dependency in B cell development increases progressively with age. IL-7Rα-mediated signals are required for the expression of many V
H
gene segments that are distal to D
H
-J
H
in the immunoglobulin heavy chain locus and for the complete diversification of the B cell antigen receptor repertoire. Therefore, we hypothesized that B cells generated in the absence of IL-7 do not recognize a wide range of antigens due to a restricted B cell antigen receptor repertoire. Compared to adult wildtype mice, young wildtype mice and IL-7-deficient adult mice generated a significantly reduced antibody response to Vi polysaccharide. Additionally, Vi polysaccharide-binding B cells in adult wildtype mice predominantly utilized distal V
H
gene segments. Transgenic expression of either IL-7, or a BCR encoded by a distal V
H
gene segment, permitted young mice to respond efficiently to bacterial polysaccharides. These results indicate that restricted V
H
gene usage early in life results in a paucity of antigen-specific B cell precursors, thus limiting anti-polysaccharide responses.
Multivalent, glycopolymer inhibitors designed for the treatment of disease and pathogen infection have shown improvements in binding correlated with general changes in glycopolymer architecture and ...composition. We have previously demonstrated that control of glycopolypeptide backbone extension and ligand spacing significantly impacts the inhibition of the cholera toxin B subunit pentamer (CT B
5
) by these polymers. In the studies reported here, we elucidate the role of backbone charge and linker length in modulating the inhibition event. Peptides of the sequence A
X
P
X
G (where
X
is a positive, neutral or negative amino acid), equipped with the alkyne functionality of propargyl glycine, were designed and synthesized via solid-phase peptide synthetic methods and glycosylated via Cu(I)-catalyzed alkyne-azide cycloaddition reactions. The capacity of the glycopeptides to inhibit the binding of the B
5
subunit of cholera toxin was evaluated. These studies indicated that glycopeptides with a negatively charged backbone show improved inhibition of the binding event relative to the other glycopeptides. In addition, variations in the length of the linker between the peptide and the saccharide ligand also affected the inhibition of CT by the glycopeptides. Our findings suggest that, apart from appropriate saccharide spacing and polypeptide chain extension, saccharide linker conformation and the systematic placement of charges on the polypeptide backbone are also significant variables that can be tuned to improve the inhibitory potencies of glycopolypeptide-based multivalent inhibitors.
Murine norovirus is genetically similar to human norovirus, and offers both an efficient
in vitro
cell culture system and animal model by which to investigate the molecular basis of replication. ...Here, we present a detailed global view of host alterations to cellular pathways that occur during the progression of a norovirus infection. This was accomplished for both
mus musculus
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 norovirus replicated in both cell types, although detected genome copies were approximately one log lower in BMDM compared to RAW cells. RAW and BMDM cells shared an IRF3/7-based IFN response that occurred early in infection. In RAW264.7 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 RAW264.7 cells compared to 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 norovirus immunity, pathogenesis, and drug development.
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