JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Distinct patterns of B-cell activation and priming by natural influenza infection versus inactivated influenza vaccination.
J. Infect. Dis.
PUBLISHED: 10-23-2014
Show Abstract
Hide Abstract
?The human B-cell response to natural influenza infection has not been extensively investigated at the polyclonal level.
Related JoVE Video
Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian children in the second year of life.
Vaccine
PUBLISHED: 08-06-2014
Show Abstract
Hide Abstract
Rotavirus gastroenteritis is one of the leading causes of diarrhea in Indian children less than 2 years of age. The 116E rotavirus strain was developed as part of the Indo-US Vaccine Action Program and has undergone efficacy trials. This paper reports the efficacy and additional safety data in children up to 2 years of age. In a double-blind placebo controlled multicenter trial, 6799 infants aged 6-7 weeks were randomized to receive three doses of an oral human-bovine natural reassortant vaccine (116E) or placebo at ages 6, 10, and 14 weeks. The primary outcome was severe (?11 on the Vesikari scale) rotavirus gastroenteritis. Efficacy outcomes and adverse events were ascertained through active surveillance. We randomly assigned 4532 and 2267 subjects to receive vaccine and placebo, respectively, with over 96% subjects receiving all three doses of the vaccine or placebo. The per protocol analyses included 4354 subjects in the vaccine and 2187 subjects in the placebo group. The overall incidence of severe RVGE per 100 person years was 1.3 in the vaccine group and 2.9 in the placebo recipients. Vaccine efficacy against severe rotavirus gastroenteritis in children up to 2 years of age was 55.1% (95% CI 39.9 to 66.4; p<0.0001); vaccine efficacy in the second year of life of 48.9% (95% CI 17.4 to 68.4; p=0.0056) was only marginally less than in the first year of life [56.3% (95% CI 36.7 to 69.9; p<0.0001)]. The number of infants needed to be immunized to prevent one episode of severe RVGE in the first 2 years of life was 40 (95% CI 28.0 to 63.0) and for RVGE of any severity, it was 21 (95% CI 16.0 to 32.0). Serious adverse events were observed at the same rates in the two groups. None of the eight intussusception events occurred within 30 days of a vaccine dose and all were reported only after the third dose. The sustained efficacy of the 116E in the second year of life is reassuring.
Related JoVE Video
Distinct cross-reactive B-cell responses to live attenuated and inactivated influenza vaccines.
J. Infect. Dis.
PUBLISHED: 03-27-2014
Show Abstract
Hide Abstract
The immunological bases for the efficacies of the 2 currently licensed influenza vaccines, live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV), are not fully understood. The goal of this study was to identify specific B-cell responses correlated with the known efficacies of these 2 vaccines.
Related JoVE Video
Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-blind, placebo-controlled trial.
Lancet
PUBLISHED: 03-12-2014
Show Abstract
Hide Abstract
Rotavirus is the most common cause of severe dehydrating gastroenteritis in developing countries. Safe, effective, and affordable rotavirus vaccines are needed in these countries. We aimed to assess the efficacy and tolerability of a monovalent human-bovine rotavirus vaccine for severe rotavirus gastroenteritis in low-resource urban and rural settings in India.
Related JoVE Video
Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation.
J. Virol.
PUBLISHED: 10-16-2013
Show Abstract
Hide Abstract
Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-?B, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.
Related JoVE Video
Permissive replication of homologous murine rotavirus in the mouse intestine is primarily regulated by VP4 and NSP1.
J. Virol.
PUBLISHED: 05-22-2013
Show Abstract
Hide Abstract
Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1.
Related JoVE Video
Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses.
J. Clin. Invest.
PUBLISHED: 02-26-2013
Show Abstract
Hide Abstract
B cell-dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-?/?-mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity.
Related JoVE Video
Lineage structure of the human antibody repertoire in response to influenza vaccination.
Sci Transl Med
PUBLISHED: 02-08-2013
Show Abstract
Hide Abstract
The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cells genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune systems clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.
Related JoVE Video
Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization.
Nat. Biotechnol.
PUBLISHED: 02-07-2013
Show Abstract
Hide Abstract
It is currently not possible to predict which epitopes will be recognized by T cells in different individuals. This is a barrier to the thorough analysis and understanding of T-cell responses after vaccination or infection. Here, by combining mass cytometry with combinatorial peptide-MHC tetramer staining, we have developed a method allowing the rapid and simultaneous identification and characterization of T cells specific for many epitopes. We use this to screen up to 109 different peptide-MHC tetramers in a single human blood sample, while still retaining at least 23 labels to analyze other markers of T-cell phenotype and function. Among 77 candidate rotavirus epitopes, we identified six T-cell epitopes restricted to human leukocyte antigen (HLA)-A*0201 in the blood of healthy individuals. T cells specific for epitopes in the rotavirus VP3 protein displayed a distinct phenotype and were present at high frequencies in intestinal epithelium. This approach should be useful for the comprehensive analysis of T-cell responses to infectious diseases or vaccines.
Related JoVE Video
The battle between rotavirus and its host for control of the interferon signaling pathway.
PLoS Pathog.
PUBLISHED: 01-24-2013
Show Abstract
Hide Abstract
Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ~450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses.
Related JoVE Video
Characterization of rotavirus RNAs that activate innate immune signaling through the RIG-I-like receptors.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
In mammalian cells, the first line of defense against viral pathogens is the innate immune response, which is characterized by induction of type I interferons (IFN) and other pro-inflammatory cytokines that establish an antiviral milieu both in infected cells and in neighboring uninfected cells. Rotavirus, a double-stranded RNA virus of the Reoviridae family, is the primary etiological agent of severe diarrhea in young children worldwide. Previous studies demonstrated that rotavirus replication induces a MAVS-dependent type I IFN response that involves both RIG-I and MDA5, two cytoplasmic viral RNA sensors. This study reports the isolation and characterization of rotavirus RNAs that activate IFN signaling. Using an in vitro approach with purified rotavirus double-layer particles, nascent single-stranded RNA (ssRNA) transcripts (termed in vitro ssRNA) were found to be potent IFN inducers. In addition, large RNAs isolated from rotavirus-infected cells six hours post-infection (termed in vivo 6 hr large RNAs), also activated IFN signaling, whereas a comparable large RNA fraction isolated from cells infected for only one hour lacked this stimulatory activity. Experiments using knockout murine embryonic fibroblasts showed that RIG-I is required for and MDA5 partly contributes to innate immune signaling by both in vitro ssRNA and in vivo 6 hr large RNAs. Enzymatic studies demonstrated that in vitro ssRNA and in vivo 6 hr large RNA samples contain uncapped RNAs with exposed 5 phosphate groups. RNAs lacking 2-O-methylated 5 cap structures were also detected in the in vivo 6 hr large RNA sample. Taken together, our data provide strong evidence that the rotavirus VP3 enzyme, which encodes both guanylyltransferase and methyltransferase activities, is not completely efficient at either 5 capping or 2-O-methylation of the 5 cap structures of viral transcripts, and in this way produces RNA patterns that activate innate immune signaling through the RIG-I-like receptors.
Related JoVE Video
Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry.
J. Virol.
PUBLISHED: 08-17-2011
Show Abstract
Hide Abstract
Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of ?-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.
Related JoVE Video
Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies.
J. Clin. Invest.
PUBLISHED: 03-03-2011
Show Abstract
Hide Abstract
During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.
Related JoVE Video
Reconciliation of rotavirus temperature-sensitive mutant collections and assignment of reassortment groups D, J, and K to genome segments.
J. Virol.
PUBLISHED: 03-02-2011
Show Abstract
Hide Abstract
Four rotavirus SA11 temperature-sensitive (ts) mutants and seven rotavirus RRV ts mutants, isolated at the National Institutes of Health (NIH) and not genetically characterized, were assigned to reassortment groups by pairwise crosses with the SA11 mutant group prototypes isolated and characterized at Baylor College of Medicine (BCM). Among the NIH mutants, three of the RRV mutants and all four SA11 mutants contained mutations in single reassortment groups, and four RRV mutants contained mutations in multiple groups. One NIH mutant [RRVtsK(2)] identified the previously undefined 11th reassortment group (K) expected for rotavirus. Three NIH single mutant RRV viruses, RRVtsD(7), RRVtsJ(5), and RRVtsK(2), were in reassortment groups not previously mapped to genome segments. These mutants were mapped using classical genetic methods, including backcrosses to demonstrate reversion or suppression in reassortants with incongruent genotype and temperature phenotype. Once located to specific genome segments by genetic means, the mutations responsible for the ts phenotype were identified by sequencing. The reassortment group K mutant RRVtsK(2) maps to genome segment 9 and has a Thr280Ileu mutation in the capsid surface glycoprotein VP7. The group D mutant RRVtsD(7) maps to segment 5 and has a Leu140Val mutation in the nonstructural interferon (IFN) antagonist protein NSP1. The group J mutant RRVtsJ(5) maps to segment 11 and has an Ala182Gly mutation affecting only the NSP5 open reading frame. Rotavirus ts mutation groups are now mapped to 9 of the 11 rotavirus genome segments. Possible segment locations of the two remaining unmapped ts mutant groups are discussed.
Related JoVE Video
The early interferon response to rotavirus is regulated by PKR and depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3.
J. Virol.
PUBLISHED: 02-09-2011
Show Abstract
Hide Abstract
In mouse embryonic fibroblasts (MEFs), the bovine rotavirus (UK strain) but not the simian rhesus rotavirus (RRV) robustly triggers beta interferon (IFN-?) secretion, resulting in an IFN-dependent restriction of replication. We now find that both rotavirus strains trigger antiviral transcriptional responses early during infection and that both transcriptional responses and IFN-? secretion are completely abrogated in MAVS/IPS-1(-/-) MEFs. Replication of UK virus could be rescued in MAVS/IPS-1(-/-) MEFs, and synthesis of viral RNA significantly increased early during virus infection. UK virus induced IFN-? secretion and transcription of IFN-stimulated genes (ISGs) in both RIG-I(-/-) and MDA-5(-/-) MEFs, and neither receptor was essential by itself for the antiviral response to UK rotavirus. However, when receptors RIG-I and MDA-5 were depleted using RNA interference, we found that both contribute to the magnitude of the IFN response. IRF3 was found to be essential for MAVS/IPS-1-directed ISG transcription and IFN-? secretion during rotavirus infection. Interestingly, absence of the double-stranded RNA-dependent protein kinase PKR led to a profound defect in the capacity of host cells to secrete IFN-? in response to virus. Both PKR and IRF3 restricted the early replication of UK as indicated by significant increases in viral RNA in fibroblasts lacking either gene. Despite the loss in IFN-? secretion in PKR(-/-) MEFs, we did not observe decreased IRF3- or NF-?B-dependent early ISG transcription in these cells. Levels of transcripts encoding IFN-?4, IFN-?5, and IFN-? were high in infected PKR(-/-) MEFs, indicating that during rotavirus infection, PKR functions at a stage between IFN gene transcription and subsequent IFN-? secretion. These findings reveal that activation of the antiviral response by rotavirus is dependent on MAVS/IPS-1 and IRF3 and involves both RIG-I and MDA-5 and that IFN-? secretion during rotavirus infection is regulated by PKR.
Related JoVE Video
Roles of VP4 and NSP1 in determining the distinctive replication capacities of simian rotavirus RRV and bovine rotavirus UK in the mouse biliary tract.
J. Virol.
PUBLISHED: 12-29-2010
Show Abstract
Hide Abstract
Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1. Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4. Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-?/? and -? receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1.
Related JoVE Video
Rhesus rotavirus entry into a polarized epithelium is endocytosis dependent and involves sequential VP4 conformational changes.
J. Virol.
PUBLISHED: 12-29-2010
Show Abstract
Hide Abstract
Rotavirus (RV) cell entry is an incompletely understood process, involving VP4 and VP7, the viral proteins composing the outermost layer of the nonenveloped RV triple-layered icosahedral particle (TLP), encasing VP6. VP4 can exist in three conformational states: soluble, cleaved spike, and folded back. In order to better understand the events leading to RV entry, we established a detection system to image input virus by monitoring the rhesus RV (RRV) antigens VP4, VP6, and VP7 at very early times postinfection. We provide evidence that decapsidation occurs directly after cell membrane penetration. We also demonstrate that several VP4 and VP7 conformational changes take place during entry. In particular, we detected, for the first time, the generation of folded-back VP5 in the context of the initiation of infection. Folded-back VP5 appears to be limited to the entry step. We furthermore demonstrate that RRV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca(2+) concentration, as bafilomycin A1 treatment as well as an augmentation of the extracellular calcium reservoir using CaEGTA, which both lead to an elevated intraendosomal calcium concentration, resulted in the accumulation of intact virions in the actin network. Together, these findings suggest that internalization, decapsidation, and cell membrane penetration involve endocytosis, calcium-dependent uncoating, and VP4 conformational changes, including a fold-back.
Related JoVE Video
Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function.
Viral Immunol.
PUBLISHED: 12-15-2010
Show Abstract
Hide Abstract
Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and "danger signals" released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-?1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-?, because they were reversed by treatment of the T cells with the TGF-?-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18?g/mL), and denser vesicles (>1.24?g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity.
Related JoVE Video
Plasmablast-derived polyclonal antibody response after influenza vaccination.
J. Immunol. Methods
PUBLISHED: 09-09-2010
Show Abstract
Hide Abstract
Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 10(5) by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.
Related JoVE Video
Rotavirus structural proteins and dsRNA are required for the human primary plasmacytoid dendritic cell IFNalpha response.
PLoS Pathog.
PUBLISHED: 04-28-2010
Show Abstract
Hide Abstract
Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNalpha and beta) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNalpha production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNalpha production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNalpha by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNalpha induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNalpha production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNalpha induction.
Related JoVE Video
Rotavirus differentially infects and polyclonally stimulates human B cells depending on their differentiation state and tissue of origin.
J. Virol.
PUBLISHED: 02-17-2010
Show Abstract
Hide Abstract
We have shown previously that rotavirus (RV) can infect murine intestinal B220(+) cells in vivo (M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, and H. B. Greenberg, J. Virol. 80:5219-5232, 2006) and human blood B cells in vitro (M. C. Mesa, L. S. Rodriguez, M. A. Franco, and J. Angel, Virology 366:174-184, 2007). However, the effect of RV on B cells, especially those present in the human intestine, the primary site of RV infection, is unknown. Here, we compared the effects of the in vitro RV infection of human circulating (CBC) and intestinal B cells (IBC). RV infected four times more IBC than CBC, and in both types of B cells the viral replication was highly restricted to the memory subset. RV induced cell death in 30 and 3% of infected CBC and IBC, respectively. Moreover, RV induced activation and differentiation into antibody-secreting cells (ASC) of CBC but not IBC when the B cells were present with other mononuclear cells. However, RV did not induce these effects in purified CBC or IBC, suggesting the participation of other cells in activating and differentiating CBC. RV infection was associated with enhanced interleukin-6 (IL-6) production by CBC independent of viral replication. The infection of the anti-B-cell receptor, lipopolysaccharide, or CpG-stimulated CBC reduced the secretion of IL-6 and IL-8 and decreased the number of ASC. These inhibitory effects were associated with an increase in viral replication and cell death and were observed in polyclonally stimulated CBC but not in IBC. Thus, RV differentially interacts with primary human B cells depending on their tissue of origin and differentiation stage, and it affects their capacity to modulate the local and systemic immune responses.
Related JoVE Video
VP5* rearranges when rotavirus uncoats.
J. Virol.
PUBLISHED: 08-19-2009
Show Abstract
Hide Abstract
Trypsin primes rotavirus for efficient infectivity by cleaving the spike protein, VP4, into VP8* and VP5*. A recombinant VP5* fragment has a trimeric, folded-back structure. Comparison of this structure with virion spikes suggests that a rearrangement, analogous to those of enveloped virus fusion proteins, may mediate membrane penetration by rotavirus during entry. To detect this inferred rearrangement of virion-associated authentic VP5*, we raised conformation-specific monoclonal antibodies against the recombinant VP5* fragment in its putative post-membrane penetration conformation. Using one of these antibodies, we demonstrate that rotavirus uncoating triggers a conformational change in the cleaved VP4 spike to yield rearranged VP5*.
Related JoVE Video
IRF3 inhibition by rotavirus NSP1 is host cell and virus strain dependent but independent of NSP1 proteasomal degradation.
J. Virol.
PUBLISHED: 08-05-2009
Show Abstract
Hide Abstract
Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1s ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation.
Related JoVE Video
Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab.
Science
PUBLISHED: 06-13-2009
Show Abstract
Hide Abstract
Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca2+) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca2+ sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.
Related JoVE Video
Broadening the age restriction for initiating rotavirus vaccination in regions with high rotavirus mortality: benefits of mortality reduction versus risk of fatal intussusception.
Vaccine
PUBLISHED: 02-27-2009
Show Abstract
Hide Abstract
Recently developed rotavirus vaccines have the potential to reduce diarrhea mortality in children in developing countries. Available data to date do not indicate risk of intussusception with these new vaccines. To avoid a potential unanticipated risk post-licensure, it is recommended that rotavirus immunization be initiated before 12 weeks of age when background intussusception rates are low. This policy could exclude a substantial number of children from vaccination, especially in developing countries where delays in vaccination are common.
Related JoVE Video
Rotaviruses: from pathogenesis to vaccination.
Gastroenterology
PUBLISHED: 02-02-2009
Show Abstract
Hide Abstract
Rotaviruses cause life-threatening gastroenteritis in children worldwide; the enormous disease burden has focused efforts to develop vaccines and led to the discovery of novel mechanisms of gastrointestinal virus pathogenesis and host responses to infection. Two live-attenuated vaccines for gastroenteritis (Rotateq [Merck] and Rotarix) have been licensed in many countries. This review summarizes the latest data on these vaccines, their effectiveness, and challenges to global vaccination. Recent insights into rotavirus pathogenesis also are discussed, including information on extraintestinal infection, viral antagonists of the interferon response, and the first described viral enterotoxin. Rotavirus-induced diarrhea now is considered to be a disease that can be prevented through vaccination, although there are many challenges to achieving global effectiveness. Molecular biology studies of rotavirus replication and pathogenesis have identified unique viral targets that might be useful in developing therapies for immunocompromised children with chronic infections.
Related JoVE Video
Innate immune response to homologous rotavirus infection in the small intestinal villous epithelium at single-cell resolution.
Proc. Natl. Acad. Sci. U.S.A.
Show Abstract
Hide Abstract
"Bulk" measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. Flow cytometry and microfluidics-based single-cell multiplex RT-PCR allowed dissection of IFN responses in single RV-infected and bystander intestinal epithelial cells (IECs). EW replicates in IEC subsets differing in their basal type I IFN transcription and induces IRF3-dependent and IRF3-augmented transcription, but not NF-?B-dependent or type I IFN transcripts. Bystander cells did not display enhanced type I IFN transcription but had elevated levels of certain IFN-stimulated genes, presumably in response to exogenous IFNs secreted from immune cells. Comparison of IRF3 and NF-?B induction in STAT1(-/-) mice revealed that murine but not simian RRV mediated accumulation of IkB-? protein and decreased transcription of NF-?B-dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of "averaged" innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.
Related JoVE Video
Heterovariant cross-reactive B-cell responses induced by the 2009 pandemic influenza virus A subtype H1N1 vaccine.
J. Infect. Dis.
Show Abstract
Hide Abstract
The generation of heterovariant immunity is a highly desirable feature of influenza vaccines. The goal of this study was to compare the heterovariant B-cell response induced by the monovalent inactivated 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) vaccine with that induced by the 2009 seasonal trivalent influenza vaccine (sTIV) containing a seasonal influenza A virus subtype H1N1 (A[H1N1]) component in young and elderly adults.
Related JoVE Video
Human rotavirus-specific IgM Memory B cells have differential cloning efficiencies and switch capacities and play a role in antiviral immunity in vivo.
J. Virol.
Show Abstract
Hide Abstract
Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2R?(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.
Related JoVE Video
Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens.
Biotechnol. Bioeng.
Show Abstract
Hide Abstract
In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the "head" domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C-terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines.
Related JoVE Video
Rotavirus immune responses and correlates of protection.
Curr Opin Virol
Show Abstract
Hide Abstract
Selected topics in the field of rotavirus immunity are reviewed focusing on recent developments that may improve efficacy and safety of current and future vaccines. Rotaviruses (RVs) have developed multiple mechanisms to evade interferon (IFN)-mediated innate immunity. Compared to more developed regions of the world, protection induced by natural infection and vaccination is reduced in developing countries where, among other factors, high viral challenge loads are common and where infants are infected at an early age. Studies in developing countries indicate that rotavirus-specific serum IgA levels are not an optimal correlate of protection following vaccination, and better correlates need to be identified. Protection against rotavirus following vaccination is substantially heterotypic; nonetheless, a role for homotypic immunity in selection of circulating postvaccination strains needs further study.
Related JoVE Video
Preparedness of the CTSAs structural and scientific assets to support the mission of the National Center for Advancing Translational Sciences (NCATS).
Clin Transl Sci
Show Abstract
Hide Abstract
The formation of the National Center for Advancing Translational Sciences (NCATS) brings new promise for moving basic science discoveries to clinical practice, ultimately improving the health of the nation. The Clinical and Translational Science Award (CTSA) sites, now housed with NCATS, are organized and prepared to support in this endeavor. The CTSAs provide a foundation for capitalizing on such promise through provision of a disease-agnostic infrastructure devoted to clinical and translational (C&T) science, maintenance of training programs designed for C&T investigators of the future, by incentivizing institutional reorganization and by cultivating institutional support.
Related JoVE Video
Rhesus rotavirus trafficking during entry into MA104 cells is restricted to the early endosome compartment.
J. Virol.
Show Abstract
Hide Abstract
Endocytosis has recently been implicated in rotavirus (RV) entry. We examined the role of Rabs, which regulate endosomal trafficking, during RV entry. Several structural proteins of neuraminidase-sensitive and -insensitive RVs colocalized with Rab5, an early endosome marker, but not Rab7, a late endosome marker. Dominant-negative and constitutively active mutants demonstrated that Rab5 but not Rab4 or Rab7 affects rhesus RV (RRV) infectivity. These data suggest that early RRV trafficking is confined to the early endosome compartment and requires Rab5.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.