PLoS Pathog

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

The Immune Response to Astrovirus Infection

Astroviruses are one of the leading causes of pediatric gastroenteritis worldwide and are clinically importantly pathogens in the elderly and immunocompromised populations. Although the use of cell culture systems and small animal models have enhanced our understanding of astrovirus infection and pathogenesis, little is known about the immune response to astrovirus infection. Studies from humans and animals suggest that adaptive immunity is important in restricting classic and novel astrovirus infections, while studies from animal models and cell culture systems suggest that an innate immune system plays a role in limiting astrovirus replication. The relative contribution of each arm of the immune system in restricting astrovirus infection remains unknown. This review summarizes our current understanding of the immune response to astrovirus infection and highlights some of the key questions that stem from these studies. A full understanding of the immune response to astrovirus infection is required to be able to treat and control astrovirus-induced gastroenteritis

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction

Swine influenza virus (H1N2) characterization and transmission in ferrets, Chile

© 2017, Centers for Disease Control and Prevention (CDC). All rights reserved. Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed

PPxY-mediated endosomal escape prevents autophagic degradation of incoming virions.

<p>(A) Left panel: U2OS cells expressing the Pi3P <i>in cellulo</i> binding probe PX-GFP were treated with vehicle (top) or with 5mM of the Pi3K inhibitor 3’MA (bottom). Middle panel: U2OS cells pre-treated with vehicle alone (black bars) or 3’MA (red bars) were transduced with WT or M1. Transgene expression was determined and normalized to vehicle treated controls to show the fold induction of infectivity upon treatment. Right panel: The same data as in the middle panel showing the level of M1 infectivity rescue compared to the normalized WT infectivity upon treatment. (B) Left panel: U2OS cells were treated with vehicle (top) or with chloroquine (CQ, 50μM, bottom) to block the autophagic flux, fixed and stained for LC3. Middle panel: Cells were treated with vehicle alone (black bars) or chloroquine (red bars) and transduced with WT or M1. Transgene expression was determined and normalized to vehicle treated controls to show the fold induction of infectivity. Right panel: The same data as in the middle panel showing the level of M1 infectivity rescue compared to normalized WT infectivity. (C) Left panel: U2OS cells depleted for ATG5 (SH-ATG5) or control depleted cells (SH-CTRL) and starved using HBSS during 4h, fixed and stained for LC3. Middle panel: Cells were transduced with WT or M1 and the relative transduction efficiency in SH-CTRL cells (black bars) and SH-ATG5 cells (red bars) was determined. Right panel: The same data as in the middle panel showing the level of M1 infectivity rescue compared to normalized WT infectivity. ATG5 expression levels were determined by western blot. (D) Left panel: Control MEFs (ATG5 +/+) and KO MEFs (ATG5 -/-) were transduced with WT or M1 as indicated and the relative transduction efficiency for the M1 (red bars) compared to the WT (black bars) was determined. Right panel: The panel shows the absolute number of transduced cells at indicated amounts of physical particles added to the cell (pp/c) for the WT (black bars) and the M1 (red bars) in ATG5 control (left) and KO (right) MEFs. ATG5 expression levels were determined by western blot.</p

Nuclear transport of AdV involves the autophagic machinery.

<p>(A) Representative panel of WT or M1 infected cells at 1hpi stained with LC3 (red signal) and pericentrin (green signal) specific antibodies. (B) Quantification of the relative distribution of autophagosomes for experiment shown in (A) essentially analyzed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.g009" target="_blank">Fig 9E</a>. (n>12 cells; NS: no significant; *: P<0.05; **: P<0.01) (C) Representative panel of WT infected cells at 1hpi depleted for ATG5 or control depleted (as indicated) and stained with AdV (red signal) and pericentrin (green signal) specific antibodies. (D) Quantification of the relative virus distribution as in (B) for the experiment shown in (C) including the distribution of M1 and WT viruses. (n>12 cells; NS: no significant, **: P<0.01) (E) Representative panel of WT infected cells at 1hpi depleted for ATG5 or control depleted (as indicated) and stained with AdV (red signal) and PVI (green signal) specific antibodies to mark PVI separation from the virus. (F) Infection time course analysis of PVI release from M1 (red line) vs. WT (black line) viruses in ATG5 depleted (dotted line) vs. control depleted cells (solid line). Shown is the percentage of PVI positive AdV at indicated time points. The errors bars are cell-to-cell variation (10 cells were analyzed for each conditions). (G) Representative panel of WT infected cells at 1hpi and stained with specific antibodies against AdV (red signal) and specific antibodies against PVII (green signal) to mark nuclear genomes. (H) Quantification of nuclear genome delivery. ATG5 and control depleted cells were infected with WT and M1 and fixed at 1 and 2hpi and stained for AdV and PVII. The number of nuclear PVII dots was calculated and normalized for virus particles at each condition as indicated below the graph (n>16 cells; **: P<0.01; ***: P<0.0001). (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.s007" target="_blank">S7 Fig</a>).</p

AdV endosomal escape is dynein dependent.

<p>(A) Cells were pretreated with 100 μM of Ciliobrevin D for 30 minutes or DMSO and transduced with GFP expressing WT vector in presence of drugs. Twenty four hours later transduction levels were determined by FACS and normalized for the vehicle control (B) Cells were infected with fluorescent viruses in presence and absence of 100μM CilioD and fixed at different time points. The number of Gal3 punctae per cell was determined (C) Cells were infected as in B. Colocalization between Gal3 and AdV signals was quantified and is displayed as percentage of colocalization. (D) Cells were infected as in B. Fluorescent viruses in control or drug treated or control cells were quantified at indicated time points in the perinuclear region vs. the rest of the cytosol. Error bars (SE) show cell-to-cell variations.</p

AdV endosomal escape is PPxY dependent.

<p>HeLa cells (A) or U2OS cells (B) were infected with varying amounts of adenoviral vector particles expressing GFP (WT in black, M1 in red) and the percentage of GFP positive cells was determined 24hpi by FACS analysis. (C) U2OS cells expressing Gal3-mCherry (red signal) were infected with WT or M1 viruses and fixed at 30 minutes post infection and stained for AdV (green signal). Note that colocalization (yellow signal) indicates membrane rupture. (D) U2OS cells were infected with fluorescent viruses fixed at different time points and stained for endogenous GAL3. At indicated time points Gal3 positive signals where quantified. The total number of Gal3 punctae is indicated. (E) U2OS cells were infected as in D and cell-associated virus was quantified at indicated time points. (F) Colocalization between Gal3 and AdV signals was quantified and is displayed as percentage colocalization of total AdV signal. **: P<0.01 and errors bars are standard error. (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.s001" target="_blank">S1 Fig</a>).</p

AdV-WT limits autophagosome maturation and antigen presentation.

<p>(A) Cells were infected with WT and M1 viruses for indicated time points and cell lysates were analyzed by western blot with LC3 specific antibodies. Specific LC3 bands and GAPDH loading control are indicated. (NI = non infected). (B) The panel shows representative confocal images of U2OS cells infected for 1h with WT or M1 as indicated to left of each row and stained with Lamp2 (red signal) and LC3 (green signal) specific antibodies. (C) Quantification of autolysosomes from the experiment shown in (B) comparing the percentage of LC3 punctae positive for Lamp2 in WT vs. M1 infected cells as indicated below the graph. (D) Cells were starved overnight in HBSS then infected for one hour with AdV as indicated below the graph. Samples were fixed and stained for LC3 and Lamp2. The total number of LC3 punctae per cell at 1hpi is shown. (NI = non infected, DMEM = non starved control cells). (E) Experiment as in (D) showing the percentage of LC3 punctae also positive for Lamp2 (n>13 cells; NS: no significant; *: P<0.05; **: P<0.01; ***: P<0.001). (F) Mice were infected with 10¹⁰ GFP expressing vector particles of WT, M1 or PBS control. 10 days post infection mice were sacrificed and splenocytes were stimulated with AdV-luc or GFP purified from <i>E</i>.<i>coli</i>. IFNγ was determined by ELIspot. (G) CD4+ T-cell clones recognizing a conserved AdV hexon epitope were incubated 3:1 with syngeneic APCs transduced with either control media or WT or M1 vectors for 24 hours. IFNγ secretion was quantified by ELISA. (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.s005" target="_blank">S5 Fig</a>).</p