Results of survey of <i>Angiostrongylus</i> worms among rodents in the Ogasawara Islands.

<p>Results of survey of <i>Angiostrongylus</i> worms among rodents in the Ogasawara Islands.</p

PfAtg8 is associated with membranes.

<p>(A) Specificity of the two independently generated anti-PfAtg8 antibodies (#1 and #2). Crude antisera and purified antibodies were used for immunoblotting of lysates of asynchronized <i>P. falciparum</i> parasites. (B) Expression of PfAtg8 increases during the erythrocytic stage of development. Highly synchronized <i>P. falciparum</i> parasites were collected at 0, 12, 24, 32, and 40 h after invasion. The duration of one cycle of the erythrocyte stage was approximately 42 h. Expression levels of PfAtg8 were analyzed by immunoblotting. An antibody against HSP70 was used as a loading control. (C) PfAtg8 exogenously expressed in mammalian cells (lane 1), endogenous PfAtg8 expressed in <i>P. falciparum</i> (lane 2), and the mixture of these two samples were subjected to immunoblot analysis using anti-PfAtg8 antibody. (D) Lysates of asynchronized <i>Plasmodium</i> were separated into low-speed (13,000×<i>g</i>) pellet (LSP), high-speed (100,000×<i>g</i>) pellet (HSP), and high-speed supernatant (HSS) fractions, and analyzed by immunoblotting using anti-PfAtg8 antibody. (E) The LSP fraction prepared in (D) was treated with 2 M urea or 2% Triton-X 100 and separated into 100,000×<i>g</i> pellet (P) and supernatant (S). (F) Infected erythrocytes were cultured in the presence of the indicated concentration of chloroquine and expression of PfAtg8 was analyzed.</p

PfAtg8 is associated with the apicoplast membrane.

<p>(A) <i>P. falciparum</i> FCR3 parasites at the schizont stage were analyzed by immunoelectron microscopy (immunogold and silver enhancement method) with an antibody against PfAtg8 (#1). (a) A schizont in an erythrocyte. (b) A magnified image of the area indicated in (a). (c) Another typical image of a PfAtg8-positive structure. (B) <i>P. falciparum</i> transfectant expressing ACP-GFP was analyzed as in panel (A) with an antibody against GFP. A, apicoplast; Mt, mitochondrion. Scale bars, (A, a) 1 μm, (A, b and c, and B) 200 nm.</p

Autophagy-Related Atg8 Localizes to the Apicoplast of the Human Malaria Parasite <em>Plasmodium falciparum</em>

<div><p>Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite <em>Plasmodium falciparum</em> (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although <em>Plasmodium</em> parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.</p> </div

PfAtg8 localizes to tubular and branched apicoplasts.

<p><i>P. falciparum</i> transfectant expressing ACP-GFP at late trophozoite and early schizont stages was stained with anti-GFP and anti-PfAtg8 antibodies and MitoTrackerRed CMXRos, and visualized by confocal microscopy. Scale bar, 1 μm.</p

PfAtg8 localizes to the apicoplast.

<p><i>P. falciparum</i> FCR3 (A–E) and <i>P. falciparum</i> 3D7 transfected with ACP-GFP (F–H) were stained with the indicated organelle markers and visualized by confocal microscopy (because ACP-GFP was not uniformly expressed, some merozoites displayed only faint GFP signals). Anti-PfAtg8 antibody #1 was used in (A–F), and anti-PfAtg8 antibody #2 was used in (G). Apical membrane antigen 1 (AMA1) as a microneme marker (A), rhoptry-associated protein 1 (RAP1) as a rhoptry body marker (B), rhoptry neck protein 2 (RON2) as a rhoptry neck marker (C), the ring-infected erythrocyte surface antigen (RESA) as a dense granule marker (D), MitoTrackerRed CMXRos as a mitochondria marker (E), ACP-GFP (F–H) and the organellar histone-like protein PfHU (H) as an apicoplast marker were used. Scale bar, 1 μm.</p

PfAtg8 localization is not affected by chloroquine or wortmannin treatment.

<p><i>P. falciparum</i> transfectant expressing ACP-GFP was treated with chloroquine (100 or 300 nM) (A), or wortmannin (10 μM) (B) for 2 h. Scale bar, 1 μm.</p

Identification of Novel Ghanaian G8P[6] Human-Bovine Reassortant Rotavirus Strain by Next Generation Sequencing

<div><p>Group A rotaviruses (RVAs) are the most important etiological agent of acute gastroenteritis in children <5 years of age worldwide. The monovalent rotavirus vaccine Rotarix was introduced into the national Expanded Programme on Immunization (EPI) in Ghana in May 2012. However, there is a paucity of genetic and phylogenetic data on the complete genomes of human RVAs in circulation pre-vaccine introduction. The common bovine rotavirus VP7 genotype G8 has been sporadically detected in Ghanaian children, usually in combination with the VP4 genotype P[6]. To investigate the genomic constellations and phylogeny of RVA strains in circulation prior to vaccine introduction, the full genomes of two unusual G8P[6] strains, GH018-08 and GH019-08, detected during burden of disease surveillance, were characterized by Illumina MiSeq sequencing. The Ghanaian isolates, GH018-08 and GH019-08, exhibited the unusual, previously unreported genotype constellation G8-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H3. Phylogenetic analyses confirmed that 10 out of the 11 genes of GH018-08 and GH019-08 were identical/nearly identical, with significant variation detected only in their VP1 genes, and clearly established the occurrence of multiple independent interspecies transmission and reassortment events between co-circulating bovine/ovine/caprine rotaviruses and human DS-1-like RVA strains. These findings highlight the contribution of reassortment and interspecies transmission events to the high rotavirus diversity in this region of Africa, and justify the need for simultaneous monitoring of animal and human rotavirus strains.</p></div

Phylograms based on the full-length open reading frame (ORF) nucleotide sequences of VP6, VP1, VP2 and VP3 encoding genome segments of RVA strains GH018-08 and GH019-08 indicating genetic relationships with cognate RVA genes.

<p>See Legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100699#pone-0100699-g001" target="_blank">Figure 1</a>.</p

Genotype constellation of GH018-08 and GH019-08 with selected reference RVA strains of known genomic constellation.

a<p>RVA strain names according to proposed nomenclature of new classification system. <b>Boldface</b> font indicates RVA strains sequenced in this study. GenBank accession numbers for reference strains are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100699#pone.0100699.s003" target="_blank">Table S2</a>.</p>b<p>DS-1-like genotypes indicated by <b>boldface</b> font;</p>†<p>Typical bovine-like RVA genotypes.</p>‡<p>AU-1-like genotype.</p