African Swine Fever Virus Is Wrapped by the Endoplasmic Reticulum

African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249–278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.African swine fever (ASF) virus is a large icosahedral enveloped DNA virus that causes a lethal hemorrhagic disease in domestic pigs. The virus is endemic in areas of southern Europe and in Africa where it causes major problems for the development of pig industries. At present there are no vaccines, and the disease is controlled through the slaughter of infected animals. The economic importance of ASF virus has made the virus the focus of much research since it was first described in 1921 (32). ASF virus is unique among animal viruses, and its classification has been controversial. ASF virus shares the striking icosahedral symmetry of iridoviruses (5, 8, 13, 34), while the presence of inverted terminal repeats and covalently linked ends in the 170-kDa genome suggests similarities with poxviruses (16). The ASF virus genome encoding at least 150 proteins has been sequenced (17, 51), and the amino acid sequences of at least 11 structural proteins are known. p73 is the major structural protein (14, 28) and has sequence similarities to the capsid protein of iridoviruses (39). The ordered proteolysis of pp220 produces p150, p37/p34 and p14 (40), which together comprise 25% of the viral proteins (3). These proteins localize to the interior of the virion (3). Three proteins, J13L/p54, I1L/p17, and p22, with membrane-spanning domains localize to the viral envelope (10, 37, 41, 43). Three other structural proteins, p14.5 encoded by E120R (30), p10 encoded by K78R (35), and p5AR encoded by A104R (7), have DNA-binding properties (51) and may be involved in DNA packaging. The virus has been the subject of several detailed electron microscopy studies (2–4, 8, 9, 11, 13, 34, 47). Electron micrographs of sections taken through ASF virus assembly sites reveal fully assembled virions as 200-nm hexagons and an ordered series of assembly intermediates with one to six sides of a hexagon. Close inspection of intracellular virions identifies multiple concentric layers of differing electron densities. According to recent models, the layers represent a central electron-dense nucleocapsid core, surrounded by an inner core shell, an inner envelope, and an outer capsid layer (3). The mechanism of formation of the inner envelope of ASF virus has not been resolved.Most viruses gain a single membrane envelope by budding into intracellular membrane compartments or from the plasma membrane, as reviewed in reference 21. When viruses bud into an intracellular compartment, the domains of the membrane proteins that are initially located in the lumen of membrane compartments are exposed on the outside of the virion after release from the cell (Fig. ​(Fig.1a).1a). A second mechanism of envelopment, described recently for poxviruses and herpesviruses (18, 20, 24, 38, 42, 46, 50), is more complex and involves the wrapping of virions by membrane cisternae derived from specific membrane compartments. Wrapping provides two membrane envelopes in one step and leaves the virion free in the cytoplasm. When compared with budding, wrapping reverses the orientation of membrane proteins within the virus such that the domains of membrane proteins located in the lumen of the wrapping organelle are confined to the interior of the virus after release from the cell, whereas cytoplasmic tails are exposed on the outside of the virus (Fig. ​(Fig.1b).1b). Given these important consequences for understanding the mechanism of assembly of the virus and for determining the final orientation of membrane proteins in virions, we have set out to determine whether ASF virus acquires its membranes by the conventional budding mechanism or whether the virus is wrapped by intracellular membrane compartments before release from the cell. Open in a separate windowFIG. 1Schematic comparison of budding and wrapping mechanisms of virus envelopment. (a) Budding. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins (|, membrane glycoproteins). Interactions between viral proteins lead to membrane curvature, and the virion gains a single membrane by budding into the lumen of the membrane compartment. When the virion is released from the cell, oligosaccharides () are exposed on the surface of the virus, and the cytoplasmic tail of the membrane glycoprotein is buried within the virion. (b) Wrapping. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins. The nucleoprotein complex is then wrapped by the membrane cisternae, and the virus gains two membranes. The particle remains in the cytosol. When the virion is released from the cell by cell lysis, oligosaccharides () are buried within the two membranes of the virion while the cytoplasmic tail of the membrane glycoprotein is exposed on the surface of the virus.In this study we have taken advantage of thin cryoelectron microscopic sections to enhance the definition of viral membranes. The micrographs show two membranes within mature intracellular virions and all structural intermediates. They also show assembly intermediates in continuity with cellular membrane compartments. Consistent with our earlier study showing that p73 was enveloped by the endoplasmic reticulum (ER) (15), immunogold labelling experiments show resident proteins of the ER within membranes found at assembly sites, in virus assembly intermediates, and in mature virions. Importantly, we have identified a protein (pXP124L) encoded by ASF virus that translocates completely into the lumen of the ER and is incorporated as a structural protein of the virus. The presence of two membranes within intracellular virions and structural intermediates and the packaging of a structural protein from the lumen of the ER into the virus, strongly suggest that ASF virus is wrapped by the ER.     

A Germination and Establishment Field Trial of Themeda australis (Kangaroo Grass) for Mine Site Restoration in the Central Tablelands of New South Wales

The current study demonstrated that Themeda australis (R. Br.) Stapf (kangaroo grass), a major understory component of the original grassy Box woodlands in the Central Tablelands of New South Wales, was suitable for use in large‐scale mine rehabilitation. The results of the trial were applied in the mine rehabilitation program. Due to extensive clearing of the woodlands and the introduction of exotic flora and fauna for agriculture, only small remnants of the original flora remain. The final land use of the gold mine is a conservation area free from agricultural pressure. Local native species adapted to the soil conditions and variable climate are highly desirable for the control of soil erosion following mining. Germination and establishment of T. australis on oxidized overburden were examined over 53 weeks. Seed‐bearing mulch was used as both the seeding material and an organic additive to the overburden. The effects of five factors were investigated: time since soil preparation, position within small contour banks, location of contour on experimental slopes, additional water, and rate of seed/mulch application. Direct seeding using the lowest rate of mulch application resulted in the establishment of more T. australis seedlings on new contours than on 15‐month‐old contours. The provision of additional water increased germination and establishment in both old and new contours, but was not essential. Seedling densities were greatest in the middle positions of contour banks up to week seven, but were destroyed during a heavy storm. At the close of the experiment, seedling densities were greater in the top and bottom positions of contours. Although significant slope × rate and age × slope interactions occurred, SNK tests did not reveal any consistent interpretable results.     

The ecology and evolution of the New World non-pollinating fig wasp communities

Abstract. We present data on several previously undescribed species from six genera of New World non-pollinating fig wasps. We show that many of these species have a negative effect on the reproductive success of both the pollinator wasps and the host figs. Our results suggest that the two most abundant genera of non-pollinating wasps, the Idarnes and the Critogaster , compete for the same pool of female flowers as the pollinating wasps in the Urostigma and Pharmacosycea figs, respectively. Wasps from the genus Aepocerus induce and develop within large galls, in the Urostigma figs. By draining resources from the fruit these wasps may have a detrimental effect on the production of pollinator wasps and viable seeds. Some of the species investigated are parasitoids of other non-pollinating species. We examine the importance of the various forms of spatial heterogeneity in the parasitism rate that can act to stabilise the host-parasitoid interaction. Finally, we discuss the factors underlying the large variation in the abundance and diversity of the non-pollinating wasps both among and within fruit crops.     

Comparative Analysis of the Full Genome of Helicobacter pylori Isolate Sahul64 Identifies Genes of High Divergence

Isolates of Helicobacter pylori can be classified phylogeographically. High genetic diversity and rapid microevolution are a hallmark of H. pylori genomes, a phenomenon that is proposed to play a functional role in persistence and colonization of diverse human populations. To provide further genomic evidence in the lineage of H. pylori and to further characterize diverse strains of this pathogen in different human populations, we report the finished genome sequence of Sahul64, an H. pylori strain isolated from an indigenous Australian. Our analysis identified genes that were highly divergent compared to the 38 publically available genomes, which include genes involved in the biosynthesis and modification of lipopolysaccharide, putative prophage genes, restriction modification components, and hypothetical genes. Furthermore, the virulence-associated vacA locus is a pseudogene and the cag pathogenicity island (cagPAI) is not present. However, the genome does contain a gene cluster associated with pathogenicity, including dupA. Our analysis found that with the addition of Sahul64 to the 38 genomes, the core genome content of H. pylori is reduced by approximately 14% (∼170 genes) and the pan-genome has expanded from 2,070 to 2,238 genes. We have identified three putative horizontally acquired regions, including one that is likely to have been acquired from the closely related Helicobacter cetorum prior to speciation. Our results suggest that Sahul64, with the absence of cagPAI, highly divergent cell envelope proteins, and a predicted nontransportable VacA protein, could be more highly adapted to ancient indigenous Australian people but with lower virulence potential compared to other sequenced and cagPAI-positive H. pylori strains.     

A role for ectophosphatase in xenobiotic resistance

Xenobiotic resistance in animals, plants, yeast, and bacteria is known to involve ATP binding cassette transporters that efflux invading toxins. We present data from yeast and a higher plant indicating that xenobiotic resistance also involves extracellular ATP degradation. Transgenic upregulation of ecto-ATPase alone confers resistance to organisms that have had no previous exposure to toxins. Similarly, cells that are deficient in extracellular ATPase activity are more sensitive to xenobiotics. On the basis of these and other supporting data, we hypothesize that the hydrolysis of extracellular ATP by phosphatases and ATPases may be necessary for the resistance conferred by P-glycoprotein.     

An outbreak of besnoitiosis in miniature donkeys

Fourteen miniature donkeys (Equus asinus) in a mid-Michigan herd of 38 animals presented with clinical signs of besnoitiosis, including the presence of typical tissue cysts in the ocular sclera, the buccal and nasal mucosa, together with characteristic dermatitis in specific areas of the body. The common histopathological change seen was the presence of many 100-200-microm diameter, thick walled, typical Besnoitia sp. tissue cysts together with a chronic cellular response associated with degenerating cysts. Microscopy of isolated scleral cysts and skin biopsies showed the presence of protozoal organisms consistent in morphology with that of Besnoitia bennetti bradyzoites. Molecular analysis of these parasites indicates that they differ from previously described coccidia, including Besnoitia sp., from rabbits and opossums. Isolated cases of infection with this agent have been reported infrequently in equids; however, this is the first report of an outbreak in a herd of donkeys in the United States.     

Situational Context and Perceived Threat Modulate Approachability Judgements to Emotional Faces

Facial expressions of emotion play a key role in guiding social judgements, including deciding whether or not to approach another person. However, no research has examined how situational context modulates approachability judgements assigned to emotional faces, or the relationship between perceived threat and approachability judgements. Fifty-two participants provided approachability judgements to angry, disgusted, fearful, happy, neutral, and sad faces across three situational contexts: no context, when giving help, and when receiving help. Participants also rated the emotional faces for level of perceived threat and labelled the facial expressions. Results indicated that context modulated approachability judgements to faces depicting negative emotions. Specifically, faces depicting distress-related emotions (i.e., sadness and fear) were considered more approachable in the giving help context than both the receiving help and neutral context. Furthermore, higher ratings of threat were associated with the assessment of angry, happy and neutral faces as less approachable. These findings are the first to demonstrate the significant role that context plays in the evaluation of an individual’s approachability and illustrate the important relationship between perceived threat and the evaluation of approachability.     

Urban gulls adapt foraging schedule to human‐activity patterns

Numerous animals are able to adapt to temporal patterns in natural food availability, but whether species living in relatively novel environments such as cities can adapt to anthropogenic activity cycles is less well understood. We aimed to assess the extent to which urban gulls have adapted their foraging schedule to anthropogenic food source fluctuations related to human activity by combining field observations at three distinct urban feeding grounds (park, school and waste centre) with global positioning system (GPS) tracking data of gulls visiting similar types of feeding grounds throughout the same city. We found that the birds' foraging patterns closely matched the timing of school breaks and the opening and closing times of the waste centre, but gull activity in the park appeared to correspond to the availability of natural food sources. Overall, this suggests that gulls may have the behavioural flexibility to adapt their foraging behaviour to human time schedules when beneficial and that this trait could potentially enable them to thrive in cities.     

Trypanosoma cruzi trypomastigotes induce cytoskeleton modifications during HeLa cell invasion

It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.