A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus into Dendritic Cells

Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation

Virus Movements on the Plasma Membrane Support Infection and Transmission between Cells

How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus–host interactions upstream of infectious entry offer new perspectives for anti-viral interference

Sanitary quality of edible bivalve mollusks in Southeastern Brazil using an UV based depuration system

The increase in seafood production, especially in mariculture worldwide, has brought out the need of continued monitoring of shellfish production areas in order to ensure safety to human consumption. The purpose of this research was to evaluate pathogenic protozoa, viruses and bacteria contamination in oysters before and after UV depuration procedure, in brackish waters at all stages of cultivation and treatment steps and to enumerate microbiological indicators of fecal contamination from production site up to depuration site in an oyster cooperative located at the Southeastern estuarine area of Brazil. Oysters and brackish water were collected monthly from September 2009 to November 2010. Four sampling sites were selected for enteropathogens analysis: site 1- oyster growth, site 2- catchment water (before UV depuration procedure), site 3 - filtration stage of water treatment (only for protozoa analysis) and site 4- oyster's depuration tank. Three microbiological indicators were examined at sites 1, 2 and 4. The following pathogenic microorganisms were searched: Giardia cysts, Cryptosporidium oocysts, Human Adenovirus (HAdV), Hepatitis A virus (HAV), Human Norovirus (HnoV) (genogroups I and II), JC strain Polyomavirus (JCPyV) and Salmonella sp. Analysis consisted of molecular detection (qPCR) for viruses (oysters and water samples); immunomagnetic separation followed by direct immunofluorescence assay for Cryptosporidium oocysts and Giardia cysts and also molecular detection (PCR) for the latter (oysters and water samples); commercial kit (Reveal-Neogee (R)) for Salmonella analysis (oysters). Giardia was the most prevalent pathogen in all sites where it was detected: 36.3%, 18.1%, 36.3% and 27.2% of water from sites 1, 2, 3 and 4 respectively; 36.3% of oysters from site 1 and 54.5% of depurated oysters were harboring Giardia cysts. The huge majority of contaminated samples were classified as Giardia duodenalis. HAdv was detected in water and oysters from growth site and HnoV GI in two batches of oysters (site 1) in huge concentrations (2.11 x 10(13), 3.10 x 10(12) gc/g). In depuration tank site, Salmonella sp., HAV (4.84 x 10(3)) and HnoV GII (7.97 x 10(14)) were detected once in different batches of oysters. Cryptosporidium spp. oocysts were present in 9.0% of water samples from site four. These results reflect the contamination of oysters even when UV depuration procedures are employed in this shellfish treatment plant. Moreover, the molecular comprehension of the sources of contamination is necessary to develop an efficient management strategy allied to shellfish treatment improvement to prevent foodborne illnesses. (C) 2011 Elsevier Ltd. All rights reserved.72SI9310

Spatially Variable Effects of Artificially-Created Physical Complexity on Subtidal Benthos

In response to the environmental damage caused by urbanization, Nature-based Solutions (NbS) are being implemented to enhance biodiversity and ecosystem processes with mutual benefits for society and nature. Although the field of NbS is flourishing, experiments in different geographic locations and environmental contexts have produced variable results, with knowledge particularly lacking for the subtidal zone. This study tested the effects of physical complexity on colonizing communities in subtidal habitats in two urban locations: (1) Plymouth, United Kingdom (northeast Atlantic) and (2) Tel Aviv, Israel (eastern Mediterranean) for 15- and 12-months, respectively. At each location, physical complexity was manipulated using experimental tiles that were either flat or had 2.5 or 5.0 cm ridges. In Plymouth, biological complexity was also manipulated through seeding tiles with habitat-forming mussels. The effects of the manipulations on taxon and functional richness, and community composition were assessed at both locations, and in Plymouth the survival and size of seeded mussels and abundance and size of recruited mussels were also assessed. Effects of physical complexity differed between locations. Physical complexity did not influence richness or community composition in Plymouth, while in Tel Aviv, there were effects of complexity on community composition. In Plymouth, effects of biological complexity were found with mussel seeding reducing taxon richness, supporting larger recruited mussels, and influencing community composition. Our results suggest that outcomes of NbS experiments are context-dependent and highlight the risk of extrapolating the findings outside of the context in which they were tested

Artrodese do quadril: estudo retrospectivo com mais de 20 anos de seguimento Hip arthrodesis: a minimum 20 year follow-up retrospective study

OBJETIVO: Avaliar os resultados a longo prazo de setenta pacientes submetidos à artrodese do quadril pela técnica original descrita por Davis. MÉTODOS: Realizamos um estudo clínico retrospectivo com 70 pacientes, operados entre 1982 e 1995. A presença de sintomas sobre a coluna lombar, joelho ipsilateral e quadril contralateral foi aferida assim como o sucesso da consolidação da artrodese, o posicionamento da mesma e a eventual indicação para conversão à artroplastia total do quadril. RESULTADOS: O tempo médio de seguimento foi 21,6 anos. Uma consolidação satisfatória foi constatada em 48 pacientes (85,7%). A dor lombar foi presente em 11 pacientes (19,6%) e osteoartrite dolorosa do joelho em quatro pacientes (7,1%). Discussão: Dois trabalhos com seguimento superior ao nosso estudo ( 35 e 38 anos) apresentaram repercussões sobre a coluna lombar mais prevalentes (57% e 62%), assim como dor no joelho homolateral (45% e 57%) e indicação para conversão em artroplastia total do quadril (17% e 28%). CONCLUSÃO: Concluímos que a artrodese do quadril pela técnica de Davis apresenta resultados satisfatórios até o vigésimo ano, porém a presença de dor lombar é mais frequente com o passar dos anos e a dor no joelho homolateral sugere associação com a posição inicial em abdução do quadril artrodesado. Nivel de Evidência III, estudo clinico.<br>OBJECTIVE: To evaluate the long-term results of seventy patients who underwent hip arthrodesis using the original technique described by Davis. METHODS: We carried out a retrospective study involving seventy patients submitted to hip arthrodesis between 1982 and 1995. The presence of symptoms involving the lumbar spine, ipsilateral knee, and contralateral hip was noted, as well as the success of the arthrodesis fusion, its positioning, and the need for conversion surgery to total hip replacement. RESULTS: The mean follow-up time was 21.6 years. A satisfactory hip fusion was found in 48 patients (85.7%). Lumbar spine pain was reported by 11 patients (19.6%) and ipsilateral knee osteoarthritis was found in four patients (7.1%). Discussion: Two works, with a longer follow-up time (35 and 38 years) present more prevalent symptoms involving the lumbar spine (57% and 62% ) and ipsilateral knee joint (45% and 57%), and higher conversion surgery rates (17% e 28%). CONCLUSION: hip arthrodesis by the Davis technique presents satisfactory results until the 20th year after surgery, however lumbar spine disease seems to become more prevalent over the years, and the pain in the homolateral knee suggests an association with the initial position in abduction of the hip submitted to arthrodesis. Level of Evidence: Level III clinical study