Neutralization Susceptibility of African Swine Fever Virus Is Dependent on the Phospholipid Composition of Viral Particles

AbstractIn this study we have investigated the generation of African swine fever (ASF) virus variants resistant to neutralizing antibodies after cell culture propagation. All highly passaged ASF viruses analyzed were resistant to neutralization by antisera from convalescent pigs or antibodies generated against individual viral proteins which neutralized low-passage viruses. A molecular analysis of neutralizable and nonneutralizable virus isolates by sequencing of the genes encoding for neutralizing proteins revealed that the absence of neutralization of high-passage viruses is not due to antigenic variability of critical epitopes. A comparative analysis of phospholipid composition of viral membranes between low- and high-passage viruses revealed differences in the relative amount of phosphatidylinositol in these two groups of viruses, independent of the cells in which the viruses were grown. Further purification of low- and high-passage viruses by Percoll sedimentation showed differences in the phospholipid composition identical to those found with the partially purified viruses and confirmed the susceptibility of these viruses to neutralization. The incorporation of phosphatidylinositol into membranes of high-passage viruses rendered a similar neutralization susceptibility to low-passage viruses, in which this is a major phospholipid. In contrast, other phospholipids did not interfere with high-passage virus neutralization, suggesting that phosphatidylinositol is essential for a correct epitope presentation to neutralizing antibodies. Additionally, the removal of phosphatidylinositol from a low-passage virus by a specific lipase transformed this virus from neutralizable to nonneutralizable. These data constitute clear evidence of the importance of the lipid composition of the viral membranes for the protein recognition by antibodies and may account in part for the past difficulties in reproducibly demonstrating ASF virus-neutralizing antibodies by using high-passage viruses

A sandwich ELISA to detect VHSV and IPNV in turbot

Abstract: The recent demonstration that reared turbot (Scophthalmus maximus L) is a natural host for salmonid rhabdoviruses has made their rapid detection relevant to these fish species. A unique protocol to select and use non-competitive monoclonal antibodies (Mabs) for two high-sensitivity sandwich ELISAs has been developed to detect both infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) in turbot kidney extracts to assess the possibility of using them in field diagnosis. For maximum sensitivity, turbot kidney extracts can be two-fold diluted with high-ionic strength buffers and assayed for the presence of the major viral proteins (VMS rhabdovirus nucleoprotein N/Nx and/or IPN birnavirus protein VP3). The use of control plates coated with irrelevant mouse antibodies (IgG1 and IgG2a) in parallel ELISAs allows for a precise estimation of possible false positives. Turbot kidney extracts with low levels of virus might now be assayed directly without using cell culture, with high precision and in a short time during the acute phase of these viral diseases in reared turbot.Acuidoro, SL; INI

Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus)

DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential use as vaccine adjuvants, antiviral treatments or markers for vaccine efficiency monitoring

Influence of a Concurrent Exercise Training Intervention during Pregnancy on Maternal and Arterial and Venous Cord Serum Cytokines: The GESTAFIT Project

The aim of the present study was to analyze the influence of a supervised concurrent exercise-training program, from the 17th gestational week until delivery, on cytokines in maternal (at 17th and 35th gestational week, and at delivery) and arterial and venous cord serum. Fifty-eight Caucasian pregnant women (age: 33.5 +/- 4.7 years old, body mass index: 23.6 +/- 4.1kg/m(2)) from the GESTAFIT Project (exercise (n = 37) and control (n = 21) groups) participated in this quasi-experimental study (per-protocol basis). The exercise group followed a 60-min 3 days/week concurrent (aerobic-resistance) exercise-training from the 17th gestational week to delivery. Maternal and arterial and venous cord serum cytokines (fractalkine, interleukin (IL)-1 beta, IL-6, IL-8, IL-10, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) were assessed using Luminex xMAP technology. In maternal serum (after adjusting for the baseline values of cytokines), the exercise group decreased TNF-alpha (from baseline to 35th week, p = 0.02), and increased less IL-1 beta (from baseline to delivery, p = 0.03) concentrations than controls. When adjusting for other potential confounders, these differences became non-significant. In cord blood, the exercise group showed reduced arterial IL-6 and venous TNF-alpha (p = 0.03 and p = 0.001, respectively) and higher concentrations of arterial IL-1 beta (p = 0.03) compared to controls. The application of concurrent exercise-training programs could be a strategy to modulate immune responses in pregnant women and their fetuses. However, future research is needed to better understand the origin and clearance of these cytokines, their role in the maternal-placental-fetus crosstalk, and the influence of exercise interventions on them

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year

Fast and efficient neural conversion of human hematopoietic cells

Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency

Proyecto de extensión universitaria: Manipulación de Alimentos, Educación y Seguridad Alimentaria (M.E.S.A) relevamiento bromatológico y control de alimentos en instituciones educativas y recreativas de la ciudad de La Plata

El objetivo de este Proyecto es el de formar Agentes Multiplicadores con criterio bromatológico que fomenten normas de control sanitario, aseguren la calidad e inocuidad de los alimentos que llegan al consumidor y apoyen la transformación, a través de Capacitación y Asistencia técnica, teniendo como punto de partida la Educación para la Salud y la Educación Ecológica.Mesa: Alimentos y Vacunas.Facultad de Ciencias Veterinaria

Proyecto de extensión universitaria: Manipulación de Alimentos, Educación y Seguridad Alimentaria (M.E.S.A) relevamiento bromatológico y control de alimentos en instituciones educativas y recreativas de la ciudad de La Plata

El objetivo de este Proyecto es el de formar Agentes Multiplicadores con criterio bromatológico que fomenten normas de control sanitario, aseguren la calidad e inocuidad de los alimentos que llegan al consumidor y apoyen la transformación, a través de Capacitación y Asistencia técnica, teniendo como punto de partida la Educación para la Salud y la Educación Ecológica.Mesa: Alimentos y Vacunas.Facultad de Ciencias Veterinaria

Development of a novel anti-CD19 chimeric antigen receptor: A paradigm for an affordable CAR T cell production at academic institutions

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdcscid Il2rdtm1Wjl/SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients

Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae

<p>Abstract</p> <p>Background</p> <p>There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in <it>E. coli</it>, yeast and baculovirus grown in insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV).</p> <p>Findings</p> <p>Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in insect larvae. Recognition of G21-465 by ß-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (<it>Oncorhynchus mykiss</it>) was demonstrated.</p> <p>Conclusions</p> <p>Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.</p