Combining multiple assays improves detection and serotyping of foot-and-mouth disease virus. A practical example with field samples from East Africa

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012–2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVβ6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%

Zebrafish Numb and Numblike Are Involved in Primitive Erythrocyte Differentiation

BACKGROUND:Notch signaling is an evolutionarily conserved regulatory circuitry implicated in cell fate determination in various developmental processes including hematopoietic stem cell self-renewal and differentiation of blood lineages. Known endogenous inhibitors of Notch activity are Numb-Nb and Numblike-Nbl, which play partially redundant functions in specifying and maintaining neuronal differentiation. Nb and Nbl are expressed in most tissues including embryonic and adult hematopoietic tissues in mice and humans, suggesting possible roles for these proteins in hematopoiesis. METHODOLOGY AND PRINCIPAL FINDINGS:We employed zebrafish to investigate the possible functional role of Numb and Numblike during hematopoiesis, as this system allows a detailed analysis even in embryos with severe defects that would be lethal in other organisms. Here we describe that nb/nbl knockdown results in severe reduction or absence of embryonic erythrocytes in zebrafish. Interestingly, nb/nbl knocked-down embryos present severe downregulation of the erythroid transcription factor gata1. This results in erythroblasts which fail to mature and undergo apoptosis. Our results indicate that Notch activity is increased in embryos injected with nb/nbl morpholino, and we show that inhibition of Notch activation can partially rescue the hematopoietic phenotype. CONCLUSIONS AND SIGNIFICANCE:Our results provide the first in vivo evidence of an involvement of Numb and Numblike in zebrafish erythroid differentiation during primitive hematopoiesis. Furthermore, we found that, at least in part, the nb/nbl morphant phenotype is due to enhanced Notch activation within hematopoietic districts, which in turn results in primitive erythroid differentiation defects

Diagnostic Performances of Different Genome Amplification Assays for the Detection of Swine Vesicular Disease Virus in Relation to Genomic Lineages That Circulated in Italy

During the last 25 years, swine vesicular disease (SVD) has occurred in Italy mostly sub-clinically. Therefore, regular testing of fecal samples from suspected holdings and high turnover premises was fundamental to identifying virus circulation and to achieve SVD eradication. In this study, we evaluated diagnostic performances of six genomic amplification methods, using positive fecal samples from 78 different outbreaks (1997–2014), which included different lineages. Comparison of three RT-PCRs, designed to amplify the same 154 nt portion of the gene 3D, demonstrated that a conventional and a real-time based on SYBR Green detection assay showed the highest diagnostic sensitivity, detecting all samples, while a real-time TaqMan-based test missed three cases, owing to two mismatches in the probe target sequence. Diagnostic and analytical specificities were optimal, as 300 negative field samples and other enteroviruses reacted negative. Three further evaluated tests, previously described, were a 3D-targeted reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and two real-time RT-PCRs targeted on the 5′UTR region. Here, the presence of multiple mismatches in probe and primers reduced the diagnostic performances, and two of the assays were unable to detect viruses from one sub-lineage. These results highlight that the choice of tests using less nucleotide targets significantly contributed to the success of the SVD eradication plan

Induced early expression of mrf4 but not myog rescues myogenesis in the myod/myf5 double-morphant zebrafish embryo

Muscle regulatory factors activate myogenesis in all vertebrates, but their role has been studied in great detail only in the mouse embryo, where all but myogenin--Myod, Myf5 and Mrf4--are sufficient to activate (albeit not completely) skeletal myogenesis. In the zebrafish embryo, myod and myf5 are required for induction of myogenesis because their simultaneous ablation prevents muscle development. Here we show that mrf4 but not myog can fully rescue myogenesis in the myod/myf5 double morphant via a selective and robust activation of myod, in keeping with its chromatin-remodelling function in vitro. Rescue does not happen spontaneously, because the gene, unlike that in the mouse embryo, is expressed only at the onset of muscle differentiation, Moreover, because of the transient nature of morpholino inhibition, we were able to investigate how myogenesis occurs in the absence of a myotome. We report that in the complete absence of a myotome, subsequent myogenesis is abolished, whereas myogenesis does proceed, albeit abnormally, when the morpholino inhibition was not complete. Therefore our data also show that the early myotome is essential for subsequent skeletal muscle differentiation and patterning in the zebrafish

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos

Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms–3 μm time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos’ vessels

In vivo imaging of zebrafish from embryo to adult stage with optical projection tomography

Optical Projection Tomography (OPT) is a three dimensional imaging technique that is particularly suitable for studying millimeter sized biological samples and organisms. Similarly to x-ray computed tomography, OPT is based on the acquisition of a sequence of images taken through the sample at many angles (projections). Assuming the linearity of the optical absorption process, the projections are combined to reconstruct the 3-D volume of the sample, typically using a filtered back-projection algorithm. OPT has been applied to in-vivo imaging of zebrafish (Danio rerio). The instrument and the protocol for in vivo imaging of zebrafish embryos and juvenile specimens are described. Light scattering remains a challenge for in vivo OPT, especially when samples at the upper size limit, like zebrafish at the adult stage, are under study. We describe Time-Gated Optical Projection Tomography (TGOPT), a technique able to reconstruct adult zebrafish internal structures by counteracting the scattering effects through a fast time-gate. The time gating mechanism is based on non-linear optical upconversion of an infrared ultrashort laser pulse and allows the detection of quasi-ballistic photons within a 100 fs temporal gate. This results in a strong improvement in contrast and resolution with respect to conventional OPT. Artifacts in the reconstructed images are reduced as well. We show that TGOPT is suited for imaging the skeletal system and nervous structures of adult zebrafis

Sphingosine-1-Phosphate Receptor-1 Controls Venous Endothelial Barrier Integrity in Zebrafish

Objective-Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P1) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P(1) ortholog in vascular development in zebrafish. Methods and Results-zS1P(1) is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P(1) downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P(1) morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P(1) morphants. Finally, S1P(1) small interfering RNA transfection and the S1P1 antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. Conclusion-The data demonstrate a nonredundant role of zS1P(1) in the regulation of venous endothelial barrier in zebrafish and identify a S1P(1)/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity. (Arterioscler Thromb Vasc Biol. 2012;32:e104-e116.

Ve-ptp Modulates Vascular Integrity by Promoting Adherens Junction Maturation

<div><h3>Background</h3><p>Endothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. <em>In vivo</em> studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs).</p> <h3>Methodology/Principal Findings</h3><p>We investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (<em>Danio rerio</em>). Whole-mount <em>in situ</em> hybridizations revealed z<em>ve-ptp</em> expression exclusively in the developing vascular system. Generation of altered z<em>ve-ptp</em> transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in z<em>ve-ptp</em> morphants but not in control embryos.</p> <h3>Conclusions/Significance</h3><p>Here we show the first <em>in vivo</em> evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system.</p> </div

Spatio-temporal expression pattern of z<i>ve-ptp</i>.

<p>(A) z<i>ve-ptp</i> and <i>β-actin</i> temporal expression analysis is performed by qualitative RT-PCR analysis on total RNAs extracted from oocytes and different embryonic and larval stages (from 2 cells to 120 hpf). Negative control is showed in the right-most line. The sizes of the obtained PCR fragments are indicated. z<i>ve-ptp</i> is expressed both maternally and zygotically during zebrafish development. (B–K) The z<i>ve-ptp</i> spatial expression was analyzed by WISH at various developmental stages. Lateral (B) and dorsal (C) view of a 26 hpf embryo with anterior to the left; (B′) higher magnification of the tail shows the expression in the DA and CV. The transverse sections at the level of the head (D) and the tail (E) of a 26 hpf embryo labelled with z<i>ve-ptp</i> probe show in detail the signal in head vessels and in the DA and CV. Lateral views of head (F) and magnification of the tail (G) of a 2 dpf embryo and lateral view of the head of a 3 dpf embryo (H). Cross sections at the level of head (I), aortic arches (J) and trunk (K) of a 3 dpf embryo. Black asterisk: AA1; black arrowhead: DA; white arrowhead: CV; red arrowhead: LDA; white arrows: vessels around the eyes; blue arrows: cranial vessels; black arrow: Se; red asterisks: aortic arches; light blue arrows: vessels around the lens; white asterisks: DLAVs.</p

Immunofluorescence analysis by confocal microscopy of endothelial intercellular junctions.

<p>Immunofluorescence experiments were performed on tg(<i>fli1</i>:EGFP)^y1 embryos at 2 dpf. Confocal images of tail vessels of std-MO (A, E) and MOb injected embryos (B, F) labeled with an anti-ZO-1 and VE-cadherin antibody (red) and the respective merged images (C, D, G, H). Se: intersegmental vessels.</p