Villalba, R. | bioRxiv Science

bioRxiv2025

Modification and validation of a reference real-time RT-PCR method for the detection of a new African horse sickness virus variant

African horse sickness (AHS) is a disease affecting equids caused by the AHS virus (AHSV). The World Organization for Animal Health (WOAH) includes AHS as a notifiable disease and, upon detection within the European Union, immediate control and eradication measures are mandated. Thus, validated diagnostic methods for rapid AHSV detection are essential. The Aguero 2008 and Guthrie 2013 rRT-PCR methods have been widely validated for detection of any AHSV strain and are included as reference rRT-PCRs in the WOAH manual. However, the WOAH reference laboratory for AHS in the Republic of South Africa (RSA), reported an AHSV variant undetected by the Aguero 2008 rRT-PCR. Therefore, a set of modified primers and probe, containing degenerate positions to avoid mismatches with the sequence of the new RSA strain, was developed. The modified-Aguero method was validated by the WOAH reference laboratories in Spain and the UK employing a broad collection of AHSV strains, clinical samples as well as a synthetic RNA mimicking the target sequence of the new RSA AHSV variant (AHSV-sRNA-RSA). Comparative assessment of the modified-Aguero versus the WOAH reference rRT-PCRs, showed that the modified method exhibited a good diagnostic performance and enabled the detection of the new RSA AHSV variant nucleic acid.

microbiology↗

bioRxiv2023

Structural Plasticity of GABAergic Pallidothalamic Terminals in MPTP-treated Parkinsonian Monkeys: A 3D Electron Microscopic Analysis

The globus pallidus pars interna (GPi) is a major source of GABAergic inhibition upon the motor thalamus. GPi neurons are endowed with properties that allow them to fire at a high rate and maintain a tonic inhibitory influence upon thalamocortical neurons. In parkinsonism, the firing rate of GPi neurons is further increased and their firing pattern switches from a tonic to a bursty mode, two pathophysiological changes associated with increased GABAergic pallidothalamic activity. At the thalamic level, GPi terminals display ultrastructural features (large diameter, multiple synapses, large number of mitochondria) that allow them to maintain tonic synaptic inhibition at high firing rate upon thalamocortical neurons in the parvocellular ventral anterior nucleus (VApc) and the centromedian nucleus (CM), the two main GPi-recipient motor thalamic nuclei in nonhuman primates. To determine if changes of GPi neurons activity are associated with neuroplastic reorganization of GPi terminals and their synapses, we used a Single Block Facing/Scanning Electron Microscopy (SBF/SEM), high resolution 3D electron microscopic approach to compare the morphometry of GPi terminals between 2 control and 2 MPTP-treated parkinsonian monkeys. Our findings demonstrate that pallidothalamic terminals in VApc and CM undergo major ultrastructural changes in parkinsonian monkeys: (1) increased terminal volume in both nuclei, (2) increased surface area of synapses in both nuclei, (3) increased number of synapses/GPi terminals in the CM, but not VApc, (4) increased total volume of mitochondria/terminals in both nuclei but not in the number of mitochondria. In contrast, the ultrastructure of putative GABAergic terminals from the reticular thalamic nucleus was not affected in both the VApc and CM of parkinsonian monkeys. Our findings also show striking morphological differences in terminal volume, number/area of synapses and volume/number of mitochondria between GPi terminals in VApc and CM of control monkeys. In conclusion, results of this study demonstrate that GABAergic pallidothalamic terminals are endowed with a high level of structural plasticity that may contribute to the development and maintenance of the abnormal increase in pallidal GABAergic outflow to the thalamus in the parkinsonian state. Furthermore, the evidence for ultrastructural differences between GPi terminals in VApc and CM suggests that Morphologically distinct pallidothalamic terminals underlie specific physiological properties of pallidal inputs to VApc and CM in normal and diseased states.

neuroscience↗