Bees can be trained to identify SARS-CoV-2 infected samples.

The COVID-19 pandemic has illustrated the need for the development of fast and reliable testing methods for novel, zoonotic, viral diseases in both humans and animals. Pathologies lead to detectable changes in the volatile organic compound (VOC) profile of animals, which can be monitored, thus allowing the development of a rapid VOC-based test. In the current study, we successfully trained honeybees (Apis mellifera) to identify SARS-CoV-2 infected minks (Neovison vison) thanks to Pavlovian conditioning protocols. The bees can be quickly conditioned to respond specifically to infected mink's odours and could therefore be part of a wider SARS-CoV-2 diagnostic system. We tested two different training protocols to evaluate their performance in terms of learning rate, accuracy and memory retention. We designed a non-invasive rapid test in which multiple bees are tested in parallel on the same samples. This provided reliable results regarding a subject's health status. Using the data from the training experiments, we simulated a diagnostic evaluation trial to predict the potential efficacy of our diagnostic test, which yielded a diagnostic sensitivity of 92% and specificity of 86%. We suggest that a honeybee-based diagnostics can offer a reliable and rapid test that provides a readily available, low-input addition to the currently available testing methods. A honeybee-based diagnostic test might be particularly relevant for remote and developing communities that lack the resources and infrastructure required for mainstream testing methods.

Enhanced virulence of sheep-passaged bovine spongiform encephalopathy agent is revealed by decreased polymorphism barriers in prion protein conversion studies.

UNLABELLED: Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to small ruminants (sheep and goats) with certain prion protein (PrP) genotypes. Polymorphisms in PrP of both the host and donor influence the transmission efficiency of transmissible spongiform encephalopathies (TSEs) in general. These polymorphisms in PrP also modulate the PrP conversion underlying TSE agent replication. Here we demonstrate that single-round protein misfolding cyclic amplification (PMCA) can be used to assess species and polymorphism barriers at the molecular level. We assessed those within and between the ovine and bovine species in vitro using a variety of natural scrapie and experimentally generated cross-species BSE agents. These BSE agents include ovBSE-ARQ isolates (BSE derived from sheep having the ARQ/ARQ PrP genotype), and two unique BSE-derived variants: BSE passaged in VRQ/VRQ sheep and a cow BSE agent isolate generated by back-transmission of ovBSE-ARQ into its original host. PMCA allowed us to quantitatively determine PrP conversion profiles that correlated with known in vivo transmissibility and susceptibility in the two ruminant species in which strain-specific molecular signatures, like its molecular weight after protease digestion, were maintained. Furthermore, both BSE agent isolates from ARQ and VRQ sheep demonstrated a surprising transmission profile in which efficient transmissions to both sheep and bovine variants was combined. Finally, all data support the notion that ARQ-derived sheep BSE points to a significant increase in virulence compared to all other tested scrapie- and BSE-derived variants reflected by the increased conversion efficiencies of previously inefficient convertible PrP variants (including the so-called "resistant" sheep ARR variant). IMPORTANCE: Prion diseases such as scrapie in sheep and goats, BSE in cattle, and Creutzfeldt-Jakob disease (CJD) in humans are fatal neurodegenerative diseases caused by prions. BSE is known to be transmissible to a variety of hosts, including sheep and humans. Based on the typical BSE agent strain signatures and epidemiological data, the occurrence of a novel variant of CJD in humans was linked to BSE occurrence in the United Kingdom. Measures, including genetic selection of sheep toward less susceptible PrP genotypes, have been implemented to lower the risk of BSE transmission into sheep, since the disease could potentially spread into a natural reservoir. In this study, we demonstrated using molecular PrP conversion studies that when BSE is first transmitted through sheep, the host range is modified significantly and the PrP converting potency increased, allowing the ovine BSE to transmit more efficiently than cow BSE into supposedly less susceptible hosts.

Prion protein self-interaction in prion disease therapy approaches.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt-Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP(Sc)), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP(C) into PrP(Sc) are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP(C) into PrP(Sc) and where the most drastic structural changes take place. Direct interactions between PrP(C) molecules and/or PrP(Sc) are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown.

Prion protein self-peptides modulate prion interactions and conversion.

BACKGROUND: Molecular mechanisms underlying prion agent replication, converting host-encoded cellular prion protein (PrP(C)) into the scrapie associated isoform (PrP(Sc)), are poorly understood. Selective self-interaction between PrP molecules forms a basis underlying the observed differences of the PrP(C) into PrP(Sc) conversion process (agent replication). The importance of previously peptide-scanning mapped ovine PrP self-interaction domains on this conversion was investigated by studying the ability of six of these ovine PrP based peptides to modulate two processes; PrP self-interaction and conversion. RESULTS: Three peptides (octarepeat, binding domain 2 -and C-terminal) were capable of inhibiting self-interaction of PrP in a solid-phase PrP peptide array. Three peptides (N-terminal, binding domain 2, and amyloidogenic motif) modulated prion conversion when added before or after initiation of the prion protein misfolding cyclic amplification (PMCA) reaction using brain homogenates. The C-terminal peptides (core region and C-terminal) only affected conversion (increased PrP(res) formation) when added before mixing PrP(C) and PrP(Sc), whereas the octarepeat peptide only affected conversion when added after this mixing. CONCLUSION: This study identified the putative PrP core binding domain that facilitates the PrP(C)-PrP(Sc) interaction (not conversion), corroborating evidence that the region of PrP containing this domain is important in the species-barrier and/or scrapie susceptibility. The octarepeats can be involved in PrP(C)-PrP(Sc) stabilization, whereas the N-terminal glycosaminoglycan binding motif and the amyloidogenic motif indirectly affected conversion. Binding domain 2 and the C-terminal domain are directly implicated in PrP(C) self-interaction during the conversion process and may prove to be prime targets in new therapeutic strategy development, potentially retaining PrP(C) function. These results emphasize the importance of probable PrP(C)-PrP(C) and required PrP(C)-PrP(Sc) interactions during PrP conversion. All interactions are probably part of the complex process in which polymorphisms and species barriers affect TSE transmission and susceptibility.

Mapping functional prion-prion protein interaction sites using prion protein based peptide-arrays.

Protein-protein interactions are at the basis of most if not all biological processes in living cells. Therefore, adapting existing techniques or developing new techniques to study interactions between proteins are of importance in elucidating which amino acid sequences contribute to these interactions. Such new insights may in turn lead to improved understanding of the processes underlying disease and possibly provide the basis for new therapeutic approaches. Here we describe the novel use of an ovine prion protein-based peptide-array normally used for determining prion-specific antibody epitopes, with the prospect that this would yield information on interaction sites between its PrP moiety and the ovine prion protein derived linear peptides. This adapted application of the peptide-array shows, by incubating the mature part of ovine (ARQ) PrPC fused to maltose binding protein (MBP), binding with between the PrP moiety and the ovine prion derived peptides occurs and indicates that several specific self-interactions between individual PrP molecules can occur; hereby illustrating that this adapted application of a peptide-array is a viable method to further specify which distinct amino acid sequences are involved in protein-protein interaction.

Transcriptome expression profiles in prenatal pigs in relation to myogenesis.

Myogenesis, the formation of muscle fibers, is a complex process. Pigs have been selected for efficient muscle growth for the past decades making them interesting to study myogenesis. We studied expression profiles of genes known to affect myogenesis, muscle structural proteins, and energy metabolism in prenatal pigs from 14 to 91 days of gestation. Primary and secondary muscle fiber formation takes place during days 30-60 and 54-90 of gestation, respectively. Differential expression and expression levels of the genes were studied using microarray technology. Gene activation and repression profiles were studied counting the number of spots with detectable signal. The number of spots for muscle tissue structural protein genes showing upregulated expression increased constantly from day 14 until day 91 of gestation indicating continued activation of genes during this period. The mRNA expression level of the genes showed a peak around day 35 of gestation. The expression levels of genes affecting myogenic differentiation (stimulating and inhibiting) showed a peak at day 35 of gestation. The number of spots for differentiation-stimulating genes showing differential expression reaches a first peak around day 35 of gestation and a nadir at day 49 of gestation while the number of spots for differentiation-inhibiting genes reaches a nadir at day 35 of gestation. Myogenic differentiation seems less a matter of the expression level of genes affecting differentiation, but depends on the balance between the number of significantly activated genes for stimulating and inhibiting differentiation. Genes stimulating myoblast proliferation showed a small peak expression prior to day 35 of gestation indicating myoblast proliferation before differentiation. The number of spots and the expression levels of genes for glycolysis and ATP-metabolism are at a nadir around days 35 and 49-63 of gestation suggesting that the energy metabolism is low during fusion of myoblasts into multinucleated muscle fibers.