Incursion of Schmallenberg virus into Great Britain in 2011 and emergence of variant sequences in 2016.

Schmallenberg virus (SBV) is a vector-borne orthobunyavirus in the family Bunyaviridae, first identified in Germany before rapidly spreading throughout Europe. To investigate the events surrounding the incursion of this virus into Great Britain (GB) and its subsequent spread, archived sheep serum samples from an unrelated field survey in 2011 were analysed for the presence of SBV specific antibodies, to determine the earliest date of seroconversion. This serological study, along with analysis of the spatial spread of the sources of samples submitted for SBV analysis after January 2012, suggests that SBV entered GB on more than one occasion and in more than one location. Phylogenetic analysis of SBV sequences from 2012 ovine samples, from a variety of counties and dates, demonstrated a non-linear evolution of the virus, i.e. there was no distinct clustering between host species, geographical locations or during the outbreak. This also supports the notion of multiple viruses entering GB, rather than a single virus incursion. Premature termination signals were present in several non-structural putative protein sequences. One SBV sequence exhibited large deletions in the M segment of the genome. After the first outbreak in 2011-2012, interest in SBV in GB waned and continuous surveillance was not upheld. The re-emergence of SBV in 2016 has raised renewed concern and ended speculation that SBV might have been eradicated permanently from GB. When SBV sequences from 2012 were compared with those from the re-emergence in 2016-2017, a second distinct clade of SBV was identified that separates recent strains from those observed during the first outbreak.

Bone toughness at the molecular scale: A model for fracture toughness using crosslinked osteopontin on synthetic and biogenic mineral substrates.

The most prominent structural components in bone are collagen and mineral. However, bone additionally contains a substantial amount of noncollagenous proteins (most notably of the SIBLING protein family), some of which may act as cohesive/adhesive "binders" for the composite hybrid collagen/mineral scaffolding, whether in the bulk phase of bone, or at its interfaces. One such noncollagenous protein - osteopontin (OPN) - appears to be critical to the deformability and fracture toughness of bone. In the present study, we used a reconstructed synthetic mineral-OPN-mineral interface, and a biogenic (natural tooth dentin) mineral/collagen-OPN-mineral/collagen interface, to measure the fracture toughness of OPN on mineralized substrates. We used this system to test the hypothesis that OPN crosslinking by the enzyme tissue transglutaminase 2 (TG2) that is found in bone enhances interfacial adhesion to increase the fracture toughness of bone. For this, we prepared double-cantilever beam substrates of synthetic pure hydroxyapatite mineral, and of narwhal dentin, and directly apposed them to one another under different intervening OPN/crosslinking conditions, and fracture toughness was tested using a miniaturized loading stage. The work-of-fracture of the OPN interface was measured for different OPN formulations (monomer vs. polymer), crosslinking states, and substrate composition. Noncrosslinked OPN provided negligible adhesion on pure hydroxyapatite, whereas OPN crosslinking (by the chemical crosslinker glutaraldehyde, and TG2 enzyme) provided strong interfacial adhesion for both hydroxyapatite and dentin using monomeric and polymeric OPN. Pre-coating of the substrate beams with monomeric OPN further improved the adhesive performance of the samples, likely by allowing effective binding of this nascent OPN form to mineral/matrix components, with this pre-attachment providing a protein layer for additional crosslinking between the substrates.

Teleost fish scales amongst the toughest collagenous materials.

Fish scales from modern teleost fish are high-performance materials made of cross-plies of collagen type I fibrils reinforced with hydroxyapatite. Recent studies on this material have demonstrated the remarkable performance of this material in tension and against sharp puncture. Although it is known that teleost fish scales are extremely tough, actual measurements of fracture toughness have so far not been reported because it is simply not possible to propagate a crack in this material using standard fracture testing configurations. Here we present a new fracture test setup where the scale is clamped between two pairs of miniature steel plates. The plates transmit the load uniformly, prevent warping of the scale and ensure a controlled crack propagation. We report a toughness of 15 to 18kJm(-2) (depending on the direction of crack propagation), which confirms teleost fish scales as one of the toughest biological material known. We also tested the individual bony layers, which we found was about four times less tough than the collagen layer because of its higher mineralization. The mechanical response of the scales also depends on the cohesion between fibrils and plies. Delamination tests show that the interface between the collagen fibrils is three orders of magnitude weaker than the scale, which explains the massive delamination and defibrillation observed experimentally. Finally, simple fracture mechanics models showed that process zone toughening is the principal source of toughening for the scales, followed by bridging by delaminated fibrils. These findings can guide the design of cross-ply composites and engineering textiles for high-end applications. This study also hints on the fracture mechanics and performance of collagenous materials with similar microstructures: fish skin, lamellar bone or tendons.

Overcoming the brittleness of glass through bio-inspiration and micro-architecture.

Highly mineralized natural materials such as teeth or mollusk shells boast unusual combinations of stiffness, strength and toughness currently unmatched by engineering materials. While high mineral contents provide stiffness and hardness, these materials also contain weaker interfaces with intricate architectures, which can channel propagating cracks into toughening configurations. Here we report the implementation of these features into glass, using a laser engraving technique. Three-dimensional arrays of laser-generated microcracks can deflect and guide larger incoming cracks, following the concept of 'stamp holes'. Jigsaw-like interfaces, infiltrated with polyurethane, furthermore channel cracks into interlocking configurations and pullout mechanisms, significantly enhancing energy dissipation and toughness. Compared with standard glass, which has no microstructure and is brittle, our bio-inspired glass displays built-in mechanisms that make it more deformable and 200 times tougher. This bio-inspired approach, based on carefully architectured interfaces, provides a new pathway to toughening glasses, ceramics or other hard and brittle materials.

Replication of hepatitis E virus in three-dimensional cell culture.

Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.

Cohesive behavior of soft biological adhesives: experiments and modeling.

Extracellular proteins play a key role in generating and maintaining cohesion and adhesion in biological tissues. These "natural glues" are involved in vital biological processes such as blood clotting, wound healing and maintaining the structural integrity of tissues. Macromolecular assemblies of proteins can be functionally stabilized in a variety of ways in situ that include ionic interactions as well as covalent crosslinking to form protein networks that can extend both within and between tissues. Within tissues, myriad cohesive forces are required to preserve tissue integrity and function, as are additional appropriate adhesive forces at interfaces both within and between tissues of differing composition. While the mechanics of some key structural adhesive proteins have been characterized in tensile experiments at both the macroscopic and single protein levels, the fracture toughness of thin proteinaceous interfaces has never been directly measured. Here, we describe a novel and simple approach to measure the cohesive behavior and toughness of thin layers of proteinaceous adhesives. The test is based on the standard double-cantilever beam test used for engineering adhesives, which was adapted to take into account the high compliance of the interface compared with the beams. This new "rigid double-cantilever beam" method enables stable crack propagation through an interfacial protein layer, and provides a direct way to measure its full traction-separation curve. The method does not require any assumption of the shape of the cohesive law, and the results provide abundant information contributing to understanding the structural, chemical and molecular mechanisms acting in biological adhesion. As an example, results are presented using this method for thin films of fibrin-a protein involved in blood clotting and used clinically as a tissue bio-adhesive after surgery-with the effects of calcium and crosslinking by Factor XIII being examined. Finally, a simple model is proposed, demonstrating how a bell-shaped cohesive law forms during the failure of the fibrin interface based on an eight-chain model whose structure degrades and changes configuration with stress.

Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR.

This study assessed the feasibility of identifying asymptomatic viral shedders using a novel TaqMan real-time PCR on trunk washes and swabs from the conjunctiva, palate and vulva of elephants. Six elephants from a UK collection were sampled weekly over a period of 11 weeks for this study. The herd prevalence of elephant endotheliotropic herpesvirus-1 (EEHV-1) was 100 per cent by PCR. The virus DNA was detected in all the sampling sites; however, the prevalence of virus DNA in the conjunctiva swabs was higher. In addition, Asian elephants from two continental European collections were sampled once and one animal tested positive on a trunk wash. The virus from this animal was phylogenetically typed as EEHV-1A based on 231 nucleotides of the terminase gene.