Virus removal from semen with a pinched flow fractionation microfluidic chip.

Nowadays pigs are bred with artificial insemination to reduce costs and transportation. To prevent the spread of diseases, it is important to test semen samples for viruses. Screening techniques applied are enzyme-linked immunosorbent assays and/or polymerase chain reaction, which are labor-intensive and expensive methods. In contrast to the current used screening techniques, it is possible to remove viruses physically from semen. However, existing methods for virus removal techniques have a low yield of spermatozoa. Therefore, we have developed a microfluidic chip that performs size-based separation of viruses and spermatozoa in boar semen samples, thereby having the potential to reduce the risk of disease spreading in the context of artificial insemination in the veterinary industry. As the head of a spermatozoon is at least twenty times larger than a virus particle, the particle size can be used to achieve separation, resulting in a semen sample with lower viral load and of higher quality. To achieve the size separation, our microfluidic device is based on pinched-flow fractionation. A model virus, cowpea chlorotic mottle virus, was used and spiked to porcine semen samples. With the proposed microfluidic chip and the optimized flow parameters, at least 84 ± 4% of the model viruses were removed from the semen. The remaining virus contamination is caused by the model virus adhering to spermatozoa instead of the separation technique. The spermatozoa recovery was 86 ± 6%, which is an enormous improvement in yield compared to existing virus removal techniques.

Polymorphic assembly of virus-capsid proteins around DNA and the cellular uptake of the resulting particles.

Virus-like particles (VLPs), i.e. molecular assemblies that resemble the geometry and organization of viruses, are promising platforms for therapeutics and imaging. Understanding the assembly and cellular uptake pathways of VLPs can contribute to the development of new antiviral drugs and new virus-based materials for the delivery of drugs or nucleic acid-based therapies. Here we report the assembly of capsid proteins of the cowpea chlorotic mottle virus (CCMV) around DNA into defined structures at neutral pH. Depending on the type of DNA used, we are able to create spherical structures of various diameters and rods of various lengths. In order to determine the shape dependency, the cellular uptake routes and intracellular positioning of these formed polymorphic VLPs in RAW264.7, HeLa and HEK 293 cells are evaluated using flow cytometry analysis with specific chemical inhibitors for different uptake routes. We observed particular uptake routes for the various CCMV-based nanostructures, but the experiments point to clathrin-mediated endocytosis as the major route for cell entry for the studied VLPs. Confocal microscopy reveals that the formed VLPs enter the cells, with clear colocalization in the endosomes. The obtained results provide insight in the cargo dependent VLP morphology and increase the understanding of shape dependent uptake into cells, which is relevant in the design of new virus-based structures with applications in drug and gene delivery.

Immobilization of catalytic virus-like particles in a flow reactor.

A functional microfluidic reactor is constructed by the immobilization of gold containing virus-based protein cages that catalyze the reduction of nitro-arenes with high efficiency.

Photoresponsive, reversible immobilization of virus particles on supramolecular platforms.

Here we report on the covalent attachment of photoresponsive azobenzene moieties to cowpea chlorotic mottle virus (CCMV). The modified virus capsids can be reversibly immobilized on cucurbit[8]uril (CB[8]) bearing surfaces via supramolecular complexation.

Pt, Co-Pt and Fe-Pt alloy nanoclusters encapsulated in virus capsids.

Nanostructured Pt-based alloys show great promise, not only for catalysis but also in medical and magnetic applications. To extend the properties of this class of materials, we have developed a means of synthesizing Pt and Pt-based alloy nanoclusters in the capsid of a virus. Pure Pt and Pt-alloy nanoclusters are formed through the chemical reduction of [PtCl4](-) by NaBH4 with/without additional metal ions (Co or Fe). The opening and closing of the ion channels in the virus capsid were controlled by changing the pH and ionic strength of the solution. The size of the nanoclusters is limited to 18 nm by the internal diameter of the capsid. Their magnetic properties suggest potential applications in hyperthermia for the Co-Pt and Fe-Pt magnetic alloy nanoclusters. This study introduces a new way to fabricate size-restricted nanoclusters using virus capsid.

Versatile post-functionalization of the external shell of cowpea chlorotic mottle virus by using click chemistry.

We present the modification of the outer protein shell of cowpea chlorotic mottle virus (CCMV) with linear and strained alkyne groups. These functionalized protein capsids constitute valuable platforms for post-functionalization via click chemistry. After modification, the integrity of the capsid and the reversible disassembly behavior are preserved.

Using viruses as nanomedicines.

The field of nanomedicine involves the design and fabrication of novel nanocarriers for the intracellular delivery of therapeutic cargo or for use in molecular diagnostics. Although traditionally recognized for their ability to invade and infect host cells, viruses and bacteriophages have been engineered over the past decade as highly promising molecular platforms for the targeted delivery and treatment of many human diseases. Inherently biodegradable, the outer capsids of viruses are composed entirely of protein building blocks, which can be genetically or chemically engineered with molecular imaging reagents, targeting ligands and therapeutic molecules. While there are several examples of viruses as in vitro molecular cargo carriers, their potential for applications in nanomedicine has only recently emerged. Here we highlight recent developments towards the design and engineering of viruses for the treatment of cancer, bacterial infections and immune system-related diseases.

Biocatalytic oxidation by chloroperoxidase from Caldariomyces fumago in polymersome nanoreactors.

The encapsulation of chloroperoxidase from Caldariomyces fumago (CPO) in block copolymer polymersomes is reported. Fluorescence and electron microscopy show that when the encapsulating conditions favour self-assembly of the block copolymer, the enzyme is incorporated with concentrations that are 50 times higher than the enzyme concentration before encapsulation. The oxidation of two substrates by the encapsulated enzyme was studied: i) pyrogallol, a common substrate used to assay CPO enzymatic activity and ii) thioanisole, of which the product, (R)-methyl phenyl sulfoxide, is an important pharmaceutical intermediate. The CPO-loaded polymersomes showed distinct reactivity towards these substrates. While the oxidation of pyrogallol was limited by diffusion of the substrate into the polymersome, the rate-limiting step for the oxidation of thioansiole was the turnover by the enzyme.