A novel mechanism of RNase L inhibition: Theiler\u27s virus L* protein prevents 2-5A from binding to RNase L

<div><p>The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L <i>in vivo</i>. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.</p></div

Advanced biosensors for detection of pathogens related to livestock and poultry

Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health and well-being of wildlife, livestock, and human populations, limit productivity and increase significantly economic losses to each sector. The pathogen detection is an important step for the diagnostics, successful treatment of animal infection diseases and control management in farms and field conditions. Current techniques employed to diagnose pathogens in livestock and poultry include classical plate-based methods and conventional biochemical methods as enzyme-linked immunosorbent assays (ELISA). These methods are time-consuming and frequently incapable to distinguish between low and highly pathogenic strains. Molecular techniques such as polymerase chain reaction (PCR) and real time PCR (RT-PCR) have also been proposed to be used to diagnose and identify relevant infectious disease in animals. However these DNA-based methodologies need isolated genetic materials and sophisticated instruments, being not suitable for in field analysis. Consequently, there is strong interest for developing new swift point-of-care biosensing systems for early detection of animal diseases with high sensitivity and specificity. In this review, we provide an overview of the innovative biosensing systems that can be applied for livestock pathogen detection. Different sensing strategies based on DNA receptors, glycan, aptamers and antibodies are presented. Besides devices still at development level some are validated according to standards of the World Organization for Animal Health and are commercially available. Especially, paper-based platforms proposed as an affordable, rapid and easy to perform sensing systems for implementation in field condition are included in this review

Phage Therapy and Photodynamic Therapy: Low Environmental Impact Approaches to Inactivate Microorganisms in Fish Farming Plants

Owing to the increasing importance of aquaculture to compensate for the progressive worldwide reduction of natural fish and to the fact that several fish farming plants often suffer from heavy financial losses due to the development of infections caused by microbial pathogens, including multidrug resistant bacteria, more environmentally-friendly strategies to control fish infections are urgently needed to make the aquaculture industry more sustainable. The aim of this review is to briefly present the typical fish farming diseases and their threats and discuss the present state of chemotherapy to inactivate microorganisms in fish farming plants as well as to examine the new environmentally friendly approaches to control fish infection namely phage therapy and photodynamic antimicrobial therapy

Use of recombinant virus replicon particles for vaccination against Mycobacterium ulcerans disease

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a necrotizing disease of the skin and subcutaneous tissue, which is most prevalent in rural regions of West African countries. The majority of clinical presentations seen in patients are ulcers on limbs that can be treated by eight weeks of antibiotic therapy. Nevertheless, scarring and permanent disabilities occur frequently and Buruli ulcer still causes high morbidity. A vaccine against the disease is so far not available but would be of great benefit if used for prophylaxis as well as therapy. In the present study, vesicular stomatitis virus-based RNA replicon particles encoding the M. ulcerans proteins MUL2232 and MUL3720 were generated and the expression of the recombinant antigens characterized in vitro. Immunisation of mice with the recombinant replicon particles elicited antibodies that reacted with the endogenous antigens of M. ulcerans cells. A prime-boost immunization regimen with MUL2232-recombinant replicon particles and recombinant MUL2232 protein induced a strong immune response but only slightly reduced bacterial multiplication in a mouse model of M. ulcerans infection. We conclude that a monovalent vaccine based on the MUL2232 antigen will probably not sufficiently control M. ulcerans infection in humans

Development of human single-chain antibodies against SARS-associated coronavirus.

The outbreak of severe acute respiratory syndrome (SARS), caused by a distinct coronavirus, in 2003 greatly threatened public health in China, Southeast Asia as well as North America. Over 1,000 patients died of the SARS virus, representing 10% of infected people. Like other coronaviruses, the SARS virus also utilizes a surface glycoprotein, namely the spike protein, to infect host cells. The spike protein of SARS virus consists of 1,255 amino acid residues and can be divided into two sub-domains, S1 and S2. The S1 domain mediates the binding of the virus to its receptor angiotensin-converting enzyme 2, which is abundantly distributed on the surface of human lung cells. The S2 domain mediates membrane fusion between the virus and the host cell. Hence two strategies can be used to block the infection of the SARS virus, either by interfering with the binding of the S1 domain to the receptor or by blocking the fusion of the virus with the cell membrane mediated by the S2 domain. Several antibodies against the S1 domain have been generated and all of them are able to neutralize the virus in vitro and in vivo using animal models. Unfortunately, point mutations have been identified in the S1 domain, so that the virus isolated in the future may not be recognized by these antibodies. As no mutation has been found in the S2 domain indicating that this region is more conserved than the S1 domain, it may be a better target for antibody binding. After predicting the immunogenicity of the epitopes of the S2 domain, we chemically synthesized two peptides and also expressed one of them using a recombinant DNA method. We screened a phage displaying library of human single-chain antibodies (ScFv) against the predicted epitopes and obtained a human ScFv which can recognize the SARS virus in vitro

Virus–host interactions: insights from the replication cycle of the large \u3ci\u3eParamecium bursaria\u3c/i\u3e chlorella virus

The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membranecontaining sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans andmice, proceeds througha bacteriophage -like infection pathway

Photocatalytic antimicrobial activity of thin surface films of TiO2, CuO and TiO2 /CuO dual layers on Escherichia coli and bacteriophage T4

TiO2 coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO2, CuO and hybrid CuO/TiO2 prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO2 prepared by a sol-gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol-gel preparations. However these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO2 which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some selfcleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO2/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries

Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune Phage Display Libraries

Broadly, neutralizing antiviral antibodies holds great promise for improving treatment opportunities for patients suffering from viral infections (e.g., human immunodeficiency virus [HIV], hepatitis B virus [HBV], cytomegalovirus [CMV], Rabies, Ebola, Zika) leading to serious health disorders or even to death without effective antiviral treatment. The potential of antibodies in host protection against lethal viral infections has been demonstrated in numerous animal models and is best exemplified by the protection conferred to neonates by maternal antibodies. Over the past few decades, virus-neutralizing human monoclonal antibodies (nAbs) have been isolated from humans successfully cured of disease using a wide range of recently developed antibody isolation technologies. In this chapter, we present an approach for isolating recombinant human nAbs from combinatorial gene libraries being cloned from individuals who have recovered from viral infections. The presented protocols describe the selection and screening of antiviral single-chain antibody fragments (scFvs) from phage display immune libraries. This technology represents a well-established, high-throughput approach allowing fast selection of broadly neutralizing, antiviral antibodies. The protocols for generating and selecting antigen-specific scFvs can be applied for the selection of scFvs against any target

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate