MicrobiologyBytes

Bluetongue is a severe disease of ruminants, notably sheep and cattle. The causal agent, the dsRNA Bluetongue virus, is spread by an insect vector and occurs in its vector’s habitat in temperate climates throughout much of the world. BTV is the type member of genus Orbivirus in the family Reoviridae, with 26 known serotypes. When bluetongue first broke out in the United Kingdom in autumn of 2007, the disease was already rapidly spreading throughout continental Europe, causing high mortality rates in sheep and having a detrimental effect on the livestock trade through trade restrictions and loss of stock. The only effective weapon against the disease is control of the spread of BTV through rigorous vaccination programmes. Currently available commercial vaccines are based on both inactivated virus and live, attenuated strains and protect against a single serotype or multiple serotypes when provided as a cocktail. However, the possibility of recombination between the live vaccine strain(s) and wild-type virus in infected animals, leading to the emergence of new infectious strains has motivated efforts to develop safer vaccines.

One approach in the development of an inherently safe vaccine has been the production of Bluetongue virus-like particles (VLPs). BTV has a nonenveloped icosahedral structure, with four main structural proteins (VP3, VP7, VP5 and VP2) arranged in concentric shells around the segmented double-stranded RNA genome and minor structural and nonstructural proteins involved in virus replication. French et al. have shown that these four structural proteins, expressed in insect cells using a baculovirus expression system, assemble into virus-like particles devoid of nucleic acid.

This paper describes plant-based high-level expression of assembled subcore-, core- and virus-like particles of BTV serotype 8. Purified preparations of the VLPs, consisting of all four structural proteins, elicited an immune response in sheep and provided protective immunity against challenge with a South African BTV-8 field isolate. This demonstrates that plant expression provides an economically viable method for producing complex VLPs, such as those of BTV, with the desired biological properties. It represents a significant advance in the use of plant-based systems for the production of complex biopharmaceuticals. The methods employed could also be applied to other situations where the expression of multiple proteins is required, such as the reconstruction of metabolic pathways.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J. 06 May 2013 doi: 10.1111/pbi.12076
Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.

The use of animal virus-like particles (VLPs) as vectors for the delivery of genes to mammalian cells has been explored for many years. Plant viruses are almost without exception “just” genetic material (DNA or RNA) surrounded by a shell composed of the capsid protein (CP) – with no membrane envelope. Plant-derived VLPs have not been used for direct gene delivery and expression. There have been no attempts to use a spherical plant viral capsid to deliver heterologous genes for expression in mammalian cells, even though there are several independent demonstrations of the internalization of plant virus by cells.

In light of there being no direct demonstration of spherical plant viruses disassembling and thereby releasing their contents in animal cells, this paper asks the question: Can heterologous genes in spherical plant VLPs be made available to a mammalian cell and their protein products synthesized?

Getting RNA into cells is a major barrier to future theraputic approaches, so robust systems are urgently needed.

Reconstituted plant viral capsids can release genes to mammalian cells. Virology. 19 April 2013 doi: 10.1016/j.virol.2013.03.001
The nucleocapsids of many plant viruses are significantly more robust and protective of their RNA contents than those of enveloped animal viruses. In particular, the capsid protein (CP) of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is of special interest because it has been shown to spontaneously package, with high efficiency, a large range of lengths and sequences of single-stranded RNA molecules. In this work we demonstrate that hybrid virus-like particles, assembled in vitro from CCMV CP and a heterologous RNA derived from a mammalian virus (Sindbis), are capable of releasing their RNA in the cytoplasm of mammalian cells. This result establishes the first step in the use of plant viral capsids as vectors for gene delivery and expression in mammalian cells. Furthermore, the CCMV capsid protects the packaged RNA against nuclease degradation and serves as a robust external scaffold with many possibilities for further functionalization and cell targeting.

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With synthetic biology racing ahead with the creation of “artificial” organisms such as Mycoplasma laboratorium, this recent review provides a nice level headed summary of the potential and difficulties of such research.

Semi-synthetic minimal cells: origin and recent developments. (2013) Curr Opin Biotechnol. pii: S0958-1669(13)00005-0. doi: 10.1016/j.copbio.2013.01.002
The notion of minimal cells refers to cellular structures that contain the minimal and sufficient complexity to still be defined as living, or at least capable to display the most important features of biological cells. Here we briefly describe the laboratory construction of minimal cells, a project within the broader field of synthetic biology. In particular we discuss the advancements in the preparation of semi-synthetic cells based on the encapsulation of biochemicals inside liposomes, illustrating from the one hand the origin of this research and the most recent developments; and from the other the difficulties and limits of the approach. The role of physicochemical understandings is greatly emphasized.

See: Mycoplasma laboratorium, the first synthetic organism

It has been known since the 1960s that lactic acid bacteria are essential for the development of cheese flavor. In the ensuing 50 years significant research has been directed at understanding the microbiology, genetics and biochemistry of this process. This review briefly covers the current status of cheese flavor development and then provides our vision for approaches which will enhance our understanding of this process. The long-term goal of this area of research is to enable technology (i.e. cultures and enzymes) that results in consistent rapid development of cheese variety-specific characteristic flavors.

As a big cheese fan, I say – more of this sort of thing!

Perspectives on the contribution of lactic acid bacteria to cheese flavor development. Curr Opin Biotechnol. 29 Dec 29 2012. pii: S0958-1669(12)00210-8. doi: 10.1016/j.copbio.2012.12.001

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses SBV is characterized by a tripartite negative sense RNA genome that encodes four structural and two nonstructural proteins. in vitro, SBV has a wide in vitro host range, and BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration.

In this paper, the three SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The open reading frame for the nonstructural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacks NSs expression was recovered. This virus had reduced capacity to shut-off host cell protein synthesis compared to wild type virus. In addition, the NSs-deleted virus induced interferon in cells, indicating that like other orthobunyaviruses, NSs functions as an interferon antagonist most probably by globally inhibiting host cell metabolism.

Development of a reverse genetic system for SBV will aid investigation of its pathogenic mechanisms as well as creation of attenuated strains that could be candidate vaccines.

Establishment of a reverse genetic system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe. J Gen Virol.19 Dec 2012

Infectious diseases are responsible for an overwhelming number of deaths worldwide and their clinical management is often hampered by the emergence of multi-drug-resistant strains. Therefore, prevention through vaccination currently represents the best course of action to combat them. However, immune escape and evasion by pathogens often render vaccine development difficult. Furthermore, most currently available vaccines were empirically designed.

This review discusses why rational design of vaccines is not only desirable but also necessary. It describes recent developments towards specifically tailored antigens, adjuvants, and delivery systems, and discuss the methodological gaps and lack of knowledge still hampering true rational vaccine design, and addresses the potential and limitations of different strategies and technologies for advancing vaccine development.

Vaccines: From Empirical Development to Rational Design. (2012) PLoS Pathog 8(11): e1003001. doi:10.1371/journal.ppat.1003001

Two recent interesting papers from Virology:

Tools from viruses: Bacteriophage successes and beyond. Virology. 11 Oct 2012, pii: S0042-6822(12)00458-8. doi: 10.1016/j.virol.2012.09.017
Viruses are ubiquitous and can infect any of the three existing cellular lineages (Archaea, Bacteria and Eukarya). Despite the persisting negative public perception of these entities, scientists learnt how to domesticate some of them. The study of molecular mechanisms essential to the completion of viral cycles has greatly contributed to deciphering fundamental processes in biology. Nowadays, viruses have entered the biotechnological era and numerous applications have already been developed. Viral-derived tools are used to manipulate genetic information, detect, diagnose, control and cure infectious diseases, or even design new structural assemblies. With the recent advances in the field of metagenomics, an overwhelming amount of information on novel viruses has become available. As current tools have been historically developed from a limited number of viruses, the potential of discoveries from new archaeal, bacterial and eukaryotic viruses may be limited only by our understanding of the multiple facets of viral cycles.

What is needed for phage therapy to become a reality in Western medicine? Virology. 08 Oct 2012,pii: S0042-6822(12)00456-4. doi: 10.1016/j.virol.2012.09.015
The current status of phage therapy approaches is reviewed and possible hurdles to a practical medical application of bacteriophages in Western countries are identified as discussed at a recent EMBO meeting on “Viruses of Microbes” in Brussels. In view of the growing antibiotic resistance crisis, a coordinated effort by the public health sector is needed to evaluate the potential of phage therapy as an adjunct to antibiotics.

It is a sad fact that a leading cause of infant mortality in developing countries continues to be from infectious diseases which are quite preventable. New technologies which can both deliver vaccines en mass and without the need for syringes, as well as induce a strong mucosal immune response, could revolutionize the accessibility of much needed vaccines in developing countries. Approaches range from the use of inhalation devices which deliver the vaccine antigen in the form of an aerosol spray, to patches containing microneedles which can deliver the desired vaccine antigen across the skin barrier. A third tactic involves the development of plant production platforms as delivery systems for oral vaccines.

Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins. (2012 ) Virology 433(1): 1-6. doi: 10.1016/j.virol.2012.06.012
Transgenic plants present enormous potential as a cost-effective and safe platform for large-scale production of vaccines and other therapeutic proteins. A number of different technologies are under development for the production of pharmaceutical proteins from plant tissues. One method used to express high levels of protein in plants involves the employment of plant virus expression vectors. Plant virus vectors have been designed to carry vaccine epitopes as well as full therapeutic proteins such as monoclonal antibodies in plant tissue both safely and effectively. Biopharmaceuticals such as these offer enormous potential on many levels, from providing relief to those who have little access to modern medicine, to playing an active role in the battle against cancer. This review describes the current design and status of plant virus expression vectors used as production platforms for biopharmaceutical proteins.