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.

Like other carnivorous plants, Nepenthes species grow on poor soil. They need to complement their mineral nutrients – primarily with nitrogen and phosphorus – from caught and digested prey. When visiting the pitfall traps, the attracted prey, mainly arthropods, falls into the trap, drowns and is digested by the enzyme cocktail of the pitcher fluid.

Due to the fact that closed Nepenthes pitchers have no direct contact with the environment, it has been widely claimed that their pitcher fluid is sterile and that all proteins and compounds identified in this pitcher fluid are solely plant-derived. But only two experiments had been conducted to demonstrate the sterility of pitcher liquid: fluid taken from a closed pitcher was plated either on plain nutrient agar (Hepburn, 1918) or on meat agar plates (Lüttge, 1964) and incubated for several days. In no case were any bacterial colonies detected and the authors concluded that the pitcher fluid is sterile. However, the presence of microbes cannot be excluded by such simple experiments because most micro-organisms cannot be grown in culture.

Researchers have now analysed the composition of Nepenthes digestive fluid from closed pitchers to reveal whether or not pitchers are really sterile inside and how these plants manage to keep microbial growth under control. Thecontent of proteins, inorganic ion compositions and secondary metabolites were studied. In addition, the effect of pitcher fluid on microbial growth was investigated. The results reveal that the fluid of closed Nepenthes pitchers is composed provides anti-microbial conditions. Thus these plants can avoid, at least to some extent, the growth of microbes that compete with the plant for the prey-derived nutrients available in the pitcher.

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth. (2013) Annals of Botany 111 (3): 375-383

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.

Plant viruses are biotrophic pathogens that need living tissue for their multiplication and thus, in the infection-defence equilibrium, they do not normally cause plant death. In some instances virus infection may have no apparent pathological effect or may even provide a selective advantage to the host, but in many cases it causes the symptomatic phenotypes of disease. These pathological phenotypes are the result of interference and/or competition for a substantial amount of host resources that can disrupt host physiology to cause disease. This interference/competition affects a number of genes, which seems to be greater the more severe the symptoms that they cause. Induced or repressed genes belong to a broad range of cellular processes, such as hormonal regulation, cell cycle control and endogenous transport of macromolecules, among others. In addition, recent evidence indicates the existence of interplay between plant development and antiviral defence processes, and that interference among the common points of their signalling pathways can trigger pathological manifestations. This review provides an update on the latest advances in understanding how viruses affect substantial cellular processes, and how plant antiviral defences contribute to pathological phenotypes.

How do plant viruses induce disease? Interactions and interference with host components. (2011) J Gen Virol. 92(12): 2691-2705

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I recently wrote about the top 10 plant viruses in molecular plant pathology. Well Ed didn’t like that list, so he’s published his own:

1. Tobacco mosaic virus (TMV)
2. Tomato spotted wilt virus (TSWV)
3. Tomato yellow leaf curl virus (TYLCV)
4. Cucumber mosaic virus (CMV)
5. Potato virus Y (PVY)
6. Cauliflower mosaic virus (CaMV)
7. African cassava mosaic virus (ACMV)
8. Plum pox virus (PPV)
9. Brome mosaic virus (BMV)
10. Potato virus X (PVX)

“I see only ONE virus in the major list – African cassava mosaic begomovirus (ACMV) – that infects and causes severe losses in one of the four major food crops grown on this planet: all the rest, excepting viruses infecting the also-ran potato, are pathogens of fruits, vegetables or horticulturally-important plants. Or hardly pathogenic at all, as in the case of BMV – and before anyone argues, I probably have the best collection of African (and other) isolates of the virus in the world, and a lot of experience of it in the field.”

I have a feeling this could go on for some time :-)

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a ‘Top 10′ based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list including, in rank order:

• Tobacco mosaic virus
• Tomato spotted wilt virus
• Tomato yellow leaf curl virus
• Cucumber mosaic virus
• Potato virus Y
• Cauliflower mosaic virus
• African cassava mosaic virus
• Plum pox virus
• Brome mosaic virus
• Potato virus X

This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

Top 10 plant viruses in molecular plant pathology. (2011) Mol Plant Pathol. 12(9): 938-954. doi: 10.1111/j.1364-3703.2011.00752.x

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Viruses infecting higher plants are typically small RNA viruses that encode only a few genes. Although small viruses have recently been discovered that infect algae, many viruses infecting eukaryotic algae are huge dsDNA viruses with genomes ranging from 160 to 560 kb with up to 600 protein-encoding genes and are the subject of this review. These large viruses (family Phycodnaviridae), are found in aqueous environments throughout the world and play dynamic, albeit largely undocumented, roles in regulating algal communities such as the termination of massive algal blooms commonly referred to as red and brown tides.

This review focuses on one genus in the Phycodnaviridae, the chloroviruses, which are large, icosahedral, plaque-forming, dsDNA-containing viruses that replicate in certain unicellular, chlorella-like green algae. Their structure, their initial stages of infection, and many of their genes resemble bacteriophages more than viruses that infect eukaryotes – i.e. they are not your everyday plant virus.

Chloroviruses: not your everyday plant virus. Trends Plant Sci. Nov 17 2011
Viruses infecting higher plants are among the smallest viruses known and typically have four to ten protein-encoding genes. By contrast, many viruses that infect algae (classified in the virus family Phycodnaviridae) are among the largest viruses found to date and have up to 600 protein-encoding genes. This brief review focuses on one group of plaque-forming phycodnaviruses that infect unicellular chlorella-like green algae. The prototype chlorovirus PBCV-1 has more than 400 protein-encoding genes and 11 tRNA genes. About 40% of the PBCV-1 encoded proteins resemble proteins of known function including many that are completely unexpected for a virus. In many respects, chlorovirus infection resembles bacterial infection by tailed bacteriophages.

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Eucaryotic organisms depend on networks of gene regulatory pathways. Small RNAs (sRNAs), are key components of these networks. sRNAs are short (21–24 nt in length), endogenously expressed, and are processed from double stranded (ds)RNAs or dsRNA-like precursors. In both plants and animals, sRNAs exert their functions upon incorporation into ribonucleoprotein silencing complexes and through their base-paring capacity. They are implicated in a variety of processes, including post-transcriptional regulation of mRNA, mRNA stability and availability for translation, establishment of heterochromatin and silencing of transposons. Different classes of sRNAs differ in the proteins required in their biogenesis, the constitution of ribonucleoprotein complexes that mediate their regulatory functions, their type of gene regulation, and the biological functions in which they are implicated. Plants display a remarkable diversity of sRNA types and sRNA pathways, likely needed for managing multiple environmental stimuli, including biotic and abiotic stresses. Several lines of evidence suggest that plant sRNAs play critical roles in plant–pathogen interactions. Indeed, upon infection, most plant pathogens can interfere with the expression of endogenous sRNAs, thus altering the expression of specific host factors implicated in the suppression or in the activation of plant defences. Evidence for these phenomena has been reported for bacterial and fungal pathogens.

Viruses are obligate infectious agents, whose life cycle (expression of viral proteins, viral genome replication and virion assembly) is integrated with host cell functions. Plant viruses can both modify the profiles of endogenous sRNAs (in common with bacteria and fungi) and induce the production of additional sRNAs derived from their own genomes (viral sRNAs; vsiRNAs). The latter gives a clear indication of the activation of RNA silencing-based responses of the plant. In some cases, this results in reduction of the titre of the invading virus and, in recovery of upper, non-inoculated leaves. To counteract RNA silencing, many plant viruses have evolved proteins (viral suppressors of RNA silencing: VSR) that target various components of the plant silencing machinery. Viruses can induce specific symptoms resembling developmental anomalies and affecting organs and tissues such as leaves, flowers and fruits. These anomalies are often reconcilable with virus-induced alterations of RNA silencing-based endogenous pathways, due to: i) the direct activity of VSRs on endogenous sRNAs or on silencing related effectors; ii) the abundance of vsiRNAs in competition with endogenous sRNAs; iii) the action of specific vsiRNAs entering into RNA silencing complexes and targeting specific host genes.

This review provides an overview of the major cellular RNA silencing pathways in plants with particular reference to those involved in antiviral functions and highlights examples of the complex interactions between viral molecular processes and host RNA processes.

Viral induction and suppression of RNA silencing in plants. Biochim Biophys Acta. Apr 30 2011
RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

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