Archive for the ‘biofarming’ Category

Your next DNA vaccine might come from tobacco

12 February, 2016

We don’t have much practice at this sort of thing, but seeing as we just got something REALLY cool published, and the man who largely made it possible is now a science writer, we decided to ask him to write a press release.  So he did.  Thanks, Paul Kennedy – take a bow, twice!


“In a pioneering step towards using plants to produce vaccines against cervical cancer and other viruses, University of Cape Town (UCT) researchers have generated synthetic human papillomavirus- derived viral particles called pseudovirions in tobacco plants.

“We’ve succeeded in making a completely mammalian viral particle in a plant – proteins, DNA, everything. That’s enormously exciting,” says Dr Inga Hitzeroth of the Biopharming Research Unit (BRU) at UCT.Dr_Inga_Hitzeroth

In an Open Access study just published in Nature Scientific Reports, BRU researchers report using tobacco plants to create a synthetic viral particle known as a pseudovirion.

A pseudovirion looks like a virus, but it contains no infectious viral DNA. A virus is usually made up of a shell surrounding the virus’s own genetic material. Pseudovirions instead carry whatever DNA the researcher wishes to include within the shell of proteins that make up the outer coating of the virus.

Until now, such particles have only ever been created in yeast or mammalian cell cultures – this is the first time researchers have successfully created pseudovirions in plants.

The BRU is part of a new movement known as biopharming, which means using plants as biological factories. Biopharming has been used to create flu vaccines, potential Ebola drugs, and an enzyme used to treat Gaucher’s Disease in humans. The technique employs the cellular machinery within tobacco plants or other plant cells to manufacture enzymes, antibodies or even the viral capsid proteins (the proteins that make up the shell of a virus), which act as vaccines.

In this research, the BRU has taken biopharming one step further by using plants to create a viral shell that encloses ‘custom’ DNA selected by researchers. “What’s unique here is that DNA that was manufactured within the tobacco plant is now being incorporated into a viral particle to form a pseudovirion,” says Hitzeroth.

The shell of this pseudovirion was that of human papillomavirus (HPV) type 16, the virus responsible for over 50% of cervical cancer cases worldwide.

The BRU team hope this new plant-based technology could one day be used to test future HPV vaccines. First author of the study, Dr Renate Lamprecht, renateexplains: “We need pseudovirions to test any new HPV vaccine candidates. At the moment it is very expensive to make pseudovirions – we need to make them in mammalian cell culture, it needs to be sterile, and the reagents are very expensive.”

All these factors contribute to the high cost of current HPV vaccines, which are actually virus-like particles. Virus-like particles (VLPs) are similar to pseudovirions, but they contain no DNA. Plant- made pseudovirions, as demonstrated by this study, could reduce the cost of testing and manufacturing such vaccines, thus helping to make HPV vaccines affordable where they are needed most: the developing world.

PsVs

Plant-made HPV pseudovirions containing geminivirus-derived DNA

The BRU team compared these new plant-made pseudovirions against the more widely-used mammalian cell culture-produced particles by using what’s known as a neutralisation assay. In this test (which is commonly used to test new HPV vaccine candidates), cells are ‘infected’ with pseudovirions, with or without pre-treatment with neutralising antibodies. The DNA inside the pseudovirion carries a ‘reporter gene’ that produces a protein that can give off a light signal. Thus, an infectious pseudovirion gets into the cell and gives off light, but one that is stopped by neutralising antibodies does not.

“I was jumping up and down the first time I saw the neutralisation results, but I repeated the experiment a few times to be sure, asking myself, ‘is everything correct, are all the controls there?’” explains Lamprecht. “It was a very exciting moment for us when we confirmed that neutralisation had occurred.”

Right now, every laboratory makes pseudovirions for such neutralisation experiments themselves. Dr Hitzeroth hopes that one day, they won’t have to: “we’re in the initial stages, but if we optimise the process and get the yield much higher, we think it’s a product that could be sold all over the world.”

ed ebola

Ed’s Ebola shirt

For Professor Ed Rybicki, Director of the BRU, this achievement was enormously satisfying, as it brought together two strands of his research interests that have co-existed for over 20 years.

“Seventeen years ago, I had the idea to combine making HPV VLPs in plants with a DNA plant virus we were working on, to see if we could make pseudovirions. It took until now for the technology to finally come together, but it shows what can happen in biotechnology if you’re willing to persevere.”

The BRU are also hoping to use this technology to create a therapeutic vaccine, which would also be a first of its kind. The idea would be to use the pseudovirion to deliver DNA that could treat an ongoing HPV infection or even a tumour.

With global acceptance and support for the biopharming movement growing rapidly, it might not be too long before the first plant-produced HPV vaccine is making a difference in Africa and around the world.”


For further enquiries, contact Dr Hitzeroth. For more info on biopharming, check out this Q&A session from Sense About Science.

“Online ‘recipes’ for bird flu virus add to bioterrorism threat!” No. No, they don’t.

10 December, 2015

The means of engineering potentially deadly avian influenza is freely available on the internet.

Despite continuing global efforts to contain avian influenza, or bird flu, the means of engineering this potentially deadly H5N1 virus to render it transmissible to humans is freely available on the internet. So too are similar instructions for engineering a virus like the “Spanish flu”, which killed some 50 million people in the pandemic of 1918-19.

The digital floodgates opened in 2011 when a peak US regulatory watchdog came down in favour of scientists seeking to publishing their work engineering the H5N1 virus. The decision to uphold such “scientific freedom” was and remains, highly contentious among the global scientific community. Its implications, however, are readily available as online “recipes” for potentially dangerous viruses, which add a new risk to the already considerable challenges of maintaining global biosecurity in the 21st century. For all the recent advances in biomedical science, drugs, vaccines and technology, this is a challenge we remain ill-equipped to meet.

Read more: http://www.theage.com.au/comment/online-recipes-for-contagious-diseases-means-australias-bioterrorism-threat-is-real-20151208-gli97v.html#ixzz3tvWn63AE ;
Follow us: @theage on Twitter | theageAustralia on Facebook

Sourced through Scoop.it from: www.theage.com.au


 

OFFS: seriously!  Again?!  Someone else has just discovered that entire virus genomes are freely available via PubMed, along with papers on gain-of-function experiments, and immediately leaps to the conclusion that this means “…the means of engineering this potentially deadly H5N1 virus to render it transmissible to humans is freely available on the internet”.

I’m sorry, this is being simple-minded to the point of parody.  I have written elsewhere – here in ViroBlogy, and in Nature Biotech’s Bioentrepreneur blog section – on how it is MOST unlikely that bearded fellows in caves in Afghanistan or remote farms in Montana are going to whip up weaponised batches of H5N1 flu or Ebola.

Yes, the papers are available; yes, the sequences necessary to make a potentially (and I say potentially advisedly) deadly virus are available online; yes, one can bypass the blocks on getting resynthesised genes in developing countries (hint: China).

But could anyone outside of a sophisticated lab environment use these to make anything nasty?

No.

Seriously, no.

Just think about what you would need to make weaponised flu, for example.  There are two ways to go here, these being the totally synthetic route (“mail order” DNA – HATE that term!), with some serious molecular biology and cell culture at the end of it, and the “natural” route – which would involve getting a natural and nasty isolate of H5N1 / H7N9 / H9N2, and being able to culture it and engineer it as well.

Both routes require a minimum of a serious 4-yr-degree-level training in microbiology / mol biol, as well as laboratory resources that would include incubators, biohazard cabinets, and disposables and reagents that are not on your normal terrorist’s priority purchase list.

In fact, the kinds of resources you’d find at a University or Institute Infectious Disease unit – or state-sponsored biowarfare lab.

Seriously, now: in order to use the information that is “freely available”, you’d have to do what amounts to an entire postgrad degree’s worth of work just to set up the kinds of reverse genetics necessary to WORK with recombinant flu, presuming you already had an isolate, and even more than that if you were to start with synthesised DNA and try to recreate infectious virus.

Again, this is the kind of work they do in biowarfare / biodefence labs (funny how they’re pretty much the same thing, isn’t it?) – because it’s finicky, expensive, laborious – and potentially dangerous to the researcher.

And it’s interesting that the only rumoured escapes of biowarfare agents have been of flu in 1977 in the old Soviet Union, and of anthrax in Sverdlovsk in the USSR in 1979. And in the US in 2001, and again in 2014.  ALL of them from official facilities, I will discreetly point out.

Oh, there have been rumours that Saddam’s Iraq weaponised camelpox; that the USSR/Russia cloned Ebola into a poxvirus; that Al-Qaeda tested anthrax – but the first two took state resources, and if the third happened at all, it’s nothing that the UK and USA and friends hadn’t already done in the 1940s.

IT IS NOT THAT EASY TO MAKE RECOMBINANT VIRUSES.

Seriously.

See on Scoop.itVirology News

“Plant cell pack” workshop

23 November, 2015

As molecular farmers, we were much impressed last year by a technology developed by the folk at the Fraunhofer IME in Aachen: this is “METHOD FOR THE GENERATION AND CULTIVATION OF A PLANT CELL PACK“, with Thomas Rademacher as sole inventor on the patent application.  Basically, this involves

  • making a “cookie” or cell pack with cultured plant cells, by suction of a suspension onto a membrane
  • drizzling recombinant Agrobacterium tumefaciens onto the cookie, then sucking away excess fluid
  • incubating the cell cookie in a humid environment for a few days, until the desired level of protein expression has been reached

There are all sorts of things one could dream up for the application of this technology, given that one can make cookies of all sorts of depths and widths, in everything from spin columns to multiwell plates – and high-throughput screening of expression constructs comes to mind immediately.

Now fortunately, Inga Hitzeroth of our Biopharming Research Unit here at UCT (the BRU) has a National Research Foundation-administered bilateral grant with the folk at the Fraunhofer IME, which has meant we have money for joint workshops and the like – so we are having a hands-on Workshop on “Plant Cell Packs for Transient Expression: Innovating the Field of Molecular Biopharming” affiliated to our “Virology Africa 2015” conference next week.  We plan to develop an illustrated manual along with a full suite of technical tips after the Workshop.

And as part of which, one has of course to feed and entertain the participants – hence our expedition to The Spice Route wine farm complex yesterday.  Hard work, this science…B-)

The BRU-IME Cookie Workshop team: from left; Romana Yanez (BRU), Tanja Holland, Susanne Bethke, Markus Sack, Juergen Drossard, Gueven Edgu all IME), Ed Rybicki (BRU)

The BRU-IME Cookie Workshop team: from left; Romana Yanez (BRU), Tanja Holland, Susanne Bethke, Markus Sack, Juergen Drossard, Gueven Edgu (all IME), Ed Rybicki (BRU)

 

 

So that’s what you lot like, is it?

21 October, 2015

My_Stats_—_WordPress_com

Virology Africa 2015: Update and Registration

19 August, 2015

REGISTRATION IS NOW OPEN – VIROLOGY AFRICA 2015

On behalf of the Institute of Infectious Disease and Molecular Medicine of the University of Cape Town and the Poliomyelitis Research Foundation, we are pleased to invite you to Virology Africa 2015 at the Cape Town Waterfront.

VENUE AND DATES:

The conference will run from Tuesday 1st – Thursday 3rd December 2015. The conference venue is the Radisson Blu Hotel with a magnificent view of the ocean. The hotel school next door will host the cocktail party on the Monday night 30th November and in keeping with Virology Africa tradition, the dinner venue is the Two Oceans Aquarium.

IMPORTANT DATES

Early Bird Registration closes – 30 September 2015
Abstract Submissions deadline – 30 September 2015

The ACADEMIC PROGRAMME will include plenary-type presentations from internationally recognised speakers. We wish to emphasise that this is intended as a general virology conference – which means we will welcome plant, human, animal and bacterial virology contributions. The venue will allow for parallel workshops of oral presentations. There will also be poster sessions. Senior students will be encouraged to present their research. We have sponsorship for students to attend the meeting and details will be announced later in the year.

A program outline has been added to the website

WORKSHOPS

Our preliminary programme includes two workshops.

There is a hands-on workshop on “Plant cell packs for transient expression: Innovating the field of molecular biopharming”, with the contact person being Dr Inga Hitzeroth – Inga.Hitzeroth@uct.ac.za. This workshop will run at UCT one day before the conference, 30th November, and a second day, 4th December, after the conference.

The second workshop is on “”Viromics for virus discovery and viral community analysis”. The workshop at UCT will be on 4 and 5 December with the contact person being Dr Tracy Meiring – tracy.meiring@uct.ac.za.

Some of the workshop presenters will be integrated into the conference programme but the practical components will be run at University of Cape Town. Separate applications are necessary for each workshop.

If you are prepared to fund an internationally recognised scientist to speak at the conference or if you wish to organise a specialist workshop as part of the conference, please contact
Anna-Lise Williamson or Ed Rybicki.

For any enquiries please contact
Miss Bridget Petersen/ Email: conference1@onscreenav.co.za or phone: +27 21 486 9111
Ms Deborah McTeer/Email: conference@onscreenav.co.za or +27 83 457 1975

Don’t fear GMOs – fear the hype!

31 July, 2015
I’m going to share a slightly disturbing exchange I just had with a dietician – because it shows that even well-educated people out there are buying into the anti-GMO frenzy.

And I will thank +Mary Mangan for pointing out some of the sites mentioned!

“Dear xxxx;

In answer to your statements and questions:

“For a while, I have had the suspicion that GMO foods might be related to the epidemic increase in allergies, worldwide”

Ummmm…there is no good evidence of a worldwide epidemic of allergies – like autism, there is better recognition of the state, rather than an increase in incidence.

“my two boys clinically react to GMO varients,”

WHICH variants?  Of what?  How do you know?  This is a dangerous path, and I have trod it with immunologists involved in this sort of research – the ONLY way you can say “it is due to a GM food” is if you have the EXACT equivalent that is NOT GM – and by that, I don’t mean “GM vs non-GM maize” – because that is not biologically equivalent unless you have the same exact variety.  The other way would be to isolate the proteins involved, and test them – which is not that difficult, and is something I have thought of doing, if only to settle this issue for once and for all.

I would strongly urge you…to look at the links I will list below: most people, and medics and non-plant scientists as well, really don’t understand what actually happens with modern GM.  What happens is that one or a very few genes are introduced into a plant, to make one or possibly two proteins – against the 40 000+ the plant already makes. The genetic modification is minimal compared to conventional or advanced breeding, which moves around whole chromosomes, and MUCH easier to track than use of irradiation, which is also used to change traits – and very often changes things you can’t see and therefore ignore, unlike GM techniques.

What is more, all of the changes induced in plants can be followed these days by techniques like whole genome sequencing and proteomics, so that we can genuinely put hand on heart and say “this is exactly equivalent to that, except for one protein”.  Seriously: the question of equivalence is no longer really up for discussion; it is subject to evidence – and I will point out that the standards expected for GM plants are FAR more stringent than for conventionally-bred plants, which may have far bigger changes in protein composition than any GM variety.

http://www.ottawacitizen.com/touch/story.html?id=8738060

http://www.sciencebasedmedicine.org/index.php/antivaccine-versus-anti-gmo-different-goals-same-methods/

http://www.scoop.it/t/virology-news/curate?q=GMOhttp://www.scoop.it/t/virology-news/curate?q=GMO

http://gmopundit.blogspot.com/2011/09/convetional-corn-is-genetically.html

http://www.healthnewsdigest.com/news/Food_and_Nutrition_690/What-You-Need-to-Know-About-GMOs-GM-Crops-and-the-Techniques-of-Modern-Biotechnology.shtml

http://www.forbes.com/sites/henrymiller/2015/07/16/the-dumbest-most-pretentious-article-ever-about-genetic-engineering/

I hope this is helpful!

Sincerely,

Ed”

Crystallising the tobacco mosaic virus

6 July, 2015

We saw last week how sulphur dioxide released from the Laki fissure system accounted for many deaths due to poisoning. We will stay with poisons this week as well, for virus has its roots in the Latin term for “poison”

Sourced through Scoop.it from: www.thehindu.com

Nice article – and from a newspaper in India, no less!  Adds to the history of virology in a very accessible way.

See on Scoop.itVirology News

The double life of a geminivirus: Bean yellow dwarf virus

29 May, 2015

Every now and then I solicit contributions for this site – but this one came without coercion, or even prompting!  I thank Romana for being so enthusiastic B-)

Romana Yanez

Biopharming Research Unit, UCT

All material copyright Romana Yanez and UCT

***

I want to tell the story of a geminivirus called Bean yellow dwarf virus that has two very distinct “lives”: one as a crop pest, infecting bean plants in South Africa and the other as a powerful molecular tool as a viral vector for recombinant protein expression in plants. As if each one of the “heads” of the twinned capsids had a life of its own. The dark side and the bright side. The yin and yang…

Introduction

Geminiviruses are small, single-stranded, circular DNA plant viruses, so called because each particle is composed of two partially assembled icosahedra joined to form a twinned capsid [1], [2]. They infect plants and are carried by insect vectors such as leafhoppers and whiteflies [2]. They are divided into seven genera: Mastrevirus, Bogomovirus, Topocuvirus, Curtovirus, Becurtovirus, Eragrovirus and Turncurtovirus; according to their genomic organization, the hosts they infect, the insect vectors by which they are transmitted and by genome-wide pairwise sequence identities [3].

Geminiviruses belonging to the genus Mastrevirus are all monopartite viruses with genome sizes between 2.5 and 3.0 kb. They have as vectors different species of leafhoppers. They infect mostly monocotyledonous plants: Maize streak virus (MSV) causes devastating crop losses in African countries, Wheat dwarf virus (WDV), but also infect dicotyledonous plants: Bean yellow dwarf virus (BeYDV), Tobacco yellow dwarf virus (TYDV) and Chickpea chlorosis virus (CpCV) [2], [4], [5].

In 1997 the production of French beans (Phaseolus vulgaris cv. Bonus) was severely reduced  in South Africa, mainly in the Northern Province and Mpumalanga District [4]. Plants presented symptoms similar to a TYDV infection, which at the time was the only mastrevirus described to infect dicotyledonous plants. These symptoms included stunted growth, brittle and leathery leaves, and leaf curling. Investigating the aetiology of this disease were Liu and co-workers. They determined it was a geminiviral infection by identifying virus-like particles (VLPs) with the characteristic twinned morphology. They then sequenced DNA samples of the virus and found it to be most closely related to TYDV, both, in nucleotide sequence (65% identity) and in genomic organization. It was similar enough to be placed under the genus Mastrevirus but distinct enough to be considered a different virus. They Then called it Bean yellow dwarf virus – BeYDV [4].

In 2007 a mild strain of BeYDV (BeYDV-m) was described by Halley-Stott et al. also isolated from P. vulgaris (cv. Top Crop). It was phylogenetically similar to BeYDV having 97% nucleotide sequence identity, but presented sufficient phenotypic differences to be a different BeYDV strain. It contained 81 nucleotide differences compared to the BeYDV type, most of which (63 changes) were found in regions of the genome that directly influenced its replication. Thus, BeYDV-m produced typical symptoms that were less severe and temporally delayed when compared to BeYDV type. The authors also suggested that P. vulgaris is not the BeYDV natural host since it is a non-indigenous plant in South Africa. Furthermore, since both strains of BeYDV were isolated in the same region, it was also suggested that their natural host may show very mild or no symptoms upon infection [6]. Subsequently BeYDV-m was renamed as Chickpea chlorotic dwarf virus (CpCDV) [3]. [However, we like BeYDV, so we’re going to keep called it that – Ed]

Molecular Characteristics and Life Cycle of BeYDV

The genome of BeYDV (Figure 1) is 2,561 nucleotides long with an organization similar to that of other mastreviruses and that replicates by rolling circle mechanism [4], [7]. Its genome is bidirectional, consisting of virion-sense open reading frames (ORFs) V1 and V2, and complementary-sense ORFs C1, C2, C3 and C4. Of these, only C3 and C4 are non-functional and non-conserved between the mastreviruses; although they are also present in TYDV [4], [8]. Within the complementary sense ORFs C1 and C2 an intron is found which is also conserved in other mastreviruses. Virion sense and complementary sense ORFs are separated by a long intergenic region (LIR) and a small intergenic region (SIR) [4]. Liu and co-workers described the functions of each component of the BeYDV genome by mutational analysis.

Figure 1. Genomic organization of Bean yellow dwarf virus. CP, capsid protein. LIR, long intergenic region. MP, movement protein. Rep, replication associated protein. SIR, short intergenic region. [9]

Figure 1. Genomic organization of Bean yellow dwarf virus. CP, capsid protein. LIR, long intergenic region. MP, movement protein. Rep, replication associated protein. SIR, short intergenic region. [9]

The LIR contains a bidirectional promoter to which host factors can bind and a stem-loop structure within the origin of replication (ori) which is required for initiation of rolling circle replication. A binding site and nicking site for the replication associated protein (Rep) are also found in this region. The SIR in turn contains a primer binding region for initiation of complementary strand synthesis as well as transcription termination elements [8]. These are the only two cis-acting elements required for BeYDV replication [8], [10].

The V1 ORF encodes for the movement protein (MP) which is associated with plasmodesmata and is important for systemic spread of the virus. It was found to be a symptom inducer as transgenic plants expressing V1 developed wild type-like infection symptoms. The putative pathogen associated molecular pattern recognized by the host plant may be within the first 17 N-terminus amino acids as plants infected with a mutant  developed wild type-like symptoms as well [8]. 

The V2 ORF encodes for the capsid protein (CP) which is important for viral movement as well and therefore for systemic infection. Thus, intracellular movement or trafficking of the viral DNA may require encapsidation. This was suggested since V2 mutants did not infect plants systemically and also, a basic domain on the N-terminal of the CP was identified which putatively binds to DNA or is involved in nuclear localization [8], [11].

From the genome of BeYDV, the complementary sense ORFs C1 and C2 are the most interesting for me. These encode two regulatory proteins involved in the replication of the virus: Rep and RepA. Their expression is regulated by alternate splicing, where spliced C1 and C2 (C1C2) mRNA is translated into Rep and unspliced C1 mRNA is translated into RepA [8]. 

Rep is responsible for initiating rolling circle replication by nicking the stem-loop structure at the ori, and for releasing nascent virion sense single stranded DNA and later ligating it to form circular ssDNA molecules [8], [12]. Rep is the only protein required for BeYDV replication, but in the presence of RepA the replication is more efficient [8], [10], [11].

RepA is a multi-regulatory protein only found in mastreviruses [2]. Even though both Rep and RepA, have a retinoblastoma related protein (RBR)-binding motif, LeuXCysXGlu, in BeYDV only RepA is able to bind to RBR proteins [10]. In mammalian cells, the retinoblastoma protein is a tumor repressor that binds to and inactivates the transcription factor E2F. By binding to RBR proteins, RepA is thought to disrupt this interaction and force the plant cell cycle into the S-phase – where DNA is replicated just before cell division. RepA is thus acting like other viruses’ oncogenic proteins, such as the human papillomavirus E7 protein and the adenovirus E1A protein. Thus, keeping conditions favorable for enhanced viral replication and proliferation [10], [11]. This could be seen when Hefferon and Dugdale mutated the RBR binding-motif of Rep and RepA to LeuXCysXGln. Only the RepA mutant showed significantly decreased replication. While the Rep mutant showed wild type-like replication [11].

Having in mind what I just described, one can picture the life cycle of BeYDV as follows:

A leafhopper (which has not been identified yet) carrying the virus infects a host plant – this will be a dicotyledonous plant such as P. vulgaris, from which it was originally isolated. The virus releases its ssDNA genome into the cytoplasm. The ssDNA enters the nucleus where host’s replication machinery synthesizes the complementary strand from the primer located in the SIR region, generating a replicative double stranded circular DNA intermediate. At this point the dsDNA serves as template for gene expression, from which Rep and RepA are expressed. RepA transactivates virion-sense gene expression and interferes with plant cell’s life cycle to produce S-phase conditions. Rep nicks the stem-loop structure located at the ori and binds to the 5’ end of the nicked strand. The 3’ end acts as a primer for the synthesis of a new virion-sense strand displacing the previous virion-sense strand. When this new strand is complete, the ori is regenerated and Rep nicks it again. Subsequent release and recircularization of the nascent virion-sense strand is also mediated by Rep. The process continues on the new circular ssDNA molecules as well. Only later, when the amount of CP is high enough, ssDNA molecules are encapsidated. The CP and MP then mediate systemic spread of the viral genome [2], [8]–[12]. When another leafhopper visits the infected plant, the virus is transferred to other plants and all starts again (Figure 2).

gv fig 2

Figure 2. The life cycle of BeYDV. Black circle, BeYDV ssDNA with the stem-loop structure. Black and green circle, BeYDV dsDNA replicative intermediate. Orange spheres, plant host’s replication machinery. Yellow spheres, Rep protein. Black line, nascent ssDNA during rolling circle replication. Purple sphere, RepA. Green sphere, plant retinoblastoma-related protein. Red spheres, BeYDV movement protein. Geminal structures, BeYDV capsid proteins. Modified from [13], [14].

Liu et al. (1997) and Halley-Stott et al. (2007) showed that BeYDV is able to infect other dicotyledonous plants besides P. vulgaris, such as: Nicotiana tabacum, N. benthamiana, Datura stramonium and Arabidopsis thaliana [4], [6]. It has also been isolated from chickpeas in Pakistan [15]. It was noted by Liu and co-workers that the intron of BeYDV (and TYDV) is not as AU-rich as intron sequences present in dicotyledonous plants, which suggested that these viruses had evolved from monocotyledonous-infecting ancestors [8]. Other thing that suggests that BeYDV (and TYDV) evolved from monocotyledonous-infecting mastreviruses is that they encode for two variants of the Rep protein while other geminiviruses infecting dicotyledonous plants encode for only one Rep protein from a continuous ORF [11].

BeYDV as a Powerful Molecular Tool

I have talked about the relatively dark side of BeYDV as a crop pest and plant cell cycle manipulator. Now I would like to introduce you to the other face of this geminivirus.

The importance of recombinant proteins in pharmaceutical, medical and research fields makes them highly demanded, which in turn requires the use efficient production systems [16], [17]. Plants provide a cheaper, faster, more efficient and highly scalable platform for the production of proteins compared to other methods [18], [19]. Vectors based on DNA viruses can be used to express complex proteins without the limitations and complexity faced by RNA viruses such as the need to use more than one virus construct, size constraint imposed on the insert and genomic instability [2], [20]. BeYDV and other geminiviruses have small and simple DNA genomes which can be rapidly amplified to very high copy numbers using mainly host factors and that can be easily manipulated. These features make them attractive viruses for the design of plant vectors for the expression of recombinant proteins [21]. BeYDV has been extensively explored as a molecular tool for the expression of mainly pharmaceutically relevant proteins, such as vaccines, antibodies and enzymes [9], [21]. And recently it has also been used as a means to deliver reagents into plant cells to genetically engineer them [22].

Hefferon and co-workers were one of the first to design a vector derived from BeYDV. They expressed a synthetic version of Staphylococcus enterotoxin B (SEB) in tobacco NT-1 cells. The synthetic SEB sequence was placed under the control of a Cauliflower mosaic virus (CaMV) 35S constitutive promoter and flanked by the cis acting BeYDV LIR and SIR. The Rep encoding gene was provided in trans from a separate construct and also constitutively expressed from the CaMV 35S promoter. Constructs were co-delivered into NT-1 cells by bombardment [11]. They obtained expression levels of ≈0.025 mg SEB / kg of NT-1 cells. They showed that expression of SEB could be enhanced by 20 times by supplying Rep in trans compared to when no Rep was supplied. Overall they showed that BeYDV-based replicon systems promised enhancement of recombinant protein expression in plants [23].

In a more deconstructed approach, Mor et al. (2003) designed a replicon system similar to that of Hefferon and Dugdale (2003) in which the BeYDV MP and CP genes were replaced by the gene of interest (GUS), controlled by CaMV 35S promoter and flanked by the LIR and SIR sequences [24]. Since the CP can sequestrate viral ssDNA, preventing dsDNA to be formed [8], by removing the CP from the viral vector, expression levels can be increased. Removing non-essential features of the virus also gives more room for larger inserts and channels energy and building blocks that would be used to synthesize these proteins into expressing the recombinant protein [20]. Mor et al. obtained expression levels 40 times higher when supplying Rep as well as RepA than when no Rep/RepA was supplied. Showing that RepA also enhances expression levels, probably by making the cell environment more favorable for replication [24]. 

Regnard et al. (2010) designed a replicon vector, pRIC, based on the mild strain of BeYDV that contained the Rep/RepA coding regions in cis rather than in trans. This allowed the vector to autonomously replicate and thus generate high levels of gene copy number and in turn enhanced protein expression. They used N. benthamiana plants and Agrobacterium tumefaciens-mediated gene delivery. They obtained higher expression levels than previously described of three unrelated proteins: enhanced GFP, Human Papillomavirus type 16 major CP, L1, and a HIV-1 p24 antigen. Yields were higher when using the replicative vector than when compared to expression from a non-replicative A. tumefaciens expression vector: 550 mg ⁄ kg fresh leaf weight (FLW) vs. 337 mg L1 ⁄ kg FLW for L1 and 3.23 mg p24 ⁄ kg FLW vs. 0.95 mg p24 ⁄ kg FLW for p24. This study showed that autonomous replication of BeYDV-based vectors dramatically increases gene expression levels [25].

Huang et al. (2009) designed a three-component replicon system that consisted of a construct derived from a deconstructed version BeYDV similar to that described by Mor et al. (2003) containing the gene of interest expression cassette, a construct encoding for the Rep/RepA under CaMV 35S promoter control and a construct expressing the posttranscriptional gene silencing suppressor protein P19. They obtained 0.34 g of Norwalk virus CP (NVCP) / kg FLW and 0.8 g of hepatitis B core antigen (HBc) / kg FLW, which were able to form VLPs. In order to simplify the replicon system, they included the Rep/RepA sequences in cis. They obtained similar expression levels when using the simplified replicon, with or without P19 supplementation as when the three-component system was used [26].  Later they designed a single vector containing multiple replicon cassettes each flanked by a LIR and a SIR. The vector also contained the Rep/RepA sequences under LIR control. Co-delivering the single-vector replicon and a P19 expression vector, they expressed the light and heavy chain of an Ebola virus-targeting monoclonal antibody (mAB), 6D8. They obtained ≈0.5 g of 6D8 mAB / kg FLW which had been assembled correctly and could bind its antigen specifically. Expression levels were comparable to those obtained by Giritch et al. (2006) [27] using two vectors based on two non-competing RNA viruses. They speculated that using this single-vector multireplicon system, even four proteins could be expressed simultaneously using two vectors or placing expression cassettes in tandem  [28].

More recently, Moon et al. (2014) were able to express Brome mosaic virus (BMV) and Cucumber mosaic virus (CMV) VLPs at 0.5 and 1.0 g / kg FLW respectively, using a BeYDV-derived single-vector replicon system. This vector included the P19 coding sequence, the gene of interest as well as the Rep/RepA coding sequences in the same backbone. In this way enhanced expression of VLPs that can be used as carriers for nano-platforms with applications in material sciences and medicine was possible with only one agroinfiltration [29].

Finally, Baltes et al. (2014) demonstrated that BeYDV-based replicon system can be also used for plant genome engineering. They were able to deliver various nucleases (TALENs and CRISP/Cas system) as well as repair templates into tobacco cells and to regenerate plantlets with the desired DNA changes within 6 weeks. This highlighted the potential of vectors derived from BeYDV and other geminiviruses to be applied in the engineering of plants for, for example,  improvement of crop characteristics, crop resistance or in fundamental biology studies [22].

In conclusion, BeYDV is a small, dicotyledonous plant-infecting mastrevirus with apparently unlimited possible molecular applications.

References

[1] W. Zhang, N. H. Olson, T. S. Baker, L. Faulkner, M. Agbandje-McKenna, M. I. Boulton, J. W. Davies, and R. McKenna, “Structure of the Maize streak virus geminate particle.,” Virology, vol. 279, pp. 471–477, 2001.

[2] E. P. Rybicki and D. P. Martin, “Virus-derived ssDNA vectors for the expression of foreign proteins in plants,” Current Topics in Microbiology and Immunology, vol. 375, pp. 19–45, 2011.

[3] A. Varsani, J. Navas-Castillo, E. Moriones, C. Hernández-Zepeda, A. Idris, J. K. Brown, F. Murilo Zerbini, and D. P. Martin, “Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus,” Archives of Virology, vol. 159, pp. 2193–2203, 2014.

[4] L. Liu, T. Van Tonder, G. Pietersen, J. W. Davies, and J. Stanley, “Molecular characterization of a subgroup I geminivirus from a legume in South Africa,” Journal of General Virology, vol. 78, pp. 2113–2117, 1997.

[5] J. Hadfield, J. E. Thomas, M. W. Schwinghamer, S. Kraberger, D. Stainton, A. Dayaram, J. N. Parry, D. Pande, D. P. Martin, and A. Varsani, “Molecular characterisation of dicot-infecting mastreviruses from Australia,” Virus Research, vol. 166, no. 1–2, pp. 13–22, 2012.

[6] R. P. Halley-Stott, F. Tanzer, D. P. Martin, and E. P. Rybicki, “The complete nucleotide sequence of a mild strain of Bean yellow dwarf virus,” Archives of Virology, vol. 152, pp. 1237–1240, 2007.

[7] K. E. Palmer and E. P. Rybicki, “The molecular biology of mastreviruses.,” Advances in virus research, vol. 50, pp. 183–234, 1998.

[8] L. Liu, J. W. Davies, and J. Stanley, “Mutational analysis of bean yellow dwarf virus, a geminivirus of the genus Mastrevirus that is adapted to dicotyledonous plants,” Journal of General Virology, vol. 79, pp. 2265–2274, 1998.

[9] Q. Chen, J. He, W. Phoolcharoen, and H. S. Mason, “Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants,” Human Vaccines, vol. 7, no. 3, pp. 331–338, Mar. 2011.

[10] L. Liu, K. Saunders, C. arole L. Thomas, J. W. Davies, and J. Stanley, “Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.,” Virology, vol. 256, pp. 270–279, 1999.

[11] K. L. Hefferon and B. Dugdale, “Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication,” Journal of General Virology, vol. 84, pp. 3465–3472, 2003.

[12] C. Gutierrez, “Geminivirus DNA replication,” Cellular and Molecular Life Sciences, vol. 56. pp. 313–329, 1999.

[13] “Adult drawing grape leafhopper,” Koppert Biological Systems 9103. [Online]. Available: https://www.flickr.com/photos/koppert/2400156751/. [Accessed: 09-Feb-2015].

[14] “Phaseolus vulgaris,” Belgium, Prelude – Royal Museum for Central Africa – Tervuren. [Online]. Available: http://www.africamuseum.be/collections/external/prelude/view_plant?pi=09910. [Accessed: 09-Feb-2015].

[15] N. Nahid, I. Amin, S. Mansoor, E. P. Rybicki, E. Van Der Walt, and R. W. Briddon, “Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan,” Archives of Virology, vol. 153, pp. 1441–1451, 2008.

[16] G. Pogue and S. Holzberg, “Transient Virus Expression Systems for Recombinant Protein Expression in Dicot-and Monocotyledonous Plants,” in Plant Science, N. K. Dhal and S. C. Sahu, Eds. InTech, 2012, pp. 191–216.

[17] F. Sainsbury, P.-O. Lavoie, M.-A. D’Aoust, L.-P. Vézina, and G. P. Lomonossoff, “Expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus RNA-2.,” Plant biotechnology journal, vol. 6, no. 1, pp. 82–92, Jan. 2008.

[18] E. P. Rybicki, “Plant-produced vaccines: promise and reality.,” Drug Discovery Today, vol. 14, no. 1–2, pp. 16–24, Jan. 2009.

[19] V. Yusibov, S. Rabindran, U. Commandeur, R. M. Twyman, and R. Fischer, “The potential of plant virus vectors for vaccine production.,” Drugs in R&D, vol. 7, no. 4, pp. 203–17, Jan. 2006.

[20] Y. Gleba, S. Marillonnet, and V. Klimyuk, “Plant Virus Vectors: Gene Expression Systems,” Encyclopedia of Virology, vol. 4, pp. 229–237, Apr. 2008.

[21] K. L. Hefferon, “DNA Virus Vectors for Vaccine Production in Plants: Spotlight on Geminiviruses,” Vaccines, vol. 2, no. 3, pp. 642–653, Aug. 2014.

[22] N. J. Baltes, J. Gil-Humanes, T. Cermak, P. a Atkins, and D. F. Voytas, “DNA replicons for plant genome engineering.,” The Plant cell, vol. 26, no. January, pp. 151–63, 2014.

[23] K. L. Hefferon and Y. Fan, “Expression of a vaccine protein in a plant cell line using a geminivirus-based replicon system,” Vaccine, vol. 23, pp. 404–410, 2004.

[24] T. S. Mor, Y.-S. Moon, K. E. Palmer, and H. S. Mason, “Geminivirus vectors for high-level expression of foreign proteins in plant cells.,” Biotechnology and bioengineering, vol. 81, pp. 430–437, 2003.

[25] G. L. Regnard, R. P. Halley-Stott, F. L. Tanzer, I. I. Hitzeroth, and E. P. Rybicki, “High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector.,” Plant biotechnology journal, vol. 8, no. 1, pp. 38–46, Jan. 2010.

[26] Z. Huang, Q. Chen, B. Hjelm, C. Arntzen, and H. Mason, “A DNA replicon system for rapid high-level production of virus-like particles in plants.,” Biotechnology and bioengineering, vol. 103, no. 4, pp. 706–14, Jul. 2009.

[27] A. Giritch, S. Marillonnet, C. Engler, G. van Eldik, J. Botterman, V. Klimyuk, and Y. Gleba, “Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 40, pp. 14701–6, Oct. 2006.

[28] Z. Huang, W. Phoolcharoen, H. Lai, K. Piensook, G. Cardineau, L. Zeitlin, K. J. Whaley, C. J. Arntzen, H. S. Mason, and Q. Chen, “High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system.,” Biotechnology and bioengineering, vol. 106, no. 1, pp. 9–17, May 2010.

[29] K. Moon, J. Lee, S. Kang, M. Kim, H. S. Mason, J. Jeon, and H. Kim, “Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.,” Applied microbiology and biotechnology, vol. 98, pp. 8281–90, 2014.

Ethical dilemma for Ebola drug trials

13 November, 2014

Public-health officials split on use of control groups in tests of experimental treatments.

With clinical trials of experimental Ebola treatments set to begin in December, public-health officials face a major ethical quandary: should some participants be placed in a control group that receives only standard symptomatic treatment, despite a mortality rate of around 70% for Ebola in West Africa?

Two groups planning trials in Guinea and Liberia are diverging on this point, and key decisions for both are likely to come this week. US researchers meet on 11 November at the National Institutes of Health (NIH) in Bethesda, Maryland, to discuss US-government sponsored trials. A separate group is gathering at the World Health Organization (WHO) in Geneva, Switzerland, on 11 and 12 November to confer on both the US effort and trials organized by the WHO with help from African and European researchers and funded by the Wellcome Trust and the European Union.

Source: www.nature.com

I have to say – faced with a deadly disease, I think it is UNethical to have control / placebo arms of any trial.

Seriously: what about comparing ZMapp and immune serum, for example, with historical records of previous standard of care outcomes rather than directly?

I know if I were an Ebola patient, and I saw someone else getting the experimental therapy and I didn’t, that I would have a few things to say.

It’s not as if these therapies have not been tested in primates, after all – in fact, both the ChAd3 and MVA-based vaccines and ZMapp have been thoroughly tested in macaques, as have the other therapeutics, with no adverse events there.

I say if people say clearly that they want an experimental intervention, that they should get one: after all, the first use of immune serum was not done in a clinical trial, but rather as a last-ditch let’s-see-if-this-works intervention – yet its use does not seem controversial?

See on Scoop.itVirology News

Virology Africa 2015: consider yourselves notified!

7 November, 2014

Dear ViroBlogy and Virology News followers:

Anna-Lise Williamson and I plan to have another in our irregular series of “Virology Africa” conferences in November-December 2015, in Cape Town.

As previously, the conference will run over 3 days or so, possibly with associated workshops, and while the venue is not decided, we would like to base it at least partially in the Victoria & Alfred Waterfront.

We also intend to cover the whole spectrum of virology, from human through animal to plant; clinical aspects and biotechnology.

We intend to make it as cheap as possible so that students can come. We will also not be inviting a slate of international speakers, as we have found that we always get quite an impressive slate without having to fund them fully.

It is also the intention to have a Plant Molecular Farming workshop – concentrating on plant-made vaccines – concurrently with the conference, in order to leverage existing bilateral travel grants with international partners. If anyone else has such grants that could be similarly leveraged, it would be greatly appreciated.

See you in Cape Town in 2015!

Ed + Anna-Lise


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