Archive for the ‘biotechnology’ Category

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

Laurie Garrett on Ebola: the recent history

18 August, 2015

20 years after I first posted something by Laurie Garrett – who has written two of the the most thought-provoking, informative and frightening books I have ever read (The Coming Plague, and Betrayal of Trust) – I see she has just published possibly the single best account of the recent Ebola virus disease outbreak in West Africa.

Seriously.  Exhaustive, deep, analytical – and like her books, throwing some harsh light on world health care systems (or the lack thereof, in the case of the WHO), while at the same time making useful suggestions.

Like this one:

“And so it comes back to money. The world will get what it pays for—and right now, that is not very much.”

Absolutely: consider that the late and haphazard and meagre response by most governments let the epidemic peak and then start to subside – without actually, in the case of the US, managing to get more than one treatment centre functional in Liberia, before they ran out of patients.  That the health systems of all three countries are in such bad shape that they can’t deal with childbirth and malaria right now.

Laurie, it’s a great piece, really it is. It’s also depressing as hell.  But that’s life!

How should we preserve old viruses?

12 August, 2015

I was reminded via Twitter by Vincent Racaniello, he of “virology blog” fame, of the problem of preserving stocks of old viruses.

Particularly, in his case, of stocks of a virus that may be eradicated in the wild in a few years, and then – according to him – will need to be destroyed.

Surely we need to at least preserve sequence information of these pathogens before we let them go into oblivion, the way variola and rinderpest viruses have already gone?

So I wrote this to him:

“Great that you have preserved these samples – but a longer-term strategy needs to be adopted, before completely irreplaceable specimens are lost forever, to you and to science in general.

tmv sedimI have the same problem: a colleagues’ samples of plant viruses; beautifully preserved in heat-sealed glass vials, dried over silica gel, dating back in some cases to the early 1960s. For that matter, I have about a thousand glass bottles of liquid plant virus samples at 4degC, dating back in some cases over 40 years – and still viable.

Surely there is a case to be made for preserving some of these viruses? Mining them for sequence in this metagenomic age is not that difficult; preserving their infectivity, however – another matter. Some of my plant viruses are probably bomb-proof; your poliovirus samples, on the other hand – probably slowly deteriorating as we watch.

A wider conversation is needed: I know of other archives, of old poxvirus collections for example, that will be lost forever in a few years. Should we not get an international effort going to log them, sequence them, preserve them?

I think so.

Want to join in?

Yours,

Ed”

If any of you out there have a similar problem, let’s hear from you – and maybe we can do something to at least preserve the genetic information in unique collections.

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”

Anyone interested? A candidate virology textbook…

28 July, 2015

I would like to test the response to a Introduction to Virology ebook that I want to develop from my extant Web-based material, given that this is likely to disappear soon with our Web renewal project here at UCT.

Virus_Picture_Book_copy_iba

Download the Virus Picture Book excerpt here. And then please tell me what you think / whether you would buy one (projected price US$15 – 20)?  Ta!

Ebola on the Web – 20 years on

21 July, 2015

I have already done a partial retrospective on having been reporting on Ebola haemorrhagic fever viruses for just over 20 years – but I totally forgot to commemorate that I have been producing Web pages for just over 21! So I’m going to go on a nostalgic ramble through the past, mainly using Ebola as the vehicle, and highlighting some of the history of virology along the way.

By the way, I HAVE to commend the Wayback Machine here: I have also previously bemoaned the fact that Web pages are NEVER preserved by their creators at regular intervals – but this is exactly what they do.  From 1997 onwards in the case of the whole of the University of Cape Town’s site and mine as part of that – and how interesting it has been to go back and look at what I thought was cool then!  But actually, what’s not to like? I mean, there’s hepatitis G, Congo fever, smallpox, Ebola, “equine morbillivirus” (aka Hendra virus) – and life on Mars. Or not B-)

What’s interesting, though, is that they have preserved almost all of my Ebola news pages – dating from May 1995, from right near the onset of the Kikwit Ebola epidemic.  There’s all sorts of interesting stuff there – though with some holes, caused by Lost Pages – ranging from a discussion of the possibility of finding Ebola in cotton plants [not!], with my old friend Murilo Zerbini, to a thread on “Candidate for the Ebola Reservoir Organism” from the late lamented bionet.virology discussion group, to whether Ebola Reston was airborne (probably not).

Great historical stuff, right there – and thank deities it is preserved via Wayback, because our upcoming Web renewal project here at UCT will kill ALL links from our Departmental site.  Get it while you can!

And while we’re at it: here’s a useful list of all Ebola-related posts on ViroBlogy since 2011.  Note when the first mention of plant-made antibodies to Ebola virus was….

Molecular evidence of Ebola Reston virus infection in Philippine bats

18 July, 2015

The Discovery of Filoviruses

Ebola virus mutating, scientists say

29 January, 2015

First Ebola case linked to bat play – really?

30 December, 2014

Ethical dilemma for Ebola drug trials

13 November, 2014

Rabies Vaccine Protects Nonhuman Primates against Deadly Ebola Virus

26 October, 2014

Packs of wild dogs spread Ebola after eating corpses!! Or…not, maybe?

13 October, 2014

Norway to get world’s last dose of ZMapp – update

8 October, 2014

8 September, 2014

20 years on, and here we are with Ebola, again

25 August, 2014

5 Viruses That Are More Frightening Than Ebola

20 August, 2014

What Would Happen if You Got Ebola?

13 August, 2014

Plant-made antibodies used as therapy for Ebola in humans: post-exposure prophylaxis goes green!

5 August, 2014

Has the Time Come to Test Experimental Ebola Vaccines?

30 July, 2014

Plant-Based Antibodies, Vaccines and Biologics 5, Part 5

3 September, 2013

Ebola Outbreak in Uganda: CDC Rushes to Contain Virus

8 August, 2012

More Ugandans Admitted with Possible Ebola

1 August, 2012

Ebola reaches Uganda’s capital

31 July, 2012

31 July, 2012

Canadian researchers thwart Ebola virus

14 June, 2012

African monkey meat that could be behind the next HIV

25 May, 2012

Current Opinion in Virology – Mass extinctions, biodiversity and mitochondrial function: are bats ‘special’ as reservoirs for emerging viruses?

5 April, 2012

When dinner could kill you: smoked chimpanzee, anyone?

14 January, 2012

Virology Africa 2011: viruses at the V&A Waterfront 2

19 December, 2011

Ebola: ex tobacco, semper a vaccine novi

6 December, 2011

Influenza virus: a short introduction

14 July, 2015

This is excerpted from the ebook “Influenza Virus. Introduction to a Killer”, which is available here for US$9.99 .

Influenza: the disease

Influenza: a disease and a virus

Influenza as a disease in humans has been known for centuries; however, its cause was only discovered in the early 20th century: this was the group of viruses now known as Influenza virus types A, B and C.

There are several influenza viruses circulating in humans at any one time; these cause “seasonal flu”, which is usually a mild disease because most people have some degree of immunity.

Influenza pandemics, however, are caused by novel viruses – which are generally derived from animals, and usually originate in birds.  Here, the disease can be much more severe.

Influenza viruses have caused some of the biggest and yet some of the most insidious disease outbreaks to have hit humankind: from 1918 to 1920, the “Spanish Flu” pandemic killed more than 60 million people across the world; subsequent pandemics in 1957, 1968 and 1977 killed millions more, and the count is still unclear on the 2009 pandemic. However, in any given year more than 400 000 people probably die of so-called “seasonal flu” – yet universal vaccination against it is still a dream.

What is Influenza?

What is Influenza?

The Centers for Disease Control and Prevention in the USA define influenza as

“…a contagious respiratory illness caused by influenza viruses that infect the nose, throat, and lungs. It can cause mild to severe illness, and at times can lead to death.”

The disease is transmitted mainly via droplets of respiratory secretions: these result from sneezing or coughing, which blows out a fine cloud of droplets or aerosol from the upper airways of infected people.  Breathing in or inhalation of these droplets – which can happen from 2 metres away – or transfer of droplets by hand from a contaminated surface to the mouth, is enough to cause infection. 

The virus initially infects cells of the upper airway, or the respiratory epithelium.  Spread to lower parts of the respiratory system, such as into the lung, depends upon the particular virus, and whether or not the individual is partially immune.

  • Fever or chills
  • Cough
  • Sore throat
  • Rhinitis, or runny nose
  • Muscle or body aches, headaches
  • Tiredness, “fuzzy head”
  • Vomiting and/or diarrhoea (more common in children than adults).

The average incubation period, or time from infection to disease, is about 48 hours.  Full recovery can take a month, although about two weeks is more common in seasonal flu.  People can pass on the virus before they show symptoms, and each infected person on average infects another 1.4 people.

While flu may be mild enough that it is hardly noticed, severe disease can also occur – especially in the elderly, the very young, heavy smokers, people who are chronically ill from other causes – and immunocompromised individuals.

While the virus can cause pneumonia directly due to damaging lung tissue, as happened in the “Spanish Flu” pandemic, severe illness with pneumonia is more usually due to secondary bacterial infections – which can be treated with antibiotics, unlike the viral pneumonia

Seasonal flu, or the disease caused by viruses circulating in the population, typically has an “attack rate” of between 5-15% of the population in annual epidemics.  Case fatality rates, or deaths among those infected, are usually between 0.1 – 0.3%. However,  pandemic flu – caused by new strains which arise spontaneously, and to which people are not immune – can attack from 25-50%, and kill 5% of those infected.  Seasonal flu also mainly infects children – because older people are often immune – but mainly causes severe disease and death in the elderly: up to 90% of victims are usually 65 or older

Conversely, pandemic strains may affect a different set of age groups: for example, the Spanish Flu affected mainly healthy young adults.

Seasonal influenza is typically a disease of the autumn and winter seasons in temperate zones – meaning October – March in the northern hemisphere, and April – August in the southern.  The CDC FluView graph shown here clearly illustrates the cyclical nature of seasonal flu, tracked in the USA over a 5 year period.  However, the exact timing is not reliable, and epidemics may peak as early as October in the north, or April in the south, or as late as the end of the season.

Tropical zones have a different epidemic profile:

here the virus may circulate year-round, typically with a peak during the one or two rainy seasons.  Because of demographic reasons incidence is severely under-reported: however, in a seasonal outbreak in Madagascar in 2002, there were more than 27 000 cases reported in 3 months, with over 800 deaths for a case-fatality rate of around 3%.  A WHO coordinated investigation of this outbreak found that there were severe health consequences in poorly nourished populations with limited access to adequate health care.

Why is influenza seasonal?

Many reasons have been invoked over the years to explain this, ranging from temperature, humidity, school schedules, increased indoor crowding during winter or rainy seasons, and even variations in host immunity due to lack of vitamin D or melatonin.  However, the same reasons cannot be given for both the increase in influenza incidence in temperate climates with the onset of winter, and the rainy season peaks in tropical regions, given the very different environmental conditions prevailing.

A recent study set out to systematically determine the interactions between relative humidity, and salt and mucus and protein content of droplets containing live flu virus, on the viability of the virus – and came up with conclusions that could explain the temperate / tropical transmission differences.

Essentially, their explanation for temperate region seasonality is that there is low relative humidity indoors in winter due to heating: this leads to increased survival of virus due to drying of particles – influenza A viruses are stabilised by being dried in the presence of salts, mucus and proteins – and leads to aerosols persisting longer in the interior environment due to smaller size, and being propagated further, meaning most transmission would be by this route.  Increased time spent indoors and increased indoor crowding due to the climate would obviously increase transmission rates under these conditions. 

Tropical environments present a very different picture: here, high temperatures would accelerate virion decay, which would tend to decrease any transmission.  However, in rainy seasons, temperatures drop and relative humidity increases to nearly 100% – conditions conducive to survival of large drops, which settle out quickly onto surfaces, where the virus remains viable.  Thus, transmission could be mainly by surface contact.  The same social factors apply as for temperate climates, with frequent rain leading to more time indoors and more crowding – and a greater opportunity for transmission.

eBook on “Influenza Virus: Introduction to a Killer”

17 June, 2015

For some five years now, I have been simultaneously writing two ebooks on viruses. The one – originally part of a longer effort not yet finished – is “A Short History of the Discovery of Viruses” which is also advertised on Virology News; the other is a labour of love on influenza.

Labour of love for me because I got more into it the more I read, and because Russell Kightley’s images were so amazing.

Both were written using Apple’s iBooks Author app; both are designed to be read by Apple’s iBooks app on iPad, iPhone or Mac.

So here it is:

Influenza Virus: Introduction to a Killer

Enjoy. Buy!

Influenza_1-6-15_sample_iba

 

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

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Tracing the bird flu outbreak in North American poultry flocks

14 May, 2015

(Reuters) – The United States is facing its worst outbreak on record of avian influenza as three deadly strains have hit North American poultry flocks since December, with the spread of infection picking

Source: www.reuters.com

Useful timeline!

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