Effect of Formaldehyde Inactivation on Poliovirus

23 September, 2014

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.


Source: jvi.asm.org

People have been treating poliovirus with formaldehyde for over 60 years – and it’s only NOW that someone thought to study in detail what happens!

I love this stuff: analytical centrifugation could have been done any time in the last fifty years (and has been, in determining structural transitions) but the newer techniques such as flow cytometry and RT-PCR could not. Analytically determining now what was empirically observed to work when polio vaccines were first made, is a historically important vindication of pioneering work that has almost made the viruses go away.

Simple and obvious findings, essentially – it is obvious that methylene bridging between amino acids would affect structural transitions; so too that HCHO treatment would kill viral ssRNA – but it hadn’t been DONE properly previously.  Great stuff!

See on Scoop.itVirology and Bioinformatics from Virology.ca

Engineering of inducible resistance in maize to Maize streak virus: a history, and a model for South-South collaboration

16 September, 2014
A Maize streak virus particle: characteristic doubled icosahedron containing a single ssDNA Graphic by Russell Kightley Media

A Maize streak virus particle: characteristic doubled icosahedron containing a single ssDNA
Graphic by Russell Kightley Media

I blogged on this paper from our group over at Virology News, but then I decided to do it again here. Because, as I said there,

“This is a big deal: seriously. It’s the culmination of some 24 years of involvement by my lab in engineering resistance in maize, and is the latest effort on top of of one unsuccessful and one partially successful construction by three top-class researchers in that time.”

…and I don’t think I did justice at the time to acknowledging the amount of effort it has taken to get to this point – which has occupied nearly 30 years of my life, and a considerable fraction of the working life of many others.  So here’s something of a chronicle of how we got here from there with some explanation of the very cool science behind it, and acknowledgement of a very valuable friendship between us and colleagues in Australia, without whom this would not have happened.

Maize streak virus is, of course, possibly the first virus described from Africa, and one famous enough to have its own web page, complete with the Proceedings of the First (and last) International Maize Streak Disease Symposium held in Hazyview in South Africa in 1997.  It is a ssDNA virus with a 2.7 kb circular genome that is encapsidated in unique geminate particles, is obligately transmitted by the leafhopper Cicadulina mbila Naude, and is probably the worst viral pathogen of maize in Africa.

The Maize streak virus investigations that led to this latest development were started by my retired colleague and former PhD supervisor Prof Barbara von Wechmar, back in the 1960s at the University of Stellenbosch.  Barbara it was who isolated MSV out of naturally infected maize, and developed home-grown methods of propagating it in sweetcorn via viruliferous Cicadulina mbila leafhoppers – and more importantly, getting clean leafhopper colonies so that new isolates could be studied.

She brought this knowledge with her to UCT when she came here with Marc van Regenmortel as the new Professor of Microbiology, and the leafhopper work and virus isolates puttered on in the background, with a couple of interesting papers coming out. The first was a collaborative work with the legendary Bob (RG) Milne, who came here on a sabbatical from the Istituto di Fitovirologia Applicata del CNR in Torino, Italy, and revolutionised our electron microscopy techniques by use of uranyl acetate instead of phosphotungstate, which meant for the first time we could see MSV particles.  This got presented at an International Maize Virus Disease Colloquium and Workshop in Wooster, Ohio in 1982, the Proceedings of which  seem to be available here.

VON WECHMAR, M.B., & MILNE, R.G. (1983). Purification and serology of a South African isolate of maize streak virus. pp. 164-166 In: Proc. Internat. Maize Virus Dis. Colloquium and Workshop. University of Ohio Press, Wooster

Maize streak virus particles isolated from maize, photographed by Robert G Milne in Cape Town

Maize streak virus particles isolated from maize, photographed by Robert G Milne in Cape Town

Another paper in J Gen Virol from 1983 was a fascinating account of using “electroinfection” to infect maize plants with MSV, published with Dr Alfred Polson: he was a quirky and idiosyncratic physical biochemist-cum-virologist who had done a PhD with The Svedberg and Ole Lamm in Uppsala in the 1930s, headed a Virus Research Unit at UCT Medical School, then retired to our Department to basically play with physical and serological techniques.  He liked nothing better than to hook plants up to kilovolt power packs, and to do large-scale electrophoresis of proteins and viruses in convoluted home-made glass contraptions – and to pull us in to help.

Also in the early 1980s, when molecular microbiology belatedly dawned in South Africa, a couple of DNA-wise colleagues and I thought it would be a great idea to sequence the MSV genome, given that it had been shown in 1977 in a Nature paper by Bryan Harrison and colleagues to be circular ssDNA.  Sadly, this never really got past the talking and wasting virus sample stage by Frank Robb and Ralph Kirby before two papers were published on the sequence of MSVs in 1984 – with the second, by Phil Mullineaux and colleagues from the John Innes Institute, drily pointing out that the first, by Steve Howell, had the sequence of the complementary, rather than the genomic strand of DNA.

I decided in 1985, after getting a PhD on a biophysical and serological investigations of small grain viruses in 1984, to change my skill set during a three-month academic leave in Belgium by learning molecular cloning and DNA handling techniques.  Thanks to the recommendation of Marc van Montagu, and the teachings of the good folks at Plant Genetic Systems in Gent (Jan Leemans and Herman Hofte, I still owe you B-), this was achieved – and the first thing I did on my return was to recruit an able Honours student in the person of Bev Clarke, cosupervised by Ralph Kirby for the DNA expertise, to clone and sequence local MSV strains that were still being maintained by Barbara von Wechmar.  Bev worked through into a Masters degree on cloning and restriction mapping of three maize isolates of MSV – sequencing was still a bit complicated and expensive in those days – and we managed to publish two babck-to-back papers out of her work, in 1989.  The first was an account of the propagation, isolation, cloning and mapping, and we in fact got the cover of that issue.

intervirol coverThe second was the start of what became a really interesting sideline for me, in evolutionary studies on viruses – because Ralph managed to use the maps we generated from our viruses and from sequenced isolates to estimate sequence divergence. It really is quite amusing, in this era of rapid and metagenomic sequencing, to read what we thought we had accomplished at the time, given that my team has just sequenced some ten geminiviruses as part of a BSc third year project:

“The aligned restriction endonuclease maps of three sequenced maize streak virus isolates, three restriction-mapped southern African maize streak virus isolates, and two other sequenced geminiviruses were used as a means of calculating the sequence divergence between these viruses. The degree of divergence was used to construct a phylogenetic tree for the viruses; this tree agrees well with predictions from sequence comparisons, and so the method can be used to study the relationship of geminivirus isolates without the labor and expense of sequencing each one. [my emphasis]”

On the strength of this and other work, I was prompted in 1988 to write a bold (and naive) lamppost-marking type of article on “Maize streak virus: an African pathogen come home?”, which I commemorated 25 years later here in ViroBlogy.  I cringed a little then and again now, seeing what we thought was so cool at the time. Ah, youth…B-)

Bev was succeeded as “the” student in my lab by Fiona Tanzer (then Hughes), who took over the MSV and other geminivirus work with verve and flair.  She produced a number of papers during her PhD, starting with an offering on “A rapid technique for typing streak virus isolates using a panel of differential hosts” at a South African Maize Breeding Symposium in 1990, and going on to characterise Sugarcane streak mastrevirus (SSV) from Natal as a distinct virus in 1991, by RE mapping, Southern hybridisation and partial sequence analysis; “Genome Typing of Southern African Subgroup-1 Geminiviruses” by the same techniques in 1992, and graduating to “Complete nucleotide sequence of sugarcane streak Monogeminivirus” in 1993.

Meantime, in what turned out to be my last stint of concentrated laboratory work, I had been investigating the potential of PCR for both detection and differentiation of mastreviruses – which led to a little detour into papillomavirology to apply the technique, which ended up being a complete change in direction, and a whole new career in vaccinology.  I used the primer design experience and PCR results I got then as the basis of an online teaching module on PCR, which is still available – and for which I actually get citations.

Degenerate Primer PCR for Mastrevirus Detection

But I digress: Fiona went on after her PhD to work as a postdoc with me and Jennifer Thomson, with whom I had been collaborating since she arrived at UCT in 1988 in the area of plant genetic engineering, on an insanely ambitious project to engineer transgenic resistance to MSV in maize.  This had in fact started in 1990, when I visited Bill Gordon-Kamm at DeKalb Genetics in Connecticut while on sabbatical at Cornell in 1990-1991: I got a suspension culture of Black Mexican Sweet (BSM) maize cells from them for us to practice biolistic techniques on with our newly-acquired BioRad helium gene gun, as well as a collaboration agreement which helped us immensely in our maize transformation and regeneration work.  Sandy Lennox it was who got that side of things working, to the extent of regenerating viable plants from supposedly non-regenerable BMS cells – which laid the foundation for all of our subsequent work in this area, using HiII embryogenic callus cultures.

Fiona, meanwhile, had managed to both clone and sequence a moderately severe isolate of MSV from Komatipoort (MSV-Kom), and then also make it agroinfectious as a partially dimeric clone in Agrobacterium tumefaciens, following the landmark 1987 example of Nigel Grimsley and colleagues in Basel.  This provided the other half of the toolkit, as we now had a means of reliably testing regenerated maize with a MSV isolate of known virulence, available for infection as a clone rather than transmitted via leafhoppers.

Fiona went on to make and test antisense RNA-expressing constructs from the Rep gene of MSV-Kom, as I had been heavily influenced by a 1991 Keystone Symposium on Antisense RNA Technology I went to while on sabbatical – and reported on here – and was convinced this was the way to go.

Inevitably, as determined by exhaustive experimentation from laboriously regenerated plants, this turned out not to be true: there was no obvious protection of the highly susceptible HiII by any of the constructs.  Fiona went off to have a baby – her second while working with me – and the MSV effort stalled for a while.  Barbara retired, and it fell to me to maintain leafhopper colonies and MSV and other mastrevirus isolates – which, very fortunately, we had begun routinely cloning and making agroinfectious, via a very able cohort of students led by Wendelin Heribert “Popeye” Schnippenkoetter, who perfected the art of the “1.1-mer” infectious clone as well as finally publishing the MSV-Kom sequence.

The value of international networking in geminivirology became apparent in 1994, while all this this was happening, when I went to the first International Geminivirus Symposium in Almeria, Spain.  There I re-made the acquaintance of  Doug Maxwell, who I had met in Ithaca in 1991 when he spoke on using PCR to detect begomoviruses.  I also recall performing “Born to be Wild” in a hotel karaoke bar with a scratch band of geminivirologists, but we shall speak no further of this.


Doug and I corresponded until the next Symposium he organised in Puerto Rico in 1998, when we heard his PhD student Steve Hanson present on the use of trans-dominant Rep gene mutants to inhibit the replication of Bean golden mosaic begomovirus in transient assays in cultured bean cells.  They sent us the PhD thesis to use as a reference document; the work was eventually published in 1999.

This kicked our efforts into a higher gear, given that we now had an exemplar in another albeit VERY distantly related geminivirus.  We also had a very bright new PhD student from Zimbabwe, Tichaona Mangwende, as the perfect guinea pig for investigation of dominant negative Rep mutants, and off we set again.  Tich quickly made a number of site-directed mutants by very ingenious methods, and we then came up to the hurdle of how to test them.  We had had in mind that this would be done in regenerated plants; however, it was quickly apparent that this would stretch his project out for several years longer than was feasible.

Here it was that the value of having students working in related projects was shown: after 1996 I had received expanded funding due to a favourable Foundation for Research Development rating, and actually had a lab full of students all working on geminiviruses.  Fortuitously, Kenneth Palmer had been doing a project on exploring the potential of MSV-based constructs as recombinant expression vectors for use in maize, and had worked out a suite of techniques for bombarding BMS suspension-cultured cells and assaying for virus genome replication and protein expression.  Even more fortuitously, Janet Willment had been investigating the minimal cis-acting control regions for replication of MSV and their sequence specificity, and following Kenneth’s example, had set up exactly the right system for biolistic introduction of DNA constructs into suspension cultured cells as well as quantitative PCR for assay of replication, that Tich required for his work.  He was able to test three different constructs in conjunction with partial dimers of MSV-Kom by transient expression in biolistically transformed cells, and prove that they significantly inhibited MSV genome replication.  He got a great PhD, and has gone on to good things in the agricultural biotech sector in SA.

The stage was now set and dressed for the final act: this was the introduction of Dionne Miles, now Shepherd, into the continuation of this project into whole plant testing.  Here also was another example of fortuitous cross-bleed between projects: it happened that Kenneth Palmer had been helping Jennifer Thomson with a PhD student who was working on a difficult project to do with regeneration of cereals from anther culture.  He helped Wusi develop a system for transformation and regeneration of a model grass species, Digitaria sanguinalis, originally sourced from the flowerbed next to the UCT Sports Centre.  As it happened, some regenerated Digitaria was in a plant room that had escaped viruliferous leafhoppers in it – and got infected with MSV-Kom, and showed splendid streak symptoms as well as stunting.  As it was easier to transform than maize, could be grown as a perennial by simply cutting it back, and went to seed inside six weeks, it was obvious that we had a wonderful model plant for MSV resistance testing.  Another vital cog in what was becoming a complicated machine was my student Darrin Martin, who had developed a truly wonderful image processing-based quantitative symptom assessment tool as a central part of his PhD project on the determinants of pathogenicity in MSV.  This also proved vital in future work on accurate determination of the degree of resistance of regenerated maize.


Dionne started what was to become her whole professional life to date in the late 1990s, by modifying Tich’s clones and testing them in maize cells for efficacy, and then introducing them into D sanguinalis cells for regeneration.  The value of the strategy was quickly apparent, when she showed that the best constructs for transient inhibition of MSV replication also either prevented regeneration of plants completely, or produced a very aberrant and infertile phenotype.


She was left with one – a truncated Rb- mutant Rep, rep1-219Rb- – that allowed regeneration of normal fertile plants and inhibited virus replication in transient assays, that has formed the basis of most of the work since. This includes the publication of her development of MSV-resistant transgenic fertile maize as “Maize streak virus-resistant transgenic maize: a first for Africain the Plant Biotechnology Journal in 2007, and getting us another cover.



What followed this was a long, painstaking grind by Dionne and team, and notably Marian Bezuidenhout who did most of the transformation and regeneration, in making as many transgenic lines as possible to provide to our maize seed producing industry partner Pannar Pty Ltd for introgression of the transgene into their elite breeding lines.  There followed much assessment of symptom development in greenhouse-tested plants, often grown from or descended from plantlets flown from our plant rooms to Greytown in an executive jet – along with boxes of wine, it does need to be said.  This has produced analyses that look like this: the product of more patient work than I think I would be capable of, and a really good example of how one should do this sort of work.  With repeats.  Many, many repeats.


Dionne followed this up in 2011 with a second-generation product, with a paper on “A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay“:

“After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicative-form-specific PCR, it was clear that… the hairpin rep construct (pHPrepΔI(662)) completely inhibited MSV replication…[and] in addition, pHPrepΔI(662) inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity.”

This is also a big deal, as it represents another, alternative strategy to confer MSV resistance on maize, that confers wider resistance, and could potentially be stacked with the previous construct.

Then at last, we come to the present work – with a sense of the history behind it.  The rationale for this was the following:

“While we have previously developed MSV-resistant transgenic maize lines constitutively expressing “dominant negative mutant” versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or “leaky” expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture.”

Backtracking slightly, this has been the product of another networking experience at a foreign conference: this time, between me and my by-then-old friend James Dale from Queensland University of Technology, at the “Virologica 2001″ conference of the Brazilian Society for Virology in Caldas Novas, Brazil.  Relaxing as invited speakers do, with a beer on a balcony, James said to me “Ed, mate, you’re going to kick yourself that you didn’t think of this!”, and proceeded to tell me about a geminivirus-based inducible expression system he and his group had just invented.  I did.  I also swore, loudly.  I then got him to offer highly reasonable terms for us to use the technology, and promptly took the idea home.  I have also described it in a recent review on ssDNA virus-derived plant expression vectors, so I take great glee in presenting my graphic here:


This has only been published recently, due to patent issues and its use in proprietary production – however, we got the full cooperation of James and Ben Dugdale of QUT early on, and

“…used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.”

msv induc res

Pretty good, you’d think?  Also an excellent example of South-South collaboration, and investigations being set up as they should: by two people having a beer, at a conference.  Thanks, James!  Thanks Benno!  The paper ends optimistically, with:

“Ultimately, the practicality of the SGCs described in this study will only be fully realised with the regeneration of phenotypically normal transgenic maize plants engineered to contain the SGC that are resistant/immune to MSV infection. To this end we have regenerated a number of transgenic maize lines containing a SGC capable of expressing the most effective Rep mutant, namely RepIII-Rb-. In contrast to lines constitutively expressing this mutant gene, SGC lines have produced T2generation offspring with normal phenotypes.

Considering that only one strain – MSV-A – causes severe disease in maize throughout the whole geographical range of MSV, and that all isolates so far discovered within this strain have a maximum divergence of only 4.62% at the nucleotide level, it is likely that this novel MSV-inducible resistance construct will be effective against the complete spectrum of severe maize streak disease-causing African MSVs.”

Sadly, and these promises notwithstanding, this has all come to an end, with the non-renewal of the funding – possibly as a result of the takeover of Pannar by Pioneer HiBred Intl, but for whatever reason, it marks literally the end of an era.

What the hell – we had fun.  And maybe we can still sell it to someone.

Ebola: “We Could Have Stopped This”

8 September, 2014

Public health officials knew Ebola was coming. They know how to defeat it. But they’re blowing it anyway.

ld, you still just don’t get it. The Ebola epidemic that is raging across West Africa, killing more than half its victims, will not be conquered with principles of global solidarity and earnest appeals. It will not be stopped with dribbling funds, dozens of volunteer health workers, and barriers across national borders. And the current laboratory-confirmed tolls (3,944 cases, with 2,097 deaths) will soon rise exponentially.

To understand the scale of response the world must mount in order to stop Ebola’s march across Africa (and perhaps other continents), the world community needs to immediately consider the humanitarian efforts following the 2004 tsunami and its devastation of Aceh, Indonesia. The U.S. and Singaporean militaries launched their largest rescue missions in history: The United States alone put 12,600 military personnel to a rescue and recovery mission, including the deployment of nearly the entire Pacific fleet, 48 helicopters, and every Navy hospital ship in the region. The World Bank estimated that some $5 billion in direct aid was poured into the countries hard hit by the tsunami, and millions more were raised from private donors all over the world. And when the dust settled and reconstruction commenced, the affected countries still cried out for more.

Source: www.foreignpolicy.com

A seriously hard-hitting article by a very good journalist with a particular interest in infectious diseases.

And she’s right: Ebola was stopped, not once, but a number of times, as long as 38 years ago, in settings that are as or even more desperate in terms of poverty and lack of medics and medical resources.

The problem is, intervention did not occur soon enough this time, or on a scale sufficient to stem the increase in infections that inevitably followed introduction of the disease into urban settings.

It is a matter of amazement to me, that with the ever-present threat of pandemic influenza AND the recent emergence of MERS, that the WHO should have its "…miniscule epidemic-response department slashed to smithereens by three years of budget cuts".

Seriously: faced with diseases that can jump out of camels, or bats, or rats literally anywhere, WHO has to have budget cuts??

See on Scoop.itVirology News

Mucosal SIV Vaccines with Bacterial Adjuvants Prevent SIV Infection in Macaques

2 September, 2014

A new paradigm of mucosal vaccination against HIV infection has been investigated in the macaque model. A vaccine consisting of inactivated SIVmac239 particles together with a living bacterial adjuvant (either the Calmette & Guerin bacillus, lactobacillus plantarum or Lactobacillus rhamnosus) was administered to macaques via the vaginal or oral/intragastic route. In contrast to all established human and veterinary vaccines, these three vaccine regimens did not elicit SIV-specific antibodies nor cytotoxic T-lymphocytes but induced a previously unrecognized population of non-cytolytic MHCIb/E-restricted CD8+T regulatory cells that suppressed the activation of SIV positive CD4+ T-lymphocytes. SIV reverse transcription was thereby blocked in inactivated CD4+ T-cells; the initial burst of virus replication was prevented and the vaccinated macaques were protected from a challenge infection. Three to 14 months after intragastric immunization, 24 macaques were challenged intrarectally with a high dose of SIVmac239 or with the heterologous strain SIV B670 (both strains grown on macaques PBMC). Twenty-three of these animals were found to be protected for up to 48 months while all 24 control macaques became infected. This protective effect against SIV challenge together with the concomitant identification of a robust ex-vivo correlate of protection suggests a new approach for developing an HIV vaccine in humans. The induction of this new class of CD8+ T regulatory cells could also possibly be used therapeutically for suppressing HIV replication in infected patients and this novel tolerogenic vaccine paradigm may have potential applications for treating a wide range of immune disorders and is likely to may have profound implications across immunology generally.


Graphic of cells involved in HIV immunity from Russell Kightley Media

Source: journal.frontiersin.org

I have heard Jean-Marie Andrieu present this work – and I can understand why there is some skepticism surrounding it, because it is almost too good to be true.

Seriously: SUPPRESSING SIV-specific CD4 T-cell activation results in immunity to challenge infection??

However, and however – if this work is found to have been done well (and there is no evidence it was not), then this really could be a simple, reliable way of immunising people against HIV

Of course, monkeys aren’t people, and SIV is not HIV, so there MAY be a problem somewhere along the line in translating these results into humans – but what if there is not?

Then we may have a vaccine, and kudos to Jean-Marie Andrieu and co-workers to persevering along a difficult road to get their idea tested.

See on Scoop.itVirology News

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

25 August, 2014

Browsing through my own web pages in an effort to clean up dead-end links, and cull tired material, I discovered that my link to an essay I wrote 19 years ago was still live – and as it referred to something written in and put up on our nascent Web server in 1994, means it has a 20-year anniversary round about now.

My essay is

The Student, the Web and the Ebola Connection


Dr Jacobson, are you going to Kikwit?”

…and it is a record of events that resulted in 1994 from (a) an Honours student essay being written on “Emerging Viruses”, and (b) me playing around with the then-very-new WWW server that UCT has enabled – but didn’t tell anyone about, because they didn’t want anyone to use it until they had sorted out policies.  Oh, and (c) – the Kikwit Ebola outbreak in 1995.

I wrote in 1995:

“The whole phenomenon has been an object exercise in the power of the Web as a tool for the wide dissemination of information: we reached not only professional virologists, but also health-care professionals, and – most importantly – the lay public on a large scale”

And of course, this is even more true now – which is why, following the benign guidance of The Guru Cann, I maintain ViroBlogy and Virology News, and heartily recommend a Web presence to anyone who feels they need to disseminate information on topics of specialist and generalist interest to the world at large.

Of course, nearly all the links out of that essay are now dead – including to the original essay, that for a while there in 1995 was the ONLY detailed information on Ebola available on the Web.  So here is Alison Jacobson’s original essay, in full, revealed by going to my teaching material and checking out essays from 1997 and thereabouts:


Of course, I also maintained a daily update on the Kikwit outbreak, and then a couple of the next ones, before the Web caught up with me and it became easier to just trawl it for news via Google and its predecessors.  It still makes interesting reading, though, to go through some of what was posted from the disease frontlines back in the 1990s – and to remember that I had the TIME to do that kind of thing!

Where we are now

Well, here we are with what is the worst outbreak of Ebola in history, and here am I – again – trying to keep up with it.  This time, by the very excellent medium of the Web news aggregator Scoop.it, where I have established Virology News as a means of quickly and easily getting news out to the public.  Again, following the very excellent example of TGC, but also Chris Upton, who babied me along by letting me co-curate his Virology and Bioinformatics site.

Of course, there is a new angle to this outbreak – and that has been the compassionate use of a plant-made monoclonal antibody cocktail (ZMapp), hitherto only tested preclinically in a primate model.  Fortuitously, this all happened while I was finishing off a review on plant-made viral vaccines, so I reported on it – with references – here on ViroBlogy.

I was also able to report on it in my Plant Molecular Farming news site, with some authoritative statements from pioneers of the technology: Charles Arntzen from the Arizona Biodesign Institute sent through a link for an interview he did, and CNN covered it quite well too.  Charlie also sent through a set of links in an email that he was happy to share:

“The original story

There is a lot of interest from the press in “why tobacco” and “how does it work”?

The other focus is on the politics of scale up of the drug — it seems that criticism of the US is mounting in some sectors of Africa, and elsewhere.   I talked to a Spanish Language radio news station this morning, and the main questions related to “why is this a Secret Drug; are you trying to hide the secret from the world?”    “Is Reynolds tobacco trying to stop the supply of this drug to Africans?”    One guy asked if it was true that the Ebola Virus had been created in a test tube.

It seems that the press is largely to blame for using terms like Secret Drug.   It appears that they are also trying to mount political pressure to make a lot more of the drug to help Africans.   [This was] a nice job answering some of this….”

And at time of writing, the outbreak was still raging, had spread to Nigeria, and airlines were banning travel to half of West Africa – and alarmist tourist firms were advising people not to come to South and East Africa, as well.  The WHO has also said the impact is probably much greater than reported.

And Alison Jacobson is alive and well, and NOT working in virology any more.  Sadly!

5 Viruses That Are More Frightening Than Ebola

20 August, 2014

By Elizabeth Palermo, Staff Writer
Published: 08/15/2014 01:58 PM EDT on LiveScience
The Ebola virus has now killed more than 1,000 people in West Africa. Although the mortality rate of the most recent outbreak isn’t as high as in previous events, it’s still the case that most people who become infected with Ebola will not survive. (The mortality rate is about 60 percent for the current outbreak, compared with 90 percent in the past, according to the National Institutes of Health.)

1. Rabies

2. HIV

3. Influenza

4. Mosquito-borne viruses

5. Rotavirus



Source: www.huffingtonpost.com

Amen!  I have a fondness for Ebola simply because it is so spectacularly nasty, but it has killed fewer people in 40 years than flu or rotavirus does in 1.

Seriously: just like charismatic animals like elephants and tigers get all of the headlines when it comes to being endangered, rather than the humble tree frog(s), so do Ebola and Marburg get all of the attention when it comes to reportage on virus epidemics / pandemics.

See on Scoop.itVirology and Bioinformatics from Virology.ca

Ill prepared for an influenza pandemic

18 August, 2014

Over the last 500 years, there have been, on average, three severe influenza pandemics in each century. The most recent pandemic was declared in 2009. Yet despite much investment in public health and many improvements in vaccine production techniques and know-how, the availability of influenza vaccines during this event was far from adequate. Six months into the pandemic, 534 million doses were available, and after one year that number had risen to 1.3 billion — enough for only 8%and 25%, respectively, of the world population. We were lucky that the pandemic declared in 2009 turned out later to be mild and that just one shot of vaccine was sufficient to protect most people. This is not usually the case during a severe influenza pandemic.


Source: www.nature.com

"As countries continue to pre-book pandemic supply, it is more and more likely that the limited vaccines available during the first months of any pandemic during the next few years will be sold out almost completely"

And what does everyone think happened in South Africa during most of 2009 and 2010?

Well, they probably don’t – because not that many of them got sick.  But THERE WAS NO VACCINE for the general population until LATE 2010 – when the chances of another round of H1N1pdm 2009 had dissipated due to summer coming on.

And the vaccine that HAD come into the the country in 2010 got used for medical personnel, and – for the 2010 World Cup staff.

Seriously, we need to do better than this – and responding QUICKLY to news of a pandemic would be the ticket.

Using plants B-)

See on Scoop.itVirology News

What Would Happen if You Got Ebola?

13 August, 2014

A secondary infection in the U.S. is highly unlikely. But here’s how the healthcare system would respond if there was one.

Source: www.theatlantic.com

Goes without saying that this would happen in a lot of other places, too.  Including our very own South Africa – where it HAS happened, with Marburg, Ebola and Lujo viruses.  Written about right here on ViroBlogy.

See on Scoop.itVirology News

How can geminiviral Rep capture the cell cycle of differentiated plant cells?

12 August, 2014

African cassava mosaic virus (ACMV) in the geminivirus family has being affected 500 million people worldwide by devastating cassava crops during the past decades. It has caused severe symptoms and reduced yield up to the complete loss of roots, the main starchy food source especially for subsistence farmers in Africa. How can a tiny virus with a small genome evoke such dramatic effects? The viral key component, the replication-initiator protein (Rep), forces differentiated plant cells in the phloem to reactivate DNA synthesis. Even more, it does the same in model cells of fission yeast. We have identified, now, a potential cyclin interaction motif, RXL, in the sequence of ACMV Rep, which may be important for cell cycle control. This motif is essential to induce rereplication in yeast and necessary for viral infection of plants.


Source: www.virologyhighlights.com

I am a sucker for geminiviruses and their replication – as can be seen in the pages published here and elsewhere over the years.  It is fascinating to me that a small protein like Rep – only ~30 kDa – can do so many things, and especially interfere in such a fundamental way with organised, differentiated cells.

What is even more interesting is that it can do it in such a wide variety of systems: it’s been shown that ACMV can replicate in maize protoplasts as well as in the dicotyledonous cassava; it can evidently function well in yeast as well – and via a pathway that no-one suspected before now.

Truly, a protein of many parts!  Congratulations to Katharina Hipp and to my old friends Bruno and Holger.

See on Scoop.itVirology News

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

5 August, 2014
Ebola virus budding from an infected cell.  Courtesy of Russell Kightley Media

Ebola virus budding from an infected cell.
Courtesy of Russell Kightley Media

Yes, I know you fans of ViroBlogy like Ebola – and just coincidentally, I was desperately trying to finish a review* on “Plant-based vaccines against viruses” against a backdrop of an out-of-control Ebola epidemic in West Africa, when three different people emailed me different links to news of use of a plant-made monoclonal antibody cocktail.  I immediately included it in my review – and I am publishing an excerpt here, for informations’ sake.  Enjoy!

Plantibodies against Ebola

The production of anti-Ebola virus antibodies has recently been explored in plants: this could yet become an important part of the arsenal to prevent disease in healthcare workers, given that at the time of writing an uncontrolled Ebola haemorrhagic fever outbreak was still raging in West Africa, and the use of experimental solutions was being suggested (Senthilingam, 2014). For example, use of a high-yielding geminivirus-based transient expression system in N benthamiana that is particularly suited to simultaneous expression of several proteins allowed expression of a MAb (6DB) known to protect animals from Ebola virus infection, at levels of 0.5 g/kg biomass (Chen et al., 2011). The same group also used the same vector system (described in detail here (Rybicki and Martin, 2014)) in lettuce to produce potentially therapeutic MAbs against both Ebola and West Nile viruses (Lai et al., 2012).

A more comprehensive investigation was reported recently, of both plant production of Mabs and post-exposure prophylaxis of Ebola virus infection in rhesus macaques (Olinger et al., 2012). Three Ebola-specific mouse-human chimaeric MAbs (h-13F6, c13C6, and c6D8; the latter two both neutralising) were produced in whole N benthamiana plants via agroinfilration of magnICON TMV-derived viral vectors. A mixture of the three MAbs – called MB-003 – given as a single dose of 16.7 mg/kg per Mab 1 hour post-infection followed by doses on days 4 and 8, protected 3 of 3 macaques from lethal challenge with 1 000 pfu of Ebola virus. The researchers subsequently showed significant protection with MB-003 treatment given 24 or 48 hours post-infection, with four of six monkeys testing surviving, compared to none in two controls. All surviving animals treated with MB-003 experienced insignificant if any viraemia, and negligible clinical symptoms compared to the control animals. A significant finding was that the plant-produced MAbs were three times as potent as the CHO cell-produced equivalents – a clear case of plant production leading to “biobetters”. A follow-up of this work investigated efficacy of treatment with MB-003 after confirmation of infection in rhesus macaques, “according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization” (Pettitt et al., 2013). In this experiment 43% of treated animals survived, whereas all controls tested here and previously with the same challenge protocol died from the infection.

In news from just prior to submission of this article, a report quoted as coming from the National Institute of Allergy and Infectious Diseases states that two US healthcare workers who contracted Ebola in Liberia were treated with a cocktail of anti-Ebola Mabs called ZMapp – described as a successor to MB-003 – developed by Mapp Pharmaceutical of San Diego, and manufactured by Kentucky BioProcessing (Langreth et al., 2014). Despite being given up to nine days post-infection in one case, it appears to have been effective (Wilson and Dellorto, 2014).

A novel application of the same technology was also used to produce an Ebola immune complex (EIC) in N benthamiana, consisting of the Ebola envelope glycoprotein GP1 fused to the C-terminus of the heavy chain of the humanised 6D8 MAb, which binds a linear epitope on GP1. Geminivirus vector-mediated co-expression of the GP1-HC fusion and the 6D8 light chain produced assembled immunoglobulin, which was purified by protein G affinity chromatography. The resultant molecules bound the complement factor C1q, indicating immune complex formation. Subcutaneous immunisation of mice with purified EIC elicited high level anti-GP1 antibody production, comparable to use of GP1 VLPs (Phoolcharoen et al., 2011). This is the first published account of an Ebola virus candidate vaccine to be produced in plants.


Chen, Q., He, J., Phoolcharoen, W., Mason, H.S., 2011. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Human vaccines 7, 331-338.

Lai, H., He, J., Engle, M., Diamond, M.S., Chen, Q., 2012. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant biotechnology journal 10, 95-104.

Langreth, R., Chen, C., Nash, J., Lauerman, J., 2014. Ebola Drug Made From Tobacco Plant Saves U.S. Aid Workers. Bloomberg.com.

Olinger, G.G., Jr., Pettitt, J., Kim, D., Working, C., Bohorov, O., Bratcher, B., Hiatt, E., Hume, S.D., Johnson, A.K., Morton, J., Pauly, M., Whaley, K.J., Lear, C.M., Biggins, J.E., Scully, C., Hensley, L., Zeitlin, L., 2012. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proceedings of the National Academy of Sciences of the United States of America 109, 18030-18035.

Pettitt, J., Zeitlin, L., Kim do, H., Working, C., Johnson, J.C., Bohorov, O., Bratcher, B., Hiatt, E., Hume, S.D., Johnson, A.K., Morton, J., Pauly, M.H., Whaley, K.J., Ingram, M.F., Zovanyi, A., Heinrich, M., Piper, A., Zelko, J., Olinger, G.G., 2013. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Science translational medicine 5, 199ra113.

Phoolcharoen, W., Bhoo, S.H., Lai, H., Ma, J., Arntzen, C.J., Chen, Q., Mason, H.S., 2011. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant biotechnology journal 9, 807-816.

Rybicki, E.P., Martin, D.P., 2014. Virus-Derived ssDNA Vectors for the Expression of Foreign Proteins in Plants. Current topics in microbiology and immunology 375, 19-45.

Senthilingam, M., 2014. Ebola outbreak: Is it time to test experimental vaccines? CNN.

Wilson, J., Dellorto, D., 2014. 9 questions about this new Ebola drug. CNN.

* = which, despite their having commissioned from me, the good folk at “Viruses” an unnamed journal decided “…may not have substantial differences with the reviews you published recently” – and rejected.  I shall have revenge.  Oh, yes…B-)


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