This Old PI….

7 March, 2014

See on Scoop.itVirology News

Ed Rybicki‘s insight:

I blame Chris Upton

VIGS in fungi – using TMV?!

5 March, 2014

See on Scoop.itVirology News

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants.

Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatumtransformant line 10 expressing GFP derived from C71.

The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

TMV graphic from Russell Kightley Media

Ed Rybicki‘s insight:

This is a nice paper for two main reasons: one, they were able to get VIGS – virus-induced gene silencing – working in a non-model fungus; two, they did it with TMV.

TMV! A plant virus in good standing, not previously shown to infect fungi productively, even if it has been studied in yeast as far as replication requirements go.

This is very interesting, not the least because it opens up the possibility that TMV NATURALLY infects some soil / leaf surface fungi.

Which could open up some investigation of just how the virus gets around, because it has always been touted as being only “mechanically” transmissible – even though we and others have shown it CAN be transmitted by aphids (reasonably inefficiently).

Mind you, Barbara von Wechmar and others in our lab showed in the 1980s that wheat stem and leaf rust fungi could transmit Brome mosaic virus and that Puccinia sorghi could transmit a potyvirus; they just did not have the techniques to look at whether or not it replicated too.

As far as my last post here is concerned, I think there is going to be a LOT of stuff coming out in the next few years on how “plant” and “insect” and “fungal” viruses are in fact considerably more promiscuous in choice of host(s) than we have hitherto been aware.

Now, just to prove what Barbara always said, that Tobacco necrosis virus is also a bacteriophage….

Thanks to Gary Foster (@Prof_GD_Foster) for pointing this out!

See on m.pnas.org

TRSV or not TRSV, that is the question. In bees, obviously.

25 February, 2014

I promised some time ago now to blog on the exciting topic of whether or not a plant virus is infecting honeybees – and here it is!  I was also contacted by the legendary Dr Adrian Gibbs about this paper, because he has read this blog, so I am including a commentary from him as well.

A little while ago, Ji Lian Li and co-workers published a paper entitled “Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera” in ASM’s Open Access journal mBio.  They stated that:

“Pathogen host shifts represent a major source of new infectious diseases. Here we provide evidence that a pollen-borne plant virus, tobacco ringspot virus (TRSV), also replicates in honeybees and that the virus systemically invades and replicates in different body parts. In addition, the virus was detected inside the body of parasitic Varroa mites, which consume bee hemolymph, suggesting that Varroa mites may play a role in facilitating the spread of the virus in bee colonies. This study represents the first evidence that honeybees exposed to virus-contaminated pollen could also be infected and raises awareness of potential risks of new viral disease emergence due to host shift events. About 5% of known plant viruses are pollen transmitted, and these are potential sources of future host-jumping viruses. The findings from this study showcase the need for increased surveillance for potential host-jumping events as an integrated part of insect pollinator management programs”.

This paper has caused all sorts of excitement, as well as coming to some possibly misleading conclusions, and leading to quite a lot of uninformed speculation – so I think it is as well to explore quite carefully what they did.

Adrian first:

“This paper reports that tobacco ringspot nepovirus, a well-known virus of plants, replicates in honeybees.  TRSV, first identified nearly a century ago in the USA, has a wide range of plant hosts, and is spread in pollen and seed, and also by many unrelated vectors, not only root-feeding nematodes, like other nepoviruses, but also insects and mites.

This report tells us that TRSV virions have been isolated from bees, and that their gene sequences are closely similar to those of TRSV.  Convincingly, biochemical tests showed that there were replication intermediates of the TRSV genome in the bees, so the virus had not merely contaminated the bees when they fed on the honey and pollen of infected plants, but had seemingly multiplied in them.

However, there are several gaps in this story.  Surprisingly, it seems that no tests were done to show whether the virus isolated from bees infected known plant hosts of TRSV; perhaps this crucial evidence will be in the sequel.  Furthermore, the reported sequences of the virus represented only around 20% of the RNA1 of typical nepoviruses, and around 50% of their RNA2, so there is still the possibility of further genetic surprises when the genome sequence is completed.

Although TRSV is a well-known and long studied virus with many distinct symptom variants, there are relatively few of its gene sequences in Genbank.  When these are compared with those of the ‘bee TRSV’ it is obvious that more sequences will be required to sort out exactly where this virus clusters; differences in the homology patterns of the RNAs 1 and 2 suggest that recombination or reassortment is active among nepoviruses.

The gene sequencing revolution is, to paraphrase Pliny the Elder, revealing that in virology the only certainty is that nothing is certain.”

Me next:

I have the same reservations as Adrian: the TRSV sequence isolated from bees and from Varroa mites is only partial (~30%); thus, it is by no means certain that the whole genome is colinear with genomes of established TRSVs or of other nepoviruses, although they assume that it is – with some justification, possibly, as sequence identities of up to 96% were found in Genbank for the putative CP fragment sequence.  They could isolate particles: why, then, in this metagenomic and NGS age, could they not sequence the whole thing??

nepo fig1

Another reservation I have concerns their methodology.  First, while I was impressed that they did strand-specific PCR to show  both the presence of viral (+ strand) and of replicative form (- strand) RNA in bee and mite tissue (see their Figure above), and did in situ hybridisation to show (-) strand presence in mites, they did not do something very simple that could have shown the same thing, AND given them genome-length +/- strand RNA to play with.

I refer, of course, to dsRNA isolation, which is a very easy and extremely clean technique that can be used to get full-length dsRNAs for many (+) strand RNA viruses from plant or insect tissues.  Moreover, the simple fact of isolating dsRNA forms for a single-strand RNA virus is indicative that replication is occurring – and was used by our group as long ago as 1988 (C Williamson, PhD Thesis, UCT) to isolate full-length ~10 kb dsRNA for two aphid picorna-like viruses.

Scan 04 Jan 2014, 16.31-page8

This means they could have had clear and simple evidence via dsRNA extraction of ALL of the coinfecting viruses present – without all of the expense of total cDNA sequencing.  And sampled more hives….

Second, I have to echo Adrian: “…no tests were done to show whether the virus isolated from bees infected known plant hosts of TRSV”.  Why ever not?  I am afraid that if I were a referee, I would have insisted on this: they did enough other work, after all, that this would not exactly have been an onerous requirement!

And here’s another thing: the authors say, in their Discussion,

“The finding from this study illustrates the complexity of relationships between plant pathogens and the pollinating insects and emphasizes the need for surveillance for potential host-jumping events as an integrated part of insect pollinator conservation.”

Ummmm…no, it doesn’t.  This is overstating the significance of their results by an order of magnitude at least.  They have simply illustrated that ONE species of honeybee may be infectable by ONE species of plant virus, and that this is ASSOCIATED with “weak” colonies.  Moreover, while the presence of TRSV was apparently associated with four weak colonies (out of only ten surveyed), it is quite possible that this is simply the emergence of a commensal-type infection against a background of known bee viruses, and in particular Israeli acute paralysis virus which was found in the same colonies (and blogged on here).  The authors also seem to take it as a given that the “emergence” of TRSV into bees is a recent jump – when it may not be recent at all.  Their statement in the abstract that

“The tree topology indicated that the TRSVs from arthropod hosts shared a common ancestor with those from plant hosts and subsequently evolved as a distinct lineage after transkingdom host alteration”

is pretty much unsubstantiated, in the absence of any investigation of the lineage in plants or in other bee colonies.  Further, they say that

“This study represents a unique example of viruses with host ranges spanning both the plant and animal kingdoms. “

Ummmmm….it doesn’t really do that, either: there are a LOT of arboviruses, with quite a few of them infecting insects and plants.  Here, for example, is an illustration from my teaching material of why it is that I think that viruses of insects and plants are an underappreciated evolutionary link for later evolution of viruses that got into mammals.

Transkingdom viruses

I note that bunyaviruses, rhabdoviruses, reoviruses and (not shown) picorna-like viruses appear linked by the fact that insects have possibly the most diverse representatives of these families, which may indicate that these originated in insects.  Which were the first complex animals to crawl out of the oceans, to join…plants on dry land?  Which explains how plants link up with the far more closely related (in evolutionary terms) insects and vertebrates: plants and insects were alone together for a long, long time before things with spines lurched up out of the water to join them.  So were their viruses.

I also said the following in the material there:

“A complicating factor in the picture of viruses co-evolving with their hosts over millennia is the fact that viruses apparently can – and obviously do – make big jumps in hosts every now and then.  It seems obvious, for example, that arthropods are almost certainly the original source for a number of virus families infecting insects and mammals - such as the Flaviviridae - and probably also of viruses infecting insects and other animals and plants - such as the Rhabdoviridae and Reoviridae - as well (see also here).  For example, picornaviruses of mammals are very similar structurally and genetically to a large number of small RNA viruses of insects and to at least two plant viruses, and – as the insect viruses are more diverse than the mammalian viruses - probably had their origin in some insect that adapted to feed on mammals (or plants) at some distant point in evolutionary time.”

Now quite a lot of interest has been shown in this paper in the blogosphere, and there have been quite a few conclusions drawn from the results that I think are largely unsubstantiated.  For example, this Sci Am blog claims

“When HIV jumped from chimpanzees to humans sometime in the early 1900s, it crossed a gulf spanning several million years of evolution. But tobacco ringspot virus, scientists announced last week, has made a jump that defies credulity. It has crossed a yawning chasm ~1.6 billion years wide.”

Again, ummmm…in light of the discussion above, not necessarily!  I am of the opinion that picorna-like viruses were shared between insects and plants, and then between insects and animals, hundreds of millions of years ago.  And TRSV is a nepovirus – and nepoviruses look like nothing more or less than a picornavirus with a divided genome.

I think TRSV represents something coming back into insects.  And I think we will probably find a lot more of them.

GM crops: European scientists descend on Africa to promote biotech

24 February, 2014

See on Scoop.itVirology News

Africa is expected to be the next target of GM food companies, as European scientists and policymakers travel to Ethiopia to boost the prospect of growing more of the controversial crops on the continent.

Anne Glover, the chief scientific adviser to the European commission, and other prominent pro-GM researchers and policymakers from European countries including Germany, Hungary, Italy and Sweden will this week meet Ethiopian, Kenyan, Ghanaian and Nigerian farm ministers as well as officials from the African Union.

Ed Rybicki‘s insight:

And that’ll go down REALLY well: European governments and policymakers pushing GM crops in Africa, when their own people don’t want them?  

Whether those reasons are stupid or not, and whether or not it is a good idea to plant GM in Africa, I can see a knee-jerk anti-Europe response could well work to completely screw up just what it is they are trying to do.

Consider: Zimbabwe and Zambia resolutely oppose even the import of GM maize as FOOD, let alone allowing the planting of it.  Africans have a history of being VERY suspicious of outsiders bearing gifts – because there is a history of dumping stuff in Africa, of everything from suspect pharmaceuticals to excess chickens.

I predict a lead balloon result for this conference and for the initiative.  Time for Europe to listen to the Kenyans, Burkinabe and South Africans about the merits of growing GM – and not the other way around!

See on www.theguardian.com

South Africa unable to produce foot-and-mouth disease virus vaccine

20 February, 2014

See on Scoop.itVirology News

Outbreak of foot and mouth disease four years ago largely due to collapse of state’s ability to manufacture vaccines

THE outbreak of foot and mouth disease four years ago, which cost South Africa R4bn a year in lost exports, was largely due to the collapse of the state’s ability to manufacture vaccines.

Agriculture, Forestry and Fisheries Minister Tina Joemat-Pettersson told a press briefing on Wednesday on the lifting the ban of red meat exports by the World Animal Health Organisation (WAHO). The removal of the boycott will not affect red meat producers significantly as South Africa usually imports the product to satisfy domestic demand. Only about 1% of red meat production, mainly venison, is exported. Other animal exports include hides and wool.

Ed Rybicki‘s insight:

This is actually horrifying, in the context of what is SUPPOSED to be Africa’s most developed economy – and especially its most developed agricultural economy.

Really: it is a fact not much appreciated outside of the agricultural sector, that we have to get our FMDV vaccines from BOTSWANA.

That’s right – the country with the largest economy in Africa gets its FMDV vaccines from the Botswana Veterinary Institute, a factory set up by the French in a country with a total population about a quarter of that of Greater Johannesburg.

So, let us get this straight: the country that owns the Onderstepoort Veterinary Institute, where the legendary Sir Arnold Theiler pioneered virus and other vaccines from the 1890s on, cannot make its own FMDV vaccines any more?

The country that USED to make a whole range of animal vaccines via the state-owned facility that is now Onderstepoort Biological Products, is now struggling to make just a few?

The country that once built a state-of-the-art BSL4 facility dedicated to FMDV now cannot operate it, or even make old-style killed vaccines?

Don’t let me get started on human vaccines, because we now don’t make ANY – but the erosion of our capacity to make vaccines means our ability to look after human and animal health is now severely under threat.

See on www.bdlive.co.za

Eat your vaccines

7 February, 2014

See on Scoop.itVirology and Bioinformatics from Virology.ca

Vaccines have been revolutionary in medicine, but why are they not used in some parts of the world and how can they be improved? …

Ouch! Wouldn’t it be great if instead of a jab with a needle, you could just eat a vaccine instead? Luckily, researchers at the University of California agree, and their attempts to use algae to produce an edible malaria vaccine is just one example of the many strides forward scientists are taking in vaccine research.

Ed Rybicki‘s insight:

I love these idealistic but naive statements about how plant-production-of-vaccines-will-let us-get-away-from-needles: very 1990s; a little out of touch with modern realities – unfortunately!

The facts are that any edible (read: oral) vaccine will have to be regulated as tightly as an injectable, in terms of dose and administration.

Really: giving too little OR too much; giving it too often or not often enough; giving a product that has not been QCed or checked for potency  after storage…is suicide, in the vaccine world.

Even if it IS safe enough to eat.

See on www.isciencemag.co.uk

RNA sequencing of 750-year-old barley virus sheds new light on the Crusades

7 February, 2014

See on Scoop.itVirology News

Scientists have for the first time sequenced an ancient RNA genome – of a barley virus once believed to be only 150 years old – pushing its origin back at least 2,000 years and revealing how intense farming at the time of the Crusades contributed to its spread.

Ed Rybicki‘s insight:

This is a good report about a VERY interesting finding – with one flaw.  They go on, apparently, about how BSMV is a "new virus": why would anyone think that?  Since the late 1980s, Adrian Gibbs and others have pointed out that tobamoviruses are probably ancient; just because RNA CAN evolve fast doesn’t mean it does, in terms of encoding functional elements.  Gibbs showed this for plant viruses; it has also been done for the HIV/SIV complex, where it is shown that a similar divergence in sequence among theses viruses to all animals since the Cretaceous, has led to NO changes in morphology, or gene function.

The simple fact is that having "plastic" genome in comparison to eukaryotic cells does NOT mean that ssRNA viruses may not be ancient.

Having said all that, it really is a tour de force to have sequenced a virus that old – aided by the fact, I am sure, that BSMV is hardy little beast, with a really stable vision.

Now, to find those hundred-year-old maize leaves put away with maize streak virus symptoms….B-)

Thanks to @Elsevier Microbiol* for pointing this out!

See on www.sciencedaily.com

Proof of principle for epitope-focused vaccine design – or is it?

6 February, 2014

See on Scoop.itVirology News

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

Ed Rybicki‘s insight:

Yes.  Um.  Well.  I have just heard a lecture by the redoubtable Marc van Regenmortel (disclosure: my former mentor) pointing out how this strategy is highly UNLIKELY to be "the" way of generating antigens that are likely to be good immunogens – and I was convinced.

Now this.

Um, he said.  And…the MHvR arguments still stand, I think: why should something that supposedly looks like the thing that supposedly elicited a strongly neutralising antibody, elicit a strongly neutralising antibody?

OK, it apparently did here – but as a general rule?  Do we KNOW what "good" neutralising epitopes look like in 3D?  Because linear epitopes are really just an approximation to what real epitopes are, and epitopes are in fact just those things that BIND antibodies – NOT necessarily what ELICITS them.

Damn, I’m sounding like Marc – must be catching!

But seriously: antibodies are the membrane receptors of B cells; they are generated by random recombinations, and then selected by adventitiously binding another molecule that is not self; they are then further selected by repeated rounds of binding, while undergoing random mutations affecting their hypervariable regions, while weak binders are selected AGAINST by Tregs.

And end up both with higher affinity, and more broad binding to related "epitopes" as a result.

Incredibly hard to duplicate as general phenomenon, I would surmise.

See on www.nature.com

Have we reached the beginning of the end of the fight against HIV/AIDS?

5 February, 2014

See on Scoop.itVirology News

The world is at a tipping point in the fight against HIV/Aids with the number of people getting treatment set to be greater than those being newly diagnosed. But have we really reached the beginning of the end?

Considering the first World Aids Day took place 25 years ago in 1988, it’s a remarkable achievement. Today, there are about 35million people with HIV and Aids in the world, with 9.7million receiving treatment that can enable a long and healthy life.

Ten years ago, just 300,000 people received such treatment so it’s clear there have been major gains, and although experts, campaigners and health practitioners are buoyed the ‘tipping point’ is on the horizon, hard work lies ahead. Erin Hohlfelder, global health policy director of the One Foundation and author of its report The Beginning Of The End?’, feels reaching the tipping point will be a landmark in the fight against HIV and Aids. ‘The disease has been outpacing us for decades, with more new infections than people being added to treatment.

‘To use an analogy, if you had five cuts on your hand, we’ve only had one plaster to treat the cuts, so you’re still bleeding,’ she said.

‘The tipping point, where we have more people living healthy lives through treatment for HIV, than newly infected people, means we’ve caught up with the disease and begun to get ahead of it, so it’s a critical moment. It’s a marker that signals for the first time HIV and Aids is on a downward curve,’ said Ms Hohlfelder.

Ed Rybicki‘s insight:

Great infographic – and potentially good news.  But as I have told my students repeatedly since the mid-1980s, this is a long. slow pandemic – and we are only just over halfway through it.

There’s still a long way to go.

See on metro.co.uk

Cases of New Deadly Bird Flu Surge in China, Experts Say

5 February, 2014

See on Scoop.itVirology News

About 300 cases of H7N9 avian influenza have been confirmed in China, with more appearing every day.

Cases of the new H7N9 avian influenza in China are surging alarmingly, flu experts warned this week.

There are now about 300 confirmed cases, with more appearing every day. Roughly a quarter of the victims have died.

The first human cases were reported only last March. By contrast, the H5N1 influenza virus, another lethal strain that jumped from birds to people, first appeared in 2003 and took almost five years to reach the 300-case mark.

Ed Rybicki‘s insight:

So THIS is the next one?  Possibly?  Good job they’re <a href="http://who.int/influenza/vaccines/virus/candidates_reagents/a_h7n9/en/index.html">already making vaccines</a>, then, isn’t it?

 

See on www.nytimes.com


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