Archive for the ‘General Virology’ Category

Giant Zombie Killer Virus Rises From Its 30 000 Year Grave To Kill Us All! Or Not?

27 March, 2014

I am not a fan of “Science By Hype”, which I think I have made abundantly clear via Virology News and elsewhere. Thus, I pour scorn on the “We found a structure which will lead to an AIDS vaccine”, and “We found an antibody that will cure AIDS” type of articles, WHILE at the same time, appreciating the ACTUAL science behind the hype. If there is any, of course. Which is why I am torn, on the subject of a giant DNA virus purportedly found in 30 000 year-old Siberian permafrost. I am also a fan of zombies, hence the title. But seriously, now: here we are, with news media and semi- and fully-serious science mags all hailing the description in PNAS, no less, of “Pithovirus sibericum“.  A giant virus, wakened from a 30 000 year sleep in Siberian permafrost, by the kiss of an amoeba. OK, by infecting an amoeba, but you see where I’m going here. Pithovirus_sibericum__Researchers_Resurrect_30_000-Year-Old_Giant_Virus___Biology___Sci-News_com So here we are, with an article from Sci.news.com, trumpeting the discovery.  And there’s more:

“The findings have important implications in terms of public health risks related to the exploitation of mining and energy resources in circumpolar regions, which may arise as a result of global warming. “The re-emergence of viruses considered to be eradicated, such as smallpox, whose replication process is similar to Pithovirus, is no longer the domain of science fiction. The probability of this type of scenario needs to be estimated realistically.””

Yeah.  Rii-ii-ght.  Giant viruses are going to erupt from the permafrost and kill us all!  Really?? No. Curtis Suttle, he of oceanic metaviromes fame, is quoted as saying the following, in Ed Yong’s Nature blog:

“…people already inhale thousands of viruses every day, and swallow billions whenever they swim in the sea. The idea that melting ice would release harmful viruses, and that those viruses would circulate extensively enough to affect human health, “stretches scientific rationality to the breaking point”, he says. “I would be much more concerned about the hundreds of millions of people who will be displaced by rising sea levels.””

Amen!  In other words, just because there ARE revivable viruses in permafrost – itself no new thing, BTW – does NOT mean they will harm humans. Think about this a moment: something locked away under the surface of the ground for 30 000+ years has to SURVIVE, first; second, it has to INFECT humans if it is to cause any harm. And what evidence do we have that anything found in Siberian permafrost can do that? None.  None whatsoever. Think again: how many humans, and how many mammals with virus that could infect humans, were there around on the Siberian plains 30 000 years ago?

Precious few.

And what likelihood is there that any viruses that COULD infect humans, got preserved? Vanishingly small. So what COULD get released from said permafrost, as it melts with inexorable global warming? Well, phages: lots and lots of phages.

Then some plant viruses, maybe: there have been previous reports of Tomato mosaic virus found in 1999 in glacial ice from Greenland, that was between 500 – 140 000 years old – that was also supposed to be a threat, as it escaped from melting icecaps. To tomatoes, possibly.  If they grew in seawater.

But there’s more: here we have “New Deadly Flu Viruses Reemerge from Melting Ice“, from 2006.  Here we have

“An international team [that] found flu viruses in the ice of Siberian lakes, fact that warns about the possibility that global warming may release germs locked in glaciers for decades or even centuries.”

Yah. Right.  But at the same time, considerably more worthy of alarm than Pandoraviruses. Because what our worthy French colleagues did NOT do, in their report in PNAS, was see what ELSE was in their permafrost samples. Seriously: they trawled melting ice from a core sample with amoebae ONLY.

This is the equivalent of the 2nd year prac I used to do, when we made students screen water obtained from the environment with E coli to see if they could amplify coliphages out of it.  Why did they not do a metagenomic sequence trawl, after filtering out bits of mammoth crap and cockroaches and bits of twigs??  What did they MISS?  HBVs that infected Denisovans?  And are we SURE that the virus came from that long ago?  Has the ice really remained frozen all that time – and is there not the possibility that water didn’t percolate down through cracks and pores in the permafrost, carrying the virus with it, from a more clement environment on the surface??

OK, OK, so it’s a great find, and reasonably worthy of SOME hype.  BUT: it is NOT a harbinger of doom, because most viruses will NOT survive 30 000 years worth of entombment in ice, and in any case, would NOT infect humans even if they did. AND I hate the name: “Pithovirus sibericum“?  Really??  Viruses are not named like that!  Except by French folk who find these strange “amphora-shaped” viruses, apparently.

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.

Legends of Virology

31 January, 2014

I have been fortunate enough this week to be in Pretoria, at the first Animal and Human Vaccine Development in South Africa Conference (Twitter #AHVDSA): partly because it is a very timeous and necessary meeting to help to establish strategies for this purpose, and partly because there is a significant presence of some legendary figures of international and South African virology.

Marc van Regenmortel – who we count as local even if he lives in Strasbourg – helped Bob Millar and others at the University of Pretoria to organise this meeting. He also used the opportunity of having a bunch of old virological friends visiting him at the University of Stellenbosch’s STIAS to bolster the conference presentations.

So it was that we have Errling Norrby of Sweden with us; we have Fred Murphy of Ebola fame; Marian Horzinek of veterinary virology repute; Marc himself, our iconoclastic viral immunologist; Jose Esparza of the BMG and an eminent poxvirologist – and Jean-Marie Andrieu, an oncologist with an interest in tolerogenic HIV vaccines.

Local legends are present too: we have Daan Verwoerd, legendary orbivirologist and former Director of the venerable and distinguished Onderstepoort Veterinary Institute; Henk Huismans, who did the first molecular work on orbiviruses in the 1970s, and is still active; Bob Swanepoel, doyen of the African haemorrhagic fever viruses.

Good people.

Oh, and of course, me and Anna-Lise Williamson; Dion du Plessis of OVI; Lynn Morris of the NICD; Albie van Dijk of UNW; Glenda Gray of the MRC, among 150 delegates

A great meeting, all in all, and very timely, given the contents of the SA Governmental Bioeconomy Strategy document released recently.

20140131-120134.jpg

Legends alive: from left, Fred Murphy; Daan Verwoerd; Bob Millar; Henk Huismans; Errling Norrby; Marc van Regenmortel
20140131-120151.jpg

Jean-Marie Andrieu; Marc van Regenmortel – at a VERY good unofficial dinner

20140131-120211.jpg

Legends and friends at supper: Marc, Fred, Eric Etter (CIRAD); Jose Esparza; Marian Horzinek; Errling, Anna-Lise Williamson

DNA Preparation Tubes Contaminated with Novel ssDNA Virus

21 September, 2013

See on Scoop.itVirology News

A novel virus thought to have come from human samples appears to have been derived from seawater during the manufacture of tubes used to extract DNA.  

Ed Rybicki‘s insight:

I have a problem with the original report, in J Virol (http://jvi.asm.org/content/early/2013/09/05/JVI.02323-13.abstract?related-urls=yes&legid=jvi;JVI.02323-13v1): not that they discovered it, because that was done well.  However, they essentially REdiscovered something that was ±100% identical to a virus already sequenced and named by Chinese researchers – who did not use the Qiagen kits, apparently – and then gave it a new name!

 

Sorry, that is simply bad practice!  It also smacks of scientific imperialism of a sort that characterised early discovery work on HTLVs and on HIV, when US researchers calmly treated earlier characterisations as if they had never happened.

 

There is another leap that I do not think is justified: the authors claim that

 

"Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture".

Really?  On the basis of presence of a sequence in a metagenomic trawl?  No resampling with specific primers on a fresh sample?   And surely the generation of the silica matrix is done under conditions that would totally destroy adventitiious DNA?

So – an interesting paper, and a valuable notification (although it might have been nicer if they’d shared their findings informally, to save other people like our Virology Diagnostics lab time and money).  But flawed, in my opinion.

See on www.the-scientist.com

Moratorium on using live rinderpest virus lifted for approved research

30 July, 2013

See on Scoop.itVirology News

Benefits of future research should be carefully balanced against potential risks

Paris, 10 July 2013 – A moratorium on using live rinderpest virus for approved research has been lifted by the Food and Agriculture Organization of the United Nations and the World Organisation for Animal Health (OIE).

The moratorium followed the adoption of a Resolution in May 2011 by all OIE Member Countries that urged members to forbid the manipulation of rinderpest virus containing material unless approved by the Veterinary Authority and by FAO and OIE.

The two organizations have now put in place strict criteria and procedures to follow in order to obtain official approval for any research proposals using rinderpest virus and rinderpest virus-containing materials. One of the most crucial requirements is that the research should have significant potential to improve food security by reducing the risk of a reoccurrence of the disease. This procedure replaces an earlier complete ban on handling the virus.

Rinderpest was formally declared eradicated in 2011, but stocks of rinderpest virus continue to exist in laboratories. In June 2012, a moratorium on handling the virus was imposed after an FAO-OIE survey found that the virus continues to be held in more than 40 laboratories worldwide, in some cases under inadequate levels of biosecurity and biosafety.

When rinderpest was officially eradicated, FAO and OIE member countries committed themselves to forbid the manipulation of rinderpest virus-containing material unless approved by the national veterinary authority as well as by FAO and OIE.

Paramyxovirus EM courtesy of Linda Stannard

Thanks to Len Bracher for alerting me to this.

Ed Rybicki‘s insight:

This is an interesting sequel to the eradication of wild rinderpest virus, which I have covered in some detail here on ViroBlogy: see here (http://rybicki.wordpress.com/2010/11/05/rinderpest-gone-but-not-forgotten-yet/) and here (http://rybicki.wordpress.com/2011/08/03/deliberate-extinction-now-for-number-3/).

The article covers an interesting prospect: that it may be possible to use attenuated, safe vaccines against the related peste des petits ruminants virus (PPRV) not only to protect against any resurgence of rinderpest, but also to eradicate this rather nasty virus.

Which is, apparently, spreading at rather an alarming rate, and is an obstacle to small ruminant production (http://www.fao.org/ag/againfo/resources/documents/AH/PPR_flyer.pdf).

So maybe this is “Now for Number 4!” time.

See on www.oie.int

Maize streak virus revisited: 25 years on

20 March, 2013
Maize streak virus: photo from 1978

Maize streak virus: photo by Robert G Milne in Cape Town from 1978

Twenty-five years ago, I wrote a brash, naïve little piece entitled “Maize streak virus virus: an African pathogen come home?” for the South African Journal of Science, laying claim to a virus that we had just started working on – Maize streak virus (MSV) – on the basis that it had first been described from this country in 1901, that it was endemic here, and that it still caused major crop losses.  I did this because research on this and related viruses seemed to have moved almost completely offshore, to Europe and the USA, and

“…the most interesting of the viruses that grow all around us have already been whisked away to foreign laboratories; [that] there they have been cloned, sequenced, and had their most intimate details exposed, far from their native shores”. [Yes, I really did write like that back then].

I asked at that time, if we should

“…perhaps be content to supply foreigners with the (pathogenic) fruits of our fields, and to marvel when the answers come filtering back from abroad?”.

I answered myself by saying that

“…prospects for worthwhile research on African geminiviruses, and on any other indigenous pathogens, are at least as good here as anywhere else.  Our facilities are the equal of those abroad, the necessary expertise is certainly not lacking, and the viruses are on our doorstep.”

I’m a little shocked now that I could have said that then: the paper quotes only three pieces of work from our lab, one of them a Masters dissertation and two papers done by my erstwhile supervisors; we had not yet sequenced any virus, let alone a geminivirus, and all we had was brashness and hope.  Indeed, I went on to say the following:

“We are, incidentally, the only research group with access to molecular biological techniques which is actually working on the virus in its natural environment: this is very useful, as with the virus in all its forms and its vector(s) literally on our doorstep, we can rapidly accumulate, identify and characterize distinct isolates for study here or elsewhere.  We hope there will be a little more of the ‘here’, and a little less of the ‘elsewhere’, from now on”.

I outlined what it was that we ambitiously wanted to do – seeing as we had no money, and only one PhD student at the time – as follows:

“…we now have distinctly different genomic maps of three isolates [!] which differ in serology and symptom expression; we have cloned genomic DNA of several more isolates, and can potentially clone and [restriction] map many more.  With this type of work now solidly established, we intend to investigate other biological variants of MSV – and other native cereal geminiviruses – in maize, cereal grains and other members of the Gramineae.  The aim is to explore the genetic diversity of naturally occurring types of MSV and related viruses, and to identify any isolates that appear unusual in terms of symptom expression, serology or transmission.  These would be interesting to map, and potentially useful in recombinational analyses for the fine mapping of determinants of pathogenicity and host range.” [see later]

The article obviously sank without trace: I can find only three citations to it; two of them mine, and the third from a South African maize breeder.  How the overseas labs that I compared us to must have sniggered…actually, I doubt that happened at all; I am sure none of them ever read it!  In retrospect, we really were regarded as a backwater, and as wannabe geminivirologists; I had at least one collaboration request rebuffed with “we don’t feel our work would be advanced by working with you”, and was told “we’re already working on that, so you shouldn’t bother” for a couple of other proposals.

My hubris was not entirely misplaced, however: we did in fact go on to develop into a world-leading MSV and geminivirus molecular virology laboratory; it just took another fifteen years or so!

So where are we, twenty-five years on from my cheeky article?  Much water has flowed under several bridges; I expanded from molecular virology in the 1990s into plant and vaccine biotechnology in the 2000s, while keeping a geminivirus research group going – and we have published and co-published something like 55 peer-reviewed journal articles and several encyclopaedia and book chapters on MSV and other “African streak viruses” alone, let alone another 14 or so articles on other geminiviruses, with some 1200 citations.  We have papers on geminivirus mapping and sequencing, virus diversity, biogeographical variation, quantitation of symptoms, molecular determinants of pathogenicity, recombination, engineering maize for resistance, the use of two of the viruses as gene expression vectors – and cover pictures for Plant Biotechnology Journal and Journal of Virology.

Cover Illustration: J Virol, October 2011, volume 85, issue 20

Cover Illustration: J Virol, October 2011, volume 85, issue 20

I started with one Honours student in 1986, who went on to do a Masters in 1988; we moved on to having one PhD student in the late 1980s to up four PhD students simultaneously in the mid- to late 1990s, and a postdoc at the same time.  The projects went from simple diversity studies of a few viruses using restriction mapping, through the application of PCR, to partial genome sequencing and studying the molecular biology of infectious clones of the viruses, with a very profitable sideline in phylogenetic analyses; we also moved – with Professor Jennifer Thomson – into a parallel track of plant biotechnology, aimed at engineering resistance to MSV in maize.  We added another track early this century, working on similar ssDNA circoviruses of parrots, using all of the expertise we had accumulated on geminiviruses.  We truly work on “circomics” now – the study of small circular genomes – with its subsets “geminiviromics” and “circoviromics”, with a library of literally hundreds of sequenced MSVs and distinct grass mastreviruses and BFDVs.

Geminivirus particle: characteristic doubled icosahedron containing a single ssDNA

Geminivirus particle: courtesy of Russell Kightley Media

The geminiviromics group has pretty much got away from me now; the folk I trained as PhD students in the late 1990s and early 2000s were enthused enough with the field that they have gradually usurped my leadership and supervisory role, and made the field their own.  I still maintain an interest in using Bean yellow dwarf mastrevirus (BeYDV) as an expression vector for “biofarming” purposes; I am also maintaining a project on Beak and feather disease circovirus (BFDV) diversity and plant-made vaccines.  I think we pretty much did what we set out to do – including the brave prediction I made about host range and pathogenicity, which led to some very interesting work on recombination and genome modularity, and the successful engineering of pathogen-derived resistance to MSV.

So I owe some thanks, in retrospect: first, to Barbara von Wechmar, who sparked the interest – and provided isolates, leafhoppers, and expertise.  Second, to Bev Clarke and Fiona Tanzer (aka Hughes), who were brave enough to blaze the trail, and clone our first MSVs – and make one infectious, in the case of Fiona.  Thanks to Wendelin “Popeye” Schnippenkoetter, for your single-minded perseverance in mixing and matching genomes; thanks Kenneth Palmer, for showing the way for transient expression assays in maize cells and engineering MSV as a vector.  Thanks Janet Willment, for mapping replication origins in MSV and expanding us into wheat viruses; thanks Jennifer Thomson for the collaboration, and Fiona and Tichaona Mangwende and Dionne Shepherd for breaking us into maize resistance engineering.  Thanks Christine Rey for the collaboration, and Leigh Berrie for your quiet competence in our detour into South African cassava mosaic virus.  Thanks Darrin (aka Darren) Martin and Eric van der Walt, for so brilliantly exploring MSV diversity, evolution and recombination – and Darrin for endless amusement in the lab, as well as for two completely distinct and invaluable software packages, for symptom quantitation and recombination analysis.  In the present generation, thanks to Suhail Rafudeen and our student Rizwan Syed (and Dionne and Darrin as supernumerary supervisors); thanks Aderito Monjane for doing such a ridiculous amount of work for a superlative PhD; thanks Dionne and Marian, for keeping the maize engineering afloat – and thanks also to Arvind Varsani, for retraining himself from a papillomavaccinologist to a circomicist, and for popping up everywhere.

Vaccines: a simple message

28 February, 2013

+MaryMangan over there on Google+ made an interesting point about simple messages to refute the kinds of nonsense promulgated by vaccine denialists, among others.

Here’s my contribution:

Vaccines!

Vaccines!

TMV in mouse lungs: more thoughts and refutations

13 February, 2013

tmv sedimhave been thinking about this paper (see last post), and it and other people’s posts (eg: Tommy Leung’s) have prompted more response.

I note the authors  say the following:

“There is other published literature that challenges the dogma of the strict boundaries between plants and vertebrates for viruses. In non-vertebrate animals, it was shown that plant pathogenic viruses displayed complex interactions with insects, and the transcription and replication of some plant viruses within insects was described [29][32]. In addition, in some cases, insects were found to be affected by plant viruses [33]. Furthermore, it was recently shown that Tomato spotted wilt virus (TSWV) could infect two human cell lines, HeLa and diploid fibroblasts, depending on the expression of a viral polymerase-bound host factor[34]. Additionally, despite plant virus replication was not observed in animals, Cowpea mosaic virus (CPMV), a plant comovirus in the picornavirus superfamily, was able to bind and enter mammalian cells, including endothelial cells, and the binding protein for the virus was identified as a cell-surface form of the intermediate filament vimentin [35]. Furthermore, CPMV was found to persist for several days post oral or intravenous inoculation in a wide panel of body tissues in mice, including in the lung and the liver [36]. Additionally, it was demonstrated that TSWV induced a strong immune response in its insect vector Frankliniella occidentalis [37] and that oral administration of Cowpea severe mosaic virus, Alfalfa mosaic virus and chimeric plant virus particles induced a durable and systemic immune response in mice [38][39]

Yes.  Um. Well.  The “dogma of the strict boundaries between plants and vertebrates for viruses”?  I have been teaching virology for 32 years, and I am not aware of actual DOGMA – as in, “that which has to be believed”.  Rather, there has been the cumulative set of OBSERVATIONS that nothing that anyone has ever isolated out of a plant – and that replicates in it – has infected a vertebrate.  I make that distinction, because there is always the possibility that, as we and others have found with insect viruses, plants can act as a “circulative, non-propagative vector” for insect viruses (for Rhopalosiphum padi aphid virus in barley, from my lab, and Leafhopper A virus in maize) – and if one realises that male mosquitoes, and often also females, feed on plants…you see where I’m going here?  As in, it might well be possible for a virus that multiplies in an insect and also in a vertebrate, to POTENTIALLY be found in a  plant?

In ay case, this is largely beside the point, because the authors get sidetracked into discussing Tomato spotted wilt – which happens to be a plant-adapted bunyavirus, most closely related to insect and vertebrate phleboviruses – “depending on the expression of a viral polymerase-bound host factor”.  Really??  And if it isn’t there?  Does the virus in fact spread?  For that matter, my lab has cell-free translated two aphid picorna-like virus genomes in rabbit reticulocyte lysates, but we made no claim that it could happen in rabbit cells.  Moreover, they make much of the fact that “a plant comovirus in the picornavirus superfamily, was able to bind and enter mammalian cells…[and] was found to persist for several days post oral or intravenous inoculation in a wide panel of body tissues in mice, including in the lung and the liver”.

Yes?  And?  A REALLY stable plant virus was able to bind and enter animal cells, and persist?  The problem with that is…?

We in the virus-like particle vaccine field RELY on the fact that VLPs will be taken up by cells of the immune system in vertebrates, and that they will elicit immune responses – so why is this regarded as a problem?  In fact, TMV has itself been tested as an RNA vaccine delivery system, due to its ability to protect a RNA payload, and get itself delivered into reticulocytes and macrophages – meaning this property has been known for some time, and has not hitherto been seen as a problem!

I think these authors have hyped something that is quite interesting into what THEY regard as a potential problem, for the purposes of getting their article accepted – and I think this needs to be recognised, and that the perceived risks need to be minimised by the knowledgeable.

PLOS ONE: Tobacco Mosaic Virus in the Lungs of Mice following Intra-Tracheal Inoculation

13 February, 2013

See on Scoop.itVirology News

“Plant viruses are generally considered incapable of infecting vertebrates. Accordingly, they are not considered harmful for humans. However, a few studies questioned the certainty of this paradigm. Tobacco mosaic virus (TMV) RNA has been detected in human samples and TMV RNA translation has been described in animal cells. We sought to determine if TMV is detectable, persists, and remains viable in the lung tissues of mice following intratracheal inoculation, and we attempted to inoculate mouse macrophages with TMV. In the animal model, mice were intratracheally inoculated with 1011 viral particles and were sacrificed at different time points. The virus was detected in the mouse lungs using immunohistochemistry, electron microscopy, real-time RT-PCR and sequencing, and its viability was studied with an infectivity assay on plants. In the cellular model, the culture medium of murine bone marrow derived macrophages (BMDM) was inoculated with different concentrations of TMV, and the virus was detected with real-time RT-PCR and immunofluorescence. In addition, anti-TMV antibodies were detected in mouse sera with ELISA. We showed that infectious TMV could enter and persist in mouse lungs via the intratracheal route. Over 14 days, the TMV RNA level decreased by 5 log10 copies/ml in the mouse lungs and by 3.5 log10 in macrophages recovered from bronchoalveolar lavage. TMV was localized to lung tissue, and its infectivity was observed on plants until 3 days after inoculation. In addition, anti-TMV antibody seroconversions were observed in the sera from mice 7 days after inoculation. In the cellular model, we observed that TMV persisted over 15 days after inoculation and it was visualized in the cytoplasm of the BMDM. This work shows that a plant virus, Tobacco mosaic virus, could persist and enter in cells in mammals, which raises questions about the potential interactions between TMV and human hosts.”

Ed Rybicki‘s insight:

Interesting paper!  Which proves…which proves…which proves TMV is seriously resistant to degradation in animals and in mammalian cells; that it can enter macrophages; and that it…what?  What, exactly, are the “…questions about the possible interactions…”?  What would TMV do in mammalian cells?  Yes, it might be uncoated and be translated; it is far less likely that it MIGHT be able to replicate its RNA – and then?  While it can apparently be taken up quite efficiently by macrophages – a property which, incidentally, has led to its being trialled as an RNA vaccine delivery system – this is a dead end, and one that is quite normal for particles of any kind being introduced into mammals.

Which is something that happens every day, as we and our cousin mammals eat: it has been shown elsewhere that animals are actually quite good spreaders of plant viruses, some of which – like TMV and the even tougher Cauliflower mosaic virus – pass right through at high survival rates, and remain infectious.  We will all probably have eaten many grams of various viruses in our lives, and derived nothing more than nutrition from them.

I also remember, even though it was very late at night, 31 years ago, and in a bar in Banff in Canada, a conversation with one Richard Zeyen.  He told me they had used ELISA to test everyone in their lab for antibodies for TMV, seeing as they worked with it, and had newly developed a test.  And everyone was immune – presumably, to aerosolised TMV that had been breathed in or otherwise ingested.  Proving…that oral vaccines based on TMV could work, and that most of us are probably immune to all sorts of viruses that don’t replicate in us – and nothing more!

Including, in the case of many people in the Eastern Cape Province of South Africa, sampled by one Don Hendry via the local blood bank, to a virus of Pine Emperor moths – because it multiples to such high levels in its host that anyone walking in the pine forests was bound to be exposed via the environment.

So this is an interesting paper – and no more.  It will, of course, lead to alarmist articles and blog posts, and people calling out for urgent surveillance of food, in which people will find many viruses.  And so what?  They have been with us for as long as we have been eating plant-derived food, and have NEVER been associated with any disease, transmissible or otherwise – so my best advice is that we ignore them.

See on www.plosone.org


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