Posts Tagged ‘MSV’

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.

A feeling for the Molechism* – revisited

10 July, 2012

This is an update of a post I did on Alan Cann’s MicrobiologyBytes back in 2007, before i started ViroBlogy: I am doing this because (a) it’s mine, (b) I want to update it – and the MB version is archived, so I can’t.  So here we are again:

I think it’s permissible, after working on your favourite virus for over 20 years, to develop some sort of feeling for it: you know, the kind of insight that isn’t directly backed up by experiment, but that may very well be right. Or not – but in either case, it would take a deal of time and a fair bit of cash to prove or disprove, and would have sparked some useful discussion in the meantime. And then, of course, the insights you have into (insert favourite virus name here) – if correct – can usually be extended into the more general case, and if you are sufficiently distinguished, people may actually take them on board, and you will have contributed to Accepted Wisdom.

I can’t pretend – at least, outside of my office – to any such Barbara McClintock-like distinction; however, I have done a fair bit of musing on my little sphere of interest as it relates (or not) to the State of the Viral Universe, and I will share some of these rambles now with whomever is interested.

I have been in the same office now, and teaching the same course, more or less, for 32-odd years. In that time I have worked on the serology and epidemiology of the bromoviruses, cucumovirus detection, potyvirus phylogeny, geminivirus diversity and molecular biology, HIV and papillomavirus genetic diversity, and expressing various bits of viruses and other proteins in plants and in insect cells. However, much of my interest (if not my effort) in that time has been directed towards understanding how grass-infecting mastreviruses in particular interact with their environment and with each other, in the course of their natural transmission cycle.

Maize streak virus

Maxwell’s Demon (left, lower) and Martian Face (right, upper) visible on a MSV virion

Fascinating little things, mastreviruses: unique geminate capsid architecture, and at around a maximum of 2.8 kb of single-strand circular DNA, we thought they were the smallest DNA genomes known until the circoviruses and then the zoo of anello- and anello-like viruses were discovered. Their genomes code for only 4 proteins – two replication-associated, one movement and one capsid – yet we have managed to work on just one subgroup of mastrevirus species for 27 years, without exhausting its interest – at least, to us… (see PubMed list here). We also showed that one could see Martian faces quite distinctly on virions – and possibly even Maxwell’s Demon. But I digress….

Maize streak

Severe symptoms of MSV on sweetcorn

We have concentrated on the “African streak viruses” – related species Maize streak virus, Panicum streak virus, Digitaria streak virus, Sugarcane streak virus and friends – for two very simple reasons:
1. They occur in Africa, near us, and nowhere else;
2. Maize streak virus is the worst viral pathogen affecting maize in Africa.

So we get situational or niche advantage, and we get to work on an economically-important pathogen. One that was described – albeit as “…not of…contagious nature” – as early as 1901, no less.

Maize streak virus

Maize streak virus or MSV, like its relatives, is obligately transmitted by a leafhopper (generally Cicadulina mbila Naudé): this means we have a three-party interaction – of virus-host-vector – to consider when trying to understand the dynamics of its transmission. Actually, it’s more complicated than that: we have also increasingly to consider the human angle, given that the virus disease affects mainly the subsistence farming community in Africa, and that human activity has a large influence on the spread of the disease. So while considering just the virus – as complicated as that is – we have to remember that it is only part of the whole picture.

So how complicated is the virus? At first sight, not very: all isolates made from severe maize infections share around 97% of their genome sequence. However, however…that 3% of sequence variation hides a multitude of biological differences, and there is a range of relatives infecting grasses of all kinds, some of which differ by up to 35% in genome sequence. Moreover, maize is a crop plant first introduced to Africa a maximum of 500 years ago, so it is hardly a “natural” host – yet, all over Africa, it is infected by only a very narrow range of virus genotypes, from a background of very wide sequence diversity available.

So here’s an insight:

the host selects the virus that replicates best in it.

And lo, we found that in the Vaalharts irrigation area in the north of South Africa that the dominant virus genotype in winter wheat was a different strain – >10% sequence difference – to the one in the same field, in summer maize. Different grass species also have quite different strains or even species of streak viruses best adapted to them.

DendrogramNot all that profound a set of observations, perhaps, but they lead on to another insight:

streak viruses travel around as a cloud of variants or virus complex.

Not intuitively obvious, perhaps…but testable, and when we did, we found we were right: cloning virus genomes back out of maize or from a grass infected via leafhoppers gave a single predominant genotype in each case, with a number of other variants present as well. Looking further, we discovered that even quite different viruses could in fact trans-replicate each other: that is, the Rep/RepA complex of one virus could facilitate the replication of the genome of a virus differing by up to 35% in DNA sequence. We have also – we think – made nonsense of the old fancy that you could observe “host adaptation” of field isolates of MSV: we believe this was due to repeated selection by a single host genotype from the “cloud” of viruses transmitted during the natural infection cycle.

So, insight number three:

there is a survival benefit for the viruses in this strategy.

This is simple to understand, really, and relates to leafhopper biology as well as to host: the insects move around a lot, chasing juicy grasses, and it would be an obvious advantage to the streak virus complex to be able to replicate as a complex in each different host type – given that different virus genotypes have differential replication potential in the various backgrounds. This is quite significantly different, incidentally, to what happens with the very distantly-related (in terms of geological time) begomoviruses, or whitefly-transmitted geminiviruses: these typically do not trans-replicate each other across a gap of more than 10% of sequence difference.

Boring, I hear you say, but wait…. Add another factoid in, and profound insights start to emerge. In recent years, the cloud of protégés or virologist complex around me has accumulated to critical mass, and one of the most important things to emerge – apart from some frighteningly effective software for assessing recombination in viral genomes, which I wish he’d charge for – was Darren Martin’s finding that genome recombination is rife among African streak viruses. This was unexpected, given the expectation that DNA viruses simply don’t do that sort of thing; that promiscuous reassortment of components between genomes is a hallmark of RNA viruses. Makes sense in retrospect (an exact science), however, because of the constraints on DNA genomes: how else to explore sequence space, if the proof-reading is too good? And if you travel in a complex anyway…why not swap bits for biological advantage?

MSV web

Linkage map of the MSV genome, showing what interacts with what

So Darren swapped a whole lot of bits, in a tour-de-force of molecular virology, to create some 54 infectious chimaeric MSV genomes – and determined that

The pathogenicity of chimeras was strongly influenced by the relatedness of their parental viruses and evidence was found of nucleotide sequence-dependent interactions between both coding and intergenic regions“.

In other words –new insight:

the whole genome is a pathogenicity determinant, and bits of it interact with other bits in unexpected ways.

At this point you could say “Hey, all his insights are in fact hypotheses!” – and you would be partially correct, except for

Profound Insight No. 1hypotheses are the refuge of the linear-thinking.

Or its variant, found on my office wall:

“**c* the hypotheses, let’s just discover something”. I also have

“If at first you don’t succeed, destroy all evidence that you tried” and a number of exotic beer bottle labels on my wall – but I digress….

As an aside here, I am quite serious in disliking hypothesis-driven science: I think it is a irredeemably reductionist approach, which does not easily allow for Big Picture overviews, and which closes out many promising avenues of investigation or even of thought. And I teach people how to formulate them so they can get grants and publications in later life, but I still think HDS is a tyranny that should be actively subverted wherever possible.

Be all this as it may, now follows

Profound Insight No. 2genome components may still be individually mobile even when covalently linked.

Now take a moment to think on this: recombination allows genes to swap around inside genetic backgrounds so as to constitute novel entities – and the “evolutionary value of exchanging a genome fragment is constrained by the number of ways in which the fragment interacts with the rest of the genome*“. Whether or not the genome is RNA, DNA, in one piece or divided. All of a sudden, the concept of a “virus genome” as a gene pool rather than a unitary thing becomes obvious – and so does the reductionism inherent in saying “this single DNA/RNA sequence is a virus”.

So try this on for size for a brand-new working definition of a virus – and

Profound Insight No. 3a virus is an infectious acellular entity composed of compatible genomic components derived from a pool of genetic elements.

Sufficiently paradigm-shifting for you? Compare it to more classical definitions – yes, including one by AJ Cann, Esq. – and see how much simpler it is. It also opens up the possibility that ANY virus as currently recognised is simply an operational assembly of components, and not necessarily the final article at all.

Again, my favourite organisms supply good object examples: the begomoviruses – whitefly-transmitted geminiviruses -

  • may have one- or two-component genomes;
  • some of the singleton A-type components may pick up a B-type in certain circumstances;
  • some doubletons may lose their B without apparent effect in model hosts;
  • some A components may apparently share B components in natural infections;
  • the A and B components recombine like rabbits with cognate molecules (or Bs can pick up the intergenic region from As);
  • in many cases have one or more satellite ssDNAs (β DNA, or nanovirus-related components) associated with disease causation;

…and so on, and on…. An important thing to note here is the lab-rat viruses – those isolated early on, and kept in model plant species in greenhouses – often don’t exhibit any of these strangenesses, whereas field-isolated viruses often do.

Which tells you quite a lot about model systems, doesn’t it?

But this is not only true of plant viruses: the zoo of ssDNA anello-like viruses found in humans and in animals – with several very distantly-related viruses to be found in any individual, and up to 80% of humans infected – just keeps on getting bigger and weirder. Added to the original TT virus – named originally for the initials of the Japanese patient from whom it was isolated, and in a post hoc exercise of convoluted logic, named Torque teno virus (TTV) [why don't people who work with human or animal viruses obey ICTV rules??] – are now Torque teno minivirus (TTMV) and “small anellovirus” SAV) – all of which have generic status. And all of which may be the same thing – as in, TTVs at a genome size of 3.6–3.8 kb may give rise to TTMVs (2.8-29 kb) and SAVs (2.4-2.6 kb) as deletion mutants as part of a population cloud, where the smaller variants are trans-replicated by the larger. Thus, a whole lot of what are being described as viruses – without fulfilling Koch’s Postulates, I might point out – are probably only “hopeful monsters” existing only as part of a population. Funnily enough, this sort of thing is much better explored in the ssDNA plant virus community, given that working with plant hosts is so much easier than with human or animal.

And now we can go really wide, and attempt to be profound on a global scale: it should not have escaped your notice that the greatest degree of diversity among organisms on this planet is that of viruses, and viruses that are found in seawater in particular. There is a truly mind-boggling number of different viruses in just one ml of seawater taken from anywhere on Earth, which leads respectable authors such as Curtis Suttle to speculate that viruses almost certainly have a significant influence on not only populations of all other marine organisms, but even on the carbon balance of the world’s oceans – and therefore of the planet itself.

Which leads to the final, and most obvious,

Profound Insight (No. 4)in order to understand viruses, we should all be working on seawater…. 

That is where the diversity is, after all; that is where the gene pool that gave rise to all viruses came from originally – and who knows what else is being

Hypolith – cyanobacteria-derived, probably – under a piece of Namib quartzite from near Gobabeb Research Station

cooked up down there?

And this is the major update: not only have I managed to get funded for a project on “Marine Viromics” from our local National Research Foundation - a process akin to winning the lottery, and about as likely to succeed - I am also collaborating with friends and colleagues from the Institute for Microbial Biotechnology and Metagenomics at the University of the Western Cape on viruses in desert soils, and associated with hypoliths- or algal growths found under quartzite rocks in extreme environments.

Thus, I shall soon be frantically learning how to deal with colossal amounts of sequence data, and worse, learning how to make sense of it.  We should have fun!

——————————————————————————————————————–

* And as a final curiosity, I find that while I – in common with the World Book Encyclop[a]edia and Learning Resources – take:mol|e|chism or mol|e|cism «MOL uh KIHZ uhm», noun. to mean any virus, viewed as an infective agent possessing the characteristics of both a living microorganism and a nonliving molecule; organule.
[molechism < mole(cule) + ch(emical) + (organ)ism; molecism < molec(ule) + (organ)ism] –
There is another meaning… something to do with sacrifice of children and burning in hellfire eternally. This is just to reassure you that this is not that.

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

19 December, 2011

We thank Russell Kightley for permission to use the images

Marshall Bloom (Rocky Mountain Laboratories, NIAID) opened the plenary session on Thursday the 1st of December, with a talk on probing the pathogen-vector-host interface of tickborne flaviruses.   Although thoroughly infected with a rhinovirus, he held our attention most ably while reminding us that while many flaviviruses are tick borne, the hard and soft body ticks that vector them are very phylogenetically different – as different as they are from spiders – meaning that if similar flaviruses replicated in them, these viruses may have much wider host range than we know.

He pointed out that while about 95% of the virus life cycle takes place in a tick, transmission to a vertebrate means suddenly adapting to a very different host.  Infection in ticks is persistent, as befits their vector role – but vertebrate infection generally is not.  It was interesting, as a sometime plant virologist, to hear that they look for dsRNA as a marker for replication, and do Ab staining for it: the technique was invented with plant viruses, and very few other virologists seem to appreciate that dsRNA can be quite easily isolated and detected.

They compared Vero and tick cells for virus replication, and saw significant differences: while tick cells could go out to 60+ days and look fine, Vero cells were severely affected at much shorter times post infection.  There was also 100-fold less virus in tick cells, and prominent tubular structures in old infected tick cells.  He noted that ticks evade host defences quite efficiently: eg they suppress host clotting during feeding, and there is huge gene activation in the tick during feeding.  In another study to envy, they are doing array work on ticks to see what is regulated and how.

 Linda Dixon (Institute for Animal Health, Pirbright, UK) recounted her lab’s work on African swine fever (ASFV), a poxvirus-like large DNA virus.  The virus is endemic to much of Africa, and keeps escaping – and there is no effective  vaccine to prevent spread, so regulation is by slaughter.  There are 3 types of isolate, with the most highly pathogenic causing up to 10% fatality and a haemorrhagic syndrome.  She described how in 2007 the virus had spread from Africa to Georgia, then in 2009 to southern Russia and all way to the far north, in wild boar.

There are more than 50 proteins in the dsDNA-containing virion; two infectious forms similar to the poxviruses with multilayer membranes and capsid layers can form, and neutralising Ab play no part in protection as a result.  They studied the interaction of viruses with cells and the immune system, and compared the genomes of pathogenic and non-pathogenic strains, in order to understand how to develop an effective vaccine.

The biggest differences were large deletions in non-virulent isolates, including genes coding for  proteins responsible for binding to RBC, and various immune evasion multicopy genes.  They planned to target regions to delete to make an attenuated virus for vaccine.  They had found non-essential genes involved in immune evasion, and ones that lower virulence, and had been systematically cutting them out.  She noted that pigs can be protected if they survive natural infection and if vaccinated with TC-attenuated virus, and can be protected by passive transfer of Abs from immune pigs – which indicated that an effective live vaccine was very possible.

Subunit vaccines were being investigated, and they had found partial protection with baculovirus-expressed proteins.  They were doing genome-wide screens for protective Ag, and were pooling Ags expressed from predicted ORFs in immunization trials – up to 47 Ags without reduction in specific  T cell responses.

Discovery One

My former labmate Dion du Plessis (Onderstepoort Veterinary Institute, OVI) made a welcome return to Cape Town, with a talk entitled “2011: A Phage Odyssey”.  He explained the title by noting the distinct resemblance of P1 coliphage to the Discovery One spacecraft dreamed up by Arthur C Clarke and Stanley Kubrick – and then went on to exuberantly and idiosyncratically recount a brief history of bacteriophages and their use in biotechnology since their discovery.  A revelation from his talk was that the first discovery of phages was probably described by a gentleman named Hankin, in 1896 in Annales de l’Institut Pasteur: he

The 1896 paper from Annales de l'Institut Pasteur

showed that river water downstream of cholera-infested towns on the Jumma river in India contained no viable Cholera vibrio – and that this was a reliable property of the water.  We were also introduced to the concept of turtles as undertakers in the Ganges….

He took us through the achievements of the Phage Group of Max Delbruck and others – where science was apparently fun, but also resulted in the establishment of modern molecular biology – through to the use of phages as exquisitely sensitive indicators immunochemistry studies in the 1960s.

All too soon we got to the modern uses of phages, with 3 types of gene library – random peptide, fragmented gene, and antibody V regions – being used to make recombinant phage tail proteins to be used for “panning” and enrichment purposes, in order to select either specific antibodies or antigens.  Dion manages a research programme at OVI aimed at developing a new generation of veterinary vaccines – and has for some years now been making significant progress in generating reagents from a chicken IgY single-chain Fv phage display library.

Carolyn Williamson (IIDMM, UCT) gave us an update on CTL epitopes associated with control of HIV-1 subtype C infections.  She said that it was now known that genome-wide association studies (GWAS) gives you certain HLAs which are associated with low viral load, and others with high – meaning that to some extent at least, control of infection was down to genetic luck.  She noted that they and others had shown that CTL escape was quick: this generally happened in less than 5 weeks in acute phase infections.

They had looked for evidence of a fitness cost of CTL escape – and shown that it exists.  She noted that this meant that even if one has “bad” HLA genes, if one was infected with a virus with fitness cost mutations from another, that one could still control infection.

It had been shown that “controllers” mainly have viruses with attenuating mutations, or have escapes in the p24 region – and it was a possible vaccine strategy to include these mutated epitopes in vaccines to help people with infections control their infections.

An interesting topic she broached was that of dual infections – there was the possibility of modelling if infection with two different viruses results in increased Ab neutralisation breadth, and if one would get different results if infections were staggered, possibly with increased nAb evolution if isolates were divergent.  She noted it was possible to track recombination events with dual virus infections too.

It was interesting that, as far as Ab responses went, there were independent responses to 2 variants and one could get a boost in Ab titres to the superinfecting virus, but not a boost to Abs reacting with the originally-infecting virus

Carolyn was of the opinion that HIV vaccines needed to include CTL epitopes where escape is associated with fitness cost.  She also reiterated that superinfection indicated that one can boost novel responses, which I take to mean that therapeutic applications are possible.

Ulrich Desselberger (University of Cambridge) is a long-time expert on rotaviruses and the vaccines against them, and it was a pleasure to finally hear him speak – and that he was mentoring young people in South Africa.  He said that more than a third of children admitted to hospital worldwide were because of rotavirus infections, meaning that the viruses were still a major cause of death and morbidity – and they were ubiquitous.

He reviewed the molecular biology and replication cycle of rotaviruses in order to illustrate where they could be targeted for prevention of infection or therapy, and noted that drugs that interfere with lipid droplet homeostasis interfere with rotavirus replication because 2 viral proteins associated proteins of lipid droplets.

He stated that there were lots of recent whole-genome sequences – we already there were many types, based on the 2 virion surface proteins; we  now know that other genes are also highly variable.  As far as correlates of immunity were concerned, VP7 & 4 were responsible for eliciting neutralising Ab.  Additionally, protective efficacy of VP6 due to elicitation of non-neutralising Ab had been shown in mice – but not in piglets, and not convincingly in humans.  Abs to VP2, and NSP2 and 4 were also partially protective in humans.  It was interesting that protection was not always correlated with high titre nAb responses.

He noted that in clinical disease primary infections partially protected against subsequent infections which are normally milder; subsequently no disease was seen even when infection occurred.  Cross-protection occurred at least partially after initial infection, and this got better after more exposure.  There was evidence one could get intracellular neutralisation by transcytosed Ab, and especially to VP6.  Ab in the gut lumen was a good indication of protection.

As far as the live modern vaccines were concerned, Merck’s Rotateq elicited type-specific nAb, with 9% of recipients shedding live virus.  GSK’s Rotarix gets elicits cross-reactive nAb and one gets 50% of recipients shedding virus.

While the vaccines seemed safe, he noted that where vaccines had been introduced, efficacy ranged from 90% in the USA and Europe, down to as low as 48% in Bangladesh, Malawi and SA, due to type mismatch, and that efficacy was correlated inversely with disease incidence and child mortality generally.  He mentioned that there had been much VLP work, but that none of the candidates was near licensure.

Johan Burger (Stellenbosch University) spoke on one of the more important non-human virus problems in our immediate environment – specifically, those affecting wine grape production in our local area.  He opened by stating that SA now produced 3.7% of the world’s wine, making grapes a nationally and especially locally important crop.  Leafroll disease was a major worldwide problem – as well as being the reason for the wonderful autumn reddening seen in grapevines, it also significantly limited production in affected vineyards.  His laboratory has done a lot of work in both characterising viruses in grapevine, and trying to engineer resistance to them.  Lately they were also investigating the use of engineered miRNAs as a response to and means of controlling, virus infection.

His group has for a couple of years been involved in “metaviromic” or high-throughput sequencing studies of grapevines, with some significant success in revealing unsuspected infections.  In this connection, he and Don Cowan pointed out that they had lots of data that they ignore – but which we should keep and study, as a resource for other studies not yet thought of.

As far as Johan’s work went, novel viruses kept popping up, including grapevine virus E (GVE), which hitherto had only been found in Japan.  They were presently looking at Shiraz disease, which was unique to SA, and was still not understood.  This was infectious, typified by a lack of lignification which led to rubbery vines, and kills plants in 5 years.  It also limits the production of the eponymous grapes – a crime when SA shirazes seem to be doing so well!

Veterinary Virology and Vaccines parallel session.

I again dodged the clinical / HIV session because of my personal biases, and was again treated to a smorgasbord of delight: everyone spoke well, and to time, and I was really gratified to see so many keen, smart young folk coming through in South African virology.  It was also very interesting to see highly topical subjects like Rift Valley fever and rare bunyavirus outbreaks being thoroughly covered, so I will concentrate on these.

P Jansen van Veeren (NICD, Johaanesburg) was again a speaker, this time representing his absent boss, Janusz Paweska.  He gave an account of the 2010 Rift Valley fever outbreak in SA, and epidemiological findings in humans – something of keen interest to me.  He said there had been some forecasting success for outbreaks in East Africa; however, there were long gaps between outbreaks, which were generally linked to abnormal rainfall and movement of mosquito and animal hosts.  RVFV isolates differed in pathogenicity but were structurally and serologically indistinguishable – because virulence was due to the NSs protein, and not a virion component.  He recounted how artificial flooding of a dambo in Kenya resulted in a population boom in the floodwater Aedes mosquitoes responsible for inititating an outbreak, and then of the Culex which maintained the epidemic.  He said there was a strong correlation between viral load and disease severity.

In terms of South African epidemiology, there had been smaller outbreaks from 2008 round the Kruger National Park (NE SA), then in the Northern Cape and KZN in 2009.  People had been infected from autopsy of animals, and handling butchered animal parts.  The 2010 outbreak started in the central Free State after an unusually wet period, and had then spread to all provinces except Limpopo and KZN.  In-house serological methods at the NICD were validated in-house too: these were HAI screening and IgM and IgG ELISAs and a virus neutralisation test.  They had got 1600+ samples of human serum, and confirmed 242 cases of disease and 26 deaths for 2010.

He noted that with winter rains there was a continuous outbreak in the Western Cape, and in 2011 the epidemic had started again in the Eastern and Western Cape Provinces, but has since tailed off.  Some 82% of human cases were people who occupationally handled dead animals, although there was some possibility of transmission by mosquitoes.

In human cases there was viraemia from 2-7 days, with IgM present transiently from 3 days at low level.  They had sequenced partial GP2 after PCR from 47 isolates, and showed some recombination occurring.  The 2010 isolates were very closely related to each other, and to a 2004 Namibian isolate.  There had been no isolation from mosquitoes yet.

Two talks on FMDV followed: Belinda Blignaut (OVI and Univ Pretoria) spoke on indirect assessment of vaccine matching by serology, and Rahana Dwarka (OVI) on a FMDV outbreak in KZN Province in 2011.  Belinda’s report detailed how 6 of 7 serotypes of FMDV occur in SA, with SAT-1 and -2 and O the most common – and that vaccines needed to be matched to emerging strains.  This was done by indirect vaccine matching tests such as serological r-value, determined by the ratio of the reciprocal serum titre to the heterologous virus against that to the homologous virus.  They had put 4 different viruses into cattle and got sera to test a range of 26 newly isolated viruses.  While they had not got sequence from the test panel viruses, indications were that topotype 3 viruses are antigenically more disparate and that a vaccine consisting of topotype 1 or 2 antigens may not be effective in the control of FMD.

In introducing Rahana’s talk, the chair (Livio Heath, OVI) mentioned that there had been 5 different major animal pathogens causing outbreaks in SA over the last 3 years – and that they had to produce reagents and validate tests for ASFV, classical swine fever (CSF) and FMDV, etc, with each outbreak.  Rahana described how they had neutralisation assays and blocking and competition ELISA for FMDV, as well as a big database of isolates from buffalo in KZN – so they were well-placed to type viruses found in cattle in the region.

C van Eeden (Univ Pretoria) had an intriguing account of their investigation of the occurrence of an orthobunyavirus causing neurological symptoms in horses and wildlife.  Horses seem to be particularly vulnerable to many of the viruses involved in such disease, and so are a useful sentinel species.  Shuni virus was first isolated from Culicoides midges and sheep and a child in Nigeria in the 1960s.  SA workers subsequently found it in some livestock and Culex mosquitoes and in horses.  The virus was shown to be a neurologic disease agent in horses and wildlife – then disappeared for some 30 years, much like Ebola.  There is apparently a new research unit at UP with a BSL3 lab, so they are well equipped to do tests with the virus.

Ms van Eeden noted that the incidence of encephalitic disease in humans and animal in SA is underreported, and the causes are mainly unknown – a revelation to me!  Horses are susceptible to many of the agents, and are useful sentinels – workers have identified flavi- and alphaviruses in some outbreaks, but many are not IDed.  They had done cell culture and EM on samples from an ataxic horse: they got a bunyavirus-like virus by EM, and did bunya-specific PCR, and got Shuni virus back.  Sequence relationships showed no linkage to type of animal or date, in subsequent samplings from horses, crocodiles,  a rhino and a warthog, and from blood, brain and spinal cord.  All positive wildlife were sampled in Limpopo Province; horses only from most other provinces.

She noted that latest cases were neurological, whereas previously these were mainly febrile.  The virus accounted for 10% all neurological cases, with a 50% fatality rate.  She noted further that vets often work without masks or gloves, and so had no protection from exposure in such cases….  There was no idea on what the vector was, but they would like to test mosquitoes, etc.  Ulrich Desselberger suggested  rodents may be a reservoir, but they don’t know if this is true.

Stephanie van Niekerk (Univ Pretoria) investigated alphaviruses as neurological disease agents in African wildlife.  The most common alphaviruses in SA are Sindbis and Middelburg viruses.  Old World alphaviruses are usually not too bad, and cause arthritic and febrile symptoms, while New World cause severe neurological diseases.  Sindbis was been found in SA outbreaks in 1974.  However, Stephanie noted that a severe neurological type had appeared since 2008 in horses.  Accordingly, they looked at unexplained cases in wildlife in the period 2009-2011: brain and spinal cord samples were investigated for all cases.  They found alphavirus in a number of rhinos, buffalo, warthog, crocodiles and jackal – and all except for one rhino were Middelburg virus.  They want to isolate viruses in cell culture, and increase the size of regions used for cDNA PCR.  Stephanie said the opinion was that the values of the animal involved justifies the development of vaccines.

 

Virology Africa 2011: viruses at the V&A Waterfront 1

12 December, 2011

We thank Russell Kightley for permission to use the images

Anna-Lise Williamson and I again hosted the Virology Africa Conference (only the second since 2005!), at the University of Cape Town‘s Graduate School of Business in the Victoria & Alfred Waterfront in Cape Town.  While this was a local meeting, with just 147 attendees, we had a very international flavour in the plenaries: of 18 invited talks, 9 were by foreign guests.  Plenaries spanned the full spectrum of virology, ranging from discovery virology to human papillomaviruses to HIV vaccines to tick-borne viruses to bacteriophages found in soil to phages used as display vectors, and to viromes of whole vineyards.  There were a further 52 contributed talks and 41 posters, covering topics from human and animal clinical studies, to engineering plants for resistance to viruses.

A special 1-day workshop on “Human Papillomaviruses – Vaccines and Cervical Cancer Screening” preceded the main event: this was sponsored by Merck Sharp & Dohme, Roche and Aspen Pharmacare, and had around 90 attendees.  Anna-Lise Williamson (NHLS & IIDMM, UCT) opened the workshop with a talk entitled “INTRODUCTION TO HPV IN SOUTH AFRICA – SCREENING FOR CERVICAL CANCER AND VACCINES”, and set  the stage for Jennifer Moodley (Community Health Dept, UCT) to cover health system issues around the prevention of cervical cancer in SA, and the newly-minted Dr Zizipho Mbulawa (Medical Virology, UCT) to speak on the the impact of HIV infection on the natural history of HPV.  This last issue is especially interesting, given that HIV-infected women may have multiple (>10) HPV types and progress faster to cervical malignancies, and HPV infection is a risk factor for acquisition of HIV.  The Roche-sponsored guest, Peter JF Snijders (VU University Medical Center, Amsterdam), gave an excellent description of novel cervical screening options using primary HPV testing, to be followed by two accounts of cytological screening in public and private healthcare systems in SA, by Irene le Roux (National Health Laboratory Service) and Judy Whittaker (Pathcare), respectively.  Ulf Gyllensten (University of Uppsala, Sweden) described the Swedish experience with self-sampling and repeat screening for the prevention of cervical cancer, especially in groups that are not reached by standard screening modalities.  Hennie Botha and Haynes van der Merwe (both University of Stellenbosch) closed out the session with talks on the effect of the HIV pandemic on cervical cancer screening, and a project aimed at piloting adolescent female vaccination against HPV infection in Cape Town.

The next part of the Workshop overlapped with the Conference opening, with a Keynote address by Margaret Stanley (Cambridge University) on how HPV evades host defences (sponsored by MSD), and another by Hugues Bogaert (HB Consult, Gent, Belgium)) on comparisons of the cross-protection by the two HPV vaccines currently registered worldwide (sponsored by Aspen Pharmacare). Margaret Stanley’s talk was a masterclass on HPV immunology: the concept that such a seemingly simple virus (only 8 kb of dsDNA) could interact with cells in such a complex way, was a surprise for all not acquainted with the viruses.  Bogaert’s talk was interesting in view of the fact that the GSK offering, which has only only two HPV types, raises far higher titre antibody responses than the MSD vaccine with four HPV types, AND seems to elicit better cross-protective antibodies: this should help inform choice of product from the individual point of view.  However, the fact that MSD seems able to respond better to national healthcare system tenders in terms of price per dose is also a major factor in the adoption stakes.

The Conference proper started with a final address by Barry Schoub, long-time but now retired Director of the National Institute of Virology / National Institute of Communicable Diseases in Johannesburg, and also long-time CEO of the Poliomyelitis Research Foundation (PRF): this is possibly the premier funding agency for anything to do with viruses in South Africa, and a major sponsor of the Conference.  He spoke on the history of the PRF, and how it had managed to shepherd an initial endowment of around 1 million pounds in the 1950s, to over ZAR100 million today – AND to dispense many millions in research project and bursary funding in South Africa over several decades.

The first session segued into a welcoming cocktail reception and registration at the Two Oceans Aquarium in the V&A Waterfront: this HAS to be one of the only social events for an academic conference where the biggest sharks are the ones in the tank, and not in the guest list!  I think people were suitably blown away – as always, in the aquarium – and the tone was set for the rest of the meeting.  The wine and food were good, too.

The first morning session of the conference featured virus hunting and HIV vaccines, as well as plant-made vaccines and more HPV.  W Ian Lipkin (Columbia University, USA) opened with “Microbe Hunting” – which lived up to its title very adequately, with discussion of a plethora of infectious agents.  As well as of the methods newly used to discover them, which include high-throughput sequencing, protein arrays, very smart new variants on PCR….  I could see people drooling in the audience; the shop window was tempting enough to make one jump ship to work with him without a second thought.  He said that probably 99% of vertebrate viruses remain to be discovered, and that advances in DNA sequencing technology were a major determinant in the rapidly-increasing pace of discovery.  He made the point that while the emphasis in the lab had shifted from wet lab people to bioinformatics, he thought it would move back again as techniques get easier and more automated – meaning (to me) that there is no substitute for people who understand the actual biological problems.  It was interesting that, while telling us of his work on the recently-released blockbuster “Contagion” – where “the virus is the star!” – he showed a slide with a computer in the background running a recombination detection package called RDP, which was designed in South Africa.  It can also be seen in the trailer, apparently.  Darren Martin will not be looking for royalties or screen credits, however.

Don Cowan (University of the Western Cape) continued the discovery theme, albeit with bacteriophages as the target rather than vertebrate viruses.  It is worth emphasising that phages probably represent the biggest source of genetic diversity on this planet – and given how even the most extreme of microbes have several kinds of viruses, as Don pointed out, it is possible that this extends to neighbouring planets too [my speculation - Ed].  He occupies an interesting niche – much like the microbes he hunts – in that he specialises in both hot and cold terrestrial desert environments, which are drastically understudied in comparison to marine habitats.  He made the interesting point that metagenome sequencing studies such as his own generate data that is in danger of being discarded without reuse, given that folk tend to take what they are interested out of it and neglect the rest.

Anna-Lise Williamson (NHLS, IIDMM, UCT) then described the now-defunct SA AIDS Vaccine Initiative vaccine development project at UCT.  It is rather sobering to revisit a project that used to employ some 45 people, and had everything from Salmonella, BCG, MVA, DNA and insect cell and plant-made subunit HIV vaccines in the pipeline – and now employs just 5, to service the two vaccines that made it into into clinical trial.  The BCG-based vaccines continued to be funded by the NIH, however, and the SA National Research Foundation funds novel vaccine approaches.  Despite all the funding woes, the first clinical trial is complete with moderate immunogenicity and no significant side effects, and two more are planned: these are an extension of the first – HVTN073/SAAVI102 – with a Novartis-made subtype C gp140 subunit boost, and the other is HVTN086/SAAVI103, which compprises different commbinations of DNA, MVA and gp140 vaccines.

It was clear from the talk that if South Africa wants to support local vaccine development, the government needs to support appropriate management structures to enable this – and above all, to provide funding.  However, all is not lost, as much of the remaining expertise in several of the laboratories that were involved in the HIV vaccine programme can now involve themselves in animal vaccine projects.

Plant-made HPV16 VLPs

Ed Rybicki made it an organisational one-two with an after-tea plenary on why production of viral vaccines in plants is a viable rapid-response option for emerging or re-emerging diseases or bioterror threats.  The talk briefly covered the more than 20 year history of plant-made vaccines, highlighting important technological advances and proofs of concept and efficacy, and concentrated on the use of transient expression for the rapid, high-level expression of subunit vaccines.  Important breakthoughs that were highlighted included the development of the Icon Genetics TMV-based vectors, Medicago Inc and Fraunhofer USA’s recent successes with H5N1 and H1N1 HA protein production in plants – and the Rybicki group’s successes with expression of HPV L1-based and E7 vaccine candidates.  The talk emphasised how the technology was inherently more easily scalable, and quicker to respond to demand, than conventional approaches to vaccine manufacture – and how it could profitably be applied to “orphan vaccines” such as for Lassa fever.

Ulf Gyllensten had another innings in the main conference, with a report on a study of a possible linkage of gene to disease in HPV infections – which could explain why some people clear infections, and why some have persistent infections.  They used the Swedish cancer registry (a comprehensive record since the end of the 1950s) to calculate familial relative risk of cancer of the cervix (CC): relative risk was  2x for a full sister, the same for a mother-daughter pair and the risk for a half sister was 50% higher while risk was not linked to non-biological siblings or parents, meaning the link was not environmental.  A preliminary study found HLA alleles associated with CC, and increased carriage of genes was linked to increased  viral load.   A subsequent genome wide association study using an Omni Express Bead Chip detecting700K+ SNPs yielded one area of major interest, on Chr 6 – this is a HLA locus.  They got 3 independent signals in the HLA region and can now potentially link HPV type and host genotype for a prediction of disease outcome.  Again, the kinds of technology available could only be wished for here; so too the registry and survey options.

Molecular and General Virology contributed talks parallel session

I attended this because of my continued fascination with veterinary and plant viruses – and because Anna-Lise was covering the Clinical and Molecular session – and was not disappointed: talks were of a very high standard, and the postgraduate students especially all gave very good accounts of themselves.

Melanie Platz (Univ Koblenz-Landau, Germany) kicked off with a description of a fascinating interface between mathematics and virology for early warning, spatial awareness and other applications.  She gave an example using a visual representation of risk using GIS for Chikungunya virus, based on South African humidity and temperature data going back nearly 100 years: this had a 3D plot model, into which one could plug data to get predictions of mosquito likelihood.  They could generate risk maps from the data, to both inform public and policy / planning.  They had a GUI for mobile devices for public information, including estimates of risk and what to do about it, including routes of escape.

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

This was followed by one of my co-supervised PhD students, Aderito Monjane – who recently got the cover of Journal of Virology with his paper on modelling maize streak virus (MSV) movement and evolution, so I will not detail more here.  However, even as a co-supervisor I was blown away by the fact that he was able to show animations of MSV spread – at  30 km/yr, across the whole of sub-Saharan Africa.

Christine Rey of Wits University provided another state-of-the-art geminivirus talk, with an account of the use of siRNAs and derivatives for silencing cassava-infecting geminiviruses.  They were using genomic miRNA precursors as templates to make artificial miRNAs containing viral sequences, meaning they got no interference with nuclear processing and there was less chance of recombination with other viruses, a high target specificity, and the transgenes would not be direct targets of virus-coded suppressors.  They could also use multiple miRNAs to avoid mutational escape.  The concept was successful in tobacco, and they had got transformation going well for cassava, so hopes were high for success there.

Dionne Shepherd (UCT) spoke on our laboratory’s 15+ year work on engineered resistance in maize to MSV.  She pointed out that the virus threatens the livelihood of 200 million+ subsistence farmers in Africa, and is thought to be the biggest disease concern in maize – which is still the biggest edible crop in Africa.  Most of the work has been described elsewhere with another journal cover; however, new siRNA-based constructs still under investigation were even more effective than the previous dominant negative mutant-based protection: the latter gave 50-fold reduction in virus replication, but silencing allowed > 200-fold suppression of replication.

2-colour surface rendition of HcRNAV

Arvind Varsani – a former UCT vaccinology PhD who is now a structural biology and virology lecturer at Univ Christchurch (NZ) – described what is probably the first 3D structure of a virus to come out of Africa.  This was of a 30 nm isometric ssRNA virus – Heterocapsa circularisquama RNA virus (HcRNAV) – infecting a dinoflagellate, which is one of the most noxious red tide bloom agents and is a major factor in killing farmed oysters.  The virus apparently controls the diatom populations.  There are two distinct strains of virus, and specificity of infection is due to the entry process, as biolistic bombardment obviates the block.  The single capsid protein probably has the classic jelly-roll β-barrel fold, but they observe a new packing arrangement that is only distantly related to the other ssRNA (+) virus capsids known.  They will go on to look at structural differences between strains that change cell entry properties.

FF Maree from the Onderstepoort Veterinary Institute and the Univ Pretoria spoke on structural design of FMDV to improve vaccine strains: they wished to engineer viruses by inserting the cell culture adapted HSPG-binding signature sequence and to mutate capsid residues to increase the heat stability of SAT-2 subtype virus vaccines.  If they put the signature sequence in a SAT1 virus, they found it could infect CHO cells – which do not express any of 4 integrins that FMDV binds to, but are far better for large-scale production of the virus than the BHK cells used till now.  It was also possible to increase hydrophobic interactions in the capsid by modeling: eg a VP2 Ser to Tyr replacement gave a considerably better thermal inactivation profile to the virus.

Daria Rutkowska (Univ Pretoria) detailed how African horsesickness orbivirus (AHSV) VP7 protein had significant potential as a scaffold that could act as a vaccine carrier.  The native protein formed as trimers assembled in a VP3+VP7 “core” particle; however, the VP7 when expressed alone could form soluble trimmers – and the “top” domain hydrophilic loop can tolerate large inserts.  The group had very promising FMDV P1 peptide responses from engineered VP7 constructs, including protection of experimental animals.

P Jansen van Veeren of the National Institute of Communicable Disease in Johannesburg finished off the session, with a description of the cellular pathology caused by Rift Valley fever bunyavirus (RVFV) in mice in acute infections.  The virus seems to have been of particular international interest recently as a potential bioterror agent; however, global warming is also responsible for its mosquito vector spreading outside of its natural base in Africa to the Arabian peninsula, and there are fears of the virus getting into Europe soon.  While there are vaccines against the virus, including a live attenuated version, none are licenced for human use.  It was interesting to hear that the viral NP appears to be the main immunogen, as there are massive amounts of NP produced in infection, and huge responses to it in infected animals – and NP immunisation protects mice.  There is a good Ab response but it is not neutralising, while NP is released independently of other proteins from infected cells.  The liver is the major target of virus infection, with a bias to apoptosis of hepatocytes and severe inflammatory responses.  Viral load is linked to these effects and is much lower in vaccinees.  Immunisation reduces liver replication markedly; that in the spleen less so.  A screen of cytokines and other gene responses showed a big down-regulation of many genes in non-vaccinated mice to do with cytokines, and down-regulation of B and T cells and NK cells.  He thinks recombinant vaccine candidates should have both the surface glycoproteins and the NP in order to be effective – and that there is a major need for proper reagents for big animal studies.


Follow

Get every new post delivered to your Inbox.

Join 436 other followers