Bandicoot Blues

Today’s post is from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

Now that the dust has begun to settle after the launch of Merck’s much-hyped Gardasil genital papillomavirus vaccine - discussed in MicrobiologyBytes here and here - people are turning again to looking at the natural history of the virus.

With some serious surprises…

There is a wonderfully-titled paper in the December issue of Journal of Virology describing a novel virus isolated from an Australian marsupial, which looks like a natural chimaera resulting from recombination between a cutaneous papillomavirus and a polyomavirus (A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae), from a collaborative group drawn from Murdoch University in Perth, WA, and the University of Leuven in Belgium.

They set out to look for papillomaviruses in lesions of a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations of the western barred bandicoot, using the relatively new technique of multiply-primed isothermal rolling-circle DNA amplification (RCA) - adapted from a natural phage replication process similar to that described here previously. It was a reasonable assumption that papillomaviruses were involved, given the type of lesions and the fact that papillomavirus sequences had previously been amplified out of other cutaneous lesions of marsupials - however, use of RCA would allow amplification of any circular DNA genome, given the non-specific nature of the technique which could show up “escapes” from even broad-spectrum degenerate primer PCR DNA amplifications. What they found was at first non-controversial: RCA products from lesions from several animals gave a ~7.5 kDa genomic DNA band after digestion with BamHI or SalI, typical of a papillomavirus; however, sequencing of the cloned DNA revealed that although the genomes did in fact contain the characteristic L1 and L2 structural protein ORFs of a papillomavirus, they also had the large-T and small-t antigen regulatory protein-coding ORFs typical of a polyomavirus - and on the opposite strand to the structural protein genes, also typical of polyomaviruses:

The natural assumption in this case would be that they had cloned an artifact of template-swapping in the RCA reaction: however, a number of checks using PCR with specific primers and sequencing of DNA from other animals confirmed the result. The newly-designated bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) DNA was subsequently found in 94.7% of fresh lesion tissue extracts, and 100% of bandicoots withe papillomatosis and carcinomatosis syndrome screened by skin swabbing, indicating it is almost certainly the causative agent.

This is a landmark finding in virology: the two families of viruses, while both classed as DNA tumour viruses, are very different indeed, and while it is possible they have a common origin, it is evolutionarily very distant indeed, given that the two groups have been cospeciating with their hosts over geological aeons. They both have circular dsDSNA genomes, and similar-looking particle morphology, but neither their structural nor their regulatory proteins have any discernible homology to one another - which means it is conceptually as likely to find a viable recombinant between them as it is to successfully cross a crocodile and a tortoise.

The authors explore a number of options in explaining just how such a tortodile or crocoise came to be, and appear to give equal credence to the possibilities of simple recombination between a papillomavirus and a polyomavirus, and that the virus is a descendant of the last common ancestor of the two groups of viruses. Their own analysis of phylogenetic relationships, however, places the putative L1 protein firmly among the beta- and gamma-papillomaviruses, which are viruses isolated from humans and associated with the condition of epidermodysplasia verruciformis in immunosuppressed people, and cutaneous lesions respectively. This indicates to me that an already-evolved cutaneous papillomavirus recombined with an as yet undiscovered representative of the family Polyomaviridae, to give a new and unexpectedly viable chimaeric offspring that has persisted in its unique biological niche.

What this result opens up is the possibility that there is a lot more of this going on than we knew about - and that techniques like RCA may be the key to unlocking a completely unsuspected world of virus diversity.

I have recently been participating in a to-and-fro evolutionary discussion in the columns of a local weekly newspaper, in the course of which one protagonist stoutly proclaimed that evolution was hokum because “like only begats like” [sic]. I would show him this virus as an example of what rubbish that statement is - if only he could understand it.

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Hurting rather than helping?

Today’s post is from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

It should not have escaped the eye of the interested bystander that there has been a most unfortunate and premature end to a HIV vaccine trial recently - and that something that had been tested as “safe” and “immunogenic” in Phase I and II trials went on to not only to not show any efficacy at all, but may actually have increased susceptibility in recipients to HIV-1 infection. The so-called STEP trial in North and South America, the Caribbean and Australia, and the Phambili trial in South Africa, were “Phase IIB” or international test-of-concept trials in at-risk populations of the efficacy of Merck & Co. Inc. Adenovirus 5-based vectors (MRKAd5) encoding subtype B-derived Gag, Pol and Nef proteins. The outcome was about as gloomy as could possibly have been predicted, and was nothing like what researchers expected from preliminary primate testing.
The trials - more formally designated as “HVTN 502 and HVTN 503 HIV Vaccine Clinical Trials” by the HIV Vaccine Trials Network - were testing a mixture of three replication-defective Adenovirus 5 vectors, containing gag, pol and nef genes respectively, aimed at stimulating mainly CD8+ T-cell responses. There had earlier been some disagreement in the research community about the ethics of trialling a subtype B-specific vaccine in a subtype C-dominated epidemic region - and in a population known to have a high incidence of high titres of antibodies against Ad5, which is after all a common cold virus. However, regulatory bodies were in no doubt that the trials were appropriate, and standard Phase I and II trial results were non-contentious, leaving the way clear for the present exercise.

The study was designed as a randomized, double-blind, placebo-controlled Phase IIB clinical trial, sponsored by Merck and the National Institute of Allergy and Infectious Diseases (NIAID), which enrolled HIV-negative volunteers between 18 and 45 years of age at high risk of HIV infection. Participants were randomly assigned to receive three injections of either the study or a placebo vaccine. Some 3000 participants were planned for each of the two trials; HVTN 502 was closed while 503 was still recruiting, when the Data and Safety Monitoring Board (DSMB) of the STEP trial (HVTN 502) - an independent committee providing oversight of the study - decided on September 18th that “… the trial as originally designed should be discontinued because the trial would not meet its efficacy endpoints”, as there was no evidence for protection. In fact, of people with low anti-adenovirus titres at enrollment who had received one vaccine/placebo dose, 24 of 741 vaccinees became HIV+, while among placebo-vaccinated people, 21 of 762 became infected. Among two-dose trialists, the figures were 19 HIV infections in 672 vaccinated volunteers, and 11 HIV infections among the 691 placebo recipients. This led to the discontinuing of the Phambili trial in South Africa as well, given no additional expectation of success.

And inevitably, things got worse: among male volunteers with high levels of antibodies against Ad5, 21 of 392 vaccinees became infected, but only 9 of 386 in the placebo group. Thus, it could be that pre-existing immunity to the vector virus actually increases susceptibility to HIV infection: one mechanism that has been proposed is that the secondary response to the Ad5 transiently boosts production of CD4+ T-cells - the preferred host cells of HIV.

The upshot of all this is that researchers worldwide are taking a hard look at Adenovirus-based HIV vaccines, even those which do not naturally occur in humans, and to which there should be no immunity: for example the Vaccine Research Center at the NIH has another Adenovirus-vectored candidate about to enter clinical trial, which may yet be delayed as the results of the STEP and Phambili trials are analysed in minute detail. The STEP volunteers are to be informed what they were inoculated with, and all Phambili volunteers have been advised to report back to the clinics for counselling. From the Merck site:

The Phambili DSMB also recommended that Phambili volunteers be told whether they received the vaccine or placebo, be strongly encouraged to return to study sites for protocol-related tests, and be counseled about the possibility that those who received the vaccine might have an increased susceptibility to HIV infections. STEP volunteers will also be counseled about this possibility, and discussions are underway to define the details of continued follow-up for STEP volunteers, including when STEP volunteers will be unblinded. Detailed analyses of the available data are being conducted, including analyses to better understand if there may be an increased susceptibility to HIV infection among those volunteers who received the vaccine.

Inevitably, there has also been political fallout in South Africa: the Minister of Health, Manto Tshabalala-Msimang, announced a moratorium last week on HIV vaccine trials, which could affect the home-grown vaccines due for trial next year.

This is not the only bad news recently on the HIV vaccine front: there seems to be evidence that another hitherto-promising HIV vaccine vector, the adeno-associated viruses (AAVs) may help to exhaust memory T-cells, by over-stimulating them with continuously-produced antigen.

All of this bad news looks, at first sight, to be a major body-blow to efforts to develop viable HIV vaccines. However, this is exactly what clinical trials are for: to test vaccines, for safety, immunogenicity, and efficacy. It is actually encouraging in a perverse sense that the trials work as they should - even though the first two HIV vaccines that have made it as far as efficacy trials are both abject failures. Lessons from the AIDSVAX debacle were that large trials can be done for an HIV vaccine; that HIV infection is a reasonable endpoint for a HIV vaccine trial; that HIV gp120 is not a good vaccine candidate. While it is still too early to be specific, lessons from this trial may be that immunogenicity / efficacy results from monkeys may not be a good predictor of human results, and that Ad5 is not a good HIV vaccine vector in pre-immune populations.

So the search goes on: the longest, most expensive vaccine development exercise in human history, with no clear end in sight.

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Related:

• HIV vaccines - bad news, good news
• The emergence of HIV/AIDS in the Americas and beyond
• How does HIV cause AIDS?

A Deeper Meaning

Today’s post is from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

I inadvertently became a published literary critic a little while ago. A long-time English Department colleague asked me for some help interpreting the collected works of possibly the most important modern poet from South Africa, and I quickly got caught up in some of the more interesting poetry I have ever read:

Douglas Livingstone was South Africa’s most important poet of the late twentieth century. He was also a practicing scientist who received a PhD for his work as a parasitologist for the CSIR in Durban, testing water purity levels along the Natal coast. Did Livingstone use poetry to reflect on his work as a scientist? Did his findings as a scientist influence his poetry? The self-reflection on the motivation for and results of a life of science come as he starts off on his early morning routine of driving along the coast to collect water for testing:

… coffee, toast, the rush for the lab
in the dark to gather up paraphernalia
load the station wagon and off again
for the river: man as hunter, Ahab
again, and Nomad, more prosaically
the quarry is microscopic Escherichias,
salmonellas, staphylococci, ascarid eggs,
coliphages, abject in the face of men,
a turning to an urge to heal the earth, its waters,
first the detection of ills which becomes
life-long non-progressive
find & measure the ills first, others
can heal with statute, exhortation,
engineering, first and for a lifetime detect. [RF: 473]

He then muses on the vision the microscope gives into the nature of life:

… Miraculous
cheek that prying probe, like some damned
gods voyeuristic telescope:
cilia spun from spirochaetes,
chloropasts from bacteria.

Billion year-old invaders
the silent mitochondria
propel our mobile towers, shared cells
sparking, colonized by vandals:
a fifth column of DNA
in interstellar sequences,
bland in their promiscuity. [RF, 287]

Heady stuff…deep thinker, Dr Livingstone. And in tune with modern microbiological thinking at a time when many biologists wondered if it were true. But he also had fun - and this is the one I printed out to stick up on the wall of the lab:

THE PASSIONATE BACTERIOLOGIST TO HIS LOVE

Come live with me & be my love
Up in the lab… first floor, above:
Where, shrouded in hygienic white.
We’ll potter through the febrile night.

Up here amid the test-tube racks
The centrifuge, the power- pack
I’ll show you botulinous meat
Mutations of a Spirochaete.

Entamoeba’s selfish mission
(Delighting in asexual fission):
And, just to elevate your hair
Some droppings from the Old Grey Mare.
Bacilli with a sunset hue
Will form a little chain for you.
And cocci on a culture plate
Will make your giddy heart gyrate.
You’ll see some eggs infected by
A Virus from a bloodshot eye;
For your delight, my lover doll,
I’ll flourish spleens in alcohol.
With dawn the roosters start to crow:
We’ll make a little fungus grow.
If you dig culture, little dove.
Why, come upstairs and be my love.

There’s obviously some culture in microbiology… B-)

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To bee or not to bee

When I’ve written previously about colony collapse disorder it’s generated a lot of interest, so today we have an update from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

A major recent mystery in US agriculture has been the phenomenon of “colony collapse disorder” (CCD) in honey bees. The phenomenon, which has manifested itself all over the US in the northern hemisphere winter of 2006-2007, has caused losses of 30-90% in individual hives - with adult worker bees mysteriously missing, with no dead bees in or around the hive. This is unusual, as a good method for sampling diseases of honey bees is simply to collect the dead bees shoveled out of hives by other workers. All sorts of reasons have been advanced to explain the problem, with cumulative insecticide / pesticide / herbicide poisoning high on the list - and meanwhile the bees continue to disappear, and the concern mounts as flowers go unpollinated, and crops may fail as a result.

Now US Dept of Agriculture researchers, working with others from Pennsylvania State and Columbia Universities, have provided very strong evidence that the disorder may be due to a single infectious agent: Israeli acute paralysis virus (IAPV) of bees, which is probably a strain of Kashmir bee virus. These are dicistroviruses: these are in a superfamily related to picornaviruses, but with two open reading frames rather than one, and the three structural proteins at the 3′ rather than the 5′ end of the ss(+)RNA genome. The virus can be transmitted between bees by the varroa mite, a common pest of honey bee hives in the US and elsewhere. Their work has just been published in Science magazine (A Metagenomic Survey of Microbes in Honey Bee Colony Collapse Disorder. Science Express Reports, September 6 2007).

The approach used by the team was a “brute force” high-throughput sequencing effort, in which total nucleic acids from honey bees collected from 30 colonies with CCD and 21 colonies with no CCD from four locations in the United States were screened. The team found genomes of six symbiotic bacteria and eight bacterial groups, 81 fungi from four lineages, and seven viruses. However, the only pathogen associated with nearly all CCD samples - 96.1% - and not with healthy bees, was IAPV. While this is not proof that the virus caused the problem - the small matter of Koch’s Postulates rears its ugly head - it seems the best candidate. As to the why and how - well, there’s going to be a lot of work and (possibly funding) for some lucky virologists.

In the words of the press release from the USDA, “Pollination is a critical element in agriculture, as honey bees pollinate more than 130 crops in the United States and add $15 billion in crop value annually. There were enough honey bees to provide pollination for U.S. agriculture this year, but beekeepers could face a serious problem next year and beyond if CCD becomes more widespread and no treatment is developed.”

They could always wait for the Africanized bees to get there …

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This is the End

This post is from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

This is the End. Or the beginning of the end. Or possibly, the end of the beginning?
To misquote the immortal Bill Shankly: “It’s not a matter of life and death: it’s much more important than that”.

Having hopefully alarmed you, let me explain: a group of mainly Thai researchers has, in the latest Journal of Virology, just published a paper entitled “An Avian Influenza H5N1 Virus That Binds to a Human-Type Receptor“. They say, nice and succinctly in their abstract, that “We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus“.

And this is a big deal, why?

Well, wild birds are the natural reservoir of just about the entire genetic repertoire of influenza viruses - and particularly of Influenza A viruses, the nastiest ones that get into humans. In fact, humans have been infected to our sure knowledge with viruses which have only 3 of 9 neuraminidase (N) and 5 of 15 or more haemagglutinin (H) gene combinations - and human pandemics in the last 100 years have only been caused by H1N1 (twice), H2N2 and H3N2 viruses. Of course, other viruses have been picked up in humans, but usually as a result of direct human-animal or human-bird contact: these include cases of H7N7, H7N3, H9N2 and H10N3 infection, which pop up, and then disappear.

And then, of course, everyone’s favourite candidate for the next Big One: H5N1 highly pathogenic avian influenza. Far from being a transient phenomenon, this has established itself as an endemic virus in chickens and other domestic fowl in Indonesia, Vietnam, Thailand and possibly Egypt, and keeps popping up in western Europe, mainly in migratory waterfowl. It has also infected over 320 people worldwide - and killed more than 190 of them, which is what makes it so sinister a threat.

One of the reasons that we are not constantly overrun with avian flu viruses popping straight out of birds and into people, and why we haven’t yet had a human H5N1 pandemic, is that bird-infecting flu virus H proteins bind to a sialic acid (SA) α2,3Gal(actose) receptor - which in birds is predominantly found in the enteric tract, which is why the virus is shed prolifically via faeces in birds, but is not much found in humans except for deep in the respiratory tract, which is difficult to reach. Thus, while humans can be infected by H5N1 viruses, this is rare, and so far onward transmission to other humans has not been reliably documented. Human-adapted flu viruses, on the other hand, bind preferentially to an SAα2,6Gal receptor - which is found predominantly in the upper respiratory tract, meaning the virus can much more easily infect and be transmitted. The frightening thing is that it takes only a few mutations in the H gene - 3 in the case of the legendary H1N1 Spanish Flu pandemic virus - to change from one type to the other, and only 2 to bind both types relatively weakly.

So Prasert Auewarakul and colleagues have isolated, from a fatal infection of a 5-year-old boy, a population of H5 HA sequences which are distributed evenly between the bird type (SAα2,3Gal-binding) and a mutant type (SAα2,3Gal- and SAα2,6Gal-binding). The mutant HA protein had two substitutions, at positions 129 and 134 (lL129V and A134V). These two residues are located close to part of the receptor binding domain, and apparently alter the binding specificity by changing the configuration of the binding pocket.

The implications of this are very worrying indeed: a lethal virus which can bind both bird and human receptors was selected for in a single bird-human transmission event - just like the Spanish Flu H1N1 HA reconstructed from archival tissue samples, which we should need no reminding, went on to kill in excess of 60 million people. In the age of the steam train and ship…. This means that we could be just one infection away from The Big One - with death tolls predicted to be in the range of tens of millions.

…of our elaborate plans, the end
Of everything that stands, the end…

Thanks to Jim Morrison and The Doors

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Related:

• Influenza: H5N1 Updates
• UK preparedness for pandemic influenza
• The Biology of Influenza
• Influenza pandemic - when?

Rolling down the road

Today’s post is from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

In my idle moments (alas, too few these days!) I often try to think up lists of rock songs with a virus theme: you know, like “Cucumo” by the Beech Boys… “I got them ol’ burnin’, hurtin’ herpesvirus blues again, mama” and “Lord, won’t you buy me some Efivarenz” by Jannie Joplin and the Zoviraxes… “Mama, weer all crazee now” by Noddy Holder and the Bornavirus Collective… but I pretty quickly run out of viable examples, and move on to more productive behaviour. But because my daughter keeps making me watch “Absolutely Fabulous”, with “This wheel’s on fire” as the theme song, which I like a lot, and I work on viruses with single-stranded circular DNA genomes, I just had to work it in somewhere…and then my Honours student Guy Regnard did a fantastic journal club paper on geminiviruses replicating in E. coli, and I saw my chance.

Geminiviruses are probably the single most important group of viral plant pathogens right now (though there is some debate about potyviruses). What is more, their continuing emergence means that there is an ever-increasing number of them in genome databases - and in people’s fields, which lab virologists tend to forget all too easily. The viruses are fascinating for a number of reasons, one being their unique virion morphology, and the other the fact that they seem to recombine so readily. The most important geminiviruses in terms of total yield losses and sheer numbers are undoubtedly the begomoviruses (named for Bean golden mosaic virus). These viruses may have one or two similarly-sized genome components, with the optional B component dependent on the A for replication, but providing movement functions which are necessary for the viruses with a B component.

Typical genomic organization of begomoviruses. ORFs are on the virion (V) or complementary (C) strand. The +-strand ori TAATATTAC-containing stemloop is shown. TrAP = transcriptional activator protein; REn = replication enhancer, MP = movement protein, NS = nuclear shuttle. [A]V2 ORF (in grey) not present in New World begomoviruses.

Replication of geminiviruses is apparently mostly via a rolling-circle mechanism (RCR), similar to many ssDNA phages and not a few bacterial plasmids: in fact, it is pretty generally accepted that the replication machinery of geminiviruses and nanoviruses from plants, circoviruses, anelloviruses and even parvoviruses from animals, and the aforementioned phages and plasmids, all has a common origin - which may extend to the mobilisation mechanism used by bacteria like Agrobacterium tumefaciens. A general scheme for ssDNA virus replication can be seen here.

Geminiviruses, like other ssDNA entities, have a rep gene, producing a Rep protein: in plants, this is expressed from a double-stranded replicative intermediate form of the genome (RF), and cleaves the genome (+) strand at a specific ori sequence, binds to the free 5′ end, and then mediates ligation of the newly-displaced (+) strand. The accumulation of ssDNA seems to depend on the production of coat protein, which probably sequesters nascent ss(+)DNA, as CP^- mutants produce no ssDNA.

There is a fair degree of specificity in all this, with viruses having a specified host range, and Reps having specificity for a narrow range of virus genomes. Thus, one might quite reasonably surmise that ssDNA viruses have been speciating with their hosts over aeons, and so would have evolved to be quite specific with regard to just what machinery they interact with.

And here’s where things get strange: apparently this is not necessarily true at all.

It has been known for many years that some geminivirus promoters work in E. coli; subsequently it has been found that some greater-than-unit-length begomovirus genomic clones seem to release viral RFs from their parent plasmids in E. coli and in A. tumefaciens, and in budding yeast cells. Lately, it is clear that the mammalian Porcine circovirus (PCV) also replicates in E. coli in the same way. Presumably, all that is required is (1) expression of functional Rep, (2) replicational or possibly recombinational release of a viral genome, (3) rolling circle replication. While this is academically fascinating, and potentially provides useful platforms outside of the natural host to study Rep-genome interactions, it is still homologous interactions (cognate Rep/DNA interactions) producing the result.

Now things have got more interesting: the Wu et al paper I started out with describes how an Ageratum yellow vein virus (AYVV) single-copy DNA A genome cloned in an M13-derived plasmid in E. coli - one RCR genome cloned into another one - produced single-stranded (+)- and (-)-sense AYVV genomes, presumably complexed with the M13 pV ssDNA-binding protein. Leaving aside the fact that this is impossible in terms of the standard models of replicational or recombinational release of unit genomes - and the models they present are largely hand-waving - it is a fascinating example of what amounts to cooperation between a prokaryote and a eukaryote virus sharing a very distant common ancestor, in a host of one of them. It is clear that M13 phage proteins are interacting with geminivirus DNA - and possibly even with the AYVV Rep - in order to effect a result not achievable with the latter alone.

The authors go on to speculate quite reasonably about what this could mean in the greater scheme of things, including whether or not it could mean that geminiviruses actually do replicate in the bacterial-descended chloroplasts, as has been claimed a couple of times: for example, in 1987, Holger Jeske’s group detected genomic ssDNA of Abutilon mosaic virus in chloroplasts of ornamental abutilon. It is also interesting that Maize streak virus (MSV) very thoroughly mangles the chloroplasts of infected maize, in the cells where virus is found.

Why this is all so interesting is that it shows that virus proteins separated by an evolutionary gulf of possibly billenia can still interact with similarly-replicating genomes. This opens up all sorts of possibilities for virus evolution and even virus survival: maybe plant viruses can survive inside endophytic bacteria; phages may be able to find refuge in plant or even animal cells; circoviruses may hide inside gut bacteria. Which means that the speculation by Mark Gibbs and Georg Weiler, that animal circoviruses derive from a complex recombination in animal cells between a plant nanovirus (ssDNA) and an animal calicivirus (ssRNA), may not be so far-fetched after all…except I think it all happened inside an insect, given that they have been associating with plants a lot longer than vertebrates have.

So what all this information means, possibly, is that there is the potential for some very deep interactions between different virus genomes and their proteins, in a very wide range of hosts. Which could make a lot of nonsense of a lot of what we think we know about how viruses and their hosts evolve.

So notify my next of kin, this wheel shall explode… current paradigms, hopefully!
Apologies to Bob Dylan and Rick Danko

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Rooting the tree

Today’s post is another from regular guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

While fossilized viruses have never been found, we can often infer probable lines of evolutionary descent by analysis of extant genomic sequences. This sort of molecular phylogenetic approach has thrown up all sorts of interesting findings for a number of important viruses: for example, it is possible that the divergence of Herpes simplex types 1 and 2 (orofacial vs genital herpes viruses) occurred during the time humans separated from our nearest chimpanzee relatives - and possibly because the development of face-to-face sex in humans reduced the frequency of facial-genital contact, although this may only be idle speculation! Herpesviruses in general seem to have evolved along with us since before the separation of invertebrate and vertebrate lineages, and possibly since a prokaryote origin: herpesvirus capsid proteins are distantly related to tailed phage proteins, and thus possibly have a common - and very evolutionarily deep - origin. Human papillomaviruses have speciated along with us primates as well, and viruses preferring particular locations are often more closely related to viruses infecting the same locus on another primate than they are to other human viruses. I have speculated in print elsewhere on the “Gondwanan” distribution of geminivirus ancestors, and how their development can be traced back for at least several hundred million years.

But while this may be academically fascinating, how is it relevant to the real world? One special-case example - that of the re-awakening of a “fossil” endogenous human retrovirus - was covered on this site recently. This has all sorts of interesting implications for the evolution of new retroviruses by recombination, and for taking a good hard look at xenotransplants. However, a more recent development in the field of HIV evolution is even more important, inasmuch as it has very important implications for HIV vaccines.

An often-quoted factoid is that the extent of variation of HIV genotypes in one individual in a year is equivalent to the worldwide variation in influenza A viruses in the same time. If one considers that the rate of flu virus antigenic change requires annual redevelopment of flu vaccines, what does this mean for HIV? James Mullins’ group in Seattle and their co-workers in Pennsylvania have gone a ways towards answering this in their just-published paper in Journal of Virology entitled “Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins”. They used phylogenetic analysis of many whole-virus sequences together with a new algorithm to computationally derive “centre of tree” (COT) ancestral protein sequences for HIV subtype B, as part of a strategy to design immunogens for use as vaccines that would elicit the broadest possible immune recognition of circulating HIV strains. The thinking behind this strategy is that use of ancestral sequences would minimise the genetic distance between a vaccine and circulating strains that could feasibly have speciated from it, compared to using any one of those strains, or even a consensus sequence.

Their Gag polyprotein sequence was capable of forming budded virus-like particles (VLPs); the ancestral Tat and Nef proteins retained appropriate function, and the proteins were immunogenic in terms of eliciting cytotoxic T-cell responses in mice. More importantly, the COT Gag elicited strong cross-subtype CTL responses when tested against peptide pools derived from subtypes A and M - and there was evidence that CD4 help was improved for these antigens. These are very important results when one considers the rate of change of HIV isolates: given that HIV phylogenetic variation can be depicted by starburst-like trees, and that the genetic distance between any two branch tips goes through a node, using the derived nodal sequences as vaccines could be a highly useful means of reducing the breadth of antigenic variation necessary to cover the current HIV spectrum - or even only part of it, given the geographic clustering of certain HIV subtypes. One could speculate that, when the correlates of protection for HIV vaccines are known (or in other words, when we finally know just what an HIV vaccine should look like), it could be possible to formulate COT-derived vaccine sequences on a yearly basis, as is routinely done for flu, based on up-to-date variation data. And given that subtype C - the target of the South African AIDS Vaccine Initiative and other vaccine development efforts - seems to be the prevalent form of HIV worldwide right now…it is possible that a single vaccine COULD protect people at risk in southern Africa, India and China. One lives in hope!

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It’s Life, Jim, but not as we know it…

Todays post is a welcome return of guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

It’s Life, Jim, but not as we know it…

Which could well apply to viruses, my very own favourite organisms - after all, they don’t respire, grow, excrete or any of those other good things that classical organisms supposedly do - but that’s not the point of this piece. Rather, I’d like to speculate on an interesting convergence of articles I’ve seen recently, on (1) extrasolar planets, (2) water on same, and (3) the possibility of novel lifeforms. Some of this is an accident of the irregular timing of receipt of my New Scientist and Nature subscriptions, but serendipity has played a large role in my life and will doubtless continue to do so.
It is amazing that, just a few years ago, the concept of planets outside of our own solar system was still a matter for debate - now we know of more than 200 of these, with an exponentially increasing number being discovered each month. Of course, most of these are weird: the very way we look for them guarantees that, with big objects very close to their parent stars being the most easily findable. Even so, we are down to about five Earth masses as the lowest yet described, and one of the newest has water - albeit as water vapour at a temperature of over 500^oC. Extrapolating from there to smaller planets with liquid water gives you…well, us, or a reasonable facsimile, galactically speaking. And given the fact that we have found so many planets in such a short time of looking augurs well for there being uncounted millions of them, in our galaxy alone - and many would look like ours.
Meaning that there would be a very good chance of similar chemistries having a similar chance of ascending the scale of negative entropy to give…well, nothing like us, probably, but quite possibly carbon-based lifeforms, dependent on water as a solvent, with some form of information storage which allows replication of itself and its accompanying phenotype. It is also highly likely that there, as here, microbiology would precede and dominate macrobiology, and single-celled or single-unit lifeforms would predominate in the biosphere. It is also quite possible that Earth itself was seeded by bacteria from Mars, given that that planet cooled down quicker than ours, had a similar early chemistry and therefore potential to develop life, and that Earth and Mars have regularly exchanged pieces of their surfaces following asteroid impacts. For the same reason, there could well be familiar sorts of organisms on Europa and other Jovian and Saturnian moons with liquid water beneath thick ice cover.
Indeed, some have speculated that there may even be more than one type of life on Earth (of course there is; viruses and everything else B-) and that we may simply have missed the other types because we don’t know what to look for yet. In fact, so-called nanobacteria could well be another type of life, for all that Alan C doesn’t like them… B-)
But why stop there? The New Scientist issue of 9th June has a most intriguing picture on its cover, of a sandy humanoid shape rising up through bricks and sand, with the caption “Why life doesn’t need DNA, carbon or water“. A case could be made for silicon-based organisms using liquid ethane or methane as solvents, which would open up all sorts of niches in our own solar system, let alone outside it. The Sirens of Titan could well be real, albeit a little slow and definitely not as we know them.
Anyone who has read any science fiction - and as I tell my classes, if you don’t then you should! - will have seen many examples of what is considered possible in terms of life. Some of the more interesting possibilities are self-aware electronic entities derived from computer programs (see Charles Stross’s “Accelerando!” for a wonderful recent example), and replicating, self-aware clouds of organised plasma.
Which is why I push my definition of life: which is “The phenomenon associated with the replication of self-coding informational systems” © E.P. Rybicki, 1996. Incidentally, I find another person with a Polish name has said something very similar, in 2001 - which means it must be true. Bernard Korzeniewski describes life as: “A network of inferior negative feedbacks subordinated to a superior positive feedback.” Which - um - means the same thing, doesn’t it?
And if there are other forms of life, there will be other forms of viruses…indeed, Steven Hawking is on record as saying that computer viruses can be considered a form of life: they are obligate parasites which exploit the “metabolism” of the host computer they infect, they replicate in the form of their source code [=genome], and they newest and nastiest can mutate while they do so.
Which means that there will be other versions of entities like Alan and I, spending our lives studying the invisible, for little reward other than simple fascination. As that late lamented student of chronosynclastic infundibula would have said, so it goes …

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A feeling for the molechism*

Todays post is a welcome return of guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

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 whoever is interested.

I have been in the same office now, and teaching the same course, more or less, for 27-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, most 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.

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 (MicrobeWiki? Who knew?) 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 22 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….

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 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.

Not 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?

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. 1: hypotheses 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, Profound Insight No. 2 follows: genome 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. 3: a 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 - wihout 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 cooked up down there?

I know what I want to do when I grow up.

* 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.

Incidentally, I can enthusiastically recommend the services of TinyURL.com: try pasting a looooooong PubMed search URL into a Web page and see how many things can go wrong compared to using a short version …

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Guest Post: Plus ça change, plus c’est … le same Web, only better?

MicrobiologyBytes is delighted to welcome our first guest blogger:

Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa.

My, how things do change… I found myself reflecting, while I was looking over the detritus on our Web server of some 13 years of posting pages on the Web. “Orphan” pages, unconnected to anything current; pages with a majority of dead links, because they are so old; pages last updated in 2000; pages left behind by the inexorable onward flow of the river that is the www; pages carried forward through several incarnations of the server… And yet, not to be deleted by the careless press of a key, because there is a sort of history there that is very hard to chronicle. A history of how the Kikwit Ebola outbreak unfolded, for example, post by email post. An account of how an Honours student inadvertently became the Web’s only Ebola expert, for a brief while in 1995… ah, what passing pleasures, now mainly gone.

Consider this: a connected set of Web pages is a network, existing as a linked series of snapshots that reflect the current update. Every single change alters the network - yet where is this recorded? If you are lucky and have a hard drive the size of the Empire State Building, or if you are disciplined enough to actually back things up as successive versions, then perhaps you have an accurate historical record of how things changed - but no-one else will. And given the fact that most normal people are not disciplined enough to do the necessary, you probably don’t either…

So how does one even approach the problem of constructing a history of any particular corpus of web-published material? We are confronted with a situation not dissimilar to the one which confronts would-be chroniclers of any ordinary human life: the only material available for research is the latest version (if still extant), and a mess of isolated snapshots and pages, if we are lucky.

I took a look back over my teaching material the other day, which I started formulating back in mid-1994, round about the time the Web came into existence for us non-professionals. I don’t have a single file dating back to that time, not one: the only thing left is a grandfathered filename (virtut1.html) that it would be too complicated to change. The earliest I can get back to - on a dusty CD-ROM backup unearthed from a bottom drawer, from a PC I gave away at least three upgrades ago - is 1998, and then only for some of the files I actually updated at the time. My first web pages are thus irretrievably gone, vanished into entropy - unless they are fossilised on some long-lived legal or illegal mirror server somewhere, like some of my outdated pages I found quite by accident on a computer in Cambridge, and only got removed by threat of copyright infringement action.

So why bother at all? Of what interest is the history of some half-baked, amateurish attempts at porting teaching material from overhead projection transparencies to the web?

Weelll… it’s not really for me to say, is it? I can’t predict who might be interested in the historiography of virology pedagogics - but it’s just a little sad to think that so much work has vanished into free electrons, wandering the universe until the inevitable heat death stills them all. I mean, look at Alan Cann: his Virology textbook is now in a fourth edition, and all three are available to anyone who wishes to compare them. I can’t even find Versions 1 - n-1 of my material, so all you’re left with is Version n, of 2007 (© Ed Rybicki). It’s paradoxical that in this electronic age, it’s still the traditional medium of print that still has the best potential for survival. I may even still have some of my original hand-printed overheads from 1981, if they survived the last office-cleaning purge!

But be that as it may…my continuous rolling upgrade of the Web pages has reached a 2006 version in most cases, and 2007 in a few - with a lot of visual material still stuck in a dark age. There is actually not that much incentive to do too much about that, frankly, given the wealth of graphics now out there in Webspace: Russell Kightley, for example, has a wealth of thoroughly professional-looking pictures of viruses, cells, and virus life cycles; I use movies of the HIV life cycle filched from Boehringer-Ingelheim’s site (as well as from Alan Cann); there are now some truly stunning cryo-EM 3D image reconstructions of virus particles available…and nearly everything is copyrighted, so putting it up on my site could be courting prosecution. Which is why linking to things via the web is the way to go…if I only had time! Aaaaarrrgghhh!!

Which is why I am impressed by this site: unlike some of us early adopters who are now hopelessly behind, he has