How papillomaviruses infect cells

28 February, 2017

We are presently hosting the 2017 Human Papillomaviirus Conference here in Cape Town, and we are experimenting with having a pre-conference Basic Science Workshop, in addition to the well-established Public Health and Clinical streams.

And it’s going…really well! We have a huge room for it, with double screens; it’s pretty full – and the kick-off was a masterful talk on HPV Entry by Michelle Ozbun.

She had a wonderfully illustrated close to 90 minute talk, with some very intriguing speculations: the theme of the Workshop is “Where Are The Gaps?”, and she pointed out a number of important gaps in our knowledge of the processes that  PVs engage with in order to get into cells.

One of the most intriguing was the fact that pseudo- or quasivirions made in and puriffied from mammalian cells, are not very infectious at all in keratinocyte raft cultures – which Martin Sapp pointed out from the audience can be hugely improved by using virions associated with cell matrix material rather than purified forms.

Michelle speculated that the natural state for PVs infecting susceptible cells – which most often probably occurs via transient wounding of cornified epithelia or mucous membranes – is in the context of squames, or exfoliated and disintegrating cells. Which means virions are associated with all of the molecules present in such a milieu, that may also serve as receptors – meaning that the virions may in fact be primed in terms of conformational changes associated with receptor binding, which could greatly facilitate binding of basal layer cells and entry into them.

How sensible is that as a concept: the natural milieu for PVs to infect other cells is in the context of debris from the cells in which they were produced.

I’m learning things by the minute B-) Michelle had a very useful set of questions (see below).

Other gaps for discussion will be posted later. #HPV2017

Purifying TMV: a blast from the archives

16 February, 2017

We have had an in-house method for purifying Tobacco mosaic virus (TMV) and its various relatives ever since I got to Cape Town – and it was propagated by copying and re-copying of what was effectively an abstract for a talk given at our local Experimental Biology Group quarterly meeting in early 1970, published in the South African Medical Journal.

Marc van Regenmortel was Professor of Microbiology at the time, and had a long history of physicochemical and serological work on TMV and strains and mutants of TMV. He also had Barbara von Wechmar, later to become my PhD supervisor, working for him as a Scientific Officer – and together they came up with an ingeniously simple, easy, high-yielding method to purify TMV out of infected tobacco.

So why do we care now? Well, we’re trying to purify some derivatised TMV [details redacted while patent is sought], and Sue Dennis in my lab could only find techniques that involved extraction with chloroform, PEG/salt precipitation x 2, high-speed centrifugation – all of which sounded unnecessarily laborious, given I knew we had a better method.

Trouble is – I cleaned up my office a while back, and seeing as “we’ll never work with TMV again, will we??”, I’d thrown out all of the old practical manuals that included it.

So I go to the old papers I could find online, and they all referred to “von Wechmar and van Regenmortel, 1970”, with no methodological details. And of course, there was no record of this paper anywhere I could find, not even using [obscure Russian language site details redacted].

Then I chanced upon the very bare bones online archive of the SAMJ, married that up with the much snazzier-looking-but-devoid-of-desired pdfs official site to find issue numbers – and there we were! Via some fascinating side trips through a history of the plague in Cape Town, among other things, but finally, a PDF of the original EBG abstract.

tmv-method

In fact, I have a big section of our coldroom with myriad bottles of purified TMV, all at 5 mg/ml concentration or higher, still infectious, and up to 40 years old – all made by this technique.

tmv sedim

So Sue is about to apply it right now, as she conveniently has a freshly mashed extract of N benthamiana ready waiting, and we have PEG and NaCl…we’ll give the charcoal/Celite a miss this time, because it can get a bit messy, but it is THE way to get pigments out of your virus preps – or even nanoparticles, @FrankBioNano & @Lomonossoff_Lab?

From plant virology to vaccinology: a personal journey

15 February, 2017

A couple of years ago now, an Editor of the journal Human Vaccines & Immunotherapeutics contacted me to say they would like to profile me as a vaccinologist. Being of a suspicious nature, I immediately inquired how much this would cost me. The encouraging answer was “Nothing!” – so I jumped straight in.

The end result is as near to a current autobiography as I will probably ever get, so I may as well put it up here. So, if you’re interested in finding out what the connections are between a swimming pool in Zambia, not doing Biochemistry (twice), plant virology and making vaccines – click below!

Fall armyworm – and how viruses could help combat the plague.

15 February, 2017

Kenneth Wilson of the Univ of Lancaster has recently written a blog post on the plagues of African and “Fall” armyworms (aka caterpillars, larvae of moth species in the genus Spodoptera) that are currently chewing their way through southern African maize and other crops. I wrote the following as a comment to his blog.

Nice article – which very ably demonstrates the perils of importing agricultural pests from elsewhere!

I am interested that you wrote:
“There are non-chemical, biological pesticides that could also be effective. These are pesticides derived from natural diseases of insects, such as viruses, fungi and bacteria.”

Some years back (OK, nearly 30) Barbara von Wechmar in the then Microbiology Dept was instrumental in our finding a number of insect viruses that were seriously lethal to aphids and green stinkbugs. These were inadvertent discoveries, which happened three times – twice with different viruses for aphids which we were investigating as wheat/barley pests, and once (with two viruses) for stinkbugs causing problems in passionfruit – and were due to observations that high density lab colonies of the insects in question often developed disease that caused rapid colony death. Barbara went on, after characterisation and publication of the viruses by me and Carolyn Williamson, to show that highly effective insecticides could be made by simply grinding up recently dead insects in some buffered saline, sieving the bits out, and spraying the juice onto plants. This worked for aphids, and was especially effective for stinkbugs.

I note that similar phenomena have been seen for a number of insects, including the spruce budworm in North America, and by Don Hendry and others in South Africa for Nudaurelia capensis, the Pine Emperor moth. In the latter case, the larvae can become literal sacs of virus, and bursting of dead caterpillars leaves viruses everywhere in the environment.

It might be a good “boer maak n’plan” type of approach for folk to gather a bucket of these things, feed ’em leaves for a while, see if they start to die – then mulch them in some half-strength (=0.075M) saline and make a spray out of it.

It couldn’t hurt, might help, and would be a pretty good biology lesson B-)

Seriously: you can find insect viruses everywhere you look, and crowding is a really good way of spreading and bringing out otherwise inapparent virus infections, just as it is with humans – with the difference being that insect viruses can reach REALLY high titres in their hosts, and are pretty stable as they are often spread by contact of live larvae with dried juices from dead ones.

References

John C. Cunningham, Basil M. Arif and Jean Percy. THE STATUS OF VIRUSES FOR SPRUCE BUDWORM POPULATION REGULATION. File Report No. 7 January 1981, Forest Pest Management Institute, Canadian Forestry Service

EP Rybicki and MB von Wechmar. Characterisation of an Aphid-Transmitted Virus Disease of Small Grains. Isolation and Partial Characterisation of Three Viruses. J Phytopathology 103, Issue 4 April 1982 Pages 306–322

Cheryl T. Walter, Michele Tomasicchio, Valerie Hodgson, Donald A. Hendry, Martin P. Hill and Rosemary A. Dorrington.  Characterization of a succession of small insect viruses in a wild South African population of Nudaurelia cytherea capensis (Lepidoptera: Saturniidae). South African Journal of Science 104, March/April 2008

C. WILLIAMSON, E. P. RYBICKI, G. G. F. KASDORF  AND M. B. VON WECHMAR. Characterization of a New Picorna-like Virus Isolated from Aphids. J. gen. Virol. (1988), 69, 787-795

Williamson C, von Wechmar MB. Two novel viruses associated with severe disease symptoms of the green stinkbug Nezara viridula. J Gen Virol. 1992 Sep;73 ( Pt 9):2467-71.

 

Welcome, MCB newbies – and let the mayhem commence!

14 February, 2017

I announced our second year of Molecular & Cell Biology Department Honours class blogs on Twitter this morning as follows:

screenshot_2017_02_14__3_05_pm

Mind you, I also put up this, so you might see a theme here…

screenshot_2017_02_14__3_06_pm

As in: blogs can be fun, as well as being serious – and we would like to see both!

So have fun, Hons of 2017 – and Vernon and I WILL be watching you…B-)

“New Virus Breaks The Rules Of Infection”! No – no, it doesn’t

31 August, 2016

I was prompted to this post by the breathless and much-hyped response to the discovery – the repeated discovery should I say; there was an earlier one that gets glossed over – of a multicomponent flavirus-like virus, this time in mosquitoes.

The actual report was published here: it is a well-done study, describing

“…a genetically distinct, segmented virus isolated from mosquitoes that also exhibits homology to viruses in the familyFlaviviridae and that appears to be multicomponent …, with each genome segment separately packaged into virions”

The authors say

“Although multicomponent genomes are relatively common among RNA viruses that infect plants and fungi, this method of genome organization has not previously been seen in animal viruses [my emphasis]

…which is why there’s all the hype, of course: claiming the virus “…breaks the rules of infection” is simply incorrect, because it is in fact related to very well characterised single-component ssRNA+ viruses of arthropods and mammals – flaviviruses – and infects its mosquito host exactly as these do, except with its genome in separate particles. Which makes it similar to quite a few plant viruses, several of which are, incidentally, probably evolutionarily related to viruses infecting insects – but more later.

Thus, a claim like “…a new study published Thursday is making researchers rethink how some viruses could infect animals” is simply hype.  But it is a sort of hype familiar to plant virologists, who after all showed that multicomponent viruses (=viruses with multipartite genomes packaged in separate particles) existed over 50 years ago – and who also showed that gene silencing was a factor in plant resistance to viruses long before their better-funded animal-researching colleagues got in on the act, but that is another story.

The way in which multicomponency was discovered with plant viruses is interesting: it relied on the fact that plants can respond with local lesions – qualitatively the same as plaques in bacterial or animal cell lawns – to mechanical infection, and that this can be used an an accurate assay of virus titre, as for phages or animal viruses (see here).  It became evident, though, that certain plant viruses produced significantly steeper lesion vs dilution curves than were expected from “one-hit” kinetics, where infection with a single virus particle sufficed to cause a lesion.

This is best shown by a plot like the one below, modified from REF Matthews’ Virology, 3rd Edn, attributed to Lous van Vloten-Doting from 1968.  This shows the curves obtained from accurate and painstaking local lesion assays with the single-component Tobacco necrosis virus (TNV), and the multicomponent Alfalfa mosaic virus (AMV): both are ssRNA+ and have isometric particles, but TNV has a single-component genome, and AMV a tripartite genome packaged in 3 particles.

multicomponent

The insect virus investigators did much the same thing:

“We used a similar approach to assay the nature of segment packaging for GCXV using cell culture plaques instead of leaf lesions. The dose-response curve for GCXV differed significantly from expectations for a single-component virus (i.e., the number of plaques decreased more quickly than expected with dilution of the inoculant)…we used our dose-response curve to estimate the presence of 3.27 ± 0.37 distinct GCXV particles required for plaque formation”

…but with the addition of rapid sequencing techniques not available in 1968, to show that indeed, the different segments were 5 distinct pieces of ssRNA, 3 mono- and 2 tricistronic (=3 ORFs), with the 2 largest monocistronic pieces being similar to flavivirus NSPs and the 3 smallest not encoding anything similar to sequences in the databases.  Four RNAs were essential for infectivity, while the smallest appeared dispensable.  Particles formed during infection of cultured cells were enveloped and 30-35 nm in diameter, considerably smaller than flavivirus virions.

This is a very interesting finding, although not unique: similar viruses were previously found in ticks in 2014, when the authors claimed that:

“To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented [flavi]viral ancestors

They were also wrong: there are a number of viruses for which this could have been said years ago, like the picornavirus superfamily-related comoviruses of plants. These have two-component genomes which both encode polyproteins, one with non-structural and the other with structural ORFs.  In fact, an evolutionary precursor to such viruses could be the more closely picornavirus-related dicistroviruses of insects, which have a classic picornavirus precursor polyprotein ORF split into two, with the structural protein ORF at the 3′ end and the regulatory or non-structural polyprotein at the 5′ end.

I got into this because it irked me mildly that such a fuss was being made of a second group of animal-infecting multicomponent ssRNA viruses, when the multicomponent plant virus precedent and history was VERY well established – but then got more interested when speculation started about what advantage multicomponency could confer on a virus.

I have thought for years that people discussing this generally have it backwards: it’s not that having a divided genome in separate particles offers advantage(s), it’s that it is not a DISadvantage in some circumstances – and particularly where there is no selection against the state.

A reason that multicomponency HAS been seen quite frequently with plant viruses could be that mechanically-transmitted viruses can reach VERY high concentrations in infected plants, and even obligately vector-transmitted viruses (eg: the bicomponent ssDNA begomoviruses, multicomponent ssDNA nanoviruses) reach quite high concentrations in the phloem tissue to and from which they are transmitted, compared to viruses in vertebrates.

This is also true for viruses of arthropods compared to vertebrate viruses: dicistroviruses in aphids can reach concentrations that are comparable to those of viruses like TMV in plants, to the point that aphids inject enough virus into plants that our lab originally mistook Rhopalosiphum padi virus for a plant virus. Moreover, plant virus virions often aggregate into quasi-crystalline arrays which can be hard to separate and which are even visible inside insect vectors, thus virtually guaranteeing that >1 virion will be present in any inoculum, even if significantly diluted.

This is most definitely NOT the case for vertebrate viruses, even where the same virus infects both an arthropod and a vertebrate host: the titre in the latter is guaranteed to be orders of magnitude lower, largely due to a more sophisticated immune system keeping viraemia in check. Thus, high inoculum concentrations relative to vertebrate viruses, and a tendency to aggregate, mean there is no DISadvantage inherent in multicomponency.

Having said this, there may be advantages to having a multicomponent genome: one such is presented in a recent article by Sicard et al. (2013), (thanks, @LauringLab and @DiagnosticChick!) in a study of the ssDNA nanovirus Faba bean necrotic stunt virus (FBNSV), which has an 8-component genome of ~1 kb/segment, encapsidated in 8 virions. They proposed:

“…that the differential control of gene/segment copy number may represent an unforeseen benefit for multipartite viruses, which may compensate for the extra costs induced by the low-frequency segments”

Thus, multicomponent viruses may achieve the sorts of gene dosage control only possible in viruses with larger genomes, by virtue of having multiple genome components rather than control elements which add genomic bulk.

Another possible advantage that I recall being touted by plant virological luminaries is the ease of reassortment compared to recombination: this is exemplified by the reo- and orthomyxoviruses, albeit in vertebrates, where they are constrained by having to have all genome components in the same capsid to guarantee infectivity.

I think Vincent Racaniello is correct in the breathless article I quoted in opening, where he is quoted as saying

“There’s so much we don’t know about viruses…We should always expect the unexpected.”

Absolutely. And I think it’s a safe bet that a LOT more multicomponent viruses will be found in arthropods – and even in some vertebrates, to which they will have been transmitted by arthropods. Because that’s the link between many of these viruses: an evolutionary history that involves plants and arthropods, or arthropods and other animals, at an early stage of life on land. Because that’s all there was for advanced eukaryotes, early on: primitive vascular plants, insects that preyed on them and on each other, and protists.

Bunyaviruses in protozoa?

25 August, 2016

A Novel Bunyavirus-Like Virus of Trypanosomatid Protist Parasites

  1. Natalia S. Akopyantsa,
  2. Lon-Fye Lyea,
  3. Deborah E. Dobsona,
  4. Julius Lukešb,c,
  5. Stephen M. Beverleya

ABSTRACT

We report here the sequences for all three segments of a novel RNA virus (LepmorLBV1) from the insect trypanosomatid parasite Leptomonas moramango. This virus belongs to a newly discovered group of bunyavirus-like elements termed Leishbunyaviruses (LBV), the first discovered from protists related to arboviruses infecting humans.

 

OK, seriously interesting viruses – BECAUSE “…The L. moramango virus thus resembles a group of related viruses discovered recently in the closely related human parasite Leishmania”…which I will note, whose phylogenetic affinity to other higher eukaryotes is via a relationship to trypanonosomes, and then – Euglena??  Which is an alga….

Seriously: a bunya-like virus found in a protozoan whose closest affinity to other hosts of bunyaviruses is via algae??
This pushes the possible evolutionary origin of bunyaviruses faaaaaar back, to possibly a billion years or so, when fungi were separating from protists from algae…..
There is another option, however: I note the Leptomonas sp. is a parasite of insects. It is of course worth noting that the Leishmania and other related parasites infecting mammals are also all arthropod-vectored – which could imply an origin in arthropods.
Which makes good sense, considering these beasties date back less than 600 million years or so – which is still pretty good for a virus?!

So: will smallpox come back to kill us, from the melting permafrost??

17 August, 2016
Variola virus, the agent of smallpox.  Image courtesy Russell Kightley Media.

Variola virus, the agent of smallpox. Image courtesy Russell Kightley Media.

There has been a lot of tweeting today about how Smallpox Will Come Back From The Grave And Kill Us All: see here, here and here for lurid examples.

This is alarmism at its insidious best: shouting out a headline, based on flimsy evidence, that says “We’re all going to die!” or similar nonsense.

Really: this IS nonsense.  Some corpses were found in the permafrost in Siberia, that MAY have had smallpox-like lesions on them, and from some of which which smallpox virus DNA could be recovered – presumably by PCR.

This does NOT constitute a threat of live virus being present, or escaping from the corpses even if it WERE there.  I have railed on about this sort of thing before, and I am as unconvinced now as I was then, albeit with SOME reservation about the possibility for smallpox.

"Pithovirus sibericum", from Jean-Michel Claverie and Chantal Abergel

“Pithovirus sibericum”, from Jean-Michel Claverie and Chantal Abergel

I can believe you could get live anthrax: those spores are incredibly tough, and can last for many years in soil, let alone in ice. I could also believe that one could find live megaviruses – the so-called pitho- and molliviruses – in permafrost, because their putative hosts are unicellular protozoans and because they are also seriously stable.

But smallpox? The virus is probably not as stable as the megaviruses mentioned; it relies for infection on its structure, which has membranes integral to it – AND it infects people, who, when they die, don’t cool down very quickly, and whose cells release all sorts of nasty enzymes (lipases, proteases) as they die. Which could be expected to chew up most things, including poxviruses.

Oh, sure, poxviruses CAN survive for years at a pinch – in the form of dried secretions or scabs, which, because they are dehydrated and full of protein, tend to stabilise virus particles. This is how the old variolators and vaccinators (literally: people who used variola or “vaccine” to vaccinate against smallpox) used to preserve their inocula, when they weren’t using fresh material.

Melting tundra is not like that, I will note: bodies with intact virions in them will thaw and rot all over again, and that rotting will reduce what little virus there may be even further.

So I am not a believer in Death From The Permafrost!

And nor should you be.  But it might not hurt for someone qualified to test whether or not there IS live virus in frozen samples, by culturing an extract?

TIJoCV honours World Hepatitis Day

28 July, 2016

IJoCV.2

Graphics by Ian Mackay and Susan Nasif.

A word from the Comics Editor

26 July, 2016

_2__Twitter___Mentions


Renaissance Virology Comics: Get the Facts with Virology Comics!

#VirologyComics (Every Tuesday)

 “The exponential increase in health-related online platforms has made the Internet one of the main sources of health information worldwide. However, online communities with greater freedom of speech have, regretfully, become a powerful platform for anti-vaccine voices and the sharing of defective medical information. Health communicators have not yet taken their responsibilities on digital media as seriously as it should be.” (quoted with a few additions)

Therefore, the main objective of Virology Comics, the winner of the Science Hero Award in 2015, is to raise public awareness, inform readers about routes to prevent the transmission of viral diseases, and to provide basic knowledge and teaching tools for health professionals and educators in an easy, enjoyable, inspirational, and informative way.

Virology Comics’ dream is big, its ability is massive and its greatest strength is determination. We realize that dreams do not become realities without sweat and hard work, but we also know that getting the significant interest & support of like-minded and generous individuals makes a huge difference. I hasten to say that I am not only an artist, but also an artist with a medical background (Ph.D. in Virology). This is a rare combination, which only adds to the uniqueness of my work.

If you enjoy this material, I ask that you consider supporting it: cartooning is expensive, unfortunately, and we need all the resources we can get!  If interested, you can click on the link to the Patreon site to provide a regular monthly financial contribution of as little as $6:  https://www.patreon.com/VirologyComics

I would like you to enjoy, via the Internet Journal of Comprehensive Virology’s site, the outstanding story of Zika Virus Comics straight from The Lancet, published some time ago, and ask you to remain in the loop for more episodes! Learn, Share & Enjoy!

https://www.youtube.com/watch?v=gWjiWUNoNQU

Thank you very much.

Susan Nasif, Ph.D.