Archive for February, 2017

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

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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:

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

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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-)