Archive for the ‘General Virology’ Category

Effect of Formaldehyde Inactivation on Poliovirus

23 September, 2014

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

 

Source: jvi.asm.org

People have been treating poliovirus with formaldehyde for over 60 years – and it’s only NOW that someone thought to study in detail what happens!

I love this stuff: analytical centrifugation could have been done any time in the last fifty years (and has been, in determining structural transitions) but the newer techniques such as flow cytometry and RT-PCR could not. Analytically determining now what was empirically observed to work when polio vaccines were first made, is a historically important vindication of pioneering work that has almost made the viruses go away.

Simple and obvious findings, essentially – it is obvious that methylene bridging between amino acids would affect structural transitions; so too that HCHO treatment would kill viral ssRNA – but it hadn’t been DONE properly previously.  Great stuff!

See on Scoop.itVirology and Bioinformatics from Virology.ca

Ebola: “We Could Have Stopped This”

8 September, 2014

Public health officials knew Ebola was coming. They know how to defeat it. But they’re blowing it anyway.

ld, you still just don’t get it. The Ebola epidemic that is raging across West Africa, killing more than half its victims, will not be conquered with principles of global solidarity and earnest appeals. It will not be stopped with dribbling funds, dozens of volunteer health workers, and barriers across national borders. And the current laboratory-confirmed tolls (3,944 cases, with 2,097 deaths) will soon rise exponentially.

To understand the scale of response the world must mount in order to stop Ebola’s march across Africa (and perhaps other continents), the world community needs to immediately consider the humanitarian efforts following the 2004 tsunami and its devastation of Aceh, Indonesia. The U.S. and Singaporean militaries launched their largest rescue missions in history: The United States alone put 12,600 military personnel to a rescue and recovery mission, including the deployment of nearly the entire Pacific fleet, 48 helicopters, and every Navy hospital ship in the region. The World Bank estimated that some $5 billion in direct aid was poured into the countries hard hit by the tsunami, and millions more were raised from private donors all over the world. And when the dust settled and reconstruction commenced, the affected countries still cried out for more.

Source: www.foreignpolicy.com

A seriously hard-hitting article by a very good journalist with a particular interest in infectious diseases.

And she’s right: Ebola was stopped, not once, but a number of times, as long as 38 years ago, in settings that are as or even more desperate in terms of poverty and lack of medics and medical resources.

The problem is, intervention did not occur soon enough this time, or on a scale sufficient to stem the increase in infections that inevitably followed introduction of the disease into urban settings.

It is a matter of amazement to me, that with the ever-present threat of pandemic influenza AND the recent emergence of MERS, that the WHO should have its "…miniscule epidemic-response department slashed to smithereens by three years of budget cuts".

Seriously: faced with diseases that can jump out of camels, or bats, or rats literally anywhere, WHO has to have budget cuts??

See on Scoop.itVirology News

Mucosal SIV Vaccines with Bacterial Adjuvants Prevent SIV Infection in Macaques

2 September, 2014

A new paradigm of mucosal vaccination against HIV infection has been investigated in the macaque model. A vaccine consisting of inactivated SIVmac239 particles together with a living bacterial adjuvant (either the Calmette & Guerin bacillus, lactobacillus plantarum or Lactobacillus rhamnosus) was administered to macaques via the vaginal or oral/intragastic route. In contrast to all established human and veterinary vaccines, these three vaccine regimens did not elicit SIV-specific antibodies nor cytotoxic T-lymphocytes but induced a previously unrecognized population of non-cytolytic MHCIb/E-restricted CD8+T regulatory cells that suppressed the activation of SIV positive CD4+ T-lymphocytes. SIV reverse transcription was thereby blocked in inactivated CD4+ T-cells; the initial burst of virus replication was prevented and the vaccinated macaques were protected from a challenge infection. Three to 14 months after intragastric immunization, 24 macaques were challenged intrarectally with a high dose of SIVmac239 or with the heterologous strain SIV B670 (both strains grown on macaques PBMC). Twenty-three of these animals were found to be protected for up to 48 months while all 24 control macaques became infected. This protective effect against SIV challenge together with the concomitant identification of a robust ex-vivo correlate of protection suggests a new approach for developing an HIV vaccine in humans. The induction of this new class of CD8+ T regulatory cells could also possibly be used therapeutically for suppressing HIV replication in infected patients and this novel tolerogenic vaccine paradigm may have potential applications for treating a wide range of immune disorders and is likely to may have profound implications across immunology generally.

 

Graphic of cells involved in HIV immunity from Russell Kightley Media

Source: journal.frontiersin.org

I have heard Jean-Marie Andrieu present this work – and I can understand why there is some skepticism surrounding it, because it is almost too good to be true.

Seriously: SUPPRESSING SIV-specific CD4 T-cell activation results in immunity to challenge infection??

However, and however – if this work is found to have been done well (and there is no evidence it was not), then this really could be a simple, reliable way of immunising people against HIV

Of course, monkeys aren’t people, and SIV is not HIV, so there MAY be a problem somewhere along the line in translating these results into humans – but what if there is not?

Then we may have a vaccine, and kudos to Jean-Marie Andrieu and co-workers to persevering along a difficult road to get their idea tested.

See on Scoop.itVirology News

20 years on, and here we are with Ebola, again

25 August, 2014

Browsing through my own web pages in an effort to clean up dead-end links, and cull tired material, I discovered that my link to an essay I wrote 19 years ago was still live – and as it referred to something written in and put up on our nascent Web server in 1994, means it has a 20-year anniversary round about now.

My essay is

The Student, the Web and the Ebola Connection

or:

Dr Jacobson, are you going to Kikwit?”

…and it is a record of events that resulted in 1994 from (a) an Honours student essay being written on “Emerging Viruses”, and (b) me playing around with the then-very-new WWW server that UCT has enabled – but didn’t tell anyone about, because they didn’t want anyone to use it until they had sorted out policies.  Oh, and (c) – the Kikwit Ebola outbreak in 1995.

I wrote in 1995:

“The whole phenomenon has been an object exercise in the power of the Web as a tool for the wide dissemination of information: we reached not only professional virologists, but also health-care professionals, and – most importantly – the lay public on a large scale”

And of course, this is even more true now – which is why, following the benign guidance of The Guru Cann, I maintain ViroBlogy and Virology News, and heartily recommend a Web presence to anyone who feels they need to disseminate information on topics of specialist and generalist interest to the world at large.

Of course, nearly all the links out of that essay are now dead – including to the original essay, that for a while there in 1995 was the ONLY detailed information on Ebola available on the Web.  So here is Alison Jacobson’s original essay, in full, revealed by going to my teaching material and checking out essays from 1997 and thereabouts:

EMERGING AND RE-EMERGING VIRUSES: AN ESSAY

Of course, I also maintained a daily update on the Kikwit outbreak, and then a couple of the next ones, before the Web caught up with me and it became easier to just trawl it for news via Google and its predecessors.  It still makes interesting reading, though, to go through some of what was posted from the disease frontlines back in the 1990s – and to remember that I had the TIME to do that kind of thing!

Where we are now

Well, here we are with what is the worst outbreak of Ebola in history, and here am I – again – trying to keep up with it.  This time, by the very excellent medium of the Web news aggregator Scoop.it, where I have established Virology News as a means of quickly and easily getting news out to the public.  Again, following the very excellent example of TGC, but also Chris Upton, who babied me along by letting me co-curate his Virology and Bioinformatics site.

Of course, there is a new angle to this outbreak – and that has been the compassionate use of a plant-made monoclonal antibody cocktail (ZMapp), hitherto only tested preclinically in a primate model.  Fortuitously, this all happened while I was finishing off a review on plant-made viral vaccines, so I reported on it – with references – here on ViroBlogy.

I was also able to report on it in my Plant Molecular Farming news site, with some authoritative statements from pioneers of the technology: Charles Arntzen from the Arizona Biodesign Institute sent through a link for an interview he did, and CNN covered it quite well too.  Charlie also sent through a set of links in an email that he was happy to share:

“The original story

There is a lot of interest from the press in “why tobacco” and “how does it work”?

The other focus is on the politics of scale up of the drug — it seems that criticism of the US is mounting in some sectors of Africa, and elsewhere.   I talked to a Spanish Language radio news station this morning, and the main questions related to “why is this a Secret Drug; are you trying to hide the secret from the world?”    “Is Reynolds tobacco trying to stop the supply of this drug to Africans?”    One guy asked if it was true that the Ebola Virus had been created in a test tube.

It seems that the press is largely to blame for using terms like Secret Drug.   It appears that they are also trying to mount political pressure to make a lot more of the drug to help Africans.   [This was] a nice job answering some of this….”

And at time of writing, the outbreak was still raging, had spread to Nigeria, and airlines were banning travel to half of West Africa – and alarmist tourist firms were advising people not to come to South and East Africa, as well.  The WHO has also said the impact is probably much greater than reported.

And Alison Jacobson is alive and well, and NOT working in virology any more.  Sadly!

5 Viruses That Are More Frightening Than Ebola

20 August, 2014

By Elizabeth Palermo, Staff Writer
Published: 08/15/2014 01:58 PM EDT on LiveScience
The Ebola virus has now killed more than 1,000 people in West Africa. Although the mortality rate of the most recent outbreak isn’t as high as in previous events, it’s still the case that most people who become infected with Ebola will not survive. (The mortality rate is about 60 percent for the current outbreak, compared with 90 percent in the past, according to the National Institutes of Health.)

1. Rabies

2. HIV

3. Influenza

4. Mosquito-borne viruses

5. Rotavirus

 

 

Source: www.huffingtonpost.com

Amen!  I have a fondness for Ebola simply because it is so spectacularly nasty, but it has killed fewer people in 40 years than flu or rotavirus does in 1.

Seriously: just like charismatic animals like elephants and tigers get all of the headlines when it comes to being endangered, rather than the humble tree frog(s), so do Ebola and Marburg get all of the attention when it comes to reportage on virus epidemics / pandemics.

See on Scoop.itVirology and Bioinformatics from Virology.ca

Ill prepared for an influenza pandemic

18 August, 2014

Over the last 500 years, there have been, on average, three severe influenza pandemics in each century. The most recent pandemic was declared in 2009. Yet despite much investment in public health and many improvements in vaccine production techniques and know-how, the availability of influenza vaccines during this event was far from adequate. Six months into the pandemic, 534 million doses were available, and after one year that number had risen to 1.3 billion — enough for only 8%and 25%, respectively, of the world population. We were lucky that the pandemic declared in 2009 turned out later to be mild and that just one shot of vaccine was sufficient to protect most people. This is not usually the case during a severe influenza pandemic.

 

Source: www.nature.com

"As countries continue to pre-book pandemic supply, it is more and more likely that the limited vaccines available during the first months of any pandemic during the next few years will be sold out almost completely"

And what does everyone think happened in South Africa during most of 2009 and 2010?

Well, they probably don’t – because not that many of them got sick.  But THERE WAS NO VACCINE for the general population until LATE 2010 – when the chances of another round of H1N1pdm 2009 had dissipated due to summer coming on.

And the vaccine that HAD come into the the country in 2010 got used for medical personnel, and – for the 2010 World Cup staff.

Seriously, we need to do better than this – and responding QUICKLY to news of a pandemic would be the ticket.

Using plants B-)

See on Scoop.itVirology News

What Would Happen if You Got Ebola?

13 August, 2014

A secondary infection in the U.S. is highly unlikely. But here’s how the healthcare system would respond if there was one.

Source: www.theatlantic.com

Goes without saying that this would happen in a lot of other places, too.  Including our very own South Africa – where it HAS happened, with Marburg, Ebola and Lujo viruses.  Written about right here on ViroBlogy.

See on Scoop.itVirology News

How can geminiviral Rep capture the cell cycle of differentiated plant cells?

12 August, 2014

African cassava mosaic virus (ACMV) in the geminivirus family has being affected 500 million people worldwide by devastating cassava crops during the past decades. It has caused severe symptoms and reduced yield up to the complete loss of roots, the main starchy food source especially for subsistence farmers in Africa. How can a tiny virus with a small genome evoke such dramatic effects? The viral key component, the replication-initiator protein (Rep), forces differentiated plant cells in the phloem to reactivate DNA synthesis. Even more, it does the same in model cells of fission yeast. We have identified, now, a potential cyclin interaction motif, RXL, in the sequence of ACMV Rep, which may be important for cell cycle control. This motif is essential to induce rereplication in yeast and necessary for viral infection of plants.

 

Source: www.virologyhighlights.com

I am a sucker for geminiviruses and their replication – as can be seen in the pages published here and elsewhere over the years.  It is fascinating to me that a small protein like Rep – only ~30 kDa – can do so many things, and especially interfere in such a fundamental way with organised, differentiated cells.

What is even more interesting is that it can do it in such a wide variety of systems: it’s been shown that ACMV can replicate in maize protoplasts as well as in the dicotyledonous cassava; it can evidently function well in yeast as well – and via a pathway that no-one suspected before now.

Truly, a protein of many parts!  Congratulations to Katharina Hipp and to my old friends Bruno and Holger.

See on Scoop.itVirology News

Plant-made antibodies used as therapy for Ebola in humans: post-exposure prophylaxis goes green!

5 August, 2014
Ebola virus budding from an infected cell.  Courtesy of Russell Kightley Media

Ebola virus budding from an infected cell.
Courtesy of Russell Kightley Media

Yes, I know you fans of ViroBlogy like Ebola – and just coincidentally, I was desperately trying to finish a review* on “Plant-based vaccines against viruses” against a backdrop of an out-of-control Ebola epidemic in West Africa, when three different people emailed me different links to news of use of a plant-made monoclonal antibody cocktail.  I immediately included it in my review – and I am publishing an excerpt here, for informations’ sake.  Enjoy!

Plantibodies against Ebola

The production of anti-Ebola virus antibodies has recently been explored in plants: this could yet become an important part of the arsenal to prevent disease in healthcare workers, given that at the time of writing an uncontrolled Ebola haemorrhagic fever outbreak was still raging in West Africa, and the use of experimental solutions was being suggested (Senthilingam, 2014). For example, use of a high-yielding geminivirus-based transient expression system in N benthamiana that is particularly suited to simultaneous expression of several proteins allowed expression of a MAb (6DB) known to protect animals from Ebola virus infection, at levels of 0.5 g/kg biomass (Chen et al., 2011). The same group also used the same vector system (described in detail here (Rybicki and Martin, 2014)) in lettuce to produce potentially therapeutic MAbs against both Ebola and West Nile viruses (Lai et al., 2012).

A more comprehensive investigation was reported recently, of both plant production of Mabs and post-exposure prophylaxis of Ebola virus infection in rhesus macaques (Olinger et al., 2012). Three Ebola-specific mouse-human chimaeric MAbs (h-13F6, c13C6, and c6D8; the latter two both neutralising) were produced in whole N benthamiana plants via agroinfilration of magnICON TMV-derived viral vectors. A mixture of the three MAbs – called MB-003 – given as a single dose of 16.7 mg/kg per Mab 1 hour post-infection followed by doses on days 4 and 8, protected 3 of 3 macaques from lethal challenge with 1 000 pfu of Ebola virus. The researchers subsequently showed significant protection with MB-003 treatment given 24 or 48 hours post-infection, with four of six monkeys testing surviving, compared to none in two controls. All surviving animals treated with MB-003 experienced insignificant if any viraemia, and negligible clinical symptoms compared to the control animals. A significant finding was that the plant-produced MAbs were three times as potent as the CHO cell-produced equivalents – a clear case of plant production leading to “biobetters”. A follow-up of this work investigated efficacy of treatment with MB-003 after confirmation of infection in rhesus macaques, “according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization” (Pettitt et al., 2013). In this experiment 43% of treated animals survived, whereas all controls tested here and previously with the same challenge protocol died from the infection.

In news from just prior to submission of this article, a report quoted as coming from the National Institute of Allergy and Infectious Diseases states that two US healthcare workers who contracted Ebola in Liberia were treated with a cocktail of anti-Ebola Mabs called ZMapp – described as a successor to MB-003 – developed by Mapp Pharmaceutical of San Diego, and manufactured by Kentucky BioProcessing (Langreth et al., 2014). Despite being given up to nine days post-infection in one case, it appears to have been effective (Wilson and Dellorto, 2014).

A novel application of the same technology was also used to produce an Ebola immune complex (EIC) in N benthamiana, consisting of the Ebola envelope glycoprotein GP1 fused to the C-terminus of the heavy chain of the humanised 6D8 MAb, which binds a linear epitope on GP1. Geminivirus vector-mediated co-expression of the GP1-HC fusion and the 6D8 light chain produced assembled immunoglobulin, which was purified by protein G affinity chromatography. The resultant molecules bound the complement factor C1q, indicating immune complex formation. Subcutaneous immunisation of mice with purified EIC elicited high level anti-GP1 antibody production, comparable to use of GP1 VLPs (Phoolcharoen et al., 2011). This is the first published account of an Ebola virus candidate vaccine to be produced in plants.

References

Chen, Q., He, J., Phoolcharoen, W., Mason, H.S., 2011. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Human vaccines 7, 331-338.

Lai, H., He, J., Engle, M., Diamond, M.S., Chen, Q., 2012. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant biotechnology journal 10, 95-104.

Langreth, R., Chen, C., Nash, J., Lauerman, J., 2014. Ebola Drug Made From Tobacco Plant Saves U.S. Aid Workers. Bloomberg.com.

Olinger, G.G., Jr., Pettitt, J., Kim, D., Working, C., Bohorov, O., Bratcher, B., Hiatt, E., Hume, S.D., Johnson, A.K., Morton, J., Pauly, M., Whaley, K.J., Lear, C.M., Biggins, J.E., Scully, C., Hensley, L., Zeitlin, L., 2012. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proceedings of the National Academy of Sciences of the United States of America 109, 18030-18035.

Pettitt, J., Zeitlin, L., Kim do, H., Working, C., Johnson, J.C., Bohorov, O., Bratcher, B., Hiatt, E., Hume, S.D., Johnson, A.K., Morton, J., Pauly, M.H., Whaley, K.J., Ingram, M.F., Zovanyi, A., Heinrich, M., Piper, A., Zelko, J., Olinger, G.G., 2013. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Science translational medicine 5, 199ra113.

Phoolcharoen, W., Bhoo, S.H., Lai, H., Ma, J., Arntzen, C.J., Chen, Q., Mason, H.S., 2011. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant biotechnology journal 9, 807-816.

Rybicki, E.P., Martin, D.P., 2014. Virus-Derived ssDNA Vectors for the Expression of Foreign Proteins in Plants. Current topics in microbiology and immunology 375, 19-45.

Senthilingam, M., 2014. Ebola outbreak: Is it time to test experimental vaccines? CNN.

Wilson, J., Dellorto, D., 2014. 9 questions about this new Ebola drug. CNN.

* = which, despite their having commissioned from me, the good folk at “Viruses” an unnamed journal decided “…may not have substantial differences with the reviews you published recently” – and rejected.  I shall have revenge.  Oh, yes…B-)

A new virus from the Namib – and a guilty secret revealed

26 June, 2014

I have to confess to a guilty secret: there is a pleasure-inducing activity I have been indulging in for a week at a time these past three years.

And not alone….

This consists of going to the Gobabeb Research & Training Centre in the Namib Desert as part of an international “scientific expedition” aimed at investigating microbial soil biodiversity in the sandy and stony desert round Gobabeb.  These were started by Professor Don Cowan when he was at the University of the Western Cape, and have fortunately continued now that he has moved to a new Institute at the University of Pretoria.

Typical quartz-associated hypolith

Typical quartz-associated hypolith

I put the scientific expedition in quotes because anything that much fun shouldn’t be called scientific, but hey, it’s already resulted in one major paper on hypolith-associated viruses that I’m a minor author on, another co-authored opinion piece in the South African Journal of Science on biodiversity assessment that got the cover, as well as me being invited to be part of the Gobabeb station’s Microbiology & Fungi research “Theme Group“.

Moreover, I am now on the Board of the Institute for Microbial Biotechnology and Metagenomics at UWC on the strength of working with Marla Trindade and Lonnie van Zyl and others on scraping bits of green stuff off rocks and then watching them ultrafilter washings of it – so I suppose that we really did do some science, even if it was sinfully enjoyable. In any case, something that happened last year took me back to my roots – as well as possibly getting me some street (or gully) cred with the biodiversity crowd.

The heavily gullied area near Homeb

The heavily gullied area near Homeb

Basically, there we were in the Welwitschia-rich gullies near Homeb, 11 km from Gobabeb, visiting said plants.  There had been some rain 3 weeks previously, apparently, and there was an amazing eruption of foliage from some kind of bulb, every plant the same age and every one frantically flowering for all they were worth.  This alone was noteworthy, as the gullies were completely devoid of any trace of such plants the previous year. As we were wandering about, looking at Welwitschias, one Olivier Zablocki from the Univ Pretoria team – who had just done a MSc in plant virology with Gerhard Pietersen at UP – said something along the lines of “I wonder if there are any viruses infecting these plants?”.

welwitschia 2012

Welwitschia down in the gully

“What, like that one?” I said, having just fortuitously noticed a plant with tell-tale streaks on its leaves.  Of course, I seem to have lost my photos – temporarily, I hope! – after a Mac Mini OSX update disaster, but Olivier was kind enough to provide the necessary:

diseased albuca

Streaky Albuca. Note how quickly the fruit has formed, just a couple of days after flowering.

This sparked a flurry of activity, with people being called to observe the plant, and going out and looking for more.

Which were not found: not one other plant, of the hundreds we saw there and nearer Homeb, had any streaks at all.  What is more, they were growing all up and down some of the more inhospitable gravelly and rocky slopes I have ever seen, meaning they had to be seriously drought-tolerant, given the unlikelihood of them ever being exposed to much water.  This meant they must be ephemeral, or putting out foliage and flowers only after rain – and I have never seen anything flower as fast; three days later they were already fruiting.

albuca 2013

Albuca growing in the gully

We made the collective decision that this was sufficiently scientifically interesting to warrant its collection, and the plant was carefully dug up – with difficulty; the onion-like bulb was deep and seriously embedded among rocks – and carefully transported back to Gobabeb, hopefully for identification and then packaging to be taken back to Pretoria.

healthy albuca

Healthy Albuca growing in gravel

And yes, we did have a permit!

Meaning the foliage could go back to Pretoria, and there be subjected by Olivier to electron microscopy, and then RNA isolation and cDNA synthesis.  And lo, it came to pass – that a new potyvirus was discovered.  Kudos, Olivier and the Cowan lab!  The ms is submitted, and we wait only for…well, acceptance would be nice, but the proof is in the sequence.  And the pictures – murky EMs done by Olivier from precious tissue extracts, to boot.

Transmission EM from diseased Albuca extract

Transmission EM from diseased Albuca extract

And it took an old plant virologist to find it.  Life in the greying dog yet!

…and now it’s published – as a Disease Note in Plant Disease.  Thanks Evelien!


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