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Influenza virus A H1N1 2009: gets to parts the other flu doesn’t reach

14 September, 2009

Flu virus life cycle. Copyright Russell Kightley Media

The September 2009 issue of Nature Biotechnology has a letter concerning the receptor specificity of AH1N1 2009 pandemic influenza virus – which accounts pretty well for why it CAN be pretty nasty, and for why it may get nastier yet.

Childs et al., in a letter entitled “Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray“, describe what amounts to a tour de force analysis of the receptor binding of a number of influenza viruses, which concludes with the statement that:

“The differences in receptor binding between the 2009 pandemic and seasonal H1N1 viruses may therefore account, at least in part, for the higher virus replication and greater pathology reported in the lungs of ferrets, mice and nonhuman primates infected with pandemic viruses, than observed with contemporary seasonal viruses.”

Which would help explain why some otherwise healthy young people are dying of the virus, while others are getting only mildly ill.  But we get ahead of ourselves: in January last year I wrote in MicrobiologyBytes about recpetor specificities of A-type influenza viruses, in the context of how H5N1 was less likely to mutate to easy human-to-human transmissibility than had origianlly been thought.

I wrote at the time:

According to a letter in the January 2008 issue of Nature Biotechnology, it is a characteristic structural topology, and not just the α2,6 linkage, that enables specific binding of HA to α2,6 sialylated glycans. The authors state:

…recognition of this topology may be critical for adaptation of HA to bind glycans in the upper respiratory tract of humans. An integrated biochemical, analytical and data mining approach demonstrates that HAs from the human-adapted H1N1 and H3N2 viruses, but not H5N1 (bird flu) viruses, specifically bind to long α2-6 sialylated glycans with this topology. This could explain why H5N1 viruses have not yet gained a foothold in the human population.

Apparently the critical shape in humans is umbrella-like, whereas the avian receptor is characteristically cone-like. Again from the paper:

The topology of α2-3 and α2-6 is governed by the glycosidic torsion angles of the trisaccharide motifs-Neu5Aca2-3Galb1-3/4GlcNAc and Neu5Aca2-6Galb1-4GlcNAc, respectively (Supplementary Fig. 3 online).

Ram Sasisekharan and colleagues showed that human-adapted viruses with mixed α2,3/α2,6 binding ability that bound the umbrella-type receptor were efficiently transmitted, whereas viruses with the same basic specificity that did not have HA binding specificity to “long” α2,6, were not.

The present paper reports the following investigation:

“We have compared directly, by carbohydrate microarray analysis, the receptor-binding characteristics of two isolates of the novel pandemic H1N1 virus, Cal/09 and A/Hamburg/5/2009 (Ham/09), with those of a seasonal human H1N1 virus, A/Memphis/14/96-M (Mem/96), as representative of a virus well adapted to humans [and a reassortant human H3N2 virus A/Aichi/2/68 x PR8 (X31)]. As the HA of the novel H1N1 pandemic virus originated from a virus similar to triple reassortant swine H1N1 viruses, we compared one such example, A/Iowa/1/2006 (Iowa/06), isolated from a human infection, and an older close relative of classical swine H1N1 viruses, A/New Jersey/76 (NJ/76), the human isolate that initiated the concern of a pandemic threat in 1976.”

This is a really comprehensive analysis – for such a short communication – which throws up a number of interesting points.  First, I was not aware it was possible to do “carbohydrate microarrays”!  Second, the paper shows quite conclusively that the swine-derived AH1N1 viruses have a significantly wider range of receptor specificities than a standard seasonal AH1N1 virus, and – but to a lesser extent – than the reassortant H3N2 virus X31.

Carbohydrate microarray analyses of the six viruses investigated.
From the following article (with permission from NBT):
Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray.
Robert A Childs, Angelina S Palma, Steve Wharton, Tatyana Matrosovich, Yan Liu, Wengang Chai, Maria A Campanero-Rhodes, Yibing Zhang, Markus Eickmann, Makoto Kiso, Alan Hay, Mikhail Matrosovich & Ten Feizi.
Nature Biotechnology 27, 797 – 799 (2009).
doi:10.1038/nbt0909-797

flu_receptor

Legend:
Numerical scores for the binding signals are shown as means of duplicate spots at 5 fmol per spot (with error bars). The microarrays consisted of eighty sialylated and six neutral lipid-linked oligosaccharide probes, printed on nitrocellulose-coated glass slides. These are listed in Supplementary Table 1 and arranged according to sialic acid linkage, oligosaccharide backbone chain length and sequence. The various types of terminal sialic acid linkage are indicated by the colored panels as defined at the bottom of the figure.

And what does all this mean, exactly?  The authors sum it up well:

These results indicate that no major change in receptor-binding specificity of the HA was required for the emergent pandemic virus to acquire human-like characteristics and become established in the human population. …

The broader specificity, namely, the ability to bind to 2-3- in addition to 2-6-linked receptors is also pertinent to the greater virulence of the pandemic virus than seasonal influenza viruses observed in animal models, and its capacity to cause severe and fatal disease in humans, despite the generally mild nature of most infections. Binding to 2-3-linked receptors is thought to be associated with the ability of influenza viruses to infect the lower respiratory tract where there is a greater proportion of 2-3- relative to 2-6-linked sialyl glycans, although long chain 2-3-linked sialyl (poly-N-acetyllactosamine) sequences are present in ciliated bronchial epithelial cells in humans where they are the receptors for another human pathogen, Mycoplasma pneumoniae.

So there you have it: the viruses can get deeper in to your lungs than the standard flu – which, if it happens, can make you seriously ill.

So what happens if it gets better at binding the 2,3-type receptors in humans?  Well, we’re only in the middle of the pandemic.  We may yet find out the hard way.

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Posted in Evolution, General Virology, Influenza viruses, Viruses | 4 Comments »

Gene discrimination

3 September, 2009

In the latest online issue of Nature, there is an article entitled “Keeping genes out of terrorists’ hands“, by Erika Check Hayden.  Like an article a little while ago in Nature Biotechnology, it makes the apparently quite reasonable point that

“the way that the industry screens orders for hazardous toxins and genes, such as pieces of deadly viruses and bacteria…could be crucial for global biosecurity”.

Yes.  Well.  They would say that, wouldn’t they??  “They” being anyone in the developed world who has a paranoid fantasy about bearded extremists in caves (or crew-cut extremists in leafy suburbs) gleefully unwrapping their couriered DNA and brewing up a nice little necrotising poxvirus, or an airborne Ebola, or possibly an H5N1 variant that spreads human-to-human better than the present versions.

I wrote the following reply to the article:

While “all right-thinking people” – for which, read “those easily scared by the unrealistic prospect of mail-order killer bugs” may agree that some kind of limitations are required on what synthetic DNA is sent out, and to whom…there is a baby being thrown out with the bathwater here.

My laboratory has just, despite many previously successful orders from the same company, been denied permission (or told to obtain clearance from the relevant government, which amounts to the same thing) to have a coat protein gene synthesised for a bluetongue virus (BTV) strain now found all over western Europe. Because, apparently, BTV is on the “Australia Group”‘s prohibited list of biological agents – and South Africa is not a signatory to this group, which started out for arms control but has apparently ramified somewhat.

This is so ridiculous as to beggar belief: the viruses are endemic to Africa; the world’s expert on cDNA cloning of their genomes is in South Africa; why would anyone want to build a BTV from synthetic DNA when they could go out and sample a sheep for some REAL virus??

A closer look at the list throws up all sorts of interesting things. It is prohibited, for example, to order genes for H5N1 influenza – although curiously, not pandemic H1N1 – and dengue viruses. This rather puts a spoke in the wheels of anyone who might want to…oh, let’s say…MAKE A VACCINE to those agents, in any country not signatory to the agreement – where the viruses happen to be endemic!!

The ways of limiting spread of genes that are being proposed are first, unnecessary; second – discriminatory in the extreme.

And may just provide a good deal of business for firms operating in developing countries who otherwise would have been ignored because of quality issues. Imagine that: a lab in Pakistan, or South Africa, or Indonesia, using home-made genes to make a vaccine.

Because that is a LOT more likely than using them to make a pathogen.

I know of a passage written some years ago in a reputable science magazine which described how easy it would be to smuggle naturally-occurring foot and mouth disease virus worldwide – with no science involved whatever.  I have enough purified material of a particular plant virus in my cold room right now to kill all the wheat grown in my country – given some carborundum and a crop sprayer.

There are enough people on this planet infected with pandemic H1N1 who live in close enough proximity to birds infected with H5N1 to make coinfection of one or the other with both a certainty – the only uncertainty remaining being what will come of it.  For that matter, where DID the H1N1 come from?  Where did Lujo virus come from?

We DON’T NEED TO MAKE VIRUSES from mail-order DNA – and only Craig Venter et al. could even dream of making whole microbes.  There are more than enough nasty agents out there that are relatively easy to obtain, and do simple kitchen-based microbiology with, to keep entire cave complexes and Montana libertarian enclaves busy for years, without resorting to complicated molecular biology.

So DO let’s keep things in perspective, shall we??  And let reputable labs doing reputable work order the materials they need to work with.

Posted in General, Vaccines: General, Viruses | 4 Comments »

H1N1 – view on a pandemic

26 August, 2009

Well, “The Big One” that we have all been waiting for since 1968 – or 1977, if you count the accidental re-release of the original humanised H1N1 as a pandemic – is well and truly here.  A nice little animated graphic for depicting how it arose, while a bit simplistic, is available here.

And what have we learned?  Has civilisation fallen; have populations been decimated?

Well, quite a lot; no, and no, in answer.  Explanations for some of this are contained in a survey just released.  Here from the News24 report published today, sourced from SAPA:

Paris – More than half the fatalities from H1N1 swine flu have been among young adults, according to one of the first surveys to gather mortality data from across the globe for the new A(H1N1) virus.

The analysis of 574 pandemic deaths from 28 countries through mid-July, released this week, also found that being diabetic or obese significantly boosted the risk of dying.

Neither children nor the elderly are as vulnerable as initial reports indicated, found the study, published by Eurosurveillance, the monitoring arm of the European Centre for Disease Prevention and Control.

“Most deaths (51%) occurred in the age group of 20-to-49 year-olds, but there is considerable variation depending on country or continent,” the researchers reported.

Only 12% of those who died were 60 or older.

All of these features – high mortality among young adults and the obese, but not the very young or elderly – are sharply different than for the seasonal flu.

More than 90% of deaths from seasonal flu – which claims 250 000 to 500 000 lives annually according to the WHO – are in people over 65.

By contrast, with the pandemic H1N1, “the elderly seem to be protected from infection to some extent, perhaps due to previous exposure to similar strains”, the study conjectured.

Persons born before 1957, other studies have suggested, were almost certainly exposed to the milder seasonal A(H1N1) viruses that evolved from the terrible pandemic of 1918, which left some 40 million dead.

With the 2009 strain, “when infection does occur, however, the percentage of deaths in elderly cases seems to be higher that in others”.

One common target across both pandemic and season strains is pregnant women, according to the study, led by Philippe Barboza of the French Institute for Public Health Surveillance….

And that’s the sinister part…here in South Africa, of 18 fatalities known to have been associated with pandemic AH1N1 infection, NINE were pregnant women, mostly in the third trimester of pregnancy.  In a report published yesterday, SA’s Minister of Health Dr Aaron Motsoaledi said the following:

“We find it very worrying that there is an increasing number of pregnant women who are succumbing to this pandemic,” Motsoaledi said.

“The directive to all health care workers… is to put pregnant women with flu-like symptoms (even if they are mild) on Tamiflu treatment.

“Doctors should not wait for any tests before such treatment is administered.”

Further on in the same report:

On Monday, the National Institute for Communicable Diseases [NICD] also said pregnancy had been identified as a particular risk factor for severe H1N1 flu.

It said that in the second and especially the third trimester, urgent treatment with antiviral drugs should be considered even before any laboratory results were received.

The institute added however that most H1N1 flu cases in South Africa remained mild and “self-limiting”.

Routine H1N1 testing for everyone with flu-like illness was still not recommended.

Nationwide, there had been 5 118 laboratory-confirmed cases of H1N1 flu, it said.

The figure is essentially meaningless, given that most suspected flu cases are not laboratory-diagnosed (it costs R700, or ~US$70, for a single test) and the pandemic flu is pretty much indistinguishable from seasonal, and may in fact have supplanted the normal flu.  It certainly has in Australia and Argentina, which remain the two worst-hit southern hemisphere countries, and probably has in South Africa too: the CDC has a very useful map illustrating this, accessible here.

International news, via the CDC site, is the following:

As of August 13, the World Health Organization (WHO) regions have reported over 182,166 laboratory-confirmed cases of 2009 H1N1 influenza virus (2009 H1N1) with 1,799 deaths. The laboratory-confirmed cases represent an underestimation of total cases in the world as many countries now focus surveillance and laboratory testing only in persons with severe illness. The 2009 H1N1 influenza virus continues to be the dominant influenza virus in circulation in the world.

One very important piece of information further down this report is the following:

There have been no significant changes detected in the 2009 H1N1 influenza virus isolated from persons in the Southern Hemisphere as compared to viruses isolated from persons in the Northern Hemisphere.

This is important because the frantic rush to make vaccines to combat the expected northern hemisphere upsurge in infections in their autumn season – October or so – depends upon the virus not having changed much from the seed material which was derived from virus isolated earlier this year.  This could negate some of theh fears that the much-anticipated “second wave” of virus infections could be a lot worse than the first.

Good news on the vaccine front – for Australians at least – is that an Australian company, CSL Ltd, has the world’s first data from human trials of a pandemic strain vaccine, and looks set to be able to provide Australia with 21 million doses of vaccine – and 2 million doses of the vaccine at the end of the month.

Other vaccine news is also fairly encouraging, notwithstanding a rather alarming report in New Scientist recently about the new strain growing only half as well in eggs as seasonal flu types: while this remains a worry, newer, faster-growing variants have been derived and distributed – though possibly not in time for a northern hemisphere autumn roll-out.

Mind you, all of this production relies on the well-proven-but-seriously-archaic 1930s technology of growing live virus in hen’s eggs: we are still trapped, in the 21st century, into having to use early 20th century methods to produce vaccines for fast-adapting pathogens.  Things ARE changing: various pharma companies are diversifying into mammalian and insect cell culture; people (including us!) are investigating making recombinant subunit vaccines in plants (see here) – and there is at least the tantalising possibility that “universal vaccines” may become available in the not-too-distant future.  These will exploit all or part of the highly conserved M2 “ion channel” protein of influenza viruses as recombinant subunit vaccines.

However, all of this is at least six months in the future for conventional vaccines, and many years hence for newer offerings.  Meantime – there is disturbing news concerning trans-species transmissions of pandemic AH1N1 viruses.

ProMED Mail (ProMED Digest V2009 #394) reports that “Chile finds H1N1 swine flu in turkeys“:

Chilean health authorities announced on Thursday night [20 Aug 2009] that they had detected and controlled an outbreak of swine flu in 2 turkey farms, according to a communication from the Agricultural and Livestock Service (SAG).

“The presence of an influenza type A virus was detected in 2 farms in the Valparaiso Region, and immediate precautionary measures were adopted to prevent the dissemination of the disease and to protect the population’s health,” said the text.

And again from ProMED on 20th August, quoting The Straits Times and AFP:

A 2nd Australian piggery was placed in quarantine due to swine flu on Wednesday [19 Aug 2009] as the number of human deaths from the virus reached 121.

Authorities ordered a biosecurity lockdown at the piggery in Victoria state amid concerns the virus could mutate and return to humans in a more deadly form.

Another piggery in New South Wales state has been quarantined since late July [2009], although the state government said most of the animals had recovered from the disease.

Victoria Agriculture Minister Joe Helper said tests confirmed the presence of influenza at the piggery after its owners reported earlier this week that the animals were not eating.

‘It is important to stress that this is not a human health issue and that national and international food authorities continue to advise that pork and pork products are safe to eat,’ he said.

Media reports said the pigs were believed to have contracted the virus from workers at the property who were suffering the human form of the disease.

Health experts fear swine flu in humans, which is easily spread but has a relatively low fatality rate, could mutate in other animals and emerge in a more virulent form. [my emphasis]

So: two independent incidents, on different continents, of pandemic AH1N1 viruses getting into different species of farmed livestock – and luckily controlled.

What would have happened if domestic fowl and/or pigs had been infected in places like Vietnam, Thailand, Indonesia, Turkey and Egypt – where highly pathogenic avian H5N1 influenza viruses appear to be endemic, and not well controlled?  Given the complex origins of the current pandemic virus – from several swine, avian and human viruses – it could be a recipe for disaster, on a scale even greater than the 1918 pandemic.

The REAL Big One.  Let’s all help get a vaccine, people!!

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Posted in Evolution, Influenza viruses, Uncategorized, Vaccines: General, Viruses | 2 Comments »

First African-developed HIV vaccine goes to trial

21 July, 2009

Finally, finally, a product of our 10-odd-year-old South African HIV vaccine development programme goes into Phase I human trial, in South Africa!

I say “our” because I was part of the overall team; however, the two vaccines which comprise the SA AIDS Vaccine Initiative (SAAVI) / HIV Vaccine Trials Network (HVTN) trial – designated SAAVI 102/HVTN 073 – were designed and developed by others.

The vaccines consist of a DNA component, consisting of an artificial “polygene” dubbed Grttn (for Gag-RT-Tat-Nef) and a truncated Env (gp150) cloned separately into vector plasmids, and the same genes recombined under the control of different promoters into the genome of a poxvirus (Modified Vaccinia Ankara, MVA).  These and their testing in mice and non-human primates have been described in published work: see here and here for relevant journal articles on the MVA and DNA components.

From the July 20th SAAVI press release:

The test vaccines – called SAAVI MVA-C and SAAVI DNA-C2 – have shown promising results in animal testing. The SAAVI DNA-C2 vaccine was constructed in South Africa using a plasmid backbone provided by the Dale and Betty Bumpers Vaccine Research Center (VRC) of NIAID, while the MVA vaccine was designed by the team at UCT and constructed and manufactured in the USA.

“Reaching this important milestone of translating our discoveries in the laboratory to testing in humans would not have been possible without the support of a large team of people from the University of Cape Town, together with national and international collaborations.  An effective vaccine against HIV/AIDS remains a top global health priority and it is our hope that the evaluation of these vaccines in clinical trial will provide some important answers that will bring us closer towards this goal,” says Prof. Anna-Lise Williamson, leader of the vaccine development team and joint staff member of UCT’s Institute for Infectious Disease and Molecular Medicine, and the National Health Laboratory Services (NHLS).

The SAAVI DNA-C2 was constructed in South Africa and manufactured in the US by Althea Technologies. The MVA vaccine was manufactured by Therion Biologics, USA. The vaccines will be tested in a prime-boost approach where the SAAVI DNA-C2 vaccine will be given to prime the immune response and the SAAVI MVA-C vaccine to boost or enhance the immune response.

National and international press got hold of the story in a big way – unsurprisingly, given as there is the 5th IAS Conference on AIDS Pathogenesis going in in Cape Town at the same time, which incidentally has its own live blog feed.

The University of Cape Town is obviously pleased with the press release (see here); however, the launch had its fair share of controversy: Associated Press reporter Michelle Faul posted a story yesterday entitled “South Africa begins AIDS vaccine trial, cuts funds“, which has been taken up by a wide spectrum of especially foreign media.  According to Faul:

“South Africa launched a high-profile trial of an AIDS vaccine created by its own researchers Monday, a proud moment in a nation where government denial, neglect and unscientific responses have helped fuel the world’s worst AIDS crisis.

After a government official lauded the project at a ceremony at Cape Town’s Crossroads shantytown, the scientist leading the research said state funding had been halted.

The contrast between Monday’s hopeful vaccine launch and the revelation of funding cuts raised questions about whether the government was backsliding on its pledge to combat AIDS.

Anna-Lise Williamson, an AIDS researcher at the University of Cape Town, told The Associated Press the clinical trial would continue with U.S. money. But she said South Africa’s Department of Science and Technology had pulled its funding in March, while the project’s other sponsor, the state electricity utility Eskom, did not renew its contract when it expired last year.

Neither government spokesmen nor Eskom immediately returned calls seeking comment about funding cuts.”

In the midst of light, there is darkness…frequently, thanks to ESKOM! 

I have blogged on my personal view elsewhere; suffice it to say that bad decisions were made, and  9 years worth of momentum has effectively been lost – along with a number of very experienced personnel, and many years worth of accumulated and very relevant experience.

For an illustration of the product pipeline which existed behind the current trial offerings – and which may now never be developed – click here  for published descriptions of our plasmid- and BCG-vectored and virus-like particle (VLP) subunit vaccines.

But who knows, this current trial may even show promise – and then it will all have been worth it.  Let’s live in hope!

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Posted in HIV, Vaccines: General, Viruses | 3 Comments »

Where the new H1N1 came from

29 June, 2009

Mixing of flu viruses to produce a new strain. copyright Russell Kightley Media

In Nature 459, 1122-1125 (25 June 2009): Smith et al. on “Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic“.

In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States1. During the first few weeks of surveillance, the virus spread worldwide to 30 countries (as of May 11) by human-to-human transmission, causing the World Health Organization to raise its pandemic alert to level 5 of 6. This virus has the potential to develop into the first influenza pandemic of the twenty-first century. [I thought a pandemic alert level of 6 meant it had already?? – Ed] Here we use evolutionary analysis to estimate the timescale of the origins and the early development of the S-OIV epidemic. We show that it was derived from several viruses circulating in swine, and that the initial transmission to humans occurred several months before recognition of the outbreak. A phylogenetic estimate of the gaps in genetic surveillance indicates a long period of unsampled ancestry before the S-OIV outbreak, suggesting that the reassortment of swine lineages may have occurred years before emergence in humans, and that the multiple genetic ancestry of S-OIV is not indicative of an artificial origin. Furthermore, the unsampled history of the epidemic means that the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic, despite the fact that we included a panel of closely related and previously unpublished swine influenza isolates. Our results highlight the need for systematic surveillance of influenza in swine, and provide evidence that the mixing of new genetic elements in swine can result in the emergence of viruses with pandemic potential in humans.
[my bolded sections – Ed]

An important paper for a number of reasons – not the least of which is pigs have

Depiction of virus mixing in a pig

been pushed to the fore as a potential source of new and dangerous human flu viruses.

Through no fault of their own, I might add: the only pigs proven to have had the new virus were probably infected by a handler who had been to Mexico!

The most important observation to emerge from this is that pigs should be surveilled systematically and worldwide – to stop yet another possible avenue for zoonotic infection for us vulnerable humans.

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Posted in Evolution, Influenza viruses, Vaccines: General, Viruses | 3 Comments »

Down, Lujo!

4 June, 2009

I am indebted to Ms Ngimezi Phiri – muli bwanji! – in the MCB2016F class, to whom I have just lectured Virology, for pointing this out – from Yahoo! News:

“Scientists identify new lethal virus in Africa

By MIKE STOBBE, AP Medical Writer  – Thu May 28, 9:15 pm ET

ATLANTA – Scientists have identified a lethal new virus in Africa that causes bleeding like the dreaded Ebola virus. The so-called “Lujo” virus infected five people in Zambia and South Africa last fall. Four of them died, but a fifth survived, perhaps helped by a medicine recommended by the scientists.

It’s not clear how the first person became infected, but the bug comes from a family of viruses found in rodents, said Dr. Ian Lipkin, a Columbia University epidemiologist involved in the discovery.

“This one is really, really aggressive” he said of the virus.

A paper on the virus by Lipkin and his collaborators was published online Thursday on in PLoS Pathogens.”

Of course, regular readers of this blog will be familiar with the Zambian arenavirus discovered under tragic circumstances last October, and covered here as follows:

Now the agent has a name – albeit an unfortunate victim of political compromise; they are calling it “Lujo” after Lusaka (where it came from, sort of) and Johannesburg, where nearly everyone who got it was treated.

The (relatively) rapid characterisation of the virus owes a lot to modern technology: in the words of the authors (MCB3019F take note – this is serious viromics…):

“RNA extracts from two post-mortem liver biopsies (cases 2 and 3) and one serum sample (case 2) were independently submitted for unbiased high-throughput pyrosequencing. The libraries yielded between 87,500 and 106,500 sequence reads. Alignment of unique singleton and assembled contiguous sequences to the GenBank database … using the Basic Local Alignment Search Tool (blastn and blastx; …) indicated coverage of approximately 5.6 kilobases (kb) of sequence distributed along arenavirus genome scaffolds: 2 kb of S segment sequence in two fragments, and 3.6 kb of L segment sequence in 7 fragments (Figure 2) [see here for depiction of arenavirus genome]. The majority of arenavirus sequences were obtained from serum rather than tissue, potentially reflecting lower levels of competing cellular RNA in random amplification reactions.”

Sequence data was used to allow primer synthesis for cDNA PCR to fill in gaps, and the whole genome is now available.  It is a novel arenavirus, with all genome segments giving the same sort of phylogenetic tree topology, which shows the virus to be near the root of Old World arenaviruses.

The authors conclude:

“To our knowledge is LUJV the first hemorrhagic fever-associated arenavirus from Africa identified in the past 3 decades. It is also the first such virus originating south of the equator (Figure 1). The International Committee on the Taxonomy of Viruses (ICTV) defines species within the Arenavirus genus based on association with a specific host, geographic distribution, potential to cause human disease, antigenic cross reactivity, and protein sequence similarity to other species. By these criteria, given the novelty of its presence in southern Africa, capacity to cause hemorrhagic fever, and its genetic distinction, LUJV appears to be a new species.”

Ex Africa, semper aliquid novi(rus)…B-)

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Posted in Evolution, General Virology, Viruses | 2 Comments »

Mimivirus unveiled

22 May, 2009

Alan Cann blogged on May 13th 2008 on mimivirus structure, in “Mimivirus and the Stargate“, following publication of a PLoS Biology paper on “Distinct DNA Exit and Packaging Portals in the Virus Acanthamoeba polyphaga mimivirus“ by Abraham Minsky’s group at the Weizman Institute in Israel.  This paper has some stunning EM images and cryoEM reconstructions, which prompt their summary statement:

“…we identified a large tunnel in the Mimivirus capsid that is formed shortly after infection, following a large-scale opening of the capsid [which they term the “stargate”]. The tunnel allows the whole viral genome to exit in a rapid, one-step process. DNA encapsidation is mediated by a transient aperture in the capsid that, we suggest, may promote concomitant entry of multiple segments of the viral DNA molecule.”

Given that PLoS Biology has an Open Access policy which  “…allow[s] anyone to download, reuse, reprint, modify, distribute, and/or copy articles in PLoS journals, so long as the original authors and source are cited”,  I HAVE to share these pictures with you.

 

Mimivirus Stargate

Mimivirus Stargate

(A) TEM image of cryo-fixed sectioned and stained extracellular Mimivirus particles revealing a star-shaped structure at a unique vertex.
(B) Cryo-TEM image of a whole vitrified fiber-less Mimivirus.
(C) SEM image of the star-shaped structure in a mature extracellular Mimivirus particle.
(D) Cryo-SEM of an immature, fiber-less particle.
(E) Tomographic slice of a mature intracellular Mimivirus particle captured at a late (12 h post infection) infection stage.
(F and G) Volume reconstruction of the particle shown in (E), revealing the presence of an outer (red) and inner (orange) capsid shells. The star-shaped structure is present in both shells but adopts partially open (dark, star-like region), and completely sealed configurations in the outer and inner shells, respectively.
(H) Superposition of the two shells in (F) and (G).
Scale bars, 100 nm in (A, B, D, and E), and 200 nm in (C).

Schematic Representation of a Mimivirus Particle at Its Final Uncoating Stage

Zauberman N, Mutsafi Y, Ben Halevy D, Shimoni E, Klein E, et al. (2008) Distinct DNA exit and packaging portals in the virus PLoS Biol 6(5): e114.   doi:10.1371/journal.pbio.0060114

 

Schematic Representation of a Mimivirus Particle at Its Final Uncoating Stage

Schematic Representation of a Mimivirus Particle at Its Final Uncoating Stage

 

The capsid (red) is opened at the stargate, allowing for fusion of the viral and phagosome membranes (light and dark blue, respectively), thus forming a star-shaped membrane conduit.

Zauberman N, Mutsafi Y, Ben Halevy D, Shimoni E, Klein E, et al. (2008) Distinct DNA exit and packaging portals in the virus PLoS Biol 6(5): e114.   doi:10.1371/journal.pbio.0060114

 

 

 I commented at the time of Alan’s blog that:

“It is becoming apparent to me – especially now as I do a 10-year revision of my Web teaching material – that there is a hitherto unsuspected level of complexity in the way big viruses get their genomes into cells – and back out into virions. Phycodnaviruses may emulate phages in dissolving their way through cell walls AND injecting DNA; now mimiviruses have special mechanisms for both loading virions and getting their DNA out.

Watch this space: a major growth area in structural biology and virology.”

And, of course, it has come to pass: Michael Rossmann’s group at Purdue University and their collaborators have just published a paper entitled “Structural Studies of the Giant Mimivirus” , also in PLoS Biology, in which they explore in greater detail aspects of the structure, particularly as this is related to getting DNA out of the particles.

Their paper has the most stunning images and reconstructions, including images which show that the “starfish” shaped portal seems to be detachable – and that the unique stargate-associated 5-fold rotational axis of symmetry also has associated with it a depression in the inner nucleocapsid, which is undoubtedly associated with delivery of the DNA within.

Stargate

(A–C) Surface-shaded rendering of cryoEM reconstruction of untreated Mimivirus. (A) Looking down the starfish-shaped feature associated vertex, (B) looking from one side, and (C) looking from the opposite side of the “starfish”-associated vertex.

(D) The “starfish”-associated vertex was removed to show the internal nucleocapsid with its concave surface facing the special vertex.

(E) Central slice of the reconstruction looking from the side of the particle showing the concave face of the nucleocapsid and the low density space beneath the “starfish”-associated vertex. A perfectly icosahedral particle is outlined in gray to show the extension of the unique vertex.

(F) Central slice of the reconstruction looking along the 5-fold axis from the starfish-shaped feature showing the enveloped nucleocapsid surrounded by a lower density space. The coloring is based on radial distance from the center of the virus. Gray is from 0 to 1,800 Å, red from 1,800 to 2,100 Å, and rainbow coloring from red to blue between 2,100 and 2,500 Å.

The scale bars in all panels represent 1,000 Å.

doi:10.1371/journal.pbio.1000092.g005

This latest paper makes summary comments as follows:

“The enveloped genome within the larger viral capsid, perhaps supported by fibers …, has some similarity to eukaryotic cells. In contrast, the external peptidoglycan component mimics bacterial cell walls …. In addition, the existence of a unique vertex in Mimivirus, possibly for genome delivery …, is reminiscent of tailed bacteriophages. These observations are consistent with other results …, implying that Mimiviruses and some other large icosahedral dsDNA viruses have gathered genes from eukaryotic, prokaryotic, as well as archaeal origins [my emphasis].

The three-dimensional cryoEM reconstruction reported here, which was made possible in part by relaxing the icosahedral symmetry, is of a virus whose volume is an order of magnitude larger than has previously been reported. Thus, the detection of a unique vertex may have been missed in other structural studies in which strict icosahedral symmetry had been imposed [my emphasis].”

There are two important points here – one of which may be wrong.

The first is that mimiviruses et alia “…have gathered genes from eukaryotic, prokaryotic, as well as archaeal origins”: given the evolutionary speculations published by Susan-Monti et al. (Virus Research 117 (2006) 145–155), who say:

“Our current hypothesis is that DNA viruses are of deep evolutionary origin close to the origin of the other domains of life.”,

and point out the virus does not seem to have exchanged much DNA (=horizontal gene transfer) with its host despite a presumably ancient association.  This builds on Suhre et al. (PNAS 102 : 14689-14693, 2005), who say:

“Our bioinformatics and comparative genomics study revealed a unique feature of Mimivirus among the eukaryotic domain [sic]: the presence of a highly conserved AAAATTGA motif in the immediate 5′ upstream region of 50% of its protein-encoding genes. By analogy with the known promoter structures of unicellular eukaryotes, amoebal organisms in particular, we propose that this motif corresponds to a TATA box-like core promoter element. This element, and its conservation, appears to be specific of the Mimivirus lineage and might correspond to an ancestral promoter structure predating the radiation of the eukaryotic kingdoms ….

Mimivirus genes exhibiting this type of promoter might be ancestral as well. [my emphasis].”

Thus, it is possibly more likely that eukaryotes and possibly prokaryotes have garnered genes from mimi- and other viruses, rather than the converse!

The second point, given that they ARE a structural biology group, is much more likely: missing unique non-icosahedral capsid structures because of averaging could mean there is a whole world of specialised machinery in large DNA viruses which has simply been missed up till now.

I reiterate, watch this research space….  Anyone interested in mimivirus basics would also be well advised to look here.

Posted in Evolution, General, Viruses | 6 Comments »

HIV Vaccine Day

19 May, 2009

And as one involved (or formerly involved, thanks to the effective demise of our funding agency…) in HIV vaccine research, it is criminal that I missed this yesterday – but the UCT Monday paper caught it, so let’s see what they said:

First human trial of UCT’s HIV vaccine
18 May 2009

 World AIDS Vaccine Day, May 18, marks the occasion in 1997 when US President Bill Clinton challenged researchers to come up with an AIDS vaccine within the following decade, stating that such a vaccine was the only way to eliminate the threat of AIDS. …

Researchers from UCT’s Institute of Infectious Disease and Molecular Medicine (IIDMM) have announced that their two new preventative HIV vaccines have reached the first stage of human clinical trials, a first for Africa.

This trial, called SAAVI 102/HVTN 073, is also a milestone for South Africa. The country is one of the few developing nations to have developed an HIV vaccine and progressed it into human clinical trials.

Professor Anna-Lise Williamson is leader of the team at the IIDMM.

The Desmond Tutu HIV Centre, based at the IIDMM, is one of three sites in the world that will conduct the trials. The others sites are in Johannesburg and Boston in the US.

These vaccines are a culmination of eight years of research by scientists at the IIDMM, UCT, and collaborators from the US National Institutes of Health and the Vaccine Research Centre. Their development and testing has been underpinned by funding from the South African AIDS Vaccine Initiative (SAAVI) and the US National Institute of Allergy and Infectious Diseases (NIAID).

…The initial human trial is being conducted jointly with the HIV Vaccine Trials Network and the NIAID, part of the US National Institutes of Health.

There is a wealth of science behind the vaccines, of course: I am listing a few of the papers giving the historical background to the DNA and the modified vaccinia Ankara virus (MVA, a smallpox vaccine strain) that are about to be used in the trial, below.

Broad, high-magnitude and multifunctional CD4+ and CD8+ T-cell responses elicited by a DNA and modified vaccinia Ankara vaccine containing human immunodeficiency virus type 1 subtype C genes in baboons.  Burgers WA, Chege GK, Müller TL, van Harmelen JH, Khoury G, Shephard EG, Gray CM, Williamson C, Williamson AL.  J Gen Virol. 2009 Feb;90(Pt 2):468-80.

A multigene HIV type 1 subtype C modified vaccinia Ankara (MVA) vaccine efficiently boosts immune responses to a DNA vaccine in mice.  Shephard E, Burgers WA, Van Harmelen JH, Monroe JE, Greenhalgh T, Williamson C, Williamson AL.  AIDS Res Hum Retroviruses. 2008 Feb;24(2):207-17.

Construction, characterization, and immunogenicity of a multigene modified vaccinia Ankara (MVA) vaccine based on HIV type 1 subtype C.  Burgers WA, Shephard E, Monroe JE, Greenhalgh T, Binder A, Hurter E, Van Harmelen JH, Williamson C, Williamson AL.  AIDS Res Hum Retroviruses. 2008 Feb;24(2):195-206.

Design and preclinical evaluation of a multigene human immunodeficiency virus type 1 subtype C DNA vaccine for clinical trial.  Burgers WA, van Harmelen JH, Shephard E, Adams C, Mgwebi T, Bourn W, Hanke T, Williamson AL, Williamson C.  J Gen Virol. 2006 Feb;87(Pt 2):399-410.

Construction and characterisation of a candidate HIV-1 subtype C DNA vaccine for South Africa.  van Harmelen JH, Shephard E, Thomas R, Hanke T, Williamson AL, Williamson C.  Vaccine. 2003 Oct 1;21(27-30):4380-9.

Characterization and selection of HIV-1 subtype C isolates for use in vaccine development.  Williamson C, Morris L, Maughan MF, Ping LH, Dryga SA, Thomas R, Reap EA, Cilliers T, van Harmelen J, Pascual A, Ramjee G, Gray G, Johnston R, Karim SA, Swanstrom R.  AIDS Res Hum Retroviruses. 2003 Feb;19(2):133-44.

The development of HIV-1 subtype C vaccines for Southern Africa.  Williamson AL.  IUBMB Life. 2002 Apr-May;53(4-5):207-8. Review.

Posted in HIV, Vaccines: General, Viruses | 1 Comment »

Are we there yet??

13 May, 2009

As I sit here in the grip of a rhino/toga/adenovirus infection [yes, acute rhinitis, just recovered from sore throat, feel like cr@p], it is hard to be optimistic about the demise of the Influenza A H1N1 “Mexico Flu” outbreak – but various media are now jumping as enthusiastically onto the “It Was All a False Alarm” bandwagon as they did onto the “We’re All Doomed” train.

Was it a false alarm?

Has the threatened pandemic gone away?

Peter Singer, the director of the McLaughlin-Rotman Centre for Global Health at University Health Network and University of Toronto, wrote this in Canada’s National Post on May 11th [bolded red comments my emphais]:

It’s been a fortnight since global attention began fixating on flu. There have been over 4,379 cases worldwide and more than 280 in Canada. We are likely past the midpoint of this episode and it’s not the “big one.” We learned lessons from SARS that we applied to this flu outbreak. This time, how have we done and what have we learned?

I would give Canadian public health authorities an A grade. …

I would give the global response a B grade. The human flu emergency turned into a mini Mad Cow-type crisis. Calling it “swine flu” initially skewered the international pork trade, even though public health authorities emphasized that you can’t catch flu from eating pork chops. In Egypt, authorities slaughtered pigs owned by a poor Christian minority group, fanning religious conflict. Afghanistan’s only known pig–in the Kabul zoo — has been quarantined. The casual musings of a World Health Organization official, and the outbreak in Alberta pigs, didn’t help.

The World Health Organization response was robust, but its pandemic scale sends the wrong signal to the public. It can reach its top level in a mild pandemic so it appears to foretell doomsday even if people around the world have only the sniffles. Meanwhile, some countries reacted to criticisms of their actions during SARS with questionable quarantines, such as with the group of healthy Quebec students [and Mexicans] quarantined in China.

Well, so far it looks like he agrees with the thesis that the “pandemic” scare was mostly hype.  But he goes on to warn us:

Did we cry wolf ? No. The flu virus is a wolf in sheep’s clothing. Flu, a cunning adversary, can mutate to be more transmissible, lethal and drug resistant. Some have argued the media is drawing attention away from other public health priorities; in fact the flu is probably drawing attention away from Paris Hilton. It is a sad coincidence, though, that while a million people died in the 1957 and 1968 flu pandemics, the same number, mostly children under five in Africa, die each year from malaria.

What is next? This flu episode will probably end like some TV shows: “to be continued.” We’ll be tuning into the flu season just starting in the southern hemisphere and our flu season here next fall.

The threat of flu is constant. It’s like the threat of terrorism. The virus needs to break through only once; we need to stop it every time. But this epidemic of H1N1 has left us better prepared for future pandemics.

So did you all register that?  Possibly, just possibly, the flu outbreak is dying away IN THE NORTHERN HEMISPHERE.  WHERE THE FLU SEASON IS OVER.  JUT IN TIME FOR THE SOUTHERN HEMISPHERE’S SEASON.  And this, we remind you, is followed in October or so by a new northern hemisphere flu season.

Singer finishes with:

In a severe pandemic, most sickness and death will be in the developing world. Unfortunately, the globalization of disease threats is greater than the globalization of health defences. Mexico’s anti-viral stockpile at the beginning of the epidemic was only one million doses for 110 million people [compared to Canada’s 55 million for ~20 million by the end], and there are concerns about future availability of flu vaccine in the developing world. Canada should help because we are compassionate and capable, and because, as this epidemic shows, we are all in this together in an interconnected world.

Amen, brother Singer…so in other words, the developing world epidemic is probably still coming, it will be worse than the outbreak we’ve just seen, and there will be way too few drugs to deal with it.

Posted in Evolution, Influenza viruses, Vaccines: General, Viruses | 2 Comments »

…And the Virus Rolled On….

4 May, 2009

ProMED – that ever-so-reliable source of breaking epidemiological news – gives us this as of yesterday.

From the WHO:

Influenza A(H1N1) – update 11 — 3 May 2009 [abridged]
As of 3 May 2009, 17 countries have officially reported 898 cases of influenza A(H1N1) infection, and 20 deaths.
Mexico has reported 506 confirmed human cases of infection, including 19 deaths. The higher number of cases from Mexico in the past 48 hours reflects ongoing testing of previously collected specimens.
The United States Government has reported 226 laboratory confirmed human cases, including one death.
The following countries have reported laboratory confirmed cases with no deaths:

 

  • Austria (1),
  • Canada (85),
  • Colombia (1),
  • China, Hong Kong Special Administrative Region (1),
  • Costa Rica (1),
  • Denmark (1),
  • El Salvador (2),
  • France (2),
  • Germany (8),
  • Ireland (1),
  • Israel (3),
  • Netherlands (1),
  • New Zealand (4),
  • Republic of Korea (1),
  • Spain (13),
  • Switzerland (1)
  • the United Kingdom (15)

Further information on the situation will be available on the WHO [/CDC] website on a regular basis.

WHO advises no restriction of regular travel or closure of borders.

 It is considered prudent for people who are ill to delay international travel and for people developing symptoms following international travel to seek medical attention, in line with guidance from national authorities.

 
And finally, pigs with the virus: 

Canada on [2 May 2009] reported the identification of the A(H1N1) virus in a swine herd in Alberta. It is highly probable that the pigs were exposed to the virus from a Canadian farm worker recently returned from Mexico, who had exhibited flu-like symptoms and had contact with the pigs.

There is no indication of virus adaptation through transfer from human to pigs at this time.

There is no risk of infection from this virus from consumption of well-cooked pork and pork products. [my bold/red]

 From South Africa’s News24:

 
Swine flu vaccine in the works
29/04/2009 14:01  – (SA)  

 

 Geneva – Four laboratories are at “various stages” of working on a seed virus that is a precursor in a future vaccine against swine flu, the World Health Organisation said on Tuesday.

“There are currently four of our reference laboratories who are working with seed virus, they are at various stages of producing seed virus needed to make the vaccines,” said WHO spokesperson Gregory Hartl.

Hartl said however that the laboratories – in Britain, Canada and the United States – have not been asked to begin production in an extensive manner.
…
WHO on Monday recommended that the UN agency “take steps to facilitate the development” of a vaccine against the swine flu virus found in the latest outbreak that has likely caused more than 150 deaths in Mexico and has spread worldwide.

But the panel stopped short of recommending a complete shift in global vaccine production capacity, warning that it would be “prudent” to continue regular seasonal vaccine production as well.

A spokesperson in Paris for Sanofi Pasteur, a subsidiary of the French pharmaceutical maker Sanofi-Aventis, had said that the time needed to make a flu vaccine is about four months.

Scaling up production of a vaccine is another hurdle. The main approach is to grow the virus samples in time-honoured fashion in embryo chicken eggs, which is slow and clumsy.

Production capacity of flu vaccines has tripled since 2007 in response to the Sars and H5N1 scares, according to a WHO-funded study published in February.

– AFP

More from News24:

Egypt works on H5N1 chicken vaccine

In news that partially redeems the very controversial decision to cull the country’s pigs – apparently based on a need to regularise the industry, rather than panic over transmission from pigs – AFP details how Egypt plans to produce its own vaccine within two years.

A useful graphic explaining how reassortant viruses occur.

Information on how the virus may have originated:

New virus may be a hybrid

Last updated: Monday, May 04, 2009

The new virus that has killed as many as 177 people and spread globally is a hybrid that appears to have mixed with another hybrid virus containing swine, bird and human bits, US researchers reported.

Raul Rabadan and colleagues at Columbia University in New York analysed the published genetic sequences from the H1N1 virus that has brought the world to the brink of a pandemic. “The closest relatives to the virus we have found are swine viruses,” Rabadan said.

“Six segments of the virus are related to swine viruses from North America, and the other two from swine viruses isolated in Europe/Asia,” they wrote in the online journal Eurosurveillance.

The US Centres for Disease Control and Prevention said last week after discovering this virus in two US children that it had four virus types – two swine, an avian and a human component. It may be even more complex than that.

‘This strain looks like another hybrid’
Influenza viruses mutate constantly, and they also swap genetic material with one another promiscuously – especially if an animal or person is infected with two strains at once.

Rabadan’s team said this particular strain looked partly like another hybrid, or what scientists call a reassortant, virus. “The North American ancestors are related to the multiple reassortants, H1N2 and H3N2 swine viruses isolated in North America since 1998,” they wrote.

“In particular, the swine H3N2 isolates from 1998 were a triple reassortment of human, swine and avian origin.”

For those in search of graphics for their webinar/presentation on flu pandemics: from the US National Archives.

http://www.archives.gov/exhibits/influenza-epidemic/records-list.html

The Influenza Epidemic of 1918 via kwout

http://www.archives.gov/exhibits/influenza-epidemic/records-list.html

And for light relief away from deadly viruses:

“Who would win in a fight: Gandalf or Darth Vader? What about Neo versus Harry Potter?”

Posted in General, Influenza viruses, Vaccines: General, Viruses | Leave a Comment »

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