Virus Musings

Two new virology labs: Pirbright's Plowright and the CVR's Sir Michael Stoker

noreply@blogger.com (Anonymous) — Sun, 14 Sep 2014 17:48:00 +0000

Walter Plowright and Sir Michael Stoker: two names inextricably linked with virology. Their names have now been given to two new buildings that have now been more or less completed.

Tomorrow our lab moves to the new Sir Michael Stoker building in Glasgow. Ultimately, the building of this new lab will see the entirety of the Centre for Virus Research based on one site, which will make life much easier for people like me who, until now, use pieces of kit from both sites.

It's gold. Very gold. But it's what's inside that counts. Expansive labs, a big ACDP 3 suite (including insectory), a next generation sequencing suite, a series of rooms for various forms of electron microscopy, and a big server to excite the bioinformaticians, it's an impressive building. Everyone should be excited about working there.

A Golden Temple of virology: the Sir Michael Stoker Building, linked directly with the Sir Henry Wellcome Building

Sir Michael Stoker was chair of virology at the Glasgow MRC virology unit. It is his work with cells for which he is most famed. Originally as a way of researching viruses causing cancer, his work yielded the hamster-derived BHK cell line. It is hard to quantify the impact that this had. Until that point, foot and mouth disease virus (FMDV) vaccines were being produced by growing the viruses in sheets of epithelium that had been peeled off of cows tongues. The BHK cell line, in addition to being generally permissive for many viruses, now enabled the mass production of FMDV vaccines.

Sir Michael Stoker (source)

Regardless of whether it was the Institute for Animal Health (now The Pirbright Institute) or Merial that was responsible for the leak of FMDV, it was already apparent by 2007 that the facilities at The Pirbright Institute were in need of an update. Plans for a redevelopment were already under way, but the outbreak simply served to focus this objective, as well as instigating a reassessment of where the institute's future lay (purely virology - at the time bacteriology and parasitology we firmly established at the sister site at Compton). Last week I went to the centenary event of the Institute, and this included a tour of the newly complete Plowright building.

In contrast to the relative compactness of the Sir Michael Stoker building, The Plowright building is vast. And it needs to be. Unlike the Stoker building, much of the Plowright building operates at negative pressure in order to allow work on the highest category of animal pathogen, including viruses such as FMDV and African Swine Fever virus. To allow this, virtually a floor of the building is taken up by the fans, filters and pipes of the air handling system. Ultimately, this results in labs that aren't actually that big, and space will be an issue, but walking around it's mighty impressive. For example, the lab I did my PhD in was made of bricks and mortar; this lab is made of a specific concrete that is designed to have fewer air bubbles.

The Plowright building is also different to most other containment labs of this level. Others, such as AAHL at Geelong, operate as a 'box within a box'; in the case of the Plowright it's been designed so that the outer surface is sufficiently robust that it acts as the outer shell, meaning that the working conditions inside are much more pleasant.

Walter Plowright (RODNEY WHITE / Associated Press)

In June 2011 the FAO declared the world free of rinderpest. It was only the second time, after smallpox, that such a disease had been wiped from the earth, at least outside the bounds of a laboratory freezer. Fundamental to the eradication of rinderpest, as for smallpox and soon hopefully polio, a vaccine was imperative. And it was Plowright's vaccine that was used for the eradication. It was a great vaccine, being both cheap to produce and resulting in lifelong immunity. The vaccine was derived 'simply' by passaging the virus multiple times in cell culture; something which wasn't quite so 'simple' during the 1950s and 1960s. It was then a huge achievement, and one without which the world may still be plagued by rinderpest.

In the end though, you can have the best building ever, but this it's useless unless it's full of good people doing good science and, with the infrastructure sorted, that's now the focus for everyone.

Where did Ebola come from? Rooting the un-rootable

noreply@blogger.com (Anonymous) — Sun, 17 Aug 2014 22:16:00 +0000

As with other emerging viruses, the manner in which Ebolavirus outbreaks appear seemingly from nowhere merely adds to their terror. What is especially intriguing about the current outbreak in West Africa is that it's the wrong virus in the wrong place: as a strain of Zaire Ebolavirus, we would expect to see this virus in central Africa. The modern way to answer the 'where did it come from' question is to compare the genomic sequences of different viruses, and this was one of the first things done for the Guinea isolates.

The problem is where to root the tree. There have been two schools of thought on why outbreaks of Ebola in Africa happen where they do. Firstly, it's simply a case that Ebola is widely distributed and it is a unfortunate event, such as butchering wild animals, that results in spillover from animals into humans. An alternative, is that Ebola is spreading in a wave across Africa, resulting in outbreaks as it progresses, breaking through at points of weakness. For the latter hypothesis we would expect all of the viruses to relate in a straightforward and logical manner.

Crest of a wave: a figure depicting the hypothesised spread of Ebolavirus in a wave-like fashion. Source

Drawing a straightforward tree with the sequences from the current outbreak along with the available genome sequences of all Ebolaviruses confirms that the virus causing the outbreak in western Africa is a divergent strain of Ebola Zaire, suggesting it arose in central Africa. However, it's out on its own relative to the main Ebola Zaire clade.

The Ebolavirus tree: assembling a tree using all species of Ebolavirus results in the Guinea sequences separated from the remainder of the Ebola Zaire clade. Source

The key, it seems, appears to be to remove the intergenic sequences that separate the coding sequences in the virus. When you do this, and concatenate the coding sequences, the sequences from the Guinea outbreak sit in the middle of the Ebola Zaire clade. If you do the same with the intergenic sequences alone you get a tree with similar topology. The issue the authors encounter is that there is no good place from which to root the tree - the other Ebolavirus species are essentially too distant. Combined with the fact that the viruses are always evolving, this all makes it difficult to see what's really happening.

One alternative is to use time, in combination with the estimated rate of evolution, as a way in which to organise the sequences. Using this molecular clock approach results in an intriguing figure whereby the Guinea sequences do indeed come out up top, where we would expect them to be.

Using a molecular clock to arrange Ebola Zaire sequences places Guinea 2014 furthest from the first isolation in 1976. Source

One thing that this approach also allows is an estimation as to when the current virus diverged from the central African sequences. Dudas and Rambaut estimate 2002, whereas a separate analysis by Calvignac-Spencer et al, also using molecular clocks to root the Ebola Zaire clade, suggest either 1999 or 2001 (depending upon the assumptions made).

The current outbreak is unprecedented in scale, and it's hard to believe that, when the outbreak is over, there will not be a large study using many sequences. This in turn should give some more clues about how the Ebola wave crashed upon Western Africa.

Dudas G, & Rambaut A (2014). Phylogenetic Analysis of Guinea 2014 EBOV Ebolavirus Outbreak. PLoS currents, 6 PMID: 24860690

A not so curious coincidence: another tick, another phlebovirus.

noreply@blogger.com (Anonymous) — Mon, 30 Jun 2014 21:43:00 +0000

In recent years there seems to have been a resurgence in Bunyaviruses. There are always incidences of certain members, for example there are thousands of cases of hantavirus each year, as well as the sporadic cases of Crimean-Congo hemorrhagic fever virus. On the other hand there are the new emergents. In Europe, the most dramatic and devastating (at least in animals) has been Schmallenberg virus, which will no doubt now be familiar to more or less all cattle and sheep farmers.

As far as 'human' viruses are concerned, there have been a couple of highly related viruses: one in the Far East, and one the US. Severe fever with thrombocytopoenia syndrome virus (SFTSV) emerged in China in 2009, and was isolated from one of a series of patients with hemorrhagic fever. The virus from the US is Heartland virus (HV), and was isolated in 2009 from two cases in Missouri. The last few weeks have seen the second death in the US from Heatland virus, this time in Oaklahoma.

SFTSV and HV are both members of the phlebovirus genus of the Bunyaviridae. Rift valley fever virus (RVFV), currently one of the great fears for Europe, is also a member of this genus and is spread by mosquitoes. However, unlike RVFV, both SFTSV and HV are associated with ixoid and lone star ticks respectively.

Distribution of the Lone Star tick in the US.

Now a paper has been published describing another tick-borne phlebovirus, named Hunter Island Group virus (HIGV, after the location from which the ticks were sampled), this time from ticks associated with shy albatrosses on an island near Tasmania. Initially an aetiological agent couldn't be found; likely viruses such as Newcastle's disease virus and avian influenza were eliminated as likely causes. Although electron micrographs indicated a virus with morphology suggestive of a bunyavirus, antibody and PCR assays still failed to find a virus.

Electron micrograph images of negatively stained virions (A) and thin sections of vero cell cultures (B). source

Several years later they performed deep sequencing on infected cultures. Although the nucleotide sequences failed to match with anything close, blast searches using the protein sequence revealed similarity with SFTSV. With the foundations of the sequence now known the remaining sequence was filled in, allowing an analysis using the whole genome, as well as the design of molecular assays for the specific detection of HIGV. Perhaps unsurprisingly considering the results from the deep sequencing, the viruses fit into the bunyavirus clade next to SFTSV and Heartland virus.

HIGV: phylogenetic trees (amino acid sequence) of the viral polymerase (A), glycoprotein (B), nucleocapsid (C) and NSs (D). source

As the ticks were associated with healthy as well as diseased birds, it was suspected that HIGV wasn't the cause of the outbreak among the shy albatrosses, something which was confirmed when the birds were tested for antibodies against, or the genome of, HIGV.

Richard Elliott, a well known figure in the bunyavirus world, says the discovery of this virus wasn't a surprise, and in a way was predicted. The simple truth is that if the ticks are there, then the viruses are there.

Discussing tick viruses with anyone always seems to leave one unanswered question; what is it that's so repulsive about ticks?

Wang J, Selleck P, Yu M, Ha W, Rootes C, Gales R, Wise T, Crameri S, Chen H, Broz I, Hyatt A, Woods R, Meehan B, McCullough S, & Wang LF (2014). Novel phlebovirus with zoonotic potential isolated from ticks, australia. Emerging infectious diseases, 20 (6), 1040-3 PMID: 24856477

Through the looking glass: Guinea, Ebola and life before germ theory

noreply@blogger.com (Anonymous) — Sun, 01 Jun 2014 12:15:00 +0000

Last week I was at a conference dedicated to viral zoonoses where the opening talk of the meeting was given by Pierre Rollin. Rollin has for a long time been based in the Viral Special Pathogens branch at the CDC in Atlanta, USA. He’s an old hand in outbreaks of savage viruses; a veteran of many Ebola outbreaks, as well as Nipah etc. All in all, he’s someone who can speak from experience. The talk itself ranged across a variety of aspects regarding Ebola virus but concentrated, unsurprisingly, upon the control of outbreaks. Part of the talk was also dedicated to describing the situation in Guinea, from where he’d just returned.

Ebola in Guinea: hot work, but preventing contact means preventing infection

It’s always more interesting to hear the voice of experience. Books such as The Hot Zone and films such as Outbreak are there to make money and must therefore offer drama; in the case of Ebola endless people with liquefied livers bleeding from every cut and orifice. Rollin pointed out that this is rubbish. Sure, a few do bleed, but only a “minority”; more usually it’s shock and multi organ failure. Gruesome, but not quite as graphic.

One particular point that resonated in the talk was how, in principle, outbreaks of Ebola were easy to control. Find the village, find and isolate those infected and suspected of being infected, plus educate the local population. As remarkably infectious as Ebola is, you need contact with the patient for transmission to occur – sleeping in the same room as an Ebola may be fine (if not recommended), but contact, such as sharing a bed, is a very bad idea. However, if reality were that simple Guinea wouldn't be staring at the prospect of 200 deaths. Speaking to Rollin the next day he confirmed that it was the small details and logistics that caused the most problems. Arriving at a hospital, for example, and finding that the water system is broken. No cleaning. No disinfection. Suddenly something as simple as making up bleach is a challenge. This, though, is a practicality that can be sorted. More difficult is the human aspect: educating the local population.

Education: a poster describing what to (and not to) do regarding Ebola for local population. Image:Medindia.

A recurring theme in Ebola outbreaks is that a lot of cases arise from two population types: healthcare workers, as a result of contact with patients, and, secondly, those involved in traditional practices, such as local ritual burial.

Ritual beliefs still hold fast in many parts of rural Africa. Some locals apparently believe that white man is bringing the disease and is deliberately infecting them. Apparently there are pockets of people in the forests of Guinea who have been hiding bodies from the doctors. In other cases there is a belief that it is spirits and spells that cause the pestilence. In general there’s little understanding of the concept of infectious disease: how many people with even limited knowledge that Ebola is caused by a virus would hug and kiss corpses who had died as a result of the infection? As strong as familial love may be, I think the rational decision may prevail. As a result of all this, there are visits to the likes of witchdoctors and herbal healers that, in turn, become infected and represent a hub of infection for many others.

Miasmas responsible for a cholera epidemic.

To many in a more developed world, this may all sound shockingly primitive. In reality however, this is simply knowledge and education. It was only around the middle of the 19^th century that the likes of Louis Pasteur and subsequently Robert Koch really established the germ theory of disease. Prior to this theories of miasmas and mysterious airs still abounded around the world; the ‘mala-aria’ (bad air) derivation of malaria perhaps being one of the most well known. In one sense then, what we are seeing in Guinea is simply life before germ theory. That’s not to say they’re in any way intellectually inferior, more that it is a demonstration that knowledge gathered in more developed nations simply hasn't found its way to other nations. Remoteness inevitably is a factor. But if ever there were an argument for open access journals freely available worldwide....

Mx2: why some species, and not others?

noreply@blogger.com (Anonymous) — Sun, 18 May 2014 21:30:00 +0000

To answer a question recently posed at a conference, some viruses are clearly good. In general though, becoming infected is something that is to be avoided. In an attempt to prevent infection, cells are armed with the interferon system. Interferon is a molecule that is made and secreted by a cell when it discovers that it's become infected. In turn other cells, yet to become infected, are alerted that an infection is present. This early warning results in the cell producing diverse collection of antiviral molecules, termed interferon stimulated genes (ISGs), each with their own mode of action. There are hundreds of genes upregulated as a result of IFN signalling, but finding out which ISG does what and how is more difficult. One way in which this has been looked at is by expressing ISGs individually and then looking to see whether it has a protective effect against a virus of choice. This approach has led to the identification of various genes such as tetherin, which has since been shown to restrict the progress of multiple viruses from several virus families. Altogether, the action of ISGs, and the various countermeasures of viruses, is a fascinating battle.

At any one point there always seems to be a fashionable ISG. Recently it's been myxovirus 2 (Mx2). the fact that Mx proteins are antiviral is not new. However, two papers came out last year revealing that Mx2 specifically is an ISG that is able to antagonise HIV-1 (so often the virus leading the way). Recently, an Mx2 paper by Busnadiego et al has been published detailing the host and virus specific factors that determine its function.

The most revealing aspect of this paper is the variation that exists between the Mx2 of different species to restrict HIV-1, in essence a 'human' virus. The authors confirmed that human Mx2 blocked HIV, then tested the Mx2 orthologues from African green monkey (which did restrict), Macaque (which did restrict), ovine (which did NOT restrict) and canine (which also did NOT restrict HIV).

Restriction of HIV-1 by Mx2 from various species: A) evidence that the Mx2 genes are expressed at similar levels. B) reduced titres in the presence of the Mx2 (grey bars) from various species.

In addition to variation at the host level, the authors also tried a variety of other retroviruses. Whilst other retroviruses weren't restricted by the Mx2 of any species, there was evidence that the Mason pfizer monkey virus (a betaretrovirus, rather than lentivirus) was sensitive, with varying degrees, to the Mx2 of macaques, African green monkeys and human Mx2. HIV-1 group O, as opposed to group M (which the previous studies had used) was also restricted by human Mx2. However, this group O virus was only partially restricted by African green monkey Mx2, clearly showing species variation. The paper goes on to describe what lies behind these difference in the action of different Mx2s against different viruses.

One way to see which part of the virus is involved in the restriction process is to passage the virus in the presence of the restriction factor. When the authors did this, and sequenced the virus, they found three substitutions in the C-terminal domain of the capsid region within the viral gag gene. By making viruses with each of these mutations in isolation, as well as screening a library of capsid mutants, it could be shown that substituting individual amino acids in the virus sequence is sufficient to direct the specificity of action of different Mx2 proteins. Clearly though, a library can only reveal so much; there are no doubt other positions within, and perhaps outside of, capsid that influence restriction.

Which parts of the Mx2 protein from different species was responsible for the variation that exists in the extent of restriction? One aspect that varied between the Mx2 of different species was its localisation within the cell. Whereas the Mx2 variants that restricted localised to the nuclear pores, those that didn't restrict, i.e. canine and ovine, failed to consistently show such a precise localisation.

Localisation to the nuclear pore of Mx2: Human Mx2 localises with nuclear pores (Nup98, red) whereas canine, and to an even greater extent ovine, Mx2 orthologs show a weaker association.

The result lack of restriction therefore makes sense, particularly when they show that several amino acids in primate Mx2 sequences, which have been shown to be important for function, (and likely nuclear localisation) are divergent in the ovine and canine versions. Making human-canine chimeric (i.e. part human, part canine) versions of Mx2 narrowed the variability down to the N-terminal end; an Mx2 with a N-terminus from human, but C-terminus from dogs essentially functioned like human Mx2 (capable of restriction, localised to nuclear pores), whereas the opposite, with a canine N-terminus, was unable to restrict, even though the other half was human. Equivalent chimeras between human and African green monkey Mx2, which have differing abilities to restrict a particular clone of HIV-1 revealed a similar story. Ultimately, all of the experiments narrowed down the capability of restriction (at least for these species), to just 3 amino acids in an 8 amino acid region within the N-terminus.

Variation in the Mx2 sequence: Top) diagram of the Mx2 layout with arrows indicating amino acids under positive selection. Bottom) dN/dS and Bayes factor values across the length of Mx2. Low values are indicative of purifying selection. Variation is clearly apparent towards the N-terminus.

The authors managed to narrow it down further by doing some sequence analyses. Using the primate sequences, they found evidence of positive selection, suggesting some form of arms race between virus and restriction factor. Of 10 amino acids identified as being under positive selection, two occurred in the 8 amino acid region identified using chimeras. By making mutants it was then possible to show that substituting amino acid 37 was sufficient to swap the specificity of human vs. African green monkey Mx2 proteins. This is, though, one example against one mutant virus. There are inevitably many more points of variation dictating species specificity.

Modern technology and methods increasingly allows us to pick apart systems in ever finer detail, and as far as viruses are concerned they don't get more significant than the interferon system; there's a reason viruses go to great lengths to prevent, avoid or overcome it. Picking apart the arsenal of ISGs, the front-line effectors of the system, may reveal more and more the variation that exists between species and, in turn, help to answer a fascinating question regarding virus host range: 'why some species, and not others?'.

Busnadiego, I., Kane, M., Rihn, S., Preugschas, H., Hughes, J., Blanco-Melo, D., Strouvelle, V., Zang, T., Willett, B., Boutell, C., Bieniasz, P., & Wilson, S. (2014). Host and Viral Determinants of Mx2 Antiretroviral Activity Journal of Virology DOI: 10.1128/JVI.00214-14

Rift Valley fever virus genome organisation: it's like it for a reason

noreply@blogger.com (Anonymous) — Mon, 10 Mar 2014 20:49:00 +0000

Following recent Bluetongue virus and Schmallenberg virus (SBV) incursions into Central and Northern Europe, Rift Valley Fever virus (RVFV) is now perceived as one of the greatest threats to Europe, crossing over the Mediterranean from Africa where it remains endemic. With good reason. Both Culex and Aedes species of mosquito are capable of transmitting the virus - West Nile virus infections in Italy have previously shown that there are conditions in Europe suitable for arbovirus transmission by such species of mosquito.
Like SBV, which has spread across Europe in the last 2-3 years, RVFV is a member of the Bunyaviridae family, (albeit in the genus Phlebovirus as opposed to Orthobunyavirus). If it were to encroach into Europe, the headline fact is that it's a zoonosis, with the potential to cause fatal disease in humans. Of equal, if not more, importance is the that fact that it can cause deaths and abortions in ruminants.

Dead cattle as a result of RVFV infection (http://www.elsenburg.com/info/els/077/077e.html)

The RVFV genome comprises three segments of (-)ssRNA. Although the small (S) segment encodes both the nucleocapsid (N) protein and the non-structural protein NSs, a peculiarity is that, whilst N is produced from RNA transcribed from the genome, the message for NSs is transcribed from the antigenome. Superficially this is a little counter-intuitive; NSs is responsible for the rapid abrogation of the interferon system and thus would, presumably, be required as early as possible upon infection. Why force the virus to produce the antigenome before NSs can be produced?

A recent paper by Brennan, Welch and Elliott has addressed this by swapping around N and NSs, such that NSs is translated from the antigenome, and N from the RNA transcribed from the antigenome.

Generation of the swap virus: the (anti)genomic strand from which N and NSs are transcribed is 'swapped'.

In addition to the virus with wild-type (vaccine strain, MP12) and 'swap' virus, they also made viruses where NSs was substituted with EGFP. Now they had a panel of viruses including ones where NSs (or EGFP) were expressed first, before N. When they tested the growth of each virus, the swap viruses all grew poorly compared to the wild-type. This was regardless of whether the cells were either interferon competent (A549), incompetent (BHK21) mammalian cells, or indeed various insect cell lines.

Growth of 'swap' vs wt MP12 RVFV: In all cases, all swap viruses (filled circles) grow with lower efficiency compared to MP12 (A:mammalian cells; C. insect cells).

The lower apparent rate of replication was reflected in the amount of protein. As time progressed, the amount of protein accumulated by the swap virus was lower, although as expected the NSs protein was on this occasion produced before the N protein; opposite to what occurs with the wt virus. The accumulations of NSs were also much more substantial than with wt. However, although there was much more NSs (and less N) than wt, significant amounts of the glycoprotein (Gn) were not detected until 48 hours after infection, compared to 18-24 in the case of wt. In the case of the swap virus with EGFP in place of NSs, Gc was virtually undetectable even at 48h.

In wt RVFV, NSs assembles into filaments that are localised to the nucleus. Rather oddly, in the case of the swap virus, these were much thicker than the wt, with additional evidence of some NSs in the cytoplasm. These alterations in NSs behaviour appeared not to affect the functions of NSs in either inhibiting both host protein synthesis and host RNA synthesis. Given its 'shut-off' function, it is intriguing that increasing the abundance of NSs had no additional effect upon the intensity or rate at which protein shut-off occurs.

Shut-off of host cell macromolecular synthesis: A: radioactive methionine/cysteine incorporation at different times post-infection; less label = less protein = shut-off. B: shut-off of RNA synthesis (newly synthesised RNA is green, red is virus).

When they looked at the targets of NSs that result in shut-off, p62 was inhibitted by the swap virus (although more slowly), just as the wt but, bizarrely, PKR appeared to be largely unaffected by the swap virus, in contrast to the wt virus where PKR levels dropped from around 5 hours onwards. Overall, it would seem that, whilst it still does, the swap virus is a bit less efficient at the shut-off. As a result, it is a little surprising that the swap virus results in the induction of less IFN than the wt virus in A549 cells.

One thing the authors did tease apart is the relative number of genome:antigenome copies in both the cell and virion fractions. The swap virus was found to transcribe much more NSs RNA compared to the wt equivalent, suggesting that increased activity of the relative promoters is responsible for the dramatic amount of NSs observed with the swap virus. Such an excess of RNA may have overwhelmed the control by RNAi in insect cells, resulting in cytopathogenic effect in infected insect cells (in contrast to wt virus, which establishes a persistent infection). One interesting finding is that more antigenome than genome copies are packaged in swap virus virions. Does this reflect the abrogation a specific packaging process, or simply the abundance of genome:antigenome copies in the cytoplasm when packaging occurs? Considering the swap virus has N transcribed from the antigenome, the increase in antigenome packaging may actually overcome some of the temporal regulation achieved by swapping the N from the genomic to the antigenomic transcipt. It seems a bit more work is required here. Virions have been shown to contain just 3 segments of RNA. If more of the virions have an antigenomic S segment, then the number of particles per infectious unit will necessarily be increased. It may be that such differences in the make-up of the viral population can explain some of the characteristics of the swap virus.

Of course such attenuating features - at least in vitro - may contribute towards the rational development of a vaccine. The most tempting feature of the swap virus described here is the fact that the virus cannot persist in insect cells and would thus be somewhat resistance to transmission; a key consideration in live arbovirus vaccines.

So, genome organisation is important. Ultimately this is not surprising: there's a reason it's like it is.

Brennan, B., Welch, S., & Elliott, R. (2014). The Consequences of Reconfiguring the Ambisense S Genome Segment of Rift Valley Fever Virus on Viral Replication in Mammalian and Mosquito Cells and for Genome Packaging PLoS Pathogens, 10 (2) DOI: 10.1371/journal.ppat.1003922

Viral polymerases and pathogenesis

noreply@blogger.com (Anonymous) — Sun, 16 Feb 2014 17:19:00 +0000

Read an introduction to viral evolution and you'll pretty quickly come across a sentence equating to 'viruses mutate and evolve fast and this is because their polymerases lack proofreading ability'. Sure, but that's a big generalisation. Large DNA viruses, for example, tend to have polymerases that are much less error-prone (again a generalisation). Nevertheless, in the world of RNA viruses it is a good rule of thumb that the polymerases tend to be error-prone when copying their genetic code. Superficially, such apparent laxity would appear to be detrimental to virus survival. However, if faithful replication of a nucleic acid was important, then the replication would indeed be faithful, but virus evolution occurs at a population level as opposed to the individual.

The viral sequences in GenBank are consensus sequences, in essence a read-out which represents the most commonly occurring base at each particular position in the genome. In a sense the concept of the consensus can be somewhat misleading: if RNA viruses have a mutation rate that is sufficiently great that it cannot copy a genome without making an error, then it's conceivable that the consensus per se does not, in reality, actually exist. Instead, a virus is a population of sequences evolving together, exploring sequence space with a great level of diversity.

Virologists tend to roll out the term quasispecies to describe this, but there remains some debate as to the validity of this theory, certainly in relation to Eigen's initial proposal.

Survival of the flattest: when mutation rates are low, all viruses accumulate together; if the peak is steep and narrow, then a low mutation error results in all of the viruses being exceptionally fit, (A outcompetes B). If mutation rates are high, then fitness is spread, in which case a spread of highly fit viruses is preferable to an extremely fit virus surrounded by viruses of low fitness (B outcompetes A, from Wilke 2005).

Having such diversity means that the virus can tackle things that get in the way of progress, most obviously an immune response; if the response impacts upon the fitness of one sequence, then there's another that is capable of preserving the virus' existence. In order to generate such diversity, a virus must necessarily make mistakes and thus viruses tend to live quite close to the maximum permitted error rate for a particular lifestyle. Too much error, and the virus suffers error catastrophe and becomes extinct, as there are insufficient fit sequences. Too little error though, and sufficient diversity becomes hard to generate.

A paper came out recently describing a variant of Chikungunya virus (CHIKV) with altered levels of fidelity. A CHIKV mutant had previously been isolated by growing the virus in the presence of the antiviral ribavirin, which resulted in a virus with increased fidelity. This virus had a single mutation in the viral polymerase. The authors systematically changed this amino acid and looked at the effect it had. Of the 19 options attempted, 12 viruses were viable.

Mutator variants: A. the impact of different amino acids on the susceptibility to ribavirin treatment. B. mutation frequencies of selected variants compared to wild-type virus; G and W result in increased rates of mutation. C. average diversity of confirmed fidelity variants at each point across the genome; anti-mutator variant with a Y results in lower diversity across the genome. D. due to the greater diversity, mutator strains are better able to escape virus neutralisation by antibody (CHK-102).

If these viruses have altered fidelity, then they should have different sensitivities to ribavirin, and indeed some viruses were less sensitive to ribavirin (antimutator strains) whereas some were more sensitive to ribavirin (putative mutator strains). When they checked for sequence diversity, they found a matching trend that sensitivity to ribavirin correlated with diversity, i.e. mutator viruses that make mistakes are susceptible to the treatment. A more highly mutated genome would in theory result in more non-viable/unfit genomes, and indeed they found that the progeny virus population of mutator strains was less infectious, even though their replication in mammalian cells was largely unaffected. When they injected some of the viruses into mice, there was a correlation between the frequency of mutation and viral loads. In line with the in vitro data, the mouse model revealed a decrease in pathogenicity associated with the increased error rates of mutator strains.

Whilst mutator strains replicated fine in mammalian cells, they replicated poorly in mosquito cells. As might be expected under such pressure, in vivo infections of mosquitoes, as well as passage in insect cells, resulted in a reversion of the mutator strains to a more wild-type level of fidelity. It would be interesting to see whether these viruses could be transmitted to subsequent hosts via mosquito bites.

Mosquito infections: Aedes albopictus (A-C) or Aedes aegypti (D-F) mosquitoes were fed blood containing wild-type (WT) or mutator versions of CHIKV. Virus levels of all viruses was equivalent in the

Another way in which they confirmed the impact of the changes associated with the polymerase fidelity and its impact upon virus phenotype was to specifically alter the equivalent position in another alphavirus: Sindbis virus (SINV). When they tested the mutated version of SINV, they found the same outcome, notably relatively little impact upon replication levels in mammalian cells, attenuation of pathogenicity in mouse models, lowered titres in insect cell cultures, and reversion to 'wild-type' in mosquito infections, thus confirming the importance of the mutator phenotype.

Mutator (and antimutator) viruses such as these may offer ways in which we can study further how viruses live close to the error threshold - perhaps with the prospect of adding evidence for or against the 'quasispecies' concept.

But replication competent virus but with attenuated phenotype? This rings the vaccine bell. At least potentially. There's more to do, and the authors highlight this, in particular with regard to how the viruses under selective pressure in vivo will behave and evolve in different hosts. But they're certainly an intriguing prospect.

Rozen-Gagnon, K., Stapleford, K.A., Mongelli, V., Blanc, H., Failloux, A-B., Saleh, M-C., Vignuzzi, M. (2014). Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models PLOS Pathogens DOI: 10.1371/journal.ppat.1003877

Dengue in Viet Nam

noreply@blogger.com (Anonymous) — Wed, 22 Jan 2014 22:02:00 +0000

In some, maybe the majority of cases, economic development tends to improve a country's situation regarding infectious disease; a proper sewerage system, for example, may decrease the incidence of diseases associated with the contamination of water sources. Where dengue virus is concerned this is not the case. Dengue is a human virus spread by mosquitoes of the genus Aedes (in particular A. aegypti), which happily breed in dirty water. Economic development tends to result in increased urbanisation and, as a result, ideal breeding conditions are generated for the mosquitoes (tin cans, old tyres etc.). Together, the result is a dense population of humans in the same location as the mosquitoes: in the absence of a vaccine or antivirals dengue has thus thrived. Whilst the conditions are favourable for dengue in general, there are inevitably more specific drivers of transmission and outbreaks.

A recently published study in PLOS Neglected Tropical Diseaes by Rabaa et al looked into what the drivers are for dengue in Viet Nam. The situation in Viet Nam can broadly be regarded as the south (tropical) region being endemic, whilst the north (sub-tropical) is not endemic, but experiences frequent introductions. In central Viet Nam the virus can persist for more extended periods of time, perhaps due to more favourable conditions for transmission and, ultimately, a higher level of immunity. As an illustration as to the impact of urbanisation, Ho Chi Minh city in the south is highly endemic and represents a large source of viruses for the rest of Viet Nam.

The authors compared the dengue serotype 1 (DENV-1) sequences of the envelope (E) gene.

Using a maximum likelihood approach to get an additional grasp of geographical relationships, they found that, perhaps unsurprisingly, all of the sequences belonged to the Southeast Asia subtype of Genotype I.

Phylogeography of DENV-1 genotype 1 in Southeast Asia, 1998-2009. A) Map of north (red) central (yellow) and south (blue) Viet Nam. The colours match in the graphs of mean min/max temperature (B, top graph) and mean precipitation (B, bottom graph), and branches in the maximum clade credibility phylogenetic tree (C). Purple branches represent Singapore sequences.

On the whole, DENV-1 seems to invade subtropical northern Viet Nam regularly, but never seems to become endemic - most likely due to the cold winter temperatures resulting in conditions that are refractory to continued transmission. Such invasions also occur in the central regions, although these persist for longer.

Interestingly, although (as may be expected) within a particular region the diversity among viruses was limited, on a broader scale Ho Chi Minh City in the south was found to act as a source of virus throughout Viet Nam.

On the other hand, despite local diversity being low, it's interesting that geographically long distance movements were observed in a time-scale that precludes the hypothesis that it's merely natural spread via vectors. Instead, it appears that the movement of infected humans is responsible for seeding at least some of the regions. This is one route by which the north can be seeded. However, because the north is sub-tropical, there comes a time in the year when the vectors die off and transmission is reduced; a familiar scenario with non-tropical arboviruses.

As interesting a piece of work as this is in itself, it arguably demonstrates something important at a more global level. Clearly DENV can be seeded in different regions by people moving around Viet Nam; if this can happen within Viet Nam, then it's not a massive step to extend Viet Nam to the world.

Maia A. Rabaa, Cameron P. Simmons, Annette Fox, Mai Quynh Le, Thuy Thi Thu Nguyen, Hai Yen Le, Robert V. Gibbons, Xuyen Thanh Nguyen, Edward C. Holmes, John G. Aaskov (2013). Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance PLOS Neglected Tropical Diseases DOI: 10.1371/journal.pntd.0002581

Bats vs. Rats

noreply@blogger.com (Anonymous) — Fri, 03 Jan 2014 11:51:00 +0000

If I had to name one book that got me interested in viruses it would be 'Virus X: understanding the real threat of the new pandemic plagues', by Frank Ryan. The book largely concentrates upon virus emergence; why it happens, where the viruses come from, and what that might mean for the future. Whether or not I now agree fully with everything that's hypothesised is a different matter, although an honest evaluation is difficult considering the advances in science since its publication (1997). Nevertheless, it got me interested at the time.

Flicking back through it last night I read a line regarding virus reservoirs that stood out. "The threat to humanity derives in particular from rodents". This was the logical conclusion derived from the fact that rodents are the most numerous mammal, which is fair enough. Nowadays it's almost all about bats. In the book Ryan does point out the suspicions of bats as reservoirs, but overall in this book the potential significance of bats is over shadowed by a focus on rodents.

With our current knowledge of virus natural reservoirs (a term itself worthy of debate), a suggestion that anything other than bats are the most important source of viruses as far as public health is concerned, is likely to be met with an element of scorn. Bats do indeed harbor a lot of viruses, as was published earlier this year. In this particular paper the authors also estimated the numbers of viruses still to be discovered (320,000), although wisely they also stated that such a calculation was based upon some rather large assumptions:

"Several important limitations must be considered in our extrapolations, including (i) the assumption that a mean of 58 viruses per species is a reasonable estimate and that host populations are panmictic with respect to viral transmission (such that expanded geographic sampling would not influence viral detections), (ii) the assumption that viruses are not shared by more than one host species, (iii) that only those viruses within the nine families are considered in this estimation, (iv) that the results are limited by the sensitivity and specificity of our tests, and (v) that a similar mean cost of sample collection is incurred across all species." (Anthony et al. 2013, mBio)

Nevertheless, it's a useful number to have.
Bats have long been suspected as reservoirs, and in the case of rabies it had been firmly established, but I'm not sure when exactly they became so popular for virus hunters. Perhaps around the time of Nipah and Hendra emergence. Nowadays everybody seems to be hunting for viruses in bats specifically.

Bats, it can safely be said, represent an important source of novel as well as known viruses. In terms of virus emergence and spread however, there is more to it. Yes, bats may harbor a lot of viruses. And perhaps yes for one reason or another those viruses may have a higher chance of being unpleasant. But there is more to epidemiology than simply the source. Spillovers in a forest/rural setting are inevitable, and in this case bats pose as much of a risk as rodents. However, the majority of people live in urban areas. And from this perspective, rodents are surely of greater importance for transmission as their populations are so intimately linked with humans. More contact means a greater likelihood of transmission. One of the worst epidemics in history, the Black Death (admittedly caused by a bacterium), was closely linked with rats. More recent, viral, examples include the Sin Nombre hantavirus in New Mexico, and the Arenaviruses (e.g. Lassa fever virus).

It could be argued that, because we've had so much interaction with rats over the years we're unlikely to find anything new. That doesn't mean they're of lesser importance; Lassa fever and Sin Nombre are responsible for the death of more people than those caused by more exotic viruses such as Ebola virus.

Global air travel: 'emergence hotspots' such as South East Asia experience more international travel than central Africa. Image: Max Planck Institute for Dynamics and Self-Organisation/ Dirk Brockmann http://www.ds.mpg.de/3004/news_publication_4406928?c=148862

It is clear that the jungles and savannas of Central Africa harbor bats with viruses of great danger to humans. But is this more important than, say, the populations of rats in densely populated urban centers in South East Asia? Human traffic to and from such urban areas is higher, enhancing the probability of an infection spreading to other parts of the world; would SARS, for example, have spread so far if it had emerged in Uganda? In the future it may be that the world is equally connected. For now though, some places remain more connected than others, and this should be remembered when people decide what's more important: bats or rats.

Anthony SJ, Epstein JH, Murray KA, Navarrete-Macias I, Zambrana-Torrelio CM, Solovyov A, Ojeda-Flores R, Arrigo NC, Islam A, Ali Khan S, Hosseini P, Bogich TL, Olival KJ, Sanchez-Leon MD, Karesh WB, Goldstein T, Luby SP, Morse SS, Mazet JA, Daszak P, & Lipkin WI (2013). A strategy to estimate unknown viral diversity in mammals. mBio, 4 (5) PMID: 24003179

Should I have published?

noreply@blogger.com (Anonymous) — Mon, 18 Nov 2013 22:00:00 +0000

Since finishing my PhD I've been faced with a dilemma. In a nutshell, having come across a (somewhat serendipitous) observation in my PhD studies, should I publish it? I decided to publish, and it was both an editor's pick and is now regarded by the journal as 'highly accessed' (the importance and possibly ephemeral nature of such labels is a completely different discussion). That implies it was worthwhile, but was it?

The study revolved around an observation in BHK (hamster) cells infected with Bluetongue virus (BTV). The cells looked very strange: rounded and with condensed DNA/chromosomes in a pattern suggestive of some stage in mitosis, albeit a rather odd looking mitosis. To try and see what's going on, we used confocal microscopy with a panel of antibodies to look at the status of various parts of the cell division machinery. In brief, we found that the centrosome, a major orchestrator of mitosis, was severely disrupted. Co-incidence or not, the BTV protein non-structural protein NS1 also located in the region.

A-D. Different BTV serotypes (16, 1 and 8) induce aberrent mitoses (although BTV-16v induces the most). Different cell types can also be affected, although BHK cells appeared to be the most susceptible.

Something that was conspicuous was the association of the viral NS2 protein with the condensed chromosomes. When we took a series of images in the z plane and analysed them it became clear that NS2 appeared to be associated with the kinetochore. Combined with the observation of its location on microtubules, it is conceivable that NS2 may be a microtubule cargo molecule (or interacting with one) that obscures the kinetochore during the initial stages of mitosis. As the microtubules polymerise though the cell, the tips don't find the kinetochore, resulting in faulty mitosis. Many viral proteins use microtubules to get around and, based on other viruses, the dynein/dynactin complex would be an interesting place to start looking for a protein that interacts with NS2.

A. NS2 expressed from a plasmid locates to microtubules (red). B. Z stack images reveal NS2 located at positions suggestive of the chromosome centromeres. C and D. Expression of NS2 from a plasmid recreates the aberrent mitotic phenotype.

To look at whether NS2 alone is capable of inducing the aberrent mitosis, we transfected cells with plasmids encoding the protein. When looked at from a confocal perspective, the transfected cells appeared to reflect the phenotype seen with virus infection. When a GFP-tagged version of NS2 was used in live cell imaging, we found that the cells were less likely to complete mitosis correctly, spent longer in mitosis, and resulted in an increased level of binucleated cells.

Transfecting HeLA cells with a palsmid expressing a GFP-tagged version of BTV NS2 resulted in a longer time spent in mitosis, a reduced level of successful mitosis, and binucleation.

So, to the options.

1) don't publish. At the end of the day it's just an observation; I have not elucidated an exact mechanism and nailed down a precise protein, as would be expected for a publication in a journal of greater 'impact'. Not taking the story to an end, followed by publishing in a prestigious journal might be viewed as poor science by some.

2) publish. Many would argue that publishing information, regardless of how seemingly insignificant, is important and, arguably, a necessity based on the fact that it is being funded by the public.

I published. Partly for the reasons outlined in scenario 2, but also because the study was at a point where other people had contributed work, in which case it would not be fair for them to have done this work only for me not to publish. Of course, continuing the project to the end would have been my (and my collaborators') preferred option, but time ran out. As it stands, this observation is in the public domain for all to see, with the option of progressing it further to try and unravel what's happening.

Should I have published? I'm satisfied that I did, but it once again highlights the question of how many other such observations are languishing in abandoned lab books around the world.

Andrew E Shaw, Anke Brüning-Richardson, Ewan E Morrison, Jacquelyn Bond, Jennifer Simpson, Natalie Ross-Smith, Oya Alpar, Peter PC Mertens and Paul Monaghan (2013). Bluetongue virus infection induces aberrant mitosis in mammalian cells Virology Journal DOI: 10.1186/1743-422X-10-319

Down on the farm with Schmallenberg virus: the full story

noreply@blogger.com (Anonymous) — Sun, 10 Nov 2013 13:27:00 +0000

I've already written a couple of posts about Schmallenberg virus (SBV), a bunyavirus that emerged in Northern Europe in 2011. What I haven't discussed is the SBV experience on my family's diary farm. Clearly an opportunity not to be missed, this has just been published. The thing that scientific publications can't convey however is the meandering thoughts and subjective observations that have little or no scientific rigour. A scientific manuscript can only report concrete and measurable results. Hence this post.

SBV is difficult to spot as the most dramatic clinical signs tend to be malformed offspring, which is a somewhat rare occurrence, at least in cattle herds. As a result there is a period whereby the virus may have been around for several months before it is discovered: when we first found SBV on the farm, the UK was more or less at this stage. In February 2012 there were only a few reported cases of SBV, all around the SE fringes of England where SBV-laden Culicoides had presumably been blown across the channel.

No doubt at least partially as a result of my ongoing comments on the phone about the SBV situation, when a cow oddly aborted close to term, "could it be SBV?" was a question that immediately arose. The cow, number 157, along with some others, was bled and the samples sent to me in Glasgow, where I tested it for SBV antibodies. The result was a clear positive for SBV.

The first ELISA result of SBV on the Bishops Farm. C2 and D2 = cow 157. A4-D4 = positive control.

And was positive again when a second sample was taken.

Another way in which to determine whether #157 was positive for SBV antibodies was to immunolabel some cells infected (or mock infected) with SBV. Serum from #157 clearly detected SBV whereas serum from the other animals didn't react.

(a) the s/p output values from the antibody ELISA of the cows tested following #157's abortion. When serum from #157 was used to immunolabel cells, green signal was only seen in cells infected with SBV.

SBV was present on the farm. At the time, this was several degrees further north than the known distribution. We tested to see whether the antibodies that were recognising the virus were IgM isotype (in which case the infection was recent) or IgG, meaning that the infection was older. Everything was IgG, so the infection had been around for a bit. How long had it been in the area?

In essence this was a 'just in time' scenario; soon virtually all of the UK's farms would be positive for SBV. I found it hard to believe that vet schools weren't already screening their flocks and herds, but here was an opportunity to look at seroprevalence at the herd level in a 'typical' UK commercial dairy farm. So every animal in the herd was sampled and tested for SBV antibodies. An important aspect is that no animals were moved onto the farm during the previous months, therefore the SBV must have arrived by some other means - clearly the likely option being midges. Only a few of the herd were positive, but clearly SBV was present.

During the summer two deformed calves were born. In over 20 years previously, only a single deformity had occurred in this herd. What's more, the dead calves had issues with joints, something that would be consistent with the deformities observed with SBV. One in particular had features that looked extremely similar to those in the literature that had been confirmed as having SBV, including fused and stunted limbs.

A dead calf with clinical features sugestive of SBV, including stunted and fused joints, most obviously a suggestion of arthrogryposis in the hind legs.

The other calf seemed quite the opposite, as if there were no joints, resulting in a floppy carcass, even if the calf otherwise (outwardly) looked fine.

A deformed calf born in the summer, with a 'bag of bones' type deformity.

Two deformed calves, knowing that SBV had been present at the crucial time, certainly seemed suggestive that these deformities were as a result of SBV. But this isn't in the paper as we can't state that they were the result of SBV without testing them for the presence of the virus. A post mortem would have been revealing.

Another thing that is rather superficial in the paper is another aspect often associated with SBV, changes in milk yields. Overall there was a depression in the milk yield, but there's no way of proving that this was not because of some other factor. What was more dramatic were sudden acute periods of no milk combined with what appeared to be severe depression, but again it's impossible to say that this was as a result of SBV. If we'd tested these animals and it had coincided with SBV viraemia, then perhaps we could say it was related. In reality, these acute episodes are the most commonly observed clinical feature of SBV, at least in cattle, with the deformed offspring representing the exception rather than the norm - there were many other calves born that were perfectly fine. Somewhat frustratingly it is the deformities aspect that people most want associated with SBV, thus that's what dominates in SBV papers and talks, including this paper. It's difficult to nail this kind of thing down though as everything is by association rather than causation, and in many cases is difficult to measure, e.g. how do you know a cow is feeling rough due to SBV?

In the paper there's a mention of high levels of diahorrea. Again, this is difficult to a) quantify and b) inextricably link to SBV circulation in the herd.

When it got colder, and the midge season was theoretically over, we tested again. This time the majority of the animals were positive for SBV antibodies. Clearly SBV had spread throughout the herd over the summer period.

The proportions of animals in the milking herd that were positive for SBV either before or after the summer period.

This is not in the least surprising. A more dramatic result would have been if there had been any other outcome. The interesting fact though is that, during the summer, the milking cows were at pasture for only a few days. Dogma until a few years ago was that midges are generally reluctant to enter livestock housing. This was based primarily upon observations in the field of Bluetongue virus (BTV), where the Afro-Asiatic species C. imicola is the key vector. It is now established that European midges are perfectly happy to enter buildings. As well as exposure to vectors, another key driver of arbovirus transmission is temperature, which affects both the biting behavior of the vector, and also the kinetics of virus replication within the midges. When the first case in #157 was found, the temperature was only around 10 degrees. The obvious caveat here being that this temperature is the outside temperature; inside it's warmer - that temperature would be very interesting to know. This is relative though: it may be warmer inside but that's relative to 10 degrees; even if it was 5 degrees warmer that's still only 15 degrees. This is still quite cool.

Overall the message would seem to be that not letting the animals spend their days and nights roaming freely at pasture is no barrier to arbovirus transmission. This perhaps shouldn't be surprising. It's warmer, the breeding habitat is textbook for Culicoides, the animals are closer together, there's no wind etc. and there is, inevitably still exposure to the outside.
I'm clearly biased, but the beauty of this study remains that it reflects a real situation. This is not a controlled vet school farm. It is not a sentinel herd kept to check for the first incursion. It is a working dairy farm that more accurately reflects the average scenario for the UK.

And lastly, in case you wondered, #157 has a name: Blossom.

A. E. Shaw, D. J. Mellor, B. V. Purse, P. E. Shaw, B. F. McCorkell, M. Palmarini. (2013). Transmission of Schmallenberg virus in a housed dairy herd in the UK Veterinary Record DOI: 10.1136/vr.101983

Filming fluorescent Marburg virus

noreply@blogger.com (Anonymous) — Mon, 07 Oct 2013 18:25:00 +0000

For some Marburg is a city in Germany. It's also the name of a virus closely related to the much more widely known Ebola virus (a name which people tend to associate with a virus as opposed to the small river it's named after). What they both have in common, beyond both being members of the Filoviridae family, is a propensity to induce highly unpleasant, and often lethal, haemorrhgagic fevers. Marburg virus (MARV) first surfaced in 1967 in laboratory workers in Marburg and Yugoslavia and, just like Ebola, has caused sporadic cases and outbreaks since then, the most horrifying of which was in 2004-2005 in Angola, where 227 of 252 (90%) of those known to be infected died.

The worm-like form of Marburg virus particles.

As per many viruses, much about the MARV lifecycle within the cell remains a mystery. A recent paper in PNAS used live cell imaging to dissect some of the events involved in making new viruses and how they shuttle to a point of release.

Live cell imaging is often based upon fluorescence, so one of the first things was to make the tools. Essentially, they made versions of structural viral proteins, VP30 and VP40, that are tagged with a fluorescent molecule. To VP30 they added green fluorescent protein (GFP) and showed that when expressed from a plasmid it behaved like the untagged VP30. Similarly, they inserted a red (RFP) version of VP40 into the genome of the virus, such that wild type (wt, = unmodified) and tagged VP40 were produced. The new virus behaved similarly to the unmodified virus, at least early during infection. In infected cells, RFP-VP40 colocalised with wt VP40, implying that this modification didn't alter its localisation. Tools made.

The first step of virus production/release they looked at was the exit of nucleocapsids from the inclusions where nascent viruses are thought to assemble. When they filmed inclusions, VP30-GFP was seen to be leaving, confirming this is where nucleocapsids are assembled, but not with RFP-VP40 (despite VP40 being present at the inclusion body), leading to the conclusion that VP40 is added elsewhere.

Nucelocapsids leaving the inclusion. Individual nucleocapsids could be seen leaving (top) and of those leaving, VP30 but not VP40 was present (bottom)

If the VP40 component of particles is being added somewhere other than the inclusion, then the obvious question to ask is 'where does VP40 get added?'. Again, they turned to microscopy. When they counted the number of nucleocapsids containing both VP40 and VP30, the number increased towards the plasma membrane, implying that it is here that VP40 associates with the nucleocapsids.

VP40 gets added near the plasma membrane: the closer a nucleocapsid is to the plasma membrane, the greater the likelihood that it also contains VP40, suggesting that it is at the cell periphery that VP40 becomes associated with nucleocapsids.

A bonus of filming an infection is that there is an additional parameter, i.e. time. This means that the speed at which things happen can be worked out. In this case the authors were able to work out that the nucleocapsids moved at up to 500 nm/sec. On top of that, they were able to figure out that the movement was quicker towards the centre of the cell, as opposed to more remote regions, possibly because nucelocapsids are using different motor proteins in different regions to surf the cytoskeleton. But which component of the cytoskeleton? Two approaches were used. First, they filmed VP30-GFP labelled nucleocapsids in cells with either red tubulin or red actin: only in the case of actin was the movement consistent with riding a particular filament .

In the second approach, treating cells with nocodazole, which disrupts microtubules, had no effect whereas cytochalasin D disruption of the actin filaments brought MARV movement to a halt. In both cases actin appears to be the answer.

Lastly, they looked at the presence of the nucleocapsids in the filopodia extruded from the infected cells. From their observations, they concluded both that VP40 must be associated with the nucleocapsids, and that the motor protein Myosin 10 (Myo10) is involved in the transport of nucleocapsids in the filopodia.

This work is impressive for many reasons. most immediately obvious is the reliance upon live cell imaging. Very often there are the (reasonable) requirements for observations in the microscope to be backed up by biochemical data. It's a great example of what can be achieved via deductions made from observations in careful experiments. The difficulty in doing this work is also easy to overlook. I get the impression that doing this project in BSL-4 would be tricky. Conveniently, they had a remote controlled microscope that they could operate from a more comfortable location, something that's rather handy if you're going to film fluorescent Marburg.

Gordian Schudt, Larissa Kolesnikova, Olga Dolnik, Beate Sodeik, and Stephan Becker (2013). Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances Proceedings of the National Academy of Science DOI: 10.1073/pnas.1307681110

Bluetongue, Schmallenberg.......Oropouche?

noreply@blogger.com (Anonymous) — Sat, 28 Sep 2013 13:08:00 +0000

Until relatively recently, Culicoides midges were probably most famed for being an annoyance to people walking, camping, fishing etc. in the Scottish hills. And this is justified - a pub I went to this summer even provided a selection of repellents for their customers. Horse owners most likely associate them with sweet itch, perhaps also that they transmit African Horse Sickness. Many UK and European cattle and sheep farmers would not have thought Cuilcoides were of any significance beyond being a nuisance to their animals.

Next to the whisky and Tennents this Highlands pub provides insect repellents for its customers.

This all changed in 2006, when a severe strain of the Cuicoides transmitted Bluetongue virus (BTV) seemingly parachuted into the middle of Northern Europe and spread wildly, with high levels of morbidity and mortality. The following year BTV arrived into the UK, probably as a result of infected midges being blown across the channel. Fortunately for UK farmers BTV didn't get very far that summer, and by the following year there was a vaccine available. Nevertheless, Culicoides were now very much in the conscience of farmers, which meant that they were fully aware of what may happen when Schmallenberg virus (SBV), another Cuicoides transmitted virus, was discovered in Germany in 2011. Sure enough, a large proportion of the UK sheep and cattle are likely to now be immune to SBV as a result of it sweeping the country in the last couple of years.

The obvious question to ask is, 'what about humans?'. There are several other livestock viruses that are spread by Culicoides, most importantly African Horse Sickness Virus, but if livestock viruses can spread so dramatically, what would happen if a Culicoides-borne virus arrived that could infect humans. The obvious candidate is Oropouche virus, which is from the same family of viruses, the Bunyaviridae, as SBV. This is discussed as an example in a recent paper by Carpenter et al discussing the impact of Culicoides on public health. In the case of OROV, C. paraensis is the primary vector involved in epidemics that occur in south America. An interesting, and potentially important, fact is that the biting patterns of C. paraensis are different from the Culicoides species found in the UK and Northern Europe. Whereas C. paraensis bites at a low rate during both day and night with relatively little impact upon human behaviour, European midges bite so aggressively that people will often seek shelter, effectively reducing exposure levels.

Where do midges occur? A) the overlap of livestock and Culicoides,
and B) the overlap of farmland (i.e. habitats for midges) with urban areas. From Carpenter et al 2013.

If you ignore the aspect of whether there's a virus that fits the requirements, the authors put forward some reasons why an outbreak is perhaps unlikely. Firstly, the biology of the vector may simply mean that not enough survive long enough to transmit an arbovirus. Secondly the habitats of European midges don't tend to overlap with humans as much, thus reducing exposure. And thirdly European midges are seasonal, as opposed to the year-round presence of C. paraensis. All of this suggests that Culucoides may be of relatively limited impact for the transmission of viruses among humans, although they may still facilitate spillover events from animals to humans. For the time being it appears that, where viruses are concerned, Cuicoides appear to be of more importance for livestock diseases. What is for sure though, is that they will remain a pain for anybody wandering the Scottish hills in summer.

Carpenter S, Groschup MH, Garros C, Felippe-Bauer ML, & Purse BV (2013). Culicoides biting midges, arboviruses and public health in Europe. Antiviral research, 100 (1), 102-113 PMID: 23933421

Hantavirus and leaky vessels

noreply@blogger.com (Anonymous) — Tue, 30 Jul 2013 19:54:00 +0000

I once saw a video in which the lung of a Bluetongue Virus (BTV) infected sheep was cut open at post-mortem. As the scalpel cut in, it was clear the lung was full of fluid. Lungs full of fluid don't work very well. As a result, sheep infected by BTV, as well as horses infected with African Horse Sickness Virus, will often die by drowning simply as a result of their vasculature leaking fluid into the lungs. I've often considered this reminiscent of what happens with Hantavirus pulmonary syndrome (HPS), caused by new world hantaviruses, including Andes virus and Sin Nombre virus (the latter of which causes sporadic outbreaks across North America). In Eurasia there are related viruses, including Hantaan virus, which cause Hantavirus haemorrhagic fever with renal syndrome (HFRS). Although different, both diseases involve vascular leakage.

A deer mouse: the wild reservoir of hantaviruses in the New World.

Endothelial cells, those that line the capillaries and other blood vessels, aren't damaged during infection, so how do the vessels become leaky? One suggestion has been that it occurs as a result of the cytokine arm of the immune response, although removing the T cells modulating cytokines appears to have limited impact upon pathology. An alternative hypothesis is that vascular endothelial growth factor (VEGF), which is reportedly elevated during hantavirus infections, degrades vascular endothelium cadherin (VE-cadherin), a molecule with importance for vessel integrity. A recent paper in PLoS Pathogens by Taylor et al contradicts some of this, and suggests a further possibility: activation of the Kallikrein-kinin system (KKS),which leads to the release of bradykinin (BK). BK in turn is an inflammatory molecule that leads to vasodilation and increased vascular permeability.

Although nothing can replicate the real thing, the authors created their own capillaries in a dish and showed that they could be infected. When they looked for a decrease in VE-cadherin (as per hypothesis 2 mentioned above), the levels appeared more or less equal regardless of infection, likewise the amount of VEGF released from the artificial capillaries did not alter significantly as a result of infection.

Home made capillaries: Hantavirus infection (indicated by the presence of nucleocapsid) appears not to alter the expression of vascular endothelial cadherin. Similarly, capillaries infected with ANDV or HTNV still produce VEGF.

When they looked for BK release as a result of activating the KSS, they found a dramatic increase when the capillaries, or the cells which are used to make the capillaries, were infected with either Hantaan or Andes virus and treated with molecules that would be found in the blood stream of infected patients (FXII, PK and HK). This implies that hantavirus infection induces permeability as a result of a more active KKS.

BK production by cells infected with hantaviruses: Cells infected with HTNV or ANDV produce more BK (and by extension enhanced permeability) relative to mock infected, although less pronounced in the case of pulmonary artery smooth muscle cells (PaSMC).

Clearly, the KKS was working OK, but more so in cells infected with the viruses. An important aspect is the cleavage of HK which ultimately leads to the release of BK. The authors looked at HK cleavage in the presence of FXII and found that cleavage was enhanced in its presence. Going further, when they added an inhibitor of FXIIa (the activated form of FXII), they found the cleavage no longer occurred; FXII is clearly therefore required in the system.

Measuring resistance: resistance is used as an indication of permeability. Infected cells have enhanced permeability when the KKS factors are applied (A). B-D show the effect on mock, HTNV or ANDV infected cells with the inhibitors CTI (blue) PKSI-527 (red) or HOE 140.

All of these, and some other, experiments suggest that the KKS and BK liberation may, at least in part, be responsible for the leaky vasculature as a result of hantavirus infection. To look at this the authors used electric cell-substrate impedance sensing to measure the resistance/permeability (leakiness) of confluent layers of endothelial cells infected (or not) with hantaviruses. Cells that were infected, i.e. effectively had an activated KKS and BK present, showed a decrease in cell resistance of up to 50%, compared to a maximum drop of only 10% observed in uninfected cells. The addition of inhibitors against the KSS altered the pattern, reducing the effect observed in infected cells, thus directly implicating alterations in the KKS as being the cause of these changes.

The KKS system and Hantavirus infection: HK and PK are enhanced on the surface of infected cells, leading to cleavage of HK (involving FXII) and subsequent release of BK, and thus enhanced permeability of the endothelial layer. Several drugs are available which target the pathway.

It remains possible, indeed likely, that there are other factors that control the vascular permeability and therefore pathogenesis of hantaviruses. Lungs filling with fluid isn't unique to hantaviruses and there are likely several mechanisms yet to be deciphered. This study does though highlight a new pathway of interest that leads to leaky vessels, and, importantly a pathway for which there are inhibitors: perhaps the leaks can be mended.

Taylor, S.L., Wahl-Jensen, V., Copeland, A.M., Jahrling, P.B., Schmaljohn, C.S. (2013). Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System PLoS Pathogens DOI: 10.1371/journal.ppat.1003470

What's Killing the Koalas?

noreply@blogger.com (Anonymous) — Fri, 05 Jul 2013 22:53:00 +0000

My current employer made his name working on a retrovirus: Jaagsiekte sheep retrovirus (JSRV). JSRV is a betaretrovirus whose greatest claim to fame is killing Dolly the sheep, but it has revealled many aspects of sheep and retrovirus biology. One of the attributes most associated with viruses is that they're obligate intracellular parasites: without a cell to replicate in, viruses are often little more than a bunch of molecules. Retroviruses take this to the extreme and insert into a cells genomic DNA in order to replicate. At it's simplest this involves the virus like any other infecting a somatic cell, intergrating into the cellular DNA, replicating and exiting. This is the 'exogenous' form.

Alternatively, if the cell happens to be a germ cell, from which sperm or eggs are produced as a precursor to another individual, the retroviral DNA will be inherited by Mendelian inheritance as for any other gene. When this happens, the virus is now regarded as being 'endogenous'.

In the last few years a type-C retrovirus, Koala retrovirus (KoRV), has been linked to the development of Koala immunodeficiency syndrome (KIDS). As the term suggests, KIDS results in a depleted immune system, resulting in enhanced vulnerability to infectious diseases and cancers. KIDS has become a prominent killer of koalas, particularly those in captivity, where the majority of studies have been performed. KoRV's closest relative appears to be gibbon ape leukemia virus (GALV) which, like KoRV, causes lymphomas and leukemia.

KoRV represents an example of a very young endogenisation event, with the intergation event thought to be only around 100 years ago, and the integrated copies are able to generate infectious viruses. A recent paper in PNAS describes the isolation of a variant of KoRV in San Diego and Los Angeles zoos (Xu et al 2013). All of the koalas at the zoos contained the endogenised form of KoRV. However, in six koalas at Los Angeles zoo, including 3 that died, they found an additional, slightly different, KoRV sequence (KoRV-B, as opposed to the original KoRV-A). The majority of the changes were in the U3 region of the long terminal repeats (LTR). Particularly striking was that parts of the virus responsible for binding to the cell receptor looked more like those of an exogenous virus as opposed to the more endogenous form possessed by KoRV-A. Indeed, when they looked at receptor usage, KoRV-B used a different receptor to KoRV-A and GALV.

Mother to Joey transmission: Joeys only become infected with KoRV-B when the dam is infected, even if the sire is positive, implying the virus is transmissible rather than inherited. (Xu et al 2013)

Further evidence of the infectious nature of KoRV-B came from the observation of infected (or not) joeys born to infected (or not) parents in a family at Los Angeles zoo. A positive joey was only born when the mother was positive; it was possible for a joey to be born negative for KoRV-B even if the father was positive for KoRV-B (as long as the mother is negative).

Koalas would appear in a bad way. However, endogenous retroviruses aren't necessarily harmful. On the contrary, some may be beneficial. A prime example is the JSRVs. The presence of endogenous JSRVs results in so-called 'late restriction'. Interference of exogenous JSRV replication by the presence of endogenous JSRVs is ultimately beneficial for the sheep.

Endogenous JSRVs: a variety of genomic arrangements of JSRVs found in the sheep genome. (Arnaud et al 2007)

Inevitably, as the sheep genome is targeted in further rounds of infection by exogenous JSRVs, a tug of war develops such that a balance exits between the late restriction imparted by endogenous JSRV(s) and the exogenous JSRV. The sheep genome has been invaded multiple times by JSRV, to the extent that the domestication of sheep can be traced based upon which endogenous JSRVs are present in the genome of a particular sheep breed (Chessa et al 2009).
Whether it's too late for something similar with the koalas, time will tell.

Wenqin Xu, Cynthia K. Stadler, Kristen Gorman, Nathaniel Jensen, David Kim, HaoQiang Zheng, Shaohua Tang,, & William M. Switzer, Geoffrey W. Pye, Maribeth V. Eiden (2013). An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1304704110

Arnaud F, Caporale M, Varela M, Biek R, Chessa B, Alberti A, Golder M, Mura M, Zhang YP, Yu L, Pereira F, Demartini JC, Leymaster K, Spencer TE, & Palmarini M (2007). A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses. PLoS pathogens, 3 (11) PMID: 17997604

Chessa B, Pereira F, Arnaud F, Amorim A, Goyache F, Mainland I, Kao RR, Pemberton JM, Beraldi D, Stear MJ, Alberti A, Pittau M, Iannuzzi L, Banabazi MH, Kazwala RR, Zhang YP, Arranz JJ, Ali BA, Wang Z, Uzun M, Dione MM, Olsaker I, Holm LE, Saarma U, Ahmad S, Marzanov N, Eythorsdottir E, Holland MJ, Ajmone-Marsan P, Bruford MW, Kantanen J, Spencer TE, & Palmarini M (2009). Revealing the history of sheep domestication using retrovirus integrations. Science (New York, N.Y.), 324 (5926), 532-6 PMID: 19390051

Agricultural Shows; a virus' dream?

noreply@blogger.com (Anonymous) — Sun, 23 Jun 2013 16:43:00 +0000

This week saw the annual event when farmers descend from all parts of Scotland to compare their animals: The Royal Highland Show. I went along and, after a while wandering around the tractors, had a look around the livestock. Looking at the plethora of pampered sheep and cattle, a few things occurred to me.

First was a half-hearted feeling of 'missed opportunity'. Sheep in particular, but also cattle, had come from across Scotland (and beyond). Imagine if a blood sample of every animal had been taken. Schmallenberg virus (SBV) is known to have reached, and circulated, in Scotland, but nobody knows to what extent. Perhaps testing these animals may have given a cross-section of SBV distribution across Scotland. Maybe even an idea of variation in breed susceptibility; although there's nothing really suggesting that such differences exist.

Show time: a Jacob sheep gets a final trim in the sheep yard prior to showing at the Royal Highland Show.

Second was considering the potential for transmission and dissemination of pathogens across wide geographical areas. In 2001 in the UK the dispersal of Foot and Mouth Disease Virus to various regions was due, at least in part, to the mixing and distribution of sheep from Hexham Market. Agricultural shows represent a similar type of mixing event, and last for days rather than hours. In the case of Foot and Mouth, the clinical signs are mild in sheep, thus allowing infected animals to go unnoticed.

Bringing animals to the show with clinical signs of disease is unlikely to happen as the idea is obviously to show the best animals. That restricts the possibility of infectious animals to those in a pre-clinical incubation stage. However, shows lasting for several days gives sufficient time in which to develop a viraemia and allow transmission, either via direct contact, fomites or vectors. Unsurprisingly there are already studies on the role of agricultural shows. One regarding UK shows (Webb, 2006) revealed competitors at shows to form a large network, with some competitors travelling to shows up to 600 km apart. Clearly there is potential, but thus far nothing seems to have been nailed down showing an outbreak resulting from mixing at agricultural shows.

The impact of time-between shows: the number of days between shows affects the network, no time limit results in a mass network (a) whereas time limits of 14 days (b), 10 days (c) or 7 days (d) reduce the number of nodes, effectively separating shows and breaking links. From Webb 2006.

Third was the zoonotic potential.
It is well established that cross-species transmission of zoonotic pathogens occurs at the interface between the animal source and humans. These shows represent perfect scenarios for such interactions. Largely absent from the Royal Highland Show were pigs, which have previously been shown to be a source of influenza A infection. Pigs may therefore represent more of a risk due to their ability to sustain various zoonotic agents. Likewise shows where chickens and ducks are present, offering the possibility for the transmission of avian influenza.

Odd choice: A sheep decides a coat is preferable to hay.

The idea of catching bugs from farm animals at these kind of events, as well as at petting farms for kids etc., is not new. And it is true, infections do happen. The knee-jerk response of a modern nanny state society, at least in the UK, would therefore dictate the banning of such scenarios, including all agricultural shows without question. But this is another case of putting risk into perspective; of all the millions of animals, and the millions of contacts with humans that have happened at these shows over the years, how many infections have occurred, or at least been serious enough to cause alarm? The simple answer: not many. For now though, the relative importance that agricultural shows play in the transmission, evolution and species transfer of viruses remains largely unknown.

Webb, C.R. (2006) Investigating the potential spread of infectious diseases of sheep via agricultural shows in Great Britain. Epidemiol Infect. 134(1)31-40. doi: 10.1017/S095026880500467X

Françoise Barré-Sinoussi visits the Centre for Virus Research

noreply@blogger.com (Anonymous) — Sun, 09 Jun 2013 22:25:00 +0000

Few stories in virology can have been more frequently or comprehensively covered as the early days of the HIV/AIDS epidemic. Randy Shilts' 'And the Band Played On' is surely one of the most detailed accounts and, whilst massive, provides insights into what went on in the first few years of the epidemic. If you don't fancy reading the book, there's always the resulting film to watch, although it concentrates much more on the heroics of the epidemiologist Don Francis.

And The Band Played On: Don Francis fights HIV in the face of politics and business.

There are so many aspects discussed both in the book/film and in general about the discovery of HIV as the cause of AIDS, but for me it is the politics which strikes the hardest. The political scene was set for the emergence of a disease like AIDS; a disease emerging largely in sections of society which a conservative government would rather not acknowledge. The original CDC Morbidity and Mortality Weekly Report MMWR) is rather poignant, now that we know what it represented - I've pasted it at the end (copied from the CDC Website).

Scientifically, politics became an issue too. A rather bitter rivalry built up between two particular virology groups as everyone raced to figure out the ultimate cause of AIDS. Now it seems obvious that it was a virus attacking T lymphocytes, but at the time things were much more unknown. As an example, one red herring in the early days was poppers/alkyl nitrates as a result of their association with homosexual bathhouses. Ultimately the rivalry ended in dispute as to who had discovered the viral cause of AIDS - something that, in theory, should have taken second place over concentrating on doing something about it. In the end, the French group lead by Luc Montagnier, (as opposed to the US group lead by Robert Gallo) are generally regarded as being the discovers of HIV - the cause of AIDS - and Montagnier won the Nobel Prize in Physiology or Medicine in 2008. Together in receiving the prize was Françoise Barré-Sinoussi. In 1983 Barré-Sinoussi published a paper in Science reporting the isolation of a retrovirus from a suspected patient - effectively reporting the viral cause of AIDS.

The first step was isolating the virus. Cells from a lymph node biopsy from the patient were cultured in growth medium. Samples were taken over several days, and tested for reverse transcriptase (RT) activity (a hallmark of retroviruses). After 15 days they detected RT activity, suggesting a retrovirus was present. When some of the 'infected' cells were mixed with healthy cells, RT activity was again detected, but only after 15 days, therefore suggesting that transmission had occurred. Media without the cells could also infect umbilical cord lymphocytes, and when these were looked at using electron microscopy, viruses could be seen budding from the cells.

HIV budding from the cell membrane

Importantly, Barré-Sinoussi showed that, in addition to isolating a virus with a predilection for human T cells, it was different to the known human T cell leukaemia viruses (HTLV) discovered to that date (largely based upon serum cross-reactivity, but also the fact that this new virus killed the T cells, as opposed to causing cancer in the case of HTLV-I and -II), backing up data that the genetic sequence was distinct from HTLV-I and -II . Altogether the data pointed towards a retrovirus as being behind AIDS.

Françoise Barré-Sinoussi receives the Nobel Prize for Physiology or Medicine in 2008.

Gallo was at times vilified, some might argue rightly so, but equally his work on T cells and viruses was fundamental towards the ability to isolate the virus in the first place. Thankfully things have placated somewhat and, when he visited the CVR, he gave an interesting talk about his views on what it is going to take to make a good HIV vaccine. My expectation is that Françoise Barré-Sinoussi's story will be equally enthralling.

The following MMRW essentially represents the first report of HIV:

Pneumocystis Pneumonia -- Los Angeles

MMWR 1981;30:250-2 (June 5, 1981)

In the period October 1980-May 1981, 5 young men, all active homosexuals, were treated for biopsy-confirmed Pneumocystis carinii pneumonia at 3 different hospitals in Los Angeles, California. Two of the patients died. All 5 patients had laboratory-confirmed previous or current cytomegalovirus (CMV) infection and candidal mucosal infection. Case reports of these patients follow.

Patient 1: A previously healthy 33-year-old man developed P. carinii pneumonia and oral mucosal candidiasis in March 1981 after a 2-month history of fever associated with elevated liver enzymes, leukopenia, and CMV viruria. The serum complement-fixation CMV titer in October 1980 was 256; in May 1981 it was 32.* The patient's condition deteriorated despite courses of treatment with trimethoprim-sulfamethoxazole (TMP/SMX), pentamidine, and acyclovir. He died May 3, and postmortem examination showed residual P. carinii and CMV pneumonia, but no evidence of neoplasia.

Patient 2: A previously healthy 30-year-old man developed P. carinii pneumonia in April 1981 after 5-month history of fever each day and of elevated liver-function tests, CMV viruria, and documented seroconversion to CMV, i.e., an acute-phase titer of 16 and a convalescent-phase titer of 28* in anticomplement immunofluorescence tests. Other features of his illness included leukopenia and mucosal candidiasis. His pneumonia responded to a course of intravenous TMP/SMX, but, as of the latest reports, he continues to have a fever each day.

Patient 3: A 30-year-old man was well until January 1981 when he developed esophageal and oral candidiasis that responded to Amphotericin B treatment. He was hospitalized in February 1981 for P. carinii pneumonia that responded to oral TMP/SMX. His esophageal candidiasis recurred after the pneumonia was diagnosed, and he was again given Amphotericin B. The CMV complement-fixation titer in March 1981 was 8. Material from an esophageal biopsy was positive for CMV.

Patient 4: A 29-year-old man developed P. carinii pneumonia in February 1981. He had had Hodgkins disease 3 years earlier, but had been successfully treated with radiation therapy alone. He did not improve after being given intravenous TMP/SMX and corticosteroids and died in March. Postmortem examination showed no evidence of Hodgkins disease, but P. carinii and CMV were found in lung tissue.

Patient 5: A previously healthy 36-year-old man with a clinically diagnosed CMV infection in September 1980 was seen in April 1981 because of a 4-month history of fever, dyspnea, and cough. On admission he was found to have P. carinii pneumonia, oral candidiasis, and CMV retinitis. A complement-fixation CMV titer in April 1981 was 128. The patient has been treated with 2 short courses of TMP/SMX that have been limited because of a sulfa-induced neutropenia. He is being treated for candidiasis with topical nystatin.

The diagnosis of Pneumocystis pneumonia was confirmed for all 5 patients ante-mortem by closed or open lung biopsy. The patients did not know each other and had no known common contacts or knowledge of sexual partners who had had similar illnesses. The 5 did not have comparable histories of sexually transmitted disease. Four had serologic evidence of past hepatitis B infection but had no evidence of current hepatitis B surface antigen. Two of the 5 reported having frequent homosexual contacts with various partners. All 5 reported using inhalant drugs, and 1 reported parenteral drug abuse. Three patients had profoundly depressed in vitro proliferative responses to mitogens and antigens. Lymphocyte studies were not performed on the other 2 patients.

Reported by MS Gottlieb, MD, HM Schanker, MD, PT Fan, MD, A Saxon, MD, JD Weisman, DO, Div of Clinical Immunology-Allergy, Dept of Medicine, UCLA School of Medicine; I Pozalski, MD, Cedars-Mt. Sinai Hospital, Los Angeles; Field Services Div, Epidemiology Program Office, CDC.

Barre-Sinoussi, F., Chermann, J., Rey, F., Nugeyre, M., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W., & Montagnier, L. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS) Science, 220 (4599), 868-871 DOI: 10.1126/science.6189183

Tick-borne encephalitis virus....in milk

noreply@blogger.com (Anonymous) — Thu, 30 May 2013 20:05:00 +0000

Based upon the name, Tick-borne encephalitis virus (TBEV) would be expected to be transmitted by ticks. And it appears it is. But it can also be spread by milk/milk products. A recent paper has reported a case in Slovenia where 3/4 people who drank the milk of a goat (later found to be positive for TBEV) became ill. TBEV is a flavivirus and is present in many parts of Europe, with incidence levels in recent years of up to 18.6 cases/100,000 people.

Unpasteurised cheese - a potential source of TBEV infection

Transmission of TBEV in unpasteurised milk hasn't previously been reported in Slovenia specifically, but it was already known that TBEV can be transmitted in raw dairy products. In a recent study, four people had drank unpasteurised colostrum from a goat, three of whom subsequently became sick, having symptoms of fever, malaise, headache, nausea and tremor. Although TBEV RNA couldn't be detected in the blood of any of the three patients, they all had antibodies. IgG and IgM isotypes were detected by ELISA, and the serum was neutralising for the virus. The fourth person, who didn't become ill, had been vaccinated against TBEV and had good antibody titres against TBEV, although IgG titres taken at different intervals suggested they'd received a boost, presumably as a result of drinking the infected milk.

Goats, not sheep seem to be the culprits when it comes to harbouring TBEV in their milk

The authors took samples from the 9 goats and 9 sheep on the farm, and tested them for evidence of TBEV.
Perhaps unsurprisingly, 5/9 goat sera and 1/4 goat milk samples were positive for TBEV antibodies. The suspect goat whose milk had been drunk prior to the patients becoming sick was also shown to have RNA (indicative of virus) in both the blood and milk, confirming that the goat had evidence of actual viral antigen.

None of the sheep samples were positive, which I find one of the most intriguing points - do ticks prefer goats, or is there species specificity when it comes to TBEV?

According to the authors, drinking unpasteurised milk is fashionable as part of a 'natural lifestyle'. Based upon this paper, the risk of contracting TBEV (as well as other pathogens) would therefore also be part of the natural lifestyle; that's an interesting choice of lifestyle.

Hudopisk, N., Korva, M., Janet, E., Simetinger, M., Grgič-Vitek, M., Gubenšek, J., Natek, V., Kraigher, A., Strle, F., & Avšič-Županc, T. (2013). Tick-borne Encephalitis Associated with Consumption of Raw Goat Milk, Slovenia, 2012 Emerging Infectious Diseases, 19 (5) DOI: 10.3201/eid1905.121442

Schmallenberg virus...where are we at: Part 3. Vaccine time

noreply@blogger.com (Anonymous) — Fri, 24 May 2013 22:00:00 +0000

As Bluetongue virus 8 was making its way to the UK in 2007 I was asked by a farmer whether or not he should vaccinate when the vaccine became available. 100% yes was the answer, and would be now. BTV-8 was a particularly virulent strain, resulting in mass death in sheep and even (unusually for BTV) causing disease and deaths in cattle. Bluetongue had never before been to the UK, so the entire herd was susceptible to the virus. The result was that, whilst East Anglia experienced a few outbreaks in 2007, the vaccine prevented recrudescence of the virus in 2008; unlike what happened on the continent after 2007, where BTV overwintered from 2006 and erupted in 2007 to cause the largest outbreak of BTV in history.

Bluetongue virus did eventually reach the UK, but vaccination stopped it from progressing

This week saw the news that a vaccine for Schmallenberg virus will be available from Merck by the summer. The obvious assumption is that every farmer will be desperate to vaccinate. That would be no surprise considering the damage it's done this past year, in particular to the sheep industry where countless lambs have been lost as a result of the disease.

But the same question now arises - should you vaccinate? This time the situation is very different. We've had it. Would vaccinating now be the equivalent of the metaphorical slamming the stable door after the horse has bolted etc.? If current reports from the continent equate to the UK, then the majority of cows and sheep will be immune, in which case a vaccine might be of limited value. There will however be some naive animals which somehow avoided becoming infected, and once maternal antibody has waned this years offspring will be open to infection.So vaccinate, right?

In the case of human diseases, the choice to vaccinate is generally simple; vaccinate. Similarly, most horse and pet owners would probably also vaccinate their animals in the face of an emerging disease (assuming a vaccine were available). Farming is different though; farming is to make money. The option to vaccinate is no longer based upon sentimentality, but business. How much do you stand to lose if there's an outbreak, relative to how much it costs to vaccinate when there's only the possibility of an outbreak? In the case of BTV the evidence from the continent was that it would be devastating. The choice to vaccinate was clear.

With SBV however, does it make sense from a business perspective to spend a lot of money to vaccinate the flock/herd? Say 90% of the animals are immune - a reasonable guess based upon studies on the continent. To begin with this level of seroprevalence already makes it more difficult for an outbreak to persist. Ignoring that, and assuming the extent of infection will be the same as last year, 9% of the flock would in theory become infected. If in turn we assume only 10-20% of those infected would be affected clinically, that's only 1-2% of the overall flock/herd.

You're fired: does it make business sense to vaccinate animals when there might not be any need?

In a flock of 1000 animals, 10 animals may be affected. Is that a sufficient number to warrant vaccinating 1000 sheep? Would you vaccinate a herd of 500 cattle on the basis there might be 5 cases of acute disease? Clearly such numbers are approximate, and may be wide of reality, but they're realistic enough to shows there's a difficult choice to make. I can't advise on whether or not to vaccinate, but I do know it's going to be a tricky choice for those involved.

poly(A) messages; lost in translation

noreply@blogger.com (Anonymous) — Fri, 17 May 2013 20:58:00 +0000

From a virus' perspective, how do you translate your own messenger RNA (mRNA), whilst not allowing your host cell to continue manufacturing its own proteins, including those that might be detrimental to virus survival? It's a problem viruses have found various ways to overcome, often by manipulating the biology of the mRNAs, which have the following structure:

The classical polyadenylated mRNA ready for translation

Simply, an eIF4F cap-binding complex binds to the cap and a poly(A) binding protein (PABP) interacts with the poly(A) tail. The PABP in turn interacts with eIF4G of the cap binding complex, thus circularising the mRNA for efficient translation to occur.

The translation complex showing circularisation enabled by PABP linking the poly(A) tail to eIF4G.

A good way in which to specifically translate viral messenger RNAs is to make the viral mRNAs different in such a way that viral mRNAs are the most efficiently translated mRNAs. Picornaviruses (e.g. polio, foot and mouth disease) and flaviviruses (e.g. West Nile, Hepatitis C), genomes contain an internal ribosome entry site (IRES), which allows ribosome attachment and subsequent translation in the absence of the 5' cap; get rid of the ability of the cell to translate capped mRNAs and suddenly the viral mRNAs are preferentially translated.

Rotaviruses target the other end. Rotavirus mRNAs all end in a specific sequence ....GACC instead of a poly(A) tail. One of the viral non-structural proteins, NSP3, has been shown to act in similar fashion to PABP; NSP3 specifically binds RNAs ending in this sequence (i.e. rotaviral mRNAs), and also binds the cap-binding complex in place of PABP, but with higher affinity than PABP. The overall result is that polyadenylated mRNAs are outcompeted by rotaviral mRNAs. NSP3 also seems to be responsible for PABP accumulating in the nucleus, where it is unable to translate cytoplasmic mRNAs. Even so, there is evidence that rotaviral mRNA translation appears to be independent of NSP3.

A paper has just come out looking into the location of poly(A), i.e. cellular, mRNAs in rotavirus infected cells.
The first question was, where do you find poly(A) mRNAs in infected cells? Using fluorescence in situ hybridisation (FISH) the authors found poly(A) containing mRNAs to accumulate in the nucleus of cells, thus preventing their translation. Removing NSP3 using RNA silencing prevented this from happening, so that poly(A) mRNAs were then found in the cytoplasm, just as in uninfected cells.

Rotavirus (and NSP3) retain polyadenylated mRNA in the nucleus. Oligo (dT) probes detects polyadenylated (i.e. cell-like) mRNA. Normally the mRNA is distributed throughout the cell (top) in infected cells, signal is only observed in the nucleus (middle row) whereas when NSP3 is silenced, the nuclear block is apparently released (bottom).

Next there is an intriguing finding that, perhaps surprisingly, the untranslated regions (UTRs) of rotaviral mRNAs do not influence how well that particular transcript is translated, as luciferase reporter RNAs with host-cell UTRs were not translated any less efficiently than reporter RNAs with rotaviral UTRs. Most strikingly though, was the fact that the overall efficiency of translation appeared to be enhanced in infected cells, implying that the translation machinery is altered upon infection. These mRNAs were directly transfected into the cytoplasm, which isn't how cellular mRNAs originate. To look at this, the authors used two ways of supplying mRNA to the translation machinery, either directly into the cytoplasm, where they again found it to be more efficiently translated in infected cells, or allowed the cell to transcribe it from a plasmid, in which case they observed a decrease in expression as as result of rotavirus infection. Silencing of NSP3 released this apparent inhibition, with the infected cells appearing more like uninfected cells. Together this all leads to the conclusion that, rather than NSP3 affecting the translation of mRNAs directly, the inhibition of poly(A)-dependent translation is due to a lack of export of newly transcribed RNA.

RNA translation in the presence of Rotavirus: (A) when RNA is transcribed in the nucleus (and needs to be exported for translation), infection suppresses translation, whereas mRNA transfected into cytoplasm implies that rotavirus infection enhances the translation efficiency of the translational machinery. (B) Suppression of rotavirus NSP3 by RNA silencing removes the block imposed upon protein expression from a plasmid.

The authors checked the location of cellular mRNAs too, and found that they too accumulated in the nucleus of infected cells, whereas in mock-infected cells the mRNAs were found in the cytoplasm. Again, when NSP3 was silenced, this block disappeared.

What happens to the polyadenylated mRNAs which accumulate in the nucleus (alongside the normally cytoplasmic PABP which is also retained in the nucleus)? By looking at the length of the RNAs, and using oligos which target the poly(A) tail, they found that the poly(A) tails were increased in length; an observation in line with data showing that PABP accumulation in the nucleus results in hyperadenylation and nuclear retention of RNAs.

Finally, the authors looked to see whether there were more cellular mRNAs in the nucleus compared to the cytoplasm in infected cells. They found that the cytoplasm of infected cells contained 50% less polyadenylated mRNAs. All of this leads to a scenario in which a translationally very active cytoplasm is (comparatively) free of cellular, polyadenylated mRNAs, into which the virus transcribes masses of its own mRNAs; essentially the viral mRNAs now have the cell's translational machinery to themselves, and all of this apparently orchestrated by NSP3.

As a strategy it makes sense; simply get rid of the host's RNAs.

Rubio, R., Mora, S., Romero, P., Arias, C., & Lopez, S. (2013). Rotavirus Prevents the Expression of Host Responses by Blocking the Nucleocytoplasmic Transport of Polyadenylated mRNAs Journal of Virology, 87 (11), 6336-6345 DOI: 10.1128/JVI.00361-13
Piron, M. (1998). Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F The EMBO Journal, 17 (19), 5811-5821 DOI: 10.1093/emboj/17.19.5811

Gold Rush: what the Hoffman crew face

noreply@blogger.com (Anonymous) — Sat, 11 May 2013 11:47:00 +0000

In the four weeks recuperating with a sling from shoulder surgery there's only so much reading and one-handed typing you can do; enter Gold Rush. I found out about this from a research fellow who's arrived from New York. For anyone not familiar (being British I clearly wasn't), this is a reality TV show on the Discovery Channel following a team of guys from Oregon who pursue the American dream by heading to Alaska aiming to mine gold, with a slight obsession for a 'glory hole'. Inspirational stuff.

Todd Hoffman (centre) and his team head to Alasksa

At one point, Todd and his crew in Alaska seemed to be having issues with mosquitoes, and these could potentially carry arboviruses, (if there were any there to begin with). The water source was rightly another point of concern; it's clearly not just pure virgin meltwater up there.

Gold mining's a dangerous business: as well as the machinery, Alaska also has plenty of inquisitive bears. There's also the added danger of being in the wilderness. One of the prime causes for virus emergence is generally accepted as being encroachment into thus far untouched environments. Disturbing forests or other

forms of wilderness bring humans into contact with nature, resulting in opportunities for spillover events to occur. As a fairly recent example, and sticking with the mining theme, miners in Uganda experienced an outbreak of Marburg haemorrhagic fever in 2007. In this case just four miners contracted Marburg virus, a very unpleasant virus related to Ebolavirus. As the authors point out though, as long as you go in the cave/mine without protection from the bat secretions (the suspected source of virus), then you'll be at risk. Is the risk worth it? That's going to very much depend on your perspective; a virologist in Scotland is inevitably going to have a different view than someone depending on the mine for their livelihood.

So Todd, if you feel the need to go prospecting in Africa, watch out for those bats.

Coke, formalin, tea...save your horse from African horse sickness!

noreply@blogger.com (Anonymous) — Tue, 30 Apr 2013 20:53:00 +0000

African horse sickness (AHS) is arguably the most lethal infectious disease of horses. Like Bluetongue virus, AHS virus is an Orbivirus in the Reoviridae family of dsRNA viruses. Also like BTV (and the bunyavirus Schmallenberg virus), it's an arbovirus that is spread between mammalian hosts by Culicoides midges.

African horse sickness: the lungs fill with fluid and the horse essentially dies by drowning. If a horse develops the pulmonary form of the disease, the likelihood is the horse will die.

After recent experiences of BTV and Schmallenberg virus, it's not an unreasonable question to ask whether AHSV would be capable of a similarly large outbreak in Europe. Spain has previously experienced AHSV, but it's never persisted and spread to the extent of the recent BTV and SBV epizootics in Northern Europe. Though an outbreak is a possibility, BTV and SBV are viruses of ruminants, particularly cattle and sheep, and whilst the midges which feed on cattle and sheep are competent to transmit these viruses, that's no guarantee that similar dynamics would be seen for AHSV. There are also many more sheep and cattle than there are horses, which would also make an AHSV outbreak harder to establish. Nevertheless, the devastating annual experience of AHSV in South Africa suggests that, given the right conditions, an outbreak could happen. Horse-lovers beware.

What can you do? Vaccination? There are both live attenuated and inactivated vaccines for AHSV, but because demand is low most pharmaceutical companies don't formulate them. The most commonly used are the South African live attenuated strains; unfortunately the efficacy isn't great, and if the horses are vaccinated during the midge season they can result in an outbreak.

That leaves therapy. Unsurprisingly people will try anything to save their horse, including some questionable as well the logical approaches (although I'm no pharmacist). I've come across the following, not all of them recommended by a vet:

Allergex tablets.
Vitamin C.
Tioctan Vet.
Phosamine or any other Vitamin B Co-injectable.
Brewers Yeast (can use 2 teaspoons Marmite 3 x per day as a substitute).
A good probiotic.
Himalayan Rock Salt.
Colloidal Silver, a homeopathy classic
Coca Cola or liquid molasses added to the water to encourage drinking.
Hydrogen Peroxide 35% in the drinking water of all horses in the yard.
DCA immune booster.
Immune boosting herbs.
The AHS herbal treatment kit.
Bute replacement herbs (containing cortisone).
Eco-Heal, Eco-Lung and Eco-heart.
Miracle mineral supplement (MMS).
Salix.
Dimethyl sulphoxide (DMSO).
Solal ribose.
Infrared lamp.
Oxygen blanket.
Bio-Electro-Magnetic_Energy_Regulation (BEMER).
Sub-cutaneous Dettol injections.
Essential oils (rubbed between the back legs).
Rooibos tea.

Coke: for AHSV maybe it works, maybe it doesn't, but it's still bad for their teeth.

The 'cure' I find most concerning though is the intravenous injection of formalin. This seems to be based on historic rumours rather than anything else; one possibility is apparently that it stops the blood vessels from becoming leaky. On the other hand, it's toxic, and I find it surprising that this would even be considered.

Someone with more knowledge of therapies than me please explain, but looking around there doesn't seem to be masses of scientific support for many of these treatments.

At the end of the day though, I really don't blame the owners. If your horse is infected with a virus which is going to kill >90% of those it infects, surely anything's worth a shot!

Schmallenberg Virus RNA in Culicoides midges

noreply@blogger.com (Anonymous) — Sat, 16 Mar 2013 20:02:00 +0000

There have generally been two views regarding the use of RT-PCR as a way to say whether Culicoides midges are vectors for arboviruses. One side argues that, if you find viral RNA, then the midges are competent. In both the bluetongue virus (BTV) and Schmallenberg virus (SBV) outbreaks in Northern Europe there were thousands of midges mashed and tested for the presence of viral RNA. Of course, midges were found containing RNA. The other side historically replies with something along the lines of, whilst a good indication, this is not evidence of virus replication and spread to the salivary glands, a necessity for transmission to a naive host during the next blood-feed. The hesitation in the latter camp is on the basis that, if you test the entire midge for viral RNA, then there is no way to determine whether any RNA detected is a result of replication, or whether it merely represents virus taken in during the original blood-meal, i.e. it is possible for a midge to be positive, without virus replication taking place.

This all relates to the very first post I wrote - having a sound understanding about what you are detecting and what can be concluded from the result. In this case, it's the fact that RNA does not equate to live virus.

There are ways to gain confidence that RNA presence = competent midge. The simplest is to just argue that the level of RNA is too high to just be a blood-meal, indicating that replication has occurred; this was the case in a recent paper regarding SBV positive midges in Denmark. In this study they also gained confidence that there was viral replication by testing for host (i.e. cow or sheep) RNA; the absence of host RNA implies that the blood-meal had been digested, and therefore the presence of SBV RNA is as a result of virus replication.

Another way is to only test the heads, as an indication that the salivary glands have been infected (a necessity for transmission). The blood-meal is in the abdomen, so any RNA detected in the head will be as a result of replicated and disseminated virus. Dissecting individual midges is a huge undertaking though, and is largely going to be impractical.

In this study by Veronesi et al, midges were either injected with virus, or allowed to feed on blood spiked with virus. After 10 days incubation to allow the virus to replicate and spread, the surviving midges were homogenised and tested by real-time 'semi-quantitative' RT-PCR for the presence of viral RNA. Quantitative RT-PCR would obviously have been preferable, but Cq values (lower value = more RNA) at least give an indication of how intense the infection is.

(A) in the figure above represents RNA levels in midges which had been injected with virus, and shows that RNA was present in the heads and even saliva (8/10 midges tested) of some midges, which may indicate that these midges would be competent to transmit the virus. The lowest values were found in the abdomen/thorax (where the virus was injected), indicating that either the replication was local or the assay was detecting the blood-meal. (B) and (C) are more interesting as they represent lines of midges that are either competent (B, C. sonorensis), or incompetent (C, C. nebeculosus) for BTV, that have been allowed to feed on blood containing virus, thus imitating more closely the 'natural' situation. The C. sonorensis infections resulted in a conspicuous bimodal distribution of Cq values, something which allows the midges to be divided into either transmissible (low Cq values) or non-transmissible (high Cq values) infections. For C. nubeculosus, this distribution was absent, indicating that this species of midge is, like for other arboviruses, non-competent.

Working out what copy numbers of RNA will equate to an infectious midge will be the next step, and this will require the adoption of quantitative RT-PCR.

Culicoides midges have proved themselves to be important vectors of arboviruses, most famously in recent years bluetongue and schmallenberg. The Pirbright lab, originally driven by the great Professor Philip Mellor, have done much work towards unpicking the precise role midges play in virus transmission, particularly in a European situation. Now Philip's one time understudy, Simon Carpenter, and his team are taking things forward, as he explains in the following video about Bluetongue virus.

For a paper looking to implicate midges as vectors of Schmallenberg though, there does seem to be something rather obvious that's missing. Pirbright are in the rare situation of having competent colonies of Culicoides - why not do the key experiment of attempting to infect sheep or cattle with blood-fed midges? RNA, or indeed virus, may be in the saliva in the midges tested for this paper, but that's not evidence that it would be sufficiently infectious to initiate infection in an animal (although the likelihood is it would).

Overall though, the paper has a slightly different focus - more about the idea of whether or not real-time RT-PCR can be used to indicate whether or not midges are competent vectors. A study tackling this shady area of RNA = infection has needed to be done for a long time; finally it has.

Rasmussen, L., Kristensen, B., Kirkeby, C., Rasmussen, T., Belsham, G., Bødker, R., & Bøtner, A. (2012). Culicoids as Vectors of Schmallenberg Virus Emerging Infectious Diseases, 18 (7) DOI: 10.3201/eid1807.120385
Veronesi, E., Henstock, M., Gubbins, S., Batten, C., Manley, R., Barber, J., Hoffmann, B., Beer, M., Attoui, H., Mertens, P., & Carpenter, S. (2013). Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR PLoS ONE, 8 (3) DOI: 10.1371/journal.pone.0057747

Make me a virus: goodbye MTAs

noreply@blogger.com (Anonymous) — Thu, 21 Feb 2013 22:50:00 +0000

Material transfer agreements - MTAs - can be infuriating. They will almost certainly exist forever, in some form or other. They say knowledge is power, so it's no surprise that people/institutions want to hold on to anything which may offer them a competitive advantage. It clearly makes sense. I've always been curious though about the amount of metaphorical wheel re-inventing which goes on across the world purely due to the fact that MTAs get in the way; considering the time and money it seems crazy.

Problems arise in the form of time delays and restrictions on what can be done with the materials, whatever they may be. Swapping the simplest of items can take what seems like forever to change hands, and even when it does eventually happen there seems to be a lot of hand tying involved.

But, the concept of virus rescue/reverse genetics means that, for a lot of viruses, the days of the MTA may be limited. Classically, cDNA clones of virus genomes were generated by cloning bits of a virus you already have. This could be extremely laborious and time consuming and meant that you were required to already possess the virus. Now though, gene synthesis is becoming cheaper and cheaper and that means, assuming the sequence is known, that infectious clones can be ordered online by labs with smaller and smaller budgets. Our lab did this recently with Schmallenberg virus. In order to get working as quickly as possible, and allow us to work on whatever aspect we wanted, we ordered the clones online and received them a few weeks later. Not long after, we had Schmallenberg virus, with no restrictions on what it could be used for.

The virus rescue strategy for Schmallenberg virus; the pUC plasmids were simply ordered online, from Varela et al 2013

For some groups of viruses, rescue systems haven't been established, for example the rotaviruses and some other members of the Reoviridae. For the majority though, there are established systems and clones, including many viruses which are under strict restrictions in laboratories. Accession number AF086833 is the full genome sequence of Ebola Zaire 1976, the original strain associated with horrifying levels of mortality. Similarly, accession number AJ539141 is the sequence of the Foot and Mouth Disease Virus from the UK in 2001 which was estimated to cost the UK government £8bn ($16bn). I'm not aware of questions being asked when ordering such sequences to be synthesised. I thought about experimenting and doing some sort of dummy orders of sequences such as these online to see whether there were any blocks in the way. In theory there should be questions from the company regarding what it is and where it's being sent once it's made. All I know for now is that we were essentially able to order a newly emerged virus online. But arguably the most positive aspect of this, is that it hasn't taken months of paperwork to formulate a restrictive MTA!

Man flu or real flu? - DIY Diagostics

noreply@blogger.com (Anonymous) — Thu, 07 Feb 2013 22:32:00 +0000

Someone in the lab emailed me today saying that she wouldn't be coming in as she was ill, and that she hoped it was a cold instead of flu. If you're in the same situation, wouldn't it be good if you could test yourself to find out? Well, you can, using a simple dip-stick style test which you can buy online.

That's great and it's easy to envisage how useful this can be but, for me, the interesting page is the one describing the test specifications - how good is the test? The most notable values are the positive (PPV) and negative (NPV) predictive values, which represent the accuracy of a result; PPV represents the proportion of positive results that are truly positive and likewise the NPV is the proportion of test negatives that are truly negative. A PPV of 62% for a nasal swab doesn't seem to be particularly high. As these values very much rely upon seroprevalence it's hard to really tell how useful this is.

These assays do work, and simple lateral flow devices worked really well during the foot and mouth outbreak in 2007 in the UK. These assays currently use antibodies and, without a good antibody, assay sensitivity can be poor. In lab settings, molecular assays based upon detecting viral nucleic acid are generally more sensitive, in particular real-time RT-PCR. Sadly, molecular methods also require much more expensive equipment, making it more difficult to convert a lab procedure into a 'point-of-care' test. Perhaps unsurprisingly, the initial major steps were taken by the military as a response to the threat of biowarfare. Now however, more commercial machines have made their way to the market, for example machines made by Smiths detection.

Smiths detection PCR machine: essentially a robot for nucleic acid extraction and a real-time PCR machine packaged in a (very heavy and expensive) briefcase.

Field based diagnosis is as close now as it has ever been. Ultimately though, the problems of hardware still exist. The machines are expensive; essentially they are the same machines that a lab has packaged into a briefcase. I think molecular assays in the field will only really take off once isothermal assays, such as loop-mediated isothermal amplification (LAMP), are more widespread. As their name suggests, isothermal assays are performed at a single temperature, so all that is required is a simple waterbath set to a particular temperature, as opposed to a block of metal heating up and cooling down.

Loop mediated isothermal amplification: modified primers loop back to prime the alternative strand. A strand displacing enzyme abrogates the need for variable temperatures.

Detection is the other problem; fluorescence as a read-out is expensive to a) achieve and b) detect, therefore the gold standard for field-based detection of amplification (PCR, LAMP etc.) is likely to be dipsticks/lateral flow devices as these are easy, more foolproof and provide a clear answer. For now, extraction of the nucleic acid probably remains the biggest obstacle. Another benefit of assays based around isothermal amplification and simple methods of detection is that it allows the use of molecular assays with minimal equipment, meaning that such assays can then be used in resource poor countries.

It is more than likely that the future will be much more sophisticated with much greater scope for what can be detected, although the requirements for simplicity are unlikely to change much. For now, imagine settings such as clinics where patients suspected of hepatitis C can get an instant result. LAMP assays are already available for many of the pathogens that would be on a list of desirable tests; I suspect it won't be long before point of care testing becomes even more widespread than it already is.