Thursday, 30 May 2013

Tick-borne encephalitis milk
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

Friday, 24 May 2013

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

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.

Friday, 17 May 2013

poly(A) messages; lost in translation
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

Saturday, 11 May 2013

Gold Rush: what the Hoffman crew face

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.