Lessons from Schmallenberg

Whilst I haven't spoken to one, being a UK sheep farmer at the moment can't be much fun. Schmallenberg virus (SBV) is robbing farmers of lambs as well as being an altogether disturbing and unpleasant experience. How long it remains an issue will only become apparent with time. It's worth looking at whether this outbreak of SBV represents a sign of what's to come.

The Hollywood films all seem to make a big issue of a virus 'going airborne'. That's unsurprising considering airbrone spread is probably one of, if not the, most efficient form of transmission when it comes to humans and their viruses, crammed together in offices and public transport. I'm not so sure it's the same for livestock viruses though, where the densities of animals may be locally high (i.e. within a farm), but with greater spaces between groups (i.e. between farms). Even though Foot and Mouth disease virus is airborne, and pig farms in particular can release sufficient amounts of virus into the air to infect other properties, the majority of the FMDV spread in 2001 in the UK was due to both direct contact between animals and indirect contact through so-called fomites. Arboviruses, on the other hand, are transmitted when insects find and bite animals, resulting in a form of transmission which is more heat seeking missile than carpet bomb. Both of the most recent two exotic livestock viruses to enter the UK (Bluetongue and Schmallenberg) have been spread by insects, more specifically, Culicoides midges. The fact that it's midge-borne viruses that have made it to the UK may be as a result of the midges being small enough to be blown in wind plumes, including across the channel from the continent where the viruses emerged. 

Wind plumes blowing across to the UK from the continent; the probable route of entry
into the UK for SBV (and a few years earlier Bluetongue virus).

Whilst wind may explain why midge-borne viruses reach the UK, it doesn't explain why it's midge borne viruses that have spread across the continent so fast. Why aren't mosquito-borne viruses going just as crazy? West Nile managed to spread from the US east coast to the west in just a few years.

Image from CDC.

Could it be insect vectors provide an efficient form of virus transmission providing the climate is right? It certainly seems so once it's introduced, although maybe not ideal for global spread when considering the relative immobility of livestock compared to humans. How Schmallenberg (or Bluetongue) arrived in the Europe to begin with is open to speculation; but the fact that it spread extremely fast across Northern Europe is undeniable. Within months of the initial observations in Germany, SBV had made its way to the UK and, within one summer, had more or less spread throughout the entire country.

The rapid spread of SBV; from covering North West Germany, Belgium and the Netherlands in the spring (top, 5th Jan 2012) to large swathes of Europe, including the UK, by the autumn (bottom, 26th October 2012).

And the future? Just like events such as the spread of West Nile virus in North America, we should perhaps regard it as a warning. Rift Valley fever seems to be the vogue virus in terms of a likely prospect for the future in Europe. Rift Valley, West Nile, as well as the majority of other candidates such as Chikungunya virus are all spread by mosquitoes; what about other Culicoides- borne viruses? First, African horse sickness; the most lethal infectious disease of horses, with up to 90% mortality, is a close relative of Bluetongue, as is epizootic haemorrhagic disease virus which, whilst still only on the fringes of Europe, already causes problems in the US. Human viruses? Oropouche virus (like SBV, an orthobunyavirus) can cause a dengue-like illness. Oropouche currently seems to be limited to South America; not many people would right now put a lot of money on it making its way to the UK. Before 2006 though, nobody thought Bluetongue virus would seriously enter Northern Europe, let alone go on to infect the vast majority of sheep and cattle in the region. Uncertain times are ahead. 

Schmallenberg...where are we at? Part 2

I have a double interest in Schmallenberg: firstly I'm a virologist involved in SBV research, and secondly my parents' dairy farm is SBV positive. Needless to say, I've been keeping an eye on what's happening.

Our first paper has just come out and seems to have been well received. It's by no means the first paper to come out on SBV, but it does establish a reverse genetics system which can be used to manipulate the virus in various ways. We're also on the way to figuring out various aspects of how the virus causes pathology in animals. Arguably just as important though are all the other papers describing the epidemiology of where the virus is and what proportion of animals have been infected etc.

How to make Schmallenberg virus using reverse genetics. From Varela et al.

As far as we can tell, SBV seems to present problems in two main forms: 1) an acute disease resulting in a drop in milk yield and a rather non-specific illness, 2) problems in utero, most strikingly deformed lambs and calves. It's only really the latter which is ever discussed, presumably as the death and deformity are such graphic and obvious losses. It can't be ignored though. Defra's stance remains that SBV is a low impact disease; try telling that to some of the sheep farmers who will end up with fewer lambs to sell this year.

A SBV induced deformed calf (Farmer's Weekly).

In terms of vaccines, there's one under assessment right now, but it's likely to be a case of closing the door after the horse has bolted. And anyway, it almost certainly won't be ready in time for next spring. The purpose of the vaccine would have been to generate immunity in ewes and cattle before pregnancy. Clearly this is now too late; the animals were naive and exposed to the circulating virus at exactly the right time for the fetus to be infected, resulting in the development of deformed young or fetal resorption, and there have been numerous reports of 'empty' cows and sheep. The true extent of the SBV impact is only now beginning to surface as the lambing season continues, and it's not good. One positive piece of news I've heard is that sheep who were infected prior to becoming pregnant (and were thus immune at the time of conception) are lambing fine. It remains to be seen for sure, but it is reassuring that long-term fertility does not appear to be too badly affected. In effect, the nation's sheep and cows have now become immune, by being infected. Assuming this will leave only limited numbers of animals which the virus can still infect (those uninfected thus far, and on the basis that the immunity is long lasting), the impact is likely to be minimal. Without sufficient naive animals the virus may not be sustained within the animal-vector system.

The overall impact will be hard to calculate: how do you tell what proportion of pregnancy failures are truly due to SBV infection? Virus isolation from every individual case will be difficult given the short lived viraemia. Furthermore, how can you really calculate the loss of milk production when there may be many confounding factors?

Where is it heading? My suspicion at this point in time is that next year won't be anywhere near as bad as we're seeing now. Unaffected newborns will initially receive maternal antibody and thus be protected from immediate infection. Even after maternal immuntiy has waned, the young animals may become infected before they reach mating age. In the case of lambs, the majority will be eaten. A vaccine should be available by then, and SBV has caused enough problems this year that it will have a wide uptake by farmers. Next year then, based on what we know now, SBV probably won't be so much of an issue. For now though, it very much is.

Varela, M., Schnettler, E., Caporale, M., Murgia, C., Barry, G., McFarlane, M., McGregor, E., Piras, I., Shaw, A., Lamm, C., Janowicz, A., Beer, M., Glass, M., Herder, V., Hahn, K., Baumgärtner, W., Kohl, A., & Palmarini, M. (2013). Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host PLoS Pathogens, 9 (1) DOI: 10.1371/journal.ppat.1003133

Reassortment among viruses (other than influenza!)

Want to read about reassortment in viruses? Sure you do. Look it up though and you'll more than likely be looking at a list dominated by influenzavirus. My guess is that in any virology course in the world influenzavirus is the example given when the topic of reassortment arises. Rightly so would be a fair argument; it's easy to relate to and is touted as the virus with the most potential to wreak havoc upon the human race. I've got nothing against influenza (scientifically), but there are many other viruses which undergo reassortment. 

If you don't already know, (and don't want to look it up), reassortment is a property of viruses with a segmented genome, and refers to the mixing of genomic segments when two or more viruses of the same species co-infect the same cell. Virus replication forms a pool of each of the virus segments such that, when it comes to the stage of forming a new virus particle, there is more than one option for each segment to make up the genome of the progeny virus. Virology blog covers it more in depth. In contrast to the accumulation of polymerase errors from copying the genome (genetic drift), reassortment (genetic shift) is a quick and simple way in which to evolve as a virus and generate increased genetic diversity. Over time with many rounds of reassortment it can become complicated - the following image is from the Nature paper describing the origin of each of the 8 segments of the H1N1 pandemic influenza strain.

Origin of the pandemic H1N1 swine influenza strain as a result of reassortment. From smith et al 2009.

Due to the complete switching of a segment of genetic code, reassortment can result in viruses with vastly altered properties, the most obvious of which is antigenicity. Say each virus had a different coloured coat, reassortment could allow a virus with a green coat to acquire a red coat. At first this might not seem much of an issue, but it can have major implications regarding vaccine efficacy - potentially allowing a virus to re-enter    a population. We published a paper recently on Bluetongue virus which included a virus found in a cow in France which was primarily of BTV-8 origin (and the characteristics this imparts), but to the immune system appeared to be BTV-1. At this point farmers were vaccinating against BTV-8 so in theory this virus, carrying genetic information of a virus successfully controlled by vaccination, could infect the vaccinated animals. BTV has 26 such 'types', and there are probably more. We engineered viruses to see whether we could work out whether segments from one virus or the other imparted specific characteristics, but (frustratingly) there wasn't really anything different between the viruses in the characteristics we looked at, apart from the difference in serotype associated with a specific protein.

FRA2008/24 is a reassortant bluetongue virus, comprising a core of BTV-8 (green) with the serotype determining protein of BTV-1 (blue) from Shaw et al 2013

A group in the Netherlands has also done this kind of work, either by adding their own segments to a replicating virus and looking for a virus which has incorporated their engineered segment or, like us, completely engineering the virus from scratch. Both approaches allowed them to make reassortant viruses in the lab. In the latter case, they were able to swap the gene responsible for the serotype of an avirulent virus (BTV-6), with that of BTV-8 (very virulent), resulting in a virus which, to the immune system appeared to be BTV-8, but had all the attenuated characteristics of the the parent virus. This is essentially a live attenuated vaccine; similar to the Sabin polio vaccine, or Plowright's Rinderpest vaccine used during the (successful) eradication programme.

Clearly, reassortment can be massively massively beneficial. On the other hand, would you use that virus in the face of a Bluetongue outbreak? Reassortment is again the issue; there would be the potential to generate never before seen viruses with unknown clinical outcomes if a wild type virus infected an animal during the time the vaccine virus was replicating. The haemorrhagic Ngari virus within the family Bunyaviridae provides a stark example whereby the viruses from which it is formed (a combination of Batai and Bunyamwera) are both relatively innocuous. Vaccinating sufficiently ahead of time or during winter in the absence of transmission would be possible ways around it, but a better scenario is to make non-reassortantable bluetongue viruses based upon attenuated strains such as the BTV-6 used by the Dutch group.

Tracking reassortable viruses in the field becomes a problem too. The more people look by full genome sequencing, the more reassortants they find; if there are sufficient variants, reassortment may actually be the norm as opposed to the exception. This creates headaches when it comes to phylodynamics and tracing the viruses, as I mentioned in a post about equine influenza. Full genome sequencing is again likely to come to the rescue, and there has been another paper recently providing a method for the detection of reassortant viruses in phylogenies; guess what, it's about influenza.....

van Gennip, R., van de Water, S., Maris-Veldhuis, M., & van Rijn, P. (2012). Bluetongue Viruses Based on Modified-Live Vaccine Serotype 6 with Exchanged Outer Shell Proteins Confer Full Protection in Sheep against Virulent BTV8 PLoS ONE, 7 (9) DOI: 10.1371/journal.pone.0044619

Smith, G., Vijaykrishna, D., Bahl, J., Lycett, S., Worobey, M., Pybus, O., Ma, S., Cheung, C., Raghwani, J., Bhatt, S., Peiris, J., Guan, Y., & Rambaut, A. (2009). Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic Nature, 459 (7250), 1122-1125 DOI: 10.1038/nature08182

Shaw, A., Ratinier, M., Nunes, S., Nomikou, K., Caporale, M., Golder, M., Allan, K., Hamers, C., Hudelet, P., Zientara, S., Breard, E., Mertens, P., & Palmarini, M. (2012). Reassortment between Two Serologically Unrelated Bluetongue Virus Strains Is Flexible and Can Involve any Genome Segment Journal of Virology, 87 (1), 543-557 DOI: 10.1128/JVI.02266-12

van Gennip, R., Veldman, D., van de Water, S., & van Rijn, P. (2010). Genetic modification of Bluetongue virus by uptake of "synthetic" genome segments Virology Journal, 7 (1) DOI: 10.1186/1743-422X-7-261

From the horse's nose: phylodynamics of equine flu

Molecular epidemiology is not new, but it is progressing rapidly.

Last month saw the publication in PLoS Pathogens of a new study of an equine influenza outbreak in Newmarket, UK, in 2003. This town is known for its high density of Thoroughbred race horses; around 3000 horses are divided among yards of 20-200 horses.

Despite vaccination against equine influenza (EI), a large outbreak of EI occurred resulting in the infection of horses on many yards. Nasal samples were taken from 19 of the 21 yards during the outbreak and the possible order of infection of the yards was determined based upon ELISA and real-time RT-PCR data. Both of these assays detect the viral antigen (which is dependent on a swab, taken only once clinical signs are detected), rather than antibodies; but it nevertheless gives an indication as to which yard was infected and when - a valuable source of information in the absence of further biological data.

Outbreak progression: the order of when yards were infected. Virus presence was detected using real-time RT-PCR, with the copy numbers obtained provided in the y axis.

The authors sequenced a huge number of clones - 2361 - of a 903bp PCR product representing a fragment of the haemagglutinin 1 (HA1) gene, with multiple clones per horse. A particularly interesting aspect is that the overall dN/dS ratio is 0.89, which implies that, in general the outbreak progressed without a massive restriction on the maintenance of mutations. When the sequences were combined to form a consensus for each horse, only two unique consensus sequences were found for the entire Newmarket outbreak. This lack of inter-horse genetic variability highlights the limitations of analysing consensus data at this level. 

When they looked further at the diversity sequences found within a horse (the term 'quasispecies' conspicuous by its absence) they found limited diversity associated with dominant sequences, with the dominant species sometimes changing in those horses sampled more than once, either due to one sequence becoming dominant, or due to co-infection.

Intrahost variation in viral populations with respect to mutations A230 and G230.  E09 and F11 are individual horses, whereas L25 and L27 are the result of horses sampled twice. E09, F11 and L25 all show a central dominant sequence, with a variety of variants surrounding it; L27 shows different dominant sequences on the different days sampled.

The classic view of transmission involves bottlenecks - something I've written about previously with regard to arboviruses. It turns out that the bottlenecks for EIV are loose, even allowing sequences with stop codons, i.e. lethal sequences, to pass between horses. This goes along with the observation of low levels of purifying selection and may tie in with a recently accepted manuscript in J Virol (not yet in press) suggesting that individual virus particles fail to express all of the proteins correctly.

Sequence data is increasingly being linked with geographical data with the aim of tracing outbreaks to a finer scale. Although 903bp might be thought of as being a bit too short for this purpose, by taking into account the within host diversity there was sufficient information within the dataset to allow such an approach here.

Figure 3. Reconstruction of EIV transmission pathways during the outbreak.

(A) Transmission network inferred from the sequences, sampling date and locations for 48 horses. Each circle represents a horse colored according to training yard as in Figure 1. The size of the circle is proportional to the intra-host mean pairwise distance. Circles with thick black edges represent horses that have the A230 mutation. Arrows between circles represent inferred transmission events from the SeqTrack analysis. Dashed arrows are for horses that only share the reference sequence. (B) Frequency distribution of the shared mutations between donor and recipient horses. (C) Distribution of the number of recipients per donor horse with the expected transmission caused by different percentage of cases (inset). The red bars represent the highly connected horses (E10 and the first sampled horse A01).

One particularly interestingly fact was that there didn't appear to be a straightforward yard-to-yard pathway. Based upon this analysis, it is possible to hypothesise that a yard may be infected by multiple yards and subsequently itself infect multiple different yards. This raises the speculation that horses within a yard were not necessarily being infected by their yard-mates, leading the authors to suggest that social networks may more fully explain the observed transmission dynamics as opposed to a model based upon proximity. Such freedom of spread is emphasised further with respect to inter-horse spread. Figure 3C shows how numerous horses may be infected by a donor horse, including one which contributed infection to 10 other horses. 

The biggest frustration is the same as that for most studies involving segmented viruses. Reassortment is a key process in the evolution of segmented viruses such as influenza, and the authors acknowledge that this aspect is missing from their analysis. As it is there's no way of saying whether two viruses which are identical in the 903bp fragment of HA are actually reassortants with their other segments derived from distinctly separate parts of the outbreak/network. When next generation sequencing methods predominate and generate full genome sequence data this should become less and less of an issue. My suspicion is that, in the not too distant future, studies like this will almost certainly be obliged to use full genome sequencing.

Equine flu might not seem the most important disease ever (although anything to do with horses = money), this study is massively extensive. They have shown how, rather than simply drawing a few phylogenetic trees, much information can be derived from what, ultimately, is a straightforward collection of sequences with some associated epidemiological data.

Hughes, J., Allen, R., Baguelin, M., Hampson, K., Baillie, G., Elton, D., Newton, J., Kellam, P., Wood, J., Holmes, E., & Murcia, P. (2012). Transmission of Equine Influenza Virus during an Outbreak Is Characterized by Frequent Mixed Infections and Loose Transmission Bottlenecks PLoS Pathogens, 8 (12) DOI: 10.1371/journal.ppat.1003081