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

The end of Koch's postulates

How do you know whether disease X is the result of infection by pathogen Y? 

In 1890 Robert Koch published a list of 'postulates' which would form the foundation of ascribing disease causation from that day - until now. Though Koch established his list based upon his studies of bacteria, more specifically Mycobacterium tuberculosis and Bacillus anthracis, (the causative agents of tuberculosis and anthrax respectively), his postulates could be applied more widely. No more would infectious diseases be merely regarded as mysterious happenings. Scientists could now pursue and nail down the cause of a disease by fulfilling Koch's postulates. There are many ways of expressing the same thing, but Wikipaedia describes them as:

1) The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms. 

2) The microorganism must be isolated from a diseased organism and grown in pure culture.

3) The cultured microorganism should cause disease when introduced into a healthy organism.

4) The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Association vs. causation will always be a key issue when it comes to diagnosis and Koch's postulates, as important as they are, soon start to fall apart under closer scrutiny. For instance, what happens in circumstances where the organism can't be isolated in culture - a not unusual occurrence in the world of virology? 

The most significant problems occur though simply due to words such as 'must'. Infectious disease is never so clear cut. Koch himself acknowledged as much during his studies of cholera; whilst the causative agent, Vibrio cholerae, could be isolated from people with cholera, it could also be isolated from healthy people. This fails the first postulate (depending on how loosely you apply 'should'). 

What about the same agent in different species? The dogma with Bluetongue virus is that it is asymptomatic in cattle, but can be lethal in sheep. Even with sheep though, only a few of those which become infected actually show bluetongue disease. The fact that the term 'case fatality' exists is an acknowledgement itself that only a fraction of animals which become infected fall ill. You could argue that this is an irrelevance as, even if it doesn't cause disease in every animal that it infects, it is still the causative agent of those which do become sick. Even though there haven't been enormous numbers of cases reported (presumably because there's no evidence of disease), I would put a lot of money on the fact that a large proportion of cows in southern England are currently seropositive for Schmallenberg virus.

In turn, when attempts are made to fulfill postulate 3, it would be perfectly normal, expected even, that an animal doesn't become sick.   

Discussions of these limitations are not new. The biggest development that has prompted the questioning of Koch's postulates has been molecular biology. The extreme sensitivity of nucleic acid-based methods means that evidence of pathogens can be detected at extremely low levels. Now it is possible to go searching for viruses with relative ease, increasing the speed and efficiency of virus detection and discovery. There are many approaches; PCR is arguably the most common, and metagenomics the most recent. I compare the methods to fishing: firstly, it’s possible to go fishing for specific viruses using PCR. Metagenomics on the other hand is more like a deep sea trawler – sequencing everything within a sample and looking within the ‘catch’ for virus sequences. Using the latter approach has allowed the identification of numerous viruses, and this is reflected in the number of publications, as revealed in a paper by Mokili et al. (2012).

Viral metagenomic studies between 2002 and 2011. From Molili et al 2012.

The paper by Mokili et al. (2012) also discusses the fact that molecular approaches remove the need for the growth of an agent in pure culture. Significantly, there is also the acknowledgement that determining causality merely by a pathogen’s presence is difficult to achieve, particularly with metagenomics approaches which are capable of finding a diverse array of agents within the same sample. The authors go on to describe a ‘metagenomic Koch’s postulates' approach whereby the metagenomes of individuals are compared. The postulates are:

1) The diseased metagenome must be significantly different from the healthy control and contain a greater abundance of the suspected metagenomic traits.

2) Inoculation of a healthy individual with a sample from the diseased individual must result in disease state.

3) Selected, specific samples containing the suspected traits from the individual infected for step 2 must cause disease when injected into another healthy individual.

Metagenomic Koch's postulates as described by Mokili et al 2012. Comparison between a diseased and healthy control animal shows a significant difference between the metagenomic libraries (depicted by the histograms of relative abundance reads). In order to fulfill the metagenomic Koch's postulates: (1) The metagenomic traits in diseased subject must be significantly different from healthy subject. For example traits A, D, E and J found in the disease animal that are not present in the healthy control; (2) Inoculation of samples from the disease animal into the healthy control must lead to the induction of the disease state. Comparison of the metagenomes before and after inoculation should suggest the acquisition or increase of new metagenomic traits (A, E and P). New traits can be purified by methods such as serial dilution or time-point sampling of specimens from a disease animal. (3) Inoculation of the suspected purified traits into a healthy animal will induce disease if the traits form the etiology of the disease

Following this procedure allows the sequence associated with disease, i.e. a biomarker of the etiological agent, to be discovered by the process of elimination.

Metagenomics will, rightly, more than likely become established as the method of choice for diagnosis. The technology is still developing, but not too far in the future I suspect metagenomics will follow a similar path to that of real-time PCR into the molecular diagnostics setting. Instead of testing a samples against some 'likely suspects' using PCR approaches, it will be possible to get a complete picture of the complex 'virome' associated with that sample.

Although the 'metagenomics Koch's postulates' are a step towards linking metagenomics to disease, there is still the issue of ‘must cause disease’; if this ‘disease’ by definition involves clinical signs, then similarly to Koch’s original postulates this may fail. However, the approach does allow the picking apart of causality in complex scenarios where multiple pathogens are present. There are still issues to be ironed out as to how molecular data is interpreted, but this is going to become increasingly important as metagenomic approaches become even more widespread. From this perspective, it appears that Koch’s postulates, landmarks and revolutionary as they may have been, may be nearing the end of their life.

Mokili, J., Rohwer, F., & Dutilh, B. (2012). Metagenomics and future perspectives in virus discovery Current Opinion in Virology, 2 (1), 63-77 DOI: 10.1016/j.coviro.2011.12.004

When is a zoonosis not a zoonosis?

Zoonosis. One of the current glam terms in virology. At it's simplest, a zoonosis refers to a disease that is transmitted to humans from animals.

But it's possible to pause and think, what actually counts as a zoonosis?

On the WHO website it's classed as "any disease or infection that is naturally transmissible from vertebrate animals to humans and vice-versa".

From Wikipedia "an infectious disease that is transmitted between species (sometimes by a vector) from animals to humans or from humans to animals".

From the medical dictionary....."refers to diseases that can be passed from animals, whether wild or domesticated, to humans".

In all of these definitions the key word is 'disease'. Look it up and disease suggests there's some sort of pathology, with overt symptoms - admittedly this too is wide open for discussion. Taking this forward, if an animal virus infects a human, but doesn't cause disease, can that then be classed as a zoonosis?

Dealing with an outbreak of Hendra virus; passed from bats to horses...and on to the handlers

I was told once that, many years ago, people who mouth pipetted viruses were seropositive for the (livestock) viruses they were working on. If there were symptoms, they weren't sufficiently serious to become part of the story, so in this case we might argue that there was no disease. That fits with the definition - no disease, therefore not a zoonosis - and indeed these viruses are not regarded as being zoonotic.  

Mouth pipetting; no longer a method of choice

An interesting paper which has recently been accepted into the Journal of Virology describing the isolation and characterisation of two novel paramyxoviruses, Achimota virus 1 and Achimota virus 2 (genus Rubulavirus) from bats (Baker et al., 2012). I enjoy a bit of virus discovery and it's nice to see a study done well with a good level of characterisation and epidemiology. The authors collected urine samples from under an Eidolon helvum bat roost and added them to cultured cells in the lab. Using the viruses recovered from the isolations they could then work out the seroprevalence, the results of which showed that there was evidence of the virus infection in E. helvum samples throughout the geographical range of this species. Variations in seropositivity among different age groups, and from year to year also showed that there has been active circulation of the virus. 

Bizarrely, one of the most intriguing things was the title, namely the "potentially-zoonotic" bit. The genus Rubulavirus contains some serious viruses, notably mumps virus, so this is an important point. The authors speculate about the zoonotic possibility based upon serology, where they found 3 people out of 442 to have evidence of prior exposure to the virus. 

But does this constitute a zoonosis? One of the positive samples was from a febrile patient, so perhaps. The authors acknowledge that whether or not these viruses are zoonotic will take some nailing down. As discussed above though, seroconversion against a livestock virus does not lead to the livestock virus becoming classed as zoonotic. So does the finding of 3/422 being seropositive for these viruses mean that they are zoonotic? Is seroconversion really sufficient to class something as zoonotic? 

Baker, K., Todd, S., Marsh, G., Crameri, G., Barr, J., Kamins, A., Peel, A., Yu, M., Hayman, D., Nadjm, B., Mtove, G., Amos, B., Reyburn, H., Nyarko, A., Suu-Ire, R., Murcia, P., Cunningham, A., Wood, J., & Wang, L. (2012). Novel potentially-zoonotic paramyxoviruses from the African straw-colored fruit bat, Eidolon helvum Journal of Virology DOI: 10.1128/JVI.01202-12

Crystal meth....a new way to treat influenza?

It wouldn't be unreasonable to suspect that smoking methamphetamine, one of the most widespread and damaging illicit drugs, would lead to enhanced susceptibility to various respiratory pathogens, such as influenza. That's what Chen et al had in mind when they set out to see whether meth had such an effect on influenza replication in cell culture; after all, it's already associated with enhanced susceptibility to other pathogens such as HIV and HCV (due to biological, as well as behavioural, factors). It seems this may not be the case. The study used levels of meth which are likely to be found in meth users blood, so in that sense it's realistic. When they tried infecting cells pre-treated with meth with influenza A virus, whilst the virus was able to replicate, it didn't reach the levels of control cells which hadn't been treated with meth. Similarly, when they looked for the expression of viral proteins in infected cells, they found that, as the concentration of meth increased, the level of viral protein decreased, further showing that meth is detrimental to influenza replication in this system.

Treating cells with increasing amounts of meth resulted in a does dependent reduction in the expression of the viral proteins M1 and NS1.

So how does meth affect the influenza virus lifecycle? Treating the virus with meth and then infecting untreated cells didn't make a difference to the number or size of the plaques which were formed, suggesting that meth doesn't affect the ability of the virus particles to infect and replicate in the cells. Therefore it's presumably downstream of entry; extrapolating to a human, meth therefore might not prevent the chances of becoming infected. Indeed, the study found the inhibitory effects to occur during the actual replication. Interestingly, the inhibitory effect doesn't appear to be significantly linked to enhancing the interferon response.

Treating influenza virus with meth didn't alter the development or size of plaques

This all leads to the tongue in cheek suggestion that if you catch influenza then smoking a bit of crystal meth may help treat the infection. Perhaps it does, and it will be intriguing to see whether the method of action can be determined and therefore less damaging drugs discovered, but that would not justify consuming meth, one of the most ravaging and repulsive drugs around. For a start, one of the stark realities about this paper is that it is using cultured cells, which doesn't really mimic the complexity of the respiratory tract. Then there are the effects of the drug: enhanced susceptibility to other pathogens, addiction, depression, heart disease, anxiety, 'meth mouth', altered heart and breathing rates, diahorrea, constipation, insomnia, hallucinations etc. etc.........

Meth mouth

......anyone fancy a cohort study???

         Chen, Y., Wu, K., & Chen, C. (2012). Methamphetamine Reduces Human Influenza A Virus Replication PLoS ONE, 7 (11) DOI: 10.1371/journal.pone.0048335

Crimean-Congo Haemorrhagic fever virus comes to Glasgow

This week saw Glasgow's first case of Crimean-Congo Heamorrhagic Fever virus (CCHFV), a virus belonging to the family Bunyaviridae which can cause (as the name suggests) a haemorrhagic disease. 

A man arrived from Dubai, having been bitten by a tick in Afghanistan, and a couple of days later went to hospital where it was confirmed that he had CCHFV. Sadly news has just surfaced that he's died, highlighting what a severe virus this can be. Headache, muscle and joint pains, vomiting, diarrhea, bruising and bleeding are all associated, to a lesser or greater extent, with CCHFV, along with a fatality rate of up to 30%.

CCHFV is a tick-borne virus which is hugely widespread; the rather bizarre name reflects its discovery in both Crimea and Congo at (roughly) the same time. Eastern Europe, large swathes of Asia and the Indian subcontinent and Africa are all affected by, or at least have some evidence of, CCHFV. 

The world distribution of Crimean-Congo Haemorrhagic fever virus

Perhaps it's their relative unfamiliarity compared with mosquitoes and midges, perhaps it's the way they lock on to a host for extended amounts of time, but there always seems something rather repulsive about ticks. They seem to be everywhere, but not all seem to be infected by CCHFV, which tends to infect species of the genus Hyalomma. If it's not via a tick bite, then an alternative way to become infected is contact with the blood of an infected animal. But if it's not endemic to western Europe and the UK, would it ever be a problem here? Can it infect the ticks here? A few years ago people thought it would be more or less unthinkable that Bluetongue virus would ever reach northern Europe and the UK, an assumption dismissed emphatically in the last few years. Similarly, Schmallenberg virus is another arbovirus that has swept across Europe (incidentally, SBV is from the same family of viruses as CCHFV).

Is it possible that it would ever establish itself here? I'd guess it's pretty unlikely right now. But with climate change, species of tick that are known to be capable of being infected by CCHFV might be on the move, and based upon the climates in other areas, the climate of western Europe might just suffice.  

Badger Herpes

For anyone reading this outside of the UK a huge uproar is currently occurring over the imminent start of a trial whereby it will be legal to intensively cull badgers. The hope is that this will reduce bovine TB (bTB) which is rife in 'hotspots' in the south west of England and Wales. The controversy is that a previous culling trial, the Randomised Badger Culling Trial (RBCT), suggested that reactive culling (that is, culling badgers in an area where a bTB breakdown has occurred) disrupted badger communities with the result that bTB incidence increased (incidentally, this itself adds further weight to the fact that badgers are important in bTB epidemiology). However, in areas where culling was proactive (whereby all badgers in an area were culled up front), bTB levels did indeed drop by a significant level (Vial and Donnelly, 2012), so culling works, right? The current trial is aiming to create regions which have been culled to a sufficient extent to reduce bTB incidence by culling within boundaries to badger movement (rivers, busy motorways etc.). Interestingly, the results of culling in Ireland are somewhat more positive, and in previous periods of culling in England (intensively using gassing) bTB levels were low. 

The large proportion of the UK population like badgers and, combined with the contrasting results, there are plenty of people who are less than keen for the culling to go ahead (hopefully they won't cause the level of culling to be insufficient and thereby cause an increase in bTB!).

Brian May is a big fan of badgers.

So what about viruses? A paper by Banks et al (2002) described the discovery of a gammaherpesvirus in a badger from Cornwall in England. The paper reports bits of sequence from the virus; nowadays the complete genome would, in theory, be relatively simple to obtain and report. The badger was negative for bTB (based on histological and immunological examinations) but was in a poor state of health and had pathology in the liver, kidneys and lungs. Of course this doesn't mean it was the virus that caused these effects. It would be interesting, and potentially important, if this virus inadvertently interferes with bTB epidemiology.
According to the phylogeny, badger herpesvirus appears to fit nicely among other herpesviruses, most closely with equine herpesvirus 2.

Bager herpresvirus (BadHV) most closely related to equine herpesvirus in either the gb  (a) or  DpoI (b) genes. From  Banks et al 2002

The virus can be propagated in mink cells so in theory could be manipulated, though my current knowledge of herpesviruses isn't that great. Perhaps a virological-fantasy would be that the virus could be modified in such a way that it induces immunity/resistance to bTB in badgers - release the virus into the wild badgers and let it spread through the population. Simple and effective. 

But releasing a genetically modified virus into the wild......would that really be any less controversial than the culling?

Banks M, King DP, Daniells C, Stagg DA, & Gavier-Widen D (2002). Partial characterization of a novel gammaherpesvirus isolated from a European badger (Meles meles). The Journal of general virology, 83 (Pt 6), 1325-30 PMID: 12029147
Vial F, & Donnelly CA (2012). Localized reactive badger culling increases risk of bovine tuberculosis in nearby cattle herds. Biology letters, 8 (1), 50-3 PMID: 21752812

Virus evolution: hitting the bottle(neck) in mosquitoes

When a virus population transmits between hosts, regardless of how this is achieved, not all of the progeny viruses make it, most likely the majority are left behind to contemplate a life of non-existence and the impeding destruction. At the animal level this is fairly straightforward; an animal becomes infected, produces virus, some of which will infect a new animal, which then becomes infected etc. etc. But transmission events impose bottlenecks. Imagine a mosquito taking a bloodmeal from a human. The virus population within that person will, more or less, be spread throughout the body. An adult human contains approximately 5 litres of blood; a mosquito will only take a few microlitres, i.e. a few millionths, of the blood, and therefore only a tiny fraction of the virus in the body. Taken to a more subtle level, of the virus which makes it to a new host, only a fraction of that virus will infect cells within the new host. In the case of the mosquito, there are many 'barriers' which are familiar to virologists studying arboviruses. The first step towards infecting a mosquito (or other biting insect such as midges) is to infect the midgut. At this point there's another bottleneck, known as the midgut-infection barrier; only a few of the viruses which were taken up in the blood-meal will be able to infect the gut cells. In the case of bluetongue virus in midges there are other described barriers, including a midgut escape barrier, a disemmination barrier, and a salivary gland infection barrier. All of these must be overcome in order to allow transmission to another mammalian host.

An array of barriers must be overcome by a virus in order to continue the virus lifecycle. Image from Black et al, 2002. 

The significance of this becomes more apparent when the virus population itself is considered. Viral RNA polymerases are notoriously error prone, resulting in a population of viruses with differences in their genomes, in other words the so-called quasispecies (itself something worthy of discussion). What happens to the population structure as the transmission cycle progresses through the various stages and bottlenecks?

An interesting talk at the SGM in Dublin this year has now been published in PLoS Pathogens. In this case, the authors made an artificial virus population of Venezuelan equine encephalitis virus (VEEV) using reverse genetics. This then allowed particular clones to be followed through the various steps of infection as the virus passes from a mouse containing the clones through to how many are injected into a fresh mouse at the end of the mosquito steps of the virus lifecycle.
If they allowed mosquitoes to feed on a mouse containing the different 'clones' of VEEV, and then looked to see how many they could find in the mosquito, they found between 6 and 8 of the clones (i.e. ~70% of all clones). After a few days however, once the infection process is under way, the number of clones had decreased, implying that not all of the clones had made it through the midgut infection process.

By the time the virus has disseminated to the legs/wings, and salivary glands, there were only 1-4 of the clones present, suggesting that yet more had been lost along the way. In the saliva, which represents the population of virus that will be passed on to the new host, they only found on average 1-3 of the clones. This contrasts to the number of clones found in the legs/wings, showing that only some of the clones from the haemocoel managed to successfully infect the salivary glands.

Interestingly, there didn't appear to be a strong bottleneck in transmission to an uninfected mouse; the same clones that were in the saliva were the same clones which infected the mice, although considering the low numbers of clones in the saliva there wasn't a massive choice.

All of this leads to the puzzling question of how does the virus survive? If at each point viruses are removed, then by chance the surviving population may be less fit. Looking for marked viruses as this study has done is an elegant way of doing it, but it is limited to signatures within the genome. A further study using deep sequencing to look at the whole population more comprehensively may offer even more detail about what's going on. Perhaps this all relates to the observation of purifying selection being popular among arboviruses, time will tell, but to begin with this study has shown that such barriers exist. How the severity of the barriers vary, and how the virus deals with these barriers, will be interesting to see.

William C. Black IVa, , , Kristine E. Bennetta, Norma Gorrochótegui-Escalantea, Carolina V. Barillas-Murya, Ildefonso Fernández-Salasb, Marı́a de Lourdes Muñozc, José A. Farfán-Aléd, Ken E. Olsona, Barry J. Beaty (2002). Flavivirus Susceptibility in Aedes aegypti Archives of Medical Research DOI: 10.1016/S0188-4409(02)00373-9