The Schmallenberg Virus and Disease

This week in the online journal club #microtwjc we will be discussing this paper titled “Schmallenberg Virus Pathogenesis, Tropis and Interaction with the Innate Immune System of the Host” (Varela M, Schnettler E, Caporale M, Murgia C, Barry G, et al.  (2013) Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host. PLoS Pathog 9(1):e1003133. doi:10.1371/journal.ppat.1003133).

I’ve written before about Schmallenberg here and here so I will only go into the background briefly before diving in to the paper.

Background

Correct as of November 2012. From FluTrackers.com

Schmallenberg virus was first discovered in November 2011 so it is still pretty new to scientists.  The virus infects ruminants and whilst it only causes mild disease in adult animals, if an animal is pregnant when she is infected then the baby can end up having both muscle and brain problems and can often be born dead.  The virus now seems to have spread across much of Europe (see the map from FluTrackers.com). The virus itself is in the Bunyaviridae family which means that the virus is surrounded by an envelope and has a genome made up of a single strand of RNA (for reference, our genome is made up of a double strand of DNA).  The virus’ genome is made of of 3 sections named (very imaginatively) small, (S), medium, (M), and large, (L).  The genome codes for 4 proteins that are important in making up the structure of the virus.  Schmallenberg virus is in a smaller subsection of the Bunyaviridae family called the Orthobunyavirus.  Orthobunyavirus members not only have the 4 structural proteins but they also have 2 extra proteins (the NS proteins).  This information will be useful for later…

So on to the paper…

Virus Growth

The first thing the authors wanted to do was to find out how Schmallenberg virus grows in various different cells – this is important because if researchers want to carry out experiments on the viruses they need to know what cells to use.  The researchers tested sheep, cow, human, dog and hamster cells lines from organs including the brain, the aorta and the kidney (all of these are cell lines that were grown in the lab – they are not being tested on live animals).  The virus grew in nearly all the cell lines but particularly well in sheep choroid plexus (part of the brain) cells so the researchers used this type of cell for all of the other experiments.

Virus growth in different cell types. Taken from the paper.

‘Rescuing’ the Virus

The next thing the researchers did was design a system to make more copies of the virus.  (Very) basically they took the S, M and L sequences of the virus genome and put the complementary sequence  into plasmids (circular loops of DNA that are found ‘naturally’ in bacteria). (So if the virus sequence was CCG the plasmid sequence would be GGC.) They then put these plasmids into another cell line and these cells replicated the plasmid genetic sequence, so making more copies of the virus.

Looking for a good animal model

Another thing the authors did was investigate whether mice could be used as an experimental model for Schmallenber virus infection.  They inoculated the virus (or a negative control) into the brains of 2 day old mice.  Those mice that were infected with the virus died within 8 days whereas those that had the negative control survived.  They then looked at other ages, using 10 day old and 18 day old mice.  Infection with the virus also killed most of the animals at these ages.  When the scientists went on to look at the brains of these infected mice they found multiple abnormalities and evidence that the virus was in many of the cells.

The authors also looked at the brains of lambs and calves that had contracted the virus ‘out in the wild’ as it were.  They found the virus in places very similar to where they had seen evidence of the virus in the mice, suggesting that the mice might be a good model.

Which bit of the virus makes it harmful

A good way to find out which proteins make a disease agent harmful is to stop the virus/bacteria from producing the protein you suspect.  You then infect this mutant back into your cell lines or animal model and look to see if disease still occurs.  If the disease is no longer seen then you know that protein is vital for the disease (although it is possibly not the only one involved).  If the disease proceeds exactly as it did before you know that you were wrong in your suspicion.

The scientists who wrote this paper did just that – they ‘knocked out’ one of the non-structural (NS) proteins and infected mice with the strain.  All the mice inoculated with the original strain died within 9 days (so very similar to the previous experiment), all the control animals survived.  Of the mice infected with the mutant strain, deaths that occurred happened later than they did in the original strain group and 40-60% survived until the end of the experiment.  These results suggest that the NS protein has an important role to play in the disease caused by the virus, although it is obviously not the only one involved.

To investigate this further the scientists first went back to previously published scientific literature – the NS proteins of other viruses in the same group have been shown to inhibit the making of host immune system proteins called interferon (IFN) alpha and beta.  The scientists speculated that this might also be true for the NS protein that they knocked out. To test this they first infected some cells that can produce IFN with the Schmallenberg virus (either the mutant or the original strain).  They then collected the supernatant (basically the fluid on top of the cells).  If their hypothesis was correct then the fluid from the original strain-infected cells would contain not contain any IFN (because the NS protein inhibits its production) but the fluid from the mutant-infected cells would.  They tested the fluid in a very clever way.  They got another cell line, this time one that could not make IFN.  They added the fluid from the first set of cells.  Then they infected these new cells with a 2nd virus.  This 2nd virus is susceptible to IFN so if the supernatant contained IFN the virus would not be able to properly infect the cells whereas if the supernatant did not contain IFN then the viruses would infect the new cell line.

The supernatant from the original strain-infected cells must not have had IFN because the 2nd set of cells got infected with the 2nd virus.  In contrast the supernatant from the mutant-infected cells must have had IFN because the cells did NOT get infected with the 2nd virus.  And so this tied in nicely with the scientists’ prediction.

Finally, to confirm this the scientists infected the 2 virus strains into mice that could not respond properly to IFN.  This time 80% of the original strain-infected mice died within 4 days and the rest died within the next 2 days.  ALL of the mutant-infected mice died within 4 days.  This further suggests that an IFN response is vital for protection against disease caused by Schmallenberg virus.

And that’s where this paper ends…

… having covered a lot.  We now know what type of cells we can grow the virus in, we have a proposed animal model of the disease and we know a little (tiny) bit about what the virus needs to cause disease and what can protect the host.  The paper also has the details of 2 different ways a synthetic virus can be produced (see the ‘rescuing’ section) which sounds like it will be of great help to researchers in the future.

I thought it was a very interesting paper and I am looking forward to discussing it with anyone and everyone who joins us at journal club on Tuesday 22nd January 8pm GMT (just follow the hashtag #microtwjc on Twitter).

Lambie!

Images

The map is kindly borrowed from FluTrackers.com

The ‘When germs go bad’ (one of my favourite microbe pictures) is by Gaspirtz (Own work) (made available under creative commons licence)

The lamb is by Evelyn Simak (made available under creative commons licence)

References

1.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

Zoonotic diseases – causing more problems than illnesses/deaths alone

 

@DiseaseMapper recently tweeted a link to a very interesting paper (which happily is also free to access so you can read it too – the link is here )

Why do I think this paper is so interesting?  Firstly, because it is a useful reminder that zoonotic infections (those that pass from animals to man and vice versa) do not just impact on our lives by causing us illness, and in the worst circumstance, death.  They have a massive economic impact as well.  In fact the paper reports that the estimated economic impact of zoonotic diseases from 1995-2008 was over 120 billion dollars.

There are many reasons for the economic burden of these diseases being so high: impact on tourism; impact on international trade agreements; impact on consumer consumption and behaviour; loss of farmed animals.  In many outbreaks the local economy is negatively impacted in multiple ways and obviously in poorer areas this can also secondarily affect people’s health.

The paper also goes on to speculate about why there should be a resurgence of zoonotic infections.  The authors split it into ‘Factors associated with human behaviour’; ‘Factors associated with pathogen characteristics’ and ‘Climate change and zoonotic resurgence’.  So, pinching their titles…

Factors associated with human behaviour

Here the authors split it down further:

Individual human practices – the authors use the example of ecotourism.  “urban citizens of the developed world who visit developing countries or rural areas of the developed world and engage in activities such as forest camping, river rafting, or bat cave exploring, are prone to zoonotic infections such as vector-borne rickettsioses, leptospirosis, and haemorrhagic fevers or lyssavirus-related illness, respectively“.  The authors also talk about how pet ownership, especially the increase in ownership of ‘exotic’ pets like reptiles is increasing people’s exposure to infections that previously they would never have been exposed to.

Socio-economic alterations – with an ever increasing global population there is an ever increasing demand for food, including meat.  It also means that as urban populations are expanding people are moving into previously uninhabited areas and so are being exposed to disease-causing agents that they had never been previously.

Political alterations - the authors talk about some countries with poorer veterinary surveillance or that have focal areas of zoonotic infections that previously were not having a global impact because they had strictly state-controlled economies but are now having a global impact because they have transitioned to allowing free  trade.  They also discuss the role that political disruption and upheaval can have on increasing the spread of zoonotic infections.

Scientific impacts – Part of the reason that we are recognising so many zoonotic infections is that we have got better at detecting them.  Infectious agents that we couldn’t have characterised decades ago can now be identified and classified.  Another scientific impact the authors mention is one that you will recognise if you are a regular reader of this blog: there have been many advances in medicine that allow us to live to an older age, but that have a negative impact on our immune system (for example, chemotherapy drugs can make us immunosuppressed; if you have an organ transplant you have to take immunosuppressing drugs, etc.)  This leaves a section of the population at a much higher risk of contracting any disease and so gives rise to some human infections with agents that would otherwise not normally infect humans.

Factors Associated with Pathogen Characteristics

The authors talk about how pathogens (disease causing organisms) that have a high genetic mutation rate (like flu viruses) can help them become zoonotic infections: in the authors’ words “ their enormous mutation rate is essentially a factory producing the species that are most potently pathogenic for humans“

The authors also talk about how biodiversity can impact zoonotic disease transmission in this section (although, personally I’m not sure why it came into this section).  They talk about how sometimes wide diversity can reduce the spread of zoonotic agents because (if I am parsing this correctly) if there are many host animals that a vector (like a mosquito) can feed off there is less chance of it coming into contact with an animal that harbours the zoonotic agent – this is called the ‘dilution effect’.

Climate Change and Zoonotic Infection Resurgence

To quote from the paper: “Global warming is an ecological emergency, but its implications for human disease caused by infectious agents remains understudied“.  We do know some of the effects it could have – increases in temperature in previously colder countries leads to the spread of insects like mosquitoes – and the diseases they carry –  into those countries.  Climate change may also affect bird migration patterns and so may affect the exposure of birds to pathogens and also the exposure of us to them via the birds.

Finally the paper finishes with Projections for the Future. The authors point to 4 issues that “need urgent clarification and further attention“.

1) Recognition of the need for pre-emptive studies on the effects of massive or smaller developmental projects on local animal fauna and local zoonotic reservoirs

2) Recognition and enhancement of the health literacy of special populations that are at increased risk for the development of zoonotic infections (meaning that those patients on immunosuppressant drugs or who are immunosuppressed for other reasons should get more information about where they might encounter zoonotic infections and hw to avoid them.

3) Recognition of the major long-term burden induced by certain of these diseases with a chronic phase. There are some diseases that take a long time for any symptoms to show.  If a person has migrated from an area where the disease is relatively common to one where the disease is rare, the clinicians may be less likely to recognise the disease (or may recognise it at a later stage than if they were practicing in a country where the disease is common).  The authors recommend that clinicians “should be prepared to recognize the long history evolving in such patients and the extreme costs, mentioned in the introductory section, that will be passed on to the host countries”

4) Planning any intervention is difficult, for financial and scientific reasons. The burden of many of these diseases remains unrecognized… any zoonosis imposes a threat to the family as a unit—exposure is likely to be common for members of a household, particularly in agricultural settings, and animal loss (owing to the disease or state regulations for sick animals) may have a significant impact on the economy of the household, which is further worsened by the often observed inadequate access to appropriate medical treatment for the human patients themselves (imagine the scenario in any impoverished or conflict-active region of Africa or Asia). … ambitious eradication campaigns are not always feasible when all of the aforementioned issues have not been taken into account, and neither are successful elimination campaigns, as these may have temporary positive results but subsequent surveillance degeneration, leading to zoonotic resurgences, usually with some twists. (So basically we don’t really fully know the burden of most zoonotic diseases and rushing in there with eradication campaigns without considering all the other factors is not necessarily the best move.)

I think sometimes it can be really easy to think of zoonotic diseases as something one human gets from one animal, but this paper was a good reminder that these diseases can have a much broader impact.  It also had a useful discussion about why the number of zoonotic infections seemed to be increasing, but as it said (and as all papers say) there is still more work to be done in this area.

Image credit

All images were released under a creative commons licence (see links for details).  Thanks to Rugby471 for the dollar sign, to Wegmann for the tourist shot and to DROUET for the virus

Reference

 
Cascio A, Bosilkovski M, Rodriguez-Morales AJ, & Pappas G (2011). The socio-ecology of zoonotic infections. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases, 17 (3), 336-42 PMID: 21175957