UPDATED: 10 things we need to find out about the #NCoV

Following a conversation on twitter on NCoV-EMC, I quickly realised that I did not know enough about this virus. But then I realised that it is that NOBODY knows a lot about it. There are very little answers to a growing list of questions (for whatever politically/funding/technical reasons).

So, here are a few questions that I think really need answered about the novel emerging coronavirus (I.E if you gave me infinite amounts of money, PhD students and post-docs this is what I would look at). If you have thoughts on them (think they're rubbish/not important/drastically important) or have ways to answer them, please comment below!

1) Is the NCoV-EMC isolated the sole causative agent of the viral pneumonia observed across the Arabian Peninsula and Europe?

2) How many humans have been exposed/infected?

3) What is the true case fatality rate?

4) What is/are the reservoir specie(s)?

5) What animal species have been exposed/infected?

6)Why has it emerged/only been detected in the last year?

7)  How efficient is human-human transmission?

8) How does NCoV-EMC induce disease in humans?

9) Is the cell-culture isolated NCoV-EMC the 'correct' wild-type viral sequence we should work on?

10) Is the virus adapting to the human population and if so, in what way and how could that impact pathogenicity/transmissibility?

BONUS question:

11) What are we going to about it apart from sit back and wait? 

Updated 2nd April 2013 from Martin Enserink, Matt Frieman and Helen Branswell

12) How similar is EMC to SARS during infection of the human airways?

13) What proteins/genes encoded by EMC inhibit - however effectively - the human innate immune response?

14) Why doesn't EMC replicate in lab mouse strains? (Apparently it doesn't)

15) What epidemiological studies are being done?

16) Is there an intermediate 'amplifying' host?

17) When did EMC first infect humans?

18) How do humans get infected?

and the clincher:

19) Why don't we know the answers to the above questions already?

Three thoughts on the novel coronavirus cell line study

Sometimes a paper is published and the real-world applicability of the study isn't easily concluded or communicated from the results. Yet despite that, these inferences spread among the media and can result in feelings of confusion, panic and dread when the public are faced with the prospect of a virus more pathogenic than the SARS coronavirus was.

This happened recently following the publication of a paper in the Journal of Infectious Diseases (Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications on disease pathogenesis and clinical manifestation) It's OA too so go have a look. There's also a very good accompanying editorial outlining the issues with drawing clinical conclusions from these data. 

A number of news storys and tweets were communicated concluding that this virus is 'more deadly' than hCoV-SARS, which could only replicate in a few cell lines or does the study even provide evidence that the virus can replicate in many different tissue types? There was however a more muted story in CIDRAP. Can they really say that from their data?

Basically the Hong Kong group used the isolated novel coronavirus (hCoV-EMC) from Ron Fouchier's lab and infected a wide range of cell lines with one infectious virus particle per cell and measured production of viral RNA (I think the genomic positive sense strand) on day 0, 1 and 3 following infection as well as nucleoprotein protein expression as markers of replication and concluding from this viral tropism in a human person. From this they showed that the virus could replicate in nearly every cell line tested and could replicate their genome up to five logs (quite a lot).

MY THREE THOUGHTS:

1) The main issue with this paper is this: these cell lines, although originally human, are all immortalized cancer cell lines characterised by markedly different biological properties when compared to normal human cells of the same tissue/cell type. They can't be readily used a surrogates for normal human tissue/cell types. None were primary cells nor were any even from recently acquired tissue samples from biopsies etc. People have infected primary human airway epithelial cultures with hCoV-EMC - so this can be done successfully - , although it would be more difficult for other tissue types as these cultures haven't been developed. Some of these cell lines used may by chance lack key viral repressors of infection present in normal primary cells, which could skew results from cell culture infection experiments. Plus, a human tissue is not just a single cell type - they are composed of diverse kinds of cells that could together behave much, much differently than cell lines in culture. 

2) The pathogenesis and spread of virus relies on the complex interaction with the human immune system in a tissue specific manner. For example, the hCoV-EMC virus may never escape the human respiratory tract because tissue-resident immune cells and the innate immune system cripple virus replication before it can spread systemically in blood or lymph.

3) Virus spread and tropism also relies on physical cell-cell interactions. For example, measles and other paramyxoviruses gain access to diverse tissues in the human body including the brain and kidneys via infection of immune cells residing in near-by draining lymph nodes or those present in sites of primary replication like the lungs. If hCoV-EMC can't do this nor survive and persist in the blood stream/lymph then how is it go systemic?

All these processes can and should be modelled in some way in the lab but certainly not only through these basic cell culture infection experiments. And I should add that this study doesn't prevent others from doing so and encourage other groups across the world to look into this. The complex interactions of emerging viruses with all cell/tissues/biological processes should be investigated! However, that will require further work in more refined models or animal studies. 

One investigation that would prove extremely useful and answer these questions would be the pathological assessment of banked autopsy material from the fatal cases in the UK (this had been done in SARS). Assessment of the distribution of viral antigen could be used to infer virus tissue/cell tropism and point us in the direction of where and what the block or inhibitory factors act to limit virus transmission/severe pathogenesis like that seen with SARS. 

N.B - the idea for this post came from below.

Over twitter I took part in this brief discussion begun by Laurie Garret's tweeting of the link to the study:

I was probably over critical saying it was 'horribly flawed' - the study and science was OK (though see Matt Frieman's - who is a coronavirus group leader in the U.S - comments below) but it is the clinical conclusions that can be drawn that would be flawed if we took this as evidence that hCoV-EMC is more pathogenic than the SARS virus (it clearly isn't).

Then it was pointed out that there was an informative editorial accompanying the article:

HIV finds a cellular door knob - the SIGLEC1 story

This provides some background/analysis of the upcoming #microtwjc week 18 paper on HIV/SIGLEC-1 interactions

Viruses are classed as 'obligate intracellular parasites' and so they have to get inside a host cell, whether they are bacterial, archaeal or eukaryotic. In the case of mammalian viruses, which I have the most experience in, this is a key aspect of how viruses infect, cause disease and transmit themselves from one host to another in a population. In fact it is probably the most important event in the virus life cycle (here's a great link describing the replication cycle of HIV).

Example: Viruses like influenza that really only get inside lung cells will be respiratory transmitted and may cause lung diseases while viruses like HIV that get inside your immune cells and find themselves rushing around your blood stream will only spread via contact with bodily fluids. This is the same for every other human virus in existence.

However one major obstacle to getting inside your cells is the cell membrane, which is impermeable to particles the size of viruses. The virus must coax or force its way into the cell cytoplasm where it can begin its replication cycle and make new virus particles.

This is what a virus has to contend with: the plasma membrane. But what molecule on the surface will it interact with?

How to grow an awkward virus like Schmallenberg vrius?

If anyone works on viruses that naturally replicate in two distinct hosts and NEED to do this, maybe you can help here.

While having a look around for work on Schmallenberg virus (SBV), I came across a paper (this paper) that compares two methods of doing experiments with this virus in animals. On one hand they have infectious serum from cattle. That is they have blood which they have taken from a single cattle that had been infected with SBV and that is packed full of infectious virus particles. These can be injected back into another cattle for infection experiments. This I will consider mammal-mammal virus.

On the other hand they have cell culture isolated virus. This virus was taken from a cattle blood sample and grown on insect cell lines then grown on mammalian cells again and then from here it can be taken into animals. I will consider this cell culture (insect-mammal) virus.

Now based on animal experiments they say that the mammal-mammal virus, grown and harvested solely from infected cattle is 'better' to use than the cell culture (insect-mammal) virus. They say it is better based on looking at the infection kinetics following inoculation of 4 animals each, in particular it seems because that it replicates to a higher rate in cattle than the cell culture one. Despite not being statistically significant I might add (!).

"this difference provides a clear indication of the pitfalls of culture-based production
of challenge inocula"

Now I really don't think this is correct to state. At least without the right evidence.

For one it is good to remember that these viruses have two natural hosts out there in the wild. At least TWO. Viruses are replicating in insects, then mammals, then insects, then mammals again... Although they can spread from insect to insect (I think) and from cow to calf (mammal to mammal), this last one is an evolutionary dead end. So 'real' SBV, whatever that is, must be that which has replicated in insects and in mammals. Growing virus in any one of these hosts for one time is bound to allow for adaptation to a single host that may prevent it from growing in the other. It is the classical evolutionary trade-off.

In experiments that will determine the development of vaccines and pathogenesis studies we want to work with what is 'real'. Not something that is essentially a lab artefact. Although the virus may replicate to a higher level in cattle, this might not be what's out their in the wild.

Now this brings me to my final point. We don't know which is best because we don't have the evidence. To determine which one is best we would have to compare it to the kinetics of a 'real' infection. Something which might be difficult to do given it's uncontrollable (in a scientific way) nature. One way to get away from this might be to sequence the genome of mammal-mammal viruses and compare them with insect-mammal viruses. They didn't do this.

Without these two pieces of evidence in hand I would want to stick with what Nature does. I would grow the virus in it's natural and medically relevant host species. Midges and cattle. This way we assure that we are working with something that is 'real'. Although it might be cheaper to sequence the damned things and see what's happening during growth on the different 'substrates'.

Notes on zoonotic rubulaviruses

I've been meaning to post about this paper for a while now and thought that nearing the end of the year would be a good time to clear my head of thoughts about it.








The paper I'm talking about is one that was published recently in the Journal of Virology (http://www.ncbi.nlm.nih.gov/pubmed/23152534), and concerns itself on the discovery, characterisation and epidemiological investigations of two bat viruses. It was carried out by a large team from the UK and around the world. The first author was Kate Baker.

These viruses, which they isolated themselves (yes! actually, physically have virus to study) were found in the urine of fruit bats (species: Eidolon hevlum, the straw coloured fruit bat - a truly magnificent animal). There's good evidence that these viruses are natural 'pathogens' of bats and there is limited evidence to suggest that there has been human exposure to them in the past. The importance of this is limited but personally I would prefer to know what is out there before it emerges.

For another take on this paper check out Andrew Shaw's Virus Musings blog: http://www.virusmusings.blogspot.co.uk/2012/11/when-is-zoonosis-not-zoonosis.html.

Antibiotic resistance found in isolated cave system

The next weeks #microtwjc paper has now been chosen and stuck up online over at http://microtwjc.wordpress.com/2012/12/09/microtwjc-week-17-christmas-edition-paper-and-discussion-points/ . If you want to discuss it, log in to twitter and join us Tuesday the 18th December at 8:00pm GMT. And if you are interested in the topic you should most definitely check this Nature review out http://handelsmanlab.sites.yale.edu/sites/default/files/AllenCalloftheWild.pdf

It's a neat paper that focusses on characterising the levels and kinds of antibiotic resistance in bacteria that live in a relatively isolated cave in New Mexico that has had extremely minimal human contact. The major point of this paper is that compared to other studies their site seems to be the most isolated microbial community, although this investigation in Alaska may be just as isolated. Although I dont think they can rule out water contamination from outside the cave system (actually from reading this article in NatGeo I think they can rule that possibility out). This they say is an 'ideal ecosystem' to study the original antibiotic resistance programs in the absence of human exposure.

To do this they employ a culture dependant approach, so obviously will only detect a small number of resistant microbes yet may be able to detect resistance mechanisms that we did not know about and so could not easily detect through purely molecular means. They do even find completely new ways that microbes have evolved to handle antibiotics.

One perhaps good thing about their results is that this cave is isolated so perhaps woudn't be such a reservoir for novel antibiotic resistance genes in a clinical setting.

A statement from their conclusion explains:

Antibiotic resistance is manifested through a number of different mechanisms including target alteration, control of drug influx and efflux, and through highly efficient enzyme-mediated inactivation. Resistance can emerge relatively quickly in the case of some mutations in target genes and there is evidence that antibiotics themselves can promote such mutations [43], [44], [45], [46]; however, resistance to most antibiotics occurs through the aegis of extremely efficient enzymes, efflux proteins and other transport systems that often are highly specialized towards specific antibiotic molecules. Such elements are the result of evolution through natural selection; this therefore implies that antibiotic resistance has a long evolutionary past.

and

The remarkable genetic diversity of the antibiotic resistome, uncovered in this and other studies has additional practical application as an ‘early warning system’ for new drugs introduced into the clinic. Resistance mechanisms in the environmental resistome can emerge in the clinics and the clinical community should be aware of them...

Some questions I had are:

How isolated is this community?

Would it have better (possible?!) to sequence everything?

Should we be worried about this resistance?

If not effected by human antibiotic use, why do they have resistance mechanisms?

Antibiotic Resistance Is Prevalent in an Isolated Cave Microbiome

Buller et al. 2012

Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.

Meningitis B and the future of vaccines

This week is a good week for vaccines. Indeed it is a good week for society, at least in Europe, for we have just got word that the European Medicines Agency has approved Novartis's Meningitis B vaccine  and it could now be available in the UK as early as next year, if licensed here. *Novartis are currently working with the US authorities at getting it approved*

This vaccine, targeting Neisseria meningitidis group B bacteria (B subgroup causes the most problems in industrialised countries) is reported to be around 70% effective against the horrible and often deadly disease. It's roll out across the UK and Europe should save the lives and prevent the permanent damage that follows meningitis and septicemia. That's great, sure, but as scientists and people interested in public health, how the heck did they achieve this feat?

Mumps in New York - it's the size that matters

Two Jewish men in New York (Flickr by Kynan Tait). The site of 2009/10's near-4,000 large mumps outbreak

It started during the middle of 2009 when an eleven-year old boy returned home to the U.S from a holiday in the UK. The UK was just experiencing an exceptionally large (about 7,500 people) outbreak of mumps that year but don't worry, that boy had been twice vaccinated with the MMR immunisation. He should be OK. Shouldn't he?

As it turns out, he wasn't and he became infected with the mumps virus. And so our three year-long story begins.

The problem was that by the time this now mumps-infected boy realised he was ill (fever, swollen glands, potentially inflammation of the testicles and meningitis) he was attending a youth camp in New York along with 400 other orthodox Jewish boys. Unluckily for us, the mumps incubation period can be over 2 weeks and you can be infectious up to one week before this, making it particularly hard to contain as we will see.

A couple of days later the camp ended and each one of those mumps-exposed, potentially infected children were seeded back into society. By the time it had subsided nearly one year later, 3502 cases of mumps had been observed across the state of New York, the biggest outbreak the US had seen in decades and it was most likely attributable to this single index patient who brought the disease in from the UK.

But what was most worrying was that 76% of them had been immunised with two doses of the MMR vaccine, our mainstay of protection against the virus. So just how could mumps get passed this defence and cause such a massive outbreak? It took the U.S Centers For Disease Control and Prevention nearly 3 years to find an answer. The investigation is published in the New England Journal of Medicine here.

HIV in High-def

Plaque outside Antoni van Leeuwenhoek's old house in Delft

The dark age of microbiology existed in the years preceding Antoni van Leeuwenhoek's most famous microscopic study of Delft's canal water and the investigative work of his contempories, Robert Hooke and Athanasius Kercher (who was most likely the first human to witness microscopic life). In these days we had recognised the effects of what we later called microbes but we had little evidence of what was causing them, for who could not wonder what induced a feverish, spluttering epidemic of 'flu? Nor who could not wonder what was controlling the geochemical processes occurring across the Earth?  We just did not have the tools then to probe their world any further in any scientific way.

Will we see Crimean-Congo Hemorrhagic Fever again?

Last Tuesday a man, flying into Glasgow, Scotland from Dubai, was admitted to the local hospital with a very rare disease in these parts - in fact it was the first reported clinical case of the disease here. It is known as Crimean-Congo Hemorrhagic Fever, or CCHF and it subsequently emerged that after being transported to a specialist centre in London he later died at the weekend, a horrible death probably characterised by rapid onset of fever, headaches, hemorrhage, intestinal damage and neurological symptoms.
 
This particular disease is caused by a virus that we know quite little about. The CCHF-virus (CCHFV) and we have nothing in our grasp to stop or prevent it but it is one that we our keeping a very, very close eye on.  Hence the big interest in the recent case in Glasgow. This virus can have a case-fatality rates of upwards of 30%, however the rate of subclinical infections is largely unknown but might be close to 90%.

CCHFV is a very geographically widespread virus found across two major continents and about 30 countries. Thankfully not yet in North-Western Europe and the UK but it is found across much of Eurasia (Eastern Europe, Asia and the Middle East) as well as Africa. It was first seen in the Crimean peninsula during the 1940's and later popped up in the Congo (hence it's name) but don't let that fool you, it's not as restricted as that might let you believe. There is evidence that its range is even increasing.

Now, CCHFV is one of those awkward viruses, it has a rather complicated lifestyle choice. It is zoonotic as in it comes from animals and is not like measles and mumps. In order to survive it has to infect both ticks (a kind of blood-eating invertebrate arachnid) and vertebrates, like domestic animals or even humans. But the major risk factor for us are tick bites. And to complicate matters even further, the young virus-laden tick particularly likes to live out its youth on the backs of smaller vertebrates like hedgehogs and only once it has matured can it jump to cattle, sheep and goats plus some species of birds (the kind of animals that we like to have around us. The virus is a two-host parasite and so is the tick. The virus can even spread from tick to tick during reproduction (sex and birth) and it can even move from vertebrate to vertebrate, given close contact with infected bodily fluids. It is this final property that makes public health workers so worried: it can really kick off around an ill-prepared hospital.

CCHFV is a bunyavirus, like schmallenberg virus.

So to understand why CCHFV is where it is, you have to understand this cycle of infection and given that the same domestic animals are found throughout the world, the major controller of CCHFV presence are the ticks. It is a pretty old and genetically diverse virus which probably spread across its range with the expansion of agriculture from the Middle East across Asia and down in Sub-Sahaharan Africa.

One of the most favourite things of CCHFV are Hyaloma species of ticks, in particularly Hyaloma marginatum. These hard-bodied ticks live for about 3 months and as described above pack that short life full of multiple host changes. These ticks are also pretty widespread across the world but they really prefer dry and open habitats full of their small and large vertebrate hosts. Worryingly these species of tick have been found in areas of South Europe surrounding the mediterranean and has even been found as far north as the UK, carried there by migratory birds. Recently, CCHFV infected ticks were even detected in Spain found on deer.However in both these places, endemic or even epidemic outbreaks of the virus have not been observed. Perhaps the virus is not completing its full life cycle there or maybe infections do occur but are sub-clinical. A recent analysis suggested that the risk to more northerly European regions is low, citing cold springs that would prevent the ticks from surviving. But that doesn't stop Europe from worrying as two years they published a report mentioning that:

 "....a rise in temperature and a decrease in rainfall in the Mediterranean region will result in a sharp increase in the suitable habitat areas for H. marginatum and its expansion towards the north, with the highest impact noted at the margins of its current geographic range"

That's right. One of the major issues with tick borne pathogens is the changing environment, whether it is climate change or agricultural growth. Here's a great review on the role of climate change on the distribution of ticks. One of their points regarding CCHFV is that very little is understood about how this virus interacts with ticks (we don't exactly know what species it infects or how) and their vertebrate hosts (what animals are infected in the wild). So to answer the question at the start: will UK ever see CCHFV again? I wouldnt be suprised if another imported case springs up from endemic regions but whether it will establish itself here is not so predictable. Certainly at the minute it cant: our weather is much too harsh for the tick. But what about in the future as climate change alters the environment in and around the Mediterannean? We're going to need more research into the virus to answer the above questions. CCHFV is an important global pathogen and an unmet medical need.