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.