Viruses and obesity: a link between the outcome of viral infection or a cause of obesity?

In both the developed and non-developed world an increase in the number of obese children has been observed since the late 1970s/early 1980s. Since then, numerous studies contributed this increase to changes in diet, physical activity, television watching, computer games and food advertising to name a few. The very same studies however also indicated that these reasons alone can not explain the increase in childhood obesity fully and consequently the pattern of the rapid spread of obesity has been (partially) attributed to other reasons such as being the result of inflammation caused by bacterial or viral infections.
Research in animal models (chicken, mice, sheep, rats, goat, dogs and hamsters) indeed led to the identification of eight viruses to cause obesity in animals and research identified both human and animal Adenoviruses -specifically human Adenovirus 36 (Ad 36)- capable to infect adipocytes, resulting in the accumulation of triglycerides in adipocytes and changes in transcription factors involved in the differentiation of pre-adipocytes into (mature) adipocytes as well as in the modulation of the inflammatory response. Indeed, a higher percentage of obese (30%) than non-obese subjects (11%) tested seropositive for Adenovirus 36 antibodies.
Moreover, adenoviral induced obesity been implicated in increased risk of Influenza virus infection and morbidity during non-pandemic and pandemic Influenza seasons. 
So how might pathogens like Adenovirus induce obesity and how might this contribute to increase risk of morbidity following infection with Influenza virus.

                              Inflammation and obesity

An important role in the antiviral response is played by pattern-recognition response factors (PRR) such as Toll-like receptor 3 (TLR-3), Retinoic acid-inducible gene I (RIG-1) and Melanoma Differentation-Associated antigen-5 (MDA5), all of them which are expressed in pre-adipocytes and mature adipocytes alike upon induction by activated IFN regulatory factor 3 (IFN3) and up regulation of pro-inflammatory factors IL-6 and TNF-α by nuclear factor kappa B (NF-κB). 
Experimentally this system can be induced by transfecting Poly (I:C), leading to the expression of leptin, resistin and adiponectin in mature adipocytes as well as the differentiation of pre-adipocytes into (mature) adipocytes. Blocking this system by using inhibitors specifically targeting IRF3 (BX795) or NF-κB (BAY11-7082) or using cells deficient in TLR-3 (TLR-3 -/-) showed that in particular RIG-1 or MDA5 are responsible for the inflammatory response since both are inducing the expression of pro-inflammatory cytokines. Notably, transfected Poly (I:C) inhibits the differ-entiation of pre-adipocytes to adipocytes and might thus contribute to increased insulin resistance. In addition, blocking this system might also be effect the expression of Interferon stimulated genes.

Poly (I:C) is considered to be mimetic of (specifically viral) dsRNA and does not occur in nature and stimulates TLR-3 in a wide variety of immune cells including dendritic cells and B-Lymphocytes.

The attentive reader however might know that Adenoviruses are double stranded (ds) DNA viruses, i.e. that their genome is consists of dsDNA (other viruses have single stranded DNA or RNA as a genome for instance) and thus are known to activate a TLR mediated antiviral respon-se. Consequently it is conceivable that the infection of Pre-adipocytes with Adenovirus might prevent the differentiation of Pre-adipocytes into mature adipocytes and furthermore induce insulin resistance. Voila, case closed or quod erat demonstrandum as the mathematician would say. Except that both experimental and causative evidence is far from certain since the disease is after all multi-causative.


What about the increased mortality due to obesity in patients in-fected with Influenza viruses? As outlined above, the activation of PPRs in pre-adipocytes downregulates the immune response and increases the expression of inflammatory cytokines. Indeed it is has been speculated that one reason of the increased morbidity in patients infected with A/H1N1/1918 was a “cytokine storm”. If however this is the case in the obese patients infected with current Influenza virus is open to speculation. 

Last but no least, in the case of Ad36, the E4orf1 gene product has been implicated to be responsible for the adipogenic effect observed in obese patients. Currently, the experimental proof is still far from certain.

What about other viruses? TLR-3 recognizes dsRNA and is part of the antiviral response of RNA viruses such as Rhinovirus and other Picornaviruses. A study from 1981 linked encephalomyocarditis virus to the development of diabetes in mice but in humans a link has not been demonstrated and the pathogenetic analysis showed only a destruction of pancreatic cells in obese mice but not in lean littermates. The inflammation was accompanied by an infiltration of macrophages. Indeed the cytokines expressed as a result of Poly (I:C) transfection into pre-adipocytes and adipocytes might also recruit Macrophages and thus induce an inflammatory response. 

Finally, other pathogens might cause similar problems since the bacteria can induce an antibacterial response akin to the antiviral response. 

Further reading:

van Ginneken V, Sitnyakowsky L, & Jeffery JE. (2009) "Infectobesity: viral infections (especially with human adenovirus-36: Ad-36) may be a cause of obesity. Medical hypotheses, 72(4), 383-8. PMID: 19138827

van Ginneken V, Sitnyakowsky L, & Jeffery JE. (2009) "Infectobesity: viral infections (especially with human adenovirus-36: Ad-36) may be a cause of obesity. Medical hypotheses, 72(4), 383-8. PMID: 19138827

Atkinson, Richard L. (2008-01--1) Could viruses contribute to the worldwide epidemic of obesity?. , 3(s1), 37-43. DOI: 10.1080/17477160801896754

Hur, Sun Jin. (2013-10--1) Effect of adenovirus and influenza virus infection on obesity. , 93(16), 531-535. DOI: 10.1016/j.lfs.2013.08.016 

Cocoros, Noelle M. (2014-01--1) Obesity as a risk factor for severe influenza-like illness. , 8(1), 25-32. DOI: 10.1111/irv.12156 Yu, Lili. (2014-02--1) Pattern recognition receptor-initiated innate antiviral response in mouse adipose cells. , 92(2), 105-115. DOI: 10.1038/icb.2013.66 

Genoni, Giulia. (2014-01--1) Obesity and infection: two sides of one coin. , 173(1), 25-32. DOI: 10.1007/s00431-013-2178-1

Emerging Influenza Viruses

Frequently media outlets are reporting the identification of a novel strain of Influenza and in recents years this includes the identification of novel strains of avian influenza. More often than not, novel strains are identified because they have shown to cause severe disease in humans infected. 
Following the conformation of a human case of Influenza A H7N9 in January of this year -and subsequent culling of chicken in Hongkong and a ban of poultry exports-, the Financial Times reported the identification of a "novel" Influenza virus, A/ H10N8 in a 73 years old female patient. The corresponding case study was published on February 5th 2014 in The Lancet and emphasizes that the virus isolated from this patient is genetically distinct from previous isolates from avian sources, thus potentially adapted to infect humans - so strictly speaking it is a virus which is already established in avian species and after crossing into the human population mutated to cause disease. Mind the words here - adapted to infect humans, which implies that so far human to human transmission has not been proven to occur. In fact, H10N8 is one of many avian Influenza viruses which have been shown the ability to infect humans. Some of them have been shown to be able to be transmitted between humans and thus have the potential to cause a worldwide pandemic. So far however this has not occurred and it may be that some of the potential viruses might have become attenuated during human to human transmission and/or that cross-immunity caused by circulating viruses might be sufficient to prevent disease. Indeed it is known that relatives from patients infected and hospitalized for "bird flu" are often seropositive for antibodies - whether they acquired the virus from the patient or from the environment is not entirely clear, but there are indications that they were infected by the same sources as the patient. 

I was delighted to read in the Financial Times that they interviewed a well known virologist and expert on Influenza from the Imperial College London, Wendy Barclay, who pointed out that we have to careful in the interpretation of the results. The caveat was indeed correctly stated in the Lancet article but might have been overlooked by less through investigation. 
Reading a well researched article in the press on a topic which can easily cause widespread fear in my opinion justifies why we should pay for newspapers instead turning to free news sites who are dependent on advertisers for funding. 

Further reading:

Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study
To KK, Chan JF, Chen H, Li L, & Yuen KY (2013). The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1: a tale of two cities. The Lancet infectious diseases, 13 (9), 809-21 PMID: 23969217

HaiYing Chen, Hui Yuan, Rongbao Gao, Jinxiang Zhang, Dayan Wang PhD, Ying Xiong, GuoYin Fan, Fan Yang, Xiaodan Li, Jianfang Zhou Shumei Zou, Lei Yang, Tao Chen, Libo Don (2014). Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study The Lancet DOI: 0.1016/S0140-6736(14)60111-2

When or if to publish

One of the questions any scientist faces at some point into her/his career is, if it is worth to publish findings which are a mere byproduct - findings which are not part of the original project and do not contribute in answering the original question.
Personally I faced this dilemma at the end of my PhD and at the time we decided not to publish the results. One or two years later however another group published almost identical results albeit in a low impact journal. Initially I was disappointed and angry of not pushing the case of publication but as time passed, I calmed down and moved on. Currently I am facing a similar dilemma but since the work was not and is not part of pet project I am relatively relaxed. Part of the problem to decide whether to publish or not is the cost associated with a publication and the time (and money!) potential additional experiments take away from more important projects. Also, these days grants may not allow to stray away too far from the proposed project. On the other hand, these "side-projects" might provide an opportunity to for new grant applications and an opportunity for postdoctoral researchers to establish their own lab.
Anyway, it was a post I read at lunchtime which caught my eye. Please find the original post by Andrew Shaw, entitled Virus Musings: Should I have published? , below:

Should I have published?

Since finishing my PhD I've been faced with a dilemma. In a nutshell, having come across a (somewhat serendipitous) observation in my PhD studies, should I publish it? I decided to publish, and it was both an editor's pick and is now regarded by the journal as 'highly accessed' (the importance and possibly ephemeral nature of such labels is a completely different discussion). That implies it was worthwhile, but was it?

The study revolved around an observation in BHK (hamster) cells infected with Bluetongue virus (BTV). The cells looked very strange: rounded and with condensed DNA/chromosomes in a pattern suggestive of some stage in mitosis, albeit a rather odd looking mitosis. To try and see what's going on, we used confocal microscopy with a panel of antibodies to look at the status of various parts of the cell division machinery. In brief, we found that the centrosome, a major orchestrator of mitosis, was severely disrupted. Co-incidence or not, the BTV protein non-structural protein NS1 also located in the region.

A-D. Different BTV serotypes (16, 1 and 8) induce aberrent mitoses (although BTV-16v induces the most). Different cell types can also be affected, although BHK cells appeared to be the most susceptible.

Something that was conspicuous was the association of the viral NS2 protein with the condensed chromosomes. When we took a series of images in the z plane and analysed them it became clear that NS2 appeared to be associated with the kinetochore. Combined with the observation of its location on microtubules, it is conceivable that NS2 may be a microtubule cargo molecule (or interacting with one) that obscures the kinetochore during the initial stages of mitosis. As the microtubules polymerise though the cell, the tips don't find the kinetochore, resulting in faulty mitosis. Many viral proteins use microtubules to get around and, based on other viruses, the dynein/dynactin complex would be an interesting  place to start looking for a protein that interacts with NS2.

A. NS2 expressed from a plasmid locates to microtubules (red). B. Z stack images reveal NS2 located at positions suggestive of the chromosome centromeres. C and D. Expression of NS2 from a plasmid recreates the aberrent mitotic phenotype.

To look at whether NS2 alone is capable of inducing the aberrent mitosis, we transfected cells with plasmids encoding the protein. When looked at from a confocal perspective, the transfected cells appeared to reflect the phenotype seen with virus infection. When a GFP-tagged version of NS2 was used in live cell imaging, we found that the cells were less likely to complete mitosis correctly, spent longer in mitosis, and resulted in an increased level of binucleated cells.

Transfecting HeLA cells with a palsmid expressing a GFP-tagged version of BTV NS2 resulted in a longer time spent in mitosis, a reduced level of successful mitosis, and binucleation.

 So, to the options. 

1) don't publish. At the end of the day it's just an observation; I have not elucidated an exact mechanism and nailed down a precise protein, as would be expected for a publication in a journal of greater 'impact'. Not taking the story to an end, followed by publishing in a prestigious journal might be viewed as poor science by some. 

2) publish. Many would argue that publishing information, regardless of how seemingly insignificant, is important and, arguably, a necessity based on the fact that it is being funded by the public.

I published. Partly for the reasons outlined in scenario 2, but also because the study was at a point where other people had contributed work, in which case it would not be fair for them to have done this work only for me not to publish. Of course, continuing the project to the end would have been my (and my collaborators') preferred option, but time ran out. As it stands, this observation is in the public domain for all to see, with the option of progressing it further to try and unravel what's happening.

Should I have published? I'm satisfied that I did, but it once again highlights the question of how many other such observations are languishing in abandoned lab books around the world.

Andrew E Shaw, Anke Brüning-Richardson, Ewan E Morrison, Jacquelyn Bond, Jennifer Simpson, Natalie Ross-Smith, Oya Alpar, Peter PC Mertens and Paul Monaghan (2013). Bluetongue virus infection induces aberrant mitosis in mammalian cells Virology Journal DOI: 10.1186/1743-422X-10-319

Again, there is no definite answer to this questions and one has to decide carefully if the work merits publication. This is a matter which the researchers involved should ask themselves always - to be frank there are  a lot of papers out which should not have published at all. Others should have been published including results from a second paper, i.e. in cases where it is evident that the group opted for two papers in order to boost the publication records.