Archaeovirology: Solving the riddles of the past

In recent years the advent of modern technology allowed researchers to amplify ancient DNA from a variant of organisms - organisms ranging from Homo (sapiens) neanderthalensis (more commonly known as the Neanderthal) to insects enclosed in amber. Common problems found is the scarcity of DNA due to the age of the specimen and problems of contamination with foreign DNA , the latter being a problem if human specimens are involved. In terms of pathogens, a recent paper in Scientific Reports researches at the Lawrence Livermore Laboratory compared samples of Vibrio Cholerae dating back to 1849 CE and Yersinia Pestis dating back to 1348 CE with modern samples; other studies identified the agent of trench fever, Bartonella quintana, in remains from Napoleon’s Grand Army.  Generally speaking the detection of ancient pathogenically DNA is hampered by false positives as well as false negatives and requires a short sequence of known DNA sequence in order to PCR amplify the DNA.

In addition to these problems, viral pathogens may contain RNA instead of DNA as their genetic material, further complicated if the genome -as in the case of Influenza virus- is segmented. 

Once the genome is sequenced however, the virus can be ‘“recreated”  by transfecting cells with the DNA/RNA and its pathogenesis studied in detail and compared with modern relatives in addition to phylogenetic studies. 

                               Case study: Influenza H1N1 1918

The classic example of a “resurrected” virus is the causative agent of the Influenza epidemic of 1918/1919,  (human) H1N1. At the time of the epidemic no virus was isolated from infected patients due to the limitations of the methods available at the time - the first swine Influenza was not isolated until 1931, followed by the isolation of the first human Influenza virus in 1933 by Andrewes, Laidlaw and Smith. In the meantime, studies using serum from patients which were exposed to and survived from infection with the 1918 suggested that the 1918 virus is similar to those circulating in swine, suggesting that either the virus crossed into the human population via swine or vice versa. Archeovirological search for the 1918 virus started in earnest in 1951 when he team including Johan V. Hultin  from the University of Iowa attempted to isolate virus particles from tissue samples taken from victims which were buried in the permafrost of Alaska. Although they did not succeed at this time, it would be samples from this very location which later allowed Jeffrey Taubenberger to isolate viral RNA corresponding to fragments of the 1918 virus by using more sophisticated technology unavailable at this time - Polymerase Chain Reaction, more commonly known as PCR.  In a similar attempt  Kirsty Duncan, a geographer then at the University of Windsor in Canada, began to search for other victims of the 1918 flu whose bodies had been buried and preserved in permafrost. She located bodies of coal miners who had died in 1918 and were buried in the cemetery of the little village of Longyearbyen  on the island of Spitsbergen (Norway); again however the team failed to isolate virus particles nor was able to isolate RNA fragments (this was in 1992 - one reason might have been that the burial ground was not frozen throughout the year). 

Following the isolation of the viral RNA segments, these were used in reverse genetic system to infect various cell lines and animals, including mice, ferrets and guinea to study the pathogenesis of the disease and help to determine the cause of the high mortality seen in patients. Ferrets were used because they are transmitting the disease easily (and are the standard animal model in Influenza virus research) and guinea pigs were reported to succumb to the 1918 Influenza in paper published in the Journal of the American Medical Association (JAMA) in 1919.  Furthermore, 1918/rec was also infectious in pigs thus supporting an earlier hypothesis that the human 1918 is able to replicate in swine and that swine might be have been a reservoir for H1N1 ever since. In this context I should mention that the A/H1N1/2009 virus is considered to have crossed into the human population from pigs.

Studies by Peter Palese from the Mount Sinai Hospital in New York suggesting that the PB1-F2 gene product might have been one of the main contributors of the massive necrosis of lung tissues observed in victims of the 1918 epidemic and in patients from other Influenza pandemics  in the twentieth century as well as in the 2009 epidemic. 

In conclusion, the recreation of the Influenza 1918 virus and subsequent studies in various animal model not only helped to understand the pathogenesis of a past epidemic but also helped to understand recent epidemics and may help to determine if one of the novel recombinants has a pandemic potential.

                            Case study: Pithovirus Sibericum

The most recent example of an ancient virus is  Pithovirus sibericum, which was recently isolated in the lab of  Jean-Michel Claverie from the University of Mediterranée in Marseille.

According to the radiocarbon dating, the sample from the Siberian permafrost was 30000 years old - so the term “ancient” is more than appropriate in this context. 

Pithovirus sibericum is a double stranded (ds)DNA virus infecting amoebae -non-infectious for humans- in their shape and size similar to Pandoraviruses and other 

Megaviruses. The latter were only recently discovered and owing to their size and shape originally not classified as viruses but “endocytobiotes”. Similar to those, Pithovirus sibericum replicates in cytoplasmic replication centers of infected hosts. 

The discovery of this novel virus highlights that as we will experience change in the climate new pathogens may emerge from the Permafrost. These might or might not be infectious for human beings. The emergence of zoonotic diseases in the past however has shown that pathogens previously associated with animals might be able to adapt to human cells.

                                 Case study: Poliovirus

Whilst Poliovirus is not an extinct virus, the creation of an artificial Poliovirus in 2002 in a cell free system caused a worldwide stir in the media. 
Based on the nucleotide sequence made publicly available and methods developed in the 1980s and 1990s, a group headed by Eckard Wimmer chemically synthesized the genome of a Poliovirus as a DNA molecule which was then converted in vitro into complete, infectious, Poliovirus particles. Although Poliovirus is a RNA virus, the researchers decided to use DNA as a template since it was not possible to synthesize stable RNA molecules of the required size. The cDNA generated was converted into viral RNA by using a specific RNA transcriptase, generating infectious  viral RNA. Although this RNA could have been used to transfect cells, the authors instead generated viral particles using a cell free extract of human cells (devoid of nuclei, mitochondria and other organelles). The resulting virus was shown to be infectious albeit less than wild-type virus.
It should be emphasized that the virus was generated -not  created- by using an existing blueprint.

Further reading:

Raoult D, Dutour O, Houhamdi L, Jankauskas R, Fournier PE, Ardagna Y, Drancourt M, Signoli M, La VD, Macia Y, & Aboudharam G (2006). Evidence for louse-transmitted diseases in soldiers of Napoleon's Grand Army in Vilnius. The Journal of infectious diseases, 193 (1), 112-20 PMID: 16323139 

Devault AM, McLoughlin K, Jaing C, Gardner S, Porter TM, Enk JM, Thissen J, Allen J, Borucki M, Dewitte SN, Dhody AN, & Poinar HN (2014). Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array. Scientific reports, 4 PMID: 24603850 

Andrewes CH (1939). Immunity in Influenza: The Bearing of Recent Research Work: (Section of Epidemiology and State Medicine). Proceedings of the Royal Society of Medicine, 32 (3), 145-52 PMID: 19991749 

Weingartl HM, Albrecht RA, Lager KM, Babiuk S, Marszal P, Neufeld J, Embury-Hyatt C, Lekcharoensuk P, Tumpey TM, García-Sastre A, & Richt JA (2009). Experimental infection of pigs with the human 1918 pandemic influenza virus. Journal of virology, 83 (9), 4287-96 PMID: 19224986 

Taubenberger JK, Hultin JV, & Morens DM (2007). Discovery and characterization of the 1918 pandemic influenza virus in historical context. Antiviral therapy, 12 (4 Pt B), 581-91 PMID: 17944266 

Zamarin, D., Ortigoza, M., & Palese, P. (2006). Influenza A Virus PB1-F2 Protein Contributes to Viral Pathogenesis in Mice Journal of Virology, 80 (16), 7976-7983 DOI: 10.1128/JVI.00415-06

Weeks-Gorospe JN, Hurtig HR, Iverson AR, Schuneman MJ, Webby RJ, McCullers JA, & Huber VC (2012). Naturally occurring swine influenza A virus PB1-F2 phenotypes that contribute to superinfection with Gram-positive respiratory pathogens. Journal of virology, 86 (17), 9035-43 PMID: 22674997 

Legendre M, Bartoli J, Shmakova L, Jeudy S, Labadie K, Adrait A, Lescot M, Poirot O, Bertaux L, Bruley C, Couté Y, Rivkina E, Abergel C, & Claverie JM (2014). Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proceedings of the National Academy of Sciences of the United States of America PMID: 24591590 

Philippe N, Legendre M, Doutre G, Couté Y, Poirot O, Lescot M, Arslan D, Seltzer V, Bertaux L, Bruley C, Garin J, Claverie JM, & Abergel C (2013). Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science (New York, N.Y.), 341 (6143), 281-6 PMID: 23869018

Wimmer E (2006). The test-tube synthesis of a chemical called poliovirus. The simple synthesis of a virus has far-reaching societal implications. EMBO reports, 7 Spec No PMID: 16819446

Poliovirus - a model for a (nearly) successful vaccination campaign

Poliomyelitis or infantile paralysis is a potentially fatal infectious disease of the spinal cord and the brain caused by Poliovirus - a small positive strand RNA virus part simliar to other Enteroviruses such as the Coxsackieviruses, Rhinoviruses and Echoviruses. Serologically three different serotypes can be distinguished, Poliovirus I, II and III, each with slightly different structures of the capsid protein. The three subtypes not only differ in the structure and properties of the capsid protein but also in their pathogenicity, with Type II being the mildest. In children, following an incubation period of about 3-20 days with minor symptoms, a second phase may occur which is characterized by meningitis and paralysis. In adults the first phase is often skipped, presenting themselves with paralysis and meningitis. Both phases are accompanied by high fever and general malaise, including diarrhea as well as vomiting and a severe headache.

Poliomyeltis was a disease very common in the western hemisphere up to the mid-1950s, declining after the introduction of the first successful vaccine, an inactivated vaccine (termed IPV) which protects vaccinated children against all three types of Poliovirus and developed by Jonas Salk at the University of Pittsburgh. Later Alfred Sabin would propagate his vaccine, a live attenuated vaccine (termed OPV for Oral Polio Vaccine) and the Salk vaccine would be replaced over time by this one. Since the 1990s however the use of OPV is discontinued in most countries since it has been shown that OPV can recombine with other Enteroviruses and cause a disease resembling poliomyelitis in non-vaccinated children and in those children which have been vaccinated but where the vaccination failed. Additionally, it also has been shown that a significant number of children which were vaccinated with OPV would shed virus in their faeces - a considerable problem in areas with poor sanitation and thus be able to infect unvaccinated members of the household. Without dwelling too much on the disease, the introduction of a mass vaccination program in the 1950s was a success story, second only to the introduction of the smallpox vaccine. Although only a relative small number of those infected with the virus required hospitalization -if showing any symptoms at all- those who were admitted often faced permanent paralysis of the extremities, the extent depending which part of the spinal cord was infected. Death was often avoided only by placing the children in iron lungs (see image above); even if the children survived often they would face paralysis and motoneuron disease later in life, often 30 to 40 years after the initial infection cleared. This disease however is not induced by the virus itself but due to the death of motoneurons during the initial infection and subsequent "rewiring" of the nervous system.

Poliovirus is transmitted via the faecal-oral route and is able to survive the acidic pH of the gastrointestinal tract. In asymptomatic patients (95% of the infections are asymptomatic), the virus can only be detected within the bloodstream followed by viral clearance. In 5% of the cases however the virus is able to infect and replicate other cells as well, such as the brown fat, mononuclear phagocyte system (which includes microglia) and muscles - causing headaches, menignitis and general malaise. It is only in 1% of these cases that the central nervous system is infected and paralysis to a varying degree occurs, including breathing difficulties. In general, this last phase of the disease (the “neurological phase”) is considered to be accidental.

So why elaborate on a disease which is close to be eradicated, with epidemics belonging to the past? First, the disease is not eradicated yet. Deadlines have been announced only to be replaced with new deadlines. According the WHO, Polio is still endemic in parts of Afghanistan, Nigeria and Pakistan. Vaccination campaigns in these countries are hampered by Muslim fundamentalists which believe that the vaccination of girls in particular is a campaign of the West to “sterilize" them or infecting them with HIV as well as causing apostasy or immoral behaviour. Aid workers are frequently targeted in attacks, fueled by suspicions that they are foreign agents, under the disguise of  providing vaccinations. 
Second, as I pointed out the Sabin vaccine was used for a long time.  Part of the reason of the introduction of the Sabin vaccine was the “Cutter incident” where a batch of the Salk vaccine was shown to cause illness in children following inoculation. This was shown to be caused by inadequate production of the vaccine; the company did not follow the guidelines, government regulators also failed to act and Jonas Salk was eventually cleared. In the early 1960s IPV nevertheless was replaced by the live attenuated vaccine developed by Alfred Sabin. This vaccine has the advantage that -since it is given orallly- it mimics the natural route of infection. Children also might prefer the sweet taste of sugar instead of the pain and scare associated with needles. In an interesting side note, this vaccine was first introduced in the Soviet Union and Czechoslovakia prior to his native US, following a visit of Mikhail Chumakov to the US in 1956. 

However, soon after the introduction of OPV in the US a phenomenon termed “Vaccine Associated Paralytic Polio” (VAPP) was reported, later to be shown to be the result of recombination with other types Enterovirus, especially Coxsackievirus, in the gut of vaccinated children or of mutations of the genome of the vaccine strains. These hybrid viruses (circulating Vaccine Derived Poliovirus or cVDPV) are highly neurovirulent and caused a smoldering outbreak in Egypt and Hispaniola; paradoxically, stringent vaccination with OPV can limit these outbreaks.

So what is to be expected in the future? Hopefully the disease can be extinguished soon, providing that the last remaining pockets vanish. The underlying question is do we need to be vaccinated? The opinions are divided, with the majority opinion be that we need to continue the vaccination for children for a while especially if cVDPV should become a problem.      

To this day, other Non-Poliovirus enteroviruses cause serious disease in humans, such as viral meningitis. A Poliovirus like disease was reported in California between August 2012 and July 2013, with symptoms similar but distinct from Poliomyelitis and not caused by Poliovirus nor cVDPV. 

What can we learn from the mass vaccination program targeting Poliovirus? The Cutter incident lead to the establishment a fund which compensates victims of vaccine failure and the FDA emerged with new powers with similar schemes in other countries. The mass vaccination campaigns highlighted a problem still prevalent in various societies. The so-called Dutch Bible Belt (“Bijbelgordel”) refused vaccinations on religious grounds. An outbreak of Poliomyelitis in 1978 left 110 children paralysed and re-introducing the disease into a community of orthodox Protestants in the US. Despite Church authorities declaring that vaccination is not contradicting Church regulations, continuing refusal by some parents lead to a renewed outbreak in 1992/1993. Regarding Muslim societies refusing the vaccine, it should be noted that vaccine tested and produced in Indonesia under the auspices of Muslim clerics has been approved by the Sharia council in Nigeria, but the damage has been done.

Others reject that Poliovirus is the causative agent of Poliomyelitis altogether; the virus is the product of the disease and despite being infectious in monkeys does not infect humans. Others deny that vaccination prevents this disease; they insists that homeopathic medicine is sufficient. 

In short, resistance against the Polio vaccination is only one example of the resistance met when vaccines are introduced into the public. Replace Polio with Smallpox, Measles or HPV and it is clear that there always people who resist modern medicine.

Viruses, red meat consumption and cancer

As reported in The Times of London on Saturday March 8th 2014, researchers are dismissing the link between the increase in beef consumption to the increase seen in colorectal cancer in the developed world.

The potential link between cancer and the consumption of red meat is no new - first reports published in 1977 linked the formation of carcinogenic polycyclic aromatic hydrocarbons and derivates of nitrosamine during processing of red meat in particular barbecuing and broiling as a risk factor for cancer. Early studies however indicated that consuming poultry or fish does not carry the same risk.

Epidemiological studies however revealed that the increased risk for red meat seems to be restricted to populations with a high consumption of beef, but virtually absent in countries in which beef consumption is low such as India or the Arabic countries. Indeed the ethnic Indian and Pakistani in England and Wales has the lower incidence rate of colorectal cancer compared to the rest of the population. Similar results have been reported from Singapore and the US, suggesting that the dietary restrictions laid down in religious rules play an important role in the prevention of colorectal cancer (it should be noted however that they have an increased risk of other cancers such as gall bladder cancer and laryngeal cancer in males). In India the exception is the state of Kerala, which has a high proportion of Christian and Muslim populations.

So far so good, where is the link to viruses? Viruses are well known to be a causative agent for a variety of cancers in humans and animals alike. One of the most examples is surely Human Papillomavirus, the causative agent of cervical and penile cancer (among others), Kaposi Sarcoma Herpesvirus, the causative agent of melanoma particularly in HIV positive patients and Epstein Barr Virus, the causative agent of Burkitts Lymphoma in wide parts of the African continent. The first virus causing cancer was identified however in chicken - Rous Sarcoma Virus. It is also known that meat can transfer diseases to humans - indeed there is some speculation that the dietary laws laid down in the Torah were enforced precisely to prevent the transmission of parasitic diseases. 

Evidence of an infectious agent wing involved in the onset of colorectal cancer comes from detailed epidemiological studies, comparing data from South Korea and Japan. Japan -traditionally known for its high consumption of seafood- shifted towards a beef dominated diet in the 1970s. From 1975 onward a rapid increase in colorectal cancer can be observed with numbers decreasing in the last years - the latter coinciding with a decrease in beef imports from the US. Korea on the other hand did not see an increase in colorectal cancer before 1995, coinciding with an increased popularity for beef and dairy products. Other countries such as Australia, Canada and the US are seeing decreased rates probably because more and more consumers decrease meat consumption for health reasons, in particular obesity and cardiovascular disease. 

Still this does not prove a link between viruses or other pathogens and colorectal cancer. In both Korea and Japan however beef is served undercooked, a link supported by observations that in Saudi Arabia that the increased consumption of beef is (so far) not linked to an increase cancer risk (here beef is served well-done). 

What about viruses? Rather than causing cancer per se, viruses might play an auxiliary role, similar to the development of Epidermodysplasia verruciformis, a hereditary form of skin cancer, where HPV types 5 and 8 are contributing factors of the disease. In this scenario, viral infection would cause a transient or latent infection which -together with inherited genetic modifications and other risk factors such as nitrosamines- would lead to the developing of colorectal (and maybe other) cancers. The viral agents are however have not been identified. Early candidates included Human or Bovine Papillomavirus but failed detection. Another group included TT virus, but again so far no conclusive evidence has been reported. The possibility of an unknown (animal) virus is not so far fetched since it has been reported that the human variant of Hepatitis C virus derived from a canine variant approximately 500-100 years ago. 

It should also be noted that Torquetenovirus  (TT) virus, although being found in colorectal tumor tissue,  is acquired by in utero infections  and prevalent in 80% of the world population. Since TT virus induces a pro-inflammatory response via Toll-like receptor-9 activated pathways, it is conceivable that this activity might contribute tot he pathogenesis of the disease. It should be noted that TT viral DNA has also been detected in bone marrow and peripheral blood mononuclear cells from cancer patients.

What is the final assessment? While the majority of researchers are dismissing the link between meat consumption and colorectal cancer a minority -in particular Harald zur Hausen- believes that viruses can be at least a contributor in the onset of various cancers. Those who follow the Paleo-diet advocate to eat meat from grass-fed cattle and free-range chicken. The answer in the end might be in the grey area of science, where undercooked meat contaminated with pathogens (either viruses or bacteria) in combination with genetic factors of the individual might be a contributing factor in the onset of colorectal cancer. Excess meat consumption plays an important role in the development of heart disease and obesity. So should we decrease the amount of meat in our diet? Absolutely! But leave the viruses out of the story.

Further reading:

Xu B, Sun J, Sun Y, Huang L, Tang Y, & Yuan Y. (2013) No evidence of decreased risk of colorectal adenomas with white meat, poultry, and fish intake: a meta-analysis of observational studies. Annals of epidemiology, 23(4), 215-22. PMID: 23375344

Egeberg R, Olsen A, Christensen J, Halkjær J, Jakobsen MU, Overvad K, & Tjønneland A. (2013) Associations between red meat and risks for colon and rectal cancer depend on the type of red meat consumed. The Journal of nutrition, 143(4), 464-72. PMID: 23427329

zur Hausen H. (2012) Red meat consumption and cancer: reasons to suspect involvement of bovine infectious factors in colorectal cancer. International journal of cancer. Journal international du cancer, 130(11), 2475-83. PMID: 22212999

Berjia FL, Poulsen M, & Nauta M. (2014) Burden of diseases estimates associated to different red meat cooking practices. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 237-244. PMID: 24491261

zur Hausen H. (2012) Q&A: On the case. Interview by Michelle Grayson. Nature, 488(7413). PMID: 22932436

de Villiers EM, Bulajic M, Nitsch C, Kecmanovic D, Pavlov M, Kopp-Schneider A, & Löhr M. (2007) TTV infection in colorectal cancer tissues and normal mucosa. International journal of cancer. Journal international du cancer, 121(9), 2109-12. PMID: 17620330

Rocchi J, Ricci V, Albani M, Lanini L, Andreoli E, Macera L, Pistello M, Ceccherini-Nelli L, Bendinelli M, & Maggi F. (2009) Torquetenovirus DNA drives proinflammatory cytokines production and secretion by immune cells via toll-like receptor 9. Virology, 394(2), 235-42. PMID: 19765789

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.