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