Skull of an orangutan in the Museum Koenig Bonn
The skull of an orangutan in the zoological research Museum Koenig Bonn, which researchers used to obtain an mpox genome. © Olivia Cheronet

Against the backdrop of the recent Covid-19 pandemic, there is great interest in better understanding the evolution of pathogens – especially those pathogens that potentially affect both animals and humans. If one could examine the genetic information of historical coronaviruses, perhaps a 100 years old, taken  from a close animal relative of humans, this would be an extremely useful source of information about the mutation rates and adaptation strategies of the virus, its potential danger and the probability of further jumps to humans.

Coronaviruses do not store their genetic information in DNA, as is the case in human cells. Like many other viruses, they are based on the much more unstable RNA molecule chains, which are important for example in the conversion of genetic information in organisms.  Compared to DNA, RNA fragments are much  more difficult to find in historical samples that have been preserved for a long time. A research team led by Martin Kuhlwilm from the Department of Evolutionary Anthropology at the University of Vienna grappled with  this demanding task and tested a new systematic approach to this problem.

In the project, which was funded by the FWF under its 1000 Ideas program, the scientists focused on museum specimens of great apes, since the probability of zoonoses – infectious diseases that can be transmitted from animals to humans – is particularly high among our closest evolutionary relatives. Skins, skulls and chemically preserved specimens from the past 100 years were examined for viral residues. Although the findings did not quite meet expectations, they did contain some surprises.

1000 ideas program funds risky research

Unlike the DNA double helix, RNA often consists of only a single strand of bases, and it also shows other chemical differences. Due to their unstable character, RNA viruses have very high mutation rates and can therefore quickly adapt to new hosts. In contrast to the more stable and slowly mutating DNA, RNA also decays much faster. “Our approach was risky in two respects. The decay rates of the RNA residues were not the only thing that made the undertaking difficult, we  even did not knowwhether the animals whose remains we examined were infected by RNA viruses at all,” Kuhlwilm says, summing up. Hence, the 1000 Ideas Program, which supports particularly unconventional, creative and risky approaches in the field of basic research, was the right funding instrument for this project.

The recent increase in successful reports of isolated RNA from historical samples has raised some hopes. According to one study, RNA molecules from the Tasmanian tiger were detected in historical samples – a species whose last known specimen died in 1936. In comparison, historical viruses with a more stable DNA genome, such as earlier variants of adenoviruses or poxviruses, are detected much more frequently.

Postdoctoral researcher Sojung Han taking samples at the Anthropological Museum Zurich.
Postdoctoral researcher Sojung Han taking samples at the Anthropological Museum Zurich. © Sojung Han

Samples from museums in Austria, Germany and Switzerland

Overall, Kuhlwilm and his colleagues examined around 100 samples of great ape museum specimens. They were sourced not only in museums in Frankfurt and Zurich, but also at the pathological-anatomical collection in the Vienna Narrenturm, the Natural History Museum Vienna and the University of Vienna's own collection. Besides skins and skulls, the team also sampled specimens preserved in formalin and ethanol, as well as specimens preserved in unidentified liquids. 

The tissue was homogenized and dissolved and the team sequenced RNA and DNA fragments, millions of which are often present in the samples, so that they could be identified using specialized databases. “In a chimpanzee, at best around 30 percent of the fragments come from the animal itself, a few percent from bacteria, a large proportion cannot be identified, and, usually, a proportion significantly lower than one percent can be traced back to viruses,” notes Kuhlwilm, giving an example of a typical finding.

Chimpanzee skin is spread out on a table
Fur of a chimpanzee in the Natural History Museum Vienna from which a sample was taken. © Olivia Cheronet

The small proportion of viruses was then concentrated and isolated in a so-called enrichment process. Once enriched, the viral RNA was again compared with known sequences in a database in order to assemble them into as large a proportion of a genome as possible – comparable to putting together a jigsaw puzzle. A positive control showed that the method basically works : the team were able to reconstruct two thirds of the genome of a non-historical sample of a measles pathogen, an RNA virus, using it . In the case of the Mpox DNA virus,  formerly known as “monkeypox”, they could even obtain the entire genetic information.

RNA needle in the DNA haystack

The result from the almost 100 samples showed how much good luck is involved in finding reconstructable RNA fragments. “We found RNA viruses that infect bacteria, so-called bacteriophages, but these are of little relevance to the research issue. Also, some results were  not very reliable and presumably produced false positive findings,” notes Kuhlwilm. “On the other hand, we were able to identify with a high degree of certainty a pathogen related to measles: the canine distemper virus, which usually infects cats and dogs. But here, too, there were only a few fragments that make up just one percent of the virus genome.”

On the other hand, they also found no more than a handful of DNA viruses. For Kuhlwilm, the findings show that museum specimens are only to a limited extent good targets for the search for RNA viruses. “Many of the old specimens do not come from the wild, but from zoos. RNA is best extracted from skins, and these items  are rather rare compared to bones,” explains the scientist. Screening with many  additional and even more specifically selected samples would probably be needed to increase the chances of success – a task for possible future research projects.

In research, it very often happens that people are looking for one thing but  find something completely different. The current project is a case in point. One research result from the project surprisingly provides insights into the origin of museum specimens. “The DNA of an Mpox virus was found in a sampled orangutang specimen from a museum in Bonn,” explains Kuhlwilm. “The database actually referred to a wild animal, but we were able to reconstruct that the animal died in the course of an Mpox outbreak that decimated the population at Rotterdam Zoo in the mid-1960s.” The discovery shows that the museum specimens have fundamental potential for the study of viral evolution. After all, non-human primates in captivity have repeatedly been the source of diseases that spread to humans.

Personal details

Martin Kuhlwilm is a biologist and evolutionary anthropologist at the University of Vienna. Previous stations in his career include the University of Halle-Wittenberg, the Max Planck Institute for Evolutionary Anthropology in Leipzig and the Universitat Pompeu Fabra in Barcelona. Since 2021, he has headed the Computational Admixture Genomics Lab at the Department of Evolutionary Anthropology at the University of Vienna. The project “Historical perspectives on RNA viruses in great apes” was awarded EUR 153,000 in funding from the Austrian Science Fund FWF under its “1000 Ideas” program.  

Publications

Llanos-Lizcano A., Hämmerle M., Sperduti A. et al: Intra-individual variability in ancient plasmodium DNA recovery highlights need for enhanced sampling, in: Scientific Reports 2025

Hämmerle M., Guellil M., Trgovec-Greif L. et al.: Screening great ape museum specimens for DNA viruses, in: Scientific Reports 2024

Hämmerle M., Rymbekova A., Gelabert P. et al.: Link between Monkeypox Virus Genomes from Museum Specimens and 1965 Zoo Outbreak, in: Emerging Infectious Diseases 2024