The tortoise and the herpesvirus

An intuition by Frédéric Gandar, a PhD student at the University of Liege and his thesis supervisor Alain Vanderplasschen to focus on a research topic that has rarely been explored in the scientific world, has paid dividends. The study of Testudinid herpesvirus 3 ( TeHV-3), a herpesvirus causing high mortality rates in several protected species of tortoises (including Hermann’s tortoise), has resulted in several discoveries and has also made front-page in the Journal of Virology(1). Up to the present, the 250 or so herpesviruses studied – these are found in oysters as well as in humans – are divided into six distinct genomic structures. All of them, that is, except Testudinid herpesvirus 3. Frédéric Gandar and Alain Vanderplasschen could not believe their eyes, but they had discovered a new herpesvirus genome structure. This would be the seventh. Virology reference books will need to be updated! And this does not even take into account immune evasion or phylogeny-related discoveries. The researchers are currently working on a vaccine against this disease that is decimating tortoise species many of which are endangered.

The renowned Journal of Virology is the cream of publications in matters relating to virology. Not only has the young doctoral student been published in this journal but he appeared on the front page of the 2015 November edition. “Being published in this journal with a herpesvirus affecting tortoises is no mean feat”! Says his thesis supervisor, Alain Vanderplasschen. “This subject was actually a handicap, which makes the achievement even greater”. The immunology and vaccinology researcher at the Faculty of Veterinary Medicine of the University of Liege has no hesitation in admitting that nobody in industry would be likely to invest in this research. The reality is that the death of tortoises is not likely to be a source of concern for many people. This subject area is not financially interesting for pharmaceutical companies so they are not interested in investing.

Fortunately for Frédéric Gandar, Alain Vanderplasschen is not an industrialist. Even though there was no likelihood of an economic dividend for his department, he invested in the project in collaboration with Professors Didier Marlier, head of the Exotic Pets Clinic and Marianne Diez, head of the Nutrition Department. Following five years of work, the investment has proved judicious: the researchers hope to perfect a vaccine that will be capable of immunising tortoises against the plague that is killing them in such great numbers.

The plague in question is Testudinid herpesvirus 3, TeHV-3. If a group of young tortoises comes into contact with the virus, the result will be fatal for 80% to 100% of them. At first, there will be no visible signs to show that they are infected. After an incubation period of around twenty days the first symptoms will appear: nasal discharge, weakness, white spots in the mouth. Secondary infections will then appear. Then the nervous system is affected avoiding tortoises from eating and moving around. Finally, the infection affects all the organs, the spleen, the kidneys and the brain. Death is inevitable after around ten days. Those that survive become “asymptomatic carriers”. Without any visible warning, the virus will be carried by them all their lives and will spread to other tortoises they meet.

When collection rhymes with propagation

The first descriptions of this disease in scientific literature goes back to the 1980s. The propagation of the disease probably accelerated at the same time as people began to collect these reptiles which live mainly in the Mediterranean Basin. Fans of these animals are prepared to spend from 200 to 250 Euros for a classic species or even up to 1,500 Euros for certain specimens. These are the types of figures that apply in the official market. But there is also a parallel unofficial market. Carefree tourists buy tortoises at the corner of the street when they are on holiday or may even pick them up on the side of the road to bring them home in their luggage, blissfully unaware that they are infected. As a result of these practices the virus can spread much more quickly than by natural means. “People are often unaware of the fact, but many species are suffering due to the fact that they have become animals of collection”, says Alain Vanderplasschen. “Certain diseases have become global whereas they would have developed much more slowly in nature, thus perhaps giving the species in question time to adapt to the disease. Because of collection, very different species can come into contact with each other artificially in terrariums…It’s like putting a crocodile and a caribou together in the same place! It is not impossible that what is happening to Testudo tortoises is the expression of the non-adaptation of a virus to a new host and therefore the consequences of a recent transfer of a virus from one species of animal to another”.

Scrambled genomes

Passionately interested in new pet animals, Frédéric Gandar decided to go against the grain of scientific disinterest with regard to this problem. When he began sequencing the genome of TeHV-3, he did not know what he would find. And, actually, what he found was meaningless. The strain he was working on – the one that all scientists use – didn’t seem to make any sense. It took him two years to understand that, in reality, it was not a strain but rather a mixture of three different strains having all an incomplete genome.

Another complication entered the mix. “When a genome is sequenced, the sequences obtained must be organised. Obviously, we always have the reflex to attach whatever we discover to what is already known. Here, we could not join all the “jigsaw pieces” that were known in established models. Nothing seemed to fit and there was always a piece of the puzzle missing”, recalls the young researcher.  All 250 herpesvirus genomes studied up to date – these include viruses that affect oysters as well as humans – are classified according to six distinct genomic structures. All, that is, except Testudinid herpesvirus 3 (TeHV-3). Frédéric Gandar and Alain Vanderplasschen could not believe what they were seeing but they had in fact just discovered a new genome structure. This structure would be the seventh.  “Virology reference books will have to be updated! Who would have believed that a virus affecting tortoises would reveal such a thing”?

The TeHV-3 had not yielded up all its secrets however. When the researchers realised that they were dealing with a mixture of three different strains, they tried to separate them from each other in order to know whether they were still capable of replication in isolation. This seemed very unlikely as these strains had been “deleted”, they were missing large sections of the genome from 12,000 to 22,000 base pairs. “It was a bit like having a car weighing 100 kilos and then removing 10 or 12 kilos of matter”, explains Alain Vanderplasschen by way of comparison.  “It is unlikely that the car would still work”.

“Surreal as far as viruses are concerned”!

And yet, the strains continued to multiply as though everything was normal. Not only did two of them appear to be capable of infecting, but also of causing death. “This is surreal as far as viruses are concerned! Large pieces of the genome are missing but this still does not prevent the virus from killing its host”. The third, however, did not seem to be faring so well. In cell-culture, it continued to proliferate without any problem but in the tortoise, on the other hand, it was unable to invade the animal!

This was fortunate. Thanks to this third strain Frédéric Gandar will probably be able to create a vaccine. The next step will be to inoculate the tortoises with this form of the virus to see whether their immune system will develop an immune response which could ultimately protect them from lethal attacks once they have been infected. “In the coming year, we will find out if we have a vaccine”.
The discoveries could have stopped there and would undoubtedly have been enough to merit publication in the prestigious Journal of Virology but Testudinid herpesvirus 3 led to other discoveries. The first of these discoveries concerned “immune evasion”. This is a characteristic that is common to all viruses: they steal genes from the animal they infect at different moments during evolution. TeHV-3 had managed to steal a gene for an interleukin-10 (IL-10), a molecule which serves to reduce inflammation. The immune system must trigger mechanisms that fight against pathogens as soon as their presence is detected. Inflammation is one of the mechanisms. It is vital for the organism to stop these anti-pathogen mechanisms as soon as they are no longer necessary. This is comparable to a firefighter who must turn off the water as soon as the fire has been extinguished in order to prevent further damage to things that were not destroyed by the fire. Interleukin-10 is a molecule that calms the response of the immune system. “The virus therefore stole this IL-10 so that when it invades the organism the immune system should be triggered but this protein inhibits its activation. This phenomenon was quite well-known but had never been described in this virus sub-family”, explains Frédéric Gandar. Even more astonishing: the structure of the interleukin-10 stolen from the tortoise is very similar to that found in humans. When one is superimposed on the other, you get a near perfect match.

Coevolution   

At the same time, the researchers made another discovery with regard to the phylogeny of virus species. A long time ago (something in the order of 156 million years), tortoises and sea turtles had a common ancestor. They then evolved into two branches, one on land and the other in the sea. While they still resemble each other physically, in reality they have become more genetically distinct from each other than a horse from a whale.

If we compare the herpesvirus that affects tortoises and another herpesvirus (chelonid herpesvirus 5) which affects their sea cousins, we can see that they are as distinct from each other as the tortoises they infect. This phenomenon is known as cospeciation. This shows that the herpesvirus of terrestrial and sea turtles probably have a common ancestor and evolved with each species. This was the last secret revealed by TeHV-3.

“When taken together, all these discoveries explain why this virus affecting tortoises made the front page of Journal of Virology”, says a smiling Alain Vanderplasschen. And yet this research almost didn’t go ahead. Indeed, this research programme which aimed to find a solution to one of the main causes of the decline of several species of endangered tortoises required the performance of animal experiments. These were only allowed to go ahead after many long discussions with the bioethics committee, the only organ which has the authority to authorise animal experiments. It is deplorable to observe that the pressure exerted by a small proportion of the society which is against the performance of animal experiments could have the consequence of preventing the development of solutions against a plague that threatens several species of endangered tortoises.

The interest shown by the international scientific community following the publication in the Journal of Virology and the common sense approach adopted by the bioethics committee succeeded in unblocking the situation. Frédéric Gandar will be able to continue to develop a vaccine. In the next year, the researchers will be able to determine whether they have produced a vaccine that will be capable to protect several endangered species of tortoise. This vaccine will be supplied by the University of Liege to any foundation wishing to contribute to the survival of species threatened by the TeHV-3 virus.

(1) Gandar F., Wilkie G., Gatherer D., Kerr K., Marlier D., Diez M., Marschang R., Mast J., Dewals B., Davison A., Vanderplasschen A., The genome of a tortoise herpesvirus (Testudinid Herpesvirus 3) has a novel structure and contains a large region that is not required for replication in vitro or virulence in vivo. Journal of Virology, 89, 22, october 2015.