Science for Saving Species Research findings factsheet Project 2.3.5

The discovery of two novel papillomaviruses in native and invasive Christmas Island geckos

In brief Context Papillomaviruses commonly infect Papillomaviruses are widespread related to a bird papillomavirus their host’s skin and mucosal in the animal kingdom. In this than other reptile papillomaviruses. membranes (mucous membranes study, we discovered two new We examined seven lizard species include the surface of the mouth, papillomaviruses in the highly for the presence or absence of the the conjunctiva, the entire digestive invasive Asian house gecko two viruses, and found it in three, tract and the reproductive tract). (Hemidactylus frenatus) and including one Critically Endangered Papillomaviruses infect all animals, mute gecko (Gehyra mutilata) species, Lister’s gecko, which now including humans, with amphibians on Christmas Island and Cocos exists only in captive breeding the sole exception. The majority (Keeling) Islands, and in the populations on Christmas Island of papillomavirus infections either Critically Endangered Lister’s and in Taronga Zoo in Sydney. present no symptoms or present as geckos (Lepidodactylus listeri) As the viruses may pose a threat benign wart-like lesions; however, in their captive breeding to this species and potentially to a minority of them can cause colony on Christmas Island. other threatened species, research cancer. A well-known example of a These two viruses are very similar into their ecology and ability to papillomavirus that causes cancer is genetically, show unique genomic cause disease in affected reptiles the virus that causes cervical cancer features, and are more closely is a high priority. in women. Unlike some other types of viruses, papillomaviruses are considered highly “host specific”, Jess in the field spotlighting geckos on which means they are rarely Christmas Island. transmitted between different species, even very closely related ones. The rate of discovery of new papillomaviruses is increasing, and more than 400 of these viruses have now been identified across all host species. The majority of these viruses have humans and other mammals as hosts. We have discovered two papillomavirus species in geckos inhabiting Christmas Island and the nearby Cocos (Keeling) Islands, and although such viruses are not unknown in reptiles, these two represent the first discovery of papillomaviruses in lizards. Image: David Phalen What the research What we did looked at We undertook a number of Our discovery that two new procedures to uncover answers species of papillomaviruses are to the aims of this research infecting lizards on Christmas project. First, we set out to look at Island and Cocos (Keeling) Islands the genetic make-up of the two has prompted investigation into viruses. To do this for a virus or any the genetics and biology of the other organism, it is necessary to viruses. To add to the urgency and determine the order of the bases importance of the research, one (part of DNA that stores information of the affected lizards, although and gives DNA its genetic code) once common on Christmas Island, that make up the DNA. This we is now a threatened species. achieved by sequencing the DNA using advanced genetic technology. To uncover as much as possible Once we had sequenced the DNA, The genome layout of the about these newly discovered papillomavirus. Image: Jess Agius we confirmed the number, type and viruses, we developed a number ordering of each gene for each virus. of diverse research aims. These included: 1. Identifying the presence and significance of the viral genes, which is important for understanding their disease- After we resolved the genetic causing mechanisms structure of each of the two new viruses, we were interested 2. Describing the evolutionary in understanding how they are relationship of these two new related to papillomaviruses that papillomaviruses to other viruses had already been described, and 3. Determining the distribution their evolutionary similarities and of these viruses in the tissues differences. This kind of study is of affected lizards known as phylogenetics. We aligned the gene sequences of a diverse 4. Determining the proportion range of papillomaviruses that are of lizards affected with distantly and closely related to those papillomavirus DNA, which is that we had discovered. When we vital for understanding how had the final alignment, we entered common the viruses are it into a statistical model to create a within the island populations. tree showing common ancestors, viral relatedness, and how the viruses evolved through time.

Simplified phylogenetic tree showing relatedness of gecko papillomaviruses to other reptile and bird papillomaviruses. Image: Jess Agius Key findings

To understand the types of tissues Our genetic analysis of both the viruses are more closely related to a the papillomaviruses were able new papillomavirus genomes papillomavirus that is hosted by a sea to infect we collected a variety of revealed that they are relatively bird, the northern fulmar (Fulmarus organs from lizards on Christmas similar to one another, sharing 89% glacialis), than to other reptile Island and Cocos (Keeling) Islands of their genetic identity. The two papillomaviruses. The evolutionary and extracted the DNA from the viruses contain the same number tree we generated also showed that organ cells. We tested DNA from and types of genes, and in identical both these lizard viruses are primitive, six species of gecko and one positions in the genome; and meaning that they evolved before species of skink for the presence they each contain the four core any other known bird or reptile or absence of papillomavirus DNA: genes that are found across all papillomavirus. papillomaviruses. In addition, they • Lister’s gecko We found papillomavirus DNA across each have one accessory gene that (Lepidodactylus listeri) all the organ types tested (liver, skin, is not found in all papillomaviruses • Asian house gecko mouth and colon), and in three of and which has been associated (Hemidactylus frenatus) the seven lizard species sampled with interference with immunity, • Mute gecko (Gehyra mutilata) (Asian house gecko, mute gecko successful establishment of infection and Lister’s gecko). The viruses • Sri Lankan house to allow the virus to replicate, and were more frequently detected gecko (Hemidactylus the transformation of cells to a in samples collected from the parvimaculatus) cancerous form. However, unlike head and skin of the lizards, • Flat-tailed gecko other papillomaviruses, which all with positive samples from the (Hemidactylus platyurus) contain at least six genes, both colon and liver less common. • Mourning gecko of the two new papillomaviruses The new papillomavirus species (Lepidodactylus lugubris) we identified contained only five. They therefore represent the were detected on both Christmas • Blue-tailed skink smallest, simplest structure of any Island and Cocos (Keeling) Islands, (Cryptoblepharus egeriae) currently known papillomaviruses. with one species more common Once we knew about the presence than the other for both locations. Our phylogenetic analysis of the or absence of disease in various The Cocos (Keeling) Islands had a viruses revealed that, as we expected, tissues from multiple animals, we higher number of lizards affected both are more closely related to could determine the proportion, with papillomaviruses (32%) than did bird and reptile papillomaviruses or prevalence of affected lizards Christmas Island (27%). Of the lizards than to those that are hosted by to non-affected lizards. We then we tested for papillomaviruses, the mammals. Interestingly, though, compared these proportions widely distributed Asian house gecko we found that both these lizard between locations and species. showed most frequent infection.

RIGHT: Asian house gecko where papillomavirus DNA was first discovered Image: Jess Agius Implications and recommendations The discovery of two novel uncertain as yet, these viruses have Additional surveillance of Lister’s, papillomaviruses in three gecko the potential to cause declines in mute and Asian house geckos species on two distinct islands Lister’s geckos and to devastate can help to assess the patterns presents multiple concerns the remaining populations. of papillomavirus disease, that is, for species conservation and whether it is declining, stable or Further work towards uncovering management. In addition to infecting increasing in the lizard populations. the ecology of these viruses two of the most invasive and Although we have not detected and their ability to cause disease widespread geckos, the Asian house papillomavirus infection in the in affected animals is urgently and mute geckos, these viruses have Christmas Island blue-tailed skink, required. The knowledge acquired also been identified in the Critically continued surveillance of these from this work can then feed into Endangered Christmas Island skinks, and the last free-ranging conservation management actions Lister’s gecko. The Lister’s gecko is Christmas Island endemic giant for the Lister’s gecko, and also the conservation-dependent, surviving gecko (Cyrtodactylus sadleiri), will Christmas Island blue-tailed skink, only in captive breeding programs be important to confirming the which is also Critically Endangered on Christmas Island and at Taronga continuing freedom from disease, and extinct in the wild, and a Zoo, Sydney. Although the ultimate and assuring the continuing health species that is in close contact effects of the papillomaviruses on of the population. with Lister’s geckos. the health of affected animals is Jess in the lab microscopically examining lizard samples for papillomaviruses. Image: Laura Pulscher

Cited material Agius, J. E., Phalen, D. N., Rose, K. & Eden, J.-S. 2019. New insights into Sauropsid Papillomaviridae evolution and epizootiology: Discovery of two novel papillomaviruses in native and invasive Island geckos. Virus Evolution, 5(2), doi: 10.1093/ve/vez051

Further Information

Jessica Agius [email protected]

This project is supported through funding from the Australian Government’s National Environmental Science Program.