Boid Inclusion Body Disease

Einschlusskörperchen-Krankheit der Boiden
(English text below)

BIBD ist eine wichtige Erkrankung von Würgeschlangen zahlreicher Familien in Gefangenschaft, vornehmlich Boas (Boa constrictor) aber auch Pythons, die ganze Schlangenbestände auslöschen kann. Die Erkrankung wird durch hoch divergente Reptarenaviren verursacht, deren natürlicher Wirt bisher unbekannt ist. Ebenso ist die Prävalenz von BIBD und Reptarenavirusinfektionen in Schlangenpopulationen in Gefangenschaft bisher nicht bekannt.

Unsere Forschungsgruppe ("The BIBD Group") konnte nachweisen, dass Schlangen mit BIBD häufig mit mehreren Reptarenaviren, oft unterschiedlicher Spezies, infiziert sind (Hepojoki et al. 2015); in einem Teil der Tiere liegen zusätzlich Infektionen mit verwandten Arenaviren, den Hartmaniviren (Hepojoki J et al., 2018), vor. Ausserdem haben wir gezeigt, dass all diese Viren von infizierten Schlangen auf ihre Nachkommen übertragen werden können (sog. vertikale Infektion), die anschliessend an BIBD erkranken (Keller et al., 2017). Diese Tatsache ist vor allem für Zuchtbestände von grosser Bedeutung. Kürzlich abgeschlossene retrospektive Studien unserer Gruppe haben gezeigt, dass wildlebende Schlangen in Brasilien und Costa Rica ebenfalls an BIBD erkranken können (Alfaro-Alarcón et al. 2022).

Zur sicheren Diagnosestellung einer BIBD (sog. “Goldstandard“) an der lebenden Schlange dient der Nachweis der typischen viralen zytoplasmatischen Einschlusskörperchen in Blutzellen (hierzu werden Blutausstriche zytologisch und/oder immunzytologisch untersucht) oder in Zellen einer Gewebebiopsie (z.B. Leberbiopsie). Tiere, die Einschlusskörperchen aufweisen, sind als erkrankt anzusehen, auch wenn sie zum Untersuchungszeitpunkt keine klinischen Symptome aufweisen. Ebenso stellen sie eine potentielle Ansteckungsquelle für andere Schlangen dar.

Der Nachweis von Serumantikörpern bei Schlangen hat nur eine sehr begrenzte Aussagekraft (Windbichler et al., 2019). Der molekulare Nachweis einer Reptarenavirus-Infektion, wie sie der BIBD zugrundeliegt, ist bei Schlangen möglich, wird jedoch durch die hohe Variabilität der Viren erschwert, was die Sensitivität dieser Herangehensweise mindert. Mit einer von uns entwickelten “multiplex-Reptarenavirus-RT-PCR“ werden alle bisher bei in Gefangenschaft gehaltenen Schlangen identifizierten Reptarenaviren nachgewiesen (Baggio et al., 2023); dies schliesst jedoch die Existenz weiterer, durch die Multiplex-PCR nicht erfasster Reptarenaviren nicht aus. Somit stellt neben einer next generation sequncing (NGS)-Vorgehensweise eine Virusisolierung in Boazellkulturen immer noch die sensitivste Methode dar. Beide Techniken sind jedoch zeitlich und finanziell aufwändig.

Boid Inclusion Body Disease

Boid inclusion body disease (BIBD) has first been described in the 1970s and is a disease of high relevance for captive boid snakes, as it has the potential to wipe out entire snake collections. The aetiology of BIBD remained enigmatic until the identification of arenaviruses in BIBD-positive snakes a few years back. The BIBD-associated arenaviruses belong to genus Reptarenavirus.

BIBD affects various boid species from different families, including boids and pythons, e.g. Boa constrictor and Python regius. In boas, the disease course is variable. Affected animals can die within weeks or months or remain asymptomatic carriers for an unpredictable length of time. In contrast, pythons more often develop severe fatal neurological symptoms within a few weeks of reptarenavirus infection. The prevalence of BIBD in captive boid populations is still unknown, however, we have recently shown that it also occurs in free-ranging boid snakes in South America (Alfaro-Alarcón et al. 2022). So far, it is unknown whether constrictor snakes are the natural host(s) of reptarenaviruses.

BIBD diagnosis (so-called “gold standard“) in a living snake requires the examination of a blood smear (cytology and/or immune cytology) or a tissue biopsy (for example from the liver) for the presence of cytoplasmic inclusion bodies. Animals that exhibit inclusion bodies are considered to suffer from BIBD, even if they do not show any clinical signs at the time of examination. They also represent potential sources of infection for other snakes.

Molecular detection of reptarenavirus infection is possible, although the genetic variability of reptarenaviruses complicates diagnosis by reducing the sensitivity of the diagnostic approaches. However, others and we have gathered evidence to suggest that a specific viral segment (so-called UGV S or S6) can be consistently detected in infected captive snakes in Europe and the USA, allowing for a generally reliable diagnosis of infection. Nonetheless, it cannot be excluded that rare reptarenaviruses would not be detected. Detection of serum antibodies against reptarenaviruses is only of limited diagnostic value (Windbichler et al., 2019).

Therefore, we have recently established a multiplex reptarenavirus RT-PCR that can detect all reptarenavirus species so far identified in captive snakes (Baggio et al., 2023); however, this does not exclude that further reptarenaviruses exist which this method would not detect. Hence, a next generation sequencing (NGS) approach or virus isolation in boid cell cultures are still the most sensitive and reliable methods. Both are (currently) no routine options, due to financial, time and/or lab constraints.

Others and we could show that snakes with BIBD often carry several reptarenavirus species (Hepojoki J et al., 2015); a proportion of these animals also exhibits hartmanivirus (another arenavirus genus) co-infection (Hepojoki et al., 2018). We have shown that all these viruses can be transmitted from infected snakes to their offspring (so-called vertical transmission), which subsequently develop BIBD (Keller et al., 2017). This fact is of major relevance for breeding colonies.

The BIBD Group

The international multidisciplinary research group with core members from the Universities of Zurich, Helsinki and Liverpool, was established to study the aetiology and pathogenesis of Boid inclusion body disease (BIBD) and related infectious diseases in snakes.

The BIBD Group was established in early 2012 by veterinary pathologists, a zoologist and virologists. It was initially based at the University of Helsinki, where all four founding members worked at that time. Our complementary expertise allows us to cover all methodological aspects relevant for our work. Since the move of some members to Zurich, the group undertakes complementary work at two sites and is growing consistently.

Our research

In early 2012, our group isolated a virus, identified as a novel arenavirus, from tissue cultures generated previously from a BIBD-positive snake in which the typical morphological features of BIBD, cytoplasmic inclusion bodies within a wide range of cells, were also observed (Hetzel et al. 2013). Coincidently, groups from the USA and the Netherlands identified several novel arenavirus sequences from BIBD-positive snakes using a different and more global methodological approach, next generation sequencing (NGS). Follow-on investigations led to the establishment of two novel genera, Mammarenavirus (the formerly known arenaviruses) and Reptarenavirus, within the family Arenaviridae. So far, more than 25 reptarenavirus species (based on the L segment sequence) have been found in snakes with BIBD.

Our group also works on other reptilian viruses, which include hepatitis D virus like agents (genus Deltatvirus, family Kolmioviridae), serpentoviruses (nidoviruses) that have recently posed a threat to python collections, and piscichuviruses found in free-ranging boas in Brazil. The group`s focus lies on the identification, characterisation and pathogen-host interactions of reptilian pathogens in general. Most recently, the group started to investigate pathological processes secondary to BIBD, such as the effect on haemolymphatic tissues (Dervas et al., 2024) and neoplastic processes (Broering Ferreira et al., 2024). 

Group members

The main investigators are PD Dr Jussi Hepojoki, a biochemist and molecular virologist, Dr Udo Hetzel, a zoologist and veterinary pathologist, and Dr. Eva Dervas, a veterinary pathologist, both with specific expertise in the pathology of reptiles, exotics, zoo and wild animals, as well as Dr Francesca Baggio, a molecular biologist. Together with Professors Olli Vapalahti and Anja Kipar, they have been developing and directing the group’s research activities.

Dr Jussi Hepojoki has almost 25 years research experience. He holds a degree in bioengineering, a master’s degree in biochemistry, and he did his PhD thesis on hantavirus structure, focusing on the interactions between the structural proteins that are essential for virus assembly and maintenance of the virion. When the BIBD Group was established, Jussi focused on preparing the crucial reagents for the BIBD and virus studies. In addition, he has been working on sequencing reptarenavirus isolates, setting up diagnostic assays for reptarenaviruses and hartmaniviruses, and identifying novel viruses by next generation sequencing approaches. Using the latter, he identified an HDV like agent, Swiss snake colony virus 1 (SwSCV1, realm Ribozyviria, family Kolmioviridae, genus Daletvirus, species: Daletvirus boae) towards which he has developed a keen interest. Recently, the team has demonstrated that SwSCV1 can use reptarenaviruses and hartmaniviruses as helpers in producing infectious particles (Szirovicza et al., 2020). Jussi Hepojoki is particularly interested in the factors enabling cross-species transmission and in the mechanisms behind viral persistence in the reservoir host with focus on (rept)arenaviruses and hantaviruses. His interests include studying the role of reptarenavirus co-infections in the pathogenesis of BIBD, including the factors directing tissue and species. In 2017, he has been granted a prestigious 5-year Academy of Finland research fellowship for the project "Immune evasion: The tool for persistent infection and cross-species infectivity of arenaviruses?". In 2022 he received a 4-year Sigrid Jusélius Senior Researcher funding from the Sigrid Jusélius foundation, and 5-year funding from Jane and Aatos Erkko foundation to study the pathogenetic association and cross species transmission of kolmiovirids. In 2023 he received, as a co-PI, a 4-year Academy of Finland consortium funding for developing bio-inspired nanoparticles for mRNA delivery.

Dr Udo Hetzel’s main research interest lies in infectious diseases with a zoonotic aspect, in particular when they cross class barriers. Udo Hetzel has extensively studied the pathological features of BIBD and, due to his links to snake breeders in Europe, has established a large archive of samples (blood, tissue cultures, tissues) from BIBD-positive boa constrictors that are an invaluable source for the group’s work. Udo has established the reptarenavirus-infected boid cell cultures and has used this unique resource to initiate and rapidly progress with the research on BIBD. He has also initiated our working group and has over the years established cell cultures of various snake species from across the world which allow him to isolate also other emerging snake viruses, such as nidoviruses in pythons, and the new snake deltavirus that the group has recently described (Hetzel et al., 2019).

Prof Anja Kipar is a veterinary pathologist with a strong interest in the (immuno)pathogenesis of infectious diseases, with a main focus on viral diseases, both in animals and in rodent models of human infections. She has extensive experience in all pathology-related technical approaches and has developed strong international collaborations through her research, a particularly interesting aspect of which is the work on infectious disease models in their natural hosts, such as wood mice, bank voles, field voles, and snakes. Anja Kipar is the Director of the Institute of Veterinary Pathology at the Vetsuisse Faculty Zurich and has established the BIBD project as one of the major research areas of the Institute.

Prof Olli Vapalahti is Professor of Zoonotic Virology at the Faculty of Medicine and the Faculty of Veterinary Medicine, University of Helsinki. His research interests include zoonotic diseases, such as tick-borne encephalitis, Pogosta disease, dengue fever, and haemorrhagic fever.

Dr Teemu Smura is a virologist at the University of Helsinki. His current interests include virus hunting, evolutionary virology, phylogenetics and NGS.

Dr Leonóra Szirovicza, MSc, PhD, undertook a PhD project with the BIBD Group. She is now a postdoctoral researcher in Helsinki and investigates the disease associations and cross species transmission potential of kolmiovirids.

MSc Annika Lintala, PhD student, investigates the molecular characteristics of reptarenavirus persistence, co-infection and their effect on BIBD pathogenesis.

Dr Francesca Baggio is a molecular biologist and a member of the team in Zurich. She holds a Master’s degree in molecular biology and a PhD in molecular biology and genetics. Francesca has extensive experience in mitochondrial biology, acquired during her first post-doctoral experience.  She has implemented and adapted her broad methodological expertise in molecular and mitochondrial biology to the reptilian system, specifically to snake-derived cell cultures. Her recent work indicates relevant interaction of the reptarenaviral nucleoprotein with mitochondria, uncovering novel infection strategies of arenavirus pathogenesis (Baggio et al., 2021). She has also recently developed a diagnostic molecular tool, i.e. a multiplex reptarenavirus RT-PCR, for the challenging detection of reptarenavirus infection in snakes (Baggio et al., 2023). She is currently investigating the cytopathic effect of reptarenaviruses, in single infections or co-infections, in in vitro models, with a focus on host mitochondria and metabolism. She is also co-supervising the projects of postgraduate students on different aspects of reptarenavirus infection and BIBD pathogenesis.

Dr Eva Dervas is a veterinary pathologist with a strong interest in the comparative study of the morphology and functions of cell types (e.g., leukocytes) and organs across various reptilian species, with a particular focus on the cytopathic effect of viral infections in snakes. Since her PhD project, she has expanded her research on the immune system of snakes, with particular emphasis on the haematological parameters, immunological functions and alterations in the haematopoetic tissues in animals with BIBD (Dervas et al., 2024).

Dr Eleni Michalopoulou is a veterinary epidemiologist who is supporting the group’s work on larger animal cohorts and the epidemiology of emerging virus infections.

Postgraduate students

The BIBD Group is completed by several postgraduate students. So far, six doctoral theses have been finalised and the results published: Dr Saskia Keller has worked on the vertical transmission of reptarenaviruses and BIBD, Dr Eva Dervas investigated nidovirus-associated pneumonia in pythons for her Dr- med. vet. dissertation and characterised immunocells and organs in her PhD thesis, Dr Katharina Windbichler’s project focussed on the reptarenavirome and immune response of snakes in breeding colonies, Dr Yegor Korzyukov worked on the cellular receptor and experimental antiviral and vaccine strategies for reptarenaviruses, Dr Leonóra Szirovicza, MSc, investigated the new snake deltavirus and its interaction with reptarenaviruses as its helper viruses and Tanja Thiele undertook an in-depth reptarenavirome investigation in a large snake colony. We currently supervise three dissertation projects. In Zurich, Iman Kriještorac Berbić investigates the in vitro growth characteristics of reptarenaviruses and Maurice Bogner’s project focusses on the expression of different reptarenaviral proteins.

Collaboration partners

The BIBD Group works closely with several international groups that are interested in infectious diseases of snakes. We currently have joint projects with veterinarians and immunologists in Brazil, the group of Professor Ricardo Portela at the Instituto de Ciências da Saúde, Universidade Federal da Bahia in Salvador, and the group of Prof. Ubirajara M. da Costa at the Center for Agroveterinary Sciences, University of the State of Santa Catarina, Lages, Santa Catarina. They previously worked with the group of Professor David Driemeier at the Department of Veterinary Pathology, Universidade Federal do Rio Grande do Sul in Porto Alegre, and with veterinary pathologists at the Departamento de Patologia, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia in Costa Rica. We also collaborate with Professor Juan Carlos de la Torre at the Scripps Research Institute, CA, USA, and Dr. Luis Martinez-Sobrido at the University of Rochester Medical Center, NY, USA on certain aspects of arenavirology, and with Dr. Karim Majzoub at the Institut de Génétique Moléculaire de Montpellier (IGMM), Université de Montpellier, France, on kolmiovirids.

Funding

Our work has received funding from the Academy of Finland, the Sigrid Jusélius Foundation, the Jane and Aatos Erkko Foundation, the Finnish Foundation for Veterinary Research, the University of Helsinki, the Stiftung für Wissenschaftliche Forschung and the North-South Cooperation scheme of the University of Zurich, the Schweizerische Vereinigung für Wild-, Zoo- und Heimtiermedizin, and the Leading House for the Latin American Region in Switzerland.

• Dervas E, Michalopoulou E, Thiele T, Baggio F, Hetzel U, Kipar A. Haemolymphatic tissues of captive boa constrictor (Boa constrictor): morphological features in healthy individuals and with Boid Inclusion Body Disease. BioRxiv: https://biorxiv.org/cgi/content/short/2024.08.26.609690v1.
• Broering Ferreira A, Fonteque JH, Withoeft JA, Casagrande RA, da Costa UM, Imkamp F, Göller P, Baggio F, Hepojoki J, Hetzel U, Kipar A, 2024. Multifocal cutaneous neoplastic vascular proliferations in a rainbow boa (Epicrates cenchria) collection with boid inclusion body disease. PLOS One 9(11):e0311015. https://doi.org/10.3389/fimmu.2024.1440324.
• Baggio F, Hetzel U, Prähauser B, Dervas E, Michalopoulou E, Thiele T, Kipar A, Hepojoki J, 2023. A multiplex RT-PCR method for the detection of reptarenavirus infection. Viruses 15(12), 2313. https://doi.org/10.3390/v15122313.
• Thiele T, Baggio F, Prähauser B, Ruiz Subira A, Michalopoulou E, Kipar A, Hetzel U, Hepojoki J, 2023. Reptarenavirus S segment RNA levels correlate with the presence of inclusion bodies and the number of L segments in snakes with reptarenavirus infection - lessons learnt from a large breeding colony. Microbiol Spectr 11(3):e0506522. https://doi.org/10.1128/spectrum.05065-22.
• Dervas E, Michalopoulou E, Liesegang A, Novacco M, Schwarzenberger F, Hetzel U, Kipar A, 2023. Haematology, biochemistry and morphological features of peripheral blood cells in captive Boa constrictor. Conservation Physiology 2023; Vol. 11; Issue1. https://doi.org/10.1093/conphys/coad001.
• Alfaro-Alarcóna A, Hetzel U, Smurad T, Baggio F, Moralesa JA, Kipar A, Hepojoki J, 2022. Boid Inclusion Body Disease Is Also a Disease of Wild Boa Constrictors. Microbiol Spectr. 2022 Sep 12:e01705-22. https://doi.org/10.1128/spectrum.01705-22.
• Lintala A, Szirovicza L, Kipar A, Hetzel U, Hepojoki J, 2022. Persistent Reptarenavirus and Hartmanivirus Infection in Cultured Boid Cells. Microbiol Spectr. 2022 Aug31; 10(4):e0158522. https://doi.org/10.1128/spectrum.01585-22.
• Szirovicza L, Hetzel U, Kipar A, Hepojoki J, 2022. Short '1.2× Genome' Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells. Viruses. 2022 Jan 8;14(1):107. https://doi.org/10.3390/v14010107.
• Baggio F, Hetzel U, Nufer N, Kipar A, Hepojoki J. 2021. A subpopulation of arenavirus nucleoprotein localizes to mitochondria. Sci Rep. 2021 Oct 26;11(1):21048. https://doi.org/10.1038/s41598-021-99887-5.