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. Unsere neuesten retrospektiven Studien zeigen, dass wildlebende Schlangen ebenfalls an BIBD erkranken können (Alfaro-Alarcón et al.2022).
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.
BIBD affects various boid species from different families, including Boa constrictor and Python spp. 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 generally develop severe fatal neurological symptoms within a few weeks. The prevalence of BIBD in captive boid populations is still unknown, and so far BIBD has not been reported in wild snakes. The natural host(s) of reptarenaviruses (RAV), if any beyond the constrictor snakes themselves, is/are not known.
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 the typical viral cytoplasmic inclusion bodies in blood cells or tissue cells. 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 high variability of the snake arenaviruses complicates the situation and reduces the sensitivity of many such approaches. However, we and others have gathered evidence to suggest that a specific viral segment (so-called UGV-1 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 some rare reptarenaviruses would not be detected. Therefore. although time consuming, expensive and difficult to establish, virus isolation in boid cell cultures is still the most sensitive and reliable method.
A recent publication from our group demonstrates the diagnostic challenges; our results have shown that the detection of serum antibodies against reptarenaviruses is only of limited diagnostic value (Windbichler et al., 2019).
We and others could show that snakes with BIBD are often infected with several reptarenaviruses, also of different species (Hepojoki J et al., 2015); a proportion of these animals also exhibits co-infection with further closely related arenaviruses, the hartmaniviruses (Hepojoki et al., 2018). We have also been able to show that all these viruses can be transmitted from infected snakes to their offspring (so-called vertical transmission) which subsequently develops BIBD (Keller et al., 2017). This fact is of major relevance for breeding colonies.
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 2011 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.
In early 2012, our group isolated and characterised a novel virus, found to be an 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. Coincidently, around the same time, 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 reptarenaviruses have been found in snakes with BIBD.
In our studies, we also fulfilled Koch’s postulates in vitro, showing that reptarenaviruses can infect boid and mammalian cells. Furthermore, we have demonstrated that reptarenavirus growth is inhibited at mammalian body temperatures, whereas they grow effectively at 30°C. These findings indicate that the reservoir hosts of reptarenaviruses are species with a lower body temperature, i.e. most likely poikilothermic animals.
For us diagnostic veterinary pathologists and virologists, the development of reliable diagnostic tools for BIBD and reptarenavirus infection is an important area of work. The variability between the different reptarenavirus isolates and lack of knowledge of their true diversity hampers the development of universal molecular, i.e. RT-PCR-based diagnostic approaches. Currently, the most efficient tool for the diagnosis of BIBD is virus isolation in boid cell cultures, however, such a technique is fairly time consuming and hard to establish for diagnostic purposes. To overcome this, we have produced virus (RAV nucleoprotein, NP) specific antibodies useful for diagnostic approaches that base on antigen detection. We have also produced antibodies against boa-IgY and -IgM which we used to detect antibodies in constrictor snakes against reptarenaviruses. While we found out that serological tests are of very limited use for diagnostic purposes, our tools for the diagnosis of BIBD in snakes also allow us to hunt for the reservoir host of reptarenaviruses in free ranging animals (rodents, bats, snakes, etc.) a broad variety of which are potential candidates for this. Such knowledge will help to understand the mechanisms of the disease process and help to develop evidence-based means of control and therapy.
Our group also works on other reptilian viruses, which also includes, for example, deltaviruses (genus Daletvirus) or serpentoviruses which are a current threat to python collections. The group`s focus lies on the identification, characterisation and pathogen-host interactions of reptilian pathogens in general.
The main investigators are Dr Jussi Hepojoki, a biochemist and molecular virologist, and Dr Udo Hetzel, a zoologist and veterinary pathologist with specific expertise in the pathology of reptiles, exotics, zoo and wild animals. Together with Professors Olli Vapalahti and Anja Kipar, they have been developing and directing the group’s research activities.
Dr Jussi Hepojoki has more than 20 years research experience. 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 Hepojoki 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 has identified a snake deltavirus towards which he has developed a keen interest. Recently, the team has demonstrated that snake deltavirus 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. He is in the final phase of his “habilitation” in Zurich where he holds an Oberassistent position. 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?".
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 Hetzel 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 (immunohistology and -fluorescence, RNA-ISH, laser microdissection etc.) 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, and has a comprehensive track record on the study of zoonotic diseases, such as tick-borne encephalitis, Pogosta disease, dengue fever, and haemorrhagic fever with renal syndrome. Olli Vapalahti’s research is focused on the development of methods for the detection and serodiagnostic of novel infectious agents as well as tracing their disease associations, ecology and epidemiology. He is also affiliated to the Hospital District of Helsinki and Uusimaa Laboratory Services, where he is responsible for diagnostics of viral zoonoses in human patients.
Dr Teemu Smura is a virologist at the University of Helsinki. His current interests include virus hunting, evolutionary virology, phylogenetics and NGS.
Dr Francesca Baggio is a molecular biologist with a background in mitochondrial biology and genetics. She is a postdoctoral scientist and member of the team in Zurich and has extensive experience in the use of the fruit fly as model organism for studies on the role of different proteins involved in the regulation of mitochondrial genome expression. She has adapted her broad methodological expertise in molecular biology to molecular virology and is currently investigating the cytopathic effect of reptarenaviruses in in vitro systems. Her most recent work indicates relevant interaction of the viral nucleoprotein with mitochondria (Baggio et al., 2020).
Dr Eva Dervas is a veterinary pathologist with a particular interest in the cytopathic and immunopathological effect of virus infections in snakes. Her PhD project aims to decipher immunological functions in snakes and their alterations with BIBD. Together with Dr. Baggio, one of her supervisors, she now tries to establish quantitative molecular methods to assess the immune response snakes.
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.
The BIBD Group is completed by several postgraduate students. So far, five 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, 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 have currently supervise three dissertation projects. In Helsinki, the PhD student Annika Linttala investigates the molecular characteristics of reptarenavirus persistence, co-infection and their effect on BIBD pathogenesis. In Zurich, Iman Kriještorac Berbić investigates the in vitro growth characteristics of reptarenaviruses and Dr Eva Dervas pursues her project on the immunopathology of BIBD.
The BIBD Group works closely with several international groups that are interested in infectious diseases of snakes. They have a joint project with veterinary pathologists and immunologists in Brazil, the group of Professor David Driemeier at the Department of Veterinary Pathology, Universidade Federal do Rio Grande do Sul in Porto Alegre, and the group of Professor Ricardo Portela at the Instituto de Ciências da Saúde, Universidade Federal da Bahia in Salvador, and with Dr Alejandro Alfaro and his colleagues at the Departamento de Patologia, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia in Costa Rica. The group has also established joint projects with wildlife veterinarians at the Department of Clinical Veterinary Studies, Faculty of Veterinary Science, University of Zimbabwe in Harare. It also works 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.
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.