Boid Inclusion Body Disease

Einschlusskörperchen-Krankheit der Boiden (English text below)

Die Einschlusskörperchen-Krankheit der Boiden (Boid inclusion body disease; BIBD) wurde in den 1970er Jahren zuerst beschrieben. BIBD ist eine zunehmend wichtige Erkrankung von Boas und Pythons in Gefangenschaft und kann ganze Schlangenbestände auslöschen. Die Ursache für BIBD war lange unbekannt, erst vor wenigen Jahren wurde bei erkrankten Schlangen eine Infektion mit Reptarenaviren nachgewiesen.

BIBD befällt Boidae zahlreicher Familien, einschliesslich Boa constrictor und Python spp. Bei Boas wird ein sehr variabler Krankheitsverlauf beobachtet. Befallene Tiere sterben oft innerhalb von Wochen oder Monaten, können aber auch symptomfreie Träger werden. Im Unterschied dazu entwickeln Pythons generell innerhalb weniger Wochen schwere neurologische Symptome, die zum Tod des Tieres führen. Die Prävalenz von BIBD in Schlangenpopulationen in Gefangenschaft ist bisher nicht bekannt. Bei wildlebenden Schlangen ist die Krankheit noch nicht beschrieben worden. Der natürliche Wirt der BIBD-assoziierten Reptarenaviren (RAV) ist ebenso unbekannt.

Unsere Forschungsgruppe ("The BIBD Group") aus Veterinärpathologen und Virologen arbeitet daran, die Pathogenese der BIBD aufzudecken und zuverlässige diagnostische Methoden für BIBD zu etablieren. Der molekulare Nachweis einer RAV-Infektion ist möglich, wird jedoch durch die hohe Variabilität der Viren erschwert, was die Sensitivität dieser Herangehensweise mindert. Derzeit stellt eine Virusisolierung in Boazellkulturen immer noch die sensitivste Methode dar. Diese Technik ist jedoch zeitlich und finanziell sehr aufwändig und kann nur schwer etabliert werden. Daher versuchen wir, kombinierte Methoden zum Nachweis der Infektion zu entwickeln.


Boid Inclusion Body Disease

Boid inclusion body disease (BIBD) has first been described in the 1970s and is a disease of increasing 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. Animals either die within weeks or months or become asymptomatic carriers. 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 not known.

Our research group ("The BIBD Group"), consisting of veterinary pathologists and virologists, is trying to reveal the pathogenesis of BIBD, but is also working on establishing reliable diagnostic tools for BIBD. Molecular detection of RAV infection is possible. However, the high variability of RAV complicates the situation and reduces the sensitivity of this approach. At present, virus isolation in boid cell cultures is still the most sensitive method. However, it is time consuming, expensive and difficult to establish. Therefore, we are trying to develop combined methods for the detection of RAV infection.

The BIBD Group

The international multidisciplinary research group with members from the Universities of Zurich and Helsinki, established to study the aetiology and pathogenesis of Boid inclusion body disease.

The BIBD Group has been established in 2011 by veterinary pathologists, a zoologist and virologists. It was initially based at the University of Helsinki, where all 4 main group 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 rapidly growing.

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.

Dr Jussi Hepojoki has 17 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. Since the BIBD Group was established, Jussi Hepojoki has focused on preparing the crucial reagents for the BIBD and reptarenavirus study. In addition, he has been working on sequencing reptarenavirus isolates, and setting up diagnostic assays (antigen and/or antibody detection) for reptarenaviruses. The main interests of Jussi Hepojoki in the project lie in the factors enabling cross-species transmission and in the mechanisms behind persistent infection of reptarenaviruses. He is currently pursuing his “habilitation” in Zurich where he holds an Oberassistent position, and has recently 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 word which allow him to isolate also other emerging snake viruses, such as nidoviruses in pythons Nidovirus Pneumonien

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. Prof. Vapalahti 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.

Also involved into our research on reptarenaviruses is Dr Teemu Smura. He is a virologist at the University of Helsinki and his current interests include virus hunting, evolutionary virology, phylogenetics and NGS.

In October 2017, Dr Francesca Baggio has joined the group in Zurich as a postdoctoral scientist. She is a molecular biologist with a background in mitochondrial biology and genetics. She has previously investigated the role of different proteins involved in the regulation of mitochondrial genome expression using the fruit fly as model organism. She has a broad expertise in molecular biology methodologies and genetic analyses that she is now adapting to molecular virology.

Postgraduate students: The BIBD Group is completed by several postgraduate students. So far, two doctoral theses have been finalised and the results published: Dr Saskia Keller has worked on the vertical transmission of reptarenaviruses and BIBD, and Dr Eva Dervas has worked on nidovirus-associated pneumonia in pythons. We currently supervise three doctoral students. The PhD project of Yegor Korzyukov focuses on the determination of the cellular receptor and experimental antiviral and vaccine strategies for reparenaviruses, and the PhD project of Leonóra Szirovicza, MSc, focuses on the molecular characterisation of reptarenavirus persistence, co-infection and their effect on BIBD pathogenesis. Both work in Helsinki. The project of Katharina Windbichler, the doctoral student in Zurich, represents an extended study on the reptarenavirome and immune response of snakes in breeding colonies.

Our research. 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 (RAVs) have been found in BIBD-positive snakes.

In our studies, we also fulfilled Koch’s postulates in vitro, showing that RAVs can infect boid and mammalian cells. Furthermore, we have recently demonstrated that RAV growth is inhibited at mammalian body temperatures, whereas they grow effectively at 30°C. These findings indicate that the reservoir hosts of RAV are species with a lower body temperature, i.e. most likely poikilothermic animals.

The temperature dependence of RAV replication is a potentially useful observation. If the replication of RAV is also temperature sensitive in vivo, housing at an optimised temperature, i.e. conditions mimicking the temperature alterations occurring naturally, could help the infected snake to clear the viral infection, as it could minimise viral replication and aid the snake’s immune system to fight the infection. However, this remains to be tested with diseased animals in a controlled way.

For us diagnostic veterinary pathologists and virologists, the development of reliable diagnostic tools for BIBD and RAV infection is an important area of work. The variability between the different RAV 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. Most recently we were able to produce antibodies against boa-IgY and -IgM, which does allow us to detect antibodies in constrictor snakes against RAV. We are currently investigating snake breeding colonies to assess the usefulness of serological tests for diagnostic purposes. The tools for the diagnosis of BIBD in snakes will also allows us to hunt for the reservoir host of RAV in free ranging animals (rodents, bats, snakes, etc.); a broad variety of which are potential candidates for this. In addition it will help to understand the mechanisms of the disease process and help to develop evidence-based means of control and therapy.

Funding: Our work has received funding from the Academy of Finland, the Finnish Foundation for Veterinary Research, the University of Helsinki, the Stiftung für Wissenschaftliche Forschung and the Norh-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.