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 wichtige Erkrankung von Boas und Pythons in Gefangenschaft, die ganze Schlangenbestände auslöschen kann. Die Ursache für BIBD war lange unbekannt, erst vor wenigen Jahren wurde bei erkrankten Schlangen eine Infektion mit Reptarenaviren nachgewiesen.

BIBD befällt Würgeschlangen zahlreicher Familien, einschliesslich Boa constrictor und Python spp. Bei Boas wird ein sehr variabler Krankheitsverlauf beobachtet. Befallene Tiere können innerhalb von Wochen oder Monaten sterben oder auch über lange Zeit symptomfreie Träger bleiben. Im Unterschied dazu entwickeln Pythons meist 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 ist ebenso unbekannt.

Unsere Forschungsgruppe ("The BIBD Group") aus Veterinärpathologen, Virologen, Molekularbiologen und Epidemiologen arbeitet daran, die Pathogenese der BIBD aufzudecken und zuverlässige diagnostische Methoden für BIBD zu etablieren. Wir konnten 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 S et al., 2017). Diese Tatsache ist für Zuchtbestände von grosser Bedeutung.

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 potentielle Ansteckungsquellen für andere Schlangen dar.

Der molekulare Nachweis einer Arenavirus-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 “multi-Reptarenavirus-RT-PCR“ werden viele, wenn auch leider noch immer nicht alle Reptarenaviren erkannt. Derzeit stellt eine Virusisolierung in Boazellkulturen immer noch die sensitivste Methode dar. Da diese Technik jedoch zeitlich und finanziell sehr aufwändig ist, versuchen wir, kombinierte Methoden zum Nachweis der Infektion zu entwickeln. Unserer jüngste Publikation zeigt allerdings, dass der Nachweis von Serumantikörpern nur eine sehr begrenzte Aussagekraft hat (Windbichler K et al., 2019).

Im Weiteren arbeiten wir an anderen Reptilienviren, wie zum Beispiel Reptilienretroviren, oder Nidoviren. Letztere sind seit einigen Jahren zunehmend von Bedeutung in Pythonbeständen. Generell liegt der Forschungsschwerpunkt der Gruppe auf der Identifikation, Charakterisierung und der Pathogen-Wirt-Beziehung von Viren bei Reptilien.

 

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.

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.

Our research group ("The BIBD Group"), comprised of veterinary pathologists, virologists, molecular biologists and veterinary epidemiologists, is trying to reveal the pathogenesis of BIBD, but is also working on establishing reliable diagnostic tools for BIBD. 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 J 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 S et al., 2017). This fact is of major relevance for breeding colonies.

Reliable 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 RAV infection is possible. However, the high variability of the snake arenaviruses complicates the situation and reduces the sensitivity of many such approaches. The group has developed a “multi-reparenavirus RT-PCR” which detects several, but unfortunately not all, reparenaviruses. Although time consuming, expensive and difficult to establish, at present, virus isolation in boid cell cultures is still the most sensitive and reliable method. Therefore, we are trying to develop combined methods for the detection of arenavirus infection in snakes. Our most recent publication demonstrates the diagnostic challenges, our results have shown that the detection of serum antibodies against reptarenaviruses is only of limited diagnostic value (Windbichler K et al., 2019).

In general, the group is working on reptilian viruses, which also includes, for example, retroviruses or nidoviruses 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 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 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.

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 around 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. Since the BIBD Group was established, Jussi Hepojoki has 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 recently 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., bioRxiv 2019). 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 was 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 U 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.

Dr Eva Dervas is a veterinary pathologist with a particular interest in the cytopathic and immunopathological effect of virus infections in snakes. Her current postdoctoral project focusses on the establishment of methodological approaches to determine the immunological function and capacity in snakes.

The most recent new member of the group is Dr Eleni Michalopoulou, a veterinary epidemiologist from the University of Liverpool, UK. Eleni Michalopoulou has previously been working with Professor Kipar on other projects and has recently developed a keen interest in the epidemiology of emerging virus infections. She is instrumental in the group’s work on larger animal cohorts.

Postgraduate students: The BIBD Group is completed by several postgraduate students. So far, three 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, and Dr Katharina Windbichler’s project has focussed on the reptarenavirome and immune response of snakes in breeding colonies. We currently supervise three doctoral students. In Helsinki, the PhD project of Yegor Korzyukov focuses on the cellular receptor and experimental antiviral and vaccine strategies for reptarenaviruses, and the PhD project of Leonóra Szirovicza, MSc, investigates the molecular characteristics of reptarenavirus persistence, co-infection and their effect on BIBD pathogenesis. In Zurich, Tanja Thiele recently started a follow-up project of Dr Windbichler’s work, an in-depth reptarenavirome investigation in large colonies.

Collaboration partners: 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 veterinary pathologists 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 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 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. We have also produced antibodies against boa-IgY and -IgM which we used to detect antibodies in constrictor snakes against RAV. This should us that serological tests are of very limited use for diagnostic purposes. However, our tools for the diagnosis of BIBD in snakes also allow 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. Such knowledge will help to understand the mechanisms of the disease process and help to develop evidence-based means of control and therapy.

In a global context, the decline of animal populations is nowadays one of the main concerns. It does not exclude the class Reptilia. Triggering factors are the loss and degradation of habitats, the introduction of invasive species, environmental pollution, diseases, unsustainable use and global climate change (Whitfield Gibbon et al 2000). More and more, indigenous reptile populations are affected by newly “emerging” pathogens, introduced through traded animals (and their “blind passenger pathogens”) worldwide. The BIBD Group aims to provide scientific and diagnostic tools to meet the requirements emerging from these recent challenges.

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 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.