Reptarenaviruses in constrictor snakes : tissue tropism and immune responses

The family Arenaviridae is a well-represented clade of RNA viruses. The genus Mammarenavirus is dominated by rodent-borne arenaviruses, several of which have been identified as the causative agents behind hemorrhagic fevers and neurological infections in humans. Despite having been studied for more than 90 years, mammarenavirus diagnostics, vaccines and antiviral compounds are only available for some mammarenaviruses. Another genus, Reptarenavirus, includes viruses linked to boid inclusion body disease (BIBD) in constrictor snakes. BIBD has been reported in captive constrictor snake species since the 1970s, but the etiological agents were only identified in 2012. BIBD can lead to the eradication of the entire affected snake populations. The range of possible host spectrum and the immune response against reptarenaviruses are not well characterized. This thesis aims to define the potential reptarenavirus host cell spectrum as well as expand understanding of the boid immune response. One of the hallmark signs of reptarenavirus infection in snakes is the formation of inclusion bodies (IB) in host cells. Snakes are poikilotherm and the replication of viruses is often susceptible to temperature variation. Reptarenavirus infection in mammalian, boid, and arthropod cells, incubated at 37 °C did not induce IB formation, whereas prominent IB formation occurred in all three phyla when incubated at 30°C. Reptarenaviruses replicated efficiently at 30°C, whereas at 37°C the replication efficiency reduced significantly. Many animal viruses take advantage of glycoproteins (GPs) to mediate binding and entry via attachment to host cell surface receptors. To study the ability of reptarenavirus GPs to mediate cell entry, a pseudovirus system based on reporter gene-bearing recombinant vesicular stomatitis virus (rVSV) was introduced. The pseudoviruses with reptarenavirus GPs served to demonstrate that the majority of arenavirus GPs could mediate entry to both mammalian and reptilian cells but at varying efficiencies. In order to validate the link between BIBD and reptarenavirus infection, constrictor snakes (Boa constrictor and Python regius) were experimentally infected. Despite transient central nervous system signs, IB were not detected in the infected snakes. The snakes were sacrificed and sera was collected to determine the magnitude of the humoral immune response. In order to assess the antibody response against reptarenaviruses it was necessary to develop reagents capable of detecting immunoglobulins in snake sera. The generated reagents were initially tested using sera from BIBD-positive snakes. IgY and IgM class antibodies binding reptarenaviruses were detected in serum samples, validating the functionality of the reagents. In the next study, these antibodies were used to detect IgM and IgY antibodies in experimentally and naturally infected snake populations. Extracted sera was further assayed using the rVSV-based pseudoviruses decorated with reptarenavirus GPs to show a neutralizing antibody response following reptarenavirus infection. This thesis adds to understanding of reptarenavirus infectivity across species barriers. The generation of novel diagnostic reagents will allow generation of serodiagnostic tools for reptarenavirus infection. Future studies of reptarenaviruses should aim to establish a virus-specific link between reptarenaviruses and BIBD that could serve in the development of effective and preventive treatment strategies.RNA-virukset, Arenaviridae-heimo mukaan lukien, muodostavat laajan ja monimuotoisen mikrobijoukon. Mammarenavirus- suvun virukset ovat pääosin jyrsijöiden viruksia, joista eräät ihmiseen tarttuessaan voivat aiheuttaa verenvuotokuumeita, toiset keskushermostotulehduksia. Vaikka mammarenaviruksia on tutkittu yli 90 vuoden ajan, diagnostiikkaa, rokotteita ja viruslääkkeitä on kehitetty vain joitain mammarenaviruslajeja vastaan. Toinen suku, Reptarenavirus, koostuu kuristajakäärmeiden viruksista, jotka on yhdistetty tautiin nimeltä Boid Inclusion Body Disease (eng.), BIBD. BIBD:tä esiintyy vankeudessa elävillä kuristajakäärmeillä ja sitä on havaittu 1970-luvulta lähtien, mutta taudinaiheuttajat tunnistettiin vasta vuonna 2012. BIBD on vaarallinen sairaus käärmeille ja se voi johtaa koko käärmekokoelman hävittämiseen. Reptarenavirusten isäntälajikirjo ja immuunivasteen kehittymisen mekanismeja tartunnan saaneilla eläimillä ei toistaiseksi tunneta yksityiskotaisesti. Tämän työn tavoitteisiin kuului reptarenaviruksen isäntäsoluspektrin tunnistaminen sekä käärmeidein immuunivasteen tutkimus. Reptarenavirukset aiheuttavat infekoituneissa soluissa inkluusiokappaleiden (IB, inclusion body, eng.) muodostumista. Aiempien raporttien mukaan IB:eiden muodostus on tyypillinen löydös tutkittaessa infektoituneita käärmeitä. Nisäkässoluille luonnollisessa 37°C:een lämpötilassa reptarenavirusinfektoituneissa soluissa ei havaittu selkeää IB:ien muodostusta, vastaavasti käärmesolujen viljelylämpötilassa (30°C) havaittiin voimakasta IB:ien muodostusta eri niveljalkais- ja nisäkässolulinjoissa. Virusten kykyä replikoitua testattiin niveljalkais-, nisäkäs- ja matelijasolulinjoilla kahdessa lämpötilassa, 30°C ja 37°C. Virukset replikoituivat tehokkaasti 30°C:een lämpötilassa, mutta heikosti 37°C:een lämpötilassa. Monet vaipalliset virukset hyödyntävät glykoproteiineja (GP) isäntäsolupinnan reseptoreihin sitoutumiseen sekä isäntäsolun ja viruskalvon välisen fuusion. Reptarenaviruksien GP:ien kykyä kuljettaa virus erilaisiin solutyyppeihin käytettiin geneettisesti muokattua vesikulaarista stomatiitti-virusta (rVSV, recombinant vesicular stomatitis virus, eng.), jonka pintarakenne korvattiin eri arenaviruksien GP:eilla. Kokeissa havaittiin eri arenaviruksien GP:ien kykenevän kuljettamaan reportterigeenillä varustetun pseudoviruksen useisiin eri kudoksista peräisin oleviin matelija- että nisäkässoluihin vaihtelevalla tehokkuudella. Kokeellista reptarenavirusinfektiota kuristajakäärmeillä (Boa constrictor ja Python regius) käytettiin työkaluna BIBD:n ja reptarenavirusinfektion välisen yhteyden todistamiseksi. Reptarenavirusinfektoiduilla eläimillä havaittiin ohimeneviä keskushermosto-oireita, mutta BIBD:een liittyvää IB:ien muodostumista ei havaittu koe-eläimillä. Käärmeet tapettiin ja kerätyistä seeruminäytteistä tutkittiin reptarenaviruksia vastaan kehittynyttä humoraalista immuunivastetta. Reptarenavirusten immuunivasteen arvioimiseksi oli välttämätöntä kehittää reagensseja, jotka kykenevät havaitsemaan immunoglobuliinit käärmeseerumista. Kehitettyjen reagenssien toimivuutta arvioitiin ensin BIBD:iä sairastavien käärmeiden seerumeilla. Seeruminäytteissä havaittiin reptarenavirusta tunnistavia IgY- ja IgM-luokan vasta-aineita, ja tämän avulla kyettiin osoittamaan luotujen reagenssien toimivan halutulla tavalla. Seuraavassa tutkimuksessa käärmeiltä löydettiin reptarenaviruksia tunnistavia IgY- ja IgM-luokan vasta-aineita sekä kokeellisen että luonnollisen reptarenavirusinfektion seurauksena. Reptarenaviruksien GP:eilla koristeltuja rVSV pseudoviruksia hyödynnettiin reptarenavirusinfektiota neutraloivien vasta-aineiden etsimiseen käärmeiden seerumista. Sekä kokeellisen että luonnollisen reptarenavirusinfektion seurauksena käärmeille oli kehittynyt reptarenavirusinfektiota neutraloivia vasta-aineita. Tämän väitöskirjan töiden ansiosta tunnemme paremmin reptarenaviruksen kykyä hyppiä lajirajojen yli. Työssä kehitettyjen uusien reagenssien avulla voidaan jatkossa kehittää testejä reptarenavirusinfektion havaitsemiseen eläviltä käärmeiltä. Tulevaisuuden tavoitteisiin reptarenavirologian alalla kuuluu muun muassa eri reptarenaviruslajien ja BIBD:n yhteyden tutkiminen, joka puolestaan voi auttaa kehittämään tehokkaita hoitoja tai menetelmiä infektioiden ennaltaehkäisyyn

Co-infecting Reptarenaviruses Can Be Vertically Transmitted in Boa Constrictor

Boid inclusion body disease (BIBD) is an often fatal disease affecting mainly constrictor snakes. BIBD has been associated with infection, and more recently with coinfection, by various reptarenavirus species (family Arenaviridae). Thus far BIBD has only been reported in captive snakes, and neither the incubation period nor the route of transmission are known. Herein we provide strong evidence that co-infecting reptarenavirus species can be vertically transmitted in Boa constrictor. In total we examined five B. constrictor clutches with offspring ranging in age from embryos over perinatal abortions to juveniles. The mother and/or father of each clutch were initially diagnosed with BIBD andor reptarenavirus infection by detection of the pathognomonic inclusion bodies (IB) andor reptarenaviral RNA. By applying next-generation sequencing and de novo sequence assembly we determined the "reptarenavirome " of each clutch, yielding several nearly complete L and S segments of multiple reptarenaviruses. We further confirmed vertical transmission of the co-infecting reptarenaviruses by species-specific RT-PCR from samples of parental animals and offspring. Curiously, not all offspring obtained the full parental "reptarenavirome". We extended our findings by an in vitro approach; cell cultures derived from embryonal samples rapidly developed IB and promoted replication of some or all parental viruses. In the tissues of embryos and perinatal abortions, viral antigen was sometimes detected, but IB were consistently seen only in the juvenile snakes from the age of 2 mo onwards. In addition to demonstrating vertical transmission of multiple species, our results also indicate that reptarenavirus infection induces BIBD over time in the offspring.Peer reviewe

Generation of Anti-Boa Immunoglobulin Antibodies for Serodiagnostic Applications, and Their Use to Detect Anti-Reptarenavirus Antibodies in Boa Constrictor

Immunoglobulins (Igs), the key effectors of the adaptive immune system, mediate the specific recognition of foreign structures, i.e. antigens. In mammals, IgM production commonly precedes the production of IgG in the response to an infection. The reptilian counterpart of IgG is IgY, but the exact kinetics of the reptilian immune response are less well known. Boid inclusion body disease (BIBD), an often fatal disease of captive boas and pythons has been linked to reptarenavirus infection, and BIBD is believed to be immunosuppressive. However, so far, the study of the serological response towards reptarenaviruses in BIBD has been hampered by the lack of reagents. Thus we set up a purification protocol for boa constrictor IgY and IgM, which should also be applicable for other snake species. We used centrifugal filter units, poly ethylene glycol precipitation and gel permeation chromatography to purify and separate the IgM and IgY fractions from boa constrictor serum, which we further used to immunise rabbits. We affinity purified IgM and IgY specific reagents from the produced antiserum, and labelled the reagents with horseradish peroxidase. Finally, using the sera of snakes with known exposure to reptarenaviruses we demonstrated that the newly generated reagents can be utilised for serodiagnostic purposes, such as immunoblotting and immunofluorescent staining. To our knowledge, this is the first report to show reptarenavirus-specific antibodies in boa constrictors.Peer reviewe

Differences in tissue and species tropism of reptarenavirus species studied by vesicular stomatitis virus pseudotypes

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), and co-infections by several reptarenaviruses are common in affected snakes. Reptarenaviruses have only been found in captive snakes, and their reservoir hosts remain unknown. In affected animals, reptarenaviruses appear to replicate in most cell types, but their complete host range, as well as tissue and cell tropism are unknown. As with other enveloped viruses, the glycoproteins (GPs) present on the virion’s surface mediate reptarenavirus cell entry, and therefore, the GPs play a critical role in the virus cell and tissue tropism. Herein, we employed single cycle replication, GP deficient, recombinant vesicular stomatitis virus (VSV) expressing the enhanced green fluorescent protein (scrVSV∆G-eGFP) pseudotyped with different reptarenavirus GPs to study the virus cell tropism. We found that scrVSV∆G-eGFPs pseudotyped with reptarenavirus GPs readily entered mammalian cell lines, and some mammalian cell lines exhibited higher, compared to snake cell lines, susceptibility to reptarenavirus GP-mediated infection. Mammarenavirus GPs used as controls also mediated efficient entry into several snake cell lines. Our results confirm an important role of the virus surface GP in reptarenavirus cell tropism and that mamma-and reptarenaviruses exhibit high cross-species transmission potential

Differences in Tissue and Species Tropism of Reptarenavirus Species Studied by Vesicular Stomatitis Virus Pseudotypes

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), and co-infections by several reptarenaviruses are common in affected snakes. Reptarenaviruses have only been found in captive snakes, and their reservoir hosts remain unknown. In affected animals, reptarenaviruses appear to replicate in most cell types, but their complete host range, as well as tissue and cell tropism are unknown. As with other enveloped viruses, the glycoproteins (GPs) present on the virion’s surface mediate reptarenavirus cell entry, and therefore, the GPs play a critical role in the virus cell and tissue tropism. Herein, we employed single cycle replication, GP deficient, recombinant vesicular stomatitis virus (VSV) expressing the enhanced green fluorescent protein (scrVSV∆G-eGFP) pseudotyped with different reptarenavirus GPs to study the virus cell tropism. We found that scrVSV∆G-eGFPs pseudotyped with reptarenavirus GPs readily entered mammalian cell lines, and some mammalian cell lines exhibited higher, compared to snake cell lines, susceptibility to reptarenavirus GP-mediated infection. Mammarenavirus GPs used as controls also mediated efficient entry into several snake cell lines. Our results confirm an important role of the virus surface GP in reptarenavirus cell tropism and that mamma-and reptarenaviruses exhibit high cross-species transmission potential

Taxonomy of the family Arenaviridae and the order Bunyavirales : update 2018

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.Peer reviewe

Differences in Tissue and Species Tropism of Reptarenavirus Species Studied by Vesicular Stomatitis Virus Pseudotypes

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), and co-infections by several reptarenaviruses are common in affected snakes. Reptarenaviruses have only been found in captive snakes, and their reservoir hosts remain unknown. In affected animals, reptarenaviruses appear to replicate in most cell types, but their complete host range, as well as tissue and cell tropism are unknown. As with other enveloped viruses, the glycoproteins (GPs) present on the virion’s surface mediate reptarenavirus cell entry, and therefore, the GPs play a critical role in the virus cell and tissue tropism. Herein, we employed single cycle replication, GP deficient, recombinant vesicular stomatitis virus (VSV) expressing the enhanced green fluorescent protein (scrVSV∆G-eGFP) pseudotyped with different reptarenavirus GPs to study the virus cell tropism. We found that scrVSV∆G-eGFPs pseudotyped with reptarenavirus GPs readily entered mammalian cell lines, and some mammalian cell lines exhibited higher, compared to snake cell lines, susceptibility to reptarenavirus GP-mediated infection. Mammarenavirus GPs used as controls also mediated efficient entry into several snake cell lines. Our results confirm an important role of the virus surface GP in reptarenavirus cell tropism and that mamma-and reptarenaviruses exhibit high cross-species transmission potential

Purification of IgM and IgY using gel permeation chromatography.

<p><b>A)</b> The IgM and IgY containing pellet from the PEG precipitation was loaded onto a 10/30 Superdex 200HR column (GE Healthcare) and the proteins were eluted with PBS at a flow rate of 0.4 ml/min. Dual wave length absorbance monitoring, A_260nm and A_280nm, was used for protein detection. <b>B)</b> Fractions collected during the gel permeation chromatography were analysed by SDS-PAGE under non-reducing conditions, the left lane shows molecular weight marker (Precision Plus Protein Dual Color, Bio-Rad) and the subsequent lanes labelled f1-f17 represent the fractions (marked with * in the chromatogram). <b>C)</b> The fractions (the left “peak” in A, corresponding to f1-f9 in B) containing IgM were pooled, concentrated using a 100 kDa centrifugal filter (Millipore), and loaded onto a 16/60 Sephacryl S-200HR column (GE Healthcare). The proteins were eluted with PBS at flow rate of 1.0 ml/min, and dual wave length absorbance monitoring, A_260nm and A_280nm, was used for protein detection. <b>D)</b> The fractions (the second “peak” in A, corresponding to f10-f17 in B) containing IgY were pooled, concentrated using a 100 kDa centrifugal filter (Millipore), and loaded onto a 16/60 Sephacryl S-200HR column (GE Healthcare). The proteins were eluted with PBS at a flow rate of 1.0 ml/min, and dual wave length absorbance monitoring, A_260nm and A_280nm, was used for protein detection.</p

Detection of anti-UHV NP antibodies in snakes with BIBD.

<p>The sera were collected from <i>Boa constrictors</i> with BIBD as characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158417#pone.0158417.ref020" target="_blank">20</a>].</p