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 and/or reptarenavirus infection by detection of the pathognomonic inclusion bodies (IB) and/or 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.

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.

e Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genus Reptarenavirus within the family Arenaviridae. We used next-generation sequencing and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus. Until very recently, arenaviruses were known as a group of mainly rodent-borne zoonotic viruses (1). The negativesense RNA genome of arenaviruses is divided into two segments, which are designated small (S, approximately 3.5 kb) and large (L, approximately 7 to 7.5 kb); both use an ambisense coding strategy (1). The S segment encodes the glycoprotein precursor (GPC) and the nucleoprotein (NP), whereas the RNA-dependent RNA polymerase (RdRp) and the Z protein (ZP) are encoded in the L segment (1). The discovery of arenaviruses in snakes with boid inclusion body disease (BIBD) by three independent groups (2-4) has expanded the family Arenaviridae by a new group of viruses. In fact, the BIBD-associated arenaviruses (BIBDAVs) have been suggested to form a new arenavirus genus called Reptarenavirus (5). At the same time, the arenavirus study group of the International Committee on Taxonomy of Viruses (ICTV) has suggested that the genus Arenavirus, harboring the "classical" Old and New World arenaviruses, be renamed Mammarenavirus (6). According to a recent proposal, the genus Reptarenavirus would contain three species: alethinophid reptarenaviruses 1 (member virus: Golden Gate virus), 2 (CAS virus, CASV), and 3 (University of Helsinki virus 1 [UHV-1], boa AV NL B3 virus). While in vitro evidence suggests a causal relationship between arenavirus infection and BIBD (4), the in vivo proof is still missing. Also, the reservoir host(s) of the reptarenaviruses has not yet been confirmed; however, our recent study suggests that these viruses preferentially grow in organisms with body temperatures close to 30°C (7).To study the diversity of reptarenaviruses, we applied nextgeneration sequencing (NGS) to characterize certain isolates described in our previous report (4). On the basis of phylogeny, we selected isolates originating from six Boa constrictor snakes and used a continuous B. constrictor kidney cell line, I/1Ki (4), for their propagation. While the virus isolate of snake 1 has already been almost fully sequenced (GenBank accession numbers KF297880.1 and KF297881.1), only partial L segment sequences were available for isolates from snakes 5 (KF564801.1), 8 (KF564796.1), 9 (KF564800.1), 11, and 41 (KF564797.1). We infected clean I/1Ki cultures with tissue homogenates and collected the viruses produced during the first passage by pelleting through a sucrose cushion as previously described (4). Viral RNAs were extracted from the pelleted viruses with the QIAmp Viral RNA minikit (Qiagen) according to the ma...

The newly discovered boid inclusion body disease-associated arenaviruses (BIBDAV) of reptiles have drastically altered the phylogeny of the family Arenavirus. Prior to their discovery, known arenaviruses were considered mainly rodent-borne viruses, with each arenavirus species having its own reservoir host. BIBDAV have so far been demonstrated in captive boid snakes, but their possible reservoir host(s) have not yet been identified. Here we show, using University of Helsinki virus as a model, that these viruses are able to infect mammalian (including human) and arthropod cells. Our results provide in vitro proof of the considerable ability of arenaviruses to cross species barriers. However, our data indicate that BIBDAV growth occurs at 30°C but is inhibited at 37°C, implying that crossing of the species barrier would be hindered by the body temperature of mammalian species.

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.