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.

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 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.

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.

Replication of boid inclusion body disease-associated arenaviruses is temperature sensitive in both boid and mammalian cells.

UNLABELLED: Boid inclusion body disease (BIDB) is a fatal disease of boid snakes, the etiology of which has only recently been revealed following the identification of several novel arenaviruses in diseased snakes. BIBD-associated arenaviruses (BIBDAV) are genetically divergent from the classical Old and New World arenaviruses and also differ substantially from each other. Even though there is convincing evidence that BIBDAV are indeed the etiological agent of BIBD, the BIBDAV reservoir hosts--if any exist besides boid snakes themselves--are not yet known. In this report, we use University of Helsinki virus (UHV; a virus that we isolated from a Boa constrictor with BIBD) to show that BIBDAV can also replicate effectively in mammalian cells, including human cells, provided they are cultured at 30°C. The infection induces the formation of cytoplasmic inclusion bodies (IB), comprised mainly of viral nucleoprotein (NP), similar to those observed in BIBD and in boid cell cultures. Transferring infected cells from 30°C to 37°C ambient temperature resulted in progressive declines in IB formation and in the amounts of viral NP and RNA, suggesting that BIBDAV growth is limited at 37°C. These observations indirectly indicate that IB formation is linked to viral replication. In addition to mammalian and reptilian cells, UHV infected arthropod (tick) cells when grown at 30°C. Even though our findings suggest that BIBDAV have a high potential to cross the species barrier, their inefficient growth at mammalian body temperatures indicates that the reservoir hosts of BIBDAV are likely species with a lower body temperature, such as snakes. IMPORTANCE: 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.

Cx46 hemichannels contribute to the sodium leak conductance in lens fiber cells.

The lens is proposed to have an internal microcirculation system consisting of continuously circulating ionic fluxes that play an essential role in maintaining lens transparency. One of the key components of this system is the sodium leak conductance. Here we investigate the contribution of Cx46 hemichannels to the basal membrane permeability of peripheral fiber cells isolated from transgenic mouse lenses lacking Cx50 or both Cx50 and Cx46 (dKO) using the whole cell patch-clamp technique. Our results show that Cx46 hemichannels were largely closed at a resting voltage of -60 mV in the presence of millimolar divalent cation concentrations. However, even though the vast majority of these channels were closed at -60 mV, a small, persistent, inward current could still be detected. This current could be mostly blocked by exposure to 1 mM La(3+) and was not observed in fiber cells isolated from dKO mouse lenses suggesting that it was due to Cx46 hemichannels. In addition, Cx50(-/-) fiber cells showed increased open channel noise and a depolarized resting potential compared with dKO fiber cells. Exposure of Cx50(-/-) fiber cells to La(3+) hyperpolarized the resting potential to -58 mV, which is similar to the value of resting potential measured in dKO fiber and significantly reduced the open channel noise. In conclusion, these results suggest that Cx46 hemichannels may contribute to the sodium leak conductance in lens fiber cells.