Experimental Reptarenavirus Infection of Boa constrictor and Python regius

Abstract

Boid inclusion body disease (BIBD) causes losses in captive snake populations globally. BIBD is associated with the formation of cytoplasmic inclusion bodies (IBs), which mainly comprise reptarenavirus nucleoprotein (NP). In 2017, BIBD was reproduced by cardiac injection of boas and pythons with reptarenaviruses, thus demonstrating a causative link between reptarenavirus infection and the disease. Here, we report experimental infections of Python regius (n = 16) and Boa constrictor (n = 16) with three reptarenavirus isolates. First, we used pythons (n = 8) to test two virus delivery routes: intraperitoneal injection and tracheal instillation. Viral RNAs but no IBs were detected in brains and lungs at 2 weeks postinoculation. Next, we inoculated pythons (n = 8) via the trachea. During the 4 months following infection, snakes showed transient central nervous system (CNS) signs but lacked detectable IBs at the time of euthanasia. One of the snakes developed severe CNS signs; we succeeded in reisolating the virus from the brain of this individual and could demonstrate viral antigen in neurons. In a third attempt, we tested cohousing, vaccination, and sequential infection with multiple reptarenavirus isolates on boas (n = 16). At 10 months postinoculation, all but one snake tested positive for viral RNA in lung, brain, and/or blood, but none exhibited the characteristic IBs. Three of the four vaccinated snakes seemed to sustain challenge with the same reptarenavirus; however, neither of the two snakes rechallenged with different reptarenaviruses remained uninfected. Comparison of the antibody responses in experimentally versus naturally reptarenavirus-infected animals indicated differences in the responses.IMPORTANCE In the present study, we experimentally infected pythons and boas with reptarenavirus via either intraperitoneal injection or tracheal instillation. The aims were to experimentally induce boid inclusion body disease (BIBD) and to develop an animal model for studying disease transmission and pathogenesis. Both virus delivery routes resulted in infection, and infection via the trachea could reflect the natural route of infection. In the experimentally infected snakes, we did not find evidence of inclusion body (IB) formation, characteristic of BIBD, in pythons or boas. Most of the boas (11/12) remained reptarenavirus infected after 10 months, which suggests that they developed a persistent infection that could eventually have led to BIBD. We demonstrated that vaccination using recombinant protein or an inactivated virus preparation prevented infection by a homologous virus in three of four snakes. Comparison of the antibody responses of experimentally and naturally reptarenavirus-infected snakes revealed differences that merit further studies.

FIG 1

Schematic representation of the first experimental infection timeline. The experiment included eight ball pythons, which were monitored for 14 days postinoculation. The vertical arrows indicate inoculation. White (mild tremor) and black (tremor) X marks in the infection timeline mark the observed CNS signs. The black crosses indicate euthanasia.

FIG 2

Schematic representation of the second experimental infection timeline. The experiment included eight ball pythons and two common boas, which were monitored up to 118 days postinoculation. The vertical arrows indicate inoculation. The observed CNS signs are marked by white (mild tremor) and black (tremor) X marks, and the cohousing of snakes is indicated by shading of the infection timeline. The black crosses indicate euthanasia.

FIG 3

Brain and spleen of Python regius euthanized 22 days after intratracheal instillation of UHV (animal 2.5). (A and C) Immunohistochemistry (IHC) showing reptarenavirus nucleoprotein in the cytoplasm of neurons (inset, arrowhead) in the brain (A) and in macrophages/dendritic cells (arrowheads) in the spleen (C). (B and D) Negative-control slides. Shown is IHC employing a broadly cross-reactive rabbit anti-pan-reptarenavirus antiserum (39) and hematoxylin counterstaining.

FIG 4

Schematic representation of the third experimental infection timeline. The experiment included 14 common boas, which were monitored up to 295 days (∼10 months) postinoculation. The inclined arrows indicate immunization, and the vertical arrows indicate inoculation time points. White (mild tremor) and black (tremor) X marks indicate the observed CNS signs, and the cohousing of snakes is indicated by shading of the infection timeline. The black crosses indicate euthanasia.

FIG 5

Antibody responses in experimentally versus naturally infected common boas. (A) Box plot of IgM class antibodies against reptarenavirus NP (using a concentrated UGV-1 lysate as the antigen). The boxes from left to right represent naturally infected snakes without IBs, naturally infected snakes with IBs, the 0-bleeds (blood samples collected prior to immunization or inoculation), samples collected following immunization, and samples collected from the experimentally infected snakes at the time of euthanasia. The y axis represents optical density at 450 nm (OD₄₅₀) values as the ELISA readout. (B) Box plot of IgY class antibodies against reptarenavirus NP (using a concentrated UGV-1 lysate as the antigen). The boxes from left to right represent naturally infected snakes without IBs, naturally infected snakes with IBs, the 0-bleeds collected prior to immunization or inoculation, samples collected following immunization, and samples collected from the experimentally infected snakes at the time of euthanasia. The y axis represents OD₄₅₀ values as the ELISA readout. (C) Box plot of neutralizing antibody (NAb) titers as studied using VSV pseudotypes with reptarenavirus glycoproteins. The boxes from left to right represent neutralizing antibodies against UGV-1 in experimentally infected snakes, neutralizing antibodies against UHV-1 in experimentally infected snakes, neutralizing antibodies against ABV-1 in experimentally infected snakes, neutralizing antibodies against UGV-1 in naturally infected snakes, neutralizing antibodies against S5-like glycoproteins in naturally infected snakes, and neutralizing antibodies against TSMV-2 in naturally infected snakes. The y axis represents the last dilution producing a 50% reduction in the number of fluorescent foci. (D) Box plot of NAb titers as studied using VSV pseudotypes with reptarenavirus glycoproteins in naturally infected snakes with and without IBs. The boxes from left to right represent neutralizing antibodies against UGV-1 in snakes with IBs, neutralizing antibodies against UGV-1 in snakes without IBs, neutralizing antibodies against S5-like glycoproteins in snakes with IBs, neutralizing antibodies against S5-like glycoproteins in snakes without IBs, neutralizing antibodies against TSMV-2 in snakes with IBs, and neutralizing antibodies against TSMV-2 in snakes without IBs. The y axis represents the last dilution producing a 50% reduction in the number of fluorescent foci.