Experimental Reptarenavirus Infection of Boa constrictor and Python regius

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.

Experimental Reptarenavirus Infection of Boa constrictor and Python regius

ABSTRACTBoid inclusion body disease (BIBD) causes losses in captive constrictor snake populations globally. BIBD associates with formation of cytoplasmic inclusion bodies (IB) 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. Herein, we report experimental infections of pythons (N=16) and boas (N=16) with three reptarenavirus isolates. First, we used pythons (N=8) to test two virus delivery routes: intraperitoneal injection and tracheal instillation. Independent of the delivery route, we detected viral RNA but no IBs in tissues two weeks post inoculation. Next, we inoculated pythons (N=8) via the trachea. During the four month following the infection snakes showed transient central nervous system (CNS) signs but lacked detectable IB at the time of euthanasia. One of the snakes developed severe CNS signs and we succeeded in re-isolating the virus from the brain of this individual, and could demonstrate viral antigen in neurons. In a third attempt, we tested co-housing, vaccination, and sequential infection with multiple reptarenavirus isolates on boas (N=16). At 10 months post inoculation all except one snake tested positive for viral RNA but none exhibited the characteristic IB. Analysis of the antibody responses demonstrated lower neutralizing but higher anti-reptarenavirus NP titers in experimentally versus naturally reptarenavirus infected boas. Our findings suggest that in addition to reptarenavirus infection, other factors, e.g. the antibody response, contribute to BIBD pathogenesis.IMPORTANCEA 2017 study demonstrated cardiac reptarenavirus injection to induce boid inclusion body disease (BIBD) in pythons and boas. In the present study, we experimentally infected pythons and boas with reptarenavirus via either intraperitoneal injection or tracheal instillation. We found both virus delivery routes to result in infection; though the latter could reflect the natural route of infection. In the experimentally infected snakes, we did not find evidence of inclusion body (IB) formation, characteristic to BIBD, in pythons or in boas. Most of the snakes (11/12) studied were reptarenavirus infected after ten-month follow up, which suggests that they could eventually have developed BIBD. We further found differences between the antibody responses of experimentally and naturally reptarenavirus infected snakes, which could indicate that the pathogenesis of BIBD involves factors additional to reptarenavirus infection. As snakes are poikilotherm, also the housing conditions could have an effect.

Identification of Reptarenaviruses, Hartmaniviruses, and a Novel Chuvirus in Captive Native Brazilian Boa Constrictors with Boid Inclusion Body Disease

ABSTRACT Boid inclusion body disease (BIBD) is a transmissible viral disease of captive snakes that causes severe losses in snake collections worldwide. It is caused by reptarenavirus infection, which can persist over several years without overt signs but is generally associated with the eventual death of the affected snakes. Thus far, reports have confirmed the existence of reptarenaviruses in captive snakes in North America, Europe, Asia, and Australia, but there is no evidence that it also occurs in wild snakes. BIBD affects boa species within the subfamily Boinae and pythons in the family Pythonidae, the habitats of which do not naturally overlap. Here, we studied Brazilian captive snakes with BIBD using a metatranscriptomic approach, and we report the identification of novel reptarenaviruses, hartmaniviruses, and a new species in the family Chuviridae. The reptarenavirus L segments identified are divergent enough to represent six novel species, while we found only a single novel reptarenavirus S segment. Until now, hartmaniviruses had been identified only in European captive boas with BIBD, and the present results increase the number of known hartmaniviruses from four to six. The newly identified chuvirus showed 38.4%, 40.9%, and 48.1% amino acid identity to the nucleoprotein, glycoprotein, and RNA-dependent RNA polymerase, respectively, of its closest relative, Guangdong red-banded snake chuvirus-like virus. Although we cannot rule out the possibility that the found viruses originated from imported snakes, the results suggest that the viruses could circulate in indigenous snake populations. IMPORTANCE Boid inclusion body disease (BIBD), caused by reptarenavirus infection, affects captive snake populations worldwide, but the reservoir hosts of reptarenaviruses remain unknown. Here, we report the identification of novel reptarenaviruses, hartmaniviruses, and a chuvirus in captive Brazilian boas with BIBD. Three of the four snakes studied showed coinfection with all three viruses, and one of the snakes harbored three novel reptarenavirus L segments and one novel S segment. The samples originated from collections with Brazilian indigenous snakes only, which could indicate that these viruses circulate in wild snakes. The findings could further indicate that boid snakes are the natural reservoir of reptarena- and hartmaniviruses commonly found in captive snakes. The snakes infected with the novel chuvirus all suffered from BIBD; it is therefore not possible to comment on its potential pathogenicity and contribution to the observed changes in the present case material.

Viruserkrankungen der Reptilien in der tierärztlichen Praxis

ZusammenfassungViren können bei Reptilien bedeutende Krankheitserreger sein. In den letzten Jahren wurden auch zunehmend neue Viren bei Reptilien beschrieben. Zudem gibt es immer mehr Möglichkeiten, Virusinfektionen bei Reptilien nachzuweisen. Dieser Übersichtsartikel bietet einen Überblick über häufige und bedeutende Virusinfektionen bei Reptilien, die damit assoziierten Krankheiten sowie deren Diagnose. Besprochen werden v. a. Infektionen bei Reptilien, die häufig als Haustiere gehalten werden: Schlangen und Echsen (Squamaten) sowie Land- und Wasserschildkröten. Themen bei den Squamaten sind die insbesondere bei Bartagamen (Pogona vitticeps) extrem häufig vorkommenden Adenoviren, ferner Paramyxo- und Tobaniviren (auch als Nido- oder Serpentoviren bekannt), die insbesondere bei verschiedenen Schlangenspezies schwere Atemwegserkrankungen verursachen können. Weitere Abschnitte beschäftigen sich mit Reptarenaviren, die die Einschlusskörperchenkrankheit („inclusion body disease“, IBD) der Boas und Pythons auslösen, sowie mit den v. a. bei verschiedenen Echsenspezies beschriebenen Iridoviren (inklusive der Rana-, Invertebraten-Irido- und Hemocytiviren). Bei den Schildkröten werden die Herpesviren der Land- und Wasserschildkröten separat diskutiert, ferner die vorkommenden Rana- und Picornaviren. Vertreter aller 3 dieser Virusfamilien können neben Erkrankungen des oberen Respirations- und Verdauungstrakts andere klinische Veränderungen hervorrufen (z. B. schwere Panzererweichung bei juvenilen Landschildkröten durch Picornaviren).

Identification of Reptarenaviruses, Hartmaniviruses and a Novel Chuvirus in Captive Brazilian Native Boa Constrictors with Boid Inclusion Body Disease

ABSTRACTBoid Inclusion Body Disease (BIBD) is a transmissible viral disease of captive snakes that causes severe losses in snake collections worldwide. It is caused by reptarenavirus infection, which can persist over several years without overt signs, but is generally associated with the eventual death of the affected snakes. Thus far, reports have confirmed existence of reptarenaviruses in captive snakes in North America, Europe, and Australia, but there is no evidence that it also occurs in wild snakes. BIBD affects both boas and pythons, the habitats of which do not naturally overlap. Herein, we studied Brazilian captive snakes with BIBD using a metatranscriptomic approach, and report the identification of novel reptarenaviruses, hartmaniviruses, and a new species in the family Chuviridae. The reptarenavirus L segments identified represent six novel species, while we only found a single novel reptarenavirus S segment. Until now, hartmaniviruses had been identified only in European captive boas with BIBD, and the present results increase the number of known hartmanivirus species from four to six. The newly identified chuvirus showed 38.4%, 40.9%, and 48.1% amino acid identity to the nucleoprotein, glycoprotein, and RNA-dependent RNA polymerase of its closest relative, Guangdong red-banded snake chuvirus-like virus. Although we cannot rule out the possibility that the found viruses originated from imported snakes, the results suggest that the viruses would circulate in indigenous snake populations.IMPORTANCEBoid Inclusion Body Disease (BIBD) caused by reptarenavirus infection affects captive snake populations worldwide, but the reservoir hosts of reptarenaviruses remain unknown. Herein, we report the identification of novel reptarenavirus and hartmanivirus species, and a chuvirus in captive Brazilian boas with BIBD. Three of the four snakes studied showed co-infection with all three viruses, and one of the snakes harbored three novel reptarenavirus L and one novel S segment. The samples originated from collections with Brazilian indigenous snakes only, which could indicate that these viruses circulate in wild snakes. The findings could further indicate that boid snakes are the natural reservoir of reptarena- and hartmaniviruses commonly found in captive snakes. The snakes infected with the novel chuvirus all suffered from BIBD; it is therefore not possible to comment on its potential pathogenicity and contribution to the observed changes in the present case material.