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Anja Kipar - One of the best experts on this subject based on the ideXlab platform.
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experimental reptarenavirus infection of boa constrictor and python regius
Journal of Virology, 2021Co-Authors: Udo Hetzel, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Saskia Keller, Leonora Szirovicza, Theresa Pesch, Jussi HepojokiAbstract: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.
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Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses-The representatives of genus Hartmanivirus, family Arenaviridae.
'Public Library of Science (PLoS)', 2018Co-Authors: Jussi Hepojoki, Olli Vapalahti, Leonora Szirovicza, Satu Hepojoki, Teemu Smura, Eva Dervas, Barbara Prähauser, Lisbeth Nufer, Elisabeth M Schraner, Anja KiparAbstract:The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular Inclusion bodies typical for the reptarenavirus-induced boid Inclusion Body Disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed
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Co-infecting Reptarenaviruses Can Be Vertically Transmitted in Boa Constrictor.
PLoS pathogens, 2017Co-Authors: Saskia Keller, Udo Hetzel, Olli Vapalahti, Anja Kipar, Tarja Sironen, Yegor Korzyukov, Jussi HepojokiAbstract: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.
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arenavirus coinfections are common in snakes with boid Inclusion Body Disease
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Udo Hetzel, Anja Kipar, Pertteli Salmenpera, Tarja Sironen, Yegor Korzyukov, Olli VapalahtiAbstract:ABSTRACT 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.
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replication of boid Inclusion Body Disease associated arenaviruses is temperature sensitive in both boid and mammalian cells
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Lesley Bellsakyi, Udo HetzelAbstract: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.
Jussi Hepojoki - One of the best experts on this subject based on the ideXlab platform.
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experimental reptarenavirus infection of boa constrictor and python regius
Journal of Virology, 2021Co-Authors: Udo Hetzel, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Saskia Keller, Leonora Szirovicza, Theresa Pesch, Jussi HepojokiAbstract: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.
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Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses-The representatives of genus Hartmanivirus, family Arenaviridae.
'Public Library of Science (PLoS)', 2018Co-Authors: Jussi Hepojoki, Olli Vapalahti, Leonora Szirovicza, Satu Hepojoki, Teemu Smura, Eva Dervas, Barbara Prähauser, Lisbeth Nufer, Elisabeth M Schraner, Anja KiparAbstract:The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular Inclusion bodies typical for the reptarenavirus-induced boid Inclusion Body Disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed
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Co-infecting Reptarenaviruses Can Be Vertically Transmitted in Boa Constrictor.
PLoS pathogens, 2017Co-Authors: Saskia Keller, Udo Hetzel, Olli Vapalahti, Anja Kipar, Tarja Sironen, Yegor Korzyukov, Jussi HepojokiAbstract: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.
-
arenavirus coinfections are common in snakes with boid Inclusion Body Disease
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Udo Hetzel, Anja Kipar, Pertteli Salmenpera, Tarja Sironen, Yegor Korzyukov, Olli VapalahtiAbstract:ABSTRACT 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.
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replication of boid Inclusion Body Disease associated arenaviruses is temperature sensitive in both boid and mammalian cells
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Lesley Bellsakyi, Udo HetzelAbstract: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.
Olli Vapalahti - One of the best experts on this subject based on the ideXlab platform.
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experimental reptarenavirus infection of boa constrictor and python regius
Journal of Virology, 2021Co-Authors: Udo Hetzel, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Saskia Keller, Leonora Szirovicza, Theresa Pesch, Jussi HepojokiAbstract: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.
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Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses-The representatives of genus Hartmanivirus, family Arenaviridae.
'Public Library of Science (PLoS)', 2018Co-Authors: Jussi Hepojoki, Olli Vapalahti, Leonora Szirovicza, Satu Hepojoki, Teemu Smura, Eva Dervas, Barbara Prähauser, Lisbeth Nufer, Elisabeth M Schraner, Anja KiparAbstract:The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular Inclusion bodies typical for the reptarenavirus-induced boid Inclusion Body Disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed
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Co-infecting Reptarenaviruses Can Be Vertically Transmitted in Boa Constrictor.
PLoS pathogens, 2017Co-Authors: Saskia Keller, Udo Hetzel, Olli Vapalahti, Anja Kipar, Tarja Sironen, Yegor Korzyukov, Jussi HepojokiAbstract: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.
-
arenavirus coinfections are common in snakes with boid Inclusion Body Disease
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Udo Hetzel, Anja Kipar, Pertteli Salmenpera, Tarja Sironen, Yegor Korzyukov, Olli VapalahtiAbstract:ABSTRACT 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.
-
replication of boid Inclusion Body Disease associated arenaviruses is temperature sensitive in both boid and mammalian cells
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Lesley Bellsakyi, Udo HetzelAbstract: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.
Udo Hetzel - One of the best experts on this subject based on the ideXlab platform.
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experimental reptarenavirus infection of boa constrictor and python regius
Journal of Virology, 2021Co-Authors: Udo Hetzel, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Saskia Keller, Leonora Szirovicza, Theresa Pesch, Jussi HepojokiAbstract: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.
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Co-infecting Reptarenaviruses Can Be Vertically Transmitted in Boa Constrictor.
PLoS pathogens, 2017Co-Authors: Saskia Keller, Udo Hetzel, Olli Vapalahti, Anja Kipar, Tarja Sironen, Yegor Korzyukov, Jussi HepojokiAbstract: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.
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arenavirus coinfections are common in snakes with boid Inclusion Body Disease
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Udo Hetzel, Anja Kipar, Pertteli Salmenpera, Tarja Sironen, Yegor Korzyukov, Olli VapalahtiAbstract:ABSTRACT 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.
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replication of boid Inclusion Body Disease associated arenaviruses is temperature sensitive in both boid and mammalian cells
Journal of Virology, 2015Co-Authors: Jussi Hepojoki, Olli Vapalahti, Anja Kipar, Yegor Korzyukov, Lesley Bellsakyi, Udo HetzelAbstract: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.
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Experimental infection of Boa constrictor with an orthoreovirus isolated from a snake with Inclusion Body Disease.
Journal of zoo and wildlife medicine : official publication of the American Association of Zoo Veterinarians, 2014Co-Authors: Sabina Darke, Rachel E. Marschang, Udo Hetzel, Manfred ReinacherAbstract:Orthoreoviruses have been associated with Disease in reptiles, but have not previously been isolated from snakes with Inclusion Body Disease (IBD). An orthoreovirus was isolated from a Boa constrictor diagnosed with IBD and then used to conduct a transmission study to determine the clinical importance of this virus. For the transmission study, 10 juvenile boas were experimentally infected with the isolated orthoreovirus and compared to 5 sham-infected control animals. Orthoreovirus was reisolated for a period of 18 wk after infection and weight gain was reduced in infected snakes. Histological examination showed a mild hepatitis in three of four virologically positive snakes up to 12 wk after infection. Results indicated that the orthoreovirus was moderately pathogenic, but, no evidence was found to indicate that it was the causal agent of IBD. In the light of the discovery of Arenaviruses in some snakes with IBD, it was proposed that orthoreoviruses may play a role in synergistic infection.
Joseph L Derisi - One of the best experts on this subject based on the ideXlab platform.
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Differential Disease Susceptibilities in Experimentally Reptarenavirus-Infected Boa Constrictors and Ball Pythons.
Journal of virology, 2017Co-Authors: Mark D Stenglein, David Sanchez-migallon Guzman, Valentina E. Garcia, Marylee L. Layton, Laura L. Hoon-hanks, Scott M. Boback, M. Kevin Keel, Tracy L. Drazenovich, Michelle G. Hawkins, Joseph L DerisiAbstract:Inclusion Body Disease (IBD) is an infectious Disease originally described in captive snakes. It has traditionally been diagnosed by the presence of large eosinophilic cytoplasmic Inclusions and is associated with neurological, gastrointestinal, and lymphoproliferative disorders. Previously, we identified and established a culture system for a novel lineage of arenaviruses isolated from boa constrictors diagnosed with IBD. Although ample circumstantial evidence suggested that these viruses, now known as reptarenaviruses, cause IBD, there has been no formal demonstration of Disease causality since their discovery. We therefore conducted a long-term challenge experiment to test the hypothesis that reptarenaviruses cause IBD. We infected boa constrictors and ball pythons by cardiac injection of purified virus. We monitored the progression of viral growth in tissues, blood, and environmental samples. Infection produced dramatically different Disease outcomes in snakes of the two species. Ball pythons infected with Golden Gate virus (GoGV) and with another reptarenavirus displayed severe neurological signs within 2 months, and viral replication was detected only in central nervous system tissues. In contrast, GoGV-infected boa constrictors remained free of clinical signs for 2 years, despite high viral loads and the accumulation of large intracellular Inclusions in multiple tissues, including the brain. Inflammation was associated with infection in ball pythons but not in boa constrictors. Thus, reptarenavirus infection produces Inclusions and Inclusion Body Disease, although Inclusions per se are neither necessarily associated with nor required for Disease. Although the natural distribution of reptarenaviruses has yet to be described, the different outcomes of infection may reflect differences in geographical origin.IMPORTANCE New DNA sequencing technologies have made it easier than ever to identify the sequences of microorganisms in Diseased tissues, i.e., to identify organisms that appear to cause Disease, but to be certain that a candidate pathogen actually causes Disease, it is necessary to provide additional evidence of causality. We have done this to demonstrate that reptarenaviruses cause Inclusion Body Disease (IBD), a serious transmissible Disease of snakes. We infected boa constrictors and ball pythons with purified reptarenavirus. Ball pythons fell ill within 2 months of infection and displayed signs of neurological Disease typical of IBD. In contrast, boa constrictors remained healthy over 2 years, despite high levels of virus throughout their bodies. This difference matches previous reports that pythons are more susceptible to IBD than boas and could reflect the possibility that boas are natural hosts of these viruses in the wild.
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identification characterization and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas candidate etiological agents for snake Inclusion Body Disease
Mbio, 2012Co-Authors: Mark D Stenglein, Chris Sanders, Amy Kistler, Graham J Ruby, Jessica Y Franco, Drury R Reavill, Freeland Dunker, Joseph L DerisiAbstract:Inclusion Body Disease (IBD) is an infectious fatal Disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the Disease is identified by the presence of large eosinophilic cytoplasmic in- clusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the dis- ease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novoassembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propa- gate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclu- sions in infected cells in culture and tissues from Diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those offiloviruses than to those of other arenaviruses. Thesefindings implicate these viruses as candidate etio- logic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens. IMPORTANCE Inclusion Body Disease (IBD) is a common infectious Disease of captive snakes. IBD is fatal and can cause the loss of entire animal collections. The cause of the Disease has remained elusive, and no treatment exists. In addition to being important to pet owners, veterinarians, breeders, zoological parks, and aquariums, the study of animal Disease is significant since animals are the source of virtually every emerging infectious human Disease. We searched for candidate causative agents in snakes diag- nosed with IBD and found a group of novel viruses distantly related mainly to arenaviruses but also tofiloviruses, both of which can cause fatal hemorrhagic fevers when transmitted from animals to humans. In addition to providing evidence that strongly suggests that these viruses cause snake IBD, this discovery reveals a new and unanticipated domain of virus biology and evolu- tion.
