Isolation and Characterization of an Antigenically Distinct 68- kd Protein from Nonviral Intracytoplasmic Inclusions in Boa Constrictors Chronically Infected with the Inclusion Body Disease Virus (IBDV: Retroviridae)

Abstract

Abstract.The relationship between a retroviral infection and the development of nonviral intracytoplasmic inclusion bodies was studied in a Boa constrictor model. Twelve juvenile age-and size-matched inclusion body disease (IBD)-negative boas were randomly divided into three groups. Each group was inoculated intraperitoneally with 1 ml of an IBD virus (IBDV)-infected liver homogenate or 1 ml of normal boa liver homogenate (sham-inoculated control) or was left untreated. All boas were monitored for development of IBD by daily examination and serial liver biopsy over 1 year. The 4 IBDV-inoculated boas became IBDV and inclusion positive by 10 weeks postinoculation. The average size and density of inclusion bodies increased with the duration of infection. Ultrastructurally, inclusion bodies Ͻ2 m in diameter consisted of intracytoplasmic aggregates of granular electron-dense material that were not membrane limited. Larger inclusions (3-6 m in diameter) were characterized as membrane-bound aggregates of amorphous to granular electron-dense material admixed with membranelike fragments. The sham-inoculated and untreated control snakes did not become inclusion or IBDV positive. Direct comparison of the protein electrophoretograms of IBDV-infected and normal boa tissues demonstrated a prominent 68-kd protein band unique to infected inclusion-positive tissues. Monoclonal antibodies directed against the 68-kd protein band specifically labeled inclusion bodies. The results of this study demonstrate that IBD inclusions represent an intracytoplasmic accumulation of an antigenically distinct IBDV-associated protein.

Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agents of Boid Inclusion Body Disease

“…Tissue samples from the central nervous, respiratory, gastrointestinal, urogenital, lymphatic, and endocrine systems of all snakes were fixed in 10% formalin and paraffin wax embedded. Consecutive sections (3 to 5 m) stained with hematoxylin-eosin (HE) and Giemsa served to confirm or exclude BIBD, based on the presence or absence of the typical intracytoplasmic IB in a wide range of cells (2,(7)(8)(9). Blood smears were stained with May-Grünwald Giemsa and examined for the presence of IB in circulating blood cells.…”

“…Affected animals usually die from secondary bacterial (salmonellosis), fungal (aspergillosis), and protozoal (amoebiasis) septicemic infections with encephalitis, pneumonia, hepatitis, enteritis, or osteomyelitis, and from neoplastic processes, such as lymphomas (6). This is probably a consequence of immunosuppression (7,8) since red and white blood cells as well as myelopoietic cells also develop the typical inclusion bodies (IB), probably impairing function. In boas, the disease outcome varies; affected animals either die within weeks or months or become asymptomatic carriers (8,9).…”

“…In contrast, pythons generally develop severe fatal neurological symptoms within a few weeks (8,9). Histologically, BIBD is characterized by typical eosinophilic to amphophilic, amorphous, intracytoplasmic IB that are ultrastructurally electron dense and consist primarily of a protein with a molecular mass of 68 kDa (7). The etiology of BIBD is not yet confirmed; however, a viral infection (i.e., retrovirus, paramyxovirus, and, more recently and convincingly, arenavirus) has been considered (10)(11)(12)(13).…”

Identification and Characterization of Two Closely Related Unclassifiable Endogenous Retroviruses in Pythons (Python molurusandPython curtus)

“…The inclusion bodies contain an antigenically distinct 68-kDa protein against which a specific monoclonal antibody has been developed (24). In all snakes with CNS disease, a nonsuppurative meningoencephalitis with neuronal degeneration and perivascular cuffing was present and viral particles resembling C-type retroviruses were detected in the brain, pancreas, and kidney, as well as in cultured primary kidney cells.…”

“…In all snakes with CNS disease, a nonsuppurative meningoencephalitis with neuronal degeneration and perivascular cuffing was present and viral particles resembling C-type retroviruses were detected in the brain, pancreas, and kidney, as well as in cultured primary kidney cells. The disease was shown to be transmissible by cell-free primary kidney cell culture supernatants from infected Boa constrictor snakes to young Burmese pythons (Python molurus bivittatus) (19) or by liver homogenates from B. constrictor (24). A polyclonal antibody raised against sucrose density gradient-purified BIBD virus produced by in vitro cocultivation of leukocytes from a BIBDpositive B. constrictor snake and VH2 viper heart cells reacted with specific proteins associated with virus particles of C-type morphology that were purified from the primary kidney cell cultures established from another BIBD-positive B. constrictor snake (13).…”