Differential Disease Susceptibilities in Experimentally Reptarenavirus-Infected Boa Constrictors and Ball Pythons

Abstract

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.

Differences in Tissue and Species Tropism of Reptarenavirus Species Studied by Vesicular Stomatitis Virus Pseudotypes

“…The finding that IBs contain reptarenavirus NP further supported the link between reptarenavirus infection and BIBD [15]. Experimental infection of boas and pythons with purified reptarenaviruses provided unequivocal evidence that reptarenaviruses can cause BIBD [14]. Interestingly, in this experiment, pythons developed strong CNS signs without evidence of IB formation, whereas seemingly healthy boas harbored large IBs in most tissues [14].…”

“…While this finding is rather surprising, it could reflect the fact that the reptilian cell lines included in the study represent quite homogenic cell populations that derive from spontaneously immortalized cultures generated from homogenates of different tissues. Given the limitation of our study, the results do not rule out the possibility of efficient viral replication in the brain nor in this particular cell line, since reptarenavirus IBs are generally frequent in neurons in the brain of boas [13][14][15]23]. The reptarenavirus GPs differentially mediated entry into boa (V/1Liv) and python (VII/1Liv) liver cells; while the boa cells were rather permissive, the python cells appeared to be non-permissive.…”

“…Experimental infection of boas and pythons with purified reptarenaviruses provided unequivocal evidence that reptarenaviruses can cause BIBD [14]. Interestingly, in this experiment, pythons developed strong CNS signs without evidence of IB formation, whereas seemingly healthy boas harbored large IBs in most tissues [14]. Moreover, we have documented vertical transmission of reptarenaviruses and hartmaniviruses in boas, and the swarm of reptarenavirus genome segments transmitted can vary between the offspring [23].…”

“…Hartmaniviruses appear to target neuronal and smooth muscle cells [22], while reptarenaviruses infect a wide range of cell types [14,48]. Interestingly, snakes with BIBD often carry more L than S segments [20,21,23], and the distribution of segments may vary between tissues of the same individual [20,23].…”

“…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.Viruses 2020, 12, 395 2 of 16 Boid Inclusion Body Disease (BIBD), initially recognized in the 1970s, affects mainly captive boas and pythons [11]. BIBD is characterized by the formation of electron dense intracytoplasmic inclusion bodies (IB) [12] in various cell types (including blood cells) and tissues [13][14][15]. BIBD spreads efficiently within collections, and the recommendation to euthanize snakes with BIBD can lead to loss of entire collections [11].…”