Serpentoviruses Exhibit Diverse Organization and ORF Composition with Evidence of Recombination.

Serpentoviruses are a subfamily of positive sense RNA viruses in the order Nidovirales, family Tobaniviridae, associated with respiratory disease in multiple clades of reptiles. While the broadest viral diversity is reported from captive pythons, other reptiles, including colubrid snakes, turtles, and lizards of captive and free-ranging origin are also known hosts. To better define serpentoviral diversity, eleven novel serpentovirus genomes were sequenced with an Illumina MiSeq and, when necessary, completed with other Sanger sequencing methods. The novel serpentoviral genomes, along with 57 other previously published serpentovirus genomes, were analyzed alongside four outgroup genomes. Genomic analyses included identifying unique genome templates for each serpentovirus clade, as well as analysis of coded protein composition, potential protein function, protein glycosylation sites, differences in phylogenetic history between open-reading frames, and recombination. Serpentoviral genomes contained diverse protein compositions. In addition to the fundamental structural spike, matrix, and nucleoprotein proteins required for virion formation, serpentovirus genomes also included 20 previously uncharacterized proteins. The uncharacterized proteins were homologous to a number of previously characterized proteins, including enzymes, transcription factors, scaffolding, viral resistance, and apoptosis-related proteins. Evidence for recombination was detected in multiple instances in genomes from both captive and free-ranging snakes. These results show serpentovirus as a diverse clade of viruses with genomes that code for a wide diversity of proteins potentially enhanced by recombination events.

Proliferative strongyloidiasis in a colony of colubrid snakes.

Strongyloides are small rhabditid nematodes primarily associated with enteric disease in a variety of animal species, including reptiles. Strongyloides spp life stages were associated with a disease outbreak in a large breeding colony of snakes. Multiple Pantherophis and Lampropeltis colubrids exhibited respiratory distress, anorexia, stomatitis, facial deformation, and waning body condition that resulted in death or necessitated euthanasia. Postmortem examinations of 13 snakes revealed epithelial hyperplasia and inflammation of the alimentary and respiratory tracts associated with varying numbers of adult and larval nematodes and embryonated or larvated ova. In a subset of snakes, aberrant nematode migration was also observed in the eye, genitourinary system, coelom, and vasculature. Histomorphology and gross examination of parasitic adult female nematodes from host tissues were consistent with a Strongyloides spp. Sedimented fecal material from 101/160 (63%) snakes housed in the affected facility was positive for nematodes and/or larvated ova. Polymerase chain reaction amplification and sequencing of portions of the 18S and 28S ribosomal ribonucleic acid (RNA) genes and the internal transcribed spacer region of adult female parasites and positive fecal samples supported the diagnosis of strongyloidiasis. Strongyloides spp possess a unique life cycle capable of alternating between parasitic (homogonic) and free-living (heterogonic) stages, resulting in the production of directly infective larvae. Commonly utilized husbandry practices in reptile collections can amplify the numbers of infective larvae generated in the captive environment, increasing the risk for rhabditid hyperinfections. This report documents morbidity, mortality, and non-enteric disease manifestations due to Strongyloides hyperinfections in a captive colubrid snake colony.

In Vitro Characterization and Antiviral Susceptibility of Ophidian Serpentoviruses.

Ophidian serpentoviruses, positive-sense RNA viruses in the order Nidovirales, are important infectious agents of both captive and free-ranging reptiles. Although the clinical significance of these viruses can be variable, some serpentoviruses are pathogenic and potentially fatal in captive snakes. While serpentoviral diversity and disease potential are well documented, little is known about the fundamental properties of these viruses, including their potential host ranges, kinetics of growth, environmental stability, and susceptibility to common disinfectants and viricides. To address this, three serpentoviruses were isolated in culture from three unique PCR-positive python species: Ball python (Python regius), green tree python (Morelia viridis), and Stimson's python (Antaresia stimsoni). A median tissue culture infectious dose (TCID50) was established to characterize viral stability, growth, and susceptibility. All isolates showed an environmental stability of 10-12 days at room temperature (20 °C). While all three viruses produced variable peak titers on three different cell lines when incubated at 32 °C, none of the viruses detectably replicated at 35 °C. All viruses demonstrated a wide susceptibility to sanitizers, with 10% bleach, 2% chlorhexidine, and 70% ethanol inactivating the virus in one minute and 7% peroxide and a quaternary ammonium solution within three minutes. Of seven tested antiviral agents, remdesivir, ribavirin, and NITD-008, showed potent antiviral activity against the three viruses. Finally, the three isolates successfully infected 32 unique tissue culture cell lines representing different diverse reptile taxa and select mammals and birds as detected by epifluorescent immunostaining. This study represents the first characterization of in vitro properties of growth, stability, host range, and inactivation for a serpentovirus. The reported results provide the basis for procedures to mitigate the spread of serpentoviruses in captive snake colonies as well as identify potential non-pharmacologic and pharmacologic treatment options for ophidian serpentoviral infections.

Piscichuvirus-Associated Severe Meningoencephalomyelitis in Aquatic Turtles, United States, 2009-2021.

Viruses from a new species of piscichuvirus were strongly associated with severe lymphocytic meningoencephalomyelitis in several free-ranging aquatic turtles from 3 coastal US states during 2009-2021. Sequencing identified 2 variants (freshwater turtle neural virus 1 [FTuNV1] and sea turtle neural virus 1 [STuNV1]) of the new piscichuvirus species in 3 turtles of 3 species. In situ hybridization localized viral mRNA to the inflamed region of the central nervous system in all 3 sequenced isolates and in 2 of 3 additional nonsequenced isolates. All 3 sequenced isolates phylogenetically clustered with other vertebrate chuvirids within the genus Piscichuvirus. FTuNV1 and STuNV1 shared ≈92% pairwise amino acid identity of the large protein, which narrowly places them within the same novel species. The in situ association of the piscichuviruses in 5 of 6 turtles (representing 3 genera) with lymphocytic meningoencephalomyelitis suggests that piscichuviruses are a likely cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles.

Divergent Serpentoviruses in Free-Ranging Invasive Pythons and Native Colubrids in Southern Florida, United States.

Burmese python (Python bivittatus) is an invasive snake that has significantly affected ecosystems in southern Florida, United States. Aside from direct predation and competition, invasive species can also introduce nonnative pathogens that can adversely affect native species. The subfamily Serpentovirinae (order Nidovirales) is composed of positive-sense RNA viruses primarily found in reptiles. Some serpentoviruses, such as shingleback nidovirus, are associated with mortalities in wild populations, while others, including ball python nidovirus and green tree python nidovirus can be a major cause of disease and mortality in captive animals. To determine if serpentoviruses were present in invasive Burmese pythons in southern Florida, oral swabs were collected from both free-ranging and long-term captive snakes. Swabs were screened for the presence of serpentovirus by reverse transcription PCR and sequenced. A total serpentovirus prevalence of 27.8% was detected in 318 python samples. Of the initial swabs from 172 free-ranging pythons, 42 (24.4%) were positive for multiple divergent viral sequences comprising four clades across the sampling range. Both sex and snout-vent length were statistically significant factors in virus prevalence, with larger male snakes having the highest prevalence. Sampling location was statistically significant in circulating virus sequence. Mild clinical signs and lesions consistent with serpentovirus infection were observed in a subset of sampled pythons. Testing of native snakes (n = 219, 18 species) in part of the python range found no evidence of python virus spillover; however, five individual native snakes (2.3%) representing three species were PCR positive for unique, divergent serpentoviruses. Calculated pairwise uncorrected distance analysis indicated the newly discovered virus sequences likely represent three novel genera in the subfamily Serpentovirinae. This study is the first to characterize serpentovirus in wild free-ranging pythons or in any free-ranging North America reptile. Though the risk these viruses pose to the invasive and native species is unknown, the potential for spillover to native herpetofauna warrants further investigation.

Oral, Cloacal, and Hemipenal Actinomycosis in Captive Ball Pythons (Python regius).

Ball pythons (Python regius) are one of the most commonly kept and bred reptiles in captivity. In a large ball python breeding colony, a unique syndrome characterized by granulomatous inflammation of the cloaca and hemipenes (phalli) was observed in 140 of 481 (29.1%) breeding males, but only one of 1,446 breeding females. Lesions were absent in virgin males (n = 201) and virgin females (n = 293). On postmortem examination (n = 13, 12 males, 1 female), numerous well-defined mucosal and submucosal granulomas were present in the hemipenes (males) and cloaca (males and female). Extension into the coelomic cavity and liver was noted in a subset of these animals. An additional small subset of breeder animals (6/2027; 0.3%) presented with oral and mandibular swellings. Postmortem examination (n = 4, all female) showed oral lesions histologically indistinguishable from the cloacal/hemipenal lesions. Aerobic bacterial culture of a hepatic granuloma of one snake resulted in the isolation of filamentous, Gram-positive bacilli; amplification, and sequencing of the 16S rRNA gene and subsequent phylogenetic analysis of the isolate identified the bacterium as a novel species of Actinomyces. Screening of cloacal and oral granulomas using a specific, heminested 16S rRNA PCR assay confirmed the presence of the agent in all 17 snakes, as well as in cloacal swabs taken at the time of necropsy in 11/13 snakes. The Actinomyces sp. was also identified by PCR of cloacal swabs of unaffected snakes (n = 94) from the affected colony and two unrelated, grossly unaffected breeding colonies. In the affected colony, 65.5% of breeding animals (n = 23) but only 11.9% of virgin animals (n = 42) tested PCR positive, with breeding status being a significant predictor of bacterium presence (P < 0.00001). This study characterizes a granulomatous mucosal disease syndrome of breeding male ball pythons associated with a novel Actinomyces. In stark contrast to male snakes, the presence of the bacterium in both breeding and virgin females was very rarely associated with clinical disease. Though additional studies are necessary, these data suggest a role for the novel bacterium in the disease process, a predilection for clinical disease in male snakes, and the potential for sexual transmission of the disease.