In Vitro Characterization and Antiviral Susceptibility of Ophidian Serpentoviruses

Abstract

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.

Serpentoviruses Exhibit Diverse Organization and ORF Composition with Evidence of Recombination

“…Samples subjected to rtPCR included extracted RNA from both oral tissue and oral swabs. One additional novel genome was generated from cell culture lysate extraction of isolated green tree python serpentovirus (GTSV) as previously described [ 14 ]. Upon receipt, samples were extracted immediately using the Zymo Quick-RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA), per manufacturer’s recommendation.…”

“…In addition to the aforementioned variability in clinical manifestations of serpentovirus infections, three isolated serpentoviruses showed strain-dependent 10 5 -fold differences in peak viral titer in in vitro infection assays on a python cell line [14]. Despite substantial Viruses 2024, 16, 310 2 of 21 sequence diversity across numerous open reading frames (ORFs) in serpentovirus genomes, thus far, genomic characterization has been primarily focused on phylogenetic relationships of the ORF1b gene [5,6], which encodes the evolutionarily conserved RNA-dependent-RNA-polymerase (RDRP), along with other replication-complex associated proteins [9].…”