Identification and Genetic Analysis of Species D Rotaviruses in Pangolin Samples

Pangolins have been found to carry severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related coronaviruses. In light of this discovery, interest has been piqued in viromes of these heavily trafficked wild animals. In this study, we performed viral metagenomic sequencing to explore viromes of both confiscated dead pangolins and captive healthy pangolins. Sequence reads of vertebrate-associated viruses in Herpesviridae, Retroviridae, Iridoviridae, Reoviridae, Arenaviridae, and Flaviviridae were detected in confiscated dead pangolins. A novel rotavirus (RV) (Reoviridae), showing a high degree of genetic similarity to the RV species D (RVD) that was previously unreported in mammals, was further confirmed by using reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing. Three out of 18 samples from the confiscated dead pangolins were positive for genomic sequences of the novel RV. Importantly, sequence alignments and phylogenetic analyses demonstrated that these RV strains genetically belonged to the RVD. Nevertheless, these novel RVD strains were divergent from known RVD strains that have been found only in Avian. They formed a separate genetic cluster. Five serial passages were attempted to isolate the RV, but no live virus was obtained. In addition, fecal samples were collected from healthy pangolins (n = 41) in our institution and screened for RVs by viral metagenomic sequencing and RT-PCR. In these fecal samples, neither species D nor previously identified species A RVs were detected. This study reported RVDs in pangolin samples for the first time to our knowledge. Identifiability disagreements between wild and captive pangolins highlight the need for further exploration into pangolin viruses to better understand their emergence and transmission potential.