1. Introduction
Pangolins, also known as scaly anteaters (family: Manidae, order: Pholidota), are unique among the mammals because of their overlapping scales (made of keratin) and have a geographic distribution that includes parts of Asia and Africa (Gaudin et al. 2009). Only eight pangolin species are present worldwide, four in Asia including China, and four in Africa. There is a growing interest in pangolin welfare, including the threats posed by poaching and the deforestation of their natural habitats (Hua et al. 2015). Most importantly, pangolins have become unfortunate icons for the international and domestic illegal trade in wildlife, and are the most trafficked mammal (Gaubert et al. 2018). Of the eight species, four are listed as vulnerable, two are endangered, and the remaining two are critically endangered on the International Union for Conservation of Nature Red List of Threatened Species (du Toit et al. 2017).
Pestiviruses (family: Flaviviridae) are enveloped RNA viruses with highly variable single-stranded positive-sense RNA genomes of ∼12.3 kb that comprises a single large open reading frame (ORF) encoding a polyprotein of about 3,900 amino acids (aa) in length (Postel et al. 2015; Tautz et al. 2015). Some pestiviruses (e.g. bovine viral diarrhea virus, border disease virus, classical swine fever virus) are well-known animal pathogens that cause severe disease including contagious hemorrhagic disease in pigs and respiratory and reproductive disease in cattle and sheep (Vilcek and Nettleton 2006; Valdazo-Gonzalez et al. 2007; Moennig and Becher 2015). Wildlife disease (e.g. wild boar and deer) due to pestiviruses has also been reported (Ridpath et al. 2008; Blome et al. 2017). Following the application of molecular and genomics methods of virus discovery, a number of novel pestiviruses have been identified in recent years, including those from bats, rodents, and harbor porpoises (Wu et al. 2012; Firth et al. 2014; Smith et al. 2017; Jo et al. 2019). Finally, pestivirus-like viruses have also identified in arthropods (Shi et al. 2016a) and arthropods collected from mammals (Harvey et al. 2018). Together, these data highlight the circulation of a diverse range of pestiviruses in a broad range of animal hosts.
Coltiviruses (genus: Coltivirus, family: Reoviridae) are double-strand segmented RNA viruses. Currently, only two viruses have been defined in the genus: Colorado tick fever virus (CTFV) found in North America, and Eyach virus (EYAV) from Europe. Their RNA genome comprises twelve segments. Both viruses can cause severe disease in humans (Attoui et al. 2005; Moutailler et al. 2016), and are transmitted by ticks (Attoui et al. 2002). Recently, several novel coltiviruses have been characterized, including Tarumizu tick virus from Haemaphysalis flava ticks in Japan (Fujita et al. 2017), Kundal virus from Hyalomma anatolicum ticks in India (Yadav et al. 2019), Shelly headland virus (SHLV) from Ixodes holocyclus ticks in Australia (Harvey et al. 2018), and Tai Forest reovirus (TFRV) from free-tailed bats (Chaereophon aloysiisabaudiae) in Côte d’Ivoire (Weiss et al. 2017). Hence, there is evidently a high diversity of coltiviruses in nature and many more are likely to be identified. To date, however, it is unknown whether coltiviruses are associated with wildlife disease.
Herein, we performed a meta-transcriptomic (i.e. total RNA Sequencing) analysis of four diseased pangolins rescued by the Jinhua Wildlife Protection Station of Zhejiang province of China in 2018. From these animals, we identified and characterized the novel viral agents—a pestivirus and a coltivirus—from two pangolins. Additionally, we analyzed the clinical features and pathological changes associated with disease in these animals.