Identification And Genome Characteristics Of Novel Pestivirus And Coltivirus In Pangolins

Illegal wildlife trade,one of the most serious criminal activities of profiteering in the world,severely damages biodiversity and the ecological environment.Smuggling and transportation of wildlife are an important transmission route for emerging and reemerging infectious diseases,which are serious threats to human public health.Despite great biosecurity risks,researches on the pathogens carried by such illegally smuggled wildlife is extremely limited.Pangolins have become unfortunate icons for the international and domestic illegal trade in wildlife,and are the most trafficked mammal.Due to some traditional concepts of "tonic" and the demand for Chinese traditional medicine,China has become one of the main destinations of smuggling pangolins.Along with the illegal smuggling and transportation of pangolins,the pathogens they carried will also be imported into China.During July to August 201 8 four sick wild pangolins(three Manis javanica and one M.pentadactyla)exhibiting a variety of clinical symptoms(i.e.anorexia)were rescued by the Jinhua Wildlife Protection Station in Zhejiang province,China.Despite the attentive care and symptomatic treatment of the staff,only the Chinese pangolin(M.pentadactyla)survived,and the remaining three Malayan pangolins(M.javanica)died two weeks after the rescue.In order to identify the pathogens that caused the four pangolins diseases,we extracted total RNA from samples collected from four sick pangolins,and constructed RNA libraries.Using meta-transcriptomics combined with RT-PCR,pestivirus and coltivirus were identified in two of the dead Malayan pangolins,1-Dongyang and 2-Lishui,respectively.Genomic analysis revealed that the two novel RNA viruses were distinct from the known pestiviruses and coltiviruses,with more than 48%and 25%sequence divergence at the amino acid level,respectively.Combined with the position of the novel viruses in the phylogenetic trees,they should be the new species of the genus Pestivirus and Coltivirus.Hence,we named them Dongyang pangolin virus(DYPV)and Lishui pangolin virus(LSPV)based on the sampling site and hosts Additionally,DYPV was also detected in nymph ticks(Amblyomma javanense)collected from pangolin 1-DongyangAs DYPV was identified in ticks(Amblyomma javanense)collected from pangolin 1-Dongyang,coltiviruses are well-known vector-borne virus,and LSPV was closely related to Shelly headland virus also identified in ticks(Ixodes holocyclus)from Australia,we collected 452 ticks at the locations where the sick pangolins were found to explore the origin and transmission route of these two viruses.Using meta-transcriptomics and RT-PCR,no homologous sequences to these two new viruses was detected in all of samples,suggesting that the origin of these viruses may not in Zhejiang.In addition,phylogenetic analysis of the mitochondrial cytochrome b(mt-cyt b)gene of the pangolins revealed that two Malayan pangolins infected with two novel viruses might be from Southeast Asia.The epidemiological investigation revealed that both novel viruses might have been imported following the illegal international trade of pangolins.In sum,these data indicate that illegal wildlife trafficking not only threatens the status of pangolin populations,but may also spread epizootic pathogens.Although there are no reports of these two viruses infecting humans or livestock,continuous surveillance is still needed due to their potential biosafety risks.