Detection of a novel Pestivirus strain in Java ticks (Amblyomma javanense) and the hosts Malayan pangolin (Manis javanica) and Chinese pangolin (Manis pentadactyla)

Abstract

Pangolins are endangered animals and are listed in the CITES Appendix I of the Convention International Trade Endangered Species of Wild Fauna and Flora as well as being the national first-level protected wild animal in China. Based on a few reports on pangolins infected with pestiviruses of the Flaviviridae family, Pestivirus infections in pangolins have attracted increasing attention. Pangolin pestivirus is a pathogen that may cause diseases such as acute diarrhea and acute hemorrhagic syndrome. To better understand the epidemiology and genomic characterization of pestiviruses carried by pangolins, we detected pestiviruses in dead Malayan pangolin using metavirome sequencing technology and obtained a Pestivirus sequence of 12,333 nucleotides (named Guangdong pangolin Pestivirus, GDPV). Phylogenetic tree analysis based on the entire coding sequence, NS3 gene or RdRp gene sequences, showed that GDPV was closely related to previously reported pangolin-derived Pestivirus and clustered into a separate branch. Molecular epidemiological investigation revealed that 15 Pestivirus-positive tissues from two pangolins individuals with a positivity rate of 5.56%, and six Amblyomma javanense carried pestiviruses with a positivity rate of 19.35%. Moreover, the RdRp gene of the Pestivirus carried by A. javanense showed a high similarity to that carried by pangolins (93-100%), indicating A. javanense is likely to represent the vector of Pestivirus transmission. This study expands the diversity of viruses carried by pangolins and provides an important reference value for interrupting the transmission route of the virus and protecting the health of pangolins.

Keywords: Amblyomma javanense; Pestivirus; molecular epidemiology; pangolin; virome.

Figure 1

(A) View of the dorsal and ventral surfaces of the tick under a stereoscope. a1 and a2 are dorsal and ventral surfaces of male adult ticks, respectively; b1 and b2 are dorsal and ventral surfaces of female adult ticks, respectively; c1, c2, d1, and d2 are dorsal and ventral surfaces of nymphs. (B) Phylogenetic trees based on nucleotide sequences of 16S rRNA. The trees were constructed via the maximum likelihood method using the MrBayes approach using the SYM + G nucleotide substitution model. Red letters represent the ticks isolated from Guangdong in the present study.

Figure 2

(A) Recombination analysis of Guangdong pangolin Pestivirus (GDPV) and other pangolin pestivirus based on the whole-genome sequences using Simplot v3.5.10. GDPV, Guangdong pangolin pestivirus isolate M1/B1; DYPV, Dongyang pangolin virus isolate DYCS; DYAJ1, Dongyang pangolin virus isolate DYAJ1; ZJ-MO1, pangolin Pestivirus 3 isolate ZJ-MO1. The analysis was performed with the Kimura model, with a window size of 11, 613 bp and a step size of 150 bp. (B) Comparison of GDPV genome structure with that of other Pestivirus genomes. GDPV, Guangdong pangolin pestivirus isolate M1/B1; DYPV, Dongyang pangolin pestivirus isolate DYCS; CSFV, Classical swine fever virus. (C) All cleavage sites of NS3 protease in GDPV. GDPV, Guangdong pangolin pestivirus isolate M1/B1; DYPV, Dongyang pangolin virus isolate DYCS; BDV, Border disease virus; and BVDV1, Bovine viral diarrhea virus 1.

Figure 3

Phylogenetic trees based on nucleotide sequences of (A) polyprotein, (B) NS3 gene, and (C) RdRp gene. The trees were constructed via the maximum likelihood method using the MrBayes approach using the GTR + G (A,B) or GTR + I + G (C) nucleotide substitution model. Red letters represent pestiviruses isolated from M1/B1 pangolins in this study. Blue letters represent pestiviruses isolated from Amblyomma javanense from Guangdong in the present study.

Figure 4

Standard curve for GDPV detection using single-plex qRT-PCR. Copies number/μl of GDPV from 1 × 10⁸ to 1 × 10² copies/μl.