Trafficked Malayan pangolins contain viral pathogens of humans

The study addresses whether trafficked Malayan pangolins harbor viruses with zoonotic potential and thus pose risks for emergence of human and animal pathogens. Pangolins are heavily trafficked for meat and scales, increasing opportunities for viral transmission. Prior reports identified SARS-CoV-2-related coronaviruses in pangolins and suggested compromised innate immunity due to pseudogenization of interferon epsilon (IFNE). The authors therefore conducted a comprehensive virome analysis of 161 pangolins smuggled into China (2018–2019) to evaluate their role in the emergence and transmission of viral pathogens.

Previous studies detected several viruses (including SARS-CoV-2-related coronaviruses, Sendai virus, canine parvovirus) in small numbers of smuggled or rescued pangolins, but the broader zoonotic risk remained unclear. Over a million pangolins have been poached in the past decade, with extensive illicit trade documented by WWF. Pangolin IFNE, key in mucosal antiviral defense in placental mammals, is pseudogenized, potentially increasing susceptibility to infections. These findings motivate a systematic, large-cohort virome characterization to assess pangolins’ roles in viral ecology and spillover.

• Samples: Archived tissues (muscle, lung, intestine, liver, spleen, heart, kidney) from 161 pangolins confiscated by Guangxi Customs (2018–2019). Tissues from each animal were pooled per individual. Species ID was inferred from mitochondrial contigs; all were Manis javanica. All 161 tested negative for SARS-CoV-2-related coronaviruses by specific RT-PCR.
• Library prep and sequencing: Homogenized pooled tissues filtered (0.45 µm). Total RNA extracted (Roche High Pure viral RNA kit), microbial nucleic acids enriched (BGI Nucleic Acid Microbes kit). cDNA synthesized (Takara PrimeScript), libraries built (QIAGEN QIAseq FX). Sequencing on MGISEQ-2000 (2×150 bp); controls (sterile water, reagent mix) co-sequenced to detect contaminants. Additional libraries prepared with NEBNext Ultra II Directional RNA kit and sequenced on Illumina NovaSeq 6000 (2×150 bp).
• Read processing and assembly: Adapters/low-quality bases removed with fastp v0.21.0. De novo assembly by MEGAHIT v1.2.9. Contigs compared to NCBI NR by DIAMOND blastx (E<1e-5); viral candidates assigned by top-hit taxonomy, then filtered by blastn to exclude host/endogenous/vector sequences. Viral genomes validated by read mapping and annotated in Geneious 2021.2.2. Species assignments followed ICTV demarcation criteria; where absent, <90% aa identity in RdRp (RNA viruses) or conserved replication proteins (DNA viruses) indicated a new species.
• Abundance estimation: Reads filtered against SILVA rRNA (v138.1) and pangolin genomes (GCF_014570555.1; GCF_014570535.1) using Bowtie2 v2.3.4.2. Family-level abundance via DIAMOND blastx (E<1e-5) and RPM normalization; species-level abundance by mapping reads to assembled viral contigs (RPM). Detection thresholds: RPM ≥1 and ≥10 reads. Controlled for index hopping using platform-specific rates (threshold 0.1%). Shannon diversity index calculated with vegan v2.5-7.
• Confirmation: Virus-specific RT-PCR on original samples with primers designed from assembled sequences (primer list provided), followed by Sanger sequencing of amplicons. For Mammalian orthoreovirus, RT-PCR filled gaps in L1 (RdRp).
• Phylogenetics: Alignments with MAFFT v7.475; trimming with TrimAl v1.4; maximum-likelihood trees with PhyML v3.1 (LG for aa; GTR+Γ for nt; 1,000 bootstraps) and Bayesian inference with MrBayes v3.2.7 (10M generations) to confirm topology. Similarity and sliding window analyses with SimPlot v3.5.1.
• Pangolin population groups: Mitochondrial variants called via GATK4 pipeline; consensus sequences built (depth≥3, Q≥30, genotype rate >0.9). Included 12 reference pangolins with known origins. Phylogeny with IQ-TREE v1.6.10; groups inferred and used to compare viral diversity.

• Sequencing output and detection: 1.9×10^10 reads generated; 12.7 million viral reads (0.07%). Viral reads spanned 42 families. After excluding likely diet/gut-associated and non-vertebrate viruses, 28 vertebrate-associated viruses were identified; 21 were previously unreported in vertebrates. Ninety-four complete/nearly complete genomes representing 24 virus species were obtained; identities confirmed by RT-PCR and Sanger sequencing.
• Pangolin-associated viruses (16 species):
  • Hunnivirus (Picornaviridae): Five pangolin hunniviruses (BIME1–5), with RdRp aa identities 62.3–69.4% to closest rat hunnivirus. Ten sequences formed a distinct clade. Seven near-complete genomes shared 73.0–77.0% nt identity among themselves and 60.5–61.7% to the closest rat strain. All pangolin hunniviruses lacked the L-protein; large deletions (82–85 aa) observed in 2B protein, suggesting adaptation to pangolins and potential effects on immune evasion and pathogenicity.
  • Pestivirus (Flaviviridae): Nine distinct pestivirus species (BIME1–9) clustered with Dongyang pangolin virus, with RdRp aa identities 69.6–88.8% among themselves. Whole genomes assembled from 22 individuals, indicating high prevalence. Protein conservation varied: capsid 64.9–76.6% aa identity; p7 highly divergent (20–30%) versus porcine pestivirus.
  • Copiparvovirus (Parvoviridae): Two new species detected in eight pangolins; six full genomes showed 80.4–81.4% identity among themselves and 49.5–50.4% to known copiparvoviruses.
• Human-associated viruses in pangolins:
  • Respiratory syncytial virus A (RSV-A; Orthopneumovirus): Detected in two samples. Pangolin RSV-A genomes were identical (100% nt), 99.98–100% to previously reported pangolin RSV-As, and 99.40–99.64% to the closest human RSV-A (Australia, 2017), suggesting recent human-to-pangolin transmission or pangolin-to-pangolin transmission. A divergent Orthopneumovirus (BIME1) shared only 65.6% RdRp aa identity with RSV-A.
  • Rotavirus A (Reoviridae): Found in seven samples. Pangolin sequences formed two clusters with near-identical sequences within clusters, consistent with shared source (same cage/market). Among 11 segments, within-segment identities were 93.37–100% nt; to known strains, 52.0–87.7%. Five segments (VP2, VP4, NSP1, VP6, NSP5/NSP6) represented new genotypes.
  • Mammalian orthoreovirus: Pangolin strain clustered with a bat strain (Plecotus auritus, Germany) at 97.7% nt identity and close to a human strain (Switzerland). Sliding-window and segment phylogenies indicated reassortment with strains from civet, pig, bat, and tree shrew across segments L1/L2/S4 (bat), M3/S2 (civet/pig), and M2/S3 (tree shrew), highlighting interspecies transmission.
• Other mammal-associated viruses:
  • Coronavirus related to HKU4 (Merbecovirus): Pangolin-CoV-HKU4-P251T showed 96.7% RdRp aa identity with Tylonycteris-bat-CoV-HKU4 and sat basally to HKU4 clade. Pangolin and bat HKU4 shared 4/10 MERS-CoV RBD key residues; pangolin DPP4 is more similar to human DPP4 (89.0–89.3% aa) than bat DPP4 (82.4–83.2%), suggesting potential human receptor usage risk.
  • Shanbavirus (Picornaviridae): Novel pangolin species most closely related to bat shanbavirus (62.2% RdRp aa identity).
  • Respirovirus (Paramyxoviridae): Four sequences clustered with Sendai virus and a known pangolin respirovirus; genome identities 93.1% (pangolin respirovirus) and 89.2% (Sendai virus).
  • Chaphamaparvovirus (Parvoviridae): New species (Pangolin chaphamaparvovirus BIME1) with 65.9% capsid aa identity to Rat parvovirus 2.
  • Protoparvovirus (Parvoviridae): Whole genomes in 23 pangolins were >99% identical to canine parvovirus from dogs in China.
• Tick-associated viruses:
  • Phlebovirus (Phenuiviridae): Two pangolins harbored phleboviruses whose L/M/S segments had 98.8–99.2% mutual nt identity and 63.2–74.0% to tick-associated Japanese strains.
  • Orthonairovirus (Nairoviridae): Pangolin orthonairovirus BIME1 clustered with Wenzhou tick and Songling viruses (associated with human febrile illness); L/M/S segment identities to Wenzhou tick virus were 67.5%, 61.1%, 64.4%.
  • Coltivirus (Reoviridae): Lishui pangolin virus sequences clustered with prior pangolin and Australian Ixodes holocyclus tick viruses.
• Population groups and diversity: Mitochondrial phylogeny grouped pangolins into five populations; groups 1–3 aligned with SE Asian islands, group 4 with inland Asia, group 5 unknown. Viral profiles showed high prevalence of pangolin-associated Pestivirus (46.0%) and Copiparvovirus (24.6%), and mammal-associated Protoparvovirus (23.0%). Group 3 exhibited higher viral diversity than group 1 (Wilcoxon P=0.006). No significant diversity difference between island (groups 1–3) and inland (group 4) groups.
• SARS-CoV-2 testing: All 161 pangolins were negative for SARS-CoV-2-related coronaviruses by RT-PCR.

This large-scale meta-transcriptomic survey demonstrates that smuggled Malayan pangolins carry a diverse set of vertebrate-associated viruses, including previously undescribed pangolin-adapted lineages and viruses closely related to known human and livestock pathogens. The consistent loss of the L-protein across pangolin hunniviruses and deletions in 2B suggest evolutionary adaptation to an IFNE-deficient host, supporting pangolins as reservoirs for certain viruses. Detection of human-associated viruses (RSV-A, Rotavirus A, Mammalian orthoreovirus) with high sequence identity to human strains indicates recent cross-species transmission, plausibly facilitated by cramped conditions during illegal trade. The identification of an HKU4-related coronavirus with RBD similarity to human DPP4 raises concern for potential receptor usage and spillover. Evidence of reassortment in orthoreoviruses across multiple hosts underscores complex multi-host transmission networks. Overall, the findings highlight pangolins’ potential role in the emergence of novel pathogens and underscore the public health risks associated with wildlife trafficking and wet markets.

The study provides a comprehensive virome characterization of 161 trafficked Malayan pangolins, uncovering 28 vertebrate-associated viruses (21 novel to vertebrates), including 16 pangolin-associated viruses and multiple human-, mammal-, and tick-associated viruses. Genomic features (e.g., loss of L-protein in hunniviruses) suggest host adaptation, while the presence of human and livestock viruses indicates frequent cross-species transmission under trafficking conditions. These results emphasize pangolins’ importance in public and veterinary health and the need to prohibit trade and consumption, and to implement routine surveillance of confiscated wildlife. Future research should: (1) determine organ/tissue tropism and pathogenicity; (2) isolate and functionally characterize novel viruses (especially HKU4-related CoV RBD usage); (3) resolve unclassified viral contigs; and (4) trace infection sources and transmission routes via ecological and trade-route data.

• Pooled tissues per individual preclude assessment of organ-specific viral distribution.
• Some assembled viral contigs remain unclassified; additional work is needed to confirm novel taxa.
• Only archived samples were available; lack of ecological and trade-route data prevents determination of infection timing, sources, and transmission pathways.
• Species identification relied on mitochondrial contigs rather than morphology due to sample constraints.