Serological evidence of sarbecovirus exposure along Sunda pangolin trafficking pathways

Abstract

Background: Early in the coronavirus disease 2019 (COVID-19) pandemic, Sunda pangolins (Manis javanica) involved in the illegal wildlife trade in mainland China were identified as hosts of severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). Although it is unconfirmed whether pangolins or other traded wildlife served as intermediate hosts for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the trafficking of pangolins presents a clear risk for transmission of viruses with zoonotic and epizootic potential regardless. We have investigated the origins of pangolin carcasses seized in Hong Kong and have evaluated their potential exposure to SARSr-CoVs, other coronaviruses, and paramyxoviruses, aiming to address a gap in our knowledge with regard to the role of wildlife trade in the maintenance and emergence of pathogens with zoonotic and epizootic potential.

Results: Using a combination of virological and wildlife forensics tools, we investigated 89 Sunda pangolin carcasses seized by Hong Kong authorities during anti-smuggling operations in the territory conducted in 2013 (n = 1) and 2018 (n = 88). Swabs, organ tissues, blood, and other body fluids were collected during post-mortem examination. Two enzyme-linked immunosorbent assays (ELISAs), which employ a double-antigen sandwich format, were used to detect antibodies reactive against SARSr-CoVs. One individual was found to be seropositive with support from both methods, while five individuals exhibited a putatively seropositive result from one ELISA method. Polymerase chain reaction (PCR) screening for coronavirus and paramyxovirus ribonucleic acid (RNA) did not yield any positives. Based on genomic data, the seropositive individual was determined to have likely originated from Java, while the putatively seropositive individuals were determined to have originated from populations in Borneo, Java, and Singapore/Sumatra.

Conclusions: While the role of pangolins in the evolution and ecology of SARS-CoV-2 is uncertain, our results suggest susceptibility and potential exposure of pangolins to SARSr-CoVs, occurring naturally or associated with the illegal trafficking of these animals. Complex dynamics between natural populations, traded individuals, and pathogen susceptibility complicate conclusions about the role of pangolins, as well as other host species, in the ecology of SARSr-CoVs and potentially zoonotic viruses with risk of future emergence.

Keywords: Conservation forensics; Coronavirus; ELISA; One Health; Pangolins; Paramyxovirus; Population genomics; SARS-related virus; Sarbecovirus; Serology.

Fig. 1

Three geographic origins of trafficked Sunda pangolins shown by the principal components analysis, STRUCTURE analysis, and fineRADstructure analyses. Analyses were based on 23,261 SNPs of 85 Sunda pangolin individuals from this study and 8 references. Color of each grid from the fineRADstructure plot corresponds to the color scale on the right, where grids towards the purple end of the spectrum show higher estimated coancestry between the two individuals. Individuals marked with a star are references from Nash et al. [32]. Possible subclusters within Borneo are marked with brackets within the fineRADstructure plot

Fig. 2

Serology results for 171 samples from 86 individuals, with all samples tested by the A WANTAI SARS-CoV-2 Ab ELISA (Wantai) targeting antibodies cross-reactive with the spike protein RBD, presenting the mean values for all samples tested in duplicate (except HKU64-PF), and for the B Platelia SARS-CoV Total Ab ELISA (Bio-Rad), targeting antibodies cross-reactive with the nucleocapsid protein (Additional file 3 for raw data from both assays). Dotted line represents seropositivity threshold of absorbance/cut-off value (A/CO) = 1.0 for both assays, while yellow shading indicates borderline seropositivity of 0.9 ~ 1.1 (inclusive) for the Wantai ELISA and represents an equivocal interpretation of 0.8 ~ 1.0 (inclusive) for the Bio-Rad ELISA. One sample was interpreted as seropositive by both assays, while six samples (representing five individuals) were interpreted as putatively seropositive (seropositive by the Wantai ELISA but equivocal or negative by the Bio-Rad ELISA). An additional eight samples were interpreted as seronegative but inconclusive with borderline or approaching-borderline results by the Wantai ELISA and/or equivocal results by the Bio-Rad ELISA (see Table 1)

Fig. 3

Summarized serological and population genomics results show complex relationships between Sunda pangolin trade and origins of pangolin SARSr-CoVs in Southeast Asia. The gray oval indicates unknown trade intermediaries where trafficked pangolins are processed. Red arrows indicate possible points where trafficked pangolins are infected with SARSr-CoVs, thereby entry points of SARSr-CoVs into the trade. Yellow arrows indicate the inferred trade flow of Sunda pangolins analyzed in this study and are proportional to number of individuals found from each population within the seizure under study. Sunda pangolin range data sourced from IUCN [16]. Blue points show results of published literature testing for SARSr-CoVs in pangolin individuals (n = total number of individuals tested), with PCR and serological results separated, wherein positive results are in red and positive (inconclusive) results are in light pink. Years in brackets indicate seizure year. Results from the literature are attributed geographically to seizure locations, while results from this study are shown according to pangolin origins (Singapore/Sumatra, Java, and Borneo). Studies used in this figure are numbered as follows: (a) Peng et al. [12]; (b) Lam et al. [1]; (c) Shi et al. [37]; (d) Lam et al. [1]; (e) Nga et al. [11]; (f) Wacharapluesadee et al. [13]; (g) Lee et al. [49]; and (h) Lee et al. [49]