Coronavirus testing indicates transmission risk increases along wildlife supply chains for human consumption in Viet Nam, 2013-2014

Abstract

Outbreaks of emerging coronaviruses in the past two decades and the current pandemic of a novel coronavirus (SARS-CoV-2) that emerged in China highlight the importance of this viral family as a zoonotic public health threat. To gain a better understanding of coronavirus presence and diversity in wildlife at wildlife-human interfaces in three southern provinces in Viet Nam 2013-2014, we used consensus Polymerase Chain Reactions to detect coronavirus sequences. In comparison to previous studies, we observed high proportions of positive samples among field rats (34.0%, 239/702) destined for human consumption and insectivorous bats in guano farms (74.8%, 234/313) adjacent to human dwellings. Most notably among field rats, the odds of coronavirus RNA detection significantly increased along the supply chain from field rats sold by traders (reference group; 20.7% positivity, 39/188) by a factor of 2.2 for field rats sold in large markets (32.0%, 116/363) and 10.0 for field rats sold and served in restaurants (55.6%, 84/151). Coronaviruses were also detected in rodents on the majority of wildlife farms sampled (60.7%, 17/28). These coronaviruses were found in the Malayan porcupines (6.0%, 20/331) and bamboo rats (6.3%, 6/96) that are raised on wildlife farms for human consumption as food. We identified six known coronaviruses in bats and rodents, clustered in three Coronaviridae genera, including the Alpha-, Beta-, and Gammacoronaviruses. Our analysis also suggested either mixing of animal excreta in the environment or interspecies transmission of coronaviruses, as both bat and avian coronaviruses were detected in rodent feces on wildlife farms. The mixing of multiple coronaviruses, and their apparent amplification along the wildlife supply chain into restaurants, suggests maximal risk for end consumers and likely underpins the mechanisms of zoonotic spillover to people.

Fig 1

Slaughtering rats at a large market (left) and a rat vendor stall displaying live rats in cages in a large market (right) in Dong Thap province, October 2013.

Fig 2. Malayan porcupine (Hystrix brachyura) farm in Dong Nai province, November 2013.

Fig 3. Bat guano farms in Soc Trang Province, October 2013.

Fig 4. Map of sampling sites by province and multi-panel plots showing individual counts of animals sampled by province, taxa, and sub-interface (rat trade) or interface.

The color of each bar represents the animal taxonomic group sampled in Dong Nai, Dong Thap, and Soc Trang provinces. Sciuridae and Rattus argentiventer were only sampled one time apiece from wildlife farms. Map was made using geoBoundaries v. 3.0.0 (https://www.geoboundaries.org; [34]) and Open Development Mekong (https://vietnam.opendevelopmentmekong.net) data under a CC BY 4.0 license.

Fig 5. Plot of the proportion of coronavirus positives in field rats by sub-interface in the live field rat trade chain.

Bars show 95% confidence intervals.

Fig 6. Phylogenetic tree of bat and rodent coronavirus sequences detected in Viet Nam.

The analysis is based on 387 bp fragment of the RdRp gene using maximum likelihood with the Tamura 3-parameter model, Gamma distributed (G), and 1000 bootstrap replicates via MEGA7. The analysis included 17 sequences from this study (red from bat hosts, blue from rodent hosts), six sequences (in gray) from a previous study in Viet Nam [26], and 26 reference sequences (in black) available in the GenBank database (S3 Table). The tree was rooted by a strain of Night-heron coronavirus HKU19 (GenBank accession No. NC_016994).

Fig 7

Median-joining networks of bat coronavirus 512/2005 RdRp sequences color-coded according to (A) host and (B) sampling location. Each circle represents a sequence type, and circle size is proportional to the number of animals sharing a sequence type. Numbers on branches indicate the number of mutations between sequence types if it was higher than one. Branches without a specified number of mutations correspond to a single mutation. Circles are colored-coded by animal host: bat (Microchiroptera), rodent (Rattus & Bandicota, Rhizomys, and Hystrix) and sampling location (Dong Thap (blue), Dong Nai (yellow), and Soc Trang (green)). Small black circles represent median vectors (ancestral or unsampled intermediate sequence types).