key: cord-0991768-refl7e32
authors: McIver, David J.; Silithammavong, Soubanh; Theppangna, Watthana; Gillis, Amethyst; Douangngeun, Bounlom; Khammavong, Kongsy; Singhalath, Sinpakone; Duong, Veasna; Buchy, Philippe; Olson, Sarah H.; Keatts, Lucy; Fine, Amanda E.; Greatorex, Zoe; Gilbert, Martin; LeBreton, Matthew; Saylors, Karen; Joly, Damien O.; Rubin, Edward M.; Lange, Christian E.
title: Coronavirus surveillance of wildlife in the Lao People’s Democratic Republic detects viral RNA in rodents
date: 2020-04-23
journal: bioRxiv
DOI: 10.1101/2020.04.22.056218
sha: fe87425ba99e7acdd576263cbc2533f53c1929a6
doc_id: 991768
cord_uid: refl7e32

Coronaviruses can become zoonotic as in the case of COVID-19, and hunting, sale, and consumption of wild animals in Southeast Asia facilitates an increased risk for such incidents. We sampled and tested rodents (851) and other mammals, and found Betacoronavirus RNA in 12 rodents. The sequences belong to two separate genetic clusters, and relate closely to known rodent coronaviruses detected in the region, and distantly to human coronaviruses OC43 and HKU1. Considering close human-wildlife contact with many species in and beyond the region, a better understanding of virus diversity is urgently needed for the mitigation of future risks.

The latest coronavirus (CoV) outbreak in humans, caused by the SARS-CoV-2 virus [1] , originated in Wuhan, Hubei Province, in the People's Republic of China in late 2019. The suspected index case contracted the virus at a local seafood and wildlife market in the city, yet the exact species of animal that hosted the virus remains unknown. Phylogenetic analysis of the SARS-CoV-2 genome indicates a strong likelihood that the reservoir species is a bat, as in the case of the related Betacoronaviruses SARS-CoV-1 and MERS-CoV [2] . An involvement of another intermediate host between bats and humans in the transmission of SARS-CoV-2 virus remains unknown. Wildlife and bushmeat markets are common across Southeast Asia, and represent a significant risk for the transfer of zoonotic pathogens between wildlife and humans. Indeed, 75% of all emerging infectious diseases in the past decades have their origin in wildlife, including highly pathogenic influenza viruses (H5N1), Ebolaviruses, Henipaviruses, Hantaviruses among others [3] .

The CoVs most closely related to SARS-CoV-2 were isolated from bats living in Yunnan province, in the south of China, not far from the 423km long border with the landlocked Lao People's Democratic Republic (Laos) [4] . Both countries were involved in the United States Agency for International Development's (USAID) Emerging Pandemic Threats PREDICT program, and surveillance of bats in wildlife markets in rural areas in Laos unveiled CoV RNA in 41 animals using family-level PCR assays [5] . In addition to bats, rodents are recognized as significant hosts of viral zoonoses, and represent an important potential host for zoonotic viral spill over in Laos, through their frequent incidental and intentional interaction with humans [6, 7] .

Multiple groups in Laos are at high risk of zoonotic viral spillover from wildlife, including from rodents, due to their occupation, economic or geographic circumstances. People contact rodents incidentally and intentionally in various ways. In traditional-style homes, especially in rural areas, rodents are often able to easily enter the houses in search of food and shelter. These 4 circumstances promote incidental contact with rodent urine and feces during everyday life, when household members clean their houses. Rodents also commonly raid food storage areas, including rice storage huts near paddies. In terms of more direct and intentional contact, some species of rodents, including the Indian giant flying squirrel (Petaurista philippensis), Finlayson's squirrel (Callosciurus finlaysonii), red-cheeked flying squirrel (Hylopetes spadiceus), and others, are hunted or trapped in rural forested areas using traps, guns, sticks, or other implements.

Designated for food or for medicinal purposes, depending on the species, the rodents are consumed within the hunter's village, or enter the value chain to reach markets. The value chain involves a series of intermediaries that transit animals from small villages to progressively larger populated areas. At the market, animals are sold to locals or to Lao people visiting from other areas of the country, and often to foreign visitors from neighbouring Thailand, China, and Vietnam [8, 9] . Even though the sales of these animals are illegal, Laos attracts many wildlife trade tourists simply because wildlife products are more available. Throughout this value chain, people are exposed to blood, viscera, feces, and saliva of rodents, and can be further exposed to these materials during the butchering process, where butchers can accidentally cut themselves with knives, allowing for efficient transmission of viruses from rodents to humans [9] . Considering the significant interactions of wildlife and especially rodents with humans in Laos, we were interested in investigating the presence of CoVs in these animals, which can be primary or intermediate as forward primers, while the reverse primer was again CoV-RVS3 [11] . Both versions amplify 387nt between the primer binding sites. CoV RNA positive samples were subjected to Cytochrome b PCR to verify the host species. The primers CytB_F (GAG GMC AAA TAT CAT TCT GAG G) and CytB_R (TAG GGC VAG GAC TCC TCC TAG T) were used to amplify a primerflanked 435nt fragment of the highly conserved mitochondrial gene [12] .

PCR products were subjected to gel electrophoresis on a 1.5% agarose gel and products of the expected amplicon sizes were excised. DNA was extracted using the Qiagen QIAquick Gel Extraction Kit and was sent for commercial Sanger sequencing (1st BASE). All results from sequencing were analyzed in the Geneious 7.1 software, primer trimmed, and consensus sequences compared to the GenBank database (BLAST N, NCBI).

All sequences were deposited in GenBank under submission numbers MT083286, MT083287, MT083291-MT083296, MT083363-MT083365 and MT083405.

Maximum likelihood phylogenetic trees were constructed including different genera (Alpha, Beta and Gamma) and species of known CoVs as well as species/sub-species detected in Laos during the PREDICT project. Only a single sequence was included for isolates with nucleotide identities of more than 95%. Multiple sequence alignments were made in Geneious (version 11.1.3, MUSCLE Alignment), and regions supported by less than 50% of the sequences were excluded.

Bayesian phylogeny of the polymerase gene fragment was inferred using MrBayes (version 3.2) with the following parameters: Datatype=DNA, Nucmodel=4by4, Nst=1, Coavion=No, # States=4, Rates=Equal, 2 runs, 4 chains of 1,000,000 generations. The sequence of a whale Gammacoronavirus served as outgroup to root the trees, and trees were sampled after every 1,000 steps during the process to monitor phylogenetic convergence [13] . The average standard deviation of split frequencies was below 0.0074 for the Watanabe PCR based analysis and below 0.0054 for the Quan PCR based analysis (MrBayes recommended final average <0.01). The first 10% of the trees were discarded and the remaining ones combined using TreeAnnotator (version 2.5.1; http://beast.bio.ed.ac.uk) and displayed with FIGTREE (1.4.4; http://tree.bio.ed.ac.uk/) [14] . CoV RNA was detected in 12 rodents, which corresponds to 1.4% of sampled rodents. All 12 of them were sampled in the south of Laos, and all but one were oral swab samples (Figure 1 Earlier, we noted a relatively high number of diverse CoVs detected in various species of bats all over Laos (Supplement 2 & 3) [5] . This corresponds to similar findings in other countries with a tropical climate, and a hypothesis has been suggested that bats may be serving as a seeding host for zoonotic CoV infections [15, 16] . The 1.4% prevalence of CoV RNA in rodents was much lower than what had been detected in bats in Laos, however such has been observed repeatedly, re-emphasizing the role of bats as a primary CoV source [5, 15, [16] [17] [18] . It is worth noting, that we targeted rodents most likely to be in contact with humans and transmit virus, and did find fewer CoV RNA positive animals than other studies of rodents in the region or elsewhere. A variety of factors may explain the lower incidence of corona virus positive animals in this study including 8 the sample types tested. Studies where CoV RNA was more frequently detected among rodents have used intestine or fecal matter for their studies, while we tested oral and rectal swab samples [18] [19] [20] . We employed swab sampling to minimize harm to live animals, and to avoid any damage to rodents possessed by hunters or market vendors during sampling. Organ collection was rarely feasible, even from dead rodents in market settings, since size and mass of the animal are used to determine the selling price. While potentially underestimating the actual CoV circulation, swab sampling has the advantage of being minimally invasive, quick, and applicable to all species.

The 12 rodent CoV sequences we found fall into two clusters, with 11 of them differing by only one nucleotide. Therefore, these 11 likely belong to the same strain that may have been circulating at that time, since they were obtained from rodents in the same southern region of Laos during December 2016 (Table 1) 

Betacoronaviruses previously detected in rodents in the region (Figure 2, Table 1 ). This suggests that the viruses had a longer evolutionary history within rodent hosts and probably did not derive from a recent cross species transmission event.

None of the CoVs detected in Laos wildlife, neither the ones described earlier in bats nor the ones described here in rodents, have a very close connection to CoVs currently known to cause human disease. The rodent coronaviruses do fall into the same cluster as human coronaviruses OC43 and HKU1 though, which are believed to be derived from a direct or indirect spill-over of rodent viruses to humans [21] . However, we still do not know enough about the molecular mechanisms and drivers of zoonotic events to determine risk or lack of risk with certainty. We conclude that Laos' wildlife does harbor diverse CoVs, and that a potential for interspecies transmission of viruses and novel diseases exists. Human contact with wildlife like bats and rodents is common throughout the country, with many rural households consuming bushmeat as a main source of protein and utilizing it as a trade commodity, this risk potential is particularly relevant. Therefore, behavioral risk reduction, vigilance, ongoing surveillance and research are important to help mitigate the risks of coronavirus zoonotic disease emergence and transmission in the region, especially in the aftermath of COVID-19.

The authors would like to thank: The government of Laos for the permission to conduct this study; 

Animal capture and specimen collection was approved by the Institutional Animal Care and Use Committee (IACUC, UC Davis) and the Ministry of Agriculture and Forestry of Lao PDR. Philippe

Buchy is currently an employee of GSK vaccines. Geographical map indicating all sampling sites within Laos (orange dots), highlighting the locations where coronaviruses in rodents were detected.

Maximum likelihood phylogenetic tree of coronaviruses presented as a proportional cladogram, based on the RdRp region targeted by the PCR by Watanabe et. al. [11] . The tree includes the sequences detected here (red boxes) and those described previously in Laos (grey boxes) and

indicates the number of isolates with less than 5% difference in brackets for isolates. GenBank accession numbers are listed for published sequences from outside of Laos, while sequences obtained during the PREDICT project are identified by cluster names (compare Table 1 

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