key: cord-0289631-uxogz5zd
authors: He, Wan-Ting; Hou, Xin; Zhao, Jin; Sun, Jiumeng; He, Haijian; Si, Wei; Wang, Jing; Jiang, Zhiwen; Yan, Ziqing; Xing, Gang; Lu, Meng; Suchard, Marc A.; Ji, Xiang; Gong, Wenjie; He, Biao; Li, Jun; Lemey, Philippe; Guo, Deyin; Tu, Changchun; Holmes, Edward C.; Shi, Mang; Su, Shuo
title: Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens
date: 2021-11-12
journal: bioRxiv
DOI: 10.1101/2021.11.10.467646
sha: 1d93b9fb4da6c0ef97603d6e3e711e771c10b634
doc_id: 289631
cord_uid: uxogz5zd

Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence. Highlights 1725 game animals from five mammalian orders were surveyed for viruses 71 mammalian viruses were discovered, 18 with a potential risk to humans Civets harbored the highest number of potential ‘high risk’ viruses A species jump of an alphacoronavirus from bats to a civet was identified H9N2 influenza virus was detected in a civet and an Asian badger Humans viruses were also identified in game animals

transcriptomic analysis of 1725 game animals, representing 16 species and five 48 mammalian orders, sampled across China. From this we identified 71 mammalian 49 viruses, with 45 described for the first time. Eighteen viruses were considered as 50 potentially high risk to humans and domestic animals. Civets (Paguma larvata) 51 carried the highest number of potentially high risk viruses. We identified the 52 transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species 53 jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We Introduction 59 Mammalian game animals are wildlife or semi-wild animals that are commonly 60 traded and consumed as exotic food in China and other Asian countries 1-3 .They 61 include rodents (such as porcupines, bamboo rats and marmots), carnivores (such as 62 civets, badgers and foxes), pangolins, hedgehogs and rabbits. These animals are 63 normally either caught and raised locally or imported illegally from neighboring 64 countries before being transferred to live animal (or "wet") markets for trading 1,3-7 , 65 and in recent decades there has been a major expansion in commercial wildlife 66 farming operations and their species diversity. The Huanan Seafood Wholesale 67 Market in Wuhan, to which many of the early COVID-19 cases were linked 8 , is a 68 notable example of a live animal market. Poor hygiene conditions and close contact 69 between animals and humans, as well as a wide mix of species within live animal 70 markets and the restaurants they serve, make them ideal breeding ground for 71 emerging infectious diseases. 72 Unsurprisingly, consuming, capturing, processing and/or trading game animals has 73 been linked to several important infectious disease outbreaks with grave public health 74 consequences. Early cases of both SARS-CoV and SARS-CoV-2 were identified in 75 animal handlers at animal markets in Guangdong 9 and Hubei provinces 8 , respectively, 76 and close relatives of SARS-CoV and SARS-CoV-2 have been identified in civets 10 , 77 raccoon dogs 10 , and pangolins [11] [12] [13] . These are the most popular exotic game animals 78 and subject to frequent trading and human consumption 14 , although their role as direct 79 hosts for the transmission of these viruses to humans remains to be confirmed. In an 80 analogous manner, Human immunodeficiency virus type I (HIV), the causative agent 81 of AIDS, likely originated from the hunting or handing of carcasses of common 82 chimpanzees in central-west Africa 15-17 . 83 As game animals are frequently associated with important human diseases, it is of 84 obvious importance to identify existing or potential pathogens within these species so 85 they can be used to trace the origins of specific epidemics and provide a risk game animal species including civets, pangolins, marmots, and badgers. Initially, 89 these studies utilized virus isolation, consensus PCR and Sanger sequencing 90 approaches 10,18-23 , while in recent years more attention has been directed toward 91 metagenomic next-generation sequencing (mNGS) [24] [25] [26] [27] . In addition to SARS-like 92 viruses, these studies have discovered several infectious agents that are of direct 93 importance for human infection, such as Rotavirus A from civets and raccoon 94 dogs 28, 29 and Hepatitis E virus in wild boar 30 . Both of these viruses are known to 95 infect humans along with a wide range of mammalian hosts [31] [32] [33] . Despite this, there 96 have been few systematic investigations of the virome in game animals, especially in 97 China where their consumption is commonplace. For example, one study identified a 98 virus commonly associated with pneumonia in rodents -Sendai virusin pangolins, 99 suggesting the epizootic potential of viruses in game animals 24 . 100 Since the emergence of SARS-CoV in 2002 China has been the focus of intense 101 viral surveillance in wildlife animals, and a number of SARS-CoV-2 related viruses 102 have been discovered in bats 34, 35 . However, with the exception of pangolins, there has 103 been little investigation of game animals, even though they have close contact with 104 humans and domestic animals and hence provide a link to other wildlife species. To 105 help fill this gap we performed a systematic meta-transcriptomic (i.e. total RNA 106 sequencing) virome investigation of 16 species of game animals representing five 107 mammalian orders collected across China. Many of the species were investigated for 108 the first time using a metagenomic framework. Our purpose was to reveal the 109 diversity and abundance of vertebrate-associated viruses in these game animals and 110 assess which species have the greatest potential for carrying viruses that could 111 eventually emerge in human populations. Tobaniviridae, Hepeviridae and Birnaviridae ( Figure 2B ). Among these, viruses of 151 the Picornaviridae, Astroviridae and Flaviviridae were the most commonly detected 152 and showed relative high abundance and prevalence in many animal species, whereas 153 other viral families were more sporadically detected ( Figure 2C ). For example, 154 coronaviruses were identified in bamboo rats, civets and hedgehogs, influenza viruses 155 were identified in civets and Asian badgers, caliciviruses were identified in bamboo 156 rats, rabbits, civets and Asian badgers, and the genera Orthorubulavirus 157 (Paramyxoviridae) and Pestivirus (Flaviviridae) were mainly identified in pangolins 158 ( Figure 2C ). Importantly, no viruses closely related to either SARS-CoV or SARS-159 CoV-2 (or other sarbecoviruses) were detected in any of animals examined.

Evolutionary history of vertebrate-associated viruses 162 Phylogenetic analyses of the viruses identified here revealed that many had close 163 evolutionary relationship (>80% nucleotide sequence identity) to virus species known 164 to cause disease in other wildlife species, domestic animals or even humans, thereby 165 greatly expanding their host range (Figure 3 and 4, Figure S1 ). reads per million total reads (RPM)). 174 We similarly identified cross-species transmission among animal viruses ( 

Dominant subtype switch in avian influenza viruses during 2016-596 2019 in China

Evolution and Prevalence of H5N6 Avian Influenza 599 Viruses in China

Poultry carrying H9N2 act as incubators for 602 novel human avian influenza viruses

Genetics, receptor binding property, and transmissibility in 605 mammals of naturally isolated H9N2 Avian Influenza viruses

Influenza B virus in seals

Domestic pigs are susceptible to infection with influenza B 611 viruses

Animals as Reservoir for 613

A new species of bat of the Hipposideros 615 bicolor group (Chiroptera: Hipposideridae) from Central Laos, with evidence 616 of convergent evolution with Sundaic taxa

MEGAHIT: an ultra-619 fast single-node solution for large and complex metagenomics assembly via 620 succinct de Bruijn graph

Sensitive protein 623 alignments at tree-of-life scale using DIAMOND

Macintosh sequence analysis software. DNAStar's LaserGene. 625 Molecular biotechnology

Geneious Basic: an integrated and extendable desktop 627 software platform for the organization and analysis of sequence data

Fast gapped-read alignment with Bowtie 2. 630

MAFFT multiple sequence alignment software 632 version 7: improvements in performance and usability. Molecular biology and 633 evolution

trimAl: a tool for 635 automated alignment trimming in large-scale phylogenetic analyses

A simple, fast, and accurate algorithm to estimate 639 large phylogenies by maximum likelihood

Mammalian orthoreovirus 2|Pig|USA|4560-2|2014 Mammalian orthoreovirus 2|Pig|USA|4560-1|2014 Mammalian orthoreovirus|Pig|China|MY01|2018

Mammalian orthoreovirus 3|Chamois|Italy|84407|2009 Mammalian orthoreovirus 2|NA|China|BYD1|2006 Pangolin orthoreovirus|Pangolin|China|ZJ-NAF2|2020

Mammalian orthoreovirus 3|Civet cat|China|MPC-04|2004

Mammalian orthoreovirus 2|Rat|Hungary|47Ma|2006

Mammalian orthoreovirus 2|Rat|Germany|TRALAU2004|2004

Mammalian orthoreovirus 3|Human|USA|Dearing|2002 Mammalian orthoreovirus 3|NA|Canada|Abney|2010 Porcine reovirus|Porcine|China|SHR-A|2011 Mammalian orthoreovirus 2|Swine|Italy|90178-3|2018 Mammalian orthoreovirus 3|Swine|Italy|52154-4|2016 Mammalian orthoreovirus 3|Swine|Italy|224660-4|2015 Mammalian orthoreovirus 2|Pig|China|sR1677|2015

Mammalian orthoreovirus 2|Pig|China|sR1590|2015 Mammalian orthoreovirus 2|Pig|USA|66848|2005 Mammalian orthoreovirus 3|Pig|USA|4476|2014

Mammalian orthoreovirus 2|Pig|USA|32755|2014

Mammalian orthoreovirus 1|Pig|USA|4543-1|2014

Mammalian orthoreovirus 3|Swine|USA|FS-03|2014

Mammalian orthoreovirus 3|Swine|USA|BM-100|2014

C B|Human|Florida|74|2019 B|Human|New York|15|2019 B|Human|Hulunbuir|1901|2019 B|Human|Hawaii|07|2019 B|Bamboo rat|China|ZJ-NA-1|2020 B|Human|Oklahoma|03|2020 B|Human|England|8092|2019 B|Human|India|Pun-1922338|2019 B|Human|Hebei|16275B|2018

B|Human|Yokosuka|NHRC OID FDX70676|2019 B|Swine|Hong Kong|2850|2016 B|Human|Boston|YGB 01039|2013 B|Human|Philippines|19|2013 B|Swine|Tainan|77-5|2014 B|Human|Guangdong Duanzhou|1766|2010 B|Human|Sao Paulo|09-218|2011 B|Human|Bolivia|1495|2010 B|Human|Japan|312|2008 B|Human|Yamagata|115|2003 B|Human|Beijing|243|97 B|Human|Victoria|02|1987 B|Human|Hong Kong|8|73 B|Human|Lee|NA|1940 B|Human|Brazil|241|96 B|Seal|Netherlands|1|99 B|Embryonated hens eggs|Wuhan|356|2000 B|Human|Malaysia|53|2005 B|Human|Egypt|2040|2004 B|Human|Taiwan|13|2004 B|Human|Xinjiang Tulufan|52|2008 B|Human|Guangxi Nanning

PPIV|Pangolin|China|ZJ-MO3-1|2019 HPIV2|Human|USA|0072|2018 HPIV2|Human|Croatia|1779|2015 HPIV2|Human|Croatia|2400|2016 HPIV2|Human|Netherlands|t146a293|2014 HPIV2|Human|USA|SC9949|2018 HPIV2|Human|USA|ACRI 0230|2017 HPIV2|Human|Croatia|1688|2014 HPIV2|Human|Croatia|1849|2015 HPIV2|Human|Argentina|ARG-001|2017 HPIV2|Human|USA|12A2|2016 HPIV2|Human|Croatia|2513|2016 HPIV2|Human|USA|2885|2019 HPIV2|Human|USA|CHLA23|2016 HPIV2|Huamn|USA|V94|2002 HPIV2|Human|USA|9B6|2016 HPIV2|Human|USA|ACRI 0185|2016 HPIV2|Human|USA|GREER|2002 HPIV2|Huamn|USA|V98|2002 HPIV2|Human|USA|9A5|2016 HPIV2|Human|USA|CHLA26|2016

Rabbit rotavirus A|Rabbit|China|JS-TNA1|2020 Rabbit rotavirus A|Rabbit|China|JS-NATF2N|2020 Lapine rotavirus|Lapine|China|N5|1992

Equine rotavirus A G3P3|Horse|Argentina|E3198|2008 Rotavirus A G3P10|Bat|China|MYAS33|2013 Rotavirus A G3P9|Human|Japan|JPNAU1115|1986 Rotavirus A G3P3|Human|Italy|PA260-97|1997

Rotavirus A G12P9|Human|Brazil|PE18974|2010 Rotavirus A G3P3|Dog|Thailand|CU20139|2017 Rotavirus A G3P9|Human|Thailand|CU365-KK|2008

Feline rotavirus G3P3|Cat|Australia|Cat97|1984 Rotavirus A G3P9|Human|China|L621|2006 Rotavirus A G3P3|Bat|China|LZHP2|2015 Rotavirus A G3P3|Bat|China|MSLH14|2011 Rotavirus A G3P3|Rat|Germany|KS-11-573|2011 Rotavirus A G8P1|Goat|Argentina|0040|2011 Bovine rotavirus G8P11|Cattle|Japan|Sun9|2008 Rotavirus A G8P14|Roe deer|Slovenia|D110-15|2015 Bovine rotavirus A G6P11|Yak|China|HY-1|2017

Simian rotavirus A G5PX|Monkey|Saint Kitts and Nevis|08979|2015 Bovine rotavirus G8P7|Bovine|Korea|KJ11|2006 Rotavirus A G4P6|Human|Congo|KisB332|2008 Porcine rotavirus A|Sus scrofa|France|001|2019

Porcine rotavirus A G9P19|Pig|Taiwan|3-20|2015

Rotavirus A G11P25|Human|Nepal|TK2620|2008 Equine rotavirus A G5P9|Horse|UK|H-1|1975

Rotavirus A G9P8|Human|China|km15119|2016 Porcupine rotavirus A|Porcupine|China|HuB-A1|2020

Coypu rotavirus A|Coypu|China|GX-T1|2020 Human rotavirus G12P6|Human|China|Z2761|2019 Porcine rotavirus A G9P23|Pig|China|SCJY-11|2017 Rotavirus A G3P13|Pig|China|SCLS-3|2018 Human rotavirus A G4P6|Human|China|E931|2008 Porcine rotavirus A G6P13|Piglet|India|HP113|2002

PIV5|Pig|South Korea|M129|2017 PIV5|Pig|South Korea|KNU-11|2011 PIV5|Snake|China|HB01|2019 PIV5|Pig|South Korea|T399|2016 PIV5|Canine|China|CC-14|2015 PIV5|Calf|China|PV5-BC14|2014

PIV5|Amur tiger|China PIV5-SR|2015 PIV5|Lesser panda|China|ZJQ-221|2015

A|Chicken|Guizhou|10.28 ZYLJJ015-C|2018 A|Asian badger|Hebei|HeB-F1|2020 A|Asian badger|Hebei|HeB-F2|2020 A|Chicken|Fujian|3.15 FZHX0013-O|2018 A|Chicken|Jiangxi|X1316|2018 A|Accipiter gentilis schvedowi|Tianjin|22|2017 A|Chicken|Heilongjiang|HL2018|2017A|Civet|Guangxi|GX-A8|2020 A|Chicken|Guangdong|LG1|2013 A|Chicken|Anhui|w107|2011A|Turtledove|Guangxi|49B6|2013 A|Chicken|Liaoning|PJXM15|2012A|AnthropoidesA|Chicken|Hong Kong|NT155|2008 A|Pigeon|Guizhou|112|2012 A|Guinea fowl|Hong Kong|NT101|2003 A|Chicken|Beijing|243|2010A|Chicken|Jiangsu|245|2004 A|Chicken|Ningxia|5|1999 A|Chicken|Neimonggu|nk|2002 A|Duck|Shantou|2134|2000 A|Turkey|Germany|R113|1995 A|mallard|Finland|13977|2010 A|Chicken|Hungary|11-459|2001 A|Northern shoveler|California|6535|2009 A|Hong Kong|1073|99A|Chicken|Jordan|11|2003 A|Houbara|United Arab Emirates|78|2006 A|Chicken|Egypt|Elfeil-26|2016