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Forum

Bat-OriginCoronaviruses ExpandTheir Host Range toPigsLiang Wang,1 Shuo Su,2

Yuhai Bi,1,3 Gary Wong,3 andGeorge F. Gao1,3,4,*

Infections with bat-origin corona-viruses have caused severe illnessin humans by ‘host jump’.Recently, novel bat-origin corona-viruses were found in pigs. Thelarge number of mutations on thereceptor-binding domain allowedthe viruses to infect the new host,posing a potential threat to bothagriculture and public health.

Coronavirus Transmission inWildlifeThe host range expansion of coronavi-ruses (CoVs) from wildlife to humans viagenetic recombination and/or mutationson the receptor-binding domain in thespike (S) gene is well established andresults in several diseases with high fatal-ity rates, such as severe acute respiratorysyndrome (SARS) and Middle East respi-ratory syndrome (MERS) [1,2]. Despitetheir bat origins, SARS-CoV and MERS-CoV have infected humans via an inter-mediate host, rather than through a directinfection from bats. Palm civets havebeen suggested, but not definitively con-firmed, to be an intermediate host forSARS-CoV, and dromedary camels areconfirmed to be the intermediate host forMERS-CoV [3]. Currently, the number ofknown intermediate hosts involved in thetransmission of bat-origin coronavirusesto humans is limited. Compared to manyother species, pigs are in frequent contactwith both humans and other animals suchas cats, dogs, horses, and aquatic birds,and theoretically possess a greater

chance to promote cross-species viraltransmission. For instance, pigs are sus-ceptible to infection with human and avianinfluenza A viruses (IAVs), such as H1N1and H3N2, which then reassort to infecthumans [4]. Thus, pigs are regarded asmixing vessels for IAVs. However, pigswere not known to be susceptible tobat-origin coronaviruses until recently,when two independent groups reportedthe detection of novel swine entericalphacoronaviruses (SeACoVs) distinctfrom known swine coronaviruses (withone group successfully isolating livevirus). The SeACoVs were found to bephylogenetically close to bat coronavirusHKU2 [5,6]. This suggests that bat-origincoronaviruses may have ‘jumped’ thespecies barrier to infect pigs.

The Emergence of Bat-OriginCoronaviruses Infecting PigsIn February 2017, outbreaks of severewatery diarrhea of suckling piglets werereported in commercial pig farms inGuangdong Province, China. The diseasehad high case fatality rates (CFRs, over35% for <10-day-old suckling piglets),and none of the animals were positivefor known pathogens responsible for por-cine diarrhea [6]. Instead, two genomes ofnovel SeACoV were detected in the ill pig-lets by two independent groups, and pre-liminary analysis showed that theSeACoVs possibly originated from batHKU coronaviruses [5,6]. It is currentlyunclear whether SeACoVs had been cir-culating undetected in pigs, if the viruseshad originated from cross-species trans-mission, or if the SeACoVs were the resultof viral recombination. To understand themolecular origin and evolution of SeA-CoVs, we performed a detailed phyloge-netic analysis at the genomic level by usingall known alphacoronaviruses and bat-ori-gin coronaviruses which are known tocause severe diseases, such as SARSand MERS. A total of 224, 312, and 778complete genomes from MERS-CoV,

SARS-CoV, and alphacoronaviruses,respectively, were used. Phylogeny wasreconstructed using conserved regionsin genomes by the maximum likelihoodmethod with 300 replicates (Figure 1A).Consistentwithpreviousstudies,phyloge-netic analysis shows that SeACoVs wereclosely related to the Rhinolophus batcoronavirus HKU2 isolated in southernChina (Figure 1A). SeACoV was found toshare a common ancestor with humancoronavirus 229E/NL63, but these virusesare distant from other known swine alpha-coronaviruses, indicating theirdifferentori-gins. Further analysis on theSgene,whichdetermines virus attachment, host cellentry, and ‘host jump’ of coronaviruses[7], showedthatdomain0 in theS1subunithas structural similarity to that of NL63.The rest of the domain on the S1 subunitwassimilar to thatofmurinehepatitis coro-navirus (betaCov) [6]. There were 11 res-idues of the receptor-binding domain(RBD, also called C-terminal domain,CTD) of SeACoV directly in contact withits receptor (angiotensin-convertingenzyme 2) that were mutated or deleted[7] (Figure1B). Sevenmutations (adeletionof four amino acids, and three substitu-tions) among the 11 sites in SeACoVwerealso found in [81_TD$DIFF]229E/NL63, indicating simi-larities in the receptor-bindingmechanismbetweenSeACoVand [81_TD$DIFF][79_TD$DIFF]229E/NL63.There-fore, SeACoV may be able to infecthumans and should be closely monitored.InfectionofSeACoV inVerocells (aprimatecell line)will provideexperimental evidenceto support this possibility [6].

Bat-Origin Coronaviruses in PigsMay Transmit to HumansAnother requirement for the host rangeexpansion of viruses is physical contactbetween different host species. The novelSeACoVs were both detected in Guang-dong Province, whereas closely relatedbat coronaviruses were also isolated fromGuangdong or Hong Kong (Figure 1A). InGuangdong Province, the high density of

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Host

Human

Bat

Pig

Virus

PRCV/TGEV

Bat coronavirus HKU10

Miniopterus bat coronavirus HKU8

Rhinolophus bat coronavirus HKU2

Human coronavirus 229E

Human coronavirus NL63

Porcine epidemic diarrhea virus

Swine enteric coronavirus

MERS

SARS

(A) (B)

Figure 1. Phylogenetic Overview of Swine Enteric Alphacoronavirus (SeACoV). (A) The phylogenetic relationship among SeACoV, other alphacoronaviruses,SARS-CoV, and MERS-CoV. Conserved genomic regions were used for phylogenetic reconstruction by using the maximum likelihood method under the GTR + Gmodel. The extended majority rules (autoMRE) bootstrapping convergence criterion was applied here to determine the most suitable number of replicates.Bootstrapping convergence was considered to be reached if over 99% permutations have low Weighted Robinson-Foulds distances (<3%). The phylogenetic treewas visualized using SARS-CoV andMERS-CoV as the outgroup. Only bootstrap values>90%were visualized as a purple circle in themiddle of the branch. The size ofthe circle is proportional to the bootstrap value. (B) Alignment of the receptor-binding domain in the S gene. Residues in direct contact with the human receptor for NL63were indicated by a ‘+’ sign. Twenty-five substitutions between bat and pig were indicated by a star. If a substitution in pigs occurred in 229E or NL63, it was marked bya red star. Sequences from bat HKU, 299E, and NL63 were indicated by a different color, consistent with those from the phylogenetic tree.

pig slaughterhouses and the wide distri-bution of bat species (Figure 2A) promotethe possibility of viral cross-species trans-mission. Additionally, bats have a widegeographical distribution in southernChina, with extensive species diversity,unique behaviors (characteristic flight pat-terns, diet, roosting, and mobility) [8], andconstant interactions with both pigs andhumans (Figure 2A).

Could Pigs Be Mixing Vessels forCoronaviruses?Pigs are well established as mixing ves-sels and as intermediate hosts for IAVs,and coronaviruses have already beenshown to possess potential for recombi-nation in animals [9]. Given that pigs are infrequent contact with human and multiplewildlife species, and that pork is one of the

2 Trends in Microbiology, Month Year, Vol. xx, No. yy

most commonly consumedmeats in non-Muslim countries, it is important to assesswhether pigs could be mixing vessels forthe emergence of novel coronaviruseswith high agricultural impact and risksto public health. It has already beenreported that pigs are susceptible toinfection with human SARS-CoV [10]and MERS-CoV [11]. Additionally, theCD26 receptor sequence alignment ofpigs and humans shows 94.5% similarity,which is sufficient for potential cross-spe-cies transmission [7]. In southern China,the unique climate, the high density ofdomestic as well as wild pigs, and exten-sive bat distribution, together with batscarrying large numbers of recombinantnovel coronaviruses [12], could lead tothe emergence of more novel coronavi-ruses in the future.

Concluding RemarksThe isolation of SeACoV from ill pigletsexpands our knowledge of the host rangeof bat-origin coronaviruses, and poten-tially poses a threat to public health.Despite considerable progress in charac-terizing cross-species transmission forcoronaviruses, several areas need to beaddressed, including: (i) whether otherunknown coronaviruses are circulatingin pigs; (ii) whether pigs are mixing vesselsfor coronaviruses; (iii) whether SeACoVinfects humans and causes severe dis-ease; and (iv) whether SeACoV vaccinesshould also be developed to control thespread of this virus in pigs. In-depth epi-demiological investigation and compre-hensive analysis of these novelcoronaviruses should be performed toanswer these urgent questions.

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Xinjiang

Tibet

Qinghai Ningxia

GansuShaanxi

Sichuan

YunnanGuangxi

Hainan

Fujian

Zhejiang

Anhui

Jiangsu

ShandongShanxi

Hebei

Taiwan

Hunan

Hubei

Henan

Liaoning

Tianjin

Heilongjiang

Jilin

Jiangxi

Beijing

ShanghaiChongqing

Guizhou

SARS-like-CoV

MERS-like-CoV

SeACoV MERS-CoV

HKU2-CoV

SARS-CoV

(A)

(B)

PteropodidaeEmballonuridaeHipposideridae

RhinolophidaeMolossidaeVesper�lionidae

<1

1-1515-3030-45

45-60>60No data

Number of slaughtered pigs in 2016 (millions)

Inner Mongolia

Hong Kong

Macau

Guangdong

?

(See figure legend on the bottom of the next page.)

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TIMI 1561 No. of Pages 4

AcknowledgmentsThis work was supported by the National Key

Research and Development Program of China

(2016YFE0205800 and 2017YFD0500101), National

Science and Technology Major Project

(2016ZX10004222-005, 2017ZX10103001-010),

the National Natural Science Foundation of China

(NSFC, 81461168030), the External Cooperation

Program of Chinese Academy of Sciences

(153211KYSB20160001), and the National Natural

Science Foundation of China International Coopera-

tion and Exchange Program (816110193).

G.F.G. is a leading principal investigator of the NSFC

Innovative Research Group (81621091). Y.B. is sup-

ported by the Youth Innovation Promotion Associa-

tion of the Chinese Academy of Sciences (CAS)

(2017122).

1CAS Key Laboratory of Pathogenic Microbiology and

Immunology, Collaborative Innovation Center for

Diagnosis and Treatment of Infectious Disease, Institute

Figure 2. Distribution of Pigs and Bats in China (Coronaviruses. (A) Pig slaughterhouse densities andcoronaviruses. The dashed line indicates potential, bu

4 Trends in Microbiology, Month Year, Vol. xx, No. yy

of Microbiology, Center for Influenza Research and Early-

warning (CASCIRE), Chinese Academy of Sciences,

Beijing 100101, China2MOE Joint International Research Laboratory of Animal

Health and Food Safety, Jiangsu Engineering Laboratory

of Animal Immunology, College of Veterinary Medicine,

Nanjing Agricultural University, Nanjing, China3Shenzhen Key Laboratory of Pathogen and Immunity,

Guangdong Key Laboratory for Diagnosis and Treatment

of Emerging Infectious Diseases, Shenzhen Third

People’s Hospital, Shenzhen 518112, China4National Institute for Viral Disease Control and

Prevention, Chinese Center for Disease Control and

Prevention (China CDC), Beijing 102206, China

*Correspondence: gaof@im.ac.cn (G.F. Gao).

https://doi.org/10.1016/j.tim.2018.03.001

References1. Su, S. et al. (2016) Epidemiology, genetic recombination,

and pathogenesis of coronaviruses. Trends Microbiol. 24,490–502

2. Lu, G.W. et al. (2013) Molecular basis of binding betweennovel human coronavirus MERS-CoV and its receptorCD26. Nature 500, 227–231

Map without the Islands in the South China Sea)the species distribution of bats in China. (B) Suspect

t unknown, transmission from pig to human.

3. de Wit, E. et al. (2016) SARS and MERS: recent insightsinto emerging coronaviruses.Nat. Rev. Microbiol. 14, 523–534

4. Brown, I.H. (2001) The pig as an intermediate host forinfluenza A viruses between birds and humans. Int. Congr.Ser. 1219, 173–178

5. Gong, L. et al. (2017) A new bat-HKU2-like coronavirus inswine, China, 2017. Emerg. Infect. Dis. 23, 1607–1609

6. Pan, Y.F. et al. (2017) Discovery of a novel swine entericalphacoronavirus (SeACoV) in southern China. Vet. Micro-biol. 211, 15–21

7. Lu, G. et al. (2015) Bat-to-human: spike features deter-mining ‘host jump’ of coronaviruses SARS-CoV, MERS-CoV, and beyond. Trends Microbiol. 23, 468–478

8. Calisher, C.H. et al. (2006) Bats: Important reservoir hostsof emerging viruses. Clin. Microbiol. Rev. 19, 531–545

9. Sabir, J.S. et al. (2016) Co-circulation of three camelcoronavirus species and recombination of MERS-CoVsin Saudi Arabia. Science 351, 81–84

10. Chen,W. et al. (2005) SARS-associated coronavirus trans-mitted from human to pig. Emerg. Infect. Dis. 11, 446–448

11. Vergara-Alert, J. et al. (2017) Livestock susceptibility toinfection with Middle East Respiratory Syndrome corona-virus. Emerg. Infect. Dis. 23, 232–240

12. Huang, C.P. et al. (2016) A bat-derived putative cross-family recombinant coronavirus with a reovirus gene. PLoSPathog. 12, e1005883

and Cross-Species Transmission of Bat-Origined routes of cross-species transmission of bat-origin