Here we go again? Emergence of a novel swine enteric alphacoronavirus (SeACov) in Southern China - Dr. Yao-Wei Huang, Zhejiang University, from the 2017 North American PRRS/National Swine Improvement Federation Joint Meeting, December 1‐3, 2017, Chicago, Illinois, USA. More presentations at http://www.swinecast.com/2017-north-american-prrs-nsif-joint-meeting

1. Emergence of a novel swine enteric
alphacoronavirus (SeACoV) in southern China
Yao-Wei Huang1
*, Yongfei Pan2
, Bin Wang1
, Pan Qin1
, Yong-Le
Yang1
, Yanhua Song2
1. Department of Veterinary Medicine, College of Animal Sciences, Zhejiang
University, Hangzhou, China
2. Hog Production Division, Guangdong Wen’s Foodstuffs Group Co, Ltd, Xinxing,
Guangdong, China

2. Outline
1. Background
2. Discovery of SeACoV in southern China (Pan et al., Vet Microbiol, 2017)
3. Update and summary

3. • Large-scale outbreaks of diarrhea with 80-100% morbidity
and 50-90% mortality in suckling piglets in China since Dec
2010
China pig industry has been suffered from outbreaks of
newborn piglet diarrhea since 2010
• Confirmed and potential enteric viruses responsible for the disease:
PEDV, TGEV, PDCoV, porcine torovirus, porcine rotavirus, et al.
1. Background

4. Porcine Coronaviruses: Discovered so far…
• TGEV/PEDV chimeric SeCoV in Europe
(SeCoV)
(SeCoV)

5. The Coronaviruses: Origin and Evolution
PEDV
TGEV
SARS-CoV
MERS-CoV PDCoV

6. Unique genetic
features at the
5’UTR between
emergent PEDV
and a bat CoV
identified in
southern China
(Huang YW et al.,
mBio, 2013)

7. Geographic locations of PEDV-like BtCoV/512 in China
• In southern China:
− Guangdong
− Guangxi
− Hainan
− Jiangxi
− Anhui
− Yunnan

8. Rhinolophus sinicus
中华菊头蝠
− An alphaCoV (Four determined full-length HKU2 sequences: one from
Guangdong, three from Hong Kong)
− Enteric tropism in healthy bats
− Shortest S protein among all CoVs and phylogenetically related to BetaCoV
− Coinfection of HKU2-alphaCoV and SARS-like-betaCoV
Bat CoV HKU2

9. BtCoV HKU2 and the other newly identified rodent
or house shrew CoVs in China harbor the S genes
divergent from AlphaCoV

10. HKU2-like BtCoV identified in
Eptesicus fuscus (big brown bat)
in Maryland, USA

11. HKU2-like BtCoV identified in
Eptesicus fuscus (big brown bat)
in Maryland, USA
A total of 45 sequence reads derived from
454 sequencing of the pool 1 sample was
assembled to the BtCoV-HKU2 genome;
Then a PCR amplification of a 2,540-nt
fragment of ORF1ab of ARCoV was used for
phylogenetic analysis.

12. Part 2:
Discovery of SeACoV in southern China
(Pan et al., Vet Microbiol, 2017)

13. Emergence of a novel swine enteric pathogen in
Guangdong, 2017
− Clinical signs indistinguishable from PEDV
− PEDV, PDCoV, TGEV and PHEV RNA negative
− Other known porcine pathogens negative

14. Detection of a batCoV HKU2-related CoV by a pan-CoV RT-PCR
Moes et al, BMC infectious diseases, 2005.
− Amplification of 251-bp in the ORF1b gene for all CoV members
− All samples 100% identical to nt positions 14024-14274 of bat enteric
AlphaCoV HKU2

15. Isolation of swine enteric alphacoronavirus (SeACoV) in cell culture
CPE IFA with a positive
sow serum
IFA with a negative
sow serum
Virus particle observed by EM:
•100-120 nm in diameter
•Surface projections typical of CoV
100 nm

16. SeACoV did not cross-react with PEDV, TGEV or PDCoV anti-N Ab
Anti-PEDV-N
SeACoV
Anti-TGEV-N
Anti-PDCoV-N
SeACoV
SeACoV
PEDV
TGEV
PDCoV
Update (2-way):
SeACoV antiserum or
anti-N Ab did not cross-
react with PEDV, TGEV
or PDCoV in vitro

17. Genomic cloning of SeACoV/CH/GD-01/2017/P2
ORF1a ORF1b S OR
F
3
M N
5’UTR 3’UTR
NS
7a
***
S1|S2
546 547
SeACoV (27,155 nt)
NTD
19-238 276-546
 6-nt longer (nsp3, S1, M) than HKU2 (27,149 nt)
 94.9% nt identity with HKU2
 75-aa substitutions plus a 2-aa insertion within S-NTD compared to HKU2
 In contrast, only 78 aa substitutions in the rest part of the S protein.

18. AlphaCoV
BetaCo
V
GammaCoV
DeltaCoV
?
AlphaCoV
BetaCo
V
GammaCoV
DeltaCoV
S geneFull-length genome

19. Structure-based S protein sequence alignment

20. TM
N C
S1 subunit S2 subunit
S2 cleavage site
BH HR2 CTCHHR1FPUHDomain B-DDomain 0
19 238 276 546 573-642 672-751 752-839
840-
890
904-
921
1029-
1057
Modeling SeACoV S protein functional domains
AlphaCoV BetaCoV

21. Another report describing the SeACoV sequence (GDS04 strain)
(Gong et al., EID, 2017)
 From the same collected samples in the same farm
 99.8% nt identity (by NGS) with GD-01/2017
 Not isolated
 GD-01/2017 and GDS04 could have the same origin

22. Duodenum
Ileum
Jejunum
SeACoV DMEM control
SeACoV was infectious and pathogenic in piglets in experimental infection
• Sows were CoVs RNA free, and SeACoV serum Ab free
• Two groups: Five 3-day-old piglets per group
• Oral infection:
− SeACoV: 1×105
PFU/ml (3 ml per pig),
− DMEM (3 ml/pig)
• Euthanized at 3 dpi (two) and 5 dpi (three)
• Performed fecal RNA detection per day, and histological
examinations in small intestine (HE staining)
Results:
Observed clinical signs characterized by acute vomiting
and watery diarrhea (NOT severe compared to PEDV
challenge)
Fecal RNA shedding started at 27 to 40 hpi and lasted
Gradual atrophy with significantly reduced VH/CD values
in jejunum and ileum but not in duodenum

23. Part 3: Update for study on in vitro species tropism of SeACoV
Generation of a rabbit anti-SeACoV-N pAb
M SeA-Vero Vero Purified N
Anti-SeACoV-N pAb(1:1000)
MARC-145
LLC-PK1
PK15
BHK-21
Huh-7
HeLa
Bat cell line
A549
Detection of SeACoV infection at 24 hpi by IFA
 Diverse species tropism and efficient infection of SeACoV in vitro

24. Update for epidemiology of SeACoV
1. Control of SeACoV in outbreak farms by feed back and autogenous vaccine
2. Does SeACoV spread to the other regions in China?
 SeACoV RNA was detected in a pig farm in Jiangsu province
of eastern China two weeks ago.

25. Summary
 A novel swine enteric alphacoronavirus (named SeACoV) was isolated from
diarrheic piglets in southern China.
 SeACoV is likely antigenetically distinct from PEDV, TGEV and PDCoV.
 Genomic and phylogenetic analysis showed that SeACoV might have originated
from the bat coronavirus HKU2.
 The extreme NTD of SeACoV S glycoprotein had an extremely high variability
compared to that of HKU2 (Cross-species transmission?)
 SeACoV is infectious and pathogenic in newborn piglets.
SeACoV represents the fifth porcine coronavirus in addition to
PEDV, TGEV, PDCoV and PHEV.

26. Acknowledgments
 The study was funded by:
 National Key R&D Program of China
(2016YFD0500102 and 2017YFD0500103)
 National Natural Science Foundation of
China (NSFC 31572518)
Thank you for your attention!
Bin Wang
Pan Qin
Yong-Le Yang