Journal name:

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Journal name: Archives of Virology

Title: Bovine Coronavirus Infections in Turkey: Molecular Analysis of the Full-Length Spike Gene Sequences of Viruses From Digestive and Respiratory Infections

Authors:

Secil Sevinc Temizkan

^*a

, Feray Alkan

^a,b

a

Ankara University, Graduate School of Health Sciences, Department of Virology, Ankara, TURKEY

b

Ankara University, Faculty of Veterinary Medicine, Department of Virology, Ankara, Turkey.

*Corresponding Author: Secil Sevinc Temizkan

Ankara University, Faculty of Veterinary Medicine, Department of Virology, Diskapi, 06110, Ankara, Turkey.

E-mail: sevinc@ankara.edu.tr Phone: +90 553 085 36 26

Supplement 1: Information about the samples used in this study.

City Sampling Date

Number of

samples Ages of Animals Samples Species

Clinical Signs

Ankara

2001 2 Faeces Diarrhea

2014 1 7 d Faeces Diarrhea

2017 3 2-4 m Nasal discharge Nasaldischarge, cough

2018 9

15 d Nasal discharge Nasaldischarge, cough

2018 4 8 m Nasal discharge Nasaldischarge, cough

2019 5 Nasal discharge Nasal discharge, dyspnea

2018 4 8 m Nasal discharge Nasaldischarge, cough

2019 7 15 d Nasal discharge Nasaldischarge, cough, depression

Aksaray 2005 3 Faeces Diarrhea, dehydration

2006 2 11-14 d Faeces Diarrhea

2008 2 5-15 d Faeces Diarrhea

Eskisehir 2006 3 15-20 d Faeces Diarrhea

2006 1 15 d Nasaldischarge Nasaldischarge, cough

Kars 2006 3 Faeces Diarrhea, dehydration

Van 2006 2 Faeces Diarrhea

Kirklareli 2011 1 70 d Faeces Diarrhea, anorexia

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Erzurum 2014 2 2 y Nasal discharge Nasaldischarge, cough

2017 3 1 m Faeces Diarrhea

S. Urfa 2014 3 8-10 d Faeces Diarrhea, dehydration

Amasya 2015 2 4 d Faeces Diarrhea

Cankiri 2016 1 5 d Faeces Diarrhea, dehydration

Izmir

2013 1 14 d Faeces Diarrhea, dehydration

2017 4 4 y Nasal discharge Nasaldischarge

2018 3

5

≥

y Nasal discharge Nasaldischarge

2019 2 15 d Nasal discharge Nasaldischarge, cough

Denizli 2017 5 7-30 d Nasal discharge Nasaldischarge, cough, anorexia

2017 9 7 d- 15 m Faeces Diarrhea, anorexia, dehydration

Burdur 2019 5 1,5- 9 m Nasal discharge Nasaldischarge

2019 5 1- 1,5 y Nasal discharge Nasaldischarge, cough

Hatay 2019 4 Nasal discharge Nasaldischarge, cough, dyspnea

Tokat 2018 5 15 d-8 m Nasal discharge Nasaldischarge, cough

2019 10

45-90 d Nasal discharge Nasaldischarge, cough

d:day; m:month; y: year

Supplement 2: The primer pairs used in this study.

Primer Name

Primer Sequence (5’→3’)

Location Target Gene Region

Annealing (°C)

Amplicon

Size References BCoV

N-F

GCAATCCAGTAGTAGAGCGT 21-40

N (first

round) 50°C 730 bp

(17) BCoV

N-R

CTTAGTGGCATCCTTGCCAA 731-750 nBCoV

N-F

GCCGATCAGTCCGACCAATG 79-98

N(second

round) 58°C 407 bp

nBCoV

N-R AGAATGTCAGCCGGGGTAG 467-485

S-AF ATGTTTTTGATACTTTTAATT 1-21

S-1 ^51°C 655bp

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S-AR AGTACCACCTTCTTGATAAA 654-635

S-BF ATGGCATTGGGATACAG 549-565

S-1 ^55°C 490 bp

S-BR TAATGGAGAGGGCACCGACTT 1039-1018

S-CF GGGTTACACCTCTCACTTCT 782-801

S-1 ^58°C 769bp

S-CR GCAGGACAAGTGCCTATACC 1550-1531 S-DF GTCCGTGTAAATTGGATGGG 1460-1479

S-1 ^55°C 827bp

S-DR TGTAGAGTAATCCACACAGT 2286-2267

S-EF TTACAAAAATCAAACACAGACAT 1855-1877 S-1 S-2 _55°C 877bp S-ER AAACTTTATTACAATCGCTTCC 2731-2710

S-FF TCAATTTTTCCCCTGTATTAGG 2680-2702

S-2 ^55°C 555 bp

(6) S-FR CMAGTCTRGATAGAATTTCTTGTAA 3234-3209

S-GF GCTACCAATTCTGCTTTAGTTA 3099-3121

S-2 ^55°C 519 bp

S-GR GTAGTAATAACCACTACCAGTG 3617-3595 S-HF TTTAGCTATGTCCCTACTAAGTA 3475-3498

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S-2 ^58°C 637 bp S-HR CCAATAAATCAAAGACGAACTTA 4112-4089

S-1F GTTTCTGTTAGCAGGTTTAA 1329-1348

S-1 ^55°C 488 bp (1)

S-1R ATATTACACCTATCCCCTTG 1797-1816

Supplement 3: The strains used in the phylogenetic tree for S gene sequences.

Strain/isolate name Sample type GenBank accession no. Source BCoV/Turkey/H1/2013 Faecal MK989633-34

This study

BCoV/Turkey/U1/2014 Faecal MK989622

BCoV/Turkey/4950/2015 Faecal MK989620 BCoV/Turkey/4945/2015 Faecal MK989621 BCoV/Turkey/G76/2015 Faecal MK989628-29 BCoV/Turkey/G83/2015 Faecal MK989630-32 BCoV/Turkey/8553/2015 Faecal MK989626

BCoV/Turkey/S8/2017 Nasal MK989624

BCoV/Turkey/T5/2018 Nasal MK989623

BCoV/Turkey/79/2018 Nasal MK989627

BCoV/Turkey/1N/2019 Nasal MK989625

BCoV/Turkey/AN4/2005 Faecal GQ259978

National Center for Biotechnology Information BCoV/Turkey/AN11/2005 Faecal GQ259979

BCoV/USA/LY138/1965 Faecal AF058942 (LY138) BCoV/USA/Mebus/1971 Faecal U00735 (Mebus)

BCoV/USA/ENT/1998 Faecal AF391541

BCoV/USA/E-AH65/2001 Faecal EF424615

BCoV/USA/7-16-23/2016 Faecal MH043955 BCoV/China/HLJ-14/2015 Faecal KM985631 BCoV/Denmark/05-2/2005 Faecal KF169917

BCoV/France/-/1979 Faecal D00731

BCoV/South Korea/KCD10/2007 Faecal DQ389641 BCoV/Sweden/C-92/1992 Faecal KF169908 BCoV/Italy/339-06/2006 Faecal EF445634 BCoV/Japan/ Kakegawa/1976 Faecal AB354579 BCoV/Vietnam/PL84/2017 Faecal MH197037 BCoV/Germany/G95/1989 Nasaldischarge M80844

BCoV/USA/LSU/1994 Nasal discharge AF058943 BCoV/USA/LUN/1998 Nasal discharge AF391542 BCoV/Denmark/05-3/2005 Nasal discharge KF169918 BCoV/France/EPI-Caen-01/2005 Nasal discharge KT318111 BCoV/South Korea/KWD1/2005 Nasal discharge AY935637 BCoV/Sweden/U-09-3/2009 Nasal discharge KF169936 BCoV/Italy/438-06-TN/2008 Nasal discharge EU814647

BCoV/Germany/V270/1983 - EF193075

BCoV/USA/DB2/1984 - DQ811784

BCoV Norden Vaccine Vaccine strain M64668

HCoV/OC43/2020 - NC006213.1

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Supplement 4: N gene gel electrophoresis results: BCoV Nested-RT-PCR 2nd round result gel image [M: DNA ladder (100 bp), 1-9: Examples, 10: Positive Control (407 bp), 11: Negative Control].

Supplement 5: S gene gel electrophoresis results: RT-PCR gel images (M: DNA ladder, 100 bp) Various primer pairs were used for the S gene amplification (4,112 bp).

Journal name:

Journal name: Archives of Virology

Title: Bovine Coronavirus Infections in Turkey: Molecular Analysis of the Full-Length Spike Gene Sequences of Viruses From Digestive and Respiratory Infections

Authors:

Secil Sevinc Temizkan

, Feray Alkan

Ankara University, Graduate School of Health Sciences, Department of Virology, Ankara, TURKEY

Ankara University, Faculty of Veterinary Medicine, Department of Virology, Ankara, Turkey.

*Corresponding Author: Secil Sevinc Temizkan

Ankara University, Faculty of Veterinary Medicine, Department of Virology, Diskapi, 06110, Ankara, Turkey.

E-mail: sevinc@ankara.edu.tr Phone: +90 553 085 36 26

Supplement 1: Information about the samples used in this study.

1

≥

d:day; m:month; y: year

Supplement 2: The primer pairs used in this study.

2

Supplement 3: The strains used in the phylogenetic tree for S gene sequences.

3

Supplement 4: N gene gel electrophoresis results: BCoV Nested-RT-PCR 2nd round result gel image [M: DNA ladder (100 bp), 1-9: Examples, 10: Positive Control (407 bp), 11: Negative Control].

Supplement 5: S gene gel electrophoresis results: RT-PCR gel images (M: DNA ladder, 100 bp) Various primer pairs were used for the S gene amplification (4,112 bp).

A. Primary SA: [2-11: Samples, 1: Positive Control (655 bp), 12: Negative Control]

B. Primary SB: [2-4: Samples, 1: Positive Control (490 bp), 5: Negative Control]

C. Primary SC: [2-5: Samples, 6: Positive Control (769 bp), 1: Negative Control]

D. Primary SD: [2-6: Samples, 1: Positive Control (827 bp), 7: Negative Control]

E. Primary SE: [2-5: Samples, 6: Positive Control (877 bp), 1: Negative Control]

F. Primary SF: [2-5: Samples, 1: Positive Control (555 bp), 6: Negative Control]

G. Primary SG: [1-2: Samples, 3: Positive Control (519 bp), 4: Negative Control]

H. Primary SH: [2-4: Samples, 1: Positive Control (637 bp), 5: Negative Control]

I. Primary S1: [1-6: Samples, 7: Positive Control (488 bp), 8: Negative Control]

4