doi:10.1007/s12250-016-3727-3

Bats		Guangdong(Year 2005)		Yunnan(Years 2012, 2013)		Tibet(Year 2013)		Liaoning(Year 2013)		Jilin(Year 2013)
Family	Species	Bat	CoV^&	Bat	CoV	Bat	CoV	Bat	CoV	Bat	CoV
Pteropodidae	Rousettus leschenaulti	6/37(16.2)	4	14/100(14.0)	4
	Cynopterus sphinx	14/51(27.5)	4
	Megaerops kusnotei			1/41(2.4)	4
Hipposideridae	Hipposideros cineraceus	0/9				1/15(6.7)
	Hipposideros pomona			0/84
	Hipposideros larvatus	0/68		0/2
	Hipposideros armiger	0/11		0/18
	Aselliscus stoliczkanus			0/33
Rhinolophidae	Rhinolophus ferrumequinum			0/42				0/16		1/30(3.3)	2
	Rhinolophus sinicus			7/78(9.0)	, 2
	Rhinolophus pusillus	0/5		0/6
	Rhinolophus affinis			0/3
	Rhinolophus hipposideros			0/37		0/5
	Rhinolophus macrotis
Vespertilionidae	Myotis daubentonii			5/95(5.3)	3
	Myotis laniger			0/8
	Myotis chinensis			0/3
	Myotis capaccinii			0/40
	Myotis ricketti							0/38		0/27
	Miniopterus schreibersi			0/8
	Murina leucogaster									1/40(2.5)
Megadermatidae	Megaderma lyra			0/1
Note: ^{positive/total bats; numbers in brackets indicate the coronavirus positive percentage. ^& CoV, α: αCoV; β: unclassified βCoV; β2: βCoV lineage 2; β3: βCoV lineage 3; β4: βCoV lineage 4.}

Table 1. Bat sample collection and coronavirus detection.

ORF	JTMC15	Rf1		Rs672		BJ01
ORF	Length	Length	% identity	Length	% identity	Length	% identity
1a	4185	4378	98.0	4190	93.8	4383	93.5
1b	2704	2704	98.1	2704	98.1	2704	98.0
S	1236	1241	86.1	1255	81.9	1241	76.7
3a	274	274	98.2	274	92.0	274	86.2
3b	114	114	97.4	114	91.3	114	90.4
E	76	76	94.8	76	96.1	76	96.1
M	221	221	99.1	221	98.2	221	97.7
6	63	63	96.9	63	92.2	63	89.1
7a	122	122	98.4	122	93.5	122	91.9
7b	52	44	83.0	44	84.9	44	79.2
8(8a)	-	122	-	121	-	39	-
-(8b)	-	-	-	-	-	84	-
N	420	421	98.1	422	96.7	422	96.2
9a	97	97	94.9	98	79.6	98	79.6
9b	70	70	94.4	70	83.1	70	84.5
Note: ^# Abbreviation and accession numbers: Rf1, DQ412042; Rs672, FJ588686; BJ01, AY278488. ^{Gene 8 in SARSr-CoVs is described as 8a and 8b in SARS-CoVs.}

Table 2. Comparison of ORF amino acid identities of JTMC15 and other SARS- and SARSr- CoVs^#

Primers	Position of first nucleotide (nt)	Sequences (5′3′)
Pan-coronavirus nested primers
CoVOF	14615	ATGGGWTGGGAYTAYCCIAARTG
CoVOR	15200	TGYTGIGARCAAAAYTCRTG
CoVF	14618	GGITGGGAYTAYCCIAARTGYGA
CoVR	15035	CCRTCATCWGAIARWATCATCAT
JTMC15
F3	2811	GCGTGTAGAYAARGTGCTTAA
R3	4799	CCACGCTTRAGAAATTCAA
F4	4648	GTKTCAGTDTCWTCACCAGA
R4	6919	AATRCTTAACAAYAAYAGCCACAT
F5	6806	CACTWCCTACRACTATWGCTAAAAAT
R5	8939	GCAGARGTRGMAAARTCACTATACT
F6	8827	CCTGGHTTACCDGGTACTGT
R6	11329	CGYCTAGCAGCATCATCATA
F10	17350	TGAGTGTYGTCAATGCTAGAC
R10	19665	CTACYTTDGTGTAAACAGCATTATT
F12	21283	GCTATACCATGCATGCTAACT
R12	22615	CGAAAAAGARGTTGAGTTGTAG
5OR	252	ATTGGCTGAAACGACACCACTTC
5IR	161	GTCGATTAAAGCACTTGGCTCCA
3OF	27341	GACATCCCAGAGTGGAGGAG
3IF	27448	AGGTGTTGATGCCTCAGGCTAT
JPDB144
F1	1	GATTTAAGWGAATAGCYTRGCTATC
R1	1749	GTVGTWCCAGAVAGWARTGC
F2	1572	GGTACTATGYACTTTRTKCCT
R2	3846	CWGCDATRCCACCRCCAT
F3	3759	GTKACHHTAGTHTTWGGTGA
R3	5978	ACTAATAGYATCACYGCCA
F4	5940	TAYWCTAATAGYTGCCTTG
R4	8244	ACATCAGAYTCCACACC
F5	796	TGGCCAGGAAARTTTAGC
R5	10071	TCACTACCAGTYTCRCTGTA
F6	9854	TACTGATGGTAARCTKAATTGTAG
R6	12438	CATAGTTTGCATAGCACT
F7	12559	TCWATGTATAAGCAAGCACGT
R7	14645	GGATCWGCKGCATACATCAT
F8	14556	TATCTTGTGGTTATCACTAC
R8	17048	ATACCTCTCTTGATTCAC
F9	16931	CGYATWGAYTATAGTGATGCTG
R9	18983	ATCCCAMTCMACACGTTC
F10	18791	TATGCCTGCTGGASTCATTC
R10	20854	ATACTGRCACAATTGCATATATT
F11	20639	CCTATTGAYTTAACWATGATTG
R11	22365	GARWAGAGRTGAACRCCTTG
F12	22338	GAGTGGTTYGGYATTACMCA
R12	24688	GAAATAGCACCRAAAGTRTTAG
F13	24267	GCWGATCCYGGYTATATGC
R13	26250	CATAACGRTTKTGYYCGAAG
F14	26140	ACTAAAGYATYAGCAAAACAAGA
R14	27969	CGTTAAACCCASTCSTCAG
F15	27842	GCTAYTMGATTATGTGTGC
R15	30232	GCCTAATCTAATTGAATAATAGC
5OR	268	GTCACACTAGCCTTGGAAAGCA
5IR	83	CAGACCACAACACAACACGCACACAACA
3OF	30061	ATCATGTTARACTTACAGTGCAAG
3IF	30151	AAAGACTGTCACCTCTGCGTGATT
JTAC2
F1	4010	CCACTATGTSACCAATWTYTATGAT
R1	6107	CTTATCAATAAGCTTAGTAGCGTCT
F2	5961	TGTYGGMCAYTATACTGTTTTTGA
R2	8460	ACACGGCAATARGTCATAGC
F3	8172	TGGTAAAACWCTTGTKTTTGC
R3	9704	ACAAGCGCCATTAATGAA
F4	9615	TTAAYATTYTGGCRTGCTATGAT
R4	11457	CYTGTTCMGCCATTCTATCAA
F5	11364	GTTCTCCACCTCAGTTGGT
R5	13349	TCCTCACCAAAWATATCACTCTT
F6	13199	GATAAYCAGGATCTTAATGGTGA
R6	15458	TGACATGRTCATAAGCRCACTT
F7	15309	ATTCWACTGCTAARTTTTGGGA
R7	17318	CCATAAASGAKATWACATGCTCATA
F8	17174	GAKGGTTGYGGTCTYTTTAAAG
R8	18608	GGTGTTGTARGCATTARCATAGC
F9	18470	TGCCMTTYTTYTTCTATGATG
R9	20451	TCRAGCACACTRTTGTAAGACATAG
F10	20301	GGACAATGTTYTGTACCAGTG
R10	22672	ACATTCTTRAAGGCKARCAACTG
F11	22567	AAYGTGTGCACCCAGTATACTAT
R11	24958	TGAMGCTTTAAACAGTGCAA
F12	24352	ATCCCAGAKTATGTYGATGTTAA
R12	26127	ACCTTATAGCCYTCKACAAGCA
F13	25921	CAGCATCCTTATGGCTTG
R13	27429	ACTTTGGCACAGTCATYTTATAG
Genome walking
R1	4161	TGGCTGTAAAGTTGGCTGAGGT
R2	4274	GCCACCACCATGAGACAAATTCT
R3	4368	CAGAGCCAACCTTAAGTTTGCCA
R4	2642	ACTTACARCTAACACCGGCCAGT
R5	2745	TAGTCAAACCGTTCTCTACWGGAAT
R6	2889	CGTCATAGAATGCATAACCATCAAC

Table 1. Primers used in this study

nsp	JTAC2^$		JTMC15		JPDB144
nsp	Length (aa)	First - last residue^Position	Length (aa)	First - last residue^Position	Length (aa)	First - last residue^Position
1	-	-	179	M¹ -G¹⁷⁹	731	M¹ -G⁷³¹
2	467	Y¹ -G⁴⁶⁷	639	G¹⁸⁰ -G⁸¹⁸	489	R⁷³² -G¹²²⁰
3 (ADRP/PL^Pro )	1637	G⁴⁶⁸ -A²¹⁰⁴	1724	A⁸¹⁹ -G²⁵⁴²	1572	G¹²²¹ -A²⁷⁹²
4	480	G²¹⁰⁵ -Q²⁵⁸⁴	500	K²⁵⁴³ -Q³⁰⁴²	512	T²⁷⁹³ -Q³³⁰⁴
5 (3CL^Pro )	302	S²⁵⁸⁵ -Q²⁸⁸⁶	306	S³⁰⁴³ -Q³³⁴⁸	306	S³³⁰⁵ -Q³⁶¹⁰
6	276	S²⁸⁸⁷ -Q³¹⁶²	290	G³³⁴⁹ -Q³⁶³⁸	292	S³⁶¹¹ -Q³⁹⁰²
7	83	S³¹⁶³ -Q³²⁴⁵	83	S³⁶³⁹ -Q³⁷²¹	83	S³⁹⁰³ -Q³⁹⁸⁵
8	195	T³²⁴⁶ -Q³⁴⁴⁰	198	A³⁷²² -Q³⁹¹⁹	199	A³⁹⁸⁶ -Q⁴¹⁸⁴
9	108	N³⁴⁴¹ -Q³⁵⁴⁸	113	N³⁹²⁰ -Q⁴⁰³²	110	N⁴¹⁸⁵ -Q⁴²⁹⁴
10	135	A³⁵⁴⁹ -Q³⁶⁸³	139	A⁴⁰³³ -Q⁴¹⁷¹	139	A⁴²⁹⁵ -Q⁴⁴³³
11	17	S³⁶⁸⁴ -D³⁷⁰⁰	13	S⁴¹⁷² -V⁴¹⁸⁴	14	S⁴⁴³⁴ -V⁴⁴⁴⁷
12 (RdRP)	927	S³⁶⁸⁴ -Q⁴⁶¹⁰	932	S^4172- Q⁵¹⁰³	934	S⁴⁴³⁴ -Q⁵³⁶⁷
13 (Hel)	597	S⁴⁶¹¹ -Q⁵²⁰⁷	601	A⁵¹⁰⁴ -Q⁵⁷⁰⁴	598	A^5368- Q⁵⁹⁶⁵
14 (ExoN)	517	A⁵²⁰⁸ -Q⁵⁷²⁴	527	A⁵⁷⁰⁵ -Q⁶²³¹	523	S⁵⁹⁶⁶ -Q⁶⁴⁸⁸
15 (XendoU)	339	N⁵⁷²⁵ -Q⁶⁰⁶³	346	S⁶²³² -Q⁶⁵⁷⁷	342	G⁶⁴⁸⁹ -Q⁶⁸³⁰
16 (2-O-MT)	301	S⁶⁰⁶⁴ -K⁶³⁶⁴	298	A⁶⁵⁷⁸ -N⁶⁸⁷⁵	302	A⁶⁸³¹ -L⁷¹³²
Note: ^$ The nsp2 of JTAC2 was partial sequence, lacking the 5’ terminal.

Table 2. Putative nonstructural proteins (nsps) of ORF1a and ORF1b (replicase) in BatCoV JTAC2, JTMC15 and JPDB144.

ORF	JTAC2	Neixiang-14		512		PEDV
ORF	Length	Length	% identity	Length	% identity	Length	% identity
1a	3700	2030	87.9	4128	68.9	4117	76.4
1b	2680	2679	92.8	2681	85.3	2680	88.8
S	1365	-	-	1371	58.6	1386	57.1
3a	224	-	-	224	56.9	224	63.1
E	76	-	-	76	83.1	76	83.1
M	226	-	-	227	79.7	226	84.6
N	307	-	-	394	62.6	441	58.8
Note: ^# Abbreviation and accession numbers: Neixiang-14: MIBtCoV Neixiang-14, KF294377; 512: BatCoV/512/2005, NC_009657; PEDV: PEDV-1C, KM609203. ^§ incomplete sequences.

Table 3. Comparison of ORF amino acid identities of JTAC2 with other three representative Alphacoronavirus strains^#

ORF	JPDB144	HKU4		HKU5		MERS
ORF	Length	Length	% identity	Length	% identity	Length	% identity
1a	4447	4445	93.8	4481	71.1	4391	64.7
1b	2699	2699	97.7	2715	89.4	2701	87.4
S	1352	1352	94.5	1352	69.6	1353	67.1
3a (3)	91	91	89.3	121	44.8	103	46.8
3b (4a)	119	119	92.5	119	53.0	109	38.3
3c (4b)	285	285	88.8	256	39.2	246	27.8
3d (5)	227	227	93.0	223	46.6	224	46.9
E	82	82	98.8	82	80.7	82	69.9
M	219	219	97.3	220	82.3	219	84.2
N	423	423	97.9	427	74.4	413	70.8
Note: ^# Abbreviation and accession numbers: HKU4: BatCoV HKU4-4, EF065508; HKU5: BatCoV HKU5-1, EF065509; MERS: MERS-CoV ChinaGD01, KT006149. ^§ ORF3a, 3b, 3c, 3d in HKU4 and HKU5 are described in MERS-CoV as ORF3, 4a, 4b and 5 respectively.

Table 4. Comparison of ORF amino acid identities of JPDB144 with other two representative strains of Betacoronavirus lineage 3 (β3)^# .