Identification and characterization of a novel rodent bocavirus from different rodent species in China

Figures & data

Detection rates of rodent bocavirus among different rodent species in Dalian, Dandong, Huludao, Manzhouli, Zhangmu, Hami, Pingxiang, Ruili and Hekou from 2011 to 2017

Scientific name	No. positive/no. tested (%) in each city									Total
	Dalian Liaoning	Dandong Liaoning	Huludao Liaoning	Manzhouli Inner Mongolia	Zhangmu Tibet	Hami Xinjiang	Pingxiang Guangxi	Ruili Yunnan	Hekou Yunnan
Rattus norvegicus	24/86 (27.9)	5/19 (26.3)	3/74 (4.1)	0/5	7/14 (50)	0	2/7 (28.6)	0	26/54 (48.1)	67/259 (25.9)
Mus musculus	1/11 (9)	0/6	0/5	0/10	0	0	0	0	1/1 (100)	2/33 (6.1)
Apodemus agrarius	0/1	4/35 (11.4)	0	0	0	0	0	0	0	4/36 (11.1)
Citellus dauricus	0	0	0	0/23	0	0	0	0	0	0/23
Meriones unguiculatus	0	0	0	0/5	0	0	0	0	0	0/5
Microtus gregalis	0	0	0	0/2	0	0	0	0	0	0/2
Cricetulus barabensis	0	0	0	1/28 (3.6)	0	0	0	0	0	1/28 (3.6)
Rattus flavipectus	0	0	0	0	16/38 (42.1)	0	2/4 (50)	8/26 (30.8)	1/4 (25)	27/72 (37.5)
Rattus rattus	0	0	0	0	4/9 (44.4)	0	0	0	0	4/9 (44.4)
Apodemus peninsulae	0	0	0	0	0/3	0	0	0	0	0/3
Cricetulus migratorius	0	0	0	0	0	0/2	0	0	0	0/2
Tamarisk Jird	0	0	0	0	0	0/1	0	0	0	0/1
Meriones meridianus	0	0	0	0	0	0/5	0	0	0	0/5
Allactaga sibirica	0	0	0	0	0	0/1	0	0	0	0/1
Rhombomys opimus	0	0	0	0	0	1/6 (16.7)	0	0	0	1/6 (16.7)
Total	25/98 (25.5)	9/60 (15)	3/79 (3.8)	1/73 (1.4)	27/64 (42.2)	1/15 (6.7)	4/11 (36.4)	8/26 (30.8)	28/59 (47.5)	106/485 (21.9)

Detection rates of rodent bocavirus in different months in in Dalian, Dandong, Huludao, Manzhouli, Zhangmu, Hami, Pingxiang, Ruili and Hekou from 2011 to 2017

Sampling time	No. positive/no. tested (%) in each city									Total
	Dalian Liaoning	Dandong Liaoning	Huludao Liaoning	Manzhouli Inner Mongolia	Zhangmu Tibet	Hami Xinjiang	Pingxiang Guangxi	Ruili Yunnan	Hekou Yunnan
February	0	0	0	0	14/34 (41.2)	0	0	0	0	14/34 (41.2)
March	0	0	0	0	1/2 (50)	0	0	0	0	1/2 (50)
April	1/7 (14.3)	0/1	0	0	0	0	0	0	15/21 (71.4)	16/29 (55.2)
May	8/34 (23.5)	3/10 (30)	0	0	0	0	0	0	0	11/44 (25)
June	1/3 (33.3)	1/27 (3.7)	0	0/19	0	1/15 (6.7)	0	0	10/20 (50)	13/84 (15.5)
July	0	2/11 (18.2)	0	1/36 (2.8)	8/12 (66.7)	0	0	0	0	11/59 (18.6)
August	4/22 (18.2)	2/7 (28.6)	3/79 (3.8)	0/18	0/6	0	4/11 (36.4)	8/26 (30.8)	3/18 (16.7)	24/187 (12.8)
September	0/12	0/2	0	0	1/4 (25)	0	0	0	0	1/18 (5.6)
October	5/10 (50)	0/1	0	0	0	0	0	0	0	5/11 (45.5)
November	6/8 (75)	1/1 (100)	0	0	0	0	0	0	0	7/9 (77.8)
Not available	0/2	0	0	0	3/6 (50)	0	0	0	0	3/8 (37.5)
Total	25/98 (25.5)	9/60 (15)	3/79 (3.8)	1/73 (1.4)	27/64 (42.2)	1/15 (6.7)	4/11 (36.4)	8/26 (30.8)	28/59 (47.5)	106/485 (21.9)

Nine isolates of rodent bocavirus identified in this study

Accession no.	Isolate name	Host	Length	GC%	Sequence type
KY927867	DL8-RN	Rattus norvegicus	5136	41.2	Nearly full-length genome
KY927868	DL9-RN	Rattus norvegicus	5138	41.1	Nearly full-length genome
KY927869	DL16-RN	Rattus norvegicus	5140	41.5	Nearly full-length genome
KY927870	ZM14-RF	Rattus flavipectus	5118	40.8	Nearly full-length genome
KY927871	ZM37-RR	Rattus rattus	5116	41.4	Nearly full-length genome
KY927872	ZM71-RN	Rattus norvegicus	5110	41	Nearly full-length genome
KY927873	DL7-RN	Rattus norvegicus	4699	41.6	Complete CDS
KY927874	DL14-RN	Rattus norvegicus	4850	41.3	Complete CDS
KY927875	DL23-RN	Rattus norvegicus	4852	41.3	Complete CDS

Fig. 1 Genomic organization of rodent bocavirus (RoBoV) compared with that of GBoV, HBoV and PBoV.

Different colored boxes represent four ORFs of bocaparvoviruses. The illustration was constructed by geneious software. (Biomatters Ltd, Auckland, New Zealand)

Amino acid and nucleotide (data in parentheses) identities of NS1, NP1, VP1 between RoBoV isolates DL9-RN, ZM71-RN and other bocaparvoviruses

Isolate	DL9-RN	ZM71-RN	PBoV- KU14	PBoV- H18	PBoV- JOA011	PBoV-2	PBoV- SX	PBoV-3	RBoV- HK1S	HBoV-1
DL9-RN
NS1	—	97.5 (94.9)	92.1 (88)	88.7 (84.9)	92 (87.7)	48.3 (54.9)	75.6 (72)	39.8 (49.5)	51.4 (56.7)	41.7 (49.4)
NP1	—	96.5 (95.2)	86.4 (86.5)	65.2 (72.7)	91.3 (88.9)	40.7 (53.5)	65.2 (73)	31.8 (45.4)	33.2 (52.6)	29.4 (53.6)
VP1	—	99.5 (94.3)	91.4 (86.8)	75.8 (73.3)	91.6 (86.9)	46.6 (52.9)	75.1 (73.1)	38 (48.7)	45.8 (52.9)	40.1 (52.1)
ZM71-RN
NS1	97.5 (94.9)	—	92.9 (87.9)	89.3 (84.9)	92.8 (87.9)	47.5 (54.1)	75.4 (71.9)	39.5 (49)	50.9 (56.7)	41.4 (49.3)
NP1	96.5 (95.2)	—	87.9 (86.3)	64.1 (72.4)	91.9 (88)	42.2 (51.9)	64.1 (72.4)	32.3 (45.1)	33.2 (52.2)	29.9 (53.4)
VP1	99.5 (94.3)	—	91.6 (87.3)	75.9 (73.8)	91.7 (87.5)	46.6 (52.6)	75.3 (73.6)	38.1 (48.7)	46 (53.3)	40.2 (52)

HBoV human bocavirus, PBoV porcine bocavirus, RBoV rat bocavirus

Fig. 2 Phylogenetic tree of NS1 amino acid sequences of RoBoV and other members belonging to the genus Bocaparvovirus.

Maximum likelihood method was used to generate the tree under the best model (LG + G + I), which was selected based on the BIC scores (Supplementary Table S2). Scale bar indicates amino acid substitutions per site. Bootstrap values obtained in 100 replicates are indicated at the nodes. The sequences obtained in this study are marked with a black diamond. The tree is midpoint rooted. Isolate’s name DL8-RN denotes that the isolate’s host is Rattus norvegicus which was captured in Dalian. Likewise, the name ZM14-RF denotes this isolate’s host is Rattus flavipectus which was captured in Zhangmu. Csl bocavirus California sea lion bocavirus, GBoV gorilla bocavirus, HBoV human bocavirus, PBoV porcine bocavirus, RoBoV rodent bocavirus, RBoV rat bocavirus

Fig. 3 Phylogenetic tree of partial VP1 nucleotide sequences (460-bp fragment) from 55 RoBoV isolates showing the genetic diversity.

The maximum likelihood tree was constructed under the best model (HKY + G), which was selected based on the BIC scores (Supplementary Table S3). Scale bar indicates nucleotide substitutions per site. Bootstrap values obtained in 100 replicates are indicated at the nodes. The nine isolates with genome or CDS sequenced are marked with a black diamond. The tree is midpoint rooted. DL, DD, HLD, ZM, HM, MZL, PX, RL and HK in isolates’ name represent samples collected from Dalian, Dandong, Huludao, Zhangmu, Hami, Manzhouli, Pingxiang, Ruili, and Hekou respectively. Suffixes of isolates’ name denote the host species: AA Apodemus agrarius, CB Cricetulus barabensis, MM Mus musculus, RN Rattus norvegicus, RF Rattus flavipectus, RO Rhombomys opimus, RR Rattus rattus