Arch Virol DOI 10.1007/s00705-014-2064-9
ANNOTATED SEQUENCE RECORD
Genomic characterization of a bovine viral diarrhea virus 1 isolate from swine Yu Deng • Tong-Ling Shan • Wu Tong • Xu-Chen Zheng • Yi-Yei Guo • Hao Zheng • San-Jie Cao • Xin-Tian Wen • Guang-Zhi Tong
Received: 14 November 2013 / Accepted: 21 March 2014 Ó Springer-Verlag Wien 2014
Abstract The SD0803 strain of the bovine viral diarrhea virus (BVDV) was isolated from a piglet in China in 2008 and has been classified as a novel subgenotype of BVDV-1. To describe the molecular features of this novel subgenotype, we sequenced and characterized the complete genome of the SD0803 virus. The genome is 12,271 bp in length and contains 50 and 30 untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a 3,898amino-acid polypeptide. The full-length genome of the SD0803 strain shares 78.8 % to 83.3 % identity with those of other BVDV-1 strains, 70.0 % to 70.7 % identity with those of BVDV-2 strains, and less than 67.6 % identity with those of other pestiviruses. The highest level of shared identity was 83.3 % between the complete SD0803 genome and that of the ZM-95 strain of BVDV-1. Phylogenetic analysis of the 50 UTR and the coding sequence for the N-terminal protease fragment of the SD0803 polyprotein indicated that the SD0803 virus is a member of the novel subgenotype BVDV-1q, isolates of which have been identified recently in dairy cattle and camels in China.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2064-9) contains supplementary material, which is available to authorized users. Y. Deng T.-L. Shan W. Tong X.-C. Zheng Y.-Y. Guo H. Zheng (&) G.-Z. Tong Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China e-mail: [email protected] S.-J. Cao X.-T. Wen College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China
Bovine viral diarrhea virus 1 (BVDV-1) belongs to the genus Pestivirus, which also includes BVDV-2, classical swine fever virus (CSFV), border disease virus (BDV), and some other viruses that have not been assigned to a species, such as giraffe, D32/00_HoBi, Bungowannah, and pronghorn viruses [9, 15, 19]. BVDV-1 has a positive-sense, single-stranded nonpolyadenylated RNA genome that is approximately 12.3 to 12.5 kb in length. The genomes contain a single open reading frame (ORF) that is flanked by 50 and 30 untranslated regions (UTRs). The BVDV ORF encodes a polyprotein that is processed co- and posttranslationally into twelve structural and nonstructural proteins, the coding sequences of which are arranged in the genome in the following order: Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, and NS5 [10]. Based on the sequences of the 50 UTR or Npro, BVDV-1 has been divided into 16 subgenotypes (BVDV-1a to BVDV-1p), and BVDV-2 has been divided into four subgenotypes (BVDV2a to BVDV-2d) [6, 8, 11, 21]. Cytopathic (CP) and noncytopathic (NCP) biotypes of BVDVs have also been recognized, based on the lytic activity of the virus in cell culture [18]. Bovine viral diarrhea virus (BVDV) infection was first described in cattle in 1946 [12]. Subsequently, BVDV was found to be epidemic worldwide on cattle farms. In mainland China, BVDV was first isolated in 1983 from aborted fetuses in an introduced cattle population [22]. To date, BVDV-1b, BVDV-1c, BVDV-1d, BVDV-1m, BVDV-2a, and the tentative subtype BVDV-1p have been detected in cattle herds in China [21, 23, 24]. Previous studies have shown that the prevalence of the BVDV in cattle reached 43 % in certain areas of China [23]. The BVDVs causes respiratory, gastrointestinal and reproductive disorders in cattle and can also infect goats, sheep, deer, and swine [1, 14, 17]. BVDV infections of pigs
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were first described from serological surveys conducted in the 1960s [13]. A strain of BVDV was first isolated from pigs in 1973, and it was etiologically demonstrated that BVDV could naturally infect pigs [4]. In Austria and some European countries, the prevalence of BVDV in pigs has increased in the last several years [17]. BVDV has also been reported in domestic swine in China. In 1995, the first BVDV-1m strain, ZM-95, was isolated from swine in Inner Mongolia, China [20]. The prevalence of the BVDV in swine in China was reported to be 23.1 % to 33.6 % from 2007 to 2010 [3]. BVDV-1a, BVDV-1b, BVDV-1m, BVDV-1o, and an unclassified subgenotype have been detected in swine [3]. Recently, six subgenotypes of BVDV-1, 1a, 1b, 1c, 1m, 1o and 1p, and a putative subgenotype, BVDV-1q, were found in camels in China [5]. Four isolates of BVDV-1 from dairy cattle in northwestern China were also considered to represent potentially novel subgenotypes [7]. In our current study, we determined the sequence of the full-length genome of the SD0803 virus and analyzed its phylogenetic relationship to other recently reported novel BVDV subgenotypes. The SD0803 strain of BVDV-1 was isolated from a piglet with hyperpyrexia in Shandong Province, China in 2008. The SD0803 virus was propagated in Madin-Darby bovine kidney (MDBK) cells, and no cytopathic effect was observed. Total RNA was extracted from virus-infected MDBK cells using a QIAamp Viral RNA Mini Kit (QIAGEN, Chatsworth, CA, USA) according to the manufacturer’s instructions. The total RNA was used for first-strand cDNA synthesis using SuperScript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and a primer complementary to nucleotide positions 12,152 to 12,172 of the ZM-95 genomic sequence (GenBank accession no. AF526381). Ten overlapping cDNA segments were amplified by polymerase chain reaction (PCR) using ten pairs of primers that were designed based on ZM-95, NADL, KE9 and SD0803 (Table S1). The sequences of the 50 and 30 ends of the SD0803 genome were determined using a 50 and 30 Rapid Amplification of cDNA Ends kit (Clontech Laboratories, Inc). The PCR products were cloned into the pMD19-T plasmid and sequenced by a commercial service provider (Invitrogen, Shanghai, China). The full-length genome of the SD0803 virus was assembled using the SeqMan program of the Lasergene software (DNASTAR, Madison, WI, USA). The ORFs of the genomes were scanned using the MapDraw program (DNASTAR). A phylogenetic analysis was performed based on complete BVDV genome sequences, partial Npro gene sequences, and 50 UTR sequences available in the GenBank database. Sequence alignments and a similarity analysis of the aligned nucleotides were performed using the MegAlign program (DNASTAR). Phylogenetic trees
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Fig. 1 Phylogenetic trees based on the nucleotide sequences of c (A) the full length genome, (B) the Npro gene, and (C) the 50 UTR. The phylogenetic trees were constructed using the neighbor-joining method. The percentage of replicate trees in which the sequences clustered together in the bootstrap test using 1000 replicates are shown next to the branches. Evolutionary distances were computed using the Kimura two-parameter method. The analyses were based on the nucleotide sequence of the12.3-kb full-length genome, the 504-nt Npro gene, and the 244-nt 50 UTR in the final dataset. The phylogenetic analyses were conducted using MEGA, version 4.1. The GenBank accession numbers of the full-length genomic sequences of reference strains belonging to the genus Pestivirus were as follows: NADL (NC_001461), 3156 (JN704144), KE9 (EF101530), 12F004 (KC963967), ZM-95 (AF526381), CP7-5A (AF220247), Singer Arg (DQ088995), 10JJ-SKR (KC757383), Oregon (AF091605), SuwaNCP (KC853440), New York ’93 (AF502399), SH-28 (HQ258810), JZ05-1 (GQ888686), 11F011 (KC963968), HLJ-10 (JF714967), D3200 HoBi (AB871953), CHKaHo/cont (JX985409), Th/04_KhonKaen (NC_012812), Shimen (AF092448), Alfort-187 (X87939), Gifhorn (KF925348), Giraffe-1 H138 (AF144617). The GenBank accession numbers of the Npro sequences of the BVDV-1 reference strains are as follows (those with the same virus strain and accession number have been omitted): 519(AF144464), Deer NZ1 (U80903), 721 (AF144463), F (AF287284), 3186V6 (AF287282), J (AF287286), W (AF287290), L (AF287287), A (AF287283), G (AF287285), 23-15 (AF287289), Deer GB1 (U80902), B440/06 (EU224257), CH-Suwa (AY894998), TR27 (EU163975), TR29 (EU163977), TJ0801 (GU120262), So CP75 (AB359929), Shitara (AB359930), IS25CP01 (AB359931), BJ0701 (GU120259), and BJ0703 (GU120261). The GenBank accession numbers of the 50 UTR sequences of the BVDV-1 reference strains are as follows (those with the same virus strain and accession number have been omitted): NADL (AJ133739), Manas-1 (EU555288), Shitara/01/05 (AB359926), Bega (AF049221), F (AF298065), 16-111 (AF298056), 20-V661-2 (AF298058), J (AF298067), W (AF298073), L (AF298069), A (AF298064), G (AF298066), KM (AF298068), 23-15 (AF298059), KS86-1ncp (AB078950), SuwaCp (AF117699), Rebe (AF299317), LZ05 (GU120241), NX0802 (GU120253), IS25CP/01 (AB359931), Shitara/02/06 (AB359930), So CP/75 (AB042661), Shihezi148 (EU159700), BJ0703 (GU120249), 890 (U18059), TJ06 (GU120246), TJ06 (GU120246), 11X80 (JX437153), 11X97 (JX437154), 11N36 (JX437156) 11N94 (JX437149)
were constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software, version 4.1, based on partial Npro genes, the 50 UTR, or the full genome [16]. The bootstrap values were based on 1000 replicates using the neighbor-joining method, and the evolutionary distances were determined using the Kimura two-parameter method. The complete genome of the SD0803 strain of BVDV-1 (GenBank accession no. JN400273) was 12,271 bp in length and contained a single ORF that was 11,694 bp in length, encoding a 3,898-amino-acid polypeptide. The 50 and 30 UTRs of the SD0803 virus were 388 and 186 bp in length, respectively. Based on the putative post-transcriptional processing sites of other BVDVs, the SD0803 genes and UTRs were mapped to the following nucleotide positions in the genome: 50 UTR, 1-388; Npro, 389-892; C, 893-1198; Erns, 1199-1879; E1, 1880-2464; E2, 2465-3586; P7, 3587-3796; NS2, 3797-5155; NS3,
Bovine viral diarrhea virus 1 from swine
5156-7204; NS4A, 7205-7396; NS4B, 7397-8437; NS5A, 8438-9925; NS5B, 9926-12085; 30 UTR,12086-12271. A phylogenetic tree was constructed based on the SD0803 genomic sequence and the complete genome sequences of 23 pestiviruses deposited in GenBank, which showed that SD0803 clustered with other BVDV-1 strains (Fig. 1a). The SD0803 genome shared nucleotide sequence identities of 78.8 % to 83.3 % with the other BVDV-1 strains, about 70.0 % with the BVDV-2 strains, about 67.0 % with HoBi-like viruses (Th/04_KhonKaen strain and D3200_HoBi), 66.6 % with the CSFV strain Alfort187, 67.2 % with the BDV strain Gifhorn, 67.6 % with the Giraffe-1_H138 strain (giraffe pestivirus), and 56.6 % with Bungowannah (Table S2) [2]. The highest degree of similarity observed was 83.3 % between the SD0803 virus and the ZM-95 strain of BVDV-1, which was isolated in 1995
from swine in China. The SD0803 genome exhibited 70.0 % sequence homology with the SH-28 strain of BVDV-2, which was also isolated from swine in China. Twelve genes of SD0803 were compared with the genes of six BVDV-1 strains and three BVDV-2 strains, respectively (Table 1). The sequences of the other BVDV-1 viruses exhibited a higher degree of similarity to those of SD0803 than to those of the BVDV-2 strains. With the exception of the Npro gene, the highest degree of shared identity was observed between the SD0803 and ZM-95 viruses. However, the ZM-95 virus is a CP strain of BVDV-1, whereas the SD0803 virus is an NCP strain of BVDV-1. In the comparison of the coding sequences of BVDV-1 and BVDV-2, the ZM-95 virus was shown to have a six-nucleotide insertion in the C gene, and NADL, CP7-5A and New York ’93 have insertions of 270, 27 and
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Y. Deng et al. Table 1 Nucleotide sequence identity (%) of the SD0803 virus, compared with the coding sequences of other isolates of BVDV-1 and BVDV-2
#
Sequences with an insertion in the C or NS2 gene were omitted
Strain
Npro
C
Erns
E1
E2
P7
NS2
NS3
NS4A
NS4B
NS5A
NS5B
ZM-95
80.8
79.8#
83.8
81.7
78.1
81.0
81.6
85.7
85.4
86.6
81.9
83.5
KE9
76.4
78.8
81.2
76.1
74.1
74.8
77.6
82.2
85.4
81.8
77.2
80.0
CP7-5A
78.6
77.8
79.9
76.6
74.1
74.3
77.1#
83.1
84.4
80.2
76.9
81.3
NADL
81.0
78.4
80.3
77.8
73.2
73.3
78.7#
84.2
81.8
83.1
77.1
79.3
Singer_Arg
80.2
77.1
81.4
77.6
73.0
74.3
78.2
83.6
82.3
82.4
77.3
79.3
SuwaNCP
78.2
76.8
80.5
77.6
71.9
79.5
79.5
82.1
85.4
81.9
76.4
79.7
JZ05-1
70.0
69.3
72.1
69.2
66.0
60.5
61.5
76.7
75.0
74.9
67.5
71.3
New York ’93
70.8
68.0
72.0
69.6
57.0
59.0
63.9#
77.8
71.9
75.5
66.3
70.9
SH-28
70.6
69.3
72.4
70.6
65.3
62.4
64.0
77.9
74.0
75.5
66.7
70.7
48 nucleotides, respectively, in the NS2 gene (Table 1) [20]. However, no insertions or deletions were revealed in the coding regions of the SD0803 virus when it was compared with the other six strains of BVDV-1 (ZM-95, NADL, CP7-5A, Singer_Arg, KE9 and SuwaNCP) and three strains of BVDV-2 (JZ05-1, New York ’93 and SH28). The shared identity of individual genes between the SD0803 virus and the other BVDV-1 strains ranged from 71.9 % to 86.6 % (Table 1). These results indicated considerable genetic divergence between the SD0803 virus and the other BVDV-1 strains. In a previous study, the SD0803 virus was classified as a BVDV-1 strain based on its 50 -UTR sequence, but it could not be further classified according to any recognized subgenotype [3]. In our current study, a phylogenetic tree was constructed based on the sequence of the Npro gene (Fig. 1b), and the SD0803 and KC207072 (camel isolate 6) viruses clustered in a branch that might represent a new subgenotype, BVDV-1q [5]. The branch of BVDV-1q was most closely related to the BVDV-1g, BVDV-1m, BVDV-1o, and BVDV-1p clusters. The Npro gene of SD0803 had 91.5 % sequence homology with the KC207072 virus. The Npro gene of SD0803 had 79.6 % to 83.5 % sequence homology with the strains classified as BVDV-1g, BVDV-1m, BVDV-1o, or BVDV-1p Four isolates of BVDVs, the 11N36, 11N94, 11X80, and 11X97 viruses, were recently isolated from dairy cattle in northwestern China. All four isolates were classified as belonging to a potentially novel subgenotype [7], and a partial 50 -UTR sequence of each isolate was obtained. To determine the genetic relationship between the SD0803 virus and these strains of a novel subgenotype, a phylogenetic tree was constructed based on of the 50 UTR sequences, which showed a single branch containing the four isolates clustered with the SD0803 virus (Fig. 1c). The 50 UTR of the SD0803 virus had a high degree of sequence homology (94.3 % to 98.7 %) to those of the 11N36, 11N94, 11X80, and 11X97 viruses, indicating that these viruses should thus be classified as strains of the BVDV-1q subgenotype. Similar to the phylogenetic tree based on the Npro sequences, the BVDV-1q branch was most closely
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related to the BVDV-1m, BVDV-1o, BVDV-1p, and BVDV-1g clusters in the 50 -UTR-based phylogenetic tree. In summary, a virus belonging to the novel BVDV-1q subgenotype was first detected in swine in eastern China, and was also detected in camel and cattle in northwestern China [3, 5, 7]. We determined the complete sequence of the genome of the swine-associated SD0803 strain of BVDV-1. Phylogenetic and sequence analyses based on the Npro gene and the 50 UTR showed that the SD0803 virus is a strain of the BVDV-1q subgenotype. This is the first report of the genomic sequence of a BVDV-1q virus isolated from swine.
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Bovine viral diarrhea virus 1 from swine 9. Kirkland PD, Frost MJ, Finlaison DS, King KR, Ridpath JF, Gu X (2007) Identification of a novel virus in pigs—Bungowannah virus: a possible new species of pestivirus. Virus Res 129:26–34 10. Meyers G, Thiel HJ (1996) Molecular characterization of pestiviruses. Adv Virus Res 47:53–118 11. Mishra N, Rajukumar K, Vilcek S, Tiwari A, Satav JS, Dubey SC (2008) Molecular characterization of bovine viral diarrhea virus type 2 isolate originating from a native Indian sheep (Ovies aries). Vet Microbiol 130:88–98 12. Olafson P, Mac CA, Fox FH (1946) An apparently new transmissible disease of cattle. Cornell Vet 36:205–213 13. Passler T, Walz PH (2010) Bovine viral diarrhea virus infections in heterologous species. Anim Health Res Rev 11:191–205 14. Ridpath JF (2010) Bovine viral diarrhea virus: global status. Vet Clin N Am Food Anim Pract 26:105–121 table of contents 15. Schirrmeier H, Strebelow G, Depner K, Hoffmann B, Beer M (2004) Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species. J Gen Virol 85:3647–3652 16. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 17. Tao J, Liao J, Wang Y, Zhang X, Wang J, Zhu G (2013) Bovine viral diarrhea virus (BVDV) infections in pigs. Vet Microbiol 165:185–189
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ANNOTATED SEQUENCE RECORD
Genomic characterization of a bovine viral diarrhea virus 1 isolate from swine Yu Deng • Tong-Ling Shan • Wu Tong • Xu-Chen Zheng • Yi-Yei Guo • Hao Zheng • San-Jie Cao • Xin-Tian Wen • Guang-Zhi Tong
Received: 14 November 2013 / Accepted: 21 March 2014 Ó Springer-Verlag Wien 2014
Abstract The SD0803 strain of the bovine viral diarrhea virus (BVDV) was isolated from a piglet in China in 2008 and has been classified as a novel subgenotype of BVDV-1. To describe the molecular features of this novel subgenotype, we sequenced and characterized the complete genome of the SD0803 virus. The genome is 12,271 bp in length and contains 50 and 30 untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a 3,898amino-acid polypeptide. The full-length genome of the SD0803 strain shares 78.8 % to 83.3 % identity with those of other BVDV-1 strains, 70.0 % to 70.7 % identity with those of BVDV-2 strains, and less than 67.6 % identity with those of other pestiviruses. The highest level of shared identity was 83.3 % between the complete SD0803 genome and that of the ZM-95 strain of BVDV-1. Phylogenetic analysis of the 50 UTR and the coding sequence for the N-terminal protease fragment of the SD0803 polyprotein indicated that the SD0803 virus is a member of the novel subgenotype BVDV-1q, isolates of which have been identified recently in dairy cattle and camels in China.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2064-9) contains supplementary material, which is available to authorized users. Y. Deng T.-L. Shan W. Tong X.-C. Zheng Y.-Y. Guo H. Zheng (&) G.-Z. Tong Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China e-mail: [email protected] S.-J. Cao X.-T. Wen College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China
Bovine viral diarrhea virus 1 (BVDV-1) belongs to the genus Pestivirus, which also includes BVDV-2, classical swine fever virus (CSFV), border disease virus (BDV), and some other viruses that have not been assigned to a species, such as giraffe, D32/00_HoBi, Bungowannah, and pronghorn viruses [9, 15, 19]. BVDV-1 has a positive-sense, single-stranded nonpolyadenylated RNA genome that is approximately 12.3 to 12.5 kb in length. The genomes contain a single open reading frame (ORF) that is flanked by 50 and 30 untranslated regions (UTRs). The BVDV ORF encodes a polyprotein that is processed co- and posttranslationally into twelve structural and nonstructural proteins, the coding sequences of which are arranged in the genome in the following order: Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, and NS5 [10]. Based on the sequences of the 50 UTR or Npro, BVDV-1 has been divided into 16 subgenotypes (BVDV-1a to BVDV-1p), and BVDV-2 has been divided into four subgenotypes (BVDV2a to BVDV-2d) [6, 8, 11, 21]. Cytopathic (CP) and noncytopathic (NCP) biotypes of BVDVs have also been recognized, based on the lytic activity of the virus in cell culture [18]. Bovine viral diarrhea virus (BVDV) infection was first described in cattle in 1946 [12]. Subsequently, BVDV was found to be epidemic worldwide on cattle farms. In mainland China, BVDV was first isolated in 1983 from aborted fetuses in an introduced cattle population [22]. To date, BVDV-1b, BVDV-1c, BVDV-1d, BVDV-1m, BVDV-2a, and the tentative subtype BVDV-1p have been detected in cattle herds in China [21, 23, 24]. Previous studies have shown that the prevalence of the BVDV in cattle reached 43 % in certain areas of China [23]. The BVDVs causes respiratory, gastrointestinal and reproductive disorders in cattle and can also infect goats, sheep, deer, and swine [1, 14, 17]. BVDV infections of pigs
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were first described from serological surveys conducted in the 1960s [13]. A strain of BVDV was first isolated from pigs in 1973, and it was etiologically demonstrated that BVDV could naturally infect pigs [4]. In Austria and some European countries, the prevalence of BVDV in pigs has increased in the last several years [17]. BVDV has also been reported in domestic swine in China. In 1995, the first BVDV-1m strain, ZM-95, was isolated from swine in Inner Mongolia, China [20]. The prevalence of the BVDV in swine in China was reported to be 23.1 % to 33.6 % from 2007 to 2010 [3]. BVDV-1a, BVDV-1b, BVDV-1m, BVDV-1o, and an unclassified subgenotype have been detected in swine [3]. Recently, six subgenotypes of BVDV-1, 1a, 1b, 1c, 1m, 1o and 1p, and a putative subgenotype, BVDV-1q, were found in camels in China [5]. Four isolates of BVDV-1 from dairy cattle in northwestern China were also considered to represent potentially novel subgenotypes [7]. In our current study, we determined the sequence of the full-length genome of the SD0803 virus and analyzed its phylogenetic relationship to other recently reported novel BVDV subgenotypes. The SD0803 strain of BVDV-1 was isolated from a piglet with hyperpyrexia in Shandong Province, China in 2008. The SD0803 virus was propagated in Madin-Darby bovine kidney (MDBK) cells, and no cytopathic effect was observed. Total RNA was extracted from virus-infected MDBK cells using a QIAamp Viral RNA Mini Kit (QIAGEN, Chatsworth, CA, USA) according to the manufacturer’s instructions. The total RNA was used for first-strand cDNA synthesis using SuperScript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and a primer complementary to nucleotide positions 12,152 to 12,172 of the ZM-95 genomic sequence (GenBank accession no. AF526381). Ten overlapping cDNA segments were amplified by polymerase chain reaction (PCR) using ten pairs of primers that were designed based on ZM-95, NADL, KE9 and SD0803 (Table S1). The sequences of the 50 and 30 ends of the SD0803 genome were determined using a 50 and 30 Rapid Amplification of cDNA Ends kit (Clontech Laboratories, Inc). The PCR products were cloned into the pMD19-T plasmid and sequenced by a commercial service provider (Invitrogen, Shanghai, China). The full-length genome of the SD0803 virus was assembled using the SeqMan program of the Lasergene software (DNASTAR, Madison, WI, USA). The ORFs of the genomes were scanned using the MapDraw program (DNASTAR). A phylogenetic analysis was performed based on complete BVDV genome sequences, partial Npro gene sequences, and 50 UTR sequences available in the GenBank database. Sequence alignments and a similarity analysis of the aligned nucleotides were performed using the MegAlign program (DNASTAR). Phylogenetic trees
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Fig. 1 Phylogenetic trees based on the nucleotide sequences of c (A) the full length genome, (B) the Npro gene, and (C) the 50 UTR. The phylogenetic trees were constructed using the neighbor-joining method. The percentage of replicate trees in which the sequences clustered together in the bootstrap test using 1000 replicates are shown next to the branches. Evolutionary distances were computed using the Kimura two-parameter method. The analyses were based on the nucleotide sequence of the12.3-kb full-length genome, the 504-nt Npro gene, and the 244-nt 50 UTR in the final dataset. The phylogenetic analyses were conducted using MEGA, version 4.1. The GenBank accession numbers of the full-length genomic sequences of reference strains belonging to the genus Pestivirus were as follows: NADL (NC_001461), 3156 (JN704144), KE9 (EF101530), 12F004 (KC963967), ZM-95 (AF526381), CP7-5A (AF220247), Singer Arg (DQ088995), 10JJ-SKR (KC757383), Oregon (AF091605), SuwaNCP (KC853440), New York ’93 (AF502399), SH-28 (HQ258810), JZ05-1 (GQ888686), 11F011 (KC963968), HLJ-10 (JF714967), D3200 HoBi (AB871953), CHKaHo/cont (JX985409), Th/04_KhonKaen (NC_012812), Shimen (AF092448), Alfort-187 (X87939), Gifhorn (KF925348), Giraffe-1 H138 (AF144617). The GenBank accession numbers of the Npro sequences of the BVDV-1 reference strains are as follows (those with the same virus strain and accession number have been omitted): 519(AF144464), Deer NZ1 (U80903), 721 (AF144463), F (AF287284), 3186V6 (AF287282), J (AF287286), W (AF287290), L (AF287287), A (AF287283), G (AF287285), 23-15 (AF287289), Deer GB1 (U80902), B440/06 (EU224257), CH-Suwa (AY894998), TR27 (EU163975), TR29 (EU163977), TJ0801 (GU120262), So CP75 (AB359929), Shitara (AB359930), IS25CP01 (AB359931), BJ0701 (GU120259), and BJ0703 (GU120261). The GenBank accession numbers of the 50 UTR sequences of the BVDV-1 reference strains are as follows (those with the same virus strain and accession number have been omitted): NADL (AJ133739), Manas-1 (EU555288), Shitara/01/05 (AB359926), Bega (AF049221), F (AF298065), 16-111 (AF298056), 20-V661-2 (AF298058), J (AF298067), W (AF298073), L (AF298069), A (AF298064), G (AF298066), KM (AF298068), 23-15 (AF298059), KS86-1ncp (AB078950), SuwaCp (AF117699), Rebe (AF299317), LZ05 (GU120241), NX0802 (GU120253), IS25CP/01 (AB359931), Shitara/02/06 (AB359930), So CP/75 (AB042661), Shihezi148 (EU159700), BJ0703 (GU120249), 890 (U18059), TJ06 (GU120246), TJ06 (GU120246), 11X80 (JX437153), 11X97 (JX437154), 11N36 (JX437156) 11N94 (JX437149)
were constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software, version 4.1, based on partial Npro genes, the 50 UTR, or the full genome [16]. The bootstrap values were based on 1000 replicates using the neighbor-joining method, and the evolutionary distances were determined using the Kimura two-parameter method. The complete genome of the SD0803 strain of BVDV-1 (GenBank accession no. JN400273) was 12,271 bp in length and contained a single ORF that was 11,694 bp in length, encoding a 3,898-amino-acid polypeptide. The 50 and 30 UTRs of the SD0803 virus were 388 and 186 bp in length, respectively. Based on the putative post-transcriptional processing sites of other BVDVs, the SD0803 genes and UTRs were mapped to the following nucleotide positions in the genome: 50 UTR, 1-388; Npro, 389-892; C, 893-1198; Erns, 1199-1879; E1, 1880-2464; E2, 2465-3586; P7, 3587-3796; NS2, 3797-5155; NS3,
Bovine viral diarrhea virus 1 from swine
5156-7204; NS4A, 7205-7396; NS4B, 7397-8437; NS5A, 8438-9925; NS5B, 9926-12085; 30 UTR,12086-12271. A phylogenetic tree was constructed based on the SD0803 genomic sequence and the complete genome sequences of 23 pestiviruses deposited in GenBank, which showed that SD0803 clustered with other BVDV-1 strains (Fig. 1a). The SD0803 genome shared nucleotide sequence identities of 78.8 % to 83.3 % with the other BVDV-1 strains, about 70.0 % with the BVDV-2 strains, about 67.0 % with HoBi-like viruses (Th/04_KhonKaen strain and D3200_HoBi), 66.6 % with the CSFV strain Alfort187, 67.2 % with the BDV strain Gifhorn, 67.6 % with the Giraffe-1_H138 strain (giraffe pestivirus), and 56.6 % with Bungowannah (Table S2) [2]. The highest degree of similarity observed was 83.3 % between the SD0803 virus and the ZM-95 strain of BVDV-1, which was isolated in 1995
from swine in China. The SD0803 genome exhibited 70.0 % sequence homology with the SH-28 strain of BVDV-2, which was also isolated from swine in China. Twelve genes of SD0803 were compared with the genes of six BVDV-1 strains and three BVDV-2 strains, respectively (Table 1). The sequences of the other BVDV-1 viruses exhibited a higher degree of similarity to those of SD0803 than to those of the BVDV-2 strains. With the exception of the Npro gene, the highest degree of shared identity was observed between the SD0803 and ZM-95 viruses. However, the ZM-95 virus is a CP strain of BVDV-1, whereas the SD0803 virus is an NCP strain of BVDV-1. In the comparison of the coding sequences of BVDV-1 and BVDV-2, the ZM-95 virus was shown to have a six-nucleotide insertion in the C gene, and NADL, CP7-5A and New York ’93 have insertions of 270, 27 and
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Y. Deng et al. Table 1 Nucleotide sequence identity (%) of the SD0803 virus, compared with the coding sequences of other isolates of BVDV-1 and BVDV-2
#
Sequences with an insertion in the C or NS2 gene were omitted
Strain
Npro
C
Erns
E1
E2
P7
NS2
NS3
NS4A
NS4B
NS5A
NS5B
ZM-95
80.8
79.8#
83.8
81.7
78.1
81.0
81.6
85.7
85.4
86.6
81.9
83.5
KE9
76.4
78.8
81.2
76.1
74.1
74.8
77.6
82.2
85.4
81.8
77.2
80.0
CP7-5A
78.6
77.8
79.9
76.6
74.1
74.3
77.1#
83.1
84.4
80.2
76.9
81.3
NADL
81.0
78.4
80.3
77.8
73.2
73.3
78.7#
84.2
81.8
83.1
77.1
79.3
Singer_Arg
80.2
77.1
81.4
77.6
73.0
74.3
78.2
83.6
82.3
82.4
77.3
79.3
SuwaNCP
78.2
76.8
80.5
77.6
71.9
79.5
79.5
82.1
85.4
81.9
76.4
79.7
JZ05-1
70.0
69.3
72.1
69.2
66.0
60.5
61.5
76.7
75.0
74.9
67.5
71.3
New York ’93
70.8
68.0
72.0
69.6
57.0
59.0
63.9#
77.8
71.9
75.5
66.3
70.9
SH-28
70.6
69.3
72.4
70.6
65.3
62.4
64.0
77.9
74.0
75.5
66.7
70.7
48 nucleotides, respectively, in the NS2 gene (Table 1) [20]. However, no insertions or deletions were revealed in the coding regions of the SD0803 virus when it was compared with the other six strains of BVDV-1 (ZM-95, NADL, CP7-5A, Singer_Arg, KE9 and SuwaNCP) and three strains of BVDV-2 (JZ05-1, New York ’93 and SH28). The shared identity of individual genes between the SD0803 virus and the other BVDV-1 strains ranged from 71.9 % to 86.6 % (Table 1). These results indicated considerable genetic divergence between the SD0803 virus and the other BVDV-1 strains. In a previous study, the SD0803 virus was classified as a BVDV-1 strain based on its 50 -UTR sequence, but it could not be further classified according to any recognized subgenotype [3]. In our current study, a phylogenetic tree was constructed based on the sequence of the Npro gene (Fig. 1b), and the SD0803 and KC207072 (camel isolate 6) viruses clustered in a branch that might represent a new subgenotype, BVDV-1q [5]. The branch of BVDV-1q was most closely related to the BVDV-1g, BVDV-1m, BVDV-1o, and BVDV-1p clusters. The Npro gene of SD0803 had 91.5 % sequence homology with the KC207072 virus. The Npro gene of SD0803 had 79.6 % to 83.5 % sequence homology with the strains classified as BVDV-1g, BVDV-1m, BVDV-1o, or BVDV-1p Four isolates of BVDVs, the 11N36, 11N94, 11X80, and 11X97 viruses, were recently isolated from dairy cattle in northwestern China. All four isolates were classified as belonging to a potentially novel subgenotype [7], and a partial 50 -UTR sequence of each isolate was obtained. To determine the genetic relationship between the SD0803 virus and these strains of a novel subgenotype, a phylogenetic tree was constructed based on of the 50 UTR sequences, which showed a single branch containing the four isolates clustered with the SD0803 virus (Fig. 1c). The 50 UTR of the SD0803 virus had a high degree of sequence homology (94.3 % to 98.7 %) to those of the 11N36, 11N94, 11X80, and 11X97 viruses, indicating that these viruses should thus be classified as strains of the BVDV-1q subgenotype. Similar to the phylogenetic tree based on the Npro sequences, the BVDV-1q branch was most closely
123
related to the BVDV-1m, BVDV-1o, BVDV-1p, and BVDV-1g clusters in the 50 -UTR-based phylogenetic tree. In summary, a virus belonging to the novel BVDV-1q subgenotype was first detected in swine in eastern China, and was also detected in camel and cattle in northwestern China [3, 5, 7]. We determined the complete sequence of the genome of the swine-associated SD0803 strain of BVDV-1. Phylogenetic and sequence analyses based on the Npro gene and the 50 UTR showed that the SD0803 virus is a strain of the BVDV-1q subgenotype. This is the first report of the genomic sequence of a BVDV-1q virus isolated from swine.
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