Virus Genes (2014) 49:339–341 DOI 10.1007/s11262-014-1067-7
Genetic characterization of a noncytopathic bovine viral diarrhea virus 2b isolated from cattle in China Wei Wang • Xinchuan Shi • Chaoyang Chen Hua Wu
•
Received: 12 February 2014 / Accepted: 1 April 2014 / Published online: 9 May 2014 Ó Springer Science+Business Media New York 2014
Abstract In January 2013, several clinical signs of cattle with diarrhea, cough, nasal discharge, and fever were reported in Jilin province, China. One virus named SD1301 was isolated and identified. Complete genome of the virus is 12258nt in length and contains a 50 UTR, one open reading frame encoding a polyprotein of 3,897 amino acids and a 30 UTR. Phylogenetic analysis of 50 UTR, Npro, E1 and E2 gene demonstrated the virus belonged to BVDV 2b, and genetically related to the BVDV strain Hokudai-Lab/09 from Japan in 2010. This bovine viral diarrhea virus displays a unique genetic signature with 27-nucleotide deletion in the 50 UTR, which is similar to the bovine viral diarrhea virus C413 (AF002227). This was the first confirmed isolation of ncp BVDV2b circulating in bovine herd of China. Keywords Bovine viral diarrhea virus type 2 Genome analysis China Bovine viral diarrhea virus (BVDV), a non-enveloped, single positive-stranded RNA virus in the genus Pestivirus of Flaviviridae family, is well known as one of pathogens resulting bovine respiratory disease complex (BRDC), a significant illness in cattle farm in the world [1]. The viral genome comprises of a 50 UTR, a single open reading frame (ORF), and a 30 UTR. The polyprotein was cleaved by the host and virus proteases into four structural proteins (C, W. Wang C. Chen Institute of Special Economic Animal and Plant Science, CAAS, No. 4899, Juye Street, Changchun 130122, China W. Wang X. Shi C. Chen H. Wu (&) Sinovet (Beijing) Biotechnology Co., Ltd., No. 5 Kaituo Street Haidian District, Beijing 100085, China e-mail: [email protected]
Erns, E1 and E2) and seven nonstructural proteins (Npro, p7, NS2-3, NS4A, NS4B, NS5A and NS5B) depending on the virus biotype [2]. In nature, two species of the virus (BVDV1 and BVDV2) are typed, based on the 50 UTR, Npro, E2 or NS3. BVDV1 can be further divided into at least 15 genetic subgroups and BVDV2 can be divided into four subgroups currently [3]. Additionally, two biotypes were classified as cytopathic (cp) or noncytopathic (ncp) depending upon the effect on cell culture [4]. In China, the first BVDV infection has been reported in 1980, when a virus strain isolated from an aborted fetus. The first BVDV2 isolate was reported in 2009 in Xinjiang [5]. Phylogeny analysis showed that the BVDV1b and BVDV2a were the predominant subgenotypes in China [6– 8]. In this study, genetic characterization of the BVDV2b virus was analyzed. Seven pair primers covering the whole genome were designed according to reference BVDV2 sequences from GenBank. Total RNA was extracted from the virus-infected MDBK cells using QIAamp viral RNA mini kit (Qiagen) according to the manufacturer’s instruction. RT-PCR was carried out using mRNA as template. The target fragment of PCR products were purified and cloned into pMD18-T vector (TaKaRa, Japan), and then transfected into E.coli DH5a cells. The positive clones were selected for sequencing by Shanghai Invitrogen Biotechnology Co., Ltd. The complete genome of the strain of BVDV2 SD1301 was of 12,258 nucleotides in length and contains a single ORF of 11694nt, a 50 UTR was of 360nt, and a 30 UTR was of 204nt. The complete genome sequence was submitted to GenBank with the accession number of KJ000672. The ORF encodes a polyprotein consisting of 3,897 amino acids and is cleaved into four structural proteins and eight nonstructural proteins. The lengths of each
123
340
Virus Genes (2014) 49:339–341
Fig. 1 Phylogenetic analysis based on 50 UTR, Npro, E1 and E2.A. Phylogenetic tree analysis of the each gene was prepared using the NeighborJoining method and the bootstrap analysis (n = 1,000) by the software MEGA v 5.0
coding sequence is as follows: C (306nt), Erns (681nt), E1 (585nt), E2 (1116nt), and eight non-structural proteins that is Npro (504nt), p7 (210nt), NS2 (1359nt), NS3 (2049nt), NS4A (192nt), NS4B (1041nt), NS5A (1491nt), NS5B (2157nt). Phylogenetic trees were constructed based on the 50 UTR, Npro, E1 and E2 (Fig. 1). Results showed that the BVDV strain SD1301 having highest identity with the strain Hokudai-Lab/09 (89.6 %) originated from Japan in 2011, belonging to BVDV 2b subgenotype. The phylogenetic tree for the Npro, E1 and E2 regions had a topology similar to that of 50 UTR, which clustered with BVDV 2b. The nucleotide coding for E2 protein of SD1301 had a highest identity of 87.6 % with Hokudai-Lab09, and the amino acid had an identity for 87.7 %, which suggested
123
that the virus has the different antigenicity with other strains. The identity values with BVDV 2 reference sequences ranged from 85.5 to 93.9 % for 50 UTR, 82.3 to 89.3 % for Npro, 80 to 89.3 % for E1 and 80.6 to 87.6 % for E2, respectively (Table 1). Comparison showed that a 27-nucleotide deletion was founded in the 50 UTR, which was similar with the strain of C413 (AF002227) which deleted 30 nucleotides. Some spots of amino acid have substitutions in NS2 and Npro proteins. This may be suggested that the virus may be a high virulent virus strain [9, 10]. Before this study, only genotype of BVDV 2a was circulating the cattle and swine in China [6, 11]. It is also possible that BVDV 2b might have been introduced from other countries through historical trade contacts.
Virus Genes (2014) 49:339–341 Table 1 Nucleotide identity of SD1301 with reference sequences
ND not determined
341
Strain
Sequence accession
Biotype
Place
Nucleotide identity (%) ORF
50 UTR
Npro
Erns
E1
E2
Hokudai-Lab/09
AB567658
NCP
Japan
89.5
93.9
89.3
90.3
89.2
87.6
HLJ-10
JF714967
CP
China
84.9
89.7
84.3
85.5
81.7
83.3
11F011
KC963968
CP
Korea
84.4
87.9
83.9
86.0
81.0
82.3
SH-28
HQ258810
NCP
China
84.6
90.7
83.3
85.6
82.6
80.6
XJ-04
FJ527854
CP
China
84.9
88.8
84.9
85.9
83.1
84.4
JZ05-1
GQ888686
CP
China
85.8
88.3
82.3
86.6
81.4
84.1
New York’93
AF502399
NCP
Germany
83.9
86.4
82.9
86.8
80.7
82.5
C413
NC_002032
Unknown
USA
84.3
86.9
83.5
86.0
81.2
81.5
1,373
AF145957
NCP
USA
83.8
85.5
83.1
86.6
80.3
82.5
p11Q#
AY149215
NCP
Canada
84.5
86.9
83.9
86.8
81.2
82.0
PI99 RNV17
JN380086 JN380090
NCP NCP
USA USA
84.8 ND
ND ND
84.1 ND
87.2 86.8
80.2 80.0
84.1 84.4
296nc
AF145969
NCP
USA
ND
ND
ND
86.9
80.5
ND
With the rapid development of livestock farm, the number of pigs reached 451 million, cattle for 100 million [12]. Emergence of the new ncp BVDV 2b was more complex and need more highlight the infection of BVDV. This new found give us a support to pay more attention to the BVDV in China. It should take appropriate measures to control the disease and take a surveillance program in a larger livestock population in vast geographical areas. Acknowledgments This work was performed under the auspices of the ‘‘Special Fund for Agro-scientific Research in the Public Interest Project No. 201003060’’and we are thankful to SINOVET (BEIJING) BIOTEHCHNOLOGY CO., LTD., for providing the necessary funds and facilities to carry out this work.
References
2. J.F. Ridpath, S.R. Bolin, Virology 212, 39–46 (1995) 3. S. Peletto, F. Zuccon, M. Pitti, E. Gobbi, L.D. Marco, M. Caramelli, L. Masoero, P.L. Acutis, Res. Vet. Sci. 92, 147–150 (2012) 4. J. Pollakova, T. Csank, I. Pilipcincova, J. Pistl, Acta Virol. 53, 287–289 (2009) 5. L.Q. Zhu, Y.Q. Lin, X.Y. Ding, M. Ren, J. Tao, J.Y. Wang, G.P. Zhang, G.Q. Zhu, Acta Virol. 53, 197–202 (2009) 6. J. Tao, Y. Wang, J. Wang, J.Y. Wang, G.Q. Zhu, Virus Genes 46, 81–87 (2013) 7. X. Gong, X. Cao, F. Zheng, Q. Chen, J. Zhou, H. Yin, L. Liu, X. Cai, Virus Genes 46, 375–376 (2013) 8. F. Xue, Y.M. Zhu, J. Li, L.C. Zhu, X.G. Ren, J.K. Feng, H.F. Shi, Y.R. Gao, Vet. Microbiol. 143, 379–383 (2010) 9. D. Deregt, R.M. Jacobs, P.S. Carman, S.V. Tessaro, Vet. Microbiol. 100, 151–161 (2004) 10. Z.C. Fan, R.C. Bird, Virus Genes 41, 218–223 (2010) 11. L.Q. Zhu, M. Ren, Y.Q. Lin, X.Y. Ding, G.P. Zhang, X. Zhao, G.Q. Zhu, Acta Virol. 53, 131–134 (2009) 12. B. Chen, H. Yuan, China Agricultural Yearbook (China Agricultural Press, Beijing, 2011)
1. H. Houe, Vet. Clin. North Am. Food Anim. Pract. 11, 521–547 (1995)
123
Genetic characterization of a noncytopathic bovine viral diarrhea virus 2b isolated from cattle in China Wei Wang • Xinchuan Shi • Chaoyang Chen Hua Wu
•
Received: 12 February 2014 / Accepted: 1 April 2014 / Published online: 9 May 2014 Ó Springer Science+Business Media New York 2014
Abstract In January 2013, several clinical signs of cattle with diarrhea, cough, nasal discharge, and fever were reported in Jilin province, China. One virus named SD1301 was isolated and identified. Complete genome of the virus is 12258nt in length and contains a 50 UTR, one open reading frame encoding a polyprotein of 3,897 amino acids and a 30 UTR. Phylogenetic analysis of 50 UTR, Npro, E1 and E2 gene demonstrated the virus belonged to BVDV 2b, and genetically related to the BVDV strain Hokudai-Lab/09 from Japan in 2010. This bovine viral diarrhea virus displays a unique genetic signature with 27-nucleotide deletion in the 50 UTR, which is similar to the bovine viral diarrhea virus C413 (AF002227). This was the first confirmed isolation of ncp BVDV2b circulating in bovine herd of China. Keywords Bovine viral diarrhea virus type 2 Genome analysis China Bovine viral diarrhea virus (BVDV), a non-enveloped, single positive-stranded RNA virus in the genus Pestivirus of Flaviviridae family, is well known as one of pathogens resulting bovine respiratory disease complex (BRDC), a significant illness in cattle farm in the world [1]. The viral genome comprises of a 50 UTR, a single open reading frame (ORF), and a 30 UTR. The polyprotein was cleaved by the host and virus proteases into four structural proteins (C, W. Wang C. Chen Institute of Special Economic Animal and Plant Science, CAAS, No. 4899, Juye Street, Changchun 130122, China W. Wang X. Shi C. Chen H. Wu (&) Sinovet (Beijing) Biotechnology Co., Ltd., No. 5 Kaituo Street Haidian District, Beijing 100085, China e-mail: [email protected]
Erns, E1 and E2) and seven nonstructural proteins (Npro, p7, NS2-3, NS4A, NS4B, NS5A and NS5B) depending on the virus biotype [2]. In nature, two species of the virus (BVDV1 and BVDV2) are typed, based on the 50 UTR, Npro, E2 or NS3. BVDV1 can be further divided into at least 15 genetic subgroups and BVDV2 can be divided into four subgroups currently [3]. Additionally, two biotypes were classified as cytopathic (cp) or noncytopathic (ncp) depending upon the effect on cell culture [4]. In China, the first BVDV infection has been reported in 1980, when a virus strain isolated from an aborted fetus. The first BVDV2 isolate was reported in 2009 in Xinjiang [5]. Phylogeny analysis showed that the BVDV1b and BVDV2a were the predominant subgenotypes in China [6– 8]. In this study, genetic characterization of the BVDV2b virus was analyzed. Seven pair primers covering the whole genome were designed according to reference BVDV2 sequences from GenBank. Total RNA was extracted from the virus-infected MDBK cells using QIAamp viral RNA mini kit (Qiagen) according to the manufacturer’s instruction. RT-PCR was carried out using mRNA as template. The target fragment of PCR products were purified and cloned into pMD18-T vector (TaKaRa, Japan), and then transfected into E.coli DH5a cells. The positive clones were selected for sequencing by Shanghai Invitrogen Biotechnology Co., Ltd. The complete genome of the strain of BVDV2 SD1301 was of 12,258 nucleotides in length and contains a single ORF of 11694nt, a 50 UTR was of 360nt, and a 30 UTR was of 204nt. The complete genome sequence was submitted to GenBank with the accession number of KJ000672. The ORF encodes a polyprotein consisting of 3,897 amino acids and is cleaved into four structural proteins and eight nonstructural proteins. The lengths of each
123
340
Virus Genes (2014) 49:339–341
Fig. 1 Phylogenetic analysis based on 50 UTR, Npro, E1 and E2.A. Phylogenetic tree analysis of the each gene was prepared using the NeighborJoining method and the bootstrap analysis (n = 1,000) by the software MEGA v 5.0
coding sequence is as follows: C (306nt), Erns (681nt), E1 (585nt), E2 (1116nt), and eight non-structural proteins that is Npro (504nt), p7 (210nt), NS2 (1359nt), NS3 (2049nt), NS4A (192nt), NS4B (1041nt), NS5A (1491nt), NS5B (2157nt). Phylogenetic trees were constructed based on the 50 UTR, Npro, E1 and E2 (Fig. 1). Results showed that the BVDV strain SD1301 having highest identity with the strain Hokudai-Lab/09 (89.6 %) originated from Japan in 2011, belonging to BVDV 2b subgenotype. The phylogenetic tree for the Npro, E1 and E2 regions had a topology similar to that of 50 UTR, which clustered with BVDV 2b. The nucleotide coding for E2 protein of SD1301 had a highest identity of 87.6 % with Hokudai-Lab09, and the amino acid had an identity for 87.7 %, which suggested
123
that the virus has the different antigenicity with other strains. The identity values with BVDV 2 reference sequences ranged from 85.5 to 93.9 % for 50 UTR, 82.3 to 89.3 % for Npro, 80 to 89.3 % for E1 and 80.6 to 87.6 % for E2, respectively (Table 1). Comparison showed that a 27-nucleotide deletion was founded in the 50 UTR, which was similar with the strain of C413 (AF002227) which deleted 30 nucleotides. Some spots of amino acid have substitutions in NS2 and Npro proteins. This may be suggested that the virus may be a high virulent virus strain [9, 10]. Before this study, only genotype of BVDV 2a was circulating the cattle and swine in China [6, 11]. It is also possible that BVDV 2b might have been introduced from other countries through historical trade contacts.
Virus Genes (2014) 49:339–341 Table 1 Nucleotide identity of SD1301 with reference sequences
ND not determined
341
Strain
Sequence accession
Biotype
Place
Nucleotide identity (%) ORF
50 UTR
Npro
Erns
E1
E2
Hokudai-Lab/09
AB567658
NCP
Japan
89.5
93.9
89.3
90.3
89.2
87.6
HLJ-10
JF714967
CP
China
84.9
89.7
84.3
85.5
81.7
83.3
11F011
KC963968
CP
Korea
84.4
87.9
83.9
86.0
81.0
82.3
SH-28
HQ258810
NCP
China
84.6
90.7
83.3
85.6
82.6
80.6
XJ-04
FJ527854
CP
China
84.9
88.8
84.9
85.9
83.1
84.4
JZ05-1
GQ888686
CP
China
85.8
88.3
82.3
86.6
81.4
84.1
New York’93
AF502399
NCP
Germany
83.9
86.4
82.9
86.8
80.7
82.5
C413
NC_002032
Unknown
USA
84.3
86.9
83.5
86.0
81.2
81.5
1,373
AF145957
NCP
USA
83.8
85.5
83.1
86.6
80.3
82.5
p11Q#
AY149215
NCP
Canada
84.5
86.9
83.9
86.8
81.2
82.0
PI99 RNV17
JN380086 JN380090
NCP NCP
USA USA
84.8 ND
ND ND
84.1 ND
87.2 86.8
80.2 80.0
84.1 84.4
296nc
AF145969
NCP
USA
ND
ND
ND
86.9
80.5
ND
With the rapid development of livestock farm, the number of pigs reached 451 million, cattle for 100 million [12]. Emergence of the new ncp BVDV 2b was more complex and need more highlight the infection of BVDV. This new found give us a support to pay more attention to the BVDV in China. It should take appropriate measures to control the disease and take a surveillance program in a larger livestock population in vast geographical areas. Acknowledgments This work was performed under the auspices of the ‘‘Special Fund for Agro-scientific Research in the Public Interest Project No. 201003060’’and we are thankful to SINOVET (BEIJING) BIOTEHCHNOLOGY CO., LTD., for providing the necessary funds and facilities to carry out this work.
References
2. J.F. Ridpath, S.R. Bolin, Virology 212, 39–46 (1995) 3. S. Peletto, F. Zuccon, M. Pitti, E. Gobbi, L.D. Marco, M. Caramelli, L. Masoero, P.L. Acutis, Res. Vet. Sci. 92, 147–150 (2012) 4. J. Pollakova, T. Csank, I. Pilipcincova, J. Pistl, Acta Virol. 53, 287–289 (2009) 5. L.Q. Zhu, Y.Q. Lin, X.Y. Ding, M. Ren, J. Tao, J.Y. Wang, G.P. Zhang, G.Q. Zhu, Acta Virol. 53, 197–202 (2009) 6. J. Tao, Y. Wang, J. Wang, J.Y. Wang, G.Q. Zhu, Virus Genes 46, 81–87 (2013) 7. X. Gong, X. Cao, F. Zheng, Q. Chen, J. Zhou, H. Yin, L. Liu, X. Cai, Virus Genes 46, 375–376 (2013) 8. F. Xue, Y.M. Zhu, J. Li, L.C. Zhu, X.G. Ren, J.K. Feng, H.F. Shi, Y.R. Gao, Vet. Microbiol. 143, 379–383 (2010) 9. D. Deregt, R.M. Jacobs, P.S. Carman, S.V. Tessaro, Vet. Microbiol. 100, 151–161 (2004) 10. Z.C. Fan, R.C. Bird, Virus Genes 41, 218–223 (2010) 11. L.Q. Zhu, M. Ren, Y.Q. Lin, X.Y. Ding, G.P. Zhang, X. Zhao, G.Q. Zhu, Acta Virol. 53, 131–134 (2009) 12. B. Chen, H. Yuan, China Agricultural Yearbook (China Agricultural Press, Beijing, 2011)
1. H. Houe, Vet. Clin. North Am. Food Anim. Pract. 11, 521–547 (1995)
123
