Infection, Genetics and Evolution 27 (2014) 230–233
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
Genetic diversity of bovine viral diarrhea viruses in commercial bovine serum batches of Chinese origin Shu-Qin Zhang, Bin Tan, Li Guo, Feng-Xue Wang, Hong-Wei Zhu, Yong-Jun Wen, Shipeng Cheng ⇑ State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 18 April 2014 Received in revised form 14 July 2014 Accepted 17 July 2014 Available online 4 August 2014 Keywords: Bovine viral diarrhea virus (BVDV) Bovine serum Genetic diversity
a b s t r a c t Bovine viral diarrhea virus (BVDV) is often detected in commercial bovine serum. BVDV genetic diversity was investigated in commercial bovine serum of Chinese origin. Twenty-two batches of bovine serum were obtained from 10 suppliers with different geographic origins in China, and 20 batches of bovine serum were positive by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. Phylogenetic reconstructions of partial 50 UTR sequences indicated that the samples examined in this work clustered within the BVDV type 1 and BVDV type 2 genotypes. Interestingly, 3 sample sequences clustered into CSFV. These results suggest a high genetic diversity in Chinese BVDV field isolates. This study will benefit epidemiological surveys of BVDV detected in China. Ó 2014 Elsevier B.V. All rights reserved.
Bovine viral diarrhea virus (BVDV), the etiological agent of bovine viral diarrhea/mucosal disease, is a worldwide pathogen in cattle that causes significant economic losses to agriculture (Xue et al., 2011). BVDV, together with classical swine fever virus (CSFV) and sheep border disease virus (BDV), belongs to the genus Pestivirus in the Flaviviridae family (Byers et al., 2009). Recently, atypical bovine pestiviruses such as Th/04_KhonKaen virus, D32/ 00-‘HoBi’ and CHKaHo/cont have been referred to as BVDV-3 (Liu et al., 2009a). Bovine viral diarrhea virus infection was first reported in Olafson et al. (1946). Subsequently, BVDV was found to be epidemic worldwide in cattle farms. In the intervening 66 years, many important advances have been made in understanding this virus and the disease it produces. However, in China, progress with BVDV has been limited. To date, no commercial BVDV vaccines are available on the market, and no management systems or control programs have been conducted in China. The genetic diversity of BVDV needs to be considered when designing and constructing effective vaccination strategies for the virus, and a vaccine must accurately reflect the genotypes and antigenic types present in the country of use (Mahony et al., 2005). The genetic and antigenic diversity of BVDV are not well known in China. Bovine serum is an important material in medicine and biological products. However, bovine serum is often contaminated by ⇑ Corresponding author. Address: State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Science, No. 4899 Juye Street, Changchun 130122, People’s Republic of China. Tel./fax: +86 0431 81919845. E-mail address: [email protected] (S. Cheng). http://dx.doi.org/10.1016/j.meegid.2014.07.021 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
bovine viral diarrhea virus (Bolin and Ridpath, 1998). Recently, atypical bovine pestiviruses were detected in many commercial fetal bovine serum samples from different geographic origins (Xia et al., 2011). Every batch of commercially available bovine serum is a mixture of collected raw serum material from different cattle farms near the supplier. Therefore, commercial bovine serum is a good material for investigating BVDV genetic diversity. The purpose of this work was to determine the genotypes of BVDV detected in commercial bovine serum in China. A total of 22 batches of bovine serum were purchased from 10 commercial suppliers. The 22 batches of bovine serum came from 8 provinces in China (Table 1). Total RNA was extracted from serum samples using TRIZOL (Invitrogen, China) according to the manufacturer’s instructions. The extracted RNA was reverse-transcribed using the M-MLV Reverse Transcriptase Kit (Invitrogen, USA) as specified by the manufacturer. The 50 UTR primers were selected as described previously (Ridpath and Bolin, 1998). The 50 UTR genomic region provides meaningful inferences as this region has the highest degree of sequence conservation and is effectively amplified by RT-PCR (Vilcek et al., 2001). The 50 UTR primer sequences were as follows: sense primer: 50 CAT GCC CAT AGT AGG AC 30 ; antisense primer: 50 CCA TGT GCC ATGTAC AG 30 , and the primers are not reliable for amplifying HoBi-like viruses. Similar 280 bp fragments of the 50 UTR region were amplified from 20 batches of bovine serum, and the amplified fragments were purified and cloned into a pMD18-T vector (TaKaRa, Japan), and 3 clones of every sample were sequenced. Sixty sequences from 20 batches of bovine serum were found, after sequence alignment,
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S.-Q. Zhang et al. / Infection, Genetics and Evolution 27 (2014) 230–233 Table 1 The batches of bovine serum tested by RT-PCR. Origin
Supplier
Sample ID
NMG
A
1 4
JL
B G
TJ
C D
BVDV-1
BVDV2 10
CSFV
9
S11, S12, S13 S41, S42, S43
1 m(S11 , S12, S13 ) 1 m(S41, S429, S433)
2 3 15 16
S21, S22, S23 S31, S32, S33 S151, S152, S153 S161, S162, S163
1 m(S22, S21, S2310) 1 m(S313) 1b(S328, S338) 1a(S1516, S152), 1p(S1534) 1 m(S1619), 1b(S1627, S1638)
7 8 9 10
S71, S72, S73 S81, S82, S83 S91, S92, S93 S101, S102, S103
1 m(S71, S72, S7312) 1 m(S8111, S8211), 1q(S832) 1a(S916, S926), 1b(S937) 1 m(S101, S102), 1b(S1037)
S51, S52, S53 S61, S62, S63
1 m(S52), 1q(S531) 1 m(S6210, S6310), 1q(S611)
SD
E
5 6
GS
F
12 13
S121, S122, S123 S131, S132, S133
1 m(S121, S122, S123) 1 m(S133), 1q (S1311, S1321)
HN
H
17 19
S171, S172, S173 S191, S192, S193
1 m(S1939), 1q (S1912, S1922)
S5113
S17213
HLJ
I
14 18
S141, S142, S143 S181, S182, S183
1 m(S1413), 1c(S1425) 1e(S182), 1c(S1835), 1a(S181)
JS
J
11 20
S111, S112, S113 S201, S202, S203
1 m(S11112, S11312) 1a(S201, S202), 1p(S2034)
S171,S17314
S143 S11214
Superscript: the same number means consensus sequence.
the consensus sequences were eliminated, thirty-four different sequences from 20 batches of bovine serum were found, containing at least one species of pestiviruses. The 34 newly determined 50 UTR sequences were deposited in GenBank with the accession numbers KF006955–KF006975 and KJ690679–KJ690691. The BVDV 50 UTR of the samples were aligned with the corresponding regions of the pestivirus reference sequences retrieved from GenBank using the Clustal W program in MegAlign of Lasergene 7.2 software (DNASTAR Inc. Madison, WI, USA). Then, phylogenetic trees were constructed based on the 251 nucleotide region of the 50 UTR. Bootstrap values were based on 1000 replicates using the neighbor-joining method (Kimura two-parameter method) with Molecular Evolutionary Genetics Analysis (MEGA version 4.1) software. Phylogenetic analysis indicated that the sequences belonging to BVDV-1 should be further classified into 8 subgenotypes: BVDV-1 m (29/60), BVDV-1b (6/60), BVDV-1a (7/60), BVDV-1c (2/60), BVDV-1p (2/60), BVDV-1e (1/60) and BVDV-1q (7/60). Of these sequences, BVDV-1 m was the most frequently identified, strain ZM-95 was the first isolated BVDV-1 m in China (Xu et al., 2006). Of these sequences, 29 sequences from 15 batches of bovine serum belonged to BVDV-1 m; the percentage of BVDV-1 m was 48.33% (29/60), and nucleotide homology among these sequences was in the range 91.1–100%. BVDV-1b is considered to be a predominant BVDV-1 strain circulating in Chinese cattle. In this study, 6 sequences from 4 batches of bovine serum belonged to BVDV-1b, with the percentage being 10% (6/60). Based on this result and a previous report (Xue et al., 2010), the percentage of BVDV-1 m identification was higher than that of BVDV-1b. Hence, BVDV-1 m is considered to be more predominant than BVDV-1b in Chinese cattle population. BVDV-1p and BVDV-1c were found in 2 sequences from 2 batches of bovine serum. BVDV-1a is the usual subtype of BVDV-1 vaccines, which is dominant in the UK and widely distributed in USA. Likewise, the BVDV isolates predominating in a neighboring country, namely Korean (Booth et al., 2013; Oem et al., 2009). BVDV-1a has been found in Chinese pig herds but has been unique reported in the Chinese cattle population. In this study, BVDV-1a was found in 7 sequences from 4 batches of bovine serum. BVDV-1q is a newly identified subgenotype from cattle, pigs and camels in China (Deng et al.,
2012; Gao et al., 2013; Gong et al., 2013). In this study, there are 7 sequences from 5 batches of bovine serum that belonged to BVDV-1q. BVDV1e was isolated in Italian Austria and UK (Booth et al., 2013). It had not yet been found in China; however, in this study, only one sequence was found in 1 batch of bovine serum. BVDV-1 h and BVDV-1f are the predominant BVDV1 strain prevalent in Australia (Mahony et al., 2005), but it was not detected in this work (see Fig. 1). To date, BVDV-2 has been grouped into at least four BVDV-2 subtypes (BVDV-2a–BVDV-2d) (Flores et al., 2002; Mishra et al., 2008; Vilcek et al., 2004).A BVDV-2 strain, SD-06, was first isolated from cattle in China (Zhu et al., 2009b). Subsequently, many BVDV2 strains were isolated from cattle and pigs in China (Liu et al., 2012; Tao et al., 2013; Zhu et al., 2009a), which belonged to BVDV-2a. Compared with the endemic situation of BVDV-1, the prevalence of BVDV-2 infection in cattle is seemingly much lower in China. In this study, 3 sequences from 3 batches of bovine serum belonged to BVDV-2, and phylogenetic analysis showed that they formed a distinct branch from other previously reported isolates. The sequences belong to BVDV-2b, with a 93.7–95% nucleotide sequence homology to BVDV-2b reference strain VS-123.4 and 34b (Flores et al., 2002). Bovine viral diarrhea virus, classical swine fever virus and border disease virus are all members of the genus pestivirus. However, in contrast with the wide host range of BVDV, CSFV only infects pig and wild boars. In this study, phylogenetic analysis showed that 3 sequences from 2 batches of bovine serum belonged to CSFV. This result is in agreement with a previous observation that BVDV strain HEN03 (KC176778) was found in cattle blood from Henan province. The Npro and E2 genomic region are important to the pestivirus genotype, attempt to amplify these genes and phylogenetic analysis. It is possible that the storage conditions and time of the collection of these samples were poor, these genes could not been amplified in many of these samples, although we did try many times (data not shown). Early in 1957, Yuan Qingzhi, the inventor of the Chinese hog cholera lapinized vaccine strain, his studies have shown that the vaccine strain can be infected and breeding in cow, and the Cattle Organization vaccine of the Chinese hog cholera lapinized vaccine strain were produced and
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S.-Q. Zhang et al. / Infection, Genetics and Evolution 27 (2014) 230–233
Fig. 1. Phylogenetic tree illustrating the subgenotypes of BVDV isolates based on the 50 UTR. The phylogenetic tree was constructed using the neighbor-joining method implemented in MEGA 4.1. Bootstrap values are the percentage of 1000 replicates and are shown below the branches. The bar indicates the substitutions per site. Accession numbers are listed in the brackets after the isolate names. The 34 newly sequence from commercial bovine serum in this study are designated with ‘‘N’’.
S.-Q. Zhang et al. / Infection, Genetics and Evolution 27 (2014) 230–233
widely applied in China. There are no commercial BVDV vaccines in the Chinese market at present. BVDV was prevented by the Chinese hog cholera lapinized vaccine strain in Tibet and Qinghai province. More studies are needed to determine whether these sequences belong to CSFV or are derived from a BVDV variant. In this study, 22 batches of bovine sera were investigated, 20 batches were positive for BVDV by RT-PCR. 3 clones of every sample were sequenced. All together 60 sequences from 20 batches of bovine serum were found, after sequence alignment, some consensus sequences were found in the same or different batch bovine serum origin. The consensus sequences were marked with superscript, the same number means same sequence (Table 1). The consensus sequences in the same batch bovine serum maybe the repeat clone. These samples were collected from 10 suppliers in 8 provinces distributed throughout different areas of China. These results imply that BVDV is highly prevalent and has considerable genetic divergence among Chinese cattle herds. But, the genetic diversity of bovine viral diarrhea viruses in every province has not yet been determined, and there are many questions that require careful investigation. For example, a possible scenario is that mixing of raw serum material from a geographical location close to the province of origin might have occurred at certain companies during manufacturing steps. Another possible scenario is that the cattle and cattle production trade in different regions could facilitate virus transmission. This could explain why many of the consensus sequences were found in serum samples with different provincial origins. This study demonstrated that commercial bovine serum products with different geographic origins in China are contaminated with at least one species of pestivirus: BVDV-1, BVDV-2 and a virus similar to CSFV. Only one BVDV-2 isolated from these samples. Nevertheless, virus was isolated from 93 of 190 lots of commercially available fetal calf serum at the National Animal Disease Center in USA in 1991 (Bolin et al., 1991). Recently, a BVDV-2 strain, HLJ-10, was isolated from contaminated fetal bovine serum (Liu et al., 2012). Contaminated bovine serum can interfere with the diagnosis of viral infection, and vaccines produced using infected cell cultures could lead to seroconversion or disease in the vaccinated animal (Bolin and Ridpath, 1998). In China, an abundance of bovine sera has been used in the production of live vaccines against PRRSV, PRV and CSFV in pig herds. CSF live vaccines have been found to be contaminated with BVDV at the China Institute of Veterinary Drug Control (Fan et al., 2010). This may be one of the reasons why both pig and cattle herds have the same predominant prevalent subgenotypes in China. Acknowledgments This study was supported by the basic scientific research funding of the Chinese academy of Agricultural Sciences (0032014016), the Project Development Plan of Science and Technology of Jilin Province (20130206024NY) and National Public Welfare of China for Agriculture Special Purpose (201003060). References Bolin, S.R., Ridpath, J.F., 1998. Prevalence of bovine viral diarrhea virus genotypes and antibody against those viral genotypes in fetal bovine serum. J. Vet. Diagn. Invest. 10, 135–139.
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Bolin, S.R., Matthews, P.J., Ridpath, J.F., 1991. Methods for detection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus. J. Vet. Diagn. Invest. 3, 199–203. Booth, R.E., Thomas, C.J., El-Attar, L.M., Gunn, G., Brownlie, J., 2013. A phylogenetic analysis of bovine viral diarrhoea virus (BVDV) isolates from six different regions of the UK and links to animal movement data. Vet. Res. 44, 43. http:// dx.doi.org/10.1186/1297-9716-44-43. Byers, S.R., Snekvik, K.R., Righter, D.J., Evermann, J.F., Bradway, D.S., Parish, S.M., Barrington, G.M., 2009. Disseminated bovine viral diarrhea virus in a persistently infected alpaca (Vicugna pacos) cria. J. Vet. Diagn. Invest. 21, 145– 148. Deng, Y., Sun, C.Q., Cao, S.J., Lin, T., Yuan, S.S., Zhang, H.B., Zhai, S.L., Huang, L., Shan, T.L., Zheng, H., Wen, X.T., Tong, G.Z., 2012. High prevalence of bovine viral diarrhea virus 1 in Chinese swine herds. Vet. Microbiol. 159, 490–493. Fan, X.Z., Ning, Y.B., Wang, Q., Xu, L., Shen, Q.C., 2010. Detection of bovine viral diarrhea virus as contaminant in classical swine fever virus live vaccine with RT-PCR. Chin. J. Vet. Med. 46, 8–10. Flores, E.F., Ridpath, J.F., Weiblen, R., Vogel, F.S., Gil, L.H., 2002. Phylogenetic analysis of Brazilian bovine viral diarrhea virus type 2 (BVDV-2) isolates: evidence for a subgenotype within BVDV-2. Virus Res. 87, 51–60. Gao, S., Luo, J., Du, J., Lang, Y., Cong, G., Shao, J., Lin, T., Zhao, F., Belak, S., Liu, L., Chang, H., Yin, H., 2013. Serological and molecular evidence for natural infection of Bactrian camels with multiple subgenotypes of bovine viral diarrhea virus in Western China. Vet. Microbiol. 163, 172–176. Gong, X., Cao, X., Zheng, F., Chen, Q., Zhou, J., Yin, H., Liu, L., Cai, X., 2013. Identification and characterization of a novel subgenotype of bovine viral diarrhea virus isolated from dairy cattle in Northwestern China. Virus Genes 46, 375–376. Liu, H., Li, Y., Gao, M., Wen, K., Jia, Y., Liu, X., Zhang, W., Ma, B., Wang, J., 2012. Complete genome sequence of a bovine viral diarrhea virus 2 from commercial fetal bovine serum. J. Virol. 86, 10233. Liu, L., Kampa, J., Belak, S., Baule, C., 2009. Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen. Vet. Microbiol. 138, 62– 68. Mishra, N., Rajukumar, K., Vilcek, S., Tiwari, A., Satav, J.S., Dubey, S.C., 2008. Molecular characterization of bovine viral diarrhea virus type 2 isolate originating from a native Indian sheep (Ovies aries). Vet. Microbiol. 130, 88–98. Mahony, T.J., McCarthy, F.M., Gravel, J.L., Corney, B., Young, P.L., Vilcek, S., 2005. Genetic analysis of bovine viral diarrhoea viruses from Australia. Vet. Microbiol. 106, 1–6. Olafson, P., Mac, C.A., Fox, F.H., 1946. An apparently new transmissible disease of cattle. Cornell Vet. 36, 205–213. Oem 1, J.K., Hyun, B.H., Cha, S.H., Lee, K.K., Kim, S.H., Kim, H.R., Park, C.K., Joo, Y.S., 2009. Phylogenetic analysis and characterization of Korean bovine viral diarrhea viruses. Vet. Microbiol. 139 (3–4), 356–360. http://dx.doi.org/ 10.1016/j.vetmic.2009.06.017. Ridpath, J.F., Bolin, S.R., 1998. Differentiation of types 1a, 1b and 2 bovine viral diarrhoea virus (BVDV) by PCR. Mol. Cell. Prob. 12, 101–106. Tao, J., Wang, Y., Wang, J., Wang, J.Y., Zhu, G.Q., 2013. Identification and genetic characterization of new bovine viral diarrhea virus genotype 2 strains in pigs isolated in China. Virus Genes 46, 81–87. Vilcek, S., Durkovic, B., Kolesarova, M., Greiser-Wilke, I., Paton, D., 2004. Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group. Vet. Res. 35, 609–615. Vilcek, S., Strojny, L., Durkovic, B., Rossmanith, W., Paton, D., 2001. Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RTPCR method. J. Virol. Methods 92, 19–22. Xia, H., Vijayaraghavan, B., Belak, S., Liu, L., 2011. Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches. PloS One 6, e28553. Xu, X., Zhang, O., Yu, X., Liang, L., Xiao, C., Xiang, H., Tu, C., 2006. Sequencing and comparative analysis of a pig bovine viral diarrhea virus genome. Virus Res. 122, 164–170. Xue, F., Zhu, Y.M., Li, J., Zhu, L.C., Ren, X.G., Feng, J.K., Shi, H.F., Gao, Y.R., 2010. Genotyping of bovine viral diarrhea viruses from cattle in China between 2005 and 2008. Vet. Microbiol. 143, 379–383. Xue, W., Mattick, D., Smith, L., 2011. Protection from persistent infection with a bovine viral diarrhea virus (BVDV) type 1b strain by a modified-live vaccine containing BVDV types 1a and 2, infectious bovine rhinotracheitis virus, parainfluenza 3 virus and bovine respiratory syncytial virus. Vaccine 29, 4657–4662. Zhu, L.Q., Lin, Y.Q., Ding, X.Y., Ren, M., Tao, J., Wang, J.Y., Zhang, G.P., Zhu, G.Q., 2009a. Genomic sequencing and characterization of a Chinese isolate of Bovine viral diarrhea virus 2. Acta Virol. 53, 197–202. Zhu, L.Q., Ren, M., Lin, Y.Q., Ding, X.Y., Zhang, G.P., Zhao, X., Zhu, G.Q., 2009b. Identification of a bovine viral diarrhea virus 2 isolated from cattle in China. Acta Virol. 53, 131–134.
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
Genetic diversity of bovine viral diarrhea viruses in commercial bovine serum batches of Chinese origin Shu-Qin Zhang, Bin Tan, Li Guo, Feng-Xue Wang, Hong-Wei Zhu, Yong-Jun Wen, Shipeng Cheng ⇑ State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 18 April 2014 Received in revised form 14 July 2014 Accepted 17 July 2014 Available online 4 August 2014 Keywords: Bovine viral diarrhea virus (BVDV) Bovine serum Genetic diversity
a b s t r a c t Bovine viral diarrhea virus (BVDV) is often detected in commercial bovine serum. BVDV genetic diversity was investigated in commercial bovine serum of Chinese origin. Twenty-two batches of bovine serum were obtained from 10 suppliers with different geographic origins in China, and 20 batches of bovine serum were positive by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. Phylogenetic reconstructions of partial 50 UTR sequences indicated that the samples examined in this work clustered within the BVDV type 1 and BVDV type 2 genotypes. Interestingly, 3 sample sequences clustered into CSFV. These results suggest a high genetic diversity in Chinese BVDV field isolates. This study will benefit epidemiological surveys of BVDV detected in China. Ó 2014 Elsevier B.V. All rights reserved.
Bovine viral diarrhea virus (BVDV), the etiological agent of bovine viral diarrhea/mucosal disease, is a worldwide pathogen in cattle that causes significant economic losses to agriculture (Xue et al., 2011). BVDV, together with classical swine fever virus (CSFV) and sheep border disease virus (BDV), belongs to the genus Pestivirus in the Flaviviridae family (Byers et al., 2009). Recently, atypical bovine pestiviruses such as Th/04_KhonKaen virus, D32/ 00-‘HoBi’ and CHKaHo/cont have been referred to as BVDV-3 (Liu et al., 2009a). Bovine viral diarrhea virus infection was first reported in Olafson et al. (1946). Subsequently, BVDV was found to be epidemic worldwide in cattle farms. In the intervening 66 years, many important advances have been made in understanding this virus and the disease it produces. However, in China, progress with BVDV has been limited. To date, no commercial BVDV vaccines are available on the market, and no management systems or control programs have been conducted in China. The genetic diversity of BVDV needs to be considered when designing and constructing effective vaccination strategies for the virus, and a vaccine must accurately reflect the genotypes and antigenic types present in the country of use (Mahony et al., 2005). The genetic and antigenic diversity of BVDV are not well known in China. Bovine serum is an important material in medicine and biological products. However, bovine serum is often contaminated by ⇑ Corresponding author. Address: State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Science, No. 4899 Juye Street, Changchun 130122, People’s Republic of China. Tel./fax: +86 0431 81919845. E-mail address: [email protected] (S. Cheng). http://dx.doi.org/10.1016/j.meegid.2014.07.021 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
bovine viral diarrhea virus (Bolin and Ridpath, 1998). Recently, atypical bovine pestiviruses were detected in many commercial fetal bovine serum samples from different geographic origins (Xia et al., 2011). Every batch of commercially available bovine serum is a mixture of collected raw serum material from different cattle farms near the supplier. Therefore, commercial bovine serum is a good material for investigating BVDV genetic diversity. The purpose of this work was to determine the genotypes of BVDV detected in commercial bovine serum in China. A total of 22 batches of bovine serum were purchased from 10 commercial suppliers. The 22 batches of bovine serum came from 8 provinces in China (Table 1). Total RNA was extracted from serum samples using TRIZOL (Invitrogen, China) according to the manufacturer’s instructions. The extracted RNA was reverse-transcribed using the M-MLV Reverse Transcriptase Kit (Invitrogen, USA) as specified by the manufacturer. The 50 UTR primers were selected as described previously (Ridpath and Bolin, 1998). The 50 UTR genomic region provides meaningful inferences as this region has the highest degree of sequence conservation and is effectively amplified by RT-PCR (Vilcek et al., 2001). The 50 UTR primer sequences were as follows: sense primer: 50 CAT GCC CAT AGT AGG AC 30 ; antisense primer: 50 CCA TGT GCC ATGTAC AG 30 , and the primers are not reliable for amplifying HoBi-like viruses. Similar 280 bp fragments of the 50 UTR region were amplified from 20 batches of bovine serum, and the amplified fragments were purified and cloned into a pMD18-T vector (TaKaRa, Japan), and 3 clones of every sample were sequenced. Sixty sequences from 20 batches of bovine serum were found, after sequence alignment,
231
S.-Q. Zhang et al. / Infection, Genetics and Evolution 27 (2014) 230–233 Table 1 The batches of bovine serum tested by RT-PCR. Origin
Supplier
Sample ID
NMG
A
1 4
JL
B G
TJ
C D
BVDV-1
BVDV2 10
CSFV
9
S11, S12, S13 S41, S42, S43
1 m(S11 , S12, S13 ) 1 m(S41, S429, S433)
2 3 15 16
S21, S22, S23 S31, S32, S33 S151, S152, S153 S161, S162, S163
1 m(S22, S21, S2310) 1 m(S313) 1b(S328, S338) 1a(S1516, S152), 1p(S1534) 1 m(S1619), 1b(S1627, S1638)
7 8 9 10
S71, S72, S73 S81, S82, S83 S91, S92, S93 S101, S102, S103
1 m(S71, S72, S7312) 1 m(S8111, S8211), 1q(S832) 1a(S916, S926), 1b(S937) 1 m(S101, S102), 1b(S1037)
S51, S52, S53 S61, S62, S63
1 m(S52), 1q(S531) 1 m(S6210, S6310), 1q(S611)
SD
E
5 6
GS
F
12 13
S121, S122, S123 S131, S132, S133
1 m(S121, S122, S123) 1 m(S133), 1q (S1311, S1321)
HN
H
17 19
S171, S172, S173 S191, S192, S193
1 m(S1939), 1q (S1912, S1922)
S5113
S17213
HLJ
I
14 18
S141, S142, S143 S181, S182, S183
1 m(S1413), 1c(S1425) 1e(S182), 1c(S1835), 1a(S181)
JS
J
11 20
S111, S112, S113 S201, S202, S203
1 m(S11112, S11312) 1a(S201, S202), 1p(S2034)
S171,S17314
S143 S11214
Superscript: the same number means consensus sequence.
the consensus sequences were eliminated, thirty-four different sequences from 20 batches of bovine serum were found, containing at least one species of pestiviruses. The 34 newly determined 50 UTR sequences were deposited in GenBank with the accession numbers KF006955–KF006975 and KJ690679–KJ690691. The BVDV 50 UTR of the samples were aligned with the corresponding regions of the pestivirus reference sequences retrieved from GenBank using the Clustal W program in MegAlign of Lasergene 7.2 software (DNASTAR Inc. Madison, WI, USA). Then, phylogenetic trees were constructed based on the 251 nucleotide region of the 50 UTR. Bootstrap values were based on 1000 replicates using the neighbor-joining method (Kimura two-parameter method) with Molecular Evolutionary Genetics Analysis (MEGA version 4.1) software. Phylogenetic analysis indicated that the sequences belonging to BVDV-1 should be further classified into 8 subgenotypes: BVDV-1 m (29/60), BVDV-1b (6/60), BVDV-1a (7/60), BVDV-1c (2/60), BVDV-1p (2/60), BVDV-1e (1/60) and BVDV-1q (7/60). Of these sequences, BVDV-1 m was the most frequently identified, strain ZM-95 was the first isolated BVDV-1 m in China (Xu et al., 2006). Of these sequences, 29 sequences from 15 batches of bovine serum belonged to BVDV-1 m; the percentage of BVDV-1 m was 48.33% (29/60), and nucleotide homology among these sequences was in the range 91.1–100%. BVDV-1b is considered to be a predominant BVDV-1 strain circulating in Chinese cattle. In this study, 6 sequences from 4 batches of bovine serum belonged to BVDV-1b, with the percentage being 10% (6/60). Based on this result and a previous report (Xue et al., 2010), the percentage of BVDV-1 m identification was higher than that of BVDV-1b. Hence, BVDV-1 m is considered to be more predominant than BVDV-1b in Chinese cattle population. BVDV-1p and BVDV-1c were found in 2 sequences from 2 batches of bovine serum. BVDV-1a is the usual subtype of BVDV-1 vaccines, which is dominant in the UK and widely distributed in USA. Likewise, the BVDV isolates predominating in a neighboring country, namely Korean (Booth et al., 2013; Oem et al., 2009). BVDV-1a has been found in Chinese pig herds but has been unique reported in the Chinese cattle population. In this study, BVDV-1a was found in 7 sequences from 4 batches of bovine serum. BVDV-1q is a newly identified subgenotype from cattle, pigs and camels in China (Deng et al.,
2012; Gao et al., 2013; Gong et al., 2013). In this study, there are 7 sequences from 5 batches of bovine serum that belonged to BVDV-1q. BVDV1e was isolated in Italian Austria and UK (Booth et al., 2013). It had not yet been found in China; however, in this study, only one sequence was found in 1 batch of bovine serum. BVDV-1 h and BVDV-1f are the predominant BVDV1 strain prevalent in Australia (Mahony et al., 2005), but it was not detected in this work (see Fig. 1). To date, BVDV-2 has been grouped into at least four BVDV-2 subtypes (BVDV-2a–BVDV-2d) (Flores et al., 2002; Mishra et al., 2008; Vilcek et al., 2004).A BVDV-2 strain, SD-06, was first isolated from cattle in China (Zhu et al., 2009b). Subsequently, many BVDV2 strains were isolated from cattle and pigs in China (Liu et al., 2012; Tao et al., 2013; Zhu et al., 2009a), which belonged to BVDV-2a. Compared with the endemic situation of BVDV-1, the prevalence of BVDV-2 infection in cattle is seemingly much lower in China. In this study, 3 sequences from 3 batches of bovine serum belonged to BVDV-2, and phylogenetic analysis showed that they formed a distinct branch from other previously reported isolates. The sequences belong to BVDV-2b, with a 93.7–95% nucleotide sequence homology to BVDV-2b reference strain VS-123.4 and 34b (Flores et al., 2002). Bovine viral diarrhea virus, classical swine fever virus and border disease virus are all members of the genus pestivirus. However, in contrast with the wide host range of BVDV, CSFV only infects pig and wild boars. In this study, phylogenetic analysis showed that 3 sequences from 2 batches of bovine serum belonged to CSFV. This result is in agreement with a previous observation that BVDV strain HEN03 (KC176778) was found in cattle blood from Henan province. The Npro and E2 genomic region are important to the pestivirus genotype, attempt to amplify these genes and phylogenetic analysis. It is possible that the storage conditions and time of the collection of these samples were poor, these genes could not been amplified in many of these samples, although we did try many times (data not shown). Early in 1957, Yuan Qingzhi, the inventor of the Chinese hog cholera lapinized vaccine strain, his studies have shown that the vaccine strain can be infected and breeding in cow, and the Cattle Organization vaccine of the Chinese hog cholera lapinized vaccine strain were produced and
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Fig. 1. Phylogenetic tree illustrating the subgenotypes of BVDV isolates based on the 50 UTR. The phylogenetic tree was constructed using the neighbor-joining method implemented in MEGA 4.1. Bootstrap values are the percentage of 1000 replicates and are shown below the branches. The bar indicates the substitutions per site. Accession numbers are listed in the brackets after the isolate names. The 34 newly sequence from commercial bovine serum in this study are designated with ‘‘N’’.
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widely applied in China. There are no commercial BVDV vaccines in the Chinese market at present. BVDV was prevented by the Chinese hog cholera lapinized vaccine strain in Tibet and Qinghai province. More studies are needed to determine whether these sequences belong to CSFV or are derived from a BVDV variant. In this study, 22 batches of bovine sera were investigated, 20 batches were positive for BVDV by RT-PCR. 3 clones of every sample were sequenced. All together 60 sequences from 20 batches of bovine serum were found, after sequence alignment, some consensus sequences were found in the same or different batch bovine serum origin. The consensus sequences were marked with superscript, the same number means same sequence (Table 1). The consensus sequences in the same batch bovine serum maybe the repeat clone. These samples were collected from 10 suppliers in 8 provinces distributed throughout different areas of China. These results imply that BVDV is highly prevalent and has considerable genetic divergence among Chinese cattle herds. But, the genetic diversity of bovine viral diarrhea viruses in every province has not yet been determined, and there are many questions that require careful investigation. For example, a possible scenario is that mixing of raw serum material from a geographical location close to the province of origin might have occurred at certain companies during manufacturing steps. Another possible scenario is that the cattle and cattle production trade in different regions could facilitate virus transmission. This could explain why many of the consensus sequences were found in serum samples with different provincial origins. This study demonstrated that commercial bovine serum products with different geographic origins in China are contaminated with at least one species of pestivirus: BVDV-1, BVDV-2 and a virus similar to CSFV. Only one BVDV-2 isolated from these samples. Nevertheless, virus was isolated from 93 of 190 lots of commercially available fetal calf serum at the National Animal Disease Center in USA in 1991 (Bolin et al., 1991). Recently, a BVDV-2 strain, HLJ-10, was isolated from contaminated fetal bovine serum (Liu et al., 2012). Contaminated bovine serum can interfere with the diagnosis of viral infection, and vaccines produced using infected cell cultures could lead to seroconversion or disease in the vaccinated animal (Bolin and Ridpath, 1998). In China, an abundance of bovine sera has been used in the production of live vaccines against PRRSV, PRV and CSFV in pig herds. CSF live vaccines have been found to be contaminated with BVDV at the China Institute of Veterinary Drug Control (Fan et al., 2010). This may be one of the reasons why both pig and cattle herds have the same predominant prevalent subgenotypes in China. Acknowledgments This study was supported by the basic scientific research funding of the Chinese academy of Agricultural Sciences (0032014016), the Project Development Plan of Science and Technology of Jilin Province (20130206024NY) and National Public Welfare of China for Agriculture Special Purpose (201003060). References Bolin, S.R., Ridpath, J.F., 1998. Prevalence of bovine viral diarrhea virus genotypes and antibody against those viral genotypes in fetal bovine serum. J. Vet. Diagn. Invest. 10, 135–139.
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Bolin, S.R., Matthews, P.J., Ridpath, J.F., 1991. Methods for detection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus. J. Vet. Diagn. Invest. 3, 199–203. Booth, R.E., Thomas, C.J., El-Attar, L.M., Gunn, G., Brownlie, J., 2013. A phylogenetic analysis of bovine viral diarrhoea virus (BVDV) isolates from six different regions of the UK and links to animal movement data. Vet. Res. 44, 43. http:// dx.doi.org/10.1186/1297-9716-44-43. Byers, S.R., Snekvik, K.R., Righter, D.J., Evermann, J.F., Bradway, D.S., Parish, S.M., Barrington, G.M., 2009. Disseminated bovine viral diarrhea virus in a persistently infected alpaca (Vicugna pacos) cria. J. Vet. Diagn. Invest. 21, 145– 148. Deng, Y., Sun, C.Q., Cao, S.J., Lin, T., Yuan, S.S., Zhang, H.B., Zhai, S.L., Huang, L., Shan, T.L., Zheng, H., Wen, X.T., Tong, G.Z., 2012. High prevalence of bovine viral diarrhea virus 1 in Chinese swine herds. Vet. Microbiol. 159, 490–493. Fan, X.Z., Ning, Y.B., Wang, Q., Xu, L., Shen, Q.C., 2010. Detection of bovine viral diarrhea virus as contaminant in classical swine fever virus live vaccine with RT-PCR. Chin. J. Vet. Med. 46, 8–10. Flores, E.F., Ridpath, J.F., Weiblen, R., Vogel, F.S., Gil, L.H., 2002. Phylogenetic analysis of Brazilian bovine viral diarrhea virus type 2 (BVDV-2) isolates: evidence for a subgenotype within BVDV-2. Virus Res. 87, 51–60. Gao, S., Luo, J., Du, J., Lang, Y., Cong, G., Shao, J., Lin, T., Zhao, F., Belak, S., Liu, L., Chang, H., Yin, H., 2013. Serological and molecular evidence for natural infection of Bactrian camels with multiple subgenotypes of bovine viral diarrhea virus in Western China. Vet. Microbiol. 163, 172–176. Gong, X., Cao, X., Zheng, F., Chen, Q., Zhou, J., Yin, H., Liu, L., Cai, X., 2013. Identification and characterization of a novel subgenotype of bovine viral diarrhea virus isolated from dairy cattle in Northwestern China. Virus Genes 46, 375–376. Liu, H., Li, Y., Gao, M., Wen, K., Jia, Y., Liu, X., Zhang, W., Ma, B., Wang, J., 2012. Complete genome sequence of a bovine viral diarrhea virus 2 from commercial fetal bovine serum. J. Virol. 86, 10233. Liu, L., Kampa, J., Belak, S., Baule, C., 2009. Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen. Vet. Microbiol. 138, 62– 68. Mishra, N., Rajukumar, K., Vilcek, S., Tiwari, A., Satav, J.S., Dubey, S.C., 2008. Molecular characterization of bovine viral diarrhea virus type 2 isolate originating from a native Indian sheep (Ovies aries). Vet. Microbiol. 130, 88–98. Mahony, T.J., McCarthy, F.M., Gravel, J.L., Corney, B., Young, P.L., Vilcek, S., 2005. Genetic analysis of bovine viral diarrhoea viruses from Australia. Vet. Microbiol. 106, 1–6. Olafson, P., Mac, C.A., Fox, F.H., 1946. An apparently new transmissible disease of cattle. Cornell Vet. 36, 205–213. Oem 1, J.K., Hyun, B.H., Cha, S.H., Lee, K.K., Kim, S.H., Kim, H.R., Park, C.K., Joo, Y.S., 2009. Phylogenetic analysis and characterization of Korean bovine viral diarrhea viruses. Vet. Microbiol. 139 (3–4), 356–360. http://dx.doi.org/ 10.1016/j.vetmic.2009.06.017. Ridpath, J.F., Bolin, S.R., 1998. Differentiation of types 1a, 1b and 2 bovine viral diarrhoea virus (BVDV) by PCR. Mol. Cell. Prob. 12, 101–106. Tao, J., Wang, Y., Wang, J., Wang, J.Y., Zhu, G.Q., 2013. Identification and genetic characterization of new bovine viral diarrhea virus genotype 2 strains in pigs isolated in China. Virus Genes 46, 81–87. Vilcek, S., Durkovic, B., Kolesarova, M., Greiser-Wilke, I., Paton, D., 2004. Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group. Vet. Res. 35, 609–615. Vilcek, S., Strojny, L., Durkovic, B., Rossmanith, W., Paton, D., 2001. Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RTPCR method. J. Virol. Methods 92, 19–22. Xia, H., Vijayaraghavan, B., Belak, S., Liu, L., 2011. Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches. PloS One 6, e28553. Xu, X., Zhang, O., Yu, X., Liang, L., Xiao, C., Xiang, H., Tu, C., 2006. Sequencing and comparative analysis of a pig bovine viral diarrhea virus genome. Virus Res. 122, 164–170. Xue, F., Zhu, Y.M., Li, J., Zhu, L.C., Ren, X.G., Feng, J.K., Shi, H.F., Gao, Y.R., 2010. Genotyping of bovine viral diarrhea viruses from cattle in China between 2005 and 2008. Vet. Microbiol. 143, 379–383. Xue, W., Mattick, D., Smith, L., 2011. Protection from persistent infection with a bovine viral diarrhea virus (BVDV) type 1b strain by a modified-live vaccine containing BVDV types 1a and 2, infectious bovine rhinotracheitis virus, parainfluenza 3 virus and bovine respiratory syncytial virus. Vaccine 29, 4657–4662. Zhu, L.Q., Lin, Y.Q., Ding, X.Y., Ren, M., Tao, J., Wang, J.Y., Zhang, G.P., Zhu, G.Q., 2009a. Genomic sequencing and characterization of a Chinese isolate of Bovine viral diarrhea virus 2. Acta Virol. 53, 197–202. Zhu, L.Q., Ren, M., Lin, Y.Q., Ding, X.Y., Zhang, G.P., Zhao, X., Zhu, G.Q., 2009b. Identification of a bovine viral diarrhea virus 2 isolated from cattle in China. Acta Virol. 53, 131–134.
