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Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
2
Letter To the Editor
4 6 5 7 8
Phylogenetic analysis of bovine viral diarrhea virus of subgenotype 1c
Dear Editor, 10 Bovine viral diarrhea virus (BVDV), a member of the family 11 Flaviviridae, genus Pestivirus, is one of the most widespread patho12 gens that cause significant economic losses to the cattle industry 13 worldwide (Ridpath, 2010). The viral isolates are segregated into 14 multiple subgenotypes (BVDV-1a -1q, BVDV-2a -2c) based on 15 divergence in the viral nucleotide sequences revealed by phylog16 eny (Vilcek et al., 2001; Tajima et al., 2001; Xue et al., 2010; Gao 17 et al., 2014). The molecular cloning of the complete BVDV genome 18 dates back to more than 2 decades ago when genomic characters of 19 several strains within BVDV subgenotype 1a and 1b were disclosed 20 (Deng and Brock, 1992; De Moerlooze et al., 1993; Colett et al., 21 Q2 1988). Since then, a great deal of progress has been made in deter22 mining the viral genomes of several additional subgenotypes. To 23 date, genome sequences of some subgenotypes including BVDV24 1a, -1b, -1d, -1k, -1j, -1m, -1q, -2a and -2b have been recorded, 25 while genome sequences of other subgenotypes remain incom26 plete, presenting major obstacles to the phylogeny of BVDV and 27 to studies on the recombination between subgenotypes. 28 Previous studies have demonstrated that BVDV-1c is one of the 29 predominant subgenotypes found in cattle and Bactrian camels 30 originated from Western China (Zhong et al., 2011; Gao et al., 31 2013). This subgenotype has also been detected in cattle, sheep, 9
buffalo, deer, goats and yaks (Bos poephagus grunniens) from other countries, including Australia (Mahony et al., 2005), New Zealand and Germany (Becher et al., 1997), India (Mishra et al., 2008, 2012), Spain (Arias et al., 2003), Mozambique (Vilcek et al., 2001), and Japan (Matsuno et al., 2007; Nagai et al., 2008), showing a wide distribution and a diverse host range. However, only partial genomic information of virus within this subgenotype was available, leaving the genome-wide evolutionary analysis impossible. In the present study, we detected BVDV RNA from a batch of fetal bovine serum of Australian origin. The non-cytopathogenic virus was recovered as described previously for genome cloning (Liu et al., 2009). Viral RNA was extracted and amplified by overlapping RT-PCR for the near full-length genome using PrimeScript™ One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China). The amplicon was sequenced in both directions with the BigDyeÒ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on an ABI PRISM3730 (Applied Biosystems) capillary DNA sequencer. Sequence obtained was assembled using the SeqManII software (DNAStar Inc. Madison, WI). A Blast search was performed with the initially obtained 50 UTR-E2 region and demonstrated nucleotide identity of 91% with the corresponding region of a BVDV-1c strain Bega that originated from Australia, therefore, the isolate was named ‘‘Bega-like’’ accordingly. Phylogenetic analysis is a useful tool in studies on BVDV, especially for epidemiological surveillance and for rapid genotyping of novel subgenotypes. A recent study has demonstrated that at least five regions of BVDV can be used for phylogenetic tree construction
Table 1 Percentage of nucleotide identity between individual coding region of BVDV-1c strain Bega-like and other pestiviruses. Species/genotype
Strain
Access. No.
50 UTR
Npro
C
Erns
E1
E2
P7
NS2
NS3
NS4A
NS4B
NS5A
NS5B
BVDV-1c BVDV-1a BVDV-1a BVDV-1b1 BVDV-1b1 BVDV-1b2 BVDV-1d BVDV-1k BVDV-1j BVDV-1m BVDV-1q BVDV-1q BVDV-2a BVDV-2b Atypical Atypical
Bega SD1 NADL Osloss VEDEVAC CP7 10JJ-SKR SuwaNcp KS86-1ncp ZM-95 camel-6 SD0803 XJ-04 Hokudai-Lab/09 Italy-1/10-1 Th/04_KhonKaen
AF049221 M96751 AJ133739 M96687 AJ585412 U63479 KC757383 KC853440 AB078950 AF526381 KC695810 JN400273 FJ527854 AB567658 HQ231763 FJ040215
95.9 91.8 94.2 89.7 89.7 89.7 90.9 81.6 85.7 84.4 89.3 88.1 74.1 75.4 55.3 54.0
92.8 83.7 83.5 79.9 80.7 79.9 80.5 79.7 80.7 79.9 80.1 78.9 68.2 66.6 65.2 63.6
92.4 81.0 79.4 82.3 80.7 81.0 79.0 78.7 79.4 78.5 78.1 79.7 69.2 69.2 67.9 67.6
91.4 85.7 84.5 82.9 82.0 83.8 80.4 81.6 81.9 79.2 79.7 80.7 70.4 70.1 70.7 71.5
91.1 83.0 80.0 78.2 78.1 78.8 77.4 76.5 76.4 76.4 77.6 77.9 71.6 70.5 68.5 68.3
88.4 80.3 80.4 74.6 74.4 75.8 76.0 74.6 76.2 75.5 74.9 74.2 64.3 64.6 60.7 60.0
85.7 79.5 75.2 74.2 78.5 74.7 73.3 77.1 76.1 74.7 79.0 78.0 64.7 64.7 60.4 60.9
– 83.8 82.1 76.6 76.3 77.9 78.7 77.4 81.2 78.5 77.4 78.2 61.7 62.2 58.5 58.6
93.3a 86.6 85.9 84.5 83.9 84.3 84.3 83.2 85.1 82.9 83.1 83.2 77.5 77.9 76.1 77.1
90.0a,b 83.8 88.0 84.8 82.2 84.3 85.9 83.8 84.3 81.7 82.8 83.3 73.9 73.9 76.0 76.5
– 84.8 85.3 79.6 80.7 81.6 80.7 82.6 83.7 81.2 81.1 81.7 72.1 72.4 70.9 71.3
– 79.7 80.8 75.2 76.2 76.0 77.2 75.5 79.2 76.0 75.1 74.9 64.8 65.1 58.8 60.7
– 83.7 80.0 85.0 81.9 82.7 82.1 81.8 80.6 83.5 80.5 79.7 71.4 71.2 70.4 70.6
–, No sequence available. a GenBank accession number is AF052303. b Identity was calculated based on alignment of the 120 nt NS4A coding fragment of BVDV strain Bega.
http://dx.doi.org/10.1016/j.meegid.2014.05.021 1567-1348/Ó 2014 Published by Elsevier B.V.
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AF049221, AF052303 Bega
100
(A)
BVDV-1c KF896608 Bega-like M96751 SD-1 BVDV-1a AJ133738 NADL
99 100
98
AB078950 KS86-1ncp BVDV-1j KC757383 10JJ-SKR BVDV-1d AF526381 ZM-95 BVDV-1m
63 72
100
KC695810 camel-6 JN400273 SD0803
100
100
BVDV-1q
U63479 CP7 AJ585412 VEDEVAC
100 100
BVDV-1b
M96687 Osloss KC853440 SuwaNcp
BVDV-1k
FJ527854 XJ-04
100
AB567658 Hokudai-Lab/09 FJ040215 Th/04 KhonKaen FJ040215 Italy-1/10-1
100
BVDV-2 Atypical Pestiviruses
0.02 KF896608 Bega-like
85 99
(B)
BVDV-1c
KF896612 GS2
82
KF896611 GS1 AJ133738 NADL
99
BVDV-1a
M96751-SD1 AB078950 KS86-1ncp
99
BVDV-1j
AB078952 KS86-1cp
99 KC853440 SuwaNcp
BVDV-1k
KC853441 SuwaCp
81
BVDV-1d
KC757383 10JJ-SKR
98
U63479 CP7
99 99
BVDV-1b
AJ585412 VEDEVAC M96687 Osloss
KC695810 camel-6
99 59
JN400273 SD0803
98
BVDV-1q
BVDV-1m 99 AB105774 IS25 CP/01
AF526381 ZM-95
89
AB105775 IS26 NCP/01 AB105773 So CP/75
99 99
BVDV-1o BVDV-1n
FJ527854.1 XJ-04 HQ258810 SH-28
BVDV-2
AB567658 Hokudai-Lab/09 FJ040215 Th/04 KhonKaen
99 99
HQ231763 Italy-1/10-1
Atypical pestiviruses
JQ612704 Italy-83/10-ncp
0.02
Q3
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
Fig. 1. Phylogenetic analysis based on complete genome (A) and NS5B (B) coding regions of BVDV. The obtained sequences were aligned using the CLUSTALW (Thompson et al., 1994) and used for bootstrap analysis (n = 1000) in MEGA 5.0 (Tamura et al., 2011). The model and parameters for complete gnome was (Test: Bootstrap method; Model: maximum composite likelihood; Substitution: transitions plus transversions; Uniform rates). Models and parameters for NS5B coding region were identical with that in a previous study (Gao et al., 2014). The bars indicate nucleotide substitutions per site.
(Nagai et al., 2004). Consistent to the branch order in the tree, pairwise sequence similarity was proved another way to evaluate the relatedness between viral strains. As the majority of genomic sequences of BVDV-1c are currently limited to 50 UTR, Npro and E2 regions, so genotyping of viral isolates within this subgenotype can be easily achieved by aligning the nucleotide sequences of such regions. Sequence alignment demonstrated that Bega-like had 90.0–95.4% nucleotide identity in the Npro with that of other BVDV-1c isolates recorded in GenBank. The highest identity was observed with an Australian strain VR999 (95.4%) followed by 2 Chinese isolates GS-2 (95.0%) and GS-1 (94.0%). The E2 coding region of Bega-like was found genetic divergent from other BVDV-1c strains, demonstrating inner subgenotype nucleotide identity of 86.2–91.5%. Comparatively, the identity was much lower for individual coding region compared with strains of other subgenotypes (Table 1). As the branch orders of BVDV-1c isolates
reflected by 50 UTR, Npro and E2 have been published elsewhere, so the phylogenetic trees based on such regions were not shown here. Besides the commonly used Npro and E2 coding regions, the complete NS3 gene was determined for a few BVDV-1c isolates in a previous study (Brown et al., 2002), but information about the NS5B nucleotide sequences was not available from GenBank. As intergenotypic recombination was previously reported in some non-cytopathogenic BVDV strains (Nagai et al., 2004), phylogenetic trees built from few sequences of NS3 and NS5B may not provide convincing phylogeny of BVDV-1c. To address this problem, we subsequently determined the 3’-half genome of Bega-like and partial nucleotide sequences of NS3 (1161 nt) and NS5B (690 nt) of two non-cytopathogenic isolates (GS1 and GS2) that were previously determined as BVDV-1c by analyzing the 50 UTR and Npro (GenBank accession number: JQ071525–JQ071528). The nucleotide sequences
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obtained were deposited in GenBank under accession number KF896608–KF896612. Consistent with genomic organization of other non-cytopathogenic pestiviruses, the near-full genomic RNA of strain Bega-like (12193 nt) contains an open reading frame (ORF) flanked by 50 and 30 NCR (278 nt and 218 nt, partially determined). The ORF (11,697 nt) comprised of 11 gene coding regions including Npro (504 nt), capsid (306 nt), Erns (681 nt), E1 (585 nt), E2 (1122 nt), p7 (210 nt), NS2–3 (3408 nt), NS4A (192 nt), NS4B (1041 nt), NS5A (1488 nt), and NS5B (2157 nt). No cellular sequence insertions were observed in the viral genome. Its nucleotide sequence showed identity of 80–84% with BVDV-1 of other subgenotypes, followed by 73% with BVDV-2 and 71% with atypical pestiviruses. The nucleotide identity varied among individual coding regions but showed a uniform distance between virus pairs (Table 1). The phylogenetic tree constructed based on complete genome unequivocally confirmed the relatedness of ‘‘Bega-like’’ with another BVDV-1c strain ‘‘Bega’’. For the strain ‘‘Bega’’, only two genomic fragments including 50 half genome (GenBank: AF049221) and NS3 coding region (GenBank: AF052303) were combined and used in the phylogenetic analysis. However, its branch order was not affected (Fig. 1, panel A). In order to confirm this phylogeny, we further analyzed the NS3 and NS5B coding regions of this subgenotype. High inner-subgenotype nucleotide identity in NS3 (92.0–94.5%) and NS5B (92.7–93.9%) was observed. Comparatively, the identity was lower for NS3 (76.1– 86.6%) and NS5B (70.4–85.0%) compared with sequences of other BVDV subgenotypes (Table 1). Such genetic distinction from other isolates suggested that the NS3 and NS5B nucleotide sequences we determined were subgenotype-specific. As we suspected, the NS5B sequences formed unique clusters in the phylogenetic trees (Fig. 1, panels B). Taken together, the data suggested that the sequences we harvested disclosed the genomic feature of BVDV-1c subgenotype exactly. In the present study, we determined the near-full length genome sequence of a BVDV-1c strain for the first time, which enabled the phylogenetic reconstruction of BVDV-1c subgenotype based on individual genomic region. The Bega-like, GS1 and GS2 isolates were determined non-cytopathogenic biotype, which can cause persistent infection of the bovine fetus and is responsible for permanent BVDV circulation in the cattle herd. As the dataset of viral genome sequences provided sufficient information, we didn’t focus on getting all the genome sequences of similar isolates identified in our laboratory. The availability of complete genomic sequence of Bega-like would facilitate the molecular studies on similar viral isolates. We believe this phylogenetic system has the potential to become a standard subgenotyping method to be carried out in many more laboratories.
138
Acknowledgments
139 141
This study was supported by ASTIP, CAAS; National Major Special Project on New Varieties Cultivation for Transgenic Organisms (2013ZX08011004-006), and NBCITS, MOA (CARS-38).
142
References
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Arias, P., Orlich, M., Prieto, M., Cedillo Rosales, S., Thiel, H.J., Alvarez, M., Becher, P., 2003. Genetic heterogeneity of bovine viral diarrhoea viruses from Spain. Vet. Microbiol. 96, 327–336. Becher, P., Orlich, M., Shannon, A.D., Horner, G., Konig, M., Thiel, H.J., 1997. Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J. Gen. Virol. 78, 1357–1366.
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Brown, L.M., Papa, R.A., Frost, M.J., Mackintosh, S.G., Gu, X., Dixon, R.J., Shannon, A.D., 2002. A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein. Virus Res. 84, 111–124. Colett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K., Purchio, A.F., 1988. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165, 191–199. Deng, R., Brock, K.V., 1992. Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1. Virology 191, 867–869. De Moerlooze, L., Lecomte, C., Brown-Shimmer, S., Schmetz, D., Guiot, C., Vandenbergh, D., Allaer, D., Rossius, M., Chappuis, G., Dina, D., Renard, A., Martial, J.A., 1993. Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 50 untranslated region. J. Gen. Virol. 74, 1433–1438. Gao, S., Du, J., Shao, J., Lang, Y., Lin, T., Cong, G., Zhao, F., Belak, S., Liu, L., Chang, H., Yin, H., 2014. Genome analysis reveals a novel genetically divergent subgenotype of bovine viral diarrhea virus in China. Infect. Genet. Evol. 21, 489–491. 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. 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. 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. Matsuno, K., Sakoda, Y., Kameyama, K., Tamai, K., Ito, A., Kida, H., 2007. Genetic and pathobiological characterization of bovine viral diarrhea viruses recently isolated from cattle in Japan. J. Vet. Med. Sci. 69, 515–520. Mishra, N., Vilcek, S., Rajukumar, K., Dubey, R., Tiwari, A., Galav, V., Pradhan, H.K., 2008. Identification of bovine viral diarrhea virus type 1 in yaks (Bos poephagus grunniens) in the Himalayan region. Res. Vet. Sci. 84, 507–510. Mishra, N., Pitale, S.S., Rajukumar, K., Prakash, A., Behera, S.P., Nema, R.K., Dubey, S.C., 2012. Genetic variety of bovine viral diarrhea virus 1 strains isolated from sheep and goats in India. Acta Virol. 56, 209–215. Nagai, M., Hayashi, M., Sugita, S., Sakoda, Y., Mori, M., Murakami, T., Ozawa, T., Yamada, N., Akashi, H., 2004. Phylogenetic analysis of bovine viral diarrhea viruses using five different genetic regions. Virus Res. 99 (2), 103–113. Nagai, M., Hayashi, M., Itou, M., Fukutomi, T., Akashi, H., Kida, H., Sakoda, Y., 2008. Identification of new genetic subtypes of bovine viral diarrhea virus genotype 1 isolated in Japan. Virus Genes 36, 135–139. Ridpath, J.F., 2010. Bovine viral diarrhea virus: global status. Vet. Clin. North Am. Food Anim. Pract. 26, 105–121. Tajima, M., Frey, H.R., Yamato, O., Maede, Y., Moennig, V., Scholz, H., Greiser-Wilke, I., 2001. Prevalence of genotypes 1 and 2 of bovine viral diarrhea virus in Lower Saxony, Germany. Virus Res. 76, 31–42. Vilcek, S., Paton, D.J., Durkovic, B., Strojny, L., Ibata, G., Moussa, A., Loitsch, A., Rossmanith, W., Vega, S., Scicluna, M.T., Paifi, V., 2001. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch. Virol. 146, 99–115. 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. Zhong, F., Li, N., Huang, X., Guo, Y., Chen, H., Wang, X., Shi, C., Zhang, X., 2011. Genetic typing and epidemiologic observation of bovine viral diarrhea virus in Western China. Virus Genes 42, 204–207.
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Shandian Gao Q1 Junzheng Du Yangfan Li Youquan Li Junjun Shao Tong Lin Guozheng Cong Furong Zhao State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Lanzhou, 730046 Gansu, PR China
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Lihong Liu Department of Virology, Immunobiology and Parasitology (VIP), National Veterinary Institute (SVA), 75189 Uppsala, Sweden
⇑
Huiyun Chang Jianxun Luo ⇑ Hong Yin State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Lanzhou, 730046 Gansu, PR China ⇑ Corresponding authors. Tel.: +86 931 8342359 (H. Chang), +86 931 8342515 (H. Yin). E-mail addresses: [email protected] (H. Chang), [email protected] (H. Yin).
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Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
2
Letter To the Editor
4 6 5 7 8
Phylogenetic analysis of bovine viral diarrhea virus of subgenotype 1c
Dear Editor, 10 Bovine viral diarrhea virus (BVDV), a member of the family 11 Flaviviridae, genus Pestivirus, is one of the most widespread patho12 gens that cause significant economic losses to the cattle industry 13 worldwide (Ridpath, 2010). The viral isolates are segregated into 14 multiple subgenotypes (BVDV-1a -1q, BVDV-2a -2c) based on 15 divergence in the viral nucleotide sequences revealed by phylog16 eny (Vilcek et al., 2001; Tajima et al., 2001; Xue et al., 2010; Gao 17 et al., 2014). The molecular cloning of the complete BVDV genome 18 dates back to more than 2 decades ago when genomic characters of 19 several strains within BVDV subgenotype 1a and 1b were disclosed 20 (Deng and Brock, 1992; De Moerlooze et al., 1993; Colett et al., 21 Q2 1988). Since then, a great deal of progress has been made in deter22 mining the viral genomes of several additional subgenotypes. To 23 date, genome sequences of some subgenotypes including BVDV24 1a, -1b, -1d, -1k, -1j, -1m, -1q, -2a and -2b have been recorded, 25 while genome sequences of other subgenotypes remain incom26 plete, presenting major obstacles to the phylogeny of BVDV and 27 to studies on the recombination between subgenotypes. 28 Previous studies have demonstrated that BVDV-1c is one of the 29 predominant subgenotypes found in cattle and Bactrian camels 30 originated from Western China (Zhong et al., 2011; Gao et al., 31 2013). This subgenotype has also been detected in cattle, sheep, 9
buffalo, deer, goats and yaks (Bos poephagus grunniens) from other countries, including Australia (Mahony et al., 2005), New Zealand and Germany (Becher et al., 1997), India (Mishra et al., 2008, 2012), Spain (Arias et al., 2003), Mozambique (Vilcek et al., 2001), and Japan (Matsuno et al., 2007; Nagai et al., 2008), showing a wide distribution and a diverse host range. However, only partial genomic information of virus within this subgenotype was available, leaving the genome-wide evolutionary analysis impossible. In the present study, we detected BVDV RNA from a batch of fetal bovine serum of Australian origin. The non-cytopathogenic virus was recovered as described previously for genome cloning (Liu et al., 2009). Viral RNA was extracted and amplified by overlapping RT-PCR for the near full-length genome using PrimeScript™ One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China). The amplicon was sequenced in both directions with the BigDyeÒ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on an ABI PRISM3730 (Applied Biosystems) capillary DNA sequencer. Sequence obtained was assembled using the SeqManII software (DNAStar Inc. Madison, WI). A Blast search was performed with the initially obtained 50 UTR-E2 region and demonstrated nucleotide identity of 91% with the corresponding region of a BVDV-1c strain Bega that originated from Australia, therefore, the isolate was named ‘‘Bega-like’’ accordingly. Phylogenetic analysis is a useful tool in studies on BVDV, especially for epidemiological surveillance and for rapid genotyping of novel subgenotypes. A recent study has demonstrated that at least five regions of BVDV can be used for phylogenetic tree construction
Table 1 Percentage of nucleotide identity between individual coding region of BVDV-1c strain Bega-like and other pestiviruses. Species/genotype
Strain
Access. No.
50 UTR
Npro
C
Erns
E1
E2
P7
NS2
NS3
NS4A
NS4B
NS5A
NS5B
BVDV-1c BVDV-1a BVDV-1a BVDV-1b1 BVDV-1b1 BVDV-1b2 BVDV-1d BVDV-1k BVDV-1j BVDV-1m BVDV-1q BVDV-1q BVDV-2a BVDV-2b Atypical Atypical
Bega SD1 NADL Osloss VEDEVAC CP7 10JJ-SKR SuwaNcp KS86-1ncp ZM-95 camel-6 SD0803 XJ-04 Hokudai-Lab/09 Italy-1/10-1 Th/04_KhonKaen
AF049221 M96751 AJ133739 M96687 AJ585412 U63479 KC757383 KC853440 AB078950 AF526381 KC695810 JN400273 FJ527854 AB567658 HQ231763 FJ040215
95.9 91.8 94.2 89.7 89.7 89.7 90.9 81.6 85.7 84.4 89.3 88.1 74.1 75.4 55.3 54.0
92.8 83.7 83.5 79.9 80.7 79.9 80.5 79.7 80.7 79.9 80.1 78.9 68.2 66.6 65.2 63.6
92.4 81.0 79.4 82.3 80.7 81.0 79.0 78.7 79.4 78.5 78.1 79.7 69.2 69.2 67.9 67.6
91.4 85.7 84.5 82.9 82.0 83.8 80.4 81.6 81.9 79.2 79.7 80.7 70.4 70.1 70.7 71.5
91.1 83.0 80.0 78.2 78.1 78.8 77.4 76.5 76.4 76.4 77.6 77.9 71.6 70.5 68.5 68.3
88.4 80.3 80.4 74.6 74.4 75.8 76.0 74.6 76.2 75.5 74.9 74.2 64.3 64.6 60.7 60.0
85.7 79.5 75.2 74.2 78.5 74.7 73.3 77.1 76.1 74.7 79.0 78.0 64.7 64.7 60.4 60.9
– 83.8 82.1 76.6 76.3 77.9 78.7 77.4 81.2 78.5 77.4 78.2 61.7 62.2 58.5 58.6
93.3a 86.6 85.9 84.5 83.9 84.3 84.3 83.2 85.1 82.9 83.1 83.2 77.5 77.9 76.1 77.1
90.0a,b 83.8 88.0 84.8 82.2 84.3 85.9 83.8 84.3 81.7 82.8 83.3 73.9 73.9 76.0 76.5
– 84.8 85.3 79.6 80.7 81.6 80.7 82.6 83.7 81.2 81.1 81.7 72.1 72.4 70.9 71.3
– 79.7 80.8 75.2 76.2 76.0 77.2 75.5 79.2 76.0 75.1 74.9 64.8 65.1 58.8 60.7
– 83.7 80.0 85.0 81.9 82.7 82.1 81.8 80.6 83.5 80.5 79.7 71.4 71.2 70.4 70.6
–, No sequence available. a GenBank accession number is AF052303. b Identity was calculated based on alignment of the 120 nt NS4A coding fragment of BVDV strain Bega.
http://dx.doi.org/10.1016/j.meegid.2014.05.021 1567-1348/Ó 2014 Published by Elsevier B.V.
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AF049221, AF052303 Bega
100
(A)
BVDV-1c KF896608 Bega-like M96751 SD-1 BVDV-1a AJ133738 NADL
99 100
98
AB078950 KS86-1ncp BVDV-1j KC757383 10JJ-SKR BVDV-1d AF526381 ZM-95 BVDV-1m
63 72
100
KC695810 camel-6 JN400273 SD0803
100
100
BVDV-1q
U63479 CP7 AJ585412 VEDEVAC
100 100
BVDV-1b
M96687 Osloss KC853440 SuwaNcp
BVDV-1k
FJ527854 XJ-04
100
AB567658 Hokudai-Lab/09 FJ040215 Th/04 KhonKaen FJ040215 Italy-1/10-1
100
BVDV-2 Atypical Pestiviruses
0.02 KF896608 Bega-like
85 99
(B)
BVDV-1c
KF896612 GS2
82
KF896611 GS1 AJ133738 NADL
99
BVDV-1a
M96751-SD1 AB078950 KS86-1ncp
99
BVDV-1j
AB078952 KS86-1cp
99 KC853440 SuwaNcp
BVDV-1k
KC853441 SuwaCp
81
BVDV-1d
KC757383 10JJ-SKR
98
U63479 CP7
99 99
BVDV-1b
AJ585412 VEDEVAC M96687 Osloss
KC695810 camel-6
99 59
JN400273 SD0803
98
BVDV-1q
BVDV-1m 99 AB105774 IS25 CP/01
AF526381 ZM-95
89
AB105775 IS26 NCP/01 AB105773 So CP/75
99 99
BVDV-1o BVDV-1n
FJ527854.1 XJ-04 HQ258810 SH-28
BVDV-2
AB567658 Hokudai-Lab/09 FJ040215 Th/04 KhonKaen
99 99
HQ231763 Italy-1/10-1
Atypical pestiviruses
JQ612704 Italy-83/10-ncp
0.02
Q3
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
Fig. 1. Phylogenetic analysis based on complete genome (A) and NS5B (B) coding regions of BVDV. The obtained sequences were aligned using the CLUSTALW (Thompson et al., 1994) and used for bootstrap analysis (n = 1000) in MEGA 5.0 (Tamura et al., 2011). The model and parameters for complete gnome was (Test: Bootstrap method; Model: maximum composite likelihood; Substitution: transitions plus transversions; Uniform rates). Models and parameters for NS5B coding region were identical with that in a previous study (Gao et al., 2014). The bars indicate nucleotide substitutions per site.
(Nagai et al., 2004). Consistent to the branch order in the tree, pairwise sequence similarity was proved another way to evaluate the relatedness between viral strains. As the majority of genomic sequences of BVDV-1c are currently limited to 50 UTR, Npro and E2 regions, so genotyping of viral isolates within this subgenotype can be easily achieved by aligning the nucleotide sequences of such regions. Sequence alignment demonstrated that Bega-like had 90.0–95.4% nucleotide identity in the Npro with that of other BVDV-1c isolates recorded in GenBank. The highest identity was observed with an Australian strain VR999 (95.4%) followed by 2 Chinese isolates GS-2 (95.0%) and GS-1 (94.0%). The E2 coding region of Bega-like was found genetic divergent from other BVDV-1c strains, demonstrating inner subgenotype nucleotide identity of 86.2–91.5%. Comparatively, the identity was much lower for individual coding region compared with strains of other subgenotypes (Table 1). As the branch orders of BVDV-1c isolates
reflected by 50 UTR, Npro and E2 have been published elsewhere, so the phylogenetic trees based on such regions were not shown here. Besides the commonly used Npro and E2 coding regions, the complete NS3 gene was determined for a few BVDV-1c isolates in a previous study (Brown et al., 2002), but information about the NS5B nucleotide sequences was not available from GenBank. As intergenotypic recombination was previously reported in some non-cytopathogenic BVDV strains (Nagai et al., 2004), phylogenetic trees built from few sequences of NS3 and NS5B may not provide convincing phylogeny of BVDV-1c. To address this problem, we subsequently determined the 3’-half genome of Bega-like and partial nucleotide sequences of NS3 (1161 nt) and NS5B (690 nt) of two non-cytopathogenic isolates (GS1 and GS2) that were previously determined as BVDV-1c by analyzing the 50 UTR and Npro (GenBank accession number: JQ071525–JQ071528). The nucleotide sequences
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obtained were deposited in GenBank under accession number KF896608–KF896612. Consistent with genomic organization of other non-cytopathogenic pestiviruses, the near-full genomic RNA of strain Bega-like (12193 nt) contains an open reading frame (ORF) flanked by 50 and 30 NCR (278 nt and 218 nt, partially determined). The ORF (11,697 nt) comprised of 11 gene coding regions including Npro (504 nt), capsid (306 nt), Erns (681 nt), E1 (585 nt), E2 (1122 nt), p7 (210 nt), NS2–3 (3408 nt), NS4A (192 nt), NS4B (1041 nt), NS5A (1488 nt), and NS5B (2157 nt). No cellular sequence insertions were observed in the viral genome. Its nucleotide sequence showed identity of 80–84% with BVDV-1 of other subgenotypes, followed by 73% with BVDV-2 and 71% with atypical pestiviruses. The nucleotide identity varied among individual coding regions but showed a uniform distance between virus pairs (Table 1). The phylogenetic tree constructed based on complete genome unequivocally confirmed the relatedness of ‘‘Bega-like’’ with another BVDV-1c strain ‘‘Bega’’. For the strain ‘‘Bega’’, only two genomic fragments including 50 half genome (GenBank: AF049221) and NS3 coding region (GenBank: AF052303) were combined and used in the phylogenetic analysis. However, its branch order was not affected (Fig. 1, panel A). In order to confirm this phylogeny, we further analyzed the NS3 and NS5B coding regions of this subgenotype. High inner-subgenotype nucleotide identity in NS3 (92.0–94.5%) and NS5B (92.7–93.9%) was observed. Comparatively, the identity was lower for NS3 (76.1– 86.6%) and NS5B (70.4–85.0%) compared with sequences of other BVDV subgenotypes (Table 1). Such genetic distinction from other isolates suggested that the NS3 and NS5B nucleotide sequences we determined were subgenotype-specific. As we suspected, the NS5B sequences formed unique clusters in the phylogenetic trees (Fig. 1, panels B). Taken together, the data suggested that the sequences we harvested disclosed the genomic feature of BVDV-1c subgenotype exactly. In the present study, we determined the near-full length genome sequence of a BVDV-1c strain for the first time, which enabled the phylogenetic reconstruction of BVDV-1c subgenotype based on individual genomic region. The Bega-like, GS1 and GS2 isolates were determined non-cytopathogenic biotype, which can cause persistent infection of the bovine fetus and is responsible for permanent BVDV circulation in the cattle herd. As the dataset of viral genome sequences provided sufficient information, we didn’t focus on getting all the genome sequences of similar isolates identified in our laboratory. The availability of complete genomic sequence of Bega-like would facilitate the molecular studies on similar viral isolates. We believe this phylogenetic system has the potential to become a standard subgenotyping method to be carried out in many more laboratories.
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Acknowledgments
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This study was supported by ASTIP, CAAS; National Major Special Project on New Varieties Cultivation for Transgenic Organisms (2013ZX08011004-006), and NBCITS, MOA (CARS-38).
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Shandian Gao Q1 Junzheng Du Yangfan Li Youquan Li Junjun Shao Tong Lin Guozheng Cong Furong Zhao State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Lanzhou, 730046 Gansu, PR China
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Lihong Liu Department of Virology, Immunobiology and Parasitology (VIP), National Veterinary Institute (SVA), 75189 Uppsala, Sweden
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Huiyun Chang Jianxun Luo ⇑ Hong Yin State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Lanzhou, 730046 Gansu, PR China ⇑ Corresponding authors. Tel.: +86 931 8342359 (H. Chang), +86 931 8342515 (H. Yin). E-mail addresses: [email protected] (H. Chang), [email protected] (H. Yin).
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