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Heterogeneity and Secondary Structure Analysis of 3′ Untranslated Region in Classical Swine Fever Viruses
Agricultural Sciences in China
January 2011
2011, 10(1): 142-148
Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses FAN Yun-feng1, 2, ZHAO Qi-zu1, ZHAO Yun1, ZOU Xing-qi1, ZHANG Zhong-qiu1, 2, WANG Qin1 and NING Yi-bao1 1 2
China Institute of Veterinary Drug Control, Beijing 100081, P.R.China China Animal Disease Control Center, Beijing 100125, P.R.China
Abstract The attenuated vaccine strains of CSFV have a 12-nucleotides (nt) insertion in the 3´-UTR of genome as compared to that of CSFV virulent strains. In this study, we found a distinct heterogeneity in the 3´-UTR of attenuated Thiverval and HCLV strains. The longest 3´-UTR of Thiverval strain was 259 base pairs (bp) with a 32-nt insertion, the shortest 3´-UTR had only 233 bp with a 6-nt insertion. The longest 3´-UTR of HCLV strain was 244 bp with a 17-nt insertion and the shortest 3´UTR was 235 bp with a 8-nt insertion. Compared with the published sequences of 3´-UTR of vaccine and virulent strains, the 3´-UTR of CSFV vaccine strains have two variable regions where insertion among the different vaccine strains were frequently found. The first is located between the second conservative TAA codon and the start of T-rich region where we found the variable length insertion in the same vaccine strain Thiveral or HCLV and the second is located between the end of T-rich region and the front of GAA codon, however, a 4-nt deletion was found in this region in the virulent Shimen strain. These two regions may represent the “hot spot” for mutation. Modeling the secondary structures of the 3´-UTR suggests that the T-rich insertion could result in the change of structure and free energy, thus affecting the stability of the 3´-UTR structure. These findings will help to understand the mechanism of attenuated vaccines and improve vaccine safety, stability, and efficacy. Key words: classical swine fever virus, 3´-UTR, heterogeneity, RNA secondary structure
INTRODUCTION Classical swine fever virus (CSFV) is a single-stranded positive-sense RNA virus. The virus belongs to the genus Pestivirus within the family Flaviviridae (Moennig and Plagemann 1992). Previous studies showed that the attenuated vaccine strains of CSFV have a 12 nucleotides (nt) insertion in the 3´-UTR of genome when compared to that of CSFV virulent strains (Vilcek and Belak 1997; Bjorklund et al. 1998; Wu et al. 2001; Xiao et al. 2004). The insertion helped the virus
adapt to the needs of cells or a sign of attenuated virulence, it is worthy of in depth study. Especially, the study of HCLV can effectively resolve the problem that no reasonable scientific explanation for attenuated mechanism of the vaccine developed in China. At the same time, exploring the biological function of RNA virus 3´-UTR insertion will help us to understand the virulence determinant of CSFV. The 3´-UTR mediates genomic replication of pestivirus. RNA-dependent RNA polymerase (RdRp) first recognizes and binds the 3´-UTR and initiates RNA synthesis to generate a minus-strand RNA from the plus-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. FAN Yun-feng, Ph D, Tel: +86-10-59194743, E-mail: [email protected]; Correspondence ZHAO Qi-zu, Professor, Tel: +86-10-6210367, E-mail: zhaoqizu@ivdc. gov.cn © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60300-X
Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses
strand genomic RNA. A progeny plus-strand RNA genome is then synthesized using the minus-strand as a template (Gong et al. 1996). The primary and secondary structures in the 3´-UTR of the pestivirus genome are closely related to the replication, multiplication, and virulence of the virus. Wang et al. (2008) constructed and analyzed two chimeras stemmed from a highly virulent strain Shimen either with introduction of the 12-nt insertion in 3´-UTR or the replacement of viral 3´-UTR by the 3´-UTR of HCLV. An animal experiment showed that the two chimeras were both dramatically attenuated in pigs, and completely protect the pigs against lethal challenge with highly virulent CSFV. These data demonstrate that the insertion in 3´-UTR are closely related to the attenuation of CSFV. In this paper, the 3´-UTR of vaccine strains Thiverval, HCLV, and virulent strain Shimen were sequenced and analyzed. The results obtained will help us to understand the influence of the insert in various position and length on replication, multiplication, and virulence of virus.
MATERIALS AND METHODS Viruses and cells CSFV Thiverval strain was kindly provided by Sanofi Corporation (France) and passaged in porcine kidney cell line 15 (PK-15) at 29-30°C. CSFV HCLV vaccine strain (HCLV, 1996, 3, 28) and Shimen virulent strain (SM/F114, 1998, 4, 25) were provided by China Institute of Veterinary Drug Control (IVDC). The PK-15 cell line was obtained from American Type Culture Collection.
Viral RNA extraction, reverse transcription, and PCR Viral RNA of Thiverval, HCLV, and Shimen was extracted by trizol (Invitrogen, USA) supplemented with 1 µL RNase inhibitor and 1.5 µL 100 mmol L-1 DTT. The extracted RNA was dissolved in 10 µL RNase-free water. A pair of primers were designed with Oligo6.0 (Molecular Biology Insights, USA) based on the sequences of Alfort/187 (accession no. X87939) and
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HCLV (accession no. AF091507). The forward primer designated as EF is 5´-GAG AAC AAG CTT CGA GAA GCT G-3´. The reverse primer designated as ER is 5´GGG CCG TTA GAA ATT ACC-3´. The cDNA was synthesized by SuperScriptTM III reverse transcriptase (Invitrogen, USA) using ER as a primer and the extracted viral RNA as a template. PCR was carried out in a total volume of 50 µL. The reaction mixture contained 2 µL cDNA, 5 µL 10 × PCR buffer, 1 µL dNTP mix (10 mmol L-1), 2.5 µL EF and ER primers (10 µmol L-1), 1 µL (2 U) PfuUltraTM highfidelity DNA polymerase (Stratagene, USA) and the final volume was adjusted with deionized water. Following a 90-s predenaturing at 94°C, PCR was done by 30 cycles of a 30-s 94°C denaturing step, a 30-s 55°C annealing step, and a 1-min 72°C extension step. A 10-min 72°C extension step was performed after the 30 cycles.
Purification, cloning and sequencing PCR product was verified by electrophoresis on 1% agarose gel and purified with PCR Purification Kit (Tiangen, China). The purified PCR fragment was treated with dATP and Taq polymerase at 72°C for 30 min and cloned into pMD18-T vector (TaKaRa, Japan). Recombinant clones were identified by PCR and restriction endonuclease digestion. The positive clones were randomly selected and sequenced with the ABI PRISM 3730 sequencer (Applied Biosystems, USA).
Primary and secondary structure analysis of CSFV 3´-UTR The sequences of positive clones were aligned with 15 CSFV strains obtained from GenBank: Shimen (accession no. AF092448), Alfort/187 (accession no. X87939), ALD (accession no. D49532), Brescia (accession no. AF091661), Glentorf (accession no. U45478), CAP (accession no. X96550), HCLV (accession no. AF091507), Chinese C (accession no. Z46258), CS (accession no. AF099102), Porcivac (accession no. AF026714), Rovac (accession no. AF026717), Russian LK vaccine (accession no. AF026718), Rimes (accession no. U45477), GPE- (accession no. D49533), and Duvaxin (accession no. AF026710). The align-
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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ment was carried out using DNAstar (DNASTAR Inc., Madison, Wisconsin, USA) and Sequencher (Gene Codes corporation, USA) software. The RNA secondary structures of different 3´-UTR were predicted and analyzed using RNAstructure 4.4 software (http://rna. chem.rochester.edu/).
RESULTS RT-PCR of CSFV Thiverval, HCLV, and Shimen strains and identification of recombinant clones Using EF and ER primers, the DNA fragments approximately 604 bp which includes the entire 3´-UTR were successfully amplified from Thiverval, HCLV, and Shimen strains after verifying by agarose gel electrophoresis. The 20 white clones from those three viruses were randomly selected from transformation plates, respectively. After further identification by PCR methods and restriction endonuclease digestion, the 13 recombinant clones from Thiveral, 12 from Shimen, and 20 from HCLV were obtained for sequencing.
Sequence and alignment analysis of CSFV strains Thiverval, HCLV, and Shimen from different recombinant clones In regard to Thiverval strain, we sequenced 13 recombinant clones from the ligation of one PCR product. The sequence alignment indicated that there were ten kinds of different length 3´-UTR among the 13 recombinant clones. The longest was 259 bp and the shortest was 233 bp. The difference in the length of the 3´UTR was resulted from the insertion of nucleotides ranging from 32 nt (ATTTTTT TTTTTTT ATTTTTTT TTT TTTTTTT) to 6 nt (ATTTTTT) (Fig. 1). The 3´-UTR of HCLV was 239 bp in length among most of the recombinant clones (17/20). The sequences were identical with those from GenBank (accession nos. AF531433, AF091507, and AY805221) and had the same 12-nt (TTTTCTTTTTTC) insertion in T-rich region when compared with CSFV virulent Shimen strain. However, three of 20 recombinant clones (HCLV4-2, HCLV4-9, and HCLVH5BC) contained different length of insertion. They were 8 nt (TTTTCTTT), 13 nt (TTT
FAN Yun-feng et al.
TCTTTTTTCT), and 17 nt (TTTTCTTTTTTT TT T TT), respectively (Fig. 1). The 3´-UTR of Shimen was 228 bp. The most of recombinant clones (11/12) were identical with Shimen sequences from GenBank (accession nos. AF092448 and AY775178). The 3´-UTR length of 23 clones of Shimen strain (Shimen-23) was 224 bp and was 4 nt (TATT) shorter than other clones (Fig. 1).
Alignment analysis of the 3´-UTR of different CSFV strains We aligned the sequences of 3´-UTR among CSFV strains Thiverval, HCLV, Shimen, and other virulent and vaccine strains. We found that the insertions among eight vaccine strains (Thiverval, HCLV, Chinese C, CS, Porcivac, Ravoc, Russian LK vaccine, and Duvaxin) were at different positions in T-rich region after stop codon. The insertions with different length were located between the second consensus TAA codon and the start of T-rich region in Thiverval strain, HCLV, and its derivative strains. The insertions were located between the end of T-rich region and the codon GAA in the CS, Porcivac, Ravoc, and Russian LK vaccine strains in which the insertions were the consensus sequence of TATTTATTTATC. However, it was 4 nt (TATT) insertion in vaccine Duvaxin strain. Interestingly, we found the Shimen strain PCR product clones Shimen-23 had a 4-nt deletion at this position (Fig. 1).
Secondary structure analysis of virulent and vaccine strains The basic model of the 3´-UTR of CSFV consisted of four serials stem-loop structures (named stem-loop I, II, III, IV). Stem-loop I was located at the 3´ terminal of 3´-UTR region and was highly conserved among all CSFV. The intervening sequence ACAGCACTTTA was conserved between stem-loop I and II. Stem-loop II was composed of part of the variable region and part of conserved region, the variable region of stem-loop II had less effect on secondary structure. Stem-loop III and IV were located in the variable region where the sequence change may affect the secondary structure of the 3´-UTR of CSFV, specially the structure of stem© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses
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Fig. 1 Alignment of nucleotide sequence of the 3´-UTR of 23 CSFV strains. “.”indicates the same nucleotides; “-” indicates gaps; vertical box indicates two conservative TAA codons and GAA codons; horizontal box indicates poly AT rich region.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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loop III (Fig. 2). To maintain the similar 3´-UTR structure, the free energy of vaccine strain was higher than that of
FAN Yun-feng et al.
virulent strain and the free energy in 3´-UTR of vaccine strain was increased with length of insert.
Fig. 2 Secondary structure and free energy of the CSFV 3´-UTR. A, Shimen strain, free energy=-55.7; B, HCLV H5BC strain, free energy =-43.6; C, HCLV 4-2 strain, free energy=-44.6; D, CS strain, free energy=-47.0
DISCUSSION Vaccination is the main measure to control CSF. At present, the routinely used vaccines are the hog cholera lapinized virus (HCLV), the Japanese guinea-pig exaltation-negative (GPE-) strain and the Thiverval strain (Blome et al. 2006; Dong and Chen 2007). The HCLV is a stable lapinized strain by adapting the virulent strain in rabbits and the Thiverval stain has been obtained by passage of the virulent strains in certain cells under low temperature (Dong and Chen 2007). Although the at-
tenuated method is different, both the 3´-UTR of Thiverval and HCLV strain contain different length of insertions as compared to that of parent strains. The insertion is longer in Thiverval strain (about 23 nt) than that in HCLV strain (about 12 nt). In addition, a significant heterogeneity of the insertion sequences is found in the 3´-UTR of both strains. For HCLV strain, besides a 12-nt insertion which has been reported previously, insertions of 8, 13, and 17 nt were found. For Thiverval strain, the heterogeneity is more significant. The lengths of insertions among 13 clones
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses
sequenced are different with the longest 32 nt, and the shortest only 6 nt. In order to further investigate the variance of the 3´UTR between virulent and vaccine strains, we compared the published sequences of the 3´-UTR of virulent and vaccine strains from different countries with the sequences determined in this work. Total eight sequences of the 3´-UTR of vaccine strains HCLV, Chinese C, CS, Porcivac, Rovac, Russian LK, Duvaxin and Thiverval were analyzed. They were divided into two groups according to site of the insertion as described by Wu et al. (2001). We found that the 3´-UTR of CSFV have two variable regions. One region is located between the second conservative TAA codon and the start of T-rich region, the other is located between the end of T-rich region and the front of GAA codon. We also found that Shimen 23 has a variance at second region, it was 4 nt shorter than other Shimen strain that had been reported. The two regions may represent the “hot spot” for mutation. Holland et al. (1992) reported that RNA viruses have a property of quasispecies. The classical swine fever virus is possible to apparent 3´-UTR length polymorphism during replication. The dominant sequence of Thiverval strain contains 23 nt insertion and HCLV contains 12 nt insertion and other insertion in two kinds of vaccine strain is secondary sequence that may be the mutated sequence in the virus evolutionary process. However, it was unpractical to purify virus through traditional plaque clone methods due to CSFV non-cytopathogenic effect in cell culture. The data imply that the heterogeneity 3´-UTR may result in the safety hidden trouble of attenuated vaccine. Previous data showed that the 3´-UTR of pestivirus contains distinctive RNA motifs, a stable stem-loop I and a considerably less stable stem-loop II. These two stem-loops are separated by a long single-stranded intervening sequence (Deng and Brock 1993; Yu et al. 1999). Based on these findings, we analyzed the secondary structure of the 3´-UTR of vaccine strains and virulent strains. We found that the length and the location of the insertion in the 3´-UTR could affect the secondary structure of the 3´-UTR. We found in vaccine strains Thiverval, CS, and HCLV, the free energy of the 3´-UTR which is higher than that of virulent strains and is increased when the length of insertion increases,
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suggesting the different length insertion may lead to the unstablization of 3´-UTR in CSFV. The phenomenon that an insertion in 3´-UTR may change viral virulence has been previously reported in other RNA viruses. Rodriguez-Cerezo et al. (1991) reported that a 58-nt insertion in 3´-UTR induced tobacco vein mottling virus (TVMV) attenuated in Nicotiana tabacum plant. Similar report was found in CSFV, while Wang et al. (2008) introduced 12 nt insertion of HCLV in virulent strain Shimen which led to virulence attenuated in swine. That data demonstrate that the 12 nt insertion in 3´-UTR are closely related to the attenuation of CSFV. However, the GPE- vaccine strain do not contain the insertion and the attenuation mechanism of that remains unknown. To further understand the relationship between the insertion in the 3´-UTR and the attenuation of CSFV, we have constructed the infectious cDNA clone of Thiverval with different length of insertions in the 3´-UTR and rescued live viruses. We found that the length of insertions in the 3´-UTR may affect virus multiplication in cell culture (unpublished data). The further studies are needed to understand the attenuation mechanism and allow rationally developing safer and more effective live-attenuated vaccine for CSF prevention and control.
Acknowledgements This work was supported by the National Natural Science Foundation of China (30571377) and the National High-Tech R&D Program of China (863 Program, 2006AA10A204).
References Bjorklund H V, Stadejek T, Vilcek S, Belak S. 1998. Molecular characterization of the 3´ noncoding region of classical swine fever virus vaccine strains. Virus Genes, 16, 307-312. Blome S, Meindl-Bohmer A, Loeffen W, Thuer B, Moennig V. 2006. Assessment of classical swine fever diagnostics and vaccine performance. Revue Scientifique et Technique, 25, 1025-1038. Deng R, Brock K V. 1993. 5´ and 3´ untranslated regions of pestivirus genome: primary and secondary structure analyses. Nucleic Acids Research, 21, 1949-1957. Dong X N, Chen Y H. 2007. Marker vaccine strategies and candidate CSFV marker vaccines. Vaccine, 25, 205-230. Gong Y, Trowbridge R, Macnaughton T B, Westaway E G, Shannon A D, Gowans E J. 1996. Characterization of RNA
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region leads to attenuation of classic swine fever virus and protects host against lethal challenge. Virology, 374, 390-
of General Virology, 77, 2729-2736. Holland J J, de la Torre J C, Steinhauer D A. 1992. RNA virus
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populations as quasispecies. Current Topics in Microbiology and Immunology, 176, 1-20.
P W, Zhao W G. 2001. Attenuated lapinized chinese strain of classical swine fever virus: complete nucleotide sequence
Moennig V, Plagemann P G. 1992. The pestiviruses. Advances in Virus Research, 41, 53-98.
and character of 3´-noncoding region. Virus Genes, 23, 6976.
Rodriguez-Cerezo E, Klein P G, Shaw J G. 1991. A determinant of disease symptom severity is located in the 3´-terminal
Xiao M, Gao J, Wang Y, Wang X, Lu W, Zhen Y, Chen J, Li B. 2004. Influence of a 12-nt insertion present in the 3´
noncoding region of the RNA of a plant virus. Proceedings of the National Academy of Sciences of the USA, 88, 9863-9867.
untranslated region of classical swine fever virus HCLV strain genome on RNA synthesis. Virus Research, 102, 191-198.
Vilcek S, Belak S. 1997. Organization and diversity of the 3´noncoding region of classical swine fever virus genome. Virus
Yu H, Grassmann C W, Behrens S E. 1999. Sequence and structural elements at the 3´ terminus of bovine viral diarrhea
Genes, 15, 181-186. Wang Y, Wang Q, Lu X, Zhang C, Fan X, Pan Z, Xu L, Wen G,
virus genomic RNA: functional role during RNA replication. Journal of Virology, 73, 3638-3648.
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January 2011
2011, 10(1): 142-148
Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses FAN Yun-feng1, 2, ZHAO Qi-zu1, ZHAO Yun1, ZOU Xing-qi1, ZHANG Zhong-qiu1, 2, WANG Qin1 and NING Yi-bao1 1 2
China Institute of Veterinary Drug Control, Beijing 100081, P.R.China China Animal Disease Control Center, Beijing 100125, P.R.China
Abstract The attenuated vaccine strains of CSFV have a 12-nucleotides (nt) insertion in the 3´-UTR of genome as compared to that of CSFV virulent strains. In this study, we found a distinct heterogeneity in the 3´-UTR of attenuated Thiverval and HCLV strains. The longest 3´-UTR of Thiverval strain was 259 base pairs (bp) with a 32-nt insertion, the shortest 3´-UTR had only 233 bp with a 6-nt insertion. The longest 3´-UTR of HCLV strain was 244 bp with a 17-nt insertion and the shortest 3´UTR was 235 bp with a 8-nt insertion. Compared with the published sequences of 3´-UTR of vaccine and virulent strains, the 3´-UTR of CSFV vaccine strains have two variable regions where insertion among the different vaccine strains were frequently found. The first is located between the second conservative TAA codon and the start of T-rich region where we found the variable length insertion in the same vaccine strain Thiveral or HCLV and the second is located between the end of T-rich region and the front of GAA codon, however, a 4-nt deletion was found in this region in the virulent Shimen strain. These two regions may represent the “hot spot” for mutation. Modeling the secondary structures of the 3´-UTR suggests that the T-rich insertion could result in the change of structure and free energy, thus affecting the stability of the 3´-UTR structure. These findings will help to understand the mechanism of attenuated vaccines and improve vaccine safety, stability, and efficacy. Key words: classical swine fever virus, 3´-UTR, heterogeneity, RNA secondary structure
INTRODUCTION Classical swine fever virus (CSFV) is a single-stranded positive-sense RNA virus. The virus belongs to the genus Pestivirus within the family Flaviviridae (Moennig and Plagemann 1992). Previous studies showed that the attenuated vaccine strains of CSFV have a 12 nucleotides (nt) insertion in the 3´-UTR of genome when compared to that of CSFV virulent strains (Vilcek and Belak 1997; Bjorklund et al. 1998; Wu et al. 2001; Xiao et al. 2004). The insertion helped the virus
adapt to the needs of cells or a sign of attenuated virulence, it is worthy of in depth study. Especially, the study of HCLV can effectively resolve the problem that no reasonable scientific explanation for attenuated mechanism of the vaccine developed in China. At the same time, exploring the biological function of RNA virus 3´-UTR insertion will help us to understand the virulence determinant of CSFV. The 3´-UTR mediates genomic replication of pestivirus. RNA-dependent RNA polymerase (RdRp) first recognizes and binds the 3´-UTR and initiates RNA synthesis to generate a minus-strand RNA from the plus-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. FAN Yun-feng, Ph D, Tel: +86-10-59194743, E-mail: [email protected]; Correspondence ZHAO Qi-zu, Professor, Tel: +86-10-6210367, E-mail: zhaoqizu@ivdc. gov.cn © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60300-X
Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses
strand genomic RNA. A progeny plus-strand RNA genome is then synthesized using the minus-strand as a template (Gong et al. 1996). The primary and secondary structures in the 3´-UTR of the pestivirus genome are closely related to the replication, multiplication, and virulence of the virus. Wang et al. (2008) constructed and analyzed two chimeras stemmed from a highly virulent strain Shimen either with introduction of the 12-nt insertion in 3´-UTR or the replacement of viral 3´-UTR by the 3´-UTR of HCLV. An animal experiment showed that the two chimeras were both dramatically attenuated in pigs, and completely protect the pigs against lethal challenge with highly virulent CSFV. These data demonstrate that the insertion in 3´-UTR are closely related to the attenuation of CSFV. In this paper, the 3´-UTR of vaccine strains Thiverval, HCLV, and virulent strain Shimen were sequenced and analyzed. The results obtained will help us to understand the influence of the insert in various position and length on replication, multiplication, and virulence of virus.
MATERIALS AND METHODS Viruses and cells CSFV Thiverval strain was kindly provided by Sanofi Corporation (France) and passaged in porcine kidney cell line 15 (PK-15) at 29-30°C. CSFV HCLV vaccine strain (HCLV, 1996, 3, 28) and Shimen virulent strain (SM/F114, 1998, 4, 25) were provided by China Institute of Veterinary Drug Control (IVDC). The PK-15 cell line was obtained from American Type Culture Collection.
Viral RNA extraction, reverse transcription, and PCR Viral RNA of Thiverval, HCLV, and Shimen was extracted by trizol (Invitrogen, USA) supplemented with 1 µL RNase inhibitor and 1.5 µL 100 mmol L-1 DTT. The extracted RNA was dissolved in 10 µL RNase-free water. A pair of primers were designed with Oligo6.0 (Molecular Biology Insights, USA) based on the sequences of Alfort/187 (accession no. X87939) and
143
HCLV (accession no. AF091507). The forward primer designated as EF is 5´-GAG AAC AAG CTT CGA GAA GCT G-3´. The reverse primer designated as ER is 5´GGG CCG TTA GAA ATT ACC-3´. The cDNA was synthesized by SuperScriptTM III reverse transcriptase (Invitrogen, USA) using ER as a primer and the extracted viral RNA as a template. PCR was carried out in a total volume of 50 µL. The reaction mixture contained 2 µL cDNA, 5 µL 10 × PCR buffer, 1 µL dNTP mix (10 mmol L-1), 2.5 µL EF and ER primers (10 µmol L-1), 1 µL (2 U) PfuUltraTM highfidelity DNA polymerase (Stratagene, USA) and the final volume was adjusted with deionized water. Following a 90-s predenaturing at 94°C, PCR was done by 30 cycles of a 30-s 94°C denaturing step, a 30-s 55°C annealing step, and a 1-min 72°C extension step. A 10-min 72°C extension step was performed after the 30 cycles.
Purification, cloning and sequencing PCR product was verified by electrophoresis on 1% agarose gel and purified with PCR Purification Kit (Tiangen, China). The purified PCR fragment was treated with dATP and Taq polymerase at 72°C for 30 min and cloned into pMD18-T vector (TaKaRa, Japan). Recombinant clones were identified by PCR and restriction endonuclease digestion. The positive clones were randomly selected and sequenced with the ABI PRISM 3730 sequencer (Applied Biosystems, USA).
Primary and secondary structure analysis of CSFV 3´-UTR The sequences of positive clones were aligned with 15 CSFV strains obtained from GenBank: Shimen (accession no. AF092448), Alfort/187 (accession no. X87939), ALD (accession no. D49532), Brescia (accession no. AF091661), Glentorf (accession no. U45478), CAP (accession no. X96550), HCLV (accession no. AF091507), Chinese C (accession no. Z46258), CS (accession no. AF099102), Porcivac (accession no. AF026714), Rovac (accession no. AF026717), Russian LK vaccine (accession no. AF026718), Rimes (accession no. U45477), GPE- (accession no. D49533), and Duvaxin (accession no. AF026710). The align-
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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ment was carried out using DNAstar (DNASTAR Inc., Madison, Wisconsin, USA) and Sequencher (Gene Codes corporation, USA) software. The RNA secondary structures of different 3´-UTR were predicted and analyzed using RNAstructure 4.4 software (http://rna. chem.rochester.edu/).
RESULTS RT-PCR of CSFV Thiverval, HCLV, and Shimen strains and identification of recombinant clones Using EF and ER primers, the DNA fragments approximately 604 bp which includes the entire 3´-UTR were successfully amplified from Thiverval, HCLV, and Shimen strains after verifying by agarose gel electrophoresis. The 20 white clones from those three viruses were randomly selected from transformation plates, respectively. After further identification by PCR methods and restriction endonuclease digestion, the 13 recombinant clones from Thiveral, 12 from Shimen, and 20 from HCLV were obtained for sequencing.
Sequence and alignment analysis of CSFV strains Thiverval, HCLV, and Shimen from different recombinant clones In regard to Thiverval strain, we sequenced 13 recombinant clones from the ligation of one PCR product. The sequence alignment indicated that there were ten kinds of different length 3´-UTR among the 13 recombinant clones. The longest was 259 bp and the shortest was 233 bp. The difference in the length of the 3´UTR was resulted from the insertion of nucleotides ranging from 32 nt (ATTTTTT TTTTTTT ATTTTTTT TTT TTTTTTT) to 6 nt (ATTTTTT) (Fig. 1). The 3´-UTR of HCLV was 239 bp in length among most of the recombinant clones (17/20). The sequences were identical with those from GenBank (accession nos. AF531433, AF091507, and AY805221) and had the same 12-nt (TTTTCTTTTTTC) insertion in T-rich region when compared with CSFV virulent Shimen strain. However, three of 20 recombinant clones (HCLV4-2, HCLV4-9, and HCLVH5BC) contained different length of insertion. They were 8 nt (TTTTCTTT), 13 nt (TTT
FAN Yun-feng et al.
TCTTTTTTCT), and 17 nt (TTTTCTTTTTTT TT T TT), respectively (Fig. 1). The 3´-UTR of Shimen was 228 bp. The most of recombinant clones (11/12) were identical with Shimen sequences from GenBank (accession nos. AF092448 and AY775178). The 3´-UTR length of 23 clones of Shimen strain (Shimen-23) was 224 bp and was 4 nt (TATT) shorter than other clones (Fig. 1).
Alignment analysis of the 3´-UTR of different CSFV strains We aligned the sequences of 3´-UTR among CSFV strains Thiverval, HCLV, Shimen, and other virulent and vaccine strains. We found that the insertions among eight vaccine strains (Thiverval, HCLV, Chinese C, CS, Porcivac, Ravoc, Russian LK vaccine, and Duvaxin) were at different positions in T-rich region after stop codon. The insertions with different length were located between the second consensus TAA codon and the start of T-rich region in Thiverval strain, HCLV, and its derivative strains. The insertions were located between the end of T-rich region and the codon GAA in the CS, Porcivac, Ravoc, and Russian LK vaccine strains in which the insertions were the consensus sequence of TATTTATTTATC. However, it was 4 nt (TATT) insertion in vaccine Duvaxin strain. Interestingly, we found the Shimen strain PCR product clones Shimen-23 had a 4-nt deletion at this position (Fig. 1).
Secondary structure analysis of virulent and vaccine strains The basic model of the 3´-UTR of CSFV consisted of four serials stem-loop structures (named stem-loop I, II, III, IV). Stem-loop I was located at the 3´ terminal of 3´-UTR region and was highly conserved among all CSFV. The intervening sequence ACAGCACTTTA was conserved between stem-loop I and II. Stem-loop II was composed of part of the variable region and part of conserved region, the variable region of stem-loop II had less effect on secondary structure. Stem-loop III and IV were located in the variable region where the sequence change may affect the secondary structure of the 3´-UTR of CSFV, specially the structure of stem© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses
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Fig. 1 Alignment of nucleotide sequence of the 3´-UTR of 23 CSFV strains. “.”indicates the same nucleotides; “-” indicates gaps; vertical box indicates two conservative TAA codons and GAA codons; horizontal box indicates poly AT rich region.
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loop III (Fig. 2). To maintain the similar 3´-UTR structure, the free energy of vaccine strain was higher than that of
FAN Yun-feng et al.
virulent strain and the free energy in 3´-UTR of vaccine strain was increased with length of insert.
Fig. 2 Secondary structure and free energy of the CSFV 3´-UTR. A, Shimen strain, free energy=-55.7; B, HCLV H5BC strain, free energy =-43.6; C, HCLV 4-2 strain, free energy=-44.6; D, CS strain, free energy=-47.0
DISCUSSION Vaccination is the main measure to control CSF. At present, the routinely used vaccines are the hog cholera lapinized virus (HCLV), the Japanese guinea-pig exaltation-negative (GPE-) strain and the Thiverval strain (Blome et al. 2006; Dong and Chen 2007). The HCLV is a stable lapinized strain by adapting the virulent strain in rabbits and the Thiverval stain has been obtained by passage of the virulent strains in certain cells under low temperature (Dong and Chen 2007). Although the at-
tenuated method is different, both the 3´-UTR of Thiverval and HCLV strain contain different length of insertions as compared to that of parent strains. The insertion is longer in Thiverval strain (about 23 nt) than that in HCLV strain (about 12 nt). In addition, a significant heterogeneity of the insertion sequences is found in the 3´-UTR of both strains. For HCLV strain, besides a 12-nt insertion which has been reported previously, insertions of 8, 13, and 17 nt were found. For Thiverval strain, the heterogeneity is more significant. The lengths of insertions among 13 clones
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Heterogeneity and Secondary Structure Analysis of 3´ Untranslated Region in Classical Swine Fever Viruses
sequenced are different with the longest 32 nt, and the shortest only 6 nt. In order to further investigate the variance of the 3´UTR between virulent and vaccine strains, we compared the published sequences of the 3´-UTR of virulent and vaccine strains from different countries with the sequences determined in this work. Total eight sequences of the 3´-UTR of vaccine strains HCLV, Chinese C, CS, Porcivac, Rovac, Russian LK, Duvaxin and Thiverval were analyzed. They were divided into two groups according to site of the insertion as described by Wu et al. (2001). We found that the 3´-UTR of CSFV have two variable regions. One region is located between the second conservative TAA codon and the start of T-rich region, the other is located between the end of T-rich region and the front of GAA codon. We also found that Shimen 23 has a variance at second region, it was 4 nt shorter than other Shimen strain that had been reported. The two regions may represent the “hot spot” for mutation. Holland et al. (1992) reported that RNA viruses have a property of quasispecies. The classical swine fever virus is possible to apparent 3´-UTR length polymorphism during replication. The dominant sequence of Thiverval strain contains 23 nt insertion and HCLV contains 12 nt insertion and other insertion in two kinds of vaccine strain is secondary sequence that may be the mutated sequence in the virus evolutionary process. However, it was unpractical to purify virus through traditional plaque clone methods due to CSFV non-cytopathogenic effect in cell culture. The data imply that the heterogeneity 3´-UTR may result in the safety hidden trouble of attenuated vaccine. Previous data showed that the 3´-UTR of pestivirus contains distinctive RNA motifs, a stable stem-loop I and a considerably less stable stem-loop II. These two stem-loops are separated by a long single-stranded intervening sequence (Deng and Brock 1993; Yu et al. 1999). Based on these findings, we analyzed the secondary structure of the 3´-UTR of vaccine strains and virulent strains. We found that the length and the location of the insertion in the 3´-UTR could affect the secondary structure of the 3´-UTR. We found in vaccine strains Thiverval, CS, and HCLV, the free energy of the 3´-UTR which is higher than that of virulent strains and is increased when the length of insertion increases,
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suggesting the different length insertion may lead to the unstablization of 3´-UTR in CSFV. The phenomenon that an insertion in 3´-UTR may change viral virulence has been previously reported in other RNA viruses. Rodriguez-Cerezo et al. (1991) reported that a 58-nt insertion in 3´-UTR induced tobacco vein mottling virus (TVMV) attenuated in Nicotiana tabacum plant. Similar report was found in CSFV, while Wang et al. (2008) introduced 12 nt insertion of HCLV in virulent strain Shimen which led to virulence attenuated in swine. That data demonstrate that the 12 nt insertion in 3´-UTR are closely related to the attenuation of CSFV. However, the GPE- vaccine strain do not contain the insertion and the attenuation mechanism of that remains unknown. To further understand the relationship between the insertion in the 3´-UTR and the attenuation of CSFV, we have constructed the infectious cDNA clone of Thiverval with different length of insertions in the 3´-UTR and rescued live viruses. We found that the length of insertions in the 3´-UTR may affect virus multiplication in cell culture (unpublished data). The further studies are needed to understand the attenuation mechanism and allow rationally developing safer and more effective live-attenuated vaccine for CSF prevention and control.
Acknowledgements This work was supported by the National Natural Science Foundation of China (30571377) and the National High-Tech R&D Program of China (863 Program, 2006AA10A204).
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