1 Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute, Beltsville Agriculture Research Center, Agricultural Research Service, United States Department of Agriculture, 10300 Baltimore Avenue, Building 173, BARC-East, Beltsville, MD 20705, USA
2 Bioinformatics, Chemical and Biological National Security Program, Computing Applications and Research Department, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA
Correspondence
M. L. Perdue
mperdue{at}anri.barc.usda.gov
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the full-length BEV sequences reported in this study are AY508696 for BEV-2 PS87 and AY508697 for BEV-2 Wye3A.
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INTRODUCTION |
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BEVs may be stable environmental contaminants on some cattle farms and, as such, might also serve as a useful surrogate for evaluating Foot-and-mouth disease virus (FMDV) farm contamination. FMDV belongs to the genus Aphthovirus (family Picornaviridae) and cannot be studied on the US mainland. As such, on-farm studies to evaluate rapid detection approaches, efficacy of disinfectants or deactivators or extraction/detection approaches for FMDV cannot be carried out directly. Utilizing BEV as a surrogate might allow optimization of extraction and/or detection approaches for bovine picornavirus contamination in the farm environment (Lund et al., 1996; Monteith et al., 1986
; Yilmaz & Kaleta, 2003
).
Study of BEV has also provided significant data of use to picornavirus virologists (Kaminaka et al., 1999; Rohll et al., 1995
; Zell & Stelzner, 1997
; Zell et al., 1999
). The crystal structure of the BEV virion was deduced from crystals of purified BEV-1 strain VG5-27 (Smyth & Martin, 2001
, 2002
; Smyth et al., 1993
) and comparisons with the capsid structure of poliovirus indicate a variety of similarities and some distinct differences in the antigenic and receptor-binding regions of the virus. We decided to compare genomic sequences of virus isolates from a local farm that is known to be endemically infected with BEV to the ATCC isolate BEV-2 PS87, originally from Pennsylvania, USA (Dunne et al., 1974
), and to evaluate the differences between these US isolates.
Further, as no full-length genome sequence for BEV-2 was available, we completed the entire genome sequence of two strains to enhance the database of sequences of members of the family Picornaviridae. Having obtained these new sequences, we were able to generate and compare structural models by using selected picornavirus templates from the Protein Database (PDB) structure coordinates database.
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METHODS |
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RNA amplification and sequencing.
RNA from infected MDBK cell supernatant was extracted with TRIZOL LS reagent (Invitrogen) according to the manufacturer's instructions, with the exception of the addition of 1 µl glycogen (20 mg ml1) at the chloroform-extraction phase. RNA pellets were resuspended in 20 µl DEPC-treated water. The presence of BEV was confirmed by RT-PCR using Titan One-Step RT-PCR (Roche) according to the manufacturer's protocol and primers corresponding to positions 126 and 9791000 of a previously published partial sequence of BEV-2 PS87 (McNally et al., 1994).
Real-time RT-PCR primers were designed to previously published partial sequences of BEV-2 (McNally et al., 1994) and BEV-2 261 (GenBank accession no. AJ250672), and to conserved regions of BEV-1 [VG5-27 (Earle et al., 1988
), SL305 and K2577 (McCarthy et al., 1999
)], BEV-2 Bot 209 (GenBank accession no. AJ250673) and BEV-1 BEV1 (GenBank accession no. AJ250671). The forward primer sequence corresponds to nt 271288, the reverse primer to nt 554569 and the 6FAM- and TAMRA-labelled probe to nt 373394. Real-time RT-PCR was performed by using Titan One-Step RT-PCR (Roche) according to the manufacturer's protocol with 1 µM primer and 0·2 µM probe. Cycling conditions run on a Stratagene Mx4000 thermal cycler were: 30 min at 48 °C preceded 40 cycles of 95 °C for 30 s, 50 °C for 30 s and 60 °C for 45 s.
Nucleotide sequence analysis.
Extracted RNA samples were amplified by using RT-PCR as described above, using primers that were initially designed to correspond to conserved regions of published BEV-1 and BEV-2 sequences and subsequently to derived sequences. Primer sequences are available from the authors upon request. Amplicons from positive reactions were purified with a High Pure PCR purification kit (Roche) and cycle-sequenced by using a Big Dye Terminator cycle sequencing kit version 3.1 and an ABI 3100 genetic analyser (both from Applied Biosystems). Three or more amplicons were used to arrive at an overlapping consensus sequence, analysed with Sequencher 4.41 software (GeneCodes). A RACE kit (Invitrogen) and a Topo TA cloning kit for sequencing (Invitrogen) were used to complete and verify the extreme 3' and 5' ends of the genomes.
Phylogenetic analysis of BEV-2 and other picornaviruses.
Nucleotide sequences were aligned and compared by using the program CLUSTAL W. The program Mfold 3.1 (Zuker, 2003) was used to generate and compare predicted secondary structures of the 5' untranslated region (UTR) RNA sequences of the BEV strains. For analysis of full-length polyprotein sequences from picornaviruses, the program PAUP version 4 (Sinauer Associates, Sunderland, MA, USA) was used to perform heuristic searches, neighbour-joining and bootstrap analyses. Phylogenetic trees were generated in Lasergene (MEGALIGN), PAUP or by the TreeView program (Page, 1996
).
Modelling of capsid proteins.
For the purpose of VP14 structure modelling, all 79 protein structures that were related to bovine enterovirus VG5-27 and deposited in PDB were considered as potential homology templates. All of these templates were examined to select the final set of structures that would be useful for modelling. Detailed structural analysis of all templates allowed definition of regions of structural conservation and regions of structural diversity between templates. The resolution of all considered templates varied from 2·15 to 3·55 Å. In addition to X-ray-solved structures, three cryo-electron microscopy (CRYO-EM) structures were also used to verify the conformation of components VP14 within the modelled protomer complex.
For modelling protein capsids VP14, the following templates were used: 1bev, BEV-1 VG5-27, X-ray 3·00 Å; 1hxs, Human poliovirus 1 (HPV-1) Mahoney, X-ray 2·20 Å; 1aym, human rhinovirus 16 (HRV-16), X-ray 2·15 Å; 1ar7, HPV-1 Mahoney (double mutant), X-ray 2·90 Å.
In addition to the structural templates above, the following protein structures were used for model templates structure comparison to detect regions of structural deviation: 1d4m, CV-A9, X-ray 2·90 Å; 1al2, HPV-1 Mahoney, X-ray 2·90 Å; 1ev1, EV-1, X-ray 3·55 Å; 2plv, HPV-1 Mahoney, X-ray 2·88 Å; 1pvc, HPV-3 Sabin, X-ray 2·40 Å; 1oop, PEV-9 (swine vesicular disease virus), X-ray 3·00 Å; 1cov, CV-B3 coat protein, X-ray 3·50 Å; 1dgi, HPV-1, CRYO-EM (fitting of X-ray 2·88 Å PDB-2plv structures); 1jew, CV-B3 (M strain), CRYO-EM (fitting of X-ray 3·50 Å PDB-1cov structures); 1m11, EV-7, CRYO-EM (fitting of X-ray 3·55 Å PDB-1ev1 structures).
Capsid proteins from BEV-2 strains PS87 and Wye3A were modelled by using the AS2TS homology modelling system (Zemla, 2003), by which protein sequences are compared against all PDB entries and those with the highest homology are evaluated as suitable templates for modelling. PDB entry 1bev (BEV-1 VG5-27) had the highest sequence identity for the BEV-2 capsid proteins, although template 1hxs_4 of poliovirus (Mahoney strain) was substituted for 1bev_4, as the former was more complete and had been solved at higher resolution (see below). In regions where residues had been omitted from the X-ray crystallography data, additional analyses using LocalGlobal Alignment (LGA) software (Zemla, 2003
) and secondary-structure prediction using PSIPRED (Jones, 1999
) were done in order to attempt completion of the templates. Backbone models consisting of main-chain atoms for the capsid proteins of BEV strains PS87, Wye3A and K2577 were then constructed by using the modified templates. Loop regions were constructed by using the LGA modeller program. The side-chain atoms for residues that were identical to those of the templates were incorporated into the models automatically and the remaining side-chain atoms were calculated by using the program SCWRL (Bower et al., 1997
).
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RESULTS |
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Table 3 illustrates sequence similarities among the completely sequenced BEV strains and other selected enteroviruses. The genomes were divided into 5' and 3' UTRs and P1, P2 and P3 subgenomic regions. Comparing the P1 region, which encodes VP4, VP2, VP3 and VP1, the two BEV-1 isolates shared 73·2 % nucleotide and 86·5 % amino acid identity and the two sequenced BEV-2 isolates shared 82·0 % nucleotide and 95·4 % amino acid identity. The BEV-2 strains shared 63·264·4 % nucleotide identity and 65·966·6 % amino acid identity with the BEV-1 strains. Whilst BEV-1 strains shared only 3233 % nucleotide identity with HPV-1 (Polio1), BEV-2 strains shared approximately 50 % nucleotide sequence identity with HPV-1; a similar nucleotide sequence identity is shared between PEV-9 and HPV-1. Although the nucleotide sequences varied, comparisons of the predicted amino acid sequences found that all of the enteroviruses compared in this analysis were equally similar, sharing approximately 45 % identity. CLUSTAL W alignments of nucleotide and amino acid sequences of the P2 and P3 regions [which encode the non-structural proteins, VPg (a structural protein), protease and polymerase] yielded similar results. The two BEV-2 isolates clearly grouped together and were separate from the BEV-1 strains, and the four BEV strains grouped together, separate from PEV-9 and HPV-1 (Table 3
).
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Template VP1.
As 13 N-terminal residues of 1bev chain 1 (VP1) were not provided in the PDB entry and temperature factors for atoms of residues 1417 were very high (above 60), additional analyses were required to model this region. In summary, we examined several PDB templates and ultimately determined that residues 417 were best modelled as an -helix by using PDB structure 1aym chain 1 as a template.
Template VP2.
Residues 14 of 1bev chain 2 (VP2) were not provided in the PDB entry. We were unable to model these residues, due to lack of a PDB homologue for which coordinates were provided for the corresponding residues. The high temperature factors (low confidence) for residues 511, however, prompted us to adjust the structural assignment of these residues. We identified 1ar7 chain 2 as the best template for modelling this region and adjusted 1bev chain 2 coordinates accordingly. This new conformation was also found to avoid clashes with the reconstructed C-terminal disordered region of 1bev chain 4.
Template VP3.
There were no missing residues in the PDB entry for 1bev chain 3 (VP3). Therefore, the VP3 proteins for the three strains were modelled completely with high confidence by homology to 1bev chain 3.
Template VP4.
It was determined that 1bev chain 4 (VP4) was a poor template for modelling VP4, due to the lack of coordinate data for 28 of 68 residues and due to very high temperature factors for most of the residues in the PDB entry. Of 25 structures of VP4 homologues that were selected from PDB, we determined that entry 1hxs of poliovirus (Mahoney strain), with 57 % sequence identity to 1bev chain 4 and solved with 2·2 Å resolution, was the most complete and would provide the best template for modelling most regions of VP4 proteins of the three strains.
Structural deviations among the capsid proteins of BEV-2 isolates and other picornaviruses
When compared with other picornaviruses, capsid proteins VP1 and VP3 of BEV-1 and -2 deviated considerably in six regions (R1R6; see Fig. 4) (residue numbering corresponds to the 1bev structure from PDB). N-terminal regions were not considered to represent significant structural deviations, as they are expected to be flexible and difficult to solve with high confidence. These six regions lie at or within the so-called canyon, which is believed to be involved in capsid proteinhost receptor binding (Smyth & Martin, 2001
; Rossmann, 2002
) and to confer antigenic variation. Four of these regions (R1: 8692, R2: 127138, R4: 226230 and R5: 266273) have various insertions or deletions with respect to the amino acid sequence of entry 1bev chain 1 and, for VP3, there was an insertion in a loop region of the BEV-2 strains relative to K2577 (R6: 180187). Region R3 (198208) exhibits structural variation among the considered PDB templates and overlaps with the region of sequence variation between the two BEV strains. Each of these regions is located on the surface of the 1bev protomer (Fig. 5
), either on a rim of the canyon or at its base. The structural models of the capsid proteins of PS87, Wye3A and K2577 farm isolates are nearly identical, as they were built by using the same modified 1bev template. However, there are clear variations in structure between BEV-1 and BEV-2 strains in insertion/deletion regions and structural variation in regions identified by sequence variation (e.g. region R3: 198208).
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DISCUSSION |
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Phylogenetically, all of the viruses that were isolated from the Wye farm were clearly shown to be BEV-2 strains, as shown here and previously (Ley et al., 2002). BEV-specific RT-PCR suggested that the virus was present in the spring in 24-month-old calves and in the environment, but the infection had probably been cleared by summer. We are interested in these wild-type isolates in terms of prevalence, pathogenesis and their potential as vectors for expressing small molecules. Studies are under way to evaluate infectious clones of various BEV-2 strains, both as vectors and as tools to study potential pathogenesis.
Analysis of the full-length and subgenomic region nucleotide and predicted amino acid sequences of PS87 and Wye3A demonstrated that they belong to the same genotypic group. The BEV genotypes segregated as the virus isolates (of known serotype) have been shown to segregate by serotype. Although we have only looked at a limited number of BEV isolates and the serotype of the Wye3A isolate has not been confirmed, the data suggest that there is a clear difference in nucleotide and amino acid sequence between BEV-1 and BEV-2 strains and that this difference probably reflects the antigenic differences that have been observed between serotypes 1 and 2.
The difference in size between the VP1 proteins of BEV-1 and BEV-2 is noteworthy. The sequence comparisons in Fig. 1 suggest that substantial nucleotide deletions have occurred near the N-terminal coding sequences of BEV-2 between positions 2550 and 2610. By using CLUSTAL W on default settings, a 5 aa deletion in BEV-2 relative to BEV-1 was detected near the N terminus (data not shown). However, the sequence-based alignment is unreliable in this region and is partly dependent on parameter settings. Therefore, a precise correspondence between the N-terminal residues of the BEV-2 and BEV-1 VP1 proteins could not be determined. Analyses using various sequence alignment search techniques (e.g. PSI-BLAST, CLUSTAL W) and homology modelling revealed structural ambiguity inasmuch as several PDB structures of VP1 consisted of
-helix in this region, whereas several others consisted of strand or disordered regions. Based on comparative analysis of PDB templates, we determined that the most reasonable solution was to model the four N-terminal residues of 1BEV_1 (NDPG) as strand and residues 517 as
-helix. However, because the BEV-2 proteins were 5 aa shorter than those of BEV-1, it was not possible to assign these residues with certainty to either the strand portion or the
-helix portion of the template. We chose to maintain continuity at the N terminus and align these residues with the
-helix portion of the modified 1BEV_1. Most data have suggested that this N-terminal region of VP1 would be internal and disordered, and may make up a protein layer that separates the external capsid from the RNA (Rossmann, 2002
). It would therefore be interesting to determine whether these differences are significant to the structure of the two BEV serotypes.
Molecular modelling of the BEV-2 capsid structure and structural comparisons with picornaviruses support the generally accepted idea that the region of VP1 that connects the eight -strands making up the wedge-shaped region of each capsid protein is part of the variable region, specifying the antigenically variable sites (Rossmann, 2002
). Most regions of structural variation detected by our structure alignment analysis (Fig. 4
) lie in this variable region around the canyon, and region R3 overlaps with an antigenic site 1 that was described previously (Smyth & Martin, 2001
). The variations described (particularly in R1 and R5) are probably the differences that specify the antigenic nature and separation of the two BEV serotypes. The significance of the difference in the structure of VP3 that appears near the bottom of the canyon is not known, but might have some influence on the receptor-binding capacity. Studies are sparse regarding the receptor specificity of BEV, but early experiments suggested that sialic acid might be a receptor (reviewed by Racaniello, 2001
).
The phylogenetic and structural differences found between BEV-1 and BEV-2 in this study support a distinct separation of the two groups, but very little is known regarding these relationships with respect to serotypic relationships. It has been suggested that antigenic properties as classification criteria for picornaviruses will become unnecessary as sequence relationships become clearer (Stanway et al., 2002). Our data would support this notion. As we know that BEVs continue to be found in US cattle and are shed into the environment, it is worthwhile to continue to collect and analyse them. As they may share a common ancestor with and form a distinct clade with some human enteroviruses (HEV-A, Fig. 3
) and as there are virtually no serological or epidemiological data on the extent of BEV infection in US cattle, it is certainly worth continuing to be proactive in the characterization of these viruses and their potential pathogenesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Dunne, H. W., Huang, C. M. & Lin, W. J. (1974). Bovine enteroviruses in the calf: an attempt at serologic, biologic, and pathologic classification. J Am Vet Med Assoc 164, 290294.[Medline]
Earle, J. A. P., Skuce, R. A., Fleming, C. S., Hoey, E. M. & Martin, S. J. (1988). The complete nucleotide sequence of a bovine enterovirus. J Gen Virol 69, 253263.[Abstract]
Jones, D. T. (1999). Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292, 195202.[CrossRef][Medline]
Kaminaka, S., Imamura, Y., Shingu, M., Kitagawa, T. & Toyoda, T. (1999). Studies of bovine enterovirus structure by ultraviolet resonance Raman spectroscopy. J Virol Methods 77, 117123.[CrossRef][Medline]
Knowles, N. J. & Barnett, I. T. (1985). A serological classification of bovine enteroviruses. Arch Virol 83, 141155.[Medline]
Ley, V., Higgins, J. & Fayer, R. (2002). Bovine enteroviruses as indicators of fecal contamination. Appl Environ Microbiol 68, 34553461.
Lund, B., Jensen, V. F., Have, P. & Ahring, B. (1996). Inactivation of virus during anaerobic digestion of manure in laboratory scale biogas reactors. Antonie van Leeuwenhoek 69, 2531.[Medline]
McCarthy, F. M., Smith, G. A. & Mattick, J. S. (1999). Molecular characterisation of Australian bovine enteroviruses. Vet Microbiol 68, 7181.[CrossRef][Medline]
McNally, R. M., Earle, J. A. P., McIlhatton, M., Hoey, E. M. & Martin, S. J. (1994). The nucleotide sequence of the 5' non-coding and capsid coding genome regions of two bovine enterovirus strains. Arch Virol 139, 287299.[Medline]
Monteith, H. D., Shannon, E. E. & Derbyshire, J. B. (1986). The inactivation of a bovine enterovirus and a bovine parvovirus in cattle manure by anaerobic digestion, heat treatment, gamma irradiation, ensilage and composting. J Hyg (Lond) 97, 175184.[Medline]
Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357358.[Medline]
Pallansch, H. D. & Roos, R. P. (2001). Enteroviruses: polioviruses, coxsackieviruses, echoviruses and newer enteroviruses. In Fields Virology, 4th edn, pp. 723775. Edited by B. N. Fields, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, S. E. Straus & D. M. Knipe. Philadelphia, PA: Lippincott, Williams & Wilkins.
Racaniello, V. R. (2001). Picornaviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 685722. Edited by B. N. Fields, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, S. E. Straus & D. M. Knipe. Philadelphia, PA: Lippincott, Williams & Wilkins.
Rohll, J. B., Moon, D. H., Evans, D. J. & Almond, J. W. (1995). The 3' untranslated region of picornavirus RNA: features required for efficient genome replication. J Virol 69, 78357844.[Abstract]
Rossmann, M. G. (2002). Picornavirus structure overview. In Molecular Biology of Picornaviruses. Edited by B. L. Semler & E. Wimmer. Washington, DC: American Society for Microbiology.
Shingu, M., Chinami, M., Taguchi, T. & Shingu, M., Jr (1991). Therapeutic effects of bovine enterovirus infection on rabbits with experimentally induced adult T cell leukaemia. J Gen Virol 72, 20312034.[Abstract]
Smyth, M. S. & Martin, J. H. (2001). Structural, biochemical and electrostatic basis of serotype specificity in bovine enteroviruses. Arch Virol 146, 347355.[CrossRef][Medline]
Smyth, M. S. & Martin, J. H. (2002). Picornavirus uncoating. Mol Pathol 55, 214219.
Smyth, M., Fry, E., Stuart, D., Lyons, C., Hoey, E. & Martin, S. J. (1993). Preliminary crystallographic analysis of bovine enterovirus. J Mol Biol 231, 930932.[CrossRef][Medline]
Smyth, M., Tate, J., Hoey, E., Lyons, C., Martin, S. & Stuart, D. (1995). Implications for viral uncoating from the structure of bovine enterovirus. Nat Struct Biol 2, 224231.[Medline]
Smyth, M., Symonds, A., Brazinova, S. & Martin, J. (2002). Bovine enterovirus as an oncolytic virus: foetal calf serum facilitates its infection of human cells. Int J Mol Med 10, 4953.[Medline]
Stanway, G., Hovi, T., Knowles, N. J. & Hyypiä, T. (2002). Molecular and biological basis of picornavirus taxonomy. In Molecular Biology of Picornaviruses. Edited by B. L. Semler & E. Wimmer. Washington, DC: American Society for Microbiology.
Yilmaz, A. & Kaleta, E. F. (2003). Evaluation of virucidal activity of three commercial disinfectants and formic acid using bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1. Vet J 166, 6778.[CrossRef][Medline]
Zell, R. & Stelzner, A. (1997). Application of genome sequence information to the classification of bovine enteroviruses: the importance of 5'- and 3'-nontranslated regions. Virus Res 51, 213229.[CrossRef][Medline]
Zell, R., Sidigi, K., Henke, A., Schmidt-Brauns, J., Hoey, E., Martin, S. & Stelzner, A. (1999). Functional features of the bovine enterovirus 5'-non-translated region. J Gen Virol 80, 22992309.
Zemla, A. (2003). LGA: a method for finding 3D similarities in protein structures. Nucleic Acids Res 31, 33703374.
Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 34063415.
Received 1 April 2004;
accepted 26 July 2004.