National Veterinary Research Institute, Al. Partyzantów 57, 24-100 Puawy, Poland1
Danish Veterinary Institute for Virus Research, Lindholm, 4771 Kalvehave, Denmark2
National Veterinary Institute, Department of Virology, Biomedical Center, Box 585, S-751 23 Uppsala, Sweden3
Veterinary Laboratories Agency (Weybridge), Addlestone, Surrey KT15 3NB, UK4
Author for correspondence: Tomasz Stadejek. Fax 48 81 886 25 95. e-mail stadejek{at}piwet.pulawy.pl
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Abstract |
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Introduction |
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PRRS is caused by a virus (PRRSV) that has been classified with lactate dehydrogenase elevating virus (LDV) of mice, equine arteritis virus (EAV) and simian haemorrhagic fever virus (SHFV) in the family Arteriviridae in the order Nidovirales (Cavanagh, 1997 ). PRRSV is a small enveloped virus with a positive-sense, single-stranded RNA genome of approximately 15 kb. The PRRSV genome contains nine open reading frames (ORFs) coding for the viral replicase (ORF1a and 1b), four membrane-associated glycoproteins (ORFs 2a to 5), two unglycosylated membrane proteins (ORF2b and ORF6) and the nucleocapsid protein N (ORF7) (Meulenberg et al., 1993
; Meng et al., 1994
; Wu et al., 2001
).
Sequence comparison has shown that there are significant genetic differences between the prototype strains from North America (VR-2332) and Europe (Lelystad virus LV) (Meulenberg et al., 1993 ; Murtaugh et al., 1995
; Nelsen et al., 1999
). These prototype strains define the two genotypes of PRRSV which are currently recognized: American (US) and European (EU) types, which are clearly distinguishable antigenically as well as by sequencing (Meng, 2000
; Dea et al., 2000
). At the beginning of the global PRRSV epidemic, EU-types were detected only in Europe, while US-types were restricted to North and Central America (Andreyev et al., 1997
) and Asia (Shibata et al., 1996
). Now, EU-type PRRSV has been found in Canada (Dewey et al., 2000
), and US-type PRRSV has been introduced to Europe through the use of a live vaccine (Bøtner et al., 1997
, 1999
; Storgaard et al., 1999
; Nielsen et al., 2001
, 2002
).
Several studies have shown that a high degree of genetic variability exists within the US-type of PRRSV (Meng et al., 1995 ; Kapur et al., 1996
; Andreyev et al., 1997
; Pirzadeh et al., 1998
; Morozov et al., 1995
; Gagnon & Dea, 1998
). While early studies suggested that a lower degree of variability might exist in EU-type PRRSV (Suarez et al., 1996
; Drew et al., 1997
; Le Gall et al., 1998
), later studies in some countries, such as Denmark and Italy, have reported a high divergence of EU-type isolates (Madsen et al., 1998
; Oleksiewicz et al., 2000
; Nielsen et al., 2000
; Forsberg et al., 2001
, 2002
). A recent study demonstrated that PRRSV isolates from the Czech republic can be very dissimilar to the prototype Lelystad virus (Indik et al., 2000
), and there are indications that PRRSV isolates from Russia may also be very different from West European PRRSV isolates (Andreyev et al., 1999
). The increase in trade foreseen with an expansion of the European Union might therefore increase the risk of exchange of new PRRSV variants between Western and Eastern Europe.
Since protection against heterologous challenge may be limited after PRRSV infection (Meng, 2000 ), and PRRSV genetic drift can influence the reactivity of monoclonal antibodies (Rowland et al., 1999
) or efficacy of PCR primers, ascertaining the true extent of variability in EU-type PRRSV is of major relevance for vaccine development as well as for diagnostics. To explore these issues, we determined the ORF5 and ORF7 sequences of several EU-type PRRSV strains, focusing on Poland and Lithuania, two Eastern European countries from which PRRSV sequences have not previously been reported. Also, the ORF5 sequences of two currently available live EU-type PRRSV vaccines, which are currently in use in Poland, were determined.
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Methods |
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Total RNA was used as template in a single-tube reverse transcription nested PCR specific for ORF5 of EU-type PRRSV. In the first step, 5 µl of 22% trehalose (Colaço et al., 1992 ) was used to store and maintain the following mixture in the lid of 0·2 ml Eppendorf tubes: 20 pmol of each of the inner primers ORF5F (5' ATGAGATGTTCTCACAAATTGGGGCG 3') and ORF5R (5' CTAGGCCTCCCATTGCTCAGCCGAAGT 3') (Suarez et al., 1996
), 1 µl of dNTPs (10 mM) and 0·25 µl of Taq Polymerase (1·25 U, Fermentas, Vilnius, Lithuania). The tubes were left to dry for 2 h at room temperature prior to storage. In the next step, RTPCR was performed in the bottom of the tubes containing the dried, trehalose-treated reagents within the lid. Amplification was carried out in 50 µl volumes containing 5 µl of RNA and the following reagents: 5 µl 10x PCR buffer [100 mM TrisHCl (pH 8·8), 500 mM KCl], 0·8% Nonidet P40 (Fermentas), 5 µl MgCl2 (25 mM, Fermentas), 2 µl dNTPs (10 mM, Sigma), 5 pmol of each of outer primers EUORF5B (5' CAATGAGGTGGGCIACAACC 3') and EUORF5C (5' TATGTIATGCTAAAGGCTAGCAC 3') (Oleksiewicz et al., 1998
), 1 µl 10% Triton X-100 (Sigma), 0·5 µl (2·5 U) Taq DNA polymerase (Fermentas), 0·25 µl (10 U) RNasin (Promega) and 0·5 µl (100 U) MMLV reverse transcriptase (Life Technologies). Mineral oil was included to act as a vapour barrier between the RTPCR reaction and the dried reagents within the lid. The tubes were then subjected to the following cycle parameters: 42 °C for 30 min, 95 °C for 5 min, and then 20 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1·5 min. The tubes were subsequently inverted several times to dissolve the dried reagents in the lid in order to initiate the nested PCR. The tubes were then centrifuged briefly before returning to the thermocycler for nested PCR, using 35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1·5 min. A single extension step of 72 °C for 10 min completed the amplification process. The RTPCR resulted in a final amplicon of 606 bp.
From selected RNAs, ORF7 was also amplified. Five µl of RNA was mixed with 8·25 µl of water, 4 µl of 5x MMLV RT buffer (Life Technologies), 1 µl dNTPs (10 mM each), 0·5 µl MMLV (Life Technologies), 0·25 µl RNasin (Life Technologies) and 1 µl random hexanucleotides (100 ng) (Pharmacia). Mineral oil was added to act as a vapour barrier. To perform reverse transcription the mixtures were incubated at 37 °C for 90 min and at 95 °C for 10 min. Five µl amounts of cDNA were PCR amplified in a total volume of 50 µl (28·8 µl water, 10 µl 5x Taq polymerase reaction buffer, 5 µl 25 mM MgCl2, 4 µl dNTP mix, 1 U of Taq Polymerase (Fermentas) and 20 pmol of each primer: 5' GCCCCTGCCCAICACG 3' and 5' TCGCCCTAATTGAATAGGTGA 3' (Oleksiewicz et al., 1998 ). Thermal cycling was done using 35 cycles of 94 °C for 15 sec, 52 °C for 30 sec and 72 °C for 30 sec. A single extension step of 72 °C for 10 min completed the amplification process. The expected size of the ORF7 amplicon was 637 bp.
Nucleotide sequencing and analysis.
Prior to sequencing, the PCR products were electrophoresed on 1·5% agarose gels. After ethidium bromide staining and desalting in ultrafiltered water, bands of the expected size were excised from the gel, and the DNA was recovered using 0·45 µm Ultrafree-MC spin filter devices (Millipore). Gel-purified (not cloned) PCR products were cycle sequenced using the BigDye Terminator Cycle Sequencing kit (v2.0, Applied Biosystems) and an ABI310 genetic analyser (Applied Biosystems). Sequences from both strands of the ORF5 PCR products were determined with the same primers as used for the nested PCR amplification. ORF7 sequences were determined using primers R133 (5' TCGCCCTAATTGAATAGGTGACTC 3') and F132 (5' GTGCTGGGCGGCAAACGAGCTGGT 3') (Drew et al., 1997 ). The sequences were assembled using SeqMan (Lasergene program package, DNASTAR Inc.). The RTPCR and sequencing strategy described above allowed the determination of partial ORF5 sequences (432 nucleotides corresponding to positions 97528 of ORF5), and complete ORF7 sequences. All sequences reported in this study have been submitted to GenBank, as summarized in Table 1
.
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Antigenic determinants in the ORF5 protein were predicted with PROTEAN (Lasergene program package). PROTEAN uses the JamesonWolf algorithm, which takes into account hydrophilicity, surface probability, chain flexibility, hydropathy and secondary structure (Jameson & Wolf, 1988 ).
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Results |
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Lithuanian PRRSV sequences are exceptionally diverse
While Spain, Denmark, the Czech Republic and Italy have previously been shown to hold the majority of PRRSV sequence diversity in Europe (Suarez et al., 1996 ; Indik et al., 2000
; Forsberg et al., 2001
, 2002
), we wished to re-examine this question in the light of the new sequences from Poland and Lithuania. Based on the simplest form of analysis, examination of percentage nucleotide identity, the two most distinct European sequences in our dataset were Nie from Poland (PL9 in Fig. 1
) and Aus from Lithuania (LT1 in Fig. 1
). Nie and Aus exhibited only 71·5% nucleotide identity between each other (not shown in detail, but see Fig. 1
). Excluding the Polish and Lithuanian sequences from the dataset, the most distinct European sequences were 2156 (IT1) and Olot/91 (ES1), which had 82·4% nucleotide identity. Thus, the Polish and Lithuanian sequences expanded the known diversity range of European-type PRRSV. Nie (Poland, 1997) and Aus (Lithuania, 2000) were temporally as well as geographically distinct. When the analysis was restricted to sequences from the year 2000, the two most distinct sequences in our dataset were Sid from Lithuania and Krz from Poland (LT2 and PL13 in Fig. 1
). Sid and Krz exhibited only 72·2% nucleic acid identity between each other (not shown in detail, but see Fig. 1
). However, the Lithuanian sequences appeared to be exceptionally different from any other European-genotype sequence previously reported (Fig. 1
, LT1LT4). Furthermore, the diversity between the Lithuanian sequences was as high as between any other European sequences (Fig. 1
and not shown).
Sequence analysis hints at common ancestor for EU and US genotypes
To see how the exceptionally different sequences from Lithuania (Fig. 1, LT14) were placed in the current EU-US PRRSV genealogy, a new phylogenetic tree was constructed, which also included a selection of the most diverse ORF5 sequences from North America and Asia (Table 1
and Fig. 2A
). All the ORF5 sequences from European countries in the current study, including the Lithuanian ones, had consistently below 64% identity at the nucleotide level and below 70% identity at the amino acid level to US ORF5 sequences, and were therefore clearly of EU-type (not shown in detail, but see Fig. 2
, large tree). However, the EUUS ORF5 tree (Fig. 2
, large tree) showed that the exceptionally diverse virus populations of Lithuania connected onto the long internal EUUS branch. This branching pattern had high bootstrap support (Fig. 2
, large tree). To investigate this finding further, we determined the complete ORF7 sequences for PL8, PL9, LT1 and LT2. A phylogenetic tree based on these ORF7 sequences (Fig. 2B
) confirmed the branching pattern found for the ORF5 genealogy, albeit with lower bootstrap support (Fig. 2A
). The same branching pattern observed for both ORF5 and ORF7 genealogies effectively excludes the possibility that the unique location of the Lithuanian samples was a result of ancestral recombination between EU and US types of PRRSV. Instead, the unique phylogenetic branching of ORF5 (Fig. 2
, large tree) as well as ORF7 (Fig. 2
, small tree) sequences, supported by high bootstrap values, suggested that the present-day European sequences are derived from common ancestral sequences that were more US-like than current European PRRSV.
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Predicted antigenic differences between vaccine and field viruses
The finding of an exceptional diversity in ORF5 sequences in Poland and Lithuania, together with the suspected role of the ORF5 protein in virus neutralization (Dea et al., 2000 ), made it interesting to compare the antigenicity profiles of selected present-day very diverse field sequences to the currently available EU-type live PRRSV vaccines (Fig. 4
). The residue 89109 region appeared to harbour the most pronounced changes in predicted antigenicity of the very diverse field sequences. Without exception, the residue 89109 region of the diverse field sequences had higher predicted antigenicity than VAC3 and VAC4 (Fig. 4
). The residue 89109 region corresponds approximately to the predicted location of the second ectodomain of the ORF5 protein (Fig. 3A
, boxed residues), and also constituted a variable segment of the ORF5 protein (Fig. 3A
, boxed residues, and Fig. 4
).
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Discussion |
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The main finding of the present study was that Lithuania harbours exceptionally diverse PRRSV strains. All four Lithuanian sequences were derived from RTPCR on porcine serum. In our opinion, this makes it likely that the Lithuanian sequences were derived from circulating, complete PRRSV virions, as opposed to being derived from, for example, intracellular defective RNA. We derived the viral sequences by direct sequencing of both strands of non-cloned PCR products to minimize Taq-induced artefacts. However, due to the highly surprising nature of the Lithuanian sequences, we performed additional controls to verify the RTPCR and sequencing strategy: First, we did repeated RTPCR and sequencing on selected serum samples and received identical DNA sequence. Second, we sequenced ORF5 as well as ORF7 for the Lithuanian viruses, and obtained identical phylogenetic clustering (Fig. 4). Furthermore, we estimate the diversity of Lithuanian PRRSV to resemble the diversity of Russian PRRSV strains, the nucleotide sequences of which were reported to differ up to 16% with Lelystad virus in ORF7 (Andreyev et al., 1999
). Thus, we consider it highly unlikely that the unique nature of the Lithuanian sequences is an artefact of the methods used. The Lithuanian sequences exhibited a highly surprising and unusual branching pattern in EUUS phylogenies, in that they appeared to be derived from ancestral sequences more US-like than previously seen in Europe (Fig. 2
). This interpretation is supported by the observation that the size of the ORF7 protein of Lithuanian PRRSV (124 residues) was intermediate between the sizes for prototypical EU-type (128 residues) and US-type (123 residues) ORF7 proteins (Fig. 3B
). Recently, Verheije et al. (2001)
found, using an infectious clone of EU-type PRRSV, that C-terminal truncation of the N protein was tolerated for up to six amino acids. However, the Lithuanian ORF7 sequences provide the first example that size polymorphism may occur in the ORF7 protein of field PRRSV isolates of EU type (Fig. 3B
). This finding has obvious implication for, for example, the use of the size of ORF7 RTPCR amplicons for typing viruses (Oleksiewicz et al., 1998
).
In the ORF5 segment sequenced by us, we found that some European PRRSV strains have as little as 71·5% nucleotide identity (Fig. 1, PL9 and LT1). For other plus-sense RNA viruses such as pestiviruses, a classification scheme based on pairwise sequence divergence of, for example, the E2 envelope glycoprotein gene has been suggested, using the categories isolate (up to 18·8% divergence), subgroups (17·433·8% divergence) and species (34·366·1% divergence) (Becher et al., 1999
). For comparison, the 71·5% nucleotide identity observed in the ORF5 envelope glycoprotein gene of PL1 and Sid (Fig. 1
) thus certainly seems to justify using the term subgroup for the Lithuanian PRRSVs, and may even place them in the subgroup/species grey zone.
Ideally, studies on the genetic diversity of European-type PRRSV should include a large number of isolates from all European countries, and preferably cover the time period from 1991 (the isolation of Lelystad virus, the first PRRSV isolate) until now. Clearly, this is not feasible. Thus, the true picture of European-type PRRSV sequence diversity will probably develop piecemeal, through successive complementary studies. The present study concerning the diversity of primarily Eastern European, EU-type PRRSV strains complemented previous studies on the diversity of EU type PRRSV (Suarez et al., 1996 ; Oleksiewicz et al., 2000
; Indik et al., 2000
; Forsberg et al., 2001
, 2002
). The current picture appears to be that the presence of very diverse PRRSV strains may not be a special attribute of any one country, but a general rule in Europe, except for countries such as Holland, Belgium, France and England where the presence of very closely related (Lelystad-like) PRRSV strains can perhaps be explained by trading links. It remains to be determined whether this hypothesis is correct, i.e. whether, for example, PRRSV strains from the remaining European states will also prove very diverse.
Isolation of European-type PRRSV requires facilities to establish primary cultures of porcine pulmonary alveolar macrophages (PPAM). Such facilities are not routinely available in many East European countries. Hence, we have not yet been able to obtain Polish and Lithuanian PRRSV isolates for functional studies. For example, it would be interesting to perform cross-neutralization studies between Lithuanian and other EU-type PRRSV strains. Also, it would be very interesting to determine whether the ORF7 protein of Lithuanian PRRSV would react as EU- or US-type with the monoclonal antibodies currently used for PRRSV detection and typing. Based on theoretical antigenicity prediction, at least some present-day Lithuanian and Polish ORF5 protein sequences are different from the ORF5 protein of the currently used live EU-type PRRSV vaccine strains (Fig. 4). The N-terminal part of the ORF5 protein, for which sequence was obtained in the present study, was originally thought to be involved in virus receptor binding and neutralization (Dea et al., 2000
; Snijder & Meulenberg, 1998
). However, recent studies have questioned the involvement of the N terminus of the ORF5 protein in receptor binding (Dobbe et al., 2001
), and in any case it is not known how the predicted antigenic differences between the ORF5 proteins of field and vaccine PRRSV isolates (Fig. 4
) relate to cross-neutralization properties. While the first ectodomain of the ORF5 protein is known to be a target for the antibody response in the related EAV and LDV (Balasuriya et al., 1997
), less is known about the antibody response to the PRRSV ORF5 protein. It was recently described that the C-terminal endodomain of the ORF5 protein is a target for the porcine antibody response (Dea et al., 2000
; Rodriguez et al., 2001
; Oleksiewicz et al., 2002
). While the complete C-terminal ORF5 sequence was not determined in our study, we observed that amino acid changes appeared to cluster in the predicted two ectodomains at the N terminus of the ORF5 protein (Fig. 3A
, boxed residues). It has previously been described that for PRRSV proteins, sequence diversity correlates with the extent of the porcine humoral antibody response (Oleksiewicz et al., 2000
, 2001
). Therefore, the clustering of amino acid changes in the putative ORF5 protein ectodomains (Fig. 3A
) suggests the presence of B-cell epitopes, which may have gone unnoticed due to, for example, a requirement for a particular ORF5 protein folding or glycosylation, as the studies by for example Rodriguez et al. (2001)
and Oleksiewicz et al. (2002)
mentioned above utilized assays that would predominantly identify linear epitopes.
In a similar vein, it has been suggested that the last 20 carboxy-terminal residues of the ORF7 protein are important for the overall conformation of the ORF7 protein of US-type PRRSV, and hence its reactivity with various monoclonal antibodies (Wootton et al., 1998 ). In contrast, Verheije et al. (2001)
found that deletion of up to six amino acids from the C terminus of the ORF7 protein of EU-type PRRSV did not affect the reactivity of the protein with various monoclonal antibodies. Also, the first 12 residues of the ORF7 protein have been shown to form a linear epitope that is conserved in European but not American PRRSVs (Fig. 3B
B, site A) (Meulenberg et al., 1998
). The Lithuanian ORF7 protein sequences exhibited radical changes in N-terminal as well as C-terminal regions (Fig. 3B
). However, the reactivity of monoclonal antibodies cannot be inferred from the linear sequence, and the antigenicity of the Lithuanian ORF7 protein remains to be investigated. It should be mentioned that sera from pigs on the four Lithuanian farms from which the LT14 sequences were obtained were positive for anti-PRRSV antibodies by ELISA (HerdChek PRRS ELISA, IDEXX Laboratories Inc., USA). Finally, it should be mentioned that the clinical signs reported on the Lithuanian farms were not different from those usually ascribed to PRRSV infection.
Nelsen et al. (1999) suggested that the EU- and US-types of PRRSV arose by the divergent evolution of related viruses on separate continents from a distant common ancestor. While the almost synchronous emergence of EU and US PRRSV in the late 1980s seems puzzling enough, our and previous results outline an even more surprising scenario, where genetically quite different EU-type PRRSV strains seem to have emerged independently in different European countries (Fig. 1
, NL1, ES1, ES2, DK1, IT1 from 19911992, and PL1 and PL2 from 1994). The reasons for the near-simultaneous emergence of genetically very different PRRSV types and strains at different geographical locations are currently unknown, but have been speculated to be due to, for example, changes in farming management factors, although no candidate factors were suggested (Nelsen et al., 1999
). A precise time estimate of the common ancestor of EU and US PRRSV might be helpful towards elucidating the reason for the split of the EU and US lineages, and the circumstances of PRRSV emergence in the late 1980s. We hope that the Lithuanian sequences, by virtue of their unique position in phylogenetic trees of EUUS sequences, may improve the precision of phylogenetic modelling of the very long branch connecting EU and US viruses (Fig. 2
), and allow precise temporal information to be extracted from PRRSV phylogenetic trees.
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Acknowledgments |
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Footnotes |
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c Present address: Novo Nordisk A/S, Novo Nordisk Park, 2760 Mløv, Denmark.
d Present address: Symphogen A/S, Elektrovej, Building 375, 2800 Lyngby, Denmark.
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Received 12 November 2001;
accepted 26 March 2002.