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Short Communication |
1 National Veterinary Research Institute, Al. Partyzantów 57, 24-100 Pu
awy, Poland
2 Novo Nordisk A/S, Virology and Molecular Toxicology, Novo Nordisk Park, 2760 Måløv, Denmark
3 S. N. Vyshelesskij Institute of Experimental Veterinary Medicine, National Academy of Sciences of Belarus, 2 Vyshelesskij Street, Minsk 223020, Belarus
Correspondence
T. Stadejek
stadejek{at}piwet.pulawy.pl
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ABSTRACT |
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Published online ahead of print on 7 April 2006 as DOI 10.1099/vir.0.81782-0.
Supplementary figures and a table showing details of the Belarusian and Polish herds from which sequences were obtained are available in JGV Online.
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), in the USA in 1985 (Zimmerman et al., 1997), in South Korea in 1985 (Shin et al., 1993), in the Asian part of the former Soviet Union in 1986 (Grebennikova et al., 2004), in Japan in 1987 (Yoshii et al., 2005), in the former German Democratic Republic in 1987 (Ohlinger et al., 2000), in the Philippines in 1987 (Thanawongnuwech et al., 2003) and in Thailand in 1989 (Thanawongnuwech et al., 2004). In western Europe, the first clinical outbreaks were reported in November 1990 in Germany, with outbreaks in the Netherlands, Spain, UK, France, Belgium and Denmark occurring through 1991–1992 (OIE, 1992).
Thus, PRRSV emerged globally through a brief time window. Surprisingly, the viruses that appeared in Europe and North America were related only distantly (55–70 % nucleotide identity). PRRSV was first isolated in the Netherlands (Wensvoort et al., 1991) and that isolate (Lelystad virus), together with the first North American isolate (VR2332), now define the two recognized genotypes of PRRSV: European (EU genotype, type I) and North American (US genotype, type II) (Snijder et al., 2004).
The cotemporal emergence of genetically very different viruses could be due to two independent species jumps in Europe and North America (triggered by as-yet-unknown factors) or a single species-jump event at an unknown location followed by very quick global spread, coupled with unusually quick evolution of the virus (Forsberg, 2005; Hanada et al., 2005). However, phylogenetic analysis places the most recent common ancestor (MRCA) for the EU and US genotypes at least 100 years back in time (Forsberg, 2005; Hanada et al., 2005), providing strong support for the hypothesis that EU and US viruses evolved in parallel in North America and Europe prior to their cotemporal species jump into pigs and emergence as clinical entities in the later 1980s.
Originally, EU genotype viruses were thought to form a very homogeneous, ‘Lelystad-like’ group (Wensvoort et al., 1991; Suarez et al., 1996; Drew et al., 1997; Le Gall et al., 1998). More recently, the view that EU genotype viruses are genetically homogeneous and Lelystad-like was challenged by the reporting of unusually diverse EU genotype PRRSV strains, first in Denmark (Oleksiewicz et al., 2000) and later in Italy (Forsberg et al., 2002), the Czech Republic (Indik et al., 2000), Poland (Stadejek et al., 2002), Spain (Mateu et al., 2003), Germany and the Netherlands (Pesch et al., 2005) and even Thailand (Thanawongnuwech et al., 2004). In a recent, groundbreaking study, by using a Lelystad-like, live-attenuated vaccine strain and an Italian isolate as challenge, it was demonstrated that the genetic diversity within EU genotype viruses is sufficient to affect vaccine efficacy (Labarque et al., 2004). The Italian challenge virus represented one of the most diverse EU genotype field isolates known at the time (Forsberg et al., 2002). Since then, new studies have demonstrated that eastern European countries such as Lithuania harbour EU genotype strains of much higher diversity than does Italy (Stadejek et al., 2002).
Because of the great importance of PRRSV diversity for vaccine development and because we wished to further explore the diversity of EU genotype PRRSV in eastern Europe, we sequenced EU genotype field strains from 11 Belarusian herds. The herds were large, ranging from 2500 to 9000 sows, and included farrow to finish and nucleus herds. All herds were infected persistently with PRRSV and presented the full range of disease conditions that could be ascribed to PRRSV. For comparison with the Belarusian sequences, we obtained sequences from four herds located in north-eastern Poland, close to the Lithuanian, Russian and Belarusian borders, and from one herd from western Poland (Fig. 1; Table 1; Supplementary Table S1, available in JGV Online). RNA purification from pig serum, RT-PCR amplification of open reading frames (ORFs) 5 and 7 and sequencing of bulk (not cloned) PCR product were done essentially as described previously (Stadejek et al., 2002). Briefly, for RT-PCR of ORF5, previously described primers were used (Stadejek et al., 2002). RT–nested PCR of ORF7 was performed by using the following primers: external, 5'-GCCCCTGCCCAICACG-3' and 5'-TCGCCCTAATTGAATAGGTGA-3' (Oleksiewicz et al., 1998), and internal, 5'-TCGCCCTAATTGAATAGGTGACTC-3' and 5'-CGAGCTGTTAAACGAGGAGTG-3' (Drew et al., 1997). The ORF5 RT-PCR was specific for EU genotype PRRSV (i.e. the primer-binding sites are not conserved in US genotype PRRSV), whereas the ORF7 primer-binding sites are conserved between EU and US genotype viruses. The three currently available EU genotype live-attenuated vaccines, Porcilis PRRS (Intervet), Amervac-PRRS (HIPRA) and Pyrsvac-183 (SYVA), were also sequenced (Table 1).
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). When analysed together with the new Belarusian EU genotype sequences, the highly diverse reference EU type sequences coalesced to form a single group, termed EU subtype 1 (Fig. 2). In contrast, the Belarusian and Lithuanian sequences were separated from EU subtype 1. Thus, the new Belarusian sequences demonstrated that EU genotype PRRSV consists of several genetic subtypes that can be separated with high bootstrap support (Fig. 2a). An identical picture was seen after phylogenetic analysis of complete ORF7 sequences (see Supplementary Fig. S1, available in JGV Online). Only one sequence, Bel-42, exhibited different positioning in the ORF5 and ORF7 trees. This could be due to recombination, which has been described previously in EU as well as US genotype field strains (van Vugt et al., 2001) and for which conserved sequence stretches between ORFs 5 and 7 are known to serve as hot spots (Forsberg et al., 2002).
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). Thus, our data show that, in most of Europe (west of the eastern Polish border), relatively closely related EU genotype strains circulate, whereas east of the eastern Polish border, a great diversity of EU genotype strains can be observed (Fig. 1). We believe that this EU genotype PRRSV distribution pattern is explained by trade: whilst Belarus imports breeding animals from the west, there is essentially no livestock trade from Belarus to Poland. Because ancestral populations are generally held to be more diverse than descendant populations, we suggest that this lack of livestock movement from east to west may have serendipitously preserved phylogenetic evidence of PRRSV emergence in eastern Europe. Other reasons also make a ‘Eurasian PRRSV origin’ hypothesis attractive: the currently available sequences place the MRCA of EU genotype PRRSV strains between 1946 and 1967, i.e. during the post-World War II development of Europe (R. Forsberg, personal communication). It seems plausible that the post-war expansion of the former Soviet Union might have created an environment that allowed a new virus to emerge or an already emerged virus to spread. Also, because Poland and other east and central European countries were influenced much less by the Soviet Union animal-breeding policies than, for example, Belarus and the Baltic states (e.g. Lithuania), it seems plausible that, if new PRRSV subtypes arose during the post-war upheavals, they would be dominant in Lithuania, Belarus and probably other countries of the former Soviet Union, but would not necessarily be present west of the Polish border (Fig. 1).
The nucleocapsid protein is one of the most conserved PRRSV proteins. Accordingly, without exception, all previous studies covering the time period 1989–2005 in western, northern, central and southern Europe, North America and Asia, yielding approximately 270 sequences deposited in GenBank of both EU and US genotypes, have so far failed to reveal size polymorphisms in the PRRSV nucleocapsid protein (Meng et al., 1995; Suarez et al., 1996; Drew et al., 1997; Le Gall et al., 1998; Forsberg et al., 2002). Accordingly, based on deduced amino acid sequence, all of the new Polish strains sequenced in the present study had ORF7 sizes prototypical for EU genotype PRRSV (128 aa). In contrast, the Belarusian PRRSV strains had ORF7 protein sizes from 124 aa, the lowest ORF7 size reported so far for EU genotype PRRSV, to 130 aa, the largest ORF7 size yet reported for any arterivirus (Snijder et al., 2004) (see Supplementary Fig. S2, available in JGV Online).
In addition to the extreme ORF7 polymorphism, the Belarusian PRRSV strains also provided a rare example of variability in the otherwise highly conserved N-46 glycosylation site of GP5 (Chen et al., 2000; Wissink et al., 2004; Mateu et al., 2005). N-46 was found to be important for infectious virion production in the context of an infectious cDNA clone of Lelystad virus (Wissink et al., 2004). In contrast, we found that viruses without N-46 were relatively common in the field in Belarus. Also, we found that, in the the same virus derived from different age groups, N-46 was present in some age groups and not in others (see Bor and Zad farm sequences in Supplementary Fig. S3, available in JGV Online). In LDV, it was found that N-46 and N-53 were always present in non-neuropathogenic strains, whereas in neuropathogenic strains, N-46 was always lacking and this correlated with a higher susceptibility to antibody neutralization (Chen et al., 2000). Similarly for PRRSV, GP5 glycosylation has recently been shown to be important for antibody neutralization (Ansari et al., 2006). Thus, based on the observation of N-46 variability in pigs of different age groups (see Bor and Zad farm sequences in Supplementary Fig. S3, available in JGV Online), it could be hypothesized that PRRSV uses variability of the N-46 glycosylation site as a genetic switch to adjust immune-system interactions to the age of its host.
In an ORF5-based phylogeny between EU and US genotype viruses, including the new Belarusian sequences, the whole EU genotype cluster exhibited clearly higher diversity than the US genotype cluster (Fig. 2b). As mentioned in the introduction, PRRSV appears to have emerged independently in Europe and North America in the 1980s (Forsberg, 2005; Hanada et al., 2005). Thus, a pre-PRRS virus must be postulated to have existed in reservoir species, making species jumps into pigs triggered by global factors that acted in Europe and North America almost simultaneously. Global candidate triggering factors can easily be conceived. For example, the global emergence in the early 1980s of porcine respiratory coronavirus, which shares cell tropism with PRRSV in the pulmonary tract (Laude et al., 1993), could be hypothesized to have helped PRRSV emergence. However, there remains the question of the origin of pre-PRRSV. Most likely, pre-PRRSV existed in either Europe or North America and spread to the other continent by means of export or migration of the unknown original host, well before the species jump into pigs the 1980s. By using arguments similar to those used to track human immunodeficiency virus emergence (Mokili & Korber, 2005), it seems plausible that the ancestral population of pre-PRRSV should exhibit a larger genetic diversity than the colonist population and that this situation would be reflected in PRRSV diversity post-emergence. Thus, because of the large diversity of EU genotype viruses revealed in the present study (Fig. 2b), a European or Eurasian origin of pre-PRRSV currently seems most likely. Further indirect support for a European origin of pre-PRRSV comes from the fact that house mice have been suggested as the most likely reservoir species for pre-PRRSV (Plagemann, 2003) and these rodents colonized North America from Europe (Tichy et al., 1994). An alternative hypothesis suggested that PRRSV was spread to the USA by wild-boar imports from Europe (Plagemann, 2003).
In summary, a quite large number of recent studies have examined the genetic diversity of EU genotype PRRSV in Europe (Suarez et al., 1996; Drew et al., 1997; Le Gall et al., 1998; Indik et al., 2000, 2005; Forsberg et al., 2002; Stadejek et al., 2002; Mateu et al., 2003, 2005; Pesch et al., 2005). On this basis, the value of performing yet more molecular-phylogeny work should be questioned. However, the current study illustrates that sampling new geographical regions in Europe remains a highly worthwhile undertaking, revealing a hitherto-unsuspected diversity of EU genotype PRRSV east of Poland (Figs 1 and 2). In contrast, all known EU genotype sequences from west of the Poland/Belarus border formed a single phylogenic cluster, operationally termed subtype 1 (Figs 1 and 2). Whilst subtype 1 appears microheterogeneous by comparison with the new Belarusian sequences (Fig. 2), the diversity within the European subtype 1 is in fact at least as large as within the total body of US genotype PRRSV (Forsberg et al., 2002) (Fig. 2b) and sufficiently large to affect vaccine efficacy (Labarque et al., 2004). Because all current EU-PRRSV vaccines, as well as all current EU genotype PRRSV ELISA antigens, belong to subtype 1 (Fig. 2 and Table 1), the new subtypes of EU genotype PRRSV described in this study are relevant for vaccine and diagnostic-assay development. In short, further molecular-epidemiology studies in the far-eastern parts of Europe and in Asia would combine applied and basic scientific gains, namely knowledge for design of second-generation diagnostic assays and vaccines, as well as unravelling the emergence of PRRSV in Europe.
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ACKNOWLEDGEMENTS |
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Received 21 December 2005;
accepted 31 March 2006.
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