4 th International Symposium on Emerging <strong>and</strong> Re-emerging Pig Diseases – Rome June 29 th – July 2 nd , 2003 26
4 th International Symposium on Emerging <strong>and</strong> Re-emerging Pig Diseases – Rome June 29 th – July 2 nd , 2003 MOLECULAR EPIDEMIOLOGY OF PRRSV T. Storgaard 1 , M. B. Oleksiewicz 1 , T. Stadejek 2 , R. Forsberg 3 , H. S. Nielsen 1 , <strong>and</strong> A. Bøtner 4 . 1 Applied Trinomics, Preclinical Development, Novo Nordisk A/S, Måløv, Denmark; 2 National Veterinary Institute, Pulawy, Pol<strong>and</strong>; 3 Department of Ecology <strong>and</strong> Genetics, University of Aarhus, Aarhus, Denmark; 4 Danish Veterinary Institute, Copenhagen, Denmark. Keywords: PRRS, epidemiology, emergence, genetic diversity, molecular clock The molecular epidemiology of <strong>porcine</strong> <strong>reproductive</strong> <strong>and</strong> <strong>respiratory</strong> <strong>syndrome</strong> virus is of major interest, both from a scientific <strong>and</strong> practical point of view. The almost simultaneous emergence in North America <strong>and</strong> Europe of genetically very distinct types of PRRSV is still a major scientific puzzle. We do not know from which reservoir(s) PRRSV emerged, nor do we know what triggered the emergence of clinical disease in pigs in the late 1980'ies. As such, we have no way of evaluating the risk of future emergence of new variants of PRRSV, <strong>and</strong> likewise, no chance of proactive development of diagnostic tests <strong>and</strong> vaccines. Maybe of more immediate practical relevance, <strong>and</strong> concern, to the swine industry is the observation that the genetic diversity of both currently known PRRSV genotypes is rapidly increasing, <strong>and</strong> has done so ever since the first complete-genome sequence of PRRSV was determined almost a decade ago. This rapid growth in the genetic diversity of the viral envelope glycoproteins is likely to be associated with reduced immunological cross protection. Examples of this may already exist, with reports that the existing vaccines appear to have poor effect against the new "acute" American-genotype PRRSV isolates. Also, with radical genetic changes being reported even in the PRRSV ORF7 (nucleocapsid) protein, which was hitherto though to be well conserved, <strong>and</strong> hence is widely used for diagnostic purposes, diagnostic laboratories may in the near future have to assure that their methods have the robustness required to h<strong>and</strong>le the genetic diversity. Of even greater concern is that the ongoing genetic changes may result in PRRSV variants with fundamentally different biological properties. At the 3 rd symposium in Ploufragan, the state of the art was that high degree of genetic diversity existed for the North American-type of PRRSV, while in contrast, all European-type isolates were remarkably similar to the first European Lelystad–isolate. Several hypotheses could have explained such a scenario. For example, PRRSV could have acquired domestic pigs as a host in North America much earlier than was the case in Europe. That would have fitted with the observation that PRRSV antibodies had been detected in Canadian serum from 1979, <strong>and</strong> the fact that clinical disease was described several years earlier in North America than in Europe. However, at the symposium in Ploufragan, the first glimpse also appeared that the situation might potentially be just the opposite. An abstract described that very different European-type PRRSV isolates were found in Russia, <strong>and</strong> that these isolates apparently became gradually more different to the prototypic Lelystad isolate the further east they were obtained in Russia (1). Unfortunately, these data were never presented at the meeting in Ploufragan, nor have they to our knowledge been published or submitted to public sequence databases since then. The data were nevertheless very intriguing, <strong>and</strong> fitted nicely with the oral presentation given by Dr. Ohlinger in Ploufragan, who had found antibodies against PRRSV in East German pig sera originating from 1988. Taken together, these observations indicated the possibility of a scenario where PRRSV had gradually spread from Eastern to Western Russia over many years, then into Eastern Europe, from where it was finally transferred to Western Europe concurrent with the events surrounding the reunification of Germany. Since the Symposium in Plufragan, several studies have been published, which support a scenario like the above. It has been shown that the genetic diversity of Europeangenotype PRRSV in some European countries, for example 27 Denmark, Pol<strong>and</strong>, Spain <strong>and</strong> Italy, is just as high as what is reported for American-genotype PRRSV from North America (2, 3, 4, 5). Even more intriguing, ORF5 <strong>and</strong> ORF7 sequences of three European-genotype PRRSVs from Lithuania have been published, that were dramatically different from all previously published European-genotype PRRSV ORF5 <strong>and</strong> 7 sequences (6). So, with our current knowledge, it seems that PRRSV either acquired domestic pigs as hosts much earlier in Europe (or potentially eastern part of Russia) than was the case in North America, or alternatively, that several independent introductions of PRRSV from an unknown reservoir species into domestic pigs has taken place in Europe. Since Plufragan, we serendipitously discovered that the vast majority of the mutations identified in the ORF3 of Europeantype PRRSV appear to be neutral, <strong>and</strong> by that, accumulated by chance alone. Thus, ORF3 of European-type PRRSV can be used as a very precise molecular clock, with potential use for molecular epidemiology in the field (2, 5). For example, for European-type PRRSV transmitted from farm A to farm B, it is possible to estimate when transmission has occurred with a precision of 1-2 months, based solely on the ORF3 sequences obtained from the two farms. Likewise, the European-type ORF3 molecular clock can be used to date the introduction of European-type PRRSV into a given area or country. By phylogenetic analysis of ORF3 sequences from Danish, British, Dutch <strong>and</strong> Italian European-type PRRSV isolates, we were able to show that the most recent common ancestor for these isolates existed in 1979, more than ten years before the emergence of clinical disease in Europe. Furthermore, this analysis allowed us to estimate the rate of nucleotide substitutions to 6x10 -3 substitutions per site per year (2). This corresponds to the fastest rate of nucleotide substitutions reported for any glycoprotein of any RNA virus. It will of course be of utmost interest to obtain ORF3 sequences of the recently reported highly divergent Lithuanian isolates, to test if they also follow a molecular clock, <strong>and</strong> if so, to determine how long back in time they will push the age of the most recent common ancestor for European-type PRRSV. Unfortunately, we have not been able to detect a molecular clock in the ORF3 of North American-genotype PRRSV. Although still controversial, the literature suggests differences in the degree of virion-association between American-type <strong>and</strong> European-type ORF3 protein, <strong>and</strong> it may thus be that the selective pressures on ORF3 differ between North American-genotype <strong>and</strong> European-genotype PRRSV. Alternatively, genetic recombination could have confounded molecular clock analysis of American-genotype ORF3. We are in the process of collecting American-genotype vaccine isolates, <strong>and</strong> sequencing ORF3 from these, in order to test for a molecular clock. This work on PRRSV molecular clocks is driven by what we consider the current "holy grail" of PRRSV molecular epidemiology: dating the split between the North American <strong>and</strong> European genotypes, which should – for the first time – allow us to build a scientifically based hypothesis for the origin of PRRSV. Knowing the history <strong>and</strong> mechanisms of PRRSV evolution might not necessarily tell us the future of this virus, but it may contribute towards proactive surveillance, serodiagnostic <strong>and</strong> vaccine development schemes, akin to what is currently practiced for influenza virus.
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