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Reprinted in the IVIS website with the permission of the meeting organizers Abstract No: P.01-04 GENETIC DIVERSITY OF AMERICAN-TYPE VACCINE-DERIVED PRRS ISOLATES IN GERMANY J Boehmer, M Homuth, K Strutzberg-Minder IVD GmbH, HANNOVER, Germany Introduction The widespread use of an american-type live attenuated vaccine for PRRS results in regular appearance of american-type PRRS positive results in routine PCRassays. These positive results are obtained from herds both with and without a history of vaccination. The aim of the present study is to genetically characterize american-type PRRS isolates from routine diagnostic samples and their relationship to the vaccine strain RespPRRSMLV and its parental strain VR-2332. Materials and Methods In our facilities routine detection of PRRSV from diagnostic samples is performed by an ORF7 PRRS-PCR discriminating between the european and american genotype. Positive samples of american- type were subjected to full length ORF5 sequencing. The amino acid sequences of ORF5 were taken to establish a phylogenetic tree by the neighbor joining method using ClustalX and TreeView© software, the alignment was constructed with BioEdit Sequence Alignment Editor© and GeneDoc© software. Results The alignment of PRRS-MLV, its parental strain VR-2332 and american-type PRRSV field isolates displayed nonsynonymous mutations predominantly in the N terminus of ORF5 and at codon 151 of routine samples. At codon 13 some of the field isolates had reverted back to the sequence of wild-type parental strain VR-2332, at codon 151 the reversion R151G is almost conserved among all isolates. Other hotspots of amino acid alterations are codon 10-16 and 24-34. Consequently phylogenetic analysis of ORF5 sequences revealed that american-type field samples clustered in groups distinct from PRRS MLV and VR-2332 strains. Figure 1 Amino acid sequence alignment of ORF5. Full length sequence of PRRS MLV is shown at top as reference. Dots indicate identities. 4 Japan 05/11498-1 05/11532-1 05/11098-3 05/10270-6 05/11096-2 05/11192-1 05/5792-1 05/6135-2 05/6135-1 VR-2332 PRRS-MLV 0.01 Figure 2 Phylogenetic tree of american-type ORF5 amino acid sequences. Sequences generated within the scope of this study are marked 05/... Discussion PRRS is caused by a virus of quasispecies character (1) with the highest evolutionary rate among RNA viruses so far reported (2). We could demonstrate here that american-type, most likely live vaccine-derived field isolates in Germany show a considerable degree of genetic plasticity within ORF5 which strikingly approves previous results including an experimental study with sequential passages of strain VR2332 in pigs (3). The N-terminal hot spots of amino acid alterations compared to PRRS MLV lie within the signal peptide and the ectodomain of ORF5 (4). Together with codon 151 codon 13 has been reported repeatedly to revert to wildtype under field conditions. These alterations represented the only two non-synonymous mutations in the ORF5 of vaccine revertants recovered from Danish swine herds with PRRS symptoms (5). Interestingly PRRS MLV (propagated on MARC cells) is not capable of replicating in primary porcine alveolar macrophages whereas vaccine revertants and VR-2332 are (6). Conserved reversion of codon 151 to wild-type has been noted just after one passage in pigs and led to the speculation it might be connected to cell tropism (3). References 1. Goldberg, T.L. et al. (2003). Virology 317, 197-207 2. Hanada, K. et al. (2005). Mol. Biol. Evol.22, 1024-1031 3. Chang, C.C. et al. (2002). J. Virol.76, 4750-4763 4. Dea, S. et al. (2000). Arch Virol 145, 659-688 5. Storgaard, T. et al. (1999). Arch Virol 144, 2389-2401 6. Botner, A. et al. (1999). Vet. Microbiol. 68, 187-195 Proceedings of the 19th IPVS Congress, Copenhagen, Denmark, 2006 · Volume 2