Download genetic diversity of american-type vaccine-derived prrs

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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