Phylogenetic Analysis of PRRSV from Danish Pigs

Introduction and Objectives Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded RNA virus belonging to the Arteriviridae family. It is the causative agent of significant respiratory and reproductive disease in swine worldwide. The virus is a recently emerged pathogen, being first identified as a cause of clinical disease in 1991.

The disease spread simultaneously in North America and Europe to gain global residence in a short time-span. Two genotypes of PRRSV are currently recognized due to profound genomic and antigenic differences: PRRSV EU type and PRRSV US type, named from their geographic origin of identification. Great diversity within the two genotypes exists, and further division of PRRSV EU type into at least 3 subtypes has been suggested (Stadejek et al. 2006, 2008).

In Denmark PRRSV EU type was first identified in 1992 and a few years later the US type also was recognized. According to serologic testing, both types are prevalent in the Danish pig population. However, the genetic drift of the virus during the past 10 years has not been determined. The objective of this study was to examine the genetic diversity and evolution of PRRSV in Danish pigs by phylogenetic analysis, in order to asses the applicability of vaccines currently used to control PRRSV infection in Danish pig herds.

Materials and methods Lung tissue from samples submitted to the National Veterinary Institute during 2003-2008 for PRRSV diagnosis were screened for PRRSV by real-time RT-PCR, essentially as described by Egli et al. 2001, on RNA extracted with RNeasy Mini Kit (QIAGEN). Complete open reading frames (ORF) ORF5 and ORF7 were PCR amplified as described (Oleksiewicz et al. 1998) and sequenced.

Sequences were aligned and Neighbour-Joining trees were constructed with ClustalX. Trees were visualized with NJ-plot software. Genbank entries of Danish PRRSV sequences from the 1990’ties were included in the phylogenetic analysis. Translated sequences were aligned with current vaccine isolates. Results Both PRRSV EU and US type viruses were isolated from material submitted from Danish pigs in the period 2003 to 2008.

Sequences were obtained from 14 viruses isolated from different herds. There was substantial sequence diversity within both types of viruses. All Danish PRRSV type EU viruses grouped with subtype EU1 viruses. Amino acid alignments of translated sequences showed that the protein sequences were highly conserved and match the vaccine strains without differences in predicted epitope regions of ORF5 and ORF7 proteins.

Discussion and conclusions PRRSV of both EU and US types currently are co-circulating in the Danish pig population. The viruses are diverse within both groups with a slightly higher degree of diversity within the EU type group of viruses. However, for both types, sequences match the corresponding vaccine strains.

Importantly, all viruses of the EU type group with subtype EU1 viruses. Subtype EU1 contains viruses from Asia and Europe, whereas the other subtypes represent viruses from East Europe only (Stadejek et al. 2006). Introduction of new subtype viruses or drift within the present viruses could potentially affect control of PRRSV infection.

Diagnostic procedures could be impaired if mutations were in primer binding sites or if they caused changes in antigenicity of viruses. Antigenic differences between EU subtypes have been demonstrated (Stadejek et al. 2008). PRRSV is a very diverse virus with a high mutation rate. It is therefore extremely important to continuously monitor and sequence the virus.

The present data suggests, that the PRRSV vaccines used for the moment are adequate for control of PRRSV infection in the Danish pig population. References 1. Egli et al. 2001 J.Vir.Methods 98:63-75. 2. Oleksiewicz et al. 1998, Vet.Microbiol. 64:7-22. 3. Stadejek et al. 2006, J.Gen.Vir. 87:1835-41. 4.

Stadejek et al. 2008, Arch.Virol. 153:1479-88.