The 23 open reading frame (ORF) 5 sequences of Korean type II porcine reproductive and respiratory syndrome virus (PRRSV) were collected from viremic sera from the (modified live vaccine) MLV-vaccinating and non-vaccinating farms from 2007 to 2008. The samples were phylogenetically analyzed with previous ORF5 sequences, including type I Korean PRRSV, and previously reported or collected sequences from 1997 to 2008. A MN184-like subgroup of type II Korean PRRSV was newly identified in the viremic sera collected from 2007 to 2008. And of the type I PRRSVs, one subgroup had 87.2~88.9% similarity with the Lelystad virus, showing a close relationship with the 27~2003 strain of Spain. The maximum parsimony tree of type II PRRSV from 1997 to 2008 showed that they had evolved to four lineages, subgroups 1, 2, 3 and 4. Most of the recently collected type II PRRSVs belonged to subgroup 4 (48%). The region of three B-cell epitopes and two T-cell epitopes of ORF5 amino acids sequences was considerably different from the MLV in subgroups 3 and 4. In conclusion, the existence of type I PRRSV, which was genetically different from Lelystad virus (Prototype of type I PRRSV), and heterologous type II PRRSVs of viremic pigs detected even in the MLV-vaccinating farms indicated the need for new vaccine approaches for the control of PRRSV in Korea.
Animals ; Epitopes, B-Lymphocyte/immunology ; Epitopes, T-Lymphocyte/immunology ; Evolution, Molecular ; Korea ; *Open Reading Frames ; Phylogeny ; Pilot Projects ; Porcine Reproductive and Respiratory Syndrome/blood/genetics/immunology/*virology ; Porcine respiratory and reproductive syndrome virus/*genetics/immunology ; RNA, Viral/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Swine ; Viral Vaccines/immunology/standards ; Viremia/genetics/immunology/virology

To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.
Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; blood ; immunology ; Binding Sites ; genetics ; Blotting, Western ; CHO Cells ; Cell Proliferation ; Cricetinae ; Cricetulus ; Female ; Glycosylation ; Mice ; Mice, Inbred BALB C ; Mutation ; Open Reading Frames ; genetics ; Porcine Reproductive and Respiratory Syndrome ; immunology ; prevention & control ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; metabolism ; Swine ; virology ; T-Lymphocytes ; cytology ; immunology ; metabolism ; Vaccines, DNA ; administration & dosage ; immunology ; Viral Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; administration & dosage ; immunology